World Research Journal of Pharmaceutical Research

Volume 1, Issue 1, 2013, pp.-01-04.

Available online at http://www.bioinfopublication.org/jouarchive.php?opt=&jouid=BPJ0000294

ANTIMICROBIAL ACTIVITY OF Camellia sinensis LEAVES AGAINST GRAM POSITIVE

AND
GRAM NEGATIVE BACTERIA

1 2 3
TARIQ A.L. *, NIRJANTHA D. AND REYAZ A.L.
1
Department of Microbiology, Sree Amman Arts and Science College, Erode- 638 003, TN, India.
2
Department of Biotechnology, KSR College of Arts and Science, Tiruchengode- 636 011, TN, India.
3
Department of Biotechnology, Periyar University Salem- 636 011, TN, India.
*Corresponding Author: Email- tariqtasin@gmail.com

Received: August 18, 2012; Accepted: July 01, 2013

Abstract- Pathogenic microorganisms are big threat for the human health as they show resistance to drugs. In order to overcome this
problem an alternative potential treatment chosen is the use of herbal medicines. Green tea leaves were identified as Camellia sinensis. The
phyto-chemical analysis showed the presence of alkaloids, flavonoids, steroids and tannins by changing the colour of the extracts when
treated with respective reagents. The absence of the terpenoid, saponins and glycosides was confirmed by no change in the colour in the
solution. The extracts of Camellia sinensis showed the presence of various functional groups when run through Fourier transform infrared
spectroscopy. The methanolic extract of Camellia sinensis showed the antibacterial activity against gram positive bacteria such as
Staphylococcus aureus, Bacillus subtilis and gram negative bacteria as Escherichia coli, Enterococcus sp and Xanthomonas sp. The highest
susceptibility was shown in Staphylococcus aureus and Escherichia coli.
Keywords- Camellia sinensis, antibacterial activity, Staphylococcus aureus, Escherichia coli, functional group analysis
Introduction
The gram negative microorganisms such as Pseudomonas species,
Proteus species, Escherichia coli, Shigella dysenteriae and
Klebsiella pneumonia were the most resistant microorganisms [1].
Microorganism like Staphylococcus aureus and Pseudomonas ae-
ruginosa are the common opportunistic pathogen of human skin [2].
Serratia marcescens and Pseudomonas aeruginosa is a pathogen
associated with pyogenic infection and urinary tract infection [3,4].
Medicinal plants are important source of pharmacological effects
that act as new anti-infectious agents [5].The most important bioac-
tive constituents of plants are steroids, terpenoids, carotenoids,
flavonoids, alkaloids, tannins and glycosides which have served a
valuable starting material for drug development [6]. Tea ( Camellia
sinensis) is consumed worldwide and is second only to water in its
popularity as a beverage and has ascribed many health benefits viz
reduction of cholesterol, protection against cardio-vascular disease
and cancer [7]. Green tea is generally safe, non-toxic and having no
side effects after use [8]. The consumption of green tea can kill
Gram positive Staphylococcus aureus and many other harmful bac-
teria [9]. Camellia sinensis possess antibiotics or antimicrobial sub-
stances like saponins, glycosides, flavonoids and alkaloids [10].
Plant materials have shown the antimicrobial activity on various
pathogenic microorganisms therefore consumption of tea has been
associated with reduced risk of major diseases [11]. Plant leaves
are the most commonly used traditionally natural antimicrobial
agents against various drug resistant microorganisms for thousands
of years by many cultures of peoples in the past decade [12].
Materials and Methods
Collection of the Sample
The fresh leaves of Camellia sinensis were collected [13] from the
dense tea state garden at Ooty, Coimbatore district, Tamil Nadu
South India.
Preparation of the Extract
The leaves of fresh samples were cleaned and washed under run-
ning tap water [14]. The samples were dried in the oven at 37C for
6 days. After drying the samples were weighed and blended with
warring blender and soaked with methanol [in ratio methanol: plant
(6:1)] for 2 days and filtered using Whatman No. 1 paper. The meth-
anol was completely removed by vacuum evaporator at 50C till it
gave a viscous mass. The crude extracts were weighed and stored
at 4C before analysis.
Preliminary Phytochemical Screening
The tests were done [15] to find the presence of the active chemical
constituents such as alkaloids, glycosides, terpenoids and steroids,
flavonoids, reducing sugar and tannin.
Alkaloid Test
The alcoholic extract was evaporated to dryness and the residue
was heated on a boiling water bath with 2% Hydrochloric acid. After
cooling, the mixture was filtered and treated with a few drops of 5%
Sodium Hydroxide solution. The samples were then observed for
the presence of turbidity or yellow precipitation
Glycoside Test
0.5grams of extract was dissolved in 2ml of glacial acetic acid and
mixed well. To this few drops of ferric chloride and concentrated
sulphuric acid were added and observed for a reddish brown color-
ation at the junction of two layers and the bluish green color in the
upper layer.
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Volume 1, Issue 1, 2013

