The Result clearly proposed that ME-Og contains high phenolic and flavonoid compound that was measured by spectrophotometric method. ME-Og contains 61.72 mg phenolic compound/g of O. gratissimum powder, and 251.83 mg flavonoid/g of O. gratissimum powder (Fig. 1).

The cytotoxic effect of the ME-Og was studied in murine peritoneal macrophages with increasing concentrations of ME-Og ranging from 0.1 µg/ml to 100.00 µg/ml using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) method. The results indicated that, treatment of peritoneal macrophages with 10, 25, 50 and 100.00 µg/ml for 24 h led to 9.75%, 11.41%, 25.37% and 46.82% reduction in cell survivability, respectively (Fig. 2). But, there is no significant difference in cell survivability among 0.1 µg/ml to 25 µg/ml of ME-Og treatment and so, it is the highest concentrations of ME-Og, which does not produce any significant damage to murine peritoneal macrophages.

Superoxide anion (O₂^.-) generation and NADPH oxidase activity in peritoneal macrophages was significantly (p < 0.05) increased in nicotine treated group by 102.71% and 117.01% respectively, as compared to their control group. Only ME-Og (25.0 µg/ml) treatment decreased the O₂^.- generation (22.97%) and NADPH oxidase activity (11.20%), as compared to their respective control group. ME-Og supplementation with nicotine could concentration dependently decreased the excess O₂^.- generation significantly (p < 0.05), when compared to nicotine treated group. Where, 1 µg/ml ME-Og supplementation could decreased the O₂^.- generation 7.14% and also decreased NADPH oxidase activity 6.12%, there 25 µg/ml ME-Og exerted maximum protective effect, and reduced the O₂^.-generation 26.05% and NADPH oxidase activity 31.93% compared with only nicotine treated group (Table 1).

MPO activity in murine peritoneal macrophages in different group is shown in Table 1. MPO is an important enzyme to produce hypochlorus acid (HOCl) in cellular system that leads to oxidative damage. So, it is an important determinant to establish the free radical generation in peritoneal macrophage. MPO activity is significantly (p < 0.05) increased in nicotine treated group by 116.71% as compared to the control group. ME-Og (1 µg/ml, 5 µg/ml, 10 µg/ml and 25 µg/ml) decrease the excess MPO activity significantly (p < 0.05) compared to their nicotine treated group. Out of the four different concentrations of ME-Og, 25 µg/ml ME-Og shows the highest protective effect to decrease the MPO activity in nicotine treated murine peritoneal macrophages.

Lipid peroxidation and protein oxidation are the two important determinants to assess the cellular damage. Lipid peroxidation and protein oxidation in peritoneal macrophages was measured in terms of malondialdehyde (MDA) and protein carbonyls (PC) respectively. MDA level and PC content was significantly (p < 0.05) increased in nicotine treated murine macrophages by 200.56% and 135.54%, respectively, as compared to their control group. Only ME-Og treatment slightly increased MDA level (14.04%) but decreased PC content (1.58%) as compared to their respective control group, but there was no significant difference. Supplementation of ME-Og can decreased the MDA level and PC content significantly (p < 0.05) in concentration dependent manner, except 25 µg/ml ME-Og in case of PC level. 7.10%, 12.89%, 37.38% and 39.81% MDA level and 1.34%, 21.81%, 24.83% and 24.14% PC content were decreased with supplementation of 1 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml ME-Og as compared with nicotine treated group (Table 2).

Glutathione is an important antioxidant in cellular system. So, to understand glutathione level, we have measured both reduced and oxidized form of glutathione. The reduced glutathione (GSH) level was decreased (66.58%) significantly (p < 0.05) in nicotine treated macrophage, as compared with control group. Supplementation with ME-Og increased the oxidized glutathione (GSSG) level in concentration dependent manner (22.72%, 59.93%, 74.40% and 77.50%), when compared with nicotine treated group (Table 3). Only ME-Og treatment (25 µg/ml) slightly increased GSH level (4.33%) as compared to their respective control group.

