Acute and Repeated Dose Toxicity Study of Clevira Syrup – A Polyherbal Formulation

This study evaluated the acute and repeated dose toxicity effects of Clevira Syrup Polyherbal formulation (CSPHF), which was prepared from ten different herbs, well known and widely used in traditional medicine for the management of viral infections and other inflammatory disease conditions. Individually these herbs (Carica papaya, Melia azedarach, Andrographis paniculata, Vetiveria zizanioides, Trichosanthes dioica, Cyperus rotundus, Zingiber officinale, Piper nigrum, Mollugo Cerviana and Tinospora cordifolia) were completely safe, but the polyherbal formulation effects were not known. Thus, this study was done for the investigation of toxicological profile of CSPHF in Wistar Albino rats. As per OECD (Organisation for Economic Co-operation and Development) guidelines 423 and 407, Acute and Repeated dose toxicity study were proceeded. In the acute toxicity study a single dose of CSPHF (2000mg/kg) was administered orally to female Wistar rats and in repeated dose toxicity study, CSPHF was administered orally in Control group and three different doses (1000, 500 and 250mg/kg body weight) to both male and female wistar albino rats for 28 days. At the end of the study, the animals were euthanized, observed the external and internal morphology (Acute Toxicity) and assessed the effect of CSPHF on histopathological and biochemical parameters (Repeated Dose toxicity study). In acute toxicity study, there were no visual signs of toxicity of CSPHF (2000mg/ kg) observed, whereas in Repeated dose toxicity study Ischaemia, inflammation and hematoma of the internal organs were observed at 1000mg/kg dose, but no such toxic features were seen at 500 and 250mg/kg dose of CSPHF. The results of the Acute and Repeated Dose toxicity study could be authenticated in future studies, which will be more useful and evidence based for the management of Viral infectionsduring pandemics.

In this modern world, the traditional medicine is more popular, curative, less toxic and with minimal side effects 1 . As per World Ethno Botanical reports, 70-80% of global population relies on traditional health care 2 . In India herbal preparations attained wide spread acceptability with lot of therapeutic value. Most of the medicinal plants in the composition showed pharmacological effects and it had been studied for years. Even today there are new objectives and efficacy profile determined and substantiated with scientific evidences for the benefits of these medicinal plants, whereas there are limitations for the safety and efficacy of the preparations 3 .

Animals
The animals were acquired from TANUVAS breeding center, Chennai. The animals were housed in a propylene cage with sterile bedding material, and fed with standard pellet feed and water, which was maintained at a room temperature of 26 ± 2°C and a relative humidity 45-55% in the Central Animal House, MGMCRI, throughout the study period.

Acute oral toxicity
Acute Toxicity testing was done as per the OECD (Organisation for Economic Co-operation and Development) guidelines 423 15,16 . Female wistar rats were randomly selected and kept in their cages for at least 5 days prior to the initiation of dosing for acclimatization to the laboratory conditions. Animals were divided into 3 groups (N=3) for testing acute oral toxicity. Following overnight fasting, the animals were weighed and the CSPHF (2000mg/kg, PO) was administered and food was withheld for a further 3-4 hours, whereas the control group only received normal saline.
All rats were allowed free access to food and water, and it was observed individually after dosing during the first 30 minutes then periodically during the first 24 hrs with special attention given during the first 4 hours and daily thereafter, for a total of 14 days. In this next 14 days, the animals were observed once daily for visual observations mortality, physical appearance, behavioural changes and any injury or illness and was recorded with individual records being maintained for each animal. Additional observations were done to see if the animals showed any signs of toxicity (Observations should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention was directed towards observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep 4/14 OECD/OCDE 423 and coma).Individual weights of animals were determined before the test substance was administered and then weekly thereafter. The weight changes were recorded. At the end of the test, animals would be weighed and humanely killed.
All test animals were subjected to gross necropsy and observed for gross pathological changes for each animal. The same procedure was repeated for the second set of test 2 (n=3) using 2000mg/kg dose (Table-1).

Repeated dose 28days toxicity study
Acute Toxicity testing was done as per the OECD guidelines 407 17 . Following the Acute Oral Toxicity study of Clevira syrup Polyherbal formulation (2000mg/kg), Wistar Albino rats of either sex (n= 5male +5female rats) were divided into four groups (n=5+5) in each group with the allocation as follows: Group I (Control group), Group II (CSPHF 250mg/kg), Group III (CSPHF 500mg/kg) and Group IV (1000mg/kg) Table-2. Other two groups of rats {n=10 (5+5)} served as Control and Satellite groups based on the 14 days of follow up of the Repeated Dose toxicity study Observations of CSPHF.
The Maximum volume of CSPHF than can be administered was at the dose 1ml/100g body weight. Based on this the dosage of administration of CSPHF was calculated and given orally through oral feeding needle. Following the period of fasting, the reproductive organs were healthy without any signs of toxicity or necrosis of tissues in Group II and Group III. But on examination of the internal organs and the orifices of the Group IV (1000mg/ kg of CSPHF) animals after their mortality, the organs were observed with necrotic tissues with hematoma of the solid organs and ischemia of the colon, with necrosis and gangrenous appearance of the reproductive tissues, etc. Hence the highest safer dose was selected for dosing in the control group and satellite group 17,18 .
In Satellite group (group V -500mg/kg CSPHF orally), the animals (n=10) were dosed with the safer highest dose from the repeated dose toxicity (500mg/kg of CSPHF) for a period of 28 days and the control group of animals (n=10) to monitor and compare any specific observations of toxic changes in the animals during the test period.
Weight changes were calculated and recorded weekly. Measurement of food consumption was made at least once weekly. At the end of the study the animals were fasted overnight prior to necropsy and blood samples for Hematological and Biochemical parameters estimation were collected from retro-orbital puncture after anaesthetising the animals with Intraperitoneal Inj. Thiopentone sodium, following which the samples for histopathological examination were collected after euthanasia of the animals.

