IPSIndian Journal of Pharmacology
Home  IPS  Feedback Subscribe Top cited articles Login 
Users Online : 326 
Small font sizeDefault font sizeIncrease font size
Navigate Here
  Search
 
  
Resource Links
 »  Similar in PUBMED
 »  Search Pubmed for
 »  Search in Google Scholar for
 »Related articles
 »  Article in PDF (498 KB)
 »  Citation Manager
 »  Access Statistics
 »  Reader Comments
 »  Email Alert *
 »  Add to My List *
* Registration required (free)

 
In This Article
 »  Abstract
 »  Materials and Me...
 » Results
 » Discussion
 » Conclusion
 »  References
 »  Article Tables

 Article Access Statistics
    Viewed808    
    Printed21    
    Emailed0    
    PDF Downloaded126    
    Comments [Add]    

Recommend this journal

 


 
 Table of Contents    
RESEARCH ARTICLE
Year : 2016  |  Volume : 48  |  Issue : 6  |  Page : 681-686
 

Anxiolytic activity of aqueous extract of Camellia sinensis in rats


1 Department of Pharmacology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
2 Department of Pharmacology, Srinivas Institute of Medical Sciences, Mangalore, Karnataka, India
3 Physician, Non Communicable Disease Clinic, District Civil Hospital, Belagavi Institute of Medical Sciences, Belagavi, Karnataka, India

Date of Submission13-Feb-2016
Date of Acceptance09-Nov-2016
Date of Web Publication29-Nov-2016

Correspondence Address:
Sheetal Dinkar Ullal
Department of Pharmacology, Kasturba Medical College, Manipal University, Mangalore, Karnataka
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0253-7613.194864

Rights and Permissions

 » Abstract 

Objectives: The present study was undertaken to evaluate anxiolytic effect of Camellia sinensis (CS) and possible mechanism on acute and chronic administration in rats.
Materials and Methods: Eight groups of rats with six in each group were used. Group I served as control. Group II received diazepam (1 mg/kg). Groups III, IV, and V received CS in doses of 3.3, 16.5, and 33 mg/kg, respectively. Three pharmacologically validated experimental models – elevated plus maze (EPM), light and dark box (LDB), and open field tests (OFT) – were employed. Each animal was tested initially in the EPM and then in the LDB, followed by the OFT in a single setting. In EMP, number of entries into, time spent in, and number of rears in each arm in a 5-min period were noted. In LDB, number of entries and time spent in bright arena, number of rears, and duration of immobility were noted. In OFT, number of peripheral and central squares crossed, time spent, and number of rears in central squares were observed for a 5-min period. One-way ANOVA followed by post hoc least significant difference test was performed.
Results: In EPM and LDB, CS at 3.3, 16.5, and 33 mg/kg (acute and chronic models) increased the number of entries and time spent and rearing in the open arms and bright arena, respectively, compared to control. In the OFT, CS at 16.5 and 33 mg/kg significantly increased the number of squares crossed, time spent, and the number of rears in the central squares compared to control. Anxiolytic effect was dose dependent in EPM and LDB and CS at 33 mg/kg showed better anxiolytic activity compared to diazepam (1 mg/kg) in all models. Flumazenil (0.5 mg/kg) and bicuculline (1 mg/kg) completely inhibited while picrotoxin (1 mg/kg) partially inhibited the anxiolytic effect of CS. Diazepam and CS at 33 mg/kg reduced the locomotor activity in rats.
Conclusion: CS has dose-dependent anxiolytic activity which is comparable to diazepam. Anxiolytic action of CS is likely mediated through GABAA-benzodiazepine receptor – Cl − channel complex – since flumazenil and bicuculline inhibited the anxiolytic effect.


