|Year : 2013 | Volume
| Issue : 2 | Page : 102-107
A study on the hematological parameters and brain acetylcholine esterase activity in immobilization induced stress and co-treatment with Centella asiatica leaves extract to wistar rats
Arumugam Sarumathi1, Nadanam Saravanan2
1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India
2 Division of Biochemistry, Rani Meyyammai College of Nursing, Faculty of Medicine, Annamalai University, Annamalai Nagar, Tamil Nadu, India
|Date of Submission||05-Jun-2012|
|Date of Acceptance||26-Jun-2012|
|Date of Web Publication||3-Jun-2013|
Division of Biochemistry, Rani Meyyammai College of Nursing, Faculty of Medicine, Annamalai University, Annamalai nagar - 608 002, Tamilnadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Our aim was to evaluate the anti-stress activities of aqueous leaf extract of Centella asiatica (CA) in immobilization-induced stress in rats. The anti-stress activities of CA were assessed by monitoring the changes in the behavior, counts of red blood cells (RBCs) and white blood cells (WBCs) (Lymphocytes, Monocytes, Neutrophils, Esinophils) and change in the level of brain acetylcholine esterase (AChEs) activity in immobilization-induced stress in male albino Wistar rats. Materials and Methods: Immobilization stress was induced in rats by placing in 20 cm × 7 cm plastic tubes for 2 h/day for 21 days. Aqueous leaf extract of CA was co-administered orally to the experimental rats at a dose of 200 mg/kg body weight daily for the 21 days. Results: Stress induced by immobilization in the rats for 21 days decreased the passive avoidance behavior, total counts of RBCs, WBCs and brain AChEs activity. Upon co-administration of CA leaves extract to the stressed animals significantly reverted the changes to near normal levels when compared to control animals. Conclusion: Aqueous extract of CA leaves significantly reverted the changes due to immobilization induced stress which may be due to the anti-stress activities of CA leaves.
Keywords: Acetyl choline esterase, Centella asiatica, immobilization stress, red blood cells, and white blood cells
|How to cite this article:|
Sarumathi A, Saravanan N. A study on the hematological parameters and brain acetylcholine esterase activity in immobilization induced stress and co-treatment with Centella asiatica leaves extract to wistar rats. Int J Nutr Pharmacol Neurol Dis 2013;3:102-7
|How to cite this URL:|
Sarumathi A, Saravanan N. A study on the hematological parameters and brain acetylcholine esterase activity in immobilization induced stress and co-treatment with Centella asiatica leaves extract to wistar rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2020 Jan 27];3:102-7. Available from: http://www.ijnpnd.com/text.asp?2013/3/2/102/112829
| Introduction|| |
Stress is a progressive degenerative neurologic disorder resulting in impaired memory and behavior. Epidemiological data indicates a potentially considerable increase in the prevalence of the disease over the next two decades.  Stress has been proven to rid in body's essential vitamins C, E and B. This leads to lethargy, lower focused mental activities. The onset of a stress response is associated with specific physiological actions in the sympathetic nervous system, both directly and indirectly through the release of epinephrine and to a lesser extent norepinephrine from the medulla of the adrenal glands.  The release is triggered by acetylcholine released from pre-ganglionic sympathetic nerves. These catecholamine hormones facilitate immediate physical reactions by triggering increases in heart rate and breathing, constricting blood vessels in many parts of the body - but not in muscles (vasodilation), brain, lungs, heart and tightening muscles. An abundance of catecholamines at neuroreceptor sites facilitates reliance on spontaneous or intuitive behaviors often related to combat or escape.  Clinically stresses cause more erratic brain activity and a reduction in short term and long term memory. The loss of memory is considered to be the result of a shortage of the nerve transmitter acetylcholine. It is possible to increase the level of this transmitter in the brain by inhibiting the activity of the enzyme acetylcholine esterase, which splits or breaks down the transmitter substance. Most treatment strategies have been based on the cholinergic hypothesis which postulated that memory impairments in patients suffering from this disease result from a deficit of cholinergic function in brain. One of the most promising approaches for treating this disease is to enhance the acetylcholine level in the brain using acetylcholinesterase (AChE) inhibitors. 
