Aliment Pharmacol Ther 2004; 19: 521527.

doi: 10.1111/j.1365-2036.2004.01874.x

Anti-inammatory effects of aloe vera gel in human colorectal mucosa in vitro

L. LANGMEAD, R. J. M AKINS & D. S. R AMPTON Centre for Adult and Paediatric Gastroenterology, Institute of Cellular and Molecular Science, Barts and the London, Queen Mary School of Medicine and Dentistry, London, UK
Accepted for publication 9 December 2003

SUMMARY

Background: Oral aloe vera gel is widely used by patients with inammatory bowel disease and is under therapeutic evaluation for this condition. Aim: To assess the effects of aloe vera in vitro on the production of reactive oxygen metabolites, eicosanoids and interleukin-8, all of which may be pathogenic in inammatory bowel disease. Methods: The anti-oxidant activity of aloe vera was assessed in two cell-free, radical-generating systems and by the chemiluminescence of incubated colorectal mucosal biopsies. Eicosanoid production by biopsies and interleukin-8 release by CaCo2 epithelial cells in the presence of aloe vera were measured by enzyme-linked immunosorbent assay.

Results: Aloe vera gel had a dose-dependent inhibitory effect on reactive oxygen metabolite production; 50% inhibition occurred at 1 in 1000 dilution in the phycoerythrin assay and at 1 in 1050 dilution with biopsies. Aloe vera inhibited the production of prostaglandin E2 by 30% at 1 in 50 dilution (P 0.03), but had no effect on thromboxane B2 production. The release of interleukin-8 by CaCo2 cells fell by 20% (P < 0.05) with aloe vera diluted at 1 in 100, but not at 1 in 10 or 1 in 1000 dilutions. Conclusion: The anti-inammatory actions of aloe vera gel in vitro provide support for the proposal that it may have a therapeutic effect in inammatory bowel disease.

INTRODUCTION

Aloe vera is a stemless, drought-resisting succulent of the lily family. It is indigenous to hot countries and has been used medicinally for over 5000 years by Egyptian, Indian, Chinese and European cultures. Aloe vera gel is the mucilaginous aqueous extract of the leaf pulp of Aloe barbadensis Miller. It contains over 70 biologically active compounds and has been claimed to have anti-inammatory, anti-oxidant, immune boosting, anti-cancer, healing, anti-ageing and anti-diabetic properties.1
Correspondence to: Professor D. S. Rampton, Endoscopy Unit, Royal London Hospital, London E1 1BB, UK. E-mail: d.rampton@qmul.ac.uk 2004 Blackwell Publishing Ltd

Aloe vera gel has been widely promoted and used by patients for the treatment of a range of inammatory digestive and skin diseases, including inammatory bowel disease.2 Although there is, as yet, little scientic evidence in support of its efcacy in these settings, a randomized trial has shown that topical aloe vera gel is superior to placebo in the treatment of plaque psoriasis.3 The pathogenesis of inammatory bowel disease has not been elucidated fully, but the over-production by the involved colorectal mucosa of reactive oxygen metabolites,4, 5 eicosanoids6 and the chemo-attractant chemokine, interleukin-8,79 is likely to play a contributory role. In this study, we have assessed the antioxidant properties of aloe vera gel in cell-free and colorectal mucosal biopsy systems, and its effect on the
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production by biopsies and a cultured colonic epithelial cell line of eicosanoids and interleukin-8, respectively.

