Anticancer and Antioxidant Property of Bunium bulbocastanum Fruits Various Fractions

Abstract

Bunium bulbocastanum (family Apiaceae) fruits are normally used as a culinary spice for years. Since antioxidant property has a significant role in preventing oxidative stress, which has been demonstrated to be a major cause in the pathogenesis of cancer, the present study was aimed to evaluate the in vitro antioxidant and cytotoxic effect of Bunium bulbocastanum fruit extracts. Standard in vitro antioxidant assays such as reducing power, super oxide scavenging, nitric oxide scavenging and lipid peroxidation were performed. Anticancer activity was demonstrated using MTT assay on human cell lines. Our results indicated that extract of ethyl acetate has more antioxidation potential than the standard (ascorbic acid). Ethyl acetate, aqueous, crude methanolic, n-hexane and chloroform fractions had 31.24, 26.07, 20.67, 15.51 and 5.72% antioxidation potential at 0.5 mg/ml respectively. Moreover, in the screening of anticancer activity MTT assay revealed the potency of ethyl acetate fraction as better anticancer agent concluding the anticancer activity is due to its antioxidant property.

Full text

RRJoPC (2016) 9-13 © STM Journals 2016. All Rights Reserved Page 9
Research & Reviews: A Journal of Pharmacognosy
ISSN: 2394-7276(online)
Volume 3, Issue 1
www.stmjournals.com
Anticancer and Antioxidant Property of Bunium
bulbocastanum Fruits Various Fractions
Iswar Hazarika1*, Anju Das2
1Department of Pharmacology, T John College of Pharmacy, Gottigere, Bannerghatta Road,
Bangalore, Karnataka, India
2Department of Pharmacology, The Oxford College of Pharmacy, Hongasandra, Bangalore,
Karnataka, India
Abstract
Bunium bulbocastanum (family Apiaceae) fruits are normally used as a culinary spice for
years. Since antioxidant property has a significant role in preventing oxidative stress, which
has been demonstrated to be a major cause in the pathogenesis of cancer, the present study
was aimed to evaluate the in vitro antioxidant and cytotoxic effect of Bunium bulbocastanum
fruit extracts. Standard in vitro antioxidant assays such as reducing power, super oxide
scavenging, nitric oxide scavenging and lipid peroxidation were performed. Anticancer
activity was demonstrated using MTT assay on human cell lines. Our results indicated that
extract of ethyl acetate has more antioxidation potential than the standard (ascorbic acid).
Ethyl acetate, aqueous, crude methanolic, n-hexane and chloroform fractions had 31.24,
26.07, 20.67, 15.51 and 5.72% antioxidation potential at 0.5 mg/ml respectively. Moreover, in
the screening of anticancer activity MTT assay revealed the potency of ethyl acetate fraction
as better anticancer agent concluding the anticancer activity is due to its antioxidant property.
Keywords: Anticancer, antioxidant, Bunium bulbocastanum
*Author for Correspondence E-mail: iswarhazarika@gmail.com
INTRODUCTION
Oxidative stress has destructive effect in our
body by damaging proteins, nucleic acid and
lipids that leads to the pathogenesis of cancer
[1]. This oxidative stress is due to the over
production of free radicals, which are
generated because of both endogenous and
exogenous sources. Endogenous include
respiratory chain and oxidative enzymes while
exogenous include smoking and other air
pollutants [2]. Since, the therapeutic effects of
several medicinal plants are attributed to its
antioxidant phytoconstituents, antioxidant
properties are one of the most important
claims for food ingredients, dietary
supplements, cosmetics and anticancer natural
products [3]. Researchers suggest an inverse
relationship between the occurrence of disease
and the intake of dietary antioxidant rich
foods. [4]. Plant based antioxidants are
preferred to the synthetic based medicines
because of multiple mechanisms of actions
and least toxic nature [5, 6]. It has been
suggested that more than 50% of drugs in the
present clinical trials studies are plant products
with a reported antioxidant property. Bunium
bulbocastanum commonly known as kala
jeera (Black cumin) belongs to the family
Apiaceae. Its fruit has been used for edible
purpose for years and are used either in raw or
cooked form to enhance flavor [7]. Bunium
bulbocastanum exhibits antioxidant property
related to its antidiabetic property, is well
understood [8]. In view of the above findings,
an attempt was made to explore the
antioxidant and anticancer activity of Bunium
bulbocastanum fruit.
MATERIALS AND METHODS
Plant Material
Fruits of B. bulbocastanum plants were
collected from Kashmir in the month of July,
2015. The sample was authenticated by
renowned botanist in the research and
development laboratory of Natural Remedies
Pvt. Ltd. Bangalore of Karnataka State, India,
by comparing the sample with authentic
sample. A voucher specimen with batch
number CP/412 has been preserved at the
laboratory for further reference.

