Abstract
Podophyllotoxin is used for the production of the anticancer drugs etoposide, etopophos, and teniposide. Currently, podophyllotoxin is extracted from the Himalayan mayapple (Podophyllum hexandrum Royle). Some junipers and other species also contain the same natural product and have been explored as a domestic source for this compound. The objective of this study was to screen junipers in the Big Horn Mountains in Wyoming for podophyllotoxin. Twenty junipers (18 accessions of Juniperus horizontalis Moench. and two accessions of J. scopulorum Sarg.) were sampled in Mar. 2012 and analyzed for podophyllotoxin. Podophyllotoxin concentration in the samples varied from 0.058% to 0.673% with five accessions having podophylloxin concentration above 0.5%. This study demonstrated wide variation of podophyllotoxin in J. horizontalis and J. scopulorum in the Big Horn Mountains. Some of the accessions had greater than 0.5% podophyllotoxin making them a feasible source for podophyllotoxin extraction.
Podophyllotoxin is used as a chemical precursor for the production of the anticancer drugs etoposide, etopophos, and teniposide, which are used for the treatment of lung cancer, testicular cancer, neuroblastoma, and hepatoma (Imbert, 1998; Koulman et al., 2004; Stahelin and Wartburg, 1991). In addition, some other derivatives of podophyllotoxin were found to be promising in the treatment of psoriasis, malaria (Leander and Rosen, 1988; Lerndal and Svensson, 2000), and also as antiviral agents (Hammonds et al., 1996). Currently, the only commercial source for podophyllotoxin is the Himalayan mayapple (Podophyllum hexandrum Royle, syn. P. emodi Wall ex. Honigberger), an endangered species in India.
Researchers identified a number of other species that contain podophyllotoxin. Much research has been focused on the American mayapple (P. peltatum L.), a native species in North America. Despite numerous studies on American mayapple in Poland, Russia (Bogdanova and Sokolov, 1973; Saraeva, 1952), and in the United States (Cushman et al., 2005; Meijer, 1974; Moraes et al., 2002; Zheljazkov et al., 2009), this species was not domesticated as a result of its limitations.
Eastern red cedar (Juniperus virginiana) has also been investigated as a source for podophyllotoxin (Canel et al., 2001; Cushman et al., 2003; Gawde et al., 2009; Hartwell et al., 1953). Rocky mountain juniper (J. scopulorum) also contains podophyllotoxin (Cantrell et al., 2013; Renouard et al., 2011) although in lower concentrations than Eastern red cedar. Our preliminary studies in the Big Horn Mountains found that some accessions of creeping juniper (J. horizontalis Moench.) had much greater concentrations of podophyllotoxin than reported for any other juniper species. Therefore, the objective of this study was to screen different accessions of creeping juniper in the Big Horn Mountains and establish the range of podophyllotoxin concentrations.
Materials and Methods
Plant collection.
In Mar. 2012, 20 different junipers (18 accessions of Juniperus horizontalis Moench. and two accessions of J. scopulorum Sarg.) were sampled in the Big Horn Mountains of Wyoming. The sampling area was chosen based on our preliminary studies that found accessions with high podophyllotoxin, and this area is characterized by a very high density of creeping junipers. Samples were taken from naturally occurring junipers situated at least 30 m away from any roads. From each plant, ≈1 kg of fresh material was collected. Only mature leaves of the juniper plants were sampled. The GPS coordinates were recorded for every plant accession that was sampled. The collected samples were dried in a shady aerated area at temperatures between 22 and 28 °C until constant weight. Subsamples from each collection were identified by Bonnie Heidel, a botanist at the Wyoming Natural Diversity Database, University of Wyoming, and representative vouchers were deposited in the University of Wyoming Rocky Mountain Herbarium.
Podophyllotoxin.
