Journal of Ethnopharmacology 102 (2005) 170–176

Bioassay-guided isolation of iridoid glucosides with antinociceptive andanti-inflammatory activities fromVeronica anagallis-aquatica L.

Esra Kupelia, U. Sebnem Harputb, Mehtap Varelb, Erdem Yesiladaa,∗, Iclal Saracoglub

a Gazi University, Faculty of Pharmacy, Etiler 06330 Ankara, Turkeyb Hacettepe University, Faculty of Pharmacy, Sihhiye 06100 Ankara, Turkey

Received 24 September 2004; received in revised form 11 May 2005; accepted 26 May 2005Available online 12 July 2005

Abstract

Extracts obtained from the herbs of variousVeronica species are used as folk remedy worldwide for the treatment of various inflammatoryailments including rheumatism. In vivo anti-inflammatory and antinociceptive activities ofVeronica anagallis-aquatica L. aerial parts wereinvestigated. Methanolic extract of the plant was shown to possess significant inhibitory activity against carrageenan-induced hind paw edema

pounds,noside (8)atalposidety as well

duced

-

ealer-

s-es.ndutere

ryl-Oty.i-nd

model and ofp-benzoquinone-induced writhings in mice. Through bioassay-guided fractionation and isolation procedures eight comaquaticoside A (1), aquaticoside B (2), aquaticoside C (3), veronicoside (4), catalposide (5), verproside (6), verminoside (7) and martywere isolated and their structures were elucidated by spectral techniques. Catapol derivative iridoid glucosides, verproside (6) and c(5), were found to possess potent antinociceptive and anti-inflammatory activities, per os without inducing any apparent acute toxicias gastric damage. Results of the present study supported the utilization of the plant in Turkish folk medicine.© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Veronica anagallis-aquatica; Scrophulariaceae; Antinociceptive; Anti-inflammatory; Iridoid glucosides; Catalpol derivatives; Carrageenan-inpaw edema;p-Benzoquinone-induced writhings

1. Introduction

SeveralVeronica species are reported to possess applica-tion in traditional medicines worldwide for the treatment of awide range of disorders; in respiratory diseases against coughor as expectorant, as antiscorbutic, as diuretics and for woundhealing (Baytop, 1984; Harput et al., 2002). In Chinese tra-ditional medicine,Veronica anagallis-aquatica L. is used forthe treatment of influenza, hemoptysis, laryngopharyngitisand hernia (Su et al., 1999). During our field expeditions onTurkish folk medicine, we have recorded that aerial parts ofVeronica anagallis-aquatica is boiled in milk to obtain poul-tice and then is applied to abdomen for abdominal pain or itswarm aqueous extract without removing the boiled herbs isused as a bath remedy to alleviate rheumatic pain in northwestAnatolia (Fujita et al., 1995).

∗ Corresponding author. Tel.: +90 312 2154468; fax: +90 312 223 5018.E-mail address: yesilada@gazi.edu.tr (E. Yesilada).

Previously, in vitro anti-inflammatory activity of fiveVeronica species (Veronica cymbalaria, Veronica hederifo-lia, Veronica pectinata var. glandulosa, Veronica persicaand Veronica polita) were investigated through evaluation the inhibitory effects on nitric oxide (NO) pro-duction in lipopolysaccharide-stimulated mouse peritonmacrophages as well as cytotoxic activity against KB epidmoid carcinoma and B16 melanoma (Harput et al., 2002).MeOH extracts of five plant materials were found to posess inhibitory effects on NO synthesis in varying degreMeOH extracts were further partitioned between water achloroform, and water fractions showed the activity withoinducing any cytotoxicity, while chloroform fractions wercytotoxic dose-dependently. Moreover, water fractions wefound to possess remarkable effect on 2,2-diphenyl-1-pichydrazyl (DPPH), suggesting that the inhibitory effect on Nproduction might be due to their radical scavenging activi

Veronica species have been known to be rich in irdoid glucosides. Mainly aucubin, catalpol, benzoic a

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2005.05.042

E. Kupeli et al. / Journal of Ethnopharmacology 102 (2005) 170–176 171

cinnamic acid esters of catalpol, mussaenoside and mus-saenosidic acid esters were reported in the investigatedVeronica species (Harput et al., 2003). Various biologicalactivities have been reported previously for the iridoid glu-cosides including antidiabetic, anti-inflammatory, anticancerand immunostimulant activities (Vijayavitthal et al., 1998;Konoshima et al., 2000; Stevenson et al., 2002; Ahmed et al.,2003).

