28-day repeated dose toxicological evaluation of Coronil in Sprague Dawley rats: Behavioral, hematological, biochemical and histopathological assessments under GLP compliance

Abstract Ayurvedic medicines are widely employed globally for prophylaxis and treatment of a variety of diseases. Coronil is a tri-herbal medicine, constituted with the traditional herbs, Tinospora cordifolia, Withania somnifera and Ocimum sanctum, with known immunomodulatory activities. Based on its proven in-vitro activity and in-vivo efficacy, Coronil has been approved as a ‘Supporting Measure for COVID-19’ by the Ministry of AYUSH, Government of India. The current study was aimed to assess the non-clinical safety of Coronil in a 28-day repeated dose toxicity study along with a 14-day recovery period in Sprague Dawley rats. This toxicity study was conducted in accordance with OECD test guideline 407, under GLP-compliance. Specific-Pathogen-Free animals of either sex, housed in Individually-Ventilated-Cages were particularly used in the study. The tested Coronil dose levels were 0, 100, 300 and 1000 mg/kg/day, orally administered to 5 males and 5 female rats per test group. In the current study, no mortality was observed in any group and in addition, Coronil did not elicit any finding of toxicological relevance with respect to clinical signs, ocular effects, hematology, urinalysis and clinical chemistry parameters, as well as macro- or microscopical changes in any organs, when compared to the control group. Accordingly, the No-Observed-Adverse-Effect-Level (NOAEL) of Coronil was ascertained to be 1000 mg/kg/day, subsequent to its 28-day oral administration to male and female rats. The acceptable safety profile of Coronil paves the way further toxicity assessments in rodents for a longer duration as well as in higher animals, and towards its clinical investigation.


Introduction
Coronil is a tri-herbal Ayurvedic formulation, which contains the enriched extracts of Tinospora cordifolia (Sapta sirik a aromapatr a, Giloy, heart-leaved moonseed), Withania somnifera (A svagandhakah : sv apakarah : , Ashwagandha, winter cherry) and Ocimum sanctum (Sumañjarik a r am a, Tulsi, holy basil). Coronil has been uniquely formulated by incorporating traditional Ayurvedic principles, as a potential prophylactic against as well as therapeutic intervention for coronavirus disease 2019 . Coronil has been approved as a 'Supportive Measure in COVID-19,' by the Ministry of AYUSH, Government of India. The constituent herbs of Coronil have been in clinical practice for centuries in India and elsewhere. The daily dose of Coronil has been approved as two tablets (650 mg each), twice a day. A brief understanding of the pathophysiology of COVID-19 is imperative to gain insights into the probable strategies amenable to pharmacological interventions. It is well established that the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with the host angiotensin-converting enzyme 2 (ACE2) receptor through its spike-protein, resulting in the entry of the virus into the host cell. Subsequently, the exaggerated host defense mechanisms are triggered leading to a 'cytokine storm,' which can ultimately result in a sustained inflammatory response and the resultant acute respiratory distress syndrome (Wiersinga et al. 2020). Hence, the central step leading to COVID-19 pathogenesis, is the entry of the pathogen in the host cell. It is naturally follows that any intervention that inhibits such access can provide prophylactic benefit, and would be of therapeutic value as well, as it will have the potential to decrease the viral load, its spread, and the concomitant exaggerated inflammatory response.
The rationale for positioning Coronil for the prophylactic and therapeutic interventions for COVID-19 originates from the scientifically determined antiviral and immune-modulatory activities of its constituent herbal extracts . Centered around these activities of the herbal components, a recent in-vitro study  evaluated the potential of Coronil to inhibit the spike protein-mediated entry of the SARS-CoV-2 virus in the host cell, wherein, in ELISA-based assay the tri-herbal formulation inhibited the interaction of the ACE2 receptor with three different spike protein variants, in a dose-dependent manner. Additionally, Coronil attenuated the spike protein-induced activation of the pro-inflammatory cytokines in A549 cells, dose-dependently . Furthermore, Coronil was also able to reduce the entry of the SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus into the host cells and could additionally prevent the pseudotyped virus-induced cytokine response in human A549 cells. In a humanized zebrafish model, generated by the xenotransplantation of A549 cells in the swim bladder of the fish (Balkrishna et al. 2020), Coronil could prevent the SARS-CoV-2 spike protein challenge-induced zebrafish mortality. In addition, Coronil also reduced the spike protein-induced behavioral fever, protected against pathological alterations in the swim bladder morphology, suppressed recruitment of inflammatory cells in the swim bladder, reduced necrosis of the renal cells and diminished skin hemorrhage (Balkrishna et al. 2020).
Given, the demonstrated in-vitro activity as well as the invivo efficacy of Coronil, it is likewise crucial to determine its non-clinical safety, ultimately directed toward assessment of its prophylactic value as well as its therapeutic potential in the human subjects infected with COVID-19. Therefore, in the current study, we evaluated the probable health hazards of Coronil after its repeated oral administration to male and female Sprague Dawley (SD) rats for a period of 28 consecutive days. A satellite group of animals that received the high dose of Coronil were also monitored for a treatment freeperiod of 14 additional days to assess the reversibility, persistence or delayed occurrence of toxic effects. The study was conducted to provide information on major toxic effects, target organs and No-Observed-Adverse-Effect-Level (NOAEL) of Coronil in rats. The study was performed according to the Organization for Economic Cooperation and Development (OECD) guideline 407 (Organization for Economic Co-Operation and Development (OECD), 'Test no. 407: repeated dose 28-day oral toxicity study in rodents,' 1995) and in conformity with the OECD Principles of Good Laboratory Practice (GLP).

