Epimagnolin A, a tetrahydrofurofuranoid lignan from Magnolia fargesii, reverses ABCB1-mediated drug resistance.

BACKGROUND: Epimagnolin A is an ingredient of the Chinese crude drug Shin-i, derived from the dried flower buds of Magnolia fargesii and Magnolia flos, which has been traditionally used for the treatment of allergic rhinitis and nasal congestion, empyema, and sinusitis. The pharmacokinetic activity of epimagnolin A remains to be evaluated. PURPOSE: In this study, we examined the possible interactions of epimagnolin A with human ATP-binding cassette (ABC) transporter ABCB1, a membrane protein vital in regulating the pharmacokinetics of drugs and xenobiotics. STUDY DESIGN/METHODS: The interaction of epimagnolin A with ABCB1 was evaluated in calcein, ATPase, and MTT assays by using Flp-In-293/ABCB1 cells and purified ABCB1 and simulated in molecular docking studies. RESULTS: Epimagnolin A inhibited calcein export by Flp-In-293/ABCB1 cells in a concentration-dependent manner in a calcein assay. ATPase assay revealed a concentration-dependent stimulation of the ATPase activity of ABCB1 by epimagnolin A. Epimagnolin A also showed saturation kinetics in the relationship between the compound-stimulated ATPase activity and the compound concentration, suggesting Michaelis-Menten kinetics similar to those of the control drug, verapamil. Km and Vmax values were calculated from Hanes-Woolf plots of (compound concentration) × (compound-stimulated ATPase activity)-1 vs. (compound concentration); the Km of epimagnolin and verapamil was 42.9 ±â€¯7.53  µM and 12.3 ±â€¯4.79  µM, respectively, and the corresponding Vmax values were 156 ±â€¯15.0  µM and 109 ±â€¯3.18  µM. Molecular docking studies on human ABCB1 showed that epimagnolin A docked to the same binding pocket as verapamil, and 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays showed that the sensitivities of Flp-In-293/ABCB1 cells against anti-cancer drugs were enhanced upon exposure to 10  µM epimagnolin A. CONCLUSION: These results strongly suggest that epimagnolin A affects the transport activity of ABCB1 as a substrate.

Acerogenin C from Acer nikoense exhibits a neuroprotective effect in mouse hippocampal HT22 cell lines through the upregulation of Nrf-2/HO-1 signaling pathways.

Oxidative stress contributes to neuronal death in the brain, and neuronal death can cause aging or neurodegenerative disease. Heme oxygenase 1 (HO-1) serves a vital role in the regulation of biological reactions, including oxidative stress associated with reactive oxygen species. In the present study, acerogenin C isolated from the Aceraceae plant Acer nikoense, which is used as a Japanese folk medicine for hepatic disorders and eye diseases. However, there have been no studies on the mechanisms underlying the antineurodegenerative biological activities of acerogenin C. In the present study, acerogenin C demonstrated neuroprotective action against glutamate­induced cell death in hippocampal HT22 cells through the upregulation of HO­1 expression. These effects were also associated with nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the activation of phosphoinositide 3­kinase/protein kinase B. Taken together of the efficacy researches, this study determines that the Nrf2/HO­1 pathways denotes a biological mark and that acerogenin C might contribute to prevention of neurodegenerative disorders.

Anti-oxidant and anti-adipogenic effects of acorn (Quercus acutissima CARR.) shell extracts via regulation of wnt signaling in 3T3-L1 cells.

Acorn (Quercus acutissima CARR.) is a nut from the Fagaceae family that has been used in traditional medicine for many years. However, shells from acorns are regarded as a by-product and are mostly discarded. Anti-adipogenic activities of acorn shells were investigated using 3T3-L1 cells and methanol shell extracts (AE-M). AE-M demonstrated Cu2+-chelation activities and anti-oxidant activities via reduction of oxidative stress levels induced using AAPH. Six days after adipocyte differentiation, 50 and 100 µg/mL AE-M completely suppressed 3T3-L1 adipogenesis and the anti-adipogenic effect was stronger than for the positive control 50 µM quercetin. Treatment with AE-M in 3T3-L1 cells reduced mRNA expression levels of adipogenic genes. AE-M-inhibition was found in pre-adipogenic, early, and intermediate stages of adipogenesis in 3T3-L1 cells. The Wnt/ß-catenin signaling pathway is required for AE-M-inhibition of 3T3-L1 adipogenesis.