As related with the above-presented data, this study dealswith the anti-inflammatory and antinociceptive effects ofVeronica anagallis-aquatica herbs, in order to evaluate thefolkloric information and isolation and chemical character-ization of the active constituent(s) through bioassay-guidedfractionation procedures.

2. Material and methods

2.1. Plant material

Aerial part ofVeronica anagallis-aquatica L. (Scrophu-lariaceae) was collected from Beytepe Campus of HacettepeUniversity, Ankara, Turkey in June, 2002. Voucher specimen(HUEF 02021) is deposited in the Herbarium of the Facultyof Pharmacy, Hacettepe University.

2a

2red

a deo ap-o eOHe icef

2

(T pres-s asr ane( n-s ted tog ctedt ye ofM Fr.B ).

2a

iump ed

on Labormatic (18.5× 352 mm) and Buchi (25× 460 mm)glass columns using reversed phase (LiChroprep RP-18;40–63�m, Merck) material with increasing concentra-tions of MeOH in H2O (0→ 100% MeOH) to yield Frs.B1–B7.

Frs. B1–B7 were further applied to a series of silica gel(Merck, Kieselgel 60, 60–230 mesh) column chromatogra-phy eluting with CHCl3/MeOH (99:1→ 65:35) and com-pound 6 (200.2 mg) from Fr. B1 (250 mg), compound5(213.1 mg) and compound2 (10.3 mg) from Fr. B2 (500 mg),compound7 (2.2 mg) from Fr. B3 (120 mg), compound3(8.0 mg) from Fr. B4 (35 mg), compound1 (15.5 mg) fromFr. B5 (64 mg), compound8 (2.5 mg) from Fr. B6 (31 mg) andcompound4 (59.5 mg) from Fr. B7 (350 mg) were isolated inpure form.

2.2.4. Structure elucidation of the compounds 1–8Structure elucidation of the isolated components1–8 from

Fr. B was carried out by spectral techniques; UV, 1D and2D NMR (1H, 13C NMR) and mass spectroscopy (HR-ESI-FTMS) and detailed data were recently published elsewhere(Harput et al., 2004). The structures of compounds1–8were as follows (Fig. 1): (1) 6′-O-benzoyl-8-epiloganic acid,aquaticoside A; (2) 6′-O-p-hydroxybenzoyl-8-epiloganicacid, aquaticoside B; (3) 6′-O-benzoyl-gardoside, aquatico-side C; (4) veronicoside; (5) catalposide; (6) verproside; (7)v

2

2rom

t ntralI fort erem Thef , buta wasu imalsw elinesf

2sus-

pc ni-m se oft lacedw heri A)( efer-e

22t e

.2. Bioassay-guided fractionation and isolation ofctive ingredients

.2.1. Preparation of H2O and MeOH extractsTwo flasks each containing 10 g of dried and powde

erial parts ofVeronica anagallis-aquatica were extracteither with MeOH and distilled water, separately, at 40◦Cvernight (3× 250 ml). Each of combined extract was evrated to dryness under reduced pressure to give 2.3 g Mxtract and 1.9 g H2O extract and was administered to mor activity assessment.

.2.2. Fractionation of the MeOH extractThe air-dried aerial parts ofVeronica anagallis-aquatica

453 g) were extracted with MeOH at 40◦C for 12 h (3× 3 l).he MeOH solution was evaporated under reducedure to give “MeOH extract” (75 g). The MeOH extract wedissolved in 90% MeOH (0.2 l) and extracted with hex10× 100 ml) to remove chlorophyl and other lipophylic cotituents. The aqueous methanolic layer was concentraive a crude extract (68 g). The extract was then subje

o polyamide (Fluka, 50–60�m) column chromatographluting with H2O, followed by increasing concentrationseOH to give six main fractions: Frs. A–F (Fr. A, 12.4 g;, 29 g; Fr. C, 2.6 g; Fr. D, 2.2 g; Fr. E 3.2 g; Fr. F. 1.54 g

.2.3. Chromatographic separation and isolation of thective constituents

Fr. B, the most active fraction, was applied to the medressure liquid chromatography (MPLC) was perform

erminoside; (8) martynoside.

