Abstract
Abstract
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 UPLCTM 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.
Introduction
Plants containing lignans have been used for about 1000 years in the folk medicine of China, Japan, and the Eastern world (Ayres & Loike, 1990). Lignans are acknowledged to have a wide variety of biological activities, including the well-documented anticancer properties of aryltetralin lignans (Gordaliza et al., 2004; Yousefzadi et al., 2010). Aryltetralin lignans, such as podophyllotoxin and its derivatives, are widely distributed throughout the plant kingdom. Some of the lignans, in particular podophyllotoxin (PDP), are known to have anticancer, antifungal, and antimicrobial properties (Gordaliza et al., 2004). Deoxypodophyllotoxin (dO-PDP), in turn, has demonstrated a wide range of relevant properties against a number of cancer cell lines (Ikeda et al., 1998; Kim et al., 2002; Masuda et al., 2002; Muto et al., 2008; Renouard et al., 2011; Xu et al., 2010). PDP was first extracted from Podophyllum peltatum L. (Berberidaceae) and from Linum sp. (Linaceae) as a resin and was used by physicians in the southern parts of the USA in the late nineteenth century. The resin was applied for the treatment of genital warts, which are associated with cancerous processes through their etiology by human papilloma virus (Koulman, 2003) (Figure 1).
Published online:
30 March 2015![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iphb20/2015/iphb20.v053.i06/13880209.2014.943246/20150611/images/medium/iphb_a_943246_f0001_b.jpg"})
Figure 1. Chemical structures of investigated compounds.
Commercially exploitable plant sources of PDP are few and this compound is currently extracted for drug use from the roots and rhizomes of two species of Podophyllum – Podophyllum hexandrum Royle and Podophyllum peltatum. PDP is a precursor for the synthesis of important antitumor drugs like etoposide and teniposide which are used in the treatment of lung cancer, testicular cancer, a variety of leukemia’s, and other solid tumors (Holthius, 1988; Imbert, 1998; Stahelin & Wartburg, 1991). NK611, TOP 53, GL 331, and azatoxin are some other promising antitumor derivatives of PDP (Majumder & Jha, 2009). The demand for PDP is ever increasing. Nevertheless, the occurrence of Podophyllum hexandrum is scarce due to its long juvenile phase and poor fruit setting ability. Overexploitation and lack of organized cultivation have made the plant “critically endangered” (CITES, 2014). Owing to its immense clinical importance, new routes for total synthesis of PDP have been discovered, however, they are not economically feasible. Thus isolation from plant sources continues to be the only viable option. A lot of effort has been put, in the past several years, to improve its isolation efficiency from different plant sources. Currently, it is assumed that the most advantageous situation is to find species which would naturally provide PDP in the desired amount (Majumder & Jha, 2009).
dO-PDP may be considered as an alternative to PDP; therefore, it is equally important to search for possible sources of dO-PDP. Stereo-selective hydroxylation of dO-PDP into epipodophyllotoxin was reported and offered as a promising alternative strategy for the large-scale production of this valuable precursor in the pharmaceutical industry (Julsing et al., 2008; Renouard et al., 2011; Vasilev et al., 2006).
The occurrence of PDP and dO-PDP in some Juniperus L. (Cupressaceae) sp. has been described in several papers, first published in 1953 by Hartwell et al. In the majority of cases, the authors reported on the low levels of these lignans in the analyzed samples. Other researchers (Canel et al., 2000; Cantrell et al., 2013; Cushman et al., 2003; Gawde et al., 2009; Kusari et al., 2011; Renouard et al., 2011; Zheljazkov et al., 2013) investigated Juniperus virginiana L. and other species as an alternative source of PDP.
The aim of this study was to examine the occurrence of PDP and dO-PDP in a number of selected Juniperus sp. collected in Poland. There is a lack of a comprehensive study on the lignans content in Juniperus sp. from this geographic region. The study aims to indicate possible, cheap natural source of PDP and dO-PDP.
