the inhibition of free radical generation by preparations of harpagophytum procumbens in vitro

Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 104–110 (2009)DOI: 10.1002/ptr

104 L. GRANT ET AL.

Copyright © 2008 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 23, 104–110 (2009)Published online 19 September 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2570

Received 4 February 2008Revised 14 March 2008

Accepted 20 March 2008

* Correspondence to: Mary Warnock, School of Health Sciences, QueenMargaret University Edinburgh, Queen Margaret University Drive,Musselburgh, East Lothian, EH21 6UU, Scotland.E-mail: [email protected]

The Inhibition of Free Radical Generation byPreparations of Harpagophytum procumbensIn Vitro

Louise Grant, Douglas E. McBean, Lorna Fyfe and A. Mary Warnock*School of Health Sciences, Queen Margaret University Edinburgh, Queen Margaret University Drive, Musselburgh, EastLothian, EH21 6UU, Scotland

Harpagophytum procumbens (Hp), commonly known as Devil’s Claw has been used as a traditional treatmentfor a variety of illnesses for centuries. Since the early twentieth century, it has become a popular antiinflammatoryand analgesic preparation amongst European herbalists for supportive or adjuvant treatment of degenerativejoint diseases. Extracts of Hp tubers have demonstrated antiinflammatory and analgesic effects in animalmodels of inflammation and in human trials. The mechanism(s) of action responsible for these attributes,however, remain to be elucidated. Reactive oxygen species generated in acute and chronic inflammatorydiseases are known to be cytotoxic and can cause tissue damage. In this study, a root tuber extract (Hp extract)and commercially available tincture (Hp tincture) were investigated for antioxidant characteristics using invitro test systems. Both preparations were found to effectively scavenge DPPH radical, inhibit nitrite levels insupernatants harvested from LPS-stimulated RAW 264.7 macrophages, and cause dose-dependent suppressionsin the detection of fMLP- and AA-induced neutrophil MPO. The antioxidant effects demonstrated for bothpreparations of Hp may contribute to the antiinflammatory and analgesic properties observed for the plant.Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: Devil’s Claw; Harpagophytum procumbens; reactive oxygen species; reactive nitrogen species; nitric oxide; superoxideanion; inflammation; arthritis.

INTRODUCTION

Harpagophytum procumbens (Hp), commonly knownas Devil’s Claw is a perennial herb belonging to thePedaliaceae family and is native to countries withinthe Southern African continent, such as Namibia andBotswana. Extracts of Hp tubers have demonstratedantiinflammatory and analgesic effects in animal modelsof inflammation and in human trials of osteoarthritis,rheumatoid arthritis and other musculoskeletal con-ditions (reviewed by Grant et al., 2007; Warnock et al.,2007). The mechanism(s) of action responsible for theseattributes, however, remain to be fully elucidated.

The generation of reactive oxygen species (ROS) andreactive nitrogen species (RNS) when unregulated cancontribute to the tissue injury involved in inflammatoryconditions, including arthritis (Babior, 2000). Reactiveoxygen species generated in acute and chronic inflam-matory diseases are known to be cytotoxic and can causetissue damage through lipid peroxidation, oxidationof amino acid side chains, protein cross-linking andfragmentation, and DNA damage (Davies, 1987; Daviesand Goldberg, 1987). Reactive nitrogen species suchas nitric oxide (NO) are not believed directly to causeoxidative damage but, in conjunction with superoxideanion (O2

−) can form the highly potent oxidizing agentperoxynitrite (PN) (Babior, 2000). Recent in vitro

studies have shown that harpagoside, one of the majorcomponents of Hp, can reduce NO release in lipopoly-saccharide (LPS) stimulated RAW264.7 cells (Huanget al., 2006). Enzymes responsible for the catalysis ofoxygen are also implicated in joint damage and inflam-mation. Myeloperoxidase (MPO) is one such enzymeand is found in higher than normal levels in the synoviumof rheumatoid arthritis (RA) patients (Torsteindóttiret al., 1999). Free radicals have also been demonstratedto contribute to the modulation of pain and some stud-ies have revealed that antioxidants can potentiate theantinociceptive responses (Penn, 1995; Yang et al., 2001).There are several sources of free radicals in inflamedarticulations, which include infiltrating neutrophils,monocytes, macrophages and articular chondrocytes.Agents that are able to interfere with the generationand/or action of free radicals in biological tissues aretherefore of therapeutic interest and it has been shownthat Hp may exert such beneficial effects (Grant et al.,2005).

