Camptothecin Production Research Articles

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.

Optimization and Kinetic Modeling of Cell-Associated Camptothecin Production from an Endophytic Fusarium oxysporum NFX06

The production of cell-associated camptothecin (CPT) from an endophytic fungus Fusarium oxysporum NFX06 isolated from Nothapodytes foetida and its kinetics studies were proposed. Response surface methodology (RSM) based on central composite design (CCD) was used to construct a model to describe the effects of substrate concentration. Three independent variables (dextrose, peptone, and MgSO4) were successfully employed to study the yield of CPT under submerged fermentation. The maximum yield of CPT obtained from CCD was about 598.0 ng/g biomass. The model-validated optimum predicted CPT yield and experimental CPT yield from the biomass were found to be 628.08 ng/g and 610.09 ng/g at the concentrations of dextrose 42.64 (g/L), peptone 9.23 (g/L), and MgSO4 0.26 (g/L) respectively. The predicted yield of CPT was 4.90% higher than the value obtained from CCD and 2.85% higher than the value obtained from experiment conducted at optimum conditions. The kinetic parameters, maximum specific growth rate μ max = 1.212 day−1, growth-associated CPT production coefficient (α = 29.35 ng/g biomass), and non-growth-associated CPT production coefficient (β = 0.03 ng CPT/g biomass-day) were obtained. The logistic model was found suitable to predict mycelial growth with a high determination coefficient (R 2). Luedeking–Piret and modified Luedeking–Piret models were employed to represent the product kinetics and substrate consumption kinetics. A good concurrence was found between the experimental and predicted values, representing that the unstructured models were able to illustrate the fermentation profile effectively.

Dependence of UV-B-induced camptothecin production on nitrate reductase-mediated nitric oxide signaling in Camptotheca acuminata suspension cell cultures

UV-B irradiation induced production of secondary metabolites in plant cells. However, the mechanisms of UV-B-induced secondary metabolite production remained largely unknown. Here we report that UV-B treatment stimulated nitric oxide (NO) generation and camptothecin (CPT) production in Camptotheca acuminata cells. To investigate the origin of the UV-B-triggered NO and the role of NO in UV-B-induced CPT production, we assayed the responses of nitrate reductase (NR) and NO synthase (NOS) activities of the cells to UV-B exposure and examined the effects of NR and NOS inhibitors on CPT production in UV-B-treated cells. The data showed that UV-B irradiation enhanced NR activities in the cells. Pretreatment with NR inhibitors tungstate and okadaic acid not only suppressed the UV-B-triggered NR activities but also inhibited the UV-B-induced NO generation and CPT production in the cells. In contrast, UV-B irradiation had no effects on NOS activity of the cells and treatment of NOS inhibitor did not suppress UV-B-induced CAT production. Together, the results demonstrated that NR activity was essential for UV-B-triggered NO generation and that NR-mediated NO signaling was involved in UV-B-induced CPT production in C. acuminata cells.

High-frequency Plant Regeneration and Accumulation of the Anticancer Alkaloid Camptothecin in Ophiorrhiza rugosa var. decumbens Deb & Mondal

An efficient protocol for obtaining high-frequency regeneration and camptothecin production from Ophiorrhiza rugosa var. decumbens (Rubiaceae) was developed. Leaves, stem, petiole, and root explants from in vitro grown plants of O. decumbens were selected, cultured on Murashige and Skoog's (MS) basal medium supplemented with benzyl adenine (5 mg.L−1) and indole acetic acid (0.1 mg.L−1). Root explants produced maximum shoot regeneration. The regenerated shoots were rooted on MS medium containing α- naphthalene acetic acid (0.1 mg.L−1) and transferred to soil. The regenerated and potted plants indicated the accumulation of camptothecin. Whole regenerated plant of O. decumbens grown in a pot for 1 month showed maximum camptothecin (0.132% d.w) compared to the regenerated shoots from other explants (0.013-0.020 % d.w).

