Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis.

Sweet basil (Ocimum basilicum) plants produce its characteristic phenylpropene-rich essential oil in specialized structures known as peltate glandular trichomes (PGTs). Eugenol and chavicol are the major phenylpropenes produced by sweet basil varieties whose synthetic pathways are not fully elucidated. Eugenol is derived from coniferyl acetate by a reaction catalysed by eugenol synthase. An acyltransferase is proposed to convert coniferyl alcohol to coniferyl acetate which is the first committed step towards eugenol synthesis. Here, we perform a comparative next-generation transcriptome sequencing of different tissues of sweet basil, namely PGT, leaf, leaf stripped of PGTs (leaf-PGT), and roots, to identify differentially expressed transcripts specific to PGT. From these data, we identified a PGT-enriched BAHD acyltransferase gene ObCAAT1 and functionally characterized it. In vitro coupled reaction of ObCAAT1 with eugenol synthase in the presence of coniferyl alcohol resulted in eugenol production. Analysis of ObCAAT1-RNAi transgenic lines showed decreased levels of eugenol and accumulation of coniferyl alcohol and its derivatives. Coniferyl alcohol acts as a common substrate for phenylpropene and lignin biosynthesis. No differences were found in total lignin content of PGTs and leaves of transgenic lines, indicating that phenylpropene biosynthesis is not coupled to lignification in sweet basil.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.

Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.

Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, ß-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

An inducible NADPH-cytochrome P450 reductase from Picrorhiza kurrooa - an imperative redox partner of cytochrome P450 enzymes.

Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH-cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V max and K m of PkCPR were found to be 5.8 ± 0.05 µmol min(-1) mg(-1), 8.1 ± 0.12 µmol min(-1) mg(-1) and 7.8 µM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa.

Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations.

BACKGROUND: Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, one of the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides. RESULTS: In an endeavor towards identification and characterization of different P450s, we here describe molecular cloning, characterization and expression analysis of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera. Full length cDNAs of WsCYP98A and WsCYP76A have open reading frames of 1536 and 1545 bp encoding 511 (58.0 kDa) and 515 (58.7 kDa) amino acid residues, respectively. Entire coding sequences of WsCYP98A and WsCYP76A cDNAs were expressed in Escherichia coli BL21 (DE3) using pGEX4T-2 expression vector. Quantitative real-time PCR analysis indicated that both genes express widely in leaves, stalks, roots, flowers and berries with higher expression levels of WsCYP98A in stalks while WsCYP76A transcript levels were more obvious in roots. Further, transcript profiling after methyl jasmonate, salicylic acid, and gibberellic acid elicitations displayed differential transcriptional regulation of WsCYP98A and WsCYP76A. Copious transcript levels of both P450s correlated positively with the higher production of withanolides. CONCLUSIONS: Two A-types P450 WsCYP98A and WsCYP76A were isolated, sequenced and heterologously expressed in E. coli. Both P450s are spatially regulated at transcript level showing differential tissue specificity. Exogenous elicitors acted as both positive and negative regulators of mRNA transcripts. Methyl jasmonate and salicylic acid resulted in copious expression of WsCYP98A and WsCYP76A. Enhanced mRNA levels also corroborated well with the increased accumulation of withanolides in response to elicitations. The empirical findings suggest that elicitors possibly incite defence or stress responses of the plant by triggering higher accumulation of withanolides.

Molecular characterization and promoter analysis of squalene epoxidase gene from Withania somnifera (L.) Dunal.

Withania somnifera is a rich reservoir of pharmaceutically active steroidal lactones known as withanolides. The plant is well characterized in terms of its chemistry and pharmacology, but very little is known about the pathway involved in the biosynthesis of withanolides. The present investigation describes the cloning, characterization and expression of squalene epoxidase (SE) gene from W. somnifera. SE (SQE; EC. 1.14.99.7) is one of the rate limiting enzymes in the biosynthesis of triterpenoids, catalyzing the stereospecific epoxidation of squalene to 2,3-oxidosqualene. A full length SE gene (WsSQE) of 1,956 bp was cloned which contained an open reading frame of 1,596 bp, encoding a protein of 531 amino acids with a predicted molecular mass of 57.67 kDa and theoretical PI of 8.48. Full length WsSQE was cloned into pGEX4T-2 vector and expressed in E.coli. Phylogenetic analysis indicated a significant evolutionary relatedness of WsSQE with squalene epoxidases of other plant species and the degree of relatedness with deduced amino acid sequences showed a significant correlation with different plant species. Using genome walking approach, a promoter sequence of 513 bp of WsSQE was isolated which revealed several key cis-regulatory elements known to be involved in various biotic and abiotic plant stresses. Comparative expression analysis of tissue specific WsSQE done by quantitative-PCR demonstrated the highest transcript levels in leaves, as compared to stalk and root tissues. This is the first report of cloning and bacterial expression of SE from W. somnifera and may be of significant interest to understand the regulatory role of SE in the biosynthesis of withanolides.