The blood is one of the major homeostatic systems of the body in humans and animals, supporting normal viability, integrity and adaptive responses. The functional state of the blood systems changes dynamically according to the nature, strength and duration of exposure to external factors. Human activity is now increasingly associated with the actions of factors creating emotional tension. Many studies performed on humans and animals have provided evidence for significant shifts in physiological and biochemical processes during the modeling of emotional tension. The nervous and blood systems are the quickest to react to emotional stimuli. This is probably among the explanations for the continuing increase in the numbers of patients with myocardial infarctions and vascular thromboses of the brain, limbs, lungs and other organs. The treatment of these and other diseases involves repeated injections of therapeutic agents.
Nature is a rich source of biological and chemical diversity. The unique and complex structures of natural products cannot be obtained easily by chemical synthesis. A number of plants in India have been used in traditional medicine remedies. For the study of drugs related to stress, we were interested in the traditional remedies known as "rejuvenating" and "neurotonic" agents which have been popular in elderly.  It is believed that these remedies can prevent forgetfulness and improve memory. The plants used in these remedies were screened for their AChE activity using Ellman's colorimetric method. , Centella asiatica (L) (CA) urban, synonym Hydrocotyle asiatica, belongs to the family (Umbelliferae) is found almost all over the world. In Ayurveda, an Indian system of medicine, this is used in the management of central nervous system, skin and gastrointestinal disorder. The extract from leaves of CA has been used as an alternative medicine for memory improvement in Indian Ayurvedic System of medicine for a long time.  The biological influences of CA include anti-inflammatory, anti-cancer  anti-ulcer  and wound healing activities.  Recently extracts from CA have been reported to improve the reduction of venous insufficiency and microangiopathy in hypertensive patients.  It also protects the myocardium from ischemic reperfusion induced myocardial infarction.  CA has a high antioxidant activity,  that is attributed to the presence of phenolic compounds (3.23-11.7 g/100 g dry sample) , and angiotensin converting enzyme inhibitor (ACE) activity (79%).
| Materials and Methods|| |
Plant collection and identification
The fresh leaves of CA leaves were collected in Thanjavur, Tamilnadu, INDIA. The accuracy of plant selection was proved and authenticated by Department of Botany, Annamalai University, Tamilnadu, India.
The fresh leaves of the plant were air-dried at 40°C and ground to powder, which was then subjected to exhaustive extraction using water in a Soxhlet apparatus. The dark green liquid extract was concentrated under vacuum and the resulting dried extract was lyophilized and preserved in a refrigerator at 4°C until use in the experiments.
Eight-week-old adult male albino rats of Wistar strain, weighing approximately 160 to 180 g, were acclimatized for 7 days at room temperature (25 ± 3°C) and relative humidity (55%) in a 12-hour light/dark cycle in a room under hygienic condition. The animals reared in Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University were used for the experiment. Male animals were used throughout the investigation to avoid complications due to the estrous cycle. The animals were allowed free access to water and standard pellet diet (Amrut Laboratory Animal Feed, Pranav Agro Industries Ltd., Bangalore, India). Animal handling and experimental procedures were approved by the Institutional Animal Ethics Committee, Annamalai University (Registration Number: 166/1999/CPCSEA, Proposal No. 543) and animals were cared in accordance with the guidelines by the "Committee for the purpose of control and supervision on experimental animals" (CPCSEA, 2004).
Source of chemicals
Chemicals and solvents were of analytical grade and purchased from Sigma-Aldrich Co. St. Louis, Missouri, USA or Himedia Laboratories Pvt. Ltd., Mumbai, India.
