A yeast-based screening system identified bakkenolide B contained in Petasites japonicus as an inhibitor of interleukin-2 production in a human T cell line

ABSTRACT

Ca²⁺ signaling is related to various diseases such as allergies, diabetes, and cancer. We explored Ca²⁺ signaling inhibitors in natural resources using a yeast-based screening method and found bakkenolide B from the flower buds of edible wild plant, Petasites japonicus, using the YNS17 strain (zds1Δ erg3Δ pdr1/3Δ). Bakkenolide B exhibited growth-restoring activity against the YNS17 strain and induced Li⁺ sensitivity of wild-type yeast cells, suggesting that it inhibits the calcineurin pathway. Additionally, bakkenolide B inhibited interleukin-2 production at gene and protein levels in Jurkat cells, a human T cell line, but not the in vitro phosphatase activity of human recombinant calcineurin, an upstream regulator of interleukin-2 production. Furthermore, bakkenolide A showed weak activity in YNS17 and Jurkat cells compared with bakkenolide B. These findings revealed new biological effects and the structure–activity relationships of bakkenolides contained in P. japonicus as inhibitors of interleukin-2 production in human T cells.

Graphical Abstract

Petasites japonicus showed inhibitory effect against Ca²⁺ signaling of mutant yeast YNS17 strain. Bakkenolide B, an active component, suppressed interleukin-2 production in Jurkat cells.

Ca²⁺ signaling, Petasites japonicus, bakkenolide B, interleukin-2

Abbreviations

Abbreviations
 
• ELISA:

  enzyme-linked immunosorbent assay

 
• EtOAc:

  ethyl acetate

 
• HPLC:

  high-performance liquid chromatography

 
• PMA:

  phorbol 12-myristate 13-acetate

 
• SD:

  standard deviation

 
• TFO:

  trifluoperazine dihydrochloride

 
• YPD:

  yeast extract-peptone-dextrose

Ca²⁺ is an important second messenger involved in various events within a living organism, such as bone metabolism, blood pressure regulation, muscle contraction, blood clotting, and cell differentiation (Clapham 1995). Thus, Ca²⁺ signaling is responsible for a variety of diseases, such as allergy, inflammation, diabetes, cancer, and hypertension (Ma and Beaven 2009). Indeed, many clinical agents targeting Ca²⁺ signaling, such as calcineurin inhibitors FK506 and cyclosporin A and the Ca²⁺-channel blocker diltiazem, have been used for the treatment of these diseases.

For the above reasons, we explored new regulators of Ca²⁺ signaling from natural resources, including microorganisms and food ingredients, using a yeast-based screening system. Recently, screening methods using yeast cells have been successfully used for drug discovery (Tucker 2002; Outeiro and Giorgini 2006; Yashiroda et al.2010; Uesugi et al.2014; Kume et al.2015; Satoh et al.2017; Zimmermann et al.2018). The YNS17 strain (zds1Δ erg3Δ pdr1/3Δ) is a Ca²⁺-sensitive mutant of Saccharomyces cerevisiae (Mizunuma et al.1998; Shitamukai et al.2000). As this strain exhibits growth-restoring activity by interrupting Ca²⁺ signaling, the inhibitors of this pathway can easily be detected as an obvious growth zone is formed around the active compounds. Using this system, we have identified many inhibitors of Ca²⁺ signaling, such as eremoxylarin A (from endophytic fungi) (Ogasawara et al.2008), falcarindiol (from Japanese parsley) (Yoshida et al.2013), and kujigamberol derivatives (from Kuji amber) (Kimura 2019), from various types of natural resources.

Petasites japonicus is an edible wild plant that is native to Japan. The flower buds of this plant are called “fukinotou,” in Japan, or “bakke,” in northeast Japan, and has been recognized as one of the representative wild plants that announce the arrival of spring. Fukinone and petasin are the main components of this plant, and have various biological effects, such as antiallergic effects (Shimoda et al.2006) and modulation of glucose metabolism (Adachi et al.2014). Additionally, as P. japonicus contains many polyphenols, such as chlorogenic acid, caffeic acid, and fukinolic acid, it is expected to have potent antioxidative effects (Hiemori-Kondo 2020). Therefore, it appears to be a promising source of novel bioactive compounds. In fact, P. japonicus has already been marketed as a functional ingredient in Japan.

In this study, we isolated and performed functional analyses of the new inhibitor of Ca²⁺ signaling identified from P. japonicus using a human cell line.