Keywords: Anxiolytic, Camellia sinensis, elevated plus maze, light and dark box, open field test


How to cite this article:
Shastry R, Ullal SD, Karkala S, Rai S, Gadgade A. Anxiolytic activity of aqueous extract of Camellia sinensis in rats. Indian J Pharmacol 2016;48:681-6

How to cite this URL:
Shastry R, Ullal SD, Karkala S, Rai S, Gadgade A. Anxiolytic activity of aqueous extract of Camellia sinensis in rats. Indian J Pharmacol [serial online] 2016 [cited 2017 Aug 19];48:681-6. Available from: http://www.ijp-online.com/text.asp?2016/48/6/681/194864


Neuropsychiatric disorders form 10.5% of global burden of disease (disability-adjusted life year) and it is suggested that this could increase to 15% by 2020.[1] It will be the third leading cause of disability in low-income countries.[2] Anxiety is a normal emotion under stressful circumstances but may become maladaptive when it constitutes a psychiatric disorder. Recent trends suggest that anxiety disorders may be more important than originally thought and have become a prominent area of research interest. Benzodiazepines are most commonly prescribed class of compounds for anxiety despite many unwanted side effects such as sedation, muscle relaxation, ataxia, amnesia, and tolerance on chronic use. These may also cause ethanol and barbiturate potentiation.[3] Many herbal products have been claimed to be less toxic than synthetic drugs [4] and have demonstrated prominent effects in psychiatric disorders. These are emerging as promising alternative therapies. In Asia, Camellia sinensis (CS), popularly known as green tea, has been widely used as a beverage for centuries for its significant health-promoting effects.[5] Various animal experiments have demonstrated CS to possesses antioxidant, antidepressant, antimutagenic, antidiabetic, anti-inflammatory, antibacterial, antiviral, and anticancer properties.[6],[7] Most of the pharmacological actions of CS have been ascribed to the presence of major constituents which include polyphenols catechin (approximately 30%–40%), gallocatechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate and flavonoids), proanthocyanidins, alkaloids (caffeine [approximately 3%], theobromine, theophylline), amino acid (theanine: γ-ethylamide of glutamic acid), polysaccharides, volatile oils, small amounts of tannin, diphenylamine, oxalic acid, trace elements, and Vitamin C.[8],[9],[10]

A polyherbal product NR-ANX-C has been reported to have anxiolytic activity in rodents.[11] CS is one of the major components (33%) of NR-ANX-C. Hence, the present study was undertaken to evaluate anxiolytic effect and its possible mechanism on acute and chronic administration of aqueous extract of CS in rats.


 » Materials and Methods Top


Experimental Animals

Adult Wistar rats of either sex, weighing 150–200 g, inbred at the institutional central animal house were housed and maintained under standard laboratory conditions, fed on commercial pelleted chow and water ad libitum. Experiments were conducted between 09:00 and 17:00 hours and in accordance with the guidelines laid down by the Committee for the Purpose of Control and Supervision on Experiments on Animals. The study protocol was approved by the Institutional Animal Ethics Committee.

Drugs

Standard drug

Diazepam (1 mg/kg, oral, using a metal gavage) was obtained from Nicholas Piramal India Ltd.

Test drug

Aqueous extract of CS (3.3, 16.5, and 33 mg/kg, oral, using a metal gavage) was obtained.[11]

The raw materials (leaves of CS) were obtained from M/S The Himalaya Drug Company, Makali, Bengaluru - 562123, Karnataka, India. Aqueous extract was prepared in our laboratory using Soxhlet apparatus. Raw material weighing 250 g underwent eight cycles of soxhlation, following which 30.8 g of investigational product was obtained. Hence, the yield was 8.1%.

Antagonists

Benzodiazepine receptor antagonist – flumazenil (0.5 mg/kg, intraperitoneally), GABAA-gated chloride channel blocker – picrotoxin (1 mg/kg, intraperitoneally), GABAA receptor antagonist – bicuculline (1 mg/kg, intraperitoneally),[12] all antagonists were obtained from Sigma-Aldrich, Bommasandra-Jigani Link Road, Bengaluru.

Each drug solution was freshly prepared before administration. Doses of diazepam and CS were selected on the basis of earlier findings with NR-ANX-C.[11]

Eight groups of animals were used and each group had six animals. Group I received normal saline, served as control. Group II received diazepam (1 mg/kg). Groups III, IV, and V received CS in doses of 3.3, 16.5, and 33 mg/kg, respectively. Group VI, VII, and VIII were cotreated with flumazenil (0.5 mg/kg), picrotoxin (1 mg/kg), and bicuculline (1 mg/kg), respectively, along with CS (33 mg/kg). In the acute study, all oral drugs were administered 60 min before the experiment. In the chronic study, drugs were administered once daily for 14 days and last dose was given on the 14th day 60 min before the experiment. Intraperitoneal drugs were given 15 min before the experiment. In Groups VI, VII, and VIII, rats were pretreated with the antagonist to identify the site/mechanism of action of CS; hence, the dose of CS which showed maximum anxiolytic activity was chosen for these groups.

