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Guduchi Ghanavati (Ayurveda medication) improves the perceived immunity in individuals at risk of SARS-CoV-2: A multicentred, controlled, before-and-after study

Introduction During the COVID-19 pandemic in India, Ayurvedic medication (Guduchi Ghanavati, GG) was prescribed by Ayurveda physicians for prevention and management. This study aimed to evaluate the prophylactic effect of GG in individuals with moderate to very high risk of SARS-CoV-2. Methods A multicenter, controlled, quasi-experimental, before-and-after study was conducted on individuals at moderate to very high risk of SARS-COV-2 exposure. In the intervention group (n = 15,992), participants received GG 1 g daily for 28 days in conjunction with standard preventive guidelines (SPG), while in the control group (n = 4953), participants were asked to follow SPG only. Outcomes were the incidence of COVID-19, perceived immune status, quality of life, and safety. The perceived immune status was assessed using a brief Likert-scale questionnaire having common immune-related complaints. Results Of the 20,945 enrolled, 20,574 completed the trial (intervention: 15,729, control: 4845). The percentage of participants who reported the incidence of COVID-19 was marginally lower in the GG+SPG group (41, 0.26%) than in the SPG group (16, 0.33%), leading to 21% (95% CI, -40% to 55%) efficacy of GG. However, the decrease in incidence percentage was statistically insignificant due to the trivial incidences reported. The scores of perceived immune status quality of life improved significantly from baseline in the GG+SPG group (p < 0.001) compared to the SPG group. Conclusion GG is safe and improves perception of immune status in individuals at risk of developing SRAS-CoV-2. However, these findings are inadequate to establish that GG lowers the incidence of COVID-19 necessitating to conduct RCTs in high-risk populations. Clinical Trial Registration CTRI/2020/06/025,525


Introduction
Coronavirus disease 2019 (COVID- 19), a global public health emergency, has affected millions of people, causing considerable morbidity and mortality [1]. As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted in its new lethal and rapidly transmissible variety, public health concerns have increased [2]. Apart from a few vaccinations, no pharmaceutical drugs have been shown to be effective in the prevention of COVID-19 [3][4][5]. Even today, the prevention and control of COVID-19 profoundly rely on physical distancing, use of personal protective equipment, and hand cleanliness [6][7].
Ayurveda, a traditional Indian medicine, has the potential to prevent and manage COVID-19. The principles of epidemiology and outbreaks, as well as its containment and management, are well described in Ayurveda, and the principle of increasing the host immune system through Rasayana drugs can be a prophylactic approach [8].
The Ministry of AYUSH (MoA, administrative body of traditional health systems in India) has published a guideline, "National Clinical Management Protocol for COVID-19 Based on Ayurveda and Yoga," which includes Ayurveda measures for the prevention and management of COVID-19 according to the severity of the disease. This guideline was developed based on the Ayurveda classics, clinical experience, empirical evidence, biological plausibility, and emerging patterns from ongoing clinical trials [9]. It is asserted that the reduced death rate due to COVID-19 in India is claimed to be due to the adoption of immunity-promoting medications by people from traditional systems such as Ayurveda. According to the protocol, Guduchi Ghanavati (also known as Samshamani Vati, prepared from the aqueous extract of Tinospora cordifolia Miers) was recommended as a prophylactic drug for high-risk populations of SARS-CoV-2 infection [9].
In addition, it is critical to understand the influence of such pandemics on mental health and other aspects of life. Psychological effects and immunological responses are linked to the COVID-19 pandemic [18,19]. Interventions with prophylactic potential against SARS-CoV-2 are required to investigate their influence on general population health and quality of life (QoL). This study aimed to evaluate the prophylactic effect of Guduchi Ghanavati (GG) in people with moderate to very high risk of SARS-CoV-2 infection through the incidence of COVID-19, perception of immunity, and QoL.

Trial design
This trial was a multicenter, prospective, controlled, before-and-after quasi-experimental study. The study was carried out in five cities (Ahmedabad, Vadodara, Bhavnagar, Gandhinagar, and Jamnagar city) in Gujarat state, India.

