INDIAN HERBS: A POSSIBLE PREVENTIVE MEASURE AND IMMUNITY BOOSTERS AGAINST CORONAVIRUS DISEASE (COVID-19)

Raminderjit Kaur, Shikha Choudhary, Sunil, Rahul and Neha Minocha K. R. Mangalam University, Sohna Road, Gurugram-122103 Haryana, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 20 April 2020 Final Accepted: 25 May 2020 Published: June 2020

In humans, coronaviruses are embraced in the span of viruses that affects the respiratory system and kidney; it shows symptoms like common cold, severe acute respiratory syndrome (SARS), fever, cough, diarrhoea etc. Transpire the infectious diseases associated with SARS became a crucial ultimatum to public health. COVID-19 proliferates quickly and is the principal reason of its wide spread in many countries. It is observed from many studies that COVID-19 predominately attacks the people with low immunity response against the virus. Immune response by acquired by COVID-19 basically have 2 phases i.e., during the incubation and non-severe phases and elimination of the virus and prohibition the development of disease to severe stages is only possible with a specific adaptive immunity. Hence, it's necessary to adopt the various strategies for boosting immune response against virus. This review focused in the use of the herbal products for strengthen the immunity of all aged groups against COVID-19. It is observed from various studies that many of the phytochemicals have the capability to inhibit lipid per-oxidation, decreases the oxidative stress, suppresses the activated T-cells (key mediators of antiviral immunity), stimulated phygocytosis of neutrophils, increases the concentration of various inflammatory mediators like cytokines, interleukin, tissue necrosis factor etc. Therefore, herbal drugs have the ability to boost immunity against antigen. In this review, we mainly discussed about the mechanism of increasing immunity by various herbal drugs like Green tea, Ashwaganda, Broccoli, Kutki, Jhandu and many more against the COVID-19.

…………………………………………………………………………………………………….... Introduction:-
Coronavirus is the crucial pathogen that predominately attacks the human respiratory system. Precursory outburst of coronavirus encompass the severe acute respiratory syndrome (SARS)-CoV and the Middle East respiratory syndrome (MERS)-CoV, these are previously distinguished as fatal negotiator that further be the reason for public health threat. It has been observed in the late December, the clump of patients was admitted in hospitals with a beginning recognition of pneumonia of an unknown etiology (1,2). These patients were epidemiologically linked to a seafood and wet animal wholesale market in Wuhan, Hubei Province, China. Preliminary outlines divined the beginning of a prospective Corona virus outbreak given the judgment of a replicating number for 2019 Novel (New) Corona virus, which is classified as COVID-19 by WHO on Feb 11, 2020 (3). More than 100 years since the 1342 outburst of 1918 influenza pandemic, we are now facing another pandemic. As the outburst of COVID-19 spreads in almost every continent, compelling us to reside with this virus for conceivably a long time. As now scientists and clinicians have grasped abundant of corona virus disease and pathogenesis (4), it has been observed that not all people exposed SARS-CoV-2 are necessarily infected. Moreover, the not all infected patients develop severe respiratory illness. From this observation, SARSCoV-2 infection can be roughly divided into three stages as below: (5) Stage I: an asymptomatic incubation period with or without detectable virus. Stage II: non-severe symptomatic period with the presence of virus. Stage III: severe respiratory symptomatic stage with high viral load.
Individuals of stage I should became the carrier and are least manageable because at some extent, they spread the virus unknowingly, as in Germany the first asymptomatic transmission was reported (6). COVID-19 infects all age groups evenly, but the patients over 65 years are at high risk and there are chances to progression of disease in severe phase (7). One of the significant queries that why only some people develop severe disease, at the same time others doesn't. Understandably, the traditional perception based on overall immunity of infected patient cannot explain this broad spectrum disease presentation. Immunity is the condition of confrontation of an organism to overwhelm biotic and abiotic pathogens and their harmful effects that prevents the spread of the disease (8). There are basically 2 components of immunity i.e. specific and non-specific. The non-specific component deed as the obstacle or eliminator of vast array of pathogens irrespective of their antigenic composition. Additional components of immune system develop pathogen-specific immunity and adapt themselves to encounter the disease (9,10). An immune system may contain innate and adaptive components. The innate system is composed of primordial bone marrow cells that are prearranged to recognise foreign substance and to fight against it (11). On the other hand, adaptive component include typical lymphatic cells that further helps in identifying self-substance and not to react. Moreover, innate immunity is also called as "Native Immunity" and it prevail by probity of an organism's configuration which is its genetic make-up, with lacking an extrinsic stimulation or primitive infection. However, it is categorised into two kinds, Non-specific which is a intensity of resistance to all infections and specific one that only resist a certain types of micro-organisms (12). At the same time, adaptive immunity can also be classified on the basis of inauguration of immunity i.e., "naturally acquired" that explains through chance contact with disease causing agent and "artificially acquired" which emerge through the vaccinations (13). Both of these immunities can further classified as "active immunity" which last for lifetime and "passive immunity" can be shortly lived, it can be adopted through the exposure (injection or infusion) of antibodies or activated T-cells from an immune host. The diagram below summarizes these divisions of immunity in Fig. 1. Activated immunity can further sub-divided on the basis of involvement of type of immune mediator; Humoral immunity is developed by release of antibodies however, cell mediated immunity includes T-lymphocytes alone. Humoral immunity is in activate phase when there is generation of its antibodies by the organism whereas transportation of antibodies between the individual classified it in passive phase. Likewise, cell-mediated immunity is active when the organisms' T-cells are stimulated and passive when T cells come from another organism (14).

