AYUSH- 64: A potential therapeutic agent in COVID-19.

Corona Virus disease (COVID-19) has become a global pandemic resulting in large scale morbidity and mortality worldwide. The management of COVID-19 has been a big challenge because of multifactorial pathophysiology and no specific treatment. AYUSH-64, a poly-herbal formulation developed by CCRAS, Ministry of AYUSH, Govt. of India through extensive pharmacological, toxicological and clinical studies has proven its safety and efficacy in infective febrile conditions such as malaria and influenza. AYUSH- 64 has four ingredients having immunomodulatory, anti-inflammatory, antipyretic, antioxidant and anti-viral activities. It arrests the extreme inflammatory responses in COVID-19 that causes progression to significant morbidity. AYUSH-64 has also been incorporated in the National COVID management protocol based on Ayurveda and Yoga by Government of India for asymptomatic and mild cases of COVID-19. Further, on the basis of tangible evidence generated through robust clinical and experimental studies on AYUSH-64, the Ministry of AYUSH has launched nation-wide campaign for mass distribution of AYUSH-64 to asymptomatic, mild to moderate COVID-19 patients in home isolation to reduce the burden on the hospital. This review will highlight the specifications of AYUSH-64, its mechanism of action, its repurposing for COVID-19, various clinical and experimental studies.

Introduction

Historically, outbreak of infectious diseases offers some model of the course of diseases and treatment such as Spanish flu, Bird flu etc. that unfold over the time. Some diseases have predictable seasonal peaks with high transmission as some pathogen may spread rapidly in dynamics of humidity, social mixing pattern leaving predictable pattern for each variant. The ‘wave’ of COVID-19 and its implications is the prominent topic of debate in current times. An epidemic wave/phase is defined as natural pattern of peaks and valleys which indicate the number of sick people and deaths in given time and then decline [1]. The first wave peaked in India in September 2020 and second wave started from 1st week of March 2021. The tragedy of second wave of COVID-19 and its impact on health care systems of our nation has been devastating and unprecedented [2]. There could be several factors responsible for the increased number of cases in the second wave. It is observed that the mutant strain of SARS-CoV-2 has higher transmission capability in addition to lesser incubation period. There has been a widespread disregard to the ‘COVID Appropriate Behavior’ by the general public, resulting in manifestation of severe illness, reduction in neutralizing antibodies and reduced effectiveness of vaccination.

The Ministry of AYUSH (MoA), Government of India has taken several public health and R&D initiatives to explore the potential of AYUSH systems to mitigate the impact of COVID-19 pandemic. Different institutions under the Ministry of AYUSH in collaboration with prominent medical and research organizations across the country to undertake research on COVID-19 through various AYUSH systems. The MoA constituted an inter-disciplinary AYUSH R&D Task Force consisting of scientists, pulmonologists, epidemiologists, pharmacologists etc., from premier organizations and research institutions to handle various aspects of clinical and experimental research through AYUSH interventions. The Ministry of AYUSH also recommended a set of self-care guidelines for preventive health measures, with special emphasis on respiratory health and improving general immunity. The Ministry of AYUSH further issued the National Clinical Management Protocol based on Ayurveda & Yoga for management of COVID -19 to enable uniform clinical management [3]. The management guidelines advised in the protocol are based on the interim trends and outcomes of AYUSH COVID-19 studies along with the published evidence related to the safety and potential benefits of Ayush interventions.

Brief about AYUSH-64

AYUSH-64, a polyherbal formulation developed by Central Council for Research in Ayurvedic Sciences, Ministry of AYUSH through extensive pharmacological, toxicological and clinical studies. It has proven saftey and efficacy in infective febrile conditions such as malaria, microfilaremia, chikungunya and influenza [[4], [5], [6], [7], [8], [9]]. Further, AYUSH 64 was found safe and non-toxic in a dose of 500 mg/kg body weight for 12 weeks in experimental studies [10,11]. Quality control and safety parameters of the ingredients of AYUSH-64 and the final formulation is in complience with the Ayurveda Pharmacopoeia limits. The constituents of AYUSH-64 viz. Saptaparna (Alstonia scholaris R. Br.), Katuki (Picrorhiza kurroa Royle ex. Benth), Kiratatikta (Swertia chirata Pexbex. Karst) and Kuberaksha (Caesalpinia crista L.) are reported to possess immunomodulatory, anti-inflammatory, antipyretic, antioxidant and anti-viral activities [[12], [13], [14], [15], [16], [17]]. These effects could inhibit the extreme inflammatory responses in COVID-19 that causes progression to significant morbidity. In a prospective, open-label, non-randomized pre-test and post-test design pilot study on AYUSH-64 in Influenza like Illness (ILI), one-week intervention of AYUSH-64 in a dose of 3 gm/day provided early clinical recovery from 'Influenza Like Illness' (ILI) symptoms with reduced frequency of usage of acetaminophen/antihistaminic [9]. The results of a Molecular Docking study revealed that the presence of Mpro–Akuammicine N-Oxide with highest Mpro binding energy along with other 34 phyto-constituents having similar anti-viral activity against SARS- CoV-2 as part of AYUSH-64 make it a suitable drug/medicine for repurposing for the COVID-19 management [18].