Another objective was to examine in vitro cytotoxic activities of PDP and two ethanolic extracts obtained from selected Juniperus species: Juniperus scopulorum Sarg. “Blue Pacific” and J. communis L. “Depressa Aurea” against a set of leukemia cell lines: J45.01, HL-60, HL-60/MX1, HL-60/MX2, U-266B1, CEM/C1, CCRF/CEM.
Materials and methods
Plant extracts
Leaves of 11 cultivated Juniperus sp. were investigated (Table 1). The plant samples were obtained from two uniform locations. Sampling location of 31 varieties was the Nursery Farm Lucjan and Grzegorz Kurowscy in the community of Końskowola. The remaining 30 varieties were collected in the Botanical Garden of Maria Curie-Sklodowska University in Lublin. These sampling locations are spaced apart a distance of 50 km and characterized with the same soil type and are under the influence of the same climate conditions. Investigated plants were collected at the same stage of their maturity: 6–8 years. In the experiment, freshly grown leaves were used. The plant material was dried at room temperature. All samples were authenticated by a botanist, Prof. Anna Bogucka-Kocka (Pharmaceutical Botany Department, Medical University of Lublin, Poland, where voucher specimens were deposited), according to morphological characteristics and based on Adams (2011) – “Keys to Juniperus”. The extraction protocol was based on Renouard et al. (2011) with minor adjustments. The chopped dried leaves were ground to powder. Typically, 5 g DW (dry weight) powder was accurately weighed. Subsequently, the plant material was extracted with 50 mL of 100% methanol for 5 h at 25 °C under stirring in a water bath. After 5 h, extracts were filtrated and the residues were rinsed by the same amount of 100% methanol, in aliquots. The filtrates were pooled to yield 100 mL. The filtrates were additionally centrifuged for 15 min at 3000 rpm and stored in a dry, dark place at 4 °C. For further ultra-performance liquid chromatography (UPLC) analyzes, supernatants were filtered through a syringe filter 0.2 μm, prior to analysis. The injection volume of the sample was 1.5 µL.
Table 1. Podophyllotoxin and deoxypodophyllotoxin content in leaves of 11 species (61 varieties) of Juniperus sp. quantified by means of ultra-performance liquid chromatography.
To determine the cytotoxic activity of Juniperus scopulorum (A) and Juniperus communis (B), 10 mL of each methanol extract was taken and evaporated to dryness under reduced pressure. The residues were dissolved in 80% EtOH to give the stock solutions (18.45 mg/mL for A and 16.55 mg/mL for B).
Chemicals
PDP standard was of analytical purity grade (≥98%) from Sigma-Aldrich Co. (St. Gallen, Switzerland). The identity of dO-PDP peak was confirmed based on the obtained chromatographic and spectral data [retention time and mass-to-charge ratio (m/z)] and their comparison with published data (Avula et al., 2011).
Acetonitrile and methanol, of HPLC purity grade and formic acid (>98%), for LC-UV-MS separations, were purchased from J.T. Baker (Phillipsburg, NJ). Water was purified in-house with a Milli-Q water purification system Simplicity-185 (Millipore Co., Billerica, MA).
Standard podophyllotoxin solution for cytotoxic assay
A stock solution containing 13.25 µg/mL of PDP was prepared by dissolving accurately weighed 26.5 mg of PDP in 500 mL 80% ethanol. Subsequently, 160 µL of stock solution was taken and transferred to a sample vial and the volume was made up to 1 mL with 80% ethanol to prepare standard solution at a concentration of 2.12 µg/mL. Sample weighting and standard dissolution steps were repeated six times resulting in six independent PDP standard solutions at uniform concentration: 2.12 µg/mL.
