Immunomodulatory Activity and Th1/Th2 Cytokine Response of Ocimum sanctum in Myelosuppressed Swiss Albino Mice
K. Narendra Babu,
B. Dinesh Kumar
P. Uday Kumar
Ocimum sanctum (O. sanctum) has gained special attention due to its biological properties, however, little is known about its immunomodulatory effects. The purpose of this study was to investigate effect of O. sanctum on inflammation and immune response and its effect on Th1/Th2 cytokine production by spleen cells of myelosuppressed mice model. Female Swiss albino mice were challenged with SRBC and then were grouped and treated with either O. sanctum methanolic extract 850 mg kg-1 or Prednisolone 5 mg kg-1 body weight for 15 days. Blood was collected on 16th day from retro orbital plexus to perform hematological and immunological tests. Bone marrow cellularity was determined and supernatants of splenocytes cultures were analyzed for Th1/Th2 cytokines by ELISA. Antioxidant activity of O. sanctum was evaluated by DPPH (2,2'-diphenyl-1-picrylhydrazyl) assay. Treatment with O. sanctum showed significant increase in bone marrow cellularity (p<0.01), total WBC count (p<0.01) and hemoglobin concentration (p<0.01). In addition, there was a significant increase in SRBC antibody titer (1:1024) compared to the control group (1:256). O. sanctum increased the production of TNF-α, IL-2, IFN- γ and IL-4 (p<0.05) significantly and decreased the production of IL-1β and NF-kB. The methanolic extracts of O. sanctum showed free radical scavenging activity at 140 μg mL-1 concentration as IC50. This study documented improved haemoglobin concentration with O. sanctum treatment. These results support the use of this herb for wound healing and infection. The results also suggest potential use of O. sanctum as adjuvant in cancer therapy, myelotoxicity and in nutritional anemia.
Received: September 06, 2010;
Accepted: December 13, 2010;
Published: May 06, 2011
Ocimum sanctum commonly known as Holy Basil or sacred tulasi, belongs
to the family Lamiaceae and is a fragrant bushy plant found in the semitropical
and tropical parts of India. From the ancient times different parts of O.
sanctum are traditionally used in Ayurveda and Siddha systems of medicine
for treating infections, skin diseases, hepatic disorders, cold, cough and malaria
fever. Many studies have shown a number of beneficial effects of O. sanctum
such as anti-inflammatory (Singh et al., 1996),
humoral immune response modulation and promotion of wound healing (Mediratta
et al., 1988; Shetty et al., 2008).
Mediratta et al. (2002) have shown the effect
of O. sanctum seed oil on some immunological parameters in both stressed
and non-stressed animals. Oil extracted from O. sanctum showed good antibacterial
activity against Staphylococcus aureus, Bacillus pumilus and Pseudomonas
aeruginosa, showed improved wound healing and increased production of TNF-α
(Singh and Majumdar, 1999; Goel
et al., 2010a). In addition to its antibacterial, antiinflammation
and wound healing properties, O. sanctum had also been studied for its
antihypersensitivity and antioxidant properties (Mediratta
et al., 1988; Shetty et al., 2006;
Uma Devi et al., 2000).
T cells play a critical role in the pathogenesis of various diseases through
the production of a variety of cytokines. Cytokines such as IL-4, IL-5 and IL-13
are known to influence a wide range of events associated with chronic allergic
inflammation in local tissues (Kay, 2000). NF-κB
controls many genes involved in inflammation and it is not surprising that NF-κB
is found to be chronically active in many inflammatory diseases such as inflammatory
bowel disease, arthritis, sepsis, gastritis, asthma, among others. Many natural
products (including anti-oxidants) that have been promoted to have anti-cancer
and anti-inflammatory activity have also been shown to inhibit NF-κB. Though
O. santum is known for its wound healing and anti-inflammatory properties,
except a few studies on humoral immune response such as SRBC antibody titers
or paw edema; studies describing the effect of O. sanctum on Th1/Th2
modulation or cytokines responses are lacking. Therefore,the present study was
carried out to investigate the effect of O. sanctum methanolic extract
on Th1/Th2 cytokine production, NFkB p65activity, heamopitic activity
and antibody response to Sheep Red Blood Cells (SRBC) in myelosuppressed mice
model. In addition, its antioxidant property was also studied.
