Indian Journal of Experimental Biology Vol. 42, February 2004, pp. 179-185

Antiinflammatory and antiulcer activities of phytic acid in rats

a M Sudheer Kumar, B Sridhar Reddy, S Kiran Babu, P M Bhilegaonkar , A Shirwaikar' & M K Unnikrishnan* Department of Pharmacology, College of Phamlaceutical Sciences, Manipal 576 119 • Department of Pharmacognosy, College of Pharmaceutical Sciences, Manipal 576 119, India Received 21 November 2002; revised 30 September 2003

Maximum antiinflammatory activity of phytic acid (PA) was seen at an oral dose of 150 mglkg in the carrageenan induced rat paw edema model. Although PA showed ability to prevent denaturation of proteins, it showed less antiinflammatory activity than ibuprofen. Ability of PA. to bring down thermal denaturation of proteins might be a contributing factor in the mechanism of action against inflammation. PA, at all the doses tested, showed significant protection from ulcers induced by ibuprofen, and cold stress, with a maximum activity at 150 mglkg. There was a significant increase in gastric tissue malondialdehyde levels in ethanol treated rats but these levels decreased following PA pretreatment. Moreover, pretreatment with PA significantly inhibited various effects of ethanol on gastric mucosa, such as, reduction in the concentration of nonprotein sulfhydryl groups, necrosis, erosions, congestion and hemorrhage. These results suggested that gastro-protective effect of PA could be mediated by its activity and cytoprotection of gastric mucosa.

Keywords: Antiinflammatory, Antiulcer, Carrageenan, Denaturation of proteins, Malondialdehyde, Phytic acid

Recognized role of phytic acid (PA) in numerous component. The possibility of antiinflammatory activity biochemical pathways and physiological processes of PA was envisaged as a part of the study. has been increasing in accordance with its ubiquity. Denaturation of proteins is a well documented Long regarded as an in due to its cause of inflammation. A number of antiinflammatory ability to chelate minerals and reduce their solubility compounds have been shown to inhibit thermally and nutritional , the value of PA in induced protein denaturation. Phenylbutazone, foods to prevent and fossibly reverse carcinogenesis salicylic acid, flufenamic acid, etc., have shown dose l is now recognized •. Biochemically, PA is an dependent ability to inhibit thermally induced protein 9 antioxidane.4, energy storeS and regulator of vesicular denaturation . As a part of the investigation on the 6 transport via binding to various proteins • PA is an mechanism of antiinflammatory activity of PA, the active ingredient of high fiber diet that has anticancer ability of PA to inhibit protein denaturation was action in both in vitro and in vivo7 . PA inhibits Fenton studied. Gastric irritation is a common side effect of reaction by complexing with Fe (II) and blocks the all NSAIDs. The activity of PA on ibuprofen, ethanol generation of OH radicalS. The antioxidant function and cold stress induced ulcers was also studied. of phytate within seeds, , nuts and pollens possibly explains its presence in all the above Materials and Methods in good quantities. Most of the present in seeds is Materials-Phytic acid, type IV lambda found to be complexed by P A. The resistance of carrageenan and albumin were obtained from Sigma unsaturated fatty acids of to oxidative stress Chemicals Co., USA. Ibuprofen was obtained from is possibly because of P A. Some seeds probably owe Astra-IDL Ltd. Famotidine was a gift from Cadila their long viability to PA, inspite of the presence of 8 Pharmaceuticals Ltd. All other chemicals were of iron and unsaturated fatty acids . Of the several analytical grade. Paw volume was measured in a chelating agents, very few displace iron-coordinated Plethysmograph by mercury displacement. water, and thereby block the formation of OH Absorbance was measured using graphicord UV-240 radicalS. PA and desferrioxamine B methane sulfonate . Shimadzu spectrophotometer. (desferal) are such compounds. However, desferal is Drugs - Phytic acid (PA) was dissolved in too toxic and expensive while PA is a nontoxic dietary distilled water and administered orally. Ibuprofen was *Correspondent author:- given orally by making suspension in 1% Fax : 91-820-2571998; E-mail: [email protected] carboxymethy1celloulose (CMC). 180 INDIAN J EXP BIOL, FEBRUARY 2004

