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Immunostimulants: Concepts, Types and Functions

Prince Jain, Pratik Darji, Bhupendra Singh Thakur, Anushree Jain, Prateek Kumar Jain, Basant Khare*

Adina College of Pharmacy, ADINA Campus Rd, Lahdara, Sagar, MP, 470001

Introduction

Humans and other vertebrates reside in a world that is occupied by a huge range of pathogenic microbes and toxic substances that menace normal homeostasis; and immunity is a specialized form of host defense mechanism that works particularly in relation to the causes and prevention of diseases¹. Manifestation of disease due to the pathogen depends on its virulence and capability of the immune system; and to achieve resistance against disease, the most important is strengthening the immune system². If the immune system fails become under or over active, or hits the wrong target it can vent a variety of adverse consequences. Under-activity of the immune system lead to loss the defensive mechanism against infections; extreme immune failure results HIV disease, certain cancer, etc. whereas over-activity can lead to autoimmune diseases, including arthritis, inflammatory bowel disease, inflammatory lung disease, connective tissue disease, autoimmune endocrine diseases, multiple sclerosis, etc. So, proper understanding and strictly regulating the immune system has become mandatory. Two main compounds are able to enhance immune responses including adjuvants and immunostimulants. An adjuvant is a substance combined with an antigen for increasing its immune response, but an immunostimulant can induce the immune response without injection with an antigen³. There are several types of stimulants with different mechanisms and functions such as bacterial products, complex carbohydrates (e.g., glucans, schizophyllan, scleroglucan, lentinan, statolon, bestatin, acemannan), vaccines, immunoenhancing drugs (e.g., Levamisole, Isoprinosine, Fluoroquindone, Avridine, Polyribonucleotides), nutritional factors (e.g., vitamins, carotenoids, lipids, trace elements, selenium), animal extracts (e.g., chitosan from shrimp), cytokines (e.g., macrophage activating factor, interferon, interleukin-2, tumor necrosis factor), and plant extracts (e.g., Lectins, mitogens such as phytohemagglutinin, concanavalin A) ⁴. Two main approaches were determined to evaluate the efficiency of an immunostimulant such as in vivo protection against pathogens, and in vitro assay of cellular and humoral immune mechanisms. In vitro tests should be performed before in vivo experiments to clarify the basic mechanisms responsible for the protection. In vitro immunostimulant evaluation is usually based on some parameters such as serum lysozyme, complement, total leucocyte count, monocyte/lymphocyte/granulocyte count, antibody titers, phagocytosis, respiratory burst and leucocyte proliferation⁴. Immunomodulation can be either specific or non-specific. Specific immunomodulation is limited to a single antigen such as vaccination, whereas non-specific immunomodulation leads to a further change in immune response both in innate and adaptive immunity causing altered host reactivity to many various antigens⁵. 

Immune system

The immune system consist of a complex network of specialized cells, tissues, molecules and biological processes within an organism that watches out the continually to protect it against attacks by foreign antigens or invaders (basically microbes-infection causing organisms such as bacteria, viruses, parasites, and fungi or any injury, and disease) ⁶. The different organs of human immune system are shown in Figure 1. Some of the potentially infectious agents includes: (a) Viruses, which are sub-microscopic non-living entities that replicates only inside the host cells (living organism) and often results in serious diseases. Examples include influenza virus, human immunodeficiency virus (HIV), herpes simplex virus (HSV, which can cause cold sores or genital ulcers), a newly discovered virus named coronavirus (causes infection in the upper respiratory tract). (b) Bacteria are single celled microbes capable of causing disease when get entry into the body through water, air, soil and also through physical contact. Examples include Staphylococcus and Streptococcus that cause acute infections such as abscesses and sore throats, Escherichia, Salmonella that cause food poisoning and Mycobacteria that cause chronic infections such as tuberculosis and leprosy. (c) Fungi, eukaryotic, nonphototrophic organisms with rigid cell walls, they can be unicellular or multicellular. Examples include Aspergillus that causes allergic disease, Candida that causes thrush, Cryptococcus that causes meningitis and meningo-encephalitis in patients with HIV infection and AIDS. (d) Parasites, which are eukaryotic organisms that live off other organisms, or host, to survive. Some are them are single-celled protozoa that cause diseases for example, malaria; others are large, multicellular organisms (metazoa) example, worms that can be seen with the naked eye. In order to prevent disease, the immune system must able to scan, recognize and attack the foreign invaders by distinguishing self from non-self substances². Self-molecules are those components that belongs to an organism's body which the immune system can distinct from foreign substances. Autoimmunity is an immune response in opposed to its own healthy cells and tissues, which may lead to various diseases⁷. Non-self-molecules are those recognized components that do not belong to an organism’s body, they are foreign invaders. One example of non-self-molecules is antigens that cause the immune system to promote the generation of antibodies against it and then combine specifically with them to induce an immune response⁸.

