Piperine mediated alterations in lipid peroxidation and cellular thiol status of rat intestinal mucosa and epithelial cells

Phytomedicine, Vol. 6(5), pp. 351-355

Phytomedicine

© Urban & Fischer Verlag 1999 http://www.urbanfischer.de/journals/phytomed

Piperine mediated alterations in lipid peroxidation and cellular thiol status of rat intestinal mucosa and epithelial cells A. Khajuria 1, R. K. johri- and U. Zutshi? 1Manitoba Institute of CellBiology, Winnipeg, Canada 2Regional Research Laboratory, Jammu, India

Summary Piperine (l-Piperoyl piperidine) is the major alkaloid of black and long peppers used widely in various systems of traditional medicine. The present study investigates the toxicity of piperine via free-radical generation by determining the degree of lipid peroxidation and cellular thiol status in the rat intestine. Lipid peroxidation content, measured as thiobarbituric reactive substances (TBARS), was increased with piperine treatment although conjugate diene levels were not altered. A significant increase in glutathione levels was observed, whereas protein thiols and glutathione reductase activity were not altered. The study suggests that increased TBARS levels may not be a relevant index of cytotoxicity, since thiol redox was not altered, but increased synthesis transport of intracellular GSH pool may play an important role in cell hemostasis and requires further study. Key words: Piperine, lipid peroxidation, cellular thiols, intestine.

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Introduction

Piperine (I-Piperoyl piperidine), the major alkaloidal constituent of black and long peppers, is widely consumed as a spice and incorporated as a therapeutic modality in various systems of traditional medicine (Charaka, 1941; Warrier, 1981) . Piperine has demonstrated various pharmacological effects on intestine, particularly enhancing bioavailability (Annamalai and Manavalan, 1990; Bano et al., 1987, 1991). In order to validate piperine as a safe bioavailabilityenhancer in therapeutic formulations, an evaluation of any cytotoxic effect was necessary. There is no conclusive evidence of toxic implications of this chemical component in animals or humans. Peppers or piperine fed at 5-20 times normal human intake did not produce any adverse effect on growth pattern or clinical pathology (Srinivasan and Satyanaryana, 1981). Acute, subacute and chronic toxicity studies also demonstrated no abnormality or clinical pathology at pharmacologically-effective doses (Zutshi et al., 1989).

The toxicity of various chemical components and xenobiotics is known to be linked with the generation of free-radical species in the presence of transition metals like ferrous and copper ions, initiating lipid peroxidation (Trush et al., 1982; Borg and Schaich, 1984; Wink et al., 1991). Some studies have suggested a cytotoxic nature for piperine through the possible involvement of lipid peroxidation (Piyachaturawat et al., 1995; Uchern et al., 1997). The present investigation aims to evaluate the influence of piperine on initiating free-radical damaging effect, if any, in the intestines.

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Materials and Methods

To achieve the objectives, in vivo and in vitro studies using various lipid peroxidation-initiating systems were undertaken. Lipid-peroxidation status was meas0944-7113/99/06/05-351 $ 12.00/0

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A. Khajuria, R. K. Johri and U. Zutshi

ured as conjugate dienes, and thiobarbituric reactive substances (TBARS) as malonaldehyde. Cellular thiols were estimated as non-protein (GSH) and protein (PSH) thiols to assess susceptibility to toxicity and functional alterations. In addition, GSH redox status was determined by measure of glutathione reductase activity (a key enzyme in maintaining thiol redox). Piperine was isolated from Piper nigrum as reported previously (Atal et al., 1975). Thiobarbituric acid (TBA); reduced nicotinamide adenosine dinucleotide phosphate (NADPH); sodium dodecyl sulphate (SDS); glutathione (GSH); oxidized glutathione (GSSG), 5,5'dithiobis (2-nitrobenzoic acid) (DTNB); bovine serum albumin (BSA), and 1,1',3,3' tetraethoxy propane were obtained from Sigma Chemical Co., USA. All other chemicals used were also of analytical grade.

Animals

Healthy, adult Charles Foster male rats (150-200 g each) were maintained under standard husbandry conditions, pellet diet and water ad libitum.

Sample Preparation for In vivo Studies

Rats fasted for 24 hours were administered piperine (piperine suspension made in 1% carboxy methyl cellulose) orally with gastric canula at 5, 10 and 20 mg/kg body wt. orally and sacrificed at 30-, 60- and 360-minute intervals after treatment. Control animals were administered 1% carboxymethyl cellulose in 0.9% saline. Each animals abdomen was opened with a midline incision, and a 25-30 ern segment (15 em beyond 10 em of the pylorus end) of the small intestine was excised. Intestines were everted over a glass rod and mucosa removed by gentle scraping on ice. Mucosa was then homogenized (10%) with an Elvez-type homogenizer for 2 min in cold distilled water and sieved through nylon mesh. An aliquot of homogenate was precipitated with equal volumes of 50% TCA and centrifuged at 3000 X g for 15 min. Supernatant thus obtained was stored at 4 °C as non-protein thiols. The pellet was washed twice with 10% TCA and solubilized using 10% SDS in Tris/HCI (pH 8.9) to estimate protein thiols.