Signs and symptoms of COVID-19:
The appearing of symptoms of COVID-19 (Fig 2) infection could be after an incubation period. The period from the onset of COVID-19 symptoms to death ranged from 6 to 41 days with a median of 14 days. The age of the patient and its immunity describes the range f period for the particular patient. The patients who are older than 70 years have less surviving rate with shorter period of days as compared to patients less than 70 years. The most common symptoms at onset of COVID-19 illness are fever, cough, and fatigue, while other symptoms include sputum production, headache, haemoptysis, diarrhoea, dyspnoea, and lymphopenia (15).

Incubation period of Corona Virus:
The incubation period (time from exposure to the development of symptoms) of the virus is estimated to be between 2 and 14 days based on the following sources (17) 1. The World Health Organization (18). 2. China's National Health Commission (NHC) had initially estimated an incubation period from 10 to 14 days (19) 3. The United States' CDC estimates the incubation period for COVID-19 to be between 2 and 14 days (20). 4. DXY.cn, a leading Chinese online community for physicians and health care professionals, is reporting an incubation period of "3 to 7 days, up to 14 days" (21).

Comparison between the incubation periods of various viruses causes SARS:
The incubation period is the time between infection and symptom onset and an important epidemiologic distribution; it is frequently involved in case definitions used to regulate appropriate quarantine periods (22). This period is extensively delineated because it is beneficial in infectious disease superintendence and control, in which the time of symptom onset may be the only indication of the time of infection. Severe acute respiratory syndrome (SARS) offers a dramatic example: by expeditiously dictate the maximum incubation period of SARS, public-health officials were able to set quarantine periods and cease a prospective pandemic without the assistance of a vaccine or treatment. The incubation period thus plays a crucial part in monitoring for healthcare-accompanying infections, and may help in detection (23). The incubation period of different viruses are reported in table 1.

Two-phase immune responses induced by COVID-19 infection:
It has been observed clinically that the immune response acquired by SARS-CoV-2 infection are of two phase i.e., during the incubation and non-severe phases, elimination of the virus and prohibition the development of disease to severe stages is only possible with a specific adaptive immunity. Therefore, we have to adopt the strategies to boost the immune response as it is the necessity of living during these phases. Our body generates specific anti-viral immunity with the development of endogen protective immune response which further depends upon the good general health and genetic background (e.g. HLA) of organism. Moreover, impairment of this protective immune response causes the invading of virus that further damages the affected tissues, especially the organs that have ACE2 receptors, e.g. lungs, intestine and kidneys. This massive destruction could be the reason for induction of inflammatory responses in the lungs that are arbitrated by the pro-inflammatory macrophages or granulocytes. Lung inflammation is one of the major etiologic reasons of life-threatening respiratory diseases at chronic stages. Therefore, efforts should be made to suppress the inflammation and to overcome the symptoms (24).

