ANTICANCER COMPOUNDS FROM MEDICINAL PLANTS


BIOLOGICA NYSSANA  4 (1-2)  December 2013: 75-79  Žabar, A. et al.  Larvicidal activity and in vitro … 

  

 75 

 

 

 

Original Article 

 

Larvicidal activity and in vitro effects of green tea (Camellia 
sinensis L.) water infusion 
 

Andrea Žabar*
1
, Vladimir Cvetković

1
, Jelena Rajković

1
, Jovana Jović

1
, Perica Vasiljević

1
, 

Tatjana Mitrović
1 

 

 

Department of Biology and Ecology, Faculty of Science and Mathematics,University of Niš, 

Višegradska 33,18000 Niš  

 

 

* E-mail: andreazabar@yahoo.com 
 

 

Abstract:  

Žabar, A., Cvetković, V., Rajković, J., Jović, J., Vasiljević, P., Mitrović, T.: Larvicidal activity and in vitro   

effects of green tea (Camellia sinensis L.) water infusion. Biologica Nyssana, 4 (1-2), December 2013: 75-

79. 
In this study green tea water infusion was tested on Drosophila melanogaster wild-type larvae in vivo, also an 

in vitro antihemolytic and hemolytic tests were performed. Three different concentrations were used 7.5 

mg/ml, 37.5 mg/ml and 75 mg/ml, the lowest dose representing the recommended dose followed by five times 

and ten times higher doses. Effect of these three concentrations was monitored and tested in vivo on 

Drosophila melanogaster (Meigen, 1830) wt (wild type) larval development and surviving. All three 

concentrations showed toxic effect for larvae, with toxicity being increased in dose – depended manner. The 

time needed for larvae to fully develop was delayed. This decrease of developmental time was in dose – 

dependent manner, too. Amount of hemolysis caused by the lowest concentration was very small when 

compared with the percent of spontaneous hemolysis. Other two higher concentrations, 37.5 mg/ml and 75 

mg/ml, showed higher hemolytic effect. During the four hour incubation period percent of hemolysis grew in 

time – dependent manner. The highest hemolytic effect was recorded for the concentration of 37.5 mg/ml. 

Antihemolytic test showed that the lowest concentration had the highest inhibitory effect to H2O2 induced 

hemolysis. The 37.5 mg/ml and 75 mg/ml concentrations had lower inhibitory effect when compared with the 

dose of 7.5 mg/ml. According to our study it can be concluded that the high concentrations of green tea water 

infusion exhibit larvicidal activity against D. melanogaster larvae, don't have protective effect to RBC 

membrane and cause greater hemolysis. 

 

Key words: green tea; Drosophila melanogaster wt; larvicidal activity; RBC; hemolytic test; antihemolytic 

test. 

 

 

Introduction 
 

One of the most consumed beverages in the 

world is green tea. Caffeine is one of the active 

compounds present in tea, and its content is around 

3 to 4 % (W i l l s o n  et al., 1992). Caffeine may 

play an important role against some pathogens (Van 

B r e d a  et al., 2012). Green tea is also a source of 

polyphenol antioxidants such as epicatechin, 

epigallocatechin, epicatechin gallate and 

epigallocatechin gallate (Z h a n g  et al., 1997, 

H o d g s o n , 2008). Catechins have similar 

backbone but differ in number and location of 

hydroxyl groups. Catechins are water – soluble, 

colorless compounds that make up to 30% of the dry 

leaf weight (G r a h a m , 1992). These natural 

antioxidants have been reported to have the ability 

of preventing cancer (L i  et al., 2008), are effective 

scavengers of free radicals (N a n j o  et al., 1996), 

inhibit HBV in vitro (X u  et al., 2008). It has been 

11
th

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4 (1-2) • December 2013: 75-79 

 



BIOLOGICA NYSSANA  4 (1-2)  December 2013: 75-79  Žabar, A. et al.  Larvicidal activity and in vitro … 

 

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reported that one cup of tea can provide 150 – 200 

mg of flavonoids. Also, lower risk of heart disease 

and stroke are related to the flavonoids intake 

(H o d g s o n , 2008). Isolated flavonoids at high 

physiological concentrations can reduce platelet 

aggregation and markers of platelet activation 

(R e i n  et al., 2000). Green tea extract can delay and 

inhibit oxidation of low-density lipoprotein LDL 

catalyzed by copper in vitro (Y o k o z a w a  et al., 

1997). Some authors report that drinking green tea 

may result in lower level of total triglyceride and 

cholesterol in serum together with deceased 

atherogenic index (I m a i  et al., 1995).  