Pellet was washed twice with 10% TCA and solubilized with 10% SDSin Tris/HCI (pH 8.9) for estimating protein thiols. Supernatant and solubilized pellet were incubated with piperine (25-100 pM) and analyzed.

Lipid Peroxidation (LPO) Estimation

• Non-enzymatic ascorbate-dependent peroxidation: LPO was assessed by measuring malonaldehyde (MDA) produced using the thiobarbituric acid (TBA) reaction (Bishayee and Balasubramanian, 1971) in a sample protein (500 pg) with 200 mM TrisIHCI, 0.3 mM ascorbic acid and 0.8 mM ferrous sulfate, to a final volume of 1 ml, which was than incubated for 60 min at 37°C. The reaction was terminated by adding 1 ml of 20% TCA and 2 ml of 0.67% TBA and heating for 15 minutes in a boiling water bath. The mixture, containing colored TBA-MDA complex, was cooled and centrifuged at 3,000 rpm for 15 min and supernatant absorbance was measured at 535 nm. The MDA produced was quantitated using a standard curve of 1,1',3,3'-tetraethoxy propane. • Enzymatic NADPH-dependent peroxidation: LPO was estimated by measuring MDA produced in a similar manner, except that 0.2 mM NADPH was used instead of ascorbic acid (Hochstein and Ernster, 1963) in a sample protein (500 ug) with 200 mM Tris/HCI, and 0.8 mM ferrous sulfate to a final volume of 1 ml, which was then incubated for 60 min at 37°C. The reaction was terminated by adding 1 ml of 20% TCA and 2 ml of 0.67% TBA and heating for 15 min in a boiling water bath. The mixture, containing colored TBA-MDA complex, was cooled and centrifuged at 3,000 rpm for 15 min and supernatant absorbance was measured at 535 nm. The MDA produced was quantitated using a standard curve of 1,1',3.3'-tetraethoxy propane. • Diene conjugates estimation: Diene conjugates were measured (Balasubramanian et al., 1988) in sample protein (500 ug) in TrisIHCI buffer incubated at 37°C for 30 min and extracted using 20 volumes of chloroform: methanol (2:1) and 5 volumes of 0.15 M NaCl. The aqueous phase was discarded and the organic phase evaporated. Dried samples were made up to 3 ml in methanol and absorbance was measured at 233 nm for the quantitation of diene conjugates.

Sample Preparation for In vitro Studies

Rat intestinal epithelial cells (4-5 mg/ml) (Lin and Williams, 1988) were characterized for viability by measuring LDH activity (Kubawitz and Ott, 1943) and using the dye exclusion method (Hank and Wallace, 1958). Cell aliquots of epithelial cells suspended in TrisIHCI were precipitated using equal volumes of 40% TCA and centrifuged at 3000 X g for 15 min. Supernatant was stored at 4 °C as non protein thiols.

DTNBAssay

Cellular thiols as GSH and protein thiols, were measured (Sedlak and Lindsay, 1968) in a sample protein (500 pg) incubated with 100 mM DTNB (final concentration), at a final volume of 3 ml for 15 min in darkness. Absorbance of the reaction mixture was measured at 412 nm. Thiols were quantitated using standard glutathione solution treated in similar manner.

Piperine-mediated alterations in lipid peroxidation

Glutathione Reductase Activity Assay mixture containing (Racker, 1955) sample protein (50 Jlg) in 100 mM phosphate buffer (50 JlI), and 0.1 ml NADPH, 0.1 ml BSA, 0.1 ml GSSG was made to 3 ml in glass with distilled water and vortexed in a cuvette. Absorbance of the reaction mixture was recorded at 340 nm at 30-sec intervals until 3 min had passed. Specific activity of the enzyme was expressed as change in absorbance (L10D) per min, per mg of protein. Protein Estimation

Protein content was estimated according to Lowry's method (Lowry et al., 1951) using bovine serum albumin as standard.

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nal epithelial cells (Figure 2). There was, however, no change in conjugate diene levels with piperine treatment (25-100 JlM) of intestinal epithelial cells. Further piperine pretreatment at 5-20 mg/kg produced an increase (p < 0.001) in GSH levels at 60- and 360-min intervals in intestinal mucosa, with only a slight increase (p < 0.05) in protein thiol levels at the 360-min interval (Figures 3a, 3b). Similarly, in intestinal epithelial cells, piperine produced an increase in GSH levels at 50- and 100-JlM concentrations, with no alteration in protein thiols (Figure 4). There was also no change in the Glutathione reductase activity of intestinal epithelial cells on piperine treatment (8.3-100 JlM).

