Nothapodytes Nimmoniana Research Articles

Critical Appraisal of Nothapodytes nimmoniana (J. Graham) Mabb. Extraction Methods, Pharmacological Activities, and Future Prospects

Nothapodytes nimmoniana (J. Graham) Mabb., commonly known as Narkya, holds immense potential as a medicinal plant, exhibiting a wide array of pharmacological activities. This review provides a comprehensive overview of the methods used for extracting bioactive compounds from different plant parts, along with an exploration of their diverse pharmacological activities. The plant’s significance stems from its high content of camptothecin (CPT), a potent anti-cancer alkaloid. We delve into the plant’s anti-microbial, antioxidant, anti-tumor, anti-helminthic, anti-malarial, and anti-inflammatory properties. Additionally, we highlight the challenges faced by N. nimmoniana (J. Graham) Mabb., due to overexploitation and its endangered status. To address these concerns, the review underscores the importance of sustainable conservation strategies and the implementation of advanced techniques like plant tissue culture and endophyte production for increased CPT production. This holistic analysis not only sheds light on the plant’s multifaceted benefits but also underscores the need for responsible utilization and conservation efforts.

Optimisation of enzyme-assisted extraction of camptothecin from Nothapodytes nimmoniana and its characterisation.

Efficient extraction of camptothecin (CPT), an anticancer agent from the commercial source Nothapodytes nimmoniana (J. Graham) Mabb in India, is of paramount importance. CPT is present in the highest concentration in the stem portion, and the stem can be readily harvested without uprooting the plant. The fluorescence microscopy mapping of the bark matrix for CPT revealed its presence in a free form within both the outer (epidermal and cortical tissues) and inner (xylem and phloem tissues) sections. The bark matrix primarily consists of cellulose, hemicellulose, and lignin, rendering it woody, rigid, and resistant to efficient solvent penetration for CPT extraction. We proposed a hypothesis that subjecting it to disruption through treatment with hydrolytic enzymes like cellulase and xylanase could enhance solvent diffusion, thereby enabling a swift and effective extraction of CPT. The present study was aimed at enzyme-assisted extraction, using cellulase and xylanase for hydrolytic disruption of the cells to readily access CPT from the stem of the plant N. nimmoniana (J. Graham) Mabb. The hydrolytic cell disruption of ground powder from the stem bark was studied using cellulase and xylanase enzymes. The enzymatically pretreated stem bark powder was subsequently recovered by filtration, dried, and subjected to extraction with methanol to isolate CPT. This process was optimised through a Box-Behnken design, employing a one-factor-at-a-time approach to assess parameters such as enzyme concentration (2-10% w/w), pH (3-7), incubation time (6-24 h), and solid-to-solvent ratio (1:30-1:70 g/mL). CPT was characterised using proton nuclear magnetic resonance (1H-NMR) and Fourier transform infrared (FTIR) spectra, and a high-performance liquid chromatography (HPLC) method was developed for quantification. The cellulase and xylanase treatment resulted in the highest yields of 0.285% w/w and 0.343% w/w, with efficiencies of 67% and 81%, respectively, achieved in a significantly shorter time compared to the untreated material, which yielded 0.18% with an efficiency of only 42%. Extraction by utilising the predicted optimised process parameters, a nearly two-fold increase in the yield, was observed for xylanase, with incubation and solvent extraction times set at 16 and 2 h, respectively. Scanning electron microscopy (SEM) images of the spent material indicated perforations attributed to enzymatic action, suggesting that this could be a primary factor contributing to the enhanced extraction. Enzyme-mediated hydrolytic cell disruption could be a potential approach for efficient and rapid isolation of CPT from the bark of N. nimmoniana.