MATERIALS AND METHODS
Plant material-Preparation and extract: Dried and powdered leaves of O. sanctum were procured from herbal stores at Hyderabad and authenticated in Heritage Bio-Natural systems pvt. Ltd. The study was conducted from 2009 to 2010. Hundred grams of dried and powdered leaves of O. sanctum were soaked in methanol for 3 consecutive days at an interval of 24 h. The fluid fraction of the three extractions was combined, concentrated and dried under vacuum. Accurately weighed quantities of the methanol extract of O. sanctum were suspended in 2% gum acacia to prepare a suitable dosage form. The dose levels of the extract were selected on the basis of the human dose and calculated for rodent dose.
Animals: The animal study protocols were approved by the Scientific Advisory Committee as well as Institutional Animal Ethics Committee of National Institute of Nutrition (NIN). Twenty four female Swiss albino mice weighing 20-25 g were obtained and acclimatized at National Centre for Laboratory Animal Sciences for 1 week and maintained at 24±20°C, 50-60% relative humidity, with a 12 h light-dark cycle. They were accommodated in individual ventilated cages with stainless steel top grill with food and water spouts and closed bottom. Autoclaved paddy husk was used for bedding with weakly changes. They were fed a casein based (20% protein) pellet control diet.
Animals treatment: Twenty four female Swiss albino mice were sensitized with 0.5 mL of 20% of fresh SRBC suspension, which was injected on day 0. The animals then were divided into four groups of six animals each. Animals in Group I received the vehicle (2% Gum acacia) and were treated as a control group. Animals in Group II received prednisolone (5 mg/Kg/b.wt.) in 2% Gum acacia. Group III animals received O. sanctum methanolic extract (850 mg/Kg/b.wt.) in 2% Gum acacia and Group IV animals received prednisolone (5 mg/Kg/b.wt.) along with O. sanctum methanolic extract (850 mg/Kg/b.wt.) in 2% Gum acacia, orally, for a period of 15 days. Blood samples were collected on 0 and 16th day of the experiment and the total White Blood Cell (WBC) count, Differential Count (DC), Red Blood Cell (RBC) count, haemoglobin concentration and platelet counts were determined using automated blood cell counter (Seimens Adna).Prednisolone was used as a standard immunosuppressant, and SRBC as an antigen at the concentration of 20% for immunization and 2% for challenge.
Determination of the bone marrow cellularity: Bone marrow was collected from femur in medium containing 2% Fetal Calf Serum (FCS). The number of bone marrow cells was determined using a haemocytometer and expressed as total live cells per femur.
T cell dependent Hemagglutinin Antibody (HA) response: All the animals in four groups were sensitized with 0.5 mL of 20% fresh SRBC suspension injected intraperitonially on day 0. Blood samples were collected in microcentrifuge tubes from retro-orbital plexus of each animal on 16th day of extract administration. The serum was separated and pooled the sera each group. Antibody titres were determined by haemagglutination technique. Two fold dilutions of pooled sera were made in 25 μL volumes of PBS in U bottomed microtitration plate and to this was added 25 μL of 2% SRBC in saline. After mixing, the plates were incubated at 37°C for 1 h and examined for haemagglutination under microscope. The reciprocal of the highest dilution of the test serum giving agglutination was taken as the antibody titre.
Estimation of cytokines (IL-1β, IL-2, IL-4, IFN-γ, TNF-α) in Con-A stimulated splenocytes: Spleen was teased and dispersed and passed through a sterilized stainless sieve (200 mesh) to obtain a single-cell suspension. Cells were washed twice with RPMI1640 medium. 1x106 cells mL-1 was dispensed into the 24-well flat bottomed plates in the absence and presence of 0.5 μg mL-1 Concanavalin A. These cells were incubated in 5% CO2 humidified incubator for 24 h. Cells were centrifuged for 5 mins at 1500 rpm and the cytokines were estimated in the cell supernatant by mouse multiplex ELISA Kit as recommended by the manufacturer. All the tests were performed in duplicates.
Estimation of NFkB in the nuclear fraction of splenocytes: Preparation of nuclear extract of splenocytes was performed as per instructions given in Trans AM Flexi kit. The NFkB was estimated by ELISA (Trans AM Flexi) Kit. All the tests were performed in duplicates.
