Simultaneous separation and analysis of camptothecin alkaloids in real samples by large-volume sample stacking in capillary electrophoresis.

Large-volume sample stacking (LVSS) is commonly used as an effective online preconcentration method in capillary zone electrophoresis (CZE). In this paper, the method LVSS combined with CZE has been proposed to analyze camptothecin alkaloids. Optimum separation can be achieved in the following conditions: pH 9.0; 25mm borate buffer containing 20 mm sulfobutylether-ß-cyclodextrin and 20 mm ionic liquid 1-ethyl-3-methyllimidazole l-lactate; applied voltage 20 kV; and capillary temperature 25 °C. The LVSS was optimized as hydrodynamic injection 4 s at 5.0 psi and the polarity switching time was 0.17 min. Under the above conditions, the analytes could be separated completely in <20 min and the detector response was increased compared with conventional hydrodynamic injection. The limits of detection were between 0.20 and 0.78 µg/L. A good linearity was obtained with correlation coefficients from 0.9991 to 0.9997. The recoveries ranged from 97.72 to 103.2% and the results demonstrated excellent accuracy. In terms of the migration time and peak area, the experiment was reproducible. The experimental results indicated that baseline separation can be obtained and this method is suitable for the quantitative determination of camptothecin alkaloids in real samples.

A high-throughput chemical screen identifies novel inhibitors and enhancers of anti-inflammatory functions of the glucocorticoid receptor.

Glucocorticoids (GCs)-ligands of the glucocorticoid receptor (GR)-are widely used to treat inflammatory diseases, but suffer from significant side effects and poor responsiveness in certain patient populations. Identification of chemical GR modulators may provide insights into the regulatory mechanisms of anti-inflammatory functions of GR and help improve GC-based therapy. Here we report the development and application of a high-throughput screening to identify compounds that either enhance or suppress the anti-inflammatory effect of GR function. Using a cell-based GR activity assay that measures Dexamethasone (Dex)-mediated NF-κB repression, we have screened ~8,000 compounds and identified several compounds that suppressed GR activity, including multiple GSK3ß inhibitors and anti-cancer agent camptothecin. Notably, we also identified two kinase IKK2 inhibitors, including TPCA-1, as GR enhancers that improve the anti-inflammatory effect of GR. In particular, TPCA-1 augmented the activity of Dex in NF-κB repression by attenuating GR down-regulation. Consistent with the observation, siRNA-mediated IKK2 knockdown decreased GR down-regulation and increased GR expression. Together, our results identified chemical compounds as novel modulators of GR and revealed an unexpected role for IKK2 in GR down-regulation. Furthermore, we have established a high-throughput screening platform for discovering GR-modulating compounds that may be repurposed to improve current GC-based therapies.

Simultaneous determination of camptothecin and 10-hydroxycamptothecine in the Camptotheca acuminate, its medicinal preparation and in rat plasma by liquid chromatography with fluorescence detection.

Camptotheca acuminata Decne is an important medicinal plant that contains various cytotoxic alkaloids, such as camptothecine (CPT) and 10-hydroxycamptothecine (HCPT). A rapid and sensitive liquid chromatography with fluorescence detection (LC-FLD) method for the quantification of CPT and HCPT is described. The separation was carried out on a DL-Cl8 column (4.6 × 150 mm, 5 µm), with the mobile phase of acetonitrile-sodium dihydrogen phosphate buffer (10 mm) using an gradient elution at the flow rate of 0.6 mL/min. The LC-FLD method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The lower detection limits of CPT and HCPT were 0.4 and 0.1 ng/mL, respectively. The precision was <1.58% and the mean recovery of the analytes was 96.0-98.6%. The LC-FLD method was successfully applied to determine CPT and HCPT in real samples including C. acuminate, HCPT injection and rat plasma.

Characterization and quantification of 10-hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography-ion trap mass spectrometry.

A rapid and sensitive method for the identification and quantification of 10-hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MS(n) spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI-MS(n) fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MS(n) in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative-ion mode at a product ion at m/z 363 → 319 by LC-MS. The LC-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC-MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time.

New plant sources of the anti-cancer alkaloid, camptothecine from the Icacinaceae taxa, India.

