Ravindra G.

Mali et al: An in vitro Study of Effect of Centella asiatica on Phagocytosis by Human Neutophils 297

An in vitro study of Effect of Centella asiatica on

Phagocytosis by Human Neutrophils
*Ravindra G. Mali, and Basavraj C. Hatapakki
Department of Pharmacognosy and Phytochemistry, K.L.E.S. College of Pharmacy, JNMC Campus, Belgaum-590 010

ABSTRACT: Present investigation was planned to evaluate the effect of ethanol extract of leaves of Centella
asiatica (CA) on neutrophil phagocytic function. The various concentrations (25, 50 and 100 mg/ml) of the
extract was subjected to study its effect on different in vitro methods of phagocytosis such as neutrophil
locomotion and chemotaxis, immunostimulant activity by phagocytosis of killed Candida albicans and
qualitative nitro blue tetrazolium test using human neutrophils. The results of this preliminary study revealed
that CA extract has stimulated chemotactic, phagocytic and intracellular killing potency of human neutrophils
at the concentration range of 25-100 mg/ml. These results suggest that the ethanol extract of CA stimulates
cell-mediated immune system by increasing neutrophil phagocytic function.

KEYWORDS: Centella asiatica, Phagocytosis, Neutrophils, Candida albicans, Chemotaxis

Introduction Centella asiatica (CA) Linn. (Umbelliferae),

In modern medicine immunology plays an important and
increasing role in understanding and diagnosis of the
diseases. The immune response is evolved I n the etiology
as well as the pathophysiologic mechanism of many
diseases. The modulation of immune response using
various agents in order to alleviate the disease has been of
interest since many years (Ballow and Nelson, 1997).
Some medicinal plants and their isolated constituents
have been reported to induce a state of non specifically
increased resistance in experimental animals and humans
(Brekhman and Dardymov, 1969). The stimulation of non-
specific defense mechanism system of the human organism
as one concept of therapy has a long tradition in medicine.
In traditional system of medicine of India, China, Korea, a
number of plants are described as ‘General tonics’ (Pallabi
and Dasgupta, 1998). In terms of modern medicine, this
approach to therapy so much similar to prohost therapy
which aim in augmenting host defense against infection by
improving immunocellular function.
Chemotherapeutic agents available today have mainly
immunosuppressive activity. Most of them are cytotoxic
and exerts a variety of side effects. This has given rise to
stimulation in the research for locating natural resources
showing immunomodulatory activity (Patil et al., 2008).
*Corresponding author: Ravindra G. Mali,
17-1, Professor Colony, Near Child Care Hospital,
Vijay Char Rasta, Navrangpura, Ahmedabad-380 009
commonly known as ‘Brahmi’ or ‘Mandukparni’ is
widely distributed in India and is of medicinal importance
(Vaidyaratnam PSV, 1994). The plant is a prostrate,
perennial, faintly aromatic herb found wild throughout
India and Sri Lanka up to an altitude of 2,000 feet. The
plant enjoys considerable reputation in Indian traditional
systems of medicine as diuretic, alterative and tonic. An
infusion of CA is used in India and Madagascar in
treatment of leprosy and is known to ameliorate the
symptoms of the disease and to improve the general health
of the patient (Anonymous, 1950). Chopra et al. (1958) has
mentioned that the plant is useful as alterative, tonic, and
also in the treatment of skin diseases, leprosy. The leaves
are taken as tonic and for improving memory and curing
syphilitic skin diseases internally as well as externally
(Kakkar, 1989). CA has been investigated scientifically
and found to possess a number of notable pharmacological
activities such as anti-psoriatic (Sampson et al., 2001),
wound healing (Shukla et al., 1999; Shetty et al., 2006),
hypoglycaemic (Mutayabarwa, 2003), hepatoprotective
(Antony et al., 2006), anti-gastric ulcer (Cheng et al., 2004),
anti-tumour (Babu et al., 1995), antimicrobial (Mamtha
et al., 2004), Antinociceptive and anti-inflammatory
(Somchit et al., 2004), anti-oxidant (Jayashree et al., 2003;
Hussin et al., 2007), nootropic and anxiolytic (Wijeweera
et al., 2006).
Neutrophils play an important role in host immune
mechanism system. The neutrophilic phagocyte system has
many advantages. They are attracted by a limited number
of stimuli, which generally signal the presence of tissue
injury of unknown reason. Even the foreign bodies,