Animals-Adult Swiss albino rats (150-200 g) Cold stress induced ulcer model-Animals fasted were used for the study. They were maintained at for 24 hr were divided into groups of six each and temperature and humidity regulated animal house, fed treated as- DW+cold stress (CS; 30 min after); on a standard diet of rat and given water ad libitum. famotidine (3.6 mg/kg) + cold stress (CS; 30 min The experiments were carried out according to the after); and PA (150 mg/kg) + cold stress (CS; 30 min institutional regulations and national criteria for after). Cold stress was induced by keeping the rats at animal experimentation. 4° to 6°C for 3 hr. These animals were sacrificed and Antiinflammatory activity-It was measured in scored ulcers as described earlier. male albino rats (I50-200g) using can'ageenan­ Ethanol induced ulcer model- Animals fasted for induced paw edema modellO. Animals fasted for 24 hr 24 hr were divided into groups of six each and treated were divided into groups of six each and treated as ­ as - DW + absolute ethanol (I ml/animal, I hr after); Distilled water control (DW); Ibuprofen (250 mg/kg) famotidine (3.6 mg/kg) + ethanol (1 mI/animal, I hr and PA (30-150 mg/kg). After 1 hr of treatment, sub­ after); and PA (150 mg/kg) + ethanol (1 ml/animal, plantar injection of 0.05 ml of 1% solution of 1 hr after). After 3 hr of ethanol treatment, animals carrageenan was given into the left hind paw. Volume were sacrificed and scored ulcers as described earlier. of the injected paw was measured by mercury Histopathological studies (ethanol induced displacement in a plethysmograph. Edema developed ulcers)-Two animals from each group were following injection of carrageenan was the index of sacrificed and the stomachs were isolated, washed acute inflammatory changes, which was determined with saline and preserved in formaldehyde solution from the differences of the paw volume measured (I 0%) for histopathological studies. The sections of immediately after carrageenan injection and after 3 hr the stomach, stained with hematoxylin and eosin, of injection. Percentage inhibition =(V c- Vt) x 100IY c; were assessed for severity of histopathological where Vc and Vt were average edema volumes of changes such as congestion, oedema, haemorrhage control and treated groups respectively. and necrosis, expressed on a comparative scale'2. Inhibition of albumin denaturation - Test solution Ulcer injury was assessed by using the following (l ml) containing different concentrations (33-132 Ilglml) criteria'3 - Grade 1: no damage; 2: shallow damage of drug was mixed with 1ml of egg albumin solution not exceeding 25% of the mucosal depth; 3: moderate (1 mM) and incubated at 27° ± 1°C for 15 min. damage reaching beyond 25% of the mucosal depth Denaturation was induced by keeping the reaction but not exceeding 75%; and 4: deep damage reaching mixture at 70°C in a water bath for 10 min. After beyond 75% of mucosal depth. The length of section cooling the turbidity was measured at 660 nm'o. with type 2, 3 or 4 damage was expressed as a Percentage inhibition of denaturation was calculated percentage of the total section length and used as from control where no drug was added. Each histological index. The ulcer depth index was experiment was done in triplicate and taken the calculated by dividing the mean mucosal thickness by average. mean ulcer depth. The ulcerated portion was measured as percentage size of the ulcer in 3 mm of Anti-ulcer studies gastric mucosa. Ibuprofen induced ulcer model: Drug dose Biochemical studies (ethanol induced ulcers)­ response - Animals fasted for 48 hr were divided Rats were divided into three groups with six animals into groups of six each and treated as­ in each group. Group I served as control while the rats DW + Ibuprofen (300 mg/kg, 1 hr after); Misoprostol in groups II and III received 1 ml of ethanol by oral (72 /-!g/kg) + Ibuprofen (300 mg/kg, 1 hr after) and PA route. Rats in group III were also given PA (150 (30-150 mg/kg) + Ibuprofen (300 mg/kg, 1 hr after). mg/kg) 1 hr before ethanol treatment. After 3 hr of Test drugs were given once orally. After 6 hr of ethanol treatment, the animals were anaesthetized ibuprofen treatment, animals were sacrificed, with ether and killed by decapitation. The stomachs removed the stomachs, opened along the greater were removed, washed with ice-cold saline and a 10% curvature, washed with saline (0.9%) and the extent (w/v) homogenate was prepared in ice-cold 0.05 M of of ulceration was scored as per the method of Rao potassium buffer (PH 7.4), which was used et ai'. Based on this study dose of 150 mg/kg, which for estimations. gave the maximum protection, was used for Lipid peroxidation _. Quantitative measuremen t of subsequent studies. lipid peroxidation in gastric ti ssue was performed by SUDHEER KUMAR e/ at.: ANTIINFLAMMATORY AND ANTIULCER ACTIVITIES 181

