Antiinflammatory effects of Cordia myxa fruit on experimentally induced colitis in rats

BASIC NUTRITIONAL INVESTIGATION

Antiinflammatory Effects of Cordia myxa Fruit on Experimentally Induced Colitis in Rats Farida M. Al-Awadi, PhD, T. S. Srikumar, PhD, J. T. Anim, MD, FRCPath, and Islam Khan, PhD From the Departments of Biochemistry and Pathology, Faculty of Medicine, Kuwait University, Safat, Kuwait Products of certain species of Cordia are reported to have antiinflammatory properties. In the present study we examined the effects of Cordia myxa fruit on experimentally induced colitis in rats. Colitis was induced by intrarectal administration of 4% acetic acid. Colitic, normal, and corresponding control animals were included. Body weight was recorded daily. All the animals were sacrificed 4 days after the fruit treatment. Colitis was monitored histologically and by activity of myeloperoxidase. Glutathione peroxidase, superoxide dismutase, as well as total antioxidant status and concentrations of zinc, copper, manganese, selenium, and iron were assayed in plasma, liver, and colon using standard methods. Histology of the colon of colitic rats showed acute colitis that was confirmed by a significant increase in the myeloperoxidase activity. Colitis was associated with significant decreases in the tissue activities of glutathione peroxidase and superoxide dismutase and lower concentrations of trace elements. Histologic examination and myeloperoxidase activity showed that the fruit treatment reversed the above findings in the inflamed colon, and in liver and plasma of colitic rats. The present results suggest that the observed antiinflammatory effect of the Cordia myxa may be attributed partly to its antioxidant property and to restoration of the levels of trace elements in the inflamed colon, liver, and plasma. Nutrition 2001;17: 391–396. ©Elsevier Science Inc. 2001 KEY WORDS: Cordia myxa, colitis, myeloperoxidase activity, antioxidants, trace elements

INTRODUCTION Diet and traditional medicines play an important role in the therapy of many diseases. However, plant remedies for treatment of colitis are uncommon. Current protocols for treatment of inflammatory bowel disease include the use of medications and colectomy. Medications vary from short term (corticosteroids), to long term (sulfasalazine). The fruit of Cordia myxa (natural order: Boraginaceae, synonym: Cordia abyssinica), has been used for the treatment of infections of urinary tract, diseases of the lung and spleen, and as an astringent, anthelminthic, diuretic, and demulcent agent.1 It has been reported that leaf extracts of certain species of Cordia such as C. myxa, C. francisci, and C. serratifolia have significant analgesic, antiinflammatory, and antiarthritic activity in the rat.2 Among other components of plant preparations, flavonoids (quercietrin or rutin) have also been considered useful in the treatment of intestinal inflammation.3,4 These flavonoids reduce the area of colonic damage, and enhance the colonic fluid absorption capacity, but their effects on biochemical markers of inflammation such as alkaline phosphatase and myeloperoxidase remain to be elucidated.5 The fruit of C. myxa, known as “bamber” (Sebestian plum) in Kuwait, has been used by the public for the treatment of asthma and lung infections, but has not been investigated scientifically. Previous studies of this fruit preparation have been restricted to investigation of its effects on the contractility of ileum and dilatation of peripheral blood vessels.6,7 In view of the reported beneficial effects on infections of the

Correspondence to: Farida M. Al-Awadi, PhD, Department of Biochemistry, Health Sciences Center, Faculty of Medicine, Kuwait University, P. O. Box 24923, Safat 13110, Kuwait. E-mail: [email protected] Date accepted: December 19, 2000. Nutrition 17:391–396, 2001 ©Elsevier Science Inc., 2001. Printed in the United States. All rights reserved.

