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|Year : 2009 | Volume
| Issue : 3 | Page : 115--119
Brahma Rasayana enhances in vivo antioxidant status in cold-stressed chickens (Gallus gallus domesticus)
V Ramnath, PS Rekha
Dept. of Veterinary Physiology, College of Veterinary and Animal Sciences, Kerala Agricultural University, Mannuthy, Thrissur-680 651, Kerala, India
Dept. of Veterinary Physiology, College of Veterinary and Animal Sciences, Kerala Agricultural University, Mannuthy, Thrissur-680 651, Kerala
Objective: To evaluate the antioxidant status of chicken during cold stress and to investigate if there are any beneficial effects of Brahma Rasayana supplementation in cold stressed chicken.
Materials and Methods: Activities of enzymatic and levels of non-enzymatic antioxidants in blood / serum and liver tissue were evaluated in chicken exposed to cold (4 ± 10C and relative humidity of 40 ± 5%, for six consecutive hours daily, for 5 or 10 days). The antioxidant properties of Brahma Rasayana (BR) supplementation (2 g/kg daily, orally) during cold stress was also studied.
Results: There was a significant (P < 0.05) decrease in antioxidant enzyme in the blood, such as, superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), and serum reduced glutathione (GSH) in cold stressed chicken. Serum and liver lipid peroxidation levels were significantly (P < 0.05) higher in cold stressed untreated chickens when compared to the treated and unstressed groups. There was also a significant (P < 0.05) increase in the antioxidant enzymes in the blood, such as, catalase (CAT) and SOD, in the liver CAT and SOD, and in GPX and GR in BR-treated cold stressed chicken, when compared to the untreated controls.
Conclusions: Results of the present study conclude that in chicken, BR supplementation during cold stress brings about enhanced actions of the enzymatic and non-enzymatic antioxidants, which nullify the undesired side effects of free radicals generated during cold stress.
|How to cite this article:|
Ramnath V, Rekha P S. Brahma Rasayana enhances in vivo antioxidant status in cold-stressed chickens (Gallus gallus domesticus).Indian J Pharmacol 2009;41:115-119
|How to cite this URL:|
Ramnath V, Rekha P S. Brahma Rasayana enhances in vivo antioxidant status in cold-stressed chickens (Gallus gallus domesticus). Indian J Pharmacol [serial online] 2009 [cited 2021 Sep 22 ];41:115-119
Available from: https://www.ijp-online.com/text.asp?2009/41/3/115/55209
Stress is a state of threatened homeostasis provoked by psychological, physiological or environmental stress. A stressful condition leads to the excessive production of free radicals, which results in oxidative stress and an imbalance in the oxidant / antioxidant system.  During environmental stress, elevation in the corticosterone level accelerates the generation of free radicals and suppresses the immune function.  Antioxidants are effective in stressful conditions, as they readily give up their own electrons to free radicals.
Breeding hens are continuously exposed to oxidative stress over their lifetime, and the cumulative stress may cause diseases.  Hence, an effective antioxidant system for breeding pullets can be of great importance. Under routine production conditions various types of stress are experienced by chickens such as heat / cold, transport, preslaughter holding, ventilation, and so on. At temperatures above or below the thermo-neutral zone, corticosteroid secretion increases in response to stress. By decreasing synthesis and secretion of corticosteroids, Vitamin C alleviates the negative side effects of stress. Although poultry can synthesize Vitamin C, it becomes inadequate under stressful conditions, such as, low or high environmental temperature, high humidity, high egg production rate, and parasite infestation. 
Brahma Rasayana (BR) is a nontoxic polyherbal preparation, derived from plant extracts, and is claimed to be a potent immunomodulator in cancer patients undergoing chemotherapy and radiation therapy.  Gooseberry ( Emblica officinalis ) and Indian gall nut ( Terminalia chebula ) are the two major ingredients of BR accounting for more than 75% w/w of the preparation.  There are studies indicating that the antioxidant activities of gooseberry are attributed to ascorbic acid and polyphenols, such as, ellagic acid, gallic acid, tannins and so on. Earlier studies revealed that BR is a potent oxygen-free radical scavenger of in vitro and in vivo models. 