Anticancer and Antioxidant Property of B. Bulbocastanum Hazarika and Das
RRJoPC (2016) 9-13 © STM Journals 2016. All Rights Reserved Page 10
Extraction
The B. bulbocastanum fruits were shade dried
and were made into powder by chopping and
grounding. The powdered materials were
soaked in methanol for 15 days at room
temperature twice. Filtrates were combined
each time the filtrate was filtered and
concentrated using rotatory evaporator at 40°C
to crude methanolic extracts.
Fractionation
The crude methanolic extract of B.
bulbocastanum fruits (137 g) was suspended in
350 ml distilled water. For further fractionation,
the suspension was partitioned with n-hexane
(3×500 ml), chloroform (3×500 ml) and ethyl
acetate (3×500 ml), respectively, to yield the n-
hexane (45 g), chloroform (30 g), ethyl acetate
(16 g) and aqueous (37 g) fractions. All the
fractions contain particular compounds based
on the solubility of these compounds from the
crude extract.
Antioxidant Activity
Nitric Oxide Method [9]
To perform the NO free radical scavenging
assay a stock solutions of different test samples
were prepared by dissolving 3 mg of the test
samples in 1 ml of methanol. Different dilutions
of test sample were made from the stock
solution viz. 0.5, 0.25 and 0.125 mg/ml and in
separate test tubes 1 ml of each dilution was
introduced along with 1 ml of sodium
nitroprusside to make the reaction mixture. This
mixture was then incubated at 27°C for 90 min.
After incubation 0.5 ml of the reaction mixture
was added to 1 ml of sulphanilic acid and
incubated for 5 min at 27°C. 1 ml of N-(1-
Napthyl) ethylene diamine dihydrochloride
(0.1% in H2O) is added to it and again
incubated at 27°C for 30 min. Results were
obtained by taking absorbance at 546 nm.
Methanol was used as blank and ascorbic acid
(Vitamin C) was used as positive control
respectively.
Percentage antioxidation potential was
calculated by the formula:
Percent inhibition=[Ao–At/Ao]×100
Where, Ao is absorbance of control reaction
mixture without extract and At is the absorbance
of test samples.
Determination of Anticancer Activity- MTT
Assay [10]
In this study, extracts were tested for their
anticancer potency against human cancer cell
lines HeLa, Hep 2, MCF 7 and NIH 3T3 mouse
embryonic fibroblasts cells using MTT assay.
For that, the cancer cells were seeded in 96-well
plates at a density of 1×104 cells/well in 100 μl
RPMI media. The medium was removed after
twenty-four hours of seeding and then the cells
were incubated for 3 days with RPMI with the
absence and/or the presence of various
concentrations of B. bulbocastanum extracts.
Extracts were added at various concentrations
ranging from 62.5–1000 μg/ml. After
incubation, 20 μl of MTT reagent was added
into each well. These plates were incubated
again at 37°C in CO2 incubator for 4 h. The
resulting MTT-products were determined by
measuring the absorbance at 570 nm using
ELISA reader. The cell viability was
determined using the formula:
Viability %=(optical density of sample/optical
density of control)×100.
IC50 values were calculated as the
concentrations that show 50% inhibition of
proliferation on any tested cell line.
RESULTS
NO Free Radical Scavenging Assay
Ascorbic acid (Vitamin C) was used as the
standard for determining the antioxidation
potential of B. bulbocastanum fruits fractions at
different concentrations.
Table 1: Percent Antioxidation Activity of B. bulbocastanum Fruits Fractions at 0.5, 0.25 and
0.125 mg/ml Concentrations.
Test Sample
Percent Antioxidation Activity
0.5 mg/ml
0.25 mg/ml
0.125 mg/ml
Ascorbic acid
25.60
25.38
23.44
Aqueous fraction
25.17
15.17
12.08
Chloroform fraction
4.82
4.34
3.68
Crude methanolic extract
19.77
16.48
10.94
Ethyl acetate fraction
30.34
23.99
20.76
n-hexane fraction
14.61
14.02
7.42

