ISCA-ISC-2015-13PCS-11-Oral

Research Journal of Recent Sciences _______________________________________________ E-ISSN 2277-2502
Vol. 5(ISC-2015), 1-5 (2016) Res. J. Recent. Sci.
International Science Community Association 1
Antioxidant property and Acetylcholinesterase inhibitory activity by Tea
Polyphenolics Grown in different Agricultural Practices
Ananya Bagchi*1
, Dillip Kumar Swain1
, Analava Mitra2
and Nairanjana Bera3
1
Agricultural and Food Engineering Department IIT Kharagpur, WB, India
2
School of Medical Science and Technology IIT Kharagpur, WB, India
3
Quality Control Section), Food Corporation of India, Regional Office (W.B.), Kolkata-700016, WB, India
bagchiananya13@gmail.com
Available online at: www.isca.in
Received rd
2014, revised th
2014, accepted th
2014
Abstract
Antioxidant capacity of Camellia sinensis (tea) variety TV25 extract grown in different agricultural practices in plain land of
IIT Kharagpur’s Agricultural and Food Engineering department were studied for free radical scavenging activities by
DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2’-azino-bis-(3- ethylbenzothiazoline)-6-sulfonic acid) and FRAP (Ferric
Reducing Antioxidant Potential) assays. DPPH, FRAP and ABTS assays showed an increase in amount of total
phenolicscontent in addition to increase in magnitude of free radical scavenging activities whenethanolic extracts of
organically grown tea is studied. The DPPH assay showed free radical scavenging activity values 75.3 for
vermicompost+vermiwash, 68.0 for vermicompost, 44.9for Inorganic fertilizer and 58.7 in control in 300µg/ml tea extract
concentration. Significant acetylcholinesterase (AChE)-inhibitory activity potential found in ethanolic extract with the AChE
enzyme activity 0.000612 U in control, 0.00812 U in Inorganic fertilizer treatment, 0.00018 in vermicompost and 0.00015 in
vermicompost + vermiwash fertilizer treatment management practices.
Keywords: Organic farming, Vermicompost, DPPH assay, Acetylcholinesterase inhibitory activity..
Introduction
Tea, beverage which stands 2nd
in popularity throughout the
world is an infusion of dried leaves of Camellia sinensis1
plants. It is a potent source of secondary metabolites like
monoterpenoids, carotenoids and catechins etc. Tea drinking
was originated in China around 4000–5000 years ago and now
more than 3 billion cups of tea are consumed everyday all over
the world. Volatile monoterpenoids and carotenoids are the
main compounds of tea aroma whereas catechins are beneficial
for health. Now-a-days, tea is cultivated in 30 countries around
the world and all varieties and cultivars of the tea plant
originated from Camellia sinensis2
, a member of Theaceae
family. Though all varieties originated from the same species,
but their distinctive taste and health benefits derived from the
differences in post-harvest processing of tea leaves as it is
found in different parts of the world, thus produces oolong tea,
green tea or black tea3
. The antioxidant activities of tea are
offered by its wide arrays of catechin content eg.epicatechin
(EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3- gallate
(ECG) and(-)-epigallocatechin-3-gallate (EGCG) along with
other minor catechinslike,(+)-catechin (C), gallocatechin
(GC), (-)-gallocatechingallate (GCG) and (-)-catechingallate
(CG)4
. They are determined to be more efficient radical
scavengers than some vitamins namely vitamin E and C5,6
. The
concentration of different polyphenols are EGCG followed by
EGC, ECG and EC in descending order7,8
and as studied in tea
extract that EGCG is more than 10% by dry weight9
.
Alois Alzheimer, a German psychiatristfirst distinguished a
neurodegenerative disorder, namely, Alzheimer’s disease in
190610
. Early symptoms are usually difficulties in
remembering recent incidents that is mistaken as
manifestations of stress or 'Age-Related' concerns11
. With the
progression of disease,long term memory loss along with
symptoms of irritability, aggression, increase in confusion and
mood swings appear11, 12
. Several hypotheses proposed so far
regarding the causes and based on which currently available
drugs and therapies are developed13
. The oldest among the
hypothesis is the cholinergic hypothesis which proposes the
disease as a cause of reduction in synthesis of a
neurotransmitter i.e. acetylcholine.
