Acta Poloniae Pharmaceutica Drug Research, Vol. 63 No. 2 pp.

117119, 2006

ISSN 0001-6837 Polish Pharmaceutical Society

AQUEOUS GINGER EXTRACT AMELIORATES PARABEN INDUCED CYTOTOXICITY

VEENA ASNANI and RAMTEJ JAYRAM VERMA Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, India, Phone: 91-079-26302362
Abstract: The present investigation is an attempt to examine the ameliorative effect of aqueous ginger extract on cytotoxicity induced by paraben (p-hydroxybenzoic acid) on red blood cells (RBC) in vitro. Blood samples collected in EDTA vials from healthy adult human beings (25-30 years) were used for preparation of RBC suspension in saline. The saline suspension of RBC was treated with paraben with and without ginger extracts. The results revealed that addition of paraben to RBC suspension caused significant increase in the rate of hemolysis. However, concurrent addition of paraben (150 g/mL) and ginger extract caused significant concentration-dependent retardation in paraben-induced hemolysis. The decrease in hemolysis was almost dosedependent. Keywords: paraben, ginger extract, cytotoxicity

Parabens are alkyl esters of p-hydroxybenzoic acid widely used in foodstuff, cosmetics, toiletries and pharmaceuticals. Parabens are carcinogenic (1) and hepatotoxic in nature. Our earlier studies have shown that addition of paraben to saline suspension of RBC caused swelling and eventual bursting of cell at higher concentrations and at lower concentration morphological alteration occurs. The effect could be due to paraben-induced oxidative stress and lipid peroxidation (2). Ginger (Zingiber officinale Roscoe, Zingiberaceae) has been widely used as a dietary spice, as well as in traditional oriental medicine and possesses potential in chemopreventive activities. Ginger extracts showed selective anticancer activity (3). It has been implicated as one of the promising chemopreventive agents against colon and skin cancer (4). Ginger is also known to possess antioxidant activity (5) and significantly lowered the lipid peroxidation by maintaining the activities of superoxide dismutase, catalase and glutathione peroxidase (6). The present investigation is an attempt to evaluate the ameliorative effect of an aqueous extract of ginger, on paraben-induced hemolysis in vitro. EXPERIMENTAL The extract was prepared according to WHO protocol CG-06 (7). Dried ginger was obtained from
* Corresponding author: e-mail: zooldeptgu@satyam.net.in

market and made to fine powder. 5 g of finely ground ginger powder and 100 mL of double distilled water was stirred with a magnetic stirrer for 1.5 h. The mixture was twice filtered with normal and then through Whatman filter paper no. 1. The supernatant was collected and allowed to dry. Human venous blood was collected in EDTA bulbs from well-nourished healthy adults (25-30 years old). It was diluted with saline (0.9% NaCl) and centrifuged at 1000 g for 10 min. RBC pellets were washed twice and suspended in fresh saline so as to get cell density of 2 104 RBC/mL. For examining hemolysis and its amelioration by aqueous extract of ginger four sets of tubes were prepared. 1. Control tubes containing 2.0 mL of RBC suspension and 2 mL of normal saline. 2. Antioxidant control tube containing 100 g/mL of aqueous ginger extract added to 2.0 mL of RBC suspension. 3. Toxin treated tubes 150 g/mL paraben and 2.0 mL of RBC suspension. 4. Tubes containing 150 g/mL of paraben and varying concentration of aqueous ginger extract (5-100 g/mL) in 2.0 mL of RBC suspension. The total volume of each tube was made up to 4 mL with additional saline. All tubes were incubated at 37OC for 4 h with intermittent shaking.

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VEENA ASNANI and RAMTEJ JAYRAM VERMA

Table 1. Reduction of paraben-induced hemolysis by ginger aqueous extract in vitro.

