Evaluation of Immunomodulatory Activity of Clerodendrum phlomidis and Premna integrifolia Root
R.H. Gokani ,
S.K. Lahiri ,
Roots of Clerodendrum phlomidis Linn. f. suppl. and Premna integrifolia Linn. Mant. (Verbanacea) are known under the common name Arni/Agnimantha. Roots of both the plants are important rasayana drugs and are considered to be useful in the treatment of variety of ailments. Roots of either of these two plants are incorporated as Arni/Agnimantha in many valued and popular Ayurvedic formulations. So the present study was aimed at evaluating the two roots for their immunomodulatory potential. Oral administration of methanol extracts of both the roots (300 mg kg-1 x 7 days) in mice prior to immunization with Sheep Red Blood Cells (SRBC) resulted in a significant increase in haemagglutinating antibody titre, plaque forming cell assay and delayed type hypersensitivity to SRBC. C. phlomidis showed higher specific immune activity as compared to P. integrifolia, C. phlomidis and P. integrifolia enhanced the non specific immune response in carbon clearance test and showed significant immunoprophylactic effect, when tested on E. coli induced abdominal sepsis. In the present study C. phlomidis showed higher response to specific immune activity as compared to P. integrifolia, where as in case of non specific immune activity both the roots showed almost equal response.
Clerodendrum phlomidis, Linn. f. suppl. and Premna integrifolia, Linn. Mant. are two different plants belonging to the family Verbanaceae, both of which are mentioned under the common names of Arni and/or Agnimantha in Ayurveda (Sharma, 1996; Bishnupriya et al., 2003; Vaidya, 1965). Their roots are important ingredient of Ayurvedic preparations like, Dashmoola kwatha, Chyanprashavleh, Haritakiavleh, Ayushyavardhaak tel, Narayan tel etc., valued for the treatment of variety of affections (Anonymous, 2001).
C. phlomidis is a large bush or a small tree, growing throughout India. Roots are valued as tonic, diuretic, febrifuge, anti-diabetic, anti-inflammatory and antitussive (Nadkarni, 1982).
P. integrifolia is a large shrub or a small tree distributed on the western sea coast from Bombay to Molucca, Srilanka and the Andaman. The root is used in the treatment of diabetes, chyluria, inflammations, swellings, bronchitis, dyspepsia, liver disorders, piles, constipation and fever (Anonymous, 1997).
Previous pharmacological studies include reports of antidiabetic (Chaturvedi et al., 1984) anti-inflammatory (Surendrakumar, 1988) and antidiahhoreal (Rani et al., 1999) activities for C. phlomidis roots. P. integrifolia is reported to possess hypolipidemic (Khanna et al., 1991), anti-inflammatory (Barik et al., 1992) and antidiabetic activity (Kar et al., 1999). Phytochemical studies include reports of presence of β-sitosterol and γ-sitosterol, ceryl alcohol, clerodin, clerosterol, clerodendrin-A in root (Joshi et al., 1979) and flavanoid pectolinarigenin, hispidulin, apigenin and luteolin in flower (Seth et al., 1982) of C. phlomidis. In P. integrifolia, alkaloids premnine (Basu and Dandiya, 1947) ganikarine (Basu and Joneja, 1949) and premnazole alkaloid (Barik et al., 1992) are reported from roots; while flavanoid luteolin (Dasgupta et al., 1984), sterols and triterpene (Debelmas et al., 1973) are reported from the leaves.
In the present study methanol extracts of C. phlomidis (CPM) and P. integrifolia (PIM) roots were evaluated for specific immune response like haemagglutinating antibody titer (HA titer), Plaque Forming Cell (PFC) assay and delayed type hypersensitivity activity (DTH response) and nonspecific immune response like carbon clearance test and E. coli induced sepsis.
MATERIALS AND METHODS
Plant material: The fresh, well-developed plants of C. phlomidis were collected from the Ayurvedic garden, Gandhinagar in the month of October 2004 and plants of P. integrifolia were collected in the month of September 2004 from the Pharmacognosy Garden of Timba Ayurvedic Pharmacy College, Timba, Gujarat. The authenticity of plants was confirmed by a taxonomist of Gujarat Ayurveda University, Jamnagar, Gujarat. Voucher specimens LM108 and LM109 were deposited in the Department of Pharmacognosy, L.M. College of Pharmacy, Ahmedabad, Gujarat. Roots of both the plants after drying in the sun were reduced to (60 #) powder separately.
Extraction: Dried root powder of each root drug (200 g) was exhaustively extracted using methanol (3x500 mL) separately. The marc and filter paper was washed with methanol each time. Methanol extract of C. phlomidis (CPM) and P. integrifolia (PIM) obtained were concentrated, dried and used for the evaluation.
