Immunostimulant Activity of n-Butanol Fraction of Root Bark of Oroxylum indicum, vent.

Abstract

In the present study, the immunomodulatory activity and the mechanism of action of the n-butanol fraction (100 mg/kg body weight, per os, once daily for 22 consecutive days) of the root bark of Oroxylum indicum, vent. (Bignoniaceae) was evaluated in rats using measures of immune responses to sheep red blood cells (SRBC haemagglutinating antibody [HA] titer) and delayed-type hypersensitivity (DTH) reactions. In response to SRBC, treatment with the n-butanol fraction caused a significant rise in circulating HA titers during secondary antibody responses, indicating a potentiation of certain aspects of the humoral response. The treatment also resulted in a significant rise in paw edema formation, indicating increased host DTH response. Additionally, the antioxidant potential of the drug was exhibited by significant reductions in whole blood malondialdehyde (MDA) content along with a rise in the activities/levels of superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH). Furthermore, histopathologic analysis of lymphoid tissues showed an increase in cellularity, e.g., T-lymphocytes and sinusoids, in the treatment group. In contrast, dexamethasone treatment caused significant reduction in the HA titer, DTH responses, and antioxidant potential. In a triple antigen-mediated immunological edema model, the extent of edema raised in drug-treated rats was greater compared to that in control rats, thus confirming enhanced DTH reactions in response to the drug treatment. Based on the above findings, the reported immunomodulatory activity of an active fraction of O. indicum might be attributed to its ability to enhance specific immune responses (both humoral and cell-mediated) as well as its antioxidant potential.

Keywords :

• immunostimulant
• oxidative stress
• O. indicum
• n-butanol fraction

INTRODUCTION

Immunomodulation is a process in which the immune system of an organism is affected by a test agent acting directly or indirectly upon system components and/or functions. If exposure to the agent results in enhancement of immune reactions, it is termed immunostimulatory; this outcome primarily implies that there is a stimulation of cellular constituents and associated cell products that comprise either/both the specific and non-specific arms of the immune system, i.e., granulocytes, macrophages, complement, certain T-lymphocytes, effector substances. In contrast, agents that give rise to immunosuppression are usually detected at the whole body level by observations of reductions in host resistance against infections and cancers, or alterations in responses to antigens. Primary examples of these types of effectors can be physical (i.e., stress, malnutrition, etc.) or chemical (i.e., occupational, environmental, and chemotherapeutic factors) in their nature.

A number of Indian medicinal plants have been claimed to possess immuno-modulatory activity. Oroxylum indicum, Vent. (Syonakh), belonging to the family Bignoniaceae, is a medicinal plant claimed to have a number of therapeutic uses and is used in different ayurvedic formulations. The plant is reported to possess anti-inflammatory, diuretic, anti-arthritic, antifungal and antibacterial activity (Warrier et al., 1997). However, the root bark of O. indicum has not been thoroughly evaluated for its immunomodulatory activity. From our earlier data examining the anti-ulcer and hepatoprotective activity of O. indicum, an n-butanol extract of this material (provided at 100 mg/kg body weight [BW], per os, single dose and once daily for 7 d for respective activities) was found to be the most potent at reducing gastric lesions and hepatotoxicity amongst all fractions obtained using various solvents (Khandhar et al., 2006, in press).

Interestingly, this hydrolysed n-butanol fraction was shown to contain a significant amount of baicalein, a major flavonoid reported by other investigators to have an immunomodulatory potential (Lien et al., 2003). Baicalein, the flavone aglycone of baicalin, is also known to be a constituent of Scutellariae Radix, a product widely used in clinical Chinese medicine as a treatment for inflammation, fever and allergic diseases. Anti-inflammatory, anti-HIV, anti-cancer, and anti-oxidant activities have all been reported for baicalein in in vitro studies using cell lines (reviewed in Lai et al., 2003). In vivo studies in rats have indicated that this agent also possesses anti-inflammatory and vasodepressor effects (Lin and Shieh, 1996; Takizawa et al., 1998).

As a result of these findings, the present study was undertaken to evaluate, more broadly, the immunomodulatory activity of this n-butanol fraction of O. indicum in an experimental animal model. In order to assess the ability of O. indicum to modulate specific immunological responses, analyses of both humoral (i.e., serum antibody titers) and cell-mediated (i.e., footpad thickness) functions in the host were performed following treatments of rodent hosts with the n-butanol extract. In order to be able to relate any potential findings here to those in the earlier anti-ulcer and hepatoprotective studies and in studies wherein the extract was shown to be effective in preventing Escherichia coli-induced peritonitis in treated hosts (Vishwas et al., 2004), the same exposure regimens as in those studies was employed. Based on the results of these studies, more traditional dose-response studies will then be undertaken to better define general toxicologic parameters (i.e., in part, by establishment of NOAEL, LOAEL, etc. values) and specifically how the bark extract (or its theorized active agent baicalein) acts at different concentrations on any of the particular immune system functions examined in the current study.

