MATERIALS AND METHODS

Chromatographic studies: Selected part of the above mentioned local plant was subjected to extraction followed by analytical studies for isolation of active constituent (Doshi et al., 2014). These extracts were stored at 8-15°C for proceeding studies related to immunomodulatory models.

Standard drugs and chemicals: Ashwagandha Churna (Dabur, India Ltd.) used as standard herbal drug was procured from local market which is reported to possess well known immunomodulatory activity (Gupta et al., 2006). Indian ink was procured from Camlin India Ltd., Mumbai and Complete Freund’s Adjuvant (CFA) from Difco Laboratories. All the chemicals used throughout the experimentation purpose were procured from local suppliers.

Animal studies: The protocol for animal studies was approved by Institutional Animal Ethics Committee of Vivekanand Education Society’s College of Pharmacy, Mumbai, Maharashtra, India (VESCOP/07/2014). Albino Wistar rats of either sex obtained from the Haffkine institute, Mumbai, each weighing between 120-150 g were used. They were divided into five groups for each model.

Experimental conditions: The animal experimental area was air-conditioned with adequate air changes per hour with a light cycle of 12 h light and 12 h dark, temperature 22±1°C and relative humidity 65±10%. They were housed in groups of three in polycarbonate cages with paddy husk bedding and fed with standard pellet food and Genpure RO water provided ad libitum. The animals were fasted for approximately 16 h before studies with free access to water.

Antigen: Fresh blood was collected from sheep sacrificed in the local slaughter house. Sheep red blood cells (SRBCs) were washed three times in large volumes of pyrogen free 0.9% normal saline and adjusted to a concentration of 0.5×10⁹ cells mL‾¹ for immunization and challenge.

Induction of arthritis: Arthritis was induced in rats by injecting 0.1 mL of CFA in to the left hind paw. The CFA used, consists of 5 mg Mycobacterium butyricum suspended in heavy paraffin oil by thoroughly grinding in mortar and pestle to give concentration of 5 mg mL‾¹.

Acute toxicity: Acute toxicity studies were undertaken as per OECD guidelines 423 (OECD., 2001). The extract was dissolved in 0.5% carboxymethyl cellulose (vehicle). Female rats were administered a single dose of the extract at 5000 mg kg‾¹ b.wt., p.o. (administration volume was 1 mL/100 g):

Animal models: Animal models were proposed by Shukla et al. (2009), Bader (1997) and Gaur et al. (2009) which are as follows:

Haemagglutination antibody (HA) titre: The rats were immunized by injecting 0.1 mL of SRBCs suspension containing 0.5×10⁹ cells intraperitoneally on day 0. Vehicle, extracts and standard were given orally to the animals for 7 days. Blood samples were collected in micro-centrifuge tubes from individual animal by retro-orbital puncture on day 7. The blood samples were centrifuged and serum was obtained. Equal volumes of individual serum samples of each group were pooled. Two-fold serial dilutions of pooled serum samples made in 25 μL volume of normal saline in microtitration plates were added to 25 μL of 1% suspension of SRBCs in saline. After mixing, the plates were incubated at 37°C for 1 h and examined for haemagglutination under microscope. Antibody levels were determined by taking the reciprocal of the highest dilution of the test serum agglutination as the antibody titre.

Delayed-type hypersensitivity (DTH) response: The rats were challenged by injection of 0.5×10⁹ cells SRBCs in right hind foot pad. Foot thickness was measured after +24 and +48 h of this challenge. The extract was administered orally on day 0 and continued till day 7 of challenge. Right hind footpad thickness was measured with micrometer screw gauge on 7th (prior to injection), 8th and 9th day of the study. Difference between prior and post injection footpad thickness was reported as DTH response.

Phagocytic response (carbon clearance test): The rats were treated from day 0-7 with different concentrations of the extracts. On day 7, all the animals of the entire groups received the treatment of an intravenous injection of 0.2 mL/animal Indian ink dispersion (pre-warmed at 37°C) through tail vein. Blood samples were collected from retro-orbital plexuses immediately at 0, 5, 10 and 15 min after the injection of Indian ink. Twenty five microliter of blood samples were added to 2 mL of 0.1% sodium carbonate solution. Absorbance of the samples was measured at 660 nm using UV visible spectrophotometer. The rate of carbon clearance (phagocytic index, K) was calculated using the following equation:

where, OD₁ and OD₂ are the optical densities and T₁ and T₂ is time in minutes, respectively.

