A flavonoid, 5-hydroxy-3,7-dimethoxyflavone, from Kaempferia parviflora Wall. Ex. Baker as an inhibitor of Ca²⁺ signal-mediated cell-cycle regulation in yeast

Abstract

Calcium (Ca²⁺) signal transduction pathways play important roles in the regulation of diverse biological processes in eukaryotes ranging from unicellular (e.g., yeasts) to complex multicellular (e.g., humans) organisms. Small-molecule inhibitors of Ca²⁺-signaling pathways in humans can be of great medical importance, as represented by the immunosuppressants FK506 and cyclosporine A. A high-throughput drug screening assay for inhibitors of Ca²⁺-signaling has been developed on the basis of the ability of test compounds to restore the severe growth defect of a Ca²⁺-sensitive zds1 null-mutant strain YNS17 of Saccharomyces cerevisiae in a medium containing a high concentration of calcium ions. A previous screening of Thai medicinal plants using this yeast-based assay indicated that the crude extract of Kaempferia parviflora Wall. Ex. Baker contains a potent inhibitory activity. The aim of this study was to isolate and characterize the pure compound(s) responsible for this inhibitory activity against Ca²⁺-mediated cell-cycle regulation in yeast. Dichloromethane and methanol extracts of K. parviflora rhizomes were subjected to bioassay-mediated chromatographic fractionation using this yeast [YNS17 (Δzds1) strain]-based assay to screen for and select positive fractions. From the dichloromethane extract, four known flavonoid compounds with significant inhibitory bioactivity were obtained: compounds 1 (5-hydroxy-3,7-dimethoxyflavone), 2 (5-hydroxy-7-methoxyflavone), 3 (5-hydroxy-3,7,4’-trimethoxyflavone) and 4 (5,7-dimethoxyflavone). The inhibitory activity of all four compounds was dose-dependent. Compound 1 exhibited the highest activity and with no observed cytotoxic activity against the yeast. The Ca²⁺ induced severe growth defect, abnormal budding morphology, and G2 cell-cycle delay of the Δzds1 yeast strain were all alleviated or abrogated by 200 μM compound 1. Therefore, we conclude that 5-hydroxy-3,7-dimethoxyflavone possesses a potent inhibitory activity against the Ca²⁺-mediated cell-cycle regulation.

Introduction

In eukaryotic organisms, elevation of the intracellular calcium ion (Ca²⁺) concentration serves as a universal second messenger that plays key regulatory roles in numerous biological processes, including cell proliferation, muscle contraction, fertilization, development, motility, memory, and apoptosis (Clapham 1995). Generally, small molecule inhibitors of Ca²⁺-dependent signaling pathways exert their physiological effects by an evolutionary conserved manner throughout eukaryotes (Mager and Winderickx 2005). Consequentially, inhibitors found to be functional in yeast may well be active in relevance to humans.

In Saccharomyces cerevisiae, Ca²⁺ signals have been implicated in the regulation of G2/M cell-cycle progression (Mizunuma et al. 1998). The Ca²⁺-sensitive zds1 null mutant (Δzds1) of S. cerevisiae can grow apparently normally in yeast-peptone-dextrose [YPD; 1 % (w/v) yeast extract, 2 % (w/v) peptone and 2 % (w/v) glucose] medium, but exhibits severe growth defects when cultivated in YPD containing a high concentration of CaCl₂ (Mizunuma et al. 1998). Accordingly, a convenient drug-screening procedure for inhibitors of Ca²⁺ signaling pathways was developed using the S. cerevisiae Δzds1 strain YNS17 on the basis of the ability of test drugs to antagonize the highly activated Ca²⁺-signals, leading to the recovery of growth of the Ca²⁺-sensitive mutant in high-calcium medium (Shitamukai et al. 2000). According to this procedure, potential targets for the inhibitors of the Ca²⁺ signaling pathway are calcineurin (potential target of immunosuppressants and anti-inflammatory agents), GSK3-family protein kinases (potential target of drugs for type 2 diabetes and Alzheimer’s disease), protein kinase C (potential target of anti-cancer drugs) and HSP90 (potential target of anti-cancer drugs) (Boonkerd et al. 2011). Therefore, small molecule Ca²⁺-signaling inhibitors obtained by such screens have high potential as drugs of medical interest.

