PLOS ONE

RESEARCH ARTICLE

Co-administration of AYUSH 64 as an adjunct

to standard of care in mild and moderate
COVID-19: A randomized, controlled,
multicentric clinical trial
Arvind Chopra ID1*, Girish Tillu2, Kuldeep Chuadhary ID3, Govind Reddy4,
Alok Srivastava5, Muffazal Lakdawala6, Dilip Gode7, Himanshu Reddy8, Sanjay Tamboli9,
Manjit Saluja1, Sanjeev Sarmukaddam ID1, Manohar Gundeti3, Ashwini Kumar Raut10, B. C.
a1111111111 S. Rao11, Babita Yadav11, Narayanam Srikanth11, Bhushan Patwardhan2
a1111111111
1 Centre for Rheumatic Diseases, Pune, India, 2 Interdisciplinary School of Health Sciences, Savitribai Phule
a1111111111 Pune University, Pune, India, 3 Central Ayurveda Research Institute, Mumbai, India, 4 Regional Ayurveda
a1111111111 Research Institute, Nagpur, India, 5 Regional Ayurveda Research Institute, Lucknow, India, 6 H.N. Reliance
a1111111111 Foundation Hospital and Research Centre, Mumbai, India, 7 Datta Meghe Institute of Medical Sciences,
Nagpur, India, 8 King George’s Medical University, Lucknow, India, 9 Target Institute of Medical Education &
Research, Mumbai, India, 10 Medical Research Centre, Kasturba Health Society, Mumbai, India, 11 Central
Council for Research in Ayurvedic Sciences, New Delhi, India

* [email protected]
OPEN ACCESS

Citation: Chopra A, Tillu G, Chuadhary K, Reddy G,

Srivastava A, Lakdawala M, et al. (2023) Co-
administration of AYUSH 64 as an adjunct to
Abstract
standard of care in mild and moderate COVID-19: A
randomized, controlled, multicentric clinical trial.
PLoS ONE 18(3): e0282688. https://doi.org/ Objective
10.1371/journal.pone.0282688 Evaluate the efficacy of AYUSH 64, a standard polyherbal Ayurvedic drug in COVID-19.
Editor: Shu-Hsing Cheng, Taoyuan General
Hospital, TAIWAN Methods
Received: August 7, 2021 During the first pandemic wave, 140 consenting and eligible hospitalized adult participants
Accepted: October 28, 2022 with mild-moderate symptomatic disease (specific standard RT-PCR assay positive) were
Published: March 16, 2023 selected as per a convenience sample, and randomized (1:1 ratio) to an open-label (asses-
sor blind) two-arm multicentric drug trial; standard of care (SOC as per Indian guidelines)
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review versus AYUSH 64 combined with SOC (AYUSH plus). Participants were assessed daily
process; therefore, we enable the publication of and discharged once clinical recovery (CR, primary efficacy) was achieved which was
all of the content of peer review and author based on a predetermined set of criteria (resolution of symptoms, normal peripheral oxime-
responses alongside final, published articles. The
try, and negative specific RT-PCR assay). Each participant was followed using an indige-
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0282688 nous software program(mobile phone) and completed a 12-week study period. The dose of
AYUSH 64 was 2 tablets oral, 500 mg each, bid for 12 weeks (AYUSH plus only). Significant
Copyright: © 2023 Chopra et al. This is an open
access article distributed under the terms of the P was <0.05 (two-sided). On randomization, the groups were found well matched.
Creative Commons Attribution License, which
permits unrestricted use, distribution, and Results
reproduction in any medium, provided the original
author and source are credited. The mean interval time from randomization to CR was significantly superior in the AYUSH
plus group [mean 6.45 days versus 8.26 days, 95% Confidence Interval of the difference
Data Availability Statement: Several relevant data
are within the manuscript and its Supporting -3.02 to -0.59 (P = 0.003, Student’s ‘t test] as per-protocol analysis (134 participants); signifi-
Information files (S8F).All relevant data on primary cant (P = 0.002) on an intention to treat analysis. 70% of the participants in AYUSH plus

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

efficacy, adverse events and withdrawals is recovered during the first week (P = 0.046, Chi-square) and showed a significantly better
available enclosed with the manuscript as a change in physical health, fatigue, and quality of life measures. 48 adverse events, mostly
Supporting Information file (S8 File). The full data
set is archived by the CCRAS, Government of India
mild and gut related, were reported by each group. There were 20 patient withdrawals (8 in
(http://www.ccras.nic.in), as an electronic data AYUSH plus) but none due to an AE. There were no deaths. Daily assessment (hospitaliza-
base and access will be provided after approval by tion) and supervised drug intake ensured robust efficacy data. The open-label design was a
Director General, CCRAS as explained below.
concern (study outcome).
Access to full data set will be available for any kind
of non-commercial purpose and without any other
restriction after approval by the sponsor (Director Conclusions
General, Central Council for Research in Ayurvedic
Sciences/ CCRAS, Government of India) six
AYUSH 64 in combination with SOC hastened recovery, reduced hospitalization, and
months after publication. The latter will require a improved health in COVID-19. It was considered safe and well-tolerated. Further clinical val-
formal request from the applicant stating the idation (Phase III) is required.
reason for access and accompanied with a CV
(applicant) and may be sent Email
([email protected]) to Arvind Chopra (first Trial registration
author) for further processing by CCRAS. CTRI/2020/06/025557.
Funding: The current study was sponsored by
Central Council of Research in Ayurvedic Sciences
(CCRAS), Ministry of AYUSH, Government of India.
CCRAS authorized the study grant vide their Order
Reference F.No.3-61/2020-CCRAS/Admn/IMR/458
dated 02 June 2020 (CCRAS website: http://www. Introduction
ccras.nic.in). The grant was distributed and
The world continues to reel under the tragic burden of the COVID-19 pandemic. The medical
supervised by the authorized CCRAS officer to the
3 CCRAS run study sites. No individual was paid
system was precariously overstretched and scarred. Several drug trials were completed and
any part of the research grant. CCRAS selected the many more are underway to unravel evidence-based medicine (EBM) for more effective and
principal investigators from the study sites. The PIs safe management [1]. However, despite several advances, the treatment of mild and moderate
selected the study site staff who were paid salary/ COVID-19 predominantly remains symptomatic and empirical, and data from drug trials is
compensation from the site grant as a-priori sparse [1–3].
approved by the CCRAS. CCRAS also appointed a
It is prudent to state upfront that most of the patients of COVID-19 suffered from asymp-
’Contract research organization (CRO)’ on contract
payment to supervise and co-ordinate the trial as tomatic or mild and moderate disease and recovered without any complication or sequel [4,
per the Government policy. CCRAS did not play any 5]. Sometimes the disease was rapidly progressive, and less than 10% of subjects reported
role in the study design, data collection and severe disease [4–6]. An exuberant and dysregulated immune response was central to the pro-
analysis, decision to publish, or preparation of the gression and severity, life-threatening complications, and fatality [6, 7]. Several drugs were
manuscript. AYUSH 64, the investigational product
repurposed and extensively used for the chemoprophylaxis and treatment of COVID-19 [8].
in this study, was a proprietary formulation of
CCRAS and directly (central procurement) supplied
The widespread use of hydroxychloroquine (HCQS) during the early pandemic in India was
to the study sites. None of the authors received any grossly restricted when drug trials failed to show unequivocal efficacy [9]. HCQS is no longer
funding for participation in the current study recommended [2, 3]. Despite limited evidence but based on good clinical experience, some
project. Amongst the authors, KC, GR, AS, ML,DG, drugs such as tocilizumab and remdesivir are still being used [2, 10, 11]. The use of steroids in
HR, MG, BCSR, BY, NS were Ayurvedic physician severe disease became pivotal following the result of a single large, controlled drug trial [12].
investigators and salaried employees of CCRAS run
The search to repurpose drugs (COVID-19) also rekindled vigorous research in the tradi-
Government medical institutions. ST was paid by
the CRO. AR and GT were Ayurvedic physician tional, complementary, and alternative systems of medicine (CAM) [13, 14]. The potential for
consultants and AR was paid a honorarium by the prophylaxis and treatment of COVID-19 in the Ayurveda medicinal system was encouraged
CCRAS. AC was a rheumatologist in practise and by the popular use of several standard herbal drugs to treat febrile respiratory disorders,
appointed as the Chief Clinical Coordinator of improve health and immunity since ancient times and the growing modern experimental evi-
AYUSH CSIR Project (research drug trials in
dence of their potent anti-inflammatory and immune modulation effects [15, 16]. Several
COVID-19). BP was the Chairman, Interdisciplinary
AYUSH R & D Task Force on COVID-19 set up
medicinal plants were considered as potential therapeutic candidates [14, 17, 18]. Despite the
Ministry of AYUSH, Government of India. MS was limited scientific evidence, a large number of Indian population used Ayurvedic and other
a research coordinator and assisted AC. AC, MS CAM drugs to prevent and treat COVID-19 from beginning of the pandemic [19, 20]. The
and BP worked in a voluntary capacity and did not deep-rooted belief in the safety and tolerability of Ayurvedic drugs was certainly an advantage
receive any remuneration from CCRAS. [21].

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Competing interests: The authors have declared India has a legal system to regulate and promote plural systems of medicine including
that no competing interests exist. The authors Ayurveda, Yoga, Naturopathy, Unani, Siddha, Sowa Rigpa, and Homoeopathy, which together
declare their relationship related to study as per the
are known as AYUSH systems. The Ministry of AYUSH established an Interdisciplinary
International Committee of Medical Journal Editors
is described in the section on ‘Financial Disclosure’ AYUSH Research and Development Task Force on COVID-19 to promote scientific research
(see above). "This does not alter our adherence to and worked closely with the Ministry of Health and Family Welfare to manage and curb the
PLOS ONE policies on sharing data and materials.” pandemic [22, 23]. The Ministry of AYUSH and its research wing namely CCRAS (Central
Council for Research in Ayurvedic Sciences) in collaboration with the Council of Scientific
and Industrial Research (CSIR) also sponsored controlled drug trial studies in April 2020 to
individually evaluate the therapeutic efficacy of 3 shortlisted Ayurvedic drugs as an adjunct to
the standard of care (SOC) in the treatment of mild and moderate symptomatic COVID-19.
Based on a common protocol, three drug trials were carried out at different sites with different
investigators [24]. A controlled drug trial of AYUSH 64, a standard proprietary poly-herbal
formulation of CCRAS, was amongst the latter studies.
The selection of AYUSH-64 was based on Ayurvedic logic and clinical experience. It was
initially developed to treat malaria [25]. Later on, it was also found useful to treat cough and
other mild respiratory tract infections and other disorders [18, 26]. Though readily available in
AYUSH medical centers, its overall use remained limited. A comprehensive description of
AYUSH 64 and other Indian medicinal plants with a therapeutic potential in COVID-19 was
recently published [18].
The current report presents the results of the AYUSH-64 drug trial.

