An integrative approach to clinical recovery for COVID-19 patients using an Ayurvedic formulation: A multicentric double-blind randomized control trial.

Traditional medicine systems such as Ayurveda contain a vast repository of naturally occurring herbs with strong antimicrobial potency. A multitude of complementary therapies have been explored for their therapeutic role to treat COVID-19 during the pandemic. There have been promising results reported from in silico, in vitro and in vivo studies that need to be further explored in humans. The present randomized placebo control trial evaluates the clinical ecacy of an integrated approach including Ayurvedic polyherbal formulation, NOQ19 in the improvement of mild to moderate category of COVID-19 infected patients. multicentric, positive COVID-19 Patients were randomized and blinded to their respective intervention, which was provided along with the standard of care treatment. Rate of recovery assessment was done on Day 3, 5 and 7 via RT-PCR test to measure the rate of recovery. Blood markers were analysed on Day 0 and Day 7. In addition, several other components of have been for their antiviral . Yashtimadhu (Glycyrrhiza glabra) contains glycyrrhizin, a strong antiviral compound. In a previous study, treatment with different concentrations of glycyrrhizin lowered SARS-CoV viral in a cell culture. At a concentration of 4000mg/ ml, Glycyrrhizin completely blocked the viral


Introduction
The COVID-19 pandemic has created an unprecedented wave of illness and loss of life compared to any other outbreak in the past few decades. Health care professionals, pharmaceutical industry and scientists from various countries have been investigating pre-existing and novel compounds as potential solutions to end this pandemic 1 . One of the major milestones to protect the population from the SARS-CoV-2 virus, was the development of safe and e cacious COVID-19 vaccines. To date, 6.34 billion vaccines have been administered globally. Almost half of the world population (45.7%) has received at least one dose and 2.68 billion are fully vaccinated 2 .
Traditional Indian systems of medicine like Ayurveda and Siddha have also emerged with pragmatic solutions during the COVID-19 outbreak. A review of the literature suggests many single drugs or compound formulations emanating from Ayurveda can boost immunity and provide antiviral activity against SARS-CoV-2 [3][4][5][6][7] . Preclinical studies also provide robust evidence of antiviral potency of various phytochemicals present in nature. In silico studies of well-known immuno-boosters like Ashwagandha (Withania somnifera), Guduchi (Tinospora cordifolia) and Tulsi (Ocimum sanctum) show strong docking properties against SARS-CoV-2 viral proteins 8 . The phytoconstituents of Ashwagandha (Withania somnifera), Withanoside V showed a binding energy of -8.96 Kcal/mol with the M pro protein of the virus 9 . A randomized clinical trial on Ashwagandha (Withania somnifera) demonstrated an improvement in markers of vaccine response, such as IgG titer, when Ashwagandha was given along with vaccine, in comparison to receiving vaccine alone 10 .
Similarly, compound sesline from Bilwa (Aegle marmelos) and several other compounds from Guduci (Tinospora cordifolia) and Tulsi (Ocimum sanctum) may serve as a potential inhibitor against the Mpro protein of SARS-CoV-2 virus, as shown in in-silico studies 8, [11][12] . Other in-silico studies demonstrate that phytochemicals present in herbs used regularly as a part of Indian diet, like Bilwa (Aegle marmelos), Khus (Vetiveria zizanioides), Drumstick (Moringa Oleifera), and Pomegranate (Punica granatum) exhibit signi cant binding with RdRp and Mpro proteins of SARS-CoV-2 virus 13 . Components of Yashtimadhu (Glycyrrhiza glabra), another well-known Ayurvedic herb, were noticed to have signi cant binding a nity with various SARS-CoV-2 proteins (main protease, spike protein and RdRp protein) and host macromolecular targets such as human (ACE2 and furin) proteins 14 . Vasaka (Adhatoda vasica), another potent Ayurvedic compound, exhibited a strong binding energy with Mpro of SARS-CoV-2 (−114.9 kcal/mol) 15 . In vivo studies among animal models showed that ayurvedic nasal formations limited the viral entry and replication 16 . These are just a few ayurvedic herbs and compounds that have shown antiviral properties against SARS-CoV-2 and can be used in the management of COVID-19.
Bhunimba (Andrographis paniculata), a well-known Ayurvedic herb, is used to treat viral and microbial infections 17 . An in-vitro study of Bhunimba (Andrographis paniculata) and Chiretta (Andrographolide) in SARS-CoV-2-infected Calu-3 cell lines (infected human lung epithelial cells) showed that these herbs signi cantly inhibit the production of infectious virions with an IC50 of 0.036 µg/mL and 0.034 µM, respectively 18 . One of the other mentioned uses of Bhunimba (Andrographis paniculata) is its antithrombotic effect. There is evidence of thrombotic pathways being implicated in COVID-19 with ample instances of thrombotic events leading to mortality in patients 19 . Due to its antithrombotic activity, Bhunimba (Andrographis paniculata) can play a signi cant role in the management of patients with COVID-19. The crude extracts of Bhunimba (Andrographis paniculata) also demonstrate an antithrombotic property in-vitro 20 . Haridra (Curcuma longa), another constituent of NOQ19, has excellent anti-in ammatory properties and thereby can regulate the cytokine release during the COVID-19 infection. Curcumin present in Haridra, regulates the Toll-like receptors, in ammatory cytokines and chemokines, which play a major role in pathophysiology and progression of the disease. Due to its phenolic nature, it also has an intensive antimicrobial effect 21,22 .
A previous in vitro study conducted on NOQ19 demonstrated its antiviral e cacy among Vero E6 SARS-CoV-2 infected cell lines. The treatment of virus infected cells with 0.9mg/ml concentration of NOQ19 resulted in a 100% viral elimination 23 . The NOQ19 was further tested in the Syrian golden hamsters animal model. A 78.2% viral load reduction in hamsters lungs was observed with NOQ19. No toxicity was reported in the animal model 24 . With the preclinical e cacy established, the drug was further tested in an open label feasibility study.
The study showed a 74% rate of recovery among the COVID-19 patients after 5 days of NOQ19 consumption 25 .
To evaluate further effectiveness and therapeutic role of NOQ19, this multicentric study was conducted with the hypothesis that NOQ19 will contribute towards early recovery of patients in mild-moderate SARS-CoV-2 infection.