The present study is based on the cholinergic hypothesis that
states the reduction in availability of acetylcholine, one of the
main neurotransmitter responsible for transmitting neuron
signal. The reduction is due to the sudden hyperactivity of
acetyl cholinesterase (AChE), enzyme that cleaves
acetylcholine into choline and acetate, a prevalent
phenomenon that occurs with the advancement of Alzheimer's
disease.
Materials and Methods
Studies on the polyphenol analysis, estimation of antioxidant
properties by DPPH method and evaluating

Research Journal of Recent Sciences ____________________________________________________________ E-ISSN 2277-2502
acetylcholinesterase inhibitory activity of fresh tea leaves of
TV 25 variety was planned.
Plant materials: The Department of Agriculture and Food
Engineering, IIT Kharagpur, provided the experimental land. it
is situatedat 22° 19’N and 87° 19’E in South-Eastern part of
West Bengal, India on an elevation of 44m above mean sea
level and at a distance of 115 km from the Bay of Bengal. The
climate is warm and humid with an annual rainfall of130cm-
150cm and 85% of total rainfall received during June-October
from South-West monsoon. During the experimental period
the annual rainfall received was in the range 111cm to 196cm.
The average maximum temperature varied from 32.1°C to
33.3°C and the mean minimum temperature varied from
22.1°C to 23.4°C. The average maximum and minimum
relative humidity ranged from 91.3% to 94.8% and 39.4% to
41.2% respectively. The duration of average daylight period
varied from 5 to 10 hours per day. The soil of the experimental
field is sandy loam in texture and acid laterite (type –
Haplustalf) in nature, having low organic Carbon and
Nitrogen(N) content and medium to low in Phosphorus and
potassium content. Fresh leaves were collected from the
Assam variety of Camellia sinensis L. namely, TV-25 grown
in the tea garden at the stage of ‘two leaves and a bud’ were
processed for preparation of green tea.
Chemicals: Analytical grade chemicals, used in sample
preparation were purchased from Merck (India). Electric
eleAcetylcholinesterase enzyme (EC 3.1.1.7) standard was
purchased from Sigma (India). Tris, bovine serum albumin, 1-
naphthyl acetate and Fast Blue B salt were purchased from
Himedia (India). HPLC grade solvents were obtained from
Merck (India). Authentic standard compounds used for HPLC
analysis were purchased from Sigma (India).
Sample preparation: Fresh tea leaves of TV 25 variety (5 g)
were taken and extraction was done with 200 ml of 80% (v/v)
methanol by soxhlet apparatus for 10 cycles. Thereafter,
excess methanol was removed from the extract by rotary
vacuum evaporator and the residue lyophilized to get the
dehydrated powdered form and stored at – 20ºC for further
requirements.
HPLC analysis: Modified HPLC analysis was done for
polyphenol content in leaves14
with HPLC Waters 600
Separation Module in addition to quaternary solvent delivery
pumps and in line degasser with a photo diode array detector
(Model: Waters 2998) set at 200-600nm wavelength.
Separation column used was XTerra RP 18 column (5 µm, 250
x 4.6 mm) and the chromatogram was detected at 274 nm. The
results were analyzed by Empower Waters software (Waters
Corporation, Singapore). A gradient linear solvent system
of2% acetic acid and acetonitrile (23:2) used with a flow rate
of 1 ml/min for 40 min to elute the phenolic compounds. The
experiment was done at room temperature, injection volumes
20 µl of the standards and sample extracts from 1mg/ml stock
solution and operating gradient was 92% of A and 8% of B at
0 min, 69% A and 31% of B at 25 min, 60% of A and 40% of
B at 35 min, 0% of A and 100% of B at 37 and 40 min.
Identification of separated constituents in eluents were
executed by comparing retention times and spectral data of
eluents with those of authenticstandards.
