Abstract
Nitidine, Chelerythrine and Sanguinarine, all these three alkaloids are benzophenanthridine alkaloids. Nitidine was used as an anti-HIV, anti-malarial and anti-cancer. Chelerythrine had anti-cancer and anti-inflammatory activities. Sanguinarine was widely used as an anti-plaquestic and anti-cancer. High performance thin layer chromatography (HPTLC) method was used for simultaneous quantification of Nitidine, Chelerythrine and Sanguinarine in callus extract of Zanthoxylum rhetsa by using Silica gel 60 F254 as stationary phase and ethyl acetate:methanol:water:diethylamine (30:5:2:0.5 v/v) as mobile phase at 280 nm. The linearity concentration range was 5 - 160 μg/band of each alkaloid. The Rf values of Nitidine, Chelerythrine and Sanguinarine were found to be 0.28, 0.49 and 0.73. The limit of detection and limit of quantification were found to be 0.026, 0.088 μg/spot and 0.010 and 0.033 μg/spot, 0.0104 and 0.035 μg/spot respectively for Nitidine, Chelerythrine and Sanguinarine. HPTLC method was developed and validated according to ICH guidelines for simultaneous estimation of Nitidine, Chelerythrine and Sanguinarine and proved to be simple, specific, accurate, robust and rapid.
Highlights
To the pre analyzed samples of callus extract, standard nitidine, chelerythrine and sanguinarine solution was added by spiking at 100 μg level and the mixture was analyzed by the proposed High performance thin layer chromatography (HPTLC) method
Different methods which were proposed by earlier authors for HPTLC individual quantification of nitidine (Praveena and Veeresham in 2014 and 2015 [19] [20], Baerhein et al, in 1983) [25], chelerythrine (Petruczynik et al, in 2008) [26], sanguinarine (Ghosh et al, in 2005, Garcia et al.,) ([27] [28]) for various mobile phases chloroform:methanol (7:1 v/v), n-butanol:pyridine: water (6:4:3 v/v), acetone:diisopropyl ether:diethyl amine (1:1:0.1 v/v), hexane: acetone:methanol (80:15:5 v/v), hexane:ethyl acetate:ammonia (25%) (6:4:0.1 v/v) respectively, simultaneous estimation of sanguinarine and chelerythrine (Bogucka-Kocka and Zalewski in 2017 [21] and Baerhein et al, in 1983 [25]) for various mobile phases “toluene:ethyl acetate:methanol (83:15:2) and benzene:methanol (6:1), chloroform:ethyl acetate:methanol (2:2:1), were tried with different modifications
Because there is no report on simultaneous estimation of proposed three benzophenanthridine alkaloids, the present study was carried out with a mobile phase of ethyl acetate:methanol:water:diethylamine (30:5:2:0.5), which gave good resolution for nitidine, chelerythrine and sanguinarine with a sharp and well defined peaks at Rf = 0.28, 0.49 and 0.73 and when the chamber was saturated with mobile phase for 20 min at room temperature (25 ̊C ± 2 ̊C) during HPTLC determination of nitidine, chelrythrine and sanguinarine from plant tissue culture extracts
Summary
Benzophenanthridine alkaloids are one of the most important sub-classes of isoquinoline alkaloids, which are the major group of pharmacologically useful. The present paper deals with simultaneous HPTLC quantification of three benzophenanthridine alkaloids namely nitidine, chelerythrine and sanguinarine. Praveena and Veeresham (2014 and 2015) were reported the HPTLC quantification of nitidine from Toddalia asiatica roots and callus cultures [19] [20]. Bogucka-Kocka and Zalewski (2017) reported the quantification of chelerythrine and sanguinarine from Chelidonium majus herb and root by using HPTLC [21]. We have undertaken this study for simultaneous quantification of these three benzophenanthridine alkaloids (nitidine, chelerythrine and sanguinarine) by densitometric HPTLC method. There are no reports on Z. rhetsa whole herb/tissue culture extract nor on simultaneous HPTLC determination of nitidine, chelerythrine and sanguinarine from the callus extracts of Z. rhetsa. The present work illustrates the denisitometric HPTLC method establishment and validation for simultaneous quantification of nitidine, chelerythrine and sanguinarine from Z. rhetsa callus extract
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