Camptothecin-producing endophytic fungus Trichoderma atroviride LY357: isolation, identification, and fermentation conditions optimization for camptothecin production

Camptothecin (CPT), the third largest anticancer drug, is produced mainly by Camptotheca acuminata and Nothapodytes foetida. CPT itself is the starting material for clinical CPT-type drugs, but the plant-derived CPT cannot support the heavy demand from the global market. Research efforts have been made to identify novel sources for CPT. In this study, three CPT-producing endophytic fungi, Aspergillus sp. LY341, Aspergillus sp. LY355, and Trichoderma atroviride LY357, were isolated and identified from C. acuminata. Most CPT produced by these fungi was found in the fermentation broth, and their corresponding CPT yields were 7.93, 42.92, and 197.82 μg l(-1), respectively. The CPT-producing capability of LY341 and LY355 was completely lost after repeat subculturing. A substantial decrease of CPT production was also observed in the second generation of LY357. However, a stable and sustainable production of CPT was found from the second generation through the eighth generation of LY357. The fermentation medium, time, pH, temperature, and agitation rate were optimized for CPT production. Methyl jasmonate and XAD16 were proven to be an optimum elicitor and adsorbent resin, respectively, in view of that CPT yield was increased 3.4- and 11-fold through their use. A 50- to 75-fold increase of CPT yield was obtained when the optimized fermentation conditions, elicitor, and adsorbent resin were combined and applied to the culture of the seventh and eighth generations of LY357, and the highest CPT yield was 142.15 μg l(-1). The CPT-producing T. atroviride LY357 paves a potential to uncover the mysteries of CPT biosynthesis.

New and Highly Efficient Column Chromatographic Extraction and Simple Purification of Camptothecin from Camptotheca acuminata and Nothapodytes pittosporoides

Camptothecin, a widely used natural anti-cancer drug, is difficult to extract and purify effectively from plants. To develop new and highly efficient extraction and purification methods for analysis and production of camptothecin from leaves and fruits of Camptotheca acuminata and Nothapodytes pittosporoides roots. Dried materials were loaded in empty columns with fivefold 60% ethanol for leaves or 70% ethanol for fruits of C. acumnata, and sixfold 70% ethanol for N. pittosporoides roots. The columns were eluted with the same solvents at room temperature. Eluent was collected as extraction solution. Extraction solution from leaves and fruits of C. acuminata was vacuum-evaporated to remove ethanol, precipitated at pH 8.0 to remove alkaline insolubles and fractionated with chloroform at pH 3.0, which yields a crude product with 70% purity. Extraction solution from N. pittosporoides roots was concentrated to 1/10 volume and precipitated at pH 3.0, which yields a crude product with 60% purity. All crude products were purified by crystallisation. All steps were monitored by HPLC. Camptothecin was extracted from the three plant materials at a 98% rate with 15- or 18-fold solvent for content analysis, or at a 97% rate with five- or sixfold solvent for production. All crude products were purified to 98%. The overall recovery rates of camptothecin from plant materials to purified products reached 92% or higher. The new procedures are simple and highly efficient, and have multiple advantages for quantitative analysis and large production of camptothecin from plants.

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.

Effect of precursors feeding and media manipulation on production of novel anticancer pro-drug camptothecin from endophytic fungus

Effect of precursors feeding and media manipulation on production of novel anticancer pro-drug camptothecin from endophytic fungus

Suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila

Camptothecin is a monoterpenoid indole alkaloid that exhibits anti-tumor activity. In Ophiorrhiza pumila, production of camptothecin and its related alkaloids was high in the hairy roots, but not in the cell suspension culture derived from hairy roots. To identify the intermediates in camptothecin biosynthesis, expression of genes encoding tryptophan decarboxylase (TDC) and secologanin synthase (SLS), the two enzymes catalyzing the early steps in camptothecin biosynthesis, were suppressed in the hairy roots of O. pumila by RNA interference (RNAi), and metabolite changes were investigated. In most TDC- and SLS-suppressed lines, accumulation of camptothecin and related alkaloids, strictosidine, strictosamide, pumiloside, and deoxypumiloside was reduced. The accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS. Some hairy root-specific chromatographic peaks detected by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) exhibited positive or negative correlation with TDC expression, suggesting their possible involvement in camptothecin biosynthesis.

Effects of over-expression of allene oxide cyclase on camptothecin production by cell cultures of Camptotheca acuminata

