Immunomodulators: traditional approach

Dr Pramod Kumar Singh¹, Dr Poonam Sharma², Dr Abhishek³, Dr Dushyant Kumar⁴, Dr Peeyush Tiwari⁵

ABSTRACT:
Immune system is the cornerstone of good health. Immunity is the balanced state of having adequate biological defense system. Immunomodulation is a very broad term which refers to any changes in the immune response and may involve induction expression and amplification in the immune response. Immunomodulation can be classified into the following three categories: immunoadjuvants, immunostimulants and immunosuppressants. The immune system dysfunction is responsible for various diseases like allergy, asthma, arthritis, cancer and other infectious diseases. Age-related changes of the immune system contribute to the increased susceptibility of elderly persons to infectious diseases, vaccine failure, and possibly autoimmunity and cancer. Immunomodulator agents increase the immune responsiveness of the body against pathogens by activating the both specific and non-specific immune system. Some of the medicinal plants valued in Ayurvedia for their therapeutic potential have been scientifically investigated with promising results. A number of plant-based principles have been isolated with potential immunomodulatory activity that can explain and justify their use in traditional medicine in the past and can form the basis for further research in the future as well. In Unani literature a large number of herbs are mentioned to promote the physical and mental health, and have been claimed to possess immunomodulatory activity. Medicinal plants are used as immunomodulator to provide alternative potential to conventional chemotherapy for a variety of diseases.

KEYWORDS: Immunomodulator, Immunity, Unani, Homeopathy, Ayurveda,  immunoadjuvants, immunostimulants and immunosuppressants

INTRODUCTION
It is an evident from the human history that medicinal plants have been the treatment regimen to cure a variety of diseases, including diseases caused by insects, fungi, bacteria, and viruses. The effects shown by the plants are due to the chemicals present in them and they work in the same manner as the conventional drugs. However, there are equally chances for these plants to have some potential harmful and toxic effects also. These undesired side effects can be reduced by processing of the plant’s crude product. Ethnobotany is the study of traditional plants for their medicinal properties and is an effective method to discover future medicines. According to 2015-16 data, more than 300 plants have been identified to have therapeutic potential.¹ Around 122 chemicals derived from plants have been identified as therapeutic substances which are also used in commercial drugs, for example, bark of willow tree is very rich in salicylic acid, which is also an active metabolite of aspirin, and this bark has been used from ancient times as a pain killer and antipyretic substance. Some of the drugs which are frequently used by the physicians are also derived from plant sources, for example, aspirin, digoxin, quinine and opium, etc.They have a long history of use as herbal drug. Currently, there is much growing interest to use these medicinal plants as modulators of the complex immune system. Through a number of researches conducted in the area have explored that many of the chemicals in the form of alkaloids, flavonoids, terpenoids, polysaccharides, lactones, and glycoside products are responsible to cause alterations in the immunomodulatory properties.^2,3

IMMUNITY
The term immunity defines body’s natural defense system against a vast array of diseases and disorders. Remarkably sophisticated and advanced among vertebrates, the complex immune system is capable to generate a limitless variety of cells and molecules to arrest enormous spectrum of infections and undesirable substances. Immunomodulators refer to those substances capable of inducing, amplifying, and inhibiting any component or phase of the immune system. Immunostimulators and immunosuppressant are two types of immunomodulators are known for use. In fact, immunopharmacology is a newer branch of pharmacology concerned with immunomodulators.⁴ Administration of immunostimulators as in the case of AIDS and use of immunosuppressor in cases of an exaggerated response of an immune system is appreciating to reconstitute the normal immune system and increase the longevity of life. Immunomodulators intake along with antigen, the process is meant to boost the immune system, and the modulator is known as immune adjuvant.⁵ Immunology is one of the rapidly developing fields of biomedical research, holds great promises concerning various diseases and disorders. The two ways of defense of an immune mechanism involving short-term mechanism which is the first line of defense and the other highly advanced adaptive immune response marked by complexity, diversity, and memory. An adaptive immune response also consists of two subtypes of immune responses, humoral immune response concerned with β-lymphocytes and cell-mediated cytotoxic response mediated by T-cells. Well, all the component cells of the immune system originate from bone marrow through hematopoiesis from bone marrow-derived stem cells. They are either develop into mature cells or migrate to other peripheral sites for migration. Besides a vast range of specialized cells of immune cells, certain molecules called cytokines which are one of the important mediators of the immune system mediate the cross talk between the specialized cells of the immune system, thereby completely integrating the behavior and action responses of the cells.⁶

