Evaluation of an Ayurvedic formulation in clinical recovery of COVID-19 patients: A placebo controlled pilot study among moderate-severe patients

Introduction: The COVID-19 pandemic is a global health crisis that has affected millions of people worldwide. Apart from measures taken to reduce the transmission of virus and provide symptomatic relief to patients, a potent drug targeted against the virus is required to manage the disease. Traditional medicines with an array of phytochemicals and potent anti-viral properties, can provide alternate solution in the therapeutic cure of COVID-19. Objective: The present study investigates the ecacy of an Ayurvedic formulation, NOQ19, on the rate of recovery and clinical improvement among moderate-severe COVID-19 infected patients who were not on a ventilator or in intensive care unit. Methods: A placebo controlled randomized study design, with 1:1 ratio between the intervention and placebo arms was adopted. The participants were provided their respective intervention along with the standard of care treatment. A follow up was conducted on Day 7 and Day 10 to assess the rate of recovery and clinical improvement. Rate of recovery was the primary outcome measured and was determined by a negative result on the RT-PCR test. The secondary outcomes included clinical improvement among patients and were measured using blood biomarkers. Results: The NOQ19 arm had a higher percentage of population who turned RT-PCR negative on Day 7 (23%) when compared to the placebo arm (11%). Further subgroup analysis demonstrated that for both moderate and severe patients, there were a greater percentage of population who were RT-PCR negative in the NOQ19 arm compared to the placebo arm at Day 7: (NOQ-19: 22.22%, Placebo: 14.29% for moderate patients), (NOQ19: 23.08% Placebo: 0% for severe patients). By Day 10, both populations depicted an almost similar percentage of the population who were RT- PCR negative. Both the groups demonstrated an improvement in blood biomarkers, although the improvement was greater in the NOQ19 arm. None of the patients in the intervention arm reported any adverse events. Conclusion: The results of this pilot RCT indicates that NOQ19 along with standard of care treatment can be considered as an effective therapy for COVID-19 and aid early recovery from the disease.


Introduction
Coronavirus disease  is an infectious disease caused by the SARS-CoV-2 virus. The rapidly growing outbreak of COVID-19 and resulting public health crisis has garnered worldwide attention1, affecting 200 nations across the world2. COVID-19 was declared a pandemic by the World Health Organization (WHO) on March 11, 20203. Supportive therapy such as ventilation to optimize oxygen saturation and uid management to intercept respiratory failure are the mainstay of current COVID-19 management4. Currently, there is a need for an antiviral drug with a targeted approach towards the virus that can reduce the viral load or cease viral replication5.
Ayurveda, an Indian traditional system of medicine, recommends several potent herbs and associated formulations for the prophylactic and therapeutic management of COVID-196. Our study focuses on an Ayurvedic polyherbal formulation containing well-known popular Ayurvedic drugs such as Ashwagandha (Withania somnifera), Yashtimadhu (Glycyrrhiza glabra), Pippali (Piper longum), and Guduci (Tinospora cordifolia). Several of these drugs have been recognized for their antiviral potency7-12. Phytochemical analysis of multiple Ayurvedic herbs used for their antimicrobial properties, demonstrates the presence of phenols, alkaloids and avonoids, which contribute towards their antimicrobial activity13. A review of literature on individual components and their phytoconstituents highlights the importance of blending selected herbs into a formulation14. In silico studies on Ashwagandha (Withania somnifera) have demonstrated its antiviral properties against SARS-CoV-215-16. Among its chief phytochemical components are Withnolides, which play an active role in inhibiting viral entry into the host cells. Potential targets of Withnolides include TMPRSS2 and Mpro receptor protein and viral proteins. These proteins play an important role in viral entry and viral replication inside the host cell17. Another molecular docking study of Ashwagandha (Withania somnifera), Guduchi (Tinospora cordifolia) and Shatavari (Asparagus racemosus) demonstrated that the combined effect of phytochemicals from the three herbs target against the spike protein, main protease and RNA dependent RNA polymerase enzyme of the virus required for replication18. Yashtimadhu (Glycyrrhiza glabra), another potent antiviral drug, has been studied in silico for its mechanism of action. In silico studies highlight glycyrrhizin as one of the best antiviral chemicals against SARS-CoV-219. An animal study also highlights that glycyrrhizin plays an important role in reducing the number of Angiotensin converting enzyme 2 (ACE2) receptors in the lung tissue, which is the main binding site of SARS-CoV-2 in humans20.
Vasaka (Adhatoda vasica) is a well-known anti-in ammatory, antiviral and antitussive herbal drug.22 Known for its antithrombotic properties, Bhunimba (Andrographis paniculata) prevents blood clotting. Blood clotting is one of the severe clinical presentation of SARS-CoV-2 virus23 and antithrombotic properties of Bhunimba (Andrographis paniculata) can reduce blood clotting. A common spice used in Indian cooking with excellent antiviral properties is Tumeric or Haridra (Curcuma longa)24. Curcumin, the chief component of Haridra is a phenolic compound which has an inhibitory effect against Toll-like receptors, in ammatory cytokines and chemokines.25-26. This is crucial in COVID-19 since it can regulate the cytokine levels and inhibit the cytokine storm.
A previous in vitro analysis of NOQ19 formulation demonstrated a 100% antiviral e cacy at 0.9mg/ml against SARS-CoV-2 virus in a Vero E6 cell based assay27. Further evaluation of NOQ19 in an in vivo model using Syrian golden hamsters demonstrated a 78.2% e cacy and no toxicity in animals. The gross lung pathology and body weight reduction improved signi cantly in the NOQ19 group of hamsters28. The present study aims to determine the clinical e cacy of the above mentioned Ayurvedic herbs in a formulation, namely NOQ19, in recovery from COVID-19. Based on the previous literature, in vitro and in vivo studies on NOQ19, we hypothesize a strong therapeutic activity of this polyherbal formulation in COVID-19 patients.