l 4 the method of Ohkawa et al • The amount of showed signific'lnt activity at a dose as low as 90 malondialdehyde formed was quantified by reaction mg/kg body wt. with thiobarbituric acid and used as an index of lipid Inhibition of albumin denaturation - PA showed peroxidation. The results are expressed as nmole significantly higher abi li ty to prevent denaturation of malondialdehyde/g of w~t tissue. egg albumin when compared with a standard viz., Non-protein sulfhydryl groups (NP-SH) estimation­ Ibuprofen (Fig. 1). The activity of PA was Gastric mucosal NP-SH was measured according to consistently better at all the tested concentrations (33- Sedlak and Lindsa/5 and the results have been 132 Ilg/ml). Consistent results were obtained in expressed as {!mole/g of wet tissue. several trails and results shown were representative Statistical analysis - Statistical evaluation of the samples drawn from raw data. data was done by one way ANOVA (Graph PAD Instar Software). Values at P<0.05 were considered Anti-ulcer activity as significant. Ibuprofen induced ulcer model: Drug dose response - To select the optimum dose, six rats per Results dose point were administered with 30, 60, 90, 120, Anti-inflammatory activity - Results on effect of and 150 mg/kg body wt of PA, Ihr before treatment PA and ibuprofen on carrageenan induced rat paw with ibuprofen (300 mg/kg). Pretreatment with PA edema are presented in Table 1. Ibuprofen produced a reduced the mean ulcer index (MUI) in a dose significant reduction in paw edema volume (73.82%). dependent manner (Table 2). At a dose level of Treatment with PA reduced the carrageenan induced 150 mg/kg in ibuprofen-induced ulcers, the gastro­ paw edema volume in a dose dependent manner. The protection afforded by PA (93.9%) was similar to that maximum reduction in the edema volume was of misoprostol (95.2%). Based on this study, 150 mglkg observed at a dose of 150 mg/kg. Though ibuprofen was selected as the optimum dose for subsequent was more powerful as an antiinflammatory agent. PA studies.

Table 1 - Effect of phytic acid (PA) and ibuprofen (Ibu) on ~~------~ canageenan induced rat paw edema 0 'j;;= .... [Values are mean ± SE of 6 animals in each group] =" ... (o(J Treatment Dose Edema volume Reduction in =.. 'C (mglkg) edema volume .... 0 (%) .::0= 40 :E Control DW 0.476 ± 0.04 :E Ibu 90 0.125 ± 0.08" 73.82 ..s PA 30 0.369 ±0.02 22.47 c~ a 20 PA 60 0.268 ± 0.02 43.65 3J (,(, JJ2 a PA 90 0.186 ± O.04 61.05 a Drug conc. ( ~g I ml) PA 120 0.163 ± 0.05 65.79 a PA 150 0.134 ± O.04 71.94 Fig. 1-Inhibition of albumin denaturation by PA (phytic acid; D ) P value: a < 0.05 compared to control and Ibuprofen (e ). [Values are expressed as mean ± SE (n = 3)].

Table 2 - Effect of phytic acid (PA) and misoprostal on Ibuprofen (Ibu) induced gastric ulcers in rats [Values are mean ± SE of 6 animals in each group] Treatment Dose Ulcer No. Ulcer Size Ulcer Index Protection (mg kg'l) (%)

Ibu 300 13.5 ± 0.9 28.2 ± 2.2 379.3 ± 32.0 Misoprostal + Ibu 0.072 2.5 ± 1.9c 3.8 ± 2.4c 18.3 ± 3.8c 95.2 a PA + Ibu 30 12.6 ± I.l 21.7 ± 1.8 273.2 ± 20.4 27.9 b PA + Ibu 60 8.4 ± 1.2 15.9 ± 1.5 133.4 ± 18.2c 64.8 c PA + Ibu 90 6.7 ± 0.6a 11.8 ± 1.2 73.0 ± lIS 80.8 PA + Ibu 120 5.3 ± LOb 7.9 ± 1.2c 45.3 ± ISS 88.1 c c PA + Ibu 150 3.7 ± 0.3b 6.3 ± 0.8 23.0 ± 3.3 93.9 P values: a < 0.05, b < 0.01 and c < 0.001 compared to ibuprofen treated control. 182 INDIAN J EXP BIOL, FEBRUARY 2004