History of Immunology:
Immunology is a science that examines the structure and function of the immune system. It originates from medicine and early studies on the causes of immunity to disease. The earliest known mention of immunity was during the plague of Athens in 430 BC. Thucydides noted that people who had recovered from a previous bout of the disease could nurse the sick without contracting the illness a second time. In the 18th century, Pierre-Louis Moreau de Maupertuis made experiments with scorpion venom and observed that certain dogs and mice were immune to this venom. This and other observations of acquired immunity were later exploited by Louis Pasteur in his development of vaccination and his proposed germ theory of disease.Pasteur’s theory was in direct opposition to contemporary theories of disease, such as the miasma theory. It was not until Robert Koch’s 1891 proofs, for which he was awarded a Nobel Prize in 1905, that microorganisms were confirmed as the cause of infectious disease. Viruses were confirmed as human pathogens in 1901, with the discovery of the yellow fever virus by Walter Reed.⁷

Immunomodulators

These are biological or synthetic substances that can stimulate, suppress or modulate any aspect of the immune system including both adaptive and innate arms of the immune system.

Classification of Immunomodulators

Clinically, immunomodulators can be classified into the following three categories:

1. Immunoadjuvants:

Immunoadjuvants are used to enhance the efficacy of vaccines and therefore could be considered specific immune stimulants. Immunoadjuvants hold the promise of being the true modulators of the immune response. It has been proposed that they be exploited as selectors between cellular and humoral helper T1 (Th1) and helper T2 cells (Th2), immunoprotective, immunodestructive, and reagenic [immunoglobulin E (IgE)] versus IgG type immune responsesdposing a real challenge to vaccine designers.⁸

2. Immunostimulants 

Immunostimulants are inherently non-specific as they are envisaged as enhancements to a body’s resistance to infection. They can act through innate as well as adaptive immune responses. In healthy individuals, the immunostimulants are expected to serve as prophylactic and promoter

agents, i.e., as immunopotentiators, by enhancing the basic level of immune response. In the individual with impairment of immune response, they are expected to act as immunotherapeutic

agents.⁹

3. Immunosuppressants 

Immunosuppressants are a structurally and functionally heterogeneous group of drugs, which are often concomitantly administered in combination regimens to treat various types of organ transplant rejection and autoimmune diseases.¹⁰

Methods for Testing Immunological Factors: 

The routine process for screening is to extract single ingredient or single distilled fraction from herbal drugs, determine its bioactivity by the classic pharmacological means. The whole animal model is the most classic pharmacological screening model, which is very important at the aspect of medicine evaluation because it can apparently respond to the efficacy, side effect and toxicity of medicines in whole. Several in vitro, in vivo methods of pharmacological screening of medicinal plants having immunomodulatory activity have been listed. ¹¹

 In vitro methods: 

Inhibition of histamine release from mast cells 

• Mitogen induced lymphocyte proliferation 
• Inhibition of T cell proliferation 
• Chemiluminescence in macrophages 
• PFC (plaque forming colony) test in vitro 
• Inhibition of dihydro-orotate dehydrogenase 

In vivo methods: 