METHODS

Materials All chemicals were provided by Sigma Chemical Co., Dorset, UK, except for 2,2-azobis-(2-amidinopropane) dihydrochloride, which was obtained from Polysciences Inc. (Warrington, PA 18976, USA), and aloe vera gel, which was produced by Forever Living Products (Jersey, Channel Islands) and kindly donated by Dr P. Atherton. Reagents Aloe vera gel was stored in an air-tight container at 4 C. It was diluted in luminol/xanthine solution (for xanthine oxidase experiments), phosphate-buffered saline (PBS) (for phycoerythrin degradation experiments) or Tyrodes medium (for biopsy incubations) to achieve nal dilutions ranging from 1 in 5 to 1 in 10 000. Luminol was made up as a stock solution (50 mg/mL) in dimethyl sulphoxide and stored at 4 C for up to 1 month. On the day of the experiments, it was diluted to 300 lmol/L in Dulbeccos PBS with glucose (5 mmol/L). A stock solution of xanthine (5 mmol/L) was made up by dissolving xanthine in 1 mol/L sodium hydroxide and diluting in PBS. It was stored for up to 1 month and added to luminol on the day of the experiments to a nal concentration of 50 lmol/L. Xanthine oxidase was stored at 4 C and diluted to a concentration of 0.017 U/mL in the nal experimental mixture. Aliquots (0.34 lmol) of 1 mL phycoerythrin were prepared by dissolving the protein precipitate in PBS, centrifuging for 4 min and re-dissolving the pellet in PBS before removing (NH4)2SO4 on a NAP-5 column (a pre-packed disposable chromatography column containing Sephadex G-25 medium of DNA grade) with Chelex-treated PBS; aliquots were stored at 4 C wrapped in foil. 2,2-Azobis-(2-amidinopropane) dihydrochloride was prepared on the day of the experiment in Chelex-treated PBS to give a nal cuvette concentration of 10 nmol/L in Tyrodes medium. Patients After written informed consent had been obtained, 810 sigmoid or rectal biopsies were taken from seven

patients (median age, 51 years; range, 2772 years; four males and three females) with active ulcerative colitis undergoing routine colonoscopy for assessment or surveillance. The diagnosis had been conrmed previously by conventional clinical, colonoscopic and histological criteria. The disease activity was determined using clinical10 and sigmoidoscopic11 scores. Four patients had left-sided disease, one had subtotal disease and two had pan-colitis. Six patients were on an aminosalicylate and one was on azathioprine; none of the patients was taking corticosteroids or smoked. The study was approved by the East London and City Health Authority Ethics Committee. Xanthine/xanthine oxidase cell-free assay The xanthine/xanthine oxidase cell-free system produces superoxide, which is subsequently detected by luminol-dependent chemiluminescence.12, 13 Pre-oxygenated luminol + xanthine solution (1 mL), with added aloe vera gel at the dilutions given above, was added to vials, adjusted to pH 7.4 and pre-counted. Xanthine oxidase was added at a nal concentration of 0.017 mol/L to start the reaction and the vials were immediately re-counted over 15 min in a repeated sequence for 0.5 s per vial in an automated LB953 luminometer (Bertholdt, Bad Wildbad, Germany).14 The nal result for each control or aloe vera dilution was recorded as the mean value of the integral count in counts per second for duplicate vials. The median coefcient of variation for duplicate assays was 16% (n 21).15 Phycoerythrin degradation cell-free assay Free radicals were generated using 2,2-azobis-(2-amidinopropane) dihydrochloride, a chemical which decomposes at a temperature-controlled rate to form carbon-centred radicals that react with oxygen to yield peroxyl radicals.16 Phycoerythrin was desalted as stated earlier. The uorescence of 1 mL of 20 nmol/L phycoerythrin, with or without the addition of dilutions (see above) of aloe vera gel, was measured in a spectrouorimeter (Kontron Instruments, Milan, Italy) with excitation at 540 nm and emission at 575 nm, thermostatically controlled to 37 C and tted with an electronic cuvette stirrer.14 Baseline uorescence in the presence of sample was measured for 23 min before the addition of 10 nmol of 2,2-azobis 2004 Blackwell Publishing Ltd, Aliment Pharmacol Ther 19, 521527

