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Attenuation of acute and chronic restraint stress-induced perturbations in experimental animals by Zingiber officinale Roscoe
Food and Chemical Toxicology 48 (2010) 530–535
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Attenuation of acute and chronic restraint stress-induced perturbations in experimental animals by Zingiber officinale Roscoe B.V.S. Lakshmi *, M. Sudhakar Department of Pharmacology, Malla Reddy College of Pharmacy, Dhulapally (via Hakimpet), Maisammaguda, Secunderabad 500014, Andhra Pradesh, India
a r t i c l e
i n f o
Article history: Received 30 August 2009 Accepted 6 November 2009
Keywords: Zingiber officinale Anoxia stress Cold restraint stress Swimming stress Adaptogenic
a b s t r a c t Ethanolic extract of rhizomes of Zingiber officinale was investigated on anoxia stress tolerance test in Swiss mice. The animals were also subjected to acute physical stress (swimming endurance test) to gauge the anti-stress potential of the extract. Further to evaluate the anti-stress activity of Z. officinale in chronic stress condition, fresh Wistar rats were subjected to cold restraint stress (4° for 2 h) for 10 days. Stimulation of hypothalamus pituitary adrenal axis in stressful condition alters plasma glucose, triglyceride, cholesterol, BUN and corticosterone levels. There is also alteration in the blood cell counts. Pretreatment with the extract significantly ameliorated the stress-induced variations in these biochemical levels and blood cell counts in both acute and chronic stress models. The extract treated animals showed increase in swimming endurance time and increase in anoxia tolerance time in physical and anoxia stress models, respectively. Treatment groups also reverted back increase in liver, adrenal gland weights and atrophy of spleen caused by cold chronic stress and swimming endurance stress models. The results indicate that ethanolic extract of Z. officinale has significant adaptogenic activity against a variety of biochemical and physiological perturbations in different stress models. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Stress basically is a reaction of mind and body against change in the homeostasis. The productive stress is called Eustress while harmful stress is called Distress. If the stress is extreme, the homeostatic mechanisms of the organism become deficit and the survival of the organism is threatened. Under these conditions, stress triggers a wide range of body changes called General Adaptation Syndrome (GAS). The stimuli, which produce GAS, are called the stressors and range from physical to psychological factors including cold, heat, infection, toxins, major personal disappointment, etc. (Selye, 1973). In the stress-filled environment we live in, successful adaptation to stress is a prerequisite for survival. In the indigenous system of medicine, there are many herbal drugs and formulations recommended to enable one to withstand stress without altering the physiological functions of the body. This, drug induced state of resistance against aversive stimuli is termed as adaptogenic activity and the drugs, named adaptogens. Stress alters the equilibrium of various hormones which have a significant
Abbreviations: Fig., figure; i.p., intra peritoneal; kg, kilogram; mg, milligram; ml, millilitre; p.o., per oral; w/w, weight/weight; w/v, weight/volume; ZO, Zingiber officinale. * Corresponding author. Tel.: +91 09885324334; fax: +91 040 23792154. E-mail address: [email protected] (B.V.S. Lakshmi). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.11.026
impact on the immune response in general. The status of immune system-immunosuppression versus immunopotentiation-will depend upon the net effect of these changes. Stress and depression have been shown to affect immune system functioning, with both immunosuppression and immune activation (Raison and Miller, 2001). Correlations between depression and elevated susceptibility for infections or mortality rates have been observed and are associated with immune suppression (Irwin, 2002). The physiological reaction to stress involves alteration in the autonomic nervous system, the endocrine system and the immune system. The secretion of glucocorticoids is a classic endocrine response to stress (Sapolsky et al., 2000). Stressful stimulation influences antigen-specific as well as non-specific reactions (Ader and Cohen, 1993). Many herbs reported in ancient literature have potent antistress activity and their utilities in current scenario need to be unveiled. Zingiber officinalis, Roscoe is a rhizome that is widely used as culinary herb and herbal remedy for some common ailments. It is used as carminative, antipyretic, antiemetic in pregnancy and as anticancer adjunct (Evans and Trease, 1979). It also ameliorates motion sickness and it is a known thromboxane synthesis and platelet aggregation inhibitors, and diaphoretic agent (Evans and Trease, 1979). It contains about 1–2% of volatile oil and 5–8% of resinous matter, starch and mucilage (Evans and Trease, 1979). The volatile oil contains monoterpenes, sesquiterpenes and sesquiterpene alcohol zingiberol (Evans and Trease, 1979), gingerol and
B.V.S. Lakshmi, M. Sudhakar / Food and Chemical Toxicology 48 (2010) 530–535
shagoals. Most of the pharmacologically active constituents reside in the volatile oils. Gingerols have cardio tonic (Kobayashi et al., 1988), analgesic, anti-inflammatory (Young et al., 2005), antipyretic (Yoshikawa et al., 1993) and antibacterial effects both in vitro and in vivo (Mascolo et al., 1989). Shagoal has antiemetic, antispasmodic, anxiolytic and anticonvulsant activity (Vishwakarma et al., 2002). Scientific reports show that it is also used for conditions such as anti-ulcerogenic (Al-Yahya et al., 1989), antidiabetic (Akhani et al., 1981), anti-oxidant (Jeyakumar et al., 1999) and anti-hepatotoxic (Omoniyi et al., 2006) activities. Ginger has been reported to possess a potent anti-oxidant activity in vitro (Jeyakumar et al., 1999) which reduces the oxidative stress in the body. Since Zingiber officinale has a number of medicinal properties and is a potent anti-oxidant, the present study was undertaken to evaluate the potential usefulness of fresh rhizomes of Z. officinale for anti-stress and adaptogenic activity in experimental animals. Withania somnifera, an established ayurvedic herb used as an adaptogen is used as reference standard (Singh et al., 2005).
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glucose, triglycerides, cholesterol, BUN, corticosterone and blood cell count (RBC, WBC and DLC). The weights of organs such as liver, adrenals, spleen were recorded after washing with alcohol. 2.5. Chronic cold restraint stress Treatment groups were similar to forced swimming endurance stress. Rats were subjected to cold stress by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days (Bhattacharya and Ghosal, 1994). Animals were sacrificed at the end of the study period and blood was collected for estimation of various biochemical parameters such as Serum cortisol, glucose levels, RBC count, total leukocyte count, differential count as well as lipid profile. Similarly the weights of organs, i.e., liver, spleen and adrenal glands were also recorded. 2.6. Statistical analysis All the values are expressed as mean ± SEM and data was analyzed by one-way ANOVA, using Graph pad INSTAT. The post hoc analysis was carried out by Dunnett’s multiple comparison test to estimate the significance of difference between individual groups.