Research & Reviews: A Journal of Pharmacognosy
Volume 3, Issue 1
ISSN: 2394-7276(online)
RRJoPC (2016) 9-13 © STM Journals 2016. All Rights Reserved Page 11
Table 1 shows the results obtained from NO
scavenging assay. It can be seen from the
Table 1 that among the different fractions of B.
bulbocastanum, ethyl acetate fraction had a
potent antioxidation (30.34%) at 0.5 mg/ml
concentration which even exceeds the
antioxidation potential of the standard
(25.60%).
Aqueous fraction at 0.5 mg/ml concentration
also showed significant antioxidation potential
of 26.07%. The rest of the three fractions
however had a lower antioxidation activity
from the standard at all the concentration.
Determination of Anticancer Activity: MTT
Assay
The results of anticancer potency of B.
bulbocastanum fruits fractions against human
cancer cell lines viz. HeLa, Hep 2, MCF 7 and
NIH 3T3 mouse embryonic fibroblasts cells
using MTT assay are given in Table 2. Table
shows that the ethyl acetate fraction and
aqueous fraction had a potent anticancer
activity against HeLa, Hep2 and MCF7 cell
lines but none of the fractions showed
cytotoxic effect for NIH 3T3 cell lines.
However, other fractions had no cytotoxic
effect.
Table 2: Cytotoxic Effect (IC50) of B. bulbocastanum Fruits Fractions in Human Cell Lines.
Test Sample
HeLa (μg/ml)
Hep2 (μg/ml)
MCF7 (μg/ml)
NIH 3T3 (μg/ml)
Aqueous fraction
368.75±0.78
314.65±0.45
321.75±0.34
>1000
Chloroform fraction
>1000
>1000
>1000
>1000
Crude methanolic extract
>1000
>1000
>1000
>1000
Ethyl acetate fraction
257.19±0.62
218.70±0.45
221.06±0.67
>1000
n-hexane fraction
>1000
>1000
>1000
>1000
HeLa: Human Cervical Cancer Cell Line; Hep2: Human Laryngeal Epithelial Carcinoma Cells;
MCF7: Breast Cancer; NTH 3T3: Mouse Embryonic Fibroblasts.
DISCUSSION
The present study was designed to provide
comparative data on the in vitro antioxidant
and cytotoxicity study of different extracts of
B. bulbocastanum fruits fractions against
human cancer cell lines viz. HeLa, Hep 2,
MCF 7 and NIH 3T3 Mouse embryonic
fibroblasts cells.
Antioxidants protect the cell constituents
against oxidative damage and hence decrease
the risk of various degenerative diseases
associated to oxidative stress [11]. Our result
for the NO free radical scavenging assay
suggest that percent antioxidation potential
increases as the concentration of test samples
increases, indicating concentration dependence
of antioxidation ability in both the test samples
and standard. The investigation suggests that
all fractions of B. bulbocastanum had
antioxidation potential with an encouraging
finding of percent antioxidation inhibition of
31.24 and 26.07% at 0.5 mg/ml for ethyl
acetate fraction and aqueous fraction. However
in particular the ethyl acetate fraction has
antioxidation ability of 30.34% as compared to
the standard (Ascorbic acid) value of 25.60%
represents its possible therapeutic use as
antioxidant. Nitric oxide (NO) itself is a
radical and it is reported that nitric oxide
molecules are directly scavenged by
antioxidants [12]. When antioxidants are used,
free radicals are scavenged and therefore can
no longer react with nitric oxide, resulting in
less damage [13].
The results in Table 2 suggest that ethyl
acetate fraction and aqueous fraction showed a
potent IC50 value for all tested cell lines except
NIH 3T3 normal cell lines (IC50>1000 µg/ml)
whereas the other fractions did not produce
any significant effect. Thereby this study
suggests that the anticancer activity of B.
bulbocastanum fruits fraction may be related
to its antioxidant property. All the reactive
oxygen species can damage DNA and cell
divisions leading to mutations with unrepaired
or mis-repaired damage. If these changes
appear crucially in critical genes, such as
oncogenes or tumor suppressor genes, they
may result in initiation or progression of
cancer. Reactive oxygen species (ROS) can
interfere directly with cell signaling and
growth. The cellular damage caused by

