Abstract
Context: Bellis perennis L. (Asteraceae) (common daisy) is a herbaceous perennial plant known as a traditional wound herb; it has been used for the treatment of bruises, broken bones, and wounds. Bellis perennis has also been used in the treatment of headache, common cold, stomachache, eye diseases, eczema, skin boils, gastritis, diarrhea, bleeding, rheumatism, inflammation, and infections of the upper respiratory tract in traditional medicine.
Objective: Antitumor activities of different fractions of B. perennis flowers at different concentrations were evaluated and through bioassay-guided fractionation and isolation procedures a saponin derivative (1) was isolated from the active fraction obtained from the n-butanol extract of flowers of the title plant by column chromatography.
Materials and methods: Antitumor activities of different fractions of B. perennis flowers at different concentrations were evaluated using Potato Disc Tumor Induction Bioassay. Structure elucidation of 1 was accomplished by spectroscopic methods [1D- and 2D-NMR, and LC-ESI(APCI)-TOF-MS(MSn)].
Results: The present study showed the antitumor activity of fractions obtained from B. perennis flowers for the first time. The most active fraction showed 99% tumor inhibition at 3000 mg/L. An oleanane-type saponin was isolated through bioassay-guided studies.
Discussion and conclusion: Through antitumoral bioassay-guided fractionation and isolation procedures, 1 was isolated from the active fraction of B. perennis. The detailed NMR data of compound 1 is given for the first time.
Introduction
Bellis perennis L. (Asteraceae) (common daisy) is a herbaceous perennial plant that grows in meadows, lawns and other grassy areas (Panda, Citation2004). It is native to western, central, and northern Europe, but is commonly found as an invasive plant in North America (Tutin et al., Citation1976). Bellis perennis is known as a traditional wound herb (Al-Douri & Al-Essa, Citation2010; Leporatti & Ivancheva, Citation2003), and it was used for the treatment of bruises, broken bones, and wounds by Crusaders in the Middle Ages (Mitich, Citation1997). Bellis perennis has also been used in the treatment of sore throat (Uysal et al., Citation2010), headache (Uzun et al., Citation2004), common cold, stomachache, eye diseases, eczema, skin boils, gastritis, enteritis, diarrhea, bleeding, rheumatism, inflammation, and infections of the upper respiratory tract in traditional medicine (Çakılcıoğlu et al., Citation2010; Panda, Citation2004). Furthermore, astringent, expectorant, diuretic, booster, purgative, and diaphoretic properties have been reported (Baytop, Citation1999; Duke et al., Citation2002; Grieve, Citation1982).
The main constituents of this plant are triterpenoid saponins (Hiller et al., Citation1988; Li et al., Citation2005; Morikawa et al., Citation2008; Schopke et al., Citation1991; Yoshikawa et al., Citation2008), essential oils (Avato et al., Citation1997; Kavalcioglu et al., Citation2010), and flavonoids (Gudej & Nazaruk, Citation2001). Antimicrobial (Avato et al., Citation1997; Kavalcioglu et al., Citation2010), antifungal (Desevedavy et al., Citation1989), antihyperlipidemic (Morikawa et al., Citation2010b), antioxidant (Kavalcioglu et al., Citation2010), postpartum antihemorrhagic (Oberbaum et al., Citation2005), pancreatic lipase inhibitor (Morikawa et al., Citation2010a), cytotoxic activities against HL-60 human promyelocytic leukemia cells (Li et al., Citation2005), anxiolytic properties (Karakas et al., Citation2011), and wound healing activity (Karakas et al., Citation2012) of B. perennis have also been investigated.
The aim of the present study was to investigate the antitumor activitiy of B. perennis and to isolate the active constituent through bioassay-guided fractionation techniques and to elucidate the structure of active principle.
Antitumor activities of different fractions of B. perennis flowers at different concentrations were evaluated using the Potato Disc Tumor Induction Bioassay modified by McLaughlin’s group (Ferrigini et al., Citation1982). In this study, the purpose was to investigate antitumoral activities of the title plant and to identify the active principle.