The objective of the study was to evaluate the antioxidant properties of BR by measuring the lipid peroxidation (LPO), enzymatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and non-enzymatic reduced glutathione (GSH) content in chickens that are exposed to cold stress, and thereby assess the benefits of BR supplementation in achieving cold tolerance in chicken.
Materials and Methods
A total of 48 one day old male egg type domestic chickens ( Gallus gallus domesticus ) were procured from the University poultry farm, at the College of Veterinary and Animal Sciences, Kerala Agricultural University, Mannuthy, and reared in a battery brooder. They were fed with commercial starter feed for the first four weeks of their life. At the end of their brooding life, the chicks were fed with commercial adult layer mash, till they attained 1 kg body weight. The body weight was recorded weekly from the day of hatch up until the experiment's completion. Before commencing the work, permission from the Institutional Animal Ethics Committee was obtained.
Brahma Rasayana is a mixture of Emblica officinalis (20%), Terminalia chebula (6.67%), Urarira pitca (0.4%), Desmodium gangeticum (0.4%), Gmelina arborea (0.4%), Solanum nigrum (0.4%), Tribulus terrestris (0.4%), Aegle marmelos (0.4%), Premna tomentosa (0.4%), Stereospermum suvaeolens (0.4%), Sida rhombilfolia (0.4%), Boerhaavia diffusa (0.4%), Ricinus communis (0.4%), Vigna vexilata (0.4%), Phaseolus adenanthus (0.4%), Asparagus racemosus (0.4%), Holostemma annulare (0.4%), Leptadenia reticulate (0.4%), Desmostachya bipinnata (0.4%), Saccharum officinarum (0.4%), Oryza malampuzhensis (0.4%), Cinnamomum iners (0.16%), Elettaria cardamomum (0.16%), Cyperus rotundus (0.16%), Curcuma longa (0.16%), Piper longum (0.16%), Aquilaria agallocha (0.16%), Santalum album (0.16%), Centella asciatica (0.16%), Mesua ferrea (0.16%), Clitoria ternate (0.16%), Acorus calamus (0.16%), Scirpus crossus (0.16%), Glycyrrhiza glabra (0.16%), and Embelia ribes (0.16%).
Brahma Rasayana was prepared as per standard recommended procedures,  and purchased from Vaidyaratnam Oushadhasala, Ollur, Thrissur, Kerala.
Administration of BR
The required quantity of BR was made into suspension using warm water (five parts) and mixed with about 30 g of poultry feed (one-third of the total daily ration) and fed to the experimental birds daily, as the first meal. The oral administration of BR at various dose levels, ranging from 0.5 to 6.0 g/kg b.wt., of the bird, daily, for 20 days, was tried, and it did not induce any drug-related toxicity. No significant weight loss or gain was observed, reflecting the effect of BR administration on the well-being of birds. Even at the highest concentration of 6.0 g/kg b.wt., the birds showed no visible toxicity as measured by loss of appetite, lack of movement or alertness. Therefore, it was concluded that BR was nontoxic. However, for all experiments the dose used was 2.0 g/kg b.w, which was at par with the dose administered to mice, that is, for 50 mg of mouse 25 g .wt. 
Cold stress regime
The protocol followed in this study was a combination of two earlier reports. , A controlled environmental unit with a holding capacity of 12 birds at a time, in six chambers (area 1800 cm 2 / chamber), with provisions for adjusting humidity and temperature was used. The chamber temperature of 4 ± 1 0 C and relative humidity (RH) of 40 ± 5% was chosen for the present study. Chickens were exposed to the above conditions in the chamber, for six consecutive hours / day for five or 10 days. During the period of cold stress in the chamber, the chickens were neither provided with drinking water nor feed.
Group I: Non-cold stressed (NCST) and untreated birds, reared randomly under an ambient temperature of 30 ± 1 0 C and 65% RH.
Group II: NCST and BR treated (2 g/kg b.wt. p.o.) for 20 days, reared under similar conditions of temperature and humidity as Group I.
Group III: Cold stressed (CST) at 4 ± 1 0 C and RH of 40 ± 5% for six consecutive hours / day for five days.
Group IV: CST at 4 ± 1 0 C and RH of 40 ± 5% for six consecutive hours / day for five days and BR treated (2 g/kg b p.o.) for 15 days (10 days prior to and five days during the experimental period of cold exposure).