Trial design
A multicentric, double blind randomized control design was adopted for this study. The study was coordinated The data was collected across the sites from June 7th to August 28th, 2021. Each patient was monitored for 7 days or until they turned RT-PCR negative.

Participants
A total of 76 participants, from the In-patient ward (IPD) or the Out-patient clinic (OPD) at the three study locations, were enrolled in the study. The patients who presented with COVID-19 symptoms and had a positive RT-PCR test were briefed about the study by research fellows in the presence of the doctor on duty, at all three locations. Based on their medical history, patients who met the eligibility criteria, mentioned below, were invited to participate in the study and informed consent was obtained.

Intervention
All the patients were provided a bottle of intervention drug or placebo based on the computer generated sequence of randomization. Each bottle contained 90 tablets. The patients were advised to take 2 tablets, under supervision, thrice a day for 14 days, after food. Both the placebo and NOQ19 bottles were packaged identically to avoid bias. Compliance was monitored by the data collector individually over the phone for OPD patients and in person for IPD patients.

Comparator group
The comparator group in this trial were given a placebo tablet. The placebo tablet was made of starch (100%).
The placebo tablets were also tested for quality control and packed in bottles identical to NOQ19.

Standard of care treatment
The standard of care treatment was administered as per the guidelines provided by the Ministry of Health and Family welfare, Government of India, during the second wave COVID-19 27 . However, the drugs were slightly different at each study location. The standard of care treatment at each location is described below:

SMVMCH Puducherry and AIIMS Rishikesh:
Patients in SMVMCH Puducherry and AIIMS Rishikesh included both mild and moderate patients and were administered the following standard of care treatment.