Methods
The protocol was approved by the following Institutional Ethics Committee at each study site:-
1. Institutional Ethics Committee, Datta Meghe Institute of Medical Sciences, Nagpur (No.
DMIMS(DU)/IEC/2020/8785)
2. Institutional Ethics Committee, Central Ayurveda Research Institute, Mumbai (No. 01/20-
21)
3. Institutional Ethics Committee, King George’s Medical University, Lucknow (No. 469/Eth-
ics/2020).
The study was a prospective, randomized, open label (assessor blind), parallel efficacy, two
arm multicentric drug trial. The protocol was registered with the Clinical Trials Registry of
India (CTRI) (registration number CTRI/2020/06/025557) [24]. The protocol is enclosed as a
S2 File. The study duration for each participant was 12 weeks. The study was carried out in the
Government approved facilities for COVID- 19 in the medical and teaching hospitals at King
George Medical University, Lucknow, Central Ayurveda Research Institute for Cancer, Mum-
bai, and Datta Meghe Institute of Medical Sciences, Nagpur. The study was carried out in
accordance with the principles of Good Clinical Practice (GCP), Declaration of Helsinki (Bra-
zil update 2013), ICMR (Indian Council of Medical Research), and CCRAS Guidelines (2018)
[27, 28] The protocol and the study report also complied with CONSORT guidelines (a check-
list is enclosed as S1 File) [29]. An independent data safety management board (DSMB) and a
monitoring committee were appointed by the sponsor. An independent accredited CRO (Clin-
ical Research Organization) was engaged by the sponsor for study oversight and regular moni-
toring checks, on-site training of personnel, implementation of study protocol, creation of a
central study database and preparation of a study report.
The overall scheme of the study, study procedures, and predetermined time points of evalu-
ation are shown in Fig 1.

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Fig 1. Study flow diagram showing study events and timelines.

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Patient and public involvement

The patients were not involved in the preparation of the protocol or in carrying out study
assessments and analyses. Numerous information bulletins on Ayurvedic remedies and
research were posted by the Ministry of AYUSH from time to time [30].

Selection, screening and eligibility, enrollment and management

During the first pandemic wave, all cases of COVID-19 including mild and suspected cases
were to be admitted in Government accredited hospitals (COVID-19) [3]. Patients could
directly access the hospital. They were first triaged by the general duty medical officer in the

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

emergency casualty department/outpatient. The diagnosis was confirmed by a standard real-

time specific (SARS-CoV-2) reverse transcriptase polymerase chain reaction (RT-PCR) assay
on a nasal and/or throat swab.
In the current study, the study investigators enrolled mild and moderate cases of COVID-
19 which was classified as per Indian guidelines and clinical judgment [3]. Following hospital
admission (study sites), the investigator explained the current study to those patients who
expressed interest. Volunteer patients signed an informed consent form. The screening was
completed within 48 hours of hospital admission as required by the protocol. Patients found
eligible were quickly randomized.
Adult patients with a typical COVID-19 illness and a confirmed diagnosis were selected
and after fulfilling the inclusion and exclusion criteria described in the protocol (enclosed, S2
File) were enrolled [24]. Patients with severe symptomatic COVID-19 were excluded if they
satisfied any two of the following criteria (protocol) (i) respiratory distress at room ambiance
(ii) Oxygen saturation (SpO2) at rest � 93% (iii) known COVID-19 complication which may
require oxygenation and/or critical care.
An Ayurvedic and a modern medicine physician in the study team supervised medical
management on daily basis and along with nursing and paramedic personnel ensured drug
compliance and reporting of all adverse events (AE). However, final decisions pertaining to
the medical management of COVID-19 and recovery were taken by an independent hospital
COVID-19 physician (blinded to treatment allocation). Patients and study personnel were
aware of the specific study intervention allocation (open label).

Randomization
Patients were randomized at each site to either of the two arms of a standard of care (SOC) or
AYUSH 64 administered along with SOC (AYUSH plus) in a 1:1 ratio on a first come first
serve basis. A central randomization schedule was prepared by the study biostatistician (SS)
using standard statistical software (WINPEPI version 4.61 for MS Windows). Permuted block
randomization was used in a group of 20 participants (strata of size 20) to ensure a number
balance. The randomization schedule was provided online with restricted access to the site
principal investigator.

Standard of care (SOC)

SOC regimen was begun in-patient by an independent COVID-19 physician according to
national and institutional recommendations [3]. However, the physician was permitted to use
clinical judgement.

Investigational drug
Each 500 mg tablet of AYUSH 64 contained aqueous extracts (100 mg each) of Alstonia scho-
laris (bark), Picrorhiza kurroa (rhizome), Swertia chirata (whole plant), and Caesalpinia crista
(200 mg seed powder). The dose was two tablets of 500 mg each and taken twice daily with a
glass of water soon after a meal, and this dosage remained fixed throughout the study. Patients
assigned to the AYUSH 64 plus arm continued the drug following clinical recovery till the
completion of the study period (12 weeks). AYUSH 64 was procured from Indian Medicines
Pharmaceutical Corporation Limited (IMPCL), Uttarakhand, India under arrangements with
CCRAS, New Delhi. The manufacturing facility was a certified ISO 9001 facility (2008) and fol-
lowed good manufacturing practices’ guidelines in the Ayurvedic Pharmacopoeia of India.
Details of composition, quality standards, and features of chemistry, manufacturing, and con-
trols are described in (Tables S3.1-S3.3, S3.1 Fig in S3 File).

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Outcome measures
The primary efficacy measure was (i) the mean duration (days) from baseline randomization
to day one of clinical recovery (CR) and (ii) the proportion of patients showing clinical recov-
ery, within a time framework of 28 days. Clinical recovery was accepted when all of the follow-
ing criteria were met for at least 48 hours under the observation of the hospital COVID-19
physician (a) normal body temperature (�36.6˚C axillae or �37.2˚C oral) (b) absence of
cough requiring regular medication (c)absence of breathlessness on routine daily self-care
activities and respiratory rate less than 30 breaths per minute without supplemental oxygen (d)
absence of any other symptom/sign attributed to COVID-19 illness and requiring continuous
treatment (e) normal SpO2 by standard peripheral oximetry device (above 95 percent)(f) nega-
tive RT-PCR assay for SARS-CoV-2 from nasal and throat swab. The checklist of symptoms
that were monitored daily for recovery was also guided by the WHO guideline [31].
There were several secondary efficacy measures pertaining to (i) timelines such as the mean
duration from onset of symptoms to CR, mean duration from hospitalization to CR (ii)
COVID-19-related blood assay biomarkers such as C-reactive protein (CRP), D-Dimer, Ferri-
tin, interleukin-6. They are described in the enclosed protocol (S2 File).

Procedures and measures

The assessment of general physical and mental health, psychosocial health, and quality of life
(QOL) were carried out by using the standard World Health Organization QOL BREF ques-
tionnaire and a recently developed Health-Related-Behavior Habit and Fitness Questionnaire
(HR-BHF CRD, Pune 2020 version) [32, S4 File]. Both questionnaires were self-reported and
administered in the local language. While the WHO QOL recorded the response on a 5-point
Likert categorical scale, the HR-BHF used a horizontal visual analog scale (VAS, 0–100 mm).
Both the scales were anchored at either end to show the worst response or the best response,
e.g., in the WHO-QOL questionnaire, category 1 was ‘very poor’ and category 5 was ‘very good
for certain questions. In the case of HR-BHF, a VAS score of 0 indicated the worst response and
a score of 100 indicated the best response for certain questions; the ascending order of better
response was reversed for few questions to facilitate understanding by the patient (such as in
case of ‘anxiety’ a VAS score of 0 meant absence of anxiety and score 100 meant maximum anx-
iety), and this was adjusted in the final score. A comprehensive description and scoring method
including pre-study validation is provided in the Text Box S4.2, Text Box S4.3 in S4 File.
The WHO QOL-BREF had 27 questions classified into 4 domains- physical health (7–35),
psychological health (6–30), social relationships (3–15), and environmental well-being (8–40);
the range of score is shown in parenthesis.
HR-BHF contained nine questions pertaining to general health, anxiety, fatigue, energy
level, bowel habits, stress, happiness, sleep, and appetite (food). Individual question score (0–
100) and the total score (0–900) was used for analysis in the current study.
Standard procedures were used to classify, monitor, record, and assign causality, in the case
of an adverse event (AE, System organ classification, clinical grade of severity, preferred terms
for recording signs, symptoms, and diagnosis). Guidelines published by ICH-GCP, ICMR
(India), MedDRA, and WHO were also followed [28, 33, 34]. All randomized participants
were assessed for safety and tolerability (AE).
Clinical evaluation included a routine physical and systemic examination. Both the clinical
and laboratory evaluation was carried out several pre-determined study time points which
included randomization baseline and study completion (Fig 2). Routine laboratory measures
included hematocrit, metabolic hepatic and renal profile, and urinalysis and were carried out
in laboratories at each study site that was a-priori endorsed by the ‘National Accreditation

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Fig 2. Patient disposition and withdrawals: A randomized controlled study to evaluate the co-administration of
AYUSH-64 with Standard of Care (SOC) in–mild-moderate symptomatic COVID-19 (CONSORT flow diagram).
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Board for Hospital and Health Care Providers’ (NABH). Further, these laboratories were nec-
essarily approved by the Indian Council of Medical Research (ICMR) to carry out real-time
RT-PCR assays for diagnosis of SARS-CoV-2 infection as per the existent Indian Government
policy. Laboratories followed standard ICMR recommendations for reagents, equipment and
procedure, and quality checks [35].
Electrocardiography was recorded during screening, hospital discharge, and on study
completion.
Standard skiagram of chest was carried out during screening, on clinical recovery/ hospital
discharge, and during any follow-up visits if clinically indicated. The radiological evaluation
described in the S5 File pertained to 86 participants at two study sites that provided digital
skiagrams of satisfactory quality according to an independent radiologist. Due to hospital pri-
orities, participants at one site were only screened if they had persistent respiratory complaints
using a mobile X-Ray unit, and skiagrams were not printed; 6 participants were reported with
mild abnormalities and none had severe disease. The latter data were not included in this
report. For the current radiographic analysis, all the available digital skiagrams were centrally
reassessed by an independent radiologist who was blinded to the allocation of study treatment.

