Anxiolytic action of Sri Lankan Orange Pekoe grade

1. Anxiolytic action of Sri Lankan Orange Pekoe grade of black tea (Camellia sinensis L.) in rats T.B.S. Muthunayake1 , W.D. Ratnasooriya1 , E.K. Indeesha1 , C.D.T. Ratnasooriya1 1 Department of Zoology, University of Colombo, Colombo 03.

2. INTRODUCTION • Besides water, Tea is the most consumed beverage in the world. • It is estimated that globally 3.5 million cups, glasses or bowls of • tea are consumed daily at present • Its per capita consumption is 2.52 – 3.1 kg/person/annum • Sri Lankan daily consumption of tea is about 3.5 cups. • It is accounting a per capita consumption of 1.38 kg/person/annum

3. Introduction to Tea cntd…… Based on the manufacturing technique; 3 main types of tea: Black (fully aerated or fermented) 2. Green (unaerated or unfermented) 3. Oolong (semi aerated or semi fermented) • Black tea accounts 78% of world’s tea production and about • 80% of global tea consumption • Sri Lanka is the 2nd main exporter of black tea in the world • and is drunk in more than 138 countries.

4. Classification of black tea Broken grade (different size tea particles) Whole leaf grade (twisted but, not broken) Eg: Orange Pekoe Broken leaf grade Eg: Broken Orange Pekoe Dust grade Eg: Dust No. 1 Fannings grade Eg: BOPF

5. Anxiety • Anxiety is an emotional state, unpleasant in nature and is • associated with uneasiness, discomfort and concern or fear • about some defined or undefined future threat • Currently, anxiety affects one eighth of the worldwide • population • It is estimated that, up to 40% of individuals would • experience an anxiety disorder in their life • There is a greater deal of popular and medicinal interest in • the use of dietary supplements in order to manage normal • “every day” stress and anxiety

6. Sri Lankan Black tea and anxiolytic property • Some reputed Sri Lankan traditional practitioners and • herbalists claim that consumption of 3-5 cups of warm black • tea help to relieve “ day to day” anxiety. • However, the grade of the black tea is not specified. • Dust grade No. 1 black tea (belongs to broken grade) has • shown to possess anxiolytic activity. • But, it is known that bioactivity of black tea depends on • particle size and agroclimatic elevation among other things. • As yet, the anxiolytic potential of whole leaf grade black tea is not scientifically tested

7. Aim of the study To investigate the anxiolytic action of Sri Lankan low grown orthodox whole leaf grade black tea (Camellia sinensis(L) O. Kuntz ; Family: Theaceae) using Orange Pekoe (O.P.) grade tea.

8. METHODOLOGY Preparation of black tea brew (BTB): • Garden fresh Orange Pekoe (O.P.) grade black tea sample was • collected from St. Jochims tea estate of the Tea Research Institute, • Hedallana, Ratnapura, Sri Lanka (29 m above mean sea level: low grown) 2g of O.P. grade Black tea + 100 ml of boiling water (ISO 3103) Brewed for 5 min 36.1 % w/v tea solids Based on this, human equivalent doses for rats were prepared as follows: 1.5 cups (223 mg/kg) Brewed for 5 min + 3 cups (446 mg/kg) 10 g of O.P. grade black tea 30 ml of boiling water 9 cups (1339 mg/kg) dose in 3 ml 9 cups (1339 mg/kg)

9. Evaluation of anxiolytic activity of O.P. grade black tea Tested in rats using three models of anxiety; Rat hole-board • Monitored parameters; (Observation time - 7.5 min) • Number of rears • Locomotary activity in terms of number of crossings • Number of head dips • Total dipping time All rats used to determine the anxiolytic activity in the rat hole-board test were closely observed for 2-3 h for any overt signs of toxicity

10. 2. Neophobic suppression of eating test Increased intake of novel food (dry fish) in an unfamiliar environment is considered as a measure of anxiolytic activity. (observation time : 30 min) 3. Neophobic suppression of drinking test Increased intake of novel drink (diluted sweetened condensed milk) in an unfamiliar environment is considered as a measure of anxiolytic activity. (observation time : 2 h) In each of above tests, anxiolytic effects were determined 1h after oral administration of O.P. tea or control (3 ml of water) / (N = 6 – 10 / group) After oral administration, rats were kept in their original cages

11. Evaluation of the GABAergic activity using the elevated plus maze model Rats were randomly divided in to 2 groups & orally administered as follows Group : 1 Control (3 ml water) N = 8 Group : 2 3 ml of BTB (446 mg/kg dose ) / N = 8 1h post treatment rats were individually placed in the neutral zone facing to an open arm of a wooden elevated plus maze model and observed for 5 min

12. Monitored parameters in elevated plus maze model: • Number of entries to open arms • Time spent in open arms • Number of entries to closed arms • Time spent in closed arms • Number of entries to the neutral zone • Time spent in the neutral zone Neutral zone Closed arm Open arm

13. Calculated indices: number of entries to open arms + number of entries to closed arms number of open arm entries  100 total number of entries time spent in open arms  100 total time spent in open arms and closed arms Total number of entries = % open arm entries = % time spent in open arms =

14. Evaluation of muscle strength and motor coordination Rats were randomly divided in to 2 groups & orally administered as follows Group : 1 Control (3 ml water) N = 9 Group : 2 3 ml of BTB (446 mg/kg dose) / N = 9 1h post treatment rats were subjected to; • Bar holding test (Plaznik, 1993) • Bridge test (Plaznik, 1993) • Righting reflex test (Martin, 1993)

15. Statistical analysis • The results are expressed as means  SEM (Standard Error • of Mean). • Statistical comparisons were made using Mann-Whitney U • test (Bluman, 1998) using Minitab 14.0 statistical package. • Significant level was set at P < 0.05.