Innate and adaptive immunity

Defence against infection is divided into two main forms namely innate immunity and adaptive immunity. Its components are shown in Figure 2. Some of the differences between innate and adaptive immunity are shown in Table 1.

Innate immunity

Innate immunity regarded as the first line of defense from both external and internal attack, also known as natural or native immunity. It is a nonspecific and antigen-independent defensive mechanism which responds immediately and within minutes or hours of meeting an antigen⁹. Its host defense mechanisms are encoded as their mature form by the germ-line gene of the host¹. This type of immune response is lack in memory to recognize the pathogen, if the same pathogen invades for second time as they cannot generate that immunologic memory. Innate immunity consists of two major components: Humoral (include complement cells) and Cellular (includes neutrophils, macrophages, adnatural killer cells).

Figure 1: Organs of the human immune system

Figure 2: Flowchart of basic components of human Immune system

The defensive barriers of  innate immunity are of four types:

1. Anatomical barriers e.g., skin and mucous membrane, the epithelial cell layers offers tight junction so there is tight cell to cell contact, the mucus layers over the respiratory, gastrointestinal and genitourinary tract, and when foreign particles are inhaled, the mucus layer get contaminated which are constantly discarded by the epithelial cilia.
2. Physiological barriers e.g., temperature, low pH and chemical mediators
3. Endocytic and phagocytic cells (neutrophils, macrophages), dendritic cells, natural killer (NK) cells and other innate lymphoid
4. Inflammatory barriers e.g., a series of events occurs in inflammation process that plays an important role to destroy or inactivate microbes.

Adaptive immunity

Adaptive immunity is also known as specific or acquired immunity which means the resistance acquired by human during their lifetime. It is antigen-dependent and antigen-specific defensive mechanism and, thus delays the time of the antigen to get expose and to produce the maximal response. The advantage of adaptive immunity is their capacity to generate memory which permits the host to elicit a more rapid, stronger and efficient immune response against consecutive exposure to the antigen¹⁰. This type of immunity provides the basis for effectual immunization facing infectious diseases. The two major components of adaptive immunity are Humoral (comprises of antibodies formed by B lymphocytes) and Cellular (mediated by T lymphocytes). B lymphocytes and T lymphocytes are two kind of lymphocytes found in this type of immunity that impart long lasting immunity against specific antigens by proliferating into memory cells. Lymphocytes are generated from the bone marrow and the type that mature in bone marrow turns into B lymphocytes whereas the type that leave the bone marrow and migrate to thymus gland get mature into T lymphocytes and based on ‘cluster of differentiation’ (CD) molecules on their surface they acquire certain genetic and immune surface characteristics which determines their different functions¹¹. B lymphocytes are responsible for formation of specific antibodies by differentiating into plasma cells while T lymphocytes get activated in presence of appropriate antigens presented by macrophages like APC and Histocompatibility Complex (MHC). The function of B lymphocytes are like military intelligence system, they find out the target and organise defensive action, while T lymphocyte perform like soldiers, they destroy the invading substance identified by the intelligence system i.e. B lymphocytes¹². Antigen specific receptors are encoded by genes that are assembled by somatic rearrangement of germ-line gene to form intact T cell receptor (TCR) and immunoglobulin (B cell antigen receptor; Ig) genes. Millions of different antigen receptors are formed from the collection of a few hundred germ-line-encoded gene elements assembly of antigen receptors, each of which are potentially unique and antigen specific¹. The advantage of this diverseness of receptors helps adaptive immunity to identify any kind of pathogen¹³. They are of two types i.e., naturally    acquired adaptive immunity and artificially acquired adaptive immunity.

