In-vitro Cytoprotection Activity of Foeniculum vulgare and Helicteres isora in Cultured Human Blood Lymphocytes and Antitumour Activity against B16F10 Melanoma Cell Line

 

Madhulika Pradhan*, S Sribhuwaneswari, D Karthikeyan, Sunita Minz, Pavani Sure, Atul N Chandu, Umesh Mishra, K Kamalakannan, A  Saravanankumar and T Sivakumar.

Department of Pharmaceutical Biotechnology. Nandha College of Pharmacy, Erode, Tamilnadu, India.

*Corresponding Author E-mail:  s.sribhu@rediffmail.com


 



ABSTRACT:

This study was designed to examine in-vitro cytoprotection activity of methanolic extract of Foeniculum vulgare and Helicteres isora against normal human blood lymphocytes by micronucleus assay and antitumor activity against B16F10 melanoma cell line by Trypan blue exclusion assay for cell viability. Lymphocyte culture treated with 70% methanolic extract of Foeniculum vulgare and 50% methanolic extract of Helicteres isora showed very less percentage of micronucleus i.e. 0.006% and 0.007% respectively as compared to standard drug doxorubicin which showed 0.018% micronucleus. On the other hand 70% methanolic extract of Foeniculum vulgare good antitumor activity at the concentration of 200μg/ml and 50% methanolic extract of Helicteres isora dislayed good antitumor activity at the concentration of 300 μg/ml. The results strengthen that the Foeniculum vulgare and Helicteres isora could be considered as a natural resource of antitumor agents as well as cytoprotective to normal cells.

 

KEY WORDS:   Antitumor, Foeniculum vulgare, Helicteres isora Cytoprotection,etc

 


 

INTRODUCTION:

The World Health Organization (WHO) has estimated that approximately 80% of the world’s population depends on traditional medicines for meeting their primary health care needs1. The areas of cancer and infectious diseases have a leading position in utilization of medicinal plants as a source of drug discovery. Among FDA approved anticancer and anti-infectious preparations drugs of natural origin have a share of 60% and 75% respectively 2.

 

Cancer is still a major cause of mortality and morbidity in developing as well as in developed countries. Overall survival rate has only improved slightly despite advances in surgery, radiotherapy, and chemotherapy. Molecular targeted agents are currently being studied in all treatment settings including that of chemoprevention, which is defined as the use of natural or synthetic non-essential dietary agents to interrupt the process of carcinogenesis and to prevent or delay tumor growth3.

 

Foeniculum vulgare is a plant belonging to the Umbelliferae (Apiaceae) family, known and used by humans since antiquity. Because of its flavor, it was cultivated in every country surrounding the Mediterranean sea. Its therapeutic and culinary utilization was so large that fennel was exported from country to country for centuries.4

 

Antimicrobial properties of the essential oil have been recognized 5. Fennel has been reported to show radical scavenging and antioxidant activity 6. Analgesic and antinflammmatory property have also been reported 7.

 

Helicteres isora Linn (Sterculiaceae) is a plant growing gregariously throughout India. The fruits of this plant are commonly called as Mrigashringa in Sanskrit. The fruits are demulscent, mildly astringent and useful in gripping and flalulence8. Its terpene derivative exhibited considerable antimicrobial and antispasmodic activity 9.

 

MATERIALS AND METHODOLOGY:

1.       Sample Collection:

Helicteres isora fruits were collected from Delawadi forest, Ratapani Abhyaran, Bhopal (M.P.) and Foeniculum vulgare fruits were collected from local market Bhopal (M.P.).

2.      Processing of sample:

Samples were washed with running tap water then rinsed with distill water to remove the dust particles adhered on   the surface of samples. The samples were dried in shade.

 

3.      Extraction:

Extraction of Helicteres isora 10: - 80g of sample was extracted with 50% methanol in soxhlet apparatus for 8 hrs at room temperature. Then the filtrate was evaporated at 40˚C till dryness. Dried extract thus obtained was collected and stored in cool place. Percentage yield obtained is 2.71% w/v.

 

Extraction of Foeniculum vulgare: - 25 g of fennel fruit was extracted with 100 ml of 70% methanol by cold maceration process. Extracts obtained were filtered through Wattman filter paper     no.1 and the filtrate was collected, methanol was removed by rotary evaporator at                 40˚C. Yield obtained is 1.3161% w/v.

 

4.    Experimental work:

In-vitro antitumor activity

4.1 Cell culture11:

The B16F10 melanoma cell line was kindly provided by Department of research Jawaharlal Nehru Cancer Hospital and Research Center, Bhopal (M.P.). Cells were cultured in Eagle's minimum essential medium (EMEM), supplemented with 10% fetal calf  serum (FCS) and streptomycin plus penicillin (100μg/ml and 100 IU/ml, respectively).Cells were cultured in a 5% CO2 humidified atmosphere at 37°C until near confluence.  All the processes were carried out in a vertical laminar flow chamber.

 

4.2 Drug treatment:

Methanolic extracts of Foeniculum vugare and Helicteres isora were prepared in increasing final concentrations, ranging from 25 to 200 μg/ml and 25 to 300 μg/ml respectively. The drug extracts were treated with plates containing confluent monolayer of B16F10 melanoma cell lines. Negative control group was B16F10 cell line only, and the positive control group was treated with doxorubicin  (0.032μg/ml). After incubating for 24 h at 37˚C, the cells were trypsinized (0.25% in PBS) and then centrifuged at 1000 rpm for 5 min, washed twice with fresh medium, and resuspended with fresh medium. Cell viability was counted for each concentrations of crude extract as well as for control.

