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J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
60
ANTIBACTERIAL ACTIVITY OF BLACK TEA AGAINST STREPTOCOCCUS MUTANS AND
ITS SYNERGISM WITH ANTIBIOTICS
Tahira Mughal1, Arifa Tahir2, Sadia Qureshi3, Taha Nazir4, Mahwish Rasheed1
1. Department of Botany, Lahore College for Women University, Lahore, Pakistan
2. Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan
3. Department of Biochemistry, Avicenna Medical College Lahore, Pakistan
4. Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan
ABSTRACT
Tea is the most common beverage after it is extracted from the leaves of Camellia
sinenesis. It is classified in to fermented tea (Black Tea) and non-fermented tea (green
tea). Methanolic extract of tea diluted in methanol and ethanol, which were tested for
antibacterial activity against Streptococcus mutans. Methanol was found to be best
antimicrobial solvent. The MIC value of methanolic extracts of Black Tea diluted in
ethanol was 0.1 mg/ml. Synergistic results of antibacterial activity of methanolic extracts
(diluted in ethanol) were found poor. Synergistic activity of Black tea sample with
antibiotics (Chloramphenicol, Tetracycline, Levofloxacin and Gentamycin) showed best
response against most of the bacteria (0.1mg/ml).
Key words: Antibacterial activity, Black Tea, Streptococcus mutans, synergistic activity
Address for Correspondence: Dr. Tahira Mughal, Assistant Professor, Department of
Botany, Lahore College for Women University, Lahore, Pakistan
E-mail: ssass85@yahoo.com
INTRODUCTION
Tea is an infusion made by steeping processed leaves, buds or twigs of tea bush, camellia sinensis, in hot
water for several minutes after, which it is drunk. The term herbal tea usually refers to infusion or tisane of
fruit or herbs that contain no Camellia sinensis. Tea came into English language from Chinese word of tea
which is pronounced in “te” in the Min Nan spoken variant .The British English slang word “char “ for tea
arose from its mandarin “ Chinese pronunciation “cha “ with its spelling affected by British English erotic
dialect pronunciation (Macfarlane). (Chow.K and Kramer.L., 1990)
Tea is one of the most consumed beverages of the world. Presently, it is cultivated in at least 30countries
around the world. Approximately 76-78% of tea produced is consumed in the form of Black Tea, 20-22%
in form of green tea and less than 2% as oolong tea. Black Tea principally consumed in Europe north
America and North Africa. While green tea in drink in China, Japan Korea and Morocco (Wu C.D, Wei
GX .2002) (Zuo et.al, 2008)
J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
61
The powerful antioxidant properties of the tea are generally attributed to its flavonoid components
theaflavins, bisflavanols and theaflavic acids. (Rice.E. C. 1999). The effect of Black tea on stomach cancer
has been studied. Out of 15 studies, five case-control studies showed a protective effect of tea on the risk of
stomach cancer (Ji B.T, et.al., in 1997)
Citral has been used for centuries for the treatment of various ailments. Citral helps to combats the
depression and bad mood and it used to treat fever cold and cough. A great remedy for stomachache
relaxes the stomach and intestine (Ming.L., et al 1996).
Streptococcus mutans is a non-motile, non-spore forming coccoid shaped, gram positive facultative
anaerobic is a part normal bacteria flora of a mouth it is an alpha hemolytic streptococci. Bacterial cells
appear in a form of chains due to cellular division in one division and incomplete cytokinesis following
mitosis. It can thrive in temperature 18-40ºC and pH below 5.5. (Smith, T. J., Blackman, S. A. & S. J
Foster 2000).
REVIEW OF LITERATURE
Friedman (2007) studied that the tea leaves produce organic compounds that may be involved in the
defense of the plants against invading pathogens including insects, bacteria, fungi, and viruses. These
metabolites include polyphenolic compounds, the six so-called catechins, and the methyl-xanthine alkaloids
caffeine, theobromine, and theophylline. These substances impart the black color to Black Tea.