• Spontaneous autoimmune diseases in animals 
• Acute systemic anaphylaxis in rats 
• Anti-anaphylactic activity (Schultz-Dale reaction) 
• Passive cutaneous anaphylaxis 
• Arthus type immediate hypersensitivity 
• Delayed type hypersensitivity 
• Reversed passive arthus reaction 
• Adjuvant arthritis in rats 
• Collagen type II induced arthritis in rats 
• Proteoglycans – induced progressive Polyarthritic in mice 
• Experimental autoimmune thyroiditis 
• Coxsackievirus B3-induced myocarditis 
• Porcine cardiac myosin-induced autoimmune myocarditis in rats 
• Experimental allergic encephalomyelitis 
• Acute graft versus host disease (GVHD) in rats 
• Influence on SLE-like disorder in MRL/lpr mice 
• Prevention of experimentally induced myasthenia gravis in rats 
• Glomerulonephritis induced by antibasement membrane antibody in rats 
• Auto-immune uveitis in rats 
• Inhibition of allogenic transplant rejection. 

Mechanism of Immuno-stimulation: 

Immunological defense is a complicated interplay between nonspecific and specific, cellular and humoral immune responses, stimulation and suppression of immunocompetent cells, and the influence of endocrine and other mechanisms upon the immune system. Primary targets of the Immunostimulant are T or B lymphocytes or the complement system, an increase in phagocytosis by macrophages and granulocytes plays a central role in immunostimulation.¹² Activation of macrophages is probably important for the stimulating agents to remain in contact with the reactive cell. The second most important role is the stimulation of T lymphocytes, which can be achieved either directly or indirectly, via macrophages.¹³ 

Immunosuppression: These agents could be used for control of pathological immune response in autoimmune diseases, graft rejection, graft versus host disease, hypersensitivity immune reaction (immediate or delayed type), and immune pathology associated with infections. Out of the list the maximum use of these agents has been for prevention of graft rejection and treatment of autoimmune diseases.

IMMUNOMODULATION BY ALLOPATHIC DRUGS: 

Immunosuppressant: Immunosuppression implies mainly the decrease in resistance against infections, stress and may occur on account of environmental or chemotherapeutic factors. 9 Clinical applications of immunosuppressant are as follows. 

• To suppress rejection of transplanted organs and tissues (kidney, bone marrow, heart, liver, etc.) 
• To suppress graft-versus-host disease (i.e. response of lymphocytes in the graft to host antigens) in bone marrow transplants. 
• To treat a variety of conditions, which, while not completely understood, are believed to have an important autoimmune component in their pathogenesis i.e. myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, psoriasis and ulcerative colitis. 
• Selective immunosuppression for prevention of Rh hemolytic disease of the immunostimulant

Immunostimulant: 

The term immunostimulation comprise a prophylactic or therapeutic concept which aims at the stimulation of our nonspecific immune system. This implies primarily the non antigen dependent stimulation of the function and efficiency of granulocytes, macrophages, complement and natural killer (NK) cells.¹⁴

Side Effects of Immunomodulator Drugs: There are various side effects are associated with the use of these drugs i.e. Pulmonary toxicity, Myelosuppression, Alopecia, Increased risk of infection, Hepatic fibrosis, Lymphoma (Epstein–Barr virus associated), Nephrotoxicity, neurotoxicity (tremor, headache, motor disturbances and seizures), GI complaints, hypertension, hyperkalemia, hyperglycemia, and diabetes, Renal dysfunction, tremor, hirsutism, hypertension, hyperlipidemia, gum hyperplasia, hyperuricemia, hyper cholesterolemia, nephrotoxicity, hypertension, diabetogenic, Elevated LDL cholesterol etc.¹⁵

IMMUNOMODULATION BY HOMEOPATHIC DRUGS:
Homoeopathy is a system of therapeutics based on the key principle of ‘Like cures like’. When homoeopathic medicines are prescribed in ultra diluted form on the basis of symptom similarity, they stimulate body’s own defense mechanisms to fight against the pathogen. Homoeopathy experiments on basophils and mast cells showed that homoeopathic medicines in ultra diluted form can influence degranulation of mast cells and basophils. Although these experiments were performed to explore the action of ultra diluted substances, they raised the possibility that homoeopathic medicines act via their action on immune system.¹⁶