Naturally acquired adaptive immunity: In naturally acquired active adaptive immunity, antigens enter the body naturally then the bodies develop antibodies and specialized lymphocytes whereas in naturally acquired passive adaptive immunity, antibodies passes from mother to foetus/infant through placenta/mother’s milk. Naturally acquired active adaptive immunity lives longer than naturally acquired passive adaptive immunity.

Artificially acquired adaptive immunity: In artificially acquired active adaptive immunity, antigens are introduced into the body through the use of vaccines then the bodies generate antibodies and specialized lymphocytes against it whereas in artificially acquired passive adaptive immunity, preformed antibodies in immune serum are introduced into the body by injection. Artificially acquired active adaptive immunity lives longer than artificially acquired passive adaptive immunity but, when there is very less time to develop active immunity then passive type is more effective as it can prevent the infection in any stage and its process is rapid.

Table 1: Differences between innate and adaptive immunity

Functioning of immune system

The immune system comprised of cells and proteins that uphold the body from the foreign invaders¹⁴. These cells emanate from the pluripotent stem cells of bone marrow. Of the two pathways - (I) the myeloid pathway, in presence of IL-3, becomes excited giving rise to the production of platelets, erythrocytes, monocytes and granulocytes. (II) the lymphoid pathway, in presence of IL-7, becomes excited giving rise to the production of innate and adaptive lymph cells (Lymphocytes). The pathways distinction relies on the chemical signals in the surrounding area^15,16. When pathogens invade the host body, innate immunity provides the first line of defense. Leukocytes like dendritic cells, monocytes, neutrophils, macrophages, eosinophils, mast cells, are allowed by the pattern recognition receptor (PRRs) to detect and react rapidly towards a large population pathogen which is structurally similar, known as pathogen associated molecular patterns (PAMPs). An example of these is the components of bacterial cell wall like lipopolysaccharides (LPS) and double-stranded ribonucleic acid¹⁷. The binding of PRPs with PAMPs triggers the release of cellular messenger called cytokines (e.g. interleukin) and causes inflammatory reaction. Inflammation leads to vasodilation, increased vascular permeability and cellular infiltration due to which the microbial cells get destroyed. Other cells, like natural killers are the critical members of innate immunity as they able to pursue and kill a vast number of pathogens along with malignant cells. All the cells of immune system are capable to suppress or induce inflammation by communicating one another over direct cell contact or through generation of cytokines. Again a complex systems of proteins known as the complement system also induces inflammatory response that aid to fight infection. This type of immunity also aid to remove dead cells or foreign substances from different organs, blood and lymph¹³. The adaptive response takes over when the innate immune response becomes ineffective to eliminate pathogen. In adaptive immunity at first antigen presenting cell (APC) like macrophages and dendritic cells recognizes, engulfs and process the antigen; and displays the specific part of antigen on its surface then present it to T- cells. T-cells receptors are there that bind with the specific antigenic sites and triggers proliferation and differentiation processes in lymphoid tissues². There are two classes of T-cells namely, helper T-cells and Cytotoxic T-cells which can be discriminate by their presence of some molecules on their surface like CD4+ and CD8+ respectively. T-helper cells aid the immune response in recognition of antigen and then activate other T and B-cells by secreting cytokines whereas Cytotoxic T-cells aid the immune response by killing pathogen infected cells or tumor cells. One other class of T-cells, known as suppressive T-cells are able to secret suppressive cytokines that can inhibit the actions of other T-cells. Antigen binding and helper T-cell can trigger the differentiation process of B-cells into plasma cells and secret antibodies which circulate in the blood and causes destruction or inactivation of the antigen¹⁸.