3. Results 2. Materials and methods
3.1. Effect of ethanolic extract in anoxia stress tolerance test 2.1. Plant material and extraction Z. officinale rhizomes were collected from the local market of Rangareddy District, Hyderabad, in the month of February–March, the botanical authentication was done by the Department of Botany, Osmania University, Hyderabad and voucher specimen (MRCP-104) is lodged in our research laboratory for the future reference. The fresh rhizomes were sliced using a home slicer and the slices obtained were shade-dried, pulverized and passed through a 20-mesh sieve. The dried, coarsely powdered plant material was extracted with 99% ethanol using Soxhlet apparatus at a temperature below 60 °C for 24 h. The solvent was evaporated under vacuum, which gave semisolid mass (yield: 26% w/w) with respect to the dried powder. Oral suspensions containing 50, 100 and 200 mg/ml of the ethanolic extract of Z. officinale were prepared in 1% w/v gum acacia.
2.2. Animals Swiss albino mice weighing 20–25 g and Albino Wistar rats weighing 150– 250 g of either sex, 4 months of age were used for this study. The experimental animals were housed in polypropylene cages and maintained under standard conditions (12 h light and dark cycles, at 25 ± 3 °C and 35–60% humidity). Standard pelletized feed and tap water were provided ad libitum. The Institutional Animal Ethical Committee (IAEC) of Malla Reddy College of Pharmacy, Hyderabad, approved the study.
2.3. Anoxia stress tolerance test in mice Swiss mice of either sex were divided randomly into five groups, each group containing six mice. Group I mice received 0.1% gum acacia in saline; (vehicle control). Group II mice were treated with W. somnifera (100 mg/kg, p.o.) and stress; Group III, IV and V mice were treated with ethanolic extract at doses of 50, 100 and 200 mg/kg, p.o. and stress. The drug treatment was carried out daily for a period of 21 days. At the end of each week, i.e., 1st, 2nd and 3rd weeks of drug treatment, the animals were exposed to the anoxia stress and anoxia tolerance time was noted. Hermetic vessel of one litre air capacity was used to induce anoxia stress (Krupavaram et al., 2007). Each animal was kept in the hermetic vessel and the time to show the first sign of convulsion was noted, and were immediately removed from the vessel and resuscitated if needed.
2.4. Forced swimming endurance test (physical stress) Rats of either sex (200–250 g) were used for forced swim endurance stress. Group I rats received 0.1% gum acacia in saline; (vehicle control). Group II mice were treated with 0.1% gum acacia in saline and stress; (negative control). Group III rats were treated with W. somnifera (100 mg/kg, p.o.) and stress; (positive control). Group IV, V and VI mice were treated with ethanolic extract at 50, 100 and 200 mg/kg, p.o. and stress. The rats were subjected to swimming stress by keeping them in propylene tank of dimension (37 37 30 cm), filled with water to a height of 25 cm. Extracts were given to rats, once daily for period of 7 days. On 8th day the rats were allowed to swim till complete exhaustion and the endpoint was taken when the animal started drowning. The mean swimming time for each group was calculated (Kannur et al., 2006). Then animals were killed and blood was collected by cardiac puncture to estimate biochemical parameters like serum
In the anoxia tolerance test (Table 1), the extract at 50,100 and 200 mg kg 1 doses statistically produced a dose dependant significant (P < 0.05) increase in mean time to convulsion in mice subjected to anoxia stress. 3.2. Effect of ethanolic extract in forced swimming endurance stress The results of the study revealed that the extract possess antistress property as it significantly (P < 0.05) increased the swimming time (Fig. 1). Swimming endurance stress resulted in significant increase in adrenal gland weight and liver weight with concomitant decrease in spleen weight in stress control group, which was significantly reverted by Z. officinale pretreatment at 50, 100 and 200 mg/kg. Similarly stress-induced elevated blood cell counts of RBC and DLC, i.e., lymphocytes, neutrophils, eosinophils and monocytes have been significantly (P < 0.01) reduced by the ethanolic extract in a dose dependant manner (Table 2). Pretreatment of animals with Z. officinale at three doses also significantly (P < 0.05) restored back forced swimming stress-induced alterations in plasma corticosterone, glucose, triglyceride, BUN and cholesterol (Table 3). 3.3. Effect of ethanolic extract in cold restraint stress In cold restraint stress, ethanolic extract at 50, 100 and 200 mg/ kg offered significant (P < 0.05) protection against the change in the weights of liver, spleen and adrenal gland when compared to stress control (Figs. 2 and 3). The extract dose dependently reduced the elevated levels of biochemical parameters (P < 0.05) (Table 4). The extract at 200 mg/kg significantly (P < 0.01) reduced all the blood cell counts (Table 5). 4. Discussion Adaptogens are the substances meant to put the organisms into a state of non-specific heightened resistance in order to better resist stressor and adapt to extraordinary challengers. They normalize body functions, strengthen systems and functions that are compromised by stress and have a protective effect against a wide variety of environmental and emotional stress. The forced swimming is the most widely used method for assessing the anti-stress property of a novel compound (Anisman and Zacharko, 1991; Subarnas et al., 1993). This paradigm is based
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Table 1 Effect of ethanolic extract of Zingiber officinale in anoxia stress tolerance in mice. Groups
Mean duration of tolerance time (min)
Control Withania somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
1st week
2nd week
3rd week
120.59 ± 0.34 162.25 ± 0.81* 131.29 ± 0.75 149.71 ± 0.61* 161.11 ± 0.73*
122.71 ± 0.58 178.91 ± 0.68* 133.16 ± 0.85* 155.08 ± 1.43* 167.95 ± 0.79*
126.57 ± 0.95 184.92 ± 1.15* 139.41 ± 1.92* 159.27 ± 0.68* 176.59 ± 1.25*
The mice were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 21 days. Control mice were given saline. Anoxia was induced at the end of 1st, 2nd and 3rd weeks and the anoxia stress tolerance time was noted. The values are expressed as mean ± SEM, n = 6. * Significance at P < 0.05 when compared to control as determined by ANOVA followed by Dunnett’s t test.