The GSSG level was increased (53.77%) significantly (p < 0.05) in nicotine treated macrophage, as compared with control group. Supplementation with ME-Og decreased the GSSG level in concentration dependent manner (3.99%, 4.09%, 13.26% and 17.59%), when compared with nicotine treated group (Table 3). But only ME-Og treatment (25 µg/ml) slightly increased GSSG level (2.650%) as compared to their respective control group.

The redox ratio was decreased (78.37%) significantly (p < 0.05) in nicotine treated macrophage, as compared with control group. Supplementation with ME-Og increased the redox ratio in concentration dependent manner (28.18%, 66.94%, 101.55% and 116.75%), when compared with nicotine treated group (Table 3). Only ME-Og treatment (25 µg/ml) slightly increased the redox ratio (1.11%) as compared to their respective control group.

The super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-s-transferase (GST) activity were measured to understand the antioxidant status of different group of macrophages. The activity of different enzymatic antioxidant (SOD, CAT, GPx, GR & GST) activity was decreased significantly (p < 0.05) when compared to their respective control. These antioxidant activities were significantly (p < 0.05) raised in ME-Og supplemented group in concentration dependent manner (except GPx and GR activity with 25 µg/ml ME-Og), as compared to their nicotine treated group. Among the different concentrations of ME-Og, 25 µg/ml is the most effective dose to play a protective role against the nicotine toxicity. Only ME-Og treatment (25 µg/ml) could enhance the SOD, CAT, GPx, GR, GST activity (Tables 4 and 5).

Hydrogen tartarate salt of nicotine, phorbol mirested aceted (PMA), quercetin, gallic acid, horse heart cytochrome-c, sodium dodecyl sulfate (SDS), 5′, 5′-dithio (bis)-2-nitrobenzoic acid (DTNB), standard reduced glutathione (GSH), NADPH Na₄, oxidized glutathione (GSSG) were obtained from Sigma, USA. RPMI 1640, fetal bovine serum (FBS), heparin, ethylene diamine tetra acetate (EDTA) were purchased from Himedia, India. All other chemicals were from Merck Ltd., SRL Pvt., Ltd., Mumbai and were of the highest grade available.

Experiments were performed using Swiss male mice 6–8 weeks old, weighing 20–25 g. Animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, and approved by the ethical committee of Vidyasagar University. All efforts were made to minimize animal suffering and to reduce the number of animals used. Macrophages were isolated by peritoneal lavage from male Swiss mice, after 24 hrs injection of 2 ml of 4% starch according to our previous lab report by Kar Mahapatra et al.²⁵ In brief, washing the peritoneal cavity with ice cold phosphate buffer saline (PBS) supplemented with 20 U/ml heparin and 1 mM EDTA performed lavage. Care was taken not to cause internal bleeding while collecting macrophages in the exudates. The cells were then cultured in 60 mm petridishes in RPMI-1640 media supplemented with 10% FBS, 50 µg/ml gentamycin, 50 µg/ml penicillin and 50 µg/ml streptomycin for 24 h at 37°C in a humidified atmosphere of 5% CO₂–95% air in CO₂ incubator. Non-adherent cells were removed by vigorously washing three times with ice-cold PBS. Differential counts of the adherent cells used for the experiments were determined microscopically after staining with Giemsa and the cell viability evaluated by Trypan blue exclusion was never below 95%.²⁵,⁴²,⁴³

O. gratissimum was collected from Egra, Puba Medinipur, West Bengal, India in September 2007, in morning. Voucher specimens were deposited at the herbarium of the Deptartment of Botany, Vidyasagar University. The fresh aerial part of O. gratissimum was dried, blended and extracted with methanol (10:1). The mixture was filtered with Whatman filter paper (No. 1) and concentrated at 38°C by a rotary evaporator, then allowed to stand at room temperature overnight. This concentrated solution was then centrifuged at 2,000 xg for 10 min and supernatant was freeze dried to obtain the crude methanol extract.