urinary tract and lung, the present study was designed to test the effect of the fruit extract on experimentally induced colitis. Inflammatory bowel disease, which includes colitis, is characterized by changes in the production of cytokines, prostaglandins, and neuromuscular activity.8,9 The etiology of these diseases is not known, and it is difficult to replicate the human conditions in experimental animals. Nevertheless, intrarectal administration of acetic acid in small rodents has served as an excellent model of colitis that permits examination of effects of various substances on this condition. We have recently reported specific decreases in the levels of trace elements and antioxidant enzymes in the colon, liver, and plasma of the colitic animal.10 Whether C. myxa has any effects on the concentration of trace elements and antioxidant enzymes in colitis induced by acetic acid, remains to be investigated. Therefore, the primary aims of this study were to examine the differences in the following parameters between fruit-treated and untreated colitic animals: 1) histopathologic changes in colonic segments; 2) activity of myeloperoxidase (a marker of inflammation); 3) total antioxidant status, and activities of glutathione peroxidase and superoxide dismutase; and 4) concentration of iron and trace elements (zinc, copper, manganese, and selenium).

ANIMALS AND METHODS Induction of Colitis and Fruit Treatment Wistar male rats weighing 150 to 200 g, bred locally and maintained by the Faculty of Medicine, Kuwait University, were used in this study. Colitis was induced by intrarectal administration of 4% acetic acid (4 mL/kg body weight) in phosphate-buffered saline (PBS) as described previously.10 –12 The rats were grouped as follows: normal colitic control (NCC) that received PBS (n ⫽ 0899-9007/01/$20.00 PII S0899-9007(01)00517-2

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Nutrition Volume 17, Number 5, 2001 Histopathologic Examination Histopathologic examinations were performed blind by one of us (J. T. A.). Two-centimeter lengths of colon (taken from the middle part of the distal 6 to 8 cm of the colon, the part that is inflamed in experimentally induced colitis) were fixed in 4% (v/v) buffered formalin. Colonic tissues were from treated and control groups of rats. Sections (4 to 5 ␮m) were stained with hematoxylin and eosin (H&E) and examined for the features of inflammation by light microscopy. Presence and severity of acute inflammation were assessed and graded semiquantitatively as follows: ● No acute inflammation (⫾ lymphoid hyperplasia) ⫽ grade 0 ● Mild acute inflammation in wall or serosa ⫽ grade 1 ● Moderate acute inflammation ⫾ small ulcers ⫽ grade 2 ● Severe acute inflammation ⫹ ulceration/necrosis of wall ⫽ grade 3

Assay of Myeloperoxidase Activity FIG. 1. Whole Cordia myxa fruit (upper left), the elasticity of the mucilage portion (upper right), and the dense central transparent mucilage portion surrounding the seed (lower left and right).

8); normal colitic controls that received PBS but were treated with the fruit (NCCFT; n ⫽ 11); colitic control (CC) that received acetic acid (n ⫽ 7); colitic group treated with C. myxa fruit (CCFT; n ⫽ 8); normal control (NC; n ⫽ 11); and normal fruit treated (NFT; n ⫽ 9). Fruit treatment was started the day after induction of colitis. Treated animals received a fruit preparation (12 g/kg body weight) orally by gastric intubation as single boluses daily in the morning for 4 d. Control groups (NCC, CC, and NC) received a water bolus each. The animals were housed in separate groups and fed normal diets (Standard Diets Services, Essex, UK) and water ad libitum. Colitis was confirmed by the measurement of myeloperoxidase activity and histopathologic examination of the colon. The study protocol was in accordance with the guidelines of the Ethical Committee, Faculty of Medicine, Kuwaiti University.

Preparation of Fruit Suspension The C. myxa fruits were washed several times with distilled water and finally with deionized water. The seeds were separated and the mucilage portion surrounding the seeds (Fig. 1) was collected and homogenized, along with the rest of the fruit, in deionized water 1:5 (w/v).

