In Vitro Comparative Study on Antineoplastic Effects of Pinoresinol and Lariciresinol on Healthy Cells and Breast Cancer-Derived Human Cells.

Background: Herbal medicines are the preferred anticancer agents due to their lower cytotoxic effects on healthy cells. Plant lignans play an important role in treating various diseases, especially cancer. The present study aimed to evaluate the effect of podophyllotoxin, pinoresinol, and lariciresinol on cellular toxicity and inducing apoptosis in fibroblasts, HEK-293, and SkBr3 cell lines. Methods: An in vitro study was conducted from 2017 to 2019 at the Faculty of Biological Sciences, Tarbiat Modares University (Tehran, Iran). The cell lines were treated for 24 and 48 hours with different concentrations of lignans. Cell viability and apoptosis were examined using MTT and flow cytometry, respectively. Expression levels of cell cycle and apoptosis regulator genes were determined using quantitative real-time polymerase chain reaction. Data were analyzed using a two-way analysis of variance followed by Tukey's HSD test. P<0.05 was considered statistically significant. Results: Podophyllotoxin significantly increased apoptosis in fibroblast cells compared to pinoresinol and lariciresinol (P<0.001). The percentage of cell viability of fibroblast cells treated for 48 hours with pinoresinol, lariciresinol, and podophyllotoxin was reduced by 49%, 47%, and 36%, respectively. Treatment with pinoresinol and lariciresinol significantly overexpressed pro-apoptotic genes and underexpressed anti-apoptotic genes in SkBr3 cells (P<0.001). SkBr3 cells treated with lariciresinol significantly reduced gene expression (P<0.001). Conclusion: Pinoresinol and lariciresinol can potentially be used as new therapeutic agents for the treatment of breast cancer.

Chemical Constituents of the Leaves of Thujopsis dolabrata and Their Cytotoxicity.

Five podophyllotoxin derivatives (1-5), two diterpenoids (6 and 7), three diterpenoid xylosides (8-10), a flavanonol glycoside (11), flavonol (12), and biflavonoid (13) were isolated from the leaves of Thujopsis dolabrata (Cupressaceae). Compounds 1, 6, and 8 were named (-)-ß-isopeltatin, epi-nootkastatin 2, and acetoxyanticopalol 15-O-ß-D-xylopyranoside, respectively. The structures of the isolated compounds were determined based on a detailed analysis of NMR spectroscopic data and through chromatographic and spectroscopic analyses following specific chemical transformations. The isolated compounds (1-5 and 7-11) were evaluated for their cytotoxicity toward HL-60 human promyelocytic leukemia cells and Caki-1 human kidney carcinoma cells. The podophyllotoxin derivatives (1-5) exhibited cytotoxicity against both HL-60 and Caki-1 cells with IC50 values ranging from 0.00069 to 5.4 µM, and the diterpenoid derivatives (7-10) demonstrated cytotoxicity against HL-60 cells with IC50 values ranging from 4.5 to 11 µM. HL-60 cells treated with 8 exhibited apoptosis characteristics, such as accumulation of sub-G1 cells and nuclear chromatin condensation.

Essential Oil Composition and Bioactivity of Two Juniper Species from Bulgaria and Slovakia.

Juniperus excelsa M. Bieb and J. sabina L. contain essential oil (EO), while J. sabina also contains podophyllotoxin, which is used as a precursor for anti-cancer drugs. Two studies were conducted. The first assessed the variability in the EO profile and podophyllotoxin concentration of the two junipers, depending on the location and tree gender. The main EO constituents of J. excelsa were α-cedrol, α-limonene and α-pinene, while the constituents in J. sabina were sabinene, terpinen-4-ol, myrtenyl acetate and α-cadinol. The podophyllotoxin yield of 18 J. sabina accessions was 0.07-0.32% (w/w), but this was not found in any of the J. excelsa accessions. The second study assessed the effect of hydrodistillation (Clevenger apparatus) and steam distillation (in a semi-commercial apparatus) on the EO profile and bioactivity. The extraction type did not significantly alter the EO composition. The EO profiles of the two junipers and their accessions were different and may be of interest to the industry utilizing juniper leaf EO. Breeding and selection programs could be developed with the two junipers (protected species) in order to identify chemotypes with (1) a high EO content and desirable composition, and (2) a high concentration of podophyllotoxin in J. sabina. Such chemotypes could be established as agricultural crops for the commercial production of podophyllotoxin and EO.

