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Oenothein B's contribution to the anti-inflammatory and antioxidant activity of Epilobium sp.

ABSTRACT

Willow herb tea or preparation are available and relatively popular in the European market, and claimed to be effective inter alia because of their anti-inflammatory activity. The present study is therefore aimed at comparing the anti-inflammatory and antioxidant activity of extracts of the three most popular Epilobium species (E. angustifolium, E. hirsutum and E. parviflorum) and at juxtaposing this activity against the dominating compounds from the following extracts: oenothein B (OeB), quercetin-3-O-glucuronide and myricetin-3-O-rhamnoside. The phytochemical analysis of the extracts has shown that OeB quantities vary between 20% and 35%, while flavonoids content does not exceed 2%. All extracts have inhibited the activity of hyaluronidase and lipoxygenase with [IC.sub.50] around 5 [micro]g/ml and 25 [micro]g/ml. The inhibition of hyaluronidase is related with the presence of OeB. a strong inhibitor of this enzyme ([IC.sub.50] 1.1 [micro]M). Additionally, the extracts inhibited myeloperoxidase (MPO) release from stimulated neutrophils. OeB inhibited MPO release similarly to the anti-inflammatory drug indomethacin with IC50 7.7 [micro]M and 15.4 [micro]M, respectively. Tested extracts significantly reduced the production of reactive oxygen species (ROS) from f-MLP and PMA induced neutrophils with IC50 5 [micro]g/ml and 25 [micro]g/ml, respectively. The flavonoids content seems to exert little influence on extracts' activity, contrary to OeB, whose high concentration explains the activity of extract obtained from Epilobium. Tested currently marketed Epilobium preparations are often wrongly assigned, but we should stress that the level of OeB in all tested herbs was high and always exceeded 2% in raw material.

[C] 2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Epilobium

Oenothein B

Inflammation

Neutrophils

Reactive oxygen species

Introduction

Epilobium (Oenotheraceae), is an extensive genus with approximately 165 species worldwide. In Europe, 28 species of the genus, divided in two sections of Epilobium and Chamaenerion, occur. Epi-lobii herba (willow herb) is used for prostate and gastrointestinal disorders, mucous membrane lesions, such as mouth ulcers, and to improve the healing of wounds, skin sores and swelling (PDR 1998; Foster and Duke 2000; Wichtl 2004). Different species are collected as plant source, including small-flowered species such as E. parviflorum, E. montanum and E. roseum, and large-flowered species: E. angustifolium and E. hirsutum (Wichtl 2004). Authentication is difficult because of species similarity and frequent interspecific hybridisation and requires a combination of TLC and morphological (trichomes and raphides) analysis (Wichtl 2004; Krajsek et al. 2006). The herb is rich in polyphenolic compounds such as flavonol-3-O-glycosides and ellagitannins (Hiermann 1995; Ducrey et al. 1995; Wichtl 2004). Flavonoid analysis has shown that quercetin glycosides, especially quercetin glucuronide, predominate in Epilobium angustifolium in contrast to other Epilobium species, where myricetin rhamnoside is the main compound (Ducrey et al. 1995). The investigation of possible active particles was initially focused on the anti-inflammatory effect of [pounds sterling] angustifolium and E. parvi-florum extracts on prostaglandin synthesis in perfused rabbit ear (Hiermann et al. 1986). From the more active extract from [pounds sterling] angustifolium a minor constituent, myricetin 3-O-glucuronide, was isolated and identified as a strong inhibitor of COX-1, COX-2 and 5-LOX activities (Hiermann et al. 1991, 1998). Interestingly, the presence of this compound in Epilobium species is limited to the large-flowered one (Hiermann 1995; Barakatetal. 1997). However, this finding does not explain the COX-1 and-2 activity inhibition by [pounds sterling] parviflorum extracts (Hevesi et al. 2008; Steenkamp et al. 2006). Simultaneously conducted studies on the anti-androgenic activity of the Epilobium extract have lead to the isolation of a dimeric ellag-itannin: oenothein B (Ducrey et al. 1997; Lesuisse et al. 1996), which seems to be widespread in Epilobium species (Ducrey et al. 1997).

Willow herb tea or preparation are available and relatively popular in the European market, and claimed to be effective inter alia because of their anti-inflammatory activity. The present study is therefore aimed at comparing the anti-inflammatory and antioxidant activity of extracts of the three most popular Epilobium species (E. angustifolium, E. hirsutum and E parviflorum) and at juxtaposing this activity against the dominating compounds from the following extracts: oenothein B (OeB), quercetin-3-O-glucuronide and myricetin-3-O-rhamnoside.