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Data were collected on a daily basis during the hospitalization phase. Subsequently, after
hospital discharge, it was collected during the predetermined follow-up schedule (4, 8, and 12
weeks) as shown in Fig 2. Participants were counseled to contact the study physicians at any
time during the follow-up if they showed any fresh symptoms or worsened or suspected a
drug-related side-effect. Patients were also provided with a specially designed software pro-
gram for mobile application called ‘COVID KAVACH’ for daily monitoring during the follow
up. The latter was used by the patient to record any AE or study-related problem which was
electronically communicated to the site investigator and the study coordinator (AC) [36].
Data were recorded in the study case report forms at the point of care and later entered into
a central electronic database using unique participant ID and study treatment code. The data-
base was handled by pre-designated study team paramedics and locked after validation for any
errors by the designated in-charge from CRO. Prior to the data analysis, a backup copy was
provided to the sponsor by the CRO. Th database was unlocked and decoded by the study bio-
statisticians (SS, ST) prior to statistical analysis.

Study withdrawal
Patients worsening clinically and likely to require prolonged oxygen and/or intensive care
were withdrawn from the study and transferred to intensive care for further management. All
patients were comprehensively evaluated at the time of withdrawal. The site personnel contin-
ued to contact withdrawn patients for further disease progress and recovery and for the occur-
rence of any AE till such time the study was completed. The latter data were not included in
the current report.

Statistical analysis
There was no prior data to use for the formal calculation of a sample size. However, we consid-
ered relevant clues for a probable medium effect size which recommended 64 participants per
group (type-I error = 0.05, power = 80%, Table 2) and was published in a classic reference
[37]. Finally, after discussion with the study group experts, the principal investigator and coor-
dinator (AC) and the chief biostatistician (SS) finalized a convenience (non-probabilistic) sam-
ple of 140 participants. This was considered adequate to address the clinical research
questions. Other factors like study logistics [mainly available time & resources including man-
power] and restrictions imposed by the pandemic were also considered.
The study data was entered by the designated personnel at each study site into a central
database and supervised by the CRO. Data were summarized using central tendencies (mean,
median), range, standard deviation, and 95% confidence interval (95% CI).
Statistical tests were carried out to compare the two treatment groups as per the distribution
(normality) and the type & level of measurement of the variable under consideration (like Stu-
dent’s ‘t-test, Mann-Whitney non-parametric test, and Chi-square test) The result of statistical
analysis was considered significant at P < 0.05 (two-sided). Both intent-to-treat (ITT) and
per-protocol/completer (PP) analyses were performed for the primary efficacy analysis and
some secondary measures. The ITT included all subjects who completed the randomized treat-
ment observation till clinical recovery. The PP analysis included all subjects from the latter
who were randomized within 48 hours of hospital admission and strictly adhered to other pro-
tocol requirements.
Participants completing the study intervention as per protocol were considered as qualified
for the primary efficacy analysis using parametric (Student’s ‘t-test) and non-parametric tests
(Mann-Whitney statistic). Categorical outcomes such as AE were compared using Chi-square
statistic. The primary efficacy measure was also analysed for the total number of participants at

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

each study site and in the study. Similarly, the two study groups were also compared for several
secondary efficacy measures (timelines, laboratory assays, Quality of life measures) at several
time points as per protocol.
A general mixed-effect linear regression model was also carried out with ‘study site’ as a
random effect and ‘group intervention ‘ as a fixed effect for the primary efficacy measure. In
the latter case, ‘Time from Randomization to Clinical Recovery’ (primary efficacy) was the
dependent variable.
Standard statistical software programs were used (GraphPad InStat Version 3.6, BMDP,
IBM SPSS Version 20, and Confidence Interval Analysis, BMJ Group, London, 2003). The
study arm of ‘AYUSH 64 plus SOC’ is referred to as ‘AYUSH plus ‘ and ‘SOC alone’ is referred
to as ‘SOC’ in the current paper.

Results
A total of 140 participants were randomized with 70 participants in each of the two study
arms- AYUSH plus and SOC (Fig 2).

Withdrawals
Three participants were withdrawn during the randomization phase -one participant withdrew
consent following randomization, one (AYUSH Plus) developed neuropathy (Guillain Barre
syndrome) and one (SOC) developed severe pneumonia with respiratory distress. 137 patients
completed the randomized treatment phase. A total of 20 (14.3%) participants withdrew (12 in
the SOC group and 8 in the AYUSH Plus group) from the study. Seventeen patients did not
wish to continue following complete recovery and hospital discharge. The latter did not report
any AE during an informal follow up. None of the withdrawals were due to a drug related AE.
There were no deaths in the study.

Randomization baseline
Both the study groups were well matched for several demographic, clinical, COVID related
timelines, SOC drugs and laboratory variables as shown in Table 1. 80% participants were clin-
ically classified as mild COVID-19 at the time of enrolment and were mostly men in the age
range 30–55 years. Several had comorbid disorders- hypertension, known diabetes, or first-
time hyperglycemia (fasting blood sugar > 120 mg/dl).
There were no significant differences between the two study groups for COVID related
timelines such as’ onset of symptom to hospital admission’ (-1.34 to 1.72),’ hospital admission
to randomization’ (-0.17 to 0.39), and ‘symptom onset to randomization’ (-1.08 to 1.98); 95%
CI of the difference (days) between means is shown in parenthesis (Table 1). Site-specific data
for selected timelines and SOC drugs, including those related to RT-PCR assay, are shown in
(Table S5.2 in S5 File).
A list of SOC drugs is shown in S5 File [R]. Most of the patients were treated with symp-
tomatic drugs, vitamins and minerals. Several patients also received Hydroxychloroquine, or
Ivermectin with Azithromycin (Table 1). At one site, anti-coagulants were empirically admin-
istered to patients with moderate COVID-19 and or with important risk factors (COVID-19).
Except for one patient, none of the trial participants were treated with steroids. Parenteral
Dexamethasone was administered to only one patient with progressive respiratory distress
who was withdrawn from the study. Importantly, both the intervention study groups were well
matched for the use of various drugs (Table 1).
62.8% of the participants showed radiographic abnormalities in the chest which were consistent
with COVID-19 and classified as mild or moderate by the radiologist (See, Table S5.6 in S5 File).

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Table 1. Randomization baseline data on demographic, clinical, COVID related timelines, laboratory variables, and Standard of Care (SOC) drugs in the study
groups.
VARIABLES AYUSH Plus (n = 69) SOC (n = 70) P-value�
Clinical
Age (years) Mean ± SD 42.87 ± 12.6 42.7 ± 12.0 0.93
Male–number (%) 54 (77.14%) 58 (82.85%) 0.52
Body Weight (kg) Mean ± SD 69.34 ±10.3 68.38 ±12.1 0.61
BMI (kg/m2) Mean ± SD 24.86 ±3.4 24.53 ±3.7 0.65
Symptom onset to randomization (days), mean ± SD 7.61 ±4.8 7.83 ± 4.5 0.51
Symptom onset to Hospitalization, mean ± SD 6.4 ± 4.64 6.5 ± 4.47 0.75
Hospitalization to Randomization, mean ± SD 1.4 ± 0.8 1.5 ± 0.9 0.55
Mild clinical disease number (%) 56 (80) 58 (82.9) 0.82
Moderate clinical disease number (%) 14 (20) 12 (17.1) 0.82
Hypertension number (%) 17 (24.29) 10 (14.29) 0.19
Diabetes mellitus-number (%) 14 (20) 06 (8.57) 0.09
Undiagnosed hyperglycemia-number (%) 9 (12.85) 14 (20) 0.36
Blood sugar level mg/dl, mean ± SD 112.50 ± 37.5 114.17 ± 35.2 0.74
ESR mm fall 1st hour, mean ± SD 50.2 ± 38.0 46.9 ± 37.4 0.79
Blood hemoglobin gm/dl, mean ± SD 13.6 ± 1.42 13.8 ±1.62 0.51
Total leucocyte count/cu mm, mean ± SD 5920.7 ± 2008 6828.3 ± 2085 0.02
Total Lymphocyte count/cu mm, mean ± SD 32.31 ± 9.1 31.07 ± 09.6 0.34
Symptoms at baseline-number of subjects (percent)
Fever 53 (75.71%) 45 (64.28%) 0.10
Sore throat 46 (65.71%) 53 (75.71%) 0.24
Cough 54 (77.14%) 54 (77.14%) 0.87
Dyspnea 24 (34.28%) 25 (35.71%) 0.90
Myalgia 48 (68.57%) 54 (77.14%) 0.31
Headache 37 (52.85%) 32 (45.71%) 0.35
Diarrhea 11 (15.71%) 12 (17.14%) 0.85
Ageusia 19 (27.14%) 19 (27.14%) 0.96
Anosmia 13 (18.57%) 14 (20%) 0.86
Drugs administered-number of subjects (percent)
Tab Azithromycin 48 (70%) 49 (70%) 0.95
Tab Doxycycline 1(2%) 0 0.31
Tab HCQS 29 (42%) 24(34%) 0.35
Tab Zinc 48(70%) 42(60%) 0.24
Tab Vitamin C 69 (100%) 69(99%) 0.32
Tab Vitamin D 3 15(22%) 18(26%) 0.58
Tab Pantoprazole 66(96%) 65(93%) 0.73
Tab Paracetamol 59 (86%) 55(79%) 0.28
Tab Cetirizine 13(19%) 15(21%) 0.70
Tab Ivermectin 3 (4%) 2 (2.9%) 0.99
Anti-coagulant 13(18.8%) 13(19%) 0.97
Oxygen intermittent (< 2 liters/min) 9(13%) 6 (9%) 0.39
�
Statistically significant (P<0.05)
NS Not statistically significant (P> = 0.05)
a. Student’s ‘t test (normative data) or Chi-Square statistic (categorical data)
b. Note- n: number of study participants; SD: standard deviation; AYUSH plus: AYUSH 64 + SOC; BMI: body mass index; ESR: erythrocyte sedimentation rate
(Wintrobe method); Other comorbid disorders: hyperlipidemia (2), cardiac disorder (1), chronic lung disease (2), thyroid disorder (5), Allergic rhinitis (3), number of
participants in parenthesis; See text for details

