Nothapodytes Nimmoniana Research Articles

Camptothecine production by mixed fermentation of two endophytic fungi from Nothapodytes nimmoniana

Two endophytic fungi isolated from the endangered plant Nothapodytes nimmoniana (Grah.) Mabb. were found to effectively synthesize CPT independent of their host plant under submerged fermentation conditions. Molecular characterization of fungi revealed their identity as Colletotrichum fructicola SUK1 (F1) and Corynespora cassiicola SUK2 (F2). Mixed fermentation experiments were carried out to study the effect of microbial signalling between the two fungal species on camptothecine production. Effect of culture parameters on CPT production was studied for both mono-cultures (F1 and F2) separately as well as for the mixed fermentation (F1 + F2). Further the most influencing ones were optimized statistically using response surface methodology. Statistical model based optimized parameters were whey (70 %), agitation rate (110 rpm), temperature (30 °C), and incubation period (7 d) for the mixed fermentation. Monocultures of the two fungal species F1 and F2 yielded CPT up to 33 ± 1.1 mg l−1and 69 ± 1.1 mg l−1, respectively; while their mixed fermentation under the optimized conditions yielded up to 146 ± 0.2 mg l−1. HPLC and LC-MS/MS techniques were used to analyze the products obtained.

Transcriptome analysis of stem wood of Nothapodytes nimmoniana (Graham) Mabb. identifies genes associated with biosynthesis of camptothecin, an anti-carcinogenic molecule.

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of DNA topoisomerase I and has applications in treating ovarian, small lung and refractory ovarian cancers. Stem wood tissue of Nothapodytes nimmoniana (Graham) Mabb. (family Icacinaceae) is one of the richest sources of CPT. Since there is no genomic or transcriptome data available for the species, the present work sequenced and analysed transcriptome of stem wood tissue on an Illumina platform. From a total of 77,55,978 reads, 9,187 transcripts were assembled with an average length of 255 bp. Functional annotation and categorization of these assembled transcripts unraveled the transcriptome architecture and also a total of 13 genes associated with CPT biosynthetic pathway were identified in the stem wood tissue. Four genes of the pathway were cloned to full length by RACE to validate the transcriptome data. Expression analysis of 13 genes associated with CPT biosynthetic pathway in 11 different tissues vis-a-vis CPT content analysis suggested an important role of NnPG10H, NnPSLS and NnPSTR genes in the biosynthesis of CPT. These results indicated that CPT might be synthesized in the leaves and then perhaps exported to stem wood tissue for storage.

Establishment and standardization of in vitro regeneration protocol in Nothapodytes nimmoniana Graham and evaluation of camptothecine (CPT) in tissue culture plants

Nothapodytes nimmoniana Graham is an important medicinal tree species occurring in the Western Ghats, a mega diversity hotspot in Southern India. The inner stem bark of the tree contains an important anti-cancer alkaloid, camptothecine (CTP), for which the natural population of the trees is heavily extracted. In this paper, we report the development of a rapid, high frequency regeneration protocol from leaf and nodal explants of N. nimmoniana. Multiple shoot induction was carried out using leaf and nodal explants on Murashige and Skoog (MS) medium supplemented with different concentration/combination of phyto-hormones. N6-benzyladenine (BA) was the most effective cytokinin for the induction of multiple shoots. The MS medium with 8.87 µM BA yielded the highest number of shoots from leaf and nodal explants, respectively. Further, proliferation and elongation of adventitious buds were observed in secondary medium containing MS supplemented with 4.44 µM BA and 0.87 µM gibberellic acid (GA3). Shoots were rooted on half strength MS medium containing 4.9 µM indole-3-butyric acid. The plantlets were acclimatized in a growth chamber at 25 °C, 60 % relative humidity, with 16/8 h light/dark photoperiod. Regenerated plants were free of any noticeable phenotypic variability and showed a survival rate of 90 %. The in vitro regenerated plants accumulated substantial amount of camptothecine (ranging from 0.08 to 0.2 %). These results suggest the possibility of using in vitro regenerated plants as a possible alternative source of CPT. This is the first report of direct regeneration in N. nimmoniana with significantly high plant regeneration frequency and with high CPT yield.