MATERIALS AND METHODS

Procurement of Plant Material and Extraction Procedure

Fresh root bark of O. indicum was collected from the Van-Aaushadhi Ektrikaran Udyan, Ahwa, Dang forest, in Gujarat, India. The voucher specimen (#404) was deposited in the Department of Pharmacognosy and Phytochemistry at the L. M. College of Pharmacy, Ahmedabad. The root bark was dried and powdered to a 60 mesh size (≈ 250 μm diameter; by grinding with a porcelain pestle a sifting through appropriate mesh screens). The powder of the root bark was first defatted using petroleum ether (remaining post-extraction material constituted 0.32% [w/w] of the original solute [i.e., root bark powder] that underwent the extraction). The residual unextracted material was then air-dried; this defatted powder was then moistened with an ammonia (NH₃) solution and then extracted with chloroform (post-extract remaining solute = 0.78% [w/w] of pre-extracted material). After drying, the remaining unextracted material was then extracted with ethyl acetate (post-extract remaining solute = 1.52% w/w). Finally, the thrice-extracted powder was then extracted with n-butanol (post-extract remaining solute = 1.68% w/w). The solvent-specific eluents recovered in each extraction regimen were then air-dried and their corresponding powdered fractions stored in airtight containers until usage. Based upon our earlier work investigating the anti-ulcer and hepatoprotective effects of this material (Khandhar et al., 2006, in press) and in studies where the extract was shown to be effective in preventing Escherichia coli-induced peritonitis in treated hosts (Vishwas et al., 2004), only the n-butanol fraction was employed in the present study.

Phytochemical Analyses

Phytochemical analyses of the butanol extract were performed using standard methods. Specifically, the extract was analyzed for the presence or alkaloids (Sim, 1969), flavanoids (Geissman, 1955), saponins (Fischer, 1952), tannins (Freudenberg et al., 1962; Robinson, 1964), and carbohydrates. Thin layer chromatography was employed to check for the presence of a baicalein (Figure 1), the aglucone version of the parent flavonoid baicalin. Quantification of the aglucone was performed using reverse phase HPLC (Khandar et al., 2006) and extrapolation against a standard curve generated using purified baicalein.

FIG. 1 The structure of balcalein (aglucone of balcalin). Adapted from Lai et al. (2003).

Drugs and Chemicals

All different organic solvents used for extraction were obtained from the S.D. Chemicals Private Limited (Mumbai, India), and were analytical grade. Fresh drug solutions were prepared in 1% carboxymethylcellulose (CMC) for oral administration. Hydrogen peroxide and Ciocalteau phenol reagent were obtained from S.D. Fine Chemicals Ltd. (Mumbai). Trichloroacetic acid, thiobarbituric acid, phosphate buffer, Tris buffer, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), bovine serum albumin, epinephrine, and dexamethasone were all obtained from Sigma-Aldrich (St. Louis, MO).

Animals

Wistar albino rats (Zydus Cadila Limited, Ahmedabad, India) of either sex weighing 175–225 g were selected for the present study. Animals were provided a standard chow diet (certified Amrut brand rodent feed, Pranav Agro Industries, Pune, India) and filtered tap water, ad libitum. The rats were maintained under standard conditions of a 12-hr dark-light cycle, 60(± 10)% humidity, and a temperature of 21.5(± 1)°C. Coprophagy (and thus re-ingestion of any drug) was prevented by keeping the animals in cages with floor gratings. The distribution of animals in the groups, the sequence of trials, and the treatment allotted to each group was randomized. Freshly prepared solutions of drugs or chemicals were used throughout the study. After completion of the experiments, animals were sacrificed by over-anesthetization with ether. All the experiments complied with the University guidelines for animal experimentation. Throughout the entire study period, the rats were monitored for growth, health status, and food intake capacity to be certain that they were healthy.

Immunologic Responses to Sheep Red Blood Cells (SRBC)

Sheep blood was aseptically collected (at a city slaughterhouse) in a sterilized bottle containing Alsever's solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid, and 0.42% sodium chloride). The SRBC were thoroughly washed with sterile normal saline and then stored in Alsever's solution in a refrigerator until needed in the study.

The rats were randomly divided (to assure equal distribution of weights) into the following groups containing six animals each.

• Group I: Control—Rats received only aqueous suspension of 1% CMC vehicle per os once daily, for 22 days.

