Inhibition of Neutrophil Elastase Activity by Cinnamic Acid Derivatives from Cimicifuga racemosa

• Abstract
• Materials and Methods
• References

Abstract

Caffeic acid, fukinolic acid as well as cimicifugic acids A, B, E and F isolated from the rhizomes of Cimicifuga racemosa (Ranunculaceae) inhibited the activity of neutrophil elastase (EC 3.4.21.37) in a dose-dependent manner. An IC₅₀ of 93 μmol/L was determined for caffeic acid and 0.23 μmol/L for fukinolic acid. Cimicifugic acid A inhibited the enzyme with an IC₅₀ of 2.2 μmol/L, cimicifugic acid B with 11.4 μmol/L, and cimicifugic acid F with 18 μmol/L. Cimicifugic acid E was only a very weak inhibitor.

The inflammatory process is connected with the release of proteinases from neutrophils and macrophages which then play an important role in the pathogenesis of inflammatory disorders. The enzymes are responsible for the neutrophil-mediated degradation of cartilage matrix proteoglycan [1]. The two main proteinases of neutrophils are elastase and cathepsin G. They are localized in the azurophil granules, together with proteinase 3 and further acidic cathepsins [2]. The neutrophil elastase contributes to the destruction of basement membranes during inflammation and is directed to the degradation of type IV procollagen [3]. One typical feature of active inflammation is an elevated plasma level of neutrophil elastase [4].

It was shown recently that caffeic acid (1, Fig. [1]) and some of its monoterpenyl esters inhibit the activity of neutrophil elastase [5]. Also recently, some rare cinnamic acid esters 3 - 7, Fig. [1]) have been isolated from the rhizomes of Cimicifuga simplex Wormsk. with inhibitory activity on α-amylase and carboxypeptidase A [6] and from the rhizomes of C. racemosa (L.) Nutt. [7] with estrogenic activity for fukinolic acid. Fukinolic acid (3) was first isolated from Petasites japonicus Maxim. [8], and later together with the cimicifugic acids (4 - 7) from C. simplex [6] and C. racemosa [7]. Fukinolic acid (3) and cimicifugic acids A and B (4, 5) possess fukiic acid, cimicifugic acid E and F (6, 7) have piscidic acid as the dicarboxylic acid moiety (Fig. [1]).

Here we describe the ability of the cinnamic acid esters fukinolic acid (3) and the cimicifugic acids (4 - 7) to influence the activity of neutrophil elastase in an in vitro assay with human leukocyte elastase; the free acids, caffeic acid (1) and isoferulic acid (2) and a synthetic peptide, elastatinal, known as selective inhibitor of the enzyme [9], were used for comparison. Figure [1] shows that the free acids as well as the esters inhibited the enzyme activity but to a very different extent. Whereas caffeic acid (1) inhibited the enzyme with an IC₅₀ of 16 μg/ml (93 μM), fukinolic acid (3) was a much stronger inhibitor in the assay with an IC₅₀ of 0.1 μg/ml (0.23 μM). The cimicifugic acids also inhibited the enzyme, especially cimicifugic acid A (4) with an IC₅₀ of 2.2 μM and cimicifugic acid B (5) with an IC₅₀ of 11.4 μM, respectively. Cimicifugic acid F (6) was a less active inhibitor with an IC₅₀ of 18 μM, whereas cimicifugic acid E (7) inhibited the elastase activity only very weakly (approximately 20 % inhibition at 50 μM). Isoferulic acid, however, showed an IC₅₀ of > 50 μM whereas ferulic acid was clearly less active with 6 % inhibition at 500 μmol/L (IC₅₀ >> 500 μM). The peptidic inhibitor of the elastase activity elastatinal inhibited the enzyme activity only weakly with an IC₅₀ of 592 μg/ml (1150 μM).

Because of the importance of neutrophil elastase in the inflammatory process and the strong inhibition of the enzyme activity, in particular, by fukinolic acid, this compound seems to be a powerful anti-inflammatory agent. By that it might participate in the therapeutic effect of extracts obtained from the rhizomes of Cimicifuga spec. which are frequently used as anti-inflammatory drug in the Chinese and Japanese Oriental medicine [10], [11]; in addition, extracts of the rhizomes of C. racemosa (black cohosh) are traditionally used in North America against rheumatism [12]. It seems that esters with less than two unsubstituted catechol units (4, 5) have reduced inhibitory activity in comparison to those with two free catechol moieties (3), and that elimination of a hydroxy group reduces the inhibitory activity of the esters more than methylation of the hydroxy groups; compounds without an unsubstituted catechol unit (e.g., 7) show very weak activity. On the other hand, larger molecules such as the esters seem to be necessary instead of the relatively small free cinnamic acids (1, 2) in order to bind to the enzyme [also cf. 5]. A possible explanation of the fact that the caffeic ester fukinolic acid inhibited the neutrophil elastase more strongly than the pure caffeic acid itself might be connected with the suggestion that the ester can easier interact with the active site of the enzyme localized not on the surface of the protein but in regions which require a high lipophilicity for interaction.

The frequent usage of medicinal plants with a variety of caffeic acid esters for the treatment of wounds on the body surface and inflammatory disorders might be connected with the inhibitory potential of these compounds against the pro-inflammatory neutrophil elastase from human leukocytes. Further studies are necessary to prove this assumption.

Materials and Methods

Fukinolic acid, the cimicifugic acids A, B, E and F, isoferula and ferula acids were isolated from Cimicifuga racemosa (L.) Nutt. as described [7]; they were of more than 95 % purity. Neutrophil elastase from human leukocytes (E.C. 3.4.21.37) was supplied by Fluka (Germany), the enzyme substrate MeO-Suc-Ala-Ala-Pro-Val-pNA was purchased from Bachem (Switzerland), caffeic acid, soybean trypsin inhibitor and the elastase inhibitor elastatinal were from Sigma (Germany).