In view of variety of reports considering 

green tea, we have conducted a study to evaluate the 

effects of high concentrations of green tea water 

infusion. Drosophila melanogaster is frequently 

used in larvacidal and insecticidal studies 

(M i y a z a w a  et al., 1998; M i y a z a w a  et al., 

2000). Our aim was to see whether these high doses 

have beneficial effects on RBC membrane and on 

Drosophila melanogaster wt larvae development. 

To evaluate green tea infusion effect RBC 

membrane the hemolytic test with Drabkin's reagent 

was used. Also, to see whether high concentrations 

of green tea water infusion could inhibit lipid 

peroxidation caused by H2O2, the antihemolytic 

method was employed. 
 

Materials and methods 
 
Test organisms 

In this experiment, the Drosophila 

melanogaster reference type 5 wt, originated from 

„Bloomington Drosophila Stock Centre”, Indiana 

University, USA was used. Fruit flies were 

cultivated on a standard Drosophila medium (9% 

sugar, 10% corn meal, 2% agar, 2% yeast, with 

addition of fungicide Nipagin
®
), in a standard 

cultivating 200 ml flasks on 25 
o
C, 60% humidity, 

with 12h/12h day/night cycle. Adults were allowed 

to oviposit eggs during the eight hour period, after 

that they were removed from the flasks. Then, after 

three days larvae which were 72±4h old, have been 

removed from the cultivating medium, washed with 

distilled water, and placed on the new medium 

containing green tea infusion. Concentrations of 

green tea in medium were 7.5 mg/ml, 37.5 mg/ml 

and 75 mg/ml. Experiment was done in triplicate. 

There were 15 larvae from the same hatch in each 

replicant flask, and there were 12 replicant flasks 

including the water control (negative control). 

Replicants were incubated in standard way, already 

described. Alive specimens were collected 10-16 

days after the egg laying.  

RBC preparation 

A modified method from Noudeh et al. (2009) 

was used. Briefly, blood was collected in 

heparinized tubes from male Wistar rats via 

cardiopuncture. Whole blood was centrifuged at 

2000 rpm for 10 minutes at 4 ºC. Plasma and the 

buffy coat were removed and an equal volume of 

PBS (pH 7.4) was added. This was repeated three 

times, to obtain washed erythrocytes. At the end 

packed erythrocytes were diluted with PBS to obtain 

4% suspension. RBC prepared in this way were 

used both for hemolytic and antihemolytic test. 

Solutions of green tea infusion and PBS 

(0.9%, pH=7.4) were made to give the final 

concentrations of 7.5mg/ml, 37.5mg/ml and 

75mg/ml. 

 

Hemolytic test 

The following method was employed: 200 μl 

of green tea water infusions (concentrations 7.5 

mg/ml, 37.5 mg/ml, 75 mg/ml) were incubated with 

200 μl of RBC suspension at room temperature for 

one, two, three and four hours. After each hour of 

incubation samples were centrifuged at 1500 rpm 

for 10 minutes at 4 ºC. Then, 200 μl of resulting 

supernatant was added to 3 ml of Drabkin's reagent 

and the absorbance was measured at 540 nm on 

spectrophotometer (Shimadzu UV-VIS 1650, 

Tokyo, Japan). Positive control consisted of 200 μl 

of distilled water and 200 μl RBC suspension and 

negative control were 200 μl of PBS and 200 μl 

RBC suspension. Everything was done triplicate. 

Percentage of hemolysis was calculated by the 

equation:  

% Hemolysis = [(Ab(sample) - Ab(negative control)) / 

Ab(positive control)] * 100%  (Gould et al., 2000). 

