The genus Peucedanum is comprised of more than 120 species that are widely distributed in Europe, Asia, Africa and North America . It contains a number of plant species used in traditional medicine for the treatment of asthma and angina , and in some areas these plants are considered potent for the treatment of diabetes  or obesity . Moreover, Peucedanum species have been found to exhibit antimicrobial, cytotoxic, immunostimulatory and antitumor [5-10] properties, which were related to the presence of furanocoumarins and pyranocoumarins.
Peucedanum luxurians Tamamsch. (Apiaceae) is an endemic plant of Armenia. Except for the detection of terpene derivatives’ in the organic extracts , little is known about this particular species. In continuation of a research interest in coumarins from plants of the Apiaceae [9, 10, 12, 13], the isolation of coumarins from P. luxurians and an evaluation of their immunomodulating activity is reported.
The immunomodulatory assessment of natural and synthetic coumarins is of significant interest, and in particular their influence on apoptosis has been well documented. Decursin, coumarin, 7-hydroxycoumarin, 6-nitro-7-hydroxycoumarin,7-hydroxy-4-methyl-coumarin, scopoletin, and esculetin are among the best-known examples of pro-apoptotic coumarin derivatives [14, 15]. However, the molecular pro-apoptotic mechanism of action is not well understood. In the search for novel metabolites to serve as candidates for further biological assessment, the coumarins from the petroleum ether extract of P. luxurians were evaluated for their immunomodulatory activity.
2.1 Plant material
The aerial parts of the cultivated P. luxurians Tamamsch. (Apiaceae) used in this study were collected from the Botanical Garden of the Department of Pharmacognosy (Medical University of Lublin, Poland). Seeds for cultivation came from the Hortus Botanicus, Universitatis Posnaniensis, Poznan, Poland (S_S047_008_0004_7973_S003), where the plant was identified by a specialist in botany. A voucher specimen is deposited in the Department of Pharmacognosy, Medical University of Lublin (number 2534).
2.2 General experimental procedures
1D and 2D NMR spectra were recorded on a Bruker DRX 400 and Bruker AC 200 spectrometers [1H (400 and 200 MHz) and 13C (50 MHz)]: chemical shifts are expressed in ppm downfield from TMS. GC-MS analysis was carried out on a Hewlett-Packard 6890-5973 system operating in the EI mode, equipped with a 30 m × 0.25 mm i.d.; 0.25 µm HP-5 MS capillary column; temperature program: 60 °C (5 min) to 280 °C at rate of 3 °C/min; injection temperature 200 °C. Medium pressure liquid chromatography (MPLC) was performed with a Büchi model 688 apparatus on columns containing silica gel 60 Merck (20-40 µm). Column chromatography was performed on columns containing Si gel 60 Merck (40-63 µm). Preparative thin layer chromatography (pTLC) was performed on plates coated with silica gel 60 F254 Merck.
2.3 Extraction and isolation of compounds
The air-dried and powdered plant material (64.9 g) was extracted in a Soxhlet apparatus for 48 h with petroleum ether and then with methanol. The organic solvents were evaporated under reduced pressure and the obtained residues were 2.2 g and 13.5 g, respectively. The petroleum ether extract was submitted to column chromatography eluting with cyclohexane:CH2Cl2:MeOH (gradient from 100:0:0 to 0:99:1) to afford 12 fractions (A1-A7). 8-Methoxypeucedanin (1) (6 mg) and stenocarpin isobutyrate (2) (22 mg) were obtained pure from fractions A7 and A9, respectively, and identified based on complete spectroscopic analysis . Fraction A5 (70 mg) was re-chromatographed eluting with cyclohexane:EtOAc (gradient from 100:0 to 20:80) to afford 10 fractions. Fraction B3 afforded peucedanin (3) (2.3 mg), and fraction B6 afforded oficinalin isobutyrate (8-demethoxystenocarpin isobutyrate) (4) (7.4 mg). Fraction A10 was re-chromatographed eluting with cyclohexane:EtOAc (gradient from 100:0 to 20:80) to afford isoimperatorin (5) (3.7 mg) and 8-methoxyisoimperatorin (6) (1.3 mg).
