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The anti-inammatory effects and the inhibition of neutrophil responsiveness by Barleria lupulina and Clinacanthus nutans extracts

Payong Wanikiat a, , Ampai Panthong d , Pacharawan Sujayanon a , Chalobon Yoosook b , Adriano G. Rossi e , Vichai Reutrakul c
a b

Department of Pharmacology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand c Department of Chemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand d Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand e MRC Centre for Inammation Research, The Queens Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UK Received 8 June 2007; received in revised form 2 November 2007; accepted 15 November 2007 Available online 5 December 2007

Abstract Aim of the study: To investigate the anti-inammatory activities of Barleria lupulina Lindl and Clinacanthus nutans (Burm.f.) Lindau extracts using two neutrophil-dependent acute inammatory models and, in order to elucidate underlying cellular mechanisms, the effects of the extracts on human neutrophil responsiveness was investigated. Materials and methods: The in vivo inammatory models examined were carrageenan-induced paw oedema and ethyl phenylpropiolate-induced ear oedema in rats. Myeloperoxidase (MPO) activity was assayed as an indicator of neutrophil migration. Human neutrophil functional responsiveness was determined by measuring fMLP-induced chemotaxis, superoxide anion generation (SAG), and release of MPO and elastase. Apoptosis was assessed morphologically and ow-cytometrically. Neutrophil viability was assessed by trypan blue exclusion and MTT cytotoxicity assays. Results: Both extracts induced powerful dose-dependent inhibitory effects in both edema models in rats. Importantly, there was a signicant inhibition of MPO activity in the inamed tissue indicating that the anti-inammatory effect of the extracts is associated with reduced neutrophil migration. Although both extracts did not affect neutrophil viability or apoptosis, treatment of neutrophils with the extracts concentration-dependently inhibited fMLP-induced chemotaxis, SAG, and MPO and elastase release. Conclusions: These ndings suggest that the powerful anti-inammatory properties of Barleria lupulina Lindl and Clinacanthus nutans (Burm.f.) Lindau extracts are mediated, in part, by inhibition of neutrophil responsiveness.Barleria lupulina Lindl, Clinacanthus nutans (Burm. f.)Lindau; Oedema formation; Neutrophil responsiveness 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Barleria lupulina Lindl; Clinacanthus nutans (Burm. f.) Lindau; Oedema formation; Neutrophil responsiveness

1. Introduction Barleria lupulina Lindl and Clinacanthus nutans (Burm. f.) Lindau, both belonging to the family Acanthaceae, are well-known medicinal plants widely used in Thai traditional medicine (Tiangburanatam, 1996) and are categorized as essential medicinal plants for primary healthcare by the Thai

Corresponding author. Tel.: +66 2 2015644; fax: +66 2 3547157. E-mail address: scpwt@mahidol.ac.th (P. Wanikiat).

Ministry of Public Health (National Drug and Committee, 2006). Important constituent of Barleria lupulina extracts are iridoid glucosides (Byrne et al., 1987; Kanchanapoom et al., 2001; Suksamrarn, 1986; Suksamrarn et al., 2003; Tuntiwachwuttikul et al., 1998) and principle constituents of Clinacanthus nutans are avonoids, stigmasterol, -sitosterol, lupeol, betulin, C-glycosyl avones, vitexin, isovitexin, shaftoside, isomollupentin, 7-O- -gluco pyranoside, orientin, isoorientin, cerebrosides monoacylmonogalactosylglycerol and sulfur-containing glucosides (Dampawan et al., 1977; Teshima et al., 1998; Tuntiwachwuttikul et al., 2004). The aerial part of

0378-8741/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.11.035

Barleria lupulina has been traditionally used for mental tension, diabetes, rheumatoid arthritis and snake bite (Chopra et al., 1968). Clinacanthus nutans extract has been widely used as an anti-hepatitis and anti-herpes agent. Extracts from the leaves of both Barleria lupulina and Clinacanthus nutans have been used as anti-inammatory agents for the treatment of insect bites and allergic responses and as remedies for herpes simplex and varicella zoster virus lesions. Clinical trials for the efcacy of topical application of Clinacanthus nutans preparation in the treatment of recurrent genital herpes simplex virus infections (Sangkitporn et al., 1993) and herpes zoster (Charuwichittratana et al., 1996) have been reported. Barleria lupulina extract exhibited high antiviral activity against HSV2 while Clinacanthus nutans showed reduced antiviral activity (Yoosook et al., 1999). The methanol extract of the aerial part of Barleria lupulina was recently shown to have a protective effect against experimental gastric and duodenal ulcer formation (Suba et al., 2004). It was reported that ethanolic extract of Clinacanthus nutans leaves at the highest dose of 1.3 g/kg given orally, subcutaneously or intraperitoneally did not produce any signs of acute toxicity in mice (Chavalittumrong et al., 1995). A subchronic toxicity study was also performed in rats by giving the extract daily for 90 days at the oral doses up to 1 g/kg and the results from histopathological examination showed no abnormalities of internal organs (Chavalittumrong et al., 1995). Despite these widely reported biological effects, many of which involve properties associated with limiting the inammatory response, understanding the mechanisms involved remain to be elucidated. Polymorphonuclear neutrophils play an important role in host defense and contribute to the propagation and maintenance of acute and chronic inammation. Dysregulated recruitment, activation or clearance of neutrophils leads to the liberation of granule products, release of toxic oxygen metabolites (e.g., O2 ) and production of various inammatory mediators which can contribute to tissue damage associated with inammatory disorders (e.g., myocardial infarction, ischaemia/reperfusion injury, adult respiratory distress syndrome, rheumatoid arthritis and atherosclerosis) (Cuzzocrea et al., 2001). Neutrophils undergo apoptosis (programmed cell death), a process that can be modulated by cytokines or other pro-inammatory agents prior to their removal by macrophages (Walker et al., 2005). Thus, apoptosis is regarded as a crucial process controlling the resolution of inammation. However, if neutrophil clearance is impaired they rapidly undergo secondary necrosis with the release of potentially toxic intracellular contents with macrophage phagocytosis of post-apoptotic cells leading to release of pro-inammatory mediators, potentially prolonging the inammatory response (Gilroy et al., 2004). In acute inammation, neutrophil inltration and the potential for inammatory neutrophils to cause tissue damage via the release of toxic reactive oxygen species (ROS) and granule enzymes is very high. Suppression of neutrophil functions would likely limit the inammatory response and this effect has been implicated in the mechanisms of action of some non-steroidal anti-inammatory agents since inhibition of constitutive and inducible cyclo-oxygenase