Materials and methods

Reagents

Cytochalasin B, HRP (horseradish peroxidase), hyaluronic acid, hyaluronidase (bovine testes, type 1-S), linoleic acid, lipoxygenase (soybean, type 1-B), luminol, f-MLP (formyl-met-leu-phenylalanine). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), NTB (nitrobluetetrazolium), PMA (4[beta]-phorbol-12[beta]-myristate-al3-acetate), SAAVNA (N-succinyl-alanine-alanine-valinine-p-nitroanilide), TMB (3,3',5,5'-tetramethylbenzidine), xanthine and xanthine oxidase were all provided by Sigma-Aldrich. Lucigenin and miricetin-3-O-rhamnoside were provided by Carl Roth. Quercetin-3-O-glucuronide and oenothein B had been isolated as described previously (Kiss et al., 2004). All substances used were of >95% purity. All solvents were analytical or HPLC grade.

Plant material

Herbs of Epilobium angustifolium L and Epilobium hirsutum L were collected in north-east Poland. The herb of Epilobium parviflorum Schreb. was collected in the Medicinal Plants Garden of the Warsaw Agricultural University and identified by M. Pete, PhD. The precise assignation of Epilobium species was based on tri-chome and raphides morphology (Krajsek et al. 2006; Wichtl 2004) and was performed in the Department of Pharmacognosy, Medical University, Warsaw (Poland). Plant materials were dried at room temperature. A specimen of each drug is available in the drug collection of the Department of Pharmacognosy, Medical University, Warsaw (Poland).

Extracts preparation and characterisation

Powdered plant materials (50 g) were extracted with water (1:10) in an ultrasonic water bath for 1 h at 40 [degrees]C. The water residue was then lyophilised.

Total flavonoid contents were determined by the Christ-Muller method. Oenothein B content was determined by HPTLC-densitometry validated method (Bazylko et al. 2007).

The phenolics of different extracts were analysed by HPLC with diode array detection. Separation was performed on a Luna C-18, 25 x 4.6 mm, 5 [micro]m column (Phenomenex,). The eluent was (A) 2.5% [CH.sub.3]COOH and (B) [CH.sub.3]CN + 2.5% CH3COOH (80:20). A gradient solvent system was used: 7-20%B (300; 20-40%B (60'). UV spectra were recorded in the range of 200-400 nm, chromatograms were acquired at 280 and 350 nm.

Cytotoxicity assay for NHDFs cell culture

Human skin fibroblast cells (NHDFs) were purchased from Lonza. NHDFs (from the third to sixth passage) were seeded on 24-well plates (1.5 x [10.sup.4] cells per well) and incubated for 3-4 days in the DMEM medium with 10% FBS, penicillin and streptomycin, in 5% [CO.sub.2] and at 37 [degrees]C. Then their medium was changed to serum-free DMEM and cells were incubated with the extracts for 24 h. Cell cytotoxicity of extracts was determined based on the measurement of the activity of lactate dehydrogenase (LDH) released from the cytosol of damaged cells into the supernatant. Extracts cytotoxicity was also assessed by the measurement of mitochondria function with the use of MTT, which is converted in live cells into insoluble formazan.

Isolation of human neutrophils

Peripheral venous blood of was collected from healthy human donors (20-35 years old) at the Warsaw Blood Donation Centre. Neutrophils were isolated with a standard method of dextran sedimentation prior to hypotonic lysis of erythrocytes and to centrifugation in a Ficoll Hypaque gradient The purity of the neutrophils preparation was >97% and viability measured by tryptan blue exclusion was >98%. Neutrophils were resuspended in a ([Ca.sup.2+])-free HSSB, ([Ca.sup.2+])-free PBS buffers at pH 7.4 or RPMI 1640 medium and were maintained at 4 [degrees]C before use.

Evaluation of ROS production by human neutrophils and cell free systems

Oxidant generation by f-MLP or by PMA-stimulated neutrophils was measured using luminol- and lucigenin-dependent chemilu-minescence.

Scavenging of superoxide anion ([O.sub.2.sup.-]) was performed using xanthine-xanthine oxidase system with nitrobluetetrazolium (NBT) reduction method. Scavenging of hydrogen peroxide ([H.sub2][O.sub2]) was performed as described by O'Dowd et al. (2004). Scavenging of hypochlorous acid (HClO) was performed by the chlorination of taurine.