Dynamics of withanolide biosynthesis in relation to temporal expression pattern of metabolic genes in Withania somnifera (L.) Dunal: a comparative study in two morpho-chemovariants.

Withania somnifera (L.) Dunal synthesizes large array of pharmacologically active secondary metabolites known as withanolides. It has been extensively investigated in terms of chemistry and bioactivity profiling. However, there exists fragmentary information about the dynamics of withanolide biosynthesis at different phenophases in concert with the expression analysis of key pathway genes. In the present study, two morpho-chemovariants of W. somnifera were harvested at five developmental stages, dissected into leaf and root tissues and assayed for three major withanolides viz. withanolide-A (WS-1), withanone (WS-2) and withaferin A (WS-3) content using high performance liquid chromatography. The present investigation also analyzed the expression pattern of five withanolide biosynthetic pathway genes namely squalene synthase, squalene epoxidase, cycloartenol synthase, cytochrome P450 reductase 1, cytochrome P450 reductase 2 to corroborate with the metabolite flux at different developmental stages. The relative transcript profiles of identified genes at various ontogenetic stages illustrated significant variation in leaf and root tissues and were largely concurrent with the alteration in withanolide pool. Comparatively, the concentrations of withanolide A, withanone and withaferin A along with expression levels of all the five genes were appreciably higher in the leaves than in roots. Relative dynamics in terms of quantitative and qualitative profiles of withanolides in leaf and root tissues revealed least correspondence between the pattern of accumulation, possibly indicting towards de novo tissue-specific biosynthesis.

Nanosensor Detection of Synthetic Auxins In Planta using Corona Phase Molecular Recognition.

Synthetic auxins such as 1-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) have been extensively used in plant tissue cultures and as herbicides because they are chemically more stable and potent than most endogenous auxins. A tool for rapid in planta detection of these compounds will enhance our knowledge about hormone distribution and signaling and facilitate more efficient usage of synthetic auxins in agriculture. In this work, we show the development of real-time and nondestructive in planta NAA and 2,4-D nanosensors based on the concept of corona phase molecular recognition (CoPhMoRe), to replace the current state-of-the-art sensing methods that are destructive and laborious. By designing a library of cationic polymers wrapped around single-walled carbon nanotubes with general affinity for chemical moieties displayed on auxins and its derivatives, we developed selective sensors for these synthetic auxins, with a particularly large quenching response to NAA (46%) and a turn-on response to 2,4-D (51%). The NAA and 2,4-D nanosensors are demonstrated in planta across several plant species including spinach, Arabidopsis thaliana (A. thaliana), Brassica rapa subsp. chinensis (pak choi), and Oryza sativa (rice) grown in various media, including soil, hydroponic, and plant tissue culture media. After 5 h of 2,4-D supplementation to the hydroponic medium, 2,4-D is seen to accumulate in susceptible dicotyledon pak choi leaves, while no uptake is observed in tolerant monocotyledon rice leaves. As such, the 2,4-D nanosensor had demonstrated its capability for rapid testing of herbicide susceptibility and could help elucidate the mechanisms of 2,4-D transport and the basis for herbicide resistance in crops. The success of the CoPhMoRe technique for measuring these challenging plant hormones holds tremendous potential to advance the plant biology study.

A Decade of Molecular Understanding of Withanolide Biosynthesis and In vitro Studies in Withania somnifera (L.) Dunal: Prospects and Perspectives for Pathway Engineering.

Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well-characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-à-vis, the exploration of genomic, transcriptomic, proteomic, metabolomics, and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude toward development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool toward enhanced withanolide production has also been attained by deciphering decisive branch point(s) as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. somnifera for understanding various molecular network interactions in entirety.

A phenylalanine ammonia-lyase ortholog (PkPAL1) from Picrorhiza kurrooa Royle ex. Benth: molecular cloning, promoter analysis and response to biotic and abiotic elicitors.

Picrorhiza kurrooa Royle ex Benth. is a highly reputed medicinal herb utilised in the preparation of a number of herbal drug formulations, principally due to the presence of novel monoterpene iridoid glycosides kenned as picrosides. Phenylalanine ammonia-lyase catalyses an important rate-limiting step in phenylpropanoid pathway and supplies precursors like cinnamic acid, vanillic acid, ferulic acid, etc., to a variety of secondary metabolites including picrosides. The imperilled status of P. kurrooa coupled with lack of information regarding biogenesis of picrosides necessitates deciphering the biosynthetic pathway for picrosides. In the present study, a PAL gene, designated PkPAL1 was isolated from P. kurrooa. The cDNA is 2312 bp in length, consisting of an ORF of 2142 bp encoding for a 713 amino acid protein having a predicted molecular weight of 77.66 kDa and an isoelectric point of pH 6.82. qRT-PCR analysis of various tissues of P. kurrooa showed that PkPAL1 transcript levels were highest in the leaves, consistent with picroside accumulation pattern. Using Genome walking, a 718 bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including TGA-element, TGACG-motif, CGTCA-motif, etc. qRT-PCR indicated up-regulation of PkPAL1 by methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations that corroborated positively with the identified cis-elements within the promoter region. Moreover, altitude was found to have a positive effect on the PkPAL1 transcript levels, driving the expression of PkPAL1 abundantly. Based on docking analysis, we identified eight residues as potentially essential for substrate binding in PkPAL1.