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Efficacy
134 participants qualified for the primary efficacy analysis and the results on per protocol anal-
ysis are shown in Table 2; three study participants were disqualified because of delay in ran-
domization. The mean duration (days) for clinical recovery (primary efficacy) from the
randomization baseline was significantly superior in the AYUSH plus (6.45, 95% CI 5.88 to
7.01 days) as compared to SOC (8.26, 95% CI 7.20 to 9.31 days); difference between means
-1.81 (95% CI—3.02 to—0.59 days) (Table 2). Significant improvement was also observed at
each of the study sites.
At this stage, we did not intend performing statistical analysis for predictor of response to
treatment. However, the results of a general linear mixed effect model using the primary effi-
cacy data as a dependent variable are shown in Table S5.4 in S5 File and indicate that the out-
come remained significantly different between the two study groups even after controlling/
removing the effect of ‘study site’.
In an intention to treat analysis (137 participants), the mean duration (days) for clinical
recovery (primary efficacy) from the randomization baseline was significantly superior in the
AYUSH plus (6.42, 95% CI 5.99 to 7.59) as compared to SOC (8.33, 95% CI 7.02 to 9.87 days);
difference between means was -1.90 (95% CI—3.11 to—0.70 days) (Table S5.5 in S5 File). This
improvement was also observed at each of the study site.
A higher proportion of patients in the AYUSH plus (69.75%) showed complete recovery as
compared to SOC (52.9% patients) during the first week following randomization (P = 0.046,
Chi-square statistic).

Table 2. Primary efficacy measure (Randomization to clinical recovery) and selected timeline in the two study groups (n = 134) in per protocol analysis; mean
(days)± standard deviation.
Time line (days) Mumbai (n = 57) Nagpur (n = 29) Lucknow (n = 48) Total Study (n = 134)
AYUSH Plus SOC AYUSH Plus SOC AYUSH Plus SOC AYUSH Plus SOC
(n = 28) (n = 29) (n = 15) (n = 14) (n = 23) (n = 25) (n = 66) (n = 68)
Randomization to Clinical Recovery (R-CR)
Mean ± SD (R-CR) 6.75± 2.14 8.45± 8.80± 1.01 11.21± 5.03 4.57± 1.56 6.40± 4.03 6.45± 2.35 8.26± 4.44
3.75
95% CI of Difference between Means -3.32 to -0.07 - 5.37 to 0.54 -3.61 to—0.06 - 3.02 to—0.59
(R-CR)
On comparison of R-CR between two P = 0.0410 (‘t’ test),0.077 P = 0.079 (‘t’ test), 0.013 P = 0.046 (‘t’ test),0.121 P = 0.003 (‘t’ test), 0.015
intervention groups at each study site and (M-W) (M-W) (M-W) (M-W)
study cohort �
Onset symptom to Clinical Recovery (S-CR)
Mean ± SD (S-CR) 15.29 ± 6.15 16.45 11.07± 1.58 14.50 ± 5.60 11.52 ± 3.26 13.48 ± 6.00 13.02 ± 4.87 14.96 ± 5.95
±5.92
95% CI of Difference between Means -4.37 to 2.05 -6.76 to -0.11 -4.75 to 0.84 -3.79 to -0.08
(S-CR)
On comparison of S-CR between two P = 0.470 (‘t’ test), 0.592 P = 0.031(‘t’ test), 0.016 P = 0.172 (‘t’ test), 0.525 P = 0.041(‘t’ test), 0.066
intervention groups at each study site and (M-W) (M-W) (M-W) (M-W)
study cohort �
�
Statistically significant (P<0.05)
NS Not statistically significant (P> = 0.05)
a. two independent samples ‘t’ test and M-W (Mann-Whitney statistic
b. Note, AYUSH plus: AYUSH 64 + Standard of Care (SOC); 134 patients qualified for primary efficacy analysis; Clinical recovery was essentially absence of COVID 19
symptoms for two successive days (with negative RT-PCR assay); See Text for detail

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

The earlier recovery in the AYUSH plus group was also observed for ‘time to clinical recov-
ery from the onset of symptom’ and this was marginally significant as compared to SOC
(Table 2).
There was a significant reduction in serum biomarkers of COVID-19 in each of the study
groups but the difference between the groups was not significant (Table 3).
Eight participants (18.6%) in the AYUSH plus and 13 participants (30.2%) in the SOC
group showed radiological features of definite COVID-19 pneumonia at the time of randomi-
zation baseline (See Table S5.6 in S5 File). A higher number of participants (84.6%) in the SOC
showed incomplete resolution as compared to AYUSH plus SOC (62.5%) at the time of clinical
recovery (Table S5.6 in S5 File). None of the patients with COVID-19 radiographic abnormali-
ties during the initial hospitalization complained of fever, persistent cough, or continuous
breathlessness on follow up till completion of the study. There were no clinically suspected
post-COVID lung complications and the skiagrams of several patients were reported normal.
In comparison to SOC, AYUSH Plus showed significant improvement in several domains
(physical health, psychological health, social relationship, and environmental well-being) in
the WHO QOL BREF and the total HR-BHF score at the time of clinical recovery and during
follow-up (Table 4). It was notable that the AYUSH Plus also showed significant improvement
in several individual item scores (fatigue, stress, anxiety, appetite, and happiness) in the
HR-BHF questionnaire as compared to SOC (See Table S4.1 in S4 File).

Safety and related issues

28 patients in the AYUSH Plus and 29 patients in the SOC group reported adverse events:
there were no statistically significant differences (Table 5). Each of the study group reported 48
AE. Additional data on AE is shown in Table S6.1 in S6 File.

Table 3. Selected biomarkers related to COVID-19 in the two study groups (n = 139); mean (days) ± standard deviation.
Variable Study groups Baseline On discharge� Week 12�
Lactose dehydrogenase (LDH) AYUSH plus 403.6 ± 131.6 338.9 ± 109.7 318.7 ± 109.6
SOC 446.7 ± 206.5 363.8 ± 115.2 381.9 ± 164.8
LDH Reference Range: 225–480 U/L
Ferritin AYUSH plus 337.8 ± 280.3 257.7 ± 226.1 84.4 ± 70.2
SOC 337.4 ± 278 201.8 ± 206 92.5 ± 89.3
Ferritin Reference Range: Male 30–350 ng/ml; Female 20–250 ng/ml
Procalcitonin AYUSH plus 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.2
SOC 0.1 ± 0.04 0.1 ± 0.2 0.1 ± 0.2
Prolactin Reference Range: <0.2 ng/ml
C-reactive protein AYUSH plus 20.83 ± 27.55 10.3 ± 19.1 6.3 ± 6.5
SOC 25.5 ± 35.3 10.7 ± 12.5 6.39 ± 8.98
C-Reactive Protein Reference Range <3 mg/L
D-Dimer AYUSH plus 462.5 ± 439.9 334 ± 224.9 297.3 ± 277.6
SOC 523.2 ± 672.8 345.3 ± 324.2 317.9 ± 418.4
D-Dimer Reference Range:0–400 ng/ml
Interleukin-6 AYUSH plus 30.6 ± 46.0 7.7 ± 12.2 8.5 ± 22.1
SOC 32.6 ± 42.2 8.5 ± 15.8 7.4 ± 10.3
Interleukin-6 Reference Range: Up to 7 pg/ml
�
Statistically significant (P<0.05, Mann Whitney statistic) change from baseline within the study group for all variables except Pro-calcitonin
��
NS Not statistically significant (P> = 0.05)
Note, Abbreviation: AYUSH plus: AYUSH 64 + Standard of Care (SOC); n = number of participants; See text for detail

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Table 4. Quality of life questionnaires and health scores (HR-BHF and WHO QOL Bref) in the two study groups (n = 139).
Variable Baseline (n = 139) Discharge (n = 137) Week 4 (n = 129) Week 8 (n = 127) Week 12 (n = 120)
Health-Related- Behavior, Habit and Fitness (HR-BHF) questionnaire: combined score
AYUSH plus 500.1 ± 89.9 667.4 ± 85.7� 690.7 ± 111� 721.6 ± 105.5� 748.1 ± 114.5��
SOC 493.4 ± 81 637.5 ± 81.1 650.6 ± 100.6 677.7 ± 89.9 682.4 ± 90.9
WHO BREF Quality of life (QOL) Domain I (Physical health)
AYUSH plus 24.6 ± 4.1 28.8 ± 2.2� 28.9 ± 2.34 30.0 ± 2.15 30.2 ± 2.07
SOC 23.05 ± 4.42 27.8 ± 2.82 28.6 ± 2.7 29.3 ± 1.8 29.4 ± 2.1
WHO BREF Quality of life (QOL) Domain II (Psychological health)
AYUSH plus 20.81 ± 3.67 23.48 ± 2.28 24.2 ± 1.51 24.6 ± 1.70� 24.7 ± 1.88
SOC 20.1 ± 4.04 23.2 ± 2.29 23.4 ± 2.21 23.9 ± 1.57 24.1 ± 1.88
WHO BREF Quality of life (QOL) Domain III (Social health)
AYUSH plus 10.21 ± 2.03 11.34 ± 1.33 11.98 ± 1.02 12.05 ± 1.19� 12.30 ± 1.22��
SOC 10.26 ± 2.21 11.52 ± 1.29 11.74 ± 1.35 11.55 ± 1.48 11.62 ± 1.25
WHO BREF Quality of life (QOL) Domain IV (Environmental health)
AYUSH plus 27.27 ± 4.68 30.81 ± 2.30 31.74 ± 2.40 32.32 ± 2.73 32.22 ± 2.52�
SOC 26.66 ± 5.21 30.38 ± 2.45 30.90 ± 2.54 31.55 ± 2.06 31.43 ± 2.32
�
Statistically significant (P<0.05, Mann Whitney statistic)
��
Statistically highly significant (P<0.01, Man Whitney statistic)
NS Not statistically significant (P> = 0.05)
a HR-BHF total score and WHO BREF QOL domain score (physical, psychological social, and environmental health) were significantly better in AYUSH plus study
group at several study time points
b. Note, See S4 File for methods and scoring of WHO QOL Bref and HR-BHF (Health Related-Behavior Health and Fitness) questionnaire; HR-BHF score range 0–900;
WHO QOL Bref domain scores vary as shown in S 4 File but higher score generally meant better outcome; n: number of participants; AYUSH 64 plus: AYUSH 64 plus
Standard of Care (SOC); See text for details

