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Pharmacologyonline 3: 651-658 (2010) Sushila et al.
651
Cytotoxic and Antibacterial Activity of Basella Alba Whole Plant
:
A Relatively Unexplored Plant
Rathee Sushila
1
, Ahuja Deepti
2
, Rathee Permender
1
, Thanki Madhavi
3
, Rathee Dharmender
2*
1
PDM College of Pharmacy, Bahadurgarh, Haryana
2
JCDM College of Pharmacy, Sirsa
3
NIPER, Ahmedabad, Gujarat
Address for Correspondence:
Dharmender Rathee
JCDM College of Pharmacy
Sirsa
Email: rathee_dharmender04@yahoo.com
Phone no: 09050352764
Summary
Aim of the study: To evaluate the Cytotoxic and antibacterial activity of Basella alba whole
plant extract.
Materials and methods: The phytochemical screening of the prepared plant extract was
carried out by chemical, thin-layer chromatography and spectroscopic methods. The
Cytotoxic activity was carried out using different dilutions of methanolic extract against
Jurkat and lung cancer cell lines. The antibacterial activity was carried out using different
dilutions of methanolic extract against gram positive strains (Staphylococcus aureus,
Micrococcus luteus, Bacillus subtilus) and gram negative ones (Pseudomonas aeruginosa) by
the cup-plate assay method and minimum inhibitory concentrations (MICs).
Results: The TLC fingerprinting reveals the presence of β-Sitosterol & Lupeol in the
methanolic extract. The BAE showed significant dose dependant cytotoxicity on Jurkat cell
lines as compared with the reference standard. The different concentrations of extract showed
moderate activity against Pseudomonas aeruginosa, Bacillus subtilis while weak response
against Staphylococcus aureus, Micrococcus luteus & Escherichia coli. The minimum
inhibitory concentration of methanolic extract was 6.25µg/ml against Staphylococcus aureus,
Micrococcus luteus, Pseudomonas aeruginosa & Bacillus subtilus and 12.5µg/ml against
Escherichia coli.
Conclusion: The methanolic extract shows the significant growth inhibition on human cancer
cell lines & momentous zone of inhibition for microorganisms studied. The overall result of
this study indicates that the methanolic extract from Basella alba have interesting anticancer
& antibacterial properties and the traditional use of this plant may also derive from its
antibacterial & anticancer properties.
Keywords: Cytotoxicity; antibacterial activity; Basella alba; Cell lines
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652
Introduction
Natural products have been used for combating human diseases for thousands of years, since
they exhibit a wide range of biological properties that can be exploited for medical
application [1]. Naturally occurring substances play an increasing role in drug discovery and
development. In fact, the majority of anticancer and antiinfectious agents are of natural
origin. In the present study we tried to investigate the anticancer and antibacterial potential of
relatively unexplored plant Basella alba Linn. Synonym- Basella rubra Roxb. commonly
known as Indian Spinach belonging to family Basellaceae is a fast growing perennial vine
found in the tropics where it is widely used as a leaf vegetable reaching 10 m in length. Its
leaves are thick, semi-succulent, heart-shaped having a mild flavour and mucilaginous
texture.
In Chinese traditional medicine, the leaves or the aerial parts of B. rubra have been used for
the treatment of constipation and also as a diuretic, toxicide, and anti-inflammatory [2].
Basella alba is an important medicinal plant in ethnoveterinary for treatment of retained
afterbirth and anaplasmosis. It is administered in gonorrhea and balanitis. The mucilaginous
liquid obtained from the leaves and tender stalks of this plant is a popular remedy for habitual
headaches. A decoction of the leaves is a good laxative for pregnant women and children [3].
It is extensively cultivated as an ornamental and the aerial parts such as the leaves, stems, and
young shoot with buds are consumed as a vegetable and health food. The roots are used in the
treatment of diarrhea, the cooked leaves and stems are used as laxatives [4-5]. The flowers
are used as an antidote to poisons and also as diuretic and febrifuge [6]. A paste of the root is
applied to swellings and is also used as a rubefacient, leaf juice is used in Nepal to treat
catarrh and is applied externally to treat boils [7]. The sap is used to anoint any part of the
body affected by acne in order to diminish the irritation. The plant is also used in treatment of
aphthae.