Cold stress induced ulcer model-PA and malondialdehyde levels as compared to ethanol famotidine produced significant reduction in ulcer treated rats. Ethanol significantly decreased NP-SH number, ulcer size and ulcer index compared with concentration. Pretreatment with PA prevented the control rats in cold stress induced ulcers. Famotidine depletion of NP-SH caused by ethanol (Table 7). was more potent in reducing MUI as compared to P A. Famotidine (3.6 mg/kg) produced a gastroprotection Discussion of 92.7%, while PA (150 mg/kg) produced a PA produced significant antiinflammatory activity protection of 70.5% (Table 3). at aU doses in carrageenan induced rat paw edema Ethanol induced ulcer model-PA and famotidine model. Though Ibuprofen was more powerful as an produced significant reduction in ulcer number, ulcer antiinflammatory agent, PA showed significant size and ulcer index as compared with control rats in activity at a dose as low as 90 mg/kg body wt. ethanol induced ulcers (Table 4). Famotidine Maximum reduction in the edema volume was produced a gastroprotection of 77.86%, while PA observed at a dose of 150 mg/kg. (150 mg/kg) produced a protection of 84.9%. Several reports point out the involvement of free l6 Histopathological studies (Fig. 2A-D) further radicals in the process of inflammation . PA inhibits confirmed that pretreatment with PA inhibited the Fenton reaction by complexation with Fe (II) and 8 ethanol induced congestion, oedema, haemorrhage blocks the generation of OH radical • Hence, the and necrosis in gastric mucosa (Fig. 2A-D; Table 5). mechanism of action of PA is likely to be mediated Histopathological examination of gastric lesion through its ability to prevent the formation of OR revealed an increase in histological and ulcer depth radicals. index (Table 6). Denaturation of proteins is a well-documented There was a significant increase in tissue cause of inflammation. Phenylbutazone, salicylic malondialdehyde levels in ethanol treated rats as acid, flufenamic acid (antiinflammatory drugs) etc., compared to normal rats. Following PA treatment, have shown dose dependent ability to inhibit 9 there was a significant reduction III tissue thermally induced protein denat!.lration • As a part of

Table 3 - Effect of phytic acid (PA) and famotidine on cold stress (CS) induced gastric ulcers [Values are mean ± SE of 6 animals in each group] Treatment Dose No. of Ulcer Ulcer Protection l (mg kg· ) ulcer size index (%) CS DW 20.7 ± 0.8 11.7±0.7 239.8 ± 12.8 Famotidine + CS 3.6 4.5 ±0.8c 3.5 ±O.s< 17.5 ± 5.7c 92.7 PA+CS 150 7.0 ± 0.6c 10.3 ±0.6 70.8 ± 3.8c 70.5

P values: c < 0.001 compared to CS control group Table 4 - Effect of phytic acid (PA) and famotidine on ethanol (AC) induced gastric ulcers in rats [Values are mean ± SE of 6 animals in each group] Treatment Dose No. of Ulcer Ulcer Protection l (mg kg· ) ulcer size index (%) AC 25.3 ± 1.8 11.8 ± 0.6 300.3 ± 30.4 Famotidine + AC 3.6 10.3 ± 1.7c 6.0 ± 0.8b 66.5 ± 15.6c 77.8 PA+AC 150 8.7 ± O.4c 5.8 ± 0.8b 45 .3 ± 2.8c 84.9

P values: b < 0.01, c < 0.001 compared to AC treated control group ------Table 5 - Effect of phytic acid (PA) and famotidine on induction of histopathological lesion in the ethanol (AC) - induced gastric ulcer model Treatment Congestion Oedema Haemorrhage Necrosis Control Ethanol (AC) ++ ++++ ++ +++ Famotidine+ AC + ++ + ++ PA 150 mg/kg+ AC + + + ++ ---: normal + : little effect ++ : appreciable effect t+++ : severe effect ++++ : very severe effect SUDHEER KUMAR et al.: ANTIINFLAMMATORY AND ANTIULCER ACTIVITIES 183 the investigation on the mechanism of antii­ The protective effect against various irritants has nflammatory activity of PA, the ability of PA to been called cytoprotective activity mediated through inhibit protein denaturation was studied. Although, prostaglandins 17. PA effectively reduced the gastric PA showed higher ability to inhibit albumin ulcer produced by both ibuprofen as well as ethanol denaturation than Ibuprofen, it showed less anti­ (irritants) indicating a possible cytoprotective action inflammatory activity. Mechanism of antiinflam­ on gastric mucosa. matory is dependent on several factors and this is not Several recent lines of evidence implicate the role in conflict with observed data. Ability of PA to bring of free radicals and lipid peroxidation in gastric down thermal denaturation of proteins is possibly a mucosal damage IS. 19. NSAIDs are known to induce contributing factor in the mechanism of action. peptic ulcer not only by denaturing mucous

