THE ROLE OF HERBAL IMMUNOMODULATORS AS ADJUVANT THERAPY FOR ASYMPTOMATIC AND MILDLY SYMPTOMATIC COVID-19: AN EXPLORATORY CLINICAL STUDY

Objectives: The objective of the study is to evaluate herbal immunomodulators (Septilin and Bresol) as a possible adjuvant therapy for the treatment of asymptomatic and mildly symptomatic COVID-19. Methods: Randomized, open-label, comparative clinical study. Subjects were randomized to either arm I [Septilin and Bresol+standard of care (SOC)] or arm II (SOC). This study was registered on CTRI (CTRI/2020/06/025801). Results: Subjects in arm I showed a greater reduction in levels of interleukin-6 tumor necrosis factor-α following treatment than in arm II. Subjects in arm I showed a greater increase in levels of interferon (IFN)-β and IFN-λ than those in arm II. There was a greater reduction in D-dimer in arm I than in arm II subjects (64.28% vs. 35.59%) and all arm I subjects had D-dimer values in the normal range compared to 70% of arm II subjects. There were statistically significant reduction in lactate dehydrogenase and neutrophil-to-lymphocyte ratio in arm I (p<0.016 and p<0.013, respectively). Clinical assessments during the post-illness convalescence period showed significant improvements in fatigue assessment scores and quality of life. Conclusion: This herbal combination as an adjuvant to SOC may provide additional long-term benefits in COVID-19 infection by reducing inflammation. This treatment may offer a good addendum for the management of post-COVID-19 illness.


Jayanthi et al.
adjuvant treatment with Septilin and Bresol (herbal immunomodulators and respiratory wellness support) compared with standard of care (SOC) alone in patients confirmed with COVID-19 and at low clinical risk (asymptomatic and mildly symptomatic). Subjects who tested positive for COVID-19 based on reverse transcription-polymerase chain reaction (RT-PCR) analyses were considered for screening (SARS-CoV-2 RT-PCR-confirmed COVID-19-positive cases within 72 h of the onset of symptoms/48 h of a positive RT-PCR result for COVID -19). The main inclusion criteria were subjects who were COVID-19 positive, age 18-60 years, asymptomatic or mildly symptomatic, and at low clinical risk as assessed by the 0-4 point National Early Warning Score (NEWS) system [23]. Overall well-being of the subject was assessed by using 0-5point WHO-5, FAS was assessed by 1-5 point Checklist Individual Strength, World Health Organization QOL assessment instrument, and Fatigue scale.
The main exclusion criteria were those with a NEWS score ≥5; acute respiratory distress presenting with a respiratory rate >24/min, oxygen saturation (SaO2/SPO2) ≤94% in air-conditioned rooms, or an arterial oxygen partial pressure to fractional inspired oxygen (PaO 2 / FiO 2 )ratio <300 mmHg; a recent significant pulmonary condition, such as asthma or chronic obstructive lung disease; inability to take oral medication; ailments related to absorption; an immunocompromised state or on immunosuppressive therapy; severe and uncontrolled metabolic, endocrinal, cardiac, renal, or hepatic disease; and pregnant or breastfeeding women.
Subjects who fulfilled the eligibility criteria were randomized into two treatment arms, arm I and arm II, at a 1:1 ratio. Subjects assigned to arm I received Septilin and Bresol orally at a dose of one tablet, twice daily, along with SOC for the specified duration of treatment (from day 1 to 14 days after hospital discharge, as applicable); subjects in arm II (the control arm) received only SOC, according to institutional guidelines. Subjects were followed up daily until their discharge from the hospital (visit 2) according to the treatment guidelines for the management of such subjects and were assessed subsequently by telephone 7±1 days after the hospital discharge (visit 3) and 14±3 days after hospital discharge (visit 4).