In-Vitro Assessment of Antioxidant and Antimicrobial Activity of Field Grown and Tissue Cultured Fruit Callus of Nothapodytes nimmoniana

This investigation aimed to evaluate in vitro antioxidant and antimicrobial activity from the methanolic extracts of tissue-cultured and field-grown parts of Nothopodytes nimmoniana. Antioxidant activity of the plant parts was measured by Di-phenyl-picryl-hydrazyl (DPPH) radical, reducing power, and hydrogen peroxide assays. Antimicrobial activity of plant extracts was evaluated by the well diffusion method and the MIC values determined against different pathogens such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, and Candida albicans. The DPPH assay showed overall strong antioxidant activities from the suspension culture and fruit callus culture with an IC50 value. Antimicrobial activity against 4 bacteria and 1 fungus was evaluated through the well-diffusion method and by determining the minimum inhibitory concentration (MIC). The suspension culture and fruit callus showed better antimicrobial activity than other plant parts. The zone of inhibition showed maximum values in suspension culture and fruit callus extract against Candida albicans. The study implies that suspension culture and fruit callus culture of plants showed the greatest values because of the enhancement of the alkaloid compound camptothecin and other compounds like phenolics and flavonoids.

Effect of abiotic elicitor on the production of camptothecin from Nothapodytes nimmoniana (J. Graham) Mabb

The plant Nothapodytes nimmoniana belongs to the Icacinaceae family, and is a significant source of camptothecin (CPT), an effective quinoline alkaloid with an array of pharmacological benefits including anti-cancer, anti-HIV, antibacterial, and antioxidant etc. The elicitation method is the most effective strategy to increase and maintain the synthesis of particular compounds in vitro culture. In this study, to understand the impact of various abiotic elicitors on CPT production by using established N. nimmoniana cell suspension culture obtained from friable leaf callus grown in Murashige & Skoog basal media with sucrose and agar augmented with 2 mg/L of 2,4-dichloro phenoxy acetic acid (2,4-D). A total of six abiotic elicitors were used such as cadmium chloride, ethanol, salicylic acid, tryptophan, copper chloride, and calcium chloride. The impact of these abiotic elicitors on the CPT yield at three different concentrations by cell suspension was evaluated for up to 15 days. High-performance liquid chromatography (HPLC) was used to quantify the CPT production. A maximum of 9.43 and 6.26-fold increment was recorded in CPT production in suspension culture elicitated with 2 mM of tryptophan and 1 mM of tryptophan on the 12th day of inoculation followed by ethanol 7.5% treated cell suspension which enhanced the CPT production up to 1.44-fold increase while compared to control. The cell viability of the suspension culture treated with calcium chloride and cadmium chloride was observed in decreased biomass as per the increment in the concentration. The findings of the current study indicate the potential use of abiotic elicitation as another possible option for boosting up the synthesis of CPT in N. nimmoniana cell suspension cultures.

Genomic and transcriptomic analysis of camptothecin producing novel fungal endophyte: Alternaria burnsii NCIM 1409

Camptothecin is an important anticancer alkaloid produced by particular plant species. No suitable synthetic route has been established for camptothecin production yet, imposing a stress on plant-based production systems. Endophytes associated with these camptothecin-producing plants have been reported to also produce camptothecin and other high-value phytochemicals. A previous study identified a fungal endophyte Alternaria burnsii NCIM 1409, isolated from Nothapodytes nimmoniana, to be a sustainable producer of camptothecin. Our study provides key insights on camptothecin biosynthesis in this recently discovered endophyte. The whole genome sequence of A. burnsii NCIM 1409 was assembled and screened for biosynthetic gene clusters. Comparative studies with related fungi supported the identification of candidate genes involved in camptothecin synthesis and also helped to understand some aspects of the endophyte’s defense against the toxic effects of camptothecin. No evidence for horizontal gene transfer of the camptothecin biosynthetic genes from the host plant to the endophyte was detected suggesting an independent evolution of the camptothecin biosynthesis in this fungus.

Genome-scale metabolic model led engineering of Nothapodytes nimmoniana plant cells for high camptothecin production.

