Comp Clin Pathol (2012) 21:1343–1347
DOI 10.1007/s00580-011-1294-4
ORIGINAL ARTICLE
Effect of methanolic extract of Mucuna pruriens seed
on the immune response of mice
James Ifeanyichukwu Eze & Sunday Ndukwe
Received: 27 January 2011 / Accepted: 12 July 2011 / Published online: 26 July 2011
# Springer-Verlag London Limited 2011
Abstract The effect of the methanolic extract of Mucuna
pruriens seed was evaluated in mice with regards to
delayed hypersensitivity reaction (DTR), primary and
secondary antibody response and in vivo inflammatory
leucocyte mobilization. The extract at 250 and 500 mg/kg
produced significant (p<0.05) inhibition of DTR in mice by
33.33% and 28.89%, respectively. The extract caused
elevation of secondary SRBCs-specific antibody titre with
antibody response being significantly (p<0.05) increased at
250 and 500 mg/kg when compared with control. The
extract at 250 and 500 mg/kg increased leucocyte mobili-
zation significantly (p<0.05) when compared with the
control and levamisole-treated groups. At 100 mg/kg, the
mobilization did not differ with the levamisole-treated
group but was significantly (p<0.05) higher than the
control. The leucocyte mobilization was more of neutro-
phils than lymphocytes. The ability of the extract to inhibit
the DTR, increase secondary antibody response and in vivo
leucocyte mobilization was an indication that the extract
probably influenced immune response in mice.
Keywords Mucuna pruriens . Delayed hypersensitivity
reaction . Antibody response . Inflammatory leucocyte
mobilization
Introduction
The immune system protects the body from antigenic
substances from bacteria, viruses, toxins, cancer cells, and
J. I. Eze (*) : S. Ndukwe
Department of Veterinary Medicine, University of Nigeria,
Nsukka, Nigeria
e-mail: jamesifeeze@yahoo.com
URL: http://www.unn.edu.ng
foreign bodies (Zinkernagel and Hengartne 2001). When the
immune system detects an antigen, it responds by producing
antibodies and stimulating cellular responses (Sherwood
2008). The host's inability to mount an effective immune
response against an invading pathogen (Robert 2009) occurs
in diseases such as human acquired immunodeficiency
syndrome (Mathew 2010), measles (Tishon et al. 1996),
trypanosomosis and tuberculosis (Oyejide et al. 1985;
Mathew 2010), and infectious bursal disease (Sharma et al.
2000). Immunosuppression therefore leads to impaired host
defenses such as disruption of the body's mechanical barriers
to invasion, defects in nonspecific and specific host defenses.
Minimizing immunosuppression is an important strategy
for success in disease control and management. The need to
maintain or rebuild a healthy defense has led researchers to
minerals, plants. and fungi in search of natural substances
with health-supporting properties (Nworu et al. 2007).
Those compounds which appear to stimulate the immune
response are being sought for the treatment of cancer,
immunodeficiency diseases, or for generalized immunosup-
pression following drug treatment; for combination therapy
with antibiotics; and as adjuncts for vaccines (Jong and
Birmingham 1992). Immunomodulatory agents of plant
origin have been shown to enhance the immune respon-
siveness of an organism against a pathogen (Fulzele et al.
2002). The main target of the immunomodulatory plant
products is primarily their action on macrophages, which
play a key role in the generation of an immune response
through increased phagocytosis, intracellular killing of
pathogens by producing effector molecules like free radical
and nitric oxide and cytokine production.
Mucuna pruriens (MP; cowhage, velvet bean) is an
important tropical medicinal plant and constituent of more
than 200 indigenous drug formulations in India, Africa, and
the West Indies (Sathiyanarayanan and Arulmozhi 2007).
1344
Among the Ibos of southeastern Nigeria, the plant (leaf) is
used as blood “booster” while the leaf and seeds are used to
rejuvenate recuperating individuals.
Phytochemical analysis of the seed of M. pruriens revealed
the presence of alkaloids, mucunine, mucunadine, mucuna-
dinine, prurienidine, nicotine, β-sitosterol, glutathione, leci-
thin, vernolic and gallic acids, tryptamine, alkylamines,
steroids, flavonoids, coumarins, cardenolides, magnesium,
copper, zinc, manganese, iron, oleic acid, linoleic acid, and
palmitic acid (Adebowale et al. 2005; Misra and Wagner
2007; Sivaraman et al. 2010).