Anticancer and Antioxidant Property of B. Bulbocastanum Hazarika and Das
RRJoPC (2016) 9-13 © STM Journals 2016. All Rights Reserved Page 12
reactive oxygen species can induce mitosis,
increasing the risk that damaged DNA will
lead to mutations, and can increase the
exposure of DNA to mutagens [14].
CONCLUSION
From the investigation we are able to conclude
that ethyl acetate and aqueous fraction of B.
bulbocastanum fruit has a noteworthy
antioxidant and anticancer activity. Although,
the components responsible for the activity are
uncertain; further investigation are required to
isolate the pure constituents and to understand
their mechanism.
REFERENCES
1. Rahimi R, Nikfar S, Larijani B, et al. A
Review on the Role of Antioxidants in the
Management of Diabetes and its
Complications. Biomed Pharmacother.
2005; 59: 365–373p.
2. Young IS, Woodside JV. Antioxidants in
Health and Disease. J Clin Pathol. 2001;
54: 176–186p.
3. Cherdshewasarta W, Sutjit W. Correlation
of Antioxidant Activity and Major
Isoflavonoid Contents of the
Phytoestrogen-Rich Puerariamirifica and
Puerarialobata Tubers. Phytomedicine.
2008; 15: 38–43p.
4. Yildrim A, Oktay M, Bilaloglu V. The
Antioxidant Activity of Leaves of Cydonia
vulgaris. Turk J Med Sci. 2001; 31: 23–
27p.
5. Akinmoladun AC, Ibukun EO, Afor E, et
al. Chemical Constituents and Antioxidant
Activity of Alstoniaboonei. Afr J
Biotechnol. 2007; 6: 1197–1201p.
6. Arun KP, Brindha P. Studies on
Antioxidant and Antiarthritic Potentials of
Jatropha tanjorensis Ellis and Saroja. Int J
Pharm Pharm Sci. 2012; 4(Supp 2): 136–
138p.
7. Clapham, Tootin, Warburg. Flora of the
British Isles. UK: Cambridge University
Press; 1962.
8. Ahmad H, Khan I, Nisar W. Antioxidation
and Antiglycation Properties of Bunium
bulbocastanum Fruits Various Fractions
and its Possible Role in Reducing Diabetes
Complication and Ageing. Vitam Miner.
2014; 3: 118p.
9. Ebrahimzadeh MA, Nabavi SF, Nabavi
SM. Antioxidant Activities of Methanol
Extract of Sambucusebulus L. Flower. Pak
J Biol Sci. 2009; 12: 447–450p.
10. Francis D, Rita L. Rapid Colorimetric
Assay for Cell Growth and Survival
Modifications to the Tetrazolium Dye
Procedure giving Improved Sensitivity and
Reliability. J Immunol Methods. 1986; 89:
271–277p.
11. Davide Grassi, Giovambattista Desideri,
Claudio Ferri. Flavonoids: Antioxidants
against Atherosclerosis. Nutrients. 2010;
2: 889–902p. doi:10.3390/nu2080889
12. Van Acker SA, Tromp MN, Haenen GR,
et al. Flavonoids as Scavengers of Nitric
Oxide Radical. Biochem Biophys Res
Commun. 1995; 214: 755–759p.
13. Shutenko Z, Henry Y, Pinard E, et al.
Influence of the Antioxidant Quercetin in
vivo on the Level of Nitric Oxide
Determined by Electron Paramagnetic
Resonance in Rat Brain during Global
Ischemia and Reperfusion. Biochem
Pharmacol. 1999; 57: 199–208p.
14. Nijveldt Robert J, Els van Nood, Danny
EC van Hoorn, et al. Flavonoids: A
Review of Probable Mechanisms of
Action and Potential Applications. Am J
Clin Nutr. 2001; 74: 418–425p.
Cite this Article
Iswar Hazarika, Anju Das. Anticancer
and Antioxidant Property of Bunium
Bulbocastanum Fruits Various
Fractions. Research & Reviews: A
Journal of Pharmacognosy. 2016; 3(1):
9–13p.