Antimicrobial and Antioxidant Activities of Tribal Medicine formulation (TMF) accomplished for Wound related remedies in Biligirirangana Hill area of Chamarajanagara district, Karnataka (India)

In the exploration, association of five important less known ethno-medicinal plants (EMP) which are practiced by Tribal Medicine Men (TMM) at Biligirirangana Hill area of Chamarajanagara district, Karnataka, India were identified. The plant drugs, Andrographis serphyllifolia, Discorea hispida , Glycosmis mauritiana, Nothapodytes nimmoniana and Rauvolfia densiflora were collected during interaction and the data on TMF drug such as, part used, mode of drug preparation, dosage and duration of the treatments essentially for wound therapeutics was analytically described. The contents and amount of EMP present in the formulations were validated scientifically to ascertain the findings of pharmacognostic parameters. Further, studies on biological activities in the vein of Antimicrobial and Antioxidant activities were attempted. The antimicrobial activity was tested against standard strains such as Escherichia coli , Bacillus subtilis , Staphylococcus aureus, Kliebsiella pneumonia and Pseudomonas aerugenosa using disc diffusion method. All the extracts have shown significant activity against tested microbes with the inhibition zones ranged 7-14mm to 15-20mm. Among various solvent extracts studied petroleum ether extract showed a highest antibacterial activity followed by ethanol, aqueous extracts of EMP and TMF. The antioxidant activity has been evaluated by DPPH radical scavenging and ABTS methods, respectively. The ethno-medicinal plants showed potent inhibition of DPPH free radicals scavenging activity, D. hispida , G. mauritiana, A. serphyllifolia are being the most potent compared to other R. densiflora and N. nimmoniana . All the EMP drugs registered significantly higher in phenolic content, individually (107.67mg GAE/g) than that of TMF (102.45 mg GAE/g). Similarly, antioxidant potential was found to be noteworthy as determined by DPPH (12.24 EC 50 g/ml) radical scavenging activity which was registered in the EMF individually. A superior ABTS radical scavenging (ranging between12.06-15.40 EC 50 g/ml), Frap assay (ranging between 8.25-21.26mol Fe 2+ /mg) and significantly higher total reduction capacity (1.28%) were realized in ethanolic extract of TMF.

Restoration of camptothecine production in attenuated endophytic fungus on re-inoculation into host plant and treatment with DNA methyltransferase inhibitor.

Fungal endophytes inhabit living tissues of plants without any apparent symptoms and in many cases are known to produce secondary metabolites similar to those produced by their respective host plants. However on sub-culture, the endophytic fungi gradually attenuate their ability to produce the metabolites. Attenuation has been a major constraint in realizing the potential of endophytic fungi as an alternative source of plant secondary metabolites. In this study, we report attempts to restore camptothecine (CPT) production in attenuated endophytic fungi isolated from CPT producing plants, Nothapodytes nimmoniana and Miquelia dentata when they are passed through their host plant or plants that produce CPT and when treated with a DNA methyl transferase inhibitor. Attenuated endophytic fungi that traversed through their host tissue or plants capable of synthesizing CPT, produced significantly higher CPT compared to the attenuated fungi. Attenuated fungus cultured in the presence of 5-azacytidine, a DNA methyltransferase inhibitor, had an enhanced CPT content compared to untreated attenuated fungus. These results indicate that the attenuation of CPT production in endophytic fungi could in principle be reversed by eliciting some signals from plant tissue, most likely that which prevents the methylation or silencing of the genes responsible for CPT biosynthesis.