Sakanaka et .al, (2000) reported that the inhibitory action of tea polyphenols towards the development and
growth of bacterial spores of Bacillus bacteria, tea polyphenols showed antibacterial effects towards
Bacillus stearothermophilus, which is a thermophilic spore-forming bacterium. The heat resistance of B.
stearother-mophilus spores was reduced by the addition of tea polyphenols. Clostridium thermoaceticum,
an anaerobic spore-forming bacterium was reduced heat resistance of its spores in the presence of tea
polyphenols. Epigallocatechin gallate, which is the main component of tea polyphenols, showed strong
activity against both B. stearothermophilus and C. thermoaceticum. The heat resistance of these bacterial
spores was more rapidly decreased by the addition of tea polyphenols at high temperatures.
Kim et.al, (2001) concluded the antibacterial potential of the methanolic extracts petals of Camellia
japonica L., against food borne pathogens in microbiological media and food. The extract showed good
bactericidal response against the pathogens Salmonella typhimurium, Escherichia coli, Listeria
monocytogenes, and Staphylococcus aureus.
J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
62
Various components of Camellia sinensis either in the form of green or Black Tea are simple catechins,
which have anti-cariogenic properties in vitro. These include a direct bactericidal effect against
Streptococcus mutans and S. sobrinus; prevented the bacterial adherence to teeth; inhibition of glucosyl
transferase, thus limiting the biosynthesis of sticky glucan and inhibition of human and bacterial amylases
(Hamilton .miller.2001)
Lakenbrink ( 2000) conducted the research on the polyphenolic, flavonoid, and caffeine compositions of
four commercial tea bag products (typical of those used in the UK, US, continental Europe, and the Middle
East) and beverages prepared from them under a range of typical consumer use conditions have been
studied. Leaf composition was determined by extraction with aqueous methanol .The absolute
compositions of all four products were remarkably similar in terms of most phenolic compounds. The
flavonoids comprised the major proportion (93-94%) of the total phenolics estimated by the Folin-
Ciocalteu method.
Tiwari et.al, (2005) studied that the synergistic antimicrobial activity of tea and antibiotics against
enteropathogens. Antimicrobial activity of boiled water tea extract and organic solvent extract were studied
against Salmonella typhimurium 1402/84, S. typhi, S. typhi Ty2a, Shigella dysenteriae, Yersinia
enterocolitica C770, and Escherichia coli determining minimum inhibitory concentration, minimum
bactericidal concentration and death rate kinetics at MBC of tea extract in presence of sub inhibitory
concentration of antibiotic. Black Tea extracts effectively inhibited the growth of S. typhimurium 1402/84,
S. typhi, S. typhi Ty2a, S. dysenteriae, Y. enterocolitica C770, and E.coli. Based on death rate kinetics
results, S.typhi Ty2a appeared to be highly sensitive and Y. enterocolitica C770 the most resistant.
Chloramphenicol and tea extract in combination inhibited the growth of S.dysenteriae at 2.5 µg/ml
chloramphenicol (MIC 5 µg/ml) and 5.094 mg/ml Black Tea extract (MIC 9.089 mg/ml). Tea extract
showed synergistic activity with chloramphenicol and other antibiotics like gentamycin, methicillin and
nalidixic acid against test strains.
MATERIAL AND METHODS
All chemicals solvents were analytical grade (Merck). Mueller-Hinton agar was used. Microbial strain
Streptococcus mutans ATCC 20572 (S. mutans) was obtained from Dental plaque centre Railway Hospital
Lahore, Pakistan growing on MSB agar.
Tea samples:
Tea samples used in the present study is Black Tea and collected from different departmental store of
Lahore, Pakistan. All the material was in the branded form. The botanical name of black tea is Camellia
sinensis Linn; family Theaceae and the part of plant used were the Leaves (Post Fermented).
J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
63
Extraction:
The purchased tea samples (Black Tea) was grinded to obtain fine powder. The sample powder were
triturated with redistilled methanol for 4-6 days by Soxhlet apparatus and process was repeated three times.
The solvent was removed in the rotary evaporator to yield the crude methanolic extracts stored at 4ºC. The
methanolic extract of all tea samples were diluted with solvent (Ethanol and Methanol) and with
combinations of different antibiotic (Chloramphenicol, Tetracycline, Levofloxacin and Gentamycin) with
different concentration (0.1,0.2,0.3, 0.4,0.5,0.6,0.7,0.8,0.9 and 1.0mg/ml).