Instrumentation and chromatographic conditions
Chromatographic conditions were based on Avula et al. (2011) with minor adjustments. Compounds of interest were analyzed using a Waters ACQUITY UPLC™ system (Waters Corp., Milford, MA), consisting of a binary pump system, sample manager, column manager, and PDA detector (also from Waters Corp., Milford, MA). Waters MassLynx software v.4.1 (Waters Corp., Milford, MA) was used for acquisition and data processing. The samples were separated on a BEH ShieldRP18 column (100 mm × 1.0 mm i.d., 1.7 µm, Waters Corp., Milford, MA), which was maintained at 40 °C. The flow rate was adjusted to 0.17 mL/min. The following solvent system: mobile phase A (0.1% formic acid in Milli-Q water, v/v) and mobile phase B (0.1% formic acid in acetonitrile, v/v) were applied. The gradient program was as follows: isocratic at 32% B for 6.0 min, 32% B to 100% B (0.1 min), hold at 100% B for 1.9 min, 100% B to 32% B (0.1 min), and hold at 32% B for 1.9 min. Samples were kept at 25 °C in the sample manager. The injection volume of the sample was 1.5 µL (partial loop with needle overfill mode) and samples were analyzed in triplicate. Strong needle wash solution (95:5, methanol:water, v/v) and weak needle wash solution (5:95, acetonitrile–water, v/v) were used. The detection wavelength was set at 210 nm at a 5 point/s rate, at 3.6 nm resolution. The separation was completed in 10 min. Peaks were assigned on the basis of their retention times and mass-to-charge ratio (m/z).
The MS analyses were carried out on a TQD mass spectrometer (Waters Corp., Milford, MA) equipped with a Z-spray electrospray interface. The following instrumental parameters were used for ESI-MS analysis of lignans (positive ionization mode): capillary voltage, 3.0 kV; cone voltage, 60 V; desolvation gas, N2650 L/h; cone gas, N250 L/h; source temp., 150 °C, desolvation temp. 350 °C, dwell time 270 ms. Compounds were analyzed in selected ion monitoring (SIM) mode; podophyllotoxin – [M + Na]+ = 437.1 m/z (retention time 1.70 min) and deoxypodophyllotoxin – [M + Na]+ = 421.1 m/z (retention time 4.25 min) (Figure 2).
Published online:
30 March 2015Figure 2. Exemplary chromatogram obtained for Juniperus scopulorum Sarg. “Moon Glow” leaf extract at 210 nm. dO-PDP, deoxypodophyllotoxin; PDP, podophyllotoxin.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iphb20/2015/iphb20.v053.i06/13880209.2014.943246/20150611/images/medium/iphb_a_943246_f0002_b.jpg"})
Figure 2. Exemplary chromatogram obtained for Juniperus scopulorum Sarg. “Moon Glow” leaf extract at 210 nm. dO-PDP, deoxypodophyllotoxin; PDP, podophyllotoxin.
Validation of UPLC method
The method was validated in compliance with the ICH guidelines in terms of the following parameters: specificity, precision, accuracy, linearity, and range (ICH Harmonised Tripartite Guideline, 2014). The method was specific for quantitation of PDP and dO-PDP in selected Juniperus sp. by means of UPLC. The method was shown to be precise (RSD <5%) both in the repeatability and intermediate precision experiments. In addition, a good linear regression model was obtained. The linearity of the proposed method was evaluated by processing the different calibration curves. The accuracy of this method was checked by conducting a recovery study at three different levels of PDP standard and the method was characterized with good accuracy.