Camptothecin from Nothapodytes nimmoniana: review on biotechnology applications

Since the first report on Camptothecin detection in Nothapodytes nimmoniana by Govindachari and Viswanathan (Phytochem 11:35–29, 1972), considerable work has been done on biotechnology and its applications on the species. Plant tissue culture techniques have applications in clonal propagation, CPT production, and conservation of N. nimmoniana. Discovery of CPT production by endophytes existing in symbiotic association with N. nimmoniana has provided new insights into finding alternative sources of the alkaloid. Development of molecular markers such as RFLP, RAPD, ISSR, and AFLP has facilitated understanding of population ecology and genetics of the species. Molecular information generated from these studies is promising in establishing strategies for conservation and sustainable use of N. nimmoniana populations under overexploitation pressure. The advances in instrumentation in the 20th century, such as desorption electrospray ionization mass spectrometry allowed CPT analysis in tissues without sample pretreatment. Other ancient techniques for qualitative and quantitative analysis such as chromatography, spectroscopy, and H1-NMR are applied in the detection of CPT due to variable sensitivity to the alkaloid. The review covers work on plant tissue culture for clonal propagation and CPT production in N. nimmoniana. Besides symbiotic endophyte sources of CPT in N. nimmoniana, population genetics studies and instrumentation analysis of the alkaloids are reviewed.

Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations

DNA topoisomerase I (Top1) catalyzes changes in DNA topology by cleaving and rejoining one strand of the double stranded (ds)DNA. Eukaryotic Top1s are the cellular target of the plant-derived anticancer indole alkaloid camptothecin (CPT), which reversibly stabilizes the Top1-dsDNA complex. However, CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila and Ophiorrhiza liukiuensis, are highly resistant to CPT because they possess point-mutated Top1. Here, the adaptive convergent evolution is reported between CPT production ability and mutations in their Top1, as a universal resistance mechanism found in all tested CPT-producing plants. This includes Nothapodytes nimmoniana, one of the major sources of CPT. To obtain a structural insight of the resistance mechanism, molecular dynamics simulations of CPT– resistant and –sensitive plant Top1s complexed with dsDNA and topotecan (a CPT derivative) were performed, these being compared to that for the CPT-sensitive human Top1. As a result, two mutations, Val617Gly and Asp710Gly, were identified in O. pumila Top1 and C. acuminata Top1, respectively. The substitutions at these two positions, surprisingly, are the same as those found in a CPT derivative-resistant human colon adenocarcinoma cell line. The results also demonstrated an increased linker flexibility of the CPT–resistant Top1, providing an additional explanation for the resistance mechanism found in CPT-producing plants. These mutations could reflect the long evolutionary adaptation of CPT-producing plant Top1s to confer a higher degree of resistance.

Enhanced in vitro Seedling Recovery in Nothapodytes nimmoniana (J. Graham) Mabberly

Nothapodytes nimmoniana is an endangered medicinal tree and regarded most convenient natural source for large scale isolation of anticancer alkaloid camptothecin. In order to meet pharmaceutical market demand for camptothecin and conserve natural population of the species, in vitro culture techniques can be employed for mass propagation and production of the alkaloid. Seed germination is one of the difficult challenges in the establishment of the aseptic in vitro cultures. Pre-germination treatments of soaking in water, GA3 solution and aseptic excision of the embryo axis were employed to enhance seed germination and seedling recovery respectively for further in vitro studies. Seed soaking in water or GA3 solution for 24 hours enhanced the germination capacity with better response obtained when seeds were soaked in GA3 (2.6 μM) solution and germinated on half strength MS medium supplemented with the GA3. Removal of the outer, inner seed coat and inoculation of the embryo axis on medium amended with or without GA3 reduced seedling recovery time to 3-4 weeks. Seedlings obtained on medium fortified with GA3 showed vigorous growth with complete plantlet development than on PGR-free medium. The strategy can be employed to reduce seedling recovery time to within 3-4 weeks compared to 7-10 weeks required for germinating the seeds in vitro. Original Research Article Isah and Mujib; BBJ, 6(1): 35-42, 2015; Article no.BBJ.2015.025 36

Evaluating Nothapodytes nimmoniana population from three localities of Western Ghats using camptothecin as phytochemical marker and selection of elites using a new-content range chart method.

