Evidence-Based Research in Ayurveda against COVID-19 in Compliance with Standardized Protocols and Practices

The COVID-19 pandemic has emerged as a global health emergency of international concern since its first identification, resulting in millions of deaths and economic disruption. The healthcare systems across the globe are at the hilt and being tested for effective management against COVID-19. Employing our ancient traditional system and integrative approach, our nation has dealt with this bolstering virus competently, which is evident from the significantly low mortality rates in India. Param Pujya Acharya Ji, with his sheer dedication, astute guidance, pragmatic approach, and deep knowledge of the ancient Indian traditional system of medicine, has written a new success story by developing an effective medicine against COVID-19. Patanjali Research Institute, Haridwar, Uttarakhand, is a lineage representative of ancient Indian Vedic and sage traditions. A team of dedicated scientists at Patanjali Research Institute has worked relentlessly to develop Ayurvedic formulations into effective medicine by screening close to 1500 phytochemicals from more than 200 medicinal plants. This was a path-finding journey with experiences shared in this book titled “Ayurveda against COVID19”. We hope that this body of work will serve as a capstone of guidance to develop a new series of Ayurvedic formulations into medicines that would be acceptable to the modern medical and scientific fraternity worldwide. By sharing our research through this book, we wish to reach a greater and wider readership and, in the process, hope to materialize our humble efforts towards ensuring the well-being of humankind worldwide. May this excellence of scientific practice in Ayurveda find its due destination!

 This chapter discusses the virtual screening of phytochemicals and computational validation of identified ones as potential antiviral agents against the SARS-CoV-2 virus. In addition, we have provided an outline of how to conduct virtual screening and computer validation to identify potential lead compounds for further studies, including their formulation, chemical characterization, validation, and licensing, which have been addressed in the next chapter. 

This chapter, besides sharing the story behind the formulation and development of Ayurvedic medications acceptable in modern medicine, provides a detailed standard operating procedure for developing Ayurvedic drugs. To fight the COVID-19 infection, caused by the SARS-CoV-2 coronavirus, Patanjali Research Institute (PRI), Haridwar, developed the Divya Swasari Coronil kit. It contains Coronil Tablet, Divya Swasari Vati, and Divya Anu Taila. Divya Pharmacy, Haridwar, India, has obtained a manufacturing license from the Ayurvedic & Unani Department of Uttarakhand, Dehradun (License No.: 13-71-72/D-431/2020-2021) for Coronil and Swasari Vati tablets. Divya Coronil tablet has been formulated from a blend of Giloy, Ashwagandha, and Tulsi, whereas Divya Swasari Vati and Divya Anu Taila were prepared according to the classical recipe mentioned in traditional Ayurvedic texts. Analytical methods have been developed to identify and quantify the active phytoconstituents present in these blends and tablets. Sophisticated techniques are used in the manufacturing of these tablets, like liquid chromatography, equipped with single quadrupole, colloisan cell and time of light (UPLC/QToF MS), high-performance liquid chromatography (HPLC) equipped with PDA detector and high-performance thin layer chromatography (HPTLC) with automatic spotting, in which developing and scanning chambers are used for the identification and quantification of herbs. The methods have been validated in-house using ICH-Q2 (R1) and pharmacopoeia guidelines to demonstrate the repeatability, reproducibility, and reliability of the data generated. There are stringent quality checks for the authentication of raw material used in the manufacturing of the tablet, along with in-process checks and the final release of the batch. The herbs used during the manufacturing of these products have been authenticated, and voucher specimens have been stored in a government-approved depositary. The synergistic effect of these tablets has been studied by Patanjali Research Institute (PRI) for their immunity-boosting properties and the restoration of health of SARS-CoV-2 infected patients.