The methanolic extract was submitted to column chromatography with CH2Cl2:MeOH (gradient from 100:0 to 50:50) as eluent to afford 16 fractions. Fractions D1-D5 (95 mg) were re-chromatographed on column chromatography with cyclohexane:CH2Cl2:MeOH (gradient from 100:0:0 to 0:99:0.5 gradient) to afford 12 fractions. Fraction E4 afforded bergapten (5-methoxypsoralen) (7) (3 mg), and fraction E8 afforded xanthotoxin (8-methoxypsoralen) (8) (6 mg). Fraction E11 was separated using pTLC eluting with CH2Cl2:MeOH (98:2 v/v) to afford stenocarpin (9) and 8-demethoxy-stenocarpin (oficinalin) (10) (6 mg).
2.4 Immunomodulating activity
The immunomodulatory activity of the isolated coumarins was examined in cultures of HL60 cells (obtained from ATCC) and peripheral blood lymphocytes (PBL). All cells were cultured in RPMI-1640 medium supplemented by 10% fetal calf serum,100 U/mL of penicillin, 100 µg/mL of streptomycin and 2 mM L-glutamine at 37 °C in an atmosphere of 5% CO2 in air. HL60 cells were re-seeded every day to maintain them in exponential and asynchronous growth phase. PBL were isolated through ficol (Gradisol, Nycomed) centrifugation, and subsequently cultured with the addition of phytohemaglutinin (PHA) (Sigma). All experiments were carried out in 4-, 6- or 12-well flasks (Nunc) for up to 5 days. All compounds were dissolved in DMSO and stored for a maximum of one month as a 1 mM stock solution. The tested concentrations were 1 µM, 10 µM, and 100 µM for all compounds. Corresponding DMSO amounts were added to the control samples. Doses of 1 µM showed no/minimal,10 µM moderate (not shown) and 100 µM maximal effects. All experiments were performed three to five times and the results of a representative are shown. Apoptosis and cell cycle distribution were evaluated using the acridine orange (AO) and flow cytometric assays. AO has unique abilities to bind stoichiometrically to both RNA and DNA, which enables the measurement of their contents by flow cytometry. Accuracy, methodological simplicity (no cell wash thereby avoiding cell loss from further analyses), and relatively low costs are the most important advantages of this assay. All methodological details are described in previous publications from this laboratory [16, 17]. The presence of apoptotic cells was also confirmed by in situ labeling DNA strand breaks by BrdUTP in the assay catalyzed by exogenous terminal deoxynucleotidyl transferase (TUNEL) using the APO-BRDU kit (Phoenix Flow System, USA). All methodological details are presented elsewhere [16, 17].
3 Results and discussion
Ten coumarins were isolated in pure form from the extracts of P. luxurians. Compounds 1-6 were obtained from the petroleum ether extract and compounds 7-10 from the methanolic extract. The structures were elucidated based on MS and NMR data and compared with those available in the literature [12, 18, 19]. Compounds 1, 2, 4, 6, 9 and 10 are very rare in nature, thus their biological properties had not been previously investigated. Given that coumarins have a distinctive ability to regulate a diverse range of cellular pathways that can be explored for selective cytotoxic activity , it was decided to assess their immunomodulatory activity.