Background:Nothapodytes nimmoniana (Grah.) Mabb. is a high valued medicinal plant endemic to Western Ghats of India, distributed in fragmented populations. The plant is valued for potent anticancer drug camptothecin (CPT).Objective:The study compares and expounds variation in CPT content from leaves and stems of N. nimmoniana obtained from three populations of Western Ghats, India. The study also describes a method for categorizing these populations using content range chart (CRC) method for percent yield of CPT.Materials and Methods:A total of 60 samples were investigated including ten each of leaves and stems from three localities. Micro-extraction method was implemented to extract CPT. reversed phase ultra-performance liquid chromatography photo diode array technique was used to quantify CPT.Results:Leaf samples of an individual collected from Joida, yielded lowest CPT content (0.002 ± 0.000 g/100 g), whereas a stem sample from Amgaon, yielded highest CPT content (0.123 ± 0.006 g/100 g). The findings suggest great variation in individuals producing and accumulating CPT. Using this data along with earlier published work, five categories of CPT yielding plants were made viz. I: Very low: <0.020, II: Low: 0.021-0.039, III: Moderate: 0.040-0.059, IV: High: 0.060-0.079 and V: Very high: >0.080. Based on CPT content in leaves, majority of individuals were under very low category (Ist) and on the other hand stem samples were in ‘II’ category. Besides, very few individuals were observed in category ‘V’.Conclusion:The study expounds use of CRC method for identifying elite population and suggests the need for its conservation.

In vitro propagation and camptothecin production in Nothapodytes nimmoniana

Nothapodytes nimmoniana is an endemic endangered tree and convenient source for large scale isolation of anticancer alkaloid camptothecin. Due to indiscriminate collection of its wood chips for camptothecin, population of the species is under heavy exploitation pressure in India. Plant tissue culture techniques offer alternative strategies for clonal propagation and camptothecin production to meet demand of the alkaloid along with conservation of the species. Several strategies are employed to enhance in vitro response through seed germination, culture medium optimisation for camptothecin production and clonal propagation. Cryopreservation of embryonic axes is an approach employed in short term preservation of the species. Scale up for the production of camptothecin from N. nimmoniana in bioreactor is a future possibility to meet pharmaceutical market demand and mitigate exploitation of natural population. In this article, we reviewed progress made so far on clonal propagation and camptothecin production in N. nimmoniana. We assessed the challenges and status of camptothecin production in N. nimmoniana by application of in vitro techniques.

Genetic Structure, Diversity and Long Term Viability of a Medicinal Plant, Nothapodytes nimmoniana Graham. (Icacinaceae), in Protected and Non-Protected Areas in the Western Ghats Biodiversity Hotspot.

Background and QuestionThe harvesting of medicinal plants from wild sources is escalating in many parts of the world, compromising the long-term survival of natural populations of medicinally important plants and sustainability of sources of raw material to meet pharmaceutical industry needs. Although protected areas are considered to play a central role in conservation of plant genetic resources, the effectiveness of protected areas for maintaining medicinal plant populations subject to intense harvesting pressure remain largely unknown. We conducted genetic and demographic studies of Nothapodytes nimmoniana Graham, one of the extensively harvested medicinal plant species in the Western Ghats biodiversity hotspot, India to assess the effectiveness of protected areas in long-term maintenance of economically important plant species.Methodology/Principal FindingsThe analysis of adults and seedlings of N. nimmoniana in four protected and four non-protected areas using 7 nuclear microsatellite loci revealed that populations that are distributed within protected areas are subject to lower levels of harvesting and maintain higher genetic diversity (He = 0.816, Ho = 0.607, A = 18.857) than populations in adjoining non-protected areas (He = 0.781, Ho = 0.511, A = 15.571). Furthermore, seedlings in protected areas had significantly higher observed heterozygosity (Ho = 0.630) and private alleles as compared to seedlings in adjoining non-protected areas (Ho = 0.426). Most populations revealed signatures of recent genetic bottleneck. The prediction of long-term maintenance of genetic diversity using BOTTLESIM indicated that current population sizes of the species are not sufficient to maintain 90% of present genetic diversity for next 100 years.Conclusions/SignificanceOverall, these results highlight the need for establishing more protected areas encompassing a large number of adult plants in the Western Ghats to conserve genetic diversity of economically and medicinally important plant species.

