Biological activities of Barleria cristata

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* Corresponding author: Tasnuva Sharmin
E-mail address: tasnuva.phr.du@gmail.com
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP |Volume 2 | Issue 2 | 2013 Research article
Biological activities of Barleria cristata
Tasnuva Sharmin1, *
Shahanaz Ahmed1
, and Farhana Islam2
1
Department of Pharmacy, State University of Bangladesh.
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka,
Dhaka -1000, Bangladesh.
ABSTRACT
The methanol extract of leaves of Barleria cristata L. as well as its hexane, carbon tetrachloride, chloroform and
aqueous soluble partitionates were subjected to screening for antioxidant, cytotoxic, thrombolytic, membrane
stabilizing and antimicrobial activities. The antioxidant potential was evaluated by DPPH and Folin-Ciocalteau
reagents using butylated hydroxytolune (BHT) and ascorbic acid as standards. The extractives of B. cristata
exhibited weak free radical scavenging activity. The carbon tetrachloride soluble fraction exhibited an IC50 value of
300.82±0.33 μg/ml which demonstrated total phenolic content of 89.22±0.12 mg of GAE / g of extractives. The
general toxicity was determined by brine shrimp lethality bioassay where the hexane (LC50 =1.52±0.34 μg/ml)
soluble fraction revealed the presence of considerable bioactive principles. Mild thrombolytic activity was discerned
by B. cristata extractives. During assay for thrombolytic activity, the aqueous soluble fraction exhibited 45.0±0.22
% of clot lysis as compared to 65.66% and 3.791% clot lysis by standard streptokinase and water, used as positive
and negative controls, respectively. The crude methanol extract and chloroform soluble fraction inhibited
55.36±0.12% & 35.7±0.05% and 48.95±0.02% & 21.77±0.87% haemolysis of RBCs induced by hypotonic solution
and heat, respectively. Here, acetyl salicylic acid (0.1 mg/ml) was used as reference showing 71.9% and 42.12%
inhibition of haemolysis of RBCs in hypotonic solution and heat induced conditions, respectively. The chloroform
soluble materials demonstrated activity against microbial growth with zone of inhibition ranging from 7.0 to 26.0
mm.
KEYWORDS: Barleria cristata L., total phenolic content, DPPH, free radical scavenging activity, cytotoxicity,
thrombolytic activity, membrane stabilizing activity, zone of inhibition.
INTRODUCTION
Barleria cristata L. (Synonyms: Barleria ciliata
Roxb., Barleria dichotoma Roxb., Barleria
napalensis Nees.; Bengali name: Raktapushpa) is a
herbaceous perennial that attains a height of 36 to 48
inches and belongs to Acanthaceae family. It is native
to a wide area ranging from Southern China to India
and Myanmar. It is cultivated as an ornamental plant
in villages and gardens [1]
. Its seeds are used as
antidote for snake bites. The plant is used as a
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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stimulant and demulcent. Root decoction is given in
anaemia. Ethanol (50%) extract of the plant is
spasmogenic and hypoglycaemic [2]
.
As part of our ongoing investigations on medicinal
plants of Bangladesh [3, 4]
, the methanol extract of
leaves of B. cristata growing in Bangladesh as well
as its organic and aqueous soluble fractions were
studied for the antioxidant potential in terms of total
phenolic content and free radical scavenging
property; cytotoxic, thrombolytic, membrane
stabilizing and antimicrobial activities for the first
time and we, here in, report the results of our
preliminary investigations.
MATERIALS AND METHODS
Plant materials
The leaves of B. cristata were collected from Dhaka,
Bangladesh, in August 2012. A voucher specimen for
this plant has been maintained in Bangladesh
National Herbarium, Dhaka, Bangladesh for future
reference.