• Group II: Drug treatment—Rats received n-butanol fraction (100 mg/kg BW, per os) once daily for 22 days.

• Group III: Rats received standard dexamethasone (0.27 mg/kg BW, per os) once daily for 22 days.

The choice of the dose of the n-butanol fraction to treat the animals with was based on earlier determinations of the impact from this material on host responses to Escherichia coli (i.e., analyses of onset of peritonitis induced by the organism) (Vishwas et al., 2004). Those studies showed that using the same regimen as employed here, treatment with the extract caused significant reductions in both the occurrence of the peritonitis and subsequent host mortality; specifically, animals that received the 100 mg/kg treatment in this regimen displayed mortality rates only 30% of that seen in infected controls that received only vehicle.

Rats in each of these treatment groups then received an intraperitoneal injection of SRBC (0.5 × 10⁹ cells/100 g BW) on Days 7 and 13 of their respective regimens in order to sensitize them for the assays below.

Humoral Immune Response

On Days 13 (pre-dose and pre-challenged with SRBC) and 20 of exposure, blood was withdrawn from the retro-orbital plexus of all SRBC-challenged rats and the serum in each sample isolated by centrifugation. Twenty-five μl of each serum was then serially diluted with 25 μl of phosphate-buffered saline (PBS, pH 7.4). SRBC (2.5 × 10⁷ cells) were then added to each of these dilutions and the mixtures incubated at 37°C for one hour. The minimum dilution that exhibited haemagglutination was considered as the antibody or haemagglutination titer (HA titer). The level of antibody titer on Day 13 of the experiment was considered as the indicator of the primary humoral immune response and that on Day 20 the indicator of the secondary response (Joharapurkar et al., 2003).

Cellular Immune Response

The cellular immune response in each rat was assayed using the footpad reaction method (Joharapurkar et al., 2003). Edema was induced in the right paw of the rats by injecting (2.5 × 10⁷) SRBC into the subplantar region on Day 20 of the treatment regimen. The increase in paw volume after 48 hrs (i.e., on Day 22) was then assessed using vernier calipers. The mean percentage increase in paw edema was considered as the indicator of delayed-type hypersensitivity (DTH) and, as such, an index of a cell-mediated immune response in the rats. The volume of the left hind paw that had been injected simultaneously with PBS served as an internal control for each animal.

Anti-Oxidant Activity

The level of antioxidant activity (i.e., reflected by antioxidant enzyme function and lipid peroxide levels) was assessed in the blood of the rats using materials withdrawn from the retro-orbital plexus on Day 22 of the treatment regimen. The total protein concentration in each blood sample was determined by the method of Lowry et al. (1951). The effects of the root bark extract on the activity of the antioxidant enzymes superoxide dismutase (SOD; in terms of mU/mg protein) and catalase (CAT; as U/min/mg protein), and on the levels of reduced glutathione (GSH; as μmole/mg protein), in the blood were assayed by the methods of Misra and Fridovich (1972), Aebi (1974), and Beutler et al. (1963), respectively. The levels of malondialdehyde (MDA) in each sample were estimated (expressed as μmole thiobarbituric acid reactive substances [TBARS]/mg protein) at 535 nm in a Shimandzu UV Spectrophotometer (Shimadzu, Japan) using the methods of Kiso et al. (1984).

SOD activity in the samples was determined by mixing 0.1 ml of sample with 0.1 ml of EDTA (1 × 10⁻⁴ M), 0.5 ml of carbonate buffer (pH 9.7), and 1 ml of epinephrine (3 × 10³ M) (Sigma). The optical density of the adrenochrome was assessed at 480 nm at 30-sec intervals for a total of 3 min. SOD activity was expressed as mU/mg of protein. One unit of activity is defined as the enzyme concentration required to inhibit the formation of the chromogen product by 50% in 1 min under the defined assay condition.

Catalase activity in each sample was measured by assessing the decomposition of hydrogen peroxide (H₂O₂) at 240 nm after addition of the whole blood. In a cuvette, 50 μl blood was mixed with 2.95 ml of reaction buffer (0.05 M phosphate buffer [pH 7.0] containing 30 mM H₂O₂) and the absorbance was measured at 15-sec intervals for 3 min. As the optical density measured reflects the peroxide concentration in the cuvette, the activity of catalase in the 3-min period was deduced and expressed as mM H₂O₂ consumed/mg tissue/min.