Seed dormancy and germination of the three tropical medicinal species Gomphandra luzoniensis (Stemonuraceae), Nothapodytes nimmoniana (Icacinaceae) and Goniothalamus amuyon (Annonaceae)

The three tropical species Gomphandra luzoniensis, Nothapodytes nimmoniana and Goniothalamus amuyon contain important cancer-fighting drugs; however, little is known about how to propagate these species from seeds. Thus, the aim of this study was to determine the germination requirements of seeds of each of these three species in order to provide an effective protocol to produce plants. Fresh seeds of G. luzoniensis germinated up to 73% at high temperatures in light in 4 weeks, and embryos were underdeveloped. Most seeds had morphological dormancy (MD), but a proportion of them had morphophysiological dormancy (MPD). Fresh seeds of N. nimmoniana germinated up to 50% in light in 4 weeks, embryo length increased by 17% before radicle emergence and ≥ 89% of the seeds had germinated after incubation for 6 weeks in light at high temperatures. Thus, about 50% of the seeds have MD and about 50% MPD. Fresh seeds of G. amuyon incubated at 30°C in light for 4 weeks germinated to 69%, whereas at the other incubation temperatures germination took longer than 4 weeks. Embryo length increased 213% before radicle emergence, and after 8 weeks of incubation at high temperatures ≥ 80% of the seeds had germinated. As in the other two species, the seed population consisted of a mixture of MD and MPD. Incubation of seeds of these three species at high temperatures (e.g. 25, 30 and 30/20°C) for up to 2 months is recommended for germination and thus seedling production.

Inhibition of Fungal Endophytes by Camptothecine Produced by their Host Plant, Nothapodytes nimmoniana (Grahm) Mabb. (Icacinaceae)

Camptothecine (CPT), a monoterpene indole alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. It is produced by a number of plants, including Notha-podytes nimmoniana (Grahm) Mabb. (Icacinaceae), occurring naturally in the Western Ghats, India. The plant is inhabited by a number of endophytic fungi, many of which have been isolated and shown to pro-duce CPT, in culture, independent of the host. In this article, we examine the sensitivity of endophytic fungi isolated from N. nimmoniana to CPT. Contrary to our hypothesis that these fungi should be resistant to CPT (as they are exposed to host CPT as well as that pro-duced by themselves), we report that these fungi are sensitive and thus inhibited by CPT. We discuss these results in the context of the role of CPT in limiting endophytic fungal growth.

English

The ethno botanical investigation of medicinal plants used by the Kurichia, Kuruma, Kattunaika, Adiya and Paniya tribes of Wayanad district, Kerala were recorded. One thousand (1000) ethno medicines derived from 500 plants used by the tribal medical practitioners were documented. Of this, 10 species were frequently used for treating various infectious diseases. An informant consensus factor was calculated for 10 species which are being frequently used. The leaf extracts of these plants were screened for antimicrobial activity against Escherichia coli, Staphylococcus aureus and Aspergillus niger. Among the plants screened Garcinia gummi gutta (Clusiaceae) was found to possess highly significant antibacterial activity and significant antifungal activity was observed in Nothapodytes nimmoniana (Icacinaceae) which possess highest antifungal activity. These two species have shown the highest informant consensus factor values. &nbsp; Key words: Medicinal plants, informant consensus factor, antibacterial activity, antifungal activity, ethno botany.