Studies that do exist have yielded positive findingsin the main, which justify further work in this area(Bhattacharya and Bhattacharya, 1998; Langmeadet al., 2002; Betancor-Fernandez et al., 2003). It couldbe hypothesized that the putative antiinflammatoryand analgesic benefits exerted by Harpagophytum areattributed to, at least in part, an antioxidant effect.The amelioration of inflammation and pain in animalmodels treated with extracts of Hp has also beendemonstrated by drugs known to possess antioxidantproperties and/or an ability to inhibit NO-synthase, suchas mercaptoethylguanidine (Cuzzocrea et al., 1998).Furthermore, it has been postulated that secondary to

INHIBITION OF FREE RADICAL GENERATION BY HARPAGOPHYTUM PROCUMBENS 105


cyclo-oxygenase (COX) inhibition the mechanism ofaction of non-steroidal antiinflammatory drugs (NSAIDs)also encompasses an antioxidant effect, thus the appar-ent equipotent effects demonstrated by Hp and someNSAIDs in animal and human trials could be a con-sequence of this capacity (Chrubasik et al., 2003, 2005).

In this study, a root tuber extract (Hp extract; BioforceAG) and commercially available tincture (Hp tincture;Bioforce UK) were investigated for antioxidant charac-teristics using in vitro test systems.

MATERIALS AND METHODS

Preparation of Hp solutions. Hp extract (no less than1.2% harpagoside; Bioforce AG, Switzerland) was madeup in dimethyl sulphoxide (DMSO; Sigma, UK) to givea top working solution of 500 mg/mL. Dilutions of theDMSO stock were made up in phosphate bufferedsaline to give working solutions of 0.05, 0.5, 5 and 50 mg/mL Hp extract. Hp tincture (Bioforce UK Ltd: 100 mg/mL in 66% ethanol) was diluted in phosphate bufferedsaline (Sigma, UK; pH 7.1–7.5) to give working solutionsof 0.05, 0.5, 5 and 50 mg/mL. Harpagoside (Extrasyn-these, France: 2 mg/mL in 66% ethanol) was diluted inphosphate buffered saline (Sigma, UK; pH 7.1–7.5).

Measurement of DPPH radical scavenging. Test agentswere added to an equal volume of 1,1-diphenyl-2-picrilhydrazyl (DPPH; Sigma, UK), to give final con-centrations of 1, 10, 100 and 1000 μg/mL, and incubatedat room temperature for 30 min in the dark (Blois et al.,1985). A 60 μM DPPH ethanol solution was prepareddaily for use. The absorbance of each solution was readin triplicate at 515 nm. Replicate cuvettes containing testagents without DPPH were created and the absorb-ance of each solution subtracted from the absorbanceof the sample solution containing DPPH. The degreeof DPPH scavenging activity was calculated as thepercentage of inhibition, where the absorbance of thecontrol solution was 100%.

LPS-stimulated nitrite generation

Cell culture of RAW 264.7 macrophages. RAW 264.7macrophages (ECACC, UK) were maintained in DMEMsupplemented with 10% fetal bovine serum (FBS), 2 mM

L-glutamine and 100 μg/mL penicillin/streptomycin at37 °C in 5% CO2. On reaching 70–80% confluence, cellswere harvested gently using a cell scraper (Fisher Sci-entific, UK) to dislodge cells from the flask.