.3. Biological activity tests

.3.1. Test animalsMale Swiss albino mice (20–25 g) were purchased f

he animal breeding laboratories of Refik Saydam Censtitute of Health (Ankara, Turkey). The animals leftwo days for acclimatization to animal room conditions waintained on standard pellet diet and water ad libitum.

ood was withdrawn on the day before the experimentllowed free access of water. A minimum of six animalssed in each group. Throughout the experiments, anere processed according to the suggested ethical guid

or the care of laboratory animals.

.3.2. Preparation of test samples for bioassayTest samples were given orally to test animals after

ending in a mixture of distilled H2O and 0.5% sodiumarboxymethyl cellulose (CMC). The control group aals received the same experimental handling as tho

he test groups except that the drug treatment was repith appropriate volumes of the dosing vehicle. Eit

ndomethacin (10 mg/kg) or acetyl salicylic acid (AS100 mg/kg and 200 mg/kg) in 0.5% CMC was used as rnce drug.

.3.3. Antinociceptive activity

.3.3.1. p-Benzoquinone-induced abdominal constrictionest in mice (Okun et al., 1963). Sixty minutes after th

172 E. Kupeli et al. / Journal of Ethnopharmacology 102 (2005) 170–176

Fig. 1. Structures of the compounds.

oral administration of test samples, the mice were intraperi-toneally injected with 0.1 ml/10 g body weight of 2.5% (v/v)p-benzoquinone (PBQ; Merck) solution in distilled H2O.Control animals received an appropriate volume of dosingvehicle. The mice were then kept individually for observa-tion and the total number of abdominal contractions (writhing

movements) was counted for the next 15 min, starting on thefifth min after the PBQ injection. The data represent averageof the total number of writhes observed. The antinociceptiveactivity was expressed as percentage change from writhingcontrols. 100 mg/kg and 200 mg/kg aspirin (ASA) was usedas reference drug.

E. Kupeli et al. / Journal of Ethnopharmacology 102 (2005) 170–176 173

2.3.4. Carrageenan-induced hind paw edema (Yesiladaand Kupeli, 2002)

Sixty minutes after the oral administration of test sampleor dosing vehicle each mouse was injected with freshly pre-pared (0.5 mg/25�l) suspension of carrageenan (Sigma, St.Louis, Missouri, USA) in physiological saline (154 nM NaCl)into subplantar tissue of the right hind paw. As the control,25�l saline solution was injected into that of the left hind paw.Paw edema was measured in every 90 min during 6 h afterinduction of inflammation. The difference in footpad thick-ness was measured by a gauge calipers (Ozaki Co., Tokyo,Japan). Mean values of treated groups were compared withmean values of a control group and analyzed using statisti-cal methods. Indomethacin (10 mg/kg) was used as referencedrug.

2.3.5. Acute toxicityAnimals employed in the carragenan-induced paw edema

experiment were observed during 24 h and mortality wasrecorded, if happens, for each group at the end of observationperiod.

2.3.6. Gastric-ulcerogenic effectAfter the analgesic activity experiment mice were killed

under deep ether anesthesia and stomachs were removed.T h theg copef

2ssed

a sb valu-a hoct*

3. Result and discussion

For the scientific evaluation of the claimed effect forVeronica anagallis-aquatica aerial parts on rheumatism andrelated disorders in Turkish folk medicine, MeOH andH2O extracts were prepared from the material and bothextracts were investigated for anti-inflammatory activityusing carrageenan-induced hind paw edema model andfor antinociceptive activity usingp-benzoquinone-inducedwrithing model in mice. Since the warm aqueous extracttogether with aerial parts was directly applied externally onthe affected joint as well as bath to reduce pain a dose calcula-tion could not be applied. Hence, extracts were administeredin a standard dose of 500 mg/kg.

As shown in Table 1, H2O extract showed a weakinhibitory effect, while MeOH extract was significantly activeagainst carrageenan-induced edema. Hence, bioassay-guidedfractionation procedures were conducted with this extract.The MeOH extract was first fractionated by polyamide col-umn chromatography after removing chlorophyll to affordsix main fractions. Each fraction was then administered tomice in doses estimated according to their ratio in MeOHextract. Since Fr. B (3–12) showed potent activity, further iso-lation studies were performed using this fraction (Table 1).Successive application of RP-MPLC and Si-gel column chro-matography techniques have led to the isolation of three new(k edc(aA aina uan-t ora d-i d as

TE nica an ice

M mm)±180

C 50.5M 33.1H 40.5F 46.5F 38.7F 41.2F 50.2F 46.7F 39.7I 33.9

*

hen, the abdomen of each mouse was opened througreat curvature and examined under dissecting micros

or lesions or bleedings.

