Radical Scavenger Activity of Phenylethanoid Glycosides in FMLP Stimulated Human Polymorphonuclear Leukocytes: Structure-Activity Relationships

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Radical scavenger activities of 21 phenylethanoid glycosides, including 15 ester derivatives of caffeic, ferulic, vanillic and syringic acid as well as 6 deacyl derivatives were determined by quantifying their effects on the production of reactive oxygen species (ROS) in a luminol-enhanced chemiluminescence assay with formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated human polymorphonuclear neutrophils (PMNs). All phenylethanoids acylated with phenolic acids showed strong antioxidant activity whereas the deacyl derivatives were more than 30-fold less active. Therefore, the antioxidant activity is mainly related to the number of aromatic methoxy and hydroxy groups and the structure of the acyl moiety (C₆-C₁ or C₆-C₃). In contrast, modification of the sugar chain or replacement of hydroxy groups by methoxy groups in the acyl or the phenylethanoid moiety is of minor importance. The position of the acyl moiety is without significance. Free caffeic, ferulic, vanillic and syringic acid are less active compared to the phenylethanoid derivatives. This points to the importance of dissociation and lipophilicity of these acids in a cellular test system.

Phenylethanoid glycosides are widely distributed in different plant families. Various plants used in traditional medicine, for example species of the genera Plantago, Phlomis or Verbascum contain significant amounts of these compounds [1], [2], [3]. The strong radical scavenger activity of some phenylethanoid glycosides in in vitro assays is already known [4], [5], [6]. Because of the small number of compounds tested and the different systems used hitherto, exact statements on structure-activity relationships and structural requirements concerning their radical scavenger activity were not possible. Therefore, we tested 15 phenylethanoid glycosides esterified with caffeic, ferulic, vanillic and syringic acid, the corresponding free acids and 6 deacyl phenylethanoids for their effects on oxygen radical production by human PMNs. The cells were stimulated with the chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP) and generated oxygen species were detected by luminol-augmented chemiluminescence measurements [7], [8]. These reactive oxygen species (ROS), e.g., superoxide anions, singlet oxygen or hydroxyl radicals, have been proposed to induce cellular damage which plays an important role in cancer, inflammatory and ageing processes.

Earlier in vitro studies on phenylethanoids postulated a dependency of radical scavenger activity on the number of free aromatic hydroxy groups [5]. In fact, the results of this ex vivo test pointed to more complex correlations. All phenylethanoid glycosides acylated with phenolic acids exhibited strong and dose-dependent inhibition of the production of oxygen radicals (see Table [1]). Calceolarioside A {1, 1-[β-(3,4-dihydroxyphenyl)-ethyl]-4-O-caffeoyl-β-D-glucopyranoside} is the most active compound, together with lugrandoside (6) and echinacoside (7). Replacement of the caffeoyl by a feruloyl moiety showed a slight but significant decrease of the radical scavenger activity [IC₅₀ values of verbascoside (4) 0.08 μM; leucosceptoside A (10) 0.18 μM or forsythoside B (8= 0.08 μM; alyssonoside (9) 0.15 μM]. In contrast, the position of the acyl moiety is without any significance [for example: martynoside (12) IC₅₀ = 0.17 μM; isomartynoside (11) 0.17 μM; or crenatoside (2)/isocrenatoside (3) both IC₅₀ = 0.17 μM]. The vanillic acid derivative (15) is less active compared to the ferulic acid derivatives and underlines the importance of the unsaturated C-3 side chain for antioxidant activity of phenolic acids. Substitution of the vanillic acid moiety with an additional methoxy group (= syringic acid) led to a significant increase of activity [phlomisethanoside (15) IC₅₀ = 1.5 μM; hattushoside (14) 0.35 μM]. Variation of the sugar backbone leads to minor changes or is devoid of any effect (calceolarioside A (1) IC₅₀ = 0.04 μM to echinacoside (7) IC₅₀ = 0.03 μM or forsythoside B (8)/verbascoside (4) both IC₅₀ = 0.08 μM]. Also, methylation of the 3,4-dihydroxyphenylethanol structure to a 3-hydroxy-4-methoxyphenylethanol moiety did not influence the activity of the acylated compounds [leucosceptoside A (10) IC₅₀ = 0.18 μM; martynoside (12) 0.17 μM; or alyssonoside (9)/leucosceptoside B (13) IC₅₀ = 0.15 and 0.17 μM, respectively].[]