Sample Preparation Animals were sacrificed 5 d after administration of acetic acid and 4 d of treatment with the fruit preparation. Corresponding control animals were sacrificed on the same day. Animals were anesthetized with phenobarbital sodium (50 mg/kg body weight, intraperitoneally). Colon (distal 6 to 8 cm of the colon) and liver were harvested after abdominal incision and blood samples (5 mL) were collected directly from the heart. Tissue samples were immediately stored at ⫺80°C and homogenized separately on the day of analysis (1/10, w/v) using a polytron homogenizer (Janke and Kuncle, Staufen, Germany) in appropriate buffers for each enzyme assay. Blood samples were collected into tubes containing EDTA and centrifuged at 3000 rpm for 10 min. Plasma was collected and stored frozen at ⫺80°C, and used for enzyme assays, determination of total antioxidants and trace element analyses. All enzyme assays and total antioxidant estimations were performed using a DU 7500 spectrophotometer (Beckman Instruments, Palo Alto, CA, USA).

Myeloperoxidase activity was estimated as described previously10,11,13 in whole colonic segments that contained both mucosa and muscle layers. Briefly, 1 g of colonic segments were minced finely in 10 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 14 mM hexa-decyltrimethyl-ammonium bromide using scissors. Tissues were homogenized for 1 min, then the lysates were frozen in liquid nitrogen, thawed once, and centrifuged for 2 min at 4°C at 15 000 g. Myeloperoxidase activity was estimated in the supernatants in presence of O-dianisidine-HCl and 0.0005% H2O2 by monitoring optical density at 415 nm using a Beckman DU700 spectrophotometer. Myeloperoxidase activity has been expressed as units 䡠 min⫺1 䡠 mg⫺1 of tissue with the enzyme unit defined as conversion of 1 ␮mol of H2O2 䡠 min⫺1 䡠 mg⫺1 of tissue at room temperature (25°C).

Assay of Glutathione Peroxidase Activity Selenium-specific glutathione peroxidase activity in plasma, colon, and liver was estimated as described previously using a Ransel kit (Randox, UK).10 Colon and liver samples were homogenized separately (1/10, w/v) using 50 mM phosphate buffer (pH 7.5) and centrifuged at 3000 rpm for 30 min at 4°C using JA20 rotor (Beckman). The supernatants were further centrifuged at 155 000 ⫻ g for 60 min at 4°C (Beckman LX-90 Centrifuge) and pellets were discarded. Aliquots (50 ␮L) of the cytosolic supernatant were mixed with 1 mL of diluent provided in the Ransel kit and the reaction was performed following the instructions supplied by the manufacturer at 37°C using a DU 700 spectrophotometer (Beckman, Geneva). The rates of reaction were calculated and enzyme activities expressed as units per gram tissue.

Assay of Superoxide Dismutase Activity The zinc/copper-dependent superoxide dismutase activity in blood hemolysate, colon, and liver samples were measured using a kit from Randox Laboratories Ltd. (Ardmore, Diamond Road, Crumlin, UK). The hemolysate was prepared by adding 1 volume of ice-cold distilled water to the separated erythrocytes and used for the superoxide dismutase assay. Tissue samples were homogenized (1/10, w/v) in deionized water and centrifuged at 3000 rpm for 30 min at 4°C, after which the supernatant was further centrifuged at 155 000 ⫻ g at 4°C for 1 h before assay. The absorbance was measured using a Beckman spectrophotometer (DU 7500) at 505 nm and 37°C. The accuracy and precision of this assay was checked using lyophilized whole blood and the coefficient of variation was less than 10%.

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Antiinflammatory Effects of Cordia myxa Fruit on Colitis TABLE I.

Assay of Plasma Total Antioxidants Plasma total antioxidant status was measured using a kit purchased from Randox Laboratories Ltd. This is an iron-dependent metmyoglobin method that measures the inhibition of an artificially generated oxidative process.14 In this protocol 2,2⬘-azino-di-(3ethylbenzthiazoline sulfonate) is oxidized in a mixture of metmyoglobin and hydrogen-peroxide to a free radical cation. When a solution containing antioxidant such as plasma is added, the absorbance of the radical cation is quenched in proportion to antioxidant concentration. Absorbance was measured at 600 nm at 37°C and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid was used as a standard.