Induction of stress
Stress was induced in rats by placing the animals in 20 cm × 7 cm plastic tubes for 2 h/day for 21 days. There will be several 3 mm holes at the far end of the tubes for breathing that allows ample air but animals will be unable to move. 
The rats were randomly divided into four groups with six rats each. CA was completely dissolved in Water to the volume of 10 ml for a required concentration.
Group I : Control
Group II : Control+ CA (200 mg/kg BW, for 21 days)
Group III : Stress (for 21 days)
Group IV : Stress + CA (200 mg/kg BW, for 21 days)
The total duration of the study was 21 days. On 19 th day onwards Passive avoidance behavior (PAB) test was started and on 21 th day, the rats were sacrificed by cervical dislocation. Blood samples were collected in a dry test tube with EDTA. The brain tissue was excised immediately, washed in chilled isotonic saline and used for the various parameters analysis.
Automatic blood analyzer was used, programmed to count red blood cells (RBCs) and white blood cells (WBCs) and the absolute numbers of lymphocytes, neutrophils, eosinophils and monocytes.
Assay of brain acetyl cholinesterase activity (AChE; E.C.220.127.116.11)
The AChE activity was estimated at 8 a.m.-11 a.m. On the 21 st day animals were euthanized by cervical dislocation. The whole brain AChE activity was measured using the Ellman's method.  The end point was the formation of the yellow color due to the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of thiocholine from acetylcholine iodide in the presence of tissue cholinesterase was measured using a spectrophotometer. The sample was first treated with 5, 50-dithionitrobenzoic acid (DTNB) and the optical density (OD) of the yellow color compound formed during the reaction at 412 nm every minute for a period of 3 min was measured. Protein estimation was carried out by Lowry  method. AChE activity was expressed as mg/protein.
On the day nineteen, passive avoidance behavior (PAB) test was started in one trial learning, step-through situation, which utilizes the natural preference of rats for a dark environment.  After 2 min habituation to the dark compartment the rat was placed on the illuminated platform and allowed to enter the dark compartment. Two more approach trials were given on the following day with a 2 min interval. At the end of the second trial unavoidable scrambled electric foot shock (0.25 mA, AC, 2 s) was delivered through the grid floor of the dark compartment (learning trial). Twenty four hours later (i.e., day 21), the animals were tested to recall the passive avoidance responses by placing the animal on the platform and measuring the latency to re-enter the dark compartment to a maximum of 300 s.
Data were analyzed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) using a commercially available statistics software package (SPSS for Windows, V. 11.0, Chicago, USA). Results were presented as means ± SD. P values <0.05 were regarded as statistically significant.
| Results and Discussion|| |
[Table 1] and [Figure 1] show that there is a significant decreased in the level of RBCs, WBCs, lymphocytes, monocytes, neutrophils and eosinophils in the stressed group when compared with control, at the same time CA alone administered group, stress induced rats co-administered with CA were shown near normal values as control. CA is well known for its antioxidant properties. Earlier studies showed that oral administration of an aqueous extract of CA reduces brain malondialdehyde levels and increases the glutathione levels in whole brain  of adult rats. Recently, CA was demonstrated to accelerate nerve regeneration in vivo and increased neurite elongation in vitro.  Notable bioactive compounds of CA are the triterpene saponins, madecassocide and asiaticoside with their respective ursane types apogenins viz., madecassic and asiatic acid. , Further, CA is reported to contain numerous caffeic acid derivatives and flavonols and in particular quercetin, kaempferol, catechin, rutin and naringin  some of which have been shown to be potent antioxidants.  The triterpenoid, sapononins which are isolated from CA has immunomodulatory effects.  At the same time it increases the RBC in blood and has anti convulsant effect.  Bagum et al. also demonstrated that Apigenin, natural dietary flavones present in common vegetables and fruit, increases the hematological parameters in gamma-radiation induced oxidative stress in Swiss albino mice. The increase in the hematological constituents in the stress loaded group co-treated with CA might be due to the antioxidant, anti-lipidperoxidation, immunostimulation and anticonvulsant effects of CA.