Cell lines and culture medium
The following human leukemia cell lines were grown in suspensions: J45.01 – human acute T cell leukemia from American Type Culture Collection (ATCC® CRL-1990™), HL-60 – human Caucasian promyelocytic leukemia from American Type Culture Collection (ATCC® CCL-240™), HL-60/MX1 – human Caucasian acute promyelocytic leukemia from American Type Culture Collection (ATCC® CRL-2258™), HL-60/MX2 – human Caucasian acute promyelocytic leukemia from American Type Culture Collection (ATCC® CRL-2257™), U-266B1 – human myeloma from American Type Culture Collection (ATCC® TIB-196™), CEM/C1 – human Caucasian acute lymphoblastic leukemia from American Type Culture Collection (ATCC® CRL-2265™), CCRF/CEM – human Caucasian acute lymphoblastic leukemia from American Type Culture Collection (ATCC® CCL-119™). Cells were cultured by ATCC® protocol in 24-well plates with area growth 2 cm2 (Sarstedt, Lower Saxony, Germany) at the concentration of 5 × 105 cells/mL. All cultures were incubated in a humidified air atmosphere containing 5% of CO2 for 24 h at 37 °C in a Galaxy R incubator (Biotech, Wiesbaden, Germany). The growing medium consisted of RPMI 1640 medium (ATCC® 30-2001™), 10% (20% only for HL-60) FBS – fetal bovine serum with 2 mm l-glutamine (ATCC® 30-2020™), antibiotics (penicillin in concentration of 100 u/mL and streptomycin in concentration of 100 μg/mL (ATCC® 30-2300™), and amphotericin B in a concentration of 2.5 μg/mL (PAA Laboratories, P11-001, Pasching, Austria). One day after seeding, the cells were exposed to the examined PDP solution and ethanol extracts, in the various concentrations. In each of the samples, the final concentration of EtOH was reduced to 1% in the assays. This concentration of ethanol did not affect cell viability at all. All tests were independently performed in triplicate.
Trypan blue assay
In vitro cytotoxicity studies were carried out using Trypan blue assay. The cell lines, at concentration 5 × 105 cells/ml, were treated separately with podophyllotoxin solutions and with the analyzed extracts. Afterwards the cell lines were incubated for 24 h at 37 °C in humidified air atmosphere containing 5% CO2. Upon completion of the incubation step, the medium, from each plate, was removed by aspiration. Then, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and centrifuged at 800 rpm for 10 min, and then DPBS was removed by aspiration. Subsequently 10 µL of cell suspension was incubated for 5 min with 10 µL 0.4% Trypan blue solution (Bio-Rad Laboratories, Inc., Hercules, CA). Cells were observed and counted using a TC10 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA) for the presence of non-viable cells, which are dark blue and for viable cells, which are excluded from staining (Bogucka-Kocka et al., 2008). Seventeen different concentrations of each of the investigated samples were used to determine the IC50 value. The highest tested concentrations of PDP, for which the cell mortality was equal or higher than 90%, were as follows: 0.0170, 0.0318, 0.0170, 0.1908, 0.1855, 0.0509, and 0.1060 μg/mL for the respective leukemia cell lines: J45.01, U266B1, HL-60, HL-60/MX1, HL-60/MX2, CRF-CEM, and CEM/C1. The highest tested concentrations of Juniperus scopulorum var. “Blue Pacific” extract for which the mortality was not lower than 90% were as follows: 73.80, 3.21, 166.05, 230.63, 166.05, 230.63, 129.15, and 156.83 μg/mL for the respective cell lines: J45.01, U266B1, HL-60, HL-60/MX1, HL-60/MX2, CCRF-CEM, and CEM/C1, while the highest tested concentrations of J. communis var. “Depressa Aurea” extract for which the mortality was approximately 90% were as follows: 157.23, 314.45, 777.85, 331.00, 587.53, 248.25, and 248.25 μg/mL for the respective cell lines: J45.01, U266B1, HL-60, HL-60/MX1, HL-60/MX2, CRF-CEM, and CEM/C1.
Results and discussion
Analysis of podophyllotoxin and deoxypodophyllotoxin content
Analysis of literature data, concerning PDP content in the leaves of various Juniperus sp., indicates that the concentration of this lignan ranged from 0.6 mg/100 g DW in J. communis var. Horstmann (Kusari et al., 2011) to 2260 mg/100 g DW in J. bermudiana L. (Renouard et al., 2011). As for the concentration of deoxypodophyllotoxin, it ranged from 0.5 mg/100 g DW in J. blaaws (Muranaka et al., 1998) to 470 mg/100 g DW in J. bermudiana (Renouard et al., 2011).