IN VIVO AND IN VITRO ANTILEISHMANIAL EFFECTS OF METHANOLIC EXTRACT FROM BARK OF BURSERA APTERA.

BACKGROUND: Cutaneous leishmaniasis lacks effective and well-tolerated treatments. The current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy. Traditional medicine offers a complementary alternative for the treatment of various diseases. Additionally, several plants have shown success as anti-leishmanial agents. Therefore, we sought to evaluate the in vitro and in vivo activity of MEBA against Leishmania mexicana. MATERIALS AND METHODS: Methanolic extract of B. aptera was obtained by macetration, after we determined in vitro anti-leishmanial activity of MEBA by MTT assay and the induced apoptosis in promastigotes by flow cytometry. To analyze the in vivo anti-leishmanial activity, we used infected mice that were treated and not treated with MEBA and we determined the levels of cytokines using ELISA. The phytochemical properties were determined by CG-MS and DPPH assay. RESULTS: We determined of LC50 of 0.408 mg/mL of MEBA for in vitro anti-leishmanial activity. MEBA induced apoptosis in promastigotes (15.3% ± 0.86). Treated mice exhibited smaller lesions and contained significantly fewer parasites than did untreated mice; in addition, we found that IFN-γ and TNF-α increased in the sera of MEBA-treated mice. GC-MS analysis showed that podophyllotoxin was the most abundant compound. Evaluation of the activity by DPPH assay demonstrated an SC50 of 11.72 µg/mL. CONCLUSION: Based on the above data, it was concluded that MEBA is a good candidate in the search for new anti-leishmanial agents.

Endophytic Fungus from Sinopodophyllum emodi (Wall.) Ying that Produces Podophyllotoxin.

The aryltetralin lactone podophyllotoxin, which exhibits pronounced antineoplastic activity, is used as the precursor of the following three clinical anticancer drugs: Etoposide™, Etopophos™ and Teniposide™. The natural occurrence of this arylnaphthalene lignan is scarce and unable to meet the ever-rising demand in the medical industry. Thus, developing alternative sources for the production of podophyllotoxin is extremely urgent. This is the first report of the production of podophyllotoxin from endophytic Alternaria tenuissima isolated from Sinopodophyllum emodi (Wall.) Ying. The identification of podophyllotoxin was performed using high-performance liquid chromatography and liquid chromatography-mass spectrometry (MS)-MS and confirmed by comparison with authentic standards.

[Effect of topographical factors on podophyllotoxin content in Sinopodophyllum hexandrum and study on ecological suitability].

In order to find the optimal topographical factor for regionslization, the content of cimetidine in 116 Sinopodophyllum hexandrum sample collected from Sichuan, Qinghai, Gansu, Tibet, Yunnan and Shaanxi provinces, was determined. Using mathematical statistics and geographical spatial analysis of GIS analysis, the relationship between content of podophyllotoxin and influencing factors including altitude gradient and gradient position was analyzed. It is found that the optimal altitude was 2 800 m to 3 600 m, the aspect of slope north or northeast and northwest and the slope 12 degrees to 65 degrees with a high suitability degree. Considering the artificial planting, the suitable planting area for S. hexandrum is comfirmed. The topographical factor is important for S. hexandrum regionalization, but has hardly effect on podophyllotoxin content. The results of the study provide an important scientific basis for S. hexandrum production development. But there are many factors which affect suitability index and podophyllotoxin content of S. hexandrum, it is necessary to consider other factors like climate and soil while exploitation and protection of S. hexandrum.

A Novel Sensitive Electrochemical Sensor for Podophyllotoxin Assay Based on the Molecularly Imprinted Poly-o-Phenylenediamine Film.

An electrochemical sensor for podophyllotoxin (PPT) based on the molecular imprinting polymer (MIP) membranes was constructed. The sensor was prepared by electropolymerizing o-phenylenediamine (o-PD) on a glassy carbon electrode (GCE) in the presence of PPT as template, and then removing the template by immersing the modified GCE in ethanol. Experimental parameters such as the types of monomer, scan cycles, concentration of o-PD and extraction condition were optimized. Under optimal conditions, the sensor exhibits a good selectivity and high sensitivity. A good linearity was obtained in the range of 4 x 10-8 mol · L(-1) to 3.2 x 10(-5) mol · L(-1) with an estimated detection limit of 4.8 x 10(-9) mol · L(-1). The sensor was applied to the determination of PPT in podophyllum hexandrum and human serum samples with satisfactory results.