A previous extensive screening study using this Δzds1 mutant yeast-based assay revealed that the crude extract of Kaempferia parviflora Wall. Ex. Baker contained a potent inhibitory activity (Boonkerd et al. 2011). K. parviflora is a member of Zingiberaceae (ginger) family, locally known in Thailand as Black Galingale or Kra-chai-dam. Members of this family, including K. parviflora, are traditionally very popular for health promotion (Putiyanan et al. 2004). The rhizomes of K. parviflora, locally known as Thai ginseng, have been used as a traditional medicine for various purposes, including the treatment of leucorrhea, oral diseases (Chomchalow et al. 2003; Sudwan et al. 2006), stomach ache, flatulence, digestive disorders, gastric ulcer as well as diuresis and tonic (Wattanapitayakul et al. 2007).

The aim of this study was to isolate pure active compound(s) from K. parviflora that display a significant ability to inhibit the Ca²⁺-induced cell-cycle regulation of yeast.

Materials and methods

Plant materials

Fresh rhizomes of K. parviflora were purchased from a Thai market in Pratumthani. A voucher specimen (BKF 152278) has been deposited at the Bangkok Forest Herbarium (BKF), Royal Forest Department, Chatuchak, Bangkok, Thailand.

Extraction and fractionation of the K. parviflora rhizome extract

Fresh rhizomes of K. parviflora were sliced and dried in the open air for a few days and then powdered into small pieces with an electrical blender. The dried powder (5 kg) was extracted with dichloromethane (CH₂Cl₂) at a 1:1 (w/v) ratio in a soxhlet apparatus and then filtered. The plant residue was then similarly extracted with a 1:1 (w/v) ratio of methanol (CH₃OH). The two solvent extracts were concentrated separately in a rotary evaporator under reduced pressure to give a brown solid residue of 470.0 g and 90.0 g [9.4 % and 1.8 % (w/w) of the rhizome mass] from the CH₂Cl₂ and CH₃OH extracts, respectively. Fractionation of the extracts was performed by sequential chromatography with increasing solvent polarity, with bioselection of positive fractions from each stage being guided by the Δzds1 yeast-based assay (rescue from Ca²⁺-mediated inhibition of proliferation), until pure compounds were obtained. Chemical structures of the pure isolated compounds were elucidated by NMR (Varian Mercury Plus 400, Palo Alto, CA) and mass spectrometry (MS) (Micromass UK Limited, Manchester, UK) and compared with those reported in the literature (Sutthanut et al. 2007).

Yeast strains, cultivation and the yeast-based screening assay

The Ca²⁺-sensitive Δzds1 strain YNS17 (MATa zds1::TRP1 erg3::HIS3 pdr1::hisG URA3 hisG pdr3::hisG), in which several genes responsible for drug resistance have been deleted (Chanklan et al. 2008), was used as the indicator cell for the bioassay. YNS17 cells were cultivated on YPD agar plates at 30 °C.

The growth-promoting and cytotoxic effects of compound 1 on the Δzds1 strain were evaluated by assays in liquid YPD and solid YPD agar culture as described by Wangkangwan et al. (2009). In brief, with respect to using solid medium, YNS17 cells (6 × 10⁵ cells) were dispersed in molten soft YPD-agar and then overlaid on top of a YPD agar plate containing 150 mM CaCl₂. Five microliters each of various concentrations of compound 1 (500, 250, 125, 62.5, 31.3 and 15.6 μM) was applied by spotting onto the surface of the agar plate. In addition, 500 nM FK506 and absolute ethanol were used as the positive and negative (solvent only) controls, respectively. Plates were then cultured at 30 °C for 2 days prior to examination for cell growth.

