Cytotoxic Activity of Glocihdium zeylanicum Leaf Extract

 

Srivalli Tripuramallu*, Srikanth Sunkara and Venkata Ramana Reddy. Maram

Gitanjali College of Pharmacy, Cheeryal, Keesara Mandal, Rangareddy District.

 

ABSTRACT

In vitro cytotoxic activity of Glocihdium zeylanicum has been investigated by tryphan blue dye exclusion method at various concentrations on Ehlirich Ascites Carcinoma cells. The ethanolic extracts of Glocihdium zeylanicum has showed the significant cytotoxic property by inhibiting the dye exclusion was evaluated by comparing with standard 5-Fluorouracil. The preliminary study indicates the ethanolic extract of Glocihdium zeylanicum has significant cytotoxic property and suggests for further investigation for its antitumor and anticarcinogenic function in different models.

 

KEY WORDS: Glocihdium zeylanicum, Cytotoxic activity, EAC cells, 5-fluorouracil

 

 

INTRODUCTION

Glochidion zeylanicum is a shrub or a small tree. It is usually found in scrub lands and along the banks of streams. Glochidion zeylanicum is natural vegetation to Sri Lanka, India, Southeast Asia and parts of East Asia. It is also called Sri Lanka Glochidion. It belongs to the family of Euphorbiaceae, evergreen tree, up to 7 m tall. Leaves simple, alternate, oblong, 5－15 cm long, 3－8 cm wide, obtuse at apex, margin entire; fruit a capsule, split when ripe, seeds red. Roots used as herbal medicine for cough and pneumonia; stems and leaves used for abdominal pain and traumatic injury. Bark containing up to 6.43% of tannin. Five megastigmane glucosides were isolated from the leaves of Glochidion zeylanicum. One of them was a known compound, blumenol C O-b -D-glucopyranoside1, and the structures of the four new compounds, glochidionionosides A—D2—5, were mainly elucidated by spectroscopic methods, including a modified Mosher’s method. In continuing work on plants growing in Okinawa, the constituents of Glochidion zeylanicum were investigated, and glochidionolactones A—F, butenolide glycosides and lignans have been isolated. Several flavanol glucosides named glochiflavanosides A—D were isolated along with several known C-glucosyl flavones, vitexin  and isoorientin have been isolated from the leaves. This paper deals with the evaluation of antioxidant activity of the ethanolic extract of leaves of Glochidion zeylanicum.

 

EXPERIMENTAL:

Collection and extraction of leaf:

The plant was collected in Ranga Reddy district, Andhrapradesh and leaf was dried under shade for 4 days and powdered in mechanical grinder. The leaf powder was extracted with ethanol by combination of maceration 12 hrs and then soxhelate for 3 hrs. The combined extracts were concentrated under rotary flash vacuum evaporator to thickness and used for the study of cytotoxic activity.

 

The Glocihdium zeylanicum, leaf organic extracts were evaluated for invitro cytotoxicity effects by tryphan blue dye exclusion method at various concentrations on Ehrlich Ascites Carcinoma cells.  The study was conducted after obtaining institution animal ethics committee approval.

Table 1: Cytotoxic activity of Glocihdium zeylanicum leafs extract on EAC Cells.

S. No.

Concentration in microgram/mL

Cytotoxic activity in Percentage of inhibition

Ethanolic extract [Test]

5-Fluoro Uracil[Standard]

1

250

44. 46%

55.46%

2

500

49.22%

58.34%

3

1000

50.26%

62.35%

4

1500

56.92%

69.72%

5

2000

58.6%

76.58%

6

IC50 [µg/mL]

1580 µg/mL.

250 µg/mL

 

 

 

 

 

 

 

 

 

Test animal:

Adult Swiss male Albino mice (25-30gm) were procured from CCMB, Hyderabad, and used throughout study. Animals were maintained at standard environmental conditions and were fed with diet and water at libitum during quarantine period.

 

Malignat Cell linings: -

Ehrlich Ascites Carcinoma cells were obtained through the courtesty of CCMB, Hyderabad, they were maintained by weekly intra peritoneal inoculation of 1x106 cells/mouse.

 

Chemicals: -

Tryphan blue dye and DMSO [AR grade].

 

Phosphate buffer saline: -

Prepared by dissolving 2.5gms of sodium dihydrogen phosphate, 2.325gms of disodium hydrogen phosphate and 8.2gms of sodium chloride in 100ml of water.