 

Antihemolytic test 

Antihemolytic test was done by modified 

method previously described (Ebrahimzadeh et al, 

2010.) 2 ml of RBC suspension was pre-incubated 

with 0.5 ml of green tea water infusion. The final 

volume was made to be 5 ml with PBS, and the final 

concentrations of green tea infusions were 7.5 

mg/ml, 37.5 mg/ml and 75 mg/ml. This mixture was 

incubated for 15 minutes at 37 ºC. Then 0.5 ml of 

H2O2 dissolved in PBS was added to the mixture. 

Concentration of H2O2 was made to give 90% of 

hemolysis after 4 hours. At the end of the incubation 

period of 4 hours, different concentration samples 

were spun at 1500 rpm for 10 min at 4 ºC. Then 200 

μl of supernatant was added to 3 ml of Drabkin's 

reagent and the absorbance was measured at 540 

nm. Positive control consisted only of PBS and 



BIOLOGICA NYSSANA  4 (1-2)  December 2013: 75-79  Žabar, A. et al.  Larvicidal activity and in vitro … 

  

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H2O2 without green tea infusion. Experiments were 

done in triplicate. Percentage of inhibition was 

calculated by equation: %Inhibition = [(Ab (H2O2) - 

Ab(sample) ) / Ab (H2O2) ] *100% .  

 
Results and Discussion 

 

The absolute mortality was monitored - 

number of hatched adult specimens that were scored 

and compared to total transferred larval specimens. 

It was observed that the mortality increases with the 

increase in green tea concentration in the medium 

(Fig. 1).  

 

 
Fig. 1. Absolute mortality of D. melanogaster larvae 

in presence of green tea infusion  

Development was also observed and number 

of pupae that were scored from ninth to sixteenth 

day of larval life (data not shown). In comparison 

with water control, developmental time from larvae 

to adults prolongs if the concentration of green tea 

infusion is higher. One of the active compounds of 

green tea is caffeine. According to the literature, 

content of the caffeine is around 3 to 4% 

(W i l l s o n  et al., 1992). It is supposed that 

caffeine, localized in the vascular bundles of young 

Camellia sinensis L. leaves plays a crucial role in 

the defense against various pathogens (V a n  

B r e d a  et al., 2012.). Itoyama and B i c u d o  (1992 

and 1997) showed harmful effects of caffeine on 

Drosophila prosaltans D., related to fecundity, egg 

lying capacity and longevity which is consistent 

with our results. On the other hand, some studies 

show that Camellia sinensis L., inhibits the 

accumulation of iron and thus extends the life span 

of Drosophila melanogaster (M a s s i e  et al., 1993). 

Also, these results are consistent with previous 

studies of other authors who showed that 

polyphenols as antioxidants affect larval 

development in Drosophila causing its time delay 

(R i c e -E v a n s  et al., 1996; A r u o m a , 2003; 

K i m  et al., 2004). 

Results for both antihemolytic and hemolytic 

test are accordant. In both experiments the lowest 

doses of green tea infusion exibited slightly 

beneficial effect on RBC membrane (Fig. 2 and 3). 

 

 

 

Fig. 2. Percentage of RBC hemolysis in presence of green tea water infusion

http://www.ncbi.nlm.nih.gov/pubmed?term=Massie%20HR%5BAuthor%5D&cauthor=true&cauthor_uid=8326745


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Percentage of hemolysis was the lowest for the 

concentration of 7.5 mg/ml, and it grew as the time 

of incubation prolonged (after first hour it was 

4.99% ± 3.28, second 4.56% ± 0.4, third 5.41% ± 

2.22, fourth 5.8 % ± 1.43). Other two higher doses 

showed an increase in RBC hemolysis also in time 

dependant manner. Hemolysis was highest for the 

37.5 mg/ml concentration. Percent of hemolysis for 

the 37.5 mg/ml concentration was 15.16% ± 2.4 

after first hour, and for the second, third and fourth 

hour of incubation it was 20.96 % ± 3.55, 20.86 % ± 

1.27 and 20.52 % ± 3.3 respectively. Percent of 

hemolysis for the concentration of 75 mg/ml was 

8.45 % ± 3.79, 10.79 % ± 0.95, 11.63 % ± 1.33, 

12.96 % ± 2.69 after first, second, third and fourth 

hour respectively.  

