Correlation between the biological activities and the chemical structures of conidendrin-related compounds: (−)-β-conidendrin inhibits degranulation of RBL-2H3 cells

Abstract

Conidendrin, a metabolite of lariciresinol (a lignan in dietary plants), has 8 stereoisomers with 3 asymmetric carbon atoms. However, the relationship between the chemical structure and biological activity of these stereoisomers remains unclear. Since strong cytotoxicity against rat basophilic cell line RBL-2H3 cells has been observed in 4 stereoisomers, the degranulation inhibitory effect of the other conidendrin isomers possessing no cytotoxicity was investigated. Significant degranulation inhibitory effect was observed on all 4 stereoisomers especially on (−)-β-conidendrin, suggesting that conidendrin exhibits stereospecific cytotoxic and degranulation inhibitory activities, and (−)-β-conidendrin is the most structurally effective isomer on antidegranulation. Additionally, (−)-β-conidendrin inhibited the antigen-induced increase in intracellular Ca²⁺ concentration and phosphorylation levels of Syk, PLCγ, and Akt, indicating that (−)-β-conidendrin inhibits Ca²⁺ influx into cells by downregulating the Syk/PLCγ and PI3K/Akt signaling pathways, thereby suppressing degranulation. Our findings suggest that conidendrin may be useful as an antiallergic functional food ingredient.

Graphical Abstract

(−)-β-Conidendrin was suggested to inhibit Ca²⁺ influx into cells by downregulating the Syk/PLCγ and PI3K/Akt signaling pathways, thereby suppressing degranulation.

lignan, antiallergy, mast cells, dietary plants, stereoisomer structure

Abbreviations

Abbreviations
 
• DMEM

  Dulbecco’s modified Eagle’s medium

 
• DNP

  dinitrophenyl

 
• HSA

  human serum albumin

 
• IgE

  immunoglobulin E

 
• PI3K

  phosphatidylinositol 3-kinase

 
• PLC

  phospholipase C

 
• Syk

  spleen tyrosine kinase

Allergy is an overreaction of the immune system to harmless substances (allergens) such as pollen, dust, and certain foods. Type I allergy, also known as immediate-type or IgE-mediated allergy, begins with the secretion of specific IgE in response to an allergen. When the allergen enters the body, it binds to the IgE that is already attached to a surface receptor called the high-affinity IgE receptor (FcεRI) on mast cells and basophils (Turner and Kinet 1999). This binding triggers the release of histamine and other chemical mediators from the cells in a process known as degranulation (Turner and Kinet 1999; Metcalfe, Peavy and Gilfillan 2009). Antihistamines are commonly used to treat allergy symptoms, but they come with side effects such as drowsiness and impaired performance. Therefore, ongoing research is focused on food-derived natural substances that are safe, have no side effects, and can inhibit allergic reactions. We have focused on food plants and reported that certain substances derived from them have antiallergic effects: (−)-matairesinol, a lignan found in flaxseed, suppresses IgE production in plasma cell myeloma cell line U266 cells (Kawahara et al. 2010); and umbelliferose contained in cumin seeds inhibits the degranulation of rat basophilic cell line RBL-2H3 cells (Ishida et al. 2022). Incorporating these foods or functional ingredients into a balanced diet may help alleviate allergic reactions and contribute to improved health and quality of life.

Lignans are a large group of plant-derived chemical compounds biosynthesized as secondary metabolites from shikimic acid, which is a biosynthetic intermediate of aromatic amino acids, including tyrosine, phenylalanine, and tryptophan. They are dimers formed by the oxidative coupling of 2 phenylpropanoids, which are natural aromatic compounds having a basic skeleton structure wherein linear propane is bonded to a benzene ring (C6-C3) (Teponno, Kusari and Spiteller 2016). In general, compounds in which C6-C3 units are dimerized at 8- and 8'-positions are called “lignans,” and others are called “neolignans” (Moss 2009; Pan et al. 2009; Teponno, Kusari and Spiteller 2016). Lignans and neolignans are abundantly present in plants and fiber-rich foods, including whole-grain products and oilseeds, such as flaxseed and sesame seed (Peñalvo et al. 2005a,b; Milder et al. 2007; Peñalvo et al. 2008). The stereochemistry of lignans and neolignans present in these plants differ among species, showing diverse enantiomeric composition, biosynthetic process, and phylogenetic distribution, in addition to structural diversity due to the carbon framework, oxidation level, and substitution pattern (Suzuki, Umezawa and Shimada 2002; Sicilia et al. 2003; Umezawa 2003). However, comprehensive determination and analysis of their structures is difficult due to the limited amount produced in plants. Therefore, to fully elucidate their effects and roles in plants, it is necessary to develop different optical isomers and stably mass-produce them.

