ASSESSMENT OF IMMUNOMODULATORY POTENTIAL OF AN AYURVEDIC FORMULATION, NIROCIL SYRUP IN WISTAR RATS

Objective: This study aims to assess the immunomodulatory potential of an Ayurvedic formulation, Nirocil syrup, in Wistar rats. Methods: The experiments were conducted on Wistar rats with prior approval from the Institutional Animal Ethics Committee. Nirocil syrup was administered for 6 weeks to experimental animals. Parameters such as hemagglutination titer, histopathology of immunological organs, complete blood count, differential leukocyte count, and immunological paw edema were recorded and compared with controlled (untreated) and becozinc treated groups. Results: Nirocil treated group significantly enhanced the antibody titer in comparison to the control group. The results are supported by the increase in blood lymphocyte count and antigenic stimulation in immunological organs (spleen). Nirocil syrup enhanced antibody formation and suppressed the immunological edema in experimental animals. Conclusions: The study concludes that the Ayurvedic formulation Nirocil syrup has immunopotentiating activity.


INTRODUCTION
Ayurveda is a pleasing dream to the traditional system of medicine, which gives a deep perception of healthy life and longevity. The term Ayurveda can be simplified as Ayu (life) and Veda (knowledge), the science of life, and knowledge [1]. The basic principle of Ayurveda is to increase the natural resistance of the body against invading agents rather than directly acting against invasions. In the Ayurvedic system of medicines, herbs are commonly used to improve the overall resistance of the body against common infections and pathogens with immunomodulatory effects [2]. The mechanism of immunomodulation is to adjust the host immune system either by immunostimulation or immunosuppression. Hence, immunomodulators are recognized as biological response modifiers, which boost the defense mechanism of the host [3,4].
This research work is an effort in the direction of analyzing the cumulative impact of different herbal components in Nirocil syrup on the immune system of Wistar rats and to revive the glitter of the ancient traditional system of medicine.

Chemicals and medicine
Nirocil syrup was obtained from Solumiks Herbaceuticals Ltd., becozinc syrup was procured from the local market (manufactured by Dr. Reddy Laboratory). Diphtheria, tetanus, and pertussis (DPT) vaccines (manufactured by Indian Immunological) were procured from the local market. Di sodium hydrogen phosphate, sodium dihydrogen phosphate, formalin, sodium acetate, sodium chloride, and sodium citrate were procured from sigma chemicals (USA).

Animals
Twenty four adult Wistar female albino rats weighing 150±15 g. were selected for the study. The animals were housed in Committee for Purpose of Control Supervision and Experimentation on Animals (CPCSEA) approved animal house facility of Shree Dhootapapeshwar Ltd., Panvel Maharashtra (MH). The animals were maintained at 22±03°C with constant humidity (50-70%) and 12 h day and night cycle. Animals were fed with Amrut brand rat pellet feed and water ad libitum. The experiments were carried out by prior approval from Institutional Animal Ethics Committee (Protocol No. SDARF/CT/2018/01).

Dose selection and schedule
The dose for experimental animals was deduced from the human dose and the drug solutions were administered using a gastric catheter affixed to a syringe [5].

Nirocil syrup
The human therapeutic dose of Nirocil syrup was determined as 10 ml thrice a day, which was equivalent to 2.7 ml/kg body weight/day for rats.

Becozinc syrup
The human therapeutic dose for becozinc syrup was determined as 5 ml a day, which was equivalent to 0.45 ml/kg body weight/day for rats.

Grouping of animals
The animals were divided into three groups having six animals each. Separate animals were utilized for humoral immunity and cell-mediated immunity study as mentioned below;

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Group 1 -Received sterile water and served as control. Group 2 -Received Nirocil syrup for 6 weeks. Group 3 -Received Becozinc syrup for 6 weeks and served as a positive control.

Effects on humoral antibody formation
The effect of Nirocil syrup on antibody formation against sheep red blood cells (SRBC) in Wistar female albino rats was studied as described by Puri et al. [6]. For antibody formation against SRBC in test animals, sheep blood was collected from a local slaughterhouse in a sterilized bottle. The collected blood was thoroughly washed with sterile saline through repeated centrifugation and made to 30% SRBC solution. This 30% SRBC solution was used as a sensitizing agent and made to 3% and injected subcutaneously in a dose of 0.5 ml/100 g of body weight to the test animals on the 35 th day of experimentation. On the 42 nd day, the blood sample was collected from test animals in a sterile tube. The serum was separated and inactivated by incubation at 56°C for 30 min in a serological water bath. Further, an antibody titer test was performed to determine the presence and the number of antibodies formed in test animals.

Estimation of antibody titer
For the estimation of antibody titer, the wells of microtiter plate were loaded with 0.1 ml sterile normal saline and 0.1 ml of two-fold serially diluted serum. The dilutions were made in the range of 1:2, 1:4, and 1:8 up to 1:1024. A 0.1 ml of SRBC (3%) was added to each well. The blood from the same animal (sheep) was used for sensitization and determination of antibody titer. The microtiter plate was shielded and then incubated overnight at 4°C in a refrigerator. Next day antibody titer (hemagglutination titer) was noted and converted to log2 values for comparison [7].