Test item, chemicals and reagents
Coronil (Batch number #A-CNT460), a light brown free-flowing powder, (produced for granulation and consequent compression into tablets) was obtained from Divya Pharmacy, Haridwar India, where it was manufactured according to the standards of WHO-Good Manufacturing Practices. The selected phytochemicals in the formulation were detected in the range of 1.017% w/w by employing high performance liquid chromatography . For administration to the animals, a suspension of Coronil was formulated by utilizing 0.5% methylcellulose as the suspending agent. Coronil was stable in this suspension for 24 hours at room temperature, as shown elsewhere ). All the other reagents and chemicals employed in the study were of the highest commercial grade.

Experimental animals and husbandry practices
The experiment was conducted on specific-pathogen-free (SPF) SD rats of both sexes (aged 7-8 weeks), procured from Hylasco Biotechnology (India) Private Limited, Telangana, India; a Charles River Laboratories bred animal supplier. The animal supplier provided a health certificate confirming that all the animals procured for the study were healthy. Rats were chosen for this study as it is one of the proposed rodent species by regulatory guidelines for the conduct of safety studies due to the availability of extensive historical data (Organization for Economic Co-Operation and Development (OECD), 'Test no. 407: repeated dose 28-day oral toxicity study in rodents,' 1995). All experimental procedures and animal husbandry practices were according the standards established by the CPCSEA (CPCSEA 2018). The experiment was approved by the Institutional Animal Ethics Committee (IAEC) of test facility (vide IAEC protocol number: IAEC-20-026). Animals of one group were together housed in sterilized Individually-Ventilated-Cages having the dimensions of 41 cm Â 41 cm Â 78 cm. These cages operate on the exhaust ventilation technology (Optirat V R Plus, Animal Care Systems, Inc., USA). Rotation of cages was performed at every week to ensure identical illumination and environment to all study animals. Sterilized corn cob was used as the standard bedding material. The temperature maintained in the animal room was 21.2 to 22.9 C; and the relative humidity ranged from 42 to 64%. The air changes in the experimentation room were set at 10 to 15 air changes per hour, and maintained throughout the in-life phase of the study. Further, the photoperiod followed was a 12-hour light-dark cycle, controlled by an automated Digital Electric Timer (Frontier Digital, India). Rats were provided ad libitum with ultraviolet light sterilized standard pelleted laboratory animal diet (PMI-Nutrition International, LLC, New York, USA), and ultraviolet light irradiated drinking water, which was purified by a reverse osmosis system, in autoclaved polypropylene bottles. Study animals were quarantined in an earmarked room for a period of five days at the test facility, post receipt from the animal supplier.