Composition and tissue specificity of endophytic fungi associated withNothapodytes nimmoniana(J. Graham) Mabberly from the Western Ghats of India

Western Ghats in India is one of the global biodiversity hot spots of the world. The objective of this study was to isolate, recognize the composition and tissue specificity of endophytic mycobiota in healthy disease free parts of an important anticancer medicinal plant, Nothapodytes nimmoniana (J. Graham) Mabberly from the forest in Amboli. Different plant parts (fresh disease free stem, leaves, leaf petiole, flowers and pedicel) were subjected to ‘3-step surface sterilization’ technique which resulted in the recovery of 192 isolates of fungi from 280 segments comprising 80 stem tissues, 120 leafy tissues, 40 leaf petiole tissues, 20 flower tissues and 20 pedicel issues segments, with infection rates of 56.25, 62.5, 50, 180 and 80 percent, respectively. The most frequent fungi were Cladosporium oxysporum, Corynespora sp., Fusarium monoliforme, one unidentified pycnidial fungus and two non sporulating mycelia (NSM 1 and 2). The composition and abundance of the endophytes varied according to the host tissues tested and was found to be dependent on the tissue type. Some fungi were specific to the various parts/tissues of the plant. They were segregated on the basis of their occurrence viz; leaf (midrib, margin, laminar), stem (outer bark, middle cortical and innermost core region), petiole, flower and pedicel.

Seasonal variation in content of camptothecin from the bark of Nothapodytes nimmoniana (Grah.) Mabb., using HPLC analysis

Objective:To study and compare seasonal variation in camptothecin (CPT) content from bark samples of Nothapodytes nimmoniana obtained from geographically and climatologically isolated populations.Methods:A standard High Performance Liquid Chromatography methodology was used to analyze and quantify CPT from bark samples of N. nimmoniana.Results:Sample collected from Amboli yielded highest CPT content 1.337 g/100 g dry bark powder during the monsoon compared to other localities in study. Monsoon (August) showed to accumulate higher levels of CPT in barks of N. nimmoniana as compared to summer (May). Amboli averaged highest accumulation of CPT compared to other localities under study.Conclusion:These findings indicate season to have control over accumulation of CPT. Locality Amboli has highest CPT content in all seasons and were the elite population during the study. The study also suggests the need for further investigation in lights of biosynthesis in the plant.

Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

The effect of culture medium nutrients on growth and alkaloid production by plant cell cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae) was studied with a view to increasing the production of the alkaloid camptothecin, a key therapeutic drug used for its anticancer properties. Amongst the various sugars tested with Murashige and Skoog (MS) medium, such as glucose, fructose, maltose, and sucrose, maximum accumulation of camptothecin was observed with sucrose. High nitrate in the media supports the biomass, while high ammonium enhances the camptothecin content. Selective feeding of 60 mM total nitrogen with a NH4+/NO3− balance of 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over the control culture (28.5 μg/g DW). Furthermore, the sucrose feeding strategy greatly stimulated cell biomass and camptothecin production. A modified MS medium was developed in the present study, which contained 0.5 mM phosphate, a nitrogen source feeding ratio of 50/10 mM NH4+/NO3− and 3 % sucrose with additional 2 % sucrose feeding (added on day 12 of the cell culture cycle) with 10.74 μM naphthaleneacetic acid and 0.93 μM kinetin. Finally, the selective medium has 1.7- and 2.3-fold higher intracellular and extracellular camptothecin content over the control culture (29.2 and 8.2 μg/g DW), respectively.