.3.7. Statistical analysis of dataData obtained from animal experiments were expre

s mean standard error (±S.E.M.). Statistical differenceetween the treatments and the control were eted by ANOVA and Students–Newman–Keuls post-

ests.p < 0.05 was considered to be significant [* p < 0.05;* p < 0.01;*** p < 0.001].

able 1ffect of H2O and MeOH extracts and fractions from MeOH extract ofVero

aterial Dose (mg/kg) Swelling thickness (×10−2

90 min

ontrol 45.2± 4.1eOH extract 500 32.8± 4.3 (27.4)*

2O extract 500 36.8± 3.2 (18.6)r. A (1–2) 222.6 41.2± 2.9 (8.8)r. B (3–12) 606.8 36.3± 2.9 (19.7)r. C (13–19) 46.2 37.0± 2.8 (18.1)r. D (20–22) 39.6 44.7± 3.2r. E (23–34) 57.2 42.8± 2.6 (5.3)r. F (35–40) 27.6 37.7± 3.7 (16.6)

ndomethacin 10 31.5± 3.1 (30.3)*

* p < 0.05 Significant from control.** p < 0.01 Significant from control.** p < 0.001 Significant from control.

1–3) and four known iridoid glucosides (4), in addition to anown phenylethanoid glycoside (8). Their structures weretermined as aquaticoside A (1), aquaticoside B (2), aquati-oside C (3), veronicoside (4), catalposide (5), verproside6), verminoside (7) and martynoside (8) by extensive 1Dnd 2D-NMR spectroscopy (Harput et al., 2004) (Fig. 1).nti-inflammatory activity of the main compounds was agssayed in the same in vivo system. Due to the minute q

ity of compounds,3 and7 were not applied to animals fctivity assessment, while1, 2 and8 were combined accor

ng to their molar ratio in the Fr. B (3–12) and administere

agallis aquatica herbs on carrageenan-induced hind paw edema in m

S.E.M. (% inhibition)

min 270 min 360 min

± 3.6 57.3± 4.1 64.0± 4.4± 3.6 (34.5)** 38.2± 2.9 (33.3)** 39.1± 2.8 (38.9)***

± 4.2 (19.8) 44.6± 4.0 (22.2) 46.8± 4.8 (26.9)*

± 3.2 (7.9) 53.2± 3.8 (7.2) 62.2± 3.6 (2.8)± 3.1 (23.4) 39.7± 2.5 (30.7)* 41.0± 3.3 (35.9)**

± 2.9 (18.4) 42.5± 3.7 (25.8) 47.0± 3.8 (26.6)*

± 3.3 54.5± 4.2 (4.9) 57.5± 5.6 (10.2)± 2.5 (7.5) 53.0± 3.1 (7.5) 58.3± 3.2 (9.4)± 3.9 (21.4) 45.2± 3.6 (21.1) 49.2± 4.2 (23.1)± 2.4 (32.9)** 37.5± 2.6 (34.6)** 38.2± 3.9 (40.3)***

174 E. Kupeli et al. / Journal of Ethnopharmacology 102 (2005) 170–176

Table 2Effect of active principles on carrageenan-induced hind paw edema in mice

Compound Dose (mg/kg) Swelling thickness (×10−2 mm)± S.E.M. (% inhibition)

90 min 180 min 270 min 360 min

Control 42.7± 3.9 49.3± 4.4 56.3± 4.7 61.3± 4.7(6) 250 41.8± 4.7 (2.1) 46.7± 5.0 (5.3) 52.5± 4.8 (6.7) 55.7± 3.5 (9.1)

500 38.8± 3.6 (9.1) 41.8± 3.5 (15.2) 45.3± 4.2 (19.5) 46.8± 3.5 (23.7)*

684 33.8± 3.0 (20.8) 38.2± 3.0 (22.5) 43.9± 2.9 (22.0)* 44.5± 2.6 (27.4)*

(5) 250 43.5± 4.9 47.5± 5.0 (3.7) 51.8± 4.8 (7.9) 54.0± 3.7 (11.9)392.8 35.7± 2.9 (16.4) 39.0± 3.9 (20.9) 42.8± 3.9 (23.9) 45.9± 3.2 (25.1)*