Immunostimulants

Immunostimulants (or immune stimulants) are biologically active substances obtained from natural or synthetic sources with different chemical characteristics and mechanism of action that modulate the immune system of host to increase resistance against various infections [19]. They interact with specific receptors and cellular components of innate and adaptive response to modulate the immune response. They used during suppressed immunity condition like cancer disease, AIDS, SARS etc. to improve the host’s resistance¹⁹.

Concept of immunostimulant

Immunostimulants known as immunostimulators are attractive substances that activate the immune system of humans and animals for prevention of diseases and improvement of the body’s natural resistance to various viral and bacterial infections. These biologically active substances are the products derived from natural sources or synthetically made with different chemical properties and mechanisms of action. In general, immunostimulants induce synthesis of specific antibodies and cytokines for treatment of infectious diseases. Two major groups of immunostimulants contain a) specific immunostimulants acting as antigen for stimulation of immune responses (e.g., vaccines), and b) non-specific immunostimulants without antigenic properties enhancing immune responses to other antigens (e.g., adjuvants and non-specific immunostimulators). Moreover, immunostimulants were classified based on their origin and mode of action²⁰.

Functions of immunostimulants

Immunostimulants activate different elements of the immune system in humans and animals. They develop the non-specific immunotherapy and immunoprevention by stimulating the major factors of the immune system including phagocytosis, properdin and complement systems, protective secretory IgA antibodies, α- and γ-interferon release, T- and B-lymphocytes, synthesis of specific antibodies and cytokines, and synthesis of pulmonary surfactant. There are several reasons for using the immunostimulants in the control of various infectious diseases including: a) antibiotic resistance of the bacteria; b) allergic reactions to antibiotics; c) immunosuppressive effects of antibiotics; and d) Poor effects of the antibiotics in viral infections¹⁹. Figure 3 shows some types of immunostimulants and their general functions.

Figure 3: Schematic representation of some types of immunostimulants and their general functions

Types of Immunostimulants

For simplification, we divided the types of immunostimulants as 7 groups such as bacterial products, complex carbohydrates, vaccines (antigens and adjuvants), cytokines, immunoenhancing drugs, plant extracts, and animal extracts as mentioned below:

Immunostimulatory drugs

A few immunostimulatory drugs (Endogenous immunostimulants or Synthetic immunostimulants) have been developed to induce humoral or cellular immune responses or both of them against bacterial or viral infections, immunodeficiency diseases, and cancer. They were classified as follows: 

a) Levamisole (Ergamisol): Levamisole is a synthetic drug inducing B and T lymphocytes, monocytes, and macrophages. It was used in adjuvant therapy with 5-fluorouracil after surgical resection in patients with Duke’s stage C colon cancer. Its disadvantages are allergy, nausea, flu, and muscle pain. Levamisole has been successfully used in combination with polymers for treatment of dermatologic disorders. For example, it was combined with cimetidine for treating recalcitrant warts, and with prednisolone for treating aphthous ulcers of the mouth^21,22. 

b) Thalidomide: Thalidomide or Immunoprin (C₁₃H₁₀N₂O₄) is an immunomodulatory drug. Thalidomide could decrease circulating TNF-α in patients with erythema nodosum leprosum. In contrast, it increased TNF-α in HIV-seropositive patients. Furthermore, its therapeutic effects were determined in severe rheumatoid arthritis and angiogenesis²¹.

c) Isoprinosine (Inosiplex/ Imunovir): Isoprinosine (C₅₂H₇₈N₁₀O₁₇) is a combination of inosine, acetamidobenzoic acid, and dimethylaminoisopropanol. Isoprinosine could enhance the levels of cytokines including IL-1, IL-2, and IFN-γ. It increased the proliferation of lymphocytes against mitogenic or antigenic stimuli. Moreover, Isoprinosine augmented active T-cells and induced T-cell surface markers on prothymocytes. It was used to treat Herpes simplex infections, Epstein-Barr, and Measles viruses. Its disadvantages are minor CNS depressant, transient nausea, and increased level of uric acid in serum and urine²¹. 