The role of Cytokine storm in lung damage:
The cytokine release syndrome (CRS) appears to influence the patients with serious diseases. As lymphocytopenia frequently observed in the patients with COVID-19, therefore CRS spawn by SARS-CoV-2 has to be arbitrating by leukocytes other than T cells and blocking IL-6 is efficacious in this condition (25). However, inhibiting the release of IL-1 and TNF-alpha may bring the beneficial results in patients with severe condition whilst many of the Chinese clinician claims the effectiveness of mesenchymal stromal/stem cells (MSCs) in severe cases with COVID-19 infection (26). One of the major challenges has been observed in the severely affected patients that T-cells are not rigorously invigorating by SARS-CoV-2 infection, hence, MSCs required to be stimulated by IFNγ to employ its anti-inflammatory action (Fig 2). Lung damage is the utmost stumbling block to recuperation in those severe patients. It has been seen from various preclinical studies that the animal models with bleomycin-induced lung injury, Vit B3 (niacin or nicotinamide) is the one of the potent treatments in halting the ling tissue damage (27).
The activation of cytokine plays a crucial role in the prevention of inflammatory response and clearance of pathogen. As alveolar macrophages are not able to inhibiting overwhelm of pathogen therefore, chemokines further activated neutrophils which causes phagocytosis due to which formation of reactive oxidative species occurred results in the necrosis of alveoli. (28)

The pathophysiology of HLA haplotypes in SARS-CoV-2 infection:
The classical aspirant in suppression of genetic susceptibility in infectious diseases is the major-histocompatibilitycomplex antigen loci (HLA). It has been suggested by Immunologists that cell antigen receptors, on CD4+ or CD8+ 1345 T cells concede the conformational structure of the antigen-binding-grove together with the associated antigen peptides (29). Hence, distinct HLA haplotypes are accompanying with different diseases susceptibilities. Therefore, it is recognised that if HLA molecules will have increased binding identification to the SARS-CoV-2 virus peptides on the cell surface of antigen of host cell could be produce the beneficial effect (30). Undeniably, the susceptibility to various infectious diseases such as tuberculosis, leprosy, HIV, hepatitis B, and influenza is auxiliary with particular HLA haplotypes. Consequently, it is imperious to research whether particular HLA loci are accompanying with the expansion of anti-SARS-CoV-2 immunity and, if so, to recognize the alleles, either class I or II, that illustrate organization of protective immunity. Upon the recognition of dominant allele only simple kits will be used for the detection. This direction is crucial for (1) strategic clinical management; (2) evaluation of the efficacy of vaccination in different individuals in the general population; (3) assignment of clinical professional and managerial teams amid interactions with COVID-19 patients (31,44) Hyaluronan: a potential cause of fatalities: Acute respiratory distress syndrome (ARDS) is associated with the activation of innate immunity against any virus which further lead to respiratory failure with the quick occurrence of inflammatory response in the lungs and that could give rise to necrosis of alveoli cells which proven fatal for patient. The symptoms of ARDS patients include short/rapid breathing, and cyanosis. The patients with emergency conditions are admitted in intensive care unit where they are treated with the mechanical supports such as extracorporeal membrane oxygenation (ECMO) and ventilators. It is observed in the CT scan that the lungs of these patients are filled with fluid in the form white patches called as "ground glass". Although, it is proven with the reports of autopsies that lungs are filled with clear liquid jelly. Moreover, hyaluronan (HA) would be the pathological reason of ARDS. The COVID patients are associated with the high level of cytokines (IL-1, TNF) which further lead to the stimulation of HA-synthase-2 (HAS2) in CD31 endothelium, EpCAM lung alveolar epithelial cells, and fibroblasts, shown in Fig 3. Markedly, hyaluronan has the capacity to absorb water up to 1000 times its molecular weight. Hence, decreasing the level or blocking the release of hyauronan will be the effective measure for helping COVID patients to breathe properly (32). 1346

Potential Immune Evasion Mechanisms:
As the incubation period of COVID-19 is longer of 2-14 days as compared with influenza because Corona virus specifically remodel to evade immune detection bedew human immune responses. SARS-CoV and MERS-CoV both of these viruses are the members belongs to betacoronacirus genus therefore, immune evasion mechanism is quite similar with each other (Fig 4). The viral proteins include membrane (M) or non-structural (NS) proteins (eg. NS4a, NS4b, NS15) are the key molecules in host immune modulation. In concurrence with the previously mentioned research that the lower response of type I interferon could lead to poor outcomes i.e. death of the patient. For adaptive immune evasion, antigen extended via MHC class I and MHC class II was downregulated when the macrophages or dendritic cells were contaminated with MERS-CoV, which would conspicuously decline T cells activation (34)  Table 2 (37). It has revealed from the clinical and pre-clinical studies that Full-length spike (S) or S1 which contains receptor binding domain (RDB) would be the effective vaccine antigen as it results in neutralizing antibodies that inhibited the host cell attachment and infection, described in Table 2 Full-length Spike, or S1im follow by electroporation Table 2:-Selected antigens for development of vaccine have been tested for SARS-CoV and MERS-CoV.

