Elsevier

Life Sciences

Volume 77, Issue 22, 14 October 2005, Pages 2760-2769
Life Sciences

A phenolic compound, 5-caffeoylquinic acid (chlorogenic acid), is a new type and strong matrix metalloproteinase-9 inhibitor: Isolation and identification from methanol extract of Euonymus alatus

https://doi.org/10.1016/j.lfs.2005.02.028Get rights and content

Abstract

A phenolic compound responsible for anti-MMP-9, which is known to be involved in tumor cell invasion and metastasis, has been isolated from methanol extracts prepared from stem barks of Euonymus alatus by assay-guided fractionation. The compound has been identified as 5-caffeoylquinic acid (chlorogenic acid; CHA) by NMR and FAB-MS. CHA showed a strong inhibitory effect of matrix metalloproteinase (MMP)-9 activity in a concentration-dependent manner on zymography. The purified CHA inhibited MMP-9 activity with the IC50 of 30–50 nM. Furthermore, the cytotoxic survival curve showed that CHA does not have cytotoxic effects on cellular proliferation, when Hep3B cells were treated with various concentrations of CHA and cell viability was measured using the XTT assay. The present data suggest a clue for possible mechanisms of cancer chemoprevention by CHA and other naturally occurring phenolic compounds. The results also imply that useful cancer chemopreventive agents can be further identified by combinations of in vitro (as a first screen) and in vivo studies.

Introduction

It was known that angiogenic enzymes (matrix metalloproteinase (MMP)-9) are directly involved in human hepatic tumorigenesis and metastasis (Chung et al., 2004a, Chung et al., 2004b). Invasion and metastasis are fundamental properties of malignant cancer cells. A number of proteolytic enzymes participate in these processes to degrade environmental barriers such as extracellular matrix (ECM) and basement membrane (Chung et al., 2004a, Chung et al., 2004b). The MMP family has been shown to play an important role in the proteolysis of various components of the ECM. MMP-9 of the MMP family has been known to degrade type IV collagen, which is a major constituent of the basement membrane in cancer invasion and metastasis (Basset et al., 1997, Nelson et al., 2000). In addition, our previous results clearly showed that the expression of MMP-9 may be associated with progression and invasion of human hepatocarcinoma cells (Chung et al., 2002, Chung et al., 2004b). These observations have led to the development of MMP-9 inhibitors to prevent tumor metastasis. Several novel MMP-9 inhibitors have been developed and are currently being investigated in clinical trials (Sugita, 1999, Wojtowicz-Praga, 1999). Since some broad-spectrum MMP inhibitors (i.e., Batimastat/BB-94 and Marimastat/BB-2516) may have serious muscloskeletal side effects such as arthralgia, myalgia, stiffness, and edema (Brown, 2000), selective (narrow-spectrum) MMP-9 inhibitors, such as BAY12-9566 (Gatto et al., 1999), KB-R7785 (Lozonschi et al., 1999), N-biphenyl sulfonyl-phenylalanine hydroxyamic acid (BPHA) (Tamura et al., 1998, Maekawa et al., 1999), and MMI-166 (Katori et al., 2002) have been developed to reduce the risk of such side effects.

It has been shown that a number of these phenolics act as antioxidants, with plural mechanisms involving free radical scavenging and metal ion chelation (Terao et al., 1993, Rice-Evans et al., 1995, Meyer et al., 1998). Their antioxidative action could prevent oxidative damages in vivo, relating to various diseases. For another activity, anticancer properties have been associated with components of various natural products, including green tea polyphenols, resveratrol, limonene, and organosulfur compounds from garlic (Kaegi, 1998). Especially, green tea polyphenols and one of its constituents (epigallocatechin gallate, EGCG) caused a strong inhibition of the gelatinolytic activity of MMP-9 (Demeule et al., 2000, Chung et al., 2004a, Chung et al., 2004b). Previously, it was suggested that hydroxycinnamic acids inhibit carcinogenesis and increase the antioxidant defense system in vivo (Tanaka et al., 1993, Nardini et al., 1997). For chlorogenic acid (5-caffeoylquinic acid), inhibitory effects on carcinogenesis in the large intestine, liver, and tongue, and protective effects on oxidative stress in vivo have been reported (Mori et al., 1986, Tanaka et al., 1990, Tanaka et al., 1993, Tsuchiya et al., 1996, Azuma et al., 2000).

The stems of Euonymus alatus (Thunb.) Siebold, commonly known as winged Euonymus, have been used in traditional medicine for cancer treatment (Lee et al., 1993). Previous phytochemical and biological studies on E. alatus have resulted in the isolation of cardenolides (Kitanaka et al., 1996). However, so far, no specific anti-metastatic molecules from E. alatus crude extract have been demonstrated. In our previous studies, we investigated the inhibitory activity of MMP-9 by the methanol extract containing several fractions, and caffeic acid has been isolated as a selective MMP-9 inhibitor from E. alatus (Cha et al., 2003, Chung et al., 2004b).