Table 7 - Effect of phytic acid (PA) on tissue malondialdehyde Table 6 - Histopathological examination of gastric lesions in the and non-protein sulphydryl levels in ethanol treated rats ethanol-induced gastric ulcer model (Fig. 2 A-D) [Values are mean ± SE of 6 animals in each group] Treatment Grade Histological Ulcer in 3 mm Ulcer depth index of gastric mucosa index Treatment Malondialdehyde Non-protein sulfhydryl (%) conc. levels (nmole/g wet tissue) (JLmole/g of tissue) Control 4 1.48 67.33 1.19 Famotidine 3 1.79 46.66 2.91 Control 23.45 ± 1.76 1.689 ± 0.045 c PA (150 mg/g) 2 6.05 15.89 3.78 Ethanol 136.93 ± 6.12< 0.765 ± 0.079 PA (150 mg/kg) 78.32 ± 2.92z 1.006 ± 0.021 x Grade 1: no damage; 2: shallow damage not exceeding 25% of the + Ethanol mucosal depth; 3: moderate damage reaching beyond 25% of the mucosal depth but not exceeding 75%; 4: deep damage reaching P values: < < 0.001 compared to control and x <0.05, Z < 0.001 beyond 75% of mucosal depth. compared to ethanol group

Fig 2 (A-D) - The stomach wall of rat-(A) Control, showing its normal appearance; (B) Treatment with 1 ml of absolute ethanol, by gavage; (C) Treatment with 3.6 mg/kg of famotidine and 1 ml of absolute ethanol, by gavage; and (D) Treatment with 150 mg/kg of PA and 1 1111 of absolute ethanol, by gavage. [Hematoxylin and eosin, x lOO] 184 INDIAN J EXP BIOL, FEBRUARY 2004 glycoproteins but a -. ~ .) by free radical formation IS. biotransformation system , sult11ydryl content 2o 29 Ethanol is known to promotc oxygen-free radicals , and lipid peroxidation in murine system . Several reduce gastric mucosa! non-protein sulfuydryI Jcvels21 dmgs are known to achieve gastric protection du e to and stimulate the formation of leukotriene C4 (LTC4), the in volvcment of NP-SH stimul ating agents2S . PA is a lipoxygenase deri ved '"'1etabolism of arachido.n ic likely to act by preventing oxidative injury rather th an 22 acid • Stress ulcers are mediated by brain gut axis scavenge free radicals, and thus may cause a and complex neural mechanism23. Stress causes an prevention of NP-SH depletion. Protection by PA ischemic condition in the gastric mucosa by activation against stress induced ulcers was comparatively less of parasympathetic and sympathetic nervous systems than the protection against ibuprofen and ethanol resulting in vasoconstriction, which in turn causes induced Illcers, suggesting that PA ~as more effective free radical generation. Ft:ther, stress has also been in preventing oxygen free radical mediated peripheral found to inactivate mucosal prostaglandin synthetase damage to the gastric mucosa. by accumulating H20 2, which in turn inhibits the Our results are substantiated by the histopathological synthesis of prostaglandins known to favour the studies, which confirmed that pretreatment with PA generation of reactive oxygen species23. The free inhibited ethanol-induced necrosis, hemorrhage, radicals produced cause lipid peroxidation, which congestion and edema in gastric mucosa. These leads to membrane fluidity. This in turn increases the findings further confirmed the cytoprotective nature influx of Ca +2 , resulting in reduced membrane of P A. Hence we concluded that PA might act as a integrity of surface epithelial cells, thereby generating cytoprotective by scavenging the reactive oxygen gastric ulcers IS, 23. metabolites in GIT. Detection and measurement of lipid peroxidation is The common NSAIDs may be given in combi­ the evidence cited to support the involvement of free nation with PA to reduce the gastric irritation. There radicals IS. Thiobarbituric assay is the most popular may be an increase in therapeutic activity as well. The method to estimate malondialdehyde level, which is anti-ulcer activity also suggests selective COX2 (H2) an indication of lipid peroxidation and free radical inhibition, which requires experimental confirmation. 4 activitl • In this study, there was significant increase Being a chelator of iron and antioxidant, the shelf life in tissue malondialdehyde levels in ethanol treated of formulations may be increased. These are areas fit rats. This is consistent with earlier study's that for future investigations. associate increased lipid peroxidation with gastric ulcers. 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