The determination of neutrophil elastase activity was performed according to [13] with human leukocyte elastase. Briefly, 250 μl substrate solution (700 μM MeO-Suc-Ala-Ala-Pro-Val-pNA in 50 μM Tris-HCl-buffer, pH 7.5) were mixed with 100 μl test solution (test substances solubilized in Tris-HCl-buffer, pH 7.5) and vortexed. After the addition of 250 μl enzyme solution (approximately 0.5 mU/L) the samples were incubated for 1 h at 37 °C. The reaction was stopped by addition of 500 μl soybean trypsin inhibitor solution (0.2 mg/ml in Tris-HCl-buffer, pH 7.5) and placed in an ice bath. After vortexing the absorbance was read at 405 nm.

All assays were performed at least three times with duplicate samples. Inhibition rates were calculated in percent to controls without inhibitors. IC₅₀ values were determined from dose-effect curves by linear regression.

References

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  PubMedSearch in Google Scholar
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• 10 Sakai  S,, Ochiai  H,, Nakajima  K,, Terasawa  K.. Inhibitory effect of ferulic acid in macrophage inflammatory protein-2 production in a murine macrophage cell line.  Cytokine. 1997;;  9 242-8
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• 12 Tyler  V E.. The Honest Herbal. New York:; Pharmaceutical Products Press, Haworth Press Inc., 1993: 45-6
• 13 Stein  R L.. Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters.  Archives of Biochemistry and Biophysics. 1985;;  236 677-80
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Prof. Dr. Matthias F. Melzig

Institute of Pharmacy Humboldt-University Berlin

Goethestr. 54

13086 Berlin

Germany

Email: matthias=melzig@pharma.hu-berlin.de

References

• 1 Keiser  H,, Greenwald  R A,, Feinstein  G,, Janoff  A.. Degradation of cartilage proteoglycan by human granule neutral proteases - a model of joint injury.  Journal of Clinical Investigations. 1976;;  57 625-32
  PubMedSearch in Google Scholar
• 2 Baggiolini  M,, Schnyder  J,, Bretz  U,, Dewald  B,, Ruch  W.. Cellular mechanisms of proteinase release from inflammatory cells and the degradation of extracellular proteins.  Ciba Foundation Symposium. 1979;;  75 105-21
  PubMedSearch in Google Scholar
• 3 Pipoly  D J,, Crouch  E C.. Degradation of native type IV procollagen by human neutrophil elastase. Implication for leukocyte mediated degradation of basement membranes.  Biochemistry. 1987;;  26 5748-54
  CrossrefPubMedSearch in Google Scholar
• 4 Fischbach  W,, Becker  W,, Mossner  J,, Ohlemuller  H,, Koch  W,, Borner  W.. Leukocyte elastase in chronic inflammatory bowel diseases: A marker of inflammatory activity?.  Digestion. 1987;;  37 88-95
  PubMedSearch in Google Scholar
• 5 Melzig  M F,, Löser  B,, Lobitz  G O,, Tamayo-Castillo  G,, Merfort  I.. Inhibition of granulocyte elastase activity by caffeic acid derivatives.  Die Pharmazie. 1999;;  54 712
  PubMedSearch in Google Scholar
• 6 Kusano  G,, Takahira  M,, Shibano  M,, Kusano  A,, Okamoto  Y,, Tsujibo  H,, Numata  A,, Inamori  Y.. Studies on inhibitory activities of fukiic acid esters on germination, α-amylase and carboxy peptidase A.  Biological and Pharmaceutical Bulletin. 1998;;  21 997-9
  PubMedSearch in Google Scholar
• 7 Kruse  S O,, Löhning  A,, Pauli  G F,, Winterhoff  H,, Nahrstedt  A.. Fukiic and piscidic acid esters from the rhizome of Cimicifuga racemosa and the in vitro estrogenic activity of fukinolic acid.  Planta Medica. 1999;;  65 763-4
  PubMedSearch in Google Scholar
• 8 Sakamura  S,, Yoshihara  T,, Katsuhiko  T.. The constituents of Petasites japonicus: Structure of fukiic acid and fukinolic acid.  Agricultural and Biological Chemistry. 1973;;  37 1915-21
  PubMedSearch in Google Scholar
• 9 Umezawa  H,, Aoyagi  T,, Okura  A,, Morishima  H,, Takeuchi  T.. Elastinal, a new elastase inhibitor produced by Actinomycetes.  Journal of Antibiotics. 1973;;  26 787-9
  PubMedSearch in Google Scholar
• 10 Sakai  S,, Ochiai  H,, Nakajima  K,, Terasawa  K.. Inhibitory effect of ferulic acid in macrophage inflammatory protein-2 production in a murine macrophage cell line.  Cytokine. 1997;;  9 242-8
  CrossrefPubMedSearch in Google Scholar
• 11 Tang  W,, Eisenbrand  G.. Chinese drugs of plant origin. Berlin:; Springer, 1992: 315
• 12 Tyler  V E.. The Honest Herbal. New York:; Pharmaceutical Products Press, Haworth Press Inc., 1993: 45-6
• 13 Stein  R L.. Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters.  Archives of Biochemistry and Biophysics. 1985;;  236 677-80
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Matthias F. Melzig

Institute of Pharmacy Humboldt-University Berlin

Goethestr. 54

13086 Berlin

Germany

Email: matthias=melzig@pharma.hu-berlin.de