Pinoresinol, secoisolariciresinol, lariciresinol, and matairesinol are the furthest upstream on the biosynthetic pathway of naturally occurring lignans. They (both as an aglycon and as a diglycoside) have various biological activities: pinoresinol and its diglucoside exhibit antitumor and anti-inflammatory activities (Zhang et al. 2018; Shuangyuan et al. 2021); secoisolariciresinol and its diglucoside inhibit adipogenesis (Tominaga et al. 2009; Tominaga et al. 2012; Kang et al. 2018); lariciresinol displays antidiabetic and antitumor activities (Saarinen et al. 2008; Alam et al. 2022); and matairesinol exerts anti-inflammatory, antioxidant, and antiosteoclastogenic effects (Choi et al. 2014; Xu et al. 2017; Wu, Wang and Li 2021). Thus, as lignans exhibit beneficial effects on maintaining human health, there is a growing interest in promoting the inclusion of lignan-rich foods into human diets.

α-Conidendrin is a lignan found in Taxus species (Dang et al. 2017; Hafezi et al. 2020). It is also produced in the human body through the metabolism of lariciresinol, which is a metabolite resulting from the reaction of secoisolariciresinol to prevent oxidation of unsaturated fatty acids present in dietary plants (Smeds et al. 2005; Peñalvo et al. 2008). α-Conidendrin has various beneficial biological activities; for example, it inhibits tumor necrosis factor-α-induced intercellular adhesion molecule-1 expression in human lung adenocarcinoma A549 cells (Vo et al. 2021). Moreover, α-conidendrin exhibits anticancer effects (Hafezi et al. 2020), and isomerization of α-conidendrin in the metabolic process produces β-conidendrin in the body. These findings suggest that dietary intake of plants would result in the accumulation of α- and β-conidendrins in the body by the metabolism of lignans. However, the biological activity of β-conidendrin has not yet been reported. Eight stereoisomers of conidendrin exist because of the presence of 3 asymmetric carbon atoms (Shirakata, Nishiwaki and Yamauchi 2020). However, no research has been conducted on the relationship between the chemical structure and the biological activity of these stereoisomers. Therefore, this study aimed to investigate the antiallergic effect of degranulation inhibition as a novel biological activity of conidendrins and the structure-activity relationship of conidendrin stereoisomers. Furthermore, as the antiallergic effect of conidendrin and the underlying action mechanism have not been reported, we analyzed this aspect at the cellular level.

Materials and methods

Reagents

Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), mouse anti-dinitrophenyl (DNP) monoclonal immunoglobulin E (IgE), DNP-human serum albumin (HSA) conjugate, and A23187 were acquired from Sigma-Aldrich (St Louis, MO, USA). All other chemicals were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan), unless otherwise indicated.

Cell and cell culture

Rat basophilic cell line RBL-2H3 cells obtained from the American Type Culture Collection (Rockville, MD, USA) were cultured in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS at 37 °C in an incubator under humidified 5% CO₂.

Sample preparation

Eight stereoisomers of conidendrin, secoisolariciresinol, lariciresinol, and cyclolariciresinol were dissolved in dimethyl sulfoxide and used at a final solvent concentration of 0.5% during testing. The optical purity of conidendrin stereoisomers was measured by employing chiral column ((−)-α-conidendrin: 99%ee, (+)-α-conidendrin: >99%ee, (−)-β-conidendrin: >99%ee, (+)-β-conidendrin: 99%ee, (7'S,8R,8'S)-conidendrin: 99%ee, (7'R,8S,8'R)-conidendrin: 99%ee, (7'S,8S,8'S)-conidendrin: 99%ee, and (7'R,8R,8'R)-conidendrin: 97%ee) (Shirakata, Nishiwaki and Yamauchi 2020).