Arylnaphthalene and aryltetralin-type lignans in hairy root cultures of Linum perenne, and the stereochemistry of 6-methoxypodophyllotoxin and one diastereoisomer by HPLC-MS and NMR spectroscopy.

INTRODUCTION: Hairy root cultures of Linum sp. are an alternative for the high production of lignans. Linum perenne is known to produce arylnaphthalene-type lignans such as justicidin B, isojusticidin and diphyllin. OBJECTIVE: To elucidate the presence of aryltetralin-type lignan diastereoisomers, besides the known arylnaphthalene-type lignans, in hairy roots of Linum perenne, and to determine the configurations of one diastereoisomer of 6-methoxypodophyllotoxin (6-MPTOX). METHODS: Lignans from hairy root cultures of Linum perenne were extracted and separated by HPLC. Arylnaphthalene-type lignans were identified by LC-MS, according to the literature. Two diastereoisomers of aryltetralin-type lignans were analysed by mass spectrometry and NMR spectroscopy. RESULTS: Numerous arylnaphthalene-type lignans (diphyllin-2-hexose-pentose, diphyllin-3-pentose and diphyllin-hexose) were identified in hairy root cultures. Methoxypodophyllotoxin, an aryltetralin-type lignan, was also identified, as well as one diastereoisomer. This aryltetralin-type lignan could be derived via 7-hydroxymatairesinol as a hypothetical biosynthetic pathway. The stereochemical configurations of aryltetralin isomers were determined. CONCLUSION: Arylnaphthalene and two diastereoisomers of aryltetralin-type lignans are produced in Linum perenne hairy root cultures. Matairesinol, the precursor of justicidin B, also seems to be converted into 6-MPTOX via 7-hydroxymatairesinol. This is the first report of the stereochemical configurations of an aryltetralin-type lignan other than podophyllotoxin (PTOX).

Purity Assessment of Aryltetralin Lactone Lignans by Quantitative 1H Nuclear Magnetic Resonance.

In the present work, a quantitative 1H Nuclear Magnetic Resonance (qHNMR) was established for purity assessment of six aryltetralin lactone lignans. The validation of the method was carried out, including specificity, selectivity, linearity, accuracy, precision, and robustness. Several experimental parameters were optimized, including relaxation delay (D1), scan numbers (NS), and pulse angle. 1,4-Dinitrobenzene was used as internal standard (IS), and deuterated dimethyl sulfoxide (DMSO-d6) as the NMR solvent. The purities were calculated by the area ratios of H-2,6 from target analytes vs. aromatic protons from IS. Six aryltetralin lactone lignans (deoxypodophyllotoxin, podophyllotoxin, 4-demethylpodophyllotoxin, podophyllotoxin-7'-O-ß-d-glucopyranoside, 4-demethylpodophyllotoxin-7'-O-ß-d-glucopyranoside, and 6''-acetyl-podophyllotoxin-7'-O-ß -d-glucopyranoside) were analyzed. The analytic results of qHNMR were further validated by high performance liquid chromatography (HPLC). Therefore, the qHNMR method was a rapid, accurate, reliable tool for monitoring the purity of aryltetralin lactone lignans.

Study of cytotoxic activity, podophyllotoxin, and deoxypodophyllotoxin content in selected Juniperus species cultivated in Poland.