(COX) does not seem to account for all their anti-inammatory effects (Kankaanranta et al., 1994). Although, Barleria lupulina and Clinacanthus nutans have been widely used in primary health care in Thailand as anti-inammatory agents, the molecular and cellular mechanisms underlying their anti-inammatory activities remain elusive. Therefore, the purpose of this study was to investigate the anti-inammatory activity of the Barleria lupulina and Clinacanthus nutans extracts using in vivo models of inammation and to investigate whether the cellular mechanism may be mediated by effects on neutrophil functional responsiveness. 2. Materials and methods 2.1. Plant materials and plant extracts The whole plants were collected in Nan and Chonburi provinces, Thailand, respectively, during September 2000. Leaves and twigs of Barleria lupulina and the whole plant of Clinacanthus nutans were collected and voucher specimens of Barleria lupulina (BKF 102216) and of Clinacanthus nutans (BKF 130056) have been deposited at the forest Herbarium, Royal Forestry Department, Ministry of Agriculture and Co-operatives, Bangkok, Thailand. The specimens were airdried at room temperature (30 C) and pulverized plants were percolated repeatedly with methanol at room temperature for 67 days. The extracts were ltered through Whatman lter paper no. 1 and evaporated using a rotary evaporator below 40 C and freeze-dried to remove any remaining trace solvent. The weights of the crude extracts were 51.2 g from 0.9 kg of Barleria lupulina and 233.5 g from 2 kg of Clinacanthus nutans. They were dissolved in acetone, solubilized by complexing with polyvinyl pyrrolidone (ratio 1:4, w/w), freeze-dried and kept at 20 C. Appropriate amounts of the extracts were reconstituted in culture medium and ltered through 0.45 m membranes before use. This procedure removed undened polyphenolic compounds, if any, from the extracts (Tan et al., 1991). 2.2. Assessment of lipopolysaccharide contamination Barleria lupulina and Clinacanthus nutans extracts were checked for bacterial LPS contamination using a commercial test kit of limulus amebocyte lysate (LAL) as indicated by the supplier (Pyrogent -5000, Biowhittaker, Inc., USA). 2.3. Animals Male SpragueDawley rats weighing 4060 g and 100120 g (Brattsand et al., 1982) purchased from the National Laboratory Animal Centre, Nakorn Pathom, Thailand, were used. Animals, with unrestricted access to water and food, were kept in a room with a temperature of 24 1 C and a 12 h light/dark cycle. All animals were acclimatized for at least 1 week before starting the experiments and experiments were performed in accordance with the General Guidelines for Methodologies

on Research and Evaluation of Traditional Medicine (WHO, 2000). 2.4. Ethyl phenylpropiolate (EPP)-induced rat ear oedema and assessment of tissue MPO activity Ear oedema was induced, as described (Brattsand et al., 1982) with some modication, by the topical application of EPP (Fluka Chemie AG, Switzerland) (dissolved in acetone and administered 1 mg/20 l/ear) to the inner and outer surfaces of both ears of male rats (4060 g) by means of an automatic microlitre pipette. Barleria lupulina extract (3, 6, 9 mg/20 l acetone per ear) or Clinacanthus nutans extract (3, 6, 9 mg/20 l acetone per ear) or indomethacin (2 mg/20 l acetone per ear) was applied topically to the inner and outer surfaces of the ear just before the irritant. The thickness of each ear was measured with vernier callipers before and at 15, 30, 60 and 120 min after oedema induction. The increase in ear thickness was compared with the control group and percent inhibition was calculated. After 2 h, animals were decapitated and equal sections of both ears were punched out and weighed immediately. Tissue samples of each ear were assessed for the neutrophil marker enzyme, MPO, as described (Bradley et al., 1982) with some modications. The entire tissue of each ear was homogenized in 50 mM phosphate buffer (Na2 HPO4 ) (pH = 5.4) containing 0.5% hexadecyl trimethyl ammonium bromide (HTAB) using an Ultra-Turrax homogenizer-dispenser. This was then taken through three cycles of freezethaw followed by centrifugation (10,000 g; 15 min; 4 C). The resulting supernatant was assayed spectrophotometrically for MPO activity. Briey, a portion of supernatant (30 l) was aliquoted into a 96 well-plate and mixed with 200 l of 0.05 M citrate phosphate buffer (pH = 5) containing 1 mg/ml 3,3 ,5,5 tetramethylbenzidine (TMB) (Sigma chemical company, USA), serving as MPO substrate, and 0.012% (v/v) hydrogen peroxide. The absorbance was measured at a wavelength of 450 nm during a 20 min period. One unit of MPO activity is dened as the quantity of enzyme degrading 1 M of peroxide per min at 25 C expressed in units per mg of tissue. 2.5. Carrageenan-induced hind paw oedema The anti-inammatory activity of Barleria lupulina and Clinacanthus nutans extracts was assessed by the carrageenaninduced hind paw oedema model in rats (Winter et al., 1962). Male SpragueDawley rats (100120 g) were used. Barleria lupulina or Clinacanthus nutans extracts (50200 mg/kg) or vehicle (5% Tween 80) was given orally to the animals (in an equivalent volume of 0.5 ml/kg) 1 h before oedema was induced on the plantar side of the right hind paw by intradermal injection of carrageenan (0.05 ml of 1% carrageenan, Sigma chemical company, USA). One group received the reference drug indomethacin (20 mg/kg). Foot volumes of animals were determined prior to and at 3 h after induction of oedema by means of a volume displacement technique using a plethysmometer.