Determination of Concentration of Trace Elements Concentrations of trace elements in C. myxa fruit, plasma, colon, and liver were determined by atomic absorption spectrometry. Approximately 100 to 200 mg of C. myxa fruit, liver, and colon samples (200 to 250 mg) were digested (in a fume hood) using a mixture of 4 mL concentrated nitric acid and 1 mL perchloric acid.10,15,16 The samples were heated on a heating block and temperature was gradually increased from 50° to 190°C until clear digests were obtained. The digested samples were quantitatively transferred to metal-free tubes and diluted (1:50) with 3% nitric acid before analysis. Concentrations of zinc and copper were measured using a flame atomic absorption spectrophotometer (Varian Spectra AA 400, Australia) with a deuterium background corrector.17 Concentrations of selenium, manganese, and iron were estimated using a graphite furnace atomic absorption spectrophotometer (GTA 100, Spectra AA 800, Australia) equipped with a deuterium background corrector. Palladium was used as a modifier while analyzing selenium. Standard curves were plotted using stock solutions of the elements.

Statistical Analysis Data were processed to obtain mean and standard deviation (SD) values. One-way analysis of variance followed by Student’s t test was used to compare the mean values of different groups. The P values were corrected according to the Bonferroni method. A value of P ⬍ 0.05 was considered to be statistically significant. The risk of inflated alpha due to multiple comparisons has been taken into consideration while interpreting the results.

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SEVERITY (GRADES) OF INFLAMMATORY ACTIVITY IN COLONIC SECTIONS OF CONTROL, COLITIC, AND FRUIT-TREATED COLITIC RATS Groups of rats NCC (n ⫽ 8) NCCFT (n ⫽ 11) CC (n ⫽ 7) CCFT (n ⫽ 8) Total

Grade 0

Grade 1

Grade 2

Grade 3

8 11 1 4 24

0 0 1 2 3

0 0 5 2 7

0 0 0 0 0

grade 0 ⫽ no acute inflammation (⫾ lymphoid hyperplasia); grade 1 ⫽ mild acute inflammation in wall or serosa; grade 2 ⫽ moderate acute inflammation ⫾ small ulcers; grade 3 ⫽ severe acute inflammation ⫹ ulceration/necrosis of wall. CC, colitic control; CCFT, colitic fruit treated; NCC, normal colitic control (PBS); NCCFT, normal control (PBS) fruit treated.

Myeloperoxidase Activity Successful induction of colitis was confirmed by a significant elevation (93%) of myeloperoxidase activity (Fig. 5). Fruit treatment caused a significant decrease (50%) in the myeloperoxidase activity. Levels of myeloperoxidase activity in the normal animals remained unchanged after treatment with the fruit (Fig. 5). Total Antioxidant Status and Activities of Antioxidant Enzymes The total plasma antioxidant status was reduced following induction of colitis. However, fruit treatment reversed these changes (Table II). The activities of glutathione peroxidase and superoxide dismutase in plasma, colon, and liver were significantly decreased in colitic animals (Table II), but were restored to normal levels by the fruit treatment, and there was a tendency for the activities to increase above normal levels (Table II). Concentration of Elements in C. myxa Fruit Analysis of zinc, copper, manganese, selenium, and iron in C. myxa fruit revealed that the fruit is a good source of these elements. Of these elements, the highest concentration found was that

RESULTS Histopathology Table I summarizes the histopathologic changes in the experimental and control animals. The H&E-stained sections of colon showed no evidence of colitis in the control normal rats (Fig. 2). However, five of the seven acetic acid-treated animals showed moderate acute colitis with areas of ulceration (Fig. 3). Treatment with the C. myxa fruit preparation significantly reversed the histopathologic changes in 50% of the animals (Fig. 4). Only mild inflammation (few inflammatory cells infiltrating in the wall of the colon) appeared in 25% of C. myxa-treated animals, and moderate inflammation persisted in the remaining 25%. Histology of the colon of normal treated animals treated with C. myxa did not show any harmful effects of the fruit on the colonic tissue, as the histopathologic features were similar to normal control animals. Histopathologic examinations were not performed in NC and NFT animals, because the aim of inclusion of these groups were to observe the effect of fruit treatment on the changes in various parameters under normal physiologic conditions.

