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.
INTRODUCTION
Tea is manufactured from top most immature leaves and buds of
Camellia sinensis (L) O. Kuntz (Family: Theaceae)
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
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:
1. Black (fully aerated or fermented)
2. Green (unaerated or unfermented)
3. Oolong (semi aerated or semi fermented)
Classification of black tea
Whole leaf grade(twisted but, not broken)
Eg: Orange Pekoe
Broken grade(different size tea particles)
Broken leaf grade
Eg: Broken Orange Pekoe Fannings
gradeEg: BOPF
Dust grade Eg: Dust No. 1
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
Anxiety
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
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
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.
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 solids
Brewed 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)
Evaluation of anxiolytic activity of O.P. grade black tea
Tested in rats using three models of anxiety;
1. 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 toxicity
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
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
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
Open arm
Closed arm
Neutral zone
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
Calculated indices:
Total number of entries =
% open arm entries =
% time spent in open arms =
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)
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.
RESULTSAnxiolytic activity in Rat hole-board test:
Control 1.5 cups 3 cups 9 cups0
5
10
15
20
25
30
35
Treatment
No.
of c
ross
ings
Effect of O.P tea on locomotory activity
Control 1.5 cups 3 cups 9 cups0
5
10
15
20
25
30
35
40
Treatment
No.
of r
ears
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
*
Results of rat hole-board test cntd…….
Control 1.5 cups 3 cups 9 cups0
2
4
6
8
10
12
14
Treatment
Tota
l dip
ping
tim
eEffect 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
Rats tested in hole-board did not show any overt signs of toxicity during the observed time period
*
Control 1.5 cups 3 cups 9 cups0123456789
10
Treatment
No.
of h
ead
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%)
Effect of O.P tea on no. of head dips
Treatment Food intake (g) Control (water/ N = 6) 0.41 ± 0.046
1.5 cups (223 mg/kg; N = 6) 0.516 ± 0.017
3 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 (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.
Treatment Milk intake (g)
Control (water/ N = 6) 15.33 ± 2.76
1.5 cups (223 mg/kg; N = 6) 20.78 ± 0.99
3 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 (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
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 *
Control
3 cups
0 10 20 30 40 50 60 70
Time spent in open arms
Trea
tmen
t
Control
3 cups
0 1 2 3 4 5 6
No. of entries to open arms
Trea
tmen
t
Control
3 cups
0 5 10 15 20 25 30 35 40 45
% open arm entries
Trea
tmen
t
**
*
the number of entries to open arms(by 111%)
% open arm entries (by 51%)
the time spent in open arms (by 129%)
% time spent in the open arms (by 94%)
Control
3 cups
0 5 10 15 20 25 30 35
% time spent in open arms
Trea
tmen
t
Control
3 cups
0 2 4 6 8 10 12
No. of entries to neutral zone
Trea
tmen
t
*
*
Mid dose of O.P. grade tea significantly (P< 0.05) decreased the time spent in closed arms (by 27%)
*
Control
3 cups
0 50 100 150 200 250
Time spent in closed arms
Trea
tmen
t
Time spent in the neutral zone and number of entries to closed arms was not significantly (P > 0.05) altered
*
Mid dose of BTB of O.P. grade black tea did not significantly (P > 0.05) affect on muscle strength and motor coordination
Effect of O.P. tea on muscle strength and motor coordination
Control 3 cups05
10152025303540
Treatment
Hold
ing
lata
ncy
of b
ar
(sec
)
Control 3 cups0
5
10
15
20
25
TreatmentLa
tenc
y in
Brid
ge te
st
(sec
)
Control 3 cups0
0.2
0.4
0.6
0.8
1
1.2
Treatment
Rith
ing
refle
x la
tenc
y (s
ec)
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.
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
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.
Increase serotonin production in brain (29).
L - theanine
Increase brain dopamine level
Possibly serotoninergic
mediated action of O.P. tea
Possibly dopaminergic
mediated action of O.P. tea
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.
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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