Materials and methods
General
1D- and 2D-NMR measurements were recorded in MeOH-d4 at room temperature on a Varian VNMRS 600 NMR spectrometer (Palo Alto, CA) (1H 600 and 13C 150 MHz). Chemical shifts are given in ppm with tetramethylsilane (TMS) as an internal standard. The MS analysis was performed on a Shimadzu LC-IT-TOF MS analyzer (Kyoto, Japan) using either electrospray (ESI) or atmospheric pressure chemical ionization (APCI). Polyamide column chromatography was applied using polyamide 6 (Fluka, Steinheim, Germany, 50–160 mm). Medium-pressure liquid chromatographic (MPLC) separations were carried out on Büchi glass column packed with LiChroprep C18 (Merck, Darmstadt, Germany, 40–63 mm), using a Büchi B-681 pump (Flawill, Switzerland). Preparative TLC (P-TLC) was carried out on Kieselgel GF254 glass plates (Analtech-Uniplate Z51303-2, Newark, NJ). Qualitative TLC analyses were carried out on pre-coated Kieselgel 60 F254 aluminum plates (Merck). Compounds were detected by UV fluorescence and spraying 1% vanillin/H2SO4, followed by heating at 100 °C for 1–2 min.
Plant material
Bellis perennis flowers and pedicels were collected from the Abant Izzet Baysal University Campus, Bolu, Turkey, in May 2009. It was identified by Arzu Ucar Turker. Voucher specimen (collection number AUT-1909) was deposited at the Abant Izzet Baysal University (AIBU) Herbarium, Bolu, Turkey.
Preparation of the B. perennis n-butanol fraction
The collected B. perennis flowers and pedicels were dried in an oven at 40 °C and then ground into a powder. Plant sample of B. perennis flower (250 g) was extracted with 2500 ml methanol (99%) at 40 °C for 18 h and then filtered. Methanol was evaporated under vacuum and the residue was dissolved in water (100 ml). First, hexane was added into the extract in a separation funnel and the hexane portion was discarded. Second, n-butanol was added into the extract in a separation funnel and the aqueous portion was discarded. The remaining fraction was soon evaporated to remove the n-butanol and the residue was suspended in water. The frozen n-butanol fraction was lyophilized using a freeze-dryer at −65 °C.
Isolation
The n-butanol fraction was subjected to C-18 silica gel vacuum liquid chromatography (H2O/MeOH→0/100%) to afford nine fractions (Fr. A–I). These fractions at different concentrations (10 000, 1000 and 100 mg/l) were evaluated using the Agrobacterium tumefaciens Potato Disc Tumor Bioassay modified by McLaughlin’s group (Al-Douri & Al-Essa, Citation2010). Complete tumor inhibition was observed with Fr. G at 10 000 mg/l similar to camptothecin(positive control). Fraction G was chosen for further phytochemical studies and this fraction was chromatographed over a polyamide column eluting with stepwise H2O–MeOH gradient (H2O/MeOH 0→100) to obtain Frs. G1–G4. Active subfraction (Fr. G3) was applied to C18-Medium Pressure Liquid Chromatography (C18-MPLC) (Flawill, Switzerland) eluting with a stepwise H2O–MeOH gradient (50–75% MeOH). The active subfraction (Fr. G3) was applied to preparative thin layer chromatography using a silica gel plate and a CHCl3:MeOH:H2O (61:32:7) solvent system to isolate 1 ()
Figure 1. Structure of compound 1.
