RESUMO
OBJECTIVE: Garden cress (Lepidium sativum L.) is an important herb in traditional medicine used to improve production of breast milk in women and semen in men. In the present research the authors evaluated its ability to destroy leukemic cancer (Jurkat E6-1) cells, using the alkaloid extract of this plant. METHODS: Constituents of the alkaloid extract were analyzed by gas chromatography-mass spectrometry (GC-MS) and their cytotoxicity in leukemic cancer cells and healthy peripheral blood mononuclear cells (PBMCs) was assessed. Cell death via apoptosis was confirmed by DNA laddering, caspase-3 activity, annexin V-fluorescein isothiocyanate and mitochondrial toxicity assays. The specific course of gene activation in treated cells was determined through quantitative polymerase chain reaction (qPCR). RESULTS: GC-MS analysis identified six alkaloids and proto-alkaloids, namely, benzyl isothiocyanate (1), 2-ethoxy-4H-3,1-benzoxazin-4-one (2), (4R)-2-(2-aminophenyl)-4-phenyloxazoline (3), 5-acetyl-1,2-dihydro-6-methyl-2-oxo-4-phenyl-3-pyridinecarbonitrile (4), benzo[b][1,8]-naphthyridin-5(10H)-one,2,4,7-trimethyl (5) and 1,4-diaminoanthraquinone (6), in the alkaloid extract of L. sativum. Of these, compound 1 was previously identified in the seeds of L. sativum. Exposure to the alkaloid extract caused death of Jurkat E6-1 cells, with median lethal concentration (LC50) of 75.25⯵g/mL. However, the alkaloid extract also showed a nontoxic and proliferative (1.6-fold) effect in healthy PBMCs. Further experiments performed with Jurkat cells at LC50 and sub-LC50 doses demonstrated DNA fragmentation, activation of caspase-3 and time-dependant phosphatidylserine translocation (apoptosis) from inner to outer cell membranes. Cell toxicity and assessment of adenosine triphosphate level, together with using qPCR to evaluate expression profile of major apoptosis genes, revealed that apoptosis may be induced by disruption in the mitochondrial outer membrane potential, through activation of extrinsic and intrinsic apoptosis pathways in Jurkat cells. CONCLUSION: The ability of the alkaloid extract of L. sativum seeds to induce apoptosis indicates a potential pharmacological use in cancer chemotherapy. The separation of individual active compounds and further in-depth exploration of the molecular mechanism of apoptosis may lead to novel chemotherapeutic compounds in our future antineoplastic research.
Assuntos
Apoptose/efeitos dos fármacos , Lepidium sativum/química , Leucemia/fisiopatologia , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Extratos Vegetais/químicaRESUMO
Present study used Aspergillus terreus strain C1 isolated from mangrove soil for itaconic acid (IA) production from potato starch waste. Fermentation parameters were optimized by classical one factor approach and statistical experimental designs, such as Plackett-Burman and response surface designs. Anionic deionization of potato waste was found to be a very effective, economic, and easy way of improving IA production. The increase in IA production by deionization was found to correlate with removal of phosphate. In our knowledge, this is the first report on application of deionization of potato waste to enhance IA production. Other parameters like inoculum development conditions, pH, presence of peptone and certain salts in the medium also significantly affected IA production. IA production by strain C1 increased 143-fold during optimization when compared with the starting condition. The optimized IA level (35.75 g/L) was very close to the maximum production predicted by RSM (38.88 g/L). Bench scale production of IA was further optimized in 3-L stirred tank reactor by varying parameters like agitation and aeration rate. The maximum IA production of 29.69 g/L was obtained under the agitation speed of 200 rpm and aeration rate of 0.25 vvm. To the best of our knowledge, it is the first report on IA production from potato starch waste at bioreactor level. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2774, 2019.
Assuntos
Biotecnologia/métodos , Proteínas Fúngicas/química , Glucana 1,4-alfa-Glucosidase/química , Solanum tuberosum/química , Succinatos/química , alfa-Amilases/química , Aspergillus/enzimologia , Reatores Biológicos , Biotecnologia/instrumentação , Fermentação , HidróliseRESUMO
The present study demonstrates apoptosis-inducing potential and mechanism of action of Tribulus terristris alkaloid extract in Jurkat E6-1 cancer cell line. Liquid Chromatography-Mass Spectrometry and High Resolution-Mass Spectrometry analysis identified the presence of four N-feruloyltyramine derivatives, namely trans-N-feruloyl-3-hydroxytyramine (1), trans-N-coumaroyltyramine (2), trans-N-feruloyltyramine (3) and trans-N-feruloyl-3-ethoxytyramine (4) in the alkaloid extract. Compounds 2 and 3 have not been yet reported in the alkaloid extract of T. terristris. In silico analysis revealed therapeutic potential of N-feruloyltyramine derivatives and strong binding efficiency to both chains of Tumor Necrosis Factor Receptor 1. Treatment of alkaloids extract to Jurkat E6-1 clone induced dose-dependent cytotoxicity (LC50 140.4 µg mL-1). Jurkat cells treated with alkaloids extract at sub-lethal concentration showed DNA fragmentation, enhancement in caspase-3 activity and phosphatidylserine translocation (apoptosis indicator) compared to control cells. Gene expression analysis using Human Apoptosis RT2 Profiler PCR Array analysis upon alkaloid treatment was found to significantly alter expression of critical genes such as TNFR1, FADD, AIFM, CASP8, TP53, DFFA and NFKB1. These genes are predicted to mediate apoptotic cell death via both intrinsic and extrinsic apoptosis pathway. In summary, we report the identification of new N-feruloyltyramine derivatives from alkaloid extract of T. terristris fruit with probable anti-leukemic and pharmacological potential.