297
298 International Journal of Pharmaceutical Sciences and Nanotechnology
thermal or chemical burns, bacterial infections and other
types of injuries can provoke an intense neutrophil
response. Moreover, neutrophils are highly effective at
killing certain bacteria and their ability to digest cellular
debris and exogenous particulate matter provides an
important step in the host defense mechanism (Berne and
Levy, 1988).
In vitro immunomodulatory activity of CA on human
neutrophils has not been documented in the literature.
Hence, in the present study an attempt has been made to
investigate cell mediated immunomodulatory potency of
leaves extract of CA using different in vitro methods for
locomotion, phagocytic and intracellular killing potency of
neutrophils, which are subsequent events involved in the
process of phagocytosis by neutrophils.
Material and Methods
Plant material
The leaves of CA were carefully collected from the fields
near Belgaum city and were authenticated at Botanical
Survey of India, Koregaon Road, Pune (Voucher specimen
No. 27047). A specimen voucher of the plant has been
deposited in the Department of Pharmacognosy, College of
Pharmacy, Belgaum. The leaves were shade dried and then
milled to coarse powder by mechanical grinder and then
used for extraction.
Preparation of extract
The powder material was then subjected to exhaustive
extraction using 95% ethanol in a soxhlet apparatus. The
dark green liquid extract so obtained was concentrated
under vacuum and the resulting dried extract was
lyophilized and preserved in a desiccator until further use.
The crude ethanol extract was subjected to preliminary
phytochemical investigation
Preparation of test sample
Samples for in vitro study were prepared by dissolving
1.0 gm of ethanol extract in 10 ml PBS (Phosphate Buffer
Solution) to get a stock solution of 100 mg/ml. From this
stock solution, different working dilutions were prepared
to get a concentration range of 25, 50 and 100 mg/ml.
Neutrophils of the blood withdrawn from normal human
volunteers were used to study the activity. PBS was used
as vehicle.
Volume 1 • Issue 3 • October - December 2008
Chemicals
All organic solvents were obtained from the S.D.Fine
Chemicals Private Limited (Mumbai, India) and all were of
analytical grade. Nitroblue tetrazolium (NBT), Phosphate
buffer saline (PBS, pH 7.2), May-Grunwald Giemsa stain,
Sodium deoxycholate, Methylene blue, Casein in Hanks
solution and Haematoxylin stain were all obtained from
Sigma Aldrich (St. Louis, MO).
Assessment of immunomodulatory activity
A. Neutrophil locomotion and chemotaxis
(Wilkinson, 1981)
Neutrophil cell suspension was prepared in phosphate
buffer saline solution (PBS) at about 106 cells/ml. The
lower compartments of the chemotaxis chamber (5 ml
beaker) was filled with appropriate chemotactic reagents
pre-adjusted to a pH of 7.2, e.g., chamber 1-PBS solution
(control), Chamber2- Casein 1 mg/ml (standard), and
chamber 3, 4 and 5 with different concentrations (25, 50
and 100 mg/ml) of test sample. The upper compartment
(1ml syringe) was filled with neutrophil cell suspension
and the wet filter (millipore) of 3mm pore size was fixed at
the bottom of the upper compartment. The upper
compartment was placed on to the lower compartment and
Incubated at 370C for 180 min. The upper compartment
was removed and inverted to empty out the fluid. The
lower surface of the filter was fixed with 70 % ethanol for
2 min and then stained with heamatoxylin dye for 5 min.
The fixed filters were observed under microscope using
100 X lens and the number of neutrophil cells reached to
the lower surface of the filter was counted.
B. in vitro immunostimulant activity studies by slide
method (Wilkinson, 1981)
Preparation of Candida albicans suspension
The Candida albicans culture was incubated in Sabouraud
broth overnight and then centrifuged to form a cell button
at the bottom and supernant was discarded. The cell button
was washed with sterile Hank’s Balanced Salt Solution
(HBBS) and centrifuged again. This was done 3-4 times.
The final cell button was mixed with a mixture of sterile
HBBS and human serum in proportion of 4:1. The cell