Statistical analyses
Statistical analyses of the data generated in this trial were performed using GraphPad Prism software, V.6.07 for Windows (San Diego, CA, USA). Means and standard deviations (SD) were calculated for continuous variables, and frequencies and percentages were calculated for categorical variables. After screening, eligible subjects were randomized into arm I or arm II using a list produced by computergenerated randomization at a 1:1 ratio. The randomization list had blocks of the same length (block size: 4), was not stratified, and was generated by a biostatistician using sealed envelope online software. The two study authors were responsible for assigning the subjects to groups according to the randomization list. Study site staff members and a study monitor were responsible for data collection and verification and ensured that the randomization was based on the sequencing.
Continuous variables with normal distributions were subjected to within-group comparisons using repeated-measures analysis of variance, followed by a post-hoc test and a paired t-test. Variables with non-normal distributions were compared using the nonparametric Friedman test, followed by a post-hoc test.
For between-group comparisons, continuous variables with normal distributions were compared using unpaired t-tests, whereas variables with non-normal distributions were compared using the MannWhitney test. The significance level for all tests was set at p≤0.05. All analyses were two-tailed.

Study subjects
A total of 59 subjects were enrolled in the study (arm I=30 subjects and arm II=29 subjects); however, 19 subjects withdrew their consent. Therefore, a total of 40 subjects (20 in each group) completed the study and were available for laboratory investigations and final analyses. The detailed baseline demographics for all subjects recruited and those who completed the study are provided in Table 1a and b. A flow diagram of the study based on the Consolidated Standards of Reporting Trials guidelines is depicted in Fig. 1.

Clinical assessments
The subjects in the study were evaluated during the time taken for the positive CoV-RT-PCR result to become negative (days) and the time taken to be discharged from hospital (days). Based on the guidelines of the institute for assessments on specific days, these parameters could not be differentiated, especially with this small sample size (Table 2). There was no increase in the severity of symptoms in any of the subjects. Further, there was no clinical failure of treatment and no recurrence of the RT-PCR-positive result for COVID-19.

Inflammatory markers (interleukin [IL]-6 and tumor necrosis factor [TNF]-α)
Slight reductions in levels of IL-6 and TNF-α were seen in both arms. This was statistically significant when compared with the baseline values. Arm I demonstrated slightly greater reductions than arm II (IL-6: 9.35% vs. 9.17%, TNF-α: 8.63% vs. 7.91%, respectively). Additionally, for the IL-6 level, 10 subjects (50%) in arm I were almost within the reference range at the time of discharge compared to six subjects (30%) in arm II (Tables 3 and 4).

Immunological markers (interferon [IFN]-β and IFN-λ)
Increased IFN levels (both β and λ) were seen in both arms. However, the increase was greater in arm I than in arm II subjects at the end of

D-dimer
D-dimer levels were evaluated at visits 1, 2, and 4. In arm I subjects, the baseline mean value was 0.56±0.91 µg/ml. This had reduced to 0.2±0.1 µg/ml at 14 days post-discharge (visit 4). In arm II, the mean value was 0.59±0.58. This had reduced to 0.38±0.39 (which is still borderline high). These results are indicative of a higher reduction in D-dimer levels in arm I than in arm II subjects by the end of the study (64.28% vs. 35.59%, respectively). All arm I subjects had D-dimer levels within the normal range at visit 4. However, 30% of arm II subjects had D-dimer levels outside the normal range at visit 4 ( Fig. 4).

LDH
In arm I, LDH reduced from 234.5±62.45 U/L at baseline to 189±36.81 U/L at 14 days post discharge (visit 4), with a significance of p<0.016. A reduction in the LDH level was also seen in arm II subjects, from 304.07±188.6 U/L at baseline to 202.75±55.25 U/L at visit 4 with a significance of p<0.026.
However, approximately 40% of subjects (8/20) still had LDH values that were borderline high or above the normal reference range in arm II, whereas only 15% (3/20) subjects had values that were borderline high or above the normal reference range in arm I (Fig. 5).