Effect of vaccination on immune system:
Multiple procedures are arrogated in the formation of COVID vaccines that principally include target antigen is surface-exposed spike (S) glycoprotein or S protein for neutralizing antibodies. Various S-protein-based procedures have been strived for thriving COVID vaccines, e.g., use of full-length S protein or S1-receptor-binding domain (RBD) and expression in virus-like particles (VLP), DNA, or viral vectors (39). The S protein molecule contains two subunits, S1 and S2. The S1 subunit has an RBD that binds with its host cell receptor, angiotensin-converting enzyme 2 (ACE2), however the S2 subunit conciliate amalgamation between the virus and host cell membranes for liberating viral RNA into the cytoplasm for replication (40). Therefore, S-protein-based vaccines should more effectively used as it releases that further inhibiting viral receptor binding as well as un-coating of virus genome. It is observed that the C-terminal domain of the S1 subunit of porcine Deltacoronavirus contains the immune-dominant region, and the immune response to this region results in most potent neutralizing effect (41). Recombinant adenovirus-based vaccine expressing MERS-CoV S protein persuade systemic IgG, secretory IgA, and lung-resident memory T-cell responses when injected intra-nasally into and yield long-lasting neutralizing immunity to MERS spike pseudotyped virus, as a consequence it is proposing that the vaccine may confer protection against MERS-CoV (42).

Herbal drugs used to boost immunity:
In the wake of COVID-19 pandemic, there have been a lot of interests in ways to strengthen the immune system; therefore, it became more important to build up the first defence against the deadly virus. Immunity cannot be built up in a day, but it can be possible by eating a well-balanced diet and being physically and mentally active and maintaining good health. There are however, many phyto-chemicals extracted from the herbal drugs which are known to boost your immunity. As from the primitive period, herbs and spices were well known for their medicinal properties. According to the World Health Organisation, around 80% of the world's population uses herbal medicines for primary health care, particularly across Europe and South Asia. Studies revealed that many of these herbs not only have anti-inflammatory properties, but also help boosting the body's natural immunity. And unlike allopathic medicines like antibiotics, which can have serious side effects, most of these herbs and spices are relatively safe. Some of the herbal drugs have been discussed below: 1) Garlic: Biological source: Allium sativum Immunity boosting mechanism: It can scavenge free Radicals and act as successful anti-oxidant. It increases the production and release of Nitric Oxide (NO) that is further responsible for enhanced release of IFN-alpha in humans, beneficial against viral disease. In addition, it can enhance Natural Killer Cells and ultimately stimulate immune response (44).

2) Mushroom: Biological source: Agaricus bisporus
Immunity boosting mechanism: These include augmenting the immune system through stimulating lymphocytes, NK cells and macrophages thus enhancing the cytokine production. (45)

4) Andrographis:
Biological source: Andrographis paniculata Immunity boosting mechanism: It significantly increased the activities of anti-oxidant defence enzymes such as catalyse, superoxide dismutase, glutathione-S-transferase and reduced glutathione content. The extract significantly inhibits lipid peroxidation by lowering the levels of thiobarbituric-acid-reactive substances in the liver and kidney of diabetic rats. A pre-treatment of andrographolide was reported to significantly attenuate the accumulation of the phorbol-12-myristate-13-acetate-(PMA-) induced formation of ROS and N-formyl-methionyl-leucyl-phenylalanine-(fMLP-) inducing adhesion of rat neutrophils (47).

6) Cordyceps: Biological source: Cordyceps militaris
Immunity boosting mechanism: It induced the highest elevation of nitric oxide production and enhanced phagocytic activity. It also significantly induced the mRNA expression of TNF-α and IL-10 and the levels of phosphorylated MAPK (mitogen-activated protein kinase) (49).

8) Broccoli: Biological source: Brassica oleracea
Immunity boosting mechanism: A chemical in broccoli switches on a set of antioxidant genes and enzymes in specific immune cells, which then combat the injurious effects of molecules known as free radicals that can damage cells and lead to disease. Free radicals are byproducts of normal body processes, such as the metabolic conversion of food into energy, and can also enter the body through small particles present in polluted air. (52) 9) Black tea: Biological source: Camellia sinensis Immunity boosting mechanism: It influences activation of transcription factors such as NFnB or AP-1 that ultimately hinder the formation of nitric oxide expression gene. It boasts immunity by enhancing the diseasefighting ability of gamma delta T cells. It causes inhibition of redox sensitive transcription factors & pro-oxidant enzymes such as xanthine oxidase or nitric oxide synthase. However, their involvement in antioxidative enzyme induction as in glutathione-S-transferases is also well documented. (53) 10) Green tea: Biological source: Camellia sinensis Immunity boosting mechanism: Green tea modulating production of immune-regulatory cytokines in stimulated dendrite cells and hence acts as suppressor of T cell activation by lowering the inflammatory reaction, reducing the lipid per-oxidation and formation of NO radicals, Thus acting as anti-oxidant (54).

