PHYTOTHERAPY RESEARCH Phytother. Res. 17, 430 – 433 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1157 Copyright © 2003 John Wiley & Sons, Ltd. Received 19 July 2001 Accepted 13 November 2001 John Wiley & Sons, Ltd. SHORT COMMUNICATION SHORT COMMUNICATION Cytoprotective Activity of Amla ( Emblica officinalis ) Against Chromium (VI) Induced Oxidative Injury in Murine Macrophages Cytoprotective Activity of Emblica officinalis M. Sai Ram*, D. Neetu, P. Deepti, M. Vandana, G. Ilavazhagan, Devendra Kumar and W. Selvamurthy Defence Institute of Physiology and Allied Sciences, Timarpur, Lucknow Road, Delhi-110054, India The cytoprotective and immunomodulating properties of Emblica officinalis (Amla) against chromium (VI) induced oxidative damage are reported. Chromium (VI) at 1 μg/mL concentration was highly cytotoxic. It enhanced free radical production and decreased reduced glutathione (GSH) levels and glutathione peroxidase (GPx) activity in macrophages. The presence of Amla resulted in an enhanced cell survival, decreased free radical production and higher antioxidant levels similar to that of control cells. Further, chromium (VI) treat- ment resulted in decreased phagocytosis and gamma-interferon (γ γ -IFN) production while Amla inhibited chromium induced immunosuppression and restored both phagocytosis and γ γ -IFN production by macro- phages significantly. Copyright © 2003 John Wiley & Sons, Ltd. Keywords: Emblica officinalis; Amla; chromium VI; cytoprotection; immunomodulation. INTRODUCTION The fruits of E. officinalis have been used for thousands of years in traditional Indian medicine for the treatment of various diseases and the therapeutic activity of this plant is believed to be due to the high content of vita- min C (Kapoor, 1990). The fruit extract was reported to inhibit clastogenicity and mutagenicity by various metals such as lead, aluminium and nickel (Dhir et al., 1991, 1993) and to protect cells against radiation (Yadav, 1987) more efficiently than vitamin C. The extracts of fruits also are reported to inhibit retroviruses such as HIV-1 (El-Mekkawy et al., 1995), tumour development (Jose et al. , 2001) and gastric ulcer (Bandopadhyay et al. , 2000). The extracts of leaf have been shown to possess antiinflammatory activity (Asmawi et al. , 1993). Although many studies have been conducted on the biological activities of Amla, little is known about its ability to modulate the immune response especially under immunosuppressive conditions. Macrophages are the first line of defence and one of the most commonly exposed cells to the oxidative stress, i.e. during pha- gocytosis of foreign organisms. Cells of the immune system are particularly sensitive to changes in oxidant– antioxidant balance because of a higher percentage of polyunsaturated fatty acids in their membranes (Meydani et al ., 1995). Therefore, these cells offer an excellent model to study the cytoprotective properties of various herbal extracts during cellular damage by free radicals. In the present study, the antioxidant and immunomodulating properties of Amla using macro- phages as model system are reported. MATERIALS AND METHODS Plant material collection and extract preparation. The fruits of Amla were collected in winter (Jan–Feb 2000) locally and air dried for 30 days. The fruit was then powdered and extraction was carried out with 70% ethanol in a soxhlet apparatus overnight. The extract was collected and dried in an oven at 100 ° C for 3 h. The powder obtained was then emulsified in saline containing 0.1% Tween 20 and 10% ethanol. Later, dilutions of the extract were made in saline containing 0.02% Tween 20. Experimental set up for macrophages. The macrophages (J-774) were grown either in 24-well plates or 96-well plates in RPMI 1640 medium for 2 days until a mono- layer was formed. The medium was replaced with fresh medium and the fruit extracts of Amla and chromium (VI) as sodium dichromate (1 μg/mL) were added to the cells and incubated for 24 h. Since the best results were obtained at 250 μg/mL of fruit extract of Amla, all the experiments were conducted using 250 μg/mL concentration. Determination of cytotoxicity. This was determined using neutral red as described by Sai Ram et al. (2000). Briefly, after incubation the cells were washed three times with sterile saline. Later, 200 μL of neutral red (0.004%) dissolved in RPMI 1640 medium was added to the cells and incubated at 37 °C for 30 min. The cells were then washed three times and the neutral red taken up by the cells was released by adding 200 μL of ethanol:acetic acid (50:1 ratio) solution and the optical density was measured at 570 nm using a microplate reader. Alternatively, the cytotoxicity was determined by adding tetrazolium salt, MTS [3-(4,5-dimethylthiazol-2- yl)-5-(3 carboxymethoxy-phenyl)-2H-tetrazolium, inner * Correspondence to: Dr M. Sai Ram, Defence Institute of Physiology and Allied Sciences, Timarpur, Lucknow Road, Delhi-110054, India. E-mail: sairam64@rediffmail.com