All of the isolates were evaluated for their immunomodulatory potential, except for compounds 6, 9 and 10 based on the paucity of material. Initially, their influence on apoptosis was tested. In this set of experiments, the promyelocytic HL60 cell line was used. All tested compounds showed dose-dependent and time-dependent pro-apoptotic abilities (maximal at 100 µM and minimal or no effect at 10 µM - data not shown). Compounds 7 and 8 were the most powerful inducers of apoptosis. The pro-apoptotic actions of the tested compounds were maximal in the 48 h culture (Table 1). The dye used for the assessment of apoptosis - acridine orange (AO) also enabled an examination of the cell cycle distribution. Surprisingly, a partial block in the G1 phase was observed with coumarin derivatives in all cultures, but only at 100 µM concentrations (Table 2). The percentage of cells blocked in the G1 phase increased with time and was maximal after 72 h. Moreover, the cultures showing the highest apoptosis rate had no, or a very limited, G1 cell block (see: the 48 h cultures of compounds 7 and 8, Table 2). It should be mentioned that the cells blocked in the G1 phase also showed a decreased percentage of cells in the S phase of the cell cycle suggesting a lower proliferation in these cultures (data not shown). To establish if these changes were reversible, the coumarin derivatives were washed out and the cells cultured for the following two days. Under those conditions, the apoptotic index was restored to the control value and the G1 block was not seen.
The percentage of apoptotic HL60 cells cultured in the presence or absence of 100 µM of coumarin derivates for up to 3 days. Two latter rows show the results obtained after washing out the coumarins.
|72 h+24 h||2.3||0.8||0.8||0.6||0.6||2.5||2.2||0.8|
|72 h+48 h||6.2||2.2||6.5||1.9||1.8||2.8||2.2||2.5|
The percentage of HL60 cells in G1 phase of cell cycle.
|72 h+24 h||52.0||48.8||44.0||46.6||51.4||55.8||60.4||47.4|
|72 h+48 h||37.6||38.3||35.1||42.4||42.6||37.9||39.0||46.0|
All of the information obtained from this set of experiments (induction of apoptosis, block in G1, decrease in the neoplastic cell line proliferation) suggested that coumarin derivatives (especially 7 and 8) could warrant more advanced biological assessment, and could be perceived as potential anti-cancer medications.
To examine this hypothesis, it was decided to repeat the experiment in a model of PHA-stimulated PBL (peripheral blood lymphocytes) (Table 3). Freshly isolated PBL were silenced in the G0 phase and PHA was applied to induce their proliferation. As soon as they reached the G1 phase (after 24 h of cultivation), the coumarin derivatives were added. In the instance of all of the compounds, except for 2, low or moderate apoptosis induction was observed. Compound 2 showed strong pro-apoptotic PBL abilities. Moreover, there were no cell blocks observed in the PBL cultures (Table 4). Surprisingly, a dose-dependent decrease of RNA content in cells in the cell cycle was observed. The use of AO in these experiments enabled an assessment of the MFI (mean fluorescent intensity) of red fluorescence, which reflects the RNA content in the cells. There were no visible changes in RNA content in the G0 cells, or in G1 cells (Table 5). The reason of this phenomenon is unknown (possibly an increased consumption or a decreased production of RNA), but perturbation in RNA synthesis seemed to be most likely explanation of the phenomenon (no changes in G0).
The percentage of apoptosis in PBL cultures.
|24 h PHA+24 h||20.8||29.5||21.6||24.5||24.6||26.8||24.9||36.6|
|24 h PHA+48 h||22.3||26.0||30.7||22.4||28.3||34.4||28.7||33.5|
The cumulative percentage of PBL in G0 and G1 phase of cell cycle stained with AO and analyzed by flow cytometry.
|24 h PHA+24 h||66.0||56.9||69.5||60.6||67.2||65.6||67.0||47.9|
|24 h PHA+48 h||62.8||66.0||62.7||61.5||68.6||62.1||64.5||58.0|
The RNA MFI (mean fluorescence intensity) of PBL in G1 phase of cell cycle stained with AO and analyzed by flow cytometry.
Based on the changes in the RNA contents in the PBL the HL60 results were re-analyzed. Surprisingly, these changes were observed in the HL60 cells also, however, they were visible only at the highest concentrations of coumarin derivatives (100 µM) (Table 6).
The RNA MFI (mean fluorescence intensity) of HL60 cells in G1 phase of cell cycle stained with AO and analyzed by flow cytometry.