11) Ashwagandha:
Biological source: Withania somnifera Immunity boosting mechanism: It increased release of various cytokines such as TNFα, IFN-γ, and/or IL-2. IFN-γ is a cytokine critical for both innate and adaptive immunity; IL-12 is primarily secreted by macrophages (55).

12) Amla:
Biological source: Emblica officinalis Immunity boosting mechanism: It is a rich source of antioxidants, including polyphenols, which confer its free radical scavenging potential (56). 1349 13) Guduchi: Biological source: Tinospora cordifolia Immunity boosting mechanism: It increases the concentration of cytokines. Cytokines regulate the intensity and duration of immune responses by stimulating or inhibiting activation, proliferation and/or differentiation of various cells and by regulating the secretion of antibodies (57).

14) Brahmi:
Biological source: Bacopa monnieri Immunity boosting mechanism: Brahmi, a traditionally reputed herbal drug, has been reported to exert antioxidant activity. There are many factors that produce free radical mediated oxidative stress. It also down regulated iNOS expression thereby inhibited nitric oxide generation (58)(59).

15) Mandukaparni:
Biological source: Centella asiatica Immunity boosting mechanism: It significantly increased proliferation and the production of IL-2 and TNF-alpha. It also inhibited mitogenesis and the production of IL-2 and TNF-alpha (60).

16) Draksha: Biological source: Vitis vinifera
Immunity boosting mechanism: The redox sensitive transcription factor nuclear factor erythroid 2-related factor-2 (Nrf2) partly regulates the expression of genes encoding antioxidant enzymes. Nrf2 is bound in the cytoplasm to its inhibitor Keap1 (Kelch-like ECH-associated protein 1). When Nrf2 is activated by electrophiles, it is released from its cytosolic protein Keap1 and binds to the antioxidant response element of the DNA in the nucleus thereby regulating the transcription of target genes including γ-glutamylcysteine synthetase (γGCS) and heme oxygenase-1 (HO-1) (61).

19) Kutki:
Biological source: Picrorrhiza kurroa Immunity boosting mechanism: It stimulates the cell-mediated immunity significantly and also stimulated the humoral immunity by regulating the function of T-cells against antigen (64).

21) Purple Coneflower: Biological source: Echinacea
Immunity boosting mechanism: It suppresses the ability of Jurkat T cells to produce IL-2 independently. It activates cellular immunity and stimulates phagocytosis of neutrophils (66-67).

24) Astragalus:
Biological source: Astragalus Immunity boosting mechanism: It causes stimulation of immune cells against stimulus-response action, elevates the tumor cell killing activity of cytotoxic T-lymphocyte (71).

Conclusion:-
Population with weak immunity is vulnerable for COVID-19, the world's pandemic disease. To strengthen and boosting the immunity natural products depict crucial role by enhancing the valuable bacteria in the body. Through the investigation it has noted that natural products like Draksha (Vitis vinifera) used in DNA synthesis and cell proliferation which synchronize innate and adaptive responses of immune system. Various herbal drugs i.e. Garlic (Allium sativum), Bleuberry (Ericaceae Vaccinium), Andrographis (Andrographis paniculata), Broccoli (Brassica oleracea), Tea (Camellia sinensis), Amla (Emblica officinalis, Brahmi (Bacopa monnieri), Haridra (Curcuma longa), Jhandu (Tagetes erecta), Vidarikand (Pueraria tuberose) have anti-oxidant and immunomodulator activity thus enhancing the innate response of immune system. Similarly, herbal drugs like Liquorice (Glycyrrhiza glabra) which increases the level of malondialdehyde (MDA), superoxide dismutase (SOD), catalase and Purple coneflower (Echinacea) and Puple coneflower (Echinacea) suppressed the ability of activated T-cells to produce IL-2 that directly influence the immune response. Ginger (Zingiber officinale) is one of the herbal drugs that inhibit NF-κB activation thus arrests cell cycle in G2/M phases. Cytokine regulates the intensity and duration of immune response stimulating or inhibiting activation, proliferation and/or differentiation of various cells and by regulating the secretion of antibodies, natural products like Ashwagandha (Withania somnifxera), Guduchi (Tinospora cordifolia), Mandukaparni (Centella asiatica) have potential to elevate the cytokine levels.