There are some medication & studies which explore the immunomodulatory action of homoeopathic medicines – Rhus toxicodendron (rhustox), Mercurius solubilis (Merc sol), Echinacea, Aconitum, Lachesis and Apis – and homoeopathic combination remedy Canova®. Canova® is a Brazilian medical formulation based on homoeopathic technique. It is composed of Aconitum napellus, Thuja occidentalis, Bryonia alba, Arsenicum album and Lachesis muta and <1% ethanol in distilled water. The medicine is also called by the name of Metodo canova (MC, Canova method). The following review was conducted using PubMed and references from the relevant articles. The keywords comprising the names of above homoeopathic medicines along with terms ‘immune function’, ‘cytokines’, etc. were used for search.

Patil et al. evaluated the immunomodulatory activity of Rhus tox, in various dilutions, through in vivo and in vitro studies. In vivo experiments the effects of Rhus tox on humoral immune response and DTH were evaluated in C57BL/6 mouse model. Following the antigenic challenge by administration of sheep red blood cells (SRBCs), the haemagglutination assay was performed to assess rise in antibody titer. SRBC suspension was also injected in mice right hind paw, and changes in paw volume measured after 24 h by a plethysmometer were recorded as the intensity of DTH. The in vitro experiment assessed the effects of Rhus tox on chemotaxis of human polymorph nuclear (PMN) cells through ‘under agarose chemotaxis assay’ method and phagocytosis of Candida albicans by PMN cells by phagocytosis assay. The results of in vivo experiments showed a greater rise in antibody titer as compared to control group which was statistically significant. Rhus tox mother tincture, 6CH and 30CH dilutions showed significant stimulation of DTH response as compared to control group. In In vitro experiments, Rhus tox mother tincture and 6CH dilution stimulated chemotaxis of PMN cells, and Rhus tox mother tincture stimulated phagocytic activity of PMN cells.¹⁷

de Oliveira et al. conducted in vivo and in vitro experiments to investigate the effects of Merc sol, in various dilutions, on mice peritoneal macrophages. The various macrophage parameters tested were change in morphology of macrophages and induction of cytokines TNF-α, IFN-γ, IL-2, IL-4 and IL-5, nitric oxide (NO), superoxide (O2) and hydrogen peroxide (H2O2). Mice received Merc sol in drinking water for 7 days. After 7 days, macrophages were harvested from mice peritoneal cavities and cultured in Dulbecco’s Modified Eagle’s medium. Two hours after plating, the cells were further treated with Merc sol or control solution. For morphological analysis and cytokine estimation, cells were treated with Merc sol 6CH, 12CH, 30CH and 200CH, and for reactive species analyses, cells were treated with Merc sol 6CH, 12CH and 200CH. Results of the experiments showed Merc sol‑treated macrophages with typical morphology of activated state, such as wider spreading, numerous cellular projections and a large and euchromatic nucleus. The spreading ability was significantly higher in Merc soltreated group which confirmed that 6CH, 12CH, 30CH and 200CH Merc sol induced macrophage activation. The cytokine quantification experiments showed that Merc sol induced IFN-γ production at lower dilutions (6CH and 12CH), while higher dilutions (200CH) stimulated IL-4 production. NO production by macrophages was evaluated in the presence or absence of lipopolysaccharide (LPS) and IFN-γ. Merc sol induced NO production at 6CH, 12CH and 200CH; however, in cells treated with LPS and IFN-γ, it led to decrease in NO production. The O2 formation was decreased whereas H2O2 production was unaltered by Merc sol at any of the tested dilutions.¹⁸

IMMUNOMODULATION BY UNANI DRUGS:

In Unani literature is has been mentioned that tabi’at is solely responsible for capacity of the body to resist the disease and pathogens. With age the function of preserving ratubaat in a body do not remain same, which leads to weakening of all qua’ of the body making tabi’at unable to struggle with the disease and pathogens. The immune system is increasingly found to be involved in the development of several chronic illnesses. Intervention by the physician is required to help tabi’at so that it can accelerate the process of elimination of disease form the body. A number of synthetic drugs like cyclosporine, corticosteroids, azathioprine etc. are being used in treating immune diseases but they have side effects like nephrotoxicity, anaemia, thrombocytopenia, bone marrow suppression etc. Medicinal plants have been used virtually in all cultures as a source of medicine for altering the immune system. In recent times, focus on plants with their extracts and active fraction has been investigated for immune response modifying activity.