The extraction and purification of podophyllotoxin from the collected juniper samples was as previously described (Canel et al., 2001). Approximately 40 mg of each dry tissue sample was incubated at 20 °C with 0.6 mL of 25 mm potassium phosphate buffer (pH of 7.0) on an Eppendorf Thermomixer R for 30 min at 750 rpm. Subsequently, 0.6 mL of ethyl acetate was added, and the incubation continued for an additional 5 min in the same manner. The aqueous and organic partitions were separated by centrifuge for 15 min (Savant speed vac, svc 200). The organic layer was removed using a Pasteur pipette and evaporated under a stream of N2, leaving the organic soluble material to be dissolved in 0.8 mL of methanol and analyzed by high-performance liquid chromatography (HPLC).
Podophyllotoxin in the purified extract was analyzed using an HPLC system (Agilent 1200 series consisting of a vacuum degasser, quaternary pump, ALS autosampler, a photodiode array detector, and an Agilent Eclipse XDB-C18, 4.6 × 150 mm, 5-μm column). The injection volume for all samples and for the podophyllotoxin standard was 10 μL and standards and samples were analyzed at room temperature. The analytical method was isocratic (28% acetonitrile:72% deionized water containing 0.1% trifluoroacetic acid) for 20 min followed by a 5-min column wash with methanol and re-equilibration. Analytes were detected at 220 nm with a reference of 450 nm by a photodiode array detector.
Quantitation was performed by means of an external standard calibration curve obtained from standard solutions of pure podophyllotoxin, purchased from Sigma-Aldrich (St. Louis, MO). Linearity was imposed by using response factors and regression coefficients independently. Response factors (RFs) were calculated using the equation RF = DR/C, where DR was the detector response in peak area (PA) and C was the analyte concentration. The target podophyllotoxin peak was confirmed by retention time and ultraviolet spectroscopy from 200 to 500 nm. The RF of the target chemical constituent was used to determine the percent for each sample using the equation: (PA/RF/C) * 100 = %.
Statistical analysis.
The effect of 20 juniper tree accessions on podophyllotoxin (%) was determined by conducting a one-way analysis of variance (ANOVA) using the GLM procedure of SAS (SAS Institute Inc., 2008). The validity of the ANOVA model assumptions, namely normal distribution and constant variance assumptions on the error terms, was verified by examining the residuals as described in Montgomery (2009). Because the effect of accession was highly significant (P < 0.01), further multiple means comparison was done using Tukey’s honestly significant difference method at the 5% level. The more conservative Tukey’s method was used because of the low experimental error and to protect the Type I experimentwise error rate from overinflation resulting from the relatively large number of accessions.
Results and Discussion
As expected, podophyllotoxin concentration in the collected samples varied in wide ranges from 0.058% to 0.673% (Table 1). Five accessions had podophyllotoxin concentration above 0.5%, two accessions had a podophyllotoxin concentration between 0.45% and 0.5%, two accessions had a podophyllotoxin concentration between 0.3% and 0.4%, four accessions had a podophyllotoxin concentration between 0.2% and 0.3%, five accessions had a podophyllotoxin concentration between 0.1% and 0.2%, and two accessions had a podophyllotoxin concentration between 0.05% and 0.1%.
Mean Podophyllotoxin concentration (%) from the 20 Juniperus accessions,z elevation, and GPS coordinates of each collection site.
In this study, we found seven accessions of creeping juniper with podophyllotoxin concentrations above 0.5%. Previously, Cantrell et al. (2013) reported 0.254% and 0.138% podophyllotoxin in two accessions of creeping juniper (J. horizontalis). In a recent study in a wider range of the Big Horn Mountains, Zheljazkov et al. (2013) found podophyllotoxin concentration range of 0.27–0.073% in J. horizontalis, 0–0.40% in J. scopulorum, and none in J. communis.
Previous research on Eastern red cedar found podophylloxin concentration to be between 0.1% and 0.34% (Gawde et al., 2009) and 0.1% to 0.18% (Cushman et al., 2003). However, Cantrell et al. (2013) in a recent study reported 0.48%, 0.044%, 0.28%, and 0.66% in four different accessions of J. virginiana.
Apparently, the range of podophyllotoxin concentrations in creeping juniper in the Big Horn Mountains in this study was either higher or similar to the ones reported for Eastern red cedar. Hence, creeping juniper accessions found in the Big Horn Mountains seem promising as a source for podophyllotoxin.
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