Evaluation of immunomodulatory activity of "Shirishavaleha"-An Ayurvedic compound formulation in albino rats.

The immunomodulatory activity of Shirishavaleha prepared from two different parts of Shirisha (Albizia lebbeck Benth), i.e., Twak (Bark) and Sara (Heartwood) as main ingredients was evaluated for humoral antibody formation and cell-mediated immunity in established experimental models. The study used Wistar rats of either sex weighing 200 ± 40 g, while the test drug was administered orally at a dose of 1.8 g/kg. Hemagglutination titer and body weight were recorded to assess effects on humoral immunity; immunological paw edema was assessed for cell-mediated immunity. Shirishavaleha prepared from heartwood shows significant enhancement in antibody formation, attenuation of body weight changes, and suppression of immunological paw edema, while Shirishavaleha prepared from bark shows weak immunomodulatory activity. The study therefore concludes that Shirishavaleha prepared from heartwood has significant immunomodulatory activity.

INTRODUCTION

Traditional and folklore medicines continue to play important roles in health services around the globe.[1] Ayurveda, the traditional medical system of India, describes thousands of herbal preparations. One, “Shirisharishta”, a well-known, fermented preparation of Albizia lebbeck Benth., is a combination of 12 ingredients, with jaggery as its base, and used for the treatment of various disorders[2]. The decoction of Shirisha bark has a protective action against bronchial asthma.[3] Recent studies found the plant to possess immunomodulatory[4] and antihistaminic activity[5] . Its extract has also been proven to be efficacious in cases of allergic rhinitis.[6] Studies on other ingredients of the formulation taken individually, have also found immunomodulatory activity: Pippali (Piper longum Linn.),[7] Haridra (Curcuma longa Linn.),[8] Kusta (Saussurea lappa C. B. Clarke)[9] Shunthi (Zingiber officinale Roscoe.),[10] and Ela (Elettaria cardamomum Maton.)[11].

However, being fermented, “Shirisharishta” may be considered inconvenient in a few ways: prolonged duration of preparation; decreased palatability for various age groups, etc. For this reason, we converted the fermented form (Arishta) to a confection (Avaleha) as instructed according to the classical texts[12]. Antitussive activity ofthe latter was evaluated.[13]

The aim of the current study was to evaluate the immunomodulatory activity of two forms of “Shirishavaleha,” viz., one obtained from the bark (SAB) and the other from the heartwood (SAH).

MATERIALS AND METHODS

Test formulations

Raw materials [Table 1] for the test formulation were procured from the pharmacy at the Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurveda University, after authentication by the pharmacognosist. Two samples of Shirishavaleha were prepared from these materials, viz. one using Shirisha bark as the main ingredient and another using heartwood as the main ingredient. The Avaleha formulation was prepared by following classical guidelines[12].

Table 1

Formulation composition of Shirishavaleha

Animals

The study employed Wistar rats of either sex weighing 200 ± 40 g, obtained from the animal house attached to the Institute's pharmacology laboratory. They were housed in spacious polypropylene cages and fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries, and tap water given ad libitum. The animals were acclimatized for at least one week in laboratory conditions before the commencement of experiment in standard laboratory conditions, 12 ± 01 hour day and night rhythm, maintained at 25 ± 3°C and 40 to 60% humidity. The Institutional Animal Ethics Committee (IAEC) approved the experimental protocol (IAEC 09-10/05MD 07), and care of the animals was conducted according the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals.

Dose fixation

Doses of the test formulations (both SAB and SAH) were calculated by extrapolating the human dose to animals (1.8 g/kg), based on the body surface area ratio referring to the standard table of Paget and Barnes (1969).[14] The test formulation was suspended in distilled water (180 mg/ml) and administered orally at a volume of 0.5 ml/100 g body weight with the help of gastric catheter of suitable size sleeved on to a syringe nozzle to overnight fasted animals.

Effect on humoral antibody formation

The effect of the test drugs on antibody formation against sheep red blood cells (SRBC) was measured as described by Doherty.[15] The selected animals were divided into four groups of six. The first group received distilled water, and served as the control group; the second group also received distilled water and served as SRBC control group. Calculated doses of the two samples of Shirishavaleha (SAB and SAH) were administered for 10 consecutive days to the third and fourth groups, respectively. On the third day, sheep blood was collected from the slaughter house in a sterilized bottle containing Alsever's solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid, and 0.42% sodium chloride) aseptically so that agglutination of blood does not take place. The collected sheep blood was thoroughly washed with sterile normal saline through repeated centrifugation until the supernatant fluid became colorless, and was made to 30% SRBC solution. This sensitizing agent was injected subcutaneously in the dose of 0.5 ml/100 g of body weight to the rats of second to fourth groups. On the 11th day, the animals were weighed again and under light ether anesthesia, blood was drawn by puncturing supraorbital plexus and collected in plain tubes. Serum was separated from it and complements were inactivated by incubating it for 30 minutes at 56°C temperature in a serological water bath.

Estimation of antibody titer[16]

The microtiter plate was filled with 0.1 ml sterile normal saline, and up to 16 serial two-fold dilutions of 0.1 ml serum in sterile saline solution were made in the microtiter plate. 0.1 ml of thrice saline-washed 3% SRBC was added to each well of the tray. Blood from the same animal (sheep) was used for both sensitization and to determine antibody titer. The trays were covered and placed in a refrigerator overnight. Antibody titer (hemagglutination titer) was noted the next day. The titer was converted to log2 values for easy between groups comparison .

