Formulation for in vivo studies
1.1 Test material
Kabasura Kudineer preparation: Kabasura Kudineer is a polyherbal formulation containing 15 herbal drugs mixed in equal quantity. They are; Chukku (Zingiber officinale), Thippali (Piper longum), Lavangam (Syzygium aromaticum), Cirukancoir ver (Tragia involvucrata), Akkirakaram ver (Anacyclus pyrethrum), Muliver (Hygrophila auriculata), Kadukkaithol (Terminalia chebula), Adathodei elai (Adhatoda vasica), Karpooravalli (Coleus amboinicus), Kostam (Saussurea lappa), Seenthil thandu (Tinospora cordifolia), Siruthekku (Clerodendrum serratum), Nilavembu (Andrographis paniculata), Vattathiruppi ver (Cissampelos pareira) and Korai kizhangu (Cyprus rotundus).
The material was procured from Sriveda Sattva Pvt Ltd, Bangalore (Sri Sri Tattva). The drug was licensed by the Ministry of AYUSH, Govt. of India. It was supplied in a powdered form and stored at 4oC until further use. All the herbs constituting Kabasura Kudineer were subjected to quality control analysis and after due approval process, ingredients were issued for production as fine powders. All the ingredients were blended with excipients followed by granulation and drying.
The animal study was conducted at Foundation for Neglected Disease Research (FNDR) in the BSL-3 laboratory. All the ethical guidelines with respect to the animal study were met in accordance.
1.2 Test Item Preparation
Stock solution of the test item Kabasura Kudineer was prepared in saline.
1.3 Animal Model: Syrian Golden Hamsters
6-8 weeks old female Syrian golden hamsters (Tata Memorial Advanced Center for Treatment, Research & Education in Cancer (ACTREC), (65/PO/ReBiBt/S/99/CPCSEA)) were included in the study. Body weight examination and other general veterinary examinations of hamsters were performed at the time of enrollment. Only healthy animals weighing around 80-100 grams were included in the study.
A total of 19 hamsters were enrolled, each group consisting of 4-6 animals.
1.4 Animal care:
All the animals were kept and familiarized in Individually Ventilated Cages (Citizen Industries, CRB-48-SS, V7E). The room conditions were maintained at 18oC-25oC temperature, 30%-70% humidity, 12-hour light and 12-hour dark. The animals were identified by body markings and therefore maintained in different groups. All the hamsters were provided with Feed and RO water ad libitum.
1.5 Animal ethics statement
The study plan for measuring the antiviral activity in COVID 19 infection model in hamsters was recommended by FNDR’s Institutional Animal Ethics Committee (IAEC), Registration Number 2082/PO/Rc/S/19/CPCSEA, on 21st December 2021 through Form-B proposal number FNDRFB-045. It was further approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. All ethical practices, as laid down in the CPCSEA guidelines for animal care, were followed during the conduct of the study. Additionally, the procedures used in this study plan were designed to conform to the accepted practices and to minimize or avoid risk of causing pain, distress or discomfort to the animals.
1.6 Viral Cell culture for infection:
The SARS-CoV-2 viral isolate was obtained by BEI resources managed by ATCC. Isolate USA-WA1/2020 was isolated from an oropharyngeal swab from a patient with a respiratory illness who had recently returned from travel to the affected region of China and developed clinical disease (COVID-19) in January 2020 in Washington, USA.
1.7 Study Duration
7 days’ acclimatization in BSL3, 1-day infection, 3 days dosing, 1-day sacrifice and plating, 7 days PFU counts.
1.8 Study Design
19 female Syrian golden hamsters, 6-8 weeks in age, were obtained for the study. Among the 19 hamsters, 4 hamsters were not infected with virus and were labelled as mock control, 5 hamsters were infected with virus and received placebo intervention and were labelled as disease control, 4 other hamsters were infected with virus and received remdesivir intervention and were labelled as positive control while remaining 6 hamsters were infected with virus and received test intervention Kabasura Kudineer and were labelled as test.
Hamsters were anesthetized with ketamine (150mg/kg) and xylazine (10mg/kg) via intraperitoneal route and inoculated with the virus intranasally with 100µL of DMEM containing 1× 106 PFU/ml. Hence, the total concentration of virus administered per hamster was 1× 105 PFU. The hamsters received respective intervention 24 hours post infection.
The respective intervention was administered via intraperitoneal route. Positive control animals were provided Remdesiver at 15mg/kg, for 3 days daily post infection. The test animals were provided Kabasura Kudineer intervention at 1000mg/kg in saline for 3 days daily post infection.
On the 4th day, the animals were euthanized by an over dosage of Isoflurane and sacrificed for viral load estimation and gross pathological examination.
1.9 Viral Load estimation
After sacrificing the hamsters, the following procedure was carried out for viral load estimation and gross pathological examination.
Sample Preparation: The whole lung was aseptically removed from the sacrificed animal. Changes in the body weight before and after clinical administration were noted. After gross pathological examination, the lung was homogenized for about 15- 30 seconds using Pro 200 homogenizer (Pro Scientific Inc. Monroe, CT. USA) in a final volume of 2 ml of sterile PBS in Wheaton Teflon-Glass tissue grinders (catalogue no. W012576). The homogenized tissue was centrifuged at 4000 rpm for 10 minutes to remove the debris and the supernatant was collected. Volume of the supernatant was measured.
Vero E6 cells preparation: A 96 well plate was coated with 200ul containing approx. 30,000 Vero E6 cells in DMEM media with 10% FBS. The plate was incubated overnight (12–18 h) at 37° C to achieve a Vero E6 cell monolayer.
Sample plating: 50 µl of samples (lung tissue) were serially diluted (10-fold) in DMEM and each dilution was plated in a different well with the pre-formed Vero E6 cell monolayers and incubated for 1 h at 37°C in a 5% CO2 incubator with shaking at 15 minute intervals.
Overlay: After 1-hour incubation, the samples were removed from the well. The cell monolayer was again overlaid with 200 µL of DMEM: CMC and incubated for 3 days at 37°C in 5% CO2. DMEM: CMC was prepared by mixing equal volume of DMEM (2x) and 2% carboxymethylcellulose.
Fixing: After the 3-day incubation, the DMEM-CMC overlay was gently removed with a pipette, the cells were washed twice with PBS and then fixed by adding 200 µL of 4% formaldehyde to each well. The plate was incubated at room temperature for 30 minutes, after which formaldehyde was removed from the wells and dispensed in an appropriate hazardous waste container.
Staining: 100 µL of 0.05% (w/v) crystal violet in 20 % methanol was added to each well and the plate was incubated for 30 minutes. Crystal violet was removed with a pipette and cells were washed twice with distilled water or until excess crystal violet was removed, and plaques are easily visualized.
Counting: The plaques (PFU) were counted for the dilution at which clear readable counts were noticed and were recorded as PFU per ml. The PFU per lung was calculated using the dilution factor and PFU per ml. Cell only control was used as a negative control.
Lung viral loads were compared for the test items and controls groups by a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison using GraphPad Prism software (Version 9).