New indication for COVID-19

AYUSH-64 was repurposed for COVID-19 based on the recommendations of Interdisciplinary AYUSH R & D Task Force constituted by the Ministry of AYUSH along with available quality standards, evidences on clinical and pre-clinical safety [[4], [5], [6], [7], [8],10,11], evidences on efficacy in Influenza-like illness [9]. The new indication was based on anti-viral and immune–modulator activity [[12], [13], [14], [15], [16]] as well as evidences drawn through molecular docking study discussed in previous section [17]. As per the clinical trial registry of India, total seven clinical studies on AYUSH-64 in asymptomatic and mild to moderate COVID-19 cases were undertaken by reputed medical institutions across the country. AYUSH-64 was administered in the management of asymptomatic & mild cases of COVID-19 as standalone treatment and for the management of mild and moderate COVID-19 as an adjunct to standard care in these clinical trials. The outcomes of these studies demonstrated that AYUSH-64 as adjunct treatment to standard care resulted in early clinical recovery compared to standard of care alone without progression of the disease to severe or critical stage [[18], [19], [20], [21]]. The mean time to negative RT-PCR assay for COVID-19 was also better in the AYUSH-64 add-on group [18,20]. Also, there was improvement in Quality of life (QoL) parameters [18]. AYUSH-64 was found to be well tolerated and safe.

Based on the leads generated through experimental and clinical studies, AYUSH-64 has been incorporated in the National COVID management protocol based on Ayurveda and Yoga for asymptomatic and mild cases of COVID-19 [3]. AYUSH-64 is further included in the “Guidelines for Ayurveda Practitioners for COVID-19 Patients in Home Isolation” issued by the Ministry of AYUSH during the second outbreak of COVID-19 in the country [22]. The Ministry of AYUSH has also launched nation-wide campaign for mass distribution of AYUSH-64 to asymptomatic, mild to moderate COVID-19 patients in home isolation to reduce the burden on the hospital-based health care delivery system [23].

The objective of this review is to scientifically explain the general therapeutic approach of AYUSH-64 to intervene in disease progression during various clinical and pathogenic stages of COVID-19.

COVID-19: window of transmission and infection dynamics

SARS-CoV-2 virus spreads through respiratory droplets of infected individuals. The average incubation period ranges from 1 to 14 days and mean is 5–6 days [24]. The infectivity is higher during initial period of infection; however patient can remain for an average of two weeks. The window of infected state varies from person to person; some individual may remain infected for several weeks and remain asymptomatic are known as long term spreader. A large proportion of individuals affected with COVID-19 are infected either with pre-symptomatic or with asymptomatic transmission. So, keeping track of these variants is very vital for this pandemic control. Nearly 80% persons have been asymptomatic or display mild symptoms, 15% display moderate to severe symptoms and require oxygen support and 5% patients have been critical with an immediate need for mechanical ventilation even during second outbreak of COVID-19. The mortality varies from country to country and is approximately 11% worldwide in hospitalized patients, although it was less than 1.1% in India [25].

The viral dynamics reveal that there is no difference between viral load and severity of COVID-19 and its clinical outcome. The infection clears by itself in early stage in mild cases, while severe cases have prolonged viral shedding. The studies so far have shown conflicting evidence in regard to the viral shedding kinetics. The patient can continue to shed the virus even after the symptom resolution. The mean duration of viral shedding is 20 days among the survivors. It has also observed that some patients with mild symptoms of COVID-19 initially can suffer from variable and debilitating symptoms for more than two months of the initial infection referred as long-term effects of COVID-19 [26].