500 34.7± 4.2 (18.4) 37.2± 3.9 (24.5) 39.5± 2.8 (29.8)* 42.3± 4.7 (30.9)*

(4) 92.8 36.7± 3.3 (14.1) 40.7± 3.4 (17.4) 44.4± 3.2 (21.1) 44.9± 3.2 (26.8)*

250 37.7± 4.6 (11.7) 43.0± 4.2 (12.8) 46.8± 4.2 (16.9) 50.2± 4.9 (18.1)500 36.3± 4.9 (14.9) 39.0± 5.0 (20.9) 40.2± 3.7 (28.6)* 41.3± 4.0 (32.6)*

(1, 2, 8) 44.1 36.7± 3.4 (14.1) 43.0± 4.3 (12.8) 46.8± 3.7 (16.9) 48.9± 4.0 (20.2)

Indomethacin 10 31.5± 2.9 (26.2) 35.1± 2.9 (28.8)* 38.5± 2.3 (31.6)** 39.7± 2.3 (35.2)**

* p < 0.05 Significant from control.** p < 0.01 Significant from control.

a test sample. The main iridoid constituents of the active frac-tion; veronicoside (4), catalposide (5) and verproside (6) arecatalpol derivative iridoid glucosides and were found to showsignificant inhibitory effect on carrageenan-induced hind pawedema in mice. These iridoids were administered in the dosesestimated according to their molar ratio in the active fraction,as well as given in standard doses of 250 and 500 mg/kg,in order to make a direct comparison of their activities. Asshown in (Table 2), significant anti-inflammatory activity wasobserved for iridoid glucosides4–6 in the estimated doses aswell as at 500 mg/kg.

To determine antinociceptive activity of the plant, theextracts, fractions and main compounds were studied usingp-benzoquinone-induced writhing model in mice. As shown inTable 3, MeOH extract, Fr. B (3–12) and Fr. C (13–19) frac-tions inhibited the writhes although not as potent as ASA.Since Fr. B (3–12) was also found to be the most activefraction in anti-inflammatory activity studies, antinocicep-tive activity of the main iridoid glucosides isolated from thisfraction were investigated. Compounds verproside (6) andcatalposide (5) were found to possess significant antinoci-ceptive activity without inducing any apparent acute toxicity

or gastric damage, while the activity of veronicoside (4) wasinsignificant and induced some gastric lesions in a higherdosage (Table 4).

Previously, anti-inflammatory effect of several iridoidderivatives has been reported by several other authors.Recioet al. (1994)studied the anti-inflammatory activity of twelveiridoid glycosides against two inflammation models in mice,carrageenan-induced paw edema and TPA-induced mouseear edema. Loganic acid and two catalpol derivatives, aucu-bin and 6′-vanilloylcatalpol, were found to possess potentinhibitory activity on the former, while catalpol derivatives,aucubin, verbenalin and loganin, were found remarkablyactive on the latter model. According to their results of exper-iments, systemic anti-inflammatory activity of iridoids wasmodest, while a higher efficacy could be observed throughtopic application as evidenced by TPA-edema model. Theyhave further suggested a structure-activity relationship fortopic anti-inflammatory activity of iridoids. Accordingly,OH-substitution as C5, unsaturation as C7–C8, methyl sub-stitution of carbonyl C11, and integrity of the cyclopentanering were essential for higher activity. Some of the results sug-gested in the study ofRecio et al. (1994)were later supported

Table 3Effect of H2O and MeOH extracts and fractions from MeOH extract ofVeronica anagallis aquatica herbs againstp-benzoquinone-induced writhings in mice

Material Dose (mg/kg) Number of writhings± S.E.M. Inhibitory ratio (%) Ratio of ulceration

CMHFFFFFFA

*

ontrol 45.7± 3.9eOH extract 500 29.3± 2.2

2O extract 500 33.9± 3.8r. A (1–2) 222.6 42.0± 2.7r. B (3–12) 606.8 31.0± 3.2r. C (13–19) 46.2 35.2± 2.8r. D (20–22) 39.6 40.0± 2.6r. E (23–34) 57.2 44.0± 3.3r. F (35–40) 27.6 34.5± 4.0SA 200 19.0± 2.1

** p < 0.01 Significant from control.** p < 0.001 Significant from control.

0/635.9** 0/625.8** 0/68.1 0/632.2** 0/622.9** 0/612.5 1/63.7 0/624.5 0/658.4*** 4/6

E. Kupeli et al. / Journal of Ethnopharmacology 102 (2005) 170–176 175

Table 4Effect of active principles againstp-benzoquinone-induced writhings in mice

