1. In vitro Anti-Inflammatory Activity of Aqueous Leaf Extract of
Vitex negundo
W.A.I.R.S. DE FONSEKA1
, K.W.J.C. KARIAWASAM2
, R.R. KUMARA2
, D.P.S.T.G. ATTANAYAKA1
E.D.
DE SILVA3
, W.D. RATNASOORIYA4
and S.M. HANDUNNETTI2
1
Department of Biotechnology, Faculty of Agriculture and Plantation Management,
Wayamba University of Sri Lanka, Makandura, Gonawila (NWP).
2
Institute of Biochemistry, Molecular Biology and Biotechnology (IBMBB),
University of Colombo, 94, Cumarathunga Munidasa Mawatha, Colombo 03.
.
3
Department of Chemistry, Faculty of Science, University of Colombo, Colombo 03.
4
Department of Zoology, Faculty of Science, University of Colombo, Colombo 03.
ABSTRACT
The immune cellular mechanisms that contribute to the anti-inflammatory activity of freeze-dried mature, leaf
extract (FLE) of Vitex negundo was investigated in this study. Inhibition of Nitric oxide (NO) production by and
cytotoxicity to rat peritoneal cells and membrane stabilising activity on rat erythrocytes were employed to study the
in vitro anti-inflammatory activity of FLE of V. negundo. In vitro cytotoxicity test using viable cell counts showed that
high concentrations (1000 µg/ml) of FLE of V. negundo were toxic to rat peritoneal cells where as the lower
concentrations (250 and 500 µg/ml) had no cytotoxicity; viable cell counts ranged from 82±1.2 to 90±1.2 %
(mean±SEM). Different FLE concentrations were used for the in vitro treatment of rat peritoneal cells and rat
erythrocytes and the respective effects on NO production and membrane stabilising activity were assessed. FLE of V.
negundo at 500 µg/ml significantly reduced NO production by rat peritoneal cells (3.2 ± 1.10 µM) compared to the
untreated cells (13.2±0.94 µM; P<0.001). In vitro treatment of FLE of V. negundo decreased the NO production by
rat peritoneal cells dose-dependently (r=0.57; P=0.002, Spearman correlation; 86.5 ± 4.9 % inhibition at 500 µg/ml).
Assay for Membrane stabilising activity showed dose-dependent percent inhibition of haemolysis by FLE of V.
negundo (r=0.91; P<0.001, Spearman correlation; 82.4±4.1% inhibition at 100 µg/ml). These results suggest that the
in vitro inhibition of NO production by immune cells and membrane stabilising activity of the freeze-dried product of
mature, fresh leaf extract of V. negundo contribute to its anti-inflammatory activity.
KEYWORDS: Anti-inflammatory activity, Cytotoxicity, Membrane stabilising activity, Nitric oxide, Vitex negundo
INTRODUCTION
About 80% of the world’s population depends
solely upon medicinal plants as a source of
medicine for the treatment of disease (Farnsworth,
1985). Most of the drugs are in fact derived from
natural sources but, as a source of novel drugs,
many of the plants remain under-studied and under-
used, especially in the ‘developed world’ (Kirby,
1996). The use of non-steroidal anti-inflammatory
drugs is associated with several adverse effects
such as gastric lesions and indigestion. Therefore,
investigations on plant-based drugs used in
Ayurvedha and traditional medicine have emerged
as an alternative since they are cheap, abundantly
available and relatively less toxic.
Vitex negundo L. (family: Verbenaceae, Nika
in Sinhala), is a large shrub with typical five foliate
leaf pattern and bluish purple flowers. This plant is
distributed in Sri Lanka, India, Malaya, The
Philippine Islands and East Africa. Flowers occur
throughout the year (Jayaweera and Senaratna,
2006). Water extracts of fresh mature leaves are
used in traditional medicine of Sri Lanka as anti-
inflammatory, analgesic and anti-itching agents
(Gunatillake, 1994). Recent study has shown that
ethanolic extracts of leaves of V. negundo can be
used orally as an adjuvant therapy along with
standard anti-inflammatory agents (Tandon, 2006).
Anti-inflammatory and analgesic activities of
mature, fresh leaf extracts of V. negundo have been
scientifically validated using the rat paw-edema
model (Dharmasiri et al., 2003). Previous study has
also shown anti-inflammatory and analgesic
activities of leaves of V. negundo using aqueous
methanol extracts (Telang et al., 1999). These
studies have indicated that anti-inflammatory
activities of V. negundo are possibly mediated via
prostaglandin synthesis inhibition, antihistamine,
membrane stabilising and anti oxidant activities
(Dharmasiri et al., 2003; Telang et al., 1999).