 
Fig. 3. Antihemolytic effect of green tea infusion 

 

According to the Fick's law, diffusion flux 

from a membrane is proportional to concentration 

difference of both sides. So, an increase in 

concentration of green tea infusion in outer 

membrane leads to diffusion to internal membrane 

until it gets to specific concentration which can 

promote membrane disruption and induce hemolysis 

(K l e s z c z y n s k a  et al., 2005.). 

Lipid peroxidation of rat RBC induced by 

H2O2 creates membrane damage and hemolysis. The 

highest percentage of inhibition was recorded for the 

7.5 mg/ml concentration (94.96 % ± 0.1). Other two 

concentrations had lower inhibitory effect, and for 

37.5 mg/ml dose it was 90.39 % ± 0.29, while for 

the 75 mg/ml it was 85.42 % ± 0.45. Certain 

polyphenols may interact with membrane, leading to 

the decrease in its fluidity and the diffusion of free 

radical into the RBC (C o s t a  et al., 2009.). In 

higher doses effects differ (Fig. 3.), percentage of 

hemolysis inhibition is lower. High concentrations 

of green tea infusion don't have ameliorative effect 

against RBC free radical hemolysis. 

 
 

Conclusion 
 

High concentrations of green tea water 

infusion exibit toxic effect to Drosophila larvae. The 

time of development was prolonged. This is 

probably due to high concentration of active 

compounds present in green tea water infusion such 

as caffeine and catechins. Also these results indicate 

that high doses of green tea infusion can cause a 

change in tonicity of the erythrocyte membrane. 

Change in tonicity is probably due to the 

interactions of polyphenols with the outer lipid 

monolayer of the erythrocyte membrane. Therefore, 

it is necessary to continue the research further. 

 
Acknowledgements. The authors acknowledge the 

financial support of the Ministry of Education, Science 

and Technological Development of the Republic of 

Serbia (Grant No.: 172047). 

 

References 
 

Aruoma, O.I. 2003: Methodological considerations 

for characterizing potential antioxidant actions of 

bioactive components in plant foods. Mutation 

Research, 523–524: 9-20.  

Costa, R.M., Magalhaes, A.S., Pereira, A.J., 

Andrade, P.B., Valentao, P., Carvalho, M., Silva, 

B.M., 2009: Evaluation of free radical-

scavenging and antihemolytic activities of quince 

(Cydonia oblonga) leaf: a comparative study 

with green tea (Camellia sinensis). Food and 

Chemical Toxicology 47: 860-865. 

Ebrahimzadeh, M.A., Nabavi, S.F., Nabavi, S.M., 

Eslami, B., 2010: Antihemolytic and antioxidant 

activities of Allium paradoxum. Central 

European Journal of Biology, 5(3): 338-345. 

Gould, L.A., Lansley, A.B., Brown, M.B., Forbes, 

B., Martin, G.P., 2000: Mitigation of surfactants 

erythrocyte toxicity by egy phosphatidylcholine. 

Journal Pharmacy and Pharmacology, 52(10): 

1203-1209. 

Graham, H.N. 1992: Green tea consumption, 

composition and  polyphenol chemistry. 

Preventive Medicine, 21:334-350. 

Hodgson, J.M., 2008: Tea flavonoids and 

cardiovascular disease. Asia Pacific Journal of 

clinical nutrition 17(S1): 288-290. 

Imai, K., Nakachi, K., 1995: Cross sectional study 

of effects of drinking green tea on cardiovascular 

and liver disease. BMJ 310:693-696. 

Itoyama, M.M., Bicudo, H.E.M.C., 1992: Effects of 

caffeine on fecundity, egg laying capacity, 

development time and longevity in Drosophila 

prosaltans. Revista brasileira de genética 15: 

303-321.  



BIOLOGICA NYSSANA  4 (1-2)  December 2013: 75-79  Žabar, A. et al.  Larvicidal activity and in vitro … 

  

 79 

Itoyama, M.M., Bicudo, H.E.M.C., 1997: Effects of 

caffeine on mitotic index in Drosophila 

prosaltans (Diptera). Revista brasileira de 

genética 20: 655-658. 