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(2-amidinopropane) dihydrochloride. Fluorescence was then monitored for 25 min. The rate of decay of phycoerythrin uorescence was calculated from the slope of the curve of uorescence intensity plotted against time. The coefcient of variation of duplicate assays was 10.1% (n 21).15 Mucosal biopsy incubations and chemiluminescence assays Four to ve paired biopsy samples from adjacent areas of similar macroscopic inammation11 from each individual patient were placed initially in pre-oxygenated incubation medium (Tyrodes medium) before transfer to a tube containing 1 mL of either Tyrodes medium as a control or Tyrodes medium with added aloe vera gel.14 Biopsies were incubated at 37 C with a continuous ow of O2/CO2 (95%/5%) bubbling through the medium. The incubation time chosen was 40 min, as this has been shown to be adequate for various specic drugs to alter the release of eicosanoids (e.g. prostaglandin E2 and leukotriene B417 and thromboxane B218), suggesting that tissue penetration is possible in this time period. At 20 min, fresh test incubation medium containing, in addition, arachidonic acid (50 lmol) as excess substrate was exchanged for old. After 40 min, incubation uid was aspirated, aliquotted and stored at ) 70 C until assay of eicosanoids (see below). Biopsies were washed to remove residual aloe vera, and transferred to pre-counted scintillation vials containing 1 mL of 300 lmol/L luminol with 5 mmol/L glucose, pH 7.4. The vials were immediately counted for 4 min each in the LB953 luminometer. After assay, biopsies were blot-dried and weighed. Chemiluminescence was recorded as the mean for paired biopsies, expressed as the number of counts per minute per milligram of tissue weight, after subtraction of background. The coefcient of variation of chemiluminescence counts for paired biopsies was 47% (n 30).15 Interleukin-8 cell culture experiments Human CaCo2 cells were obtained from the European Collection of Cell Cultures (www.ecacc.org.uk; UK). Cells were grown in 75-cm2 culture asks (Corning, NY, USA) containing 25 mL of complete growth medium [consisting of Dulbeccos modied Eagle medium supplemented with 10% foetal calf serum, 1% 100X non-essential amino acids, penicillin (250 U/mL), streptomycin (50 lg/mL) and glutamine (2 mmol/L)]. The
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medium was changed every 23 days. At 9095% conuence, the medium was removed and the cell monolayer was washed twice with 25 mL of PBS. Cells were then incubated in 2 mL of 10% trypsin/ethylenediaminetetra-acetic acid for 10 min at 37 C, 5% CO2. A further 25 mL of complete medium was added to the culture ask and agitated to ensure complete cell layer elevation. The cell suspension was then transferred to a 50-mL Falcon centrifuge tube (Corning) and centrifuged at 1000 r.p.m. for 10 min. The resultant cell pellet was suspended in 25 mL of complete medium and the cell concentration was determined manually with a haemocytometer. Medium containing (12) 105 cells was then added to each well of a 24-well tissue culture plate (Corning) in addition to a further 2 mL of complete medium. Cells were assessed daily for conuence by visual inspection and, when this was achieved, were serum-starved for 1218 h. Following starvation, the medium was replaced by complete medium containing either no aloe vera (control) or aloe vera at dilutions of 1 in 10, 1 in 100 or 1 in 1000. Cells were incubated for 24 h and the supernatant was removed and stored at ) 70 C until assay for interleukin-8. Eicosanoid assays Concentrations in the biopsy supernatant of prostaglandin E2 and thromboxane B2, the stable derivative of thromboxane A2, were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Abingdon, UK). Samples of biopsy incubation uid were diluted 1 in 5 and analysed in duplicate. The inter-assay coefcient of variation for prostaglandin E2 was 16% and for thromboxane B2 was 18%. The intraassay coefcient of variation for prostaglandin E2 was 5% and for thromboxane B2 was 7%.15 Serial dilution of selected samples revealed parallelism with assay standards. Prostaglandin E2 and thromboxane B2 standards made up in Tyrodes medium with aloe vera gel diluted 1 in 10 also revealed parallelism with standards made up with assay buffer. Interleukin-8 assay Interleukin-8 was measured using a commercial ELISA kit (BD OptEIA ELISA Kits, BD Biosciences Pharmigen, San Jose, CA, USA). Samples of cell culture supernatant were diluted 1 in 20 and analysed in duplicate. The inter-assay coefcient of variation for interleukin-8 was

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18%15 and the intra-assay coefcient of variation was 7%. Serial dilution of selected samples revealed parallelism with assay standards. Calculations and statistics For the chemiluminescence studies, the results of duplicate experiments were averaged and expressed as a percentage of the mean result of the control pair for that experiment. Except for the doseresponse data shown in Figure 1, results are shown as the median (interquartile range, IQR) and were compared using the Wilcoxon signed rank test for paired data, Mann Whitney U-test for unpaired data and KruskalWallis test for analysis of variance, using two-tailed P values and taking P < 0.05 as statistically signicant. As all comparisons were planned prospectively, no correction was made for multiple comparisons.19 Doseresponse curves and ELISA standard curves were drawn using linear regression analysis. The 50% inhibition value (IC50) was obtained from the line of best t.