Chemical constituents: Plant is reported to have Betacyanins, Carotenoids and Organic acids
[8-10]. Triterpene oligoglycosides, Basellasaponins A, B, C, and D [11] having a
dioxolanetype substituent, along with Betavulgaroside I [12-16], Spinacoside C [17],
Momordins IIb, and IIc, [18] had been isolated from the aerial parts of B. rubra. The leaves
also contain carotenoids, organic acids and water soluble polysaccharides, bioflavonoids and
vitamin K [19]. β-Sitosterol & Lupeol are also reported to be present in the plant and are
reported to have anticancer, anti-inflammatory and antioxidant activities [20-30].
Material and Methods
Selection of plant: The plant was selected on the basis of traditional claims for its anti-
inflammatory and anti diarrhoeal activity. As inflammation plays a significant role in cancer
metastasis, so based on this hypothesis we tried to found out the cytotoxic potential of the
plant having anti-inflammatory activity. Chemical constituents (Betacyanins [31],
Carotenoids, Sterols & Triterpenes) present in this plant were also reported to have
antioxidant, anti-inflammatory & anticancer activities. Since the plant is extensively used
traditionally for diarrhoea by locals, this further strengthens our selection for the work on this
plant.
Collection of plant material: The Basella alba whole plant was collected freshly from
Jamnagar (Gujarat) in the month of August-September, 2008 depending upon its easy
availability. It was authenticated by our taxonomist against voucher specimen BA-1. The
plant was subjected to shed drying and further crushed to powder and then the powder was
passed through the mesh 40.
Pharmacologyonline 3: 651-658 (2010) Sushila et al.
653
Preparation of extract: The dried and ground plant material (1.0 kg) was first defatted with
petroleum ether and then successively extracted with methanol using Soxhlet apparatus for 12
hours and filtered to yield the extract. The extract was then concentrated in rotavapour and
finally dried to a constant weight. The extract obtained was stored in a refrigerator at 4
0
C
until use. The dried extract was used for the evaluation of cytotoxic and antibacterial activity.
Phytochemical screening: The Liebermann-Burchard’s and Salkowski reactions were carried
out to detect the sterols nucleus. It was further confirmed by TLC fingerprinting and UV-
Spectrophotometry. The extract was examined for β-Sitosterol & Lupeol by TLC on silica gel
60 F254 pre-coated TLC glass plates (Merck) developed with Toluene: methanol (9:1). The
spraying reagents used were anisaldehyde-H
2
SO
4
reagent and heated at 105
0
for 5 min. The
Rf values and colour of the resolved bands were noted and matched with the standards.
Further HPTLC was performed to estimate the percentage of β–Sitosterol & Lupeol in the
methanolic extract by calibration curve using peak height and peak area ratio. In addition,
since both, β-Sitosterol & Lupeol possess a characteristic UV absorption (λ max at 206 &
202, 320 nm respectively in Methanol); the UV absorbance of extracts for β-Sitosterol &
Lupeol was measured.
Cytotoxicity studies
Cell lines and culture: Jurkat and lung cancer (A549) cell lines were procured from National
Centre for Cell Science (NCCS), Pune. Cells were maintained in DMEM (Dulbecco’s
Modified Eagle Medium, Himedia) and 10% FBS with Penicillin (100 U/ml) and
Streptomycin (100 lg/ml) at 37
0
C, 5% CO
2
.
Preparation of sample: Immediately before assays were performed, the stored extract was
dissolved in small amount of dimethylsulfoxide (DMSO, Qualigens) upto 0.5% before
diluting with medium.
Cytotoxicity assay (MTT assay): The cytotoxicity of Basella alba whole plant extract (BAE)
was determined by tetrazolium (MTT, Sigma) assay [32]. Cells (2 × 10
3
/well) were plated in
100 µL of medium/well in 96- well plates. After overnight incubation, BAE extract was
dissolved in DMSO and the concentrations were adjusted to 25µg/ml, 50µg/ml, 75µg/ml &
100µg/ml and were added to each well. Paclitaxel was used as positive control in
concentration (5µM). After treatment for 48 hours 20 µl of 5 mg/ml MTT (pH 4.7) was added
to each well and cultivated for another 4 hours. The supernatant was removed and 100 µl
DMSO was added per well. Samples were then shaken for 15 min. The absorbance at 570 nm
was measured with microplate reader (Bio-Rad). All experiments were performed in triplicate
and all tests were performed 5 times individually. The effect of BAE on the proliferation of
cancer cells was expressed as relatively cell viability, using the following formula: Percent
viability = OD of drug treated sample/OD of none treated sample) × 100 [33].