Camptothecin (CPT) is an anticancer and antiviral monoterpene-derived indole alkaloid which can be induced by plant hormone, jasmonates. To improve the production of the pharmaceuticals, the jasmonate biosynthesis related gene allene oxide cyclase from Camptotheca acuminate was transferred back into C. acuminate using the method of Agrobacterium -mediated genetic transformation and over expressed. The results of high performance liquid chromatography (HPLC) analysis indicated that the camptothecin content of transgenic callus was higher than that of non-transgenic callus. The highest camptothecin content in transgenic callus was 3.9310 mg/g DW. However, camptothecin content in both transgenic and non-transgenic calli significantly decreased after further extrinsic methyl-jasmonate’s (MeJA's) induction, whereas the content of CPT in transgenic callus was still higher than in non-transgenic one. All the results indicate that endogenic jasmonate's accumulation may be promoted after allene oxide cyclase gene was transformed into C. acuminate and over expressed. In this way, jasmonates can affect second metabolism pathway genes' expression and then the camptothecin content improved. Inexpectantly, the mechanism of extrinsic jasmonate to secondary metabolism of C. acuminate was different from that of endogenic jasmonates. Key words : Agrobacterium tumefaciens , Allene oxide cyclase, Camptotheca acuminata, camptothecin, methyl-jasmonates, transformation.

Micropropagation and production of camptothecin form in vitro plants of Ophiorrhiza mungos

ObjectiveTo explore the biotechnological potential of Ophiorrhiza mungos for micropropagation and camptothecin (CPT) production from in vitro grown plants. MethodsSurface sterilized explants of O. mungos were transferred aseptically in MS media containing various combinations of phytohormones for callus initiation and multiple shoot proliferation. The content of CPT was quantified in the methanolic extract of O. mungos plants and in in vitro grown plants by using high performance liquid chromatography (HPLC). ResultsMaximum fresh weight and dry weight biomass of O. mungos callus was obtained from MS medium supplemented with IAA (2 ppm) + BAP (2 ppm) + GA (1 ppm). The maximum shoot proliferation (25) and elongation (6.5 cm) was found in MS medium supplemented with Picloram + Thidiazuron + Gibberellic Acid in 1:2:1 ratio after four weeks of incubation. The maximum content of CPT (0.0768 % w/w) was found in whole in vitro plants whereas the minimum CPT was observed in adventitious buds (0.0026 % w/w) as compared to the naturally grown O. mungos plants (0.0030% w/w). ConclusionsPresent findings indicate that O. mungos plants respond favourably for in vitro propagation and also produce higher amount of CPT as compared to naturally grown plants.

Effect of precursors feeding and media manipulation on production of novel anticancer pro-drug camptothecin from endophytic fungus.

We have established methodology for the isolation and characterization of a novel endophytic fungus from the inner bark of medicinal plant Nothapodytes foetida, which produced camptothecin in Sabouraud broth (SB) under shake flask conditions. Camptothecin and its related compounds are at present obtained by extraction from intact plants, but fungal endopytes may be an alternative source of production. In present study we have observed the effect of different nutrient combinations and precursors (tryptophan, tryptamine, geraniol, citral, mevalonic acid and leucine) on the accumulation of camptothecin by endophytic fungus Entrophospora infrequens. The precursors were fed either alone or in combinations (tryptophan and geraniol, tryptophan and citral, tryptophan and mevalonic acid, tryptophan and leucine). The highest camptothecin content was observed in the range of 503 ± 25µg/100g dry cell mass in Sabouraud medium. Camptothecin content in the medium was increased by 2.5 folds by the presence of tryptophan and leucine whereas the production with trytophan was also significantly different from other treatments. Furthermore, the effect of fungal camptothecin on the morphology of human cancer cell lines was also studied. The treated cells showed reduction in size, condensation of nucleus and the protoplasmic extensions were reduced. All these characteristics are found in apoptotic cells.

Production of camptothecin by hairy roots and regenerated transformed shoots of Ophiorrhiza rugosa var. decumbens

Camptothecin (CPT), the derivatives of which are used clinically for the treatment of metastatic colon cancer, is isolated from intact plants that can be subjected to environmental fluctuations. In vitro cultures may be an alternate and continuous source for year-round production of CPT. Since CPT production by undifferentiated cell cultures is low, differentiated tissues such as root cultures may be a viable alternate source for CPT production. Hairy roots were induced in Ophiorriza rugosa, a source of CPT, using Agrobacterium rhizogenes strain LBA9402. The hairy roots, when cultured in light, showed spontaneous regeneration of shoots. Analysis of CPT levels in the hairy roots and in vitro-grown transformed shoots revealed 0.009% d.w. and 0.012% d.w., respectively.

Micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin production

An efficient system was developed for the in vitro micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin (CPT) production. Shoot multiplication on leaf and node explants from germinated seeds of O. alata was successful on half-strength Murashige and Skoog medium supplemented with varying amounts of kinetin and α-naphthaleneacetic acid. Node explants grown in vitro were successfully infected by Agrobacterium rhizogenes TISTR 1450 for the establishment of hairy root culture. The amount of CPT in various parts of O. alata was analyzed by HPLC. The accumulation of CPT in transformed hairy roots was twice that in soil-grown plants (785 ± 52 and 388 ± 32 μg/g dry wt, respectively). In the presence of a polystyrene resin (Diaion HP-20) that absorbed CPT, the CPT content in the culture media increased sevenfold compared with controls (1,036 and 151 μg per 250 ml medium, respectively). These results enable the feasible production of CPT of O. alata by means of a cell culture strategy. These measures can help safeguard the plant from extinction.

Nitrate reductase-mediated nitric oxide generation is essential for fungal elicitor-induced camptothecin accumulation of Camptotheca acuminata suspension cell cultures

Secondary metabolite accumulation and nitric oxide (NO) generation are two common responses of plant cells to fungal elicitors, and NO has been reported to play important roles in elicitor-induced secondary metabolite production. However, the source of elicitor-triggered NO generation in plant cells remains largely unknown. To investigate the origin of elicitor-triggered NO, we examined nitrate reductase (NR) activities and the expression levels of NIA1 and NIA2 genes of Camptotheca acuminata cells treated with PB90, a protein elicitor from Phytophthora boehmeriae. The data show that PB90 treatment stimulates NR activity and induces upregulation of NIA1 but does not affect NIA2 expression in the cells. Pretreatment of the cells with NR inhibitors tungstate and Gln abolishes not only the fungal elicitor-triggered NR activities but also the PB90-induced NO generation. Treatment of PB90 enhances camptothecin contents of the cells, suggesting that the fungal elicitor might stimulate camptothecin biosynthesis. Furthermore, application of tungstate and Gln suppresses the fungal elicitor-induced camptothecin accumulation of the cells and the suppression of NR inhibitors on PB90-induced camptothecin production can be reversed by NO via its donor sodium nitroprusside. Together, the results suggest that NIA1 is sensitive to PB90 and the fungal elicitor-induced upregulation of NIA1 may lead to higher NR activity. Furthermore, our data demonstrate that NR is involved in the fungal elicitor-triggered NO generation and the fungal elicitor induces camptothecin production of C. acuminata cells dependently on NR-mediated NO generation.

Correlation of Camptothecin-producing Ability and Phylogenetic Relationship in the GenusOphiorrhiza

Camptothecin (CPT) is an essential precursor of semisynthetic chemotherapeutic agents for cancers throughout the world. In spite of the rapid growth of market demand, CPT raw material is still harvested by extraction from Camptotheca acuminata and Nothapodytes foetida because its total synthesis is not cost-effective. In this study, we examined eight species of the genus Ophiorrhiza (Rubiaceae) from Thailand as novel alternative sources of CPT. CPT and/or 9-methoxy camptothecin (9-MCPT) were detected at different amounts in the leaf and root extracts of five species. We found that the CPT production ability of Ophiorrhiza spp. in Thailand was related mainly to species, not habitat. Chloroplast MATK and nuclear TOPI genes of eight species were investigated and compared with those of other Ophiorrhiza sequences from GenBank in order to classify and study the evolution in this genus. The molecular phylogenetic trees of both separated and combined MATK and TOPI nucleotide sequences revealed a major clade of Ophiorrhiza taxa correlated with production of CPT and its derivatives. Several amino acid markers of CPT- or 9-MCPT-producing Ophiorrhiza plants were also suggested from the alignment of TopI amino acid sequences. Our findings suggest that genetic factors play an important role in determining the CPT- and 9-MCPT-producing properties of Ophiorrhiza plants. Consequently, MATK and TOPI gene sequences could be utilized for the prediction of CPT and 9-MCPT production ability of members of Ophiorrhiza.

Production of camptothecin in cultures of Chonemorpha grandiflora.