The growth-promoting effect of 200 μM compound 1 was confirmed in liquid culture in YPG medium containing 75 mM CaCl₂. YNS17 cells were seeded at 5 × 10⁶ cells/mL in YPD broth and cultured at 30 °C with or without compound 1 (or FK506 or DMSO as positive and negative controls) for 30 min before the addition of CaCl₂ to 75 mM (preincubation was to ensure the effect of the drug) and cultured at 30 °C. Samples were taken at 2 h intervals for measurement of the cell density with a hemacytometer under light microscopy following suitable dilution.

Flow cytometric analysis

Analysis of DNA content in the cells was performed by flow cytometric evaluation of cell DNA content as described by Mizunuma et al. (1998), using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ). The yeast cell suspensions were fixed, permeabilized (Mizunuma et al. 1998), and then stained with propidium iodide for DNA analysis.

Results and discussion

Purification of the extract from K. parviflora

Fresh rhizomes of K. parviflora were extracted with CH₂Cl₂ and the 300.0 g of the CH₂Cl₂ extract was further fractionated using solvents with increasing polarity to obtain four pure compounds, designated as 1, 2, 3 and 4, that are likely to be known flavonoids.

Compound 1 was obtained as yellow solid (6.27 g; 2.09 % yield). The molecular formula was suggested as C₁₇H₁₄O₅ and confirmed by ¹H-NMR. The ¹H-NMR spectrum (Table 1) clearly showed two sets of methoxy group resonating at δ _H 3.87 (3H, s) and 3.88 (3H, s) while a methylene group appears at δ _H 6.37 (1H, d, J = 2.0 Hz), 6.46, (1H, d, J = 2.4 Hz), aromatic protons at δ _H 7.52 (3H, m), 8.07 (2H, m) and hydroxyl group at δ _H 12.59 (1H, s). The ¹H-NMR spectral data of compound 1 showed a pattern similar to that of 5-hydroxy-3,7-dimethoxyflavone (Table 1, see Sutthanut et al. 2007).

Table 1 Comparison of the ¹H-NMR data between 5-hydroxy-3,7-dimethoxyflavone and compound 1

Compound 2 was isolated as a yellow crystal (1.02 g; 0.34 % yield). The molecular formula was suggested as C₁₆H₁₂O₄, and confirmed by ¹H-NMR, which showed an identical pattern to that of 5-hydroxy-7-methoxyflavone (Sutthanut et al. 2007).

Compound 3 was obtained as a yellow crystal (3.45 g; 1.15 % yield). The molecular formula was suggested as C₁₈H₁₆O₆ and its ¹H-NMR spectral data was identical to that of 5-hydroxy-3,7,4′-trimethoxyflavone (Sutthanut et al. 2007).

Compound 4 was isolated as a yellow crystal (7.92 g; 2.64 % yield). The molecular formula was suggested as C₁₇H₁₄O₄ and its ¹H-NMR spectrum showed the pattern expected from that of 5,7-dimethoxyflavone (Sutthanut et al. 2007).

Accordingly, the likely structures of the four flavonoids (1–4) are shown in Fig. 1. When the biological activities of the serial dilutions of each compound were compared, compound 1 showed the highest activity against the Ca²⁺-induced growth arrest of the YNS17 strain yeast (Table 2). Therefore, compound 1 was chosen for further characterization.