FIG. 2. Representative section of colon from normal rat showing no inflammatory changes (hematoxylin and eosin ⫻177.5).

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Nutrition Volume 17, Number 5, 2001 activity. Treatment with C. myxa fruit preparations resulted in less ulceration in this model of colitis. Fewer ulcers were evident and only minor collections of inflammatory cells (neutrophils, lymphocytes, and plasma cells) remained in the colonic walls after treatment. The antiinflammatory effect of C. myxa fruit preparation was further evident from a reduction in myeloperoxidase activity in the colon. One of the advantages of the fruit treatment was that it did not cause any side effects. Total Antioxidant Status and Activities of Antioxidant Enzymes

FIG. 3. Representative section of colon from rat treated with acetic acid to induce experimental colitis. Note the extensive ulceration (arrowheads) with underlying heavy inflammatory changes (hematoxylin and eosin ⫻71).

of iron (19 mg/kg) followed by zinc (7 mg/kg), copper (3 mg/kg), manganese (2 mg/kg), and selenium (0.08 mg/kg). Trace Element Status of Experimental Animals The concentrations of manganese and selenium in plasma (Table III), those of zinc, copper, manganese, selenium, and iron in the colon (Table IV), and those of manganese, selenium, and iron in the liver (Table V) were significantly decreased in the colitic rats. Among the elements analyzed, selenium was decreased the most in all the tissues, and the colon seemed to be the most severely affected. However, treatment with the C. myxa fruit restored the levels of these elements close to normal values in the colitic animals and led to even higher levels in normal rats (Tables III–V).

DISCUSSION Histopathology and Myeloperoxidase Activity The degree of colonic inflammation was assessed and confirmed by quantification of microscopic damage and myeloperoxidase

FIG. 4. Representative section of colon from rat treated with Cordia myxa fruit preparation after induction of colitis with acetic acid. Note the presence of only small collections of inflammatory cells (neutrophils, lymphocytes, and plasma cells) in the wall (arrowheads) (hematoxylin and eosin ⫻177.5).

The total antioxidant capacity measures ability of tissues to inhibit free-radical-mediated processes. We have previously reported that total antioxidant status is dependent on the concentration of the individual antioxidants and activity of scavenging enzymes such as glutathione peroxidase, superoxide dismutase, catalase, and glutathione reductase.10 Oxidative activity of free radicals is an important cause of tissue injury in inflammation.18 Oxidants are also believed to regulate the expression of genes involved in the inflammatory response and promote intestinal epithelial cell apoptosis.3 We have previously reported that the antioxidant defense system is impaired in two models of experimentally induced colitis.10 Low glutathione peroxidase levels indicated that protection from free radicals was insufficient and that the colon was open to damage by free radicals. In the present study, the oxidant stress in colitis was further confirmed by a significant decrease in the total antioxidant status, which may indicate that oxidant stress is overwhelming during progression of inflammation/ulceration under the experimental condition used in this study. Our findings demonstrated that C. myxa preparation exerts an inhibitory effect on the oxidant stress factors that lead to progression of colitis, giving not only an improvement of total antioxidant status level, but its restoration to normal levels. Treatment with C. myxa led to increases in glutathione peroxidase activity in colon and plasma in experimental colitis. The effect of the fruit treatment on the superoxide dismutase was less pronounced; an increase in activity was evident in the colon of colitic but not with the normal animals. The present data confirmed our previous report10 that an effect of colitis on antioxidant status is extended to tissues such as the liver, where there was a significant decrease in the activities of antioxidant enzymes and in total antioxidant status. The wide extent of the antioxidant effect of C. myxa was evident in the liver where a significant increase in the total antioxidant status and in

FIG. 5. Activity of myeloperoxidase (antiinflammatory marker) in colon of normal colitic control (NCC), colitic control (CC), colitic fruit-treated (CFT), and normal control (phosphate-buffered saline) fruit-treated (NCCFT) animals. aSignificantly higher than NCC; bsignificantly lower than CC.