Adjuvant arthritis: Vogel (2002), Krensky et al. (2005), Petchi et al. (2013), Subash et al. (2012), Kuncha et al. (2014) and Chattopadhyay et al. (2004) stated the adjuvant arthritis methods. The rats, after induction of Complete Freund Adjuvant (CFA) were administered with extracts and standard from day 1 and continued dosing till 12 days.

Paw volume: Paw volumes of both the sides was measured with plethysmometer and body weights were recorded on the day of injection. Rats were not dosed with extracts or the standard from 13th to 21st day. Rat paw volume was measured on 1st, 3rd, 5th, 9th, 13th and 21st day in order to record the primary and secondary lesions. Mean changes in injected paw edema with respect to initial paw volume, were calculated using the equation as:

Primary and secondary lesions: Primary lesions refer to edema formation in the injected hind paw that peaks 3-5 days after injection of the phlogistic agent and is measured on day 5 by calculating percent inhibition of the edema volume of the injected left paw over the vehicle control. Secondary lesions are immunologically mediated changes characterized by inflammation of the noninjected sites (hind leg, forepaws, ears, nose and tail) paw over the vehicle control. Percent reduction in body weight is calculated using the equation:

where, B₀ and B₂₁ is body weight recorded on day 0 and 21, respectively.

Arthritic index: An arthritic index is calculated as the sum of the scores as shown in Table 1 for each animal. The average of treated animals is compared with the control group.

Biochemical parameters: Parasuraman et al. (2010) analysed the biochemical parameters. At the end of the 21st day, blood samples were withdrawn from all groups through retro-orbital plexus puncture by anaesthetizing the animals with diethyl ether and the biochemical parameters such as hemoglobin content, total WBC count and RBC count were analyzed.

Histological determination: Kilimozhi et al. (2009) determined the historical parameters on 21st day, the rats were subjected to appropriate euthanasia. The hind paws were amputated above the knee joint, fixed in 7.4% formalin, decalcified using 10% nitric acid, embedded in paraffin and sectioned in a mid-sagittal plane. The Transverse Section (TS) of knee joint of tarsals were stained with haematoxylin and eosin and examined microscopically for bone destruction, presence of mononuclear infiltration and pannus formation.

Table 1:	Arthritic index for each animal

Statistical analysis: The statistical significance was assessed using one way analysis of variance (ANOVA) followed by Dunnett test. The values were expressed as Mean±SEM and *p<0.05, **p<0. 01, ***p<0.001 when compared with control group was considered significant.

RESULTS

Acute toxicity
Mortality/viability: No mortality was reported up to 5000 mg kg‾¹ b.wt.

Body weights: All surviving animals had gained body weight by day 14 as compared to day 0.

Clinical signs: All the animals appeared normal throughout the experimental period.

Macroscopic findings: No abnormalities were detected for any of the animals at terminal sacrifice.

Animal models (Immunomostimulatory effects)
Haemagglutination antibody (HA) titre: The PL extract showed mild (100 mg kg‾¹) and significant immunostimmulation (250 and 500 mg kg‾¹) as depicted in Table 2.

Delayed-Type Hypersensitivity (DTH) response: The results interpreted showed dose dependent decrease in paw edema after 24 h of challenge which was observed at all dose levels of PL extract when compared to control group. The PL extracts (100, 250 and 500 mg kg‾¹) after 48 h lowered the paw thichness depicting good immunostimulatory activity (Table 2).

Phagocytic response (carbon clearance test): The model showed prominent phagocytic index (0.0125, 0.0143 and 0.0180 at 100, 250 and 500 mg kg‾¹ b.wt., respectively) suggesting sufficient immunostimulatory activity of the extract as compared to control group (Table 3).

Adjuvant arthritis
Paw volume: The percentage of inhibition expressed that of PL extract (100 mg kg‾¹ mild and 250, 500 mg kg‾¹ moderate activity) and standard (100 mg kg‾¹ significant) b.wt., p.o reduced paw swelling induced by CFA when compared to the control group from days 5-21 (Table 4).

Primary and secondary lesions: Polyalthia longifolia extract at 250 and 500 mg kg‾¹ b.wt., p.o and standard (100 mg kg‾¹) b.wt., gained body weights as compared to control group (Table 5).

Arthritic index: The PL extract showed good arthritic index as compared to control arthritic group at 250 and 500 mg kg‾¹ b.wt. (Table 5).