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AE were generally mild in nature and pertained to episodic fever, myalgias, fatigue, occa-
sional breathlessness, loss of taste and/or smell and were mostly reported during the post-hos-
pitalization follow-up. Several AE were possibly symptoms of COVID-19 rather than due to
any study drug. A probable or definite causality of AE with AYUSH 64 could not be confirmed
in any of the study participants. However, based on a-priori knowledge and experience of the
Ayurvedic physicians in the study, some of the gut-related AE, albeit mild, which were present
in the AYUSH plus may have been due to AYUSH 64 medication. Most of the time, AE did
not require any specific treatment. Three participants reported serious AE and all recovered
without any complications. Moderate AE was treated symptomatically. Those suspected of
severe AE were referred to a specialist for an opinion. Participants with naïve hyperglycemia
and/ or dyslipidemia were managed by a specialist physician.
Clinically, none of the AE was related to a drug interaction.
Repeated routine laboratory assays remained within normal limits in the two arms and
there were no significant differences between the treatment arms Table S7.1 in S7 File. Electro-
cardiography of all participants was reported normal at baseline, hospital discharge, and on
study completion.

Discussion
This randomized controlled multicentric study showed that a combination regimen of
AYUSH 64, a standard Ayurveda drug, and SOC was significantly superior to SOC in the treat-
ment of mild and moderate COVID-19. 140 eligible participants were randomized for study
intervention and monitored under direct physician observation in an in-patient COVID

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Table 5. Adverse events (AE) in the two study groups� (n = 139).

Adverse Events AYUSH plus (n = 69) OC (n = 70)
Participant Events Participant Events
Summary
Total 28 48 29 48
Mild 14 27 14 33
Moderate 12 19 13 14
Severe 2 2 2 2
Serious AE 1 1 2 2
Causality
Unrelated - 45 - 47
Unlikely - 03 - 2
AE is classified according to System Organ Classification and Preferred Term/Diagnosis (according to the investigator)
Cardiac 1 1 0 0
Transient hypertension 1 1 0 0
Ear and labyrinth 0 0 1 1
Ear ache 0 0 1 1
GIT 10 10 5 5
Gastritis 0 0 1 1
Dyspepsia 1 1 1 1
Diarrhea 4 4 1 1
Constipation 2 2 1 1
Epigastric pain 1 1 0 0
Vague pain 0 0 1 1
Hyperacidity 2 2 0 0
Hepatic 1 1 0 0
Transaminitis 1 1
Infections & infestations 5 6 6 9
Episodic Fever 5 5 2 2
Malaria 0 0 2 2
Cellulitis 0 0 1 1
Sore throat 1 1 3 4
Skin 2 2 1 1
Itch 1 1 0 0
Eczema 1 1 0 0
Non-specific 1 1
Respiratory 6 7 7 8
Cough 1 1 2 2
Episodic Breathlessness 5 6 4 5
Non-specific 0 0 1 1
Nervous system 3 3 0 0
Neuropathy 1 1 0 0
Vertigo 2 2 0 0
Renal 0 0 1 1
Dysuria 0 0 1 1
Endocrine 6 6 6 6
Hyperglycemia 6 6 6 6
Investigation (Laboratory) 0 0 1 1
Hyperlipidemia 0 0 1 1
(Continued )

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

Table 5. (Continued)

Adverse Events AYUSH plus (n = 69) OC (n = 70)

Others 12 13 12 15
Weakness 2 2 5 6
Chills 0 0 1 1
Myalgia 5 6 6 8
Arthralgia 2 2 0 0
Headache 3 3 0 0

(1) Abbreviations: AYUSH Plus: AYUSH 64 plus Standard of Care (SOC); n: number of participants; GIT: gastrointestinal
(2) Clinical grading as per WHO classification
(3) Causality in the AYUSH plus pertained to AYUSH 64 drug while causality in the SOC arm was not specified to any particular drug
(4) Transaminitis: raised serum glutamic oxalacetate and or pyruvate
(5) No AE recorded for Disorders of blood and lymphatic, immune system, metabolism and nutrition, psychiatric, reproductive system and breast, eye, vascular system,
congenital familial and genetic, injury poisoning and procedural complications, and surgical and medical procedures
(6)See Text and S6 File for further detail

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hospital setting. The 95% confidence interval of the difference in the mean duration (days) of
clinical recovery (a-priori definition) from randomization baseline was—3.02 to—0.59 days
(Table 2) as per the protocol analysis and -3.11 to -0.71 as per the intention-to-treat analysis
Table S5.5 in S5 File in favor of the AYUSH plus intervention. The latter was also shown at
each study site. A significantly higher proportion of AYUSH plus participants (69.7% versus
51.7%) achieved clinical recovery within the first week after randomization. AYUSH Plus also
showed substantial, and often significant, improvement in several secondary efficacy and qual-
ity of life measures (Tables 2, 4).
AYUSH 64 was well tolerated and found safe over 12 weeks of use in the dosage prescribed
in the current study. There were no differences in the AE between the two study groups. AE
were generally mild, and none caused the withdrawal of participants. Only 3 serious AE were
reported (2 in SOC). 20 participants withdrew from the study and mostly after clinical recov-
ery as per the personal preference not to continue in the study. There were no deaths in the
study.
Though enrolled with mild and moderate COVID-19, several participants in the current
trial also suffered from chronic co-morbid disorders (Table 1) that have been reported to be
risk factors for severe, progressive, and fatal disease [2–4, 6, 7]. Several naïve participants
showed hyperglycemia on enrolment (Table 1) which has been reported to complicate recov-
ery [38]. One participant in the AYUSH Plus developed Guillain Barre Syndrome which has
been uncommonly reported as a COVID-19 neurological complication [39]. Over 60% of par-
ticipants showed radiographic abnormalities consistent with COVID-19. Following recovery,
none of the participants complained of persistent respiratory symptoms or were diagnosed
with pulmonary fibrosis during the prolonged follow up. It is prudent to add that the current
study protocol did not recommend CT scan of the chest for the diagnosis of an asymptomatic
pulmonary sequel. Respiratory disorders including pulmonary fibrosis, and which are often
asymptomatic, have been reported as an important COVID-19 complication [40].
COVID-19 is a dreadful disease with a huge burden of psychosocial disorders [41]. A meta-
analysis from India reported several psychological comorbidities ranging from 26% (anxiety
and depression) to 40% (poor sleep quality) of study participants [42]. In the current study,
WHO QOL Bref and HR-BHF questionnaires were used. The significantly superior improve-
ment in both the physical (including fatigue) and mental health (such as reduction in anxiety,

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

and stress) shown in the AYUSH Plus was clinically important and needs to be emphasized
(Table S4.1 in S4 File). Several Ayurvedic medicines including the herbal ingredients of
AYUSH 64 are reported to improve mental health [15, 17, 18, 20]. Several other QOL measures
also showed a better improvement in the AYUSH Plus group (Table 4). In the passing, we
wish to add that our study participants found it easier to answer visual analog scale-based
questions in the HR-BHF questionnaire as compared to the somewhat cumbersome but popu-
lar WHO QOL BREF questionnaire (S4 File) [32].
More participants in the SOC arm showed definite radiographic pneumonitis (30% versus
19%) and failed resolution of radiographic abnormalities (85% versus 63%) at the time of clini-
cal recovery/hospital discharge. One patient with mild radiographic disease in the SOC arm
developed acute onset of progressive respiratory distress and required oxygenation and inten-
sive care for recovery. All of this may suggest a more serious form of disease in the SOC arm,
but this does not seem to be the case as shown by several other clinical variables, serum bio-
markers, and overall clinical progress and response to standard of care treatment (Tables 1, 3
and S5 File). No uniform protocol was followed for radiological evaluation in the current
study. Radiographic abnormalities often persist beyond clinical recovery and take a longer
time for resolution, and often do not conform to the clinical severity of symptoms or disease
[40]. Also, there is insufficient data on a prospective evaluation of radiographic abnormalities
shown by conventional skiagrams in COVID-19 [40].
This study was exploratory in design and carried out during the first year of the pandemic.
There were several concerns while the preparing the protocol. The pandemic and the stringent
lock down imposed unique challenges for enrollment, physical and other examination, and
monitoring of study participants. Our overall experience was consistent with that described
recently in a report on drug trials in COVID-19 [43]. People were intensely scared and reluc-
tant to participate. All treatment was mostly empirical and based on repurposed drugs [1–3].
There was no uniform protocol for standard care [3]. There were ethical issues with the use of
placebo and blind study design.