Clerodendrin-A was isolated from C. phlomidis and was estimated in both the roots by HPTLC method, data are published elsewhere.
Animals: BLAB/c albino mice of either sex (22-25 g) were used. The animals received a standard pellet diet (Lipton, Bombay), water ad libitum and were maintained in regulated temperature and humidity conditioned rooms.
In preliminary experiments both the extracts were found to induce significant response in the animals after oral administration for 5-7 days and the optimum response was served at 300 mg kg-1 dose.
Specific immune response: BLAB/c albino mice were administered with CPM and PIM (300 mg kg-1 b.wt.) in water for 7 days, on the 8th day mice were immunized by injecting 0.25 mL of fresh sheep red blood cell suspension (SRBCs) (108 cells mL-1 suspended in normal saline) intraperitoneally. Four days later blood was collected from the retro-orbital plexus for the HA titre assay (Joharapurkar et al., 2003; Puri et al., 1994). The animals were then killed by cervical dislocation and spleen was taken out for PFC assay (Puri et al., 1992; Jerne and Nordin, 1963). In another group, the DTH response to SRBC was determined (Joharapurkar et al., 2003; Puri et al., 1994).
Haemagglutinating Antibody (HA) titre: Serum was separated from the
blood samples and antibody levels were determined by the microtitre hemagglutination
technique. Twenty five microliter aliquots of two-fold diluted sera were prepared
in 0.15 M phosphate buffer saline (pH- 7.2) and dispensed in U bottom microtitre
plates (Polylab); 1% SRBC suspension (25 μL) in PBS was dispensed into
each well and mixed thoroughly. After 1 h of incubation at room temperature
the highest dilution of test serum giving agglutination was observed and the
reciprocal of the highest dilution observed was considered as antibody titre.
Plaque Forming Cell (PFC) assay: The assay was done according to technique of Jerne and Nordin (1963). Spleen cells were separated in RPMI-1640 medium; washed with the same and suspended in the same to a concentration of 1x106 cells mL-1. A bottom layer was prepared with 1.2% agarose in 0.15 M NaCl in glass petridishes. A mixture of 2 mL 0.6% agarose in RPMI-1640 medium (at 42°C), 0.1 mL suspension of 20% SRBC (in normal saline) and 1x105 spleen cells were poured over the bottom layer and petridishes were incubated at 37°C for 90 min. After that 2 mL of 1:10 diluted fresh Guinea pig serum in PBS was added as a source of complement in each petridish and further incubated for 60 min. The plaques were counted immediately and expressed as count per 105 spleen cells.
Delayed type hypersensitivity (DTH) response: On 11th day, the thickness of the right hind footpad was measured using vernier caliper. The mice were then challenged by injection of 20 μL of 1% SRBCs (suspended in normal saline) in right foot pad. Foot thickness was again measured after 24 h of this challenge (Saiki et al., 1981). The difference between the pre and post challenge foot thickness expressed in cm was taken as a measure of delayed type hypersensitivity.
Non-specific immune response
Carbon clearance test (phagocytic activity): Phagocytic index was
determined by a reported method (Gonda et al., 1990; Bafna and Mishra,
2004). Swiss albino mice were given CPM and PIM (300 mg kg-1 b.wt.)
in water in treated groups and control group received vehicle (water) for 5
days orally. After 48 h of the last dose on 5th day, mice were injected via
the tail vein with 0.1 mL Indian ink. Blood samples were drawn from orbital
vein at 3, 7 and 15 min from each mouse. 25 μL of each blood sample was
mixed with 2 mL of 0.1% w/v Na2CO3. The carbon clearance
i.e., the rate of elimination of carbon from the blood was determined by turbidometric
spectroscopy at 650 nm with UV spectrophotometer (Schimadzu 1601 Japan). The
graph of absorbance vs. time was plotted. The rate of carbon clearance, termed
as Phagocytic Index (PI), is the ratio of slope of time-concentration curve
of sample and standard.
||Slope of regression line.
E. coli induced abdominal sepsis: E. coli induced abdominal sepsis was done by a method reported by Pallable et al. (1998). The strength of E. coli (2.5x108 cells mL-1) was standardized to induce 100% mortality. Swiss albino mice were divided into two groups, treatment groups received CPM and PIM (300 mg kg-1 b.wt.) and control group received water orally for 15 days. On 15th day, 3 h after the last dose of extracts, E. coli were injected intraperitoneally to all mice. Percentage mortality was observed for 24 h in the treated groups and control group.
Statistical analysis: All the data were statistically analysed by one way Analysis of Variance (multiple comparison procedure: Tukeys test).