2.6. DPPH scavenging activity Reduction of the stable free radical was determined as described (Cavin et al., 1998) with some modications. 2,2-diphenyl-1-pycryl-hydrazyl (DPPH) (Sigma chemical company, USA), in methanol (1.8 ml; 0.15 mM) was added to 200 l of Barleria lupulina or Clinacanthus nutans extracts (1400 g/ml). Absorbance at 517 nm was determined spectrophotometrically for 10 min and the scavenging activity was calculated as a percentage of radical reduction. Trolox (Sigma chemical company, USA), a known radical scavenger, at the concentration of 3.125400 g/ml was used as a reference compound. 2.7. Electron paramagnetic detection of superoxide anion The possible direct scavenging effect of Barleria lupulina or Clinacanthus nutans extracts was determined using electron paramagnetic resonance (EPR) technique. Spin-trapping experiments were performed in PBS containing a superoxide generator [pyrogallol (10 M)], the chemical spin trap [1-hydroxy2,2,6,6-tetramethyl-4-oxo-piperidine. HCl (Tempone-H) 1 mM] (Dikalov et al., 1997) prepared in water containing EDTA (10 mM) and in the presence or absence of Barleria lupulina or Clinacanthus nutans extracts. Reaction mixtures were incubated at 37 C throughout the experiments and the intensity of the EPR signal corresponding to the formation of the stable radical adduct 4-oxo-Tempo (triplet centred at 3365G) was measured (arbitrary units) at 10 and 60 min (Magnettech Miniscope MS100 Xband spectrometer) with the following parameter settings: eld sweep 51.2G, microwave frequency 9.5 GHz, microwave power 20 mW, modulation amplitude 1500 mG. In control experiments in the absence of pyrogallol, small increases in the EPR signal corresponding to the auto-oxidation of Tempone-H to 4-OxoTempo were subtracted from the corresponding experimental signals. 2.8. Preparation of human neutrophils Human neutrophils were isolated by PolymorphprepTM (Axis-Shield) density gradient centrifugation. Briey, venous blood obtained from healthy donors, using heparin as an anticoagulant, was mixed with an equal volume of polymorphprepTM , and the mixture centrifuged at 400 g for 35 min at room temperature. After centrifugation, PMN were harvested and washed with phosphate-buffered saline (PBS). Any contaminating red cells were removed by hypotonic lysis. The cells were >99% viable as determined by trypan blue exclusion and were resuspended as required (see below). 2.9. Cytotoxicity assay Barleria lupulina and Clinacanthus nutans extracts were tested for their cytotoxicity of cultured neutrophils by a colourimetric assay (Mosmann, 1983) prior to the investigation of their effects on human neutrophil functional responsiveness and apoptosis. Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyl tetrazolium bromide (MTT) (Sigma chemical company, USA) was used as a substrate which is metabolically reduced by mitochondrial dehydrogenase enzymes to yield a soluble, coloured formazan product. Human neutrophils (3 106 cells/ml in RPMI 1640 with 10% autologous serum) were incubated with Barleria lupulina or Clinacanthus nutans extracts (11000 g/ml) in a 96 well-plate for 45 min or 4 h at 37 C, and then MTT (5 mg/ml) was added and incubated at 37 C for an additional 4 h. The reaction was stopped by adding 0.04 N HCl in isopropanol. The formazan product of MTT is measured spectrophotometrically at a reference wavelength of 630 nm and a test wavelength of 570 nm using a microplate reader (SpectraCountTM , PACKARD). Results are expressed in absolute absorbance readings; a decrease in absorbance indicating a reduction in cell viability. The cytotoxicity of the herb extracts was expressed as 50% cytotoxic concentration (CC50 ). All experiments were done at least six times with each treatment performed in triplicate. 2.10. Chemotaxis assay An in vitro assay for chemotaxis of neutrophils was performed using a 96 well chemotaxis chamber (Neuroprobe, Cabin John, MD, USA) as described previously (Wanikiat et al., 1997). In brief, neutrophils (3 106 cells/ml in RPMI 1640) were treated with Barleria lupulina or Clinacanthus nutans extract (0.1100 g/ml) or indomethacin (0.01100 g/ml) for 10 min at 37 C. The bottom wells of the chamber were lled with N-formyl-methionyl-leucyl-phenylalanine (1 M fMLP) (Sigma chemical company, USA). The top plate with the installed polyvinylpyrrolidone-free polycarbonate lter (3 m) was placed onto the lled bottom plate, and upper wells were lled with neutrophils. After incubation for 45 min at 37 C under a 5% CO2 atmosphere, the lter removed, washed, xed and stained with DiffQuikTM (Baxter Diagnostics AG, U.K.). Chemotaxis was quantied spectrophotometrically by measuring absorbance at 550 nm and the magnitude of the absorbance taken as being directly proportional to the number of cells which migrated and were trapped in the lter. Basal absorbance was taken as cells without fMLP. Each treatment was carried out in triplicate. 2.11. Chemokinesis assay In order to study the effects of Barleria lupulina or Clinacanthus nutans extract on neutrophil chemotaxis and chemokinesis, an in vitro assay for neutrophil chemokinesis was also performed using a 96 well chemotaxis chamber. For the study of chemotaxis, fMLP (0.1 M) in 30 l chemotaxis buffer was added to the bottom wells of the chamber. For the study of chemokinesis, fMLP in 30 l chemotaxis buffer was added to the bottom wells of the chamber and in 30 l chemotaxis buffer to the upper wells of the chambers. Neutrophils (3 106 cells/ml in RPMI 1640) were treated as mentioned above and were added to the upper wells. In both cases, the chamber was incubated for 45 min at 37 C under a 5% CO2 atmosphere. The number of cells that migrated to the bottom wells was quantied microscopically

using a Neubauer hemocytometer. Viability (assessed by trypan blue exclusion) at the end of the assay in the lower chamber remained >95 2%, even in the presence of both extracts. 2.12. Superoxide anion generation (SAG) Neutrophil SAG was determined by spectrophotometric evaluation of the SOD-inhibitable reduction of ferricytochrome C to ferrocytochrome C (A 550 nm) as described (Talpain et al., 1995). Briey, aliquots of neutrophils (1.5 106 cells/ml) were pre-incubated with Barleria lupulina or Clinacanthus nutans extracts, indomethacin (0.01100 g/ml) or the vehicle (PBS) for 10 min at 37 C followed by addition of fMLP (100 nM) and incubated for further 10 min at 37 C. SAG was measured by the reduction of cytochrome C at 550 nm. Basal absorbance was taken as cells without fMLP. Each treatment was carried out in triplicate. 2.13. Myeloperoxidase (MPO) production Human neutrophil MPO production was determined spectrophotometrically using 3,3 ,5,5 -tetramethylbenzidine (Sigma chemical company, USA) as a substrate. The assay is based on the oxidation of TMB by MPO in the presence of H2 O2 (Suzuki et al., 1983) with some modication. Briey, cells (2.5 106 cells/ml) were pre-incubated with Barleria lupulina extract, Clinacanthus nutans extract, indomethacin (0.01100 g/ml) or vehicle for 10 min at 37 C and then stimulated with cytochalasin B (5 g/ml) and fMLP (100 nM) for 10 min at 37 C. After centrifugation (320 g; 10 min; 4 C), supernatants were incubated with the reaction mixture (1 mg/ml TMB in 0.05 M citrate phosphate buffer (pH = 5.0) supplemented with 0.012% (v/v) H2 O2 ). Oxidized TMB formed a soluble chromophore. The reaction was terminated after 5 min by the addition of 4 M H2 SO4 and the absorbance was measured spectrophotometrically at a wavelength of 450 nm. The possible direct inhibitory effects of the extracts on MPO activity were also measured. Briey, a sample of Barleria lupulina or Clinacanthus nutans extracts was aliquoted into a 96 well-plate and mixed with 0.05 M citrate phosphate buffer (pH = 5) containing 1 mg/ml TMB and 0.012% (v/v) H2 O2 . The absorbance was measured at a wavelength of 450 nm during a 20 min period. One unit of myeloperoxidase activity is dened as the quantity of enzyme degrading 1 M of peroxide per min at 25 C expressed in units per mg of tissue. 2.14. Elastase release by human neutrophils Neutrophil elastase activity was determined as described (Barrett, 1981) using N-tert-butoxy-carbonyl-l-alanine pnitrophenyl ester (t-Boc) (ICN Biomedicals Inc., USA) as a substrate and p-nitrophenol release was measured spectrophotometrically. Briey, cells (2.5 106 cells/ml) were pre-incubated with Barleria lupulina extract, Clinacanthus nutans extract, indomethacin (0.01100 g/ml) or vehicle for 10 min at 37 C and then stimulated with cytochalasin B (5 g/ml) and fMLP (100 nM) for 10 min at 37 C. After centrifugation (2000 g;

10 min; 4 C) supernatants were incubated with t-Boc (200 M) for 20 min at 37 C and the extent of p-nitrophenol release was measured spectrophotometrically at a wavelength of 414 nm in a microplate reader. Possible direct inhibitory effects of the extracts on elastase activity were also assessed (Barrett, 1981).