Bioassay:
MIC and Bactericidal activity was determined by agar well diffusion method Norrel and Messley,
(1997). This test was performed in triplicate by spreading 18-24 hour old pathogenic bacterial
cultures containing approximately 106 -1010 colony forming units (CFU/ml) on the surface of
MSB agar medium (Mitis-Salivarius Bacitiracin Agar) plates. Wells (4 mm) were dug in the
media with the help of sterile metallic borer. Test samples of different concentrations
prepared in different solvents (Merck) were added (50 µl) in their respective wells. Pure
methanol was used as negative control (3 mm). Other wells were supplemented with reference
compound i. e. Chloramphenicol, Tetracycline, Levofloxacin and Gentamycin serving as positive
control. Synergistic activity against bacterial strain was determined by taking equal amounts (0.1mg/ml) of
antibiotics (Chloramphenicol, Tetracycline, Levofloxacin and Gentamycin)
RESULT AND DISCUSSION
It is observed that methanol was the best solvent for extracting antimicrobial substances from tea samples
based on the number of organisms inhibited and the diameter of inhibitory zones produced. It could also be
seen that different extracts were different in their antimicrobial effectiveness depending on the extractive
solvent used. These results favor Oloke and Kolawole (Oloke and Kolawole, 1988) that bioactive
components of any medicinal plant may differ in their solubility depending on the extractive solvents used.
The water extract was least bactericidal as compared to other solvent extracts and showed medium activity
against S.mutans.
J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
64
Table-1 Antibacterial Activity of Methanol Extract of black Tea samples (Dilution in Methanol)
Sr.
No
Concentration
mg/ml
Zone of inhibition (mm) Black Tea
Crude Methanol
Extract
Crude Ethanol
extract
Synergistic antimicrobial activity of crude
methanol extract (diluted in ethanol )
1 0.01 _ - 15
2 0.2 _ - 20
3 0.3 _ - 17
4 0.4 _ - 18
5 0.5 _ - 25
6 0.6 25 - 23
7 0.7 13 10 20
8 0.8 16 - 18
9 0.9 13 - 20
10 1.0 20 - 17
The crude methanolic extract of Black Tea showed full growth of bacteria between the concentrations 0.1-
0.5 mg/ml while it showed moderate activity at the concentrations of 0.6- 1.0mg/ml. The crude methanolic
extract of Black Tea, which was diluted in ethanol, showed good bactericidal activity nearly at all
concentrations. (Table 1)
J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
65
Table-2 Synergistic Antibacterial activity of crude methanol extract of black tea samples with
chloramphenicol, tetracycline, levofloxacin and gentamycin antibiotics
Sr.
No.
Concentrations mg/ ml Zone of inhibition (mm) of black tea with antibiotics
Chloramphenicol Tetracycline Levofloxacin Gentamycin
1 0.1 12 25 18 18
2 0.2 _ 28 18 18
3 0.3 _ 26 12 12
4 0.4 _ 27 17 17
5 0.5 13 20 19 19
6 0.6 12 25 15 15
7 0.7 15 20 18 18
8 0.8 15 25 20 20
9 0.9 14 25 20 20
10 1.0 17 28 18 18
Synergistic activity with different antibiotics:
The crude methanolic extract of black tea mixed with chloramphenicol in same ratio (1:1) diluted in
ethanol showed good activity against S. mutans at all concentrations. Mixing of extracts with
chloramphenicol enhance the activities of tea samples. (Table-4)
The crude methanolic extract of Black Tea when mixed with tetracycline showed moderate results at the
concentrations of 0.5-1.0 mg/ml but it gave poor results at other concentrations. The synergistic activity of
crude methanolic extract of Black Tea and Levofloxcin diluted in ethanol showed best activity against
S.mutans at all concentrations. The synergistic combination showed better result as compared to the black
tea alone. (Table-1& 4)
J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
66
Table 3- MIC values and Statistical analysis of Methanolic extract of Tea samples
Sr.