Acerogenin A from Acer nikoense Maxim Prevents Oxidative Stress-Induced Neuronal Cell Death through Nrf2-Mediated Heme Oxygenase-1 Expression in Mouse Hippocampal HT22 Cell Line.

Oxidative cell damage contributes to neuronal degeneration in many central nervous system (CNS) diseases such as Parkinson's disease, Alzheimer's disease, and ischemia. Inducible heme oxygenase (HO)-1 acts against oxidants that are thought to play a key role in the pathogenesis of neuronal diseases. The stem bark of Acer nikoense Maxim (Aceraceae) is indigenous to Japan; it has been used in folk medicine as a treatment of hepatic disorders and eye diseases. Acerogenin A, a natural compound isolated from Japanese folk medicine A. nikoense, showed neuroprotective effects and reactive oxygen species (ROS) reduction on glutamate-induced neurotoxicity by inducing the expression of HO-1 in mouse hippocampal HT22 cells. Furthermore, acerogenin A caused the nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) and the activation of the PI3K/AKT signaling pathways. In this study, we demonstrated that acerogenin A effectively prevents glutamate-induced oxidative damage, and HO-1 induction via PI3K/Akt and Nrf2 pathways appears to play a key role in the protection of HT22 cells. Therefore, this study implies that the Nrf2/HO-1 pathway represents a biological target and that acerogenin A might be a candidate for the prevention of neurodegeneration.

Long-term supplementation of honokiol and magnolol ameliorates body fat accumulation, insulin resistance, and adipose inflammation in high-fat fed mice.

SCOPE: This study investigated the effect of honokiol (HON) and magnolol (MAG), phenolic compounds in Magnolia plants, on adiposity and adiposity-related metabolic disturbances in mice fed high-fat diet (HFD), and the potential underlying mechanisms focusing on the lipid metabolism and inflammatory response. METHOD AND RESULTS: C57BL/6J mice were fed HFD (45 kcal% fat) with or without HON (0.02%, w/w) or MAG (0.02%, w/w) for 16 wk. Despite no changes in body weight, food intake, and hepatic fat accumulation, HON and MAG significantly lowered the weight of white adipose tissue (WAT) as well as adipocyte size and protected against insulin resistance induced by HFD. These effects were associated with increases in energy expenditure and adipose fatty acid oxidation and decreases in fatty acid synthase activity and expression of genes related to fatty acid synthesis, desaturation, and uptake, as well as adipocyte differentiation in WAT. Moreover, HON and MAG significantly lowered the expression of proinflammatory genes in WAT and elevated the plasma IL-10 level. Particularly, HON significantly decreased the plasma resistin level and increased the plasma adiponectin level compared to the control group. CONCLUSION: HON and MAG have potential as novel agents for amelioration of adiposity and associated insulin resistance and inflammation.

Dehydroglyasperin C, a component of liquorice, attenuates proliferation and migration induced by platelet-derived growth factor in human arterial smooth muscle cells.

Liquorice is one of the botanicals used frequently as a traditional medicine in the West and in the East. Platelet-derived growth factor (PDGF)-BB is involved in the development of CVD by inducing abnormal proliferation and migration of vascular smooth muscle cells. In our preliminary study, dehydroglyasperin C (DGC), an active compound of liquorice, showed strong antioxidant activity. Since phytochemicals with antioxidant activities showed beneficial effects on chronic inflammatory diseases, the present study aimed to investigate the effects of DGC on PDGF-induced proliferation and migration of human aortic smooth muscle cells (HASMC). Treatment of HASMC with DGC for 24 h significantly decreased PDGF-induced cell number and DNA synthesis in a dose-dependent manner without any cytotoxicity, as demonstrated by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide test and thymidine incorporation. Upon cell cycle analysis, DGC blocked the PDGF-induced progression through the G0/G1 to S phase of the cell cycle, and down-regulated the expression of cyclin-dependent kinase (CDK); 2, cyclin E, CDK4 and cyclin D1. Furthermore, DGC significantly attenuated PDGF-stimulated phosphorylation of PDGF receptor-b, phospholipase C-g1, AKT and extracellular-regulated kinase 1/2, and DGC inhibited cell migration and the dissociation of actin filaments by PDGF. In a rat vascular balloon injury model, DGC suppressed an excessive reduction in luminal diameters and neointimal formation compared with the control group. These results demonstrate the mechanistic basis for the prevention of CVD and the potential therapeutic properties of DGC.