Participants
The target population for the trial was people who had a medium to very high risk of exposure to SARS-CoV-2 infection of any sex in the age group of 18 to 68 years, such as health care personnel directly or indirectly involved in COVID-19 management, front-line city/community workers, family members of COVID-19 cases (direct contact), people who inhabit quarantine, containment, and high flow/density population areas. Participants who were less exposed to SARS-CoV-2 or confirmed cases of COVID-19; pregnant and lactating females, known cases of uncontrolled diabetes, and hypertension or any other systemic uncontrolled conditions; an immune-compromised condition, such as HIV, hepatitis, tuberculosis, or cancer; any medical or surgical condition requiring immediate medical or surgical intervention; and those receiving any type of immunosuppressive therapy were excluded at the time of screening. A four-point ordinal scale (lower, medium, high, and very high) was developed that measures the risk of exposure according to the Occupational Safety and Health Administration's risk classification guidelines for occupational exposure to SARS-CoV-2 [20], the details of which are mentioned in the Supplemental File S1.
Individuals from the target population were recruited from the community-based public settings of five cities in Gujarat state, India, from June 9, 2020, to October 22, 2020. Initially, the team received a list of some individuals at high risk from the local health administration employee roster, and the recruitment process was initiated. To escalate recruitment further, the research team under local health administration guidance spotted high-risk areas and identified potential individuals such as quarantine personnel, dweller of COVID-19 hotspot zones, and close contact with COVID-19 patients. Thus, a small fraction of the screened potential individuals at high risk to SARS-CoV-2 were received from the government, and the research team themselves listed remaining out. All accessible individuals of high-risk areas were contacted door-to-door at their homes or places of employment and interviewed face-to-face for eligibility.
Individuals who met the criteria were invited to participate in the study and informed consent was obtained. Those who opted to participate were further examined to determine the baseline status of the outcome variables. Participants were assigned to a trial arm based on their choices. Participants who chose to take GG were assigned to the intervention group and received an entire course of trial drug with instructions, while those who opted out of receiving prophylaxis medicine were recruited as controls. Both groups were instructed to follow standard preventive guidelines (SPG). Patient's adherence to the protocol was recorded through the patient diary. Patients were instructed to complete a diary on a daily basis for dose and time of medication. At fortnightly intervals, all participants were contacted by telephone to assess adherence. The adherence percentage (number of pills taken during a specific period divided by the number of pills prescribed during the same period) was then computed using the patient diary and transformed to an ordinal scale [poor (50%), moderate (50 to 80%), and good (>80%). At the end of the trial, the outcome data were obtained by telephone contact with the registered participants.
Study team members were trained in community-based recruitment strategies and activities before commencing the trial. The study was prospective in which baseline data on individuals were obtained first, followed by intervention and follow-up over time to evaluate the effects on the intended outcome.

Sample size
The effect size of the trial drug could not be estimated for the primary outcome, as this was the first study of its kind. Considering that it was a population-based trial, the sample size was fixed at 20,000 participants. Due to the high demand for Ayurveda medicines in the Indian population during the pandemic, a 3:1 enrollment ratio was assumed for the intervention to control.

Intervention and control
The study drug "Guduchi Ghanavati" was obtained from the Indian Medicines Pharmaceutical Corporation Limited (IMPCL) Almora, Uttarakhand, India. Each tablet consisted of 250 mg of dry concentrated aqueous extract of the stem of Guduchi (Tinospora cordifolia). The trial drug was administered in a dose of two tablets (total 500 mg) twice daily, that is, 1 g/day orally after food for 28 days from baseline, and participants were instructed to follow the standard SARS-CoV-2 infection prevention guidelines (frequent hand washing, physical distancing, and covering the mouth and nose). The control group was advised to adhere to SPG only. This was performed for a duration of 28 days.

Incidence of COVID-19
The primary outcome was the incidence of SARS-CoV-2 infection (COVID-19 test-positive) in participants through self-reporting. The diagnostic policy for COVID-19 in Indian governments prevalent at that time is provided in Supplementary File S1. Considering the lack of reverse transcription-polymerase chain reaction (RT-PCR) test availability and administrative policy, the rapid antigen test was also used to confirm the diagnosis of COVID-19.
2.6. Secondary outcomes 2.6.1. Immune status (ISQ score) [21] The status of the perception of immune was determined using the immune status questionnaire (ISQ). The ISQ consists of seven items (common cold, diarrhea, sudden high fever, headache, muscle and joint pain, skin problems, and coughing). The items were classified on a 5level Likert scale ranging from) to 4 (0, never; 1, sometimes; 2, regularly; 3, often; 4, almost [always]), stating how often the participants experienced these complaints. The converted ISQ score ranged from 0 to 10, and higher scores corresponding to better immune functioning were used for the analysis. A single item question ranging from 0 (very poor) to 10 (excellent) was used for perceived immune functioning and perceived overall health status.