Materials and methods

Chemicals and strains

The yeast strains used in the present study were W303-1A and YNS17 (MATa zds1::TRP1 erg3::HIS3 pdr1::hisG URA3 hisG pdr3::hisG) (Ogasawara et al.2008). Difco^® yeast extract–peptone–dextrose (YPD) broth and agar were obtained from Becton Dickinson (Franklin Lakes, NJ, USA). FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (now Astellas Pharma Inc., Tokyo, Japan) and purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Trifluoperazine dihydrochloride (TFO), bakkenolide B, and bakkenolide A were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), BioBioPha Co., Ltd. (Yunnan, China), and ChemFaces Biochemical Co., Ltd. (Hubei, China), respectively. Unless otherwise stated, the chemicals used were of the best commercially available grade.

Extract library

Various materials, including agricultural, forestry, and fishery products, were obtained from Iwate prefecture (Japan). They were freeze-dried and powdered in a mill. The powder was extracted overnight with either methanol or hexane. The extracts were filtered using filter paper and dried using a rotary evaporator. The concentrated methanol and hexane extracts were brought to a concentration of 50 mg/mL by dissolving in methanol and ethanol, respectively, and then stored at −35 °C as an extract library.

Screening of Ca²⁺ signaling inhibitors

This procedure was performed on Petri dishes containing YPD agar (Becton Dickinson) and CaCl₂. The media were inoculated with the mutant YNS17 strain (zds1Δ erg3Δ pdr1/3Δ) according to a previously described procedure (Ogasawara et al.2008). Samples dissolved in MeOH were spotted on the surface of YPD agar plates containing the YNS17 strain and 0.3 m CaCl₂. The plates were incubated at 28 °C for 3 days. The inhibitory effect on Ca²⁺ signaling was determined by examining the strength and/or distinction of the yeast growth zone. The immunosuppressive drug FK506 was used as the positive control (Ogasawara et al.2008).

Isolation of an active compound from Petasites japonicus

Dried flower buds of P. japonicus (63.00 g) was powdered and extracted with MeOH at room temperature. The MeOH extract (11.34 g) showed growth-restoring activity against the YNS17 strain, and it was diluted with water and extracted 3 times with 1 volume of hexane. Only the hexane layer exhibited activity in the obtained organic layer and water layer. The hexane layer (1.79 g) was subjected to silica gel column chromatography (hexane and ethyl acetate (EtOAc)) and separated into 16 fractions (hexane:EtOAc = 1:0, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, and MeOH). In these fractions, Fr. 7-13 (hexane:EtOAc = 3:1-1:2) showed activity. Since Fr. 8 (228.7 mg) and Fr. 9 (177.8 mg; hexane:EtOAc = 2:1) exhibited potent activity, these 2 fractions were combined and concentrated under reduced pressure. The active compound, which was a colorless oil, was purified (85.5 mg) by high-performance liquid chromatography (HPLC) with the following parameters: mobile phase, MeOH:H₂O (75:15); flow rate, 15 mL/min; and column, InertSustain C18 (20 mm i.d. × 250 mm; GL Sciences Inc., Tokyo, Japan).

Liquid chromatography-mass spectrometry analysis

Samples were analyzed by liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (Triple TOF 5600+, SCIEX). The isolated compound and purchased standard of bakkenolide B were separated using an InertSustain AQ-C18 column (2.1 mm diameter × 50 mm, 1.9 µm; GL Sciences Inc., Fukushima, Japan) with gradient elution (water containing 0.1% formic acid/acetonitrile containing 0.1% formic acid, 0-2 min: 90/10; 2-14 min: 90/10 to 20/80; 14-18 min: 20/80) at a flow rate of 0.2 mL/min. The column temperature was maintained at 40 °C. The pressures of the nebulizer gas, turbo gas, and curtain gas were 50, 50, and 30 psi, respectively. The temperature of the turbo gas was 550 °C. The voltage of the ion spray was 5500 V. Samples were ionized by the electrospray ionization method using the positive ion mode, and bakkenolide B was detected at a retention time of 14.03 min using the above method.

Li⁺ sensitivity

Li⁺ sensitivity was measured using wild-type S. cerevisiae (W303-1A) as described previously (Ogasawara et al.2008). Five microliters aliquots of samples dissolved in MeOH was spotted on the surface of plates containing wild-type yeast with or without 0.16 m LiCl. The plates were incubated at 28 °C for 2 days.