The percentage of inhibition was calculated in comparison to the control without inhibitors, taking into account the absorbance of light by test extracts/compounds.

Elastase and myeloperoxidase (MPO) release by human neutrophils

Neutrophil elastase release was determined using N-succinyl-alanine-alanine-valinine-p-nitroanilide (SAAVNA) as a substrate and p-nitrophenol was measured spectrophotometrically. Neutrophil myeloperoxidase release was determined using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The assay is based on the oxidation of TMB by MPO in the presence of [H.sub.2][O.sub.2] (Suzuki et al., 1983).

The percentage of inhibition was calculated in comparison to the control without inhibitors, taking into account the absorbance of light by test extracts/compounds.

Lipoxygenase, leukotriene [B.sub.4], cyclooxygenase and hyaluronidase assays

Lipoxygenase and hyaluronidase assays were performed as described by Ling et al. (2003). COX-1 and COX-2 were performed using enzyme-linked immunoabsorbent assay ELISA kit, following the indications of the manufacturer (Cayman).

Statistical analysis

The results were expressed as mean [+ or -]SD of the indicated number of experiments. Statistical significance of differences between means was established by ANOVA with Tukey post hoc test. P values below 0.05 were considered statistically significant. All analyses were performed using Statistica 8.

Results and discussion

The phytochemical analysis of extracts has shown that oenothein B is a dominating compound, whose quantity varies from 20% to 35%, while flavonoids contents do not exceed 2% (Table 1). E. angustifolium contains quercetin-3-O-glucuronide as a main flavonoid, while E.. hirsutum and E. parviflorum contain myricetin-3-O-rhamnoside and higher than E. angustifolium oenothein B quantity (Fig. 1). None of the tested extracts at the concentration of 3.125-50 [micro]g/ml has shown cytotoxicity, when tested on human skin fibroblast cells. This is of importance because of the frequent external use of those extracts. We have demonstrated that all extracts inhibited the activity of hyaluronidase and lipoxygenase with [IC.sub.50] 3.3-6.5 [micro]g/ml and 16-28 [micro]g/ml, respectively. The inhibition of hyaluronidase is related with the presence of oenothein B, a strong inhibitor of this enzyme ([IC.sub.50] 1.1 [micro]M).This might partly explain the use of Epilobiurn as a wound healing factor (Girish and Kemparaju 2007). Extracts also inhibited myeloperoxidase (MPO) release from stimulated neutrophils, but were without effect on elastase release (Table 2). It is noteworthy that oenothein B inhibited MPO release similarly to the anti-inflammatory drug indomethacin with [IC.sub.50] 7.7 [micro]M and 15.4 [micro]M, respectively. All extracts significantly attenuated the [LTB.sub.4] release (Fig. 2). This effect may be due to the inhibition of lipoxygenase activity, however E hirsutum and E. parviflorum extracts have shown more significant effect on LTB4 release than only on lipoxygenase activity. The activity of oenothein B appears to be related with the lipoxygenase inhibition. Extracts have shown weak activity against cyclooxy-genases (Fig. 3). Steenkamp et al. (2006) observed COX-1 activity inhibition by water extract of E. parviflorum but in 10-time higher concentration. However, the effect of E. parviflorum and other Epilobiurn extracts may be expected at the level of prostaglandin [E.sub.2] release (Hevesi et al. 2008). Oenothein B appears as specific COX-1 inhibitor comparable in activity to indomethacin (Fig. 3).
Table 1

Quantity of oenothein B and flavonoids in aqueous extract of Epilobium
sp.

   Extracts                  Oenothein B                   Flavonoids

                 Mean [+ or -]SD (a)  CV% (a)  Mean[+ or -]SD (a)   CV%
                    [[mgg.sup.-1]]                 [mgg.sup.-1]     (a)

E.angustifolium  225.8 [+ or -] 7.6     3.38   13.4 [+ or -] 0.5   4.07
E.parviflorum    326.7 [+ or -] 6.2     1.91    7.1 [+ or -] 0.3   3.49
E.thirsutum      333.6 [+ or -] 24.8    7.45   19.2 [+ or -] 0.5   2.63

(a) n = 3

Table 2
Summary of the [IC.sub.50] values for inhibiting isolated enzymes
activity and enzymes released from human neutrophils by Epilobium
aqueous extracts, oenothein B, quercetin-3-glucuronide (GQ) and
myricetin-3-rhamnoside (MR).

