Immunomodulatory Activity of Imanol Tablets

Vador N, Vador B and Jagtap A

Published on: 2020-05-30

Abstract

Various chemical agents have been used to modulate immune system. Ayurvedic medicines constitute a rich source of active substances. Various plants have been shown to modulate immune system either by stimulating specific or nonspecific immunity. Hence combination of various plants would therefore have diverse actions on various aspects of the immune system [1]. Earlier study shown that Immunol Tablet Simulated Phagocytic and Haemtopoietic activity. Hence current study was designed to investigate the immunomodulatory activity of an Ayurvedic formulation, Immunol Tablets. Effect of Immunol Tablet was evaluated on hematological profile in normal and myelosuppressed mice, humoral and cell mediated responses and prophylactic activity in E.coli induced peritonitis. Results were analyzed by students‘t’-test at p<0.05. It produces significant leucocytosis with predominant neutrophilia and prevented myelosuppressive activity of cyclophosphamide. Immunol at the dose of 750 mg/kg produced significant increase in antibody titre and potentiated DTH reaction against sheep erythrocytes. Immunol at the dose of 750 mg/kg showed 85.72 % protection against E.coli induced peritonitis. Immunol tablet enhanced B and T cell proliferation as well as protected mice from E. coli induced abdominal peritonitis.

Keywords

Immunity; Immunol; Antibody; Ayurved

Introduction

Charaka or Sushruta suggested that various properties of plants such as jeevaniya, balya, vayasthapaniya or rasayana might have immunological effects. Jeevaniya means life promoters, balya means strengtheners and vayasthapaniya means increase life span [2]. The term rasayana includes all these activities. Rasayana is one of the therapeutic strategies in ayurvedic medicines which increase the body’s own resistance power to the disease causing agents. Different agents of plant origins are reported to interact with immune system in a complex way and modulate the pathophysiological process [3].

Immunol Tablet an Ayurvedic formulation has following composition. Each Tablet contains aqueous extract derived from the following Amrita (Tinospora cordifolia), Gokhru (Tribularis terristris), Amala (Emblica officinalis), Vidang (Embelli ribes), Tulsi (Ocimum sanctum), Shatavari (Asparagus racemosus), Katuki (Picrorrhiza kurroa), Ashwaghandha (Withania somnifera), Trikatu (Piper longum, Piper nigrum, Zingiber officinalis). All these plants are used ethnomedically and have been proven to have immunomodulatory activity [4-7]. But the combinations of these plants have not been studied earlier. Hence it was decided to evaluate Immunol for its immunomodulatory activity.

Materials

List of chemicals, drugs and solvents

Cyclophosphamide injection Ledoxan, Dabur Pharmaceuticals, Carbon ink Rotring, black, Germany, ERBA haemolyses Transasia, ERBA diluent Transasia, MacConkeys agar, Hi- media laboratories, SRBC were brought from Bombay Veterinary College, Parel in Alsevers solution and stored in the same. Immunol Tablet was provided as a gift sample by Ayurchem Products, Mumbai.

Animals

Mice

Healthy Swiss albino mice of either sex were housed in the animal house of Bombay College of pharmacy were used. Healthy female Balb/c mice were brought from Glen mark laboratories, Mumbai and housed in the animal house of Bombay College of Pharmacy were used.

Animal feed

Animals were fed with commercially available Am rut rat and mice feed, manufactured by Nav Maharashtra Chaka Oil Mill Ltd, Pune. The animal feed contained crude protein, crude fiber, and crude oil. Animal housing: animals were maintained under standard conditions of temperature (25 C±5 C) and relative humidity (55± 10%), and 12h/12h light /dark cycle. They were housed in standard polypropylene cages with wire mesh top and husk bedding. The research project was approved by institutional animal ethics committee vide 242/PO/RE/S/2000/CPCSEA; 01/08/2000 vide protocol approval number as CPCSEA-BCP-/206/ 2002.

Experimental Models

Selection of the dose of Immunol

The dose of Immunol was calculated from the human dose. The human dose was 2.3 grams i.e. 4 Immunol tablets. This dose was for a 60 kg individual and hence considering the conversion factor 12.3 for a mouse, the dose calculated was 500 mg/kg. Preliminary studies were carried using 500 mg/kg. This dose was found to show activity and hence this dose was selected for the rest of the study. Studies of Immunol with 500 mg/kg did not show significant effect on humoral, cell mediated responses and E.coli peritonitis hence it was increased to 750 mg/kg.