Sub-acute toxicity study
The sub-acute toxicity potential assessment of Coronil was conducted in accordance with OECD test guideline number 407.

Animal acclimatization
Subsequent to conclusion of the quarantine, 64 specificpathogen-free SD rats (32 males and 32 females) were issued by Animal Research Facility for the present toxicity study. Animals were allowed to acclimatize in the experimental room for further period of five days before their allocation to the various treatment groups. Throughout the acclimatization period, rats were distinguished by markings made towards the tip of the tail, by utilizing a nontoxic marker pen. Animals were monitored minimum once daily for clinical signs and twice daily for morbidity and mortality.

Randomization of animals
Prior to randomization, a detailed clinical examination was conducted by a qualified staff veterinarian who ensured that all animals were healthy. following which, 30 male and 30 female animals were randomly allocated to the various treatment groups, on the basis of their body weights, as outlined in Table 1. Each group included five male and five female rats. After randomization, the animals were assigned a permanent animal number and were marked accordingly using a nontoxic marker pen. At the initiation of vehicle/Coronil administration, the body weight variation of animals did not exceed ± 20% of the mean weight of each gender. Post-randomization, ophthalmoscopic examination of all the study animals was conducted by employing an ophthalmoscope (HEINE, Germany) by a qualified staff veterinarian trained to perform the procedure.

Preparation of the formulations of Coronil and their oral administration
The test item was weighed on an analytical balance (CONTECH, India) and was then transferred to a mortar, followed by trituration with a pestle. Subsequently, a small volume of 0.5% Methyl Cellulose (Loba Chemie Private Limited, India) solution was dispensed into the mortar with continuous stirring to ensure proper wetting. Subsequently, the remaining volume of methylcellulose was added in dropwise manner by utilizing a syringe and with uninterrupted stirring to acquire a stable suspension. The homogeneity of the Coronil suspension was maintained by constantly triturating it in mortar and pestle prior to its administration to the animals. The formulations were freshly prepared, on each day of the administration of the test item.
Animals allocated to group G2 (low dose), G3 (mid dose), G4 (high dose) and G4R (high dose) received a dose of 100, 300, 1000 and 1000 mg/kg body weight, respectively by oral gavage. Control (G1) and Control Recovery (G1R) group animals were given the vehicle (0.5% Methyl Cellulose) alone by oral gavage, at the highest dose volume based on dose volume of high dose group. The dose volume was maintained at 10 mL/kg body weight.