Cell line and culture

Jurkat cells (RCB3052, RIKEN BioResource Center, Tsukuba, Japan) were cultured in RPMI 1640 medium (Sigma Aldrich Corp., St. Louis, MO, USA) supplemented with 10% heat-inactivated FBS (Gibco, Invitrogen Corp., Carlsbad, CA, USA) and penicillin (100 U/mL)–streptomycin (100 µg/mL)–amphotericin B (0.25 µg/mL) (Sigma-Aldrich Corp.) at 37 °C in a humidified atmosphere of 5% CO₂.

Real-time PCR

Jurkat cells were seeded in 24-well plates (5 × 10⁵ cells/well). The cells were treated with samples dissolved in MeOH for 30 min, added with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL ionomycin for 3 h, and harvested and centrifuged at 3000 rpm for 5 min at 4 °C. The obtained pellets were subjected to total RNA extraction using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany), and RNA was reverse-transcribed to cDNA using the PrimeScript RT reagent kit (Takara Bio, Otsu, Japan). All procedures were performed according to the supplier's protocol. Real-time PCR analysis was performed using the Fast SYBR Green master mix (Applied Biosystems, Foster City, CA, USA) and a QuantStudio 3 real-time PCR system (Applied Biosystems). The PCR primer sequences were as follows: IL-2 forward primer 5′-AGACCCAGGGACTTAATCAGC-3′, reverse primer 5′-AATGGTTGCCTCATCAGC-3′; 18S (housekeeping gene) forward primer 5′-TAAGTCCCTGCCCTTTGTACACA-3′, reverse primer 5′-GATCCGAGGGCCTCACTAAAC-3′. FK506 was used as the positive control.

ELISA

Jurkat cells were seeded in 24-well plates (5 × 10⁵ cells/well). The cells were treated with samples dissolved in MeOH for 30 min, and then with 10 ng/mL PMA and 500 ng/mL ionomycin for 24 h. The medium was collected in a tube and centrifuged at 1500 rpm for 10 min at 4 °C. The supernatant was subjected to enzyme-linked immunosorbent assay (ELISA) using the human IL-2 ELISA Ready-SET-Go! kit (eBioscience, San Diego, CA, USA) according to the manufacturer's protocol. FK506 was used as the positive control.

Calcineurin assay

Calcineurin activity was measured using a commercial calcineurin phosphatase assay kit (Enzo Life Sciences, Farmingdale, NY, USA) with slight modification of the manufacturer's protocol, as described previously (Ogasawara et al.2008). The free phosphate ions released from the substrate phosphopeptide (DLDVPIPGRFDRRVpSVAAE) was quantified by colorimetric analysis (650 nm) using the malachite green method. The calmodulin antagonist, TFO, was used as the positive control.

Statistical analysis

Statistical tests were performed using the statistical software R version 3.6.1. Statistically significant differences between the experimental groups were determined by one-way ANOVA and Tukey's post hoc tests. Data are expressed as mean ± SD.

Results and discussion

Growth-restoring activity of Petasites japonicus extracts against the YNS17 strain

We screened for Ca²⁺ signaling inhibitors from our extract library consisting of agricultural, forestry, and fishery products from Iwate prefecture using the YNS17 strain. Methanol and hexane extracts of the flower buds of P. japonicus showed growth-restoring activity in a dose-dependent manner (Figure 1). The hexane extract exhibited obvious activity compared to the MeOH extract. However, the aqueous layer of the hexane extract had no activity. These results suggested that the P. japonicus extract contained Ca²⁺ signaling inhibitors, and the polarity of the active compound might be hydrophobic.

Figure 1.

Growth-restoring activity of MeOH and hexane extracts of P. japonicus against the YNS17 strain. Growth-restoring activity of MeOH (left) and hexane (right) extracts of P. japonicus against the YNS17 strain in the presence of 0.3 m CaCl₂. 1, 50 µg/spot; 2, 25 µg/spot; 3, 12.5 µg/spot; 4, 6.25 µg/spot; 5, 3.125 µg/spot; 6, 1.563 µg/spot; 7, FK506 (2.5 ng/spot).