                  Hyaluronidase  Lioxygenase  Elastase  MPO release
                                              release

E. angustifolium  3.3(a)         24 (a)       >50 (a)     34 (a)
E. parviflorum    4.0 (a)        28 (a)       >50 (a)     >50 (a)
E. hirsutum       6.5 (a)        16 (a)       >50 (a)     26 (a)
Oenothein B       1.1 (b)        15 (b)       n.in        7.7 (b)
QG                n.in           67 (b)       n.in        n.in
MR                n.in           54 (b)       n.in        50 (b)
Heparin           62 (a)         -            -           -
Fisetin           -              180 (b)      _           -
Quercetin         -              -            20 (b)      -
Indomethacin      -              -            -           15.4 (b)

Data were calculated as 50% inhibitory concentration (IC50).
Experiments were performed in duplicate in three independent
experiments. Experiments with neutrophils were performed using cells of
different donors, n.in. - no inhibition.
(a) [micro].g/ml.
(b) [micro]-M.


[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Neutrophils activation results in a rapid increased formation of reactive oxygen species: [O.sub.2.sup.-]. [H.sub.2][O.sub.2], HClO extracellularly, as well as proteolitic enzymes (e.g. elastase) and other inflammatory mediators (e.g. myeloperoxidase, leukotriene B4) released, increased cell mobility and adhesion. It has been shown that the excessively activated neutrophils can contribute to a variety of chronic diseases such as colon and bowel inflammation (D'Odorico et al. 2000), as well as affect wound healing (Dovi et al. 2004; Schafer and Werner 2008). Tested extracts also significantly reduced the production of reactive oxygen species (ROS) from f-MLP and PMA induced neutrophils with [IC.sub.50] 5 [micro]g/ml and 25 [micro]g/ml, respectively (Table 3). The f-MLP induced ROS production was strongly inhibited by all extracts. This may be due to the scavenging properties of the extract and also because of inhibition of myeloperoxidase release. There is no really significant difference between species and the extracts activities reflected in oenothein B activity (Tables 2 and 3), which appears as a strong scavenger of [O.sub.2.sup.-] and [H.sub.2][O.sub.2]. with IC50 below 1 [micro]M. The flavonoids content seems to have little influence on extracts' activity, unlike oenothein B, whose high activity and concentration explain the efficiency of the extract obtained from Epilobium. We also tested Epilobium preparations available on the market and we determined that often species were wrongly assigned, especially in the case of products allegedly containing E. parviflorum. Yet it should be emphasized that the levels of oenothein B in all currently marketed tested herbs, as well as other investigated species: E montanum and E. roseum, were high and always exceeded 2% in raw material.
Table 3
Summary of the [IC.sub.50] values for inhibiting f-MLP- and PMA-induced
reactive oxygen species production on human neutrophils and scavenging
selected radicals in cell free systems by Epilobium aqueous extracts:
E. angustifolium (EA), E. hirsutum (EH), E. parviflorum (EP) and
compounds: oenothein B. quercetin-3-glucuronide (GQ) and
myricetin-3-rhamnoside (MR).

                    EA [[micro]g/ml]  EH ([micro]g/ml]        EP
                                                        [[micro]g/ml]

f-MLP induction             5                5                5
PMA induction              30               25               24
[O.sub.2.sup.-]             5                3.5              4
[H.sub.2][O.sub.2]          3.6              2.3              2.2
HCIO                       25               26               33

                    OeB [[micro]M]  QG [[micro]M]  MR [[micro]M]

f-MLP induction           0.3             46            6.5
PMA induction             5             >100           82
[O.sub.2.sup.-]           0.9           >100            3.7
[H.sub.2][O.sub.2]        0.7             42            5.4
HCIO                     10             >100           43

Data were calculated as 50% inhibitory concentration ([IC.sub.50]).
Experiments were performed in duplicate in three independent
experiments. Experiments with neutrophils were performed using cells of
different donors.


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Anna K. Kiss *, Agnieszka Bazylko, Agnieszka Filipek, Sebastian Granica, Edyta Jaszewska, Urszula Kiarszys, Anita Kosmider, Jakub Piwowarski

Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland

* Corresponding author. Tel.: +48 22 572 09 85; fax: +48 22 572 09 85. E-mail address: akiss@wum.edu.pl (A.K. Kiss).

doi: 10.1016/j.phymed.2010.10.016
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Title Annotation:Short communication
Author:Kiss, Anna K.; Bazylko, Agnieszka; Filipek, Agnieszka; Granica, Sebastian; Jaszewska, Edyta; Kiarszy
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:May 15, 2011
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