Behavioral Testing

Three pharmacologically validated experimental models, elevated plus maze (EPM),[13] light and dark box (LDB),[14] and open field test (OFT),[15] were employed.

Each animal was tested initially in the EPM and then in the LDB, followed by the OFT in a single setting. Each animal was placed in the central square of EPM, facing one of the open arms. Number of entries into and time spent in the open and closed arms and number of rears in each arm in a 5-min period were noted. In the LDB, animals were placed at the center of brightly lit arena and number of re-entries into the bright arena; time spent in the bright arena, number of rears, and duration of immobility were noted. In the OFT, each animal was placed in one of the peripheral corner squares of the open field, and number of peripheral and central squares crossed, time spent and number of rears in central squares were observed for a 5-min period. Following each trial, apparatus was cleaned with spirit to mask the odor left by the animal in the previous experiment.

Statistical Analysis

Data obtained were coded and analyzed in a blind manner by a statistician who was not aware of the coding. One-way ANOVA was used with drug treatment as the independent factor. Post hoc comparisons were performed by applying least significant difference test. P < 0.05 was considered statistically significant.


 » Results Top


Acute Study

In the EPM [Table 1], diazepam and CS at the dose of 33 mg/kg showed a significant increase in the entries (P = 0.004 and P < 0.001, respectively), time spent (P < 0.001), and rears (P = 0.025 and P < 0.001, respectively) in the open arm compared to the control group. CS at the dose of 33 mg/kg also significantly increased the time spent (P < 0.001) and number of rears (P = 0.003) in the open arm compared to the diazepam treated group.
Table 1: Effect of drugs on behavior of rats in elevated plus maze on single dose administration (acute study)

Click here to view


Coadministration of flumazenil and bicuculline decreased the number of entries (P < 0.001), time spent (P < 0.001), and rears (P < 0.001) in the open arms as compared to CS at 33 mg/kg alone. Picrotoxin coadministration reduced the time spent (P = 0.03) and rears (P = 0.002) but had no effect on the number of entries into the open arm compared to CS at 3 mg/kg alone.

In the LDB [Table 2], diazepam and CS at all the three doses (3.3, 16.5, and 33 mg/kg) significantly increased the number of entries (P < 0.001, P = 0.014, P < 0.001, and P < 0.001, respectively), time spent (P < 0.001, P = 0.008, P < 0.001, and P < 0.001, respectively), and rears (P < 0.001, P = 0.002, P < 0.001, and P < 0.001, respectively) in the bright arena compared to control. Moreover, for each, CS at the dose of 33 mg/kg significantly increased the number of entries (P < 0001) and time spent (P = 0.038) in the bright arena compared to the diazepam-treated group. Diazepam-treated rats showed significant increase in the duration of immobility (P < 0.001) compared to control. CS-treated rats at all the three doses (3.3, 16.5, and 33 mg/kg) significantly decreased immobility compared to control (P = 0.004, P = 0.009, and P = 0.012, respectively) and diazepam-treated group (P < 0.001).
Table 2: Effect of drugs on behavior of rats in light and dark box on single dose administration (acute study)

Click here to view


Coadministration of flumazenil and bicuculline reduced the number of entries (P < 0.001), time spent (P < 0.001), and rears (P < 0.001) in the bright arena compared to CS at 33 mg/kg alone. Picrotoxin coadministration reduced the number of entries (P = 0.002) into the bright arena but had no effect on the time spent and rears in the bright arena. None of the antagonists had any effect on immobility.