Hp treatment. Cells were resuspended at a concentrationof 2 × 106 cells/mL using antibiotic-free DMEM supple-mented with 10% FBS and 2 mM L-glutamine. 500 μLof cell suspension was added to wells of a 24-well, flat-bottomed tissue culture plate (Fishers, UK). Cells wereeither: (1) pre-treated (1 h) with test agents followedby the removal of Hp before addition of lipopolysac-charide (LPS) (Pseudomonas aeruginosa serotype 10;Sigma); (2) treated concurrently, i.e. 1 h pre-treatmentwith Hp (one dose) and subsequent removal of Hpbefore addition of LPS for 20 h; or (3) both treatments(1 h pre-treatment with Hp (first dose), removal of first

dose and addition of second dose plus LPS for 20 h).Following incubation at 37 °C, 5% CO2, the plates werecentrifuged at 300 × g for 20 min. 100 μL of supernatantwas removed from each well and added to a 96-well,flat-bottomed tissue culture plate for the immediateassay of NO content. 100 μL Griess reagent (Sigma)was added to each well and the nitrite content derivedfrom sodium nitrite standards.

Sodium nitrite standards (0.1–100 μM) were made upin PBS and 100 μL of each standard added in triplicateto a 96-well plate. 100 μL of Griess reagent was added toeach well and the absorbance read at 550 nm (Ohshimaet al., 1991). A 7-point standard curve was plottedusing the mean absorbencies of the sodium nitrite stand-ards and a best-fit line placed through the points. Theequation of the line was used to calculate the nitritecontent of each sample well and the degree of NO scav-enging activity of each text extract/tincture was calcu-lated as a percentage of inhibition, where the absorbanceof the control solution was 100%.

Formyl methionyl leucine phenylalanine (fMLP)- andarachidonic acid (AA)-stimulated neutrophilsuperoxide anion generation (SAG)

Isolation and purification of human neutrophils fromwhole blood. 50 mL of fresh human blood was collectedand added to 2 mL heparin and 50 mL of 3% dextran(Fisher Scientific, UK) in 0.9% saline. Erythrocytes wereleft to sediment for 45 min after which the leukocyterich fraction was removed and centrifuged at 280 × gfor 10 min. Platelet rich supernatant was discarded andthe remaining leukocyte pellet was resuspended in 55%Percoll (Sigma, UK) and layered over a discontinuousPercoll gradient (70% and 81% v/v). The gradients werecentrifuged at 650 × g for 30 min, and the neutrophillayer below the 70%:81% interface was removed andresuspended in 0.9% saline.

Treatment and stimulation of neutrophils. Neutrophilswere resuspended at a concentration of 1.5 × 106 cells/mL in PBS without Ca2+/Mg2+, supplemented withcytochalasin B (10−5 M) and cytochrome C (0.5 mg/mL).1 mL aliquots of cell suspension were treated withHp extract or tincture (0.001–1 mg/mL final concentra-tion) in triplicate and incubated at 37 °C for 10 min.

At 10 s intervals, fMLP (1–1000 nM), or AA (10–100 μM in PBS) was added to each set of tubes, vortexedand incubated at 37 °C. The reaction was stoppedby placing on ice for 10 min. The samples were thencentrifuged at 320 × g for 10 min.

Quantification of neutrophil SAG. 200 μL of superna-tant was aliquoted into a 96-well, flat-bottomed tissueculture plate and read at 550 nm. The mean valueswere calculated and neutrophil SAG was determinedfrom the absorbance of each sample as nanomolesper million cells using a modification of that describedby Talpain et al. (1995).

fMLP- and AA-stimulated neutrophil MPO

Treatment and stimulation of neutrophils. Humanneutrophils were collected, counted and resuspended


106 L. GRANT ET AL.

at a concentration of 1.5 × 106 cells/mL, in cytochalasinB buffer as described previously. The method for MPOdetection was adapted from Bozeman et al. (1990) andSuzuki et al. (1983). 1 mL aliquots of cell suspensionwere treated with Hp extract and tincture (0.001–1 mg/mL final concentration). Samples were incubated for10 min at 37 °C in a water bath. At 10 s intervals, 50 μLof PBS (control), fMLP (1–1000 nM) or AA (10–100 μM)was added to respective tubes, vortexed and then incu-bated at 37 °C for 5 min. Samples were placed on icefor 10 min and then centrifuged at 320 × g for 10 min at4 °C to pellet cells. 30 μL was taken from each sampleand added to a 96-well plate.