WHOQOL-BREF [22]
QoL was measured using the World Health Organization Quality of Life Instrument, Short Form (WHOQOL-BREF) at baseline and the end of the trial. It consists of 24 items (Likert scale score) to assess the perception of the QoL in four domains: physical health, psychological, social relationships, and environment. Individual domain scores were converted to a linear scale between 0 and 100, with a higher score indicating better QoL. Safety: The incidences adverse events encountered during the clinical study were recorded on Case Record Form. Severity of events were graded on a three-point scale (mild, moderate, severe) and its relationship to treatment was categorised in six categories (certain, probable, possible, unlikely, unclassified, unassessable) and assessed [23].

Statistical methods
Data were analyzed using IBM SPSS version 27. The primary outcome was analyzed using the risk ratio (RR), odds ratio (OR), adjusted odds ratio, and relative risk reduction (efficacy%). Continuous variables were evaluated using the paired t-test and analysis of covariance (ANCOVA) test for within-group and between-group analyses. Nonparametric tests, such as the Wilcoxon signed-rank test and the Mann− Whitney U test, were used to evaluate ordinal data for comparisons within and between groups. The effect size on secondary outcomes was represented by Cohen's d. Through bivariate logistic regression, the primary outcome was adjusted for age, sex, education, risk of exposure to SARS-Cov-2, and morbidity. Additionally, a subgroup analysis was performed for age category, exposure level, morbidity status, sex, economic status, religion, habitat and marital status. Correlations between perceived immune status and QoL were analyzed using Pearson's product moment coefficient (r). The confidence level was set at 95%, and p-values of less than 0.05 were considered significant.

Ethical approval and registration
The study was approved by the Institutional Ethics Committee, IPGT&RA, Jamnagar (approval no. PGT/7/-A/Ethics/2020-21/239 dated May 28, 2020), registered in the Clinical Trial Registry of India (CTRI/2020/06/025,525), and conducted according to the guideline of the Indian Council for Medical Research of good clinical practice.

Results
From June 9 to October 22, 2020, among 21,888 participants screened, 943 were found not to satisfy eligibility criteria and therefore, were excluded. Further, as 452 participants declined to participate, remaining 20,945 were included. Of these, 15,992 participants received GG with SPG (GG+SPG), whereas 4953 participants received SPG only. In the GG+SPG group, 89 participants dropped out, 18 terminated due to adverse events, and 156 were lost to follow-up; in the SPG group, 108 patients dropped out. Data from the remaining participants (GG+SPG, 15,729; SPG, 4845) were analyzed for the outcomes (Fig. 1). Recruitment was stopped once a predefined sample size was reached.
The baseline characteristics obtained from the participants in both groups are shown in Table 1. The mean age of the participants was 38.7 ± 12.1 years and 37.2 ± 12.4 years in the GG+SPG and SPG groups, respectively. The male: female ratio was 2.6:1 in the GG+SPG group and 1.88:1 in the SPG group. In the GG+SPG group, 58.7%, 31.3%, and 10% of the participants had a medium, high, and very high risk of exposure to COVID-19, respectively. Meanwhile, in the control group, 74%, 23.1%, and 2.9% participants had medium, high, and very high risk exposure to COVID-19, respectively. A total of 6.4% of the participants reported morbidity in the interventional group and 3.8% in the control group. A total of 9878 (48%) participants were recruited from the study site in Ahmedabad, 4878 (23.7%) from Vadodara, 2709 (13.2) from Gandhinagar, 1497 (7.3%) from Bhavnagar, and 1612 (7.8%) from Jamnagar (Table 1). At baseline, demographic characteristics was uniformly distributed between groups.