The sun dried and powdered leaves (800 g) were
macerated in 2.5 L of methanol for 7 days. The
extract was filtered through fresh cotton bed and
finally with Whatman filter paper number 1 and
concentrated with a rotary evaporator at reduced
temperature and pressure. An aliquot (5 g) of the
concentrated methanol extract was fractionated by
modified Kupchan [5]
partition protocol and the
resultant partitionates were evaporated to dryness
with rotary evaporator to yield hexane (HXSF, 1.5 g),
carbon tetrachloride (CTCSF, 1.5 g), chloroform
(CSF, 1 g) and aqueous (AQSF, 0.5 g) soluble
materials. The residues were then stored in a
refrigerator until further use.
Total phenolic content:
The total phenolic content of the extractives was
determined with Folin-Ciocalteau reagent by using
the method developed by Harbertson and Spayd
(2006) [6]
.
DPPH free radical scavenging assay:
Following the method developed by Brand-Williams
et al. (1995) [7]
, the antioxidant activity of the test
samples was assessed by evaluating the scavenging
activities of the stable 1,1-diphenyl-2-picrylhydrazyl
(DPPH) free radical by using synthetic antioxidants,
butylated hydroxytoluene (BHT) and ascorbic acid as
positive controls.
Brine shrimp lethality bioassay:
This technique was applied for the determination of
general toxic properties of the DMSO solutions of
plant extractives against Artemia salina in a single
day in vivo assay [8]
. Vincristine sulphate was used as
positive control.
Thrombolytic activity:
The thrombolytic activity was evaluated by the
method developed by Prasad et al. (2006) [9]
by using
streptokinase (SK) as positive control.
Membrane stabilizing activity:
The membrane stabilizing activity of the extractives
was assessed by evaluating their ability to inhibit
hypotonic solution and heat induced haemolysis of
human erythrocytes following the method developed
by Omale et al. (2008) [10]
.
Antimicrobial screening:
Antimicrobial activity was determine by disc
diffusion method [11]
.
Statistical analysis:
For all bioassays, three replicates of each sample
were used for statistical analysis and the values are
reported as mean ± SD.
RESULTS AND DISCUSSION
The present study was undertaken to evaluate the
antioxidant potential in terms of total phenolic
content and free radical scavenging property;
cytotoxic, thrombolytic, membrane stabilizing and
antimicrobial activities of different organic and
aqueous soluble materials of the crude methanol
extract of B. cristata leaves.
In DPPH free radical scavenging assay, all the
fractions demonstrated weak free radical scavenging
activity. The carbon tetrachloride soluble fraction
exhibited an IC50 value of 300.82±0.33 μg/ml that
could be correlated to its phenolic content of
89.22±0.12 mg of GAE / g of extractives. (Table 1).

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Table 1: Total phenolic content, free radical scavenging and cytotoxic activities of B. cristata
Samples/
Standards
Total phenolic content (mg of
GAE/ g of dried extract)
Free radical scavenging
activity IC50 (μg/ml)
Brine shrimp
lethality bioassay
LC50 (µg/ml)
ME 53.30±0.55 498.97±0.22 47.62±0.53
HXSF 0.525±0.85 728.58±0.91 1.52±0.34
CTCSF 89.22±0.12 300.82±0.33 78.45±0.87
CSF 85.65±0.25 407.73±0.27 15.18±0.50
AQSF 4.35±0.87 660.94±0.18 340.83±0.17
VS - - 0.451±0.04
BHT - 27.5±0.54 -
Ascorbic acid - 5.8±0.21 -
ME= Methanolic crude extract; HXSF= Hexane soluble fraction; CTCSF= Carbon tetrachloride soluble fraction; CSF= Chloroform
soluble fraction; AQSF= Aqueous soluble fraction; BHT= Butylated hydroxytolune; VS= Vincristine sulfate
In case of brine shrimp lethality bioassay, all the
fractions demonstrated significant cytotoxic potential
against A. salina with LC50 values ranging from 1.52
to 340.83 µg/ml. The hexane soluble fraction
revealed the highest cytotoxic activity with LC50
value of 1.52±0.34 μg/ml as compared to 0.451 μg/ml
for Vincristine sulphate (Table 1).