Reduced glutathione (GSH) content in each sample was measured after initial precipitation of proteins with 10% chilled trichloroacetic acid. After a 30 min incubation, the samples were then centrifuged at 1000 g for 10 min at 4°C. The GSH levels in the supernatant were then determined by mixing 0.5 ml of the material with 2 ml 0.3 M phosphate buffer (pH 7.0) and 0.25 ml DTNB reagent (40 mg/l00 ml in 1% sodium citrate buffer), and then measuring the absorbance at 412 nm. Standard solutions containing different concentrations of GSH were prepared in parallel to generate a standard curve. Results are expressed as μmoles of GSH/mg of protein.

The levels of malondialdehyde (MDA, representative of peroxidative damage to cell membranes) were measured by mixing 2 ml of a 5% suspension of recovered, separated red blood cell samples (in 0.1 M phosphate-buffered saline [pH 7.4]) with 2 ml of a 28% trichloroacetic acid solution. After thorough mixing, the mixture was then centrifuged at 10,000 g at 4°C for 5 min and the supernatant was recovered for estimation of MDA. For the latter, 4 ml supernatant was mixed with 1 ml of 1% thiobarbituric acid solution (TBA) and heated at 100°C for 60 min. The mixture was then cooled to room temperature and the absorbance measured spectrophotometrically at 532 nm. After accounting for background absorbance using buffer blanks, the total TBARS (TBA-reactive substrates) concentration in each sample was derived from the TBA extinction coefficient ε = 1.56 × 10⁵ M⁻¹ cm⁻¹. The level of MDA in each sample was calculated and data expressed in terms of nmoles of MDA/mg of protein in each sample.

Histopathological Studies

On Day 22, the final day of the treatments, all rats were sacrificed by overdose of ether anesthesia and several tissues recovered during necropsy for use in histopathological analyes. The spleens, thymi and axillary lymph nodes from both control and treated animals were preserved in 10% formalin solution. Thereafter, after embedding in paraffin, 6 μm thick sections were cut and then stained with haematoxylin to permit histological examination. All tissues were then assessed for any morphological changes under a photomicroscope. Photomicrographs of representative sections were taken at 10X magnification using a Trinocular Research Carl Zeiss Microscope (Gottingen, Germany).

Alum Adjuvant-Induced Hind Paw Edema in Pre-Sensitized Rats

A separate group of Wistar albino rats of either sex (150–250 g) was randomly divided into the following groups containing six animals each.

• Group I: Control—Rats received only aqueous suspension of 1% CMC per os once daily for 5 days.

• Group II: Drug treatment—Rats received n-butanol fraction (100 mg/kg BW, per os) once daily for 5 days.

Immunological inflammation was then produced by injection of triple antigen (a vaccine composed of a mixture of Diphtheria toxoid, Tetanus toxoid, and killed organisms of strains of Bordetella pertussis) with alum precipitates in the following proportion: 1 ml of Triple antigen, 4 ml of normal saline (0.9% NaCl), and 1 ml of 10% potash alum (Bhattacharya et al., 1981). The pH of the solution was adjusted to 5.6–6.8 using 10% sodium carbonate solutions. Initially, rats were sensitized by injecting the triple antigen with alum precipitates subcutaneously into the nape of the neck at a dose of 0.5 ml/100 g BW. Test drug (or vehicle) administration began on the day of sensitization (30 min after sensitization) and continued for the next 5 days. On the last day, 1 hr after administration of the test drug (or vehicle), rats were injected with 0.1 ml triple antigen with alum precipitates beneath the plantar aponeurosis in the left hind paw. Initially and after 48 hr of the triple antigen challenge, the paw volume was measured using vernier calipers. The percentage increase in paw volume after alum adjuvant injection in comparison to the initial value was recorded and used for subsequent statistical comparisons between the treatment groups.

Statistical Analyses

All results were expressed as the mean ± SEM. The significance of difference between mean values for the various treatments was tested using the Student's t-test. One-way analysis of variance (ANOVA), followed by Tukey's multiple range test (Bolton, 1997), was used wherever applicable to assess the statistical significance of differences between groups. Differences between treatment groups were considered as statistically significant at p < 0.05.

RESULTS

Phytochemical Analyses

On preliminary phytochemical screening, the n-butanol extract of the root bark was positive for the presence of alkaloids, flavonoids, tannins, and anthraquinones. Thin layer chromatography was employed to specifically check for the presence of a a single flavonoid, baicalein. Using reverse phase-HPLC analysis to quantify the baicalein present in the extract (Khandar et al., 2006), the results (Table 1) show that the n-butanol fraction used in these particular studies contained 11.56% (w/v) baicalein.