Isolation and identification of active compounds

To clarify the active compound, we purified and isolated the flower buds of P. japonicus obtained from Iwate prefecture. Dried P. japonicus (63.00 g) was powdered and extracted with MeOH. The MeOH extract (11.34 g) showed growth-restoring activity against the YNS17 strain and partitioned with hexane. In the hexane and aqueous layers, only the hexane layer exhibited activity. The hexane layer (1.79 g) was subjected to silica gel column chromatography using hexane and EtOAc. The active fractions obtained (Fr. 7-13), specifically Fr. 8 (228.7 mg) and Fr. 9 (177.8 mg), exhibited potent activity (Figure S1). These 2 fractions were combined (406.5 mg) and purified by preparative HPLC, and a compound (85.5 mg), which was a colorless oil, was isolated.

The ammonium adduct molecular ion of the isolated active compound gave an m/z of 408.2373, suggesting the molecular formula to be C₂₂H₃₀O₆⋅NH₄ (calcd for [M + NH₄]⁺ 408.2386). The molecular formula of bakkenolide B is also C₂₂H₃₀O₆. Thus, we compared the MS/MS spectra of the isolated active compound and the commercial standard of bakkenolide B. The MS/MS spectrum of the isolated compound was identical to that of the standard of bakkenolide B (Figure 2a). In addition, their retention times were also identical, as confirmed by coinjection of these compounds (Figure 2b). These results revealed that the active compound isolated from P. japonicus using the yeast-based assay was bakkenolide B (Figure 2c). Bakkenolide B is one of the major components of P. japonicus and a sesquiterpene lactone (Lee et al.2013).

Figure 2.

Identification of bakkenolide B. (a) LC-ESI-MS/MS spectrum of bakkenolide B and the isolated active compound. (b) HPLC chromatogram of bakkenolide B and the isolated active compound. (c) Chemical structure of bakkenolide B.

Growth-restoring activity of bakkenolide B against the YNS17 strain

Bakkenolide B showed growth-restoring activity against the YNS17 strain in a dose-dependent manner (Figure 3). To clarify whether the effect of this compound is dependent on Ca²⁺ signaling, we also assessed its activity in CaCl₂-free medium. Bakkenolide B did not show growth-restoring activity in the absence of CaCl₂ (Figure 3), indicating that the effect of bakkenolide B on the YNS17 strain was mediated by regulating Ca²⁺ signaling. Bakkenolide B exhibited growth-restoring activity at doses greater than 0.3125 µg/spot. On the other hand, the MeOH extract showed activity at doses greater than 12.5 µg/spot (Figure 1). The content of bakkenolide B in the MeOH extract was determined to be 21.2 mg/g; thus, 12.5 µg of the MeOH extract contains 0.26 µg of bakkenolide B. This amount was comparable to the minimum effective dose of the bakkenolide B standard (0.3125 µg). Thus, we concluded that bakkenolide B is the main active component responsible for the inhibitory effect on Ca²⁺ signaling in P. japonicus.

Figure 3.

The Ca²⁺-dependent growth-restoring activity against the YNS17 strain. Growth-restoring activity of bakkenolide B against the YNS17 strain in the presence (left) or absence (right) of 0.3 m CaCl₂. 1, 5 µg/spot; 2, 1.25 µg/spot; 3, 0.3125 µg/spot; 4, 0.078 µg/spot; 5, 0.020 µg/spot; 6, 0.005 µg/spot; 7, FK506 (2.5 ng/spot). The position of each sample is identical with that indicated in Figure 1.

Additionally, we discussed the possibility of chemical interactions between bakkenolide B and Ca²⁺. If bakkenolide B has a Ca²⁺-chelating effect as well as EDTA and EGTA, Ca²⁺ is trapped in the medium and cannot enter the cells. In a previous study, bakkenolide B (10 and 50 µg/mL = 25.6 and 128.1 µm, respectively) did not affect the increase in intracellular Ca²⁺ concentration after antigen exposure in RBL-2H3 cells (Lee et al.2013). This finding clearly indicates that bakkenolide B does not have a Ca²⁺-chelating effect. Therefore, the activity of bakkenolide B against the YNS17 strain appears to be the result of biological regulation of Ca²⁺ signaling.

Target prediction based on Li⁺ sensitivity

Based on the above results, we evaluated the target of bakkenolide B in Ca²⁺ signaling. Mutation in the regulatory subunit of calcineurin causes a defect in tolerance to salts such as LiCl and NaCl (Nakamura et al.1996). Indeed, inhibitors of the calcineurin pathway induced growth inhibition against wild-type yeast cells in the presence of 0.16 m LiCl. Thus, we can suspect that bakkenolide B affects the calcineurin pathway by this phenomenon. Moreover, bakkenolide B inhibited the growth of yeast cells in the presence of LiCl and was more potent than that in the absence of LiCl (Figure 4), and was similar to that observed for FK506, a clinical calcineurin inhibitor. These findings demonstrated that bakkenolide B might inhibit the calcineurin pathway in S. cerevisiae.