In the open field paradigm [Table 3], both diazepam and higher doses of CS (16.5 and 33 mg/kg) significantly increased the number of squares crossed (P < 0.001), rears (P < 0.001), and time spent (P < 0.001) in the central squares compared to the control group. CS at 33 mg/kg also significantly increased the time spent (P < 0.001) and number of rears (P = 0.001) in the central squares compared to the diazepam-treated group. Treatment with diazepam and CS at 33 mg/kg significantly reduced the total number of squares crossed (P < 0.001) compared to control. CS at 33 mg/kg showed significantly higher open arm entries compared to CS at 3.3 mg/kg (P < 0.001). Time spent in the open arms was significantly higher in the CS at 33 mg/kg treated group compared to CS at 16.5 and 3.3 mg/kg (P < 0.001). Time spent in the open arms was also significantly higher in the CS at 16.5 mg/kg treated group compared to 3.3 mg/kg (P = 0.008).
Table 3: Effects of drugs on behavior of rats in open field paradigm on single dose administration (acute study)

Click here to view


Coadministration of flumazenil, bicuculline, and picrotoxin significantly reduced the number of squares crossed (P < 0.001), rears (P < 0.001), and time spent (P < 0.001) in the central squares compared to the CS 33 mg/kg treated group. All three antagonists also significantly increased the total number of squares crossed compared to the CS 33 mg/kg treated group (P < 0.001).

Chronic Study

In the EPM [Table 4], diazepam and CS at the dose of 16.5 and 33 mg/kg showed a significant increase in the entries (P = 0.001, P = 0.012, P < 0.001, respectively), time spent (P < 0.001) and rears (P = 0.026, P = 0.04, P < 0.001, respectively) in the open arm compared to the control group. CS at the dose of 33 mg/kg also significantly increased the number of entries (P = 0.017) and time spent (P < 0.001) in the open arm compared to the diazepam-treated group.
Table 4: Effect of diazepam and Camellia sinensis on behavior of rats in elevated plus maze on administration for 14 days (chronic study)

Click here to view


In the LDB [Table 5], diazepam and CS at all the three doses significantly increased the number of entries, time spent, and rears in the bright arena compared to control. Moreover, CS at the dose of 33 mg/kg significantly increased the number of entries (P < 0.001) and time spent (P = 0.013) in the bright arena compared to the diazepam-treated group. The diazepam-treated rats showed significant increase in duration of immobility compared to control. CS-treated groups in all the three doses (3.3, 16.5, and 33 mg/kg) significantly reduced the immobility (P < 0.001) compared to the diazepam-treated groups. Number of entries to the bright arena was significantly higher in the CS at 16.5 and 33 mg/kg treated groups compared to CS at 3.3 mg/kg (P = 0.03, <0.001). Number of entries to the bright arena was also significantly higher in the CS 16.5 mg/kg compared to CS at 33 mg/kg treated groups (P < 0.001). Time spent in the bright arena was significantly higher in the CS 33 mg/kg treated group compared to CS 3.3 at mg/kg and 16.5 mg/kg treated groups (P < 0.001).
Table 5: Effect of diazepam and Camellia sinensis on behavior of rats in light and dark box on administration for 14 days (chronic study)

Click here to view


In the open field paradigm [Table 6], both diazepam and higher doses of CS (16.5 and 33 mg/kg) significantly increased the number of squares crossed (P < 0.001), rears (P < 0.001, P < 0.001, and P = 0.014, respectively), and time spent (P < 0.001) in the central squares compared to the control group. CS at 33 mg/kg also significantly increased the number of squares crossed (P = 0.001) and time spent (P < 0.001) in the central squares compared to the diazepam-treated group. Treatment with diazepam and CS at 33 mg/kg significantly reduced the total number of squares crossed compared to control. However, the total number of squares crossed in the CS at 3.3 and 16.5 mg/kg treated groups was significantly more than the diazepam-treated group (P < 0.001).
Table 6: Effects of diazepam and Camellia sinensis on behavior of rats in open field paradigm on administration for 14 days (chronic study)

Click here to view



 » Discussion Top


In the present study, anxiolytic effects of CS have been studied in three experimental models of anxiety – EPM, LDB, and open field paradigm. These models are based on the fact that an unfamiliar, brightly lit, and open environment induces stress which provokes anxiety, thereby inhibiting normal behavior in rats.[16]