Mild Patients:
OPD patients with mild disease were prescribed Tablet Azithromycin (500 mg, one tablet, OD 5 days), Vitamin C (one tablet, BD 7 days), Zinc (one tablet, OD 7 days), Tablet Ivermectin (12 mg OD 3 days), Cap. Doxycycline The Primary Outcome measured was the rate of recovery among patients in NOQ19 and Placebo arms, using the RT-PCR negative test. One nasal swab and one throat swab was collected and subjected to analysis for viral load reduction at Day 3, 5 and 7.

Secondary Outcomes:
The secondary outcomes included assessment of clinical markers and blood biomarkers, both of which served as an indication of clinical improvement post intervention. The clinical markers of signi cance for C OVID-19 infected patients, especially moderate patients, measured in this study included fever, dyspnoea (breathlessness), SpO2 levels and need for oxygen support. Blood biomarkers included levels of total lymphocyte count, IL-6 (cytokine marker) and CRP (in ammatory marker). The study also recorded any adverse events or side effects from the drug.

Follow up and Assessment:
The RT-PCR tests were conducted on Day 0, Day 3, Day 5 and Day 7. Blood biomarkers were tested on Day 0 and Day 7. Other clinical markers were assessed on Day 0, Day 3, Day 5 and Day 7. Patients were monitored for a maximum of 7 days, or until RT-PCR negative result, whichever was earlier. Information about any adverse events reported by the patients following the intervention was also collected.

Sample calculation
Based on the sample size formula 28 , a sample size of 380 was calculated for the overall study.
z is the z score ε is the margin of error N is the population size p is the population proportion However, due to the decrease in the cases of COVID-19, a total of 76 patients with positive COVID-19 infection were enrolled, 39 in the NOQ19 arm and 37 in the placebo arm and the data is presented as an interim report.

Randomization and blinding
A computer-generated sequence of randomization was adopted for the study. Based on the in ow of patients at each study site, the participants were randomized to the two study groups in a 1:1 ratio. The randomization was conducted by a research associate who was not involved in the study. The patients were assigned the respective intervention by a nurse who was not a part of the study. Along with the patients, the lab technician, data collector, data operator and statistician were blinded to the randomization group.

Statistical analysis
An Intention to Treat (ITT) approach was utilized for data analysis. The patient data was pooled from the three sites. The baseline characteristics were reported as proportion/ mean (SD) for both the study groups.
Compilation and analysis of the data was performed in Microsoft Excel 2019 (16.0.12026.20334) 32-bit. Data was tested for normality using the IBM SPSS statistics software version 2.0. The blood parameters were evaluated by descriptive statistics. Association between variables of qualitative nature was tested by Student's T Test (paired and unpaired) wherever appropriate at signi cance of 5%. Quantitative data not normally distributed in nature was analyzed using Mann-Whitney Test (for two groups) and Independent t test was used for comparison of normally distributed data between two groups at a 95% con dence interval.

Sub-group analysis
The data was further separated into mild and moderate patients based on Government of India guidelines 26 .
The categorization as per the guidelines was on SpO2 levels or respiratory rate. The clinical improvement in moderate patients was further evaluated.

Results
The present study evaluates the rate of recovery and clinical improvement in SARS-CoV-2 infected patients following an Ayurvedic intervention called NOQ19. The study enrolled a total of 76 patients. Patients who turned RT-PCR negative before day 7 were not considered as drop outs.
A comparison of demographic characteristics demonstrated similarities in both study arms, across the three sites (Table1). No signi cant variation was noticed in the age, height, weight, or severity of illness of the study population at the time of enrollment. Clinical parameters such as temperature, respiration rate, SpO2 and pulse rate of recruited subjects were also collected. Majority of the patients in the study were diagnosed with mild infection, and a few as moderate.  experiencing breathlessness was lesser in the placebo arm at baseline, and the placebo arm showed a gradual improvement in breathlessness, slower than the NOQ19 arm. A correlating trend was also observed in the percentage of people requiring oxygen between both arms. The percentage of patients requiring oxygen rapidly decreased in the NOQ19 arm and none of the patients required oxygen support by day 7, however, the number of patients requiring oxygen support in the placebo arm remained constant throughout the time points. Although both arms experienced an improvement in the blood biomarkers, no signi cant changes were noted in IL6, CRP or TLC values between the two study groups at day 0 or day 7. None of the patients in the NOQ19 arm reported any adverse event, allergies or discomfort after taking NOQ19 daily.