d) Immunocynin: Immunocynin is a stable form of haemocynin, a copper-containing protein, found in molluses and arthropods. It was used to treat urinary bladder cancer with poor side effects such as rare-mild fever²¹. 

e) Bestatin: Bestatin, a dipeptide [(2S, 3R)-3-amino-2- hydroxy-4-phenylbutanoyl]-L-leucine, is an immunostimulant with low toxicity which binds to the cell surface of lymphocytes and macrophages and enhances both humoral and cellular immune responses. It is a leucine aminopeptidase and aminopeptidase-B inhibitor. Bestatin possesses antitumor activity and also increase the antitumor activity of bleomycin and adriamycin. Bestatin efficiently prevented the metastasis of P388 leukemia when the antibiotic was constantly injected after tumor inoculation²³. The dipeptide was immunorestorator in the elderly and cancer patients and HIV-infected subjects. It stimulated granulocytopoiesis and thrombocytopoiesis in vitro and could restore them in myelo-hypoplastic man²⁴.

Bacterial products

The immunostimulatory effects of bacteria and bacterial products are due to the release of cytokines. Live bacillus Calmette-Guerin (BCG) is an attenuated, live culture of the bacillus of Calmette and Guerin strain of Mycobacterium bovis. Its mechanism of action includes: a) induction of a granulomatous reaction at the site of administration, and b) prevention and treatment of carcinoma types. Furthermore, BCG enhances both B and T cell-mediated responses leading to phagocytosis and resistance to infection. Its disadvantages are hypersensitivity, fever, shock, and immune complex disease²¹. 

Recombinant cytokines

Several interferons and interleukins are suggested to stimulate effective immune responses. Interferons could be obtained from trout leucocytes after stimulation with mitogens. It was able to cause an in vitro resistance against pancreatic necrosis virus in trout cells. In mammalian, low doses of interferon could induce stable positive results without side effects. On the other hand, vaccination of animals with the recombinant IL-2 against different infections increased the protective effects. However, IL-2 was a very toxic compound in high doses causing side effects such as fever and diarrhea. The purified cytokines showed unsatisfactory results in clinical trials, because the immune responses were produced by a mixture of cytokines generated by the immune cells, but not against a single cytokine. Thus, the enhancers of nonspecific cytokine synthesis may improve immune responses and solve this problem⁴. Thus, recombinant cytokines are produced recently in different expression systems (e.g., plants) and used in clinical trials such as interferons, TNF-α and IL-2²⁵. Complex carbohydrates

Several types of the complex carbohydrates were described as follows:

a) Glucans: An important class of immunostimulants is the β-(1→3)-linked chain of glucose units. The main chain has β-(1→6)-branched glucose units. The β-glucans were obtained from highly conserved structural components of cell walls in fungi, algae, yeast, and have a broad range of molecular weights from 5 to 200 kDa. The length and frequency of these branches vary depending on different sources. β-glucan was used to stimulate anti-tumor mechanisms (e.g., increased macrophage activity) and to enhance host resistance to a variety of microbial pathogens in mammalian. Glucan might also be helpful to prevent the carcinogenic effects of aflatoxin. β-glucan was consideredas a stimulator of cellular immunity. Indeed, mammalian macrophages or monocytes have specific receptors for β- glucans and produce mediators such as cytokines (e.g., IL-1, IL-9, TNF-α) and prostaglandins in the presence of glucans^{26, 27}. In Japan, the β-glucans such as Lentinan derived from the Shiitake mushroom and Polysaccharide-K derived from Coriolus versicolor were licensed as anti-cancer drugs²⁸. Lentinan could induce protective Th1 immune responses to control the proliferation of malaria parasites red blood cells by stimulating maturation of DCs, increasing the expression of MHCII, CD80/CD86, Toll-like receptors (TLR2/TLR4) and the level of IL-12, and preventing the adverse effects of Tregs^29,30. The main roles of glucans were detected in cancer treatment, infection immunity, stress reduction, and restoration of damaged bone marrow. A mixture of polysaccharides isolated from the cell walls of Saccharomyces cerevisiae named as zymosan could potently stimulate macrophages and induce the release of cytokines from neutrophils. Indeed, β-glucan in zymosan was identified as its effective component for non-specific immunomodulation. In addition, β-glucan could reverse myelosuppression generated by chemotherapeutic drugs via targeting the C3 fragment of complement and circulating antibodies. The recent studies have shown that daily therapy with soluble or insoluble β-glucan led to a 70%-95% reduction in tumor size. Indeed, after the binding of antibodies on the surface of cancer cells, C3 fragments of complement could coat the cancer cells. Then, β-glucan-primed cells, such as blood neutrophils, macrophages, and NK cells specifically recognized these complement-antibody complexes and killed the tumor cells. In fact, the cooperation of β-glucan with anti-tumor antibodies is an effective approach in combination therapy²⁸. 