Fargesin, a component of Flos Magnoliae, stimulates glucose uptake in L6 myotubes.

Flos Magnoliae (FM) is a commonly used Chinese medicinal herb for symptomatic relief of allergic rhinitis, sinusitis and headache. Although several FM species have been used as substitutes or adulterants for clinical use, possible differences in their pharmacological actions have not been reported. To confirm the effects of FM on skeletal muscle glucose metabolism, we tested the effects of several compounds isolated from FM on glucose uptake by L6 myotubes. We found that fargesin, a component of FM, dose-dependently stimulated glucose consumption in L6 myotubes, which was accompanied by enhanced glucose transporter (GLUT)-4 translocation to the cell surface. Fargesin-stimulated glucose uptake was blocked by wortmannin, a phosphatidylinositol-3 kinase (PI3 K) inhibitor. Fargesin stimulated Akt phosphorylation, a key component in the insulin signaling pathway, which was completely inhibited by wortmannin. Here, we demonstrated that fargesin, a bioactive component of Flos Magnoliae, increases basal glucose uptake and GLUT4 translocation in L6 myotubes by activating the PI3 K-Akt pathway.

Honokiol and magnolol stimulate glucose uptake by activating PI3K-dependent Akt in L6 myotubes.

Honokiol and magnolol, ingredients of Magnolia officinalis, which is used in traditional Chinese and Japanese medicines, have been reported to have antioxidant, anticancer, and antiangiogenic effects. Effects of these compounds on glucose metabolism in adipocytes have also been reported. However, their effects on skeletal muscle glucose uptake and the underlying molecular mechanisms are still unknown. Here, we investigated the direct effects and signaling pathways activated by honokiol and magnolol in skeletal muscle cells using L6 myotubes. We found that honokiol and magnolol dose-dependently acutely stimulated glucose uptake without synergistic effects of combined administration in L6 myotubes. Treatment with honokiol and magnolol also stimulated glucose transporter-4 translocation to the cell surface. Honokiol- and magnolol-stimulated glucose uptake was blocked by the phosphatidylinositol-3 kinase inhibitor, wortmannin. Both honokiol and magnolol stimulated Akt phosphorylation, a key element in the insulin signaling pathway, which was completely inhibited by wortmannin. These results suggest that honokiol and magnolol might have beneficial effects on glucose metabolism by activating the insulin signaling pathway.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells.

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 µM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Honokiol attenuates vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway in rat aortic rings.

OBJECTIVES: Honokiol is a small-molecule polyphenol isolated from the species Magnolia obovata. We hypothesized that honokiol attenuated vascular contractions through the inhibition of the RhoA/Rho-kinase signalling pathway. METHODS: Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. Phosphorylation of 20kDa myosin light chains (MLC(20) ), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase (MLCP) of 17kDa (CPI17) were examined by immunoblot. We also measured the amount of guanosine triphosphate RhoA as a marker for RhoA activation. KEY FINDINGS: Pretreatment with honokiol dose-dependently inhibited the concentration-response curves in response to sodium fluoride (NaF) or thromboxane A(2) agonist U46619. Honokiol decreased the phosphorylation levels of MLC(20) , MYPT1(Thr855) and CPI17(Thr38) as well as the activation of RhoA induced by 8.0mm NaF or 30nm U46619. CONCLUSIONS: These results demonstrated that honokiol attenuated vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway.

Osteogenic activity of diphenyl ether-type cyclic diarylheptanoids derived from Acer nikoense.

Osteogenic activity of six diarylheptanoids, acerogenin A (1), (R)-acerogenin B (2), aceroside I (3), aceroside B(1) (4), aceroside III (5) and (-)-centrolobol (6) and two phenolic compounds; (+)-rhododendrol (7) and (+)-cathechin (8), isolated from the stem bark of Acer nikoense (Nikko maple) was evaluated using alkaline phosphatase (ALP) activity as a marker for early osteoblast differentiation. We found that the diphenyl ether-type cyclic diarylheptanoids 1-5 promoted ALP activity in mouse preosteoblastic MC3T3-E1 cells without affecting cell proliferation, but linear-type diarylheptanoid 6 and phenolic compounds 7 and 8 did not. Diphenyl ether-type cyclic diarylheptanoids 1-4 also increased protein production of osteocalcin, a late stage maker for osteoblast differentiation, and induced osteoblastic mineralization. Structure-activity relationships of these compounds demonstrated that the stimulative efficacy of aglycones was higher than that of its glycosides. Taken together, diphenyl ether-type cyclic diarylheptanoids promote early- and late-stage osteoblastogenesis, which may open the possibility for the development of novel osteogenic agents.

Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes.

Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10 µM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.

Effects of a Citrus depressa Hayata (shiikuwasa) extract on obesity in high-fat diet-induced obese mice.

Citrus depressa Hayata (commonly known as shiikuwasa) is cultivated in the northern areas of Okinawa, Japan, and used as a juice. In this study, we examined the anti-obesity effects and mechanism of action of shiikuwasa peel extract (SE) using high-fat diet (HFD)-induced obese mice. Mice were fed a low-fat diet (LFD), HFD or HFD containing 1% or 1.5% (w/w) SE (HFD+1 SE and HFD+1.5 SE, respectively) for 5 weeks. The body weight gain and white adipose tissue weight were significantly decreased in the HFD+1.5 SE group compared with the HFD group. The plasma triglyceride and leptin levels were also significantly reduced in the HFD+1.5 SE group compared with the HFD group. Histological examinations showed that the sizes of the adipocytes were significantly smaller in the HFD+1.5 SE group than in the HFD group. The HFD+1.5 SE group also showed significantly lower mRNA levels of lipogenesis-related genes, such as activating protein 2, stearoyl-CoA desaturase 1, acetyl-CoA-carboxylase 1, fatty acid transport protein and diacylglycerol acyltransferase 1, than the HFD group. These results suggest that the anti-obesity effects of SE may be elicited by regulating the expressions of lipogenesis-related genes in white adipose tissue.

Harmine, a ß-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo.

Bone homeostasis is controlled by the balance between osteoblastic bone formation and osteoclastic bone resorption. Excessive bone resorption is involved in the pathogenesis of bone-related disorders such as osteoporosis, arthritis and periodontitis. To obtain new antiresorptive agents, we searched for natural compounds that can inhibit osteoclast differentiation and function. We found that harmine, a ß-carboline alkaloid, inhibited multinucleated osteoclast formation induced by receptor activator of nuclear factor-κB ligand (RANKL) in RAW264.7 cells. Similar results were obtained in cultures of bone marrow macrophages supplemented with macrophage colony-stimulating factor and RANKL, as well as in cocultures of bone marrow cells and osteoblastic UAMS-32 cells in the presence of vitamin D(3) and prostaglandin E(2). Furthermore, harmine prevented RANKL-induced bone resorption in both cell and bone tissue cultures. Treatment with harmine (10 mg/kg/day) also prevented bone loss in ovariectomized osteoporosis model mice. Structure-activity relationship studies showed that the C3-C4 double bond and 7-methoxy group of harmine are important for its inhibitory activity on osteoclast differentiation. In mechanistic studies, we found that harmine inhibited the RANKL-induced expression of c-Fos and subsequent expression of nuclear factor of activated T cells (NFAT) c1, which is a master regulator of osteoclastogenesis. However, harmine did not affect early signaling molecules such as ERK, p38 MAPK and IκBα. These results indicate that harmine inhibits osteoclast formation via downregulation of c-Fos and NFATc1 induced by RANKL and represses bone resorption. These novel findings may be useful for the treatment of bone-destructive diseases.

Palmatine attenuates osteoclast differentiation and function through inhibition of receptor activator of nuclear factor-κb ligand expression in osteoblast cells.