Fig. 1

Structure of the four flavonoid compounds obtained from Kaempferia parviflora (compounds 1–4), shown as the 7-methoxyflavone backbone and the three variable substituent groups (R₁–R₃). In addition the ring numbering of the cyclic atom positions is shown for reference

Table 2 Relative activity of the four isolated compounds (1–4) from Kaempferia parviflora rhizomes in suppressing the growth defect due to Ca²⁺-induced G2 cell-cycle arrest of the YNS17 (Δzds1) yeast cells. Relative activity of each compound is indicated by +, ++, +++, and ++++ in increasing order of activity as estimated by the diameter of the cell-growth zone in the yeast-based assays (as shown in Fig. 2a for compound 1). The result shown are representative of those seen in three independent assays

Effect of compound 1 on growth of the Δzds1 mutant yeast strain YNS17

The growth-promoting effect of compound 1 on the Ca²⁺-sensitive Δzds1 strain YNS17 was evaluated by assays in solid and liquid YPD culture. For solid medium, the assay cells suspended in molten soft agar containing CaCl₂ were solidified. The samples were spotted on the surface of the medium. After 2 days of incubation, no visible growth of the YNS17 yeast cells on the agar plate was evident, but their growth was restored in a dose-dependent manner around the spot where compound 1 was applied (Fig. 2a). In the liquid culture assay, YNS17 yeast cells grew well in YPD medium and the addition of 200 μM compound 1 showed no discernible inhibition of this growth (Fig. 2b). In the presence of 75 mM Ca²⁺ the YNS17 cells hardly grew at all, as expected, but this was reversed in a dosage-dependent manner by the addition of compound 1 (400, 200 and 100 μM, respectively) or the calcineurin inhibitor FK506 (500 nM), to a similar extent as each other (Fig. 2b). These results suggest that compound 1 alleviated the Ca²⁺-induced growth defect of Δzds1 cells, whilst compound 1 itself had no significant toxic effect on the growth of YNS17 yeast cells (Fig. 2b).

Fig. 2

Effect of compound 1 on the Ca²⁺-induced growth inhibition of the Ca²⁺-sensitive YNS17 yeast strain (Δzds1) in a solid-plate and b liquid culture assays. a 1–6 represent the application of 5 μL of 500, 250, 125, 62.5, 31.3 and 15.6 μM compound 1, respectively, whilst 500 nM FK506 (P) and absolute ethanol (N) were the positive and negative controls. Plates were evaluated after 2 days at 30 °C for YNS17 growth, and the image shown is representative of two independent trials. b YNS17 cells (seeded at 5 × 10⁶ cells/mL) were cultivated in YPD broth at 30 °C and samples were taken at 2 h intervals for measurement of cell density with a hemacytometer. Data are shown as the mean ± 1 SD, and are derived from three independent repeats. Symbols: ♦ YPD medium; − YPD medium with 200 μM compound 1; ▲ YPD medium with 75 mM CaCl₂ and 250 nM FK506; ■ YPD medium with 75 mM CaCl₂; --*-- YPD medium with 75 mM CaCl₂ and 400 μM compound 1; --●-- YPD medium with 75 mM CaCl₂ and 200 μM compound 1; --▲-- YPD medium with 75 mM CaCl₂ and 100 μM compound 1

Effect of compound 1 on YNS17 cell morphology, nuclear division and cell-cycle progression

The hyperactivation of Ca²⁺ signaling in YNS17 cells by external CaCl₂ leads not only to a severe growth defect, but also to the G2/M cell-cycle arrest/delay and polarized bud growth (Mizunuma et al. 1998). Accordingly, the ability of compound 1 to overcome these physiological effects induced by Ca²⁺ was examined in liquid YPD broth containing 100 mM CaCl₂. YNS17 cells grown in YPD plus 100 mM CaCl₂ medium mostly (> 70 %) displayed an abnormal morphology with an elongated bud (Fig. 3, left) and defects in nuclear division (Fig. 3, right), due to the hyperactivation of Ca²⁺ signaling. In contrast, when cotreated with 200 μM compound 1, only 25 % of the cells showed an abnormal morphology (Fig. 3, left) with an equal distribution of nuclei between mother and daughter cells (Fig. 3, right). These results were broadly similar to those obtained with 500 nM FK506 (Fig. 3), and revealed that compound 1 could suppress Ca²⁺-induced abnormal Δzds1 yeast cell morphology and the defect in nuclear division.