In this study, the production of camptothecine and its derivatives, in thirteen species of the family Icacinaceae, namely, Apodytes dimidiata, Codiocarpus andamanicus, Gomphandra comosa, Gomphandra coriacea, Gomphandra polymorpha, Gomphandra tetrandra, Iodes cirrhosa, Iodes hookeriana, Miquelia dentata, Miquelia kleinii, Natsiatum herpeticum, Pyrenacantha volubilis and Sarcostigma kleinii is reported. Seeds of M. dentata were found to produce the highest content of camptothecine (1.0-1.4% by dry weight of seeds). Full scan LC-MS and ESI-MS/MS analysis of M. dentata revealed, besides camptothecine, a number of other derivatives, namely, 10-hydroxycamptothecine, 9-methoxycamptothecine, 20-deoxycamptothecine. Crude extract preparations of the seeds of M. dentata were effective against a breast cancer cell line (IC50=3.82 µg/ml for MDA MB273 cell lines) and two ovarian cancer cell lines (IC50=2.8 µg/ml for NCI/ADR-RES and 4.5 µg/ml for SKOV). These results are the first reports of camptothecine and its derivatives in these species and offer rich alternative plant sources for the anticancer compound, camptothecine.

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.

[Fluorimetric analysis of camptothecin in Chinese herbal medicine common Camptotheca fruit].

Three-dimensional (3D) fluorescence spectra and thin layer fluorescence chromatogram of common Camptotheca fruit (CCF) crude drug, camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) have been studied, and a novel fluorimetric method for determination of CPT in CCF crude drug has been established. In 3D fluorescence spectra, CPT presented 3 fluorescence peaks with excitation wavelengths lambdaex of 215, 255 and 365 nm, separately, and all peaks with emission wavelength lambdaem of 430 nm. HCPT presented 4 fluorescence peaks with lambdaex of 220, 265, 325 and 375 nm, separately, and all peaks with lambdaem of 555 nm. The fluorescence of CPT is much stronger than that of HCPT. Comparison of 3D fluorescence spectra and analysis of thin layer fluorescence chromatogram revealed that the main fluorescent component of CCF is CPT. HCPT and other components basically do not interfere with the fluorescence of CPT. Under the condition of pH 3.0-6.7, CCF aqueous solution can produce strong and steady fluorescence. Using methanol as solvent, the extracting solution of CCF was prepared, and diluted properly with water, then measured fluorescence intensity at lambdaex/lambdaem = 365/430 nm to determine the content of CPT. A linear calibration curve covered the concentration range 0.002 35-0.235 microg x mL(-1). The regression equation was IF = 9 + 30,844 c, with correlation coefficient r = 0.999 (n=9). The method has been applied to the analysis of CPT in a CCF sample, with a result of 0.127% and a spiked recovery rate of 102%. The reliability of the method has been verified by a thin layer chromatography-fluorescence scanning method. Experimental results demonstrated that this method can be used for quality evaluation of CCF crude drug.

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.

Camptothecin distribution and content in Nothapodytes nimmoniana.

Nothapodytes nimmoniana is an important anti-cancer medicinal plant which possesses various bioactive substances, of which camptothecin (CPT) is the most important. CPT is mainly obtained by extraction from plants. However, extraction is problematic because of the lack of reference information, which leads to a large number of trees being felled. In this study, CPT was detected in different parts (shoots, two- and three-year-old stems, tender leaves, mature leaves, senescent leaves, roots and bark) of N. nimmoniana. A high performance liquid chromatographic (HPLC) method was used to determine the CPT content. All parts analyzed contained CPT, but concentrations depended on the different growth phases of the stems and leaves. The highest CPT content was 1.87 mg/g from roots, followed by the shoots, with a content of 1.07 mg/g. With a view to resource utilization, we concluded that the shoots were the best tissues for exploitation. Suggestions for shoot picking are also provided.

Development and characterization of a novel lipid nanocapsule formulation of Sn38 for oral administration.

The purpose of this work was to encapsulate 7-Ethyl-10-hydroxy-camptothecin (Sn38) in lipid nanocapsules (LNCs) using phase inversion-based method in order to deliver Sn38 by oral route. LNCs were prepared by a low-energy emulsification method and were characterized for size, polydispersity index (PDI), surface charge, drug payload, in vitro drug release, and storage stability. Moreover, in view of an oral administration, in vitro stability in gastrointestinal fluid and permeability across Caco-2 cells were tested. Sn38-loaded LNCs with a mean particle size of 38±2 nm were obtained. The particles displayed a narrow size distribution and a drug payload of 0.40±0.07 mg/g of LNC dispersion. In vitro stability in simulated gastric and intestinal media was also observed. Finally, Sn38-loaded LNCs improved permeability of Sn38 across Caco-2 cells (5.69±0.87×10(6) cm s(-1) at 6h vs 0.31±0.02×10(6) cm s(-1)) and intracellular concentration compared with free Sn38. In conclusion, Sn38 nanocarriers have been developed and display a strong potential for oral administration.

Metabolite fingerprinting of Camptotheca acuminata and the HPLC-ESI-MS/MS analysis of camptothecin and related alkaloids.

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC-MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.

HPTLC densitometric evaluation of tissue culture extracts of Nothapodytes foetida compared to conventional extracts for camptothecin content and antimicrobial activity.