The extractives of B. cristata demonstrated mild to
moderate thrombolytic activity. The aqueous soluble
fraction demonstrated 45.0±0.22% of clot lysis as
compared to 65.66 % clot lysis by standard
streptokinase (Table 2).
Table 2. Thrombolytic activity of B. cristata
Samples % of lysis of RBC
ME 13.16±0.12
HXSF 10.41±0.13
CTCSF 1.96±0.28
CSF 4.61±0.08
AQSF 45.0±0.22
Water 3.791±0.55
SK 65.66±0.36
ME = Methanolic crude extract; HXSF= Hexane soluble fraction; CTCSF = Carbon tetrachloride soluble fraction; CSF = Chloroform
soluble fraction; AQSF = Aqueous soluble fraction; SK =Streptokinase.
At concentration 1.0 mg/ml, the extractives of B.
cristata significantly protected the haemolysis of
RBC induced by hypotonic solution and heat as
compared to the standard acetyl salicylic acid (0.10
mg/mL). The methanolic crude extract inhibited
55.36±0.12% and 35.7±0.05% haemolysis of RBCs
induced by hypotonic solution and heat as compared
to 71.9% and 42.12 % by acetyl salicylic acid,
respectively (Table 3).

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Table 3: Effect of different extractives of leaf of B. cristata on heat and hypotonic solution induced
haemolysis of erythrocyte membrane
Samples/Standard % Inhibition of haemolysis
Heat induced Hypotonic solution induced
Hypotonic medium - -
ME 35.7±0.05 55.36±0.12
HXSF 10.08±0.23 35.83±0.57
CTCSF 13.28±0.11 36.06±0.41
AQSF 7.97±0.87 33.39±0.27
CSF 21.77±0.87 48.95±0.02
ASA 42.12±0.38 71.9±0.78
ME = Methanolic crude extract; HXSF= Hexane soluble fraction; CTCSF = Carbon tetrachloride soluble fraction; CSF = Chloroform
soluble fraction; AQSF = Aqueous soluble fraction; ASA= Acetyl salicylic acid.
The antimicrobial activity of B. cristata test samples
was evaluated against five gram positive and eight
gram negative bacteria and three fungi and the results
were compared with standard antibiotic,
ciprofloxacin. Among the test samples of B. cristata,
only the chloroform soluble fraction revealed
antimicrobial activity with zone of inhibition ranging
from 7.0 to 26.0 mm. The highest zone of inhibition
(26.0 mm) was showed against Staphylococcus
aureus (Table 4).
Table 4: Antimicrobial activity of B. cristata
Test microorganisms
Diameter of zone of inhibition (mm)
CSF Ciprofloxacin
Bacillus cereus 11.0±0.22 45.0±2.01
B. megaterium 17.0±0.01 42.0±1.17
B. subtilis 15.0±0.40 42.0±0.73
Staphylococcus aureus 26.0±017 42.0±0.23
Sarcina lutea 17.0±0.18 42.0±0.56
Escherichia coli 9.0±0.26 42.0±0.43
Pseudomonas aeruginosa 12.0±0.63 42.0±1.11
S. typhi 16.0±0.08 47.0±2.33
Salmonella paratyphi 7.0±0.30 45.0±0.73
Shigella boydii 16.0±0.54 34.0±0.58
S. dysenteriae 9.0±0.47 42.0±0.22
Vibrio mimicus 15.0±0.65 40.0±0.45
V. parahaemolyticus 19.0±0.87 35.0±0.44
Candida albicans 16.0±014 38.0±0.49
Aspergillus niger - 37.0±0.33
Sacharomyces cerevacae 12.0±0.05 38.0±0.11
CSF= Chloroform soluble fraction

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It is clearly evident from the above findings that the
test samples of B. cristata possess significant
cytotoxic and membrane stabilizing activities. The
test samples are found to have mild to moderate
thrombolytic and antimicrobial activities as well.
Therefore, the plant is a good candidate for further
systematic, chemical and biological studies to isolate
the active principles.
Acknowledgement
The authors wish to acknowledge the phytochemical
research laboratory of State University of
Bangladesh.
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