Group V: CST at 4 ± 1 0 C and RH of 40 ± 5% for six consecutive hours /day for 10 days.
Group VI: CST at 4 ± 1 0 C and RH of 40 ± 5% for six consecutive hours /day for five days and BR treated (2 g/kg b. p.o.) for 20 days (10 days prior to and 10 days during the experimental period of cold exposure).
Blood and tissue collections from birds
Blood samples (3 ml) were collected from the wing vein of birds with and without anticoagulant (heparin) from groups III and V after five days of CST, and from birds of groups IV and VI after 10 days of CST. Blood collection was done on day 20, from all birds of group I and II controls. Immediately after collection, the serum was separated, aliquoted, and used for assays. On the same day of blood collection, the birds were sacrificed by cervical dislocation. The liver was excised and thoroughly washed in ice cold saline (0.9%). It was perfused with ice cold saline via the portal vein before homogenization.  A liver homogenate (10%) was made in an ice cold Tris-HCl buffer (pH 7.4) and a cytosolic sample of liver homogenate was obtained by centrifuging at 10,000 g for 30 minutes, at 4 0 C.
The catalase (CAT) activity in fresh blood (erythrocytes) and liver was assessed by the decomposition of hydrogen peroxide, according to Aebi's method and the activities expressed as Kat/g Hb and Units /mg protein.  The superoxide dismutase (SOD) activity in fresh blood (erythrocytes) and liver was determined by the photoreducion of nitroblue tetrazolium (NBT), according to the McCord and Fridovich method.  One unit of SOD activity was defined as the amount of protein that inhibited the rate of reduction of NBT by 50% and was expressed as Units/g Hb and Units/mg protein. Serum- and liver-reduced glutathione (GSH) content were determined by their reaction with 5-5'dithiobis (2-nitrobenzoic acid), according to Moron et al ., and the concentration in the serum and liver was expressed as nmol/ml and nmol/mg protein.  Liver Glutathione peroxidase (GPx) activity was assessed by degradation of hydrogen peroxide (H 2 O 2 ) in the presence of reduced glutathione, according to Paglia and Valentine's method, and the activity expressed as Units/mg protein.  Liver cytosolic glutathione reductase (GR) activity was determined by the amount of NADPH consumed in the conversion of oxidized glutathione (GSSG) to GSH, by following the Racker's method and the activity expressed as nmol of NADPH consumed/min/mg protein.  Serum and liver lipid peroxidation levels were assessed by determining small amounts of malondialdehyde (MDA) produced during peroxidation, following Ohkawa et al ., and the concentration in serum and liver was expressed as nmol of MDA formed per ml and per mg protein.  Protein concentration in the liver was estimated by Lowry's method and expressed in terms of mg protein. 
Data were expressed as mean ± standard error (SE). Statistical analysis was done by one-way classification of ANOVA followed by Duncan's Multiple Range Test (DMRT).  P Cold stress influenced levels of blood antioxidant enzymes
Untreated CST birds of Group III and V exhibited significantly ( P ± 0.87 K/g Hb, respectively) [Table 1] and blood SOD (236.50 ±1.65 U/g Hg and 246.63 ±3.41 U/g Hg, respectively) [Table 1] when compared with NCST - BR treated controls of Group II birds (100.25 ± 1.05 K/g Hb and 362.13 ± 2.46 U/g Hg, respectively).
Untreated birds showed increase in non-enzymatic antioxidants during cold stress
Untreated CST birds in group III and V exhibited significantly ( P b ± 0.45 nmol/ml). However, the liver GSH level of untreated birds belonging to group III and V exhibited significantly ( P ± 0.03) [Table 2].