Background:Chonemorpha grandiflora (Syn. Chonemorpha fragrans (Apocynaceae) is an endangered medicinal plant. It is used in different preparations, such as sudarsanasavam and kumaryasavam used in Kerala Ayurvedic system. C. grandiflora is used for the treatment of fever and stomach disorders. Phytochemical investigations have revealed the presence of steroidal alkaloids, such as chonemorphine and funtumafrine in C. grandiflora. Camptothecin, a well-known anticancer alkaloid has been detected in ethanolic extracts of stem with bark and callus cultures derived from C. grandiflora.Methods:Callus cultures of C. grandiflora were raised on Murashige and Skoog’s medium supplemented with 2, 4-D. Stem with bark and callus were used for phytochemical analysis mainly the alkaloids. Detection and identification of camptothecin was carried out using thin-layer chromatography (TLC), high-performance thin-layer chromatography, (HPTLC) and high-performance liquid chromatography (HPLC).Results:An important anticancer alkaloid, camptothecin was detected in ethanolic extracts of stem with bark and callus cultures of C. grandiflora. camptothecin content was 0.013 mg/g in stem with bark and 0.003 mg/g in callus.Conclusion:This is the first report on in vivo and in vitro production of camptothecin in C. grandiflora. Camptothecin is known to occur only in six plant sources so, alternative sources for camptothecin are needed. Thus of C. grandiflora could be a new promising alternative source of camptothecin.

Endophytic fungal strains of Fusarium solani, from Apodytes dimidiata E. Mey. ex Arn (Icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin

Camptothecin and 10-hydroxycamptothecin are two important precursors for the synthesis of the clinically useful anticancer drugs, topotecan and irinotecan. In recent years, efforts have been made to identify novel plant and endophytic fungal sources of camptothecin and 10-hydroxycamptothecin. In this study we have isolated endophytic fungi strains from Apodytes dimidiata (Icacinaceae), a medium sized tree from the Western Ghats, India. The fungi were identified as Fusarium solani using both ITS rDNA sequencing and spore morphology. Two strains, MTCC 9667 and MTCC 9668 were isolated, both of which produced camptothecin and 9-methoxycamptothecin in their mycelia; one of the strains, MTCC 9668 also produced 10-hydroxycamptothecin, though in small amounts. The yields of camptothecin in MTCC 9667 and MTCC 9668 were 37 and 53 μg/100 g, respectively, after 4 days of incubation in broth culture. The yields of 10-hydroxycamptothecin and 9-methoxycamptothecin in MTCC 9668 were 8.2 and 44.9 μg/100 g, respectively. Further research in optimizing the culture conditions of these fungal strains might permit their application for the production of camptothecin and 10-hydroxycamptothecin.

Separation by Macroporous Resins of 10-Hydroxycamptothecin from the Remainder Extracts in the Production of Camptothecin

In the present study, a method for the separation by macroporous resins of 10-hydroxycamptothecin (HCPT) from the extracts remaining from the production of camptothecin (CPT) has been developed. The adsorption/desorption characteristics of HCPT on eight macroporous resins were compared. Of these, the X-5 resin offered the best adsorption/desorption densities for HCPT, with its adsorption data being best fitted by the Langmuir and Freundlich isotherms. Dynamic adsorption/desorption experiments were carried out in a column packed with X-5 resin in order to optimize the separation process. The optimum parameters for adsorption were as follows: sample solution concentration = 0.3156 mg/mℓ, processing volume = 5 BV (bed volume), flow rate = 4 BV/h, temperature = 313 K, pH = 5; for desorption, the corresponding parameters were: 10–60% ethanol as eluent, processing volume = 2 BV, flow rate = 4 BV/h. After being treated with X-5 resin, the content of HCPT in the dry product increased from 0.30% to 7.19%, i.e. a 23.9-fold increase when compared to the untreated extract, with the recovery yield of HCPT being 82.1%.

Micropropagation and production of Camptothecin from in vitro plants of Ophiorrhiza rugosa var. decumbens

Camptothecin and its derivatives are used as anticancer alkaloids. At present, they are obtained by the extraction from the Chinese tree Camptotheca acuminata and the indigenous tree Nothapodytes foetida. Identification of alternate species of plants like Ophiorrhiza species and development of tissue culture methods may be a suitable alternative for large-scale micropropagation as well as for the production of camptothecin. Plantlets were successfully regenerated from shoot cultures of Ophiorrhiza rugosa initiated from axillary meristems on medium containing benzyladenine (BA) (4.0 mg L−1) + α-naphthalene acetic acid (NAA) (0.05 mg L−1). Rooting was initiated in half concentration of Murashige and Skoog's (MS) basal medium devoid of growth hormones. Tissue culture derived plants of O. rugosa were similar to the normal plants in their morphological characteristics and chemical constitution. Ten O. rugosa plants that were obtained through micropropagation showed when analysed a higher alkaloid content compared to the normal plant. Chemical analysis of the different organs of the tissue culture plant of O. rugosa established in soil indicated 0.002% dry weight of camptothecin in the roots, 0.011% dry weight in the stems, 0.090% dry weight in the leaves and 0.015% in the floral parts.