Biochemical parameters: PL extract at 500 mg kg‾¹ b.wt., significantly enhanced the levels of RBCs, WBCs and Hb as compared to the control arthritic group which showed decreased levels (Table 6).

Histological determination: Histological studies on transverse section of knee joint reports showed severe bone erosion with inflammatory infiltration in control group whereas PL extract (100 mg kg‾¹ showed the synovial tissue consists of focal loss of continuity in the synovial cells with inflammatory infiltration (Long-Arrow) with vascular spaces (Short-arrow), 250 mg kg‾¹ section studied shows bone trabeculae covered with intact articular hyaline cartilage (Long-arrow) and adjacent synovial tissue (Short-arrow), 500 mg kg‾¹ depicted that the synovial tissue consists of focal loss of continuity in the synovial cells with inflammatory infiltration (Long-arrow) and edema separating the fibrous tissue with vascular spaces (Short-arrow) in dose dependent manner and the standard showed section of the synovial tissue consists of intact synovial membrane with proliferation of synovial cells (long-arrow) and outer layer shows increased fibrous tissue (short-arrow) with vascular spaces and scattered inflammatory cells (Fig. 1a-e).

Table 2:	In vivo assessment of immunomodulatory activity using HA titre and DTH
Values are expressed as Mean±SEM (n = 6), *p<0.05, **p<0.01, ***p<0.001 when compared with control group. One-way ANOVA followed by Dunnett test

Table 3:	Phagocytic index (PI)
Values are expressed as Mean±SEM (n = 6), *p<0.05, **p<0.01, ***p<0.001 when compared with control group using one-way ANOVA followed by Dunnett test

Fig. 1(a-e):	TS of knee joint, (a) Control, (b) PL-100, (c) PL-250, (d) PL-500 and (e) STD

Table 4:	Mean changes in paw volume using plethysmometer in adjuvant induced arthritis
Values are expressed in Mean±SEM (n = 6), when compared with control, *p<0.05, **p<0.01, ***p<0.001, One-way ANOVA followed by Dunnett test.() means % inhibition

Table 5:	Changes in body weight in adjuvant induced arthritis
Values are expressed in Mean±SEM (n = 6), when compared with control, *p<0.05, **p<0.01, ***p<0.001, One-way ANOVA followed by Dunnett test

Table 6:	Effect of hematological parameters
Values are expressed in Mean±SEM (n = 6), when compared with control, *p<0.05, **p<0.01, ***p<0.001, One-way ANOVA followed by Dunnett test

DISCUSSION

Leukocytes regarded as guard cells consists of two major divisional sections viz., granular and non-granular cells. The former encompasses of neutrophils, basophiles and eosinophiles responsible for macrophage phagocytic mechanism whereas, later includes lymphocytes (B-cells-Bone marrow and T-cells-Thymus) and monocyctes participate in specific and nonspecific responses. They are found in higher concentration in lymph nodes, spleen, bone marrow and thymus (Barar, 2009; Krensky et al., 2005; Pelczar, 1993). In present immunomodulatory studies, rats were selected which contains Ox49 antigen equivalent to functionally important protein as CD₂ in SRBC’s receptor to which these antigens bind in an antigen-specific manner, catalyze cell activation and finally on incubation with SRBC’s lead to rosettes formation (Bader, 1997).

Polyalthia longifolia Thwaites (Mast tree) cultivated as ornamental and noise pollution prevention tree in urban cities by residents and equally known for its vast pharmacological significance with many constituents isolated as cited in literature by many researchers guided us to select these easily available local tree for immuno studies which could serve as herbal remedy to various immune system related disorders either stimulation or suppression (Warrier et al., 1994; Wagner, 1984). The present results reveal that Polyalthia longifolia can act as potent immunostimulator acting on T and B cells by specific and nonspecific immune mechanisms in the host.

In humoral immunity, B-cells on their cell surface receptors stimulate cells with antigen specific antibodies. Short lived plasma cells recognize circulating soluble antigens in the host and under the chemical influence of T-cells secrete antibody molecules which are a product of B-lymphocytes and plasma cells. IgG and IgM are the major immunoglobulins which are involved in complement activation, opsonization, neutralization of toxins, etc. against the antigens (Miller and Peacock, 1991). In addition, long lived memory cells respond to the antigen if encountered subsequently. In case of ethanolic extract of PL leaves (Table 5) high values indicated by haemagglutination antibody titre immuno model suggested enhanced responsiveness of T and B lymphocyte subsets involved in the antibody synthesis (Benacerraf, 1978) and thereby stating the probable role of PL extract as an immunostimulator.