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Tariq A.L., Nirjantha D. and Reyaz A.L. (2013) Antimicrobial Activity of Camellia sinensis Leaves against Gram Positive and Gram Negative Bacteria.
World Research Journal of Pharmaceutical Research, Volume 1, Issue 1, pp.-01-04.
Terpenoid Test
Four mg of extract was treated with 0.5 ml of acetic anhydride and
0.5 ml of chloroform. Then concentrated solution of sulphuric acid
was added slowly and red violet color was observed for the pres-
ence of terpenoid.
Steroid Test
Four mg of extract was treated with 0.5 ml of acetic anhydride and
0.5 ml of chloroform. Then concentrated solution of sulphuric acid
was added slowly and green bluish color was observed for pres-
ence of steroids.
ous concentrations (100g, 200g and 300g) of extracts. The
plates were incubated at 37C for overnight. The results were ex-
pressed in terms of the diameter of the inhibition zone and Metha-
nol used as control.
Result
Identification of Leaves
The leaves belongs to the Kingdom-Plantae, Order-Ericales, Family
-Theaceae, Genus-Camellia, Species-sinensis is shown in [Fig-1].

Flavonoid test
2ml of extract solution was treated with 1ml of lead acetate solution
and white colour was observed for the presence of flavonoids.

Gallic Tannin Test

0.5 ml of extract was dissolved in 1 ml of water, mixed uniformly
and then 2 drops of ferric chloride solution were added and blue
color was observed for presence of gallic tannin.