Camptothecin (CPT) is a vital monoterpene indole alkaloid used in anti-cancer therapeutics. It is primarily derived from Camptotheca acuminata and Nothapodytes nimmoniana plants that are indigenous to Southeast Asia. Plants have intricate metabolic networks and use them to produce secondary metabolites such as CPT, which is a prerequisite for rational metabolic engineering design to optimize their production. By reconstructing metabolic models, we can predict plant metabolic behavior, facilitating the selection of suitable approaches and saving time, cost, and energy, over traditional hit and trial experimental approaches. In this study, we reconstructed a genome-scale metabolic model for N. nimmoniana (NothaGEM iSM1809) and curated it using experimentally obtained biochemical data. We also used in silico tools to identify and rank suitable enzyme targets for overexpression and knockout to maximize camptothecin production. The predicted over-expression targets encompass enzymes involved in the camptothecin biosynthesis pathway, including strictosidine synthase and geraniol 10-hydroxylase, as well as targets related to plant metabolism, such as amino acid biosynthesis and the tricarboxylic acid cycle. The top-ranked knockout targets included reactions responsible for the formation of folates and serine, as well as the conversion of acetyl CoA and oxaloacetate to malate and citrate. One of the top-ranked overexpression targets, strictosidine synthase, was chosen to generate metabolically engineered cell lines of N. nimmoniana using Agrobacterium tumefaciens-mediated transformation. The transformed cell line showed a 5-fold increase in camptothecin production, with a yield of up to 5 µg g-1.

Chloroplast genome sequence of Nothapodytes nimmoniana (Icacinaceae), a vulnerable reservoir of camptothecin from Western Ghats

Nothapodytes nimmoniana belongs to family Icacinaceae and is a major source of compound Camptothecin. The global demand for Camptothecin has caused large-scale exploitation of N. nimmoniana from its wild habitat in Western Ghats of India, thereby making it vulnerable. The species is known to exhibit genetic diversity among the populations in Western Ghats. In this study, we report plastome sequence of N. nimmoniana, first for the genus. For the study, the species was collected from Western Ghats of Maharashtra. The plastome of N. nimmoniana was 150,726 bp in length and exhibited typical quadripartite structure with 83,771 bp LSC, 18,513 bp SSC and 24,221 IR region. The plastome was characterized by presence of 124 unique genes, 87 protein coding genes, 29 tRNA genes and four rRNA genes. Further, the plastome was compared with the available basal lamiid plastomes for gene order and composition. N. nimmoniana plastome exhibited SSC region in an inverted configuration. Phylogenomic study placed N. nimmoniana sister to Mappia mexicana. The SSR markers identified in this study, might help to distinguish genetically diverse populations, prioritizing the populations which need immediate conservation effects as well as for checking adulteration.

Phylogenomics of transcriptionally active AP2/ERF and bHLH transcription factors and study of their promoter regions in Nothapodytes nimmoniana (J.Graham) Mabb.

Nothapodytes nimmoniana is a medicinally important plant producing anticancer monoterpene indole alkaloid (MIA), camptothecin (CPT). The CPT is synthesised through the strictosidine intermediate following the MIA pathway; however, transcriptional regulation of CPT pathway is still elusive in N. nimmoniana. Biosynthesis of MIA is regulated by various transcription factors (TFs) belonging to AP2/ERF, bHLH, MYB, and WRKY families. The present study identified transcriptionally active full-length 105 AP2/ERF and 68 bHLH family TFs from the N. nimmoniana. AP2/ERF TFs were divided into three subfamilies along with a soloist, while bHLH TFs were divided into 10 subfamilies according to their phylogenetic similarities. Three group IXa ERFs, Nn-ERF22, Nn-ERF29, and Nn-ERF41, one subfamily IVa TF Nn-bHLH7, and three subfamilies IIIe Nn-bHLH33, Nn-bHLH51, and Nn-bHLH52 clustered with the TFs regulating alkaloid biosynthesis in Catharanthus roseus, tomato, tobacco, and Artemisia annua. Expression of these TFs in N. nimmoniana was higher in roots, which is a primary CPT accumulating tissue. Moreover, genome skimming approach was used to reconstruct the promoter regions of candidate ERF genes to identify the cis-regulatory elements. The presence of G-boxes and other jasmonic acid-responsive elements in the promoter suggests the regulation of ERFs by bHLHs. The present study effectively generated and used genomics resource for characterisation of regulatory TFs from non-model medicinal plant.

