An improved method for toxicological profiling of chemical substances.

Toxicity profiling is an integral part of the drug discovery pipeline. The 3Rs principle-Replacement, Reduction, and Refinement, is considered a golden rule in determining the most appropriate approach for toxicity studies. The acute toxicity study with proper estimate of median lethal dose (LD50) is usually an initial procedure for the determination of most suitable test doses for preclinical toxicological and pharmacological profiling. Several methods, which have been devised to determine the LD50, are faced with the challenge of using a large number of animals and time constraints. Despite the inherent advantage of the newer OECD Test Guidelines, the increasing concerns among toxicologists, the regulatory authorities and the general public, on the need to adhere to 3Rs principle, necessitated the need for an improved approach. Such an approach should not only minimize the time and number of animals required, but also take into cognizance animal welfare, and give accurate, comparable, and reproducible results across laboratories. While taking advantage of the inherent merits of the existing methods, here is presented the mathematical basis and evaluation of an improved method for toxicity profiling of test substances and estimation of LD50. The method makes use of the generated Table of values for the selection of appropriate test doses. Our proposed method has capacities to optimize the time and number of animal use, ensure more reliable and reproducible results across laboratories, allow for easy selection of doses for subsequent toxicity profiling, and be adaptable to other biological screening beyond toxicity studies.

In vivo and in silico studies of Dennettia tripetala essential oil reveal the potential harmful effects of habitual consumption of the plant seed.

Dennettia tripetala G. Baker (Annonaceae), is a plant with nutritional, social economy, and medicinal values. Its rising medicinal profile makes this plant a prospect in drug discovery. However, the reported strong addictive potential among habitual consumers makes the need to establish its safety imperative. In this report, we evaluated the safety profile of the essential oil of the seed of D. tripetala (EODS) in nulliparous female Wistar rats using in vivo single and repeated dose toxicity profiling, as well as in silico toxicity profiling of its known seed oil derived phytoconstituents. Our results showed consistent significant dose-dependent alterations in relative body weights, organ-body and organ-brain weight ratios, haematological and biochemical indices, as well as liver and kidney histoarchitectures, following single and repeated oral administrations. Significant alterations in liver and kidney histoarchitectures were consistent with the observed significant increase in AST/ALT ratio, suggesting deleterious effects of EODS on the kidney and liver. However, the lack of alterations in the histoarchitectures of the hippocampus and hypothalamus suggests that the brain may not have been adversely affected. Also, the in silico analysis suggests that hepatotoxic effects of EODS may be linked to Benzylnitrile, Humulene, Linalool, (Z)-ß-Ocimene. In addition, the failure of ß-Phenylnitroethane, the most abundant phytoconstituent of EODS, to pass phases I and II in silico toxicity screening, and the presence of Caryophyllene oxide, a known toxic compound, coupled with the predicted binding of both to DNA and protein, low LD50 and high percent mortality at 250 mg/kg of repeated doses, further confirmed the potentially toxic nature of EODS. We concluded that based on our in vivo and in silico observations, there is an urgent need for public education to regulate the excessive consumption of the seeds of D. tripetala.

Psychoneuropharmacological activities and chemical composition of essential oil of fresh fruits of Piper guineense (Piperaceae) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper guineense Schum & Thonn (Piperaceae) is a medicinal plant used in the Southern States of Nigeria to treat fever, mental disorders and febrile convulsions. AIMS OF THE STUDY: This study aims at determining the chemical composition and the central nervous system (CNS) activities of the essential oil obtained from the plant׳s fresh fruits in order to rationalize its folkloric use. MATERIALS AND METHODS: Essential oil of P. guineense (EOPG) obtained by hydrodistillation was analysed by GC/MS. EOPG (50-200mg/kg, i.p.) was evaluated for behavioural, hypothermic, sedative, muscle relaxant, anti-psychotic and anticonvulsant activities using standard procedures. RESULTS AND DISCUSSION: Analysis of the oil reveals 44 compounds of which 30 compounds constituting 84.7% were identified. The oil was characterized by sesquiterpenoids (64.4%) while only four monoterpeneoids (21.3%) were found present in the oil. Major compounds identified were ß-sesquiphellandrene (20.9%), linalool (6.1%), limonene (5.8%), Z-ß-bisabolene (5.4%) and α-pinene (5.3%). The EOPG (50-200mg/kg, i.p.) caused significant (p<0.01) inhibition on rearing {F(4,20)=43}, locomotor {F(4,20)=22} activity and decreased head dips in hole board {F(4,20)=7} indicating CNS depressant effect; decreased rectal temperature {F(4,20)=7-16}, signifying hypothermic activity; decreased ketamine-induced sleep latency {F(4,20)=7.8} and prolonged total sleeping time {F(4,20)=8.8}, indicating sedative effect; reduced muscular tone on the hind-limb grip test {F(4,20)=22}, inclined board {F(4,20)=4-49} and rota rod {F(4,20)=13-106}, implying muscle relaxant activity; induced catalepsy {F(4,20)=47-136}, inhibited apomorphine-induced climbing behaviour {F(4,20)=9} and inhibited apomorphine-induced locomotor {F(4,20)=16}, suggesting anti-psychotic effect; and protected mice against pentylenetetrazole-induced convulsions, indicating anticonvulsant potential. CONCLUSION: The most abundant component of the fresh fruits essential oil of P. guineense was ß-sesquiphellandrene (20.9%); and the oil possesses CNS depressant, hypothermic, sedative, muscle relaxant, antipsychotic and anticonvulsant activities, thus providing scientific basis for its ethnomedicinal applications.

Hypnotic, anticonvulsant and anxiolytic effects of 1-nitro-2-phenylethane isolated from the essential oil of Dennettia tripetala in mice.

This study investigated the hypnotic, anti-convulsant and anxiolytic effects of 1-nitro-2-phenylethane (BPNE) obtained from the oil of Dennettia tripetala G. Baker (Annonaceae) and established its mechanism of action. The essential oil (EO) from the leaf, fruit and seed was obtained by hydrodistillation, followed by isolation of BPNE purified to 99.2% by accelerated gradient chromatography on silica, and identified by NMR and GC-MS. The pure BPNE and EO of the dried seed (93.6%) were comparatively evaluated for hypnotic, anticonvulsant and anxiolytic effects in mice. The acute toxicity of BPNE was determined and the LD50 was 490 mg/kg, intrapritonealy. The hypnotic activities of the EO and BPNE (50-400 mg/kg, i.p.) were assessed by loss of righting reflex, while sodium pentobarbitone (PBS) and diazepam (DZM) were used as positive controls. The anticonvulsant and anxiolytic effects of the EO and BPNE were evaluated in mice. Both BPNE and EO at doses ≥100 mg/kg induced spontaneous hypnosis with loss of righting reflex, significantly decreased sleep latency (SL) and also increased total sleeping time (TST) dose-dependently. They had comparable activity with NAP in TST. The BPNE exhibited higher hypnotic potency than EO at the same dose levels. The EO and BPNE offered comparable dose-related protections against PTZ- and strychnine-induced convulsions. Flumazenil (2 mg/kg) blocked the hypnotic and anticonvulsant (PTZ-convulsions) effects of both EO and BPNE. The essential oil at 5-20 mg/kg dose levels significantly (p<0.05) increased the percentage time spent and number of entries into the open arms. While at the same dose range BPNE significantly (p<0.05) increased the percentage time spent and the number of entries into the open arms respectively. The study concluded that 1-nitro-2-phenylethane exhibited dose dependent significant hypnotic, anticonvulsant and anxiolytic effects and it is the compound largely responsible for the neuropharmacological effects of the oil.