Damage to the liver in cold stress was averted in BR treated birds
Untreated CST birds in group III and V exhibited a significant ( P P P BR improved enzymatic and non-enzymatic antioxidants in blood and liver
When compared to untreated CST birds (Group III and V), BR treated birds of NCST and CST groups (groups II, IV, and VI) exhibited significantly ( P P Lipid peroxidation (LPO) levels in serum and liver increased in untreated CST birds
All untreated CST birds of group II and V showed significant ( P P P in vivo resulting in an imbalance in the antioxidant status due to excessive formation of free radicals like peroxides, singlet oxygen, and free radicals.  Lower levels of plasma antioxidant such as vitamin C, vitamin E, zinc and chromium, and increased oxidative damage, were observed in stressed poultry. ,
Exposure to cold is a direct threat to the body. When the surrounding temperature drops below 8 0 C, the body may not be able to warm itself, and hence, serious cold-related illnesses, permanent tissue damage, and even death may result.  Effective in vivo antioxidant mechanisms include vitamins A, C, and E, glutathione, and antioxidant enzymes, are essential for protecting the organism against damage resulting from ROS. Rich source of gallic acid is found in Terminalia chebula and vitamin C in Emblica officinalis . The endogenous antioxidant system counteracts the ROS and reduces the oxidative stress. Vitamin C, well known as a potent water soluble, non-enzymatic antioxidant effectively intercept oxidants in the aqueous phase before they attack and cause detectable oxidative damage. Reduced glutathione is considered to be the master antioxidant of the body and is found in almost all living cells. The body utilizes GSH chiefly for reducing oxidized vitamin C and E back to their reduced state, to detoxify many toxins, to maintain the cellular redox potential, to maintain erythrocyte membrane integrity and so on. Glutathione peroxidase (GPx) and glutathione reductase (GR) are enzymes associated with conversion of GSH to the oxidized form and back.  The liver is the chief organ concerned with the detoxification process and availability of an increased amount of GSH in the liver ensures this function. Depletion in the activity of these enzymatic and non-enzymatic antioxidants can be the result of enhanced radical production during stress conditions.
In this study it was found that untreated birds exposed to cold for five or 10 days invariably showed reduced activities of blood CAT and SOD as well as serum GSH levels when compared to both control and CST - BR treated birds. Earlier reports supported the present findings that BR supplementation alone at 2 g/kg b.wt. p.o. for 20 days in NCST birds (group II) facilitated an increased activity of CAT and SOD.  This could be beneficial for the birds, as increased antioxidant activity in erythrocytes ensured proper and rapid elimination of ROS that could be formed during cold stress, thereby protecting the integrity of the erythrocyte membrane.The BR supplementation to CST / NCST birds resulted in increased serum GSH content, when compared to their untreated counterparts.
Results, herein, revealed that the GSH content in the liver of NCST / CST - BR treated birds became significantly elevated over the NCST untreated group, concomitant with activities of GPx and GR enzymes. The rate of GSH formation and the rate at which it was oxidized by antioxidant liver enzymes did not match that in the CST - untreated birds, while in NCST / CST - BR treated birds an appreciable balance between activities of oxidizing and reducing enzymes of glutathione was observed, at least by day 10 of the cold treatment. Thus it could be considered as one of the markers of cold tolerance / acclimatization.
Lipid peroxidation is regarded as one of the basic mechanisms of tissue damage caused by free radicals, as observed in aging, atherosclerosis, and other pathological disorders. LPO of serum lipids, especially that of HDL is highly unfavorable, which may result in many vascular disorders. Results of this study revealed that LPO levels in the serum and liver of NCST / CST - BR treated birds were significantly lower than those encountered in CST - untreated birds. These results are suggestive of the beneficial aspects of BR supplementation, 10 days prior to CST, to be continued during the period of CST in birds; thereby, successfully neutralizing the undesired side effects produced by the free radicals that were generated.
On account of the increased activity and concentration of enzymatic and non-enzymatic antioxidants that resulted from oral BR supplementation, the birds remained healthy and maintained body weight. It may be concluded that BR treatment facilitates earlier adaptation of chickens to stressful cold conditions.