Incidence of COVID-19
COVID-19 incidence was reported by 41 (0.26%) participants in the GG+SPG group and 16 (0.33%) participants in the SPG group. Although the incidence was higher in the control group, the difference was not statistically significant between the two groups (RR and OR, 0.79; 95% CI, 0.44− 1.40; adjusted OR, 0.67; 95% CI 0.37− 1.21). The efficacy (relative risk reduction) of GG was 21% (95% CI, − 40% to 55%) higher than that of SPG, but the difference was not statistically significant ( Table 2).

Changes in immunity
Data on the perceived immune status of participants are presented in Table 3. The mean score of ISQ increased significantly (p < 0.001) from baseline to EOT in both groups. The perceived immune function score of 1 item was significantly (p < 0.001) improved in the GG+SPG interventional group only. Perceived general health also improved in both arms (p < 0.001 in GG+SPG, p = 0.04 in SPG). Compared to SPG, GG+SPG significantly improved the mean score of immune score, immune function, and general health (p < 0.001). The effect size (Cohen's d) of GG+SPG compared to SPG was small [ISQ (d): − 0.24, immune function; (d): − 0.13, general health]. Among the participants in the GG+SPG group, QoL scores improved significantly (p < 0.001) in all four domains of their baseline scores, whereas in the SPG group, mean scores increased in two domains, psychological (p < 0.001) and environmental (p = 0.007). At the end of the trial, the mean score improvement from baseline was considerably higher in the GG+SPG group than in the SPG group for all four domains  Table 5 illustrates the baseline correlations between perceived immune status, general health, and QoL. There was a positive correlation between perceived immune status and all domains of QoL. Immune status was strongly associated with general health (r = 0.826) and weakly associated with QoL. Moderate to strong associations were observed among all QoL domains. The physical, psychological, and environmental domains were strongly associated with each other. The social domain was also moderately associated with other QoL domains.

Adverse events reported
No serious adverse events were reported among the participants of the test drug group. In the GG+SPG group, of 18 non-serious adverse events reported, 17 events were mild and only 1 event had moderate severity. These events included gastric discomfort, diarrhea, and headache, common cold, throat pain, stomatitis, cough, weight gain, piles, weakness, and fever. Causality assessment showed that 'gastric discomfort' was drug-related adverse event, whereas diarrhea may have possibly relation to test drug. Other events were unlikely related to GG. However, the proportion of participants who reported adverse events was negligible (Table 6).

Drug adherence
Among all participants, 89.7% had good adherence to the trial drugs, followed by moderate adherence (6.7%). The incidence of COVID-19 was lower in moderate-to-good adherence to the trial drug than in poor adherence, implying that adherence to GG is related to a lower incidence of COVID-19. However, it needs further confirmation, as the frequency of the outcome is less (Table 7).

Subgroup analysis
Subgroup analyses were performed for the primary outcome on binary variables of age (age, ≤50 and >50), exposure level (medium, high to very high), status of morbidity (present or absent), sex (male, female), economic status (APL, BPL), religion (Hindu, Muslim), habitat (urban, other) and marital status (married, unmarried). The efficacy of GG+SPG compared to SPG did not change remarkably for variables such as age, morbidity, sex, economic status, religion, habitat and marital status in the subgroup analysis. However, when participants were stratified according to their risk of exposure, the efficacy was considerably altered, as the incidence of COVID-19 was significantly lower in GG+SPG than in SPG in participants with high to very high risk. In the high-risk subgroup, the efficacy of GG+SPG was 59% (CI 16% to 80%) greater than that of SPG. Due to the small number of incidents, these findings should be considered with caution ( Table 2 in Supplementary File S1). Similarities in outcome measures among sub-groups validates the study finding and ensures the generalizability among Indian population having diverse characteristics.