In the EPM, animals find the open arms to be unprotective and more anxiogenic than the closed arms. Animal hence prefers to spend more time and shows normal rearing behavior in the closed arm. Anxiolytic drugs increase the number of entries, time spent, and rearing in the open arms.[16] Following both acute and chronic treatment, CS in all three doses increased the number of entries, time spent, and rearing in the open arms compared to control. There was no difference between the acute and chronically treated groups. Behavioral changes were comparable to diazepam and CS at 33 mg/kg increased the time spent and rears in open arm more than diazepam-treated group. This suggests that anxiolytic effect of CS was comparable to diazepam at low doses and better than diazepam at a higher dose. A dose-dependent anxiolytic action was also observed in this model.

In the LDB, rodents tend to avoid entry into and reduce spontaneous exploratory behavior in the brightly illuminated area; a natural tendency when a rat is exposed to an unfamiliar environment.[13] Anxiolytics tend to increase the number of entries, time spent, and rears in the bright arena. In the present study, CS, at different doses on both acute and chronic therapy, increased the number of entries, time spent, and rears in the bright arena compared to control indicating anxiolytic effect. There was no difference in the acute and chronically treated groups. A better anxiolytic effect of CS than that of diazepam was observed at a higher dose. A dose-dependent anxiolytic effect was observed in this model also.

The open field paradigm is used with the dual purpose of assessing not only the anxiolytic activity but also the locomotor activity in rodents.[17] After both acute and chronic treatments, CS at 16.5 and 33 mg/kg showed a significant increase in the number of squares crossed, time spent, and the number of rears in the central squares compared to control. At 33 mg/kg, a better anxiolytic effect than that of diazepam was also observed. Treatment with diazepam reduced the locomotor activity. This was observed at CS 33 mg/kg but not at 16.5 mg/kg. Hence, CS at 16.5 mg/kg showed anxiolytic activity without affecting the locomotor activity.

An earlier study by Heese et al.[18] using L-theanine which is one of the components of CS did not show anxiolytic activity when used alone but showed anxiolytic activity in combination with midazolam. However, anxiolytic effect of CS, observed in the present study, suggests the presence of other components in CS extract which may be responsible for anxiolytic effect. Our findings are similar to the study done by Tabassum et al.,[19] who found that CS increased number of entries and time spent in the open arm in EPM and increased ambulation in OFT; however, the dose (100 mg/kg) used was much higher than in our study (33 mg/kg). Indian traditional medicine too is a proponent of the idea that whole parts of plants are better than individual constituents since the combined synergistic action of constituents produces final beneficial effect.

Anxiolytics such as benzodiazepines act through the GABAA-benzodiazepine receptor – Cl channel complex. CS also appears to mediate its anxiolytic action predominantly through this complex since flumazenil and bicuculline completely inhibited while picrotoxin partially inhibited the anxiolytic effect of CS. Flumazenil, bicuculline, and picrotoxin pretreatment also antagonized the effect of CS on locomotor activity. Both these effects appear to be mediated through the GABAA-benzodiazepine receptor – Cl channel complex. Further studies are required to confirm site of action of CS.

Foodstuff can be considered as functional if it satisfactorily demonstrates beneficial action on target functions in the body. Tea is the most consumed beverage in the world. Beneficial health effects of green tea are increasingly proved, and it should be considered in the group of beverages with functional properties. Anxiolytic effect of CS extract needs further evaluation in large-scale studies.


 » Conclusion Top


CShas dose-dependent anxiolytic potential, likely mediated through the GABAA-benzodiazepine receptor – Cl channel complex. Anxiolytic effect is comparable to diazepam and needs evaluation in further studies.

Financial Support and Sponsorship

Nil.

Conflicts of Interest

There are no conflicts of interest.