Among the immune cellular mechanisms,
nitric oxide (NO) produced by the phagocytic cells
play an important role in the inflammatory
response (Nathan, 1997). Inhibition of NO
production by immune cells is therefore considered
as another procedure for screening for anti-
inflammatory activity of plant extracts (Tezuka et
al., 2001).
The objective of this study was to further
investigate the immune cellular mechanisms that
contribute to the anti-inflammatory activity of
mature leaves of V. negundo. In the present study,

2. DE FONSEKA ET AL.
a freeze-dried preparation of a mature, fresh leaf
extract was used to assess its in vitro effects on (a)
inhibition of nitric oxide production by rat
peritoneal cells, (b) cytotoxicity and (c) membrane
stabilizing activity.
MATERIALS AND METHODS
Preparation aqueous leaf extract of V. negundo
Mature, fresh leaves of V. negundo were
collected from a tree in the campus garden of the
Faculty of Science, University of Colombo Sri
Lanka, between March and April 2008. Leaves
from the same tree have been used by Dharmasiri
et al, (2003) on the previous study. The leaves
were washed under running water and air-dried for
1h. The leaves were cut into small pieces and 500
g was blended with 1 L of distilled water in an
electric blender for 10 min. The extract was filtered
through a muslin cloth and freeze-dried using a
freeze dry system (Labconco Corporation, USA).
The freeze-dried leaf extract (FLE) was stored at
-20º
C in an air-tight container.
Animals
Healthy, adult, Wistar rats (150 - 200 g) were
obtained from the Medical Research Institute,
Colombo 8, Sri Lanka. The rats were housed under
standardized animal house conditions (temperature:
28-31º
C; photoperiod: 12 h natural light and 12 h
dark; humidity: 50 - 55%) and all animals had free
access to pelleted food (Finisher Feed, Ceylon
Grain Elevators, Colombo, Sri Lanka) and water
ad libitum.
Isolation of rat peritoneal cells
Rat peritoneal cells were isolated as described
previously with some modifications (Nacife et al,
2004). One ml of 0.1% carrageenan in Phosphate
Buffered Saline (PBS) pH 7.4 (5 mg/kg) was
injected intraperitoneally under ether anesthesia.
After two hours, 40 ml of sterile PBS was injected
into the peritoneal cavity. Five minutes later, 30 ml
of fluid from the peritoneal cavity was drained by
using 18 G cannular and centrifuged at 150 g for 5
min at 4
°
C. Supernatant was discarded and cells
were resuspended in 1 ml of complete culture
medium (CCM) RPMI 1640 medium (GIBCO BRI,
Life Technologies) supplemented with 0.2%
sodium bicarbonate and 1% bovine serum albumin
(Sigma Chemicals, USA) and gentamycin (50
µg/ml).
An aliquot of 20 µl of the cell suspension was
mixed with 4 µl of 1 % neutral red to visualize the
phagocytic cells and cell counts were made using a
haemocytometer (Karl Hecht, Germany).
In vitro cytotoxity test
Peritoneal cells resuspended in a dilution series
of FLE, i.e., (125, 250, 500, 1000 µg/ml) in CCM
were assessed for viability by Trypan blue
exclusion assay (Maccioni et al, 2003) after 0, 0.5
and 24 h of incubation at 37ºC in a 5% CO2
incubator. Peritoneal cells resuspended in CCM,
were used as the negative control.
Assay for (NO) production by rat peritoneal cells
Assay for production by rat peritoneal cells
was performed as described previously by
Maccioni et al, (2003).
a) Treatment of peritoneal cells with V. negundo
Peritoneal cells were resuspended in a dilution
series of FLE, i.e., (125, 250, 500, µg/ml) in CCM.
Peritoneal cells were also treated with 1 mM N-
monomethyl-L-arginine acetate salt (NMMA; nitric
oxide synthase inhibitor) (Sigma Chemicals, USA)
as the positive control. Peritoneal cells cultured in
complete culture medium, was used as the negative
control. All samples were incubated at 37ºC in a
5% CO2 incubator (Sanyo Electric CO. Ltd., Japan)
for 30 min. Cells were then centrifuged (150 g for 2
min) and resuspended in fresh complete culture
medium.
b) Assay for Nitrite production
To assess the NO production by rat peritoneal
cells that were treated in vitro, plated in 96 well
tissue culture plates at 1 × 106
cells/ml in complete
culture medium. Cells were plated in wells (n=6)
and incubated at 37ºC in a 5% CO2 incubator. After
24 h, culture supernatant was aspirated from each
well, centrifuged at 10,000 g for 10 min and clear
supernatant was assessed for nitrite production.
