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Activity-guided chemo toxic profiling of Cassia occidentalis (CO) seeds: detection of toxic compounds in body fluids of CO-exposed patients and experimental rats.

Chemical research in toxicology (2015-04-29)
Gati Krushna Panigrahi, Ratnasekhar Ch, Mohana K R Mudiam, Vipin M Vashishtha, S Raisuddin, Mukul Das
ABSTRACT

Our prior studies have shown an association between the deaths of children and consumption of Cassia occidentalis (CO) seeds. However, the chemicals responsible for the CO poisoning are not known. Therefore, the present study was designed to identify the key moieties in CO seeds and their cytotoxicity in rat primary hepatocytes and HepG2 cells. Activity-guided sequential extraction and fractionation of the seeds followed by GC-MS analysis identified the toxic compounds in the CO seeds. These identified compounds were subsequently detected and quantified in blood and urine samples from CO-exposed rats and CO poisoning human study cases. GC-MS analysis of different fractions of methanol extracts of CO seeds revealed the presence of five anthraquinones (AQs), viz. physcion, emodin, rhein, aloe-emodin, and chrysophanol. Interestingly, these AQs were detected in serum and urine samples from the study cases and CO-exposed rats. Cytotoxicity analysis of the above AQs in rat primary hepatocytes and HepG2 cells revealed that rhein is the most toxic moiety, followed by emodin, aloe-emodin, physcion, and chrysophanol. These studies indicate that AQ aglycones are responsible for producing toxicity, which may be associated with symptoms of hepatomyoencephalopathy in CO poisoning cases.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Pyridine, ≥99%
Sigma-Aldrich
Trypan Blue solution, 0.4%, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Chlorotrimethylsilane solution, 1.0 M in THF
Sigma-Aldrich
Pyridine, anhydrous, 99.8%
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N,O-Bis(trimethylsilyl)trifluoroacetamide, ≥99%
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Emodin, from Frangula bark, ≥90% (HPLC)
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Thiazolyl Blue Tetrazolium Bromide, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥97.5% (HPLC)
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Thiazolyl Blue Tetrazolium Bromide, 98%
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Sodium pyruvate, Hybri-Max, powder, suitable for hybridoma
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Sodium pyruvate, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99%
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Sodium pyruvate, powder, BioXtra, suitable for mouse embryo cell culture
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Sodium pyruvate, BioXtra, ≥99%
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Sodium pyruvate, ReagentPlus®, ≥99%
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Chlorotrimethylsilane, ≥98.0% (GC)
Sigma-Aldrich
Physcion, ≥98.0% (TLC)
Sigma-Aldrich
Chlorotrimethylsilane, produced by Wacker Chemie AG, Burghausen, Germany, ≥99.0% (GC)
Sigma-Aldrich
Trypan Blue, powder, BioReagent, suitable for cell culture
Supelco
N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane, with 1% trimethylchlorosilane, for GC derivatization, LiChropur
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Trypan Blue, Dye content 60 %
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1,8-Dihydroxy-3-methylanthraquinone, 98%
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Chlorotrimethylsilane, purified by redistillation, ≥99%
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Sodium pyruvate, anhydrous, free-flowing, Redi-Dri, ReagentPlus®, ≥99%