Evaluation of immune potentiating effect of Immunol tablets Cyclophosphamide induced myelosuppression in mice [8]

Male Swiss albino mice were divided in to four groups of 5 mice each. Group I control (Distilled water). Group II Immunol treated (500 mg/kg for 11 days). Group III cyclophosphamide treated (200 mg/kg on 11th day of vehicle treatment). Group IV Immunol treated (500 mg/kg) + cyclophosphamide (200 mg/kg) on 11th day, Group III & IV received a single dose of cyclophosphamide (200 mg/kg). On 12th day, blood was collected from retro-orbital plexuses of the individual mice from all the groups. RBC, Total WBC, % Neutrophil count was determined. Total cell values were determined using Erma PC-607 cell counter. Dry smear of the blood on the slides were stained using Field A and Field B stain for % Neutrophil count of each animal using compound microscope [9].

Humoral response (Antibody Production) in normal and cyclophosphamide treated mice [10]

Female Swiss albino mice were divided in to 4 groups of 5 mice each. Group I control group (distilled water). Group II Immunol treated (750mg/kg for 1-5 days). Group III Cyclophosphamide treated 25 mg/kg i.p. 2 hours prior to sensitization with sheep erythrocytes. Group IV Immunol treated (750mg/kg for 1-5 days) + Cyclophosphamide 25 mg/kg i.p. 2 hours prior to sensitization with sheep erythrocytes Sensitization: All the mice were primed with 2 x 108 cells of SRBC on day 0. On 4th day blood was withdrawn from animal by retro-orbital plexus, serum was separated. 25 μl of two fold-diluted serum was challenged with 25 μl of 0.1% v/v of SRBC suspension in titre plates. The plates were incubated at 37  C for 1 hour and then observed for haemagglutination. The highest dilution was taken as antibody titre. The antibody titres were expressed in a graded manner, the minimum dilution to be taken rank 1.

Delayed type hypersensitivity in mice [10]

Animals from humoral responses were continued for the Delayed type hypersensitivity reaction. Sensitization and challenge: Delayed hypersensitivity was induced in mice using sheep red blood cells (SRBC) as antigen in Alseviers solution. Animals were primed with 2 x 108 SRBC (day 0) and challenged on day 5 with 2 x 108 SRBC subcutaneously in the hind footpad. The right hind paw received saline alone. Paw thickness measurement were made with Mitutoyo dial caliper at 20, 22, 24, 26, 28, 48, 72 and 96 hours after challenge. The results were expressed as the percentage increase in hind paw volume as compared to the initial hind paw volume and % edema was calculated.

Evaluation of Immunoprophylactic effect of Immunol: E.coli induced abdominal peritonitis [11]

Female Balb/c mice were divided into 3 groups of 7 mice each. Group I control group (distilled water). Group II Immunol treated (750 mg/kg for 15 days). Group III Positive control (plain nutrient broth i/p). E.coli induced abdominal peritonitis was carried out in two parts. In the first part the strength of E.coli was standardized to induce 100% mortality. In the second part the effect of E.coli injection in mice and protection by the drug was evaluated. On day 15th, 3 hours after the last dose of Immunol, E.coli (1x 10 8 cells) was given intraperitoneally to all the groups of mice and percentage mortality was observed from 16-19 hours as that was the expected time of mortality.

Evaluation: % protection of Immunol treated group with respect to control group was calculated.

Results

Evaluation of immune potentiating effect of Immunol Cyclophosphamide induced myelo suppression in mice

Immunol treated group showed significant (P< 0.05) increase in total WBC and % Neutrophil count as compared to control animals, CYP treated group and (CYP+ Immunol) treated group (Table no: 1). Cyclophosphamide (200mg/kg) showed significant (P< 0.05) decrease in the total RBC and WBC count as compared to control and immunol treated group. Significant decrease in % Neutrophil count was observed as compared to immunol treated group and (CYP+ Immunol) treated group Table 1. When Cyclophosphamide (200 mg/kg) was given along with Immunol (500 mg/kg), significant (P< 0.05) increase in total WBC count and % Neutrophil as compared to CYP treated group (Table 1).

Results are expressed as the mean ± s.d. of 5 observations.
*: significant difference at p< 0.05 as compared to control group by student’s t-test.
ã: significant difference at p< 0.05 as compared to drug treated by students t-test.
?: significant difference at p< 0.05 as compared to CYP group by student’s t-test.

Table 1: Effect of Immunol Tablets on hematological profile in normal and myelosuppressed mice.

Treatment Groups

Dose(mg/kg)

RBC(million/mm3)

WBC(1000/mm3)

%Neutrophils

Control

-

7.73 ± 1.3

8.78 ± 0.4

22.53 ± 3.7

Immunol

500

8.18 ± 0.8

14.66 ± 1.5*

33.22 ± 3.3*

CYP

200

5.73 ± 0.3* ã

6 ± 0.7* ã

21.39 ± 1.58 € ¥

CYP+ Immunol

200, 500

7.96 ± 1.4 ?