2.17. Annexin V binding Assessment of neutrophil apoptosis was also performed by ow cytometry using FITC-labelled recombinant human annexin V (Bender Med Systems, Vienna, Austria) that binds to phosphatidylserine exposed on the surface of apoptotic cells (Koopman et al., 1994). FITC-annexin V (180 l) diluted 1:2000 in HBSS plus 5 mM CaCl2 , was added to 20 l of neutrophils, which were incubated either in the presence or absence of Barleria lupulina or Clinacanthus nutans extracts or dexamethasone (used as a reference drug) for 20 h of culture, and incubated (4 C, 10 min) before ow cytometric analysis using a FACS Calibur (Beckton Dickinson, Oxford, U.K.). 2.18. Statistical analysis Results were expressed as mean S.E.M. Statistical analysis of data was performed by one-way analysis of variance (ANOVA) followed by the Dunnetts test for multiple comparisons of paired data. P values <0.05 were considered signicant. Percentage of inhibition was calculated from the difference between drug-treated group and control group. 3. Results

2.15. In vitro culture of neutrophils for assessment of apoptosis In vitro culture of neutrophils for assessment of apoptotic neutrophils was performed (Ward et al., 1999a). Briey, neutrophils (5 106 cells/ml) were re-suspended in Iscoves DMEM (Life Technologies, Paisley, U.K.) containing 100 U/ml penicillin and 100 U/ml streptomycin and supplemented with 10% autologous serum. They were cultured in at-bottomed 96 well polypropylene plates (37 C, 5% CO2 ) for 20 h in the presence of either Iscoves DMEM (control) or Barleria lupulina or Clinacanthus nutans extracts at various concentrations (10500 g/ml). Dexamethasone (Sigma chemical company, USA) (1 M) was used as a reference drug. All experiments were done at least three times with each treatment performed in triplicate.

2.16. Morphological assessment of neutrophil apoptosis After 20 h of culture either in the presence or absence of Barleria lupulina or Clinacanthus nutans extracts at various concentrations (10500 g/ml), neutrophils were gently mixed and 100 l harvested from each well, cytocentrifuged (Shandon Cytospin II, Shandon U.K.) and the slide preparations air-dried, xed in methanol and stained with DiffQuikTM . Cell morphology was examined by 100 objective oil immersion light microscopy to determine the proportion of apoptotic neutrophils (Ward et al., 1999b). For each treatment, slides were prepared in duplicate, a total of at least 500 neutrophils were counted over a minimum of ve high power elds. The data were expressed as the mean percent apoptosis S.E.M. Cell viability was determined in parallel by assessing the ability of neutrophils to exclude the vital dye trypan blue, determined by light microscopy.

3.1. Lipopolysaccharide contamination The LPS concentrations in the Barleria lupulina and Clinacanthus natans extracts determined by the LAL test were 2.4 104 and 4.87 104 EU/ g, respectively. 3.2. EPP-induced rat ear oedema and measurement of MPO activity The average basal values of rat ear thickness (t = 0) of control animals, indomethacin-treated animals, and Barleria lupulina and Clinacanthus nutans extracts treated animals were 353 8, 350 10, 348 8 and 349 5 m, respectively. Barleria lupulina and Clinacanthus nutans extracts were found to signicantly inhibit ear oedema formation after topical application of EPP in a dose-dependent manner (Table 1). The most potent inhibition of both extracts at all concentra-

Table 1 Inhibitory effects of Barleria lupulina and Clinacanthus nutans extracts on EPP-induced ear oedema in rats Group Dose (mg/ear) Control Indomethacin Barleria lupulina 2 3 6 9 3 6 9 Oedema thickness ( m) 15 min 63 3 20 5*** 37 3*** 10 7*** 13 4*** 37 3*** 29 4*** 13 7*** 30 min 163 3 90 4*** 103 6*** 70 4*** 53 8*** 117 3*** 90 4*** 77 3*** 1h 150 7 90 4*** 107 4*** 63 6*** 50 7*** 110 4*** 77 3*** 77 3*** 2h 143 3 90 4*** 87 4*** 50 4*** 50 9*** 103 6*** 60 5*** 70 4*** Oedema inhibition (%) 15 min 68 41 84 79 41 54 79 30 min 45 37 57 67 28 45 53 1h 40 29 58 67 27 49 49 2h 37 39 65 65 28 58 51

Clinacanthus nutans

Results are expressed as mean S.E.M. of six animals. *** P < 0.001 indicates statistically signicant difference from control group.

Table 2 Effects of Barleria lupulina and Clinacanthus nutans extracts on MPO activity in EPP-induced rat ear oedema Group Control Indomethacin Barleria lupulina Clinacanthus nutans Dose (mg/ear) 2 9 9 MPO (U/mg tissue) 2.7 1.2 1.3 1.5 0.1 0.08* 0.05* 0.1* Inhibition (%) 55.6 51.8 44.4

Results are expressed as mean S.E.M. of six animals. * P < 0.05 indicates statistically signicant difference from control group.

tions was observed at 15 min. Indomethacin, a non-steroidal anti-inammatory agent, at the dose of 2 mg/ear, signicantly inhibited the ear oedema formation. The two extracts at a dose of 9 mg/ear, when separately performed to measure the effect on MPO, signicantly inhibited MPO activity after 120 min of ear oedema induction by EPP (P < 0.05, ANOVA, Table 2). 3.3. Carrageenan-induced rat paw oedema The average basal foot volumes (t = 0) of control animals, indomethacin-treated animals, Barleria lupulina and Clinacanthus nutans extracts treated animals were 139 4, 133 1, 132 3 and 136 5 ml, respectively. As shown in Table 3, carrageenan-induced oedema in the hind paw was signicantly reduced after oral administration of Barleria lupulina or Clinacanthus nutans extracts at doses of 50, 100 and 200 mg/kg. Indomethacin at a dose of 20 mg/kg exerted pronounced inhibition of the oedema formation of the paw. 3.4. DPPH scavenging activity Both extracts of Barleria lupulina (EC50 of 500 21.2 g/ ml) and Clinacanthus nutans (EC50 of 240.1 15.3 mg/ml) exhibited very low radical scavenging activity compared to Trolox (EC50 of 7.5 2.3 g/ml) which showed high radical scavenging activity.
Table 3 Effects of Barleria lupulina and Clinacanthus nutans extracts on carrageenaninduced hind paw oedema in rats Group Control (5% Tween 80) Indomethacin Barleria lupulina Dose (mg/kg) 20 50 100 200 50 100 200 Paw oedema (ml) 3h 0.63 0.03 0.15 0.02*** 0.46 0.02*** 0.38 0.02*** 0.33 0.01*** 0.44 0.02*** 0.32 0.01*** 0.26 0.01*** Inhibition (%) 3h 76 27 40 48 30 49 59