In the present study, a method established by Avula et al. (2011) was applied for the determination of two lignans in selected Juniperus species and varieties. The results clearly showed that the analyzed samples differ in the content of the determined lignans (Table 1). The concentration of PDP ranged from 2.24 to 486.7 mg/100 g DW (0.002–0.487% DW), while the concentration of dO-PDP was found to be from 0 to 726.8 mg/100 g DW (0–0.727% DW) (Table 1).
In the present study, J. scopulorum sp. showed the highest concentration of PDP – 0.429%, 0.469%, and 0.487% in all tested varieties – Moon Glow Variegata, Moon Glow, and Blue Pacific, respectively. That is contrary to Renouard et al. (2011), who found no PDP in J. scopulorum.
Fairly curious is the fact that Renouard et al. (2011) also found no dO-PDP in J. scopulorum, while the amount of this compound, in J. scopulorum samples collected in Poland, was relatively high (0.223%, 0.262%, and 0.299%). The literature survey shows that the content of dO-PDP has never exceeded 0.6% DW (Cairnes et al., 1980; Hartwell et al., 1953; Kusari et al., 2011; Muto et al., 2008). In our study: J. davurica, J. sabina L. “Variegata”, and J. sabina var. “Mas” were identified as characterized with the highest dO-PDP concentration ever mentioned: 0.727%, 0.721%, and 0.618%, respectively.
Renouard et al. (2011), Gawde et al. (2009), Cantrell et al. (2013), and Zheljazkov et al. (2013) reported that PDP concentration in Juniperus sp. varied significantly depending on the geographic origin of the samples. To the best of our knowledge, this is the first time that PDP and dO-PDP contents have been determined as samples collected in Poland.
PDP has been reported to be present in J. scopulorum, as first confirmed by Hartwell et al. (1953) in amount of 0.17% and then confirmed by two groups in 2013. Different varieties of J. horizontalis, investigated by Zheljazkov et al. (2013) and Cantrell et al. (2013), showed concentration of PDP, ranging from 0% to 0.726%. In the present study, the investigated varieties of J. horizontalis were characterized with the PDP content ranging from 0.003% to 0.305%. These values were found to be higher when compared with the data reported by Cantrell et al. (2013).
Renouard et al. (2011) did not find PDP in J. conferta Parl. Contrary to this report, our study indicates that J. conferta collected from Polish flora contains PDP in trace amounts – approximately 0.005%.
Renouard et al. (2011) and Kusari et al. (2011) reported only traces of PDP in J. squamata Buch. Ham. The content of PDP in J. squamata, analyzed in the present study varied from 0.008% to 0.116%.
Almost all examined varieties of Juniperus sp., grown in Poland, have been investigated for the first time. To the best of our knowledge, there are no previous reports on quantification of PDP and dO-PDP in J. depressa Raff and J. davurica. This is also the first time that dO-PDP was quantified in the following species: J. chinensis, J. conferta, J. horizontalis, J. scopulorum, and J. virginiana. It has to be stressed that all the analyzed Juniperus samples were found to contain PDP and there were only 10 varieties without dO-PDP.
Analysis of cell viability
PDP as well as dO-PDP are known for their anticancer properties. Both lignans demonstrated a wide range of relevant properties against a number of cancer cell lines (Ikeda et al., 1998; Kim et al., 2002; Masuda et al., 2002; Muto et al., 2008; Renouard et al., 2011; Xu et al., 2010).
Seven leukemic cell lines: J45.01, HL-60, HL-60/MX1, HL-60/MX2, U-266B1, CEM/C1, and CCRF/CEM, were treated with PDP solution and EtOH extracts of the leaves of two Juniperus sp. Selection of the extracts was based on the PDP content, previously determined using the UPLC method.
The investigated cell lines represent different types of leukemia, namely acute leukemia and myeloma. It was decided to use a set of different leukemia cell lines, as there are considerable differences between these types, what may result in various performances in the cytotoxicity test.