Cellular localisation and quantification of Camptothecin in different plant parts of Nothapodytes nimmoniana (J. Graham) Mabberley of Sri Lankan origin.

Nothapodytes nimmoniana (Icacinaceae) is a rich source of Camptothecin (CPT), an anti-cancer prodrug. Efficient extraction of CPT from various plant parts is crucial for better recovery of this pre-drug. To investigate the distribution of CPT in plant parts and to compare the methods of extraction on CPT yield to evaluate how cellular localisation affects the efficiency of extraction methods. Transverse sections of plant parts were observed under a ultraviolet (UV)-fluorescence microscope for the fluorescence that the CPT molecule emits when exposed to UV radiation. Dried plant parts were extracted using 90% methanol with ultrasonic assistance, hot ethanol (61% ethanol at 60°C), and chloroform-methanol (4:1, v/v). The CPT in plant parts were detected by thin-layer chromatography (TLC) and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). Quantification was carried out by HPLC. Blue fluorescence indicated a prominent accumulation of CPT in roots compared to leaf with petiole, twigs, and stembark. This accumulation was observed in upper and lower epidermis of the leaf, isolated strands of fibres in the phloem in the petiole, and groups of idioblast cells in the cortex. The ultrasonic-assisted extraction with 90% methanol showed the highest CPT yield in the root (1.91 ± 0.02 mg/g of dry weight), followed by stembark and the least in leaves [0.02 ± 0.01 mg/g (dry weight)] irrespective of the method of extraction. However, hot ethanol extraction gave the highest CPT yield for twig and leaf, indicating the necessity of tissue-specific extraction methods for better recovery of CPT.

Microscopic Studies and Preliminary Pharmacognostical Evaluation of Leaves and Roots of Ophiorrhiza eriantha Wight

Death and suffering caused by cancer have been a major concern throughout the history of mankind. Extensive research has been undertaken in the plant kingdom to find out an effective drug for the treatment of various cancer diseases. Drugs derived from plants and microorganism plays a major role in the chemotherapy of cancer. Monoterpene indole alkaloid camptothecin (CPT) is a potent anticancer agent first isolated and reported from extracts of the Chinese tree Camptotheca acuminata (Nyssaceae). Thereafter camptothecin was isolated from plants of different families mainly Ophiorrhiza mungos and Nothapodytes nimmoniana. The genus Ophiorrhiza is a potent natural source of camptothecin. The presence of fortyseven species and nine varieties of Ophiorrhiza was reported in the Indian subcontinent. Ophiorrhiza eriantha Wight is an erect subshrub species, its anticancer potential and detailed phytochemical analysis are yet to be studied. Our aim is to evaluate the pharmacognostic parameters of leaves and roots of Ophiorrhiza eriantha Wight. Our objective is to identify characteristic microscopic features of leaves and roots of the plant and to study the quantitative microscopy, ash value, loss on drying, phytochemical screening, fluorescence analysis, and powder analysis of leaf and roots of Ophiorrhiza eriantha Wight. The thickness of the midrib, lamina, leaf margin, and epidermal trichome is measured. It showed the presence of paracytic stomata. Non-glandular epidermal trichomes are common in leaf powder. Ophiorrhiza eriantha Wight is a promising anticancer plant; this is a pioneer work to explore the anatomical features of Ophiorrhiza eriantha Wight. Keywords: ,, , , And