Strengths and limitations

The study data was captured using a pragmatic protocol. Presuming a modest effect size, a
sample size of 128 subjects was suggested [37]. However, as there was no prior data to guide
the latter assumption (see section above on statistical analyses), a convenience sample of 140
participants was agreed by the study experts. The concern of a selection bias due to a conve-
nience sample size and an open label design seemed to have been nicely addressed by the ran-
domization process and the study drug administration under direct medical observation
during the hospitalization phase. Encouragingly, the two intervention groups were found to be
well matched for several variables at randomization baseline (Table 1). The daily diligent clini-
cal observation in the hospital ensured good compliance to the study intervention and robust
efficacy data. The latter was also crucial for capturing AE and any obvious drug interaction.
The tolerability and safety profile of AYUSH 64 was good and reassuring.
The confirmation of ‘Clinical Recovery’ (CR) in the current study may seem to be unduly
subjective but was based on a pre-determined set of stringent criteria which included clinical
and investigation measures (normal peripheral oximetry and a negative standard RT-PCR
assay). Importantly, 48 hours of observation was mandatory to declare the resolution of symp-
toms and the total assessment was performed in a blinded manner. To the best of our knowl-
edge, we did not find use of a similar set of criteria in any other interventional drug trial in
COVID-19 during a search for relevant literature in the current study [44–46]. We did not
measure ‘viral load’ as was performed in most of the drug trials [44]. The viral load may not

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

correlate with symptoms in mild and moderate disease, and during recovery [45]. It is notable
that none of the study participants reported any clinical post COVID complication and actu-
ally improved their general physical and mental health during the follow-up period (Table 4).
There were other limitations that may have influenced the study outcome. Enrolment of
subjects with early illness was a complex issue as was observed in several other COVID drug
trials [43–45]. The delay was about a week from the onset of symptoms (Table 1 and S5 File).
Investigations were not carried out in a central lab due to difficult logistics, but all study site
laboratories were accredited (national standards) and compelled to strictly adhere to the guide-
lines on molecular testing for SARS-CoV-2 and quality control [35].
We were concerned about the surreptitious use of Ayurvedic drugs in the current drug
trial. Ayurveda drugs and other popular traditional home remedies were extensively used in
India during the pandemic and AYUSH 64 was available in the market [19, 20, 23]. None of
the study participants admittedly used Ayurvedic drugs prior to hospital admission. It is
unlikely that any medicine other than that permitted in the current study was taken by the par-
ticipants during the in-patient treatment phase. Patients were counseled regarding medication
by the study physician at the time of hospital discharge. Only the AYUSH plus participants
were to continue AYUSH 64 drug till study completion. A special mobile software application
(see methods) was used to maintain regular contact with the study participants. It is notewor-
thy, that several participants who continued AYUSH 64 showed better improvement in physi-
cal and mental health during the prolonged follow-up (Table 4).
The current study dealt with mild and moderate COVID-19 and no extrapolation of the
outcome can be made to progressive and or severe disease. By the last quarter of 2020, the
management of mild and moderate COVID-19 was rapidly shifted to a domiciliary or a quar-
antine facility in India [47]. Though the current trial participants were treated in a hospital set-
ting (current study), it seemed fair to recommend the use of AYUSH 64 in a domiciliary or a
quarantine setting under appropriate medical supervision [48].

Mechanism of action
The human host, and not the microbe, is the therapeutic focus in Ayurveda while treating
infections. The primary objective is to strengthen immunity. Ayurvedic physicians use a holis-
tic approach to treat and heal which includes assessment of the individual constitution (called
Prakruti and Doshas in Ayurveda) and several lifestyle changes [15, 21]. The pharmacological
and therapeutic properties and experimental evidence (non-clinical) of AYUSH 64 and its
ingredient medicinal plant extracts were recently published [18]. Some of the purported thera-
peutic properties were antipyretic, anti-infective, anti-inflammatory, anti-allergic, and immu-
nomodulatory (called Rasayana in Ayurveda) [18, 26, 49].
Several experimental studies (animal, cell culture, and in-vitro model) of individual plant
extract ingredients of AYUSH 64 have provided a wide array of evidence to explain the reduc-
tion in inflammation and modulation of immune response (anti-oxidant effect, increased
phagocytosis and altered inflammatory pathways- Nuclear Factor Kappa B, p 65), direct inhi-
bition of pro-inflammatory biochemical mediators and cytokines (prostaglandins, tumour
necrosis factor alfa, Interleukin (IL)-1 beta, IL-6, and IL- 8), and suppression of inflammatory
and allergic response in airways (cellular and cytokines) [18, 26, 49–53]. Interestingly, several
inhibitory effects were also shown against viral protein R (HELA cells and plasmids) and some
specific viruses (such as Herpes Simplex Type I, Coxsackie B2, Adenovirus, Poliovirus, and
Chikungunya) [25, 50–53]. In a more recent in-silico molecular docking study, several ingredi-
ents of AYUSH 64 (and especially Akuammicine N-Oxide from Alstonia scholaris) showed
good binding with the main protease enzyme of the SARS-CoV-2 [54]. AYUSH 64 showed

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

uncomplicated recovery with lesser requirement of symptomatic drugs and good safety when
administered along with standard symptomatic treatment to 38 patients suffering from influ-
enza-like illness in a prospective uncontrolled study of about one week duration [26].

Other selected studies

Several Ayurvedic drug trials in COVID-19 were registered during the pandemic and the
results of few published studies were encouraging [55, 56]. A uniform wholesome Ayurvedic
regimen showed a reduction in the viral load in asymptomatic and early COVID-19 patients
in a randomized placebo-controlled drug trial study but did not provide sufficient clinical data
[57].
A meta-analysis of 18 randomized controlled drug trials showed the clinical benefit of co-
administration of Chinese Herbal Medicine (CHM) with conventional Western Medicine
(WM) in treating COVID-19; two trials showed that CHM plus WM significantly reduced
hospital stay (95% CI of the mean difference -3.28 to -0.70 days) [14]. There were several meth-
odological differences between the latter and the current study but intriguingly, the outcome
of a mean reduction in hospital stay was almost similar. Superior cure rates and amelioration
of individual symptoms were reported in a more recent systematic review and meta-analysis of
controlled drug trials which used a combination of CHM and WM in mild to moderate
COVID-19 [58].
Of late, monoclonal antibodies (MAB) are at the forefront of treating COVID-19 although
they are presently contraindicated in severe and progressive disease [59]. However, MAB are
specific for a particular SARS-CoV-2 variant and are recommended for use in subjects with
early disease and risk factors (COVID-19). However, several issues connected with cost and
logistics are hurdles in their clinical use in the Indian context [45]. Oral drugs like AYUSH 64
hold a greater appeal.
Despite extensive clinical use during the pandemic, CAM therapies such as Ayurveda and
Traditional Chinese medicine seemed under-reported. [14, 20, 58]. A recent report based on a
large cross-sectional survey of in-patients treated for COVID-19 described the use of repur-
posed and adjuvant modern medicines but failed to make mention of herbal drugs or other
CAM therapies [60].

Study implications and dissemination of results

In view of the lack of evidence for effective and safe drug therapy in COVID-19, several poten-
tial Ayurveda drugs and CAM were selected for repurpose and accelerated research and devel-
opment [1, 2, 8, 9, 42]. Overall, the data from mild and moderate COVID-19 drug trials was
sparse [43–45]. The current drug- trial of AYUSH 64 ought to be viewed from this perspective.
In our experience, the success of the current study provided a substantial boost to the ongoing
research efforts in Ayurveda and CAM. We believe that it will also encourage an integrative
medicinal approach to treating difficult diseases like COVID-19.
It is prudent to add that several medicinal plant ingredients of AYUSH 64 have been used
to promote health and treat diverse medical disorders for several centuries by physicians and
traditional healers in India, China, Southeast Asia, Europe, and North America [18, 26, 49–
51].
Study participants were informed about the current study results telephonically and/or
through small virtual meetings by investigators and coordinators. A widely circulated Govern-
ment press release in May 2021 announced the core study results and promoted the use of
AYUSH 64 in COVID-19 [58]. A national education program was launched by the Ministry of
AYUSH to disseminate information about AYUSH 64 and other drugs [61]. Along with the

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

latter, a nationwide distribution campaign (AYUSH 64) was also launched [61]. Simulta-
neously, the Ministry of AYUSH launched an evidence-based management protocol for Ayur-
veda and Yoga for the management of COVID-19 which contained a reference to the current
study [48].

Future research
AYUSH 64 ought to be further evaluated for the treatment of mild and moderate COVID-19,
both as mono and a combination therapy (modern medicine), in a phase III drug trial. Studies
should also evaluate the potential of AYUSH 64 to block progression of COVID-19 to severe
disease and reduce post-COVID-19 complications. Experimental evidence is required to vali-
date its anti-viral and other health benefits.

Conclusion
AYUSH 64 (a standardized polyherbal Ayurveda drug) was shown to be a significantly effec-
tive and safe adjunct in the treatment of mild and moderate COVID-19 in a prospective, ran-
domized controlled drug trial. Open-label study design and other limitations necessitate
judicious interpretation and extrapolation of the current study data and outcome. AYUSH 64
hastened clinical recovery, reduced hospitalization period, and showed early persistent health
benefits with minimal/ absent drug-related side effects.

Supporting information
S1 File. CONSORT check list.
(DOC)
S2 File. Study protocol.
(DOCX)
S3 File. Composition, chemistry, manufacturing, and controls of AYUSH 64.
(DOCX)
S4 File. Health and quality of life questionnaires (WHO-QOL & HR-BHF).
(DOCX)
S5 File. Additional data–standard of care drugs, site specific drugs & timelines, general lin-
ear model output, intention to treat analysis, and radiological data.
(DOCX)
S6 File. Additional data- adverse events.
(DOCX)
S7 File. Additional data- laboratory results.
(DOCX)
S8 File. Selected raw data—efficacy, withdrawals, adverse events.
(DOCX)

Acknowledgments
A special thanks to Vaidya Dr. Rajesh Kotecha, Secretary, Ministry of AYUSH, Government of
India, for his invaluable guidance and encouragement towards the current study project and
preparation of study publication. We are grateful to senior Vaidya KS Dhiman, former DG
CCRAS, GOI for the speedy completion of the drug trial. We thank several research colleagues

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PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

in the CCRAS—Dr Ravindra Singh, Dr Shruti Khunduri, and Dr B S Sharma. We acknowl-

edge with gratitude several colleagues from each trial site- Dr Manish Deshmukh, Dr Swati
Munde, Dr Pratap Makhija, Dr Alia Rizvi, Prof Wahid Ali, Dr. Neeta Warty, Dr. Parth Dave
and Mr Akash Saggam. We make a special mention of Dr Manesha Talekar, an Ayurvedic phy-
sician, who volunteered to be a co-investigator despite pregnancy and contracted COVID-19
during her medical duty. Dr Vinay Pawar played an important role in statistical analysis.
Finally, we thank all the patients with folded hands for their participation and wholehearted
support.

Author Contributions
Conceptualization: Arvind Chopra, Girish Tillu, Manjit Saluja, Sanjeev Sarmukaddam, Nar-
ayanam Srikanth, Bhushan Patwardhan.
Formal analysis: Arvind Chopra, Girish Tillu, Sanjeev Sarmukaddam.
Funding acquisition: Narayanam Srikanth, Bhushan Patwardhan.
Investigation: Kuldeep Chuadhary, Govind Reddy, Alok Srivastava, Muffazal Lakdawala,
Dilip Gode, Himanshu Reddy, Sanjay Tamboli, Manohar Gundeti, Ashwini Kumar Raut.
Methodology: Arvind Chopra, Girish Tillu, Manjit Saluja, Sanjeev Sarmukaddam, Ashwini
Kumar Raut, Narayanam Srikanth, Bhushan Patwardhan.
Project administration: Arvind Chopra, Sanjay Tamboli, Manjit Saluja, B. C. S. Rao, Babita
Yadav.
Resources: B. C. S. Rao, Babita Yadav, Narayanam Srikanth.
Software: Sanjay Tamboli.
Supervision: Arvind Chopra, Narayanam Srikanth, Bhushan Patwardhan.
Validation: Arvind Chopra, Kuldeep Chuadhary, Govind Reddy, Alok Srivastava, Muffazal
Lakdawala, Dilip Gode, Himanshu Reddy, Sanjay Tamboli, Manjit Saluja, Manohar Gun-
deti, Ashwini Kumar Raut, B. C. S. Rao, Babita Yadav.
Visualization: Arvind Chopra.
Writing – original draft: Arvind Chopra, Girish Tillu.
Writing – review & editing: Girish Tillu, Kuldeep Chuadhary, Govind Reddy, Alok Srivastava,
Muffazal Lakdawala, Dilip Gode, Himanshu Reddy, Sanjay Tamboli, Manjit Saluja, Sanjeev
Sarmukaddam, Manohar Gundeti, Ashwini Kumar Raut, B. C. S. Rao, Babita Yadav, Nar-
ayanam Srikanth, Bhushan Patwardhan.

References
1. Khheirabadi D, Haddad F, Mousavi-Roknabadi RS, Rezaeisadrabadi M, Dehghan H, Fazlzadeh A. A
complementary critical appraisal on systematic reviews regarding the most efficient therapeutic strate-
gies for the current COVID-19 (SARS-CoV-2) pandemic. J Med Virol. 2021; 93: 2705–2721. https://doi.
org/10.1002/jmv.26811 PMID: 33463727
2. NIH Coronavirus Disease 2019 (COVID-19) Treatment Guidelines. NIH. 2020. Available from: https://
files.covid19treatmentguidelines.nih.gov/guidelines/covid19treatmentguidelines.pdf
3. Guidelines on Clinical Management of COVID-19 issued by Ministry of Health and Family Planning,
Government of India (17 Mar 2020). Available from https://www.mohfw.gov.in/pdf/
GuidelinesonClinicalManagementofCOVID1912020.pdf
4. Richardson S, Hirsch JS, Narasimhan M, Crawford JM, McGinn T, Davidson KW, et al. Presenting
Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in

PLOS ONE | https://doi.org/10.1371/journal.pone.0282688 March 16, 2023 20 / 23

PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

the New York City Area. JAMA 2020; 323: 2052–2059. https://doi.org/10.1001/jama.2020.6775 PMID:
32320003
5. Bi Q, Wu Y, Mei S, Ye C, Zou X, Zhang Z et al. Epidemiology and transmission of COVID-19 in 391
cases and 1286 of their close contacts in Shenzhen, China: a retrospective cohort study. Lancet Infect
Dis 2020; 20: 911–19. Available from: https://doi.org/10.1016/S1473-3099(20)30287-5 PMID:
32353347
6. Berlin DA, Gulick RM, Martinez FJ. Severe Covid-19. N Engl J Med 2020; 383: 2451–2460. https://doi.
org/10.1056/NEJMcp2009575 PMID: 32412710
7. Williamson EJ, Walker AJ, Baskaran K, Bacon S, Bates C, Morton CE, et al. Factors associated with
COVID-19-related death using Open SAFELY. Nature. 2020; 584: 430–436. https://doi.org/10.1038/
s41586-020-2521-4 PMID: 32640463
8. Yousefi H, Mashouri L, Okpechi SC, Alahari N, Alahari SK. Repurposing existing drugs for the treatment
of COVID-19/SARS-CoV-2 infection: A review describing drug mechanisms of action. Biochem Phar-
macol. 2021; 183: 114296. https://doi.org/10.1016/j.bcp.2020.114296 PMID: 33191206
9. Hernandez A V, Roman YM, Pasupuleti V, Barboza JJ, White CM. Hydroxychloroquine or Chloroquine
for Treatment or Prophylaxis of COVID-19: A Living Systematic Review. Ann Intern Med. 2020 [cited 4
Jun 2020]. https://doi.org/10.7326/M20-2496 PMID: 32459529
10. Grein J, Ohmagari N, Shin D, Diaz G, Asperges E, Castagna A, et al. Compassionate use of remdesivir
for patients with severe Covid-19. N Engl J Med 2020, 382, 2327–2336. https://doi.org/10.1056/
NEJMoa2007016 PMID: 32275812
11. Fu B, Xu X, Wei H. Why tocilizumab could be an effective treatment for severe COVID-19? J Transl
Med. 2020; 18: 164. https://doi.org/10.1186/s12967-020-02339-3 PMID: 32290839
12. Horby P, Lim WS, Emberson JR, Mafham M, Bell JL, Linsell L, et al. Dexamethasone in Hospitalized
Patients with Covid-19. N Engl J Med. 2021; 384: 693–704. https://doi.org/10.1056/NEJMoa2021436
PMID: 32678530
13. Rastogi S, Pandey DN, Singh RH. COVID-19 pandemic: A pragmatic plan for ayurveda intervention. J
Ayurveda Integr Med. 2020. https://doi.org/10.1016/j.jaim.2020.04.002 PMID: 32382220
14. Xiong X, Wang P, Su K, Cho WC, Xing Y. Chinese herbal medicine for coronavirus disease 2019: A sys-
tematic review and meta-analysis. Pharmacol Res. 2020; 160: 105056. https://doi.org/10.1016/j.phrs.
2020.105056 PMID: 32622723
15. Payyappallimana U, Patwardhan K, Mangalath P, Kessler CS, Jayasundar R, Kizhakkeveettil A, et al.
The COVID-19 Pandemic and the Relevance of Ayurveda’s Whole Systems Approach to Health and
Disease Management. J Altern Complement Med. 2020; 26: 1089–1092. https://doi.org/10.1089/acm.
2020.0370 PMID: 33121250
16. Dilip Pandkar P, Sachdeva V. Pathophysiology of COVID-19 and Host centric approaches of Ayurveda.
J Ayurveda Integr Med. 2020. https://doi.org/10.1016/j.jaim.2020.11.010 PMID: 33519134
17. Tillu G, Chaturvedi S, Chopra A, Patwardhan B. Public Health Approach of Ayurveda and Yoga for
COVID-19 Prophylaxis. J Altern Complement Med. 2020; acm.2020.0129. https://doi.org/10.1089/acm.
2020.0129 PMID: 32310670
18. Ahmad S, Zahiruddin S, Parveen B, Basist P, Parveen A Gaurav, et al. Indian Medicinal Plants and For-
mulations and Their Potential Against COVID-19-Preclinical and Clinical Research. Front Pharmacol.
2020; 11: 578970. https://doi.org/10.3389/fphar.2020.578970 PMID: 33737875
19. Srikanth N, Rana R, Singhal R, Jameela S, Singh R, Khanduri S, et al. Mobile App-Reported Use of Tra-
ditional Medicine for Maintenance of Health in India During the COVID-19 Pandemic: Cross- sectional
Questionnaire Study. JMIRx Med. 2021; 2: e25703. https://doi.org/10.2196/25703 PMID: 34032815
20. Debnath R, Bardhan R. India nudges to contain COVID-19 pandemic: A reactive public policy analysis
using machine-learning based topic modelling. PLoS ONE 2020; 15(9): e0238972. https://doi.org/10.
1371/journal.pone.0238972 PMID: 32915899
21. Chopra A, Doiphode V V. Ayurvedic medicine. Core concept, therapeutic principles, and current rele-
vance. Med Clin North Am. 2002; 86: 75–89. https://doi.org/10.1016/s0025-7125(03)00073-7 PMID:
11795092
22. Kotecha R. The journey with COVID-19: Initiatives by Ministry of AYUSH. J Ayurveda Integr Med 2021;
12(1): 1–3. https://doi.org/10.1016/j.jaim.2021.03.009 PMID: 33812534
23. Patwardhan B, Sarwal R. Significance of AYUSH: India’s first line of defense against COVID-19. J Ayur-
veda Integr Med. 2021; 12: 227–228. https://doi.org/10.1016/j.jaim.2021.05.007 PMID: 34082895
24. Trial registration in Clinical Trial Registry of India -CTRI/2020/06/025557 [Registered on: 02/06/2020].
Available from: http://ctri.nic.in/Clinicaltrials/showallp.php?mid1=43812&EncHid=&userName=025557.
25. Kazim M, Puri SK, Dutta GP, Narasimham M V. Evaluation of Ayush-64 for blood schizontocidal activity
against rodent and simian malaria parasites. Indian J Malariol 1991; 28: 255–258. PMID: 1824361

PLOS ONE | https://doi.org/10.1371/journal.pone.0282688 March 16, 2023 21 / 23

PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

26. Gundeti MS, Bhurke LW, Mundada PS, Murudkar S, Surve A, Sharma R, et al. AYUSH 64, a polyherbal
Ayurvedic formulation in Influenza-like illness—Results of a pilot study. J Ayurveda Integr Med. 2020.
https://doi.org/10.1016/j.jaim.2020.05.010 PMID: 33446377
27. National guidelines of Ethics Committees reviewing biomedical and health research during COVID-19
pandemic (2020). Available from: https://main.icmr.nic.in/sites/default/files/guidelines/EC
28. Ministry of AYUSH. Government of India. Interdisciplinary AYUSH R & D Task Force. Guidelines for
AYUSH Clinical Studies in COVID-19 (April 2020). Available from: https://www.bing.com/search?q=
Ministry+of+AYUSH.+Government+of+India.+Interdisciplinary+AYUSH+R+%26+D+Task+Force.
+Guidelines+for+AYUSH+Clinical+Studies+in+COVID-19+(April+2020).&cvid=
0ce49902f6d04b32a00d3c5f79266890&aqs=edge..69i57.2235j0j1&pglt=297&FORM=ANSPA1&PC=
U531
29. Gagnier JJ, Boon H, Rochon P, Moher D, Barnes J, Bombardier C. Recommendations for reporting ran-
domized controlled trials of herbal interventions: Explanation and elaboration. J Clin Epidemiol. 2006;
59: 1134–1149. https://doi.org/10.1016/j.jclinepi.2005.12.020 PMID: 17027423
30. Ministry of AYUSH. Government of India. Available at http://main.ayush.gov.in.
31. Rubio-Rivas M, Mora-Luján JM, Formiga F, Arévalo-Cañas C, Lebrón Ramos JM, Villalba Garcı́a MV
et al. WHO Ordinal Scale and Inflammation Risk Categories in COVID-19. Comparative Study of the
Severity Scales. J Gen Intern Med. 2022; 37(8):1980–1987. https://doi.org/10.1007/s11606-022-
07511-7 PMID: 35396659
32. WHOQOL-BREF Introduction, Administration, Scoring and Generic Version of The Assessment Field
Trial Version December 1996 Programme on Mental Health. 1996. Available from: https://www.who.int/
mental_health/media/en/76.pdf
33. Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Event (Cor-
rected Version 2.1, July 2017). Available from: https://rsc.niaid.nih.gov/sites/default/files/corrected-
grading-table-v-2-1-with-all-changes-highlighted.pdf.
34. Maintenance of the ICH Guidelines on Clinical Safety Data management (CH2R, 2001). Available from:
http://admin.ich.org. Accessed on 14 Mar 2020.
35. https://www.icmr.gov.in/pdf/covid/labs/SARS_CoV2_using_TaqPath_COVID19_ComboKit.pdf
36. Covid Kavach—Apps on Google Play. [cited 26 Jun 2021]. Available from: https://play.google.com/
store/apps/details?id>=com.moa.covidrecorder&hl=en
37. Cohen J. “A power primer”. Psychological Bulletin, 1992; 112: 155–159. https://doi.org/10.1037//0033-
2909.112.1.155 PMID: 19565683
38. Bode B, Garrett V, Messler J, McFarland R, Crowe J, Booth R, et al. Glycemic Characteristics and Clini-
cal Outcomes of COVID-19 Patients Hospitalized in the United States. J Diabetes Sci Technol. 2020;
14: 813–821. https://doi.org/10.1177/1932296820924469 PMID: 32389027
39. Harapan BN, Yoo HJ. Neurological symptoms, manifestations, and complications associated with
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 19 (COVID-
19). J Neurol. 2021; 1–13. https://doi.org/10.1007/s00415-021-10406-y PMID: 33486564
40. Mallia P, Meghji J, Wong B, Kumar K, Pilkington V, Chhabra S, et al. Symptomatic, biochemical and
radiographic recovery in patients with COVID-19. BMJ Open Resp Res 2021; 8:e000908. https://doi.
org/10.1136/bmjresp-2021-000908 PMID: 33827856
41. Torales J, O’Higgins M, Castaldelli-Maia JM, Ventriglio A. The outbreak of COVID-19 coronavirus and
its impact on global mental health. Int J Soc Psychiatry 2020; 66: 317–320. https://doi.org/10.1177/
0020764020915212 PMID: 32233719
42. Krishnamoorthy Y, Nagarajan R, Saya GK, Menon V. Prevalence of psychological morbidities among
general population, healthcare workers and COVID-19 patients amidst the COVID-19 pandemic: A sys-
tematic review and meta-analysis. Psychiatry Res. 2020; 293: 113382. https://doi.org/10.1016/j.
psychres.2020.113382 PMID: 32829073
43. Chen Z, Chen L, Chen H. Effect of COVID-19 on Clinical Drug Trials. PLoS ONE 2021; 16(5):
e0251410
44. Dodd LE, Follmann D, Wang J, Koenig F, Korn LL, Schoergenhofer C et al. Endpoints for randomized
controlled clinical trials for COVID-19 treatments. Clin Trials. 2020; 17(5):472–482. https://doi.org/10.
1177/1740774520939938 PMID: 32674594
45. Chopra A. Monoclonal antibodies to treat COVID 19 in rheumatoid arthritis: A case report and a clinical
appraisal of selected drug trials. Indian J Rheumatol 2022; 17 (3): 300–305. https://doi.org/10.4103/injr.
injr_5_22
46. Kim JY, Săndulescu O, Preotescu LL, Rivera-Martı́nez NE, Dobryanska M, Birlutiu V et al. A Random-
ized Clinical Trial of Regdanvimab in High-Risk Patients With Mild-to-Moderate Coronavirus Disease

PLOS ONE | https://doi.org/10.1371/journal.pone.0282688 March 16, 2023 22 / 23

PLOS ONE Adjunct treatment with AYUSH 64 in mild and moderate COVID-19

2019. Open Forum Infect Dis. 2022 8; 9(8):ofac406. https://doi.org/10.1093/ofid/ofac406 PMID:

36043180
47. Revised guidelines for home isolation of mild /asymptomatic COVID-19 cases by Ministry of Health and
Family Welfare, Government of India. 2021. Available from: https://www.mohfw.gov.in/pdf/
RevisedguidelinesforHomeIsolationofmildasymptomaticCOVID19cases.pdf
48. National Clinical Management Protocol Based on Ayurveda and Yoga for the management of COVID-
19 (2021). Available from: https://yoga.ayush.gov.in/public/assets/ayush-Protocol-covid-19.pdf
49. Gogte VM. Ayurvedic pharmacology and therapeutic uses of medicinal plants (Dravyagunavidnyan-
Translation). Mumbai: The Academic Team of Bharatiya Vidya Bhavan’s SPARC; 2000.
50. Natesh S. Remarkable Trees on NII Campus-Saptaparna. 2010. Available: http://nitte.edu.in/journal/
March2013/TSHFP.pdf.
51. Kumar V, Van Staden J. A Review of Swertia chirayita (Gentianaceae) as a Traditional Medicinal Plant.
Front Pharmacol. 2015; 6:308 (1–14). https://doi.org/10.3389/fphar.2015.00308 PMID: 26793105
52. Upadhyay P, Joshi BC, Sundriyal A, Uniyal S. Caesalpinia crista L.: A review on traditional uses, phyto-
chemistry and pharmacological properties. Curr Med Drug Res. 2019; 3:135–40.
53. Zhao Y-L, Shang J-H, Pu S-B, Wang H-S, Wang B, Liu L, et al. Effect of total alkaloids from Alstonia
scholaris on airway inflammation in rats. J Ethnopharmacol 2016; 178: 258–265. https://doi.org/10.
1016/j.jep.2015.12.022 PMID: 26707569
54. Ram TS, Munikumar M, Raju VN, Devaraj P, Boiroju NK, Hemalatha R, et al. In silico evaluation of the
compounds of the ayurvedic drug, AYUSH-64, for the action against the SARS-CoV-2 main protease. J
Ayurveda Integr Med 2022 Jan-Mar; 13(1):100413. https://doi.org/10.1016/j.jaim.2021.02.004 PMID:
33654345
55. Bhapkar V, Sawant T, Bhalerao S. A critical analysis of CTRI registered AYUSH studies for COVID- 19.
J Ayurveda Integr Med. 2022; 13(1):100370. https://doi.org/10.1016/j.jaim.2020.10.012 Epub 2020 Nov
26 PMID: 33262559
56. Rao MVV, Juneja A, Maulik M, Adhikari T, Sharma S, Gupta J, et al. Emerging trends from COVID- 19
research registered in the Clinical Trials Registry—India. Indian J Med Res. 2021; 153: 26–63. https://
doi.org/10.4103/ijmr.IJMR_2556_20 PMID: 33818466
57. Devpura G, Tomar BS, Nathiya D, Sharma A, Bhandari D, Haldar S, et al. Randomized placebo- con-
trolled pilot clinical trial on the efficacy of ayurvedic treatment regime on COVID-19 positive patients.
Phytomedicine. 2021; 84: 153494. https://doi.org/10.1016/j.phymed.2021.153494 PMID: 33596494
58. Du X, Shi L, Cao W, Zuo B, Zhou A. Add-on effect of Chinese herbal medicine in the treatment of mild to
moderate COVID-19: A systematic review and meta-analysis. PLoS One. 2021; 16(8):e0256429.
https://doi.org/10.1371/journal.pone.0256429 PMID: 34415962
59. Anti-SARS-CoV-2 Antibody Products. COVID 19 Treatment Guidelines Panel. Coronavirus Disease
2019 (COVID 19) Treatment Guidelines. National Institutes of Health. Available from: https://www.
covid19treatmentguidelines.nih.gov/=171672.
60. Prats-Uribe A, Sena AG, Lai LYH, Ahmed W-U-R, Alghoul H, Alser O, et al. Use of repurposed and
adjuvant drugs in hospital patients with covid-19: multinational network cohort study. BMJ. 2021; 373:
n1038. https://doi.org/10.1136/bmj.n1038 PMID: 33975825
61. Press Information Bureau Government of India. Ayush Ministry launches nationwide distribution cam-
paign of AYUSH 64 & Kabasura Kudineer. 2021May 07.Available from: https://pib.gov.in/
Pressreleaseshare.aspx?PRID55.

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