Tissue culture technique is becoming popular because of its well-known ability to enhance the content of secondary metabolites in plants. Callus tissue cultures of Nothapodytes foetida were developed using 250 different medium compositions to optimize this procedure. Methanolic extracts of callus (MEC) and of various parts of N. foetida were comparatively analyzed for camptothecin content, and a high performance thin layer chromatography method was developed for its quantitation. Chloroform-ethylacetate-methanol (4 : 5 : 0.5 v/v) was used as the mobile phase. The method was validated for linearity, precision (interday and intraday), repeatability, limit of detection (LOD), limit of quantitation (LOQ), and accuracy. The relationship between the concentration of standard solutions and the peak response was linear within the range of 80 to 480 ng/spot with a correlation coefficient of 0.998 +/- 0.020. Instrumental precision was evaluated as 0.54 (% CV). Repeatability of sample and standard were estimated to be 1.08 and 1.01 (% CV), and LOD and LOQ were found to be 40 and 80 ng/spot, respectively. The accuracy of the method was checked out by a recovery study and the average percentage recovery was calculated as being 99.13 %. The methanolic extract of callus grown in tissue culture with medium composition picloram + thidiazuron + gibberellic acid (1 : 1 : 4; MEC-PTG) showed a higher percentage of camptothecin (5.74 % w/v) than the methanolic extract of fruits (3.56 % w/w), leaves (1.56 % w/w), stem (1.19 % w/w), and root (1.11 % w/w). The results of the antimicrobial screening indicate that MEC-PTG exhibited maximum activity against all microorganisms. Among the fungi tested, MEC-PTG showed maximum activity against A. niger and C. albicans (MIC value 10 microg/mL) whereas among bacteria strains, its activity was highest against B. subtilis and S. lutea (MIC 20 microg/mL).

[The study of dynamic acccumulation of camptothecin in Camptotheca acuminata fruit by TLCS].

OBJECTIVE: To establish TLC scanning method for the determination of camptothecin in Camptotheca acuminata fruit, and analysis the dynamic accumulation of camptothecin in Camptotheca acuminata fruit to find out the best collection period. METHODS: Silica gel H-CMC-Na thin layer plate was adopted in the determination with chloroform-acetone (7 : 3) used as deeloper, Single-wavelength and linear scanning of TLC was used, and the detection wavelength was 360 nm. RESULTS: There was a good linear relationship for Comptothecin within the range of 0.0542 - 0.3252 microg, the average recovery was 97.13%, RSD was 1.76%. CONCLUSION: The method is accurate, simple and reliable, and can be used for the determination of camptothecin in Camptotheca acuminata fruit and dynamic accumulation research.

Determination of camptothecins in DMSO extracts of Nothapodytes foetida by direct injection capillary electrophoresis.

A rapid capillary electrophoresis procedure was developed for determining the anti-cancer components, camptothecins, in Nothapodytes foetida. The hydrophobic compound was extracted from plant tissue (ca. 1 mL of DMSO for 100 mg of dried plant tissue) with a water-miscible organic solvent, DMSO, at elevated temperature (60 degrees C). The extract was directly injected into the separation capillary (untreated fused silica, 34 cm in length, 75 microm i.d.) and analysed in MEKC mode (369 nm). Within 5 min of migration, camptothecins were successfully separated and quantified by adding organic modifiers to the running buffer (20% DMSO, 90 mm SDS in 10 mm borate buffer, pH 8.60). The linear dynamic range for camptothecin was from 5 to 400 microg/mL. This method was proven to be very suitable for monitoring the amount of camptothecins during the cultivation of the medicinal plant.

Immunomodulatory activity of an extract of the novel fungal endophyte Entrophospora infrequens isolated from Nothapodytes foetida (Wight) Sleumer.

A novel camptothecin-producing endophytic fungus viz., Entrophospora infrequens was isolated from an important Indian medicinal plant Nothapodytes foetida. The present study reports evaluation ofbioactivities of two novel extracts viz., chloroform (CEEI) and methanolic (MEEI) extracts of Entrophospora infrequens with respect to their immunomodulatory potential in vitro and in vivo (in Balb/c mice). The endophyte E. infrequens was found to synthesize camptothecin, which tested positive in CEEI. The immunomodulatory potential of CEEI and MEEI was compared with standard camptothecin (CPT). Doses of the chloroform extract (CEEI) ranging from 12.5-100 mg/kg body weight, significantly (p < 0.05) stimulated the humoral and cell-mediated immune responses in a dose-dependent manner. MEEI on the other hand significantly (p < 0.05) stimulated the delayed type hypersensitivity (DTH) reaction (by nearly 80%), plaque forming cell (PFC) assay (33%), phagocytic response (38%) and haemagglutination antibody (HA) titre [IgM by 79.07% and IgG by 62.05%] at a dose of 12.5 mg/kg body weight. The present study is the first report of the immunomodulatory potential of this neoteric camptothecin-producing endophyte from Nothapodytes foetida.