Review on Camptothecin Producing Medicinal Plant: Nothapodytes Nimmoniana

Nothapodytes nimmoniana is an endangered medicinal plant widely distributed throughout the Western Ghats of India. The plant contains camptothecin (CPT) which is renowned anticancer drugs. Though, CPT found in many plant species but maximum amount of CPT has been reported from N. nimmoniana. Due to very good source of CPT, this plant has been explored for its Phytochemical, Biotechnological and Pharmacological aspects. Looking to the huge global demand for CPT, overexploitation of N. nimmoniana, unplanned deforestation, and lowest production of CPT from intact plant, reduction of seed germination, high market cost and not have economically feasible process of production has optimistic us to investigate this plant in a systematic manner. The proposed article can be utilized for the establishment of extraction methods and analytical protocol for CPT. Also, Bioreactors production of CPT using high yielding cell line of N. nimmoniana. The pharmacological data will be applicable for discovery of new Drug and development lead to novel compounds which are safe and effective.

Biotechnology of camptothecin production in Nothapodytes nimmoniana, Ophiorrhiza sp. and Camptotheca acuminata.

Cancer is probably the deadliest human disease in recent years. In the past few years, rapid clinical progress has been made in the field of anticancer drug development. Plant secondary metabolites have been noted as extremely efficacious as promising natural source for anticancer therapy for many years. Camptothecin (CPT) is one of the popularly used anti-tumor drugs possessing clinically proven properties against a plethora of human malignancies that include ovarian and colorectal cancers. For the first time, CPT was obtained from the extracts of a Chinese medicinal tree, Camptotheca acuminata Decne. from the family Cornaceae. Subsequently, CPT was also isolated from the bark of Nothapodytes foetida (Wight) Sleumer (Icacinaceae). However, the availability of enough natural sources for obtaining CPT is a major constraint. Due to overexploitation and harvesting, loss of habitat, excessive trading, and unfavorable environmental factors, the natural source of CPT has become extinct or extremely limited and hence they are red listed under endangered species. Conventional propagation has also failed to meet the ever-expanding demand for CPT production. With this, biotechnological toolkits have constantly been used as a boon to produce sustainable source, utilization, and ex situ conservation of medicinal plants. The approaches serve as a supplement to traditional agriculture in the mass production of plant metabolites with potent bioactivities. Non-availability of enough anticancer medicine and the requirement to satisfy current demands need a sustainable source of CPT. With this background, we present a comprehensive review on CPT discovery, its occurrence in the plant kingdom, biosynthesis, phytochemistry, pharmacological properties, clinical studies, patterns of CPT accumulation, and biotechnological aspects of CPT production in three plants, viz., N. nimmoniana, Ophiorrhiza species, and C. acuminata.Key points• Biotechnological approaches on production of camptothecin from Nothapodytes nimmoniana, Ophiorrhiza species, and Camptotheca acuminata• In vitro propagation of camptothecin-producing plants• Genetic diversity and transgenic research on camptothecin-producing plants.

In vitro elicitation of Camptothecin by challenging with biotic elicitors in Nothapodytes nimmoniana (J.Graham) Mabb

Camptothecin (CPT) and its analogs are gaining prominence for its promising anti-cancer activity and are used throughout the world for the treatment of colorectal, breast, and lung cancers in particular. Nothapodytes nimmoniana (J. Graham) Mabb. (Olacaceae), a plant native to India's Western Ghats, has been harnessed for the production of CPT. The endangered nature of its wild population has urged plant tissue culture and engineering techniques to increase CPT production in multifold levels by adapting elicitation methods using chemical stress-inducing substances called elicitors. The main objective of this study is to increase the CPT production through in vitro cell suspension culture of N. nimmoniana by treating it with 5 different biotic elicitors such as chitin, chitosan, pullulan, glutathione, and jasmonic acid. The preliminary quantification of CPT in methanolic extracts of wild N. nimmoniana revealed that the bark extracts showed higher level of CPT than the leaf extracts. Further, column chromatography technique was employed to obtain CPT with 99.25 % purity. The elemental composition and structure of the purified compound was confirmed through HR-MS and Proton NMR spectroscopy. Shake flask in vitro elicitation of CPT was observed at a maximum level in 10 % chitin treated elicited culture with a yield of 15.06 μg/150 ml culture followed by 5 % jasmonic acid amounting to 6.025 μg/150 ml of culture as compared with other elicitors and control. LC-MS/MS fragmentation analysis was utilized for the structural confirmation of CPT. In conclusion, the biotic elicitor chitin 10 % treated suspension cells of N. nimmoniana showed maximum level (11.48-fold) of CPT elicitation followed by 4.65-fold in 5 % jasmonic acid treatment as compared with other elicitors and control suspension cells.