Aluminum chloride colorimetric method was used for flavonoid determination in O. gratissimum.⁴⁴ Plant methanol extracts (0.5 ml of 1:10 g/ml) were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1.0 M potassium acetate and 2.8 ml of distilled water. It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm with a double beam Hitachi U2001 UV/Visible spectrophotometer (USA). The calibration curve was prepared by preparing quercetin solutions at concentrations 10 to 100 µg/ml in methanol.

Total phenols were determined by Folin Ciocalteu reagent.⁴⁵ A dilute ME-Og (0.5 ml of 1:10 g ml) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5 ml, 1:10 diluted with distilled water) and aqueous Na₂CO₃ (4 ml, 1.0 M). The mixtures were allowed to stand for 15 min and the total phenols were determined by Hitachi U2001 spectrophotometer at 765 nm. The standard curve was prepared using 0, 50, 100, 150, 200, 250 mg/L solutions of gallic acid in methanol:water (50:50, v/v). Total phenol values are expressed in terms of gallic acid equivalent (mg/g of dry mass), which is a common reference compound.

Cell cytotoxicity assay was performed by 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method according to Mosmann.⁴⁶ Murine peritoneal macrophages were treated with ME-Og at concentrations ranging from 0.1 µg/ml to 100.0 µg/ml were further cultured in RPMI-1640 supplemented with 10% FBS for 24 h. Thereafter, the medium was replaced with fresh RPMI (without Phenol Red and FBS) containing 0.5 mg/ml of MTT. After additional 3 h incubation at 37°C, HCl-isopropanolic solution was added to each culture plate. After 15 min of incubation at room temperature, absorbance of solubilized MTT formazan product was spectrophotometrically measured at 570 nm.

Hydrogen tartarate salt of nicotine, obtained from Sigma was dissolved in normal saline (0.9% NaCl) to get the required concentration. Then pH of the nicotine solution was adjusted to 7.4 by NaOH.²⁵

The peritoneal macrophages were divided into seven groups. Each group contained six petridishes (4 × 10⁶ cells in each). The cells of each petridishes of control and experimental groups were maintained in RPMI 1640 media supplemented with 10% FBS, 50 µg/ml gentamycin, 50 µg/ml penicillin and 50 µg/ml streptomycin at 37°C in a 95% air/5% CO₂ atmosphere in CO₂ incubator.

The following groups were considered for the experiment and cultured for 12 hrs:

• Group 1: Control i.e., culture media
• Group 2: 10 mM Nicotine in culture media
• Group 3: 25 µg ME-Og/ml culture media
• Group 4: 10 mM Nicotine + 1 µg ME-Og/ml culture media
• Group 5: 10 mM Nicotine + 5 µg ME-Og/ml culture media
• Group 6: 10 mM Nicotine + 10 µg ME-Og/ml culture media
• Group 7: 10 mM Nicotine + 25 µg ME-Og/ml culture media

After the treatment schedule, the treated cells are subjected to stain with Giemsa and morphological analysis has been done. The concentration of nicotine was selected according to our previous lab report.²⁵ After the treatment schedule the cells were collected from the petridishes separately and centrifuged at 2,200 rpm for 10 min at 4°C. Then the supernatant was collected in separate micro centrifuge tube and the cells were washed twice with 50 mM PBS, pH 7.4. The pallets were lysed with hypotonic lysis buffer (10 mM TRIS, 1 mM EDTA and Titron X-100, pH 8.0) for 45 min at 37°C and then processed for the biochemical estimation.⁴⁷ Intact cells were used for superoxide anion generation and NADPH oxidase activity.

Assessment of superoxide anion (O₂^.-) generation. Superoxide anion generation was determined by a standard assay.⁴⁸ Briefly, 0.1 µg/ml of PMA (Sigma), a potent macrophage stimulant, and 0.12 mM horse heart cytochrome-c (Sigma) were added to isolated cell suspensions after treatment schedule, and washing with PBS. Cytochrome-c reduction by generated superoxide was then determined by spectrophotometric absorbance at a 550 nm wavelength. Results are expressed n mol of cytochrome-c reduced/min, using extinction-coefficient 2.1 × 10⁴ M⁻¹ cm⁻¹.