β-Hexosaminidase release assay

Antigen-induced degranulation of RBL-2H3 cells was evaluated by determining the activity of extracellularly released β-hexosaminidase stored in granules as previously described (Ishida et al. 2022). Briefly, RBL-2H3 cells sensitized with anti-DNP IgE were treated with the prepared samples, followed by stimulation with DNP-HSA to induce degranulation. The β-hexosaminidase release rate was calculated as: [(A _supernatant − A _{blank of supernatant})/{(A _supernatant − A _{blank of supernatant}) + (A _{cell lysate} − A _{blank of cell lysate})}] × 100, where “A” is the absorbance at 415 nm measured using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

A calcium ionophore A23187 was used to induce degranulation without antigen-antibody reaction. RBL-2H3 cells were seeded and cultured overnight without anti-DNP IgE. After treatment with conidendrin as described earlier, the cells were stimulated with A23187 (10 μm) for 30 min to induce degranulation. The β-hexosaminidase release rate was then measured as described earlier.

Cell viability

Cytotoxicity was assayed by the WST-8 Cell Count Reagent (Nacalai Tesque) according to the manufacturer’s instructions. RBL-2H3 cells were seeded, sensitized with anti-DNP IgE, and stimulated as described earlier. After washing with DMEM, a fresh medium containing 10% Cell Count Reagent SF was added to each well, and the plate was incubated at 37 °C for 20–40 min, followed by measuring absorbance at 450 nm using iMark microplate reader (Bio-Rad Laboratories).

Monitoring intracellular Ca²⁺ concentration

Intracellular Ca²⁺ concentrations were monitored using a fluorescent calcium indicator Fluo 3-AM (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. RBL-2H3 cells were seeded, sensitized with anti-DNP IgE, and stimulated as described earlier. The cells were incubated for 1 h in the loading buffer supplemented with Fluo 3-AM (both prepared with reagents provided in the kit). After washing with phosphate-buffered saline (pH 7.4), the cells were incubated in the recording buffer containing (−)-β-conidendrin (500 μm) for 10 min at 37 °C. The cells were subsequently stimulated with DNP-HSA, and the fluorescence intensity was immediately monitored using Infinite 200 PRO (Tecan, Switzerland) at excitation and emission wavelengths of 480 and 530 nm, respectively.

Immunoblot analysis

The preparation of cell lysates and immunoblotting was performed as described previously (Kitamura et al. 2022). Denatured proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Buckinghamshire, UK). Primary antibodies for β-actin (13E5), Lyn (C13F9), phosphorylated Lyn (Tyr507), Syk (D3Z1E), phosphorylated Syk (Tyr525/526), PLCγ1 (D9H10), phosphorylated PLCγ1 (Tyr783), PI3K p85 (19H8), phosphorylated PI3K p85 (Tyr458)/p55 (Tyr199), Akt (pan) (C67E7), and phosphorylated Akt (Ser473) (D9E) were purchased from Cell Signaling Technology (Danvers, MA, USA). As the secondary antibody, anti-rabbit IgG, horseradish peroxidase-conjugated antibody from Cell Signaling Technology was used. The bands were visualized using a ChemiDoc XRC system (Bio-Rad Laboratories), and the amount of target proteins was normalized with loading controls in the same blots.

Statistical analysis

The obtained data were expressed as means ± SEM. GraphPad Prism version 8.4.3 (GraphPad Software, San Diego, CA, USA) was used to analyze the results using a 1-way analysis of variance (ANOVA) followed by the post hoc Dunnett’s test. Statistical significance is reported at *P < .05, **P < .01, ***P < .001, and ^****P < .0001.

Results

Eight conidendrin stereoisomers exhibited different cytotoxicity against RBL-2H3 cells

Since conidendrin has 3 asymmetric carbon atoms at 7'-, 8-, and 8'-positions, it has 8 stereoisomers as shown in Figure 1. The cytotoxicity of 8 conidendrin stereoisomers against RBL-2H3 cells at the same concentration was examined using WST-8. (−)-α-, (+)-α-, (−)-β-, and (+)-β-Conidendrin did not affect the cell viability, and no differences were observed among these 4 stereoisomers. On the other hand, the cell viability of RBL-2H3 cells was significantly decreased by the treatment with the other 4 stereoisomers (Table 1). Particularly, the (7'R,8R,8'R)-form showed the strongest cytotoxicity.

Figure 1.

Chemical structure of conidendrin stereoisomers.

Table 1.