TABLE 1 The percentage of baicalein in isolated fractions of O. indicum

Extraction solvent	Percent baicalein (w/v)
Petroleum ether	4.59%
Chloroform	0.48%
Ethyl Acetate	0.79%
n-Butanol	11.56%

Immunological Responses to SRBC

In order to assess the ability of O. indicum to stimulate specific immunological responses, measurements of serum antibody titer and footpad thickness were conducted to evaluate the effect of the n-butanol fraction of the root bark (at 100 mg/kg BW, per os, daily for up to 22 days) on humoral and cell-mediated immune responses, respectively.

The humoral antibody response was determined after immunization of the animals with SRBC and assessed via a haemagglutination (HA) test. Treatment with the n-butanol fraction caused a significant increase in the secondary response anti-SRBC titers as compared to those found in rats treated with the vehicle control (7.51 [± 0.41] vs. 5.89 [± 0.29]; p < 0.01) over the treatment period (Table 2). Dexamethasone-treated rats showed marked decreases in the secondary HA titer (3.12 [± 0.29]) as was expected for this immunosuppressant.

TABLE 2 Effect of the n- butanol fraction (100 mg/kg/day per os) of root bark of O. indicum on antibody (Ab) formation against SRBC in sensitized rats (humoral immune response)

Group	Primary Ab titer on Day 13 (1° HA titer)	Secondary Ab titer on Day 20 (2° HA titer)
Control	3.58 ± 0.27	5.89 ± 0.29
n-Butanol	4.39 ± 0.22	7.51 ± 0.41
Dexamethasone	2.31 ± 0.29	3.12 ± 0.29

All values represent mean ± SEM, n = 6 in each group.

*p < 0.05, when compared with the control group (ANOVA, followed by Tukey's multiple range test), F_tab = 3.68; F_cal (2,15) = 15.22 (1°HA titer), 42.19 (2°HA titer). Dexamethasone treatment was at 0.27 mg/kg BW.

The data pertaining to the effect of the n-butanol treatment on SRBC-induced hind paw edema in pre-sensitized rats are presented in Table 3. The n-butanol fraction treatment caused marked enhancement of delayed-type hypersensitivity (DTH) in the rats. The DTH response (expressed in terms of percentage edema) was 87.01 [± 9.84] for the drug-treated rats as compared to 49.04 [± 4.97] for the control hosts (p < 0.05). Dexamethasone-treated rats did not evince statistically significant decreases in this specific endpoint (percentage edema = 40.04 [± 4.20]) compared to the controls.

TABLE 3 Effect of n- butanol fraction (100 mg/kg/day per os) of root bark of O. indicum on SRBC-induced hind paw edema in pre-sensitized rats

Group	0 hr (on Day 20) Edema (mm)	48 hrs (on Day 22) Edema (mm)	% Edema
Control	2.50 ± 0.06	3.72 ± 0.11	49.04 ± 4.97
n-Butanol	2.50 ± 0.12	4.65 ± 0.25	87.01 ± 9.84
Dexamethasone	2.40 ± 0.06	3.16 ± 0.19	40.06 ± 4.20

All values represent mean ± SEM, n = 6 in each group.

*p < 0.05, when compared with the control group (ANOVA, followed by Tukey's multiple range test), F_tab = 3.68; F_cal(2,15) = 15.03 (% Edema). Dexamethasone treatment was at 0.27 mg/kg BW.

Anti-Oxidant Activity

The effect of the n-butanol treatment on marker parameters of oxidative stress was also analyzed in the rats. While treatment with the test extract caused a significant decrease in the MDA content of the host blood, it induced increases in both SOD and CAT activities, as well as in GSH levels, as compared to those corresponding values in the blood of the respective control rats (after immunization with SRBC). In contrast, dexamethasone-treated rats displayed marked increases in MDA content and decreases in SOD levels as compared to the controls (Table 4). This suggested that the n-butanol fraction of O. indicum possessed significant anti-oxidant potential.

TABLE 4 Effect of the n- butanol fraction (100 mg/kg/day per os) of root bark of O. indicum on MDA content, anti-oxidant enzyme activities, and reduced glutathione levels

Treatment group	MDA content (μmole/mg protein)	SOD (mU/mg protein)	CAT (U/min/mg)	GSH (μmole/mg protein)
Control	0.682 ± 0.031	0.091 ± 0.004	0.655 ± 0.005	0.016 ± 0.000
n-Butanol	0.530 ± 0.029	0.220 ± 0.013	0.806± 0.028	0.095 ± 0.007
Dexamethasone	1.038 ± 0.040	0.064± 0.003	0.535 ± 0.016	0.025 ± 0.002

All values represent mean ± SEM, n = 6 in each group.

