Studies of the membrane stabilizing activity of V.

Negundo Linn
(Verbenaceae).
Md. Shohel Rana1 , Mohammad A. Kaiser 2 , and Mohammad A. Rashid2
1
Department of Pharmacy , State University of Bangladesh, Dhaka-1205, Bangladesh
2
Department of Pharmaceutical Chemistry , Faculty of Pharmacy, University of Dhaka, Dhaka-1000,
Bangladesh.
ABSTRACT
Vitex negundo Linn. is a large aromatic shrub distributed throughout India. Herbal medicine, rather than merely
curing a particular disease, aims at returning the body back to its natural state of health. It has been used since
ancient times as a female remedy and also for pains in Ayurveda and also in Roman medicine. It became known
as the chaste berry tree. This species is globally distributed in Indo-Malesia, cultivated in America, Europe, Asia
and West Indies.
The methanolic extract of the whole plant V. Negundo (MEF) and it different partitionates i.e N-hexane (NHSF),
Carbon tetrachloride (CTCSF), Dichloromethane (DMSF) and aqueous (AQSF) soluble fractions are highly
effective in the membrane stabilizing activity as the extractives prevented the lyses of erythrocyte induced by
hypnotic solution.
INTRODUCTION
Vitex negundo Linn. is a large aromatic shrub distributed throughout India. Herbal medicine, rather than merely
curing a particular disease, aims at returning the body back to its natural state of health. It has been used since
ancient times as a female remedy and also for pains in Ayurveda and also in Roman medicine. It became known
as the chaste berry tree. This species is globally distributed in Indo-Malesia, cultivated in America, Europe, Asia
and West Indies. Within India, it is found throughout the greater part of India, in the outer Himalayas. Myriad
medicinal properties have been ascribed to Vitex negundo Linn. and the plant has also been extensively used in
treatment of a plethora of ailments as traditional medicine, folk medicine and pharmacological evidence.
Traditionally the leaves of Vitex negundo Linn. are documented to possess antibacterial, antitumor, astringent,
febrifuge, sedative, tonic and vermifuge. It has been reported to posses potent pharmacological properties like
anti-inflammatory, anti-rheumatic, antibiotic, Hepatoprotective, antioxidant, anticonvulsant, oxidative stress,
anti-androgen, snake venom neutralization and anti-allergic activities. The various chemical constituents like
flavonoids, flavones glycosides, volatile oil, triterpenes, tannins and many others were identified in this plant.
This review gives a bird’s eye view mainly on the pharmacognistic characteristics. traditional uses,
phytochemistry and pharmacological actions of Vitex negundo Linn.
Materials and Methods
Plant materials: The whole plants of V. negundo were collected from the department of Botany, University of
Dhaka in the month of February 2011 and voucher specimens for each of the collections have been deposited in
Bangladesh National Herbarium (BNH) for future references. The plants were first cleaned, cut into small pieces
and air- dried for several days. Then they were dried in oven at 400 C to facilitate size reduction through
grinding.
Extraction: The air-dried and powdered plant parts (300 gm each) were separately soaked in methanol (1.0 L
each) for 15 days at room temperature with occasional shaking and stirring. They were then filtered through a
fresh cotton plug and finally with Whatman No.1 filter paper. The volume of each of the filtrate was reduced
using a Buchii Rotary evaporator at low temperature and pressure and subsequent evaporation of solvents
afforded extracts of V. Negundo (1.0 kg)
Thrombolytic activity: Whole blood was drawn from healthy human volunteers without a history of oral
contraceptive or anticoagulant therapy and 1.0 ml of blood was transferred to the previously weighed
microcentrifuge tubes and was allowed to clot. The thrombolytic activity of all extracts was evaluated by the
method developed by Daginawala (2006) using streptokinase (SK) as the standard substance. The extract (100
mg) from each plant was suspended in 10 ml of distilled water and it was kept overnight. Then the soluble
supernatant was decanted and filtered through a 0.22-micron syringe filter. For clot lysis venous blood drawn
from healthy volunteers was distributed in different pre-weighed sterile icrocentrifuge tube (1 ml/tube) and
incubated at 37°C for 45 minutes. After clot formation, the serum was completely removed without disturbing
the clot and each tube containing the clot was again weighed to determine the clot weight (clot weight = weight
of clo containing tube – weight of tube alone).