Effect on cell-mediated immunity

Effect on cell-mediated immunity was evaluated using the procedure of Bhattacharya.[17] The selected animals were divided into three groups of six animals in each group. First group received distilled water and served as normal control group. To the second and third groups, calculated doses of SAB and SAH were administered. All the animals were sensitized subcutaneously (0.5 ml/100 g body weight) on the first day of drug administration using the following solution: triple antigen (DPT) - 1 ml, normal saline (0.9%) - 4 ml, and potash alum (10%) - 1 ml. The solution pH was maintained between 5.6 and 6.8 using 10% sodium carbonate. The drug was administered for seven consecutive days. On the seventh day, one hour after drug administration, the initial volume of the left hind paw was noted, and 0.1 ml of the above solution was injected into plantar aponeurosis of same paw. The volume of immunological edema thus produced was measured by the volume displacement method[18] 24 and 48 hours after the injection, using a plethysmograph. From this the percentage increase in paw volume, the index of edema formation, was calculated.

Statistical analysis

Results were presented as Mean ± SEM, statistical significance of between groups difference was determined using unpaired Student's ‘t’ tests.[19]

RESULTS

A normal range of body weight increase was observed in control group rats. In contrast, an apparent decrease in body weight was observed in SRBC control rats. Pretreatment with SAB and SAH significantly attenuated body weight changes compared to the SRBC control group [Table 2].

Table 2

Effect on body weight

Pretreatment with SAH significantly (P<0.05) enhanced antibody titer [Table 3] in SRBC presensitized rats in comparison with the SRBC control group. Treatment with SAB also enhanced antibody titer; however, the observed increase did not reach statistical significance.

Table 3

Effect on antibody formation

In the SAB group, a decrease in paw volume was observed for both the 24- and 48-hour recordings, but did not reach statistical significance. In the SAH group, a statistically significant decrease in immunological edema was observed both after 24- and 48-hours [Table 4].

Table 4

Effect on immunological paw edema

DISCUSSION

Gain in body weight indicates normal progressive health status of an organism while decrease is indicative of degenerative changes in the body or certain organs. In the present study, presensitization of animals with SRBC led to significant decreases in body weight in 10 days. Both SAB and SAH formulations significantly attenuated SRBC-induced body weight changes. The observed effects may be attributed to prevention of SRBC- (antigen) induced degenerative changes in the body.

Hemagglutination antibody titer is a primary parameter for studying the humoral response. Antigen and antibody reaction results in agglutination. Antibody molecules secreted by plasma cells mediate the humoral immune response. SAH showed significant increase in hemagglutination titer, indicating immunostimulant activity. Such stimulation of humoral response to SRBC indicates enhanced responsiveness of macrophages, T and B lymphocyte subsets, involved in antibody synthesis.[20] SRBC may therefore stimulate lymphocyte proliferation, in turn leading to production of cytokines activating other immune cells such as B cells.

Cell-mediated immunity (CMI) involves effector mechanisms carried out by T lymphocytes and their products (lymphokines). The Th-1 T-lymphocyte pathway controls cell-mediated immunity.[21] Results show that SAH pretreatment significantly suppresses immunological edema suggesting that it may suppress that pathway. At the same time it seems to influence the Th-2 pathway, as indicated by enhanced antibody formation. Such effects could be produced by promoting formation and release of IL-4, while at the same time, decreasing formation of IL-12, the cytokine favoring Th-1 mediated immune response. Exact mechanism can only be elucidated by specific studies measuring different types of cytokines both in vivo and in vitro.

Immunomodulatory activity of the compound formulation may be understood using the concept of Rasayana. This indicates that components of the formulation help promote nutrition at Rasa, Agni, and Srotas levels, by the virtue of which immunomodulatory effects become possible.

In earlier research, Visha is understood as free radicals or antigens. The Prabhava of Shirisha, the main component of the formulation, is Vishahara (antagonistic to Visha). Other ingredients also possess the Vishahara property eg Naga kesara, Haridra, and Nilini. By the virtue of this, immunomodulatory activity could probably be anticipated. The components, Pippali, Sunthi, Priyangu, Kusta, Daru haridra, and Naga kesara, possess the properties Dipana and Pachana, which are known to promote digestion and revitalize metabolic activity resulting in improved nutritional status at the Dhatu level, and preventing further formation of Ama in the body. Guda (jaggery), a major ingredient of the formulation, contains bulk quantities of carbohydrates and other minerals, which, when administered, directly enter into the pool of nutrition. This in turn helps improve tissue nourishment leading to subsequent Rasayana effects. These reasons may help explain why, in the presence of jaggery, the polyherbal combination possesses immunomodulatory activity.

CONCLUSION

From the present study, it is observed that both the test formulations possess immunomodulatory activity, that of SAH being more significant. Overall results suggest that both parts of Shirisha (bark and heartwood) can be used in the formulation. Indeed, since bark collection does not involve destructive collection practices, it should generally be used. However, where heartwood is abundantly available, it may be preferred. Even then a mixture of both would be useful.