Compound 1: [α]D 20: −58 (c 0.33 MeOH), 1H and 13C NMR data: , HPLC-ESI-MS [M − H−] (m/z 1393.66)
Antitumor activity assay
Antitumor activity of all extracts was assessed with the potato disc method as modified by McLaughlin’s group (Ferrigini et al., Citation1982). Agrobacterium tumefaciens (ATCC® 23341) was cultured on a Yeast Extract Media (YEM) for 2–3 days at 28 °C. Camptothecin (Sigma, St. Louis, MO) (tumor suppressant) served as a positive control and water was used as a negative control. Suspensions of A. tumefaciens in phosphate-buffered saline (PBS) were standardized to 1.0 × 109 Colony-Forming Units (CFU) as determined by an absorbance value of 0.96 ± 0.02 at 600 nm. All fractions were dissolved in water to a final concentration of 100, 750, 1000, 1500, 3000, 5000 or 10 000 mg/ml. All extracts and control solution were filter-sterilized (sterile 0.22 µm filter, Millex®, Billerica, MA). The test solutions consisted of 600 µl of extract or control solution, 150 µL of sterile distilled water and 750 µl of the standardized A. tumefaciens in PBS.
Potatoes (Solanum tuberosum L.) were washed and scrubbed with a brush under running water and surface-sterilized by immersion in 10% commercial bleach (Domestos®, Konya, Turkey) for 20 min. Tubers were then placed on sterile paper towels and cut along either side revealing the largest surface area available. The trimmed tubers were then immersed in 20% commercial bleach (Domestos®) for 15 min. Cylinders (10 mm diameter) were cut from the center of potato tissue (skin portion was eliminated) using a cork borer on sterile paper towels and placed in sterile distilled water with lactic acid (pH = 4.0). Cylinders were rinsed twice using sterile distilled water with lactic acid. Each cylinder was cut into 0.5 cm discs after excluding 1 cm end pieces. These discs were transferred to 24-well culture plates containing water-agar (15 g/l). Each disc was overlaid with 50 µL of appropriate inoculum. The potato discs where inoculated within 30 min after cutting them. Plates were incubated at 28 °C in the dark for 2 weeks. After 2 weeks, discs were stained with Lugol’s reagent (I2KI; 5% I2 plus 10% KI in distilled water) and tumors on each disc were counted. Lugol’s reagent stains the starch in potato tissue to dark blue to dark brown color, but the tumors do not get stained and appear creamy to orange. Experiments were repeated three times. Percent inhibition of tumors was calculated using the formula: “% inhibition = [(solvent control mean-tested extract mean)/solvent control mean] × 100” (McLaughlin, Citation1991; McLaughlin et al., Citation1993, Citation1998).
Bacterial viability testing
Standardized bacterial suspension (1 × 109 CFU of A. tumefaciens in PBS) was serially diluted with PBS to 1 × 103 CFU. Bacterial viability was determined by incubating 1 ml of each plant fraction with 1 ml of bacterial suspension (1 × 103 CFU of A. tumefaciens in PBS) in microcentrifuge tubes (4 tubes per extract) and left for 30 min. After 30 min of inoculation, 0.1 ml of inoculum (bacteria + fraction) was removed and inoculated on YEM media with the spread plate technique. After 24 h incubation of inoculated plates at 28 °C, colonieswere counted. Also, bacterial growth was observed by growth across the plates (Coker et al., 2003).
Results and discussion
Nine different fractions (Frs. A–I) obtained from the n-butanol fraction of B. perennis flowers were prepared at different concentrations (100, 1000 or 10 000 mg/l). Generally, all the tested fractions at all concentrations showed tumor inhibition. Among the tested fractions, the best tumor inhibition was observed with Fr. G at 10 000 mg/l similar to the positive control camptothecin (100 % tumor inhibition) ().
Table 1. Mean number of tumors and % tumor inhibitions of fractions (Fr. A, Fr. B, Fr. C, Fr. D, Fr. E, Fr. F, Fr. G, Fr. H and Fr. I) obtained from the n-butanol fraction of B. perennis flowers at 100, 1000 or 10 000 mg/l and controls (water and camptothecin).
Then, Fr. G was separated to four different fractions (Frs. G1–G4). Especially, fraction G3 showed very good activity at 1500 mg/l as well as 3000 mg/l compared to camptothecin that was used as a positive control. The best antitumor activity (no tumor formation) was observed with Fr. G3 at 3000 mg/l (). Later, Fr. G3 was purified and compound 1 was obtained. These findings make the title plant very interesting for the search for new therapeutic agents. Our results suggest that further investigations are necessary to determine the medicinal properties of the plant in vivo and in vitro.
