Background
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease (COVID-19) has emerged as a global pandemic [
1‐
3]. Most SARS-CoV-2 patients manifested mild-to-severe respiratory symptoms between 2 and 14 days of exposure, along with additional symptoms such as fever, coughing, and shortness of breath. In severely ill COVID-19 patients, the dysregulated, and uncontrolled production of pro-inflammatory cytokines such as higher levels of interleukins-6 (IL-6), Tumor necrosis factor α (TNF-α), ferritin, and C-reactive protein (CRP) leading to hypoxic respiratory failure are the major contributor to acute respiratory distress syndrome (ARDS) and are linked to a severe deterioration in health [
4‐
6]. Earlier research has found an association between elevated circulating D-dimer levels pointing to a specific coagulation pathology linked with COVID-19 [
7,
8]. This finding is also supported by other studies in which COVID-19 patients show substantial extracellular fibrin deposition, and fibrin thrombi within dilated small arteries, and capillaries [
9]. The link between hypoxia and coagulation is well documented, and studies have shown that HIF-1α, and vWF play key roles in the development of thrombosis, and bleeding susceptibility in response to hypoxia [
10]. To eliminate the virus in a non-severe state or prevent development to a severe state during incubation, a specific humoral, and cell-mediated adaptive immune response is essential. Thus, in such states, measures required to increase immunological responses become significant [
11,
12]. Since corticosteroids have been shown to improve clinical outcomes in COVID-19 patients with severe inflammation, and pneumonia, therefore, it is possible that other, more targeted immunomodulatory, anti-hypoxic, and anti-thrombotic medications will have similar effects [
13]. Thus, recommended supportive therapies for acute hypoxia-induced respiratory failure were included in COVID-19 treatment [
6].
Adhatoda vasica (AV)
(Nees.) also known as
Vasa/
Adulsa in Ayurveda has
pitta kapha balancing activity with bitter taste, and cooling effect. It is useful in the treatment of chronic bronchitis, asthma, colds, cough, fever, dyspnea, phthisis, blood disorders, jaundice, and diarrhea [
14‐
16]. In earlier studies, we have demonstrated that water-soluble extract of AV significantly decreases the severity of airway inflammation caused by an enhanced hypoxic response in asthmatic mice who are resistant to other treatments [
17]. We particularly observed inhibition of HIF-1α levels (hypoxia-inducible factor-1Alpha) in vivo, which was further confirmed in in vitro cellular model of hypoxia. Additionally, AV prevented hypoxia-induced mitochondrial dysfunction and reversed oxidative phosphorylation and ATP synthesis in lung epithelial cell lines. In severe COVID-19 cases, altered bioenergetic profiles of monocytes in response to elevated HIF-1α are a relevant molecular marker. Further, AV attenuates the increased levels of TGF-β1, IL-6, and HIF-1α in the mouse models of pulmonary fibrosis, and sepsis. AV treatment rescues the PHD2 siRNA-induced hypoxia, inflammation, and thrombosis phenotypes in mice. In addition to the potential to modify the host response, AV was also shown to inhibit the SARS-CoV2 virus load in infected Vero cell culture [
18]. In silico studies have shown that along with other chemical constituents of AV, the alkaloid compound vasicine has a very good binding affinity against SARS-CoV-2 viral proteins such as 3CL-pro and RdRp, and host receptors such as angiotensin converting enzyme (ACE-2) and TMPRSS2 [
18,
19].
Similarly, the second herb,
Tinospora cordifolia (Thunb.) Miers (TC), also called
Guduchi in Ayurveda, is known for its diverse applications in various diseases.
Guduchi has
Rasayana effect/homeostatic potential with all three
Dosha balancing activities. It also has a bitter and astringent taste with hot potency. Numerous researches on TC have demonstrated its potent antioxidant, hepatoprotective, cardiovascular protective, anti-inflammatory, antipyretic, thrombolytic, anti-microbial, and anti-cancer effects [
20,
21]. It has been demonstrated that TC modulates the activity of a variety of immune response cells, which is either directly or indirectly essential for enhancing immunity. According to reports, TC stimulates macrophages' phagocytic activity [
22]. It also activates the cytotoxic T cells and promotes the B cell differentiation [
23]. All these effects are relevant in producing the anti-viral impact, and eradicating viruses such as SARS-CoV2 in the early phase of infection [
20,
24]. M/o AYUSH national clinical management protocol based on Ayurveda and Yoga also includes
Guduchi ghan tablet for managing asymptomatic and mild cases of COVID-19 [
25]. Additionally, virtual screening and molecular docking studies of the phytochemical components of TC can reduce or prevent SARS-CoV-2 entrance and consequent infectivity by blocking the interaction between the receptor binding domain (RBD), and ACE2 [
26]. Compounds like xanosporic acid, tinosponone, cardiofolioside B, berberine, and tembetarine of TC were identified as potential lead compounds to combat SARS-CoV-2 [
27].
In the present study, the clinical efficacy of AV, TC, and a combination of both in terms of primary outcomes such as management of COVID-19 symptoms, prevention from progression to severe stages and viral clearance was examined. Additionally, we also studied the effects of treatment on inflammatory cytokines as well as the makers of hypoxia and thrombosis. To evaluate the systemic effects of the disease, as well as that of trial medication, parameters such as CBC, LFT, KFT, and lipid profile were also tested as secondary outcomes. Here we report the results of a randomized, preclinical lead-based, open-labeled three-armed clinical trial aimed at evaluating the direct effects of AV (Vasa Ghan) and TC (Guduchi Ghan) individually, and their combination’s effect on SARS-CoV2-positive patients with no or mild symptoms. We observed overall these herbal formulations exhibited no adverse effects, all the patients recovered clinically and did not progress to a severe form of the disease. In addition to other anti-inflammatory and anti-thrombotic markers, preclinical observations of Adhatoda vasica on HIF-1α inhibition were also observed. To the best of our knowledge, this is the first investigation into the therapeutic effects of the herbal formulation administered standalone in COVID-19 positive cases with regular monitoring of the symptoms as well as viral clearance and blood parameters done at three time points. This study suggests the inclusion of AV and TC as standard care for COVID-19 positive patients.
Discussion
The majority of SARS-CoV-2 infections had either no or mild symptoms. But there is also a significant proportion of infected individuals who suffer from a severe hyper-inflammatory and hypoxic response that entails the development of a potent and effective treatment for COVID-19. The result of this clinical trial demonstrated that the early-onset of oral treatment with Vasa (Group A), and Guduchi (Group B) with a dosage of 500 mg each, and a combination of these two drugs (Group C) with a dosage of 250 mg each twice a day, effectively prevented progression of COVID-19 disease from mild to moderate and/or severe state and was shown to be beneficial in the recovery process.
In our study, the average viral clearance observed was 13.92 days (95% CI 12.85–14.99) in Group A, 13.44 days (95% CI 12.14–14.74) in Group B and 11.86 days (95% CI 10.62–13.11) days in Group C. Furthermore, we also observed the median time taken to normal body temperature was 4 days in Group A and C, and 3 days in Group C. On the 7th day, 88% of patients in Group A and 90% of patients in Groups B and C had achieved normal temperature below 98.6 °F after treatment. This observation is comparable with another study in which avifavir, an RNA polymerase inhibitor, the treated group had a median time of two days to normalize body temperature and 4 days in the standard of care group [
29]. Studies have reported a median viral clearance time to be 13 days after a 10-day treatment of hydroxychloroquine (HCQ) administration regardless of the severity of the sickness [
30]. Another study that involved 396 non-severe COVID-19 patients found that the median time for viral shedding in the asymptomatic group was 14.5 days in contrast to 18.0 days in the symptomatic group [
31]. Since our study does not involve any standard treatment control group, we compared it with the published data. Among the clinical symptoms, we observed improvement in fever, sore throat, headache, loss of smell, and loss of taste after treatment. Very few patients reported the persistence of some symptoms at the end of the intervention period, that too with lower grades. According to WHO Clinical Progression Scale [
32], all the patients recruited in the study were mild (scores ≤ 2). During the clinical trial, two patients progressed to the moderate stage. At the end of the trial, all the patients in each group reverted to score of zero, i.e., healthy, asymptomatic, and uninfected.
Daily monitoring of oxygen saturation (SpO
2) and respiratory rate revealed that none of the patients clinically progressed to higher stages or developed any complications. The heart rate, systolic, and diastolic blood pressure of patients remained normal throughout the trial period signifying the protective effect of
Vasa and
Guduchi on the cardiovascular system health during COVID-19. The leaves of AV are administered orally for bronchial asthma, colds, cough, whooping cough, dyspnea, fever, headache, phthisis, jaundice, and diarrhea [
14]. The decoction prepared from
Adhatoda leaves possesses a soothing effect that helps to clear throat irritation and can also act as an expectorant [
33].
In addition to the clinical symptoms and viral clearance, we wanted to test the effect of
Vasa,
Guduchi, and a combination of
Vasa and
Guduchi on COVID-19-associated progression markers such as hsCRP, D-dimer, serum ferritin as well as inflammatory cytokines. We overall observed a decline in hsCRP in Group B and C, and serum ferritin in Group A. D-dimer levels were lowered in Group A and C but not statistically significant. In COVID-19, a high serum ferritin level is linked to worse outcomes and more severe illness, [
34] and in the current study, we noted that the median ferritin level decreased from baseline 98.4 (65.93–164) to 75.6 (29.88–141) (ng/ml) at the end of the
Vasa treatment in Group A. Among the inflammatory cytokines IP-10 decreased in Group A and C, whereas MCP-1 decreased significantly in Group C although before Bonferroni correction and MCP-3 remained statistically significant even after Bonferroni correction in Group C. Chen et al. have shown that critically ill patients had considerably greater serum IP-10, MCP-1, and MCP-3 levels than severe patients [
35,
36]. In this study, AV treatment exhibited a beneficial effect on IP-10 and a combination of AV and TC reduced MCP-1 and MCP-3 levels. TNF-α was slightly increased in Group B.
SARS-CoV-2 is a systemic disorder that involved multiorgan effects to some extent. To estimate the overall safety of these drugs, we evaluated systemic health parameters such as CBC, ESR, PT, PT/INR, liver function, kidney function, and lipid profile. In our patient’s group, most parameters were within normal limits and were largely unaffected or altered within normal limits in all three groups. Almost half of SARS-CoV-2 infected individuals showed some impairment in their liver function and elevated liver enzymes [
37]. Interestingly we observed a decrease in SGOT in Group A during the trial period which signifies that
Vasa seemed to be effective in attenuating liver injury caused by the virus and other conditions. Patients with severe forms of COVID-19 had greater levels of bilirubin than those with milder ones [
38]. In Group B, treatment did not exhibit any effect on slightly increased liver enzymes; instead, we observed a marginal increase in bilirubin levels that are reported to be raised in general in COVID-19 patients. Our results are in the line with a recent study in which TC was used as a part of the AYURAKSHA kit [
39]. Group C also showed a decrease in ALP, serum protein, and globulin. However, in the middle of the treatment a decrease in Hb, TRBC, and HCT levels were observed although within the normal range. Hb and HCT values were within normal range and were not affected during the course of treatment in Group A and B.
The overall decline in the systemic health parameters has been observed and reported in literature during the course of the disease [
40]. In a cohort of 127 patients with COVID-19, monocytosis (51.97%), lymphocytopenia (25.20%), eosinopenia (37.80%), and anemia (51.97%) were the most prevalent hematologic abnormalities observed [
40]. Qin et al. also reported based on data from 452 patients that more severe cases had lymphocytopenia and greater leukocyte counts and lower percentages of monocytes, basophils, and eosinophils [
41]. Overall, lung involvement, oxygen demand, and disease activity are related to the levels of alterations in leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, basophils, and platelets as well as Hb, MCV, and MCHC. Furthermore, eosinophils of moderate patients recovered faster than those of severe, suggesting that dynamical eosinophils may be the key to COVID-19 recovery [
40,
42]. During our study, we observed eosinophil levels considerably increased in both A and C Groups. It is also reported that eosinophils also play a crucial role in viral infections, such as HIV infection and respiratory syncytial virus [
43,
44]. The monocyte levels were decreased from baseline to the end of the
Vasa treatment. A meta-analysis by Ulloque-Badaracco et al. reported that a total of 11,356 patients from 31 cohort studies had lower AGR in severe COVID-19 and non-survivors than non-severe COVID-19 patients [
45,
46]. Our clinical trial findings also suggest that
Vasa and combination of
Vasa and
Guduchi treated groups showed an increase in AG ratio and subsequently decreased Globulin levels.
It is reported that without any indications of respiratory discomfort or dyspnea, patients with COVID-19 are frequently shown to have life-threatening hypoxemia which is termed as Silent hypoxia. The viral ORF3a protein is shown to increase the production of HIF-1α during SARS-CoV-2 infection, which in turn promotes the inflammatory responses and subsequently the infection. Since HIF-1α is a critical promoter of both SARS-CoV-2 infection and inflammatory response, it is suggested as a viable therapeutic target for COVID-19 and virus-induced inflammatory infection [
47]. Since COVID-19 hypoxemia is linked to a higher risk of death, early detection and rapid treatment are crucial to avert any complications [
48]. In the current study, hypoxia markers were investigated to understand the effect of trial drugs on them, and a variability in the levels of HIF-1α at baseline were observed. A significant decrease in HIF-1α levels was detected during the trial after treatment with
Vasa in Group A. Our earlier preclinical study on AV by Gheware et al. showed that in mouse models of hypoxia-hemostasis, pulmonary fibrosis, and sepsis, in addition to other parameters, HIF-1α levels were reduced with oral treatment of AV [
18]. Another interesting aspect of SARS-CoV-2 infection is “hypoxithrombosis” [
49]. Elegant studies have shown a significant increase in vWF antigen and its activity in patients with COVID-19, which could be a factor in the higher risk of thrombosis [
50]. In our study, a large number of patients had undetectable values of vWF before and/or after the treatment because it was conducted on mildly symptomatic patients. In Group B we observed decreased of vWF levels although not statistically significant. Further, VEGFA levels significantly lowered in Group A. In Group A, we also observed PT/INR significantly increased within normal limits which is also an indicator of AV having preventive effects on thrombotic outcomes of hypoxia and COVID-19. In mice, it is shown that AV extract reverse the inflammatory and blood coagulation characteristics [
18].
Although this analysis provided in-depth understanding of the clinical effects of tested formulation in COVID-19 patients, it has two limitations. One, due to the highly contagious nature of the disease, the patient's willingness to comply with blood sample collections was adversely affected. As a result, this left us with less than half of the patients from whom three-timepoint sample workups. Second, the study did not include a placebo control group. In the context of the COVID-19 pandemic, the absence of established therapeutic interventions necessitated the implementation of symptomatic management as the prevailing standard of care. The Government of India, Ministry of AYUSH, subsequently issued guidelines for the management of COVID-19, incorporating Ayurvedic medicines, with Tinospora cordifolia extract serving as a designated Ayurvedic standard of care intervention for mild cases. In this study, we adopted this Ayurvedic approach as one arm of our investigation, while Adhatoda vasica was considered as another distinct arm. Additionally, our research aimed to assess the potential synergistic effects arising from the concurrent administration of both Ayurvedic drugs to determine if any supplementary benefits could be observed.
This study demonstrated the effects of Vasa ghan, Guduchi ghan, and a combination of Vasa and Guduchi Ghan on mild COVID-19 patients. This is the first clinical trial study based on preclinical leads involving traditional Indian Ayurvedic medicine treatment with effects observed on multiple clinical, biochemical, and molecular parameters at different timepoints.
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