Table 1. Effect of ethanolic extract of Fumaria officinalis Linn. (FO) on immune response in rats (mean±SEM, n=6).

Groups	Treatments	Humoral immune response (Mean antibody response titer levels)		Cellular immune response (% increase in paw volume)	Phagocytosis percentage	Digestion index	Total index	Leukocyte count (cells/mm³)
Groups	Treatments	Primary	Secondary	Cellular immune response (% increase in paw volume)	Phagocytosis percentage	Digestion index	Total index	Leukocyte count (cells/mm³)
Control	Vehicle	8.33±0.47	11.27±0.25	34.37±2.65	89.75±4.98	2.90±0.33	0.93±0.02	6714.33±53.77
	FO	7.66±0.51	11.90±0.9	35.27±2.7	87.50±1.64	2.89±0.37	0.99±0.06	6887.67±37.15
	Vitamin C and E	7.85±1.06	11.50±0.67	36.2±3.2	79.67±2.68	2.75±0.52	0.90±0.05	6695.21±23.56
Ethanol-treated	Vehicle	4.33±0.47*	8.10±0.3*	12.14±1.28*	57.60±4.7*	0.9±0.1*	0.21±0.01*	4572.17±19.38*
	FO	7.33±0.37#	10.80±0.5#	30.50±1.1#	80.89±2.82#	2.79±0.56#	0.87±0.02#	6644.17±74.88#
	Vitamin C and E	7.31±0.51	10.30±0.4	28.85±2.33	75.64±6.82	2.48±0.69	0.79±0.09	6564.67±46.82
One-way ANOVA	F	5.48	6.13	14.79	7.11	2.74	32.17	353.89
	df	5,30	5,30	5,30	5,30	5,30	5,30	5,30
	P	0.0011	0.0005	<0.0001	0.0002	0.039	<0.0001	<0.0001

* P<0.05 when compared with respective control group. #P<0.05 when compared with vehicle treated experimental group.

Table 2. Effect of ethanolic extract of Fumaria officinalis Linn. (FO) on oxidative stress in rats (mean±SEM, n=6).

Groups	Treatments	Lipid peroxidation Glutathione nmMDA /g Hb	Glutathione µm DTNB conjugated/g Hb	Superoxide dismutase units/mg protein	Catalase units units/mg protein
Control	Vehicle	70.79±1.64	4.69±0.25	29.37±0.62	283.44±6.32
	FO	65.65±2.45	5.02±0.45	29.24±0.35	278±5.65
	Vitamin C and E	69.36±3.89	4.98±0.98	29.32±0.65	291±10.52
Ethanol-treated	Vehicle	170.38±1.89*	2.59±0.16*	16.12±0.83*	192.87±4.25*
	FO	72.65±1.68# \	4.71±0.24#	19.89±0.65#	232.56±8.67#
	Vitamin C and E	99.30±2.82@	4.18±0.17	20.12±0.32	252.64±9.32
One-way ANOVA	F	258.61	3.78	100.02	23.17
	df	5,30	5,30	5,30	5,30
	P	<0.0001	0.0090	<0.0001	<0.0001

*P< 0.05 when compared with respective control group. # P< 0.05 when compared with vehicle treated experimental group. @P<0.05 when compared with FO treated experimental group.

Animals:

The Institutional Animal Ethics Committee approved the animal studies. The Sprague-Dawley rats of both sex and average weight (150-200 g) were housed at controlled temperature (25±2°C) with food and water ad libitum.

The experimental group was subdivided in such a manner that all subgroups concurrently received ethanol (20% w/v, 2 g/kg)⁸ and either the alcoholic extract of FO (100 mg/kg each, orally) or the vitamin C (100 mg/kg, orally) and E (100 mg/kg, orally)⁹ or vehicle. The above treatments were given daily for four weeks. The control groups received vehicle (distilled water) instead of ethanol.