Agricultural and soil characteristics for tea plantation:
TV-25 variety cultivated in fallow land, in the Agriculture and
food engineering department, IIT Kharagpur which hadnot
been cultivated for years. The soil of experimental site was
acid lateritic, sandy‐loam in texture, with pH ~5.1 and low in
organic carbon content 0.3% and available NPK content
72.7ppm, 11.8ppm and 47ppm respectively. Plants were raised
in a control plot, two organic fertilization treatments i.e.,
Vermicompost (VC), Vermicompost + Vermiwash (VC+Vw)
and Inorganic Fertilizer (IF) treatments during
experimentation. The experiment was executed in four
replications with TV-25 in split‐plot design (TV-25 in main
plot and fertilizer source in sub-plot) with subplots of 20m×20
m. The treatments given on yearly basis with NPK at a ratio of
10:3:6 in the form of urea, SSP and MOP. Contamination
between the treatments and leaching was prevented by
inserting plastic sheetsupto a depth of 4 m within the soil and
0.5m above the surface soil. The vermicompost was applied @
833 gm/pit for all VC and VC+VW treatments. In the VC+Vw
treatment, in addition to vermicompost application, vermiwash
was sprayed @ 650 Lt per ha, which supplied approx. 0.0325
kg N/ha (calculation made to supply 200 kg N/ha through
Vermicompost treatment taking into consideration its N
content of 1.8% and in case of vermiwash N equivalent of
0.005%). The control plot was treated with water only and
without any fertilizer.
Evaluation of antioxidant activity
DPPH (2, 2 diphenyl -1 picrylhydrazyl) radical scavenging
assay: The antioxidant property of fresh tea leaf extract of
TV25 variety was measured in terms of hydrogen ion donating
or radical scavenging activity, with the free radical DPPH15
.
The ethanolic solution of lyophilized tea extracts of
concentration 1 mg/ml was used as stock solution to monitor
radical scavenging activity. The free radical scavenging ability
of lyophilized tea extracts at concentrations of 50, 100,200,
and 300 µg/mL as described in Table-2 were evaluated. A
higherradical scavenging activity in tea leaf extracts of the
VC+Vwtreatment was found, followed by VC, Control and IF
for TV 25 variety. Radical scavenging capacity of the tea
extracts were steadily rose up to the concentration of 300
µg/mL.
Evaluation of acetylcholinesterase (AChE) inhibitory
activity: Continuous spectrophotometric assay were made for
determination of acetylcholinesterase activity

Enzyme preparation: AChE enzyme (500 U) solution was
made by adding 1 mg lyophilized enzyme in 1 ml of miliQ
water.
Sample preparation: Lyophilized extract of 1mg tea sample
was dissolved in 200 µl of ethanol and kept at – 20 ºC for
further requirements.
Enzyme Assay: AChE activity was measured using 1-
naphthyl acetate as a substrate for the reaction16
. Generation of
1-naphthol was measured with a spectrophotometrically Firstly
continuous spectrophotometric assay of normal enzyme
catalyzed reaction was carried out and product formation was
monitored. Absorbance was measured at 320 nm at for 5 min
at 0.5 min intervals as absorption maxima of the product 1-
naphthol is 320 nm. The control reaction mixture (1 ml)
contained 710 µl of 50 mM potassium phosphate buffer (pH
7), 250 µl of 2 mM substrate and 40 µl of enzyme solution.
Then 50 µl of tea extract solution was introduced along with
660 µl of buffer, 250 µl of 2 mM substrate and 40 µl of
enzyme solution. Again the product formation was monitored
keeping all the conditions same. The enzyme activity was
calculated by using following formula:
1U= (∆A X 1) / (Є X 0.04)
∆A= (A2 –A1)/T
Where, A2 = Final absorbance value at time = 5 min. A1 =
Initial absorbance value at time = 0 min. ∆A = Change in
absorbance. T = Total time (5 min). Є = Molar extinction
coefficient of 1 naphthol = 2,222 M -1
cm-1
. 1 = Volume of
total reaction mixture (1 ml) = dilution factor. 0.04 = Volume
of enzyme solution in the reaction mixture (0.04 ml)
Statistical Analysis: Quantitative results were expressed as
mean values and standard deviation (SD). Standard error was
shown in some cases where SD was too low. Mean was
calculated by dividing the sum of all the observations (X) by
the total number of observations (n) from the following
formula:
Standard deviation (S.D): The standard deviations were
calculated by the following formula:
A spectral analysis was also performed with and without the
addition of the inhibitor (keeping the same volume of buffer,
substrate, enzyme and inhibitor as mentioned above) in a wide
range of wavelength starting from 190 nm to 600 nm for 5
min.