NADPH-cytochrome P450 reductase: molecular cloning and functional characterization of two paralogs from Withania somnifera (L.) dunal.

Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis. Present investigation so far is the only report of characterization of CPR paralogs from W. somnifera.

Molecular characterization of UGT94F2 and UGT86C4, two glycosyltransferases from Picrorhiza kurrooa: comparative structural insight and evaluation of substrate recognition.

Uridine diphosphate glycosyltransferases (UGTs) are pivotal in the process of glycosylation for decorating natural products with sugars. It is one of the versatile mechanisms in determining chemical complexity and diversity for the production of suite of pharmacologically active plant natural products. Picrorhiza kurrooa is a highly reputed medicinal herb known for its hepato-protective properties which are attributed to a novel group of iridoid glycosides known as picrosides. Although the plant is well studied in terms of its pharmacological properties, very little is known about the biosynthesis of these important secondary metabolites. In this study, we identified two family-1 glucosyltransferases from P. kurrooa. The full length cDNAs of UGT94F4 and UGT86C4 contained open reading frames of 1455 and 1422 nucleotides, encoding polypeptides of 484 and 473 amino acids respectively. UGT94F2 and UGT86C4 showed differential expression pattern in leaves, rhizomes and inflorescence. To elucidate whether the differential expression pattern of the two Picrorhiza UGTs correlate with transcriptional regulation via their promoters and to identify elements that could be recognized by known iridoid-specific transcription factors, upstream regions of each gene were isolated and scanned for putative cis-regulatory elements. Interestingly, the presence of cis-regulatory elements within the promoter regions of each gene correlated positively with their expression profiles in response to different phytohormones. HPLC analysis of picrosides extracted from different tissues and elicitor-treated samples showed a significant increase in picroside levels, corroborating well with the expression profile of UGT94F2 possibly indicating its implication in picroside biosynthesis. Using homology modeling and molecular docking studies, we provide an insight into the donor and acceptor specificities of both UGTs identified in this study. UGT94F2 was predicted to be an iridoid-specific glucosyltransferase having maximum binding affinity towards 7-deoxyloganetin while as UGT86C4 was predicted to be a kaempferol-specific glucosyltransferase. These are the first UGTs being reported from P. kurrooa.

Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal.

Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC 2.5.1.21) from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1mM IPTG at 37°C after 1h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.

A cyano analogue of boswellic acid induces crosstalk between p53/PUMA/Bax and telomerase that stages the human papillomavirus type 18 positive HeLa cells to apoptotic death.

The p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV) in over 90% of cervical cancers. HPV E6 protein promotes the degradation of p53 thereby inhibiting its stabilization and activation. This study demonstrates that treatment with a novel cyano derivative of 11-keto-ß-boswellic acid, i.e. butyl 2-cyano-3, 11-dioxours-1,12-dien-24-oate (BCDD) reduced the viral E6 mRNA expression and lead to the accumulation of transcriptionally active p53 in the nucleus of HPV18 HeLa cells following DNA damage. Western blot analysis showed that BCDD robustly up regulated time-dependent expression of p53/PUMA/p21 whereas it deprived cells essentially of p-AKT and NF-κB cell survival signalling cascade. BCDD appeared to gear up PUMA activation through p53 pathway and that both p53 and p21 translocated heavily into the nucleus. Simultaneously, it inhibited anti-apoptotic Bcl-2, augumented Drp-1 expression, disrupted mitochondrial functions causing the activation of proapoptotic proteins and caspases activation. Additionally, BCDD inhibited telomerase expression that's likely to result in a marked reduction of the tumorigenic potential of high-grade cervical cancers. Consequently BCDD caused apoptotic death in cervical cancer cells as evidenced by DNA fragmentation and PARP-cleavage. Further, BCDD did not affect the extrinsic signalling transduction pathway as depicted by its null effect on caspase-8. The in vivo anticancer activity of BCDD was investigated in Ehrlich Ascites carcinoma model where it exhibited tumor regression by 48% at 30 mg/kg, i.p., in mice. These findings indicated that BCDD is a potential candidate that may be found useful in the management of cervical cancer.