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Discussion

StatisticalAnalysis

Student's unpaired 't' test was employed to determine the significance of difference between different groups.

Results Pretreatment with piperine (5-20 mg/kg) at 30- and 60-min intervals induced TBARS accumulation (p < 0.01) in intestinal mucosa, which declined thereafter as measured at 360-min intervals (Figure 1). Nonenzymatic ascorbate-dependent and enzymatic NADPH-dependent LPO were increased (p < 0.01) only at 100-JlM concentrations of piperine in the intesti-

Cytotoxicity caused by chemicals can be understood as a consequence of simultaneous or sequential alterations in several cellular processes, among which lipid peroxidation and alteration in thiol status are of relative importance. In the present study, we have investigated piperinemediated alterations (piperine is a pure chemical principle and major alkaloidal constituent of Piper nigrum L.) in lipid peroxidation and thiol status, to evaluate its cytotoxicity. Results showed that piperine-induced accumulation of TBARS in both in vivo and in vitro studies at higher concentrations in the presence of radical-initiating species. However, conju-

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Figure 1. Effect of piperine on lipid peroxidation levels in intestinal mucosa, Piperine was administered orally at 5-20 mg/kg body wt, at 30-, 60- and 360-min intervals before the experiment. Lipid peroxidation levels were measured as TBARS as stated in Materials and Methods. Values are expressed as Mean e SEM; n =6 for each group. ns =non significant (p> 0.1); "p < 0.05; ':"'p < 0.01; "':'*p < 0.001 versus control (1% carboxy methyl cellulose in 0.9% saline).

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Figure 2. Effect of piperine on lipid peroxidation levels in intestinal epithelial cells measured as TBARS content. Cells were treated with piperine at 25-100 pM concentrations and TBARS content was measured as stated in Materials and Methods. Values are expressed as Mean e SEM; n = 6 for each group. ns =non significant (p> 0.01); *"p < 0.01; versus control (1% carboxy methyl cellulose in 0.9% saline).

354

A. Khajuria, R. K. johri and U. Zutshi (b) Protein Thlols

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Figure3. Effectof piperine on cellularthiollevels in intestinal mucosa. (a) Non Protein Thiols; (b) Protein Thiols. Piperine was administered orally at 5-20 mg/kg body wt. at 30-, 60- and 360-min intervals before the experiment. Cellular thiols were measured using the DTNB reaction as stated in Materials and Methods. Values are expressedas Mean ± SEM; n = 6 for each group. ns = non significant (p > 0.1); *p < 0.05; ""p < 0.01; **"p < 0.001 versus control (1% carboxy methyl cellulose in 0.9% saline).

gate dienes (initial peroxidation products) were not altered on piperine treatment, suggesting that piperine may influence lipid peroxidation at the propagating rather than at the initiating stage. TBARS levels observed were not cytotoxic, as LDH leakage in response to increasing concentrations of piperine was not substantial « 10%) and correlated poorly with TBARS levels at increasing concentration-time points (data not shown) (Khajuria, 1996). Chemical toxicity via reactive species is known to

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Figure4. Effectof piperine on cellular thiollevels in intestinal epithelial cells. Cells were treated with piperine at 25-100 11M concentrations and cellular thiols were measured using the DTNB reaction. Values are expressed as Mean ± SEM; n =6 for each group. ns = non significant (p> 0.1); *p < 0.05; **p < 0.01; versus control (1% carboxy methyl cellulose in 0.9% saline).

produce GSH depletion and also loss of protein sulphydryl groups, causing cell injury (Drew and Miners, 1984; Kretzschmar and Klinger, 1990). Our results, however, demonstrated a significant increase in GSH levels with no alteration in protein thiols. Glutathione reductase activity was also not altered on piperine treatment, i.e, thiol redox was not affected, suggesting that piperine is not toxic to cells. Enhanced levels of GSH can be related to an increased influx or intra-organ synthesis of GSH (Meister and Anderson, 1983). Piperine is known to increase membrane permeability (Khajuria and Zutshi, 1998) and may therefore increase organ influx due to the increased activity of GSH transporters located in brushborder and basolateral membranes (Vincenzini et aI., 1992) or to produce enhancement of intracellular synthesis by increasing y-glutamyl transpeptidase activity in the intestinal epithelium (Johri et aI., 1992). Further studies are needed to understand the increased GSH levels. In conclusion, piperine inclusion induced lipid peroxidation in the presence of radical-initiating species, but simultaneously affected the increase in GSH levels significantly. These data suggest a protective role of piperine in the cellular environment. Additional studies are necessary to explain the mechanism of GSH synthesis and/or transport and its cytoprotective potential.

Acknowledgement

Authors are grateful to Talwar Research Foundation for providing financial support for this study.

Piperine-mediated alterations in lipid peroxidation

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References