M. pruriens extracts are associated with pharmacological
activities that are antiparkinson (Gupta et al. 1997), antioxi-
dant (Tripathi and Upadhyay 2001), neurorestoratative
(Manyam et al. 2004), learning and memory enhancing
(Poornachandra et al. 2005), antidiabetic (Akhtar et al. 1990;
Grover et al. 2002), aphrodisiac (Amin et al. 1996; Shukla et
al. 2010), antivenomic (Guerranti et al. 2002), antimicrobial
(Rajeshwar et al. 2005), antiprotozoal (Ekanem et al. 2004),
and analgesic and anti-inflammatory (Hishikar et al. 1981).
This study was conducted to determine the effect of
methanolic extract of M. pruriens seed on the immune
response of mice as it relates to antibody response and
inflammation.
Materials and methods
Collection, preparation, and extraction of plant seeds
Dry pods containing seeds of M. pruriens were obtained
from a bush in Nsukka, Enugu State, Nigeria in February
2009. The pods were opened to remove the seeds. The pods
and seeds were identified and authenticated by a plant
taxonomist in the Department of Botany, University of
Nigeria, Nsukka. The seeds were air-dried and pulverized to
fine powder using a laboratory mill (Model 4, Thomas
Wiley, USA). Cold extraction was done with 70% methanol
for 72 h at room temperature with intermittent shaking.
After filtration through Whatman's No. 1 filter paper,
solvent was removed using rotary evaporator and the
extract stored at −4°C in sterile universal bottles until use.
The seed yielded a creamy brownish black sticky extract
and with a percentage yield of 5.7% (w/w). The extract at
100, 300, 600, 1,000, and 2,000 mg/kg did not cause death
or produce any signs of illness in mice during the acute
toxicity test which is an indication that the LD50 is above
2,000 mg/kg.
Experimental animals
Adult albino mice weighing (28–35 g) with mean weight of
both sexes were used for the study. The animals were
Comp Clin Pathol (2012) 21:1343–1347
housed in a fly-proof laboratory animal house and given
pelleted chick grower feed (Grand Feeds, Jos, Nigeria) and
water ad libitum.
Red blood cell antigen
Fresh sheep red blood cells (SRBCs) used as antigen
were obtained from blood collected by venipuncture of a
15-month-old female west African dwarf sheep. Three
milliliters of sheep blood was collected. Before use, the
red blood cells were washed three times with 15 ml of
phosphate-buffered saline (PBS), pH 7.2, by centrifugation
at 3,000×g for 10 min on each occasion using desktop
centrifuge (Labofuge 1®; Haraeus Christi, Germany).
After the final wash, the SRBCs were suspended in PBS
as a 2% suspension (based on packed cell volume) for the
serological tests and as a10% suspension for immunization
of the rats. A 1-ml amount of the 2% suspension contained
approximately 109 red blood cells (Nworu et al. 2007).
Delayed hypersensitivity reaction
Delayed-type hypersensitivity was induced in mice using
SRBCs as antigen (Nworu et al. 2007). Thirty adult male
mice divided into five groups of 6 mice each were used for
the study. Groups A, B, and C were given 100, 250, and
500 mg/kg of the extract, respectively, while group D
received 7.5 mg/kg body weight of levamisole and group E
received 0.02 ml distilled water as control. SRBCs (0.02 ml
of 109 cells ml−1) were administered (subcutaneous) on the
planter region of right hind footpad on day 0 to sensitize the
animals. On day 5 subcutaneous injection of the same
amount of antigen was administered on the left hind pad as
challenge. Oedema was produced by antigenic challenge
in the left footpad and was measured as the difference in
the paw thickness before and 24 h after the challenge.
This was done with a vernier calliper (Naved et al. 2005).
MP extract (100, 250, and 500 mg/kg) was administered
3 days prior to sensitization and continued till challenge
(Naved et al. 2005).
Humoral antibody response
Thirty adult male mice divided into five groups of 6 mice
each were used for the study. Groups A, B, and C were
given 100, 250, and 500 mg/kg of the extract, respectively,
while group D received 7.5 mg/kg body weight of
levamisole and group E received 0.1 ml distilled water as
control. SRBCs (0.1 ml−1 of 109 ml−1) were used to
immunize mice by an intraperitoneal (IP) injection on
day 0 and challenged by similar IP injection of the same
amount on day 5 postimmunization (PI). Primary antibody
titre was determined on day 5 PI and secondary titre on
Comp Clin Pathol (2012) 21:1343–1347 1345