Antibacterial studies
Microbial strains & culture media: Four strains of bacteria were procured from the
Microbial Type Culture Collection (MTCC, IMTECH), Institute of Microbial Technology,
Chandigarh, and were tested: Pseudomonas aeruginosa (MTCC 1688), Staphylococcus
aureus (MTCC 737), Bacillus subtilis (MTCC 441), Micrococcus luteus (MTCC 106),
Escherichia coli (MTCC 443). All the strains were stored at freeze temperature until use.
Nutrient agar (NA, Himedia) containing bromocresol purple was used for the activation of
Bacillus species, while NA was used for other bacteria. The NA was used in sensitivity assay.
Nutrient broth was used for MIC determination.
Pharmacologyonline 3: 651-658 (2010) Sushila et al.
654
Chemicals for antimicrobial assay: Ciprofloxacin (Central Drug House (P). LTD., New
Delhi) was used as positive reference standard for all bacterial strains. The DMSO was used
as solvent for the tested samples.
Preparation of inoculums: Bacterial inoculums were prepared by growing freeze-dried cells
in Nutrient Broth for 24 hours at 37
0
C. Slants were prepared by streaking of these cell
suspensions and sub culturing was done by using the same broth to provide initial cell counts
of about 104 CFU/ml and then sub culturing was done, incubated at 37
0
C for required time.
Preparation of test sample: The methanolic extract was dissolved in 10% aq. DMSO to
obtain the different concentrations (10 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml). 10% aq.
DMSO was used as negative control (solvent control). Ciprofloxacin was used as positive
reference standard having a concentration of 5µg/ml for all bacterial strains.
Antibacterial assay: Antibacterial activity was determined by cup plate method. Petri plates
were prepared by pouring 30 ml of Nutrient Agar Medium for all the bacteria. The test
organism was inoculated on solidified agar plate with the help of micropipette and spreaded
and allowed to dry for 10 min. Three wells or cavities were made in agar containing each
petri dish by a sterilized steel borer. To these cavities standard and test solutions were filled.
All the work was carried out under aseptic conditions for microbial assay. The plates for the
bacteria were incubated at 37
0
C ± 1
0
C for 24 hours. The antibacterial potential of test solution
was determined on the basis of mean diameter of zone of inhibition around the wells in
millimeters. Each assay was carried out in the form triplicate three times.
Minimum Inhibitory Concentration (MIC): The experiment was according to two fold
serial dilution method. The stock solution of test solution (extracts) was prepared at
concentration of 100µg/ml in nutrient broth and serially diluted up to five times. Six assay
tubes were taken for screening of minimum inhibitory concentration of each strain. In the
first tube 1ml of the sterilized nutrient broth was inoculated and then 1ml of the test solution
was added and thoroughly mixed to concentration of 50µg/ml. Further dilutions of this
solution were made by inoculating 1ml from first tube into second assay tube serially and 0.1
ml of each test inoculums were added in each tube and were done in duplicate. The
procedures were conducted under aseptic conditions. The inoculated tubes were kept at 37
0
C
± 1
0
C at 24 hours for bacterial assay during the incubation period. After the incubation
period, tubes were removed and observed for any deposits or turbidity in the solution and
shaken to suspend bacteria that might have been settled down. These concentrations were
observed & assumed as minimum inhibitory concentration (MIC).
Results and Discussion
TLC fingerprinting profile: The colorimetric reactions and UV absorption spectrum
confirmed the presence of β- Sitosterol & Lupeol in the extract. In the TLC plates one blue
(visible) and one purple (UV 525 nm) spots were present at Rf value of 0.37 & 0.60 after
spraying with anisaldehyde- H
2
SO
4
reagent, respectively. The colours and Rf values above
mentioned were the same as those obtained for standard reference compounds under the same
conditions. Though a TLC densitometric method was reported for the quantification of
lupeol, we modified the method so that both the compounds (β-Sitosterol & Lupeol) could be
quantified simultaneously. The contents estimated by HPTLC were found to be 0.07 & 0.05%
w/w respectively.
Pharmacologyonline 3: 651-658 (2010) Sushila et al.