Previously, the cytotoxicity of PDP against human leukemia cell lines has been tested only against HL-60 and CEM/C1 cells (Chia-Jen et al., 2002; Neung-Ju et al., 2006). Determination of cytotoxicity against other leukemia cell lines would extend our knowledge about PDP cytotoxicity. Investigated extracts have not been tested before towards a set of different leukemia cell lines and this is the first time when the cytotoxicity of the extracts is reported.
One of the selected species – Juniperus scopulorum was chosen to check its activity against the set of cell lines, as it was characterized by the highest PDP content. The activity of the other one, J. communis, was investigated due to the lowest PDP content, when compared with other species (Table 1). It was decided to examine these two varieties to check whether the amount of PDP influences cytotoxicity of Juniperus extracts. The viable cells were counted using Trypan blue dye exclusion assay. The percent of cell viability was determined by dividing the number of viable cells by the number of total cells. The percentage of viable cells in controls was higher than 97%.
The results of the cytotoxic assays showed that the solution of PDP and the extracts of two selected Juniperus species were active against all examined cell lines. High activity of the investigated samples was observed against some of the cancer cell lines. The cytotoxicity values obtained for the extracts were obviously lower than those obtained for pure PDP. As shown in Table 2, PDP solution and EtOH extracts of the investigated species produced a dose-dependent inhibition of cell growth at various concentrations (0.0040–0.0286 µg/mL for PDP solution and 0.369–24.825 µg/mL for Juniperus extracts). The IC50 values were calculated from the graph for each of the EtOH extracts. Previously, Neung-Ju et al. (2006) determined the IC50 value for podophyllotoxin, which equaled 0.0014 µg/mL against the CEM/C1 cell line, which was lower the value obtained in the present study (IC50 value for CEM/C1 cells was 0.0286 µg/mL). Chia-Jen et al. (2002) reported 0.2 nM as the minimal dose of PDP, which induced the apoptosis of HL-60 cells.
Table 2. IC50 values obtained for two Juniperus ethanol leaf extracts and podophyllotoxin solution against the set of leukemia cell lines (values represent the mean ± SD of three replicates).
This is the first time that PDP cytotoxic activity against a set of leukemia cell lines has been reported. Upon the exposition to PDP solution, the highest leukemia cell mortality was observed in J45.01 cell line while the lowest mortality was observed in CEM/C1 (Table 2).
In case of the examined extracts, high cell mortality was observed after cell exposition to Juniperus scopulorum ethanolic extract for almost all cancer cell lines used in the study. The highest mortality of cells was observed in the J45.01 cell line after exposition to EtOH leaf extract of Juniperus scopulorum. The lowest mortality of cells was observed in the U-266B1 cell line after exposition to EtOH leaf extract of J. communis.
It was demonstrated for the first time that the examined Juniperus ethanolic leaf extracts were active against a set of leukemia cell lines. The obtained results indicate that there is a possible correlation between the level of PDP and the exerted cytotoxic effect of Juniperus leaf extracts.
Conclusion
It is the first time that PDP and dO-PDP were quantified in a number of Juniperus sp. cultivated in Poland. Juniperus scopulorum Sarg. “Blue Pacific” contained the highest amount of PDP (486.7 mg/100 g DW) from among all the analyzed species and varieties. The highest level of dO-PDP was confirmed in Juniperus davurica: 726.8 mg/100 g DW. These relatively high contents of active lignans may stimulate further research aiming to use selected Juniperus sp. as a cost-effective source of cytotoxic metabolites.