day 10 PI by the hemagglutination technique (Nelson Table 1 Effect of methanolic extract of M. pruriens seed on delayed
hypersensitivity reaction in mice estimated as paw swelling
and Mildenhall 1967); also, total and differential leuco-
cytes were determined on days 5 and 10 PI, respectively. Treatment Dose (mg/kg) Paw swelling (mm) Inhibition (%)
About 0.5 ml of blood was collected from the retrobulbar
plexus of the medial canthus of mice. Of the blood, 0.2 ml Extract 100 0.52±0.07b 6.0
was put into an anticoagulant bottle for total and Extract 250 0.37±0.04a 33.5
differential leucocyte count while the remainder was put Extract 500 0.25±0.04a 55.8
in Eppendorf tube, allowed to clot, and later centrifuged at Levamisole 7.5 0.28±0.04a 48.9
15,000 rpm for 10 min to separate the serum for Control – 0.56±0.04b –
determination of antibody response to SRBC. The MP
Means±SEM with different lowercase letters are significantly different
extract (100, 250 and 500 mg/kg) was administered 3 days (p<0.05)
prior to immunization and continued daily for 5 days
after challenge.
by levamisole but are significantly (p<0.05) higher than
In vivo leucocyte mobilization the control.
Also, the extract caused elevation of secondary SRBCs-
Using the method of Ribeiro et al. (1991), the effect of the specific antibody titre and cellular response (Table 2).
MP extract on in vivo leucocytes migration induced by The secondary antibody responses caused by the extract
inflammatory stimulus was investigated. Thirty adult at 100–500 mg/kg were significantly (p <0.05) higher
female mice divided into five groups of 6 mice each were than the control but were significantly lower than the
used for the study. Groups A, B, and C were given 100, levamisole group. At 100 mg/kg antibody titre did not
250, and 500 mg/kg of the extract, respectively, while differ significantly (p >0.05) with the control. The leuco-
group D received 7.5 mg/kg body weight of levamisole gram showed that the TLC increased significantly (p<
and group E received 0.5 ml distilled water as control. 0.05) in the extract-treated groups when compared with
Oral administration of the MP extract (100, 250, and the control (Table 3). Differential leucocyte count showed
500 mg/kg) was given to the mice. One hour later, each that the increase in total leucocyte count in the treated
mouse in the group (A, B, C, D, and E) received groups could be attributed to increase in neutrophil and
intraperitoneal injection of 0.5 ml of 3% (w/v) agar lymphocyte.
suspension in normal saline. Four hours later, the mice The effect of the extract on in vivo leucocyte mobiliza-
were sacrificed under anaesthesia and the peritoneum tion is shown in Table 4. The extract at 250 and 500 mg/kg
washed with 5 ml of phosphate buffer saline containing increased the total leucocyte count of the perfusate and
0.5 ml of 10% EDTA. The peritoneal fluid was recovered increased significantly (p<0.05) when compared with the
and total leucocyte counts (TLC) determined with hemo- control and levamisole-treated group. At 100 mg/kg, the
cytometer, and the differential cell count was determined by mobilization did not differ with levamisole-treated group
microscopic counting of Giemsa-stained perfusate smear on but was significantly (p<0.05) higher than in the control.
glass slide. The differential leucocyte mobilization involved more
neutrophils than lymphocytes.
Statistical analysis