Quantification of neutrophil MPO. The cell pellets fromthe control samples were lysed by 1 mL hexadecyl-trimethyl-ammonium bromide (HTAB; 0.5% solutionin sodium phosphate buffer, pH 6; Sigma, UK) andvortexed. After 45 min at room temperature, 15 μLof control suspension was added to the plate. 50 μL oftetramethylbenzidine (TMB) was added to all wellsfollowed immediately by the addition of 100 μL of 0.5 M

sulphuric acid (H2SO4) to each well. The plates wereread at 450 nm. The mean values were calculated fortriplicate sets of data and MPO release was expressedas a percentage of the maximum response determinedfor control cells treated with HTAB.

Cell viability. Cell viability was assessed using the trypanblue exclusion method.

Statistics. All experiments were conducted in triplicatewith a minimum of three repetitions. Data are expressedas the mean ± the standard error of the mean (SEM) ofthe averaged value. Paired Student’s t-tests (two-tailed)were used to investigate differences between means andANOVA (one-way between groups) for differencesbetween groups, where p < 0.05 was taken as significant.Percentage inhibition data was calculated for test agentsas a percentage of control values. IC50 values were cal-culated as the concentration of an agent to produce50% of the maximum response using regression analysis.

RESULTS

DPPH radical scavenging

Figure 1 shows the comparative inhibitory effects onDPPH radical for Hp extract, Hp tincture, and the main

Figure 1. DPPH radical scavenging activity of 1–1000 μg/mL Hpextract, Hp tincture and harpagoside (n = 3). Data expressedas the mean percentage inhibition of DPPH ± SEM followingincubation of DPPH (60 μM) with ethanol (control), in the ab-sence of test substance. * Indicates significantly greater inhibi-tion than harpagoside at the same dose (ANOVA; p < 0.05).Significance markers (*) shown above the data lines are rep-resentative of Hp extract, those below the data lines arerepresentative of Hp tincture.

Table 1. Percentage inhibition of nitrite (±±±±± SEM) produced by RAW 264.7 macrophages treated with Hp extract or Hp tincture (300–1000 μμμμμg/mL) (n ===== 3)

1 h pre-treatment 20 h treatment Both treatments

Extract Tincture Extract Tincture Extract Tincture

300 μg/mL 9.02 ± 3.19 7.64 ± 0.99 11.48 ± 3.56 17.16 ± 3.77 16.23 ± 4.95 16.02 ± 2.8500 μg/mL 14.08 ± 5.24 13.95 ± 5.22 16.11 ± 5.35 26.57 ± 4.43 34.95 ± 5.37 30.39 ± 5.931000 μg/mL 28.81 ± 3.12 21.17 ± 1.98 35.59 ± 3.74 39.26 ± 2.00 53.73 ± 6.82 43.80 ± 3.09

Effects of test substances are displayed for all treatment types; 1 h pre-treatment, 20 h treatment, both treatments (1 h pre-treatmentfollowed by 20 h concurrent treatment with LPS) compared with control. Both preparations were found to exert a significant dose-dependent inhibition of nitrite levels in the supernatants under all treatment regimes, except following 1 h pre-treatment with 300 μg/mL Hp extract which did not reach significance (p = 0.05).

constituent of Hp, harpagoside. A dose-dependentinhibition of DPPH can be observed for all test com-pounds. Ascorbic acid was used as a positive controland the scavenging capacity of this compound was foundto be signficantly superior to that of all other com-pounds (data not shown). Both Hp preparations wereineffective at 0.001–1 μg/mL. At the maximal dose of1000 μg/mL, Hp extract and Hp tincture achievedscavenging capacities (SC) of 91.75 ± 1.40% and 89.95± 2.80% DPPH inhibition. Hp extract and Hp tinctureachieved IC50s of 49.87 μg/mL and 33.45 μg/mL, respec-tively. At a maximal concentration, harpagoside dis-played minimal antioxidant activity with an SC of 25.49± 0.97%. This was significantly lower than the SCvalues for Hp extract and tincture (ANOVA; p < 0.05).At 100 μg/mL, a slight scavenging activity was observed(2.43 ± 0.62%) but 1 and 10 μg/mL harpagoside werefound to have no significant effects on DPPH levels.