The cytotoxic activity of PDP and two ethanolic leaf extracts obtained from Juniperus scopulorum and Juniperus communis against investigated leukemia cell lines was measured and the IC50 values were given for the first time. Juniperus scopulorum extract was found to be the most active against the cell lines used in the study.
| Voucher specimen number | Species | Varieties | Podophyllotoxin mg/100 g DW | Deoxypodophyllotoxin mg/100 g DW | |
|---|---|---|---|---|---|
| 1 | Juniperus chinensis L. | “Blue and Gold” | 208.3 ± 6.2 | 204.2 ± 4.6 | |
| 2 | “Robust Green” | 60.6 ± 0.9 | 224.3 ± 5.5 | ||
| 3 | “Stricta” | 4.7 ± 0.2 | ND | ||
| 4 | “Hirta” | 3.0 ± 0.1 | ND | ||
| 5 | “Blauuw” | 3.5 ± 0.5 | ND | ||
| 6 | “Blue Alps” | 15.7 ± 1.8 | 79.1 ± 12.1 | ||
| 7 | “Kuriwao Gold” | 176.0 ± 8.7 | 152.6 ± 3 | ||
| 8 | “Mountbatten” | 3.4 ± 0.3 | ND | ||
| 9 | “Variegata” | 6.3 ± 0.8 | ND | ||
| 10 | Juniperus communis L. | “Gold Cave” | 5.0 ± 0.8 | 37.1 ± 3.8 | |
| 11 | “DepressaAurea” | 2.2 ± 0.1 | 64.7 ± 13.1 | ||
| 12 | “Horstmann” | 12.5 ± 0.6 | 78.1 ± 9.2 | ||
| 13 | “Pendula” | 3.9 ± 0.4 | 54.9 ± 10.7 | ||
| 14 | Juniperus conferta Parl. | “Blue Pacific” | 6.9 ± 0.5 | ND | |
| 15 | “Emerald Sea” | 5.4 ± 0.3 | ND | ||
| 16 | Juniperus davurica Pall. | 14.6 ± 0.5 | 726.8 ± 28.6 | ||
| 17 | Juniperus depressa Raf. | 13.4 ± 1.3 | 56.2 ± 4.2 | ||
| 18 | Juniperus horizontalis Moench | “Blue Forest” | 27.5 ± 0.9 | 227.5 ± 9.7 | |
| 19 | “Golden Carpet” | 120.0 ± 3.6 | 154.3 ± 16 | ||
| 20 | “Hughee” | 305.1 ± 4.4 | 178.8 ± 4.7 | ||
| 21 | “Icee Blue”® | 117.3 ± 5.3 | 111.3 ± 2.9 | ||
| 22 | “Jukon Belle” | 162.0 ± 3.8 | 187.0 ± 13.3 | ||
| 23 | “Prince of Wales” | 164.2 ± 4.8 | 232.2 ± 6 | ||
| 24 | “Winter Blue” | 192.3 ± 4.5 | 535.1 ± 9.9 | ||
| 25 | “Alpina” | 128.5 ± 7.4 | 300.2 ± 15.5 | ||
| 26 | “Blue Chip” | 143.7 ± 4.8 | 108.7 ± 11.4 | ||
| 27 | “Douglasii” | 120.2 ± 9.1 | 376.4 ± 25.1 | ||
| 28 | “Parsonii” | 3.0 ± 0.1 | 57.6 ± 2.4 | ||
| 29 | “Plumosa” | 156.0 ± 0.9 | 407.6 ± 15.2 | ||
| 30 | “Wiltonii” | 183.0 ±19.5 | 170.6 ± 20.1 | ||
| 31 | Juniperus sabina L. | “Variegata” | 61.3 ± 2.9 | 721.2 ± 22.9 | |
| 32 | “Mas” | 45.0 ± 1.2 | 618.4 ± 28.6 | ||
| 33 | “Tamariscifolia” | 349.0 ± 12.7 | 209.3 ± 24.4 | ||
| 34 | Juniperus scopulorum Sarg. | “Blue Pacific” | 486.7 ± 19.8 | 262.2 ± 4 | |
| 35 | “Moon Glow Variegata” | 428.7 ± 7.4 | 299.0 ± 8 | ||