Discussion
To the best of our knowledge, this is the first study of its kind that has evaluated the prophylactic effect of GG in people with moderate to high risk exposure to COVID-19 by reporting the incidence of COVID-19, perceived immunity, and QoL. This study showed that oral administration of 1 g of GG daily for 28 days marginally reduced the incidence of COVID-19 compared to those who did not receive the prophylaxis drug. This statistically insignificant reduction can be attributed to a trivial occurrence reported by the participants, which can be further explained by the reduction in cases of COVID-19 at the study sites during the study period and the registration of the moderate risk group. In the subgroup analysis, the incidence of COVID-19 was significantly lower in the GG+SPG group than in the SPG group among high-risk subjects.
Guduchi is an essential "Rasayana" [24] drug that exhibits anti-depressive, anxiolytic [25,26], and immune-modulatory effects [12] through stimulation of nonspecific immune mechanisms [27]. Borse et al. established the mechanism by which GG modulates several immune pathways through bioactive target associations and showed that GG has the potential to inhibit the replication of SARS-CoV-2 using network pharmacology and in silico approaches [14,15]. Such properties of Guduchi, which modulate several immune pathways, are likely to explain the significant improvement in perceived immunity status and QoL reported in the present study. Improved QoL following SPG alone may be associated with increased awareness and education about the new pandemic situation.
For clinical significance, efficacy (%) (relative risk reduction) for the primary outcome and Cohen's d effect size for other outcomes were calculated. Furthermore, the relatively small effect size (Cohen's d) of the intervention on secondary outcomes might be due to the relatively shorter duration of the trial or the comparatively healthy status of most participants since baseline. In an earlier study in GG, the therapeutic  benefit in patients with mild COVID-19 was evident by reducing the duration of hospital stay and averting disease progression [28]. No serious adverse events were reported during the study. It was generally well tolerated, and only very few nonserious adverse events were reported, primarily with gastrointestinal symptoms, indicating the relative safety of the drug. Furthermore, the cost of herb-based drugs is very low (2 USD or 150 INR) compared to certain expensive conventional drugs. In India, GG may be considered a cost-saving drug compared to conventional care, as per-day hospitalization costs range from 77 to 136 USD (5000− 10,000 INR). However, a detailed study on the cost-benefit analysis of the drug through an economic model considering diverse outcomes and other related factors is needed.

Study limitations
This study had several limitations. We were unable to randomly assign participants to arms due to the short duration of the study; therefore, some unavoidable baseline imbalances were observed. To minimize the impact of imbalances, the primary outcome was adjusted for important prognostic factors/confounders. Furthermore, due to the subjective nature of secondary outcomes and unmasked participants, the risk of measurement bias was inevitable. However, it was minimized using validated tools, trained-team for data collection, and monitoring. The incidence of COVID-19 was self-reported by the participants, and asymptomatic cases that were not tested or reported were likely missed. The sensitivity of rapid antigen testing is comparatively low in illness, leading to a lower detection of incidence. The cohort largely comprised   Value: Correlation coefficient (r); QoL, quality of life. *The correlation is significant at the 0.01 level (two-tailed).
relatively healthy young and middle-aged populations; therefore, further studies are warranted to extrapolate the findings to other populations. It is also limited to the geographical and cultural groups of the study site.

Conclusion
Evidence gathered from a multicenter, controlled, before-after trial suggests that GG increases the perception of immune status and QoL; hence, it may be used to improve general health in individuals at high risk of SARS-CoV-2. The data findings are inadequate to show that GG reduces the incidence of COVID-19 in people with moderate to high risk exposure to SARS-CoV-2 due to the reported trivial incidence. Therefore, RCT is recommended in populations at high risk of SARS-CoV-2 for a longer duration to confirm the trends observed in this study.

Financial support
This work was supported by Institute of Teaching and Research in Ayurveda, Jamnagar, Gujarat, India, 361,008 (Grant order no. PGT/ COVID19/Research/15)

Ethical approval
The study protocol was approved by the Institutional Ethics Committee, Institute for Post Graduate Teaching and Research in Ayurveda (Approval no. PGT/7/-A/Ethics/2020-21/239).

Data availability
Data linked to this study is available in supplementary file S1. Extended data associated to this study is available at Zenodo: Clinical Dataset of project 'prophylaxis effect of Ayurveda intervention 'Guduchi Ghanavati' on COVID-19 ′ https://doi.org/10.5281/zenodo.4584445

Declaration of Competing Interest
The authors declare that there are no conflicts of interest. Testing drug (GG) was procured from Government of India owned pharmaceutical company 'Indian Medicines Pharmaceutical Corporation Limited (IMPCL)' and investigators did not receive any funds or commercial benefits from pharmaceutical company in this trial.