*p < 0.05, when compared with the control group (ANOVA, followed by Tukey's multiple range test), F_tab = 3.68; F_cal(2,15) = 59.35 (MDA), 111.30 (SOD), 51.79 (CAT), 96.81 (GSH). Dexamethasone treatment was at 0.27 mg/kg BW. Units for each enzyme are defined in the Methods section.

Histopathological Studies

The histologic analyses of the rats indicated that in the spleen, there was an increased proportion of white pulp, cellularity, and presence of lymphocytes following the treatments with the n-butanol fraction as compared to within the tissues obtained from the control and dexamethasone-treated hosts (representative tissue sections shown in Figure 2). Examination of the isolated thymic tissues indicated that the treatment with extract also caused an increase in the cellularity in this organ as compared to what occurred in the thymi of rats from the other two groups (representative tissue sections shown in Figure 3). Finally, in the lymph nodes recovered from the extract-treated rats, there was an increase in the cellularity within the medulla and an increase in the number of lymphoid follicles in the cortex relative to the observations made in the tissues isolated from rats in either other treatment group (representative tissue sections shown in Figure 4).

FIG. 2A Representative photomicrograph of splenic tissues recovered from rats on Day 22 of the respective indicated treatment regimens (WP = White pulp and RP = Red pulp)—Control rats. Left image at 10×, Right image at 40×.

FIG. 2B Representative photomicrograph of splenic tissues recovered from rats on Day 22 of the respective indicated treatment regimens (WP = White pulp and RP = Red pulp)—n-Butanol extract-treated rats. Left image at 10×, Right image at 40×.

FIG. 2C Representative photomicrograph of splenic tissues recovered from rats on Day 22 of the respective indicated treatment regimens (WP = White pulp and RP = Red pulp)—Dexamethasone-treated rats. Left image at 10×, Right image at 40×.

FIG. 3A Representative photomicrograph of thymus recovered from rats on Day 22 of the respective indicated treatment regimens (L = Lymphocytes, S = Sinusoid, HB = Hassel's Body. Left image at 10×, Right image at 40×.

FIG. 3B Representative photomicrograph of thymus recovered from rats on Day 22 of the respective indicated treatment regimens (L = Lymphocytes, S = Sinusoid, HB = Hassel's Body. (B) n-Butanol extract-treated rats; Left image at 10×, Right image at 40×.

FIG. 3C Representative photomicrograph of thymus recovered from rats on Day 22 of the respective indicated treatment regimens (L = Lymphocytes, S = Sinusoid, HB = Hassel's Body. (C) Dexamethasone-treated rats. Left image at 10×, Right image at 40×.

FIG. 4A Representative photomicrograph of axillary lymph node tissues recovered from rats on Day 22 of the respective indicated treatment regimens (C = Cortex, CP = Capsule, M = Medulla, and LF = Lymphoid follicles)—Control rats. Left image at 10×, Right image at 40×.

FIG. 4B Representative photomicrograph of axillary lymph node tissues recovered from rats on Day 22 of the respective indicated treatment regimens (C = Cortex, CP = Capsule, M = Medulla, and LF = Lymphoid follicles)—n-Butanol extract-treated rats. Left image at 10×, Right image at 40×.

FIG. 4C Representative photomicrograph of axillary lymph node tissues recovered from rats on Day 22 of the respective indicated treatment regimens (C = Cortex, CP = Capsule, M = Medulla, and LF = Lymphoid follicles)—Dexamethasone-treated rats. Left image at 10×, Right image at 40×.

Alum Adjuvant-Induced Hind Paw Edema in Pre-Sensitized Rats

The data pertaining to the effect of the n-butanol fraction of the root bark of O. indicum on alum-adjuvant-induced immunological paw edema formation in rats indicated that the treatments caused a significantly marked enhancement of the DTH response (p < 0.05; Table 5). The response (measured in terms of percentage increase in edema) was found to be 80.81 (± 9.97) in rats that had received the drug treatment in comparison to a value of 40.04 (± 8.20) for the control rats.

TABLE 5 Effect of the n-butanol fraction (100 mg/kg/day per os) of root bark of O. indicum on alum adjuvant-induced hind paw edema in pre-sensitized rats

Treatment	0 hr (on Day 5) Edema (mm)	48 hrs (on Day 7) Edema (mm)	% Edema
Control	2.40 ± 0.07	3.37 ± 0.25	40.04 ± 8.82
n-Butanol	2.43 ± 0.08	4.37 ± 0.21	80.81 ± 9.97

All values represent mean ± SEM, n = 6 in each group.

*p < 0.05, when compared with the control group (Unpaired Student's t-test).