Figure 4.

Li⁺ sensitivity of wild-type yeast cells. Growth inhibition of wild-type yeast by bakkenolide B in the presence (left) or absence (right) of 0.16 m LiCl. 1, 10 µg/spot; 2, 2.5 µg/spot; 3, 0.625 µg/spot; 4, 0.156 µg/spot; 5, 0.039 µg/spot; 6, 0.010 µg/spot; 7, FK506 (0.1 µg/spot). The position of each sample is identical with that indicated in Figure 1.

Effects of bakkenolide B on IL-2 expression

These above results suggested that bakkenolide B inhibits calcineurin pathway. Although it was reported that bakkenolide B has antiallergic and anti-inflammatory effects in mouse mast cells and mouse peritoneal macrophages (Lee et al.2013) or mouse microglial cells (Park et al.2018), its effects against human-derived cell lines have not yet been elucidated. As bakkenolide B inhibits the calcineurin pathway, we evaluated its effects on Jurkat human T cells, a mammalian cell system. Jurkat cells are commonly used to study the calcineurin pathway (Ishiguro et al.2007; Suauam et al.2015; Ngo et al.2017). In this cell line, calcineurin signaling is activated by costimulation with PMA (a protein kinase C activator) and ionomycin (a Ca²⁺ ionophore). Bakkenolide B did not show significant growth inhibition at concentrations less than 40 µm against Jurkat cells (Figure 5a). The upregulation of intracellular Ca²⁺ concentration activates calcineurin, which in turn causes dephosphorylation of the transcription factor NF-AT. The dephosphorylated NF-AT localizes to the nucleus, promoting the transcription of cytokines, such as IL-2 (Liu et al.1991; Rothenberg and Ward 1996; Trevillyan et al.2001). Calcineurin inhibitors FK506, which suppress IL-2 expression, have been used for the treatment of atopic dermatitis. Previously, clausmarin A, a component of Clausena harmandiana (Pierre), was identified as an inhibitor of the calcineurin pathway using a nearly similar yeast-based assay (Suauam et al.2015). They found that clausmarin A inhibits IL-2 gene expression and production in Jurkat cells. We performed real-time PCR and ELISA to determine the effects of our compounds on IL-2 expression in human Jurkat cells. In our experiments, bakkenolide B inhibited IL-2 gene expression (Figure 5b) and production (Figure 5c; IC₅₀ value of 6.3 µm) in a dose-dependent manner. In one of the previous studies reported that bakkenolide B weakly suppressed antigen-induced degranulation in mouse RBL-2H3 cells and LPS-induced expression of COX-2 and iNOS in mouse peritoneal macrophages at the concentration about 10 µg/mL (=25.6 µm) (Lee et al.2013). Furthermore, the other report showed that bakkenolide B inhibits LPS-induced production of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) at the IC₅₀ values about 20-40 µm in mouse microglial cells (Park et al.2018). These results indicated that bakkenolide B inhibits IL-2 production in human-derived Jurkat cells at lower dose compared with previously reported antiallergic and anti-inflammatory effects in mouse-derived cells.

Figure 5.

Effects of bakkenolide B on Jurkat cells. (a) Cytotoxicity. Jurkat cells were treated with bakkenolide B for 24 h. The cytotoxicity was measured by MTT assay. (b) IL-2 mRNA expression. Jurkat cells were treated with bakkenolide B for 3 h. The mRNA expression levels of IL-2 were measured by real-time PCR. (c) IL-2 protein expression. Jurkat cells were treated with bakkenolide B for 24 h. FK506 (100 n m) was used as the positive control. The protein expression levels of IL-2 were measured by human IL-2 ELISA assay. Values are presented as mean ± SD of 3 samples. **P < .01, ***P < .001.