% control

Log AV dilution

% control

RESULTS

AV dilution

Anti-oxidant effects of aloe vera gel Xanthine/xanthine oxidase cell-free system. Aloe vera had a dose-dependent effect on superoxide detection by chemiluminescence (Figure 1a), with almost 100% inhibition of oxidant release at high concentrations; 50% inhibition (IC50) occurred at 1 in 100 dilution. Phycoerythrin degradation assay. Aloe vera gel produced a dose-dependent peroxyl radical scavenging effect (Figure 1b), with an IC50 of about 1 in 1000. Chemiluminescence with colorectal biopsies. In comparison with paired controls, chemiluminescence was signicantly reduced in biopsies incubated with 1 in 50, 1 in 10 and 1 in 5 dilutions of aloe vera (Figure 1c), with 50% inhibition of reactive oxygen metabolite release in the range of 1 in 1050 dilution of aloe vera gel. Effects of aloe vera on eicosanoid release from colorectal biopsies Prostaglandin E2 release [median (IQR)] from biopsies incubated in aloe vera gel at 1 in 50 dilution [984 pg/mL per milligram of tissue (6442575), n 6] was

% control

AV dilution
Figure 1. Anti-oxidant effects of aloe vera gel in vitro. (a) Xanthine/xanthine oxidase cell-free system; (b) phycoerythrindegradation assay; (c) chemiluminescence of biopsies after incubation with AV. Results are expressed as a percentage of paired control experiments with mean [S.E.M.] shown. n 5 for each data point in the cell-free assays, and n 7 for the biopsy experiments. The concentration [expressed as dilution in parts per volume (ppv)] required to produce 50% inhibition of reactive oxygen metabolite detection (IC50) for the xanthine/xanthine oxidase system was 1 in 100 (a), for the phycoerythrin degradation assay was 1 in 1000 (b) and for biopsy incubations was in the range of 1 in 1050 (c). AV, aloe vera gel.

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reduced by about 30% compared with paired controls [1415 (8607475)] (P < 0.05). No signicant effect was seen at 1 in 10 [1307 (8053675), n 8] or 1 in 5 [1639 (11264316), n 7] dilutions of aloe vera in comparison with paired controls. Thromboxane B2 production was not signicantly altered by incubation with aloe vera gel at these dilutions [1 in 50: 150 pg/ mL per milligram of tissue (63388); 1 in 10: 136 (105219); 1 in 5: 100 (87289)] compared with paired controls [136 (112496), n 7 in each case]. Effect of aloe vera gel on interleukin-8 production from CaCo2 cells At 1 in 100 dilution, aloe vera gel inhibited interleukin-8 production by 20% [1280 pg/mL (12501300), n 5] in comparison with controls [1660 (15901750), n 5] (P < 0.05). At 1 in 10 and 1 in 1000 dilutions, aloe vera had no signicant effect [1740 (16751990), n 5 and 1520 (14901590), n 5, respectively].
DISCUSSION