b) Trehalose: Trehalose dimycolate (TDM), Muramyl dipeptide (MDP), and Lipopolysaccharides (LPS) as the bacterial products promote the production of antibody, stimulate activation of lymphocytes, and elicit specific immunity against different bacterial infections. Trehalose dimycolate, a glycolipid present in the cell wall of Mycobacteria is a potent immunostimulant that limits tumor growth and enhances resistance against bacterial, parasitic, and viral infections. It can interact with membranes due to its amphipathic properties. TDM primes murine macrophages to generate nitric oxide (NO) and to develop anti-tumoral activity. As an adjuvant, TDM enhances both cellular and humoral immunity, but elicits a stronger cellular response. TDM could induce potent immune responses against malaria antigens in mice as compared to groups immunized with malarial antigens and Freund’s adjuvant. The reports showed that the protective effect of TDM is reduced in macrophage-depleted mice injected with silica particles indicating the role of macrophages. T lymphocytes were not necessary for TDM to prime peritoneal macrophages. Trehalose diesters could induce IL-12p40 and IFN-γ mRNA^31,32.

 c) Prebiotics: Prebiotics are indigestible fibers that increase beneficial gut commensal bacteria resulting in improvement of the host's health. Prebiotics, such as fructooligosaccharide, mannanoligosaccharide, inulin, or β-glucan, are called immunosaccharides. They directly enhance innate immune responses including phagocytic activation, neutrophil activation, activation of the alternative complement system, and increased lysozyme activity. Immunosaccharides directly activate the innate immune system by interacting with pattern recognition receptors (PRR) expressed on innate immune cells. They can also associate with microbe associated molecular patterns (MAMPs) to activate innate immune cells. Indeed, probiotics activate the innate immune system in two ways: a) by directly stimulating the innate immune system and b) by enhancing the growth of commensal microbiota³³. 

Immunostimulants used in vaccines

Vaccines contain a wide range of immunostimulants. For example, an adjuvant heat-labile enterotoxin from Escherichia coli (LT), administered as an immunostimulant (LT-IS) patch on the skin may further enhance immune responses to influenza vaccine in the elderly³⁴. Also, the immune activation mediated by LT-IS improved the potency of generating Alzheimer's disease (AD)-specific vaccination responses as an adjuvant in the clinical trial³⁵. Co-administration of a potent adjuvant in IS patches containing heat-labile enterotoxin from E. coli placed on the skin at the site of DNA vaccination significantly increased anti-influenza antibody immune response³⁶. Adjuvants enhance and modulate immune responses to antigens. This is important when the purified antigens do not elicit the effective innate or adaptive immune systems. Adjuvants are different in the types and levels of immune responses. Expected advantages of adjuvants contain stronger immune priming, effective immune responses in low-response populations (e.g., the elderly or immuno-compromised patients), the use of smaller amounts of the antigen, and safety profile³⁷. New adjuvants have already applied to more efficient influenza vaccines, as well as vaccines targeting hepatitis B (HBV) and human papillomavirus (HPV) ³⁸. On the other hand, CpG oligonucleotides and imiquimod drugs (an antiviral agent) could activate dendritic cells, induce in situ maturation and migration of DCs, and augmented both humoral and cellular immune responses³⁹. The unmethylated CpG motif in bacterial DNA was identified as a B-cell stimulating adjuvant, and synthetic oligodeoxynucleotides (ODNs) containing the CpG motifs were shown to induce potent therapeutic activities in different infections and tumor animal models. Imiquimod was topically used for patients with anogenital warts as well as basal-cell carcinoma. The studies indicated that CpG ODNs and imiquimod (resiquimod) drugs act as synthetic ligands for TLR9 and TLR7, respectively, and both stimulate efficiently DC maturation³⁹. 