Post-illness convalescence symptoms
This study evaluated symptoms commonly seen during convalescence from COVID-19. A statistically significant improvement was seen in energy levels, appetite, and ability to concentrate in arm I subjects at visit 2 and 3 as compared with these factors at baseline. There was also a significant difference between these improvements in arm I and arm II subjects. A significant improvement was also seen in body pain, joint movement, sleep quality, recurrent respiratory tract infection, and shortness of breath in arm I subjects. This was also true for arm II, but the improvements were greater in arm I (Table 5).

QOL
This study evaluated QOL using the WHO-5 Well-Being Index (WHO-5) and the FAS. The FAS included a questionnaire comprising 10 questions on fatigue, individual strength, and QOL. Answers to the questions are scored on a 5-point scale ranging from 1 to 5 (where 1=never, 2=sometimes, 3=regularly, 4=often, and 5=always). A total FAS score <22 indicates no fatigue; a score ≥ 22 indicates fatigue. Arm I subjects demonstrated significant improvements in FAS scores between baseline and later assessments. Improvements in arm I subjects were also significantly greater than those in arm II subjects. All (100%) arm I subjects were relieved of fatigue after treatment, whereas 35% of arm II subjects continued to experience fatigue (Tables 6-7 and Fig. 7).
The QOL was further evaluated using the WHO-5, which utilizes a 6-point Likert scale (ranging from 0 to 5). The well-being of subjects was evaluated at visits 1, 2, and 4. Subjects in arm I demonstrated a substantially greater improvement in all the parameters evaluated than those in arm II (Tables 8-9 and Fig. 8).

Laboratory investigations
Most of the hematology and blood biochemistry parameters were within the normal reference ranges. Values outside the reference range were not considered clinically significant.

Adverse event (AE) profile
A total of five AEs occurred in the study. Two mild AEs were reported in arm I (indigestion). These subjects recovered from the AE without treatment or any sequelae. There were three mild AEs (sore throat) reported in arm II. However, none of these were serious enough for subjects to be withdrawn from the study. No serious AEs were reported or observed in any of the subjects. Neither group raised any significant safety concerns, indicating the safety profile of Septilin and Bresol tablets.

