A total of 150 patients diagnosed with COVID-19 using a RAT or RT-PCR positive test were enrolled among them 101 were males and 49 females with mean age 38.59 ± 11.74 years. Fourteen patients were considered to drop out of the trial. Among 136 patients, 50 patients were in Group A (Vasa treated group), 43 patients were in each Group B (Guduchi treated group), and in Group C (Vasa + Guduchi treated group) (Fig. 1).

All the groups had similar demographic and illness characteristics (Table 1). The proportions of patients who complained of fever were 60% in Group A, 55% in Group B, and 53.49% in Group C at the time of enrollment, and with cough in 54%, 57.5%, and 37.21% in Group A, B, and C, respectively (Table 1). There was an improvement in fever, sore throat, headache, loss of smell, and loss of taste in all the groups after the treatment.

Table 1 Baseline demographics and clinical features of COVID-19 patients in each groupViral clearance

Patients were mostly home quarantined and opted to come for follow-up RT-PCR at their convenience. Hence this is more indicative of the confirmation date rather than the actual time to negativity which could be earlier. The mean time taken to become COVID-19 negative was 13.92 days (95% confidence interval [CI] 12.85–14.99) in Group A (n = 50), 13.44 days (95% confidence interval [CI] 12.14–14.74) in Group B (n = 43), and 11.86 days (95% confidence interval [CI] 10.62–13.11) in the Group C (n = 43). The difference of 2.06 days was the time reduced by Group C in comparison to Group A alone, and that was significant (p = 0.048). From the results, it was also clear that Group A and B both had taken nearly equal time to COVID-19 negativity (Fig. 2A). Using Kaplan–Meier survival analysis, the percent of patients with a positive RT-PCR test was 80% in Group C, and 90% in Group A and B on the 7th day of the trial period. It was 20% in Group C, 30% in Group B and 36% in Group A on the 15th day of the trial period.

Fig. 2

Kaplan–Meier survival analysis of the proportion of patients: A positive RT-PCR test and B time to normal body temperature

Time to normal body temperature

At the time of recruitment, 40% of patients had a temperature ≤ 98.6°F (normal body temperature) in Group A and < 50% of patients in Group B and C. Patients had higher body temperature recovered with the median day of 4 (95% confidence interval [CI] 3.86–6.72), 3 (95% confidence interval [CI] 2.66–4.41), 4 (95% confidence interval [CI] 2.86–5.94) in Group A, B, and C, respectively. On the 7th day, nearly 90% of patients had a temperature ≤ 98.6 °F in Group B, C, and A (Fig. 2B).

Primary clinical outcome

The average morning and evening data of each individual were assessed to compare the trend in their vital parameters for 14 consecutive days. There was a significant decline in mean temperature over 14 days from 98.92 ± 1.28 to 98.04 ± .80 in Group A; n = 42, from 98.49 ± 1.33 to 97.7 ± .89 in Group B; n = 32, and from 98.68 ± 1.29 to 98.01 ± .89 in Group C; n = 33 (Fig. 3A). However, between-group variations were insignificant. Initially, on Day 1, the levels of oxygen saturation (SpO2) (Fig. 3B), heart rate (HR) (Fig. 3C), blood pressure (BP) (Fig. 3D, E), and respiratory rate (RR) (Fig. 3F) were within the normal range, and it remained the same during the trial period. At baseline mean SpO2 was 97.95 ± 0.81% in Group A (n = 45), 98.15 ± 0.91% in Group B (n = 33), and 97.79 ± 0.83% in Group C (n = 35), mean HR was 76.12 ± 6.76 bpm in Group A (n = 43), 75.52 ± 6.85 bpm in Group B (n = 32), and 75.12 ± 5.47 bpm in Group C (n = 34), mean systolic BP was 123.64 ± 4.24 mmHg in Group A (n = 27), 123.28 ± 3.95 mmHg in Group B (n = 21), and 123.60 ± 7.25 mmHg in Group C (n = 19), and mean diastolic BP was 84.22 ± 2.91 mmHg in Group A (n = 27), 84.04 ± 4.48 mmHg in Group B (n = 21), and 83.76 ± 4.51 mmHg in Group C (n = 19). Further mean RR was 14.23 ± 1.40 breath per minute in Group A (n = 42), 14.60 ± 1.15 breath per minute in Group B (n = 33), and 14.29 ± 1.37 breath per minute in Group C (n = 34) on Day 1. No significant differences or linearity in trend was observed in any of the three groups or between groups in these parameters.

Fig. 3

Trend in mean clinical symptoms in three study Groups over 14 days of observations: A temperature (℉), B oxygen saturation (%), C heart rate (bpm), D systolic blood pressure (mmHg), E diastolic blood pressure (mmHg), F respiratory rate (breath\min). ℉, degree Fahrenheit; bpm, beats per minutes; mmHg, millimeters of mercury; %, percent

Secondary clinical outcomeBiochemical and hematological parameters

The various hematological parameters such as CBC, ESR, and PT/INR were observed in the normal range and remained within the normal range even after treatment in all three groups. Although mild changes were observed in some parameters which were statistically significant after Bonferroni post hoc correction. In Group A, the percentage of eosinophil significantly increased (treated with Vasa) during treatment whereas monocyte decreased, this led to a slight increase in NMR from 6.57 ± 1.89 to 7.71 ± 2.14 (Additional file 1). Further, the PT/INR was increased from baseline to the end of the treatment although remained within the normal range. Considering liver function parameters, serum glutamic oxaloacetic transaminase (SGOT) was slightly higher than normal at baseline which decreased significantly at the end of the treatment period in Group A. Kidney function parameters were in the normal range at baseline and remained within range; slight decrease in globulin was observed. The lipid profile was in the normal range and remained unchanged (Additional file 1). Biochemical markers related to COVID-19 progression and severity such as serum ferritin levels were significantly decreased from baseline to end of the treatment (Table 2 and Fig. 4A, B). Levels of D-dimer decreased during the course of treatment, however, not statistically significant.

Table 2 Group-wise comparison of immunological and molecular markers at baseline (before treatment) and after treatment as well as timepoint-wise comparison within groupFig. 4

The levels of biochemicals and molecular markers: A hsCRP, B serum ferritin, C D-dimer, D IL-1β, E MCP-3, F TNF-α, G HIF-1α, H vWF, and I VEGFA at three timepoints in all the groups. HIF-1α, hypoxia-inducible factor-1 alpha; hsCRP, high-sensitivity C-reactive protein; ng/ml, IL, interleukin;, MCP-3, monocyte chemotactic protein-3; mg/ml, milligrams per millimeters; ng/ml, nanograms per milliliter; pg/ml, picograms per millimeters; TNF-α, tumor necrosis factor-alpha; VEGFA, vascular endothelial growth factor A; vWF, von Willebrand factor; star, In Group A Friedman, p = 0.013; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

In Group B (treated with Guduchi) (n = 19) hematological parameters such as CBC, ESR, and PT/INR were observed in the normal range and remained within the normal range even after treatment. A slight increase in NMR was observed (Additional file 1). As regards, liver enzymes such as SGOT, and SGPT were slightly on the higher side at baseline and remained nearly the same after the treatment. Serum bilirubin (total bilirubin, and direct bilirubin) levels increased significantly although remained within normal limits till the end of the treatment. Kidney function parameters such as blood urea, serum creatinine, uric acid, total protein, albumin, and globulin were normal at baseline and remained so even after treatment. Serum creatinine levels significantly increased albeit within the normal range. The lipid profile was nearly within the normal limit and remained unchanged. We also observed that the median hsCRP significantly decreased from baseline 6.8 to 5.15 to 4.2 mg/l at the end of the treatment. Change in D-dimer and serum ferritin was not statistically significant in Group B. In Group C hematological parameters such as CBC, ESR, and PT/INR were observed in the normal range at baseline and remained within the normal range even after treatment. However, marginal decrease in hemoglobin and hematocrit values such as TRBC and HCT were observed to be statistically significant. Similarly, we observed a significant rise in eosinophil levels in Group C (Additional file 1) but well within normal limits. Liver functions parameters SGOT, SGPT, ALP, and bilirubin (Total, direct, and indirect) were within the normal limit and remained in the normal range except for ALP levels which decreased significantly. Kidney function parameters blood urea, creatinine, and uric acid remained unchanged. Total protein and globulin levels were significantly decreased at the end of the trial although within normal limits. The lipid profile was nearly at the normal limit and remained unchanged. There was no significant change observed in Group C with respect to COVID-19 disease activity specific parameters such as hsCRP, D-dimer, and serum ferritin. Although levels of D-dimer and hsCRP were lowered during the treatment, they were not statistically significant (Table 2, and Fig. 4).

Pro-inflammatory and anti-inflammatory cytokines

A total of 15 pro-inflammatory and anti-inflammatory cytokines levels were examined using their serum samples. The cytokine levels in Group A (n = 28) (treated with Vasa) did not change significantly except for VEGFA levels, which decreased significantly during the treatment (Table 2).

On the other hand, in Group B (n = 19) (treated with Guduchi) the level of TNF-α were observed significantly increased during the trial period (Table 2). Changes in all other cytokines were not statistically significant.

In Group C (n = 20) (combination of Vasa + Guduchi), decreased MCP-3 while increased IL-1β levels were observed. IL-8 levels decreased from baseline, but after treatment, it restored to the baseline level. The values of some cytokine were below the detectable range in many samples such as IL-4 and IFN-γ (Table 2).

Molecular biomarkers for hypoxia and thrombosis

The levels of hypoxia and thrombosis biomarkers, HIF-1α and vWF were tested. The level of HIF-1α significantly decreased in Group A after treatment (AT) (Table 2 and Fig. 4G). Overall, the median of HIF-1α levels at baseline was similar in all groups; however, it would be worthwhile to highlight that there were few individuals with very high values in Group A, and their levels were lowered after treatment and came closer to the median. No significant change in HIF-1A levels in Group B and C was observed.

vWF, a molecular marker of thrombosis induced by HIF-1α, was undetectable in a large number of patients either before and/or after the treatment. The samples included in the analysis were Group A, n = 12; Group B, n = 6; Group B, n = 8. A decrease in the levels of vWF was observed in all three groups although not statistically significant. Overall, Group A showed a lowering effect on hypoxia and thrombosis markers HIF-1α, VEGFA (Table 2 and Fig. 4G, I) and PT/INR (Additional file 1).

Furthermore, to see the difference between groups we conducted a comparative analysis of the biochemical, hematological parameters, and molecular markers at baseline (referred to as "Before Treatment") and After Treatment. We observed that MCP-1 was significantly higher in Group C as compared to Group B at baseline (Table 2 and Additional file 1). However, we did not find any significant differences between groups in any other examined parameters.

Management of complication

One patient from Group A and another from Group B with diabetes were on oxygen supplementation for 4 days since their condition deteriorated from mild to moderate stage, antipyretic, and antibiotics were also given along with intervention drugs. One of them continued with the trial medication and hence included in the analysis, whereas the other discontinued the medication and was considered a dropout. However, both were followed up till recovery and tested negative for RT-PCR during the trial period only.