Effects of Hp extract and tincture on nitriteproduction from RAW 264.7 cells

Table 1 displays the percentage inhibition of nitriteincurred by 1 h pre-treatment, 20 h treatment and bothtreatments, respectively, with 300–1000 μg/mL Hp extractand tincture. The inhibitory/toxic effects measured forrespective DMSO or ethanol controls were subtractedto ensure true comparisons between doses, Hp prepa-rations and treatment types.



1 h pre-treatment. When cells were treated for 1 h withall doses (300–1000 μg/mL) of Hp extract or tincture,then washed and stimulated for 20 h with LPS, signifi-cant reductions in nitrite release were incurred (p <0.05), excluding 300 μg/mL Hp extract which did notreach significance (p = 0.05). No significant differencewas observed between the inhibitory abilities of theextract and tincture and both preparations exerted adose-dependent inhibition. Maximum nitrite inhibitionwas observed following treatment with 1000 μg/mL withreductions of 28.81 ± 3.12% (extract) and 21.17 ± 1.98%(tincture) from control nitrite levels.

20 h treatment. A similar trend was noted when thecells were treated with 300–1000 μg/mL extract ortincture for 20 h whilst being concurrently stimulatedwith LPS. Hp extract and tincture exerted significantdose-dependent inhibitions of nitrite, reaching maxi-mum reductions of 35.59 ± 3.74% and 39.26 ± 2.00% at1000 μg/mL, respectively.

Both treatments. When the cells were pre-treated for1 h with Hp extract and tincture (300–1000 μg/mL),washed, and then concurrently treated with a seconddose of extract plus LPS for 20 h, nitrite levels weresignificantly attenuated in a dose-dependent manner(p < 0.05). Maximum reductions of 53.73 ± 6.82% of43.80 ± 3.09% nitrite were measured following treat-ment with 1000 μg/mL Hp extract and Hp tincture,respectively.

Cell viability

Trypan blue exclusion was used to determine cellviability following Hp treatment and LPS stimulation.After each incubation, cell viability was confirmed tobe >93% by the trypan blue dye exclusion method.

Effect of Hp extract and Hp tincture on neutrophilSAG

Following the confirmation that neither Hp prepara-tion interfered with the absorbance of the cytochromeC buffer, the technique was repeated in the presence ofneutrophils. Neither preparation (1–1000 μg/μL) exertedany significant effect on the amount of SAG producedby neutrophils following inducement with fMLP or AA(p > 0.05; data not shown).

Effects of Hp extract and Hp tincture on thedetection of fMLP-stimulated neutrophil MPO

Following the confirmation that neither Hp preparationwas able to oxidize the TMB substrate, the techniquewas repeated in the presence of neutrophils. Treatingcells for 10 min with 1–100 μg/mL Hp extract or Hptincture induced no significant alterations in the detec-tion of fMLP-stimulated neutrophil MPO (p > 0.05) (datanot shown).

The effect of increasing the dose range of Hp extractand Hp tincture to 300, 500 and 1000 μg/mL is displayedin Fig. 2. At 300 μg/mL, Hp tincture had no significanteffect on the detection of MPO (p > 0.05). An equiva-

lent dose of Hp extract significantly reduced the detec-tion of MPO compared with control levels by 27.00 ±5.10% (p < 0.05). At 500 μg/mL, both Hp preparationssignificantly attenuated fMLP-stimulated MPO detec-tion from control levels by 55.32 ± 5.02% (extract) and35.47 ± 6.40% (tincture). Levels/activity of MPO weresignificantly suppressed from control levels by 88.24 ±4.25% and 70.61 ± 2.31% following treatment with1000 μg/mL Hp extract and Hp tincture, respectively(p < 0.05). Both extract and tincture suppressed thedetection of MPO to a significantly greater degree thantheir respective DMSO and ethanol controls (p < 0.05).