NADPH oxidase activity. After the treatment schedule, the macrophages of different groups prewarmed in Krebs ringer buffer (KRB) with 10 mM glucose at 37°C for 3 min. PMA (0.1 µg/ml) prewarmed at 37°C for 5 min was added, and the reaction was stopped by putting in ice. Centrifugation was carried out at 400 g for 5 min and the resultant pellet was resuspended in 0.34 M sucrose. The cells were then lysed with hypotonic lysis buffer. Centrifugation was carried out at 800 xg for 10 min and the supernatant used to determine enzyme activity. NADPH oxidase activity was determined spectrophotometrically by measuring cytochrome c reduction at 550 nm. The reaction mixture contained 10 mM phosphate buffer (pH 7.2), 100 mM NaCl, 1 mM MgCl₂, 80 µM cytochrome c, 2 mM NaN₃ and 100 µl of supernatant (final volume 1.0 ml). A suitable amount of NADPH (10–20 µl) was added last to initiate the reaction.⁴⁹

Myeloperoxidase (MPO) activity. 200 µl of cell lysate was reacted with 200 µl substrate (containing H₂O₂ and OPD) in dark for 30 min. The blank was prepared with citrate phosphate buffer (pH 5.2) and substrate, in absence of cell free supernatant. The reaction was stopped with addition of 100 µl 2(N) sulfuric acid and reading was taken at 492 nm in a spectrophotometer.⁵⁰

Determination of lipid peroxidation (MDA). Lipid peroxidation was estimated by the method of Ohkawa et al. in cell lysate.⁵¹ Briefly, the reaction mixture contained Tris-HCl buffer (50 mM, pH 7.4), tert-butyl hydroperoxide (BHP) (500 µM in ethanol) and 1 mM FeSO₄. After incubating the samples at 37°C for 90 min, the reaction was stopped by adding 0.2 ml of 8% sodium dodecyl sulfate (SDS) followed by 1.5 ml of 20% acetic acid (pH 3.5). The amount of malondialdehyde (MDA) formed during incubation was estimated by adding 1.5 ml of 0.8% TBA and further heating the mixture at 95°C for 45 min. After cooling, samples were centrifuged, and the TBA reactive substances (TBARS) were measured in supernatants at 532 nm by using 1.53 × 10⁵ M⁻¹ cm⁻¹ as extinction coefficient. The levels of lipid peroxidation were expressed in terms of n mol/mg protein.

Protein carbonyls contents (PC). Protein oxidation was monitored by measuring protein carbonyl contents by derivatization with 2, 4-dinitrophenyl hydrazine (DNPH).⁵² In general, cell lysate proteins in 50 mM potassium phosphate buffer, pH 7.4, were derivatized with DNPH (21% in 2 N HCl). Blank samples were mixed with 2 N HCl incubated at 1 h in the dark; protein was precipitated with 20% trichloro acetic acid (TCA). Underivatized proteins were washed with an ethanol:ethyl acetate mixture (1:1). Final pellets of protein were dissolved in 6.0 N guanidine hydrochloride and absorbance was measured at 370 nm. Protein carbonyls content was expressed in terms of µ mol/mg protein.

Activity of super oxide dismutase (SOD). SOD activity was determined from its ability to inhibit the auto-oxidation of pyrogalol according to Mestro Del and McDonald.⁵³ The reaction mixture considered of 50 mM Tris (hydroxymethyl) amino methane (pH 8.2), 1 mM diethylenetriamine penta acetic acid, and 20–50 µl of cell lysate. The reaction was initiated by addition of 0.2 mM pyrogalol, and the absorbance measured kinetically at 420 nm at 25°C for 3 min. SOD activity was expressed as unit/mg protein.