Osteoclasts are the only cell type capable of resorbing mineralized bone, and they act under the control of numerous cytokines produced by supporting cells such as osteoblasts and stromal cells. Among cytokines, receptor activator of nuclear factor-κB ligand (RANKL) was found to be a key osteoclastogenetic molecule that directly binds to its cognate receptor, RANK, on osteoclast precursor cells. In turn, RANKL, which is an essential factor for differentiation and activation of osteoclasts, is one of the major targets of anti-resorptive agents. In this study, we found that palmatine, an isoquinoline alkaloid originally isolated from Coptis chinensis, had an inhibitory effect on osteoclast differentiation and function in vitro. Palmatine inhibited osteoclast formation in the co-culture system with mouse bone marrow cells (BMC) and osteoblasts in the presence of 10 nM 1α,25-(OH)(2)D(3). Palmatine did not affect osteoclast formation induced by RANKL in the BMC cultures. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that palmatine significantly inhibited the expression of 1α,25-(OH)(2)D(3)-induced expression of RANKL mRNAs in stromal cells without loss of cell viability. Moreover, palmatine suppressed resorption pit formation by mature osteoclasts on dentin slices and induced disruption of actin ring formation in mature osteoclasts with an impact on cell viability. Taken together, these results suggest that palmatine attenuates osteoclast differentiation through inhibition of RANKL expression in osteoblast cells, and its inhibitory effect on bone resorption is due to its disruptive effect on actin rings in mature osteoclasts. Therefore, palmatine might be an ideal candidate as an anti-resorptive agent for the prevention and treatment of bone disorders such as osteoporosis.

Honokiol inhibits osteoclast differentiation and function in vitro.

Honokiol, a neolignan, is a physiologically active component of kouboku (Magnolia obovata), a herb used in traditional Chinese medicine. This study investigated the effects of honokiol on the differentiation and function of osteoclasts induced by receptor activator of nuclear factor-kappaB ligand (RANKL). Honokiol markedly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP-positive multinucleated cells in both bone marrow-derived monocytes and RAW264 cells. In experiments to elucidate its mechanism of action, honokiol was found to suppress RANKL-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The RANKL-induced expressions of c-Fos and nuclear factor of activated T cells-c1 (NFATc1), which are crucial transcriptional factors for osteoclastogenesis, were also reduced by treatment with honokiol. Furthermore, honokiol induced disruption of the actin rings in mature osteoclasts (mOCs) without affecting the cell viability and suppressed osteoclastic pit formation on dentin slices. Taken together, these results suggest that honokiol inhibits osteoclast differentiation by suppressing the activation of MAPKs (p38 MAPK, ERK and JNK), decreasing the expressions of c-Fos and NFATc1, and attenuates bone resorption by disrupting the actin rings in mOCs. Therefore, honokiol could prove useful for the treatment of bone diseases associated with excessive bone resorption.

Large-scale and effective screening of Korean medicinal plants for inhibitory activity on matrix metalloproteinase-9.

Matrix metalloproteinase-9 (MMP-9) degrades type IV collagen constituting the major structural component of the basement membrane and extra cellular membrane. The enzymatic activity is found to be elevated in tumor tissues. With the aim of finding novel MMP-9 inhibitors from natural products, 87 extracts of oriental medicinal herbs, which are used as prescriptions for cancer treatment in traditional Korean medicine, were screened for their inhibitory activities towards MMP-9. It was found that most of the hexane and chloroform fractions as well as water extracts showed a weak inhibitory effect on MMP-9 activity at a concentration of 100mug/ml. However, a strong inhibition was found in the butanol fractions of Cinnamomum cassia PRESL, Magnolia obovata THUEB., Magnolia officinalis REHD. et WILS., Magnolia officinalis REHD. et WILS. var. biloba REHD. et WILS., and Euonymus alatus (THUNB.) SIEB. with inhibitory activity (>90%) at a concentration of 100 microg/ml.

Inhibitory effect of methanol extract of Euonymus alatus on matrix metalloproteinase-9.

Various extracts prepared from stems of Euonymus alatus were tested for cytotoxic activity on human hepatocellular carcinoma cell line, Hep3B cells using the XTT assay method. Also, the extracts were investigated the inhibitory effects on matrix metalloproteinase (MMP)-9 activity using gelatin zymography. The methanol extract, hexane and ethyl acetate fraction exhibited weak cytotoxic activity (IC(50) of >100 microg/ml). However, butanol (IC(50)=65 microg/ml) and chloroform (IC(50)=85 microg/ml) fraction exhibited strongly cytotoxic activity. Gelatin zymography showed that the Hep3B cells secreted matrix metalloproteinase (MMP), probably including MMP-9, which may be involved in tumor cell invasion and metastasis. The butanol fraction showed stronger inhibitory effect of proteolytic activity than other fractions. Also, the butanol fraction was able to decrease the proteolytic activity of MMP-9 in a concentration-dependent manner on zymography. These results suggest that the butanol fraction from E. alatus has highly inhibitory effect on MMP-9 in comparatively low cytotoxicity.