Effect of bakkenolide B on calcineurin activity

As in the case of FK506, inhibitors of the calcineurin pathway suppress IL-2 production. Thus, we assessed the inhibitory effect of bakkenolide B on the phosphatase activity of calcineurin using the malachite green method (Ogasawara et al.2008). Bakkenolide B showed very weak activity against human recombinant calcineurin in vitro (Figure 6), suggesting that bakkenolide B inhibits IL-2 production without directly suppressing calcineurin activity, as in the case of clausmarin A (Suauam et al.2015). Since it has been suggested that bakkenolide B does not suppress Ca²⁺ influx as described above (Lee et al.2013), this compound might affect the phosphorylation and/or localization of NF-AT, a transcription factor of IL-2. For example, acetic acid and sodium acetate (Ishiguro et al.2007) have been reported to inhibit IL-2 production and the interaction between NF-AT and importin β1 (Ishiguro et al.2007). They are expected to suppress inflammation of the skin and bowel. Furthermore, we considered the possibility that bakkenolide B might be metabolized to produce acetic acid by intracellular esterases. Since acetic acid is also an inhibitor of IL-2 production, we quantified the content of bakkenolide B in treated Jurkat cells. There was no significant decrease in bakkenolide B content after treatment with bakkenolide B for 1, 3, 5, and 24 h (Table S1). Although bakkenolide B inhibited IL-2 production at an IC₅₀ value of 6.3 µm, Ishiguro et al. reported that acetic acid and sodium acetate inhibit IL-2 production in the order of mM in Jurkat cells (Ishiguro et al.2007). Thus, these results indicate that bakkenolide B is stable in cells and inhibits IL-2 production in the intact form.

Figure 6.

Effects of bakkenolide B on calcineurin activity in vitro. Assays were performed as described in the Materials and methods section. TFO (50 µm) was used as the positive control. Values are expressed as mean ± SD of 3 samples. **P < .01, ***P < .001.

Effects of bakkenolide A, an analog of bakkenolide B

We confirmed that bakkenolide B isolated from P. japonicus inhibits IL-2 production in a human T cell line. However, although bakkenolide B is one of the major components of this plant, the molecular mechanisms involved in its biological effects have not been fully elucidated. Bakkenolide A, an analog of bakkenolide B lacking 2 ester moieties, was also found in P. japonicus (Figure 7a). Thus, we investigated the substructures that are responsible for the activities of bakkenolide B in comparison with those of bakkenolide A. Bakkenolide A did not show any obvious growth-restoring activity against the YNS17 strain (Figure 7b). Additionally, this compound weakly suppressed IL-2 production in Jurkat cells at non-toxic doses (Figure 7c and d) with an IC₅₀ value of 24.6 µm. The effects of bakkenolide A on the YNS17 strain and human Jurkat cells indicated that the 2 ester moieties of bakkenolide B are critically important for biological activities.

Figure 7.

Effects of bakkenolide A. (a) Chemical structure of bakkenolide A. (b) Growth-restoring activity of bakkenolide A against the YNS17 strain in the presence of 0.3 m CaCl₂. 1, 5 µg/spot; 2, 1.25 µg/spot; 3, 0.3125 µg/spot; 4, 0.078 µg/spot; 5, 0.020 µg/spot; 6, 0.005 µg/spot; 7, FK506 (2.5 ng/spot). The position of each sample is identical with that indicated in Figure 1. (c) Cytotoxicity. Jurkat cells were treated with bakkenolide A for 24 h. The cytotoxicity was measured by MTT assay. (d) IL-2 protein expression. Jurkat cells were treated with bakkenolide A for 24 h. FK506 (100 n m) was used as the positive control. The protein expression levels of IL-2 were measured by human IL-2 ELISA assay. Values are presented as mean ± SD of 3 samples. *P < .05, ***P < .001.

Conclusion

In conclusion, we isolated a Ca²⁺ signaling inhibitor, bakkenolide B, from P. japonicus using a yeast-based screening system. Bakkenolide B was found to inhibit the production of IL-2 in Jurkat cells. Further studies are necessary to identify the primary molecular target and its efficacy against diseases in which IL-2 is involved, such as atopic dermatitis, in vivo. We have demonstrated that bakkenolide B is a promising lead compound for functional components.

Acknowledgments

We would like to thank Editage (www.editage.jp) for English language editing.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Author contribution

S.U. designed the research; S.U., M.H., Y.Ka., H.T., and Y.Ku. conducted the research; S.U. analyzed the data; S.U. wrote the manuscript; K.K., H.Y., and A.Y. performed critical revision and edited the manuscript. All authors reviewed and approved the final manuscript.

Funding

This work was supported by funds from the Basic Biotechnology Project of Iwate Prefecture, Japan, and JSPS KAKENHI (Grant Number JP19K14046).

Disclosure statement

No potential conflict of interest was reported by the authors.