Soil microbial community structure and microbial activities in the root zone of Nothapodytes nimmoniana

Camptothecin (CPT) is a bioactive secondary metabolite of Nothapodytes nimmoniana (Graham) Mablerley and has strong antitumor activity due to its ability to inhibit DNA topoisomerase I. In this study the changes of soil chemical and microbiological properties in the root zone of N. nimmoniana under application of two different nitrogen (N) fertilizers with different rates were investigated. There were seven treatments consisting of four rates of chemical N fertilizer, two rates of organic fertilizer, and a control where there was no fertilization and planting. Two-year-old N. nimmoniana plants were used in the study. Ten months later the plants were harvested and the soils were collected for analyses of chemical properties, microbial carbon (MBC), microbial nitrogen (MBN), respiration rate (basal respiration), phospholipid fatty acids of microorganisms, and six soil enzyme activities. Results showed that higher rates of N fertilizer resulted in greater dry matter yield of N. nimmoniana, while N rates had no significant effect on the CPT concentrations of roots and the whole plants except for lower CPT concentration in the N4 (applying 10.0 g N per pot in total) treatment. There was no difference in chemical properties, MBC, MBN, respiration rate, and N mineralization rate of soils among chemical N fertilizer treatments. N. nimmoniana decreased the soil microbial communities as compared with the control. Compared with bacteria and actinomyctes, fungi were more affected by N. nimmoniana cultivation. In contrast, N. nimmoniana had no effect on the activities of soil enzymes. Different rates of chemical N fertilizer had the same effect on the microbial communities. However, the application of organic fertilizer demonstrated a significant amelioration on the adverse effect of the soil microbial communities resulting from the cultivation of N. nimmoniana.

Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semisolid and liquid cultures and estimation of camptothecin in the regenerated plants

Nothapodytes nimmoniana (Icacinaceae) yields camptothecin (isoquinoline alkaloid) which is a potent anti-cancer drug. The major objectives of the present study were to develop an efficient protocol for mass propagation of N. nimmoniana using liquid medium and to compare regeneration with semisolid cultures; as also to quantify the amount of camptothecin in regenerated plants. Adventitious shoots were induced from the callus derived from nodal explants on semisolid and liquid Murashige and Skoog (MS) medium supplemented with 1.0, 2.0, 5.0 and 10.0 μM 6-benzylaminopurine or kinetin or 2-isopentenyl adenine (2-iP). The highest number of adventitious shoots was regenerated on medium supplemented with 2.0 μM BAP. Compared to semisolid medium (41.9 shoots per explant), liquid medium (165.9 shoots per explant) was found suitable for shoot induction and shoot multiplication. Shoots were rooted on MS semisolid medium of one-fourth strength containing IBA (2.4 μM) and IAA (5.7 μM). The plantlets were acclimatized in a growth chamber at 25°C, 60% relative humidity, with 16-h photoperiod (40 μmol m−2 s−1). The camptothecin content was determined in ex vitro plants using HPLC. The analysis revealed that the leaves and stems of ex vitro plants had a considerable amount of camptothecin and these plants could be used as a raw material for camptothecin extraction.

A simple, rapid and sensitive spectrofluorimetric method for the determination of camptothecin

Camptothecin (CPT) represents a clinically useful class of anticancer agent. Proper identification and quantitation of the CPT in the plant extracts and in-vitro cell culture extracts is fundamental to assess the CPT content and its biosynthetic potential in plants. A simple, sensitive and rapid, spectrofluorimetric method has been developed and validated for the quantitative estimation of camptothecin. The method was validated in terms of linearity (2–20 ng/ml), precision (intra-day variation below 0.15, inter-day variation below 1.2), and accuracy (98.0 to 100.2%). The limit of detection and limit of quantification for CPT were found to be 0.10 ng/ml and 0.36 ng/ml, respectively. The developed spectrofluorimetric method provides a rapid and cost effective method for the routine analysis of CPT in plant extracts and tissue culture samples. The developed method was successfully used for the estimation of CPT in natural plant extracts and cell culture extracts. The Nothapodytes nimmoniana callus cells having nearly 3-fold higher CPT content over the leaf (0.005%) explant of the plant. The highest CPT content was found in the stem part (0.092%) followed by the fruit (0.088%). The method is simple, sensitive and precise; it can be used for the routine quality control testing of formulations containing CPT.