Paraben g/mL) 0 0 150 150 150 150 150 150 150 150 150 150 150 150

Aqueous ginger extract g/mL) 0 100 0 5 10 20 30 40 50 60 70 80 90 100

% Hemolysis

% Retardation

2.110.356 1.780.355 78.182.00a* 50.580.860b* 41.511.731b* 31.121.60b* 23.001.03b* 19.440.86b* 18.910.62b* 12.571.51b* 40.851.25b* 50.950.64b* 26.831.89b* 22.521.80b*

35.30 46.90 60.19 70.58 75.13 75.82 83.92 47.74 34.82 65.68 71.19

Values are mean S.E.M.; n = 7 a* = p < 0.001 or as compared to control b* = p < 0.001 or as compared to treated

Required concentration of aqueous ginger extract and paraben were prepared in saline. Absorbance of the supernatants obtained after centrifuging the incubated tubes at 1000 g for 10 min, were read spectrophotometrically at 540 nm (8). Percent hemolysis was calculated by the formula:
Percent Hemolysis = Absorbance of individual tubes Absorbance with 100 % hemolysis 100

100% hemolysis was prepared by adding 2 mL of distilled water to 2 mL of RBC suspension (9). Percent retardation with different concentration of antioxidant was calculated with the formula (10):
Percent Retardation = AB A 100

spheres or biconcave discs. Incubation of RBC suspension with 150 g/mL of paraben causes pronounced swelling and cell lysis (79% hemolysis). Incubation of RBC suspension with paraben (150 g/mL) in the presence of 5-100 g/mL of aqueous ginger extract caused reductions in paraben-induced hemolysis. The effect was concentration dependent with maximum retardation at 40-60 g/mL concentration of ginger extract. Further increase in concentration caused lesser retardation as compared to 40-60 g/mL concentration, however, retardation was significant as compared to toxin treated one (Table 1). DISCUSSION AND CONCLUSION The results shown in Table 1 clearly indicate that 150 g/mL concentration of paraben caused swelling and eventual bursting of cells. It might be due to the direct action of paraben on the plasma membrane causing lipid peroxidation, membrane permeability alteration and cell lysis. Nakagawa and Moore (11) have also reported that p-hydroxybenzoate ester induces cytotoxicity in rat hepatocytes due to mitochondrial membrane permeability transition. The results shown in Table 1 also indicate that concurrent addition of ginger extract in paraben significantly retards paraben-induced hemolysis. It could be due to antioxidative properties of ginger extracts. Ginger contains antioxidant component(s)

Where A = paraben induced hemolysis B = hemolysis caused by concurrent addition of paraben and antioxidant. Student t test was used for statistical analysis of the data. RESULTS In control and antidote treated tube the cells remain settled in the bottom of the tubes with clear ambient supernatant and RBC appeared as intended

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that act within the cell membrane and slow lipid peroxidation in situ (12). The aqueous extract of ginger was also found to inhibit the formation of diene, triene and tetraene conjugates in human erythrocyte membrane (13). In conclusion, ginger aqueous extract is an effective reductant for percentage reduction of hemolysis and cytotoxicity of paraben. REFERENCES 1. Dabre P.D., Aljarrah A., Miller W.R., Coldham N.G., Sauser M.J., Pope G.S.: J. Appl. Toxicol. 24, 5 (2004). 2. Verma R.J., Asnani V.: J. Bull. Environ. Contam. Toxicol. submitted. 3. Braga M., Sato N., Carvalho E., Marques M., Meireles M.: J. Agric. Food Chem. 51, 2520 (2003). 4. Nakamura Y., Yoshida C., Murakami A., Ohigashi H., Osawa T., Uchida K.: FEBS Lett. 572, 245 (2004).

5. Kikuzaki H., Nakatai N.: Food. Sci. 58, 1407 (1993). 6. Rafat S., Vandana S., Banerjee B: Indian. J. Exp. Biol. 38, 604 (2000). 7. WHO protocol CG-06.1983. APJF/IP 1001A, Geneva; World Health Organisation (1983). 8. Mukherjee K.: Medical Laboratory Technology: A procedure manual for routine diagnostic tests. Tata Mc Graw-Hill Ltd, New Delhi 1988. 9. Verma R.J., Raval P.J.: Bull. Environ. Contam. Toxicol. 47, 428 (1991). 10. Raval P.J., Verma R.J.: Indian J. Exp. Biol. 31, 780 (1993). 11. Nakagawa Y., Moore G.: Biochem. Pharmacol. 58, 811 (1999). 12. Chung W., Yow C., Benzie I.: Redox-Report. 8, 31 (2003). 13. Sujatha R., Srinivas L.: Toxicol. In-Vitro 9, 231 (1995). Received: 9.01.2006