This chapter deals with the scientific authentication of the formulated Ayurvedic medicine. . Scientific research for mechanistic insights into the functionality is not a regulatory requirement for developing a herbal drug. Nevertheless, the outcomes from such scientific research works have been included in this chapter. Such scientific evidence on modes-of-actions of the herbal medicines helps in generating awareness among the end users, who could be from both scientific and non-scientific backgrounds. In this chapter, we have shared our scientific observations from the laboratory validations of the medicines, Coronil and Divya Swasari Vati,that have been developed. We have also discussed the modes of action of these medicines against the SARS-CoV-2 virus, as gathered from in-vitro experiments. Biochemical studies have shown that the medicines formulated by Patanjali Research Foundation Trust against the SARS-CoV-2 virus are capable of inhibiting the physical interactions between viral spike (S) protein and host ACE2 receptor protein. This interaction between S protein and ACE2 receptor is critical for COVID-19 infection. Our medicines were found to be effective in disrupting this interaction regardless of the initial mutation, like, D614G, that the SARS-CoV-2 virus has undergone to increase its infectivity. These medicines could also rescue the lung epithelial cells from S protein- and pseudovirus-induced cytokine storms. Pseudoviruses are non-pathogenic study viruses used for experimental purposes to understand the host entry mechanisms in viruses. In this case, the non-pathogenic viral genome was encased with SARS-CoV-2 S protein so that we can follow the S protein and ACE2 interactions. Besides, these pseudoviruses also had reporters inside them that helped us to monitor their entry into host cells. We found that cells, when treated with our medicines, showed lesser internalization of the viruses, suggesting that the medicines are preventing the virus entry. During COVID-19 pathogenesis, the pro-inflammatory cytokines put the immune response into an overdrive by inducing each other. We tried to mimic this in-vivo condition in-vitro by inducing inflammation in the lung epithelial cells with one pro-inflammatory cytokine and then checked the levels of others and how the treatment with our medicines altered this response. We observed that cells, when exposed to one pro- inflammatory cytokine showed an increase in the levels of others and interestingly when these cells were treated with Ayurvedic medicines, the cytokine levels reduced. Taken together, these in-vitro observations revealed that these Ayurvedic medicines disrupted physical interaction between viral S protein and host ACE2 receptor and attenuated the cytokine storm, implicating their potential in managing acute respiratory distress syndrome (ARDS), one of the prime causes of COVID-19 associated mortality. 

This chapter deals with the in vivo preclinical studies involving the COVID19 zebrafish model conducted on the Ayurvedic medicines mentioned in the last chapter to further validate their efficacy against COVID-19. Animal models are needed in order to understand the disease's progress and associated symptoms. While it is possible to understand the disease characteristics based on historical evidence and previous research on similar disease-causing organisms, newer species of diseasecausing agents have been recently discovered. These newer organisms without any previous history, pose the biggest challenge in drug discovery and development. In these cases, the use of relevant animal models of disease becomes important in order to understand the disease progression as well as the interaction of the body with the disease-causing agent. In the current SARS-CoV-2 infections, the virus is potentially lethal in humans. In such cases, the danger of using humans to test new drugs becomes ethically unacceptable unless the drug has been tested in animal models against the virus. The use of higher primates, like monkeys, or small animals like dogs and rodents, which are generally accepted pre-clinical models of drug discovery, has a myriad of ethical concerns. Despite this, several different models of SARS-CoV-2 infection are currently in use, ranging from non-human primates, such as rhesus macaques (Rhesus monkey), and rodent models, such as transgenic mice and hamsters. While it is difficult to incorporate all the different pathological features of the disease in a single model, it is important to choose the correct model animal in order to answer the primary question that the investigator seeks. For example, rodents lack the coagulopathy component, which is often seen in severe SARS-CoV-2 infections. By the same token, the narrow spectrum of viral infectivity and the inability to the crossspecies barrier by the virus is an important consideration while studying the disease pathology. This was seen in a rhesus monkey model where no overt clinical signs were detected even though prolonged viral shedding was detected in the upper respiratory tract of animals. With these issues in mind, we developed a humanized zebrafish model to test the efficacy of Coronil and Divya Swasari Vati in decreasing the pathogenic characteristics associated with SARS-CoV-2 spike protein expression. Zebrafish has proven to be a solid model system for investigating human viral pathophysiology, and various human viruses, including chikungunya and influenza, can colonize zebrafish, making it an appealing and alternative model system. Zebrafish have well-defined innate and adaptive immune systems that are strikingly comparable to those of humans. Unlike mouse models, zebrafish have swim bladders as buoyancy organs, and human cells could be transplanted into swim bladders to create xeno-transplanted humanized models for respiratory disorders, such as SARS-CoV-2 infection. The implantation of human lung cells into the zebrafish's air bladder increases the model's relevance and gives human-equivalent methods of inquiry. Different groups have successfully employed this strategy to replicate lung cancer in zebrafish as well as COPD and Pseudomonas aeruginosa pathogenesis. Results obtained from treating the humanized zebrafish model injected with the S protein of SARS-CoV-2 with either Coronil or Divya Swasari Vati are shared in this chapter, along with a proposed mode of action for both of these Ayurvedic formulations.