The present investigation is a part of the Adhoc ICAR project implemented through the Department of Physiology. The authors are thankful to the Dean, College of Veterinary and Animal Sciences, Mannuthy, for the facilities provided to conduct the work, and to the Indian Council of Agricultural Research, Government of India, New Delhi, for funding the project.
|1||Maier SF, Watkins LR. Cytokines for psychologists, Implications for bidirectional immune to brain communication for understanding behaviour, mood and cognition. Psychol Rev 1998;105:83-107.|
|2||McIntosh LJ, Saposlsky RM. Glucocorticoids increase the accumulation of reactive oxygen species and enhance adriamycin induced toxicity in neuronal culture. Exp Neurol 1996;141:201-6.|
|3||Enkvetchakul B, Bottje W, Anthony N, Moore R, Huff W. Compromised antioxidant status associated with ascites in broilers. Poult Sci 1993;72:2272-80. |
|4||Brown KI, Nedtor KE. Some physiological responses of turkeys selected for high and low adrenal response to cold stress. Poult Sci 1973;52:1948-54.|
|5||Sykes AH. Vitamin C for poultry some recent research. In: Proceedings of the Roche Symposium. 1978. p. 5. |
|6||Joseph CD, Praveen Kumar V, Kuttan G, Kuttan R. Myeloprotective effect of nontoxic indigenous preparation Rasayana in cancer patients receiving chemotherapy and radiation therapy. J Exp Clin Cancer Res 1999;18:3.|
|7||Vaidya Jadavaji Trikanji Acharya. Rasayana part (Chapter 1). In: Charakasamhitha -Chilsasthanam. 4 th ed. Munshiram Manoharlal Publishers; 1981. p. 41. |
|8||Rekha PS, Kuttan G, Kuttan R. Antioxidant activity of Brahma Rasayana. Indian J Exp Biol 2001;39:447-52. |
|9||Anonymous. The Ayurvedic Formulatory of India. Ministery of health and Family Planning. New Delhi: 1978.|
|10||Thaxton P. Influence of Temperature on the immune responses of birds. Poult Sci 1978;57:1430-40.|
|11||Shrikant Kulkarni, Varshney VP. Enhanced adrenocorticotropic hormone and nitric oxide production from caprine peripheral lymphocytes during heat stress. In Compendium of National Symposium on Recent advances in cryopreservation of livestock germplasm. XIV th Annual Conference of Society of Animal Physiologists of India. 2005. p. 348. |
|12||Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. |
|13||Aebi H. Catalase. In: Bergmeyer HU, editor. Methods in Enzymatic Analysis. 1983. p. 276. |
|14||McCord JM, Fridovich I. The utility of superoxide dismutase in studying free radical reaction I. Radicals generated by the interaction of sulfite, dimethyl sulfoxide, and oxygen. J Biol Chem 1969; 244:6056-63. |
|15||Moron MA, DePierre JW, Mannervick B. Levels of glutathione, glutathione reductase, glutathione-s-transferase activities in rat liver. Biochem Biophys Acta 1979; 582: 67-9. |
|16||Paglia DE, Valentine WN. Studies on the qualitative and quantitative characterisation of erythrocytes glutathione peroxidase. J Lab Clin Med 1967;70:158-9. |
|17||Racker E. Glutathione reductase (liver and yeast).In: Sidney PC, Nathan OK, editors. Methods in enzymology. Academic Press; 1955. p. 722-5. |
|18||Lowry DH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biol Chem 1951;193:265-75. |
|19||Snedecor GW, Cochran WG. Statistical Methods. Oxford and IBH Publishing Corporation; 1994. |
|20||Bollengier-Lee S, Mitchell MA, Utomo DB, Williams PEV, Whitehead CC. Influence of high dietary vitamin E supplementation on egg production and plasma characteristics in hens subjected to heat stress. Brit Poult Sci 1998;39:106-12.|
|21||Kafri I, Cherry JA. Supplemental ascorbic acid and heat stress in broiler chicks. Poult Sci 1984;63:S125-6.|
|22||Sahin N, Sahin K, Onderci M, Ozcelik M, Smith MO. In vivo antioxidant properties of vitamin E and chromium in cold stressed Japanese quails. Arch Anim Nutr 2003;57:207-15.|
|23||NIOSH (National Institute for Occupational Safety and Health). Safety notes. Vol. 2. 2002. p. 22. |
|24||Yu BP. Cellular defences against damage from reactive oxygen species. Physiol Rev 1994;74:139-62. |
|25||Dhanalakshmi S, Srikumar R, Manikandan S, Parathasarathy NJ, Sheeladevi R. Antioxidant property of Triphala on cold stress induced oxidative stress in experimental rats. J Health Sci 2006;52:843-7.|
|26||Charles B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527-605.|