Plant-derived immunostimulants

Natural plant product promote various activities such as anti-stress, growth promotion, appetite stimulation, immunostimulation, aphrodisiac and antimicrobial properties, due to the active substances such as alkaloids, flavanoids pigments, phenolics, terpenoids, steroids, and essential oils. Medicinal plants have been known as immunostimulants, growth promoters, immune enhancers, where they act as antibacterial and antiviral agents to the host immune system. Unfortunately, the mechanisms were not understood⁴⁰. Some medicinal plants were described as following:

a) Ocimum sanctum (Tulsi): Leaves of O. sanctum containing water-soluble phenolic compounds and various other constituents may act as an immunostimulant. Leaves extract of O. sanctum affected both specific and nonspecific immune responses. It stimulated both antibody response and neutrophil activity^5,41.

b) Phyllanthus emblica (Amla): P. emblica has antioxidant, anti-fungal, anti-microbial, and anti- inflammatory activities. Amla fruit pulp contains a large amount of vitamin C as an immunostimulant^5,41.

c) Azadirachta indica (Neem): A. indica possesses antihuman immunodeficiency virus, anti-tumor, and antimicrobial activities. Azadirachtin, a triterpenoid derived from A. indica, enhanced respiratory burst activities, the leukocyte count and the primary and secondary antibody responses against SRBC (sheep erythrocytes) in tilapia^5,41.

d) Solanum trilobatum (Purple Fruited Pea Eggplant): The herbal extract of S. trilobatum possesses a broad spectrum of antibiotic, antibacterial and anticancer activities. A study showed that the water-soluble fraction of S. trilobatum significantly enhanced the production of reactive oxygen and decreased the percentage of mortality following a challenge with Aeromonas hydrophila^5,41.

e) Eclipta alba (Bhringraj): E. alba possesses several medicinal properties. The methanol extracts of E. alba significantly increased the phagocytic index, antibody titer and WBC count in mice^5,41. 

f) Zingiber officinale (Ginger): The extracts of Z. officinale contain polyphenol compounds which have a high antioxidant activity. Moreover, it showed a significant increase in proliferation of neutrophils, macrophages, and lymphocytes, as well as it enhanced phagocytic, respiratory burst, lysozyme, bactericidal and antiprotease activities^5,41.

g) Echinacea (purple coneflowers) and Allium sativum (garlic): Echinacea and A. sativum improved the gain in body weight, survival rate and resistance against challenge infection of Aeromonas hydrophila. Both compounds developed resistance to cold stress during the winter season^5,41.

h) Camellia sinensis (Green tea): Green tea extracts possess biological activity including antioxidant, antiangiogenesis, and anti-proliferative activities that are related to the prevention and treatment of various forms of cancer^5,41.

i) Aloe vera: Oral administration of A. vera could enhance the specific and non-specific immune responses and increase lysozyme activity, serum bactericidal potency, and the total protein and IgM levels^5,41. 

j) Cynodon dactylon (Bermuda Grass): The antiviral activity of C. dactylon was confirmed to prevent white spot syndrome virus (WSSV) infection with no mortality and no signs of WSD (White spot disease) ^5,41.

k) Achyranthes aspera (Prickly Chaff Flower): A. aspera showed both specific and non-specific immunity revealed by higher levels of serum antibody and also serum antiproteases in fish. Moreover, the level of serum globulin and RNA/DNA ratio of the spleen were also significantly enhanced in the fish fed with A. aspera^5,41.