For quantification of nitrite, 100 µl of culture
supernatant was mixed with equal volume of Griess
reagent (equal mixture of 1% Sulphanilamide in
5% phosphoric acid and 0.1% N-(1-naphthyl)
ethylenediamine hydrochloride in distilled water),
kept at room temperature for 15 min and optical
density (OD) was read at 540 nm in a ELISA plate
reader (ELX 800, Bio-Tek Instruments INC, USA).
The nitrite concentration was calculated using
standard curve between 0.78 – 100 µM NaNO2
(Sigma Chemicals, USA).
Assay for membrane stabilizing activity
This assay was performed using heat-induced
haemolysis of rat erythrocytes as described
previously (Dharmasiri et al, 2003) with some
modifications (Nanayakkara, 2007). A ten fold
dilution series of FLE was prepared with PBS, in
triplicates for concentrations from 100 µg/ml to
0.001µg/ml. Similar dilutions of aspirin (State
Pharmaceutical Corporation) were also prepared in
triplicates and used as the positive control. PBS
was used as the negative control. Rat blood was
drawn using EDTA as the anticoagulent and 10 µl
of blood was added to each tube containing 500 µl
of control samples and dilutions of FLE. All tubes
were incubated at 37ºC for 15 min and centrifuged

3. IN VITRO ANTI-INFLAMMATORY ACTIVITY OF VITEX NEGUNDO
at 1500 g for 3 min. Supernatants were removed
and the cells were resuspended in 500 µl of PBS.
The samples were then incubated at 54ºC for 25
min and then centrifuged at 150 g for 3 min. Two
hundred µl of supernatant from each tube was
transferred into an ELISA plate and the OD was
measured at 540 nm. Percent inhibition of
haemolysis was calculated compared to controls.
Percent inhibition = (1-[OD] test / [OD]control) × 100
of haemolysis
Statistical analysis
Data were expressed as the mean and the
standard error of the mean (SEM). Statistical
analysis was performed using SPSS version 15.0.
Independent sample t-test for two independent
variables was used to test for the difference
between groups, at a 95% confidence interval.
Spearman correlation coefficients were calculated
to determine the dose-dependency.
RESULTS AND DISCUSSION
In Vitro Cytotoxicity of FLE of V. negundo on
peritoneal cells
As shown in the Table 1, the viable cell count
of rat peritoneal cells in the highest concentration
(1000 µg/ml) of FLE of V. negundo had reduced
markedly after 30 min incubation indicating that
this concentration was toxic to rat peritoneal cells.
The viable cell counts in the other two
concentrations (250 and 500 µg/ml) ranged from
82 to 90 % and were comparable to that of CCM,
indicating that these concentrations were well
tolerated by the rat peritoneal cells in vitro.
Therefore, FLE concentrations of 125, 250 and 500
µg/ml were used to assess the effects of FLE on
NO production by rat peritoneal cells and to assess
the membrane stabilising effect of FLE of V.
negundo.
Table 1. Viability of peritoneal cells cultured
with different concentrations of FLE of V.
negundo
Incub
-ation
Time
(h)
Percentage of living cells in culture medium
containing FLE (µg/ml)*
1000 500 250 0#
0 84 ± 0.10 90 ± 1.15 85 ± 1.91 90 ± 1.15
0.5 20 ± 1.63 85 ± 1.91 90 ± 1.15 90 ± 1.15
24 13 ± 1.15 82 ± 1.15 88 ± 1.00 88 ± 1.15
* Values are mean percentage of living cells ± SEM.
(n = 4) in the Trypan blue test. # - CCM
Inhibition of NO production by rat peritoneal
cells in vitro
Production of NO by rat peritoneal cells in
vitro was calculated using the standard curve of
NaNO2 (Figure 1).
y = 0.0146x+ 0.0518
R
2
= 0.9989
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 20 40 60 80 100 120
NaNO2 concentration (µM)
OD540nm
Figure 1. Standard curve for nitrite
Treatment of rat peritoneal cells with FLE had
significantly reduced the production of NO by rat
peritoneal cells (3.2 ± 1.1µM) of nitrite at 500
µg/ml (Figure 2). Percent inhibition of NO
production by V. negundo increased dose-
dependently from -0.2 ± 2.6 to 86 ± 4.9% for the
FLE concentration tested from 125µg/ml to 500
µg/ml (r=0.57; P=0.002, Spearman correlation)
(Figure 3). The percent inhibition observed at 500
µg/ml of FLE was comparable to that of the
positive control, NMMA (P=0.22). These results
indicated that the FLE contains compounds which
are potent NO inhibitors.