9.02 ± 1.4 ã ?

38.58 ± 1.9*

Table 2: Effect of immunol on humoral responses in normal and cyclophosphamide treated mice.

Treatment Groups

Dose(mg/kg)

Mean antibody titre

Control

-

8.8 ± 0.72

Immunol

750

10.2 ± 0.32*

Cyclophosphamide

25

9.2  ± 0.64

Cyclophosphamide+ Immunol

25, 750

9.2 ± 0.64

Table 3: Effect of Immunol on mean % edema in normal and cyclophosphamide treated mice.

Treatment Groups

Dose(mg/kg)

Mean % edema

24 hrs

48 hrs.

72 hrs.

96 hrs.

Control

-

74.44 ± 10.00

55.73± 9.44

31.21 ± 4.06

9.89 ± 2.7

Immunol

750

89.74 ± 2.56 *

59.31± 3.21

34.34 ± 8.49

10.85 ± 2.30

Cyclophosphamide

25

109.71 ± 4.43 * ¥

58.88± 3.42

40.19 ± 6.79

20.85 ± 4.37

Cyclophosphamide+ Immunol

25, 750

96.44± 2.16 * ¥ ã

51.59± 4.89

38.86 ± 8.98

17.76 ± 4.03

Table 4: Effect of immunol on E.coli induced abdominal peritonitis in mice.

Treatment Groups

Dose(mg/kg)

Percentage mortality

Control

-

100

Positive control(plain broth)

-

0

Immunol

750

14.28

 

Humoral response (Antibody Production) in normal and cyclophosphamide treated mice

Studies of Immunol with 500 mg/kg did not showed significant effect on humoral and cell mediated responses hence it was decided to increase the dose to 750 mg/kg. From table no: 2 it can be seen that the antibody titre of Immunol treated animals is significantly (p < 0.05) more than control, cyclophosphamide and (Cyclophosphamide + Immunol) treated animals (Table 2).

Results are expressed as the mean ± s.d. of 5 observations.

*: significant difference at p <0.05 as compared to control, cyclophosphamide and (Cyclophosphamide + Immunol) by student’s t-test.

Delayed type hypersensitivity reaction in mice

The mean percent edema at 24 hrs for control animals was 74.44% and that of Immunol treated group 89.74% which is significantly (p< 0.05) higher than the control group (Table no: 3). Low dose of cyclophosphamide given prior to sensitization showed potentiation of DTH (Mean percent edema 109.71% at 24 hrs.) which is significantly (p< 0.01) high as compared to control and Immunol treated group (Table no: 3). (CYP + Immunol) treated animals, showed a significant (p<0.01) reduction in mean percent edema as compared to cyclophosphamide treatment alone. But the mean percent edema of (CYP + Immunol) treated group was significantly (p< 0.01) higher than control and Immunol treated animals (Table 3).

Results are expressed as the mean ± s.d. of 5 observations.

*: significant difference at p< 0.05 as compared to control by students t-test.

¥: significant difference at p< 0.01 as compared to immunol treatment by student’s t-test.

ã: significant difference at p< 0.01 as compared to CYP group by students t-test.

Evaluation of Immunoprophylactic effect of Immunol: E.coli induced abdominal peritonitis in mice

100% mortality observed in control animals. Immunol at the dose of 750 mg/kg/day orally for 15 days reduced percentage mortality to 14.28 % as compared to control animals (Table no: 4). Positive control i.e. only plain broth was given and there was no mortality observed. Thus any mortality observed was due to E. coli infection.

Results are expressed on the basis of 7 observations (Table 4).

