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Pharmacologyonline 3: 947-952 (2011) Karchuli et al.
947
CURCUMA AMADA ROXB. RHIZOME EXTRACT MODULATES CELLULAR A'D
HUMORAL IMMU'E SYSTEM
Manvendra Singh Karchuli
1*
, Debasish Pradhan
1
University Department of Pharmaceutical Sciences, Utkal University, Vanivihar, Bhubaneshwar,
751004, Orissa, India
*Corresponding author – karchulims@gmail.com
Summary
Immunomodulatory effect of Curcuma amadaRoxb.was assessed using In-vitro
Phagocytosis activity, delayed type hypersensitivity test and Hem agglutination
test. Extraction of plant rhizome was performed using petroleum ether,
chloroform, acetone and alcohol. Alcoholic extract showed maximum effect in in-
vitro assay thus it was used for in-vivo investigation at 400 mg/kg, 200 mg kg and
100 mg/kg. Extract showed significant immunomodulatory effect at all selected
dose.
Key words: Curcuma amada Roxb., Phagocytosis, Hemaggluination,
immunomodulatory
Introduction
Any components which can alter immunity are considered as immunomodulators. They may
either amplify or inhibit immunity. Medicinal plants and their active components have been
shown to be an important source of immunomodulators. Effect of components from herbal origin
on immunity is well documented
[1]
. Many herbs, e.g. Hibiscus rosasinensislinn.
[2]
, Cleome
gynandralinn
[2]
, Pouteriacambodiana
[3]
, Clausenaexcavate
[4]
, Acacia catechu
[5]
,
Moringaoleifera
[6]
, Tinosporacordifolia
[7]
Terminaliabellerica
[8]
,Alternantheratenellacolla
[9]
are
reported to possess significant immunomodulatory activity.
Curcuma amada Roxb. is commonly known as mango ginger. It is a perennial, rhizomatous,
aromatic herb belonging to the family Zingiberaceae. It is reported to possess therapeutic
potential for its antioxidant activity
[10]
, antibacterial activity
[11]
, antifungal activity
[12]
, anti-
inflammatory activity
[13]
, platelet aggregation inhibitory activity and cytotoxicity
[14]
. It consist of
various bioactive chemical components like starch, phenolic acids, volatile oils, curcuminoids
and terpenoids like difurocumenonol, amadannulen andamadaldehyde
[15]
. It is reported that
Curcuma amada is used traditionally in Chandaka denudated forest patches of Bhubaneswar,
Orissa, India, for treatment of various disease
[16]
.
Pharmacologyonline 3: 947-952 (2011) Karchuli et al.
948
Materials and methods
Chemical and reagents
The minimum essential medium (MEM), bovine serum albumin and FicollHypaque were
procured from Hi-Media Lab Pvt. Ltd., Mumbai. Candida albicans (ATCC-10231) was used for
bioassay. All chemical and reagents were of analytical grade.
Sheep Red Blood Cells (SRBC)
SRBC was collected in Alsevier’s solution, and were used for immunization in a concentration
of 5 x 10
9
cells/ml.
Animals
Albino wistar rats weighing 200 ± 30 of either sex were selected at random from animal house of
PBRI, Bhopal, India. Animals were further randomly divided into various treatment groups and
kept in propylene cage with sterile husk as bedding. Animals were housed in relative humidity of
30.7 % at 22 ± 2 °C and 12:12 light and dark cycle. Animals were fed with standard pellets
(Golden feeds, New Delhi, India) and water was available ad libitum. All animal experiments
were approved by Instutional Animal Ethics Committee (IAEC) of PBRI, Bhopal (CPCSEA Reg
No. - 1283/c/09/CPCSEA).
Polymorphonuclear leucocytes (PM' cells)
Healthy rats weighing 200 ± 30 were used for collection of PMN cells. Presence of any
microbial contamination was strictly avoided.
Extract preparation
Rhizomes of C.amada was collected locally near Bhubaneswar region. Herbarium of plant was
submitted, and authenticated by Dr. Ziaul Hasan, Botanist, Department of Botany, Safia Science
College. Rhizome was washed with water and shade dried. Dried rhizomes were crushed and
extracted with petroleum ether (CPEE), chloroform (CCE), acetone (CAE) and ethanol (CEE)
successively in soxhlet apparatus. The extract was dried in rotary vacuum evaporator and
collected in air tight container for further use.