Aloe vera gel has been applied topically by ancient and modern cultures throughout the world for its antiinammatory and wound healing properties. More recently, in the west, it has been widely used as an oral preparation for its supposed anti-inammatory effects. Many patients with inammatory bowel disease, in particular, use aloe vera gel orally as a complement or even alternative to conventional therapy.2 In the present in vitro experiments, we have found that aloe vera gel has anti-oxidant properties and inhibitory effects on colorectal prostaglandin E2 and interleukin-8 production. At the outset, two general points should be made. The rst relates to the relevance of the concentrations of aloe vera gel used in these in vitro studies to those likely to be found in the digestive tract after its oral ingestion in the doses commonly used by the general public (up to 100 mL twice daily). Aloe vera gel contains a large number of components of varying chemical composition and biological activity.1 An approximate calculation, assuming an ileal efuent of about 1 L/day, suggests that the concentrations of gel used in this study, in relation to its indigestible and non-absorbable phytic components, may roughly resemble those likely to be present in the colonic lumen. Clearly, however, it is not possible to estimate the colonic concentrations of the absorbable and biologically active components of orally
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ingested aloe vera. In respect of the concentrations used, therefore, it is difcult to extrapolate directly from the results obtained in vitro to what may occur in the human colon in vivo. Second, the preparation of aloe vera given in the present work has been reported to contain a very high proportion (> 95%) of the active ingredient, namely the pulp of the leaf of the aloe vera plant; some commercially available preparations contain far less20 (International Aloe Science Council; http://www.iasc.org). In relation to doseresponse, the results might have been different with other preparations of aloe vera. In two cell-free systems and on incubation with inamed colorectal mucosal biopsies, aloe vera gel had a dose-dependent anti-oxidant action. The cell-free techniques allow the assessment of the scavenging of both superoxide21 and peroxyl16 radicals. In colonic biopsies, the mechanisms by which aloe vera reduces the production of reactive oxygen metabolites are likely to be more complex, but the results obtained may resemble more closely that which occurs in vivo. Any residual traces of aloe vera gel were removed from the biopsies before they were placed in the chemiluminometer. Therefore, the results obtained in this system indicate biological and chemical events in the tissue rather than either a pure scavenging effect or a direct effect of aloe vera on the chemistry of chemiluminescence. The observation that aloe vera gel was a less potent anti-oxidant in relation to colonic tissue than in cell-free systems may indicate a lack of penetration of important constituents of the gel into the tissue during the short incubation period used. The present results conrm and extend previous in vitro studies of the anti-oxidant effects of aloe vera, although none has involved gastrointestinal mucosa specically. Various fractions of aloe vera, as well as the unfractionated whole gel, have been shown to have anti-oxidant effects.2225 Aloe vera gel contains glutathione peroxidase activity,26, 27 seven superoxide dismutase enzymes28 and a phenolic anti-oxidant.23 Which of these factors predominates in accounting for our results is not known. Aloe vera gel at a dilution of 1 in 50 inhibited prostaglandin E2 production from inamed colorectal biopsies, but had no effect at higher concentrations or on thromboxane B2 release. Whether the small inhibition of prostaglandin E2 production by aloe vera (1 in 50) is of biological signicance is uncertain. Furthermore, the explanation for the U-shaped doseresponse

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relationship between aloe vera and mucosal prostaglandin E2 production is not immediately clear. Previous studies have suggested that aqueous and glycoprotein extracts have inhibitory effects on cyclo-oxygenase and thromboxane synthase activity.25, 29 In our experiments using unfractionated aloe vera gel, it is possible that, at high concentrations, one or more of the multiple components of aloe vera has a stimulatory effect on prostaglandin E2 production which outweighs the inhibitory effect of other components seen at lower concentrations. The absence of any effect of aloe vera gel on thromboxane B2 production suggests that the observed inhibition of prostaglandin E2 release by aloe vera (1 in 50) is mediated through a target specic to the prostaglandin synthesis pathway. Our results with colonic epithelial cell cultures and interleukin-8 production resemble those found with the biopsies and prostaglandin E2 in that a small but statistically signicant reduction of interleukin-8 production occurred during incubation with a 1 in 100 dilution of aloe vera, but not with stronger or weaker concentrations. We are aware of only one previous study of the effect of aloe vera on interleukin-8 production: this showed no inhibition by the herb of the production of interleukin-8 by bacterially stimulated monocytes.30 The possible explanation for the U-shaped doseresponse found in the present work mirrors that for prostaglandin E2 release from biopsies, and is again likely to reect the complexity of the composition of aloe vera gel. Further exploration of the effects of aloe vera on the production of reactive oxygen metabolites, eicosanoids and interleukin-8 requires the separation and purication of its multiple, individual, potentially bioactive components. However, in view of the paucity of controlled trials indicating its efcacy in inammatory bowel disease or other inammatory diseases, this timeconsuming and expensive exercise may be premature. Furthermore, it would contradict the concept of the use of whole plant extracts by herbalists and could prove to be counterproductive if the efcacy of aloe vera in inammatory diseases depends on the actions of several of its components, be they synergistic or separate. Whatever the mechanisms by which aloe vera inhibits the production of reactive oxygen metabolites, prostaglandins and interleukin-8 by colonic mucosal cells, their probable involvement in the pathogenesis of inammatory bowel disease49 suggests that the evaluation of aloe vera gel as a potential novel therapy is

worthwhile. Furthermore, these results provide support for the proposal that aloe vera gel could prove to be benecial in other inammatory conditions of the gastrointestinal tract and elsewhere.
ACKNOWLEDGEMENTS

We are grateful to the National Association for Colitis and Crohns Disease (NACC) for nancial support and to Dr Peter Atherton and Forever Living Products (Jersey, Channel Islands) for the provision of aloe vera gel.

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