Deacylation of the phenylethanoids led to a 30-fold decrease of activity and resulted in compounds with moderate antioxidative effects. Among the deacyl derivatives stepwise methylation of the 3,4-dihydroxyphenyl structure led to a significant decrease of activity [IC₅₀ of ferruginoside B (16) 1.9 μM, darendoside B (17) 3.6 μM and deacylverbascoside dimethyl ether (18) 8.5 μM]. Again, modification of the sugar chain showed insignificant effects [IC₅₀ of deacylverbascoside dimethyl ether (18) 8.5 μM, deacylforsythoside-B dimethyl ether 8.6 μM (19), deacyllavandulifolioside dimethyl ether (20) 9.3 μM]. The lack of antioxidant activity of darendoside A (21) confirms in accordance to other studies the special importance of catechol functionalities for antioxidant effects of phenolic compounds [9]. The results indicate that the radical scavenger activity of phenylethanoids is mainly related to the structure (C₆-C₁ or C₆-C₃) and the number of aromatic methoxy and hydroxy groups of the acyl moiety. Interestingly enough, phenylethanoids showed in the same test system higher activity than the corresponding free phenolic acids, and much higher activity than their esters with lipophilic components [for example: lugrandoside (6), echinacoside (7) IC₅₀ = 0.04 and 0.03 μM, respectively > caffeic acid IC₅₀ = 0.3 μM > (-)-bornyl caffeate IC₅₀ = 1.9 μM [8]; leucosceptoside A (10) IC₅₀ = 0.18 μM, martynoside (12) IC₅₀ = 0.17 μM > ferulic acid IC₅₀ = 0.5 μM > (-)-bornyl ferulate IC₅₀ = 3.4 μM [8]; phlomisethanoside IC₅₀ = 1.5 μM > vanillic acid IC₅₀ = 2.5 μM > 27-O-vanilloylbetulinic acid, unpublished results: not active]. In order to correlate the obtained data with other effects in the field of antioxidant and radical scavenger activity of phenylethanoids, further examinations in different cellular and enzymatic assays are in progress.

Materials and Methods

Test compounds: Compounds 4, 8, 9, 14 and 15, were isolated from Phlomis grandiflora H.S. Thompson var. fimbrilligera Hub.-Mor. [10]. Compounds 11 and 12 were obtained from Galeopsis pubescens Besser [11]. Compound 13 was isolated from Marrubium alysson L. [12]. Compounds 2, 3 and 5 were isolated from Globularia trichosantha Fisch. & Mey. [13]. Compound 1 was obtained from Globularia orientalis L. [14], compounds 7 as well as 10 from Pedicularis comosa var. acmodonta Boiss. [15]. Compounds 6 and 16 were isolated from Digitalis ferruginea ssp. ferruginea L. (syn. D. aurea Lindley) [16]. Compound 17 was obtained by deacylation of 12, compounds 18, 19 and 20 by methylation with diazomethane and subsequent deacylation of 4, 8 and lavandulifolioside [17]. Compound 21 was isolated from Scutellaria orientalis ssp. pinnatifida Edmondson [18]. The identity of all compounds was confirmed on the basis of MS, ¹H-, and ¹³C-NMR data, as well as by determination of their optical rotation. The purity has been proven by TLC and HPLC.

Chemiluminescence assay with PMNs: The neutrophils were isolated from 4 different healthy donors. 50 μl PMNs suspension (5 × 10⁶ ml^-1) in HBSS were incubated for 30 min at 37 °C with 50 μl luminol (2 × 10^-4 mol), 50 μl HBSS and 50 μl of the solution of the test compound. The reaction was initiated by adding 50 μl N-formyl-methionyl-leucyl-phenylalanine (FMLP) (5 × 10^-7 mol). For a detailed description of the isolation procedure and test conditions: see [7], [8]. Cytotoxic effects were excluded observing the viability of the cells with trypan blue after testing.

Statistics: Maximum observed standard deviation (absolute) was about 10 %. Positive control measurements were performed with 0.5 μM quercetin. IC₅₀ values were expressed as means ± standard deviation of four experiments and five different concentrations. Student's t test was done to compare the calculated means (p < 0.05) [7].

Acknowledgements

We thank Ms. B. Bosilij for technical assistance and Prof. J. Tarnow as well as Dr. C. M. Schulte for using their laboratory equipment (Med. Einrichtungen der Heinrich-Heine-Universität Düsseldorf).

References

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• 14 Çalis  I,, Kirmizibekmez  H,, Sticher  O.. manuscript in preparation. 
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• 18 Çalis  I,, Saracoglu  I,, Basaran  A A,, Sticher  O.. Two alcohol glycosides from Scutellaria orientalis subsp. pinnatifida. .  Phytochemistry. 1993;;  32 1621-3
  CrossrefPubMedSearch in Google Scholar