Phagocytic assay (PM' function test)
FicollHypaque density gradient method was used for isolation of neutrophils from blood
collected in heparinised tube from peripheral vein of healthy rats. Afterward dextral
sedimentation was performed and supernatant with more than 90 % PMN cells were collected
and used cell density was 1 x 10
6
cells/ml in MEM.Candida albicans in concentration of 1 x 10
6
cells/ml was used for present study.PMN cells and C.albicans were mixed and incubated in 5 %
CO
2
at 37°C with different extract at different concentration. After incubation cytosmears were
prepared and fixed in methanol and stained with Giemsa stain. Prepared slides were observed
under 100X magnification for determination of phagocytic activity. Hundred neutrophil were
analyzed and it was how many cells ingested microorganism. Percentage of PMN cells involved
in phagocytosis (percentage phagocytosis) and ratio of number of microorganism engulfed to the
total number of neutrophils (phagocytic index) was calculated for ascertaining in-vitro
phagocytic activity.
Pharmacologyonline 3: 947-952 (2011) Karchuli et al.
949
Acute toxicity study
Acute oral toxicity study was performed as per OECD 423 guidleines. Extract (CEE) was given
upto the maximum dose of 2000 mg/kg and animals were observed for mortality.
Delayed type hypersensitivity test
To measure cellular immunity delayed type hypersensitivity test was performed. SRBC was used
as antigen in present study. In group I (six rats) vehicle was administered orally for five days. In
group IV, III and IICEE was administered orally at the dose of 400 mg/kg, 200 mg/kg and 100
mg/kg respectively for five days. CEE and vehicle was administered on each two day before
immunization, on the day of immunization and on each two day after immunization (i.e. -2,-
1,0,+1+2). Rats were immunized by injecting 0.1 ml of SRBS subcutaneously into the right hind
paw on day zero. Animals were challenged seven day later with same amount of SRBC into the
left hind paw. Change in paw thickness was measured using digital caliper at 4
th
and 24
th
hour
after challenge.
Hemagglutination reaction
To measure humoral immunity hemagglutination test was performed. In group I (six rats) vehicle
was administered orally for five days. In group IV, III and II CEE was administered orally at the
dose of 400 mg/kg, 200 mg/kg and 100 mg/kg respectively for five days. CEE and vehicle was
administered on each two day before immunization, on the day of immunization and on each two
day after immunization (i.e. -2,-1,0,+1+2). Rats were immunized by intraperitonealinjection of
0.5 ml SRBC. On the tenth day after immunization blood sample was collected by retro orbital
puncture. Hemaggluination titer assay was performed for antibody level determination. Serial
dilution of serum and 0.1% bovine serum albumin (100 µl: 100 µl) was prepared in sterile saline.
One volume (100 µl) of 0.1 % SRBC in saline was added and mixed. They were allowed to settle
at room temperature for 90 min till control tube showed a negative pattern (a small button
formation). The value of highest serum dilution showing visible hemagglutination was
considered as antibody titer.
Biostatical analysis
All data were analysed by One Way ANOVA followed by Dunnet’s test. P<0.05 was considered
as level of significance.
Results
Different extracts of Curcuma amada was investigated for its effect on PMN associated
phagocytosis, observations are presented in Table 1. It was observed that chloroform extract of
Curcuma amada (CCE) produced significant effect (p<0.05) on percentage phagocytosis at 2.0
and 3.0 mg/ml concentration, but at 1.0 mg/ml concentration effect was non significant (p>0.05)
as compared to that of control group. Ethanolic extract of Curcuma amada (CEE) produced
significant effect (p<0.05) on percentage phagocytosis at all selected concentration. When
extracts were evaluated on the basis of Phagocytic index it was found that chloroform extract
(CCE) at all selected concentration produced significant effect on Phagocytic index as compared
to that of control group. Acetone extract (CAE) also produced significant effect at 3.0 mg/ml
concentration (p<0.05) on phagocytic index. Ethanolic extract (CEE) also produced significant
effect al all selected concentration of 1.0. 2.0 and 3.0 mg/ml on phagocytc index as compared to
that of control group. Thus ethanolic extract showed best effect as compared to other exracts, so
it was selected as test sample for further investigations.
Pharmacologyonline 3: 947-952 (2011) Karchuli et al.
