Research · Pre-Clinical

Pre-Clinical Research

Three years of in vitro and in vivo investigation examining antioxidant activity, anxiety-related behaviour, seizure response and preliminary safety.

Pre-clinical research established the scientific foundation for investigating Bacosane® in cognitive health, neuronal protection and stress management. Over three years, Bacosane® was evaluated throughin vitro assays and animal models. These findings provide mechanistic and safety data that can inform future human clinical research.

Anticonvulsant Activity

Albino rats and mice — MES and PTZ models

Saponin, a key compound in Bacosane®, offers seizure protection by enhancing GABA production, modulating neurotransmitter activity, and inhibiting sodium channels—effectively improving brain function and reversing epilepsy-related changes.

Objective: Investigate whether Bacosane® could influence experimentally induced seizure activity and provide protection against convulsions.
Method: Bacosane® at 50 mg/kg and 100 mg/kg, with phenytoin 25 mg/kg as the standard comparator. Researchers measured seizure phases, convulsion onset, recovery and survival.
Findings: In the MES model, Bacosane® reduced flexion, extension, clonus and stupor duration versus control, more strongly at 100 mg/kg. In PTZ, Bacosane® produced 67% protection at 50 mg/kg and 84% protection at 100 mg/kg (phenytoin: 100%).
Relevance: Saponin, a key compound in Bacosane®, offers seizure protection by enhancing GABA production, modulating neurotransmitter activity, and inhibiting sodium channels—effectively improving brain function and reversing epilepsy-related changes.

Anxiolytic Activity [Effects of Bacosane® on Actophotometer method]

Male and female Wistar rats — Actophotometer test

Effects of Bacosane® on Actophotometer method: Dose-dependent reduction in anxiety-related locomotor scores in male and female Wistar rats.

Objective: Evaluate Bacosane®'s effect on behavioural measures of anxiety, exploration and locomotor activity using the actophotometer model.
Method: Bacosane® administered at 50 mg/kg and 100 mg/kg compared against vehicle control and standard diazepam (1 mg/kg) in male and female Wistar rats.
Findings: The anxiolytic effect of Bacosane® was evaluated using the Actophotometer in both male and female Wistar rats, where it significantly reduced immobility time and demonstrated strong, dose-dependent anti-anxiety effects (activity fell from 301.50 → 158.17 → 110.83; diazepam 79.00).
Relevance: Demonstrates strong, dose-dependent anti-anxiety effects in behavioural rodent models.

Anxiolytic Activity [Bacosane® and diazepam on behavior of rats in Elevated Plus Maze test (n=6)]

Male and female Wistar rats (n=6 per group) — Elevated Plus Maze (EPM)

Bacosane® and diazepam on behavior of rats in Elevated Plus Maze test (n=6): Bacosane® demonstrated strong, dose-dependent anxiolytic effects in both male and female Wistar rats by significantly reducing immobility time in the Elevated Plus Maze test.

Objective: Evaluate the effects of Bacosane® and standard reference diazepam on open-arm exploration and anxiety behaviour in rats.
Method: Bacosane® at 50 and 100 mg/kg with diazepam 1 mg/kg as reference; n=6 per group in the Elevated Plus Maze test.
Findings: Bacosane® demonstrated strong, dose-dependent anxiolytic effects in both male and female Wistar rats by significantly reducing immobility time in the Elevated Plus Maze test. Open-arm entries increased from 18.5% (vehicle) to 49.21% (50 mg/kg) and 47.82% (100 mg/kg). Time in open arms rose from 8.3% to 26.3% and 28.4%.
Relevance: Combined results indicate dose-responsive anxiolytic activity in animal models; provides preclinical rationale for stress-resilience applications.

In Vitro Antioxidant Activity

DPPH free-radical scavenging assay

In-vitro antioxidant activity (DPPH assay): Bacosane® demonstrated strong antioxidant activity with an IC50 of 150.50 µg/ml, compared to 87.25 µg/ml for standard BHT.

Objective: Determine the antioxidant capacity and free-radical scavenging potential of standardized Bacosane® under laboratory conditions.
Method: DPPH assay evaluated across concentrations with BHT (butylated hydroxytoluene) as reference antioxidant.
Findings: Bacosane® demonstrated strong antioxidant activity with an IC50 of 150.50 µg/ml, compared to 87.25 µg/ml for standard BHT. Bacosane® and its bioactive compounds have also been extensively studied for reducing oxidative stress by boosting antioxidant enzymes like SOD, CAT, GPX, and GSR.
Relevance: Provides a mechanistic basis for neuronal protection against oxidative stress and membrane lipid peroxidation.

Acute and Sub Acute Toxicity Study

Albino rats — Acute oral toxicity (5,000 mg/kg) & 28-day sub-acute (up to 1,000 mg/kg/day)

Acute and sub-acute toxicity study: The acute and sub acute toxicity of Bacosane® were evaluated by using albino rats. There was no significant difference (p > 0.05) observed in the relative organs, body weights, hematological, biochemical parameters, and gross abnormalities, compared to the control.

Objective: Evaluate preliminary safety following single-dose (acute) and 28-day repeated oral administration in albino rats.
Method: Acute: 5,000 mg/kg single oral dose. Sub-acute: 28 days at 250, 500 and 1,000 mg/kg/day. Monitored mortality, toxicity signs, body weight, organ weights, haematology, biochemistry and gross abnormalities.
Findings: The acute and sub acute toxicity of Bacosane® were evaluated by using albino rats. There was no significant difference (p > 0.05) observed in the relative organs, body weights, hematological, biochemical parameters, and gross abnormalities, compared to the control. No mortality or apparent toxicity reported.
Relevance: Favourable preliminary safety profile in experimental animals with no adverse effects observed at the highest tested doses.

In-silico Molecular Docking

Computational binding-affinity study

Molecular docking results showing binding affinities for Jujubogenin (−8.25 kcal/mol), ebelin lactone (−8.16 kcal/mol) and diazepam (−7.98 kcal/mol). Lower (more negative) binding-energy values indicate stronger predicted binding.

Objective: Assess the binding affinity of selected Bacosane® compounds against a target associated with anxiolytic activity, using molecular docking.
Method: Molecular docking of Jujubogenin, ebelin lactone and the reference standard diazepam.
Findings: Jujubogenin showed the highest binding affinity (−8.25 kcal/mol), followed by ebelin lactone (−8.16 kcal/mol) and diazepam (−7.98 kcal/mol). The two Bacosane® compounds exceeded the binding energy of the standard anxiolytic reference.
Relevance: These in-silico findings provide a computational basis for continued preclinical investigation and validate target interaction potential.

Summary

Key pre-clinical findings

• Dose-responsive activity in MES and PTZ anticonvulsant models (67% and 84% protection).
• Multiple behavioural models (Actophotometer & Elevated Plus Maze) indicated anxiolytic effects at 50–100 mg/kg.
• Measurable free-radical scavenging Activity in the DPPH assay (IC50 150.50 µg/mL).
• No mortality or significant therapy-related abnormalities (p > 0.05) in acute (5,000 mg/kg) or 28-day sub-acute studies.

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