 

4.3 Trypan blue exclusion assay: growth and viability test12  :

The percentage of viable and non-viable cells was determined, using trypan blue exclusivity stain. Cell growth and viability was measured by adding 0.4% trypan blue in 0.9% saline to a 50% dilution, and cells were counted using the hemocytometer. Cells were examined and counted in duplicates under light microscope at 100 x (Olympus, Japan). Percentage cell viability was calculated by the formula:

 

Cell viability  =  No of viable cells (unstained cells) x l00

Total no of cells (stained and unstained)

4.  In vitro cytoprotection activity:

It is based on the assessment of genotoxicity by “Micronucleus assay”.

 

Micronucleus assay13,14:

Micronucleus cytochalasin-B test used in this study is based on the observation of small extranuclear formations (micronuclei) of genetic material in cytoplasm. The expression of micronuclei is the consequence of chromosome breaks or spindle disruption.

 

4.2.1 Sample collection:

Fresh blood from normal healthy individual was collected by vein puncture and

transferred into sterile, heparinized vacutainers.

 

4.2.2 Application of crude extracts:

For the study methanolic extract of Foeniculum vulgare and  Helicteres isora was prepared in the concentration of 200 μg/ml and 300 μg/ml respectively where as positive contol was 0.032 μg/ml. Four groups taken for the study are as follows:         

 

I)        Negative control group.

II)       Doxorubicin treated group.

III)     Foeniculum vulgare extract treated group.

IV)     Helicteres isora extract treated group.

 

4.2.3  Cultivation and slide preparation 15:

Blood (0.4 ml) was added to 5 ml of RPMI 1640 medium with L glutamine, 20 %  of foetal bovine serum, 10 μg/ml of phytohaemaglutinin and 200 IU/ml of penicillin streptomycin solution. At the same time the lymphocytes culture was treated with crude extracts, and chemotherapy drugs  as per the group divided above. Positive and negative control were also taken. On the 44th hour of phytohaemaglutinin stimulation, cytochalasin B  was added to the cultures to the final concentration of 4.5 μg/ml. Cells were harvested on the 72nd hour of cultivation. After centrifugation, cultures were briefly treated with a hypotonic solution (0.56 % KCl). In further processing, cold and fresh fixative (3:1, methanol : acetic acid) was added to the cell suspensions. After the third fixation stage, cell pellet was gently resuspended once more in a few drops of fresh fixative and the suspension was dropped on clean, cold and dry microscope slides. Dry slides were stained with a 5 % Giemsa stain for 8 minutes and then washed in distilled water.

 

4.2.5           Microscopic analysis:

Air-dried and coded slides were analysed using a light microscope at 400x    magnification. Micronuclei were scored in no less than 1000 binucleated cells per sample.

 

RESULTS:

1. Antitumor activity:

Antitumor activity of above crude extracts was evaluated on the basis of “Trypan blue exclusion assay” for cell viability.

1.1    Cell viability:

Percentage viability of methanolic extract of Foeniculum vulgare and Helicteres isora is shown in the fig1 and fig 2

 

Fig No. 1 Effect of different conc. of Foeniculum vulgare on  percent cell viability;   Concentration Vs Percent viability

 

Fig No. 2 Effect of different conc. of Helicteres isora on  percent cell viability; Concentration Vs Percent viability

 

2. In Virto Cytoprotection activity:

2.1 Micronucleus assay:

Micronucleus count per 1000 binucleated cells is given in the table o 3.

Groups

Total binucleated cells scored

Disrtibution of micronucleus

Total MN/

1000 binucleated cells

MN/

100 cells

1 MN

2

MN

3

MN

 

Control

1000

 

--

--

--

--

Dox + blood

1000

11

2

1

18

0.018

FV + blood

1000

6

 

--

6

0.006

Dox     =     Doxorubicin                 MN      =    Micronucleus

FV      =     Foeniculum vulgare     HI        =    Helicteres isora    

 

DISCUSSION:

Treatment of Foeniculum vulgare  extracts against B16F10 melanoma cell line,  in all concentration range  showed decrease in percent cell viability, as compared to that of negative when examined by “Trypan blue exclusion assay”. Also, Helicteres isora in all concentration range showed decrease in the percent cell viability as compared to that of   melanoma cell line control. In overall variation of test samples, Foeniculum vulgare extract  showed its best activity in the conc. of 200 μg/ml. However Helicteres isora extract showed its best activity in the concentration of 300 μg/ml which was approximately equal to the activity of standard drug doxorubicin. Therefore, we concluded from cell viability assay that 70% methanolic extract of Foeniculum vulgare and 50% methanolic extract of Helicteres isora  exhibited very good antitumor activity

 

Cytoprotection against normal human lymphocytes was based on the comparative toxicity caused by chemotherapy drug doxorubicin  alone and the toxicity caused by our crude extracts. Micronucleus assay revealed that percentage of micronucleus was increased in doxorubicin and blood treated group. However, the normal lymphocytes when subjected methanolic extracts of Foeniculum vulgare and  Helicteres isora, there was decrease in the percentage of micronucleus as compared to that of doxorubicin treated group. Since the presence of micronucleus is indirect evidence of DNA damage,  our results suggests that methanolic extract of Foeniculum vulgare and Helicteres isora showed more cytoprotection against normal human lymphocytes in comparision to that of doxorubicin.

 

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Received on 27.08.2008       Modified on 28.08.2008

Accepted on 15.09.2008      © RJPT All right reserved

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008; Page 450-452