The ability of Hp extract to either inhibit the re-lease of fMLP-stimulated neutrophil MPO or reducethe peroxidation activity of the enzyme exceeded thatof the tincture and this is reflected in respective IC50

values of 547.69 μg/mL (extract) and 915.55 μg/mL (tinc-ture), but this difference was not significant (p > 0.05).

Effect of Hp extract and Hp tincture on AA-inducedneutrophil MPO production/peroxidation activity

Figure 3 shows the effects of Hp extract and Hp tincture(300–1000 μg/mL) on the detection of neutrophil MPOinduced by 100 nM AA. Both preparations were unable

Figure 2. Percentage reduction in the detection of fMLP-inducedneutrophil MPO following treatment with Hp extract and Hptincture (300–1000 μg/mL) (n = 3). Control neutrophils werestimulated with fMLP in the absence of test compounds. Dataexpressed as the mean percentage reduction in MPO ± SEMproduced by control cells stimulated with fMLP, in the absenceof test substance (p < 0.05). * Indicates significant inhibitioncompared with control (p < 0.05).

Figure 3. Percentage reduction in the detection of AA-inducedneutrophil MPO following treatment with Hp extract and Hptincture (300–1000 μg/mL) (n = 3). Control neutrophils werestimulated with AA in the absence of test compounds. Dataexpressed as the mean percentage reduction in MPO ± SEMproduced by control cells stimulated with AA, in the absenceof test substance (p < 0.05). * Indicates significant inhibitioncompared with control (p < 0.05).


108 L. GRANT ET AL.

to significantly affect MPO levels/activity at 300 μg/mL.At 500 μg/mL, the Hp extract and Hp tincture signifi-cantly suppressed the detection of MPO compared withcontrol levels by 45.99 ± 5.02% and 27.55 ± 8.34%,respectively (p < 0.05). At 1000 μg/mL, the inhibitoryactivities increased to 85.99 ± 4.25% (extract) and 57.05± 6.12% (tincture) (p < 0.05). Both extract and tincturesuppressed MPO detection to a significantly greaterdegree than their respective DMSO and ethanol con-trols (p < 0.05).

The extract of Hp induced significantly greater inhi-bition of MPO release or activity than the Hp tinctureat 1000 μg/mL (p < 0.05) but not at 300 and 500 μg/mL(p > 0.05). The IC50 values determined for Hp extractand Hp tincture were calculated as 601.49 μg/mL and776.49 μg/mL, respectively.

Cell viability

Using the trypan blue exclusion method, the cellviability of human neutrophils remained above 93%following treatments with all doses of Hp extract andHp tincture, and was not significantly different tothe viability determined for the control neutrophils(p < 0.05).

DISCUSSION

In this study, the DPPH radical scavenging methodwas utilized as a rudimentary gauge of the free radicalscavenging ability of the Hp preparations in a cell freesystem. The ability of both Hp preparations effectivelyto scavenge DPPH concurs with a similar study thatinvestigated the efficacy of a commercially available Hpextract to scavenge ABTS-derived radicals (Betancor-Fernandez et al., 2003). In the present study harpagosidedisplayed minimal DPPH scavenging activity whichcould be attributed to the presence of hydroxyl groupsin the bicyclic ring; a structural feature required forradical scavenging (Chen et al., 1996). However, thiswas only achieved at doses above 100 μg/mL. Onthe basis of the content of harpagosides provided bythe manufacturer, harpagoside comprises 1.3% of theHp extract and 0.12– 0.15% of the Hp tincture, thus forevery 1000 μg of extract/tincture just over 10 μg and1 μg harpagoside is present, respectively. The apparentinactivity of harpagoside as a scavenger at these levelsimplies that this constituent does not account for thepronounced scavenging activity of Hp extract andtincture observed at 1000 μg/mL. The potent anti-oxidant characteristics of the flavonoids, luteolin andkaempferol, which are both constituents of Hp, are morelikely to be responsible for free radical quenching bythe whole extract (Betancor-Fernandez et al., 2003).