Data were summarized as mean±standard error of means. Table 2 Effect of methanolic extract of M. pruriens seed on primary
and secondary antibody response in mice
Statistical significance between the groups was analyzed by
the Student's t test, and p<0.05 was considered to be Treatment Dose (mg/kg) Antibody response titre
statistically significant (Steel and Torrie 1980).
Primary Secondary

Extract 100 12.40±5.46 20.00±5.37a

Results
Extract 250 13.60±5.00 25.60±3.92ab
Extract 500 16.00±4.38 36.00±12.26b
The result of the delayed hypersensitivity response in mice
Levamisole 7.5 31.20±13.53 48.00±20.24 b
(Table 1) showed that the extract produced inhibition of
Control – 12.80±5.28 14.60±3.92c
paw swelling at all doses tested. This inhibition was dose
dependent with 500 mg/kg producing the highest level of Means±SEM with different lowercase letters are significantly different
inhibition. The inhibition produced at 250 and 500 mg/kg (p<0.05)
did not differ significantly (p>0.05) from that produced MPE M. pruriens extract
1346 Comp Clin Pathol (2012) 21:1343–1347

Table 3 Effect of M. pruriens

on total and differential 100 mg/kg 250 mg/kg 500 mg/kg Levamisole Control
leucocyte counts following
primary and secondary cellular TLC 1° 8.26±0.64 7.89±1.76 10.46±2.57 8.77±0.82 7.19±1.05
immune response in mice 2° 9.38±1.43 9.88±1.40 14.59±0.47 10.44±2.02 8.01±0.53
Neut 1° 4.68±0.33 4.80±1.09 5.26±1.98 4.16±0.53 3.80±0.53
2° 5.49±1.67 4.78±1.27 8.57±0.42 4.32±0.45 5.16±0.35
Lymp 1° 2.58±0.24 2.20±0.45 2.94±0.70 2.49±0.58 2.15±0.51
TLC total leucocyte counts, Neut 2° 2.91±0.43 2.32±0.10 4.25±0.52 2.70±0.19 2.64±0.17
neutrophil, Lymp lymphocyte, Others 1° 1.16±0.22 0.89±0.13 1.30±1.18 0.89±0.14 0.91±0.25
Others eosinophil, monocytes, 2° 0.51±0.08 0.83±0.18 1.18±0.18 0.61±0.04 1.66±1.15
and basophils

Discussion differentiate to become plasma cells that make and secrete

The manifestation of the delayed hypersensitivity reaction
(DTR) induced by SRBCs was inhibited by the extract at all
the doses tested. Cell-mediated immune response by helper
T cells has been reported to be augmented under the
influence of immunostimulants (Sainis et al. 1983). The
ability of the extract to inhibit DTR in this experiment
could be due to its anti-inflammatory properties (Hishikar
et al. 1981) and antioxidative properties (Tripathi and
Upadhyay 2001; Shukla et al. 2010). Excessive amounts of
reactive oxygen species (particularly hydrogen peroxide)
may have adverse effects on lymphocytes, which play a key
role in immune response (McDowell 2000). DTR requires
specific recognition of the given antigen by activated T
lymphocytes, which subsequently proliferate and release
cytokines (Kannan et al. 2007). These, in turn, increase
vascular permeability, induce vasodilatation, macrophage
accumulation and activation, promoting increased phago-
cytic activity and increased concentrations of lytic enzymes
for more effective killing (Kuby 1997; Descotes 1999).
This may be responsible for the lesion characterized by
induration and erythema seen in this experiment.
The anti-SRBC antibody titre was raised in extract-
treated groups. When mice are sensitized with SRBCs, the
SRBC antigens are taken up by macrophages and are
processed. Subsequently, B cells are triggered to proliferate,
giving rise to clones of a large numbers of daughter cells,
some of which serve as memory cells while others
large quantities of specific antibodies (Dale and Formanj
1989; Goldsky et al. 2003). In the present study, assessment
of humoral immunity was carried out using the humoral
antibody titre as described by Nworu et al. (2007). Effect of
enhancement of the antibody production in the present
experiment may be associated with effect of the extract on
the lymphoid organs. This indicates enhanced responsive-
ness of macrophages and T- and B-lymphocyte subsets
involved in antibody synthesis (Benacerraf 1978).
The extract increased the total leucocyte count of the
perfusate significantly (p<0.05) when compared with the
control group. Leucocyte migration is important for the
transport of immunological information between the different
compartments of the immune system. The chemotactic
movement of neutrophils towards a foreign body is the first
and most important step in phagocytosis (Ganachari et al.
2004). This activity may help to increase the general
resistance of the body against microbial infections. The
neutrophils which engulf and eliminate invading micro-
organisms are the most mobilized leucocytes into the
peritoneum in this experiment. For immunosurveillance to
be maintained throughout the body, lymphocytes must
recirculate among the blood, organs, and tissues via the
lymphatic system (Hay and Andrade 1998).
The results of this study suggest that the methanolic seed
extract of M. pruriens reduced inflammatory swelling, but
increased antibody production and inflammatory leucocyte
mobilization in mice.

Table 4 The effect of M.

pruriens extract on in vivo Treatment Dose (mg/kg) TLC (103 cell/mm) Leucocytes DLM
leucocytes mobilization mobilization (%)
Neut (103) Lymp (103)

Extract 100 8.18±0.71a 3.10 5.65±0.04a 2.06±0.27

Means±SEM with lower letters
are significantly different Extract 250 9.67±1.00a 4.59 6.78±0.01a 2.63±0.43
(p<0.05) Extract 500 9.14±1.00a 4.06 6.18±0.52a 2.85±0.54
TLC total leucocyte count, Levamisole 7.5 7.10±0.87a 2.02 5.01±0.96ab 2.61±0.64
DLM differential leucocyte Control – 5.08±0.96b – 3.11±0.54b 1.94±0.42
mobilization
Comp Clin Pathol (2012) 21:1343–1347
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