Fig. 3

Effect of compound 1 on Ca²⁺-induced abnormal YNS17 cell morphology and defect of nuclear division. YNS17 cells in YPD were pretreated for 30 min with either DMSO as a negative (solvent) control, 500 nM FK506 as a positive control, or 200 μM compound 1. Then, 100 mM CaCl₂ was added and the treated cells were cultivated for 6 h. Cell morphology was examined after Hoechst 33342 staining under a phase contrast light (left) and a fluorescence microscope (right). Images shown (× 40 magnification) are representative of those seen in at least 4–5 such fields of view (>100 cells) per sample and three independent samples

We further examined the effect of compound 1 on Ca²⁺-induced G2 cell-cycle arrest/delay by flow cytometric analysis of propidium iodide-stained YNS17 cells. YNS17 cells grown for 3 h in YPD plus 100 mM CaCl₂ revealed a 2.5-fold higher 2C DNA content than those with a 1C DNA content, indicating that a G2 phase delay was induced by Ca²⁺ (Fig. 4). In contrast, in the presence of 200 μM compound 1, the proportion of cells with a 2C DNA content decreased to about 0.9-fold that of those with a 1C DNA content (Fig. 4). A similar 1C / 2C cell DNA content profile was obtained with YNS17 cells treated with FK506 (Fig. 4). Thus, compound 1 alleviated the G2/M cell-cycle delay caused by hyperactivation of Ca²⁺-signaling.

Fig. 4

Effect of compound 1 on Ca²⁺-induced G2 cell-cycle delay. YNS17 yeast cells grown in YPD were treated as described in Fig. 4, except that cells were treated with the compounds for 3 h. The harvested cells were fixed, stained with propidium iodide, and subjected to flow cytometric analysis. The percentages shown are the proportion of cells with a DNA content of 1C and 2C, respectively, after exclusion of populations of < 1C and > 2C). Images shown are based upon 5,000 gated events (cells) and are representative of three independent repeats

Previously, flavonoids (flavan-3-ols) and chalcones (phenyl styryl ketones) have been isolated from K. parviflora extract without the use of a bioassay-guided fractionation and purification (Jaipetch et al. 1983; Panthong et al. 1994; Yenjai et al. 2004). Subsequently, several studies on the biological activities of those flavonoids have been reported, including studies showing that compound 2 (5-hydroxy-7-methoxyflavone) has anti-mutagenic and anti-allergic activities (Azuma et al. 2011; Tewtrakul et al. 2008), and that compound 4 (5,7-dimethoxyflavone) exhibits an anti-inflammatory effect, in terms of the carrageenan-induced hind paw edema in rats (Panthong et al. 1994), and anti-mutagenic and anti-butyrylcholinesterase activities (Azuma et al. 2011; Sawasdee et al. 2009). However, to the best of our knowledge, the biological activities of compounds 1 (5-hydroxy-3,7-dimethoxyflavone) and 3 (5-hydroxy-3,7,4’-trimethoxyflavone) have not yet been clarified. Here, we showed that compound 1 from K. parviflora exhibited an inhibitory activity against the Ca²⁺-induced G2 cell-cycle arrest, but a detailed mechanism for the action of compound 1 in yeast still needs to be investigated.

Conclusion

Four flavonoids, compounds 1, 2, 3 and 4, were isolated from the CH₂Cl₂ extract of K. parviflora rhizomes through bioassay guided fraction selection, using inhibition of the Ca²⁺-mediated inhibition of proliferation of Δzds1 yeast cells, to detect pure compound inhibitors of the Ca²⁺-induced G2 cell-cycle arrest. Compound 1 (5-hydroxy-3,7-dimethoxyflavone) exhibited the highest activity against the Ca²⁺-induced phenomena in YNS17 yeast cells, such as the growth defect, polarized bud growth, defect of nuclear division and the G2/M phase delay, without any significant toxic effect on the cell growth. We are currently investigating its molecular target in yeast.