Effect of conidendrin stereoisomers on the viability of RBL-2H3 cells

Conidendrin stereoisomers		Concentration (μm)	Relative cell viability (% of control)	P-value (vs control)
(−)-α-Conidendrin	7'S,8R,8'R	250	101.9 ± 5.8	–
		500	119.9 ± 4.5	–
(+)-α-Conidendrin	7'R,8S,8'S	250	124.4 ± 8.1	–
		500	119.4 ± 4.2	–
(−)-β-Conidendrin	7'R,8R,8'S	250	111.0 ± 4.9	–
		500	115.8 ± 1.6	–
(+)-β-Conidendrin	7'S,8S,8'R	250	103.5 ± 4.0	–
		500	109.4 ± 5.9	–
	7'S,8R,8'S	250	85.2 ± 2.5	.115
		500	62.8 ± 2.5	.001
	7'R,8S,8'R	250	77.1 ± 7.6	.092
		500	63.3 ± 0.7	.008
	7'S,8S,8'S	250	72.3 ± 3.4	.012
		500	65.0 ± 1.5	.006
	7'R,8R,8'R	250	65.3 ± 11.0	.158
		500	30.8 ± 19.7	.005

Data with (−)-α-, (+)-α-, (−)-β-, and (+)-β-conidendrin are shown as means ± SEM of triplicate wells from a representative experiment from 2 independent experiments. Data with other conidendrin are shown as means ± SEM from 3 independent experiments, and statistical analysis was performed using a 1-way ANOVA with Dunnett’s post hoc test.

(−)-β-Conidendrin most strongly inhibited the antigen-induced degranulation of RBL-2H3 cells

The degranulation inhibitory activity of the 4 conidendrin stereoisomers with no cytotoxicity on RBL-2H3 cells was examined. Previously, the effect of these conidendrins on β-hexosaminidase activity was examined. As a result, they did not affect the enzymic activity of β-hexosaminidase, meaning no effect on the degranulation assay system. As shown in Figure 2, these 4 conidendrins significantly inhibited degranulation. Particularly, (−)-β-conidendrin showed the strongest inhibitory activity among these 4 conidendrins. The degranulation inhibition rate of (−)-β-conidendrin was 58.9% ± 2.2% at 500 μm, while those of (−)-α-, (+)-α-, and (+)-β-conidendrins were 15.5% ± 2.1%, 14.6% ± 2.1%, and 13.3% ± 2.2%, respectively.

Figure 2.

Effects of 4 conidendrin stereoisomers on degranulation of RBL-2H3 cells. (−)-α-Con, (+)-α-Con, (−)-β-Con, and (+)-β-Con represent (−)-α-conidendrin, (+)-α-conidendrin, (−)-β-conidendrin, and (+)-β-conidendrin, respectively. Data are shown as means ± SEM of triplicate wells from a representative experiment from 2 independent experiments. Statistical analysis was performed using a 1-way ANOVA with Dunnett’s post hoc test, ***P < .001 and ^****P < .0001 versus control.

As shown in Figure 3a, (−)-cyclolariciresinol ((7'R,8S,8'S)-CLar) is metabolized to (−)-β-conidendrin via (+)-α-conidendrin, and then to (7'R,8R,8'S)-CLar. The degranulation inhibitory effects of secoisolariciresinol, lariciresinol, and cyclolariciresinol were investigated and compared with that of (−)-β-conidendrin. None of the tested compounds affected cell viability (Figure 3b and c); however, (−)- and (+)-secoisolariciresinol did not inhibit the antigen-induced degranulation of RBL-2H3 cells, and (−)- and (+)-lariciresinol significantly inhibited the degranulation at 500 μm (the inhibition rate was 36.3% ± 6.9% and 39.3% ± 6.8%, respectively) (Figure 3d). In addition, (7'R,8S,8'S)-CLar significantly inhibited the antigen-induced degranulation (Figure 3e), and its degranulation inhibition rate was 26.1% ± 4.0%. The degranulation inhibition rate of (−)-, (+)-lariciresinol, and (7'R,8S,8'S)-CLar was lower than that of (−)-β-conidendrin (57.8% ± 5.9%), suggesting that (−)-β-conidendrin most strongly inhibited antigen-indued degranulation of RBL-2H3 cells among the tested lignans and their metabolites.

Figure 3.