Fig. 1. Direct scavenging activity of Barleria lupulina and Clinacanthus nutans extracts. Representative EPR spectra (A) and relative intensities (arbitrary units) (B) showing the EPR signals of the stable radical adduct 4-oxo-Tempo generated after incubation of pyrogallol (100 M) and Tempone-H (1 mM) in the absence or presence of Barleria lupulina or Clinacanthus nutans extracts (100 g/ml) at 37 C for 10 min (B) and 60 min (A and B). Results are mean S.E.M. (n = 3). * P < 0.05 indicates statistically signicant difference from control. The value of control spectra (Tempone-H) was subtracted from each treatment (B).

3.5. Direct scavenging activity of Barleria lupulina and Clinacanthus nutans extracts assessed by EPR The direct scavenging activity of Barleria lupulina and Clinacanthus nutans extracts was not observed when the two extracts were incubated with Tempone-H (the spin trap) and pyrogallol (a superoxide generator) for 10 min (Fig. 1B), but they showed signicant direct scavenging activities when the incubation time was extended to 60 min (Fig. 1A and B, P < 0.05, ANOVA).

Clinacanthus nutans

Results are expressed as mean S.E.M. of six animals. *** P < 0.001 indicates statistically signicant difference from control.

Fig. 2. Inhibitory effects of Barleria lupulina extract (0.1100 g/ml), Clinacanthus nutans extract (0.1100 g/ml) and indomethacin (0.01100 g/ml) on fMLP-induced human neutrophil chemotaxis. Chemotaxis was quantied spectrophotometrically at A550 nm . Results are mean S.E.M. of seven experiments using cells from different donors.

3.6. Cytotoxicity Reconstituted extracts of Barleria lupulina and Clinacanthus nutans were assessed for their cytotoxicity on neutrophils prior to the determination of their effects on neutrophil functional responsiveness and apoptosis. Barleria lupulina and Clinacanthus natans extracts at the concentration up to 500 g/ml did not affect neutrophil viability both during, 45 min and 4 h incubation periods. Only a slight cytotoxic effect was observed at the concentration of 1000 g/ml (IC50 > 1000 g/ml). 3.7. Neutrophil chemotaxis and chemokinesis When cells were pre-incubated with Barleria lupulina or Clinacanthus nutans extracts at concentrations of 0.1100 g/ml for 10 min at 37 C, neutrophil chemotaxis induced by fMLP (1 M) was signicantly suppressed in a concentrationdependent manner (P < 0.05, ANOVA) (Fig. 2), with an IC50 of 1.8 0.2 g/ml (n = 8) and an IC50 of 2.7 0.6 g/ml (n = 8), respectively. The inhibitory effect of the two extracts did not differ signicantly (P > 0.05), whereas indomethacin more potently (IC50 of 56.3 3.5 ng/ml; 0.16 0.01 M, n = 8) inhibited fMLP-induced neutrophil chemotaxis. Barleria lupulina and Clinacanthus nutans extracts (0.1100 g/ml) also exerted signicant inhibitory effects on neutrophil migration (control) in the absence of fMLP where Barleria lupulina extract exerted stronger inhibitory effect than that of Clinacanthus nutan extract (P < 0.05; IC50 = 24.4 6.4 and > 100 g/ml, n = 3, respectively, Fig. 3A and B) and their inhibitory effects were signicantly different from each other (P < 0.05, ANOVA). fMLP-induced (0.1 M) neutrophil chemotaxis which was represented as 100% maximal control (6.1 0.7 104 cells/ml, n = 3) and caused neutrophil chemokinesis which was represented as 100% maximal control for chemokinesis (1.5 0.05 104 cells/ml, n = 3). Pre-incubation
Fig. 3. Inhibitory effects of Barleria lupulina extract (0.1100 g/ml) (A) and Clinacanthus nutans extract (0.1100 g/ml) (B) on fMLP-induced human neutrophil chemotaxis and chemokinesis induced in the absence of an fMLP gradient. Neutrophil migration was quantied microscopically using a Neubauer hemocytometer. Results are mean S.E.M. of three experiments using cells from different donors.

of neutrophils with Barleria lupulina or Clinacanthus nutans extracts were found to attenuate fMLP-induced neutrophil chemotaxis in a concentration-dependent manner with IC50 of 2.9 0.5 g/ml, n = 3 (Fig. 3A) and 5.5 0.6 g/ml, n = 3 (Fig. 3B), respectively. Neutrophil chemokinesis induced in the absence of an fMLP gradient was also attenuated by the two extracts with an IC50 of 2.4 0.4 g/ml for Barleria lupulina (Fig. 3A) and 5.0 0.5 g/ml for Clinacanthus nutans, n = 3 (Fig. 3B). The inhibitory effects of the two extracts on chemokinesis were comparable. 3.8. Neutrophil superoxide anion generation Upon stimulation with fMLP (100 nM), neutrophils released O2 into the extracellular medium, as determined by SODinhibitable cytochrome C reduction. When the cells were pre-incubated with Barleria lupulina or Clinacanthus nutans extracts at the concentration of 0.1100 g/ml for 10 min at

Fig. 4. Inhibitory effects of Barleria lupulina extract (0.1100 g/ml), Clinacanthus nutans extract (0.1100 g/ml) and indomethacin (0.01100 g/ml) on fMLP-induced O2 generation in human neutrophils. Superoxide anion generation was measured by the reduction of cytochrome c at A550 nm . Results are mean S.E.M. of eight experiments using cells from different donors.

Fig. 5. Inhibitory effects of Barleria lupulina extract (0.1100 g/ml), Clinacanthus nutans extract (0.1100 g/ml) and indomethacin (0.01100 g/ml) on fMLP-induced MPO production in human neutrophils. MPO production was determined spectrophotometrically by the measurement of oxidized TMB formed at A450 nm . Results are mean S.E.M. of seven experiments using cells from different donors.