Dr. Jörg Heilmann

Institute of Pharmaceutical Sciences, ETH Zurich

Winterthurerstrasse 190

8057 Zürich

Switzerland

Email: heilmann@pharma.ethz.ch

Phone: ++4116356049

Fax: ++4116356882

References

• 1 Murai  M,, Tamayama  Y,, Nishibe  S.. Phenylethanoids in the herb of Plantago lanceolata and inhibitory effect on arachidonic acid induced mouse ear edema.  Planta Medica. 1995;;  61 479-80
  PubMedSearch in Google Scholar
• 2 Klimek  B.. 6′-O-Apiosyl-verbascoside in the flowers of mullein (Verbascum species).  Acta Poloniae Pharmaceutica. 1996;;  53 137-46
  PubMedSearch in Google Scholar
• 3 Baytop  T.. Therapy with Medicinal Plants in Turkey (Past and Present). 216-217; Publications of Istanbul University, Instanbul,; 1984
  Search in Google Scholar
• 4 Facino  R M,, Carini  M,, Aldini  G,, Saibene  L,, Pietta  P,, Mauri  P.. Echinacoside and caffeoyl conjugates protect collagen from free radical-induced degradation: a potential use of Echinacea extracts in the prevention of skin photodamage.  Planta Medica. 1995;;  61 510-4
  PubMedSearch in Google Scholar
• 5 Wang  P,, Kang  J,, Zheng  R,, Yang  Z,, Lu  J,, Gao  J,, Jia  Z.. Scavenging effects of phenypropanoid glycosides from Pedicularis on superoxide anion and hydroxyl radical by the spin trapping method (95)02255-4.  Biochemical Pharmacology. 1996;;  51 687-91
  CrossrefPubMedSearch in Google Scholar
• 6 Xiong  Q B,, Kadota  S,, Tani  T,, Namba  T.. Antioxidative effects of phenylethanoids from Cistanche deserticola. .  Biological and Pharmaceutical Bulletin. 1996;;  19 1580-5
  PubMedSearch in Google Scholar
• 7 Heilmann  J,, Merfort  I,, Weiss  M.. Radical scavenger activity of different 3′,4′-dihydroxyflavonols and 1,5-dicaffeoylquinic acid studied by inhibition of chemiluminescence.  Planta Medica. 1995;;  61 435-8
  PubMedSearch in Google Scholar
• 8 Lobitz  G O,, Heilmann  J,, Zschocke  S,, Tamayo-Castillo  G,, Bauer  R,, Merfort  I.. Bornyl cinnamate derivatives with anti-inflammatory activity from Verbesina turbacensis.  Pharmaceutical and Pharmacological Letters. 1998;;  8 115-8
  PubMedSearch in Google Scholar
• 9 Limasset  B,, Ojasoo  T,, le Doucen  C,, Dore  J-C.. Inhibition of chemiluminescence in human PMNs by monocyclic phenolic acids and flavonoids.  Planta Medica. 1999;;  65 23-9
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Çalis  I,, Heilmann  J,, Harput  S,, Schühly  W,, Sticher  O.. Joint Meeting of GA, AFERP, ASP and PSE, Abstract No. 212, July 26 - 30,. Amsterdam; 1999
• 11 Çalis  I,, Lahloub  M F,, Rogenmoser  E,, Sticher  O.. Isomartynoside, a phenylpropanoid glycoside from Galeopsis pubescens. .  Phytochemistry. 1984;;  23 2313-5
  CrossrefPubMedSearch in Google Scholar
• 12 Çalis  I,, Hosny  M,, Khalifa  T,, Rüedi  P.. Phenylpropanoid glycosides from Marrubium alysson. .  Phytochemistry. 1992;;  31 3624-6
  CrossrefPubMedSearch in Google Scholar
• 13 Çalis  I,, Krimizibekmez  H,, Rüegger  H,, Sticher  O.. Phenylethanoid glycosides from Globularia trichosantha. .  Journal of Natural Products. 1999;;  62 1165-8
  CrossrefPubMedSearch in Google Scholar
• 14 Çalis  I,, Kirmizibekmez  H,, Sticher  O.. manuscript in preparation. 
• 15 Akdemir  Z,, Çalis  I.. Iridoid and phenylpropanoid glycosides from Pedicularis comosa var. acmodonta Boiss.  Doga-Turkish Journal of Pharmacy. 1992;;  2 63-70
  PubMedSearch in Google Scholar
• 16 Çalis  I,, Tasdemir  D,, Sticher  O,, Nishibe  S.. Phenylethanoid glycosides from Digitalis ferruginea subsp. ferruginea (= D. aurea Lindley) (Scrophulariaceae).  Chemical and Pharmaceutical Bulletin. 1999;;  47 1305-7
  PubMedSearch in Google Scholar
• 17 Basaran  A A,, Çalis  I,, Anklin  C,, Nishibe  S,, Sticher  O.. Lavandulifolioside: a new phenylpropanoid glycoside from Stachys lavandulifolia. .  Helvetica Chimica Acta. 1988,;  71 1483-90
  CrossrefPubMedSearch in Google Scholar
• 18 Çalis  I,, Saracoglu  I,, Basaran  A A,, Sticher  O.. Two alcohol glycosides from Scutellaria orientalis subsp. pinnatifida. .  Phytochemistry. 1993;;  32 1621-3
  CrossrefPubMedSearch in Google Scholar

Dr. Jörg Heilmann

Institute of Pharmaceutical Sciences, ETH Zurich

Winterthurerstrasse 190

8057 Zürich

Switzerland

Email: heilmann@pharma.ethz.ch

Phone: ++4116356049

Fax: ++4116356882