Kim, D.O., Lee, C.Y., 2004: Comprehensive study 

on vitamin C equivalent antioxidant capacity 

(VCEAC) of various polyphenolics in 

scavenging a free radical and its structural 

relationship. Critical Reviews in Food Science 

and Nutrition, 44: 253 - 273.  

Kleszczynska, H., Bonarska, D., Luczvnski, J., 

Witek, S., Sarapuk, J., 2005: Hemolysis of 

erythrocytes and erythrocyte membrane fluidity 

change by new lysosmtropic compounds. 

Journal of Fluorescence, 15(2): 137-141. 

Li, F., Wang, F., Yu, F., Fang, Y., Xin, Z., Yang, F., 

Xu, J., Zhao, L., Hu, Q., 2008: In vitro 

antioxidant and anticancer activities of ethanolic 

extract of selenium-enriched green tea. Food 

Chemistry, 111: 165-170. 

Massie, H.R., Aiello, V.R., Williams, T.R., 1993: 

Inhibition of iron absorption prolongs the life 

span of Drosophila. Mechanisms 

of Ageing and Development, 67(3):227-37. 

Miyazawa, M., Nakamura, Y., Ishikawa, Y., 2000: 

Insecticidal sesquiterpene from Alpinia 

oxyphylla against Drosophila melanogaster. 

Journal of agricultural and food chemistry, 48: 

3639-3641. 

Miyazawa, M., Yoshio, K., Ishikawa, Y., Kameoka, 

H., 1998, Insecticidal alkaloids against 

Drosophila melanogaster from Nuphar 

japonicum DC. Journal of agricultural and food 

chemistry, 46: 1059-1063. 

Nanjo, F., Goto, K., Seto, R., Suzuki, M., Sakai, M., 

Hara, Y., 1996: Scavenging effects of tea 

catechins and their derivatives on 1,1-diphenyl-

2-picylhydrazyl radical. Free Radical Biology 

and Medicine, 21: 895-902. 

Noudeh, G.D., Sharififar, F., Khatib, M., Behravan, 

E., Afzadi, M.A., 2009: Study of aqueous extract 

of three medicinal plants on cell membrane-

permeabilizing and their surface properties. 

African Journal of Biotechnology, 9(1): 110-116. 

Rein, D., Paglieroni, T.G., Pearson, D.A., Wun, T., 

Schmitz, H.H., Gosselin, R., Keen, C.L. 2000: 

Cocoa and wine polyphenols modulate platelet 

activation and function, Journal of Nutrition 

130:2120S-6S. 

Rice-Evans, C.A., Miller, N.J.,  Paganga, G., 1996: 

Structure-antioxidant activity relationships of 

flavonoids and phenolic acids. Free Radical 

Biology and Medicine, 20: 933 - 956.  

Van Breda, S.V., Van der Merwe, C.F., Robbertse, 

H., Apostolides, Z., 2012. Immunohistochemical 

localization of caffeine in young Camellia 

sinensis (L.) O. Kuntze (tea) leaves. Planta, 

DOI: 10.1007/s00425-012-1804-x 

Willson K.C., Clifford, M.N., 1992: Tea. 

Cultivation to consumption. - Chapman and Hall, 

London. 

Xu, J., Wang, J., Deng, F., Hu, Z., Wang, H., 2008: 

Green tea and its major component 

epigallocatechin gallate inhibits hepatitis B virus 

in vitro. Antiviral Research 78: 242-249. 

Yokozawa, T., Dong, E., 1997: Influence of green 

tea and its three major components upon low-

density lipoprotein oxidation. Experimental and 

toxicologic pathology, 49(5): 329-335. 

Zhang, A., Zhu, Q.Y., Luk, Y.S., Ho, K.Y., Fung, 

K.P., Chen, Z.Y., 1997: Inhibitory effect of 

jasmine green tea epicatechin isomers on free 

radical induced lysis in red blood cells. Life 

Science, 61(4): 383-394.