37 C, fMLP-induced SAG by human neutrophils was signicantly suppressed in a concentration-dependent manner (P < 0.05, ANOVA), where Barleria lupulina extract (IC50 of 8.8 1.1 g/ml, n = 8, Fig. 4) exerted stronger inhibition than that of the extract from Clinacanthus nutans (IC50 of 23.4 3.1 g/ml, n = 8, Fig. 4). Indomethacin (0.01100 g/ml) caused strong inhibition of fMLP-induced SAG in human neutrophils, with IC50 of 0.82 0.2 g/ml (2.3 0.17 M), n = 8. 3.9. Neutrophil MPO production The MPO production induced by 100 nM fMLP was 1.74 0.5 (OD 450 nm). The extracts of Barleria lupulina and Clinacanthus nutans (0.1100 g/ml) caused signicant inhibition of fMLP-induced MPO production in human neutrophils (P < 0.05, ANOVA) with an IC50 of 84.0 8.9 g/ml, n = 8 and IC50 of 219.5 25.7 g/ml, n = 8, respectively (Fig. 5). Barleria lupulina extract exerted a stronger inhibition than that of the extract from Clinacanthus nutans on human neutrophil MPO production (P < 0.05). Indomethacin exerted strong inhibitory effect on fMLP-induced human neutrophil MPO production with and IC50 of 7.1 1.6 g/ml (19.8 4.4 M), n = 8. Direct inhibitory effects of both extracts on MPO activity were not observed (data not shown). 3.10. Neutrophil elastase release Pre-incubation of isolated human neutrophils with Barleria lupulina or Clinacanthus nutans extracts (0.1100 g/ml) elicited a weak, but signicant inhibition of human neutrophil elastase release induced by fMLP (100 nM) (P < 0.05, ANOVA) with an IC50 for Barleria lupulina of 89.8 10.8 g/ml (n = 8) and for Clinacanthus nutans of 186.8 20.5 g/ml (n = 8) (Fig. 6). Indomethacin (1100 g/ml) also caused inhibition of

fMLP-induced human neutrophil elastase release with an IC50 of 69.0 9.5 g/ml (192.5 26.5 M), n = 8. Direct inhibitory effects of both extracts on elastase activity were not observed (data not shown). 3.11. Neutrophil apoptosis Neutrophil apoptosis is characterised by dramatic morphological changes where apoptotic neutrophils exhibit hyperchromatic pyknotic nuclei. Cellular necrosis at 20 h was negligible with >98% cell viability, assessed by trypan blue exclusion. After 20 h culture, neutrophils underwent approximately 71.9 and 73.5% apoptosis as assessed by morphology

Fig. 6. Inhibitory effects of Barleria lupulina extract (0.1100 g/ml), Clinacanthus nutans extract (0.1100 g/ml) and indomethacin (0.01100 g/ml) on fMLP-induced elastase release in human neutrophils. Results are mean S.E.M. of seven experiments using cells from different donors.

and analyses of annexin V binding, respectively. Both Barleria lupulina and Clinacanthus nutans extracts up to the concentration of 500 g/ml showed no signicant effect on neutrophil apoptosis as assessed by both methods. Dexamethasone (1 M) signicantly inhibited neutrophil apoptosis with 54.5% being apoptotic. 4. Discussion The methanolic crude extracts of Barleria lupulina and Clinacanthus nutans were studied for their anti-inammatory activities in models of EPP-induced ear oedema and carrageenan-induced paw oedema in the rat. EPP-induced ear oedema has a good predictive value for the screening of antiinammatory agents and these extracts dose-dependently and signicantly inhibited oedema formation in this model at all time points examined. The methanolic extract of Barleria lupulina however possessed more efcacious inhibitory effects than did Clinacanthus nutans extract. As EPP causes the release of many inammatory mediators such as kinin, serotonin and prostaglandins, it might be possible that these extracts also affect the release and/or synthesis of these mediators. The most potent inhibition of both extracts at all concentrations was observed at 15 min suggesting that both extracts strongly inhibit the release and/or the effect of histamine and serotonin which are the rst released inammatory mediators of inammation in this model (Brattsand et al., 1982). The two extracts also signicantly inhibited MPO activity in the rat ear (Table 2) indicating the likely involvement of neutrophils in their cellular mechanism of action. However, they were found to be less potent than the COX inhibitor indomethacin. Carrageenan-induced rat paw oedema has also been successfully used for the evaluation of anti-inammatory drugs, since relative potency estimates obtained from anti-inammatory agents tend to reect clinical experience (Winter et al., 1962). Paw oedema formation is a result of a synergism between inammatory mediators that increase blood ow and microvascular permeability (Ialenti et al., 1992). The carrageenan-induced rat paw oedema is characterised by an early phase (1 h) caused by the release of histamine, 5-hydroxytryptamine and bradykinin followed by a late phase (2 h) mainly sustained by prostaglandin release which causes oedema dependent on mobilisation of neutrophils (Di Rosa et al., 1971). Thus this model has also been successfully used for identifying inhibitors of COX (Winter et al., 1962). In this model, both extracts showed signicant inhibitory effects on oedema formation at 3 h after intradermal injection of carrageenan (Table 3) suggesting that the extracts may be affecting the synthesis and/or release of mediators during both phases of the response. Our results conrm previous reports of Clinacanthus nutans extract exhibiting anti-inammatory activity (Satayavivad et al., 1987). The leaf extracts of Clinacanthus nutans (5401300 mg/kg) showed no acute or subacute toxicity in rats when given daily for 6 weeks (Satayavivad et al., 1987). However, the cytotoxic effects of these herbs have never been investigated. In our study, both Barleria lupulina and Clinacanthus natans extracts (11000 g/ml) at concentrations up to 500 g/ml did not affect the viability of neutrophils

both during the 45 min and 4 h incubation periods. A number of factors, including cytokines, chemical mediators and bacterial products such as LPS, have been shown to induce neutrophil functional responsiveness and to prolong the functional lifespan of neutrophils (Walker et al., 2005; Ward et al., 1999b). It is possible that the herb extracts may be contaminated with small amount of LPS. To ascertain that the effects of both Barleria lupulina and Clinacanthus nutans extracts on neutrophil functions are not related to the actions of contaminating LPS, we determined the amounts of LPS in both herb extracts before other assessments. The results showed that these extracts contained negligible quantities of LPS. Furthermore, since the extracts did not induce neutrophil responses per se and inhibited neutrophil function, not delaying neutrophil apoptosis (see below), rules out the possibility that LPS is a biologically signicant contaminant. During inammation neutrophils migrate to sites of trauma (e.g., injury or infection) and can release superoxide anions and tissue destructive enzymes such as MPO and elastase; a process that can exacerbate the inammatory response and leads to tissue destruction (Fujie et al., 1999). Formylated peptides such as fMLP are liberated from bacteria and are powerful neutrophil agonists (Ferrante and Thong, 1980; Liang et al., 1990). Pre-treatment of neutrophils with Barleria lupulina or Clinacanthus nutans extracts (0.1100 g/ml) signicantly inhibited fMLP-induced chemotaxis in a concentration-dependent manner. Barleria lupulina and Clinacanthus nutans extracts were also found to attenuate neutrophil chemokinesis. Activation of neutrophils also results in both the extracellular release of O2 and the intracellular production of the radical, which may be attributed to distinct pools of NADPH oxidase (Karlsson et al., 2000). In this study, fMLP concentration-dependently triggered neutrophil O2 generation; an effect signicantly inhibited by both herb extracts, where Barleria lupulina showed stronger inhibitory effects than Clinacanthus nutans. The two extracts possess very low radical scavenging activity as observed by the DPPH scavenging activity test. In addition, the inhibitory effects of the two extracts on fMLP-induced O2 generation in neutrophils are not related to their direct scavenging activity as the extracts showed no direct activity as assessed by EPR. Neutrophil activation also results in degranulation with consequent release of several enzymes such as MPO and elastase. MPO, an abundant chloride peroxidase found in neutrophil azurophilic granules, is involved in microbial killing and inammatory tissue damage (Kettle and Winterbourn, 1991), and the anti-inammatory activities of certain drugs have been attributed to the inhibition of leukocyte MPO activity (Ramos et al., 1995). Barleria lupulina and Clinacanthus nutans extracts were found to signicantly inhibit fMLP-induced MPO production in a concentration-dependent manner where Barleria lupulina exerted stronger inhibitory effects than did the Clinacanthus nutans extract. In response to divergent stimuli, recruited neutrophils at the site of infection or inammation, also release elastase from the gelatinase granules. Neutrophil elastase promotes inammation and inhibits the healing process in chronic inammatory processes. The effects of the methanolic extracts of Barleria lupulina and Clinacanthus nutans on fMLP-induced elastase release in neutrophils was also assessed and the results

