Substance P inhibitory activity of Azadirachta Indica bark extract-in vitro analysis
Introduction: Substance P is a neuropeptide – but only nominally so, as it is ubiquitous. Through cytoplasmic and nuclear membranes of many cell types of various tissues and organs, its receptor – the neurokinin type 1 – is distributed. Neem bark is taken from the neem tree which predominantly grows in India and Asia. It can be grounded into fine powder and can be used as organic pesticide. It has very strong antibacterial and spermicidal activity. Prior to the advent of toothpaste, the astringent qualities of the bark were used for prevention of bleeding gums, tooth decay and foul smell. Materials and methods: Azadirachta Indica bark extract was obtained from green chem Bangalore. SH-SY5Y neuronal cell lines were obtained from the NCCS, Pune with Passage no 11. Cell culture was maintained at sub confluence in a 95% air, 5% CO2 humidified atmosphere at 37oC using MEM HAM in a 1:1 mixture supplemented with 10% fetal bovine serum supplemented with 100units/ml penicillin and 100µg/ml streptomycin. Differentiation of SH-SY5Y was performed by adding 10μM of all-trans-retinoic acid to the culture medium for 6 days. The differentiated SH-SY5Y cells were incubated with LPS for 2 h. The medium containing the appropriate agents for each condition was replaced every other day. Cells were washed with DMEM + HAM F-12 medium and detached with 0.25% trypsin-EDTA. Results: The short – term exposure of differentiated SH-SY5Y neuronal cells to LPS (1µg/ml) resulted in secretion of inflammatory marker Substance P, indicated by increased level of Substance P in LPS alone induced group. Treatment with different concentrations of neem extract has significantly (p<0.001) prevented LPS induced neuro-inflammation in concentrations from 3µg/ml – 100µg/ml. Conclusion: Substance P can be potentially used for pain relief and anti-inflammatory effects.
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