 

Principle:

Tryphan blue is recommended in dye exclusion procedure for viable cell counting based on the principle that live cells (viable) actively pump out the dye by efflux mechanism where as dead cells (non viable) do not.

 

Method:-

Male albino mice were inoculated with 1x106 EAC cells/mouse through the courtesy of CCMB till the desired growth of the tumor in mice was notice6,7 .Cytotoxicity was assessed by incubating 1ml of peritoneal fluid (consisting of 1x106 EAC cells with a cell viability beyond 60%) in 1 ml phosphate buffer saline and 1ml of varying concentrations (250,500,1000,1500,2000µg/ml)of the test extracts of Glocihdium zeylanicum (Ethanolic) at 37oC for 3 hr in CO2 atmosphere, using Mclntosh field jar.  The viability of the cells was determined by tryphan blue dye exclusion method.  The equal proportion of incubated EAC cells and tryphan blue dye (10%in water) were mixed and kept aside.  After 10 min the total number of viable and non viable were (dead cells take up the dye whereas viable cells exclude the dye) counted by using newbaurs chamber 5-Fluorouracil was used as standard drug and Ethanol as a control.  The percentage of dead cells was calculated using the formula from which IC50 concentration was determined.

 

RESULTS AND DISCUSSIONS:

A number of natural products have been studied for anticancer activity in various experimental models.  A reliable criteria for judging of anticancer activity in prolongation of life span and decrease of WBC from blood 8,9 .  A preliminary   attempt was made to assess the invitro cytotoxic properties of Glocihdium zeylanicum extracts on Ehrlich Ascites Carcinoma (EAC) cells at varying concentrations 250, 500,1000, 1500and 2000 µg/ml. 5-fluorouracil is used as a reference standard and Ethanol is used as control.  The number of viable and nonviable cells was counted by using Tryphan blue exclusion method.

 

The table 1-illustrates the activity of Glochidium zeylanicum leaf extract against EAC cells. The activity of 5-fluorouracil at varying concentrations is comparatively very high with respect to Glochidium zeylanicum leaf extract. The decreasing % inhibition [IC50] cytotoxicity activity is in order of 5-flourouracil [250 µg/mL]> Ethanolic extract of Glochidium zeylanicum [1580 µg/mL]. These preliminary study results indicate that ethanolic extract suggests for further investigation for its antitumour and cytotoxic properties in different experimental models.

 

CONCLUSION:

The present study revealed that the leaf extract of Glochidium zeylanicum possess the significant cytotoxic activity and the further pharmacological investigations are required for evaluation of potential benefits as natural anticancer agent in different experimental models.

 

REFERENCES:

1.       Nakata F., Nakata K., “Medicinal Plants,” Shinsei Press Ltd., Naha, Okinawa, 1991; Tawada S., Ohta F., “Encyclopedia of Medicinal Plants in Okinawa,” Shinsei Press Ltd., Naha, Okinawa, 1985.

2.       Otsuka H., Hirata E., Takushi A., Shinzato T., Takeda Y., Bando M., Kido M., Chem. Pharm. Bull., 48, 547—551 (2000).

3.       Morita N., Arisawa M., Yoshikawa A., Yakugaku Zasshi, 96, 1180—1183 (1976).

4.       Komatsu M., Tomimori T., Makiguchi Y., Chem. Pharm. Bull., 15,1567—1572 (1967).

5.       Hideaki OTSUKA,* Hidehiko KIJIMA, Eiji HIRATA, Takakazu SHINZATO, Anki TAKUSHI, Masahiko BANDO, and Yoshio TAKED, Chem. Pharm. Bull. 51(3) 286—290 (2003) Vol. 51, No. 3.

6.       Sheeja KR, Kuttan G, and Kuttan R, Amala ResBull, 17, 1997, 3

7.       Rajkapoor B, Jayakar B, and Murugesh N, Ind.J Pharmacology, 36, 2004, 38.

8.       Clarkson BDand Burchenal JH. Prog.Clin Cancer, 1, 1965, 6259

9.      Oberling C and Guerin M. Adv. Cancer Res, 2, 1954, 353.

 

 

Received on 27.10.2009

Accepted on 18.11.2009     

© A&V Publication all right reserved

Research J. Pharmacology and Pharmacodynamics 1(3) Nov - Dec.  2009; 145-146