| 36 | “Moon Glow” | 469.1 ± 16 | 223.2 ± 11.5 | ||
| 37 | Juniperus squamata Buch. Ham | “Blue Slow” | 7.8 ± 1.5 | ND | |
| 38 | “Dream Joy” | 105.1 ± 2.1 | 53.2 ± 3.5 | ||
| 39 | “Floreant” | 4.6 ± 0.5 | ND | ||
| 40 | “Gold Tip” | 115.6 ± 11.5 | 58.6 ± 7.2 | ||
| 41 | “Hunnetorp” | 71.3 ± 0.8 | 59.6 ± 2.9 | ||
| 42 | ex D. Don “Blue Carpet” | 26.0 ± 1.3 | 83.2 ± 6.2 | ||
| 43 | ex D. Don “Holger” | 66.3 ± 1 | 41.9 ± 1.9 | ||
| 44 | Juniperus virginiana L. | “Blue Clouds” | 384.5 ± 18.2 | 106.6 ± 2.4 | |
| 45 | “Golden Spring” | 248.9 ± 10.6 | 91.2 ± 2.2 | ||
| 46 | “Cinerascens” | 156.6 ± 5.3 | 196.0 ± 16.9 | ||
| 47 | “Hetz” | 206.7 ± 10.4 | 231.2 ± 9.7 | ||
| 48 | “Kosteri” | 4.3 ± 0.3 | 94.1 ± 8.3 | ||
| 49 | “Pyramidiformis” | 245.8 ± 11.2 | 390.2 ± 6.7 | ||
| 50 | “Reptans” | 196.8 ± 5.8 | 216.8 ± 6.1 | ||
| 51 | “Tripartita” | 169.5 ± 7.3 | 165.9 ± 6 | ||
| 52 | Juniperus x-media Melle | “Gold Slow” | 140.2 ± 3 | 133.8 ± 4.3 | |
| 53 | “Gold Sovereign” | 182.1 ± 4 | 127.3 ± 8.8 | ||
| 54 | “Mordigan Aurea” | 194.4 ± 21.9 | 260.0 ± 31.8 | ||
| 55 | “Saybrook Gold” | 121.4 ± 4.5 | 112.3 ± 4 | ||
| 56 | “Mint Julep” | 157.8 ± 12.8 | 205.8 ± 11 | ||
| 57 | “Old Gold” | 153.6 ± 12.6 | ND | ||
| 58 | “Pfitzeriana Glauca” | 194.4 ± 3.6 | 110.7 ± 8.4 | ||
| 59 | “Pfitzeriana” | 152.7 ± 3.9 | 149.1 ± 16.6 | ||
| 60 | “Snow” | 253.6 ± 4.8 | 152.1 ± 3.9 | ||
| 61 | Pfitzeriana “Dierks Gold” | 140.2 ± 7 | 129.2 ± 6.1 |
Quantitation was performed using a PDA detector, the detection wavelength was set at 210 nm (values are the mean ± SD (standard deviation) of three replicates, ND, not detected).
| Ethanol extracts µg/ml | |||
|---|---|---|---|
| Cell line | Juniperus scopulorum Sarg. “Blue Pacific” (%) | Juniperus communis L. var. communis “Depressa Aurea” (%) | Podophyllotoxin solution (µg/ml) (%) |
| J45.01 | 0.369 ± 2.27 | 3.310 ± 3.22 | 0.0040 ± 4.31 |
| U-266B1 | 4.612 ± 4.19 | 24.825 ± 4.62 | 0.0064 ± 5.16 |
| HL-60 | 4.612 ± 4.27 | 8.275 ± 4.47 | 0.0085 ± 4.27 |
| HL-60/MX1 | 1.107 ± 2.85 | 4.799 ± 4.26 | 0.0078 ± 5.15 |
| HL-60/MX2 | 9.225 ± 3.62 | 5.461 ± 2.21 | 0.0106 ± 4.16 |
| CCRF-CEM | 0.646 ± 3.31 | 4.137 ± 3.54 | 0.0064 ± 4.09 |
| CEM/C1 | 1.107 ± 4.04 | 5.461 ± 4.82 | 0.0286 ± 5.38 |
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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