Table 2. Mean number of tumors and % tumor inhibitions of fractions (Fr. G1, Fr. G2, Fr. G3 and Fr. G4) obtained from Fr. G fraction of B. perennis flowers at 750, 1500 or 3000 mg/l and controls (water and camptothecin).
Compound 1 was isolated as an amorphous powder. High-resolution HPLC-ESI-MS in negative ionization mode revealed a small peak (m/z 1393.66) belonging to [M − H−] ion and two much stronger peaks (m/z 1439.6563 and 742.3341) belonging to [M + HCOO]− and [M + 2(HCOO)]2−, respectively, all corresponding to the molecular mass 1394.6718 Da. The 1H and 13C chemical shifts of 1 were assigned by various NMR experiments. Data are shown in . Signals were assigned to six methyls [δ 0.80, 0.88, 0.91, 0.95, 1.33, 1.39 (3H each, all s H-26, 29, 24, 30, 25, 27)], a methylene and three methines bearing an oxygene function [δ 3.26, 3.33 (each 1H, both d, J = 11.1 Hz, H-23); δ 3.68 (1H, d, J = 3.5 Hz, H-3), 4.21 (1H, m, H-2), 4.46 (1H, m, H-16)], an olefin [δ 5.43 d, J = 3.4 Hz, H-12] arising from aglycone moiety. The NMR data of aglycone moiety of 1 are in good agreement with those reported for polygalacic acid, which is an oleanane-type saponin (Yoshikawa et al., Citation2008). Furthermore, signals belonging to a fucopyranosyl (δ 5.43–1.06), a xylopyranosyl (δ 4.49–3.22), three rhamnopyranosyls [(δ 5.14–1.24 Rha-1), (δ 5.14–1.32 Rha-2), and (δ 5.14–1.24 Rha-3)], an arabinofuranosyl (δ 5.05–3.59), moieties together with an acetyl group [δ 2.14 (3H, s)] were also observed in the 1H spectrum of 1. The chemical shifts of all the individual protons of the sugar units were ascertained from the combination of 2D TOCSY and 2D ROESY spectral analysis, and the 13C chemical shifts of their relative attached carbons were assigned unambiguously from the HSQC spectrum. HMBC correlations between the 4-proton in the fucopyranosyl moiety [δ 5.34 (d, J = 3.4 Hz) and the acetyl carbon (δ 171.1) suggested the presence of an acetyl group at C-4 of fucopyranosyl. The determination of the aglycon skeleton and linkage sites of sugar units was done from the HMBC spectrum, which showed key correlation peaks between the proton signal at δ 4.86 (H-1Rha1) and the carbon resonance at δ 81.1 (C-3), δ 5.43 (H-1Fuc) and δ 175.8 (C-28), δ 3.81 (H-2Fuc) and δ 100.6 (C-1Rha2), δ 3.53 (H-4Rha2) and δ 105.5 (C-1Xyl), δ 3.47 (H-3Xyl) and δ 101.1 (C-1Rha3), δ 3.89 (H-3fuc) and 110.2 (C-1Ara). The fragmentation analysis performed by HPLC-APCI-IT-TOF MS1-MS3 spectra, measured mostly in negative ionization mode, showed the presence of fragments corresponding to the mass of the aglycone and expected sugar units. The structure of compound 1 was determined on the basis of all the above information (Muraoka et al., Citation2009).
Table 3. 1H, 13C-NMR of 1 (1H: 600 MHz, 13C: 150 MHz, MeOH-d4).
Conclusions
The present study showed the antitumor activity of fractions obtained from B. perennis flowers for the first time. Also, an oleanane-type saponin was isolated through bioassay-guided studies, and spectral data of 1 was given for the first time. Perennisaponin (A–M) are also isolated from the title plant so far (Morikawa et al., Citation2010a; Yoshikawa et al., Citation2008). Their structures are closely related to compound 1.
Declaration of interest
The NMR/LC-MS part of this work was supported by the VEGA grant no. 1/0972/12 and 1/1305/12.
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