Catecholic Tannin Test

0.5 ml of extract was dissolved in 1 ml of water, mixed uniformly
then 2 drops of ferric chloride solution were added and green black Fig. 1- The morphological appearance of green leaves shown
colour was observed for presence of catecholic tannin. it belongs to Kingdom-Plantae, Order-Ericales, Family-
Theaceae, Genus-Camellia, Species-sinensis
Saponins Test
0.5ml of extract was treated with 5ml of distilled water and frothing Qualitative Analysis of Phytochemicals
persistence indicate the presence of saponins. The extract showed the presence of phytochemicals namely alka-
loids, flavonoids, steroids, gallic tannins and catecholic tannin by
Fourier Transform Infrared Spectroscopy Qualitative Analysis changing the colour of the solution to yellow, white, green bluish,
Camellia sinensis extracts was subjected to Fourier Transform In- blue, green black respectively. But indicated the absence of terpe-
frared Spectroscopy Qualitative analysis [16] by using Perkin Elmer noid, saponins, and glycosides as there was no colour change in
Fourier Transform Infrared Spectroscope (Model-spectra 100) in- the solution with respect to them.
strument and the obtained spectra for the product was analyzed
and interpreted with a chart for characteristics infrared absorption FT-IR Qualitative Analysis
frequencies of organic functional groups and carbonyl containing The Fourier Transform Infrared Spectroscopy (FT-IR) Qualitative
functional group. analysis of Camellia sinensis obtained was analyzed and interpret-
ed with a chart for characteristics infrared absorption frequencies of
Antimicrobial activity of Camellia sinensis organic functional groups and carbonyl containing functional group
Leaves Preparation of Inoculum which showed the presence of alkene, alcohol, ester, amine acid,
The inoculum was prepared by [4] culturing the microorganisms in alkane, aromatic alkane, nitro compounds, aromatic amide, alkene
nutrient broth at 37C for 12 hours to a concentration of approxi- amide, carbonyl anhydride, alkane hydroxyl group ketone, aromatic
mately 105 CFU/ml was used for the antimicrobial analysis. amine, alcohol amine and alcohol [Table-1].
Table 1- The Fourier Transform Infrared Spectroscopy analysis
Preparation of Extracts of Camellia sinensis are presented below
The dried powder of leaves was prepared by [14] taking 5g of S. Wave number Functional groups Type of vibration Intensity
leaves extracts and dissolved in 25ml of methanol and incubated for No. (absorptions) (cm -1)
24 hours at room temperature on constant shaking. After incubation 1 675.09 -1037.7 =C-H (Alkene) Bending 8.252915
the solution was filtered twice through Whatman no. 1 filter paper. 1060.85 (C-O) alcohol, Stretch 0.317464
2 (C- O) Ester
The filtrate was evaporated to dryness at 50C and then crude ex-
3 1037.7 (C-O) Ester, Stretch 0.120728
tracts were used for analyzing the antimicrobial activity of Camelia 1087.85-1095.57 (C-O) alcohol, Stretch 0.441905
sinensis leaves. 4 (C-N) amine
5 1244.09 (C-O) Acid Stretch 0.206284
Agar-well Diffusion Method 6 1386.82 (-C-H) alkane Bending 0.351405
The agar-well diffusion assay was adopted [14] for the present as- 1402.25 -1510.26 (C=C) aromatic Stretch Bending 0.57121
say. Each bacterial suspension was spread over the surface of 7 (-C-H) alkane
Mueller-Hinton agar (Himedia, India) plates containing 4 wells hav- 1525.69-1535.34 (C=C) aromatic, Stretch 0.773965
8 (N-O) Nitro compounds
ing 6 mm diameter. The wells were filled with 30l each of the vari-
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Tariq A.L., Nirjantha D. and Reyaz A.L. (2013) Antimicrobial Activity of Camellia sinensis Leaves against Gram Positive and Gram Negative Bacteria.
World Research Journal of Pharmaceutical Research, Volume 1, Issue 1, pp.-01-04.
Table 1- Continues concentration was increased. Similarly in case of gram negative
S. No. Wave number Functional groups Type of vibration Intensity bacteria such as Enterococcus sp, Xanthomonas sp Escherichia
(absorptions) (cm -1 ) coli [Fig-3] the zones of inhibition were measured in millimeter (mm)
1583.56-1595.13 (C=C) aromatic Stretch Bending 0.521811 and compared with antibiotic standard chart thus were found sus-
9 (N-H) amide
1635.64-1641.42 (C=C) alkene Stretch Bending 0.05785
ceptible.
10 (C=O)) Amide The susceptibility increased when the concentration was increased.
11 1730.15-1795.73 (C=O) carbonyl Stretch 9.729339
12 1820.8 (C=O) anhydride Stretch 12.00783
The more susceptible bacteria were found Staphylococcus aureus
13 2856.58 -2922.16 (C-H) alkane stretch 0.222975 and Escherichia coli.
14 3412.08 (O-H) hydroxyl group Stretch 0.026572
15 3439.08 Ketone Stretch 0.036351 Discussion
16 1508.33 (C=C) aromatic Stretch 0.793156 Camellia sinensis is the species of plant leaves and leaf buds are
3387 (N-H) Amine, Stretch 0.02208
used to produce Chinese tea [17]. The presence of phytochemical
17 (O-H) alcohol
3402.43 (N-H) Amine, Stretch 0.022399 namely alkaloids, flavonoids, steroids, gallic tannins, catecholic
18 (O-H) alcohol tannin plays the vital role in the plant defense mechanisms [5,10].
The active substance found in tea is supposed to reduce growth
Antimicrobial Activity and development of microorganisms [18]. The highest antimicrobial
The Camellia sinensis leaf extracts of various concentrations activity of tea is due to presence of catechins and polyphones which
(100g/ml, 200g/ml and 300g/ml) concentrations showed the damages bacterial cell membrane [19,20]. They also serve in plant
zone of inhibition against gram positive bacteria such as , Staphylo- defense mechanisms to counteract reactive oxygen species in or-
coccus aureus, Bacillus subtilis [Fig-2]. der to survive and prevent molecular damage and caused by micro-
organisms, insects, and herbivores [6]. The antibacterial activity of
Camellia sinensis leaf against Listeria monocytogenes by disc diffu-
sion method, the methanolic extract had greater antibacterial prop-
erty as compared to the water extract [13]. In this work, methanolic
extract of Camellia sinensis had greater antibacterial activity against
Staphylococcus aureus and Escherichia coli [21]. These observa-
tions are likely to be the result of the differences in cell wall struc-
ture between Gram-positive and Gram-negative bacteria [22]. In the
Gram-negative outer membrane acting as a barrier to many envi-
ronmental substances including antibiotics [23,24]. Spices in the
past decade confirmed that the growth of both Gram-positive and
Gram-negative food borne bacteria, yeast and molds can be inhibit-
ed [25]. Green tea leaf extracts tested in current study have also
shown varying activities against environmental bacteria [7]. The
Fig. 2- The antibacterial activity of Camellia sinensis agaainst green sorts of tea have shown higher antimicrobial activity than the
F1. black ones [26. The antibacterial activity of Camellia sinensis tea
Staphylococcus aureus and F2. Bacillus subtilis where A-100g/ml, extracts was selective and depends upon the concentration, type of
B-200g/ml, C-300g/ml of Camellia sinensis leaves and D-Control the extracts and bacterial species [12,19]. In this work different
wells. A zone of inhibition around the colonies are measured in concentration of leaf extracts were used against different pathogen-
millimeter (mm) ic bacteria and highest zone of inhibition was observed against
E.coli and Staphylococcus [9]. According to the antibacterial assay
done for screening purpose, all the gram positive microorganisms
such as Staphylococcus aureus, Bacillus subtilus, Bacillus cereus
and Bacillus thuringiensis were the most susceptible bacteria to all
plant extracts, whereas only one of the gram negative microorgan-
isms, i.e. Salmonella typhi was susceptible to one of the plant ex-
tracts [5,27].