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TABLE II. EFFECT OF TREATMENT OF EXPERIMENTALLY INDUCED COLITIS USING CORDIA MYXA FRUIT ON THE CHANGES IN TOTAL ANTIOXIDANT STATUS (TAS) AND ACTIVITIES OF GLUTATHIONE PEROXIDASE (GSHPX) AND SUPEROXIDE DISMUTASE (SOD) IN PLASMA, LIVER, AND COLON OF RATS Plasma Groups of animals NCC (n ⫽ 8) NCCFT (n ⫽ 11) CC (n ⫽ 7) CCFT (n ⫽ 8) NC (n ⫽ 11) NFT (n ⫽ 9)

Liver

Colon

TAS (mmol/L)

GSHPx (U/mL)

Hemolysate SOD (U/L)

GSHPx (U/g)

SOD (U/kg)

GSHPx (U/mg)

SOD (U/mg)

1.1 ⫾ 0.2 0.9 ⫾ 0.06 0.6 ⫾ 0.1a 1.0 ⫾ 0.07b 1.2 ⫾ 0.3 1.0 ⫾ 0.6

6.9 ⫾ 1.6 4.3 ⫾ 2.2 5.4 ⫾ 1.3a 6.6 ⫾ 1.2 6.3 ⫾ 1.9 6.8 ⫾ 1.0

0.26 ⫾ 0.02 0.25 ⫾ 0.10 0.14 ⫾ 0.05a 0.21 ⫾ 0.02b 0.27 ⫾ 0.05 0.31 ⫾ 0.03

74 ⫾ 16 81 ⫾ 12 69 ⫾ 18a 101 ⫾ 30b 75 ⫾ 16 97 ⫾ 17c

1.71 ⫾ 0.3 1.40 ⫾ 0.3 1.10 ⫾ 0.3a 1.62 ⫾ 0.4b 1.59 ⫾ 0.2 1.63 ⫾ 0.3

10 ⫾ 1.7 13 ⫾ 1.8 6.9 ⫾ 0.9a 9.4 ⫾ 1.1b 10.2 ⫾ 1.0 10.7 ⫾ 0.9

264 ⫾ 30 249 ⫾ 21 196 ⫾ 20a 241 ⫾ 14b 228 ⫾ 20 246 ⫾ 24

a Significantly lower than NCC; b significantly higher than CC; c significantly higher than NC. CC, colitic control; CCFT, colitic fruit treated; NC, normal control; NCC, normal colitic control (PBS); NCCFT, normal control (PBS) fruit treated; NFT, normal fruit treated. Figures are mean ⫾ SD.

the activities of glutathione peroxidase and superoxide dismutase was observed both in the treated colitic and normal animals. It is likely that the therapeutic property of the fruit preparation is multifactorial and may involve activity of antioxidant factors other than the glutathione peroxidase and superoxide dismutase, such as antioxidant vitamins (A, C, and E), glutathione, and urate. These factors will be investigated in our future studies. Trace Elements The present study attempted to investigate whether a trace element component of C. myxa plays any role in the beneficial effects of this fruit in the treatment of colitis. Trace elements such as selenium, copper, and zinc are essential for maintaining adequate levels of activity of antioxidant enzymes implicated in oxygen free-radical generation, selenium-dependent glutathione peroxidase, and zinc/copper-containing superoxide dismutase. The results presented in this study confirm our previous report that colitis induces decreases in selenium, zinc, copper, and manganese in the