Trial design
The present study was a pilot, single centre, double blind, randomized control trial. The study was approved by the Institutional ethics committee of Atharva multi-speciality hospital and research, with the registration number IEC/04/17/21/01. The study protocol was in compliance with the Helsinki Declaration and Good Clinical Practice. Adults presenting with moderate to severe COVID-19 infection, without requiring ventilation, at the OPD or IPD were screened for eligibility and invited to participate in the study. Informed consent was obtained from patients willing to participate in the trial. The study was conducted from 19th April to 28th May, 2021. Each patient was monitored for 14 days from the time of enrollment. The study has been registered at Clinical Trial Registry India after the completion of this pilot study with registration number CTRI/2021/10/037423 Patients enrolled in the study were provided respective intervention along with the standard of care treatment.

Participants
The patients received a bottle containing 90 tablets each, as per their intervention. They were advised to take two tablets (500mg each) TID (thrice a day), under supervision (if IPD), along with the standard of care treatment. Compliance was monitored by the study team daily for each patient individually, in person for the patients admitted in the hospital while out patients were monitored telephonically.

Comparator group
The placebo control group was provided with the placebo tablets (made of only starch), which were packed in bottles identical to the NOQ19 bottle.

Standard Treatment of Care
Standard of care treatment included allopathic medicines as prescribed by the Government of India29.
Moderate patients admitted in the hospital were given Inj. Doxycycline, Inj. Azithromycin, Antipyretics and Supportive care i.e Fluids, Intravenous Steroids like Dexamethasone, Supportive oxygen therapy, Nebulization, Vitamin C and Zinc. Patients who were clinically stable were treated on OPD basis and did not require hospitalization. They were given Tab Doxycycline 100mg BD for 5 Days, Azithromycin (500mg)-one tablet, OD, for 5 days, Vitamin C one tab TID, Tab Ivermectin 12mg OD for 3 days, Zinc, one tablet, OD and Paracetamol (500 mg) when required.

Outcomes
After enrollment, the patients were monitored for 14 days. All patients were followed up for RT-PCR test on Day 0, Day 7, Day 10 to monitor viral load changes. One nasal and one throat swab were obtained for RT-PCR analysis.

Primary Outcome:
The primary outcome was the rate of recovery from COVID-19 based on RT-PCR negative test results.

Secondary Outcomes :
Secondary outcomes included clinical improvement among the patients as evaluated by blood biomarkers and chest CT score. In ammation markers (CRP), antithrombotic activity (D-Dimer), cytokine levels (IL-6), HR-CT chest score and S-Ferritin were measured. The blood parameters and immune markers were measured on Day 0 and Day 10 to observe any signi cant changes.

Sample size calculation
Being a pilot study, as a rule of thumb, the sample size was restricted to 50 in number30. However, due to the decrease in the number of patients in the IPD/OPD ward, the study was discontinued at 40 patients.