Study observations
Mortality and clinical signs. All the animals were observed at least once a day throughout the study period for clinical signs, and at least twice a day for morbidity and mortality. Following cessation of the vehicle/test item administration, the observation period was extended for an extra 14 days for control-recovery (G1R) and high-dose-recovery (G4R) groups. Onset, severity, duration and the reversibility of clinical signs were documented.
Detailed clinical observations. All the animals were scrutinized for detailed clinical observations once a week up to the termination of the experiment. Examination included, changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and basic observations of autonomic activity (e.g., lacrimation, piloerection, pupil size, and unusual respiratory pattern).
Body weights. Rats were weighed on a balance (CONTECH, India) and their respective body weights were recorded on Days 1, 8, 15, 22 and 28 for the main group and were continued on days 35 and 42 for the recovery groups. In addition, the body weights before randomization and fasting body weights on the day of necropsy were also recorded.
Food consumption. Food consumption were recorded by employing a pan balance (CONTECH, India) once every week up to the end of observation period. The amount of food consumed by each animal per cage in a week was computed by using the following formula: Food consumption per week g ð Þ ¼ Food offered g ð Þ ÀFood leftover ðgÞ Number of animals Ophthalmoscopic examination. Ophthalmoscopic examination was performed by utilizing an ophthalmoscope (HEINE, Germany) during the last week of treatment for vehicle control (G1) and high dose (G4) group animals. Since, ophthalmoscopic examinations conducted during last week did not show any treatment related ophthalmic changes, the inspection was not conducted for low and mid-dose groups. Animals were subjected to examination after inducing mydriasis with 1% Tropicamide (Sunways India Private Limited, India).
Clinical pathology observations. After the conclusion of the vehicle/Coronil administration period on day 28, all the main group animals and after completion of recovery period at day 42, all the recovery group animals were subjected to overnight fasting. The blood samples were collected from retro-orbital sinuses under mild isoflurane anesthesia, on day 29 from main study groups and on day 43 from recovery group animals for hematological and clinical chemistry analysis.
Hematology. For hematology analysis, blood was collected in tubes containing 1% ethylene diamine tetra acetic acid, dipotassium salt (HIMEDIA, India). The following parameters were measured by utilizing a Sysmex XP-100 Auto Analyzer Coagulation. For coagulation analysis, the whole blood was collected in vials containing 3.2% Sodium Citrate (Merck KGaA, Germany). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) coagulation parameters were measured using an Erba ECL-105 Coagulation Analyzer (Erba Mannheim, Germany).
Clinical chemistry. Whole blood samples were collected into vials containing heparin (250 IU/mL) (Samarth Life Sciences Private Limited, India) for plasma and without anticoagulant for serum. Plasma was separated by centrifugation at 3000 rpm for 10-15 minutes at room temperature. Serum samples were used for electrolyte analysis and plasma samples were processed for the remaining clinical chemistry analysis. Serum samples were processed using Sensacore Electrolyte Analyzer ST-200CL (Sensacore, India), whereas plasma samples were processed using Erba EM Destiny 180 Auto Analyzer (Erba Mannheim, Germany). The following clinical chemistry parameters were assessed: Glutamate Oxaloacetate Transaminase ( Urinalysis. In the last week of treatment and recovery periods, animals from each group were subjected to overnight urine collection by employing metabolic cages. Urine samples were analyzed for appearance (color and clarity), bilirubin (BIL), glucose (GLU), protein (PRO), pH, and specific gravity (SG) on Erba LAURA SMART urine analyzer (Erba Mannheim, Germany).
Necropsy and gross pathology. On day 29, all animals from main group and on day 43, all animals from recovery group were euthanized with carbon dioxide asphyxiation. All the animals underwent detailed gross pathological examination, which included a thorough examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities with their contents.
Organ collection and organ weight. Liver, kidneys, adrenals, testes/ovaries, thymus, spleen, brain and heart of all animals were trimmed off to remove any adherent tissue and their wet weights were recorded. Paired organs were weighed together on a weighing balance (CONTECH, India). Relative organ weights were computed for each animal by using following formula: The following organs/tissues of all animals were collected at termination and fixed in 10% Neutral Buffered Formalin for the subsequent histopathological examination: Adrenals, Aorta, Bone (Bone marrow) Histopathology. The preserved organs from all control (G1) and high dose group (G4) animals were routinely processed and embedded in paraffin by utilizing a Tissue Embedding Station (Especialidades M edicas MYR, S.L., Spain). Sections of 3-5 mm thickness were prepared by employing a CUT 5062 microtome (SLEE medical GmbH, Germany), those were then stained with hematoxylin-eosin (Merck KGaA, Germany) and examined microscopically by using an LX 300 microscope (Labo America, Inc., USA).