suspension of concentration 1x 108 was used for the
experiment.
 Ravindra G. Mali et al: An in vitro Study of Effect of Centella asiatica on Phagocytosis by Human Neutophils
Slide preparation
Human blood (0.2 ml) was obtained by finger prick
method on a sterile glass slide and incubated at 370C for 25
min to allow clotting. The blood clot was removed very
gently and slide was drained slowly with sterile normal
saline, taking care not to wash the adhered neutrophils
(invisible). The slide consisting of polymorphonuclear
neutrophils (PMNs) was flooded with predetermined
concentration of test sample and incubated at 370C for 15
min. The PMNs were covered with Candida albicans
suspension and incubated at 370C for 1 h. The slide was
drained, fixed with methanol and stained with Giemsa
stain. Positive control was tested by preparing the slide in a
same way with pooled normal human serum.
Phagocytosis evaluation
The mean number of Candida cells phagocytosed by
PMNs on the slide was determined microscopically for 100
granulocytes using morphological criteria. This number
was taken as phagocytic index (PI) and was compared with
basal PI of control. This procedure was repeated for
different concentrations (25, 50 and 100 mg/ml) of test
sample). Immunostimulation in % was calculated by using
following equation:
Stimulation (%) = PI (test) - PI (control) x 100/ PI (control)
C. Qualitative nitroblue tetrazolium (NBT) test
(Wilkinson, 1981)
A suspension of leucocytes (5 x 106/ml) was prepared in
0.5 ml of PBS solution in 5 tubes. 0.1 ml of PBS solution
(control) and 0.1 ml of endotoxin activated plasma
(standard) is added to the 1st and 2nd tube respectively and
to the other 3 tubes 0.1 ml of different concentrations
(25, 50 and 100 mg/ml) of test sample were added. 0.2 ml
Table 1. Effect of extract of Centella asiatica on neutrophil locomotion and chemotaxis.
SI. No. Group
1 Control (PBS)
2 Standard (Casein)
3 C.asiatica extract
4 C.asiatica extract
5 C.asiatica extract
Values are mean ± SEM (n=3), * p<0.001 compared to control group
299
of freshly prepared 0.15 % NBT solution was added to
each tube and incubated at 37oC for 20 min. Centrifuged at
400g for 3-4 min. to discard the supernatant. The cells
were resuspended in a small volume of PBS solution.
A thin film was made with the drop on a slide, dried
and fixed by heating, counterstained by dilute carbol-
fuchsin for 15 sec. The slide was washed under tap water,
dried and observed under 100X oil emulsion objective. 200
neutrophils were counted for the % of NBT positive cells
containing blue granules/lumps.
Statistical analysis
The values are expressed in mean ± SEM (n=3). The
results were analyzed by using one way analysis of
variance (ANOVA) followed by Dunnett’s t-test to
determine the statistical significance.
Results
The preliminary phytochemical screening of the ethanol
extract of leaves of CA showed the presence of
triterpenoids, saponins, glycosides, sterols and alkaloids.
The results shown in Table 1. illustrates that the ethanol
extract of CA has caused significant increase in movement
of number of neutrophils from the upper compartment to
lower surface of filter in a dose dependant manner. The
mean numbers of Candida cells phagocytosed by
neutrophils, in presence of extract were significantly
increase (p<0.001) as compared to the control group
(Table 2) and also depicted increase in percentage of NBT
positive cells containing the reduced NBT dye when
compared with control samples containing PBS solution
(Table 3). In case of neutrophil locomotion and qualitative
NBT test the results obtained with CA extract were
comparable with that of standard.
Concentration Mean number of
(mg/ml) neutrophil/Field
- 5.26 ± 0.45
01 72.21 ± 1.25
25 46.38 ± 1.32*
50 49.79 ± 1.48*
100 54.12 ± 1.25*
300 International Journal of Pharmaceutical Sciences and Nanotechnology
Table 2. Effect of extract of Centella asiatica on neutrophil phagocytosis
SI. No. Group
1 Control (pooled
plasma serum)
2 C.asiatica extract
3 C.asiatica extract
4 C.asiatica extract
Values are mean ± SEM (n=3), * p<0.001 compared to control group
Table 3. Effect of extract of Centella asiatica on qualitative NBT test