DISCUSSION
COVID-19 is a highly contagious infection, with no definitive treatment available. Currently available treatments are largely supportive. In the absence of evidence-based modern interventions, it is possible that herbal ayurvedic medicines can aid value in the overall management of COVID-19. Rasayana ayurvedic botanicals have been used for centuries to strengthen immunity and may be considered for COVID-19 prophylaxis and as an add-on treatment [2]. This study affirmed the potential of Ayurveda as a useful adjuvant treatment in the management of COVID-19. Integrating Ayurveda with modern medicine will offer a novel and effective means of prevention, cure, and rehabilitation from COVID-19 [24].
This study was an open-label, comparative clinical study that explored the efficacy and safety of Septilin and Bresol tablets as adjuvants to SOC, according to applicable guidelines related to the management of the COVID-19 pandemic. Clinical assessments were conducted to measure factors (hospital stay, conversion to negative test, severity) relevant to the treatment of COVID-19 infection. However, these assessments could not demonstrate large differences, given the small sample size.
The inflammatory markers IL-6 and TNF-α are known to be produced in response to COVID-19 infection. Severity is believed to be due to inflammatory storms caused by an over-reaction of cytokines [25]. Several studies have found inflammation to be an important indicator of COVID-19, which is closely related to its severity. Therefore, IL-6 might be a promising therapeutic target [26,27]. In this study, subjects treated with Septilin and Bresol (arm I) demonstrated a greater reduction in IL-6 levels after treatment than subjects in the control arm (arm II). More subjects in the experimental group than in the control group had IL-6 values close to the reference range at the time of discharge (50% vs. 30%, respectively). This indicates that adjuvant therapy with Septilin and Bresol could confer both short-and long-term benefits by reducing inflammation, thus contributing to recovery from COVID-19 and its associated symptoms.   Other markers of significance to COVID-19 include D-dimer, LDH, and NLR levels, which were also evaluated in this study. There are numerous reports that coagulopathy can develop in subjects with COVID-19, LDH is an enzyme found abundantly in the heart, liver, lungs, muscles, kidneys, and blood cells. It is a general indicator of acute or chronic tissue damage and an inflammatory marker [34]. LDH levels are known to increase in response to acute and severe lung damage and elevated LDH levels are seen in other interstitial lung infections [35].
In subjects who are COVID-19 positive, LDH levels may be used to measure abnormal inflammation status, which can lead to lung damage and respiratory distress [36]. In this study, a significant reduction in LDH levels (p<0.016) was observed in individuals treated with Septilin and Bresol. Despite the small sample size, it was encouraging that approximately 40% of the subjects (8/20) in arm II showed LDH levels that were borderline high or outside the normal range in arm  NLR is are liable prognostic tool and marker of inflammation and subsequent COVID-19 complications [37,38]. A greater reduction in NLR was observed in arm I subjects (p<0.013) than arm II subjects during the critical inflammation period (until the RT-PCR result for COVID-19 turns negative). This indicates that the Septilin and Bresol combination has effective anti-inflammatory properties that may be helpful in the hyperinflammatory stage of COVID-19. A lower NLR has been shown to correlate with a more favorable COVID-19prognosis, which is established and proved in multiple studies [37,38].
The emerging evidence of trouble some residual symptoms (e.g., cough, sputum, sore throat, disorders of the gastrointestinal tract, fatigue, and dyspnea, along with reduced mental well-being) after discharge from the hospital (post-illness convalescence symptoms of COVID-19 infection) are of great concern and were therefore clinically assessed and analyzed in this study after the subjects became negative on the COVID-19 test [39][40][41][42]. Statistically significant improvements were observed in energy levels, appetite, and ability to concentrate/ focus, supporting the use of Septilin and Bresol for full recovery from COVID-19.
Overall well-being was assessed in this study using the FAS and the WHO-5. Arm I subjects demonstrated significant improvements in their FAS scores between baseline and later evaluations. There was also a significantly greater improvement in FAS scores in arm I subjects than in arm II subjects. All (100%) arm I subjects were assessed as free of fatigue after treatment, whereas 35% of subjects in the control arm still experienced fatigue. Arm I subjects demonstrated significant and substantially greater improvements in all WHO-5parameters than those in arm II subjects. This indicates that a combination of Septilin and Bresol may be beneficial in over coming a range of troubling residual symptoms in the post-illness convalescence period of COVID-19. The favorable data during the active infection phase may be correlated with a positive outcome of the post-illness convalescence phase.
From the encouraging safety profile in this study, it can be inferred that the combination of Septilin and Bresol is a safe and well-tolerated adjuvant in patients with confirmed asymptomatic and mildly symptomatic COVID-19. This is in line with the safety profile of both the products, which have been on the market for decades. In the absence of a standard treatment and management regimen for COVID-19 infections (including active treatment and post-COVID-19 illness), we hope that the results of this exploratory study provide a ray of hope to the scientific community. Adjuvant therapy with Septilin and Bresol for the management of COVID-19 may be a promising addendum to modern medicine armament. However, the sample size is in this study was too small to offer conclusive evidence. A sample size-estimated study may provide a more convincing and acceptable conclusion. Unfortunately, there was a limitation in determining a statistically justified sample size during the initial phase of the pandemic. After many months since the conceptualization of this study, it is still challenging to derive a sample size with numerical assumptions from a large spectrum of assessments. This study was designed to use the herbal treatments tested as open-label and adjuvant in mild cases. The herbal composition of the proprietary drugs, the need to avoid increasing risk in severe cases, and the further ethical need not to use these products as a standalone therapy for acute COVID-19 were all kept in mind in the design of this study. As the understanding of the disease course developed over the study period, less impact is expected in clinical scenarios with asymptomatic and mildly symptomatic subjects. However, we could still identify some favorable effects on inflammatory and immunological markers known to be related to the severity of the disease. Considering the intricacies of this condition, this study was planned as an open-label study. Therefore, variations due to subjective assessments cannot be ruled out.
Besides limitations beyond our control during study conceptualization, this research has strong credentials: (i) The study was conducted at a reputed medical institute (the only dedicated COVID-19 treatment facility in the state at the time), after approval from the ethics committee of the institute and registration with the Clinical Trials Registry in India; (ii) Documentation and verification of data were performed according to ICH guidelines, and (iii) data management was performed in compliance with 21 CFR 11 EDC (Oracle Clinical) for data collection (with SDTM-CDISC standards). This ensured the highest level of data credibility. The laboratory involved in the immunological and inflammatory markers assessment is ISO 9001:2008 and NABLaccredited. For all other laboratory assessments, the hospital's