In case of Cell Mediated Immunity (CMI), T-lymphocytes with T specific-cell receptor (TcR) with antigen-specific molecules on their surface similar to antibodies recognize antigens when they are presented by another cell in combination with proteins of Major Histocompatibility Complex (MHC). By functioning as molecular markers they discriminate between cells that are self and nonself. CD₄ and CD₈ lymphocytes are the two major subpopulations of T-cells based on their functions and surface proteins. T-lymphocytes and their products (lymphokines) serve to carry out defense mechanisms against infectious organisms, infectious foreign grafts, tumor immunity and delayed-type hypersensitivity reactions involving effectors mechanisms due to release of mediator such as histamine (Miller and Peacock, 1991). Diagnosis of vast number of cell mediated hypersensitivities and auto-immune diseases rely on T-cell population scrutinization. Therefore, in PL extract the reaction was notified by elevation in response to T-cell dependent antigen supported the same underlying role of the PL extract as in humoral immunity related to B-cells.

Phagocytosis is a process in which phagocytes ingests and removes micro-organisms, malignant cells, inorganic particles and tissue debris. The concept of swift clearance of Indian ink has been correlated with the enhanced phagocytic activity (Miller and Peacock, 1991) further suggested by linear growth in the statistical data which interpreted that PL extract is supporting B and T-cells concepts of the immunity with more focus on macrophage activity.

Patients on steroid therapy suffer from associated side effects and hormonal imbalance due to synthetic nature of the compounds. Hence, researchers urge for plant derived drugs in order to reduce these complications. In CFA induced arthritic model, since paw swelling is one of the major factor, it has been used to assess the degree of inflammation and therapeutic efficacy (Krensky et al., 2005; Vogel, 2002; Habila et al., 2010) of the PL extract and standard groups at different time intervals and dose levels with respect to control group. Animals under the experimental study conditions developed severe inflammation and typically, hind limbs became intensely red and oedematous within 16 to 24 h time course with peak severity of arthritic condition within 3-4 day after the onset of disease. PL extract demonstrated moderate (100 mg kg‾¹ Day 9-21) and significant (250 and 500 mg kg‾¹ Day 5-21) inhibition in paw volume on respective days in comparison to the control. The mechanism suggests probable suppression of T cell-mediated immune functions involving the release of inflammatory mediators due to induction of CFA during the phase of primary lesions. Increase in body weight on 21st day and inhibition of secondary lesions suggested significant immunomodulatory activity of the extract and standard groups. The condition of arthritis was severe in the control whereas the extract at various dose levels and standard groups gained b.w. Mean arthritic index results suggested good role of the selected part of the plant in arthritic condition. In arthritic control patients, there is a mild to moderate increase in the WBC count due to a release of IL-IB which increases the production of both granulocyte and macrophage colony stimulating factors (Parasuraman et al., 2010; Kumar et al., 2008). Researchers over the years have drawn common conclusion that migration of leukocytes produces a significant reduction in the WBC count. The model depicts the involvement of pathway in which prostaglandins, cyclooxygenase products and various cells are involved in significant inflammatory changes (Parasuraman et al., 2010). Hence, our research studies reveal that the arthritic rats exhibited decreased RBC, WBC counts and Hb levels correlated to indicate an anaemic condition, which is a common diagnostic feature in patients with chronic arthritis (Tiwari et al., 2004; Sharma and Kumar, 2009). The treatment with the extract and standard improved the RBC, WBC counts and Hb levels closer to normal suggesting significant recovery from anaemic condition and arthritic progress. Improved knee joint results indicated that the extract has dose dependent response in arthritic condition as compared to 100 mg kg‾¹ of standard.

CONCLUSION

The present investigation suggests that ethanolic extract of Polyalthia longifolia Thwaites leaves exert immunostimulatory effect on B and T lymphocyctes. Further studies on the constituents identification as well as different immunomodulatory models on the selected plant extract could be explored.

ACKNOWLEDGMENT

We would like to acknowledge the college Management and Principal, Dr. Supriya Shidhaye who provided us all the facilities to screen the plant for immunomodulatory activity and Radiant Research Services for all the pathological examination.