|Figure 1: Effect of Centella asiatica (CA) leaves extract on the number of RBCs and WBCs in immobilization induced stress in Wistar rats. Values are means ± SD of six rats from each group. Values not sharing a common alphabet as superscripts are signifi cantly different from each other at the level of P < 0.05 (ANOVA followed by DMRT)|
Click here to view
|Table 1: Effect of Centella asiatica leaves extract on the hematological parameters in immobilization induced stress in Wistar rats|
Click here to view
[Figure 2] shows the AChE activity. AChE activity was significantly increased in stress condition when compared with control group. When the stressed animals were co-administered with CA the AChE activity was near normal. In CA alone administered group there is no significant change in the level of AChE. There are also few reports which suggest that CA significantly increase the AchE level.  Other workers have also observed the alteration in cholinergic system due to stress because of the reduction of choline up take and elevation in the number of Muscarinic binding sites at the cholinergic terminals and septo-hippocampal cholinergic system. CA has been used to treat dementia, mental disorders and anxiety, as it acts in synergistic manner.  Published data suggest that CA leaves extract has neuroprotective effect which promotes nerve growth and neuronal dendritic arborization.  In this present study stress caused a significant increase in AChE activity thereby leading to memory defects. CA significantly ameliorated the stress induce AChE activity which may be due to its neuroprotective effects.
|Figure 2: Effect of Centella asiatica (CA) leaves extract on the brain acetylcholine esterase in immobilization induced stress in Wistar rats. Values are means ± SD of six rats from each group. Values not sharing a common alphabet as superscripts are signifi cantly different from each other at the level of P < 0.05 (ANOVA followed by DMRT)|
Click here to view
[Figure 3] shows the PAB behavior change in experimental rats. Passive-avoidance response (PAR) is extensively used for the screening of stress affect learning and memory.  The hippocampus is one of the brain structures that are involved in many kinds of learning and memory like PA latency. The PA paradigm is widely utilized for test the learning and memory in rats and mice.  Thus, PA paradigm might be viewed as measuring explicit memory, to the point that terms such as 'declarative' or 'explicit' can be applied to experiments using rodents, the task is learned very rapidly and memory can be easily and reliably assessed.  Immobilization induced stress loaded rats were loosed their memory and entered the darker compartment. Significance was observed that co-administration of immobilization mediated stressed rats with CA did not enter the dark compartment, when compared to stressed rats. Stress is significantly effective in PA i.e., re-entering of rats to the dark compartment of the apparatus earlier than controls rats and rats receiving CA only. In fact a complete recovery from the post-stress memory deficits assessed by this test was observed in all animals co-administered with CA. Tertamethylpyrazine which is a alkaloid used in traditional Chinese medicine has an neuroprotective and beneficial effect on memory impairments in bilateral common carotid artery occlusion induced global ischemia-reperfusion injury in rats.  Aegle marmelos leaves which contains phytochemicals such as alkaloids, coumarins, flavonoids, steroids, triterpenes and essential oils is established to have anti stress activity in our lab.  The present study exploits rat model of psychosocial stress-induced memory deficits in which CA seems to be more effective in the stress groups, which may be due to the presence of alkaloids in CA.
|Figure 3: Effect of Centella asiatica (CA) leaves extract on the Passive avoidance behaviour change in immobilization induced stress in Wistar rats. Values are means ± SD of six rats from each group. Values not sharing a common alphabet as superscripts are signifi cantly different from each other at the level of P < 0.05 (ANOVA followed by DMRT)|
Click here to view
| Conclusion|| |
The present findings present findings indicate, that administration of Centella asiatica leaves extract restore the counts of RBCs and WBCs and the activity of AchEs during stress condition. Apparently it may offer safe and inexpensive means of preventing decline of immunity, anemia and memory impairments caused by stress.