Randomization and blinding
The participants were randomized into respective study groups. A computer-generated sequence of randomization was adopted for the study. Based on their in ow, the patients were allocated numbers sequentially and thereafter randomized into groups. All the subjects were randomized in a 1:1 ratio between the intervention and placebo arm. The appropriate intervention was administered by the duty doctor who was not involved in the study. The data collector, operator and lab technicians were blinded to the intervention along with the subjects.

Statistical analysis
The baseline characteristics of both study groups were reported as proportion/ mean (SD). Data analysis was conducted using the intention to treat (ITT) approach. Data was compiled and analyzed using Microsoft Excel

Subgroup analysis
Based on the Government of India guidelines, the patients were categorized as moderate or moderate-severe without ventilation based on SpO2 rate. This was further con rmed with Chest CT score, since all the patients also underwent a chest CT to assess the severity of illness31.

Results
The study compared the COVID-19 recovery, as measured by RT-PCR and blood biomarkers, between the intervention NOQ19 arm and placebo arm. Attrition rate in both the study groups was zero. Table 1 demonstrates the demographic characteristics of the patients enrolled in the study. No signi cant differences in age or gender distribution were observed between the study groups. The two groups were also similar in height, weight, blood pressure and pulse rate at the time of enrollment. Vaccination status of the patients was also captured to analyse any change in response of the intervention between the partially and fully vaccinated groups. A signi cant difference was noted in the HR-CT score, SpO2 values, temperature and respiratory rate between the two groups at the time of enrollment.. According to Berkson's bias, due to the increased rate of exposure during the study enrollment and likelihood of patients being admitted in the hospital, the intervention arm had more patients with severe or advanced symptoms than the placebo arm from the start of the study.32 This resulted in a higher number of patients being severe in the NOQ19 arm than in the placebo arm. Although patients were classi ed as severe, none of Page 7/16 them were in ICU or on ventilation. At baseline, the NOQ19 arm had 41% moderate patients and 59% patients classi ed with severe disease, none of whom were on ventilation, while the placebo group consisted of 78% moderate and 22% patients classi ed with severe disease.    Table 3 depicts the blood biomarker progression in both the study arms. Signi cant differences in temperature, immune markers and blood parameters were noted between the two groups on the day of admission. The values for IL-6, a cytokine marker, were signi cantly higher in the NOQ19 group as compared to placebo group at baseline (p=0.02). IL-6 showed a signi cant reduction from day 0 to day 10 in both the arms (intra group p<0.001). However, the mean reduction was greater in the NOQ19 arm (2.2 units) as compared to placebo (1 unit). Similar trends were observed for S. Ferritin, CRP and D-Dimer. The baseline values for all the markers were much higher in the NOQ19 arm than the placebo arm. The intragroup p values were signi cant for all markers in both arms, except D-Dimer, signifying that both the arms showed an improvement. However for all clinical markers, NOQ19 showed a greater improvement as seen by the mean difference.
Due to the differences in severity of disease between the two arms, the data was further divided into moderate and severe groups. It was analysed individually for both the arms because the sample size in each sub-group varied between the arms. Table 4 demonstrates the difference in blood parameters in severe patients among the NOQ19 and placebo arm. Except D-dimer, all the blood markers signi cantly improved by Day 10 in the NOQ19 arm. However, a signi cant improvement was not noted in the placebo arm, probably because of small sample size. Therefore, the data was not presented.   Ayurvedic medicines are usually tested in mild cases for their e cacy. Our study is the rst of its kind to examine the effect of NOQ19 in moderate-severe patients who were not on ventilation or ICU.
In addition to objective marker analysis, we were interested in evaluating any side effects or adverse events that may require discontinuation of the drug in humans. There were no dropouts, side effects or adverse events in the NOQ19 arm.
In an earlier open label feasibility study on the NOQ19 polyherbal formulation, similar results were noticed with respect to RT-PCR conversion to negative. Out of 161 patients enrolled in the study, 74% of the patients turned RT-PCR negative by Day 534. In vitro and in vivo studies27-28 on the effectiveness of the polyherbal formulation, NOQ19 also showed signi cant viral load reduction. In the in vitro analysis of NOQ19, a 100% viral load reduction was noted in the Vero E6 SARS-CoV-2 infected cell line. Declarations Figure 1 CONSORT Flow Diagram