Bikas C. Pal

Mahanine Reverses an Epigenetically Silenced Tumor Suppressor Gene RASSF1A in Human Prostate Cancer Cells

It is becoming clear that frequent epigenetic silencing of tumor suppressor genes could be responsible for the development of cancer in various organs. Several recent reports suggest that suppression of RASSF1A is associated with the advanced grade and stage of prostate cancer and many other cancers. In this investigation, we demonstrated that, mahanine, a plant derived carbazole alkaloid, induced RASSF1A expression in both androgen-responsive (LNCaP) and androgen-negative (PC3) prostate cancer cells by inhibiting DNA methyltransferase (DNMT) activity. Mahanine-induced expression of RASSF1A in turn significantly reduced cyclin D1 but not other cyclins. To understand the inverse relationship between RASSF1A and cyclin D1, we observed that mahanine treatment down-regulates cyclin D1 and addition of RASSF1A siRNA prevented this inhibition. This study show for the first time that mahanine can reverse an epigenetically silenced gene, RASSF1A in prostate cancer cells by inhibiting DNMT activity that in turn down-regulates a key cell cycle regulator, cyclin D1. Mahanine therefore, promises an encouraging therapeutic choice for advanced prostatic cancer.

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl+ CML cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl(+) chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl(+) cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl(+) cells.

Anti-leukemic activity of Dillenia indica L. fruit extract and quantification of betulinic acid by HPLC

The methanolic extract of Dillenia indica L. fruits showed significant anti-leukemic activity in human leukemic cell lines U937, HL60 and K562. This finding led to fractionation of the methanolic extract, on the basis of polarity, in which the ethyl acetate fraction showed the highest anti-leukemic activity. A major compound, betulinic acid, was isolated from the ethyl acetate fraction by silica gel column chromatography and was identified and characterized. Betulinic acid could explain the anti-leukemic activity of the methanolic extract and the ethyl acetate fraction. Hence the quantitative estimation of betulinic acid was approached in methanolic extract and fractions using HPLC.

SPERM IMMOBILIZING EFFECT OF TRITERPENE SAPONINS FROM ACACIA AURICULIFORMIS

A mixture of two partially triterpenoid saponins (Tg), isolated from Acacia auriculiformis was tested for sperm immobilizing activity by using in vitro system. The lowest concentration (ED) required for an obvious immobilization of human sperm by using a modified Sander-Cramer test was found to be 0.35 mg/ml in physiological saline. The ability of the compound as a sperm immobilizing agent was compared with that of Triton X-100 and found to be more potent. Cervical mucus penetration test was also performed and the ED successfully prevented sperm entry in human cervical mucus. Supra-vital staining with eosin-nigrosin indicated death of the treated sperm. Electron microscopic study of Tg-treated sperm showed plasma membrane disintegration and dissolution of acrosomal cap which is presumably the cause for the spermicidal effect of the saponins. No permanent lesion was observed after application of 1.25 mg/ml Tg solution in physiological saline to the eye of rabbits for ten consecutive days.

Saponins from Albizia lebbeck

Three main saponins named albiziasaponins A, B, and C were isolated from the barks of Albizia lebbeck. Their structures were established through spectral analyses as acacic acid lactone 3-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)- beta-D-glucopyranoside, 3-O-beta-D-glucopyranosyl-(1-->2)-O-[alpha-L-arabinopyranosyl-(1-- >6)]- beta-D-glucopyranoside and 3-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)- O- [beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranoside.

Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function

AIM OF THE STUDY Cajanus cajan Linn. (Leguminosae) is a nontoxic edible herb, widely used in Indian folk medicine for the prevention of various liver disorders. In the present study we have demonstrated that methanol-aqueous fraction (MAF2) of Cajanus cajan leaf extract could prevent the chronically treated alcohol induced rat liver damage. MATERIALS AND METHODS Chronic doses of alcohol (3.7 g/ kg) orally administered to rats for 28 days and liver function marker enzymes such as GPT, GOT, ALP and anti-oxidant enzyme activities were determined. Effect of MAF2 at a dose of 50mg/kg body weight on alcohol treated rats was noted. RESULTS Alcohol effected significant increase in liver marker enzyme activities and reduced the activities of anti-oxidant enzymes. Co-administration of MAF2 reversed the liver damage due to alcohol; it decreased the activities of liver marker enzymes and augmented antioxidant enzyme activities. We also demonstrate significant decrease of the phase II detoxifying enzyme, UDP-glucuronosyl transferase (UGT) activity along with a three- and two-fold decrease of UGT2B gene and protein expression respectively. MAF2 co-administration normalized UGT activity and revived the expression of UGT2B with a concomitant expression and nuclear translocation of Nrf2, a transcription factor that regulates the expression of many cytoprotective genes. CONCLUSION Cajanus cajan extract therefore shows a promise in therapeutic use in alcohol induced liver dysfunction.