| References|| |
|1.||Johnson N, Davis T, Bosanquet N. The epidemic of Alzheimer's disease. How can we manage the costs? Pharmacoeconomics 2000;18:215-23. |
|2.||Lambert MJ. Bergin and Garfield's handbook of psychotherapy and behavioral change. New York: Wiley; 2004. |
|3.||Creamer M, O'Donnell ML, Pattison P. Acute stress disorder is of limited benefit in predicting post-traumatic stress disorder in people surviving traumatic injury. Behav Res Ther 2004;42:315-28. |
|4.||Enz A, Amstutz R, Boddeke H, Gmelin G, Malonowski J. Brain selective inhibition of acetylcholinesterase: A novel approach to centellasaponins B, C, and D, from Centella asiatica cultivated in Sri Lanka. Chem Pharm Bull 1993;4:1368-71. |
|5.||Loliger J. The use of antioxidants in foods. In: Aruoma IO, Halliwell, editors. Free Radical and Food Additives. London: Taylor and Francis; 1991. p. 121. |
|6.||Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95. |
|7.||Ingkaninan K, De Best CM, Irth H, Van der Heijden R, Hofte AJP, Karabatak B, et al. High performance liquid chromatography with on-line coupled UV-mass spectrophotometric-biochemical detection for identification of acetylcholinesterase inhibitors from natural products. J Chromatogr A 2000;872:61-73. |
|8.||Shinghal AK, Naithani V, Bangar OP. Medicinal plants with a potential to treat Alzheimer and associated symptoms. Int J Nutr Pharmacol Neurol Dis 2012;2:84-91. |
|9.||Babu TD, Kutten G, Padikkala J. Cytotoxic and antitumour properties of certain taxa of Umbelliferae with special reference to Centella asiatica (L.) Urban. J Ethnopharmacol 1995;48:53-7. |
|10.||Lue YH, Sinha Hikim AP, Swerdloff RS, Im P, Khay ST, Bui T, et al. Single exposure to heat induces stagespecific germ cell apoptosis in rats: Role of intra testicular testosterone on stage specificity. Endocrinology 1999;140:1709-17. |
|11.||Sampson JH, Raman A, Karlsen G, Navsaria H, Leigh IM. In vitro keratinocyte antiproliferant effect of Centella asiatica extract and triterpenoid saponins. Phytomedicine 2001;8:230-5. |
|12.||Shetty BS, Udupa SL, Udupa AL, Somayaji SN. Effect of Centella asiatica L (Umbelliferae) on normal and dexamethasone- suppressed wound healing in Wistar Albino rats. Int J Low Extrem Wounds 2006;5:137-43. |
|13.||Mutayabarwa CK, Sayi JG, Dande M. Hypoglycaemic activity of Centella asiatica (L) Urb. East Cent Afr J Pharm Sci 2003;6:30-5. |
|14.||Antony B, Santhakumari G, Merina B, Sheeba V, Mukkadan J. Hepatoprotective effect of Centella asiatica (L) in carbon tetrachloride-induced liver injury in rats. Indian J Pharm Sci 2006;68:772-6. |
|15.||Cheng CL, Guo JS, Luk J, Koo MW. The healing effects of Centella extract and asiaticoside on acetic acid induced gastric ulcers in rats. Life Sci 2004;74:2237-49. |
|16.||Ullah MO, Sultana S, Haque A, Tasmin S. Antimicrobial, cytotoxic and antioxidant activity of Centella asiatica. Eur J Sci Res 2009;30:260-4. |
|17.||Marcilhac A, Dakine N, BourHim N, GuillaumeV, Grino M, Drieu K, et al. Effect of Ginko Bilba Extract or Ginko lode on hypothalamic extract or ginkolide on Hypothalamic-pituitary-adrenal axis in the rats. Life Sci 1998;62:2329-40. |
|18.||Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement using folin-phenol reagent. J Biol Chem 1951;193:265-75. |
