Anxiolytic - PowerPoint Presentation

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Anxiolytic action of Sri Lankan Orange Pekoe grade of black tea (Camellia sinensis L.) in ratsT.B.S. Muthunayake1 , W.D. Ratnasooriya1 , E.K. Indeesha1 , C.D.T. Ratnasooriya11 Department of Zoology, University of Colombo, Colombo 03.Slide2

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/annumSlide3

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.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)Slide4

Classification of black teaWhole leaf grade(twisted but, not broken)Eg: Orange PekoeBroken grade(different size tea particles)Broken leaf gradeEg: Broken Orange PekoeFannings gradeEg: BOPFDust grade Eg

: Dust No. 1Slide5

Anxiety is an emotional state, unpleasant in nature and is associated with uneasiness, discomfort and concern or fear about some defined or undefined future threatAnxiety 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 anxietySlide6

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 testedSlide7

Aim of the studyTo 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.Slide8

METHODOLOGYPreparation 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 36.1 % w/v tea solidsBrewed for 5 min(ISO 3103) Based on this, human equivalent doses for rats were prepared as follows:+10 g of O.P. grade black tea

Brewed for

5 min

9 cups

(1339 mg/kg) dose in 3 ml

30 ml of boiling water

1.5 cups (223 mg/kg)

3 cups (446 mg/kg)

9 cups (1339 mg/kg)Slide9

Evaluation of anxiolytic activity of O.P. grade black teaTested in rats using three models of anxiety; Rat hole-boardMonitored 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 toxicitySlide10

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 cagesSlide11

Evaluation of the GABAergic activity using the elevated plus maze modelRats were randomly divided in to 2 groups & orally administered as followsGroup : 1 Control (3 ml water)N = 8Group : 2 3 ml of BTB(446 mg/kg dose ) / N = 81h 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 minSlide12

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 zoneOpen armClosed armNeutral zoneSlide13

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 armsCalculated indices: Total number of entries =% open arm entries =% time spent in open arms =Slide14

Evaluation of muscle strength and motor coordinationRats were randomly divided in to 2 groups & orally administered as followsGroup : 1 Control (3 ml water)N = 9Group : 2 3 ml of BTB(446 mg/kg dose) / N = 91h post treatment rats were subjected to; Bar holding test (Plaznik, 1993) Bridge test (Plaznik

, 1993) Righting reflex test (Martin, 1993)Slide15

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. Slide16

RESULTSAnxiolytic 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 rearsThese two effects were dose dependent; r2 = 1.0, P < 0.05*Slide17

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 < 0.05Rats tested in hole-board did not show any overt signs of toxicity during the observed time period*All the test doses moderately increased the number of head dips (low dose by 49%, mid dose by 31% and high dose by 33%)Effect of O.P tea on no. of head dips Slide18

TreatmentFood intake (g) Control (water/ N = 6)0.41 ± 0.0461.5 cups (223 mg/kg; N = 6)0.516 ± 0.0173 cups (446 mg/kg ; N = 6)

0.784 ± 0.069 *

9 cups (1339 mg/kg ; N = 6)

0.474 ± 0.026

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 (r

2

= 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. Slide19

TreatmentMilk intake (g) Control (water/ N = 6)15.33 ± 2.761.5 cups (223 mg/kg; N = 6)20.78 ± 0.993 cups (446 mg/kg ; N = 6)

25.5 ± 1.4 *

9 cups (1339 mg/kg ; N = 6)

19.17

± 1.89

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 (r

2

= 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 doseSlide20

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 number of entries to open arms(by 111%)% open arm entries (by 51%) the time spent in open arms (by 129%)Slide21

% time spent in the open arms (by 94%) **Slide22

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*Slide23

Mid dose of BTB of O.P. grade black tea did not significantly (P > 0.05) affect on muscle strength and motor coordinationEffect of O.P. tea on muscle strength and motor coordinationSlide24

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 Also, this study scientifically justify the claims made by some Sri Lankan traditional practitioners that black tea has anxiolytic potentials. Since the anxiolytic activity of O.P. tea was dose-dependent, it indicates that the effect is genuine, specific and possibly receptor mediated. Slide25

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 anxietySlide26

Increasing of the above mentioned parameters in elevated plus maze model indicates GABAergic action of O.P. grade tea in this study. L-theanine passes 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. Slide27

Increase serotonin production in brain (29). L - theanineIncrease brain dopamine level Possibly serotoninergic mediated action of O.P. teaPossibly dopaminergic mediated action of O.P. teaSlide28

CONCLUSIONThis 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. Slide29

ACKNOWLEDGEMENTThis study received financial support from the National Science Foundation of Sri Lanka under the grant number NSF/Fellow/2011/01.Slide30

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