m) Nyctanthes arbortristis (Night-flowering Jasmine): N. arbortristis possesses hepatoprotective, anti-leishmanial, antiviral and antifungal activities. The extract of N. arbortristis significantly enhanced serum lysozyme, complement activities and cellular reactive oxygen species (ROS), reactive nitrogen intermediate (RNI) and myeloperoxidase (MPO) production^5,41.

n) Fermented vegetable product (FVP): The phagocytic activities, the activity of lysozyme, and superoxide generation of peritoneal leukocytes enhanced in fish fed with the FVP supplemented diet^5,41.

o) Saffron: Saffron, a spice derived from the flower of Crocus sativus, is rich in carotenoids. Carotenoids are lipophilic molecules accumulating in lipophilic compartments including lipoproteins and/or membranes. Two main natural carotenoids of saffron, crocin, and crocetin, are responsible for its color⁴². Saffron and its components were suggested as promising candidates for cancer prevention⁴³. The mechanisms underlying cancer chemopreventive activities of carotenoids contain modulation of carcinogen metabolism, regulation of cell growth and cell cycle progression, inhibition of cell proliferation, antioxidant activity, immune modulation, enhancement of cell differentiation, stimulation of cell-tocell gap junction communication, apoptosis and retinoid dependent signaling. The immunomodulatory activity of saffron was determined on driving toward Th1 and Th2 limbs of the immune system⁴². Carotenoids increase the proliferative response of T and B lymphocytes to mitogens, the activity of natural killer cells, the number and activity of cytotoxic T-cells, macrophage tumor-killing activity and also induce the secretion of TNF-α in an animal model. These effects are involved in preventing tumor growth, killing tumors and lowering tumor burden. Different carotenoids were used as main phytonutrients to inhibit the development of tumors in vitro and in vivo⁴⁴. For example, a single treatment with crocin significantly decreased tumor size in a mouse model⁴³.

Animal originated immunostimulants

There are some immunostimulants derived from animals. For example, chitin and chitosan are the non-specific immunostimulators which are protective against infections for a short time. Also, fermented products of chicken egg (EF203) containing immunoactive peptides showed immunomodulatory effects when administered orally to rainbow trout, Oncorhynchus mykiss. Fish treated with EF203 displayed an increased resistance to both natural and experimental β-haemolytic streptococcal infection⁴¹. Moreover, chitosan, the deacetylated derivative of chitin, has shown strong anti-microbial activity depending on its degree of deacetylation and molecular weight. Both oligomers of chitin and chitosan were effective in enhancing the migratory activity of macrophages. Furthermore, chitosan could activate the production of cytokines such as IL-1β, TNF-α, and reactive oxygen intermediates to promote the defense system against microbial infections⁴⁵. On the other hand, glycated chitosan (GC) as an immunoadjuvant was used in combination with phototherapy for cancer treatment in animal models. In vitro studies also showed that after incubation of GC with macrophages, it could significantly stimulate the secretion of TNF-α⁴⁶.

Conclusion

Immunomodulators are divided into several groups including physiological products, cytokines, host defense peptides, microbial products, probiotics, synthetic chemical compounds, herbal products, adjuvants, and polysaccharides. Immunostimulants represent a promising class of drugs for the treatment of infectious disorders and cancer. Herbal extracts and animal originated product have a potential application as an immunostimulant, because they can be easily obtained, are not expensive and act against a broad spectrum of pathogens. Most of the herbs and herbal extracts can be given orally, which is the most convenient method of immunostimulation in a dose dependent approach. Recently, carbohydrate-based immunostimulants that target Toll-like receptor 4 (TLR-4) and cluster of differentiation 1D (CD1d) receptors as vaccine adjuvants are underway. Also, the incorporation of immunostimulants into nanomaterials has shown a novel approach to enhance the immunostimulation properties. However, further studies are needed to identify effective immunostimulants without adverse side effects and determination of their mechanism of action.

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