Activity of catalase (CAT). Catalase activity was measured in the cell lysate by the method of Luck.⁵⁴ The final reaction volume of 3 ml contained 0.05 M Tris-buffer, 5 mM EDTA (pH 7.0), and 10 mM H₂O₂ (in 0.1 M potassium phosphate buffer, pH 7.0). About 50 µl aliquot of the cell lysates were added to the above mixture. The rate of change of absorbance per min at 240 nm was recorded. Catalase activity was calculated by using the molar extinction coefficient of 43.6 M⁻¹ cm⁻¹ for H₂O₂. The level of CAT was expressed in terms of m mol H₂O₂ consumed/min/mg protein.

Determination of reduced glutathione (GSH). Reduced glutathione estimation in the cell lysate was performed by the method of Moron et al.⁵⁵ The required amount of the cell lysate was mixed with 25% of trichloroacetic acid and centrifuged at 2,000 xg for 15 min to settle the precipitated proteins. The supernatant was aspirated and diluted to 1 ml with 0.2 M sodium phosphate buffer (pH 8.0). Later, 2 ml of 0.6 mM DTNB was added. After 10 minutes the optical density of the yellow-colored complex formed by the reaction of GSH and DTNB (Ellman's reagent) was measured at 405 nm. A standard curve was obtained with standard reduced glutathione. The levels of GSH were expressed as µg of GSH/mg protein.

Oxidized glutathione level (GSSG). The oxidized glutathione level was measured after derevatization of GSH with 2-vinylpyidine according to the method of Griffith.⁵⁶ In brief, with 0.5 ml cell lysate, 2 µl 2-vinylpyidine was added and incubates for 1 hr at 37°C. Then the mixture was deprotenized with 4% sulfosalicylic acid and centrifuged at 1,000 xg for 10 min to settle the precipitated proteins. The supernatant was aspirated and GSSG level was estimated with the reaction of DTNB at 412 nm in spectrophotometer and calculated with standard GSSG curve.

Redox ratio (GSH/GSSG). Redox ratio was determined for all the seven groups by taking the ratio of reduced glutathione/oxidized glutathione.

Activity of glutathione peroxidase (GPx). The GPx activity was measured by the method of Paglia and Valentine.⁵⁷ The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reductase and 1 mM reduced glutathione. The sample, after its addition, was allowed to equilibrate for 5 min at 25°C. The reaction was initiated by adding 0.1 ml of 2.5 mM H₂O₂. Absorbance at 340 nm was recorded for 5 min. Values were expressed as n mol of NADPH oxidized to NADP by using the extinction coefficient of 6.2 × 10³ M⁻¹ cm⁻¹ at 340 nm. The activity of GPx was expressed in terms of n mol NADPH consumed/min/mg protein.

Activity of glutathione reductase (GR). The GR activity was measured by the method of Miwa.⁵⁸ The tubes for enzyme assay were incubated at 37°C and contained 2.0 ml of 9 mM GSSG, 0.02 ml of 12 mM NADPH, Na₄, 2.68 ml of 1/15 M phosphate buffer (pH 6.6) and 0.1 ml of cell lysate. The activity of this enzyme was determined by monitoring the decrease in absorbance at 340 nm. The activity of GR was expressed in terms of n mol NADPH consumed/min/mg protein.

Activity of glutathione-s-transferase (GST). The activity of GST activity was measured by the method of Habig et al.⁵⁹ The tubes of enzyme assay were incubated at 25°C and contained 2.85 ml of 0.1 M potassium phosphate (pH 6.5) containing 1 mM of GSH, 0.05 ml of 60 mM 1-chloro-2, 4-dinitrobengene and 0.1 ml cell lysate. The activity of this enzyme was determined by monitoring the increase in absorbance at 340 nm.

Protein estimation. Protein was determined according to Lowry et al. using bovine serum albumin as standard.⁶⁰

The data were expressed as mean ± standard error, n = 6. Comparisons of the means of control, nicotine and nicotine with different concentration of ME-Og treated group were made by two-way ANOVA test (using a statistical package, Origin 6.1, Northampton, MA) with multiple comparison t-tests, p < 0.05 as a limit of significance.