Data analysis
Data was compiled from all the groups, and mean and standard deviation were calculated. The body weight, hematology, clinical chemistry, urinalysis (for specific gravity and urinary pH) and relative organ weight data was analyzed using oneway ANOVA followed by Dunnett's post-hoc multiple comparison test for main groups, and Student's t-test for recovery groups using Graph Pad Prism (Version 7.03). All analysis and comparisons were evaluated at the 5% (p < 0.05) level in comparison with their respective controls.

Mortality and clinical signs
As corroborated by the daily clinical examinations, oral administration of Coronil did not result in any mortality, morbidity, or aberrant clinical signs, in any of the animals of either sex, from both the main and recovery groups, throughout the study duration (Supplementary Table S1). Additionally, the weekly detailed clinical examinations did not reveal any clinical abnormalities, which could be attributed to the formulation, in any animal during the course of the experimentation (Supplementary Tables S2 and S3).

Effects of Coronil on body weights and food intake
Administration of Coronil did not ensue in any statistically significant changes, in the body weights of the animals from both the main and the recovery groups, throughout the experimental period, when compared to their respective control groups (Figure 1(A,B)). Similarly, the weekly food consumption in the rats from the Coronil-administered groups was equivalent to that of their respective controls (Figure 2(A,B)).

Ocular effects of Coronil
The rats from the main study, which were allocated to the control and high dose (1000 mg/kg/day) groups were assessed for abnormal ophthalmic findings during the last week of treatment. Coronil did not elicit any detectable ocular-anomaly, in any animal on the examination day, when compared to the control group (Supplementary Table S4). The rats which were administered with the low and mid-dose of the tri-herbal formulation, were not subjected to ophthalmoscopic examination, as no abnormality was detected in the high-dose administered group.

Effect of Coronil on hematological parameters
The hematological parameters in the rats, from both the main as well as the recovery groups, that received Coronil, were found to be comparable to their respective control groups (Tables 2 and 3). Nevertheless, amongst the animals assigned to the main study groups, a statistically significant  decrease in total erythrocyte count (p < 0.05) was noted in female rats, which were administered the mid-dose of Coronil (300 mg/kg/day), when compared to the control group. This observed effect, however, cannot be directly attributed to Coronil, as it is not dose-related and considered as the incidental finding. Further, a significant increase in MCH was observed in female animals that received the high dose of Coronil (1000 mg/kg/day). However, this change is within the historical reference ranges for SD rats at the site of the study and within the normal ranges defined for SD rats (Matsuzawa et al. 1993), and therefore may not be of any clinical relevance.

Effects of Coronil on coagulation parameters
The effect of Coronil on coagulation parameters namely, prothrombin time and activated partial thromboplastin time was assessed in this study. Both the parameters were, largely, unaffected subsequent to Coronil administration in rats in both the main and recovery groups (Tables 2 and 3). However, as compared to the control group, a minor but statistically significant increase in activated partial thromboplastin time (p < 0.05) was observed rats of either sex from the main study group, which received the high dose of Coronil (1000 mg/kg/day). This increase was well within the historical reference range for SD rats at the study site and within the normal ranges defined for SD Rats (Matsuzawa et al. 1993). Accordingly, this observation is unlikely to be of any clinical significance.

Effect of Coronil on clinical chemistry parameters
Most of the clinical parameters were unaffected by Coronil administration (Tables 4 and 5). Nonetheless, as compared  to the control group, the serum Na þ and K þ levels were decreased (p < 0.05) in the male animals that received the low (100 mg/kg/day) and the mid-dose (300 mg/kg/day) of Coronil from the main study group and additionally, the serum Cllevels were elevated (p < 0.05) in male animals that were administered with the high-dose (1000 mg/kg/ day). These effects, however, cannot be ascribed to Coronil, largely as the observed variance was within the experimental limits. Further, in female animals from the main study group, a minor elevation (p < 0.05) was detected in the bilirubin levels in the plasma of rats treated with the high dose of Coronil (1000 mg/kg/day), which is unlikely to have a clinical relevance as the value was well within the historical reference ranges for SD rats, at the site of the study and within the normal ranges defined for SD Rats (Matsuzawa et al. 1993). Additionally, the serum chloride levels were also noted to be significantly elevated in rats that received the mid (300 mg/kg/day) and high dose (1000 mg/kg/day) of Coronil, which nevertheless was not dose-related.