Biomass and camptothecin production in the calcium chloride elicited and liquid medium overlayed Nothapodytes nimmoniana (J. Graham) Mabberly callus cultures

Biomass and camptothecin production in the calcium chloride elicited and liquid medium overlayed Nothapodytes nimmoniana (J. Graham) Mabberly callus cultures

Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila

BackgroundThe anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined.ResultsIn this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis.ConclusionsIn our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.

Sustainable production of camptothecin from an Alternaria sp. isolated from Nothapodytes nimmoniana

Camptothecin the third most in demand alkaloid, is commercially extracted in India from the endangered plant, Nothapodytes nimmoniana. Endophytes, the microorganisms that reside within plants, are reported to have the ability to produce host–plant associated metabolites. Hence, our research aims to establish a sustainable and high camptothecin yielding endophyte, as an alternative source for commercial production of camptothecin. A total of 132 endophytic fungal strains were isolated from different plant parts (leaf, petiole, stem and bark) of N. nimmoniana, out of which 94 were found to produce camptothecin in suspension culture. Alternaria alstroemeriae (NCIM1408) and Alternaria burnsii (NCIM1409) demonstrated camptothecin yields up to 426.7 ± 33.6 µg/g DW and 403.3 ± 41.6 µg/g DW, respectively, the highest reported production to date. Unlike the reported product yield attenuation in endophytes with subculture in axenic state, Alternaria burnsii NCIM1409 could retain and sustain the production of camptothecin up to ~ 200 μg/g even after 12 continuous subculture cycles. The camptothecin biosynthesis in Alternaria burnsii NCIM1409 was confirmed using 13C carbon labelling (and cytotoxicity analysis on different cancer cell lines) and this strain can now be used to develop a sustainable bioprocess for in vitro production of camptothecin as an alternative to plant extraction.

Transcriptome-wide identification, characterization, and phylogenomic analysis of cytochrome P450s from Nothapodytes nimmoniana reveal candidate genes involved in the camptothecin biosynthetic pathway.

The plant Nothapodytes nimmoniana is an important source of camptothecin (CPT), an anticancer compound widely used in the treatment of colorectal, lung, and ovarian cancers. CPT is biosynthesized by the combination of the seco-iridoid and indole pathways in plants. The majority of the biosynthetic steps and associated genes still remain unknown. Certain reactions in the seco-iridoid pathway are catalyzed by cytochrome P450 enzymes. Hence, identifying transcriptionally active cytochrome P450 genes becomes essential in the elucidation of the CPT biosynthetic pathway. Here, we report the identification of 94 cytochrome P450s from the assembled transcriptomic data from leaf and root tissues of N. nimmoniana. The identified cytochrome P450 genes were full length and possessed all four conserved characteristic signature motifs of cytochrome P450 genes. Phylogenetic analysis of the protein sequences revealed their evolution and diversification and further categorized them into A-type (52.12%) and non-A-type (47.87%) cytochrome P450s. These 94 sequences represent 38 families and 63 subfamilies of cytochrome P450s. We also compared the transcriptional activity of identified cytochrome P450s with the expression of their homologs in the CPT-producing plant Ophiorrhiza pumila. Based on expression profiles and quantitative PCR validation, we propose NnCYP81CB1 and NnCYP89R1 as candidate cytochrome P450 genes involved in camptothecin biosynthesis in N. nimmoniana.