DPPH radical assay: Different aliquots of standard and test were mixed with Tris HCl buffer and made the volume to 1 mL. To this added 1 mL of 0.3mM DPPH (2,2'-diphenyl-1-picrylhydrazyl) in methanol and made the final volume to 2 mL. These mixtures were shaked well and kept in dark for 30 min. The absorbance was measured at 517 nm using ethanol as blank. One milliliter of 0.3 mM DPPH was diluted in ethanol, which was used as a control. Inhibition of DPPH radical was calculated using the equation: IC50 (%) = 100 x (A0-As)/A0, where A0 is absorbance of the control, As is absorbance of the sample and IC50 is concentration of the test extract that caused 50% inhibition.
Statistical analysis: Data are presented as mean±Standard Error (SE). Analysis of variance (ANOVA) was used to estimate the main effects and interactions. P values <0.05 were considered significant. Duncans test was used to identify the groups that are homogenous with respect to mean. Analysis was performed using SPSS (version 11.5, SPSS Inc, Chicago, IL).
Effect of O. sanctum methanolic extract on the haematological parameters, T cell dependent antibody response and bone marrow cellularity : There was a significant increase in haemoglobin concentration (p<0.01), RBC and WBC count in O. sanctum treated mice compared to controls. However, there was no change in lymphocyte population with O. sanctum, though, there was a significant (p<0.01) decrease in lymphocyte population in Prednisolone treated mice compared to the control (Table 1). O. sanctum treated mice showed significant (p<0.01) increase in bone marrow cellularity compared to the control mice. Furthermore, O. sanctum prevented the suppressive effect of prednisolone on bone marrow cellularity (Table 2). O. sanctum treatment increased the production of SRBC antibody titre (1:1024) compared to the control mice and prevented the suppressive effect of prednisolone on antibody response to SRBC (Fig. 1).
Effect of O. sanctum methanolic extract on Th1 and Th2 cytokine profile
and NFkB P65 activity: O. sanctum treatment significantly
increased the production of Th1 cytokines (p<0.01) IL-2, TNF-α and IFN-γ
and Th2 cytokine (p<0.05) IL-4, compared to control mice. O. sanctum
and prednisolone suppressed the production of IL-1β significantly (p<0.01)
compared to controls.
|| Effect of O. sanctum on haematological parameters
|All values are Mean±SE; 95% CIs in parentheses; p<0.01.
Means bearing similar superscripts in each row do not differ significantly.
|| Effect of O. sanctum on Bone Marrow Cellularity
|| Effect of O. sanctum on heamagglutinin antibody response
|| Effect of Antioxidant potential of O. sanctum
Significant (p<0.05) increase in TNF-α levels was observed in mice
treated with prednisolone compared to control group (Fig. 3a-e).
There was a significant decrease (p<0.01) in NFkB P65 activity
with O. sanctum treatment and combined treatment with Prednisolone compared
to control group. However, prednisolone enhanced significantly (p<0.01) NFkB
P65 activity when given alone compared to controls (Fig.
Antioxidant activity of O. sanctum methanolic extract: Free radical
activity of O. sanctum methanolic extract and ascorbic acid activities
are depicted in Fig. 2. Though O. sanctum free radical
activity was not comparable to ascorbic acid, it showed a modest free radical
scavenging activity at 140 μg mL-1 as compared to ascorbic acid
which showed at 14 μg mL-1.
||Effect of O. sanctum on Th1 and Th2 cytokine response
All values are Mean±SE; 95% CIs in parentheses; p *<0.05 **<0.01
Means bearing similar superscripts in each bar do not differ significantly
||Effect of O. sanctum on the production of p65 (Rel
A) of NF-κB All values are Mean±SE; 95% CIs in parentheses;
p *<0.05 **<0.01 Means bearing similar superscripts in each bar do
not differ significantly
In the present study, inflammation and immunomodulation activity of O. sanctum,
a popular traditional plant in medicine was investigated with special reference
to Th1 and Th2 phenotype response and NFkB activity. Ocimum sanctum is
an extensively used medicinal plant in the Ayurvedic system of medicine and
is known for its beneficial effects on human health. Ocimum sanctum treatment
resulted in increased haemoglobin concentration in line with that observed by
other investigators (Goel et al., 2010b). The
production of SRBC antigen-specific antibodies represents a major defense mechanism
to assess T-cell-dependent antibody responses. Similar to earlier reports, we
documented enhanced SRBC agglutinin titers with O. sanctum treatment
(Mediratta et al., 1988). The mechanism for the
enhanced SRBC antibody response has not been defined in this study. However,
heightened IL2 response observed in this study could have contributed to enhance
SRBC antibody response (Callard et al., 1991).