Effect of Coronil on qualitative urinalysis parameters
Coronil administration in rats from both the main and recovery groups, did not largely have a significant effect on the urinalysis parameters when compared to their respective control groups (Tables 6 and 7). The specific gravity of urine was, however significantly decreased in male animals from the main study group that were treated with the low and high dose of Coronil; the effect being non dose-related. A similar decrease was also observed in males from the recovery group that received Coronil. In female animals, however, an increase in the urinary specific gravity was detected in animals from the main group, which received the low dose of Coronil. Accordingly, these observations can be considered to be incidental findings due to random biological variation.

Relative organ weights in Coronil-administered rats
The relative organ weights in male rats, both from the main as well as the recovery groups, that received Coronil were  similar to those of their corresponding controls (Tables 8).

Gross pathological findings
Gross pathological examination of the Coronil-administered animals, in both the main as well as the recovery groups, did not demonstrate any lesions of pathological significance, by and large, when compared to their respective control groups (Table 10). Nevertheless, red spots of minimal intensity were observed in the thymus of two male rats treated with the high dose of Coronil. Based on the gross pathological observation, the thymus from the animals in question was subjected to histopathological analysis, wherein, no aberrant changes were detected. Accordingly, this observation can be regarded to be an incidental one.

Histopathological analysis
Histopathological investigations were conducted in animals of the main study group which received the vehicle and the high dose of Coronil (1000 mg/kg/day) respectively. When compared to the control group, the animals which received the high dose of Coronil, did not demonstrate any aberrant histopathological alterations that can be directly attributed to the tri-herbal formulation (Table 11, Figures 3 and 4). As no treatment related changes were observed in high dose group, and no treatment related gross pathology changes were observed in any organ system in low (G2) and mid (G3) dose group and recovery group (G1R, G4R) animals, no tissue from animals of these group was processed for histopathological examination.

Discussion
A wide body of prevailing scientific evidence suggests that the herbal components of Coronil possess anti-viral as well as immunomodulatory activities, attributed to their phytoconstituents. One of the plausible mechanisms of their demonstrated activity against SARS-CoV-2 virus is the prevention of the entry of the virus in the host cells through the inhibition of the host-pathogen interaction. Lately, in an in-vitro experiment the formulation, per se, has been demonstrated to attenuate the spike-protein mediated entry of SARS-CoV-2 in human alveolar epithelial cells by inhibiting the spike protein-ACE2 receptor interaction, as well as the ensuing proinflammatory cytokine release, in a dose-related manner . Furthermore, Coronil has also demonstrated credible in-vivo efficacy in a humanized zebrafish model, wherein, it ameliorated the SARS-CoV-2 spike protein induced mortality, behavioral fever and several pathological features triggered by the spike protein challenge (Balkrishna et al. 2020). Owing to its proven in-vitro activity as well as invivo efficacy, the tri-herbal formulation holds promise to be added to the prophylactic as well as a therapeutic armamentarium against COVID-19 and consequently, Coronil is a candidate for a comprehensive clinical scrutiny. Accordingly, we evaluated the non-clinical safety of Coronil in a sub-acute toxicity potential assessment, wherein,