227.95**
250 Time in mins
200
183.61
228** 198.82**
186.79 164.15*
150 100 50 0 Control
Stress control
Withania somnifera
ZO extract1
ZO extract2
ZO extract3
Groups Fig. 1. Effect of ethanolic extract of Zingiber officinale in forced swimming endurance test in rats (swimming time). The forced swimming endurance time was measured in rats. The mice were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 7 days. Control mice were given gum acacia. Three hours after the final treatment, the rats were exposed to forced swimming endurance test. The mean increase in duration of swimming time was noted. The values are expressed as mean ± SEM, n = 6 in each group. *P < 0.05 significant as compared to control, **P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnet’s t test.
on the observation that animals forced to swim in water eventually assumed a characteristic immobile posture, devoid of any activity (Weiss et al., 1981). The appearance of immobility therefore, reflects a state of tiredness, fatigue, reduced stamina with the end point being the moment when the rat could not swim further and started drowning (Bargava and Singh, 1981). However, increased swimming time has been observed in rat pre-treated with ethanolic extract and has enhanced the physical performance long-
er than untreated (control) group confirming their adaptogenic nature. In present study, in acute and chronic stress models, the significant increase in blood glucose level was observed because; under stressful conditions adrenal cortex secretes cortisol in man and corticosterone in rats. Hyper secretion of cortisol helps in maintenance of internal homeostasis through the process of gluconeogenesis and lipogenesis (Krupavaram et al., 2007). Pretreatment with
Table 2 Effect of ethanolic extract of Zingiber officinale on organ weights and blood cell counts in forced swimming endurance stress in rats. Treatment
Organs weight Spleen (mg/ 100 g)
Control Stress control W. somnifera 100 mg/kg p.o. ZO extract 50 mg/ kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
Blood cell counts Liver (g/ 100 g)
Adrenal glands (mg/ 100 g)
RBC in millions
WBC (no. of cells/mm3)
DLC no. of cells/Cumm
L
N
E
M
250.01 ± 2.25 196.24 ± 1.58 263.21** ± 1.58
3.71 ± 1.58 5.43 ± 1.23 3.85** ± 2.54
8.76 ± 0.57 15.93 ± 1.46 9.87** ± 0.85
8.02 ± 0.44 13.6* ± 2.52 8.27** ± 1.25
6439 ± 23.98 9650* ± 21.23 6200** ± 24.23
4537 ± 14.89 6439* ± 14.56 4236** ± 18.85
1836 ± 21.24 3121* ± 15.65 1889** ± 14.89
63.24 ± 2.36 79.96* ± 1.98 66.42** ± 4.56
9.21 ± 0.54 10.82* ± 0.63 9.34** ± 0.87
204.24 ± 2.54
4.95 ± 1.69
11.96 ± 1.65
11.54* ± 2.25
7650* ± 26.25
5923* ± 17.89
1646* ± 16.74
71.26* ± 4.75
10.62* ± 1.48
219.95** ± 2.65
4.29* ± 2.58
10.84** ± 1.58
9.59* ± 1.23
7100** ± 14.25
5426** ± 19.48
1595** ± 17.36
69.84** ± 6.02
9.87* ± 1.52
234.61** ± 1.97
4.05** ± 3.35
10.21** ± 1.35
8.76** ± 1.79
6700** ± 18.64
4727** ± 28.54
1897** ± 18.58
66.91** ± 5.23
9.56* ± 0.89
The rats were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 8 days. Control rats were given gum acacia. Swimming stress was induced at the end of treatment period. The animals were sacrificed, the organs were isolated from the animals and the weights were noted. Blood was collected for the estimation of cell counts. The results are expressed as mean ± SEM, n = 6 in each group. * P < 0.01 significant as compared to control. ** P < 0.01, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
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B.V.S. Lakshmi, M. Sudhakar / Food and Chemical Toxicology 48 (2010) 530–535 Table 3 Effect of ethanolic extract of Zingiber officinale on biochemical parameters in swimming endurance stress in rats. Groups
Corticosterone (lg/dl)
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
BUN (mg/dl)
Control swimming Stress control W. somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
98.60 ± 2.56 165.73 ± 2.95 101.62 ± 2.57** 136.39 ± 1.85* 119.48 ± 3.87** 105.64 ± 3.35**
91.98 ± 1.58 198.23 ± 1.96 99.78 ± 0.42** 134.23 ± 2.98* 126.59 ± 3.02** 92.36 ± 1.85**
89.41 ± 3.63 184.62 ± 1.89 84.62 ± 3.59* 146.15 ± 4.05* 92.30 ± 3.82** 88.46 ± 1.56**
63.22 ± 2.85 95.87 ± 5.02 66.86 ± 0.89* 89.42 ± 2.64* 72.56 ± 3.98* 67.89 ± 2.63**
28.18 ± 2.69 58.63 ± 1.24 36.42 ± 0.78* 48.37 ± 2.58* 41.92 ± 3.58* 38.29 ± 3.25*
The rats were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 7 days. Control rats were given gum acacia. Swimming stress was induced at the end of treatment period. The animals were sacrificed; the blood collected and the biochemical parameters were estimated. The values are expressed as mean ± SEM, n = 6 in each group. * P < 0.05 significant as compared to control. ** P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
**
350
*
300
**
* Weight in mg
250
*
Control Cold Stress control
200
Withania somnifera ZO extract1
150
ZO extract2 ZO extract3
100 * 50
* **
*
**
0 Spleen
Adrenal glands Groups
Fig. 2. Effect of ethanolic extract of Zingiber officinale on organ weights in cold restraint stress in rats. The rats were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed, the organs were isolated from the animals and the weights were noted. The values are expressed as mean ± SEM, n = 6 in each group. *P < 0.05 significant as compared to control, **P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnet’s t test.
Liver weight in gms
7
*
*
6
*
**
**
5 4 3 2 1 ZO extract3
ZO extract2
ZO extract1
Withania somnifera
Cold Stress control
Control
0
Fig. 3. Effect of ethanolic extract of Zingiber officinale on organ weights in cold restraint stress in rats. The rats were pretreated with Withania somnifera (100 mg/ kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed, the organs were isolated from the animals and the weights were noted. The values are expressed as mean ± SEM, n = 6 in each group. *P < 0.05 significant as compared to control, **P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnet’s t test.
the Z. officinale as well as reference standard drug W. somnifera significantly (P < 0.05) reduced the elevated glucose levels indicating their suppressant effect on hyper activity of adrenal cortex and maintained the homeostatic mechanism.