16. RESULTS Anxiolytic activity in Rat hole-board test: Effect of O.P tea on locomotory activity Effect of O.P tea on no. of rears * * * * * * O.P tea significantly (P<0.05) increased the number of crossings (low dose by 43%, mid dose by 54% and high dose by 50% ) Mid dose (by 58%) and high dose (by 58%) of O.P tea significantly (P < 0.05) increased the number of rears These two effects were dose dependent; r2 = 1.0, P < 0.05

17. Results of rat hole-board test cntd……. Effect of O.P tea on total dipping time * O.P tea significantly (P < 0.05) increased the total dipping time (low dose by 123%, mid dose by 65% and high dose by 114%) * * * This effect was dose dependent; r2 = 1.0, P < 0.05 Effect of O.P tea on no. of head dips All the test doses moderately increased the number of head dips (low dose by 49%, mid dose by 31% and high dose by 33%) Rats tested in hole-board did not show any overt signs of toxicity during the observed time period

18. Effect of O.P. tea on food intake in a novel environment: As shown in the Table , all three doses of BTB of O.P. grade black tea dose dependently (r2 = 1) increased the food intake (low dose by 25%, mid dose by 91% and high dose by 16%). However, the effect was significant (P < 0.05) only with the mid dose.

19. Effect of O.P. tea on milk intake in a novel environment: As shown in the Table, all three doses of BTB of O.P. grade black tea dose dependently (r2 = 1) increased the milk intake (low dose by 35%, mid dose by 66% and high dose by 25%). Like with food intake, significant (P < 0.05) increase in the milk intake was evident only with the mid dose

20. Effect of O.P. grade black tea on activity in elevated plus maze model The mid dose of BTB of O.P. grade black tea markedly and significantly (P < 0.05) increased * * * the time spent in open arms (by 129%) the number of entries to open arms (by 111%) * % open arm entries (by 51%)

21. * % time spent in the open arms (by 94%) *

22. * Mid dose of O.P. grade tea significantly (P< 0.05) decreased the time spent in closed arms (by 27%) * Time spent in the neutral zone and number of entries to closed arms was not significantly (P > 0.05) altered

23. Effect of O.P. tea on muscle strength and motor coordination Mid dose of BTB of O.P. grade black tea did not significantly (P > 0.05) affect on muscle strength and motor coordination

24. DISCUSSION • The results show, for the first time, that BTB of Sri Lankan • low grown orthodox O.P. grade black tea possesses • marked anxiolytic activity with no any observable side effects • It is almost comparable to Dust grade No.1 black tea • This BTB induced anxiolytic activity had a quick onset • (within 1h) indicating that the action is mediated via a • phytoconstituent/s already present in the BTB • Since the anxiolytic activity of O.P. tea was dose-dependent, • it indicates that the effect is genuine, specific and • possibly receptor mediated. • Also, this study scientifically justify the claims made by • some Sri Lankan traditional practitioners that black tea has • anxiolytic potentials.

25. GABAergic activity of O.P. grade black tea • The elevated plus maze model is based on rodents' natural • aversion to open spaces. • Therefore, they typically avoid the open arms of the maze • and spend most of their time in the two enclosed arms. • Anxiety reduction in the plus-maze is indicated by; • an increase in the open arm entries, time spent in the open • arms, % open arm entries and reduction in the time spent in • closed arms compared to the control and it is related with • GABAergic mechanism. • It is well recognized that GABA pathways play a critical role • in the etiology of and treatment of anxiety • Low levels of GABA in the brain are associated with anxiety

26. Increasing of the above mentioned parameters in elevated • plus maze model indicates GABAergic action of O.P. grade • tea in this study. • L-theaninepasses the blood-brain barrier and has been • shown to potentiate the release of GABA on GABAergic • synapses and increases GABA level in rat brains. • Flavanoids(such as flevanols, theaflavins and thearubigins • that are rich in tea) are shown to bind to GABAa receptors • and this mechanism may also play a role in modifying the • GABA signaling pathway to induce an anxiolytic action in this • study.

27. L - theanine Increaseserotonin production in brain (29). Increase brain dopamine level Possibly serotoninergic mediated action of O.P. tea Possibly dopaminergic mediated action of O.P. tea

28. CONCLUSION This study demonstrate, for the first time, that BTB made from Sri Lankan low grown orthodox O.P. grade black tea has fast acting oral anxiolytic activity with no observable side effects. Anxiolytic activity is possibly mediated via GABAergic, serotoninergic and dopaminergic mechanisms. As claimed in Sri Lankan traditional medicine, regular consumption of at least 3 cups of black tea may be a useful strategy to alleviate day to day anxiety resulting from present day stressful lifestyles.

29. ACKNOWLEDGEMENT This study received financial support from the National Science Foundation of Sri Lanka under the grant number NSF/Fellow/2011/01.

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