One study has demonstrated, using the same pro-prietary tincture of Hp that was used in this study,that 7 days of i.p. treatment with the tincture was ablesignificantly to attenuate the extent of brain lipidperoxidation in rats (Bhattacharya and Bhattacharya,1998). This finding, in conjunction with the resultsobtained from the DPPH assay shown here, indicatethat the ability of the tincture directly to scavenge freeradicals may, at least in part, contribute to the protec-

tion of critical biomolecules against radical-mediateddamage. In addition, the authors reported significantaccentuation in levels of antioxidant enzymes (super-oxide dismutase, SOD; catalase, CAT; and glutathioneperoxidase, GPx) in the brains of Hp-treated rats(Bhattacharya and Bhattacharya, 1998). In active in-flammatory conditions, levels of serum antioxidantssuch as glutathione and malondialdehyde are often sup-pressed below normal levels (Jaswal et al., 2003; Paredeset al., 2002). Agents which are able to enhance con-stitutive antioxidants, or quench ROS that oxidize theenzymes, have been shown to be of benefit as adjunctsto conventional drugs in the treatment of arthritis(Jaswal et al., 2003).

In this study, Hp preparations were also investigatedfor their inhibitory activity on nitrite released fromLPS-stimulated RAW 264.7 macrophages, in vitro. BothHp preparations were found to dose-dependently(300–1000 μg/mL) inhibit the levels of nitrite presentin cellular supernatants under all treatment regimes,except following 1 h pre-treatment with 300 μg/mL Hpextract. In order of the greatest to the lowest inhibitoryactivity, the treatment regimes are arranged as follows:both treatments > 20 h concurrent Hp treatment withLPS > 1 h pre-treatment with Hp. This implies theoccurrence of a cumulative dosing effect by Hp in vitroand since no differences between the viability of stimu-lated Hp treated cells and stimulated control cellswere observed, this reinforces the occurrence of aninhibitory rather than a cytotoxic effect by Hp. Thecumulative dosing effect may have some relevance toin vivo dosing conditions since it is recommended thatextracts of Hp are taken repeatedly throughout the day.

The relatively moderate ability of Hp to inhibitnitrite released from LPS-stimulated murine macrophagesmay therefore be a contributing factor to the observedantiinflammatory and analgesic properties of the plant.Agents that are able to modulate NO synthesis or scav-enge its effect have demonstrated therapeutic benefitsfor cases of arthritis when given prophylactically(Stefanovic-Racic et al., 1995). Levels of nitrite, a break-down product of NO, are elevated in the serum andsynovial fluid of rheumatoid and osteoarthritis patients(Westacott and Sharif, 1996; Farrell et al., 1992) andinhibitors of inducible nitric oxide synthase (iNOS)have been found to suppress the onset of arthritis inanimals (Stefanovic-Racic et al., 1994). In addition, theover-expression of iNOS-derived NO by inflammatorycells, such as macrophages and neutrophils, as well aschondrocytes and synoviocytes, is implicated in jointdestruction (Chapman et al., 1989).

Of additional interest is the fact that 1 h pre-treatment,and the subsequent removal of Hp from cells beforethe addition of LPS, was found significantly to attenu-ate nitrite levels. This would not be supportive ofthe occurrence of a scavenging effect alone and itmay be possible that Hp could exert a dual inhibitoryeffect via a direct scavenging of nitrite in addition toan intracellular inhibitory mechanism. Studies havedemonstrated significant reductions in iNOS in vitrofollowing treatment with Hp which would support thefindings shown here. Using the reverse transcriptional-polymerase chain reaction, Jang et al. (2003) foundthat an aqueous extract of Hp caused a significant dose-dependent (100–1000 μg/mL) inhibition of LPS-inducediNOS expression in L929 murine cells. Similarly, Kaszkin



et al. (2004) demonstrated significant concentration-dependent (0.3–1 mg/mL) suppressions in nitrite releaseand iNOS expression from Hp-treated IL-1β-stimulatedrat mesangial cells. It was further demonstrated thatHp treatment inhibited the translocation of NF-κB fromthe nucleus which most probably explains the suppres-sion of iNOS and thus nitrite release.