The obtained data corresponded with the G1 block, anti-proliferative, and pro-apoptotic activities described for other coumarin derivates [14, 15, 21]. Similar to previous results, mainly tested in cancer cell lines rather than in normal epithelial cells, decursin induced G1 arrest, strongly inhibited growth, and also induced apoptosis . It should be emphasized that the isolated compounds were characterized by relatively strong pro-apoptotic activities (especially 7 and 8). Moreover, compound 2 acted significantly in normal cells. In other studies, although the anti-proliferative abilities of the tested coumarins (6-nitro-7-hydroxycoumarin, 3,6,8-trinitro-7-hydroxycoumarin) were mentioned, the S or G2M phase cell accumulation was described, and not the G1 phase accumulation . The reason for these cell changes is not simplistically explained, although some changes in the expression of cyclins and cyclin-dependent kinases have been previously confirmed . According to the literature, coumarin derivatives may effect caspase activation [14, 22, 23], and 6-nitro-7-hydroxycoumarin decreases the expression of cyclin D . Esculetin induces cytochrome c release from mitochondria to cytosol, reduces BCL-2 protein expression  and up-regulates the p27 expression , while scopoletin upregulates NF-κB translocation to the nucleus by increasing of IκBα degradation . As well as the caspase-dependent pathway, the caspase-independent pathway is also involved in the induction of apoptosis by decursin . Coumarin derivatives exhibit pro-apoptotic actions; for example osthole prevents the anti-Fas antibody-induced apoptosis in vivo in mice through an effect upstream of caspase-3 activation . Dicoumarol and warfarin also inhibit wild-type p53-mediated apoptosis . Other well-established abilities of coumarin derivatives are their anti-proliferative properties [14, 15, 21, 24, 27-29]. Most coumarins induce the G1 cell cycle block [14, 15, 21, 30], while some of them act at the S stage [14, 28, 29], or even arrest the cell cycle at G2M . Among other immunomodulatory abilities of coumarins, the induction of ubiquitin-independent p53 degradation by dicumarol, esculetin and warfarin , and the redifferentiation of human hepatoma cells increasing their malignancy by 7-hydroxy-4-methylcoumarin have been reported .
This is the first time that the immunomodulatory properties of simple and furanocoumarin derivatives have been evaluated. Previously, xanthotoxin was reported to induce cell apoptosis in HepG2 cells in a modest way when used alone (IC50 =124.07 ± 6.89 µM for 24 h treatment), and to inhibit hepatocellular carcinoma cell invasion and migration properties [31, 32]. Similarly, bergapten (0.5-50 µM) appears to exert its anticarcinogenic properties through a cytotoxic effect, inducing apoptosis and inhibiting proliferation in the human hepatocellular carcinoma cell line by killing cells directly and inducing apoptosis by arresting cells at the G2/M phase of the cell cycle . Additionally, a weak influence on apoptosis induction and heat-shock proteins expression in HeLa cells has been noticed .
All of the investigated coumarins expressed immunomodulatory abilities. They induced apoptosis, blocked the G1 phase of the cell cycle, and reduced the proliferation of HL60 cells. Compounds 7 and 8 exhibited the strongest activity among the tested coumarins. Taken together, these actions suggested that selected coumarin derivatives could be considered for further investigation as possible candidates for anti-cancer drugs. This hypothesis was supported by the weaker actions of most of the tested compounds on PBL (no cell blocks, weak apoptosis induction). On the other hand, the strong reduction of RNA content in PBL, and a relatively strong induction of apoptosis by 2, indicated that this compound warrants further investigation and could be successfully used in the treatment of autoimmune diseases. Considering the above data, it is important to explore these hypotheses through in vivo studies.
The authors would like to express gratitude to the Director and the employees of the Botanical Garden, UAM, Poznań for the plant material. The authors would also like to thank Professor Geoffrey A. Cordell, University of Florida, for his review of the manuscript.
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