655
Cytotoxicity assay: The results of preliminary screening showed that the BAE exhibited
cytotoxic activity against both cancer cell lines but the activity was very much significant
against Jurkat cell lines. The data also showed that the BAE exhibited cytotoxicity
comparable with positive control Paclitaxel on Jurkat cell lines. The percentage of growth
inhibition of BAE at various concentrations on human cancer cells were determined as the
relatively cell viability of viable treated cells in comparison with viable treated cells of
positive control. It showed a dose dependent inhibitory effect on cell growth (Fig. 1 & Table
1).
Fig. 1: % Growth inhibition of BAE on Jurkat & lung cancer cell lines
Table 1: Effect of different doses of BAE on cell viability of human cancer cell lines
% Cell Viability* Sample Concentration
A549 Jurkat
Paclitaxel (5 µM) 34.09 ± 0.93 40.72 ± 2.34
(25 µg/mL) 88.33 ± 1.20 53.32 ± 0.70
(50 µg/mL) 78.35 ± 0.99 48.70 ± 0.79
(75 µg/mL) 69.73 ± 5.51 43.28 ± 0.72
Basella alba
whole plant
Extract (different
conc.) (100 µg/mL) 57.12 ± 1.11 41.78 ± 1.25
*Mean ± S.D. (n=3)
Antibacterial assay: Methanolic extract was screened for antibacterial activity. The different
concentrations of extract showed moderate activity [34] against Pseudomonas aeruginosa,
Bacillus subtilis while weak response [34] against Staphylococcus aureus, Micrococcus
luteus & Escherichia coli [Table 2]. The results of different concentrations of extract were
correlated with standard drug and activity was found to be dose dependant against all
bacteria. The minimum inhibitory concentration of methanolic extract against bacterial
strains was found to be 6.25µg/ml for Staphylococcus aureus, Micrococcus luteus,
Pseudomonas aeruginosa and Bacillus subtilus and 12.5µg/ml for Escherichia coli [Table 3]
which clearly indicates its strong inhibition potential [35].
Pharmacologyonline 3: 651-658 (2010) Sushila et al.
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In summary, the present results provide evidence that the extracts of Basella alba contains
substances with cytotoxic & antibacterial activity, and, therefore, suggest that the traditional
use of this plant for the treatment of diarrhoea and anti-inflammatory properties can be linked
to cytotoxic & antibacterial properties. Anyway, further studies are necessary to isolate and
characterize the active constituents of the plant to evaluate their modes of action and render
this species interesting for future research.
Table 2: Antibacterial activity of BAE on various strains
Cup-plate method (inhibition zone, mm) Extracts Conc.
(mg/ml)
S. A. M. L. B. S. P. A. E. Coli
Basella alba
extract
10
25
50
100
-
5.0 ± 0.1
10.3 ± 0.058
16.3 ± 0.058
-
3.3 ± 0.058
7.7 ± 0.12
10.7 ± 0.12
3.32 ± 0.28
6.02 ± 0.036
13.67 ± 0.058
21.02 ± 0.1
5.3 ± 0.058
10.7 ± 0.12
15.1 ± 0.1
22.2 ± 0.1
-
4.0 ± 0.1
7.7 ± 0.058
15.3 ± 0.058
Ciprofloxacin
5 µg/ml 26 ± 0.051 14 ± 0.068 32 ± 0.024 25 ± 0.035 22 ± 0.056
*Mean ± S.D. (n=3)
S. A. -Staphylococcus aureus, M. L. - Micrococcus luteus, B. S. - Bacillus subtilus,
P. A. - Pseudomonas aeruginosa, E. coli - Escherichia coli;
- Sign shows no zone of inhibition
Table 3: The MIC values of BAE on various bacterial strains
Serial dilution (µg/ml)
Microorganisms
50
25
12.5
6.25
3.12
1.56
Staphylococcus aureus
- - - - + +
Micrococcus luteus
- - - - + +
Pseudomonas aeruginosa
- - - - + +
Bacillus subtilus
- - - - + +
Escherichia coli
- - - + + +
- No growth; + Growth; Stock solution = 100 µg/ml
Acknowledgement
Authors are highly thankful to Dabur India Ltd. for providing the free gift sample of
Paclitaxel and to Dr. Reena Agrawal for her kind and timely assistance during the work.
Pharmacologyonline 3: 651-658 (2010) Sushila et al.
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