The leukocyte responses of respiratory burst anddegranulation are additional factors thought to con-tribute directly to inflammatory tissue damage (Neal et al.,1987). Studies have demonstrated that antiinflammatorydrugs can modulate the responses of neutrophils tofMLP. For example, phenylbutazone, which has demon-strated equipotent antiinflammatory and analgesiceffects to Hp in animal models, is a potent inhibitor ofboth SAG and neutrophil degranulation (Neal et al.,1987; Grant et al., 2007). The capabilities of fMLP- andAA-stimulated human peripheral neutrophils obtainedfrom healthy donors (aged 18–29 years who had nottaken any NSAIDs for 2 weeks prior to blood dona-tion), to generate O2

− (measure of respiratory burst)and MPO release (measure of degranulation) in thepresence of Hp were therefore investigated in vitro.

Although Hp treatment was found to exert nosignificant effect on neutrophil degranulation byvirtue of SAG measurement, pre-treating neutrophilswith Hp extract (300–1000 μg/mL) and Hp tincture(500–1000 μg/mL), respectively, demonstrated dose-dependent suppressions in the detection of fMLP- andAA-induced neutrophil MPO. This is the first evidenceto suggest that extracts of Hp are able to interfere withthe MPO/hydrogen peroxide (H2O2)/halide system. Theresults appear to indicate that the effect of Hp on MPOrelease/peroxidation activity is not dependent on thetype of stimulation used as the extracts were able tosuppress fMLP- and AA-stimulated MPO to a similarmagnitude. Since fMLP and AA activate neutrophilsvia different mechanisms, it is more likely that the treat-ment effect exerted by Hp is linked to free radical scav-enging than interference in signal transduction. It couldbe postulated that the levels of phenols present in theHp extract (in doses of 300–1000 μg/mL) and in the Hptincture (in doses of 500–1000 μg/mL) were sufficient

to either directly scavenge hypochlorous acid (HOCl)or to suppress the production of HOCl by inhibitingthe chlorinating ability of MPO (Ximenes et al., 2005).Future studies into the effects of Hp extracts on MPOwould be of worth to confirm that the inhibitory effectis attributed to an extracellular scavenging mechanism.

The ability of Hp to interfere with the MPO–H2O2–halide system in neutrophils, in addition to the reportedinhibitory effect of Hp on neutrophil elastase (Bojeet al., 2003) may be worthy of future study to investi-gate the therapeutic relevance to arthritic conditions.Neutrophils constitute over 90% of cells found in thesynovial fluid of rheumatoid arthritis patients and arefound in great numbers at the site of bone erosion, thushaving implications for both inflammatory and degen-erative arthropies (Weissmann and Korchak, 1984).Stimulated neutrophils isolated from patients diagnosedwith rheumatoid arthritis and osteoarthritis have alsobeen shown to generate greater levels of free radicals,and in rheumatoid arthritis the neutrophils tend to existmore prevalently in degranulated form than neutrophilsobtained from healthy subjects (Biemond et al., 1986).

In summary, the antioxidant effects demonstrated forboth preparations of Hp may contribute to the anti-inflammatory and analgesic properties observed for theplant. However, the efficacy of antioxidant therapy alonein the treatment of arthritis is questionable. Resultsfrom ex vivo studies indicate a necessity for therapeuticco-administration of antioxidants along with conven-tional drugs to patients with arthritic conditions (Jaswalet al., 2003). Further confirmation of whether the dosesused in this study are relevant to physiological systemsmerits further investigation. A potential free radicalscavenging effect was shown, however, this should beviewed with some caution as natural products oftenshow non-specific reactions ex vivo. It would also beof value to investigate the treatment effects of Hpon other ROS pertinent to inflammatory conditions,such as peroxynitrite (PN) and HOCl. It may be thatthe moderate antioxidant activities of the herb onindividual free radicals, as displayed here, is amplifiedby an ability to scavenge or inhibit a wide variety ofpro-inflammatory and destructive radicals.

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