Distribution of anticancer drug camptothecin in Nothapodytes foetida.

The topoisomerase I-DNA inhibitor alkaloid camptothecin has been evaluated from the various parts of Nothapodytes foetida. The bark contained 0.27% dry wt of camptothecin and 0.11% dry wt 9-methoxycamptothecin followed by the root, stem, and leaves. Immature seeds contained higher concentrations of camptothecin (0.32% dry wt) and 9-methoxycamptothecin (0.16% dry wt) compared to mature seeds. Various parts of mature and immature seeds were analysed to determine the content of the major alkaloids. Zygotic embryos of immature seeds contained 0.11% dry wt of camptothecin and 0.04% dry wt of 9-methoxycamptothecin. The highest concentration of camptothecin (0.42% dry wt) and 9-methoxycamptothecin (0.18% dry wt) were accumulated in the cotyledons of immature seeds.

Quantitative analysis of camptothecin derivatives in Nothapodytes foetida using 1H-NMR method.

A quantitative analysis using (1)H-NMR has been developed for the determination of camptothecin derivatives and trigonelline in Nothapodytes foetida root, stems and leaves. In the region of delta 9.5-5.5, the signals of H-7 of camptothecin (1), H-10 of 9-methoxycamptothecin (2), H-19 of pumiloside (3) and H-2 of trigonelline (4), were well separated from each other in DMSO-d(6). The quantity of the compounds was calculated by the ratio of the intensity of each compound to the known amount of internal standard 3,4,5-trimethoxybenzaldehyde. These results were compared with the conventional HPLC method. The advantages of the method are that no reference compounds are required for calibration curves, the quantification could be directly realized on a crude extract, an overall profile of the preparation could be directly obtained, and a very significant time-gain could be achieved, in comparison to conventional HPLC methods, for instance.

[Improved camptothecin production by cell lines of Camptotheca acuminata].

The concentration of camptothecin was determined in different tissues of Camptotheca acuminata seedling. The concentrations of camptothecin in new leaves and roots were significantly higher than in other tissues. However, the concentration of camptothecin declined with leaves becoming old. The induction of callus and cell suspension cultures from younger leaves of Camptotheca acuminata was observed. Cell lines were selected with improved camptothecin production as 0.02%.

Rapid micro-assay of camptothecin in Camptotheca acuminata.

A micro-assay has been developed to extract and rapidly quantify the anticancer alkaloid, camptothecin (CPT), from two leaf disks of Camptotheca acuminata Decaisne (Nyssaceae). This assay utilizes thin-layer chromatography in conjunction with fluorescence imaging to obtain reproducible measurements in the nanogram range. A large number of trees can be screened using this procedure to identify high producers of CPT in a relatively short period of time.

Yeast cells expressing differential levels of human or yeast DNA topoisomerase II: a potent tool for identification and characterization of topoisomerase II-targeting antitumour agents.

PURPOSE: To identify and characterize the specificity and potency of topoisomerase II-interacting antitumour drugs in an in vivo model utilizing the yeast Saccharomyces cerevisiae. METHODS: Four yeast transformants were selected for the expression of either human or yeast DNA topoisomerase II at different, biologically relevant, levels under the tight control of promoters of various strengths. RESULTS: Analyses of 24 drugs permitted their classification into three distinct groups, depending on whether they induced topoisomerase II-related cytotoxicity (etoposide), showed nonspecific cytotoxicity (camptothecin), or exerted no cytotoxicity at all (vinorelbine). Within the first group different patterns of action were distinguishable: (1) classical topoisomerase II expression-dependent cytotoxicity for both species of enzyme (e.g. etoposide, amsacrine, doxorubicin, actinomycin D), although amsacrine and TOP 53 were more active, respectively, on human and yeast topoisomerase II; and (2) compounds that appeared to poison only one species of topoisomerase II with, for example, genistein and the bisdioxopiperazine ICRF-193 lethally targeting only the human type, and mitoxantrone only the yeast isozyme. Three of the 16 known topoisomerase II inhibitors tested were not correctly identified with this assay, possibly owing to restricted cell wall permeability or to the absence of correct processing pathways such as, for example, in the case of the prodrug etopophos. CONCLUSION: This methodology, in vivo in yeast, selected for a large range of potent topoisomerase II-targeting anticancer agents. Of particular interest in this yeast model, and in contrast to yeast topoisomerase II, human topoisomerase II was shown to confer dominant sensitivity in the presence of the series of bisdioxopiperazine derivatives tested. This assay therefore has the potential easily to identify and characterize the potency and specificity of synthesized anticancer drugs with modified original chemical structures or those present, for example, in natural plant extracts or marine organisms.