Specific immune response:
Haemagglutinating antibody titre: Increase in the humoral antibody
titer was evident on the 3rd day post immunization in treated mice in comparison
with untreated mice (n = 8). Anti-SRBC-haemagglutination antibody titer with
the use of the CPM and PIM treated mice were found to be 768±90.5 and
384±106.13, respectively, while the HA titre value for control animals
was 104±11.711. The difference was statistically significant (F = 14.09,
p = <0.05) (Table 1).
Plaque forming cell assay: Effect of CPM and PIM on PFC count (356±5.96, 252±5.36) was significantly higher than that of control animals (100.75±3.89) (F = 544.1, p = <0.001) (Table 1).
Delayed type hypersensitivity response: The mean value of the DTH response to SRBC was 0.118±0.0088 for the CPM and 0.107±0.0118 for the PIM treated animals as compared to 0.039±0.0024 for the control animals, which was significantly different (F = 27.77, p = <0.001) (Table 1).
Non-specific immune response
Carbon clearance test (Phagocytic activity): CPM and PIM possessed
macrophage stimulatory activity as evidenced by increased phagocytic index in
carbon clearance test. The phagocytic activity of reticuloendothelial is generally
measured by the rate of removal of carbon particles from the blood stream. CPM
and PIM at the dose of 300 mg kg-1 showed significant clearance of
carbon particles. Phagocytic index and rate of clearance are shown in the Fig.
1 and Table 2.
E. coli induced abdominal sepsis: In this assay, after challenging
animals with fatal dose of E. coli significant bacteraemia was found
to be induced. All (n = 6) animals of control group died within 15 h while CPM
and PIM treated animals showed 20 and 25% mortality, respectively in 24 h and
remaining animals showed survival without any symptoms of peritonitis.
||Effect of C. phlomidis and P. integrifolia extract
on rate of carbon clearance
||Effects of C. phlomidis and P. integrifolia
on antigenic specific responses on BLAB/c albino mice
|The values are mean±SEM (n = 8) *: p< 0.05; **:
||Effects of C. phlomidis (CPM) and P. integrifolia
(PIM) extracts on macrophage phagocytic activity in vivo on BLAB/c
|The values are mean±SEM (n = 6), *: p<0.001
The results of the present study provide an evidence of the immunostimulant activity in C. phlomidis and P. integrifolia, capable of inducing both antigen specific and nonspecific immune responses. In specific immune system, lymphocyte derived cells like T and B lymphocytes play active role (Pallabe et al., 1998). It has become evident from the present study that CPM and PIM produced significant enhancement of the antigen specific response against humoral as well as cell mediated immune response. CPM and PIM induced about nine-fold and four-fold increase respectively in the antibody formation as compared to that of the control group as evident from respective HA titre values. This result is also supported by the in vitro PFC assay, where nearly three-fold and two-fold increase in the IgM antibody plaque formation was observed in the spleen cells of the CPM and PIM treated mice as compared to the control animals.
Furthermore, in DTH assay, the cell-mediated immune response via helper T cells was found to be augmented under the influence of the extracts (Sainis et al., 1983). CPM and PIM treated animals showed similar responsiveness towards SRBC, which were higher than the control animals.
Since PI is greater than one, stimulation of reticulo endothelial system and activity of macrophages is evident from carbon clearance assay (Ponkshe and Indap, 2002). In this, in vivo assay macrophages are known to secrete a number of cytokines, which in turn stimulates other immunocytes. This may enhance the defense ability to counter the infectious stress. CPM showed higher PI than PIM, which reflects a marked increase in the rate of carbon clearance suggests a significant enhancement in the phagocytic function of the macrophages and thus, non-specific immunity. Phagocytosis by macrophages is important against the smaller parasites and its effectiveness is markedly enhanced by opsonization of the parasites with the antibody and complement C3b, leading to more rapid clearance of parasites from the blood (Sagle et al., 2004).
In E. coli induced sepsis, mice of treated and untreated group when subjected to the fatal dose of E. coli, treated groups showed significantly lesser percentage of mortality as compared to untreated group, when observed for 24 h after E. coli challenge. Though the difference between the two treated groups was minor, CPM showed somewhat better survival pattern the PIM.
These observations are suggestive of immunomodulatory activity of C. phlomidis and P. integrifolia in different immunomodulatory modules of specific and nonspecific immune responses. It would also be interesting to notice that C. phlomidis gave marginally higher response in most of the models. The methanol extracts (CPM and PIM) were found to be rich in diterpenoids and flavonoids. Both the types of components are reported to exhibit Immunomodulatory activity in various experimental models (Puri et al., 1993; Patwardhan et al., 1990; Chiang et al., 2003). Presence of these compounds in both the extracts might be contributing towards the Immunomodulatory activity.
Further, present study substantiates the claims made in Ayurveda regarding the use of both the roots as rasayana drugs under the common name of Arni or Agnimantha.
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