Discussion

Results of earlier studies indicate that immunol had an immuno-stimulating effect in normal mice. Immunol stimulated monocyte-macrophage lineage as well as stimulated haematopoiesis. So the potentiating effect of immunol was evaluated against myelosuppression induced by cyclophosphamide in mice and against hypersensitivity induced by antigen. Bone marrow is a site of continued proliferation and turnover of blood cells and is a source of cells involved in immune reactivity. A high degree of cell proliferation renders bone marrow a sensitive target, particularly to cytotoxic drugs [12]. Cytotoxic drugs like cyclophosphamide and azathioprin act at various levels on cells involved in defence against foreign invaders. The suppression of bone marrow activity reflecting myelosuppression by cyclophosphamide was significant as can be seen by decrease in blood cell counts and % Neutrophils table no: 1. Combined treatment of myelosuppressive drug and Immunol resulted in a restoration of bone marrow activity as compared to cyclophosphamide alone. Immunol treated animals showed significant increase in bone marrow activity as compared to cyclophosphamide alone and even control animals. Hence Immunol might have prevented myelosuppressive activity of cyclophosphamide and could be used as an adjuvant in cancer therapy. In specific immunity macrophages regulate both humoral and cellular immune responses i.e. the regulation of B and T cells. As the results of our study showed stimulation of monocyte-macrophage lineage by immunol, it was decided to study the effect on humoral responses i.e. on B cells. Humoral responses were initiated by sensitizing and challenging mice with sheep erythrocytes as foreign antigens. Antibody titres were determined using haemagglutination method [13]. Studies of immunol with 500 mg/kg did not showed significant effect on humoral and cell mediated responses hence it was decided to increase the dose to 750 mg/kg. The haemagglutination antibody titre test is indicative of the degree of humoral responses. The humoral immunity involves interaction of B-cells with antigen and their subsequent proliferation and differentiation into antibody secreting plasma cells. The augmentation of the humoral response to sheep erythrocytes indicates the enhanced responsiveness of the macrophages and T and B lymphocyte subsets involved in the antibody synthesis [14]. Macrophages also play a pivotal role in coordinating the processing and presentation of antigen to T and B cells [15]. The present study showed stimulation of monocyte-macrophage lineage by Immunol and thus enhancement of humoral effect by facilitating such responses. Control of disease by immunologic means has two objectives: the development of immunity and the avoidance of undesired immune reactions. Modification of immune functions by pharmacological agents is emerging as a major area of therapeutics in those cases where undesired immunosuppression is the result of therapy. Such efforts were previously being carried out by using glucocorticoids in combination with cytotoxic drugs like cyclophosphamide. On other hand, undesired immunostimulation (i.e. hyper-reactivity) is a common side effect with drugs like quinine, salicylates, indomethacin, etc. Whether or not experimentally induced hyper reactivity is restored back to normal was checked by using immunol in the delayed type hypersensitivity animal models [16].                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             

Delayed type hypersensitivity (DTH) is a typical T-cell mediated immune response in the skin. DTH begins with the first exposure of specific antigen to macrophages, resulting in antigen specific CD4+ T-cell activation, expansion and differentiation into effector and memory T-cells. Upon second exposure of same antigen, the specific memory T-cells are stimulated to proliferate and to release lymphokines. The continual release of lymphokines from sensitized T-cells results in the accumulation of large number of activated macrophages that become epitheloid cells. Immunol showed a potentiating effect on DTH in normal mice whereas the potentiating effect on DTH of cyclophosphamide was reduced by Immunol. Cyclophosphamide at the dose of 25mg/kg showed maximum potentiation of DTH because cyclophosphamide damaged short lived suppressor T cells in immune regulatory system. This is also in accordance with earlier reports. Thus Immunol shows modulating effect on the immune system. It stimulates immune system in normal conditions whereas it suppresses immune system in hypersensitized conditions. Thus it can be concluded that Immunol stimulated cell mediated immunity, by having stimulating effect on T lymphocytes and accessory cell types required for the expression of the DTH reaction. The intestinal tract harbours a large number of bacteria which under normal condition are not able to invade the peritoneal cavity. However, if the defense barriers get broken down under diseased conditions or trauma, bacteria have an access into the peritoneal cavity and produce sepsis. Intraabdominal sepsis continues to be a major cause of morbidity and mortality following trauma and abdominal surgery for bowel perforations. Treatment of this condition has always been focused on appropriate surgery supplemented with antibiotics and good nutritional support. In spite of such therapy fatal complications often occur. Nowadays stimulating cellular immune function and increase in resistance to infection has been more emphasized.  Immunol has stimulated macrophage count and its phagocytic capability. It also produced significant leucocytosis along with predominant neutrophilia, which probably occurs due to secretion of IL-1 and GM-CSF from activated macrophages. Thus immunol showed stimulation of non-specific defense system which appears to be the underlying mechanism of protection against E. coli induced peritonitis [16].

Conclusion

From this study it can be concluded that Immunol Tablets have an immunomodulatory activity from the following effects:

  • Enhanced macrophages and its phagocytic capability.
  • Stimulated hematopoiesis and bone marrow.
  • Stimulated B cell proliferation.
  • Stimulated T cell and accessory cells types.
  • Enhanced responsiveness of macrophages and T and B lymphocytes subsets involved in antibody synthesis.
  • Can be used as an adjuvant in cancer chemotherapy.
  • Also as an adjuvant in vaccination.
  • Can be used with antibiotics for treating infections.

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