No
Tea samples MIC values in
mg/ml
*Statistical analysis
at 0.5mg/ml
1 Black Tea(Methanolic extract diluted in methanol) 0.6 -
2 Black Tea(Methanolic extract diluted in ethanol) 0.1 22 (±0.5)
*Good activity > 22, Moderate activity > 15 and Poor activity < 15
Table 4- MIC values and Statistical analysis of Methanolic extract of Black Tea samples (Diluted in
Ethanol) in synergism (With tea samples and antibiotics)
Sr.
No
Tea samples MIC values in mg/ml *Statistical analysis
1 Black Tea+ Chloramphenicol 0.1 22 (±0.5)
2 Black Tea+ Tetracycline 0.5 18 (±0.5)
3 Black Tea+ Levofloxacin 0.1 18 (±0.5)
4 Black Tea+ Gentamycin 0.1 15 (±0.5)
*Good activity > 22, Moderate activity > 15, Poor activity < 15
CONCLUSION
It is concluded that methanol was the best solvent for extracting antimicrobial substances from Black tea
samples. The synergistic activity of all methanolic extracts Black tea samples diluted in ethanol showed
good activity against S.mutans at all concentrations. In synergism, the Black Tea activity was enhanced
with Chloramphenicol, Levofloxacin and Gentamycin. It is suggested that Black tea samples used in the
study could be explored for possible antimicrobial agents for various infections of the oral cavity.
J App Pharm 2 (2) : 60-67 (2010) Tahira et al., 2010
Journal of Applied Pharmacy (ISSN 19204159)
17-3825 Luther Pl Saskatoon SK Canada S7H4B1
67
REFERENCES
1. Chow.k and Kramer.L .1990 All the Tea in China,China Book & Periodicals Inc ISBN 0-8351-
2194-1
2. Friedman,M. 2007 .Over view of antibacterial ,antitoxin, antiviral and antifungal activities of tea
flavonoids .Molecular Nutrition & food Research.51(1):116-134
3. Hamilton-miller, JMT.2001 Anticarcinogenic properties of tea (Camellia sinensis) .J.Med.
Microbiology .50;299-302
4. Ji,BT., Chow,W.H., Hsing ,A.W,Mclaughling,Dai,Q., Gao,Y.T., Blot,w.J., and Fraumeni, J.R.
1997 green tea consumption and the Risk of Pancreatic and Colorectal Cancer.Int .J.Cancer .70:
255-258
5. Kim ,K.Y., Davidson,P.M and Chung .H.J.2001 Antibacterial effect of water-soluble tea extracts
on food borne pathogens in Laboratory medium and in afood model .Journal of food protection .67
(11) : 2608-2612
6. Lakenbrink, C : Lapczynski, S : Maiwald, B : Engelhardt, U H., 2000 Flavonoids and other
polyphenols in consumer brews of tea and other caffeinated beverages. Journal of Agriultur Food
Chemistry 48(7): 2848-52
7. Ming .L., Figueiredo,R., Machado,S. and andrede,R.1996.Yield of essential oil and citral (D.C).
Proceedings of international symposium on medicinal and aromatic plants .ISHS .555-559
8. Norrel, S.A and Messley,k.E 1997. Micrbiology Laboratory Manual Principles and Applications
Prentice Hall.Upper saddle River .New jersey USA.
9. Rice-Eveans, C.1999 Implications of the mechanisms of action of tea poly phenols as antioxidants
in vitro for chemoprevention in humans. Proc.Soc.Exp.Bio.Med. 220 :262-266
10. Smith,T.J., Blackman,S.J. and Foster .,S.J 2000 Micribiology ,146:249-262
11. Sakanaka S., Jumneja,L.R and Taniguchi,M,2000 Antimicrobial effects of green tea polyphenols
on thermophilic sore forming bacteria . Journal of Bioscienceand Bioengineering.
Biotech.Biochem. 90 (1) : 81-85
12. Tiwari, R.P., Bharti,S,k., Kaur,H.D., Dikhshit,R.P. and Hondal,G.S. 2005.Synergistic
antimicrobial activity of tea and antibiotics. J.Ind.Med.Res.122 :80-84
13. Wu, C.D, and Wei,G.X. 2002 Tea as a functional food for oral health .Nutrition, 18; 443-444