0
2
4
6
8
10
12
14
16
Nitriteconcentration(µM)
Cell free medium Carrageenan (CAR) CAR + FLE 125 µg/ml
CAR + FLE 250 µg/ml CAR + FLE 500 µg/ml 1mM NMMA
*
*
*
*
Figure 2. Production of NO by rat peritoneal
cells treated in vitro with FLE of V. negundo
Values represent mean ± SEM. *P<0.01 compared to
control group.
0
20
40
60
80
100
0 100 200 300 400 500
FLEConcentration (µg/ml)
Inhibitionof
NOproduction(%)
Figure 3. Inhibition of NO production by FLE of
V. negundo
Values represent mean ± SEM

4. DE FONSEKA ET AL.
Membrane stabilising activity
In this assay, increasing inhibition of
haemolysis of rat erythrocytes was observed with
increasing concentration of FLE and reached
82.4% ± 4.1% inhibition at 100 µg/ml (Figure 4).
Similar membrane stabilizing activity was observed
at the highest concentrations of the reference drug,
(80 ± 1.3%) at 100 µg/ml. However, FLE showed
lower stabilizing activity than the reference drug at
concentrations lower than 1µg/ml showing a sharp
decrease in the membrane stabilizing activity.
Membrane stabilizing activity of aspirin showed a
persisting higher levels at the lower concentration
(0.01, 0.1, 1 µg/ml). Both FLE and aspirin showed
significant dose-dependent membrane stabilizing
activity, r = 0.91; P<0.001 and r = 0.95; P<0.001
respectively, (Spearman correlation).
0
20
40
60
80
100
0.01 0.1 1 10 100 1000
Concentration (µg/ml)
Inhibition
ofHaemolysis(%)
Aspirin V. negundo
Figure 4. Membrane stabilizing activity of FLE
of V. negundo
Values represent mean ± SEM
A previous study has reported inverse
relationship of membrane stabilising activity of V.
negundo (Dharmasiri et al., 2003). This was
thought to be due to interference of the color of the
extract. Therefore, this assay was modified to have
an additional centrifugation step after the initial
incubation at 37ºC and prior to the heat-induced
haemolysis. The color detected at 540 nm,
following incubation at 54ºC was indeed due to the
heat-induced hemolysis. Using this modified assay,
dose-dependent membrane stabilising activities of
FLE of V. negundo were observed. These data also
indicated that the anti-inflammatory activity is
retained after the processing (freeze-drying) of the
mature, fresh leaf extract of V. negundo.
CONCLUSION
In conclusion, this study has shown that the in
vitro inhibition of NO production by immune cells
and membrane stabilizing activity contribute to the
anti-inflammatory activity of fresh mature leaf
extract of V. negundo.
ACKNOWLEDGEMENTS
Authors wish to thank Professor Kamani H.
Tennakoon, Director, Institute of Biochemistry,
Molecular Biology and Biotechnology (IBMBB)
for giving permission to conduct this research at
IBMBB. Deep appreciation is extended to Ms. C.
Nannayakkara and all the staff members of the
IBMBB. This project was carried out with the
assistance from NRC 05-52 and NSF 2005/HS/15
grants awarded to Dr S M Handunnetti. Sincere
gratitude is extended to Dr. S Jayasekara at the
Medical Research Institute for providing training
on handling rats.
.
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5. IN VITRO ANTI-INFLAMMATORY ACTIVITY OF VITEX NEGUNDO
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6. IN VITRO ANTI-INFLAMMATORY ACTIVITY OF VITEX NEGUNDO
therapy to standard anti-inflammatory drugs.
Indian Journal of Medicine 124, 447-450.
Telang, R. S., Chatterjee, S., and Varshneya C.
(1999). Studies on analgesic and anti-inflammatory
activities of Vitex negundo Linn. Indian Journal of
Pharmacology 31, 363-366.
Tezuka, Y., Irikawa, S., Kaneko, T., Banskota, A.
H., Nagaoka, T., Xiong, Q., Hase, K., and
Kadota, S. (2001). Screening of Chinese
herbal drug extracts for inhibitory activity
on nitric oxide production and
identification of an active compound of
Zanthoxylum bungeanum. Journal of
Ethnopharmacology 77, 209-217.