Immunological studies:

Blood was withdrawn from the jugular vein of a sheep (Government Veterinary College, Nagpur) and RBCs were preserved in Alsever solution. It was then suspended in phosphate buffered saline for further use. All rats were antigenically challenged twice with sheep RBC (0.5x 10⁹ cells/ 100 g, i.p.), with first challenge on day 14 and second on 20th day of the experiment.

Humoral immune response:

On day 20 and 27, the blood was withdrawn from retro-orbital plexus of all antigenically challenged rats. 25 µl of serum was serially diluted with 25 µl of phosphate-buffered saline. Sheep RBC (0.025 x 10⁹ cells) were added to each of these dilutions and incubated at 37°C for one hour. The rank of minimum dilution that exhibited hemagglutination was considered as the antibody titer. The level of antibody titer on day 20 of the experiment was considered as the primary humoral immune response whereas the one estimated on day 27 of the experiment was the secondary humoral immune response.

Cellular immune response:

This was assayed by footpad reaction method in rats. The increase in the paw volume induced by an injection of sheep RBC (0.025 x 10⁹ cells), in the sub plantar region of right hind paw on day 27, was assessed after 48 h i.e. on day 29. The mean percent increase in paw volume was considered as delayed type of hypersensitivity reaction and considered as an index of cell-mediated immunity. The volume of left hind paw, injected similarly with phosphate buffered saline, served as control.

Phagocytosis:

On day 28, 3 ml of HBBS containing 10% bovine serum albumin (BSA), was injected into the peritoneal cavity of the animal and the same was recovered by gentle aspiration. The macrophages (3x 10⁵ / 600 µl), present in the aliquots, were incubated on glass plates at 37°C for 30 min in a humidified chamber. The cells, adhered to the glass, were incubated with live cells of Candida albicans, previously opsonized in autologous plasma (2x10⁶ /250 µl), at 37°C for 30 min, then washed with HBSS, again incubated for 30 min with autologous plasma and finally stained with Wright’s dye. A total of 300 cells were counted under microscope and results are reported as: a) Phagocytosis percent, b) Phagocytosis index, and c) Digestion index ¹⁰.

Blood parameters:

Following parameters were assessed in the blood, withdrawn from the retro-orbital plexus, at the end of 4th week i.e. on 29th day.

Total leukocyte count (TLC):

TLC was assessed by routine hematological method.

Lipid peroxidation (LPO):

To 2 ml, 5% suspension of separated RBC in 0.1 M phosphate buffered saline, 2 ml of 28% trichloroacetic acid was added and centrifuged. One ml of 1% thiobarbituric acid was added to 4 ml of supernatant, heated in boiling water for 60 min and cooled immediately. The absorbance was measured spectrophotometrically at 532 nm. The lipid peroxidation was calculated on the basis of the molar extinction coefficient of malondialdehyde (MDA) (1.56x 10⁵), and expressed in terms of nanomoles of MDA/g Hb¹¹

Superoxide dismutase (SOD):

It was estimated in the erythrocyte lysate prepared from the 5% RBC suspension. To 50 µl of the lysate, 75 mM of Tris-HCl buffer (pH 8.2), 30 mM EDTA and 2 mM of pyrogallol were added. An increase in absorbance was recorded at 420 nm for 3 min by spectrophotometer (Shimadzu 1601, Japan). One unit of enzyme activity is 50% inhibition of the rate of auto oxidation of pyrogallol as determined by change in absorbance/min at 420 nm. The activity of SOD is expressed as units/mg protein ¹².

Catalase (CAT):

Catalase activity was determined in erythrocyte lysate using Aebi’s method with some modifications. 50 µl of the lysate was added to a cuvette containing 2 ml of phosphate buffer (pH 7.0) and 1ml of 30 mM H₂O₂. Catalase activity was measured at 240 nm for 1 min using spectrophotometer. The molar extinction coefficient of H₂O₂, 43.6 M cm^-1 was used to determine the catalase activity. One unit of activity is equal to one millimoles of H₂O₂ degraded per minute and is expressed as units per milligram of protein ¹³.