Discussion
Ayurveda, an ancient Indian system of medicine contains a vast repository of plants and herbs with therapeutic properties. In the ght against COVID-19, herbal extracts with robust scienti c evidence of therapeutic e cacy against SARS-CoV-2 can support rapid recovery and reduce mortality due to COVID-19.
Our study investigates the effectiveness of NOQ19, an Ayurvedic polyherbal formulation, in clinical improvement and rate of recovery from the SARS-CoV-2 infection. This multicentric trial demonstrates an early recovery among patients who took NOQ19 along with standard of care treatment, compared to those who received standard of care alone.
The study was conducted during the second wave of COVID-19 in India. Along with other upper respiratory symptoms, the chief complain presented by patients during the second wave was shortness of breath associated with low SpO2 levels, requiring oxygen support 27,29 . In our study, a signi cant clinical improvement was noted in patients among the NOQ19 arm who had experienced lower oxygen saturation and breathlessness at baseline. Patients who received NOQ19 with the standard of care treatment showed an improvement in terms of oxygen requirement, breathlessness and SpO2. Thus, despite lack of intergroup statistical signi cance, the clinical signi cance of these ndings cannot be ignored.
The second wave of COVID-19 in India was twice as severe as the rst wave and created immense pressure on the health infrastructure and economy of the country 30 . Of the total deaths reported in India, 59% deaths were reported during the second wave 31 .The study was conducted in a hospital, where as per current guidelines, only patients with signi cant symptoms or considerable comorbidities were included. In the current study, an early clinical recovery was observed among moderate patients. With improvement in oxygen levels and clinical markers, it was noted that patients in the NOQ19 arm were feeling much better than the placebo arm. Also, a lesser number of patients in the NOQ19 arm required hospitalization on day 7. Therefore, this ayurvedic drug can aid early discharge, allowing rational utilization of limited resources, reduced burden on the healthcare system and support management of the illness at home.
Previous studies on the same formulation NOQ19 have shown promising results with respect to rate of recovery and antiviral e cacy [23][24][25] . A review of Ayurvedic literature presents robust preclinical evidence for e cacy of multiple NOQ19 ingredients such as Ashwagandha (Withania somnifera) and Guduchi (Tinospora cordifolia), along with Amalaki (Phyllanthus emblica) in proliferation of B and T cells and activation of nonspeci c immunity 32 . In addition, several other components of NOQ19 have been highlighted for their antiviral properties previously 8, 33,34 . Yashtimadhu (Glycyrrhiza glabra) contains glycyrrhizin, a strong antiviral compound. In a previous study, treatment with different concentrations of glycyrrhizin lowered SARS-CoV viral antigen in a cell culture. At a concentration of 4000mg/ ml, Glycyrrhizin completely blocked the viral replication 35 .
A key observation of our study was the improvement in oxygen levels and reduction in breathlessness among The authors acknowledge the limitations of the study and present further scope to improve the limitations.
One of the key limitations of the study was the small sample size. Due to the small sample size, several clinically signi cant ndings were not statistically signi cant. Secondly, although the blood biomarkers were captured, eg. IL-6 and D-dimer, no signi cant difference was noticed among both the groups. A possible reason for this could be that the duration between the two measurements was not long enough to notice any signi cant difference. Further studies with larger sample size and relatively longer duration can investigate in depth e cacy and mechanism of action of NOQ19 in patients with COVID-19. The ndings of this study create an interesting backdrop for exploring the role of the NOQ19 in moderate to severe COVID-19 patients on oxygen therapy.