Effect of ferulic acid from Hibiscus mutabilis on filarial parasite Setaria cervi: molecular and biochemical approaches.

In the reported work the in vitro activity of a methanolic extract of leaves of Hibiscus mutabilis (Malvaceae) against bovine Setaria cervi worms has been investigated. Bioassay-guided fractionation led to isolation of ferulic acid from ethyl acetate fraction. The crude extract and ferulic acid, the active molecule, showed significant microfilaricidal as well as macrofilaricidal activities against the microfilaria (L(1)) and adult of S. cervi by both a worm motility and MTT reduction assay. The findings thus provide a new lead for development of a filaricidal drug from natural products. To examine the possible mechanism of action of ferulic acid, the involvement of apoptosis in adult worms of S. cervi was investigated. We found extreme cellular disturbances in ferulic acid-treated adult worms characterized by chromatin condensation, in situ DNA fragmentation and nucleosomal DNA laddering. In this work we are reporting for the first time that ferulic acid exerts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants (GSH, GST and SOD) of the filarial nematode S. cervi. Our results have provided experimental evidence supporting that ferulic acid causes an increased proapoptotic gene expression and decreased expression of anti-apoptotic genes simultaneously with an elevated level of ROS and gradual dose dependent decline of parasitic GSH level. We also observed a gradual dose dependent elevation of GST and SOD activity in the ferulic acid treated worms.

Structure of acaciaside, a triterpenoid trisaccharide from Acacia auriculiformis

Abstract The structure of a new triterpenoid trisaccharide isolated from the seeds of Acacia auriculiformis has been elucidated as acacic acid lactone-3- O -β- d -glucopyranosyl (1 → 6)-[α- l -arabinopyranosyl (1 → 2)]-β- d -glucopyranoside based on its spectral properties and some chemical transformations.

Structure elucidation of two acylated triterpenoid bisglycosides from acacia auriculiformis cunn.

Abstract Acaciasides A and B, two novel acylated triterpenoid bisglycosides isolated from the fruits of Acacia auriculiformis, were respectively defined to be 3- O -[β-D-glucopyranosyl (1→6) &{;α-L-arabinopyranosyl (1→2)&};- β-D-glucopyranosyl]-21- O -&{;6′S)-2′-trans-2′,6′-dimethyl-6′- O -β-D-glucopyranosyl-2′,7′-octadienoyl&}; acacic acid 28- O -α-L-rhamnopyranosyl (1→6) [β-D-xylopyranosyl (1→6) &{;α-L-arabinopyranosyl (1→2)&};-β-D-glucopyranosyl]-21- 0 -[(6′S)-2′-trans-2′,6′- O -&{;β-D-xylopyranosyl (1→2)-β-D-glucopyranosyl&};- 2′,7′-octadienoyl] acacic acid 28- O -α-L-rhamnopyranosyl (1→6) [β-D-xylopyranosyl (1→2)]-β-D-glucopyranoside (2). The structural details were elucidated by a combination of fast-atom-bombardment mass spectrometry, 1H-, and 13C NMR spectroscopy, and some chemical transformations.

New triterpenoid saponins from Corchorus acutangulus

Abstract Four new triterpenoid glycosides, corchorusins C 1 , D 1 , D 2 and D 3 isolated from the aerial parts of Corchorus acutangulus were respectively identified as saikogenin C, 3- O -β- d -galactopyranoside, saikogenin B, 3- O -β- d -glucopyranosyl (1 → 2)-β- d -galactopyranoside, longispinogenin, 3- O -β- d -glucopyranosyl (1 → 2)-β- d -galactopyranoside and saikogenin C, 3- O -β- d -glucopyranosyl (1 → 2)-β- d -galactopyranoside ( 6 ) based on their spectroscopic properties and some chemical transformations.