|19.||Ader R, Weijnen WM, Moleman P. Retention of passive avoidance responses as a function of the intensity and duration of electric shock. Psychon Sci 1972;26:125-9. |
|20.||Veerendra Kumar MH, GuptaYK. Effect of different extracts of Centella asiatica on cognition and oxidative stress in rats. J Ethnopharmacol 2002;79:253-60. |
|21.||Soumyanath A, Zhong YP, Gold SA, Yu X, Koop DR, Bourdette D, et al. Centella asiatica accelerated nerve regeneration upon oral administration and contains multiple active fractions increasing neurite elongation in vitro. J Pharmacy Pharmacol 2005;57:1221-7. |
|22.||Mangas S, Bonfill M, Osuna L, Moyano E, Tortoriello J, Cusido RM, et al. The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants. Phytochemistry 2006;67:2041-9. |
|23.||Wijeweera P, Arnason JT, Koszycki D, Merali Z. Evaluation of anxiolytic properties of Gotukola (Centella asiatica) extracts and asiaticoside in rat behavioral models. Phytomedicine 2006;13:668-76. |
|24.||Zainol MK, Abdul-Hamid A, Yusof S, Muse R. Antioxidative activity and total polyphenolic compounds of leaf, root and petiole off our accessions of Centella asiatica (L.) urban. Food Chem 2003;81:575-81. |
|25.||Hussin M, Abdul-Hamid A, Mohamad S, Saari N, Ismail M, Bejo MH. Protective effect of Centella asiatica extract and powder on oxidative stress in rats. Food Chem 2007;100:535-41. |
|26.||Jayathirtha MG and Mishra SH. Preliminary immunomodulatory activities of methanol extracts of Eclipta alba and Centella asiatica. Phytomedicine 2004;11:361-5. |
|27.||Visweswari G, Prasad KS, Chetan PS, Lokanatha V, Rajendra W. Evaluation of anticonvulsant effect of Centella asiatica in pentylenetetrazol-induced seizures with respect to cholergic neurotransmission. Epilepsy Behav 2010;17:332-5. |
|28.||Begam N, Prasad NR, Thayalan K. Apigenin protects gamma-radiation induced oxidative stress, hematological changes and animal survival in whole body irradiated Swiss albino mice. Int J Nutr Pharmacol Neurol Dis 2012;2:45-52. |
|29.||Sulochana B, Rao M, Chetana P Uma D. Centella asiatica treatment during postnatal period enhances learning and memory in mice. Physiol Behav 2005;86:449-57. |
|30.||Upadhyay SK, Saha A, Bhatia BD KulKarni K. Evaluation of the efficacy of mentat in children with learning disability placebo Controlled Double Blind Clinical trial. Neurosci Today 2002;4:184-8. |
|31.||Mohandas Rao KG, Muddanna Rao S, Guromadhana Rao S. Centella asiatica (L) Leaf extract treatment arborization in rats. Evid Based Complement Alternat Med 2006;3:349-57. |
|32.||Porsolt RD, LePichon M, Jalfre M. Depression: A new animal model sensitive to antidepressant treatments. Nature 1977;266:730-2. |
|33.||Izquierdo I, Souza DO, Carrasco MA, Dias RD, Perry ML, Eisinger S, et al. B-endorphin causes retrograde amnesia and is released from the rat brain by various forms of training and stimulation. Psychopharmacology (Berl) 1980;70:173-7. |
|34.||Undale VR, Desai SS, Sangamnerkar SK, Upasani CD. Neuroprotective effect of cow colostrums and tetramethyl pyrazine against global cerebral ischemia reperfusion injury. Int J Nutr Pharmacol Neurol Dis 2012;2:111-20. |
|35.||Anusha C, Sarumathi A, Shanmugapriya S, Anbu S, Ahmad RS, Saravanan N. The effects of aqueous leaf extract of Aegle marmelos on immobilization-induced stress in male albino Wistar rats. Int J Nutr Pharmacol Neurol Dis 2013,3:1. |
[Figure 1], [Figure 2], [Figure 3]