The marked increase in serum cholesterol, triglycerides and BUN levels in stress-induced animals is due to stimulation of hypothalamo–pituitary axis (HPA) and sympathetic system, resulting in, liberation of catecholamines and glucocorticosteroids, which inhibits the immune system at multiple sites like liver, kidney (Schimmer and Parker, 2006). Z. officinale as well as reference standard drug W. somnifera significantly (P < 0.05) reduced the elevated serum cholesterol, triglycerides and BUN levels, which may be due to inhibition of stimulation of sympathetic nervous system. The increase in weight of adrenals in stressed animals is due to the stress-induced adrenomedullary response leading to increased production of corticotropic hormone that leads to increase in weight of adrenals (Krupavaram et al., 2007). Z. officinale and W. somnifera has significantly (P < 0.01) reduced the liver, adrenal gland weight, this may be due to the reversal of the stress-induced adrenomedullary response and hence decreased production of corticotropic hormone. The decrease in weight of spleen may be due to recruitment of lymphocytes to blood from spleen which results in squeezing of the spleen (Rai et al., 2003). The pretreatment with the Z. officinale and reference standard W. somnifera significantly (P < 0.01) increased the spleen weight. This may be due to inhibition of recruitment of lymphocytes to blood from spleen. During stress, heart rate, blood pressure and blood flow rate increases. To meet these extra demands RBC and WBC counts will be increased. In the present study the extract has decreased the elevated levels of RBC and WBC in both swimming endurance and cold restraint stress models.
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Table 4 Effect of ethanolic extract of Zingiber officinale on biochemical parameters in cold restraint stress. Groups
Corticosterone (lg/dl)
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
BUN (mg/dl)
Control Cold stress control W. somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
98.60 ± 2.56 162.71 ± 1.95* 102.83 ± 2.95** 121.63 ± 0.98 109.58 ± 1.87* 101.56 ± 2.35**
91.42 ± 2.35 152.25 ± 2.16* 96.49 ± 1.52** 122.28 ± 1.48* 109.54 ± 3.32* 99.87 ± 2.84**
89.41 ± 3.63 163.33 ± 2.14* 92.86 ± 4.02** 132.38 ± 2.32* 93.33 ± 4.52* 82.38 ± 1.89**
63.22 ± 2.85 106.80 ± 3.65* 67.36 ± 1.32** 94.89 ± 3.58* 78.30 ± 4.05* 68.04 ± 2.25**
28.18 ± 2.69 49.25 ± 2.83* 30.01 ± 1.54** 40.38 ± 2.04* 36.13 ± 1.25* 31.56 ± 3.01**
The rats were treated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed; the blood collected and the biochemical parameters were estimated. The values are expressed as mean ± SEM, n = 6 in each group. * P < 0.05 significant as compared to control. ** P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
Table 5 Effect of ethanolic extract of Zingiber officinale on Blood cell counts in Cold Restraint stress in rats. Treatment
Blood cell counts RBC in millions
Control Cold stress control Withania somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
8.02 ± 0.44 10.11* ± 0.52 8.29** ± 0.48 9.87 ± 0.65 9.03* ± 1.23 8.89** ± 0.79*
WBC (no. of cells/mm3)
6439 ± 23.98 9854* ± 18.56 6832** ± 21.96 8100** ± 19.12 7833** ± 22.87 6539** ± 15.24
DLC no. of cells/Cumm L
N
E
M
4537 ± 19.89 7436* ± 15.89 4963** ± 14.56 5325* ± 14.23 5104** ± 9.84 4624** ± 58.25
1836 ± 15.35 2327* ± 24.21 1794** ± 23.11 2689** ± 14.24 2649* ± 18.58 1839** ± 25.23
63.24 ± 2.25 79.86* ± 2.36 65.82** ± 3.35 74.85* ± 5.23 69.41** ± 5.36 66.32** ± 4.65
9.21 ± 0.23 11.34* ± 0.58 9.43** ± 0.48 11.02 ± 1.02 10.56* ± 0.57 9.84** ± 0.47
The rats were treated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed; the blood was collected for the estimation of cell counts. The values are expressed as mean ± SEM, n = 6 in each group. * P < 0.01 significant as compared to control. ** P < 0.01, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
This study has also shown that the extract prolonged mean time to convulsion, which therefore demonstrate anti-stress property. The prolongation of mean time to convulsion could be attributed to its powerful anti-oxidant and free radical scavenging activities (Oke and Hamburger, 2001). A variety of biological activities including adaptogenic activity were reported with flavonoids, tannins and phenolic glycosides (Krupavaram et al., 2007). Z. officinale contains biologically active chemicals that include flavonoids, volatile oils which contain monoterpenes, sesquiterpenes and sesquiterpene alcohol zingiberol, gingerol and shagoals (Evans and Trease, 1979). The adaptogenic activity may be due to these constituents where as standard drug W. somnifera an established adaptogenic drug too contains glycosides, steroids and flavonoids (Krupavaram et al., 2007). Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments The authors are thankful to the management of Malla Reddy College of Pharmacy, for providing the chemicals and required facilities to carry out the research work. We are thankful to Dr. Ram Chandra Reddy, H.O.D, Dept. of Botany, Osmania University, Hyderabad for authentication of the plant. References Ader, R., Cohen, N., 1993. Psychoneuroimmunology: conditioning and stress. Annu. Rev. Psychol. 44, 53–85.
Akhani, S.P., Vishwakarma, S.L., Goyal, R.K., 1981. Anti-diabetic activity of Zingiber officinale in streptozotocin-induced type I diabetic rats. J. Pharm. Pharmacol. 56 (1), 101–105. Al-Yahya, M.A., Rafatullah, S., Mossa, J.S., Ageel, A.M., 1989. Gastroprotective activity of ginger Zingiber officinale rosc., in albino rats. Am. J. Chin. Med. 17 (1– 2), 51–56. Anisman, H., Zacharko, R.M., 1991. Multiple neurochemical and behavioural consequences of stressors: implication for depression. In: Psychopharmacology of Anxiolytics and Antidepressants. Pergamon Press, New York, pp. 57–82. Bargava, K.P., Singh, N., 1981. Effect of Ocimum sanctum on noise induced changes in neutrophil functions. Ind. J. Med. Res. 73, 143–151. Bhattacharya, S.K., Ghosal, S., 1994. Experimental evaluation of anti stress activity of Zee stress. Indian J. Indg. Med. 2, 1–8. Evans, W.C., Trease, G.E., 1979. Volatile Oils and Resins, Pharmacognosy, 11th ed. Balliere, Tindale. p. 475. Irwin, M., 2002. Psychoneuroimmunology of depression: clinical implication. Brain Behav. Immunol. 16, 1–16. Jeyakumar, S.M., Nalini, N., Menon, V.P., 1999. Antioxidant activity of ginger (Zingiber officinale Roscoe) in rats fed with a high fat diet. Med. Sci. Res. 27 (5), 341–346. Kannur, D.M., Hukkeri, V.I., Akki, K.S., 2006. Adaptogenic activity of caesalpinia bonduc seed extracts in rats. J. Ethano. Pharmacol. 108, 327–331. Kobayashi, M., Ishida, Y., Shoji, N., Ohizumi, Y., 1988. Cardiotonic action of (8)gingerol, an activator of Ca2+ – pumping adenosine triphosphate of sarcoplasmic reticulum, in guinea pig atrial muscle. J. Pharmacol. Exp. Ther. 246 (2), 667–673. Krupavaram, B., Venakat Rao, N., Nandakumar, K., Gowda, T.S., Shalam, M.D., Shantakumar, S., 2007. Study on adaptogenic activity of root extracts of Boerhaavia diffusa (Linn). Indian Drugs 44 (4), 264–270. Mascolo, N., Jain, R., Jain, S.C., Capasso, F., 1989. Ethno pharmacologic investigation of ginger (Zingiber officinale Roscoe). J. Ethnopharmacol. 27 (1–2), 129–140. Oke, J.M., Hamburger, 2001. Screening of some Nigeria medicinal plants for antioxidants activity using 2,2, diphenyl picryl-hydrazyl radical. Afr. J. Biomed. Res. 5, 77–79. Omoniyi, K. Yemitan, Matthew, C. Izegbu, 2006. Protective effects of Zingiber officinale (Zingiberaceae) against carbon tetrachloride and acetaminopheninduced hepatotoxicity in rats. Phytother. Res. 20(11), 997. Rai, D., Bhatia, G., Patil, G., Pal, R., Singh, S., Singh, H.K., 2003. Adaptogenic effect of Bacopa monniera (Bramhi). Pharmacol. Biochem. Behav. 75, 823–830. Raison, C.L., Miller, A.H., 2001. The neuroimmunology of stress and depression. Semin. Clin. Neuropsychiatry 6, 277–294.
B.V.S. Lakshmi, M. Sudhakar / Food and Chemical Toxicology 48 (2010) 530–535 Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress response? Integrating permissive, suppressive, stimulatory and preparative actions. Endocrinol. Rev. 21, 55–89. Schimmer, B.P., Parker, K.L., 2006. Adrenocortical steriods and their synthetic analogues. In: The Pharmacological Basis of Therapeutics 11th ed. The McGrawHill Medical Publishing Division, New York, pp. 1655–1662. Selye, H., 1973. The evolution of the stress concept. Am. Sci. 61, 693–699. Singh, B., Chandan, B.K., Sharma, N., Singh, S., Khajuria, A., Gupta, D.K., 2005. Adaptogenic activity of glyco-peptido-lipid fraction from the alcoholic extract of Trichopus zeylanicus Gaerten (part II). Phytomedicine 12 (6), 468–481. Subarnas, A., Tadano, T., Nakahata, N., Arai, Y., Kinemuchi, H., 1993. A possible mechanism of antidepressant activity of beta-amyrin palmitate isolated from Lobelia inflata leaves in the forced swimming test. Life Sci. 52, 289–296. Vishwakarma, S.L., Pal, S.C., Kasture, V.S., Kasture, S.B., 2002. Anxiolytic and antiemetic activities of Z. officinale. Phytother. Res. 16 (7), 621–626.
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Attenuation of acute and chronic restraint stress-induced perturbations in experimental animals by Zingiber officinale Roscoe B.V.S. Lakshmi *, M. Sudhakar Department of Pharmacology, Malla Reddy College of Pharmacy, Dhulapally (via Hakimpet), Maisammaguda, Secunderabad 500014, Andhra Pradesh, India
a r t i c l e
i n f o
Article history: Received 30 August 2009 Accepted 6 November 2009
Keywords: Zingiber officinale Anoxia stress Cold restraint stress Swimming stress Adaptogenic
a b s t r a c t Ethanolic extract of rhizomes of Zingiber officinale was investigated on anoxia stress tolerance test in Swiss mice. The animals were also subjected to acute physical stress (swimming endurance test) to gauge the anti-stress potential of the extract. Further to evaluate the anti-stress activity of Z. officinale in chronic stress condition, fresh Wistar rats were subjected to cold restraint stress (4° for 2 h) for 10 days. Stimulation of hypothalamus pituitary adrenal axis in stressful condition alters plasma glucose, triglyceride, cholesterol, BUN and corticosterone levels. There is also alteration in the blood cell counts. Pretreatment with the extract significantly ameliorated the stress-induced variations in these biochemical levels and blood cell counts in both acute and chronic stress models. The extract treated animals showed increase in swimming endurance time and increase in anoxia tolerance time in physical and anoxia stress models, respectively. Treatment groups also reverted back increase in liver, adrenal gland weights and atrophy of spleen caused by cold chronic stress and swimming endurance stress models. The results indicate that ethanolic extract of Z. officinale has significant adaptogenic activity against a variety of biochemical and physiological perturbations in different stress models. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Stress basically is a reaction of mind and body against change in the homeostasis. The productive stress is called Eustress while harmful stress is called Distress. If the stress is extreme, the homeostatic mechanisms of the organism become deficit and the survival of the organism is threatened. Under these conditions, stress triggers a wide range of body changes called General Adaptation Syndrome (GAS). The stimuli, which produce GAS, are called the stressors and range from physical to psychological factors including cold, heat, infection, toxins, major personal disappointment, etc. (Selye, 1973). In the stress-filled environment we live in, successful adaptation to stress is a prerequisite for survival. In the indigenous system of medicine, there are many herbal drugs and formulations recommended to enable one to withstand stress without altering the physiological functions of the body. This, drug induced state of resistance against aversive stimuli is termed as adaptogenic activity and the drugs, named adaptogens. Stress alters the equilibrium of various hormones which have a significant
Abbreviations: Fig., figure; i.p., intra peritoneal; kg, kilogram; mg, milligram; ml, millilitre; p.o., per oral; w/w, weight/weight; w/v, weight/volume; ZO, Zingiber officinale. * Corresponding author. Tel.: +91 09885324334; fax: +91 040 23792154. E-mail address: [email protected] (B.V.S. Lakshmi). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.11.026
impact on the immune response in general. The status of immune system-immunosuppression versus immunopotentiation-will depend upon the net effect of these changes. Stress and depression have been shown to affect immune system functioning, with both immunosuppression and immune activation (Raison and Miller, 2001). Correlations between depression and elevated susceptibility for infections or mortality rates have been observed and are associated with immune suppression (Irwin, 2002). The physiological reaction to stress involves alteration in the autonomic nervous system, the endocrine system and the immune system. The secretion of glucocorticoids is a classic endocrine response to stress (Sapolsky et al., 2000). Stressful stimulation influences antigen-specific as well as non-specific reactions (Ader and Cohen, 1993). Many herbs reported in ancient literature have potent antistress activity and their utilities in current scenario need to be unveiled. Zingiber officinalis, Roscoe is a rhizome that is widely used as culinary herb and herbal remedy for some common ailments. It is used as carminative, antipyretic, antiemetic in pregnancy and as anticancer adjunct (Evans and Trease, 1979). It also ameliorates motion sickness and it is a known thromboxane synthesis and platelet aggregation inhibitors, and diaphoretic agent (Evans and Trease, 1979). It contains about 1–2% of volatile oil and 5–8% of resinous matter, starch and mucilage (Evans and Trease, 1979). The volatile oil contains monoterpenes, sesquiterpenes and sesquiterpene alcohol zingiberol (Evans and Trease, 1979), gingerol and
B.V.S. Lakshmi, M. Sudhakar / Food and Chemical Toxicology 48 (2010) 530–535
shagoals. Most of the pharmacologically active constituents reside in the volatile oils. Gingerols have cardio tonic (Kobayashi et al., 1988), analgesic, anti-inflammatory (Young et al., 2005), antipyretic (Yoshikawa et al., 1993) and antibacterial effects both in vitro and in vivo (Mascolo et al., 1989). Shagoal has antiemetic, antispasmodic, anxiolytic and anticonvulsant activity (Vishwakarma et al., 2002). Scientific reports show that it is also used for conditions such as anti-ulcerogenic (Al-Yahya et al., 1989), antidiabetic (Akhani et al., 1981), anti-oxidant (Jeyakumar et al., 1999) and anti-hepatotoxic (Omoniyi et al., 2006) activities. Ginger has been reported to possess a potent anti-oxidant activity in vitro (Jeyakumar et al., 1999) which reduces the oxidative stress in the body. Since Zingiber officinale has a number of medicinal properties and is a potent anti-oxidant, the present study was undertaken to evaluate the potential usefulness of fresh rhizomes of Z. officinale for anti-stress and adaptogenic activity in experimental animals. Withania somnifera, an established ayurvedic herb used as an adaptogen is used as reference standard (Singh et al., 2005).
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glucose, triglycerides, cholesterol, BUN, corticosterone and blood cell count (RBC, WBC and DLC). The weights of organs such as liver, adrenals, spleen were recorded after washing with alcohol. 2.5. Chronic cold restraint stress Treatment groups were similar to forced swimming endurance stress. Rats were subjected to cold stress by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days (Bhattacharya and Ghosal, 1994). Animals were sacrificed at the end of the study period and blood was collected for estimation of various biochemical parameters such as Serum cortisol, glucose levels, RBC count, total leukocyte count, differential count as well as lipid profile. Similarly the weights of organs, i.e., liver, spleen and adrenal glands were also recorded. 2.6. Statistical analysis All the values are expressed as mean ± SEM and data was analyzed by one-way ANOVA, using Graph pad INSTAT. The post hoc analysis was carried out by Dunnett’s multiple comparison test to estimate the significance of difference between individual groups.
3. Results 2. Materials and methods
3.1. Effect of ethanolic extract in anoxia stress tolerance test 2.1. Plant material and extraction Z. officinale rhizomes were collected from the local market of Rangareddy District, Hyderabad, in the month of February–March, the botanical authentication was done by the Department of Botany, Osmania University, Hyderabad and voucher specimen (MRCP-104) is lodged in our research laboratory for the future reference. The fresh rhizomes were sliced using a home slicer and the slices obtained were shade-dried, pulverized and passed through a 20-mesh sieve. The dried, coarsely powdered plant material was extracted with 99% ethanol using Soxhlet apparatus at a temperature below 60 °C for 24 h. The solvent was evaporated under vacuum, which gave semisolid mass (yield: 26% w/w) with respect to the dried powder. Oral suspensions containing 50, 100 and 200 mg/ml of the ethanolic extract of Z. officinale were prepared in 1% w/v gum acacia.
2.2. Animals Swiss albino mice weighing 20–25 g and Albino Wistar rats weighing 150– 250 g of either sex, 4 months of age were used for this study. The experimental animals were housed in polypropylene cages and maintained under standard conditions (12 h light and dark cycles, at 25 ± 3 °C and 35–60% humidity). Standard pelletized feed and tap water were provided ad libitum. The Institutional Animal Ethical Committee (IAEC) of Malla Reddy College of Pharmacy, Hyderabad, approved the study.
2.3. Anoxia stress tolerance test in mice Swiss mice of either sex were divided randomly into five groups, each group containing six mice. Group I mice received 0.1% gum acacia in saline; (vehicle control). Group II mice were treated with W. somnifera (100 mg/kg, p.o.) and stress; Group III, IV and V mice were treated with ethanolic extract at doses of 50, 100 and 200 mg/kg, p.o. and stress. The drug treatment was carried out daily for a period of 21 days. At the end of each week, i.e., 1st, 2nd and 3rd weeks of drug treatment, the animals were exposed to the anoxia stress and anoxia tolerance time was noted. Hermetic vessel of one litre air capacity was used to induce anoxia stress (Krupavaram et al., 2007). Each animal was kept in the hermetic vessel and the time to show the first sign of convulsion was noted, and were immediately removed from the vessel and resuscitated if needed.
2.4. Forced swimming endurance test (physical stress) Rats of either sex (200–250 g) were used for forced swim endurance stress. Group I rats received 0.1% gum acacia in saline; (vehicle control). Group II mice were treated with 0.1% gum acacia in saline and stress; (negative control). Group III rats were treated with W. somnifera (100 mg/kg, p.o.) and stress; (positive control). Group IV, V and VI mice were treated with ethanolic extract at 50, 100 and 200 mg/kg, p.o. and stress. The rats were subjected to swimming stress by keeping them in propylene tank of dimension (37 37 30 cm), filled with water to a height of 25 cm. Extracts were given to rats, once daily for period of 7 days. On 8th day the rats were allowed to swim till complete exhaustion and the endpoint was taken when the animal started drowning. The mean swimming time for each group was calculated (Kannur et al., 2006). Then animals were killed and blood was collected by cardiac puncture to estimate biochemical parameters like serum
In the anoxia tolerance test (Table 1), the extract at 50,100 and 200 mg kg 1 doses statistically produced a dose dependant significant (P < 0.05) increase in mean time to convulsion in mice subjected to anoxia stress. 3.2. Effect of ethanolic extract in forced swimming endurance stress The results of the study revealed that the extract possess antistress property as it significantly (P < 0.05) increased the swimming time (Fig. 1). Swimming endurance stress resulted in significant increase in adrenal gland weight and liver weight with concomitant decrease in spleen weight in stress control group, which was significantly reverted by Z. officinale pretreatment at 50, 100 and 200 mg/kg. Similarly stress-induced elevated blood cell counts of RBC and DLC, i.e., lymphocytes, neutrophils, eosinophils and monocytes have been significantly (P < 0.01) reduced by the ethanolic extract in a dose dependant manner (Table 2). Pretreatment of animals with Z. officinale at three doses also significantly (P < 0.05) restored back forced swimming stress-induced alterations in plasma corticosterone, glucose, triglyceride, BUN and cholesterol (Table 3). 3.3. Effect of ethanolic extract in cold restraint stress In cold restraint stress, ethanolic extract at 50, 100 and 200 mg/ kg offered significant (P < 0.05) protection against the change in the weights of liver, spleen and adrenal gland when compared to stress control (Figs. 2 and 3). The extract dose dependently reduced the elevated levels of biochemical parameters (P < 0.05) (Table 4). The extract at 200 mg/kg significantly (P < 0.01) reduced all the blood cell counts (Table 5). 4. Discussion Adaptogens are the substances meant to put the organisms into a state of non-specific heightened resistance in order to better resist stressor and adapt to extraordinary challengers. They normalize body functions, strengthen systems and functions that are compromised by stress and have a protective effect against a wide variety of environmental and emotional stress. The forced swimming is the most widely used method for assessing the anti-stress property of a novel compound (Anisman and Zacharko, 1991; Subarnas et al., 1993). This paradigm is based
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Table 1 Effect of ethanolic extract of Zingiber officinale in anoxia stress tolerance in mice. Groups
Mean duration of tolerance time (min)
Control Withania somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
1st week
2nd week
3rd week
120.59 ± 0.34 162.25 ± 0.81* 131.29 ± 0.75 149.71 ± 0.61* 161.11 ± 0.73*
122.71 ± 0.58 178.91 ± 0.68* 133.16 ± 0.85* 155.08 ± 1.43* 167.95 ± 0.79*
126.57 ± 0.95 184.92 ± 1.15* 139.41 ± 1.92* 159.27 ± 0.68* 176.59 ± 1.25*
The mice were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 21 days. Control mice were given saline. Anoxia was induced at the end of 1st, 2nd and 3rd weeks and the anoxia stress tolerance time was noted. The values are expressed as mean ± SEM, n = 6. * Significance at P < 0.05 when compared to control as determined by ANOVA followed by Dunnett’s t test.
227.95**
250 Time in mins
200
183.61
228** 198.82**
186.79 164.15*
150 100 50 0 Control
Stress control
Withania somnifera
ZO extract1
ZO extract2
ZO extract3
Groups Fig. 1. Effect of ethanolic extract of Zingiber officinale in forced swimming endurance test in rats (swimming time). The forced swimming endurance time was measured in rats. The mice were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 7 days. Control mice were given gum acacia. Three hours after the final treatment, the rats were exposed to forced swimming endurance test. The mean increase in duration of swimming time was noted. The values are expressed as mean ± SEM, n = 6 in each group. *P < 0.05 significant as compared to control, **P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnet’s t test.
on the observation that animals forced to swim in water eventually assumed a characteristic immobile posture, devoid of any activity (Weiss et al., 1981). The appearance of immobility therefore, reflects a state of tiredness, fatigue, reduced stamina with the end point being the moment when the rat could not swim further and started drowning (Bargava and Singh, 1981). However, increased swimming time has been observed in rat pre-treated with ethanolic extract and has enhanced the physical performance long-
er than untreated (control) group confirming their adaptogenic nature. In present study, in acute and chronic stress models, the significant increase in blood glucose level was observed because; under stressful conditions adrenal cortex secretes cortisol in man and corticosterone in rats. Hyper secretion of cortisol helps in maintenance of internal homeostasis through the process of gluconeogenesis and lipogenesis (Krupavaram et al., 2007). Pretreatment with
Table 2 Effect of ethanolic extract of Zingiber officinale on organ weights and blood cell counts in forced swimming endurance stress in rats. Treatment
Organs weight Spleen (mg/ 100 g)
Control Stress control W. somnifera 100 mg/kg p.o. ZO extract 50 mg/ kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
Blood cell counts Liver (g/ 100 g)
Adrenal glands (mg/ 100 g)
RBC in millions
WBC (no. of cells/mm3)
DLC no. of cells/Cumm
L
N
E
M
250.01 ± 2.25 196.24 ± 1.58 263.21** ± 1.58
3.71 ± 1.58 5.43 ± 1.23 3.85** ± 2.54
8.76 ± 0.57 15.93 ± 1.46 9.87** ± 0.85
8.02 ± 0.44 13.6* ± 2.52 8.27** ± 1.25
6439 ± 23.98 9650* ± 21.23 6200** ± 24.23
4537 ± 14.89 6439* ± 14.56 4236** ± 18.85
1836 ± 21.24 3121* ± 15.65 1889** ± 14.89
63.24 ± 2.36 79.96* ± 1.98 66.42** ± 4.56
9.21 ± 0.54 10.82* ± 0.63 9.34** ± 0.87
204.24 ± 2.54
4.95 ± 1.69
11.96 ± 1.65
11.54* ± 2.25
7650* ± 26.25
5923* ± 17.89
1646* ± 16.74
71.26* ± 4.75
10.62* ± 1.48
219.95** ± 2.65
4.29* ± 2.58
10.84** ± 1.58
9.59* ± 1.23
7100** ± 14.25
5426** ± 19.48
1595** ± 17.36
69.84** ± 6.02
9.87* ± 1.52
234.61** ± 1.97
4.05** ± 3.35
10.21** ± 1.35
8.76** ± 1.79
6700** ± 18.64
4727** ± 28.54
1897** ± 18.58
66.91** ± 5.23
9.56* ± 0.89
The rats were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 8 days. Control rats were given gum acacia. Swimming stress was induced at the end of treatment period. The animals were sacrificed, the organs were isolated from the animals and the weights were noted. Blood was collected for the estimation of cell counts. The results are expressed as mean ± SEM, n = 6 in each group. * P < 0.01 significant as compared to control. ** P < 0.01, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
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B.V.S. Lakshmi, M. Sudhakar / Food and Chemical Toxicology 48 (2010) 530–535 Table 3 Effect of ethanolic extract of Zingiber officinale on biochemical parameters in swimming endurance stress in rats. Groups
Corticosterone (lg/dl)
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
BUN (mg/dl)
Control swimming Stress control W. somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
98.60 ± 2.56 165.73 ± 2.95 101.62 ± 2.57** 136.39 ± 1.85* 119.48 ± 3.87** 105.64 ± 3.35**
91.98 ± 1.58 198.23 ± 1.96 99.78 ± 0.42** 134.23 ± 2.98* 126.59 ± 3.02** 92.36 ± 1.85**
89.41 ± 3.63 184.62 ± 1.89 84.62 ± 3.59* 146.15 ± 4.05* 92.30 ± 3.82** 88.46 ± 1.56**
63.22 ± 2.85 95.87 ± 5.02 66.86 ± 0.89* 89.42 ± 2.64* 72.56 ± 3.98* 67.89 ± 2.63**
28.18 ± 2.69 58.63 ± 1.24 36.42 ± 0.78* 48.37 ± 2.58* 41.92 ± 3.58* 38.29 ± 3.25*
The rats were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 7 days. Control rats were given gum acacia. Swimming stress was induced at the end of treatment period. The animals were sacrificed; the blood collected and the biochemical parameters were estimated. The values are expressed as mean ± SEM, n = 6 in each group. * P < 0.05 significant as compared to control. ** P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
**
350
*
300
**
* Weight in mg
250
*
Control Cold Stress control
200
Withania somnifera ZO extract1
150
ZO extract2 ZO extract3
100 * 50
* **
*
**
0 Spleen
Adrenal glands Groups
Fig. 2. Effect of ethanolic extract of Zingiber officinale on organ weights in cold restraint stress in rats. The rats were pretreated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed, the organs were isolated from the animals and the weights were noted. The values are expressed as mean ± SEM, n = 6 in each group. *P < 0.05 significant as compared to control, **P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnet’s t test.
Liver weight in gms
7
*
*
6
*
**
**
5 4 3 2 1 ZO extract3
ZO extract2
ZO extract1
Withania somnifera
Cold Stress control
Control
0
Fig. 3. Effect of ethanolic extract of Zingiber officinale on organ weights in cold restraint stress in rats. The rats were pretreated with Withania somnifera (100 mg/ kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed, the organs were isolated from the animals and the weights were noted. The values are expressed as mean ± SEM, n = 6 in each group. *P < 0.05 significant as compared to control, **P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnet’s t test.
the Z. officinale as well as reference standard drug W. somnifera significantly (P < 0.05) reduced the elevated glucose levels indicating their suppressant effect on hyper activity of adrenal cortex and maintained the homeostatic mechanism.
The marked increase in serum cholesterol, triglycerides and BUN levels in stress-induced animals is due to stimulation of hypothalamo–pituitary axis (HPA) and sympathetic system, resulting in, liberation of catecholamines and glucocorticosteroids, which inhibits the immune system at multiple sites like liver, kidney (Schimmer and Parker, 2006). Z. officinale as well as reference standard drug W. somnifera significantly (P < 0.05) reduced the elevated serum cholesterol, triglycerides and BUN levels, which may be due to inhibition of stimulation of sympathetic nervous system. The increase in weight of adrenals in stressed animals is due to the stress-induced adrenomedullary response leading to increased production of corticotropic hormone that leads to increase in weight of adrenals (Krupavaram et al., 2007). Z. officinale and W. somnifera has significantly (P < 0.01) reduced the liver, adrenal gland weight, this may be due to the reversal of the stress-induced adrenomedullary response and hence decreased production of corticotropic hormone. The decrease in weight of spleen may be due to recruitment of lymphocytes to blood from spleen which results in squeezing of the spleen (Rai et al., 2003). The pretreatment with the Z. officinale and reference standard W. somnifera significantly (P < 0.01) increased the spleen weight. This may be due to inhibition of recruitment of lymphocytes to blood from spleen. During stress, heart rate, blood pressure and blood flow rate increases. To meet these extra demands RBC and WBC counts will be increased. In the present study the extract has decreased the elevated levels of RBC and WBC in both swimming endurance and cold restraint stress models.
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Table 4 Effect of ethanolic extract of Zingiber officinale on biochemical parameters in cold restraint stress. Groups
Corticosterone (lg/dl)
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
BUN (mg/dl)
Control Cold stress control W. somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
98.60 ± 2.56 162.71 ± 1.95* 102.83 ± 2.95** 121.63 ± 0.98 109.58 ± 1.87* 101.56 ± 2.35**
91.42 ± 2.35 152.25 ± 2.16* 96.49 ± 1.52** 122.28 ± 1.48* 109.54 ± 3.32* 99.87 ± 2.84**
89.41 ± 3.63 163.33 ± 2.14* 92.86 ± 4.02** 132.38 ± 2.32* 93.33 ± 4.52* 82.38 ± 1.89**
63.22 ± 2.85 106.80 ± 3.65* 67.36 ± 1.32** 94.89 ± 3.58* 78.30 ± 4.05* 68.04 ± 2.25**
28.18 ± 2.69 49.25 ± 2.83* 30.01 ± 1.54** 40.38 ± 2.04* 36.13 ± 1.25* 31.56 ± 3.01**
The rats were treated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed; the blood collected and the biochemical parameters were estimated. The values are expressed as mean ± SEM, n = 6 in each group. * P < 0.05 significant as compared to control. ** P < 0.05, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
Table 5 Effect of ethanolic extract of Zingiber officinale on Blood cell counts in Cold Restraint stress in rats. Treatment
Blood cell counts RBC in millions
Control Cold stress control Withania somnifera 100 mg/kg p.o. ZO extract 50 mg/kg p.o. ZO extract 100 mg/kg p.o. ZO extract 200 mg/kg p.o.
8.02 ± 0.44 10.11* ± 0.52 8.29** ± 0.48 9.87 ± 0.65 9.03* ± 1.23 8.89** ± 0.79*
WBC (no. of cells/mm3)
6439 ± 23.98 9854* ± 18.56 6832** ± 21.96 8100** ± 19.12 7833** ± 22.87 6539** ± 15.24
DLC no. of cells/Cumm L
N
E
M
4537 ± 19.89 7436* ± 15.89 4963** ± 14.56 5325* ± 14.23 5104** ± 9.84 4624** ± 58.25
1836 ± 15.35 2327* ± 24.21 1794** ± 23.11 2689** ± 14.24 2649* ± 18.58 1839** ± 25.23
63.24 ± 2.25 79.86* ± 2.36 65.82** ± 3.35 74.85* ± 5.23 69.41** ± 5.36 66.32** ± 4.65
9.21 ± 0.23 11.34* ± 0.58 9.43** ± 0.48 11.02 ± 1.02 10.56* ± 0.57 9.84** ± 0.47
The rats were treated with Withania somnifera (100 mg/kg), ZO (50, 100, and 200 mg/kg, p.o.) once daily for 10 days. Control rats were given gum acacia. Cold restraint stress was induced by exposing them to 4 ± 1 °C, daily for 2 h for a period of 10 days. The animals were sacrificed; the blood was collected for the estimation of cell counts. The values are expressed as mean ± SEM, n = 6 in each group. * P < 0.01 significant as compared to control. ** P < 0.01, significant as compared to stress control, statistical test employed is ANOVA followed by Dunnett’s t test.
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