Thymus
Minimal focal hemorrhages 0/5 0/5 0/5 2/5 0/5 0/5 No other gross pathological lesions were observed in other organs. the formulation was orally administered to SD rats of either sex for a period of 28-days, together with a 14-day treatment-free recovery period.
In the present study, although we did identify statistically significant changes in certain parameters, those were not regarded as adverse and furthermore those were not directly attributed to Coronil treatment, due to either a non-existence of an evident dose-relationship or owing to the absence of the consequent alterations in the gross organ or histopathology, which could expound such outcomes. Besides, the studied parameters in which the statistically significant variations were noticed were in the normal laboratory ranges described for rats.
Consequently, as inferred from the study, the NOAEL for Coronil is 1000 mg/kg. Taken together, the hitherto established in-vitro activity  as well as in-vivo efficacy of Coronil (Balkrishna et al. 2020), along with an exceptional safety demonstrated in the current study encourage the further clinical evaluation of the tri-herbal formulation. Sparse information exists in the public domain regarding the toxicity assessments of the extracts prepared from the individual components of Coronil. Much like the pharmacological effects, adverse effects or toxicological findings of herbal medicines also depends on several factors like, the time and site of plant collection, weather and altitude of the site, manufacturing process and variances in extractive values etc. Therefore, it may be difficult to ascertain a specific toxicological profile to a given herbal extract; nevertheless, it is relevant to outline the published non-clinical safety profiles of the constituents of Coronil.
The principal component of Coronil is the stem extract of Tinospora cordifolia. With regard its safety evaluation, the aqueous extract of Tinospora cordifolia has been reported to be non-toxic up to a dose of 2000 mg/kg in an acute toxicity evaluation in Wistar rats (Ghatpande et al. 2019). In an alternative acute toxicity assessment, no remarkable adverse effect and no mortality was reported in rats that were orally administered a dose of 3000 mg/kg Tinospora cordifolia extract (Agarwal et al. 2002). Further, in one sub-acute toxicity assessment a Siddha formulation, made from the stem of Tinospora cordifolia, using a traditional method, was administered to Wistar rats of either sex at the doses of 360, 1800 and 3600 mg/kg/day for 28 days (Uma et al. 2016). This study concluded that the preparation was devoid of any major toxic effects up to the highest dose, which was nearly ten times of the intended therapeutic dose. Additionally, in another study (Sharma et al. 2011) a hydro-alcoholic extract of the stem bark of the herb was administered to female albino rats at the doses of 250, 500 and 1000 mg/kg/day for three weeks, wherein, the extract did not demonstrate any untoward effect, except some observational effects like initial excitement, followed by mild depression, dullness, decreased respiration and reduced spontaneous motor activity only at the high dose. Based on its proportion in Coronil, the daily doses of the extract of Tinospora cordifolia, which the animals would have received in our study is calculated to be approximately 47, 141 and 470 mg/kg/day respectively and even the highest dose administered to the animals is a fraction of the dose received by the animals in the above mentioned subacute toxicity studies.
Coronil, additionally contains a hydro-alcoholic extract of Withania somnifera. Several studies have reported the nonclinical safety profiles of the extracts prepared from the roots of the plant. In an acute toxicity study assessment, a standardized root extract was administered to female Wistar rats by oral route at the doses of 500, 1000 and 2000 mg/kg, wherein, the LD 50 was greater than 2000 mg/kg and no toxic, behavioral or gross organ pathologies were noticed (Patel et al. 2016). In the same report, the root extract was additionally assessed for its sub-acute toxicity potential, subsequent to oral administration of the extract to rats of either sex at the doses of 500, 1000 and 2000 mg/kg. In this experiment the extract did not exhibit any major toxic effect and the variations noted in some of the hematology or clinical chemistry parameters were well with in the laboratory reference ranges, and no gross or histopathological changes were evident. Accordingly, the NOAEL was adjudged to be 2000 mg/kg. In another similar study, a hydroalcoholic extract of the roots of Withania somnifera was evaluated for its acute and sub-acute toxicity potentials (Prabu et al. 2013) and the outcomes were exactly similar to the previously