Effects of secoisolariciresinol, lariciresinol, and cyclolariciresinol on degranulation of RBL-2H3 cells. (a) Metabolic scheme. (b) Effects of secoisolariciresinol, lariciresinol, and their stereoisomers on the viability of RBL-2H3 cells. (c) Effect of (7'R,8S,8'S)- and (7'R,8R,8'S)-cyclolariciresinol on the viability of RBL-2H3 cells. (d) Effect of secoisolariciresinol, lariciresinol, and their stereoisomers on antigen-induced degranulation of RBL-2H3 cells. (e) Effect of (7'R,8S,8'S)- and (7'R,8R,8'S)-cyclolariciresinol on antigen-induced degranulation of RBL-2H3 cells. The Seco, Lar, CLar, and (−)-β-con represent secoisolariciresinol, lariciresinol, cyclolariciresinol, and (−)-β-conidendrin, respectively. Data are shown as means ± SEM from 3 independent experiments. Statistical analysis was performed using a 1-way ANOVA with Dunnett’s post hoc test, *P < .05 and ***P < .001 versus antigen (+).

(−)-β-Conidendrin inhibited the elevation of intracellular Ca²⁺ concentration and the phosphorylation of signal molecules in the degranulation signaling pathway

Ca²⁺ is an intracellular secondary messenger, and the Ca²⁺ influx into cells is involved in degranulation response (Turner and Kinet 1999; Metcalfe, Peavy and Gilfillan 2009). The increase in intracellular Ca²⁺ concentration after antigen stimulation was measured. The increase in intracellular Ca²⁺ concentration was found to be significantly suppressed by (−)-β-conidendrin (Figure 4a). At 10 s after antigen stimulation, the increase in intracellular Ca²⁺ was almost completely inhibited, and the inhibition rate was approximately 50% until 120 s, suggesting that (−)-β-conidendrin suppressed degranulation by inhibiting Ca²⁺ influx into cells.

Figure 4.

Effects of (−)-β-conidendrin on the elevation of intracellular Ca²⁺ concentrations and phosphorylation of signal molecules. RBL-2H3 cells sensitized with IgE were treated with (−)-β-conidendrin (500 μm), and then the cells were stimulated with antigen. (a) Relative fluorescence intensity of Fluo 3 reflects intracellular Ca²⁺ concentration. Open square: cells not treated with (−)-β-conidendrin nor stimulated with antigen; gray square: cells not treated with (−)-β-conidendrin but stimulated with antigen (control); closed circle: (−)-β-conidendrin. Data are shown as means ± SEM of triplicate wells from a representative experiment from 3 independent experiments. (b) Lyn/Syk/PLCγ and (c) PI3K/Akt signaling pathways. The cells were lysed 10 min after stimulation, and the cell lysate was subjected to immunoblot analysis. The p-Lyn, p-Syk, p-PLCγ, p-PI3K, and p-Akt represent phosphorylated Lyn, phosphorylated Syk, phosphorylated PLCγ, phosphorylated PI3K, and phosphorylated Akt, respectively. (−)-β-Con represents (−)-β-conidendrin. The result of densitometric analysis is expressed as the ratio of phosphorylated protein amount to whole protein amount. A representative blot from 3 independent experiments is shown. Data are shown as means ± SEM from 3 independent experiments. Statistical analysis was performed using a 1-way ANOVA with Dunnett’s post-hoc test, *P < .05, **P < .01, and ***P < .001 versus the control.

The effect of (−)-β-conidendrin on the phosphorylation of signal molecules involved in the degranulation signal pathway was examined by immunoblotting. No effect was observed on the phosphorylation of Lyn (P = .4), which is located most upstream of the degranulation signal pathway, but the phosphorylation levels of Syk and PLCγ1, downstream of Lyn, were decreased (Figure 4b). Additionally, the phosphorylation level of Akt, which is located downstream of PI3K, was significantly decreased (Figure 4c). These results suggested that (−)-β-conidendrin suppressed the increase in intracellular Ca²⁺ concentration by downregulating the phosphorylation of Syk and Akt, resulting in the inhibition of degranulation.

(−)-β-Conidendrin inhibited degranulation induced by A23187 without affecting Ca²⁺ influx into the cells

The calcium ionophore A23187 bypasses the antigen-IgE cross-linking through FcεRI and the early tyrosine phosphorylation events, allowing the Ca²⁺ influx into cells to induce the release of granules (Siraganian et al. 1975; Kim et al. 2014). After treatment with (−)-β-Conidendrin, RBL-2H3 cells were induced degranