Journal of Ethnopharmacology 108 (2006) 327331

Adaptogenic activity of Caesalpinia bonduc seed extracts in rats

D.M. Kannur a, , V.I. Hukkeri b , K.S. Akki b
a

Department of Pharmacognosy & Phytochemistry, MAEERs Maharashtra Institute of Pharmacy, Kothrud, Pune 411038, India b Department of Pharmacognosy & Phytochemistry, K.L.E.Ss College of Pharmacy, Vidyanagar, Hubli 580031, India Received 26 July 2005; received in revised form 13 March 2006; accepted 12 May 2006 Available online 26 May 2006

Abstract Caesalpinia bonduc Roxb. (Caesalpiniaceae) seed extracts were screened for adaptogenic activity using cold stress model and swim endurance model, the seed coat as well as kernel extracts showed signicant antistress activity when administered orally at a dose of 300 mg/kg. The extracts signicantly increased the swim endurance time. Stress induced animals exhibited hypoglycaemia as well as depletion in serum cortisol level and increased total leukocyte count, the extracts showed a signicant action in overcoming these imbalances. It was also found that extracts proved efcient in controlling the hyperlipidaemic condition due to stress. 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adaptogenic; Swimming endurance; Blood glucose level; Caesalpinia bonduc

1. Introduction Caesalpinia bonduc Roxb. (Caesalpiniaceae) is a large scandant prickly shrub found throughout the interior parts of India, Sri Lanka and West Indies. It is common in southern parts of India and is often grown as a hedge plant (Sutaria, 1969). This plant has profound medicinal use and is a proved antiinammatory (Jethmalani et al., 1966), anthelminitic and antimalarial (Jain et al., 1992) drug. It has also been an effective stomachic, digestive and is used as liver tonic in the treatment of jaundice and various liver disorders. It is attributed to be an aphrodisiac and general tonic helping in the rejuvenation of body (Shrikantha Murthy, 2000). The roasted seed powder is used as an anti-leprotic. The seeds are useful as antidiabetic, antiperiodic, antipyretic, etc. The seeds are found to contain various chemical constituents (Williamson, 2002) such as furanoditerpenes caesalpin, caesalpin, caesalpin, caesalpin, caesalpin, and caesalpin-F. Fatty acidspalmitic, stearic, octadeca-4-enoic and octadeco-24-dienoic, lignocenic, oleic and linoleic acids, phytosterinin, sitosterol. Homoisoavone, bonducellin, amino acidsaspartic acid, arginine, and citrulline. Carbohydrates starch, sucrose, carotene, glycoside-bonducin, gums and resins.

Stress, according to late Dr. Hans Seyle (Seyle, 1973), an early guru on stress, is the non-specic response of the body to any demand made upon it. Stress is the bodys physical, mental or chemical reaction when we get excited, confused or we otherwise feel unsafe or threatened. Stress is the outcome of people reacting with one another. If daily demands are easy and well balanced an individual is ne. It is when one decides the pressure is unreasonable or the situation is upsetting, that the potential for damage occurs. That is when one feels stressed. Stress reactions begin in our minds. 1.1. Adaptogens Adaptogens (Bhattacharya et al., 2000) are the plant derived biologically active substances, which appear to induce a state of non-specic increase of resistance of the organism to diverse aversive assaults which threaten internal homeostasis and which improve physical endurance for doing work even in adverse circumstances and in difcult environmental conditions. These agents are basically preventive rather than curative in action and appear to function best when the resistance of the body is diminished, as seen in the case of prolonged illness, chronic stress and old age. They increase tolerance to change in environmental conditions and resistance to noxious stimuli such as exposure to cold, heat, pain, general stress and infectious organisms. Such agents have been claimed to arrest ageing process and age induced deterioration in physical and mental performance.

Corresponding author. Tel.: +91 9860143343. E-mail address: dmkannur@rediffmail.com (D.M. Kannur).

0378-8741/$ see front matter 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.05.013

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2. Materials and methods 2.1. Caesalpinia bonduc (Caesalpiniaceae) The seeds of Caesalpinia bonduc were collected from the local areas of Kannur (Bijapur), Karnataka, India and were submitted and the Herbarium (Specimen Voucher No. DMK281002) was preserved and authenticated by Dr. P.S.N. Rao, Deputy Director, Botanical Survey of India, Pune, India. 2.2. Preparation of the extracts The seed coat was broken and the kernels and the seed coat were separated and size reduced to a coarse powder. The seed coat was extracted with 95% ethanol. The ethanol was recovered on rotary ash evaporator. The seed coat extract was further concentrated and dried over anhydrous sodium sulphate under vacuum to get brownish black sticky residue (12%) (seed coat extract, SE). The kernel powder was then loaded in a soxhlet extractor in a batch of 200 g and was defatted with petroleum ether (6080). The pet ether was distilled off on a rotary ash evaporator and was air-dried further to get yellow oil (22%, w/w) (pet ether extract, PE). The marc was dried and further extracted with 95% ethanol. The ethanolic extract was concentrated on rotary ash evaporator and vacuum dried over anhydrous sodium sulphate. The residue was reddish brown sticky mass (16%, w/w) (ethanolic extract, EE). 2.3. Animals Healthy adult Swiss albino mice of either sex weighing between 25 and 30 mg were used for acute toxicity studies and swim endurance test. Healthy Wistar albino rats of either sex were used for the evaluation of adaptogenic activity in cold stress model. The animals were fed ad libitum with commercial pellet diet (Lipton India Ltd., Mumbai) and had free access to water. 2.4. Extracts and standard drug The petroleum ether extract (PE), ethanolic extract (EE) of the seed kernels and the ethanolic extract of seed coat (SE), were used in all the models. The extracts were formulated into an emulsion using gum acacia (2%). Geriforte tablets (Himalaya drugs) 43 mg/kg was used as a standard adaptogenic drug which is a multi-constituent ayurvedic drug with 35 herbal and natural constituents like Withania somnifera, Asparagus racemosus, Glycyrrhiza glabra, Centella asiatic, Terminalia chebula, Piper longum, Shilajit, etc. 2.5. Acute toxicity studies Acute toxicity studies were carried out using up and down Staircase method (Gosh, 1984). Healthy adult Swiss albino mice of either sex weighing between 25 and 30 mg were used for the study.

2.6. Swim endurance test Greater swimming endurance has been reported in mice when pretreated with antistress agents (Singh et al., 1979) and the test has been utilized to investigate the adaptogenic activity of different agents, based on the fact that swim endurance reects physical endurance (Bhargava and Singh, 1981). Swiss albino mice (1520 g) of either sex were used for swim endurance. The normal animals were treated with 2% acacia and other three groups were treated with EE, PE and SE extracts (300 mg/kg b.w.), respectively; after drug administration the animals were forced to swim in glass chambers 30 cm 30 cm 15 cm containing water at room temperature. The mice were allowed to swim till they got exhausted and the moment they drowned was considered as the endpoint (Nimbakar et al., 2001). The time was noted. The data obtained were subjected to statistical analysis. 2.7. Cold stress model In the present study induction of stress was done using the cold stress model. The animals were divided in to six groups of six animals each of either sex. The stress was induced by exposing animals (ve groups) to 4 C for 4 h. The animals were taken from their home cages and individually placed in polymer containers with partition to separate individual animal. The containers were placed inside refrigerator such that the temperature to which the animals were exposed was 4 C, they were returned to their home cages after 4 h. This procedure was repeated for 10 days at a specic time period between 10:00 a.m. to 2:00 p.m. Group 1 animals served as normal control were administered vehicle and 2% acacia and were not exposed to stress, Group 2 animals served as negative control as untreated stress induced, Group 3 animals were administered standard (Geriforte) (43 mg/kg), Group 4 animals were administered ethanolic extract (EE), Group 5 animals were administered pet ether extract (PE), Group 6 animals were administered seed coat extract (SE). All the extracts were administered orally at a dose of 300 mg/kg in 2% acacia. The animals were treated and were subjected to stress for 10 days and were free to food and water, on the 11th day animals were sacriced by decapitation, blood was collected from the arterial jugular and serum was separated. The serum was used for the estimation of various biochemical parameters (Lalitha et al., 1988; Nimbakar et al., 2001). 2.8. Statistical analysis Comparison between control and drug treated groups were made by Students t-test, P values of less than 0.05 were considered to be signicant. 3. Results 3.1. Acute toxicity test The acute toxicity study was carried out using Swiss albino mice of either sex weighing between 25 and 30 g. All the three

D.M. Kannur et al. / Journal of Ethnopharmacology 108 (2006) 327331 Table 1 Effect of caesalpinia bonduc seed extracts on swimming endurance in minutes Animals Normal Stress + standard Stress + EE Stress + PE Stress + SE Mean 225.0 22.5 306.3 23.0** 315.0 13.2** 311.2 17.4** 282.5 14.5**

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Seed coat extract (SE) and the standard drug were more effective in preventing the alterations in the blood glucose levels due to stress. Comparatively the ethanolic extract (EE) and the pet ether extract (PE) showed a less preventive action against stress induced hypoglycemia as compared to the standard drug and the seed coat extract (Table 2). 3.4. Effect of Caesalpinia bonduc seed extracts on serum cortisol level in rats exposed to cold stress Estimation of serum cortisol levels was done using standard VIDAS diagnostic kit (bio Merieux). The assay principal combines an enzyme immunoassay sandwich method with a nal uorescent detection (ELFA). Stress induced animals showed a lot of variation as compared to the normal animals. It was observed that the serum cortisol levels were depleted in the stress induced group. All the three extracts showed a remarkable preventive action against the alterations in the serum cortisol levels due to stress (Table 2). The seed coat extract (SE), pet ether extract (PE) and the standard drug were more effective as compared to the ethanolic extract EE in keeping the cortisol levels near to normal. 3.5. Effect of Caesalpinia bonduc seed extracts on total leukocyte count and differential leukocyte count (TLC and DC) level in rats exposed to cold stress Total leukocyte count was determined by using standard Neubauers chamber (an area of 9 mm2 and a depth of 0.1 mm) and WBC diluting uid method and differential count was determined by using standard Leishmans staining method. In the stress induced animals total leukocyte count was signicantly increased in comparison to that of normal animals (Table 2). In differential count the percentage of neutrophils was increased signicantly, where as the eosinophils were slightly increased, the lymphocytes were decreased and the monocytes were not affected much (Table 3). All the three extracts exhibited preventive action against stress induced alterations in the TLC as well as DC. The ethanolic extract (EE) was more effective as compared to the standard, pet ether extract (PE) and seed coat extract (SE) in controlling the increased level of total leukocyte count. In the differential count the seed coat extract (SE) was more effective as compared

Values are mean S.E.M. ** P < 0.05 when compared with stress control. PE, the petroleum ether extract of the seed kernels; EE, ethanolic extract of the seed kernels; SE, ethanolic extract of seed coat.

extracts: the pet ether extract (PE), ethanolic extract (EE) and seed coat extract (SE) were administered orally. All the extracts showed 3000 mg/kg as the maximum non-lethal dose. Hence 1/10th of the dose, i.e. 300 mg/kg was considered as the effective dose for the screening of the activity for all the extracts 3.2. Effect of Caesalpinia bonduc seed extracts on swim endurance The mice were forced to swim in water at room temperature till exhaustion and death. It was observed that all the three extracts increased the physical endurance as compared with untreated animals. The swimming time was signicantly increased in all the treated groups as compared to the untreated animals. The ethanolic extract (EE) and the pet ether extract (PE) effects were at par with that of the standard drug. The seed coat extract (SE) too possessed capability of increasing the physical endurance (Table 1). 3.3. Effect of Caesalpinia bonduc seed extracts on blood glucose level in rats exposed to cold stress The extracts of Caesalpinia bonduc were screened for the adaptogenic activity using the cold stress model. The blood glucose level was determined using the (GODPOD method) glucose oxidaseperoxidaseaminoantipyrine and phenol method where the quinonemimine dye formed is estimated spectrophotometerically at 500 nm (ERBA TESTTransasia Biomedicals, Diagnostic Kit). In the stress induced animals the blood glucose level was found to be below the baseline indicating hypoglycemia. All the three extracts showed a moderately signicant activity in preventing the alterations in the blood glucose levels as compared to the untreated animals.

Table 2 Effect of caesalpinia bonduc seed extracts on blood glucose level and serum cortisol in cold stress (mg%) Animals BGL (mg%) SC (ng/dl) TLC Normal 82.7 2.14 5.78 1.28 4925 561.8 Stress induced 60.0 3.39 3.26 0.93 7500 404.1 Stress + standard 77.7 3.43*** 5.29 2.24*** 5725 232.3** Stress + EE 69.5 3.12** 4.86 1.48** 4775 517.0*** Stress + PE 72.5 1.71** 5.41 1.16*** 6075 319.8** Stress + SE 83.0 4.02*** 6.92 0.89*** 6300 208.2**

Values are mean S.E.M. *** P < 0.001 when compared with stress control, ** P < 0.05 when compared with stress control. PE, the petroleum ether extract of the seed kernels; EE, ethanolic extract of the seed kernels; SE, ethanolic extract of seed coat; SC, serum cortisol; TLC, total leukocyte count; NS, not signicant.

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Table 3 Effect of Caesalpinia bonduc seed extracts on differential count in cold stress Normal Neutrtophils Monocytes Lymphocytes Eosinophils 22.5 6.1 70.1 2.0 0.4 0.1 0.5 0.1 Stress induced 31.6 6.3 67.2 3.2 0.6 .0.04 1.1 0.2 Stress + standard 25.3 6.2 71.3 2.3 0.1 NS 1.5** 0.1** 0.9*** Stress + EE 25.6 6.3 69.2 2.4 0.1 NS 0.8** 0.1** 0.9*** Stress + PE 24.8 6.2 68.2 1.8 0.1 NS 0.9 NS 0.1*** 0.8*** Stress + SC 25.9 6.1 69.7 2.1 1.2** 0.2 NS 0.5** 0.1***

Values are mean S.E.M. *** P < 0.001 when compared with stress control, ** P < 0.05 when compared with stress control. PE, the petroleum ether extract of the seed kernels; EE, ethanolic extract of the seed kernels; SE, ethanolic extract of seed coat; NS, not signicant. Table 4 Effect of Caesalpinia bonduc seed extracts on lipid prole in cold stress Normal TC (mg%) TTG (mg%) HDL (mg%) LDL (mg%) VLDL (mg%) 73.0 57.0 43.0 18.6 11.4 8.43 5.84 2.25 2.66 2.18 Strees induced 92.0 43.0 50.0 33.4 8.6 2.12 6.25 5.56 2.66 6.32 Stress + standard 74.0 56.0 42.0 20.8 11.2 2.77*** 1.14*** 4.36*** 4.98*** 4.17*** Stress + EE 83.0 50.0 46.0 27.0 10.2 2.61*** 4.44** 7.32** 10.55** 6.91** Stress + PE 85.0 47.0 48.0 27.6 9.4 4.26** 8.36** 5.69** 1.87** 7.28 NS Stress + SC 87.0 52.0 45.0 32.8 9.2 3.67** 5.46*** 1.57** 12.66 NS 6.37 NS

Values are mean S.E.M. *** P < 0.001 when compared with stress control, ** P < 0.05 when compared with stress control. PE, the petroleum ether extract of the seed kernels; EE, ethanolic extract of the seed kernels; SE, ethanolic extract of seed coat; TC, total cholesterol; TTG, total triglycerides; HDL, high density lipoproteins; LDL, low density lipoproteins; VLDL, very low density lipoproteins; NS, not signicant.

to the standard and other two extracts in maintaining the DC level near to the normal. 3.6. Effect of Caesalpinia bonduc seed extracts on lipid prole level in rats exposed to cold stress The cholesterol was estimated using (Modied RoeschlausCHOD-PAP method) cholesterol esterase aminoantipyrinephenol method where the quinonemimine dye formed and was estimated spectrophotometerically at 510 nm (ERBA TESTTransasia Biomedicals, India, Diagnostic Kit). HDL cholesterol was determined using phosphotungstic acid method. The triglycerides were determined (GPO-PAP method with lipid clearing factor) after enzymatic hydrolysis with lipases and formation of quinonemimine, which was proportional to the triglycerides concentration in the sample when measured at 510 nm spectrophotometerically (ERBA TESTTransasia Biomedicals, India, Diagnostic Kit). In the stress induced group the total cholesterol and the LDL levels were increased signicantly and the triglyceride levels were depleted to some extent, the % of HDL of total cholesterol was found to be decreased as compared to the normal. There was no signicant action on the VLDL levels. Stress induced animals thus had a slight hyperlipidaemic condition as compared to the normal animals (Table 4). The standard, ethanolic extract (EE), pet ether extract (PE) and the seed coat extract (SE) showed an overall preventive effect in varying lipid levels due to stress. The standard was comparatively more efcient in controlling any alterations than different extracts. The ethanolic extract (EE) was signicantly effective in controlling the rise in total cholesterol level and the LDL level as compared to the other two groups, the % increase in the HDL levels to the total cholesterol was highest in the ethanolic extract treated group.

4. Discussion and conclusions The studies conducted so far indicate that all the three extracts ethanolic extract (EE), pet ether extract (PE) and the seed coat extract (SE) had a protective action on the animals against the alterations inicted due to cold stress, such as changes in the serum cortisol and blood glucose levels, total leukocyte count, differential count as well as the lipid balance. The swim endurance test results indicate clearly that the extracts have the properties whereby they increase the physical endurance as well as the overall performance in rats. The enhanced swimming endurance in mice as compared to the normal animals may be attributed to the steroids and the triterpenoids (Lyder et al., 1998), which are found in all the extracts. Exposure of the animals to the cold caused severe imbalance in the normal physiological conditions and might have resulted in a slight pathological condition, as indicated by the increased total leukocyte count. The extracts have shown a remarkable preventive action against these alterations. The stress induced hypoglycaemia and depleted levels of serum cortisol are unknown conditions and are being reported for the rst time, and the mechanism is non-conclusive, but the extracts have shown promising results in controlling the hypoglycaemia as well as restoring the depleted cortisol levels, thus indicating the ability to prevent the alterations in the HPA system due to stress and helping in maintaining the homeostasis and the normal functioning of the body. Stress has proven to be a triggering factor for hyperlipidaemia (Mungatiwar et al., 1997) as such in our studies again it was proved that cold stress leads to hyperlipidaemic conditions, both the ethanolic and pet ether extracts have shown signicant antihyperlipdaemic action which can be attributed to the presence of phytosterols and that to specically -sitosterol, which has

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shown to be responsible for maintenance of lipid balance (Cicero et al., 2002). Caesalpinia bonduc seed extracts at a dose of 300 mg/kg have shown signicantly that they possess the ability to prevent the alterations due to stress. The above results are promising and further dose variant studies would be helpful in substantiating the claim of adaptogenic action of this drug. Thus with all the above results it can be claimed and concluded that the seeds of Caesalpinia bonduc have adaptogenic properties. Acknowledgements The authors are grateful to Dr. P.S.N. Rao, Deputy Director, Botanical Survey of India, Pune, India, for authenticating the plant material. We would like to thank Dr. Ramesh Godbole, Director, Godbole Laboratories, Pune for his help in carrying out the biochemical investigations. References
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