COVID-19: clinical features and immune response

The clinical features vary from patients to patient depending on the individual's immune response. The clinical category of COVID-19 include asymptomatic (positive RT-PCR without symptoms), mild to moderate (positive with clinical manifestations) or severe and critical (positive with high degree of manifestations). The common symptoms of COVID-19 include fever (83–98%), fatigue (70%), dry cough (82%), headache (34%), dyspnea (50%), sore throat (14%), rhinorrhea (7%), anosmia (Loss of smell) and ageusia (Loss of taste) (<7%), diarrhea (20%), vomiting (14%) and weakness (70%) in the hospitalized patients [[27], [28], [29]] along with a significant increase of C- reactive protein (>20 mg/dl). The CRP concentration is worse in severe (>40 mg/dl) and critical cases (>100 mg/dl) compared to mild cases (<18 mg/dl) [30]. The raised absolute lymphocyte count (<100 cells/μl) is also found in severe patients [31].The increased respiratory rate >30/min and SpO 2 <90% are the indicators of severe COVID-19. Some patients proceed to critical stage as observed by systemic inflammation due to cytokine storm and widespread platelet aggregation leading to multi-organ failure as the end stage of COVID-19.

An infected person initially moves through stage of replication over first few days followed by a stage of adaptive immunity over the next few days. In the replication stage, the virus replication leads to flu-like illness characterized by mild symptoms due to direct cytopathic effect (structural changes in host cells) of the virus. In the stage of adaptive immunity, virus levels decline as immune system takes over, but in inflammatory phase, there is a possibility of cytokine storm leading to tissue destruction and clinical deterioration [32].

Pathological progression of COVID-19

To develop effective therapeutics and preventive measures against COVID-19, an accurate and precise understanding of its pathogenesis at the molecular level is needed. Based on the most recently published literature, the overall COVID-19 pathogenesis process can be summarized as three clinically distinct and potentially overlapping phases. As shown in Fig. 1, its pathological progression can be classified in chronological order as viral replication or early infection, pulmonary, pro-inflammatory, and pro-thrombotic phases [33].

Fig. 1

Clinical stages of COVID-19.

Mechanism of action of AYUSH-64

The chemical constituents and therapeutic indications of the four ingredients of AYUSH-64 are given in Table 1. As per the published evidence, the ingredients of AYUSH-64 have the potential to control the initial symptoms of COVID-19, mitigate the cytokine storm through the inhibition of cytokines, inhibition of Angiotensin-Converting Enzyme II (ACE2) and Reactive Oxygen Species (ROS).

Table 1

Chemical constituents and indications of four constituents in AYUSH-[64].

Name of the Medicinal Plant	Major chemical constituents	Indication as per Ayurveda
Saptaparna (Alstonia scholaris R. Br.)	Akuammicine N-Oxide, Akuammiginone, Echitaminic acid Echitamidine N-oxide, Alstonides, alpha -amyrin	Jvara (disease conditions associated with pyrexia), Kasa(cough), Swasa (dyspnea), Pravahika (dysentry)
Katuki (Picrorhiza kurroa Royle ex. Benth)	Picroside I and II, kutkoside, cucurbitacins, vanillic acid, cinnamic acid, ferulic acid apocynin, saptoparna, amarogentin, eufoliatorin, kutkin	Visamajvara (disease conditions associated with recurrent pyrexia), Kasa (cough), Swasa(dyspnea)
Kiratatikta (Swertia chirata Pexbex. Karst)	Xanthine, Xanthine glycoside, flavonoids, iridoid glycocide, triterpenoid	Visamajvara (disease conditions associated with recurrent pyrexia), Kasa (cough), Swasa(dyspnea)
Kuberaksha (Caesalpinia crista L.)	Diterpines, caesalpinin N, Diterpenoids, Casalmin, NonCasalpine-E, caesalpinins	Jvara (disease conditions associated with pyrexia), Kasa(cough), Swasa(dyspnea), Sopha(inflammation)

Anti viral activities isolated molecules of AYUSH-64

The isolated molecules of medicinal plants present in AYUSH-64 have antiviral activities against COVID-19 through potentiate the production of arginine (Amino acid) to inhibit the activity of spike glycoprotein of SARS-CoV-2 as well as ACE2. The α-amyrin and Alstonides present in saptoparna, amarogentin, eufoliatorin, kutkin present in Katiki and caesalpinins in Kuberakshya (Table- 1) are the top ranked molecules with highest affinity towards both the spike glycoprotein and ACE2 [34]. In addition AYUSH-64 as add on treatment with conventional care is reported to be effective in reducing the duration of symptometic stage [35].