SI. No. Group
1 Control (PBS)
2 Endotoxin activated plasma
3 C.asiatica extract
4 C.asiatica extract
5 C.asiatica extract
Values are mean ± SEM (n=3), * p<0.001 compared to control group
Discussion
Immunomodulators are biologic response modifying
(BRM’s) compounds that affect the immune response in
either a positive or negative fashion. If it results in an
enhancement of immune reactions, is named as
immunostimulation and primarily implies stimulation of
non-specific system such as stimulation of the function and
efficiency of granulocytes, macrophages, complement,
certain T-lymphocytes and different effector substances.
Immunosuppression implies mainly to reduce resistance
against infections, stress and may be because of
environmental or chemotherapeutic factors (Hennessey and
Baker, 1994). Immunostimulation and immunosuppression
both need to be tackled in order to regulate the normal
immunological functioning. Both the immunostimulative
and immunosuppressive agents have their own standing
and search for better agents exerting these activities is
becoming the field of major interest all over (Wierda and
Reasor, 1986).
A number of disorders can be treated by BRM’s. These
include immunodeficiency diseases and autoimmune
disorders. These drugs may work on cellular or humoral
immune system or both (Claman, 1992).
In earlier immunological studies CA has shown
promising immunomodulatory activity. In one study, Patil
et al. (1998) evaluated aqueous suspension of CA for its
immunostimulant property using humoral
(Haemagglutinating antibody titre) and cell mediated
Volume 1 • Issue 3 • October - December 2008
Concentration % stimulation
(mg/ml)
- 4.26 ± 0.75
25 27.33 ± 1.12*
50 31.54 ± 1.58*
100 36.12 ± 1.45*
Concentration % NBT
(mg/ml) positive cells
- 22.21 ± 1.04
- 74.21 ± 0.82
25 61.28 ± 0.32*
50 66.49 ± 1.18*
100 79.12 ± 1.35*
(Delayed type hypersensitivity) immunity responses and
compared the activity with recombinant interferon alfa-2b
which is used as supportive medicine in the treatment of
cancer to increase immunity of patient. The results
demonstrated good activity, that is comparable to about
60% that of alfa-2b interferon. In another study, the pectin
and its degraded product, isolated from CA showed
immunostimulating activity to different extent in vitro
(Wang et al., 2005). Further, Jayathirtha and Mishra (2004)
also evaluated immunomodulatory potential of methanol
extract of CA using carbon clearance, antibody titer and
cyclophosphamide immunosuppression parameters and
claimed for significant activity of CA extract.
In our present study extract of leaves of CA
significantly increased the phagocytic function of human
neutrophils when compared to control indicating the
possible immunostimulating effect. The extract has
significantly increased the neutrophil chemotactic
movement as indicated by the increase in number of cells
reached the lower surface of filter. The ingestion of micro-
organism after coming in contact with them studied by
slide method which provides a rapid and simple means of
assessing the overall phagocytic process by the
neutrophils. The CA extract significantly increased
ingestion of Candida albicans by neutrophils. A
significant increase in the intracellular reduction of NBT
dye to form formazan (deep blue compound) was observed
confirming the intracellular killing property of
phagocytosing neutrophils.
 Ravindra G. Mali et al: An in vitro Study of Effect of Centella asiatica on Phagocytosis by Human Neutophils
Triterpenoid saponins widely distributed in plant
kingdom have been reported to exert immunomodulatory
activity (Plohmann et al., 1994; Mali et al., 2006). CA has
also been found to contain triterpenoid saponins. From the
results reported here, it can be concluded that the ethanol
extract of CA has significant effect on phagocytosis by
human neutrophils and chemotactic locomotion of
neutrophils and suggest further studies to evaluate its
potential as adjuvant to chemotherapeutics in the treatment
of infectious diseases with an added advantage of
stimulation of neutrophils for phagocytosis.
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