Fig. 3- The antibacterial activity of Camellia sinensis agaainst F3.
Enterococcus sp., F4. Xanthomonas sp. and F5. Escherichia coli,
where A-100g/ml, B-200g/ml, C-300g/ml of methanolic extracts
Camellia sinensis leaves and D-Control wells. A zone of inhibition
around the colonies was measured in millimeter (mm)
The zones of inhibition of gram positive bacteria were measured in
millimeter (mm) and then compared with antibiotic standard chart
thus were found susceptible. The susceptibility increased when the
Conclusion
Camellia sinensis leaves are having the duel benefits as medicinal
values and food value. In this study we found that leaf extracts were
found to be potential antibacterial agents against the gram positive
Staphylococcus aureus and Escherichia coli as shown the maxi-
mum zone of inhibition. Thus Camellia sinensis leaves can be used
an alternative medicine against the bacterial infection.
Reference
[1] Odugbemi T. (2006) Nigeria University of Lagos press, 53-64.
[2] Souza E.L., Stamford T.L.M., Lima E.O., Trajano V.N. and Filho
 World Research Journal of Pharmaceutical Research
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 Tariq A.L., Nirjantha D. and Reyaz A.L. (2013) Antimicrobial Activity of Camellia sinensis Leaves against Gram Positive and Gram Negative Bacteria.
World Research Journal of Pharmaceutical Research, Volume 1, Issue 1, pp.-01-04.