Effect of AYUSH-64 in SARS-CoV-2 induced pro-inflammatory process

The majority of COVID-19 patients have subnormal or reduced leucocyte count, lymphocytopenia, increased concentration of interleukins and tumour necrosis factor α. Interleukin (IL-6) is a pleiotropic, proinflammatory cytokine produced by a variety of cell types, including lymphocytes, monocytes, and fibroblasts. Infection by SARS-CoV induces a dose-dependent production of IL-6 from bronchial epithelial cells. SARS-CoV-2 virus enters through ACE2 via TLR-7 (Tell like Receptor-7) which activates the pro-inflammatory kinases. AYUSH -64 is rich in phytoacids and has acidic pH. The anti-viral activities of AYUSH-64 may be achieved by increasing pH of intra-cellular vacuoles and decreased endosomal activities similar to other anti-malarial drugs [34]. The immediate immune responses to COVID-19 are deregulation of central metabolism for mobilization of energy, cells and biomolecules [[35], [36], [37], [38]]. This deregulation of central metabolism is called Amadosha (undigested byproducts of digestion and metabolism) in Ayurveda. Saptaparna and Katuki have Deepana and Pachana properties (enhancing digestion and metabolism) which clear the Amadosha. So, AYUSH-64 may be reprogramming the host metabolism and regulate the enzymatic activities and biosynthesis to generate antiviral defense response. Moreover, anti-inflammatory and antioxidant activities of S. chirata Pexbex. Karst potentiates its action [39].

Effect of AYUSH-64 on potential agents for pulmonary impairment

COVID-19 begins in upper respiratory tract but replicates quickly in ACE2 receptor rich respiratory mucosa. After reaching the alveoli, virus begins to replicate fast in pneumocytes causing cell death. This cellular damage accelerates the appearance of multinucleated giant cells and fibrin rich hyaline membrane. All the phytoconstituents of AYUSH-64 have cough and dyspnea relieving properties which reverse the inflammatory process and reduce fibrosis. The concept of Pramathi (clearing property of cell debrides) is extremely useful for downward regulation of fibrin deposition. The alkaloids from A. scholaris R. Br. inhibit Influenza A virus replication and lung immunopathology by regulating the innate immune response [40,41]. Research studies found that Neutrophil Extracellular Traps (NET) is the main culprit of pulmonary dysfunction and AYUSH-64 decreases the total neutrophil counts of COVID-19 patients. The clinical indicators of COVID-19 patients (fever, cough and dyspnea) decreased in AYUSH-64 group indicating that AYUSH-64 has a promising efficacy in preventing lung impairment [[42], [43], [44], [45]]. C. crista L. has excellent therapeutic potential to reduce fever, cough and clears the lungs [46].

Effect of AYUSH-64 in SARS-CoV-2 induced hyperinflammatory and prothrombin phase

The inflammatory phase of COVID-19 passes through various mediators activating haemocytic system through endothelial dysfunction, platelet activation in addition to micro and macro vascular thrombosis. Human platelets express through ACE2 and TMPRSS2 receptors. SARS-CoV-2 and its Spike protein directly enhance the platelet activation by binding to these receptors [47]. P. kurroa Royle ex. Benth in AYUSH-64 possess thrombolytic action as described in Ayurveda. Another view is that there is an elevation of D-dimer in critical patients of COVID-19 and AYUSH-64 decrease the levels of D-dimer significantly. Immunosuppressive effect of S. chirata Pexbex. Karst may reduce the inflammatory process [48].

Conclusion

The ingredients of AYUSH-64 have the potential to control the initial symptoms of COVID-19 and the cytokine storm, inhibition of the Angiotensin-Converting Enzyme II (ACE2) and Reactive Oxygen Species (ROS) and thereby prevention of the progression of COVID-19. Taking leads from the outcomes of clinical study on influenza-like illness and molecular docking study, AYUSH-64 has been repurposed for the management of asymptomatic and mild to moderate cases of COVID-19 as standalone therapy or adjunct to standard care in several clinical trials with promising results. Thus, AYUSH- 64 could play a significant role in reducing the large-scale morbidity associated with COVID-19 and reduce the burden on the hospital-based health care delivery system by effectively managing the home-isolated cases.

Source of funding

Nil.

Conflict of interest

None.

Author contributions

Contribution of Dr. A.K. Panda - Create a concept to write a review article and prepare the model. Creation of research group and preparation of initial draft. Worked as corresponding author after all the authors agreed for it. Contribution of Dr. S. Kar - Help in preparation of initial draft. Oversight of the manuscript. Contributions of A.K. Rai - Collection of evidence and computing the data and writing of final manuscript. Contribution by BCS Rao - supervision the manuscript and help in writing the final draft. Contributions of N. Srikanta - Mentorship and leadership of the core idea, supervision of the manuscript, set the title and approve the final draft.