In this study, we have isolated CHA as another selective MMP-9 without cytotoxicity inhibitors from E. alatus. However, the direct inhibitory effect of CHA on MMP-9 catalytic activity has not been reported yet. Hence, our present study is attempted for the first time to show whether CHA has a strong MMP-9 inhibitory activity.

Section snippets

Materials

(−)-Epigallocatechin gallate (EGCG) and the tissue inhibitor of metalloproteinase-1 (TIMP-1) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Chlorogenic acid was purchased from Nacarai Tesque Co. (Kyoto, Japan) for comparison with the purified CHA. Other chemicals were of analytical or HPLC grade.

Cell culture

A human hepatocellular carcinoma cell line (Hep3B) was obtained from the Korean cell line bank and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, USA) containing 10%

FAB-MS and NMR

The FAB-MS data were obtained with a JEOL JMS-SX102A system. 1H and 13C NMR, 1H–1H COSY, 1H–13C COSY, and HMBC data were obtained with JEOL JNM-LA500 and JNM-EX270 systems in DMSO-d6 at 35 °C.

Identification of phenolic compound CHA from E. alatus

In a preliminary study, MeOH extract from E. alatus showed the strong MMP-9 inhibitory activity (IC50 = 8.0 μg/ml) in a concentration-dependent manner when compared to the established MMP-9 inhibitor such as EGCG. Therefore, to show the compound responsible for this inhibitory activity, in the present study, using HPLC and FAB-MS and NMR, an active phenolic compound was obtained from the acidic n-BuOH fraction of the MeOH extract. FAB-MS: m/z 354: 1H NMR (270 MHz, DMSO-d6): δ 7.42(1H, d, J = 16 Hz,

Discussion

Anticancer properties have been associated with components of various natural products including green tea polyphenols, resveratrol, limonene, and organosulfur compounds from garlic. Especially, green tea polyphenols and one of its constituents (EGCG) caused a strong inhibition of the MMP-9 activity (Demeule et al., 2000). Understanding the molecular mechanisms by which the major compound from winged Euonymus interacts with and inhibits these enzymatic activities, thus restraining matrix

Acknowledgments

This work was supported by the Dongguk University Research Fund and NRL Program, KOSEF, Korean Government (2004–2007).

References (44)

  • P.D. Brown

    Ongoing trials with matrix metalloproteinase inhibitors

    Expert Opinion on Investigational Drugs

    (2000)
  • J. Buckingham

    Dictionary of Organic Compounds

    (1984)
  • N.S. Choi et al.

    Comparison of three substrates (casein, fibrin, and gelatin) in zymogram

    Journal of Biochemistry and Molecular Biology

    (2001)
  • Y. Chuda et al.

    Contents and cooking loss of three quiinic acid derivatives from garland (Chrysanthemun corronarium L.)

    Journal of Agricultural and Food Chemistry

    (1998)
  • T.W. Chung et al.

    Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential

    FASEB Journal

    (2004)
  • T.W. Chung et al.

    Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism

    FASEB Journal

    (2004)
  • M.N. Clifford

    Chlorogenic acids

  • J.L. Donovan et al.

    Phenolic composition and antioxidant activity of prunes and prune juice (Prunus domestica)

    Journal of Agricultural and Food Chemistry

    (1998)
  • C. Gatto et al.

    BAY 12-9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity

    Clinical Cancer Research

    (1999)
  • F. Hayase et al.

    Antioxidative components of sweet potatoes

    Journal of Nutritional Science and Vitaminology

    (1984)
  • K. Herrmann

    Occurrence and content of hydroxycinnamic and hydroxybenzoic acid compounds in foods

    Critical Reviews in Food Science and Nutrition

    (1989)
  • Y. Jiang et al.

    Inhibition of chlorogenic acid-induced cytotoxicity by CoCl2

    Anticancer Research

    (2001)
  • Cited by (201)

    • Microwave-assisted polyethylene glycol-based aqueous two-phase extraction of gallic acid and ellagic acid from Euonymus alatus: Process optimization, quantification analysis and antioxidant activity

      2022, Chemical Engineering and Processing - Process Intensification
      Citation Excerpt :

      Modern pharmacological researches have demonstrated its outstanding curative effect in anti-hyperglycemia, anti-inflammation and immune-modulating due to containing biological components such as flavonoids, sesquiterpenes, phenolic acids and alkaloids [6–8]. For example, the phenolic compounds in the methanolic extracts of Euonymus alatus were responsible for effective inhibitory activity on matrix metalloproteinase (MMP)−9 and NO production [9–11]. Influentially, gallic acid and ellagic acid as main phenolic compounds in Euonymus alatus have exhibited pharmaceutical effects of antibacterial, anti-inflammatory and anti-apoptotic [12].

    View all citing articles on Scopus
    1

    These two authors have equally contributed to the study.

    View full text