J.B. (2005) Brazilian Arch. Biol. Technol., 4, 549-558.

[3] Islam G.M.R., Iqbal M., Quddus K.G. and Ali M.Y. (2005) Jour-
nal of Pakistan Academic Science, 42, 305-315.
[4] Tariq A.L. and Prabakaran J.J. (2011) Journal of Microbiology
and Antimicrobials, 3(6), 130-135.
[5] Ushimaru P.I., Mariama T.N., Luiz C., Di Luciano D. and Ary F.J.
(2007) Brazilian Journal of Microbiology, 38, 717-719.
[6] Akowuah G.A., Ismail Z, Norhayati I., Sadikun A. (2005) Journal
of Food Chemistry, 93, 311-317.
[7] Wang Z.Y., Huang M.T., Chang R., Ma W. and Ferraro T. (1992)
Cancer Research, 2, 6657-6665.
[8] Erturk Y., Ercisli S. and Tosun M. (2009) International Journal of
Plant Production, 3, 89-92.
[9] Catherine A.F. (2007) Tropical Biomedicine, 22(2), 165-170.
[10] Padmini E., Valarmathi A. and Rani Usha M. (2011) Asian Jour-
nal of Experimental Biological Science, 4, 772-778.
[11] Robinson E.E., Maxwell S.R.J. and Thorpe G.H.G. (1997) Free
Radical Research, 26, 291-302.
[12] Tiwari R.P., Bharti S.K, Kaur H.D., Dikshit R.P. and Hoondal
G.S. (2005) Journal of Medical Research, 122, 80-84.
[13] Mbata T.I., Debiao L.U. and Saikia A. (2008) African Journal of
Biotechnology, 7, 1571-3.
[14] Tariq A.L. and Reyaz A.L. (2012) Universal Journal of Medicine
and Dentistry, 1(4), 051-055.
[15] Zuo Y., Chen H. and Deng G. (2002) Journal of Talanta, 57,
307-316.
[16] Johny J.M., Kulandhaivel M., Palaniswamy M. and Reeta J.
(2011) International Journal of Pharmaceutical and Biological
Archives, 2(4), 1202-120.
[17] Zakaria M. (1991) Asia Pacific Journal of Pharmacology, 6, 15-
20.
[18] Sarkar A. (2001) Biochemical and Biophysical Research Com-
munication, 284(1), 173-178.
[19] Hsu C.L., Chen W.L., Weng Y.M. and Tseng C.Y. (2003) Jour-
nal of Food Chemistry, 83, 85-92.
[20] Thangapazham R.L., Singh A.K., Sharma A., Warren J., Gad-
dipati J.P. and Maheshwari R.K. (2007) Cancer Letters, 245,
232-241.
[21] Yam T.S., Shah S. and Hamilton-Miller J.M.T. (1997) FEMS
Microbiology Letters, 152, 169-174.
[22] Toda M., Okubo S., Ikigai H., Suzuki T. and Shimamura T.
(1991) Journal of Applied Bacteriology, 70, 109-112.
[23] Smith D.M. (2001) International Journal of Molecular Medicine ,
7(6), 645-52.
[24] Kaneria M., Baravalia Y., Vaghasiya Y. and Chanda S. (2009)
Indian Journal of Pharmaceutical Sciences, 71, 406-412.
[25] Rathee J.S., Hassarajani S.A. and Chattopadhyay S. (2007)
Journal of Food Chemistry, 103, 1350-135.
[26] Chou C.C., Lin L.L. and Chung K.T. (1999) International
Journal of Food Microbiology, 48, 125-130.
[27] Salvet A., Antonnacci L., Fortunado R.H., Suarez E.Y. and Go-
doy H.M. (2001) Journal of Ethnopharmacology, 32, 293-297.

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