DISCUSSION

Phytochemical analysis of the root bark of Oroxylum indicum, vent. (Bignoniaceae) has previously revealed the presence of alkaloids, flavonoids, tannins, and anthra-quinone. In the present study, preliminary screening using thin layer chromatrographic techniques revealed the presence of the flavonoid baicalein, an agent reported to possess immunomodulatory activity (Lien et al., 2003), in an n-butanol extract prepared from the intact bark. Furthermore, reverse phase-HPLC analysis was performed to develop a complete chemoprofile and to quantify the baicalein in the active fraction; the results indicated a significant quantity of this flavonoid in the isolates. Therefore, any results in the current studies that demonstrate broad immunomodulatory activities of this n-butanol fraction might be attributed to the baicalein present in the O. indicum root bark.

Perturbations of the immune system are known to be involved in the etiology as well as pathophysiologic mechanisms of many diseases. Immunological defense is complex as it involves a well-regulated interplay between non-specific and specific components of immunity, the cellular and humoral arms of responses, as well as the stimulation and suppression of immunocompetent cells. Occupational, environmental, pharmacologic, or chemotherapeutic agents that have been shown to modify one or more of these processes in exposed hosts are deemed immunomodulants (Chirigos, 1992). Along these lines, different agents of plant origin—and their polyherbal formulations—have been reported to interact with the immune system in a complex way and thereby modulate pathophysiological processes (Atal et al., 1986). Considering the variety of cellular and humoral functions of the immune system and the condition-dependent effects of immunomodulatory agents, it is unlikely that a single test model would be satisfactory for the screening/characterization of any new immunomodulant.

The present study critically evaluated the immunomodulatory effects of the n-butanol fraction of O. indicum on some non-specific and specific aspects of immunity in rats. For example, effects on humoral immunity were assessed during treatment with the extracts by monitoring the host responses to a foreign antigen. Upon antigen entry, an antigen-specific immune response is mounted during the humoral response. This response involves the interaction of B-lymphocytes (or other types of antigen-presenting cells) with the antigen and, in the case of the B-cell, their subsequent proliferation and differentiation into antibody-secreting plasma cells. The product antibody then functions as the effector of the humoral response by binding to the antigen and neutralizing it, or facilitating its elimination by crosslinking to form clusters that are readily ingested by phagocytic cells.

The magnitude of the humoral response depends on both endogenous factors and cellular interactions. During the response, recruitment of macrophages is essential for antigen processing and presentation; at the same time, T-lymphocytes are essential for transformation of B-lymphocytes to antibody-secreting plasma cells during T-dependent antigen processing. Sheep red blood cells (SRBC) used in the present study are an example of a T-dependent antigen. In the study reported here, daily treatment with the n-butanol fraction of O. indicum (100 mg/kg BW, per os) caused a significant rise in secondary antibody titers against SRBC. The extract significantly enhanced the secondary immune response in treated hosts. The secondary response was both much quicker and more intense than the primary response. These two observations suggest that the drug treatment significantly enhanced the responsiveness of macrophages and both T- and B-lymphocyte subsets involved in antibody synthesis, leading to an enhanced antibody production.

The delayed-type hypersensitivity (DTH) response is a direct correlate with the integrity of cell-mediated immune function in a host. During a DTH reaction, sensitized T-lymphocytes challenged by antigen are converted into lymphoblasts that secrete lymphokines. These then induce vasodilatation, increase vascular permeability, cause macrophage accumulation and activation, promote phagocytic activity, and increase the concentration of lytic enzymes in order to assure more effective killing (if a viable pathogen is present). Due to vasodilatation, increased permeability, and macrophage accumulation, there is a local inflammation and concomitant formation of edema. Using the latter as an endpoint, cell-mediated immunity can be routinely evaluated by the local induction of antigen-mediated edema. In the current studies, treatment of rats with the O. indicum n-butanol fraction caused a significant rise in paw volume (and in the percentage of) edema as compared to that observed in the paws of control (vehicle-dosed) counterparts. In rats that received treatment with the known immunosuppressant dexamethasone, these values were both decreased, albeit that the extent of difference compared to the control values failed to reach statistical significant. These results thus suggest that the root bark drug caused an enhancement of cell-mediated immunity, again indicating that the extract is a potent immunostimulant.