showed that Barleria lupulina and Clinacanthus nutans extracts also signicantly suppressed elastase release in agreement with a previous report (Gil et al., 1995). Flavonoids (Dampawan et al., 1977) and glucosides (Teshima et al., 1998) are known as important constituents of Clinacanthus nutans and previous studies have reported that glucosides and avonoids exert inhibitory effects on human neutrophil elastase release (Melzig et al., 2001). Therefore, it is possible that inhibitory effects of Clinacanthus nutans on neutrophil elastase release might be related to its avonoids and glucoside content. Apoptosis provides a granulocyte clearance mechanism that would tend to limit tissue injury and promote resolution of inammation (Walker et al., 2005). Failure to remove neutrophils and their toxic products from tissues would likely result in chronic persistent inammation. Thus, in this study, we determined whether or not Barleria lupulina and Clinacanthus nutans extracts affected constitutive neutrophil apoptosis. In contrast to the glucocorticoid dexamethasone (Meagher et al., 1996), both Barleria lupulina and Clinacanthus nutans extracts did not affect neutrophil apoptosis or viability. In conclusion, we have demonstrated that the methanolic extracts from Barleria lupulina and Clinacanthus nutans possess signicant anti-inammatory properties as seen in both the rat paw oedema model induced by injection of carrageenan and the EPP-induced rat ear oedema model and the mechanism is clearly related to the neutrophil migration as conrmed by the reduction of MPO activity in EPP-induced rat ear oedema model. Both Barleria lupulina and Clinacanthus nutans extracts exerted their in vitro inhibitory effects on neutrophil functional responsiveness without having a signicant cytotoxic effect. In addition, these herb extracts caused no effect on neutrophil apoptosis. The anti-inammatory activity of both herb extracts may be attributed to their inhibitory effects on neutrophil functions other than neutrophil migration. However, further investigation to fully identify the biologically active ingredients and to dene the underlying molecular mechanisms of the inhibitory effects of these extracts on neutrophil functional responsiveness is required. Acknowledgements This work was supported by an Initiative Research Scholarship to Payong Wanikiat from the Faculty of Science, Mahidol University. The partial support by the Thailand Research Fund for the award of a Senior Research Scholarship to Prof. Vichai Reutrakul and the Center for Innovation in Chemistry: Postgraduate Education and Research Program in Chemistry, Higher Education Development Project of the Commission on Higher Education, are also gratefully acknowledged. References
Barrett, A.J., 1981. Leukocyte elastase. Methods in Enzymology 80 (Part C), 581588. Bradley, P.P., Priebat, D.A., Christensen, R.D., Rothstein, G., 1982. Measurement of cutaneous inammation: estimation of neutrophil content with an enzyme marker. Journal of Investigative Dermatology 78, 206209.

Brattsand, R., Thalen, A., Roempke, K., Kallstrom, L., Gruvstad, E., 1982. Inuence of 16 alpha, 17 alpha-acetal substitution and steroid nucleus uorination on the topical to systemic activity ratio of glucocorticoids. Journal of Steroid Biochemistry 16, 779786. Byrne, L., Sasse, J., Skelton, B., Suksamrarn, A., White, A., 1987. The minor iridoid glucosides of Barleria lupulina: isolation, crystal structure and plant growth-inhibiting properties of 6-O-acetylshanzhiside methyl ester. Australian Journal of Chemistry 40, 785794. Cavin, A., Hostettmann, K., Dyatmyko, W., Potterat, O., 1998. Antioxidant and lipophilic constituents of Tinospora crispa. Planta Medica 64, 393 396. Charuwichittratana, S., Wongrattanapasson, N., Timpatanapong, P., Bunjob, M., 1996. Herpes zoster: treatment with Clinacanthus nutans cream. Internal Journal of Dermatology 35, 665666. Chavalittumrong, P., Attawish, A., Rungsamon, P., Chuntapet, P., 1995. Toxicological study of Clinacanthus nutans (Burm.f.) Lindau. Bulletin of the Department of Medical Services (Thailand) 37, 323338. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1968. Glossary of Indian Medicinal Plants (Suppl.). Academic Publishers, New Delhi, India, pp. 20. Cuzzocrea, S., Riley, D.P., Caputi, A.P., Salvemini, D., 2001. Antioxidant therapy: a new pharmacological approach in shock, inammation, and ischaemia/reperfusion injury. Pharmacological Review 53, 135159. Dampawan, P., Huntrakul, C., Reutrakul, V., 1977. Constituents of Clinacanthus nutans and the crystal structure of LUP-20(29)-ENE-3-ONE. Journal of Science Society of Thailand 3, 1426. Dikalov, S., Skatchkov, M., Bassenge, E., 1997. Spin trapping of superoxide radicals and peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine and the stability of corresponding nitroxyl radicals towards biological reductants. Biochemical Biophysical Research Communications 231, 701704. Di Rosa, M., Giroud, J.P., Willoughby, D.A., 1971. Studies on the mediators of the acute inammatory response induced in rats in different site by carrageenan and terpentine. Journal of Pathology 104, 1521. Ferrante, A., Thong, Y.H., 1980. Optimal conditions for simultaneous purication of mononuclear and polymorphonuclear leucocytes from human blood by the Hypaque-Ficoll method. Journal of Immunological Methods 36, 109117. Fujie, K., Shinguh, Y., Inamura, N., Yasumitsu, R., Okamoto, M., Okuhara, M., 1999. Release of neutrophil elastase and its role in tissue injury in acute inammation: effect of the elastase inhibitor, FR 134043. European Journal of Pharmacology 374, 117125. Gil, B., Ferrandiz, M.L., Sanz, M.J., Terrencio, M.C., Ubeda, A., Rovirosa, J., San-Martin, A., Alcaraz, M.J., Paya, M., 1995. Inhibition of inammatory responses by epitaondiol and other marine natural products. Life Science 57, 2530. Gilroy, D.W., Lawrence, T., Perretti, M., Rossi, A.G., 2004. Inammatory resolution: new opportunities for drug discovery. Nature Reviews Drug Discovery 3, 401416. Ialenti, A., Ianaro, A., Moncada, S., Di Rosa, M., 1992. Modulation of acute inammation by endogenous nitric oxide. European Journal of Pharmacology 211, 177182. Kanchanapoom, T., Kasai, R., Yamasaki, K., 2001. Iridoid glucosides of Barleria lupulina. Phytochemistry 58, 337341. Kankaanranta, H., Moilanen, E., Vapaatalo, H., 1994. Effects of non-steroidal anti-inammatory drugs on polymorphonuclear leukocyte functions in vitro: focus on fenamates. Naunyn-Schmiedebergs Archives of Pharmacology 350, 685691. Karlsson, A., Nixon, J.B., McPhail, L.C., 2000. Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidyl-inositol 3-kinase. Journal of Leukocyte Biology 67, 396404. Kettle, A.J., Winterbourn, C.C., 1991. Mechanism of inhibition of myeloperoxidase by anti-inammatory drugs. Biochemical Pharmacology 41, 14851492. Koopman, G., Reutelingsperger, C.P., Kuijten, G.A., Keehnen, R.M., Pals, S.T., van Oers, M.H., 1994. Annexin V for ow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 14151420.