Compound Dose (mg/kg) Number of writhings± S.E.M. Inhibitory ratio (%) Ratio of ulceration

Control 45.3± 4.2 0/6(6) 250 34.0± 4.2 24.9 0/6

500 29.0± 2.3 35.9** 0/6684 31.7± 3.4 30.0* 0/6

(5) 250 36.8± 3.0 18.8 0/6392.8 32.9± 2.8 27.4* 0/6500 30.8± 2.6 32.0* 0/6

(4) 92.7 34.1± 2.1 24.7 0/6250 32.3± 3.1 28.7 0/6500 33.0± 4.7 27.2 1/6

(1, 2, 8) 44.1 37.9± 4.4 16.3 0/6

ASA 100 21.6± 1.9 52.3*** 2/6

* p < 0.05 Significant from control.** p < 0.01 Significant from control.

by other authors as well; i.e. picroside II, a catalpol derivative,was only active topically and was also found to possess mod-erate topic anti-inflammatory activity in mouse ear-swellingassay byJia et al. (1999).

Shapoval et al. (1998)isolated a potent anti-inflammatoryiridoid, ipolamiide, fromStachytarpheta cayennensis (Ver-benaceae) against carrageenan-induced paw edema (70.2%inhibition). A structurally related compound, lamiide (lackof C7-OH group), was also reported to possess a dose-dependent and potent anti-inflammatory activity (79%)against carrageenan-induced edema (Delaporte et al., 2002)in similar experimental protocols and at the same dose lev-els. However, these results were not in agreement with theresults ofRecio et al. (1994), i.e. lamiide was reported topossess a weak activity on oral administration at the samedose (100 mg/kg), while a moderate activity when appliedtopically.

Iridoid glucosides, harpagoside B, 8-O-acetyl harpagideand, a catalpol derivative, koelzioside fromScrophulariadeserti were shown to possess a moderate inhibitory activityagainst carrageenan-induced paw edema in rats at 10 mg/kgdoses, decreased edema 30, 23 and 26%, respectively (Ahmedet al., 2003). Authors suggested that substitution of cin-namoyl moiety may enhance the activity. Harpagoside (C6-demethyl derivative) was also reported to possess a moderateactivity byRecio et al. (1994)on oral and topic applications.

atao iri-d s iso-l hb alpold -s ntat nan-i CO ved,w

vided a higher activity. However, in our study esterificationfrom C6 with benzoic acid (as in veronicoside), or phenolicacids (p-hydroxybenzoic acid in catalposide and protocat-echic acid in verproside) provided a significant increase inanti-inflammatory activity in higher doses (Fig. 1). On theother hand, the activity of a mixture of loganic acid deriva-tives, aquaticoside A, aquaticoside B with a phenylpropanoidderivative, martynoside, was weak.

In a reference survey, no more reports about the antinoci-ceptive activity of iridoid glucosides have been found so far.The catalpol derivative iridoid glucosides, catalposide (5) andverproside (6), were shown to possess a significant antinoci-ceptive activity in addition to their anti-inflammatory activitywithout inducing any apparent gastric lesion. Further studiesare required in order to reveal the possible pharmacokineticfactors contribute to the activity of these compounds.

Furthermore, the conclusion suggested byRecio et al.(1994) reporting that a higher efficacy could be observedthrough topic application of iridoid glucosides than oraladministration provides a further support to folkloric utiliza-tion in Turkey, where people applied boiled herbs ofVeronicaanagallis-aquatica on their extremities, while taking bath inwarm aqueous extract to alleviate rheumatic pain (Fujita etal., 1995).

A

GaziU l toP sity,A en.

R

A d,doid

According to the results of present study, further dn the anti-inflammatory and antinociceptive activity ofoid glucosides were presented. Among the compound

ated fromVeronica anagallis-aquatica aerial parts througioassay-guided fractionation procedures, the main caterivative iridoid glucosides; veronicoside (4), catalpoide (5) and verproside (6) were found to possess potenti-inflammatory activity.Recio et al. (1994)reported

hat catalpol was completely inactive against carrageenduced edema, and through vanilloyl substitution from6-

H (picroside II) only a small increase could be obserhile substitution of vanilloyl from glucose C′6 moiety pro-

cknowledgements

This study was supported by the Research Fund ofniversity (GUBAP-EF 02/2003-16). Authors are gratefurof. H. Duman of the Faculty of Sciences, Gazi Univernkara, Turkey, for the authentication of the plant specim

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