Reduced glutathione (GSH):

Blood glutathione was measured by addition of 0.2 ml of whole blood to 1.8 ml distilled water followed by 3.0 ml of precipitating mixture (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl to make 100 ml of solution). It was centrifuged at 5000 X g for 5 min and 1 ml of the filtrate was added to 1.5 ml of the phosphate solution, followed by the addition of 0.5 ml of DTNB reagent. The optical density was measured at 412 nm using spectrophotometer ¹⁴.

Statistical analysis:

All data were analyzed with oneway ANOVA followed by Dunnett’s multiple comparison test. The intergroup difference was considered significant when P<0.05. The correlation between oxidative stress and immunological parameters was checked by Pearson correlation analysis.

RESULTS:

Table 1 exhibits that prolonged oral treatment of rats with ethanol (2 g/kg, 20% w/v) significantly (P<0.05) decreased both, the antibody titer and the increase in paw volume. Similarly, there appears to be a parallel decrease in the percentage and index of phagocytosis, digestion index and total leukocyte count. It was further observed that the daily treatment with alcoholic extract of FO (100 mg/kg) significantly (p<0.05) prevented these effects of ethanol. The oxidative stress marker studies (Table 2) revealed that the chronic administration of ethanol increased the levels of LPO, decreased the activities of SOD and CAT and reduced the content of GSH. Pearson correlation analysis revealed that the immunotoxic and oxidative stress-generating effects of ethanol possess a significant correlation with each other (Humoral immunity Vs Lipid peroxidation, r = -0.961; Cellular immunity Vs Lipid peroxidation r =-0.982; and Phagocytosis percentage Vs Lipid peroxidation r = - 0.948, all at p<0.05). The concurrent treatment of ethanol-administered rats with FO prevented the above ethanol-induced changes in the markers of oxidative stress.

Influences of FO on ethanol-induced changes were more or less similar and comparable with the vitamin E and C treatment.

DISCUSSION:

The present study has shown that the administration of ethanol, over a period of four weeks, not only impaired the immune responses but also produced oxidative stress, in rats. Since, the mixture of vitamin E and C significantly attenuated these ethanol-induced changes; it appears that the immunotoxic effects of ethanol may be due to oxidative stress. The literature has documented free radical generation during the metabolism of ethanol. The level of the markers of oxidative stress, observed in ethanol-treated rats, substantiate the possibility of extensive generation of free radicals. It is further observed that administration of alcoholic extract of FO, prevented the ethanol induced changes of immunological and oxidative stress parameters, and the effect was comparable to that of vitamin E and C. Hence, the immunomodulatory effect of FO may be subsequent to the antioxidant activity, which it possesses. The observed in vivo antioxidant activity of FO is in substantiation of its earlier reported activity in vitro studies.

The mechanism of free radical-induced impairment of immune system is not yet properly delineated. Reduced glutathione, a free radical scavenger, plays a key role in the activation of T cells and macrophages¹⁵. The present investigations have revealed that chronic treatment with ethanol depleted the glutathione, reduced the digestion index and impaired phagocytosis. It is further observed that alcoholic extract of FO or the mixture of vitamin E and C prevented the above influences of ethanol on glutathione levels, digestion index and phagocytosis. Hence, the observed impairment of phagocytosis by ethanol may be subsequent to the depletion of glutathione. Since the antioxidant therapies could reverse these influences of ethanol, the free radicals appear to be involved in this effect of ethanol. In fact, free radicals are the signaling entities in T cell activation. However, continued generation of free radicals, over a long period was shown to down regulate the activation of T cells and NF-kappa B ^16. Similar down regulation of T cell activation has been observed when nitric oxide, a free radical generator, is constantly generated by macrophages ¹⁷. It has also been shown that NO synthase inhibitor attenuates this downregulation. Ethanol is also known to generate nitric oxide. Hence, the reduction in oxidative stress, by the antioxidant effect of ethanolic extract of FO or the mixture of vitamin E and C, appear to be the underlying mechanism for the observed immunomodulation.

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Received on 18.03.2009 Modified on 14.04.2009

Research J. Pharm. and Tech.2(2): April.-June.2009,;Page 405-408