colon,10 and decreased manganese and selenium in the plasma. It is noteworthy that the colonic levels of all these elements were significantly elevated in the tissues of normal and colitic animals after the fruit treatment. A similar increase was observed in the plasma, but only for selenium and manganese. The increase in the level of selenium in colon and plasma parallels the increase in the activity of selenium-dependent glutathione peroxidase in both the normal and colitic animals. C. myxa raised the zinc concentration in the colon, a change that was concordant with the zinc/copperdependent superoxide dismutase activity, but only in the colitic group. It has been reported that lack of zinc may result in damage to mucosal linings of the gastrointestinal and pulmonary tract.19 It can be suggested that glutathione peroxidase is not only selenium dependent, but its activity may be induced by elevated concentrations of this element. However, superoxide dismutase activity is dependent on zinc/copper but may not be induced by increasing their levels. The levels of zinc and copper in plasma in normal and colitic animals, and in the colon of normal animals, remained unchanged. TABLE IV.

TABLE III. EFFECT OF TREATMENT OF EXPERIMENTALLY INDUCED COLITIS USING CORDIA MYXA FRUIT ON THE CHANGES IN THE CONCENTRATION OF TRACE ELEMENTS IN COLON OF RATS

EFFECT OF TREATMENT OF EXPERIMENTALLY INDUCED COLITIS USING CORDIA MYXA FRUIT ON THE CHANGES IN THE CONCENTRATION OF TRACE ELEMENTS IN PLASMA OF RATS Groups of animals NCC (n ⫽ 8) NCCFT (n ⫽ 11) CC (n ⫽ 7) CCFT (n ⫽ 8) NC (n ⫽ 11) NFT (n ⫽ 9) a

Zinc (␮g/mL)

Copper (␮g/mL)

Manganese (ng/mL)

Selenium (␮g/mL)

2.2 ⫾ 0.08 2.1 ⫾ 0.1 1.9 ⫾ 0.07 2.0 ⫾ 0.04 1.9 ⫾ 0.06 2.3 ⫾ 0.07

1.78 ⫾ 0.3 1.83 ⫾ 0.4 1.69 ⫾ 0.02 1.77 ⫾ 0.3 1.66 ⫾ 0.3 1.71 ⫾ 0.5

6.8 ⫾ 0.2 7.0 ⫾ 0.3 5.9 ⫾ 0.3a 7.2 ⫾ 0.4b 5.7 ⫾ 0.3 6.7 ⫾ 0.2c

0.66 ⫾ 0.05 0.72 ⫾ 0.04 0.51 ⫾ 0.06a 0.62 ⫾ 0.04b 0.60 ⫾ 0.03 0.71 ⫾ 0.04c

Significantly lower than NCC; b significantly higher than CC; c significantly higher than NC. CC, colitic control; CCFT, colitic fruit treated; NC, normal control; NCC, normal colitic control (PBS); NCCFT, normal control (PBS) fruit treated; NFT, normal fruit treated.

Groups of animals

Zinc (␮g/g)

Copper (␮g/g)

Manganese (␮g/g)

Selenium (␮g/g)

Iron (␮g/g)

NCC (n ⫽ 8) NCCFT (n ⫽ 11) CC (n ⫽ 7) CCFT (n ⫽ 8) NC (n ⫽ 11) NFT (n ⫽ 9)

22 ⫾ 2 24 ⫾ 2

2.5 ⫾ 0.2 2.7 ⫾ 0.2

2.4 ⫾ 0.2 2.6 ⫾ 0.1

0.23 ⫾ 0.03 0.29 ⫾ 0.02

16 ⫾ 3 19 ⫾ 3

19 ⫾ 2a 23 ⫾ 3b 28 ⫾ 5 26 ⫾ 4

1.6 ⫾ 0.2a 2.2 ⫾ 0.1b 1.9 ⫾ 0.2 2.1 ⫾ 0.3

2.1 ⫾ 0.2b 2.4 ⫾ 0.1a 3.1 ⫾ 0.2 4.2 ⫾ 0.1c

0.17 ⫾ 0.02a 0.22 ⫾ 0.03b 0.19 ⫾ 0.02 0.25 ⫾ 0.03c

13 ⫾ 2a 17 ⫾ 3b 14 ⫾ 2 18 ⫾ 3c

a

Significantly lower than NCC; b significantly higher than CC; c significantly higher than NC. CC, colitic control; CCFT, colitic fruit treated; NC, normal control; NCC, normal colitic control (PBS); NCCFT, normal control (PBS) fruit treated; NFT, normal fruit treated.