Hematology and histopathology
To determine the immunomodulatory effects of Nirocil syrup on experimental animals, hematological analysis was carried out. Blood samples were collected from all the surviving animals a day before initiation of dosing (basal), on completion of 5 th week (interim), and finally on 9 th week (terminal). Approximately 0.5 ml of blood sample was collected from each experimental animal by puncturing the orbital plexus with the help of a fine glass capillary tube under the influence of light isoflurane (anesthetic). The blood samples were stored in sterile vials containing 4% EDTA.
After 6 weeks, all the surviving animals belonging to different groups were euthanized by using carbon dioxide asphyxiation. Physical examinations of animals were carried out externally.
Following this, animals were dissected and gross examination of all the internal organs was carried out. Spleen tissue of all the animals was collected and processed for histopathological (microscopic) observations.

Effect on cell-mediated immunity
The effect of Nirocil syrup on cell-mediated immunity in Wistar albino rats was evaluated by following the procedure of Bhattacharya [8]. Animals were divided in three groups as previously described. All the animals were sensitized subcutaneously (0.5 ml/100 g body weight) by 1 ml triple antigen solution (DPT), 4 ml normal saline (0.9%), and 1 ml potash alum (10%) on 35 th day of drug administration. The pH of the solution was adjusted 5.6-6.8 by 10% sodium carbonate. The drug dosing was continued for next 7 days. On the 42 nd day, 0.1 ml of triple antigen solution was injected into plantar aponeurosis of paw after measuring of initial volume of the left hind paw. Triple antigen produced the immunological edema in rat hind paw and the paw volume of edema was measured by volume displacement method [9] at 24 h and 48 h of injection. The percentage increase in paw volume, over initial paw volume, was calculated. Cell-mediated immune response was analyzed by comparing the values of the control group versus the test drug administered group.

Effects on body weight
The effect of Nirocil syrup on the body weight of experimental animals was also studied. The bodyweight of experimental animals of different groups was recorded on every 7 th day till the completion of the study.

Statistical analysis
Results were presented as mean±SEM, difference between the groups was statistically determined by one-way ANOVA with post hoc Tukey HSD test with the level of significance set at p<0.05. The significance of level was noted and interpreted accordingly.

Body weight
The effect of Nirocil syrup on the body weight of experimental animals of different groups is presented in Table 1. On comparison to the control group of animals, there was a significant increase (p<0.01) in body weight in Nirocil treated group and becozinc treated group.

Hematology
The results of hematological analysis of all the experimental groups at different time intervals are presented in Table 2A-C. A slight increase in total WBC count (10.33-11.38/µl) and absolute lymphocyte count (8586.30±340.00 cells/µl) was observed in Nirocil treated groups after 5 weeks in comparison to 0 days (7884.30±198.04). However, no major changes were observed in other hematological parameters. A significant increase in total WBC count and absolute lymphocyte count (12630.3±488 cells/µl) was observed only after 9 weeks in Nirocil treated groups, compared to the control group. Whereas, the becozinc treated group showed similar results (12234.5±550.0 cells/µl) (Table 2C), suggesting increased immune response in both Nirocil and becozinc treated groups.

Humoral antibody formation
The effect of Nirocil on antibody titer formation against SRBC in Wistar female albino rats is presented in Table 3. Nirocil treated groups showed a significant (**p<0.01) increase in antibody titer formation in comparison to the control group. The increase of antibody titer formation in the Nirocil treated group was observed 48.22%, which was comparable to the titer formation in the becozinc treated group (48.21%). On comparing the titer formation between the groups (Nirocil vs. Becozinc) the result was not significant at p<0.05, as both the groups showed a similar increase in titer formation.
Lymphocytes are the cells involved in adaptive immunity and subdivided into B, T, and NK lymphocytes [10]. Leukocyte count analysis showed a significant increase (*p<0.05), specifically total lymphocytes count in Nirocil and becozinc treated groups. This suggests the increased immune stimulation in the treated group of animals in comparison to the control group. These results are in agreement with the findings of Ketema et al. [11] indicated a synergism between increased lymphocyte counts and elevated immune response.

Histopathology
The microscopic examination of the spleen tissue of the control group of animals did not show any lesion of pathological significance (Fig. 1a).

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While the spleen tissue of Nirocil treated, group showed various lesions of multifocal mild to the moderate category in four out of six animals (Fig. 1b). Whereas, the spleen tissue of the becozinc treated control group showed multifocal moderate extramedullary hematopoiesis in two out of six animals (Fig. 1c). The presence of splenic extramedullary hematopoiesis suggested the antigenic stimulation in treated groups as it is reported for the production of antigen-presenting cells and phagocytes in the spleen [12][13][14][15].

Effect on cell-mediated immunity
The effect of Nirocil syrup on immunological paw edema in triple antigen sensitized rats is shown in Table 4. A gradual increase in paw volume was observed in all the groups starting from 0 h to 48 h. After 48 h, the paw volume was found significantly lower in Nirocil treated group (1.508 ml) and becozinc (1.41 ml) treated group in comparison to the control group (1.673 ml). The results indicate that Nirocil is involved in lowering edema/inflammation induced by cell-mediated immune response.