 
 » References Top

1.
World Bank. World Development Report 1993: Investing in Health. Washington, DC: World Bank; 1993.  Back to cited text no. 1
    
2.
Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 2006;3:e442.  Back to cited text no. 2
    
3.
Lader M, Morton S. Benzodiazepine problems. Br J Addict 1991;86:823-8.  Back to cited text no. 3
    
4.
Pari L, Maheswari JU. Hypoglycaemic effect of Musa sapientum L. in alloxan-induced diabetic rats. J Ethnopharmacol 1999;68:321-5.  Back to cited text no. 4
    
5.
Balentine DA, Wiseman SA, Bouwens LC. The chemistry of tea flavonoids. Crit Rev Food Sci Nutr 1997;37:693-704.  Back to cited text no. 5
    
6.
Shastry R, Gopalakrishna HN, Ullal SD, Sindhu KC, Ravindran N, Chowta MN. Anti-depressant activity of Camellia sinensis in mice. Am J Pharm Tech Res 2012;2:539-44.  Back to cited text no. 6
    
7.
Cabrera C, Artacho R, Giménez R. Beneficial effects of green tea – A review. J Am Coll Nutr 2006;25:79-99.  Back to cited text no. 7
    
8.
Chopade VV, Phatak AA, Upaganlawer AB, Tankar AA. Green tea (Camellia sinensis): Chemistry, traditional, medicinal uses and its pharmacological activities – A review. Pharmacogn Rev 2008;2:157-62.  Back to cited text no. 8
    
9.
Rogers PJ, Smith JE, Heatherley SV, Pleydell-Pearce CW. Time for tea: Mood, blood pressure and cognitive performance effects of caffeine and theanine administered alone and together. Psychopharmacology (Berl) 2008;195:569-77.  Back to cited text no. 9
    
10.
Sumpio BE, Cordova AC, Berke-Schlessel DW, Qin F, Chen QH. Green tea, the “Asian paradox,” and cardiovascular disease. J Am Coll Surg 2006;202:813-25.  Back to cited text no. 10
    
11.
Gopala Krishna HN, Sangha RB, Misra N, Pai MR. Antianxiety activity of NR-ANX-C, a polyherabal preparation in rats. Indian J Pharmacol 2006;38:330-5.  Back to cited text no. 11
    
12.
Anuradha H, Srikumar BN, Shankaranarayana Rao BS, Lakshmana M. Euphorbia hirta reverses chronic stress-induced anxiety and mediates its action through the GABAA receptor benzodiazepine receptor – Cl - channel complex. J Neural Transm 2008;115:35-42.  Back to cited text no. 12
    
13.
Pellow S, Chopin P, File SE, Briley M. Validation of open: Closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14:149-67.  Back to cited text no. 13
    
14.
Costall B, Domeney AM, Gerrard PA, Kelly ME, Naylor RJ. Zacopride: Anxiolytic profile in rodent and primate models of anxiety. J Pharm Pharmacol 1988;40:302-5.  Back to cited text no. 14
    
15.
Bhattacharya SK, Satyan KS. Experimental methods for evaluation of psychotropic agents in rodents: I – Anti-anxiety agents. Indian J Exp Biol 1997;35:565-75.  Back to cited text no. 15
    
16.
Rai S, Pai PG, Shastry R, Ullal SD, Nishith RS, Belagali Y. Evaluation of anxiolytic effect of chronic administration of Mucuna pruriens in wistar albino rats. Am J PharmTech Res 2014;4:611-9.  Back to cited text no. 16
    
17.
Bailey KR, Crawley JN. Anxiety-related behaviors in mice. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd ed., Ch. 5. Boca Raton (FL): CRC Press; 2009. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5221/. [Last accessed on 2015 Feb 13].  Back to cited text no. 17
    
18.
Heese T, Jenkinson J, Love C, Milam R, Perkins L, Adams C, et al. Anxiolytic effects of L-theanine – A component of green tea – When combined with midazolam, in the male Sprague-Dawley rat. AANA J 2009;77:445-9.  Back to cited text no. 18
    
19.
Tabassum I, Siddiqui ZN, Rizvi SJ. Effects of Ocimum sanctum and Camellia sinensis on stress-induced anxiety and depression in male albino Rattus norvegicus. Indian J Pharmacol 2010;42:283-8.  Back to cited text no. 19
[PUBMED]  Medknow Journal  



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

Top
Print this article  Email this article
 

    

Site Map | Home | Contact Us | Feedback | Copyright and Disclaimer
Online since 20th July '04
Published by Wolters Kluwer - Medknow