These results provide evidence supporting O. sanctums role as anti-bacterial
and anti-viral compound (Gupta and Charan, 2005). Furthermore,
O. sanctum reduced the adverse effects of predinosolone and increased
bone marrow cellularity, haemopoetic activity considerably as reported earlier
with Ashwagandha (Ziauddin et al., 1996).
Transcription factor NFkB controls the expression of genes involved in immune
responses, apoptosis and cell proliferation. P50/P105, P52/P100 (NFkB2), P65
(Rel A), Rel B and C-Rel are the five subunits of NFkB, that exist in unstimulated
cells as homo or hetero dimer bound to IkB family proteins (Matthew
and Ghosh, 2004). P65 or Rel A exists in a wide variety of cell types and
is a key molecule in the classical pathway. The classical pathway is typically
triggered by ligand binding to tumor necrosis factor type 1/2 receptors (TNFR1/2),
T-Cell Receptors (TCR), B-Cell Receptors (BCR), or the Toll-Like Receptor (TLR)
- interleukin-1 receptor (IL- 1R) super family members. This pathway terminates
in the increased transcription of target genes encoding chemokines, cytokines,
and adhesion molecules, perpetuating inflammatory responses and promoting cell
survival and thus protects cells from apoptosis during TNFα signaling (Beg
et al., 1995). However, overexpression of NF-κB, together with
COX-2 and LOX5 is frequent in cancer. The anti-inflammatory property of O.
sanctum has been atributed to decreased CoX-2 and LOX-5 enzymes activity
(Singh et al., 1996), but suppression of NF-κB
classical pathway as observed in the present study could be yet another mechanism
by which O. sanctum might act as anti- inflammatory agent. O. sanctum
contains Ursolic acid and Carnosol components which have been shown to down
regulate NFκB and also possess anticancer activity (Lo
et al., 2002). O. sanctum contains a number of compounds such
as carnosol, ursolic acid, rosmarinic acid, apigenin, eugenol, cirsilineol and
cirsimaritin, all of which have shown to have potent redox/anti-oxidant properties
as well as COX-2 inhibitory effects (Kelm et al.,
2000). Thus apart from anti-inflammatory role, O. sanctum could be
a potential anti cancer agent.
Furthermore, O. sanctum reduced NF-κB activity even in prednisolone treated animals wherein prednisolone per se has increased NF-κB activity.
In the present study we observed upregulation of IL-2, IFN-γ and TNF-α
with O. sanctum. Similarly to that observed elsewhere (Goel
et al., 2010b). Additionally, the present study also demonstrated
down regulation of IL-1 β, which had not been reported earlier. Studies
on cancer immunity have demonstrated enhanced anti-tumor immunity and reduction
of tumor growth with specific adjuvants (Dredge et al.,
2002). Many cancer vaccines, particularly in combination with immune adjuvants,
elicit strong cellular immune responses leading to the production of Th1 type
cytokines such as IL-2, IFN-γ, TNF- α (Dalgleish,
2000). However, one major adverse effect of most of these adjuvants is increased
IL-1β production, which is now known to contribute to invivo angiogenesis
and invasiveness of different tumor cells (Voronov et
al., 2007). The interesting aspect of the present study is, upregulation
of IFN-γ and TNF-α on a backdrop of low IL-1β. O. sanctum
suppressed IL-1β production, whereas it increased IL-2 , IFN-γ
and TNF-α (Mondal, 2010).
Taken together, these results suggest a potential adjuvant role of O. sanctum in cancer therapy. The present study also suggests that O. sanctum might modulate inflammation and immune response by modulating NFkB activity. However, more studies are required to delineate the role of O. sanctum on inflammation and mechanism there of.
The authors would like to thank the Director, National Institute of Nutrition for his unstinted support throughout the study and Officer-Incharge, NCLAS and animals. The authors declare no conflict of interest whatsoever.
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