CONTEXT: The demand for podophyllotoxin and deoxypodophyllotoxin is still increasing and commercially exploitable sources are few and one of them, Podophyllum hexandrum Royle (Berberidaceae), is a "critically endangered" species. OBJECTIVE: The first aim was to quantify the amount of podophyllotoxin and deoxypodophyllotoxin in 61 Juniperus (Cupressaceae) samples. Cytotoxic activity of podophyllotoxin and ethanolic leaf extracts of Juniperus scopulorum Sarg. "Blue Pacific" and Juniperus communis L. "Depressa Aurea" was examined against different leukemia cell lines. MATERIALS AND METHODS: Ultra-performance liquid chromatography (UPLC) analysis was performed with the use of a Waters ACQUITY UPLC(TM) system (Waters Corp., Milford, MA). The peaks of podophyllotoxin and deoxypodophyllotoxin were assigned on the basis of their retention data and mass-to-charge ratio (m/z). Trypan blue assay was performed to obtain IC50 cytotoxicity values against selected leukemia cell lines. RESULTS: Juniperus scopulorum was characterized with the highest level of podophyllotoxin (486.7 mg/100 g DW) while Juniperus davurica Pall. contained the highest amount of deoxypodophyllotoxin (726.8 mg/100 g DW). Podophyllotoxin IC50 cytotoxicity values against J45.01 and CEM/C1 leukemia cell lines were 0.0040 and 0.0286 µg/mL, respectively. Juniperus scopulorum extract examined against J45.01 and HL-60/MX2 leukemia cell lines gave the respective IC50 values: 0.369-9.225 µg/mL. Juniperus communis extract was characterized with the following IC50 cytotoxity values against J45.01 and U-266B1 cell lines: 3.310-24.825 µg/mL. CONCLUSIONS: Juniperus sp. can be considered as an alternative source of podophyllotoxin and deoxypodophyllotoxin. Cytotoxic activity of podophyllotoxin and selected leaf extracts of Juniperus sp. against a set of leukemia cell lines was demonstrated.

Mucor fragilis as a novel source of the key pharmaceutical agents podophyllotoxin and kaempferol.

CONTEXT: Podophyllotoxin, a pharmaceutically important bioactive compound of Podophyllum sps. (Berberidaceae), is in great demand worldwide as an anticancer and antivirus drug precursor. However, the source of podophyllotoxin is very limited due to the endangered status of the Podophyllum plant. OBJECTIVE: The aim of this study was to isolate podophyllotoxin-producing endophytic fungi from Sinopodophyllum hexandrum (Royle) Ying (1979) (Berberidaceae) plants of the Taibai Mountains of China in order to obtain bioactive compounds. MATERIALS AND METHODS: The strains producing kaempferol and podophyllotoxin were screened by thin-layer chromatography (TLC) analysis. The presence of kaempferol and podophyllotoxin in extracts of these strains was further confirmed by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) analyses. RESULTS: Among six endophytic fungi isolated from the rhizomes of S. hexandrum, one strain was able to produce kaempferol. Another strain, named TW5, was able to produce both kaempferol and podophyllotoxin simultaneously according to the TLC, HPLC, and NMR results. The podophyllotoxin yield of TW5 was calculated to be 49.3 µg/g of mycelial dry weight after 7-d fermentation. Strain TW5 was identified morphologically and phylogenetically to be Mucor fragilis Fresen. (Mucoraceae). These results suggest that the podophyllotoxin-synthesizing ability is obtained by uptaking genes involved in the podophyllotoxin synthesis from the host plant into endophytic fungal genomes. CONCLUSION: Our results showed, for the first time, that the endophytic fungus M. fragilis is able to produce simultaneously the same two bioactive metabolites, podophyllotoxin and kaempferol, as its host plant. Furthermore, the relatively high podophyllotoxin yield obtained may improve the industrial production of podophyllotoxin, which may help protect this endangered plant.

[Determination of podophyllotoxin and total lignans in Sinopodophyllum emodi].

OBJECTIVE: To determine the content of podophyllotoxin and total lignans in Sinopodophyllum emodi Ying from different areas and evaluate the resource utilization of this endangered medicinal plant. METHOD: HPLC and UV spectrophotometry were used to determine the content of podophyllotoxin and total lignans in 126 samples from different habitats and the total lignans content was determined by the reference wavelength method. RESULT: According to the results, the highest content was determined from the samples from Yongdeng Nature Reserve in Gansu province, and the lowest ones was found in the samples from Tibet. The former's podophyllotoxin and total lignans contents were 7.40% and 20.66%, respectively, which were 19 times and 4 times more than those of the latter. The content of podophyllotoxin and total lignans in S. emodi were significantly positively related, meanwhile, samples from both low altitude and high latitude showed the higher content. CONCLUSION: The two determination methods are simple, rapid, accurate and repeatable. It is more scientific and rational to evaluate the resource utilization of S. emodi with two indicators, those are the content of podophyllotoxin and the content of total lignans. This paper is instructive to the collection of wild resources and the establishment of production bases.

Identification of lignans and related compounds in Anthriscus sylvestris by LC-ESI-MS/MS and LC-SPE-NMR.