Results and Discussion
Polyphenols, a group of secondary metabolitesare vital for
tea quality, aroma and antioxidant properties. The effect of
different fertilizer treatments i.e. organic with vermicompost
and vermiwash as well as inorganic fertilizer with SSP and
MOP on synthesis of polyphenolic antioxidants in tea plants
are presented in Table-1. Total catechin content in tea leaves
grown under inorganic fertilizer treatments was significantly
lower as compared to the organic treatments, i.e., VC,VC+Vw
and Control with Gallic Acid Equivalent of 195.44 mg/gm,
281.05 mg/gm, 287 mg/gm, 245.34 mg/gm respectively. It
wasfound that the total concentration of (-)-EGCG, (-)-GCG
and (-)-ECG were in greater amount in VC and VC+Vw than
in IF in Table-1. Increase in polyphenol concentration of
organically grown fieldcrops has also been described in other
lituratures17
. Tea catechins are described as
potentantioxidants with the efficiency to regulatein vivo key
biological pathways in mammals17, 18
. For the determination
of the radical scavenging or antioxidant activity of tea, a
rapid antioxidant activity measurement by DPPH staining
method19
was done. The assay is performed in presence of
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and the
scavenging ability of the antioxidants were performedby
transforming it tothe hydrazine20, 21
. The DPPH converted
violet to pale yellow in reaction with hydrogendonors and
according to the radical scavenging property of the
antioxidant22
. The radical scavenging ability of lyophilized
tea extracts at different concentrations is presented in Table-
2. Higher radical scavenging ability in tea leaf extracts of the
VC+Vw treatments were determined, followed by VC,
Control, and IF for TV25.
Detection of acetylcholinesterase inhibitory activity
measurement by continuous spectrophoto metric assay
method: The formation of 1-naphthol with the substrate 1-
naphthyl acetate byacetylcholinesterase enzyme catalyzed
reaction was continuously studied for 5 minutes at 320 nm
during the experiment. Acetylcholinesterase enzyme activity
was measured in presence and absence of the test tea
samples. The amount of product formed, increased with time
as determined in 320 nm. With the inclusion of the different
concentration of tea extracts from different treatments, the
increment in the product formation was decreased gradually.
Table-1 showed that the enzyme activity of control was
0.000612 U while that of the reaction mixture containing
ethanolic solution of lyophilized extract of TV 25 fresh tea
leaves grown in IF treatment, TV 25 fresh tea leaves grown
in Vermicompost, TV 25 fresh leaves grown in
vermicompost +vermiwash were 0.00812 U, 0.00018 U,
0.00015 U. The comparison is depicted in Table 1.

Table-1
Polyphenolic concentration (mg/gm) in tea leaves as influenced by fertilizer sources
Variety Treatments
EGCG mg/gm tea
leaves
GCG mg/gm
tea leaves
ECG mg/gm
tea leaves
Total catechin content
mg/gm tea leaves
TV25
C 8.00 4.50 3.40 15.90
IF 1.95 3.50 1.81 7.26
VC 10.11 4.84 2.95 17.90
VC+VW 9.00 7.00 3.85 19.85
Table-2
Free radical scavenging property for DPPH radical (% inhibition) of tea leaf extracts as influenced bydifferent fertilizer
sources
Treatments
Experimental concentration of tea extracts
50µg/ml 100µg/ml 200µg/ml 300µg/ml
C 23.00 35.00 58.00 58.70
IF 19.00 30.50 44.40 44.90
VC 24.00 39.40 68.00 68.00
VC+VW 26.90 43.00 75.00 75.30
LSD(p=0.05%) 2.10 2.50 4.10 4.30
0.000612
0.00812
0.00018 0.00015
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
control IF V V+VW
enzyme activity (U)
enzyme activity (U)
Figure-1
Acetylcholinesterase enzyme activity (U) measurement in tea grown in different ferlizer treatment

Conclusion
Organically grown tea has shown improvement in quality
parameter i.e. increase in antioxidants as well as catechin levels.
Organic tea cultivated in the experimental site has shown higher
antioxidant properties because of high concentration of
catechins. The tea grown in Vermicompost+ Vermiwash has the
highest AChE enzyme inhibitory activity because the enzyme
activity is lowest in this treatment in comparison to other
treatments. Thus, this study showed that organically grown tea
having betterpotential to inhibit AChE enzyme by scavenging
free radicals those areresponsible for Alzheimer’s disease.
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