Unani herbs with Immunomodulatory Potential

Asgand (Withania somnifera L.)

Standardized aqueous extract of Asgand was evaluated in laboratory animals immunized with DPT vaccine. Treatment of immunized animals with extract of Asgand 100 mg/kg/day for 15 days resulted in significant increase of antibody titers to B. pertussis. The main constituents of Asgand responsible increased antibody titer are steroidal Lactones (Withanolides), polysaccharides, lectins, proteins and peptides.

Elwa (Aloe vera)

Pyrogallol-induced suppression of humoral as well as cell mediated immune response was significantly attenuated by daily oral treatment with elwa extract at the dose of 100 mg/kg on the albino mice. It produces stimulatory effect on the humoral and cell mediated immune response in the experimental animals. This suggests its therapeutic usefulness in disorders of immunological origin. Immunostimulant effect of elwa could be attributed to the alkaloids content.

Fowah (Rubia cordifolia L.)

Administration of ethanolic extracts of the leaves of fowah 100 mg/kg, b.w. orally to CP- exposed Swiss albino rats resulted in enhanced immune responses. There was an increase in total

counts of WBC and RBC cell count which was significantly high in immunocompromised animals. Responsible chemical constituents probably were alkaloids, cardiac glycosides, tannins, flavonoids and phenols.

Kalonji (Nigella sativa)

Ethanol extract of kalonji seeds 1.5 g/kg, b.w. was orally administered in dexamethasone-induced immune-suppressed male rabbits for 42 days. It is reported to increase the phagocytic activity by stimulating the immune cells and increase the activity of immune potential. The immunomodulatory action may be due to its main constituent’s thymoquinone, nigellone and d-limonene through their antioxidant and anti-inflammatory activities.

Gudhal (Hibiscus Rosa sinensis L.)

The assessment of immunomodulatory activity of hydroalcoholic extract of flowers of gudhal (75, 150 and 300 mg/kg p.o.) was done for non-specific immunity, humoral immunity and cell mediated immunity on wistar albino rats. Gudhal was found to possess significant immunostimulatory action on immune system. The chemical constituents reported in plant were cyanidin, quercetin, flavonoids, hentriacontane, thiamine, riboflavin, niacin and ascorbic acid.

Haldi (Curcuma longa)

Aqueous extract of haldi rhizome at a dose of 50 mg/kg bw is given to carbon tetra chloride intoxicated Swiss albino mice for 15 days. Administration of haldi aqueous extract in that carbon tetra chloride intoxicated mice renders the macrophages to acquiring abnormal surface morphology so that they can function normally. The mechanism involving this protection by haldi extract probably related to the production of monocyte colony stimulating factor or granulocytemonocyte stimulating factor. Curcumin is a main active constituent of haldi.

Neem (Azadirachta indica)

Aqueous extract of Neem flowers were given at doses of 200 and 400 mg/kg, p.o. to albino mice may stimulate both specific and non-specific immune responses by stimulating macrophages, humoral and cell mediated response. It can therefore be concluded that aqueous extract of Neem flowers is a potential immunostimulant against cytotoxic drugs and can be used as a complimentary therapeutic agent.¹⁹

Immunomodulation by Ayurvedic Drugs:

Ayurveda has propounded the concept of immunity as “Vyadhikshamatwa”. Acharya Chakrapanidatta has interpreted the term Vyadhi-ksamatwa as Vyadhi bala Virodhitwa i.e., antagonistic to the strength and virulence of the disease and Vyadhyutpada Pratibandhakatwa i.e., the capacity to inhibit and bind the causes and factors of the disease. In fact, one of the therapeutic strategies in Ayurvedic medicines is to enhance the body’s overall natural resistance to the disease causing agent rather than directly neutralizing the agent itself. The use of herbs for improving the overall resistance of body against common infections and pathogens has been a guiding principle of Ayurveda. Such herbs possessing immunomodulatory effects are referred to as Rasayana in Ayurvedic classics. They are supposed to have the ability of protecting the body against external factors that induce disease. This implied resistance against disease may represent the modern concept of immunity. Ayurveda describes a number of drug as rasayana and ojovardhak which are claimed to posses immunomodulatory effect. Some of the rasayanas which have possess immunomodulatory effect are Aswagandha (Withania somnifera), Amalki (Emblica officinalis), Tulsi (Ocimum sanctum), Guduchi (Tinospora cardifolia), Pippali (Piper longum), Punarnava (Boerhavia diffusa), Yashtimadhu (Glycyrrhiza glabra), Brahmi (Bacopa monnieri), Bhringraj (Eclipta alba), Gold, of which Guduchi, Tulsi and Brahmi have been extensively studies. Bhringraj have free oxygen radical scavenger mechanism. The active principles of Guduchi have been found to possess anticomplementery and immunostimulating activities. Yashtimadhu (Glycyrrhiza glabra) another important Rasayana drug has been found to be immunostimulating which accelerates lymphocytic transformation activation of macrophage and increases the leucocyte count. It also have anti-allergic, anti-inflammatory and antioxident activity. A controlled clinical study with combination of the Rasayana drugs- Amalki, Vidang and Atibala have shown an increase in immunoglobulin levels in infants which is significant greater than that of multivitamin used cases. A combination of four important Rasayana drugs – Guduchi, Ashwagandha, Amalki and Tulsi in equal amounts was found to potentiate both the cellular and humoral components of immunity.²⁰

CONCLUSION:
Due to economic constraints, providing modern medical healthcare in developing countries such as India is still a far reaching goal. The most commonly used drugs of modern medicine such as aspirin, anti-malarials, anti-cancers, digitalis, etc. have originated from plant sources. Out of an estimated 2,50,000 higher plants, less than 1% have been screened pharmacologically and very few in regard to immunomodulatory. Therefore, it is prudent to look for options in herbal medicine as immunomodulatory as well. We have been working continuously towards establishing the scientific basis of use of certain plants in immune disease. Immunomodulatory drugs are agents that could alter immune system of an organism, if it increases the immune response are called as immunostimulants or if it decreases immune response are called as immunosuppressant.

The results obtained from pre-clinical studies show that homoeopathic drugs, in various potencies, can influence mice bone marrow cells, macrophages, lymphocytes and PMN cells. The medicines produced modulation of immune function at multiple levels. It is well recognized fact that there is high incidence of drug related unexpected side effects in allopathic medicine for Immunomodulation in elderly persons. It is where role of Unani medicine come into play. Modulation of immune system with herbal drugs may offer novel approach in treatment of variety of diseases. To conclude, Ayurvedic immunomodulators are effective growth and development enhancer and considered now as one of the most potent tools in the management of health and disease.

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Dr Pramod Kumar Singh¹, Dr Poonam Sharma², Dr Abhishek³, Dr Dushyant Kumar⁴, Dr Peeyush Tiwari⁵

1Professor and HOD of Homeopathic Pharmacy,Dr. Madan Pratap Khunteta Homoeopathic Medical College, Hospital & Research Centre, Homeopathy University, Jaipur, Rajasthan.

2,3,4. MD Scholars, Department of Homeopathic PharmacyDr. Madan Pratap Khunteta Homoeopathic Medical College, Hospital & Research Centre, Homeopathy University, Jaipur, Rajasthan.

5. MD Scholar, Department of Repertory Dr. Madan Pratap Khunteta Homoeopathic Medical College, Hospital & Research Centre, Homeopathy University, Jaipur, Rajasthan.