950
Table 1. PMN function test for different extracts of Curcuma amada
Sample Concentration (mg/ml) Percentage phagocytosis Phagocytic index
Control -- 28.66±3.66 1.6±0.09
1.0 31.67±2.58 1.69±0.11
2.0 32.5±3.15 1.76±0.12
CPEE
3.0 33.33±4.23 1.78±0.13
1.0 32.67±3.39 1.93±0.10
a
2.0 36.17±1.94
a
2.05±0.24
a
CCE
3.0 38±4.56
a
2.12±0.32
a
1.0 29.67±5.32 1.74±0.09
2.0 29.83±3.97 1.85±0.10
CAE
3.0 31±4 1.99±0.28
a
1.0 36.67±3.33
a
2.04±0.31
a
2.0 37.5±5.79
a
2.12±0.21
a
CEE
3.0 52.83±8.57
a
2.19±0.40
a
*All data presented in mean ± SD
a-P<0.05 as compared to control group
In delayed type hypersensitivity (DTH) test it was observed that ethanolic extract (CEE) showed
significant effect (p<0.05) at 100 mg/kg, 200 mg/kg and 400 mg/kg as compared to that of
vehicle treated group. Results are mentioned in Table 2. Effect of extract was significant at 4
th
hour as well as on 24
th
hour.
Table 2. Effect of Ethanolic extract of Curcuma amada (CEE) in hypersensitivity reaction test
hypersensitivity reaction on
#
Group No Treatment*
4
th
hour 24
th
hour
01 Vehicle 0.38 ± 0.06 0.17 ± 0.08
02 CEE (100 mg/kg, p.o.) 0.70 ± 0.09
a
0.37 ± 0.05
a
03 CEE (200 mg/kg, p.o.) 0.79 ±0.07
a
0.46 ± 0.06
a
04 CEE (400 mg/kg, p.o.) 0.89 ±0.1
a
0.69 ± 0.12
a
* Each group consist of six animal
#
Data presented in Mean ± SD
a
P<0.05 as compared to vehicle treated group
Ethanolic extract of Curcuma amada (CEE) was evaluated for its immunomodulatory by using
Hemagglutination antibody titer method. CEE produced significant effect (p<0/05) at 100 mg/kg,
200 mg/kg and 400 mg/kg as compared to that of vehicle treated group. Observation is
mentioned in Table 3.
Pharmacologyonline 3: 947-952 (2011) Karchuli et al.
951
Table 3. Effect of ethanolic extract of Curcuma amada (CEE) on Hemagglutination
Group No Treatment* Hemagglutination antibody titer
#
01 Vehicle 193.33 ± 44.96
02 CEE (100 mg/kg, p.o.) 248.5 ± 59.82
03 CEE (200 mg/kg, p.o.) 595.83 ± 252.87
a
04 CEE (400 mg/kg, p.o.) 966.33 ± 137.654
a
* Each group consist of six animal
#
Data presented in Mean ± SD
a
P<0.05 as compared to vehicle treated group
Discussion
In fighting against various diseases immune system plays an important role. Modulation of this
immune response may help in treating and preventing many diseases. Agents that can modulate
immunity in the presence of an impaired immune responsiveness can provide supportive therapy
inmany treatment approaches
[1]
.
Phagocytosis is an important part of immunity. It was observed that CEE elevated percentage
phagocytosis at all selected dose thus for investigation on humoral immune
responsehaemagglutination antibody titre (HAT) methodology was used. Augmentation of the
humoral immune response to SRBCs by extract (CEE)evidenced by increase in the antibody
titres in the blood of rats. B lymphocytes,plasma cells, IgG and IgM are the components involved
in the complement activation, opsonization, and neutralization of toxins
[17]
. Thus they become an
integral part of humoral immune system. Thus from HAT test it can be postulated that CEE
showed its effect by modulating any of these factors involvedin humoral immune system.
SRBC-induced delayed type hypersensitivity was used to assess the effect of thefraction on cell-
mediated immunity. Cell mediated immunity involves effector mechanisms carried out
by T lymphocytes and lymphokines. Cell mediated immunity responses are critical to defence
against infectiousorganisms, infection of foreign grafts, tumor immunity and delayed-type
hypersensitivity reactions
[17]
. Thus stimulatory effect of CEE on delayed type hypersensitivity
test revelaed that extract was having stimulatory effect on T lymphocytes.
Conclusion
Study concludes that ethanolic extract Curcuma amadaRoxb. Possess significant
immunomodulatory effect and this effect is mediated through stimulation of phagocytosis, B
Cells and T cells.
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