cited study (Patel et al. 2016). Furthermore, one study has assessed the safety of a pure extract of Withania somnifera upon its repeated oral administration to rats of either sex for a period of 90 days at the doses of 100, 500 and 1000 mg/kg/day (Antony et al. 2018). In this study the NOAEL was considered as 1000 mg/ kg/day as no observable toxicities were reported up to this dose with reference to body weight gain, feed consumption, hematology and clinical chemistry parameters, organ weights as well as gross organ and histopathology. In our study, the dose of Withania somnifera extract which the rats would have been administered with are computed to be approximately 39, 117 and 390 mg/kg/day which is again a fraction of the NOAEL reported in the above discussed sub-acute and sub-chronic studies.
Finally, Coronil comprises of the hydro-alcoholic extract of the leaves of Ocimum sanctum. Few studies have evaluated the acute and sub-acute safety of the extracts of the herb. The acute oral toxicity of a hydro-methanolic extract of the whole plant has been evaluated by administering the extract to female Wistar rats at a limit dose of 5000 mg/kg (Chandrasekaran et al. 2013). In this study, no mortality or abnormal clinical signs were observed post-extract administration and no effect on body weight gain, major gross or histopathological changes were observed 14 days after the administration of the extract. Hence, the extract was deemed to be safe up to 5000 mg/kg upon acute administration. In another study, the hydro-ethanolic extract of the leaves of the plant were orally administered to Swiss albino mice of either sex at doses of 200, 600 and 2000 mg/kg and this study also reported no mortality or toxic signs in the animals (Gautam and Goel 2014). The sub-acute safety assessment of a hydromethanolic extract of the whole Ocimum sanctum plant has been reported (Raina et al. 2015), wherein, the extract was orally administered to Wistar rats of either sex at the doses of 250, 500 and 1000 mg/kg/day for 28 days. In this study, the extract did not adversely impact body weight gain, food and water consumption, behavioral, neurological, hematology and clinical parameters and furthermore it did not elicit any macro-or microscopic changes in the vital organs. Hence, the NOAEL was adjudged to be 1000 mg/kg/day. In another experiment, a hydro-ethanolic extract of the leaves of the plant were administered to Wistar rats of either sex, for a period of 28 days, at the doses of 200, 400 and 800 mg/kg/ day (Gautam and Goel 2014), wherein, no treatment-related toxic effects were evident and accordingly the NOAEL was considered to be 800 mg/kg/day. Based on its proportion in Coronil, the dose of the extracts which the animals would have received in our study are calculated to be approximately 8, 24 and 80 mg/kg and in light of the published data these are unlikely to have any bearing on the safety of Coronil.
One of the limitation of this study is the toxicokinetic correlation, which is an essential component for the toxicological characterization of small molecule based pharmaceuticals. In the case of poly-herbal formulations, it is rather challenging. This is primarily due to the complex phytochemical constituents present in such formulations, which belong to a wide array of chemical classes in small quantities, making it an arduous exercise for the toxicokinetic assessment.
Taken together, observed safety profile of Coronil seems to be in line with the reported safety of its constituent herbs. This would pave the way for the non-clinical Toxicity assessment in non-rodents or for longer duration in rodents. Finally, the detailed clinical assessment of Coronil in human patients could also be conducted with appropriate regulatory approvals.

Conclusion
The sub-acute toxicity potential evaluation of Coronil was conducted according to OECD test guideline 407, in compliance with OECD Principles of GLP, by orally administering it to male and female SD rats for a period 28 consecutive days, at the doses of 100, 300 and 1000 mg/kg/day. Furthermore, our study design also included satellite groups that were either administered the vehicle or the high dose of Coronil (1000 mg/kg/day) for 28 days followed by a 14-day treatment free recovery period. In the present study, Coronil did not demonstrate any clinically relevant changes in the assessed parameters as compared to the vehicle-treated control group. Based on the above findings, the NOAEL for Coronil was adjudged to be 1000 mg/kg in either sex and consequently the findings support its further safety evaluations in rodents for a longer duration and additionally in non-rodents. This study serves as a starting point toward the clinical investigations, from the non-clinical safety perspective.