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ACKNOWLEDGMENTS

TABLE V. EFFECT OF TREATMENT OF EXPERIMENTALLY INDUCED COLITIS USING CORDIA MYXA FRUIT ON THE CHANGES IN THE CONCENTRATION OF TRACE ELEMENTS IN LIVER OF RATS Groups of animals

Zinc (␮g/g)

NCC (n ⫽ 8) NCCFT (n ⫽ 11) CC (n ⫽ 7) CCFT (n ⫽ 8) NC (n ⫽ 11) NFT (n ⫽ 9)

Copper (␮g/g)

Selenium (␮g/g)

Iron (␮g/g)

30 ⫾ 2 4.1 ⫾ 0.3 3.9 ⫾ 0.1 33 ⫾ 3 5.2 ⫾ 0.5 4.1 ⫾ 0.2

0.22 ⫾ 0.01 0.26 ⫾ 0.02

68 ⫾ 7 70 ⫾ 4

28 ⫾ 2 29 ⫾ 4 29 ⫾ 5 32 ⫾ 4

0.13 ⫾ 0.02a 0.19 ⫾ 0.01b 0.29 ⫾ 0.03 0.34 ⫾ 0.02c

56 ⫾ 3a 62 ⫾ 4b 73 ⫾ 3 81 ⫾ 4c

5.1 ⫾ 0.3 5.8 ⫾ 0.4 4.9 ⫾ 0.5 4.7 ⫾ 0.4

Manganese (␮g/g)

3.0 ⫾ 0.2a 4.3 ⫾ 0.2b 3.1 ⫾ 0.2 3.9 ⫾ 0.1c

a

Significantly lower than NCC; b significantly higher than CC; c significantly higher than NC. CC, colitic control; CCFT, colitic fruit treated; NC, normal control; NCC, normal colitic control (PBS); NCCFT, normal control (PBS) fruit treated; NFT, normal fruit treated.

So these trace elements may be effective only locally, in the injured colon tissues. It appears that among the trace elements studied, selenium plays a major role in the induction and recovery of tissue from oxidative stress through its association with glutathione peroxidase. In any case, components other than trace elements of C. myxa fruit might play a major role in its antiinflammatory properties. However, the 30% to 40% increase in the trace element content of colon and liver of fruit-treated colitic animals may be regarded as having potential clinical importance. This hypothesis may be true for groups having subnormal trace element status, such as individuals living in low selenium areas and those having anemia. In view of the growing interest in alternate medicine, use of a cheaply available fruit like C. myxa for treatment of colitis would definitely be advantageous. Furthermore, it seems from the present study that C. myxa fruit is a good source of several elements with respect to their bioavailability, a finding that needs to be confirmed. Mechanisms of therapeutic efficacy of C. myxa through activities other than antioxidation are worth considering. It has been reported that alterations to colonic blood flow ameliorate tissue injury due to trinitrobenzene sulfonic acid induced colitis in rabbits.20 Previous studies have shown that a C. myxa fruit preparation activates parasympathetic ganglia and causes dilatation of peripheral blood vessels.6 This finding suggests that the therapeutic effect of the C. myxa fruit also could be mediated through increasing colonic blood flow. In conclusion, we demonstrated the antiinflammatory properties of the C. myxa fruit preparation in the treatment of experimental colitis. The mechanism appears to be mediated through the fruit’s antioxidant effects.

The authors thank Drs. A. Owunwanne and J. Craik for suggestions, and Ms. Leyla Jamaleddine, Ms. Mona Al-Rustom, Ms. Fatima Sequeira, Ms. Nancy Thomas, and Mr. Mohammed Ejaz for providing excellent technical assistance. The study was supported by grant MB027 from the Kuwait University Research Administration.

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