Blood Analysis
For haematological analysis different parameters such as Hemoglobin

histopathological Examination
After blood collection, the animals were sacrificed by Inj. Thiopentone Sodium i/p and different organs such as Ovary, Testes, Liver, Kidney, Heart, Adrenals and Pancreas were collected from each rat in all groups to observe for any changes is histopathology. The organs were then fixed in 10% neutral buffered formalin for 18hrs at 4 degree Celsius and processed by conventional techniques. Paraffin sections were stained with hematoxylin and eosin, following the standard laboratory procedures. The stained sections were examined under Oil Immersion Microscope under magnification for any change in morphology and for any cellular damage.

RESuLtS And dISCuSSIon
Acute Toxicity Study of Clevira Syrup Polyherbal Formulation (Table:3and

4) on observation
The animals in each test group (Test 1 and Test 2) after overnight fasting were observed for their activity and looked for signs of toxicity. The Orifices and internal organs were carefully examined and observations recorded on Individual basis. During the study none of the animals showed signs of toxicity or had a moribund status. After Inj. Thiopentone Sodium IP the animals were sacrificed and necropsy was done. There was no gross pathological changes noted in any of the groups. Hence from the data obtained after the acute toxicity study the Repeated dose toxicity study of CSPHF was done in the Group of Male and Female Wistar Rats for a period of 28 days.
The animals from each group were monitored from day one of the study and the observations were done on the external features for signs of toxicity. All systems were carefully examined and recorded on individual basis on every seventh day (Day 0, 7, 14, 21 and 28 days). During the study period none of the animals showed signs of toxicity or had a moribund status in the dose of  At the end of the study animals were anasthetizedby Inj. Thiopentone sodium I.P Blood samples were collected for Biochemical and Hematological parameters, animals were sacrificed and necropsy was done Table (7 and 8). There were no gross pathological changes of the internal organs noted in group 1 (250mg/kg) and group 2 (500mg/ kg). All the orifices, cranial, thoracic, pelvic and abdominal cavity and the internal organs including the reproductive organs were In case of utilization of polyherbal formulations we should be clear with some merits and demerits of the combined use of multiple components to demarcate the salient features of each component in the formulation. From the present compound CSPHF during the Acute Toxicity study period the animals in each group were observed for their activity and looked for signs of toxicity from Day 0 to Day 14 once in every 7 days. Every system was carefully examined and recorded on individual basis for each animal.
In this group of animals there were none of the animals which showed signs of toxicity or had a moribund status during the acute toxicity study period.
At the end of the study after gross pathological examination there was no changes noted in any of the groups. Hence from the data obtained the CSPHF was evident to be non-toxic up to single high oral dose of 2000mg/kg body weight without any mortality. Acute toxicity studies showing toxicity signs and mortality for a polyherbal formulation in Wistar Albino rats were meager in the past. [19,20] .Based on this data we further proceeded for Repeated dose toxicity study of CSPHF.
During the repeated dose toxicity study of CSPHF the animals from each group we monitored from day one of the study and the animals were observed for any external features of signs of toxicity. All systems were carefully examined and recorded on individual basis on every seventh day (Day 0, 7, 14, 21 and 28) for 28 days. During the study period at the dose of 250mg/kg and 500mg/ kg, none of the animals showed signs of toxicity or ended up with moribund status. But to mention in specific at the dose of 1000mg/kg PO of CSPHF the animals started showing the signs of toxicity (Conjunctival and paw edema, distension of abdomen and hemoptysis, blood stained oral and rectal orifices, increased respiratory rate) from day 15 onwards and then mortality occurred within 27 days, for all the animals in this group with high dose of CSPHF (1000mg/kg).
At the end of the study, animals on examination for gross pathological changes after necropsy the changes noted in Group 1 (250mg/kg, PO, CSPHF) and Group 2 (500mg/ kg, PO, CSPHF) were healthy without any signs of inflammation or any other signs of toxicity of the internal organs and normal orifices. But in animals from Group 3 (1000mg/kg, PO, CSPHF), the morbidity and mortality of animals were high even before completion of the study period with inflamed organs on gross pathological examination immediately after the animal mortality.

ConCLuSIon
Hence CSPHF(Clevira Syrup Polyherbal formulation) was safe as a single dose at the maximum level (2000mg/kg, PO) in acute toxicity testing, whereas at repeated dosing of CSPHF the dosage of 500mg/kg and below was found to be safe without any signs of toxicity, morbidity and mortality. We hence plan to continue with chronic toxicity testing of CSPHF to provide more evidence for management with CSPHF without any further signs of toxicity or morbidity on further investigation of the formulation, so it can be useful in chronic viral infections like Hepatitis B, HPV etc, in near future.