* Correspondence to: Mohammad A. Rashid, Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

University of Dhaka, Dhaka-1000, Bangladesh. E-mail: rashidma@univdhaka.edu
To each microcentrifuge tube with the pre-weighed clot, 100 μl aqueous solution of different partitionates and
crude extract was added separately. Then, 100 μl of streptokinase (SK) and 100 mg of distilled water were
separately added to the control tube as positive and negative controls respectively. All the tubes were then
incubated at 37 °C for 90 minutes and observed for clot lysis. After incubation, the released fluid was removed
and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in
weight taken before and after clot lysis was expressed as percentage of clot lysis as shown below:
% of clot lysis = (wt of released clot /clot wt) × 100
Streptokinase (SK): Commercially available lyophilized Altepase (Streptokinase) vial (Beacon pharmaceutical
Ltd) of 15, 00,000 I.U., was collected and 5 ml sterile distilled water was added and mixed properly. This
suspension was used as a stock from which 100 μl (30,000 I.U) was used for in vitro thrombolysis.
Membrane stabilizing activity: The erythrocyte membrane resembles to lysosomal membrane and as such, the
effect of drugs on the stabilization of erythrocyte could be extrapolated to the stabilization of lysosomal
membrane (Omale, 2008). The membrane stabilizing activity of the extractives was assessed by using hypotonic
solution-induced and heat-induced mice erythrocyte haemolysis (Shinde et al., 1999). To prepare the
erythrocyte suspension, whole blood was obtained from mice and was taken in syringes (containing
anticoagulant EDTA) through cardiac puncture. The blood was centrifuged and blood cells were washed three
times with solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4) through centrifugation for 10
min at 3000g.
Hypotonic solution- induced haemolysis: The test sample consisted of stock erythrocyte (RBC) suspension (0.50
mL) mixed with 5 mL of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffered saline (pH
7.4) containing either the extract (1.0 mg/mL) or acetyl salicylic acid (0.1 mg/mL). The control sample
consisted of 0.5 mL of RBCs mixed with hypotonic-buffered saline alone. The mixture was incubated for 10
min at room temperature, centrifuged for 10 min at 3000 g and the absorbance of the supernatant was measured
at 540 nm. The percentage inhibition of either haemolysis or membrane stabilization was calculated using the
following equation-
% inhibition of haemolysis = 100 x (OD1-OD2/OD1),
where, OD1 = optical density of hypotonic-buffered saline solution alone (control) and OD2 = optical density of
test sample in hypotonic solution.
Heat- induced haemolysis: Isotonic buffer containing aliquots (5 ml) of the different extractives were put into
two duplicate sets of centrifuge tubes. The vehicle, in the same amount, was added to another tube as control.
Erythrocyte suspension (30 μL) was added to each tube and mixed gently by inversion. One pair of the tubes
was incubated at 54oC for 20 min in a water bath, while the other pair was maintained at 0-5oC in an ice bath.
The reaction mixture was centrifuged for 3 min at 1300 g and the absorbance of the supernatant was measured at
540 nm. The percentage inhibition or acceleration of hemolysis in tests and was calculated according to the
equation:
% Inhibition of hemolysis = 100 x [1- (OD2-OD1/OD3-OD1)]
where, OD1 = optical density of unheated test sample, OD2 = optical density of heated test sample and OD3 =
optical density of heated control sample
Result and discussion:
The different methanolic extracts of V. Negundo at concentration 2.0 mg/mL significantly protect the lysis of
human erythrocyte membrane induced by hypotonic solution, as compared to the standard acetyl salicylic acid
(0.10 mg/mL) (Table-1). At concentration of 2.0 mg/mL, the methanolic extract (MESF) produced 61.61% and
N – hexane soluble fraction (NHSF) produced 47.191% inhibition of haemolysis of RBC as compared to
65.543% produced by acetyl salicylic acid (0.10 mg/mL).
Table-1: Effect of different extractives of V. Negundo on hypotonic solution induced haemolysis of
erythrocyte membrane.
Sample code Concentration Absorbance % inhibition of
haemolysis
Hypotonic medium 50 mM 2.136 ---
MESF 2 mg/mL 0.820 61.61
NHSF 2 mg/mL 1.128 47.191
CTCSF 2 mg/mL 1.315 38.436
DMSF 2 mg/mL 1.362 36.235
AQSF 2 mg/mL 1.44 33.473
Acetyl salicylic acid 0.10 mg/mL 0.736 65.543

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