Research J. Pharm. and Tech. 1(4): Oct.-Dec.

2008,
,

ISSN 0974-3618 www.rjptonline.org

RESEARCH ARTICLE

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: Foeniculum vulgare is a plant belonging to the

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.
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
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.).
450
 Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008,
,
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.
451
 Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008,
,
1.1 Cell viability: doxorubicin. Therefore, we concluded from cell viability
Percentage viability of methanolic extract of Foeniculum assay that 70% methanolic extract of Foeniculum vulgare
vulgare and Helicteres isora is shown in the fig1 and fig 2 and 50% methanolic extract of Helicteres isora exhibited
very good antitumor activity
Fig No. 1 Effect of different conc. of Foeniculum vulgare on
percent cell viability; Concentration Vs Percent viability Cytoprotection against normal human lymphocytes was
based on the comparative toxicity caused by
Control
std chemotherapy drug doxorubicin alone and the toxicity
200 µg
/ ml 25 µg / ml caused by our crude extracts. Micronucleus assay
150 µg
/ ml
100 µg
/ ml
50 µg / ml
revealed that percentage of micronucleus was increased in
.

100 µg / ml
50 µg
/ ml 150 µg / ml doxorubicin and blood treated group. However, the
25 µg
/ ml
std
200 µg / ml normal lymphocytes when subjected methanolic extracts
Control of Foeniculum vulgare and Helicteres isora, there was
0 50 100 150 decrease in the percentage of micronucleus as compared
% Ce ll Viability
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
Fig No. 2 Effect of different conc. of Helicteres isora on
vulgare and Helicteres isora showed more cytoprotection
percent cell viability; Concentration Vs Percent viability
against normal human lymphocytes in comparision to that
300 µg / ml Control
of doxorubicin.
250 µg / ml Std
200 µg / ml
150 µg / ml
25 µg / ml REFERENCES:
50 µg / ml
100 µg / ml
1. Jing S, Bao RL, Wen JH., Li-Xia Y and Xiao PQ. In vitro
.

100 µg / ml
50 µg / ml anticancer activity of aqueous extracts and ethanol extracts of
150 µg / ml
25 µg / ml 200 µg / ml
fifteen traditional Chinese medicine on human digestive tumor
Std
250 µg / ml
cell line. Phytother. Res. 2007; 21: 1102- 1104.
Control
300 µg / ml 2. Thi-Shan D, Fu-Sheng J, Ni-Pi C, Gui-Yuan L and Cheng GZ. Isolation
0 50 100 150 and identification of an antitumor promoter component from leaves of
Impatiens balsamia. Molecules. 2008; 113: 220-299.
% Cell Viability
3. Shukla Y and Pal SK. Dietary cancer chemoprevention: An
overview. Int. J. Hum Genet. 2004; 4(4): 265-276.
2. In Virto Cytoprotection activity: 4. Muckenstrum B, Foechterlen D, Reduron JP, Danton P and Hildenbrand M.
Biochemical Systematics and Ecology. 1997; 25(4): 353-358.
2.1 Micronucleus assay: 5. Kawther FA. Antimicrobial activity of essential oil of some medicinal plants
Micronucleus count per 1000 binucleated cells is given in from Saudi Arebia. Saudi J. Biological Sci. 2007; 14 (1): 53-60.
the table o 3. 6. Oktay M, Gulchin I and Kufrevioglu IO. Determination of in
Groups Total Disrtibution of Total MN vitro antioxidant activity of fennel seed extract. Lebensn-
binuclea micronucleus MN/ / Wiss.U-technol. 2003; 36: 263-271.
ted cells 1000 100 7. Rosemayre S, Freire S, Sclene M and Francisco E. Synthesis
1 2 3 and antioxidant, anti-inflammatory and gastroprotector
scored MN MN MN binuclea cells
ted cells activities of anethole and related compounds. Fitoterapia.
2005; 75: 216-221.
Control 1000 -- -- -- -- 8. Kirthikar KR and Basu BD. An ICS. Indian medicinal plants, VI,
Dox + 1000 11 2 1 18 0.01 II, Lalit Mohan Basu, Allahabad, India,1981; 370-372.
blood 8 9. Sandhya P and Grampurohit ND. Isolation and characterization of
FV + 1000 6 -- 6 0.00 bioactive terpene from the fruits of Helicteres isora Linn.
blood 6 Pharmacognosy magazine ISSN. 2001; 0973-1296.
Dox = Doxorubicin MN = Micronucleus 10. Kamiya K, Saiki Y, Hama T, Fujimoto Y, Endang H, Umar M and Satake T.
FV = Foeniculum vulgare HI = Helicteres isora Flavonoid glucuronides from H. isora Phytochem. 2001; 57: 297-301.
11. Rodrigueg J, Yanez J and Lazona JA. Effects of several flavonoids on the
growth of B16f10 and SK-MEL-1 melanoma cell lines: relationship
DISCUSSION: between structure and activity, Melanoma research. 2002; 12: 99-107.
Treatment of Foeniculum vulgare extracts against 12. Jame KDN, Guyen V and Butter A. Cytotoxic potential of the
B16F10 melanoma cell line, in all concentration range phytochemical genistein isoflavone and certain environmental
showed decrease in percent cell viability, as compared to chemical compound on testicular cells, Biology of the cell.
1999; 91: 515-523.
that of negative when examined by “Trypan blue
13. Novakoric T, Olivera DM, Dqrko G and Slobodan J. Effect of
exclusion assay”. Also, Helicteres isora in all intratumoral application of methotrexate in vivo, on frequency
concentration range showed decrease in the percent cell of micronuclei in peripheral blood lymphocyte. Archive of
viability as compared to that of melanoma cell line Oncology. 2003; 11(1): 1-4.
control. In overall variation of test samples, Foeniculum 14. Jerzyrchive S, Grazyna B, Urszulam M, Maria W and Monika S.
vulgare extract showed its best activity in the conc. of Cytokinesis sblock micronucleus assay in human cells exposed to
radiation. Image anal Sterol . 2004; 23: 159-165.
200 μg/ml. However Helicteres isora extract showed its 15. Krunic A, Haveric S and Iburulj S. Micronuclei frequency in
best activity in the concentration of 300 μg/ml which was peripheral bloodof individual exposed to uranium. Arh Hig
approximately equal to the activity of standard drug Rada Tokshikol.2005; 56: 227-232.
452
Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008,
,

453