During the process of macrophage phagocytosis, there is generation of reactive oxidative species (ROS) that accompany a cellular respiratory burst. Free radicals generated among the ROS cause iron to be released from cellular ferritin; in turn, this now-liberated iron is capable of inducing lipid peroxidation events in/on the cells via Fenton or Haber Weiss reactions (Li et al., 2000). The peroxidation products that are formed on the cell membrane are highly cytotoxic and can cause suppression of both humoral and cell-mediated responses. Corticosteroids are a good example of a class of agents known to generate free radicals in situ and thereby cause oxidative stress (Knight, 2000; Hirokawa and Utsuyama, 2002) and subsequently, immunosuppression. To prevent these pathologies from developing or being overly drawn out, endogenous antioxidant systems that effectively neutralize these oxidative stresses are often present (or activated) and thus help to preserve immune function. Our observations in the present study showed that there was an inverse relationship between malondialdehyde (MDA) content and that in the levels/activities of several endogenous antioxidant defenses (e.g., SOD, CAT, and GSH) in cells or in the blood. Specifically, treatment of the rats with the n-butanol fraction caused significant reductions in MDA content and a rise in the activities of SOD and CAT, as well as in levels of reduced GSH, in the blood. In comparison, dexamethasone caused a significant rise in MDA content along with decreases in SOD; however, in these hosts, CAT activity and GSH levels were not significantly impacted. Thus, it seems that the n-butanol fraction of O. indicum possesses a significant anti-oxidant potential that, in turn, might reduce oxidative stress and so lead to some immunomodulation.

Normal splenic parenchyma consists of both white and red pulp. While white pulp is involved in the formation of white blood cells (WBC), red pulp is involved in the destruction of worn-out and/or defective WBC. As such, when an antigen such as SRBC is administered, there is an increase in white pulp due to destruction of antigen by WBC. In case of root bark-treated rats, due to the immunostimulatory nature of the drug, there was both a proliferation of white pulp and the white pulp proportion. In contrast, in immunosuppressed dexamethasone-treated rats, the white pulp was destroyed and there was a concomitant increased in the proportion of red:white pulp in isolated tissue sections.

The cytoarchitecture of the normal thymus includes the capsule, cortex, medulla, sinusoids, T-lymphocytes, and Hassel's bodies. These latter bodies are nonviable and therefore do not respond to stimuli; in addition, they are not regenerated and as such, they are more often indicative of degenerative structures. Both antigenic properties and immunological reactions are only present in the early growth stages of Hassel's bodies when the cells are often hypertrophic. The component parts of the lamellated corpuscle are subject to involutive phenomena; as such, they cease to function and to supply structural material until they finally disappear. Thus, the Hassel's bodies are a characteristic of only the thymus gland and are utilized to identify this gland. In the study here, exposure to the n-butanol-treated materials did not result in any effect on Hassel's bodies.

The thymus is associated with the production of mainly the T-lymphocytes that are responsible for cell-mediated immunity in the body. In the current study, an examination of the thymii from the root bark-treated rats revealed increases in cellularity such as T-lymphocytes and sinusoids, as compared to what was seen in the organs recovered from the control rats. In normal axillary lymph nodes, the cytoarchitecture includes a capsule, cortex and medulla. Lymphoid follicles of the cortex mainly contain T-lymphocytes while lymphoid follicles of the medulla contain B-type and plasma cells. The examination of the lymph nodes recovered from the extract-treated rats revealed increases in cellularity. Specifically, due to a proliferation of lymphocytes, the lymphoid follicles became enlarged and thickened. On the other hand, in dexamethasone-treated rats, there was a marked destruction of lymphoid follicles that led to a decrease in cellularity overall.

The studies with the alum were meant to assess the effects of the treatment with the root bark extract on delayed type hypersensitivity reactions. Here, instead of SRBC, triple antigen with alum precipitates was used as the antigen. The change in the percentage edema was found to be greater in treated rats when compared with the controls, indicating enhanced DTH reactions. This, in conjunction with the earlier finding in this study, confirmed that the n-butanol fraction of O. indicum possessed immunostimulant properties.

In summary, the results of these studies have demonstrated the potential effects of the n-butanol fraction of O. indicum on immune regulation. These findings confirm earlier ones regarding the general immunopotentiating role of this plant extract. These studies have also begun to lay the groundwork for defining the possible mechanisms underlying the immunomodulating activity of this active fraction. Specifically, the effects of O.indicum are likely to be attributed to enhancement of specific immune responses (i.e., both humoral and cell-mediated) as well as to its ability to act as an anti-oxidant. Whether the latter is a direct effect or attributable to stimulation of anti-oxidant mechanisms already in place remains to be determined. In any case, based on the phytochemical analyses, it is more likely than not that many of the observations reported in these studies are associated with the presence of baicalein, a major flavonoid found in the n-butanol fraction of this plant. Follow-up studies using the flavonoid alone or extracts that have this agent selectively removed, as well as studies using more traditional dose-response approaches, will help further define precisely how—and at what levels of dosage—this plant extract is exerting its effects in treated hosts.

ACKNOWLEDGMENTS

The authors are thankful to GUJCOST for financial assistance by providing a minor research scheme.