Proteomic and metabolomic analysis of Nothapodytes nimmoniana (J. Graham) extracts’ treatment on HeLa cells

Nothapodytes nimmoniana is a natural source of camptothecin, a known anticancer drug. Stem and leaf extracts of Nothapodytes nimmoniana were studied to assess its in vitro action on cancer progression events in a cervical cancer cell line, the HeLa. As a well-studied ideal model system, the HeLa has been chosen and the effects of extracts on cancer progression events have been traced out. HeLa cells and media samples were analyzed for differentially expressed proteins and metabolites respectively on RRLC–ESI-QTOFMS. The CBFA2T1, cysteine-rich protein 2-binding protein, Zinc finger protein 788, transcription factor RFX3 and angiomotin-like protein 1 were significantly expressed proteins while 3-hydroxysuberic acid, indole-3-carboxylic acid, N8-acetylspermidine and l-octanoylcarnitine were significantly expressed metabolites. Aminoacyl-tRNA biosynthesis, purine metabolism, and valine, leucine, arginine, proline metabolism were the most prominent pathways. The multi-omics approach has the greatest metabolites and proteins profiling potential, hence was applied here to find signatures of N. nimmoniana extracts’ treatment of cervical cancer.

Inhibition of the collar rot fungus, Sclerotium rolfsii Sacc. by an endophytic fungus Alternaria sp.: implications for biocontrol

Sclerotium rolfsii is a necrotrophic, soil-borne pathogenic fungus responsible for serious crop losses worldwide. It is one of the major phytopathogens infecting chilli and causes yield losses ranging from 16 to 80%. Among the various approaches to manage S. rolfsii, biological control is an environmentally acceptable alternative over other chemical methods. In this study, 120 endophytic fungal isolates obtained from a medicinal plant, Nothapodytes nimmoniana were evaluated for their in vitro and in vivo antagonism towards S. rolfsii. Among these, one isolate, Alternaria sp. exhibited 46.62% inhibition against S. rolfsii in dual culture assay on PDA. The inhibition was found to be mediated by the mycotoxin, tenuazonic acid, produced by the endophyte. Pre-germinated chilli seeds treated with mycelial suspension of Alternaria sp. reduced the seedling mortality due to S. rolfsii infection. Pretreatment with the endophyte also enhanced seedling growth and biomass compared to untreated seedlings under greenhouse conditions. These results suggest that the endophytic fungus, Alternaria sp. has potential implication for use as a biocontrol against S. rolfsii as well as for promoting growth of chilli.

Molecular characterization and overexpression analyses of secologanin synthase to understand the regulation of camptothecin biosynthesis in Nothapodytes nimmoniana (Graham.) Mabb.

Camptothecin is a high-value anti-cancerous compound produced in many taxonomically unrelated species. Its biosynthesis involves a complex network of pathways and a diverse array of intermediates. Here, we report the functional characterization and regulation of secologanin synthase (NnCYP72A1), a cytochrome P450 involved in camptothecin biosynthesis from Nothapodytes nimmoniana. It comprises an open reading frame of 1566bp in length. Heterologous expression in Saccharomyces cerevisiae and in vitro enzymatic assays using loganin as substrate confirmed the formation of secologanin. In planta transient overexpression analysis of NnCYP72A1 resulted in 4.21- and 2.73-fold increase in transcript levels of NnCYP72A1 on days 3 and 6 respectively. Phytochemical analysis of transformed tissues revealed ~ 1.13-1.43- and 2.02-2.86-fold increase in secologanin and CPT accumulation, respectively. Furthermore, promoter analysis of NnCYP72A1 resulted in the identification of several potential cis-regulatory elements corresponding to different stress-related components. Methyl jasmonate, salicylic acid, and wounding treatments resulted in considerable modulation of mRNA transcripts of NnCYP72A1 gene. Chemical analysis of elicitor-treated samples showed a significant increase in CPT content which was concordant with the mRNA transcript levels. Overall, the functional characterization and overexpression of NnCYP72A1 may plausibly enhance the pathway intermediates and serve as prognostic tool for enhancing CPT accumulation.