This chapter highlights the requirements for conducting an adverse effect study involving laboratory animals. Though this study is not required for Ayurvedic formulations, we are conducting these studies to follow the requirements in order to make the medicinal formulation acceptable by modern medical practitioners. The maximum tolerance limit for the formulation needs to be tested in toxicology studies using a rat and a rabbit model under the ‘New Drugs and Clinical Trials Rules,’ Ministry of Health and Family Welfare, Government of India, and the ‘Organization for Economic Co-operation and Development (OECD)’ guidelines. These two animals are the species accepted by regulatory agencies for conducting safety and toxicity studies. The acute toxic class method is a stepwise procedure using 3 animals of a single-sex per step. Depending on the mortality and/or the moribund status of the animals, on average, 2-4 steps may be necessary to allow judgement on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals, and can rank substances based on their toxicity. The acute toxic class method is based on biometric evaluations with fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The objective is to determine the possible health hazards of the formulation after repeated daily oral administration for 28 consecutive days. In a second set, the animals were allowed to recover for a further 14 days after a 28-day drug administration to test for reversibility, persistence, or delayed occurrence of toxic effects. The study will provide information on major toxic effects, target organs, if any, and determine the No-Observed- AdverseEffect-Level (NOAEL) of the Ayurvedic formulation. We tested Coronil and Divya Swasari Vati in both rats and rabbits. Preliminary studies showed that either formulation did not show any toxic effects for 28-day administration followed by a 14- day recovery period. We will present data obtained in the toxicity studies in an appropriate forum once the study is completed. This chapter mentions the protocols and standard procedures to be followed while conducting such studies. This is critical since the clearance for clinical studies is based on these toxicological observations, as is described in the next chapter.

This chapter is a guide on designing and executing clinical trials for traditional medicines. It includes the guidelines to be followed during protocol designing and study execution. Patanjali Research Institute combines deep therapeutic and scientific knowledge of Ayurvedic medicines with unmatched clinical trial design execution. Every patient who participates in a clinical trial plays a critical role in conquering disease and discovering cures for COVID-19 on behalf of all of us. Thus, with an in-depth understanding of the issues related to the clinical trial, Patanjali Research Institute has conducted human clinical trials and studies as per the ethical codes of biomedical research. 

This chapter essentially discusses the contribution of Patanjali Research Institute, Haridwar, in conducting research for human ailments in general and COVID 19 in particular. We strive to enhance the quality and understanding of healthcare and traditional medicine globally. We are committed to harnessing the power of Ayurvedic knowledge resources in order to discover, comprehend, and resolve unmet public health requirements. It can be assumed that by harnessing the power of Human Data Science, we can create new approaches to solving the world's most challenging health problems.