Liang, S.L., Woodlock, T.J., Whitin, J.C., Lichtman, M.A., Segel, G.B., 1990. Signal transduction in N-formyl-methionyl-leucyl-phenylalanine and concanavalin. A stimulated human neutrophils: superoxide production without a rise in intracellular free calcium. Journal of Cellular Physiology 145, 295302. Meagher, L.C., Cousin, J.M., Seckl, J.R., Haslett, C., 1996. Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes. Journal of Immunology 156, 44224428. Melzig, M.F., Loser, B., Ciesielski, 2001. Inhibition of neutrophil elastase activity by phenolic compounds from plants. Pharmazie 56, 967 970. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 5563. National Drug Committee., 2006. List of Herbal Medicinal Products A.D. 2006, ISBN: 974-244-217-7, Chuoomnoom Sahakorn Karnkaset Publisher, Bangkok, Thailand, pp. 5961. Ramos, C.L., Pou, S., Rosen, G.M., 1995. Effects of anti-inammatory drugs on myeloperoxidase-dependent hydroxyl radical generation by human neutrophils. Biochemical Pharmacology 49, 10791084. Sangkitporn, S., Polchan, K., Thawatsupa, P., Bunchob, M., Chawalitthumrong, P., 1993. Treatment of recurrent genital herpes simplex virus infection with Clinacanthus nutans extract. Bulletin of the Department of Medical Services (Thailand) 18, 226231. Satayavivad, J., Bunyapraphatsara, N., Kittisiripornkul, S., Tanasomwong, W., 1987. Analgesic and anti-inammatory activities of extract of Clinacanthus nutans (Burm. F.) Lindau. Thailand Journal of Phytopharmacy 3, 7 17. Suba, V., Murugesan, T., Pal, M., Mandal, S.C., Saha, B.P., 2004. Antiulcer activity of methanol fraction of Baleria lupulina Lindl. in animal models. Phytotherapy Research 18, 925929. Suksamrarn, A., 1986. Iridoid glucosides from Barleria lupulina. Journal of Natural Products 49, 179. Suksamrarn, A., Wongkrajang, K., Kirtikara, K., Suksamrarn, A., 2003. Iridoid glucosides from the owers of Barleria lupulina. Planta Medica 69, 877879. Suzuki, K., Ota, H., Sasagawa, S., Sakatani, T., Fujikura, T., 1983. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Analytical Biochemistry 132, 345352.

Talpain, E., Armstrong, R.A., Coleman, R.A., Vardey, C.J., 1995. Characterisation of the PGE receptor subtype mediating inhibition of superoxide production in human neutrophils. British Journal of Pharmacology 114, 14591465. Tan, G.T., Pezzuto, J.M., Kinghorn, A.D., 1991. Evaluation of natural products as inhibition of human immunodeciency virus type 1 (HIV-1) reverse transcriptase. Journal of Natural Products 54, 143154. Teshima, K., Kanako, T., Ohtan, K., Kasai, R., Lhieochaiphant, S., Pichensoonthon, C., Yamasaki, K., 1998. Sulfur-containing glucosides from Clinacanthus nutans. Phytochemistry 488, 831835. Tiangburanatam, W., 1996. Dictionary of Thai Medicinal Plants, fourth ed. Prachoom Karn Pim, Bangkok, Thailand, pp. 788789. Tuntiwachwuttikul, P., Pancharoen, O., Taylor, W., 1998. Iridoid glucosides of Barleria lupulina. Phytochemistry 49, 163166. Tuntiwachwuttikul, P., Pootaeng-on, Y., Phansa, P., Taylor, W.C., 2004. Cerebrosides and a monoacylmonogalactosylglycerol from Clinacanthus nutans. Chemical and Pharmaceutical Bulletin 52, 2732. Walker, A., Ward, C., Taylor, E.L., Dranseld, I., Hart, S.P., Haslett, C., Rossi, A.G., 2005. Regulation of neutrophil apoptosis and removal of apoptotic cells. Current Drug Targets. Inammation and Allergy 4, 447454. Wanikiat, P., Woodward, D.F., Armstrong, R.A., 1997. Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils. British Journal of Pharmacology 122, 11351145. Ward, C., Chilvers, E.R., Lawson, M.F., Pryde, J.G., Fujihara, S., Farrow, S.N., Haslett, C., Rossi, A.G., 1999a. NF- B activation is a critical regulator of human granulocyte apoptosis in vitro. Journal of Biological Chemistry 274, 43094318. Ward, C., Dranseld, I., Chilvers, E.R., Haslett, C., Rossi, A.G., 1999b. Pharmacological manipulation of granulocyte apoptosis: potential therapeutic targets. Trends in Pharmacological Sciences 20, 503509. Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-induced oedema in hind paw of the rat as an assay for anti-inammatory drugs. Proceedings of the Society for Experimental Biology and Medicine 111, 544547. WHO, 2000. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine. World Health Organization, Geneva. Yoosook, C., Panpisutchai, Y., Chaichana, S., Santisuk, T., Reutrakul, V., 1999. Evaluation of anti-HSV-2 activities of Barleria lupulina and Clinacanthus nutans. Journal of Ethnopharmacology 67, 179187.

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