The aryltetralin lignan deoxypodophyllotoxin is much more widespread in the plant kingdom than podophyllotoxin. The latter serves as a starting compound for the production of cytostatic drugs like etoposide. A better insight into the occurrence of deoxypodophyllotoxin combined with detailed knowledge of its biosynthestic pathway(s) may help to develop alternative sources for podophyllotoxin. Using HPLC combined with electrospray tandem mass spectrometry and NMR spectroscopy techniques, we found nine lignans and five related structures in roots of Anthriscus sylvestris (L.) Hoffm. (Apiaceae), a common wild plant in temperate regions of the world. Podophyllotoxone, deoxypodophyllotoxin, yatein, anhydropodorhizol, 1-(3'-methoxy-4',5'-methylenedioxyphenyl)1-ξ-methoxy-2-propene, and 2-butenoic acid, 2-methyl-4-[[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy]-, (2E)-3-(7-methoxy-1,3-benzodioxol-5-yl)-2-propen-1-yl ester, (2Z)- were the major compounds. α-Peltatin, podophyllotoxin, ß-peltatin, isopicropodophyllone, ß-peltatin-a-methylether, (Z)-2-angeloyloxymethyl-2-butenoic acid, anthriscinol methylether, and anthriscrusin were present in lower concentrations. α-Peltatin, ß-peltatin, isopicropodophyllone, podophyllotoxone, and ß-peltatin-a-methylether have not been previously reported to be present in A. sylvestris. Based on our findings we propose a hypothetical biosynthetic pathway of aryltetralin lignans in A. sylvestris.

Podophyllotoxin and deoxypodophyllotoxin in Juniperus bermudiana and 12 other Juniperus species: optimization of extraction, method validation, and quantification.

The lignans podophyllotoxin and deoxypodophyllotoxin are secondary metabolites with potent pharmaceutical applications in cancer therapy. However, the supply of podophyllotoxin from its current natural source, Podophyllum hexandrum, is becoming increasingly problematic, and alternative sources are therefore urgently needed. So far, podophyllotoxin and deoxypodophyllotoxin have been found in some Juniperus species, although at low levels in most cases. Moreover, extraction protocols deserve optimization. This study aimed at developing and validating an efficient extraction protocol of podophyllotoxin and deoxypodophyllotoxin from Juniperus species and applying it to 13 Juniperus species, among which some had never been previously analyzed. Juniperus bermudiana was used for the development and validation of an extraction protocol for podophyllotoxin and deoxypodophyllotoxin allowing extraction yields of up to 22.6 mg/g DW of podophyllotoxin and 4.4 mg/g DW deoxypodophyllotoxin, the highest values found in leaf extract of Juniperus. The optimized extraction protocol and HPLC separation from DAD or MS detections were established and validated to investigate podophyllotoxin and deoxypodophyllotoxin contents in aerial parts of 12 other Juniperus species. This allowed either higher yields to be obtained in some species reported to contain these two compounds or the occurrence of these compounds in some other species to be reported for the first time. This efficient protocol allows effective extraction of podophyllotoxin and deoxypodophyllotoxin from aerial parts of Juniperus species, which could therefore constitute interesting alternative sources of these valuable metabolites.

Seasonal variations in the deoxypodophyllotoxin content and yield of Anthriscus sylvestris L. (Hoffm.) grown in the field and under controlled conditions.

Deoxypodophyllotoxin (DPT) is the main lignan in Anthriscus sylvestris . For this study two sets of experiments with 16 plants and seeds, collected from a wide geographical range, were carried out. The DPT content in roots was significantly lower (p < 0.05) when the plants were cultivated in a non-native environment. For field-grown plants the highest DPT content was found in March (second year): 0.15% w/w (dry weight) in roots; 0.03% w/w in aerial parts. For plants grown in the climate room, the highest concentration (0.14% w/w) was observed in April (second year) in the roots and in July (first year) in the aerial parts (0.05% w/w). For the isolation of DPT, roots are the most suitable part. The best harvest times are March (second year) for outdoor plants and April (second year) for indoor plants when height content and adequate biomass give the optimal DPT yield.

Development and characterization of molecularly imprinted polymers for the selective enrichment of podophyllotoxin from traditional Chinese medicines.

In the present work, microwave heating initiated precipitation polymerization was developed to prepare podophyllotoxin (PPT) molecularly imprinted polymers (MIPs), resulting in much shorter polymerization time and better particle morphology. Prior to the polymerization, ultraviolet and FTIR spectroscopy were used to study the interactions between PPT and the functional monomers. The synthesized parameters were respectively optimized and the optimal conditions for the efficient adsorption property were template: PPT, 1 mmol; functional monomer: acrylamide, 6 mmol; bi-crosslinker: ethylene glycol dimethacrylate, 20 mmol and divinylbenzene, 20 mmol; porogen: acetonitrile, 40 mL; initiator: azobisisobutyronitrile, 0.01mol L⁻¹; polymerization temperature: 60°C. FTIR spectroscopy, SEM and thermal analysis were used to characterize the MIPs. The results of the equilibrium rebinding experiments and the competitive adsorption experiments showed that these imprinted polymers exhibited good adsorption ability for the PPT. Scatchard analysis illustrated that two and one types of binding sites were generated in the MIPs and non-imprinted polymers (NIPs), respectively. Using the prepared MIPs as the solid phase extraction (SPE) sorbent, PPT was extracted selectively and efficiently from Dysosma versipellis, Sinopodophyllum hexandrum and Diphylleia sinensis. The regression equation was y=5.873×106x+17075.659 with the correlation coefficient of 0.9994 in the concentration range of 0.005-0.4 mg mL⁻¹. After washing and eluting the SPE column with methanol and MeOH/acetic acid solution (v/v, 9:1), the limits of detection were 0.12-0.18 µg mL⁻¹ and their recoveries were in the range of 89.5-91.1% with all RSDs lower than 3.7.

Application of ionic liquids in the microwave-assisted extraction of podophyllotoxin from Chinese herbal medicine.

A micelle-mediated extraction technique, i.e. ionic liquid-based microwave-assisted extraction (ILs-MAE) technique has been developed for the effective extraction of podophyllotoxin from three Chinese medicinal plants. Several operating parameters were successively optimized by single-factor and L(9) (3(4)) orthogonal array experiments. 1-Butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)]), 1-decyl-3-methylimidazolium tetrafluoroborate ([demim][BF(4)]) and 1-allyl-3-methylimidazolium tetrafluoroborate ([amim][BF(4)]) were selected as the optimal surfactants for Dysosma versipellis, Sinopodophyllum hexandrum and Diphylleia sinensis, respectively. Compared with other extraction techniques, such as ionic liquids-based maceration extraction (ILs-ME), heat extraction (ILs-HE) and ultrasound-assisted extraction (ILs-UAE), the ILs-MAE technique not only took a shorter time but also afforded a higher extraction rate of podophyllotoxin from the herbs. Reversed phase high performance liquid chromatography was employed for the analysis of podophyllotoxin. The results showed that the linearity for analyzing podophyllotoxin in all three herbs was in the concentration range of 0.005-0.4 mg mL(-1) with the correlation coefficient between 0.9993 and 0.9996. LODs were 2.05-2.58 µg mL(-1) and RSDs of inter-day stability were less than 5.8%. Repeatability and intermediate precision were separately lower than 3.3% and 6.3%. The recoveries for podophyllotoxin extracted with the ILs-MAE technique were in the range of 97.1-102% and all RSDs were lower than 3.0%. Furthermore, the mechanism of ILs-MAE was preliminarily studied by means of kinetic mechanism, surface structures and chemical compositions of samples before and after different extraction techniques. On the basis of the destruction of herb surface microstructures and high solubility of ILs, the ILs-MAE technique eventually got the maximum yield value.

Rapid analysis of lignans from leaves of Podophyllum peltatum L. samples using UPLC-UV-MS.

A new rapid UPLC-UV-MS method has been developed that permits the analysis of four lignans (4'-O-demethylpodophyllotoxin, podophyllotoxin, α-peltatin and ß-peltatin) in P. peltatum L. Podophyllotoxin is a natural lignan that is being used as a precursor for the semi-synthetic anti-cancer drugs etoposide, teniposide and etopophos. The chromatographic separation was achieved using a reversed-phase C18 column with a mobile phase of water and acetonitrile, both containing 0.05% formic acid. Analyses of P. peltatum leaves collected from different colonies within a single site indicated a significant variation in 4'-O-demethylpodophyllotoxin, α-peltatin, podophyllotoxin and ß-peltatin content. Within 3.0 min four main lignans could be separated with detection limits of 0.1, 0.3, 0.3 and 0.2 µg/mL, respectively. 4'-O-demethylpodophyllotoxin and α-peltatin appeared most prominently among the lignans obtained. The podophyllotoxin content was found in the range of 0.004-0.77% from 16 samples collected from 6 colonies within the same site. The content of podophyllotoxin is directly proportional to the content of 4'-O-demethylpodophyllotoxin and inversely proportional to α-peltatin and ß-peltatin content. LC-mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of four lignans in various populations of plant samples. By applying principal component analysis and hierarchical cluster analysis, Podophyllum samples collected from various colonies within a location were distinguished.

Determination of picropodophyllin and its isomer podophyllotoxin in human serum samples with electrospray ionization of hexylamine adducts by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of the new anticancer agent picropodophyllin (AXL1717) and its isomer podophyllotoxin levels in human serum has been developed. Monitoring of hexylamine adducts rather than proton adducts was used to optimize sensitivity. The chromatography system was an Acquity BEH C18, 2.1 mm × 50 mm 1.7 µm column with gradient elution (mobile phase A: 2.5 mM hexylamine and 5 mM formic acid in Milli-Q water and mobile phase B: methanol). The retention times were 1.4 min for picropodophyllin, 1.5 min for podophyllotoxin and 1.9 min for internal standard deoxypodophyllotoxin. The isomers were base-line separated. The analytes were detected after electrospray ionization in positive mode with selected reaction monitoring (SRM) with ion transitions m/z 516→102 for picropodophyllin and podophyllotoxin and m/z 500→102 for internal standard. The sample preparation was protein precipitation with acetonitrile (1:3) containing internal standard followed by dilution of the supernatant with mobile phase A (1:1). The limit of quantification (LOQ) was 0.01 µmol/L for picropodophyllin and podophyllotoxin. The limit of detection (LOD) at 3 times the signal to noise (S/N) was estimated below 0.001 µmol/L for picropodophyllin and podophyllotoxin. The quantification range of the method was between 0.01 µmol/L and 5 µmol/L for both isomers. The accuracy was within ±15% of the theoretical value for both picropodophyllin and podophyllotoxin and inter-assay precision did not exceed ±15%, except for the 0.016 µmol/L level of podophyllotoxin, which was 18%. The selectivity of the method was verified by analysis of two different product ions for each analyte and by analysis for interference of seven different batches of blank human serum. The combined recovery and matrix effects were about 83% for picropodophyllin and podophyllotoxin. The new LC-MS/MS method showed sufficient sensitivity and selectivity for determination of picropodophyllin and its isomer podophyllotoxin levels in human serum from subjects receiving therapeutic doses of AXL1717.

Chemometric evaluation of the anti-cancer pro-drug podophyllotoxin and potential therapeutic analogues in Juniperus and Podophyllum species.

INTRODUCTION: Podophyllotoxin, deoxypodophyllotoxin, demethylpodophyllotoxin and podophyllotoxone are four therapeutically potent secondary metabolites. There is a dearth of information on the holistic analysis of their distribution pattern in both phylogenetic and ecological contexts. OBJECTIVES: To analyse the continuum of the above metabolites in Juniperus and Podophyllum species collected from natural populations in Himalayan environments and the botanical gardens of Rombergpark and Haltern (Germany) using multi-component LC-ESI-MS/MS, coupled with statistically relevant chemometric assessment. METHODOLOGY: We evaluated the individual and holistic metabolite profiles and chemometrically correlated the phytochemical loads between various species (infraspecific), organic and aqueous extracts, and populations of the same species from different locations, different species from same location, different species from different locations and infrageneric populations from same and different locations. RESULTS: Multivariate analysis revealed Juniperus x-media Pfitzeriana as a suitable alternative to Podophyllum hexandrum for commercial exploitation. A significant positive correlation of podophyllotoxone with both podophyllotoxin and demethylpodophyllotoxin, and a negative correlation of podophyllotoxin with both deoxypodophyllotoxin and demethylpodophyllotoxin (infraspecific among Podophyllum), were observed by Kruskal's multidimensional scaling and corroborated by principal component analysis, indicating probable similarity and/or difference between the biosynthetic pathways, and synergistic and/or antagonistic principles, respectively. Finally, linear discriminant analysis and hierarchical agglomerative cluster analysis revealed considerable infrageneric and infraspecific variability in secondary compound spectra and load of the different populations under study. CONCLUSION: Such holistic studies of plants and their therapeutic metabolites ought to assist in selecting plants, geographical areas and environmental conditions for bioprospecting and global-scale phytochemical and phylogenetic diversity studies in the future.