CONCLUSION
This clinical study shows that a combination of Septilin and Bresol as adjuvant therapy may provide long-term benefits by reducing inflammation, as demonstrated by the reduction and normalization of key COVID-19 inflammatory markers (IL-6, TNF-α, D-dimer, and LDH). The normalization of the levels of these inflammatory markers may prevent further physical damage, contribute to recovery from COVID-19, and minimize symptoms, even after recovery. The enhanced IFN-β and IFN-λ activity observed in the experimental arm of the study indicates an improved immune response to COVID-19.
Reductions were observed in post-COVID-19 symptoms and improvements in QOL after treatment with the polyherbal products tested in this study. Psychological distress and depression can have negative effects on immune responses, so helping those with COVID-19 to overcome these aspects and could contribute greatly to disease recovery. The data obtained in this research using various validated scales support the adjuvant role of these products in individuals struggling to overcome a range of troubling residual symptoms (especially fatigue and weakness) in post-illness convalescence from COVID-19.
Considering the favorable effects on inflammatory and immunological markers, and the improvements in post-illness COVID-19 symptoms and overall well-being (including QOL and fatigue) found in this study, the addition of Septilin and Bresol would seem a good addendum to the treatment of COVID-19 and for those suffering from post-COVID syndrome.
This study has shown that conventional clinical trial parameters may be adopted for research into traditional ayurvedic and herbal medicines that may assist in the control of the COVID-19 pandemic, for which no modern medicines are available (especially for post-COVID illness). In light of the strong credentials of this study, we are confident that these products will help those struggling with this disease by controlling inflammation and reducing overwhelming residual symptoms from post-COVID syndrome. Further research is warranted to corroborate our findings.

ACKNOWLEDGMENTS
We acknowledge Dr. Archana. R. Shetty, Ms. Pallavi K R for their support in the preparation of this manuscript, Mr. Umesh Kumar for data management, and Mr. Abdul Rehman for statistical analysis. We extend our thanks to Dr. Palaniyamma D (Medical expert) for assisting during the study conceptualization and data interpretation and Dr. Soorya Narayan for clinical operation support. The authors also thank all subjects who participated in this study.

AUTHORS CONTRIBUTIONS
Dr. C. R. Jayanthi has contributed to conducting clinical study, preparation, and review of manuscript. Dr. Avinash HR, Dr. Swetha Sridhar, Dr. Sriya Sridhar and Dr. Akhila K contributed to assisting the clinical study and preparation of manuscript. Dr. Rajesh Kumawat contributed to the revisions and finalization of manuscript.

CONFLICTS OF INTEREST
Dr. C.R. Jayanthi has received clinical trial support from Himalaya Wellness Company. Dr. Rajesh Kumawat is an employee of Himalaya Wellness Company. The authors declare that the financial interests or personal relationships have not influenced the work reported in this paper. Dr. Avinash H. R, Dr. Swetha Sridhar, Dr. Akila K, and Dr. Sruthi Nambiar have no known competing financial interests or personal

Summary of overall well-being (WHO-5) [n (%)]
Over the past 2 weeks Response Arm I (n=20) (%) Arm II (n=20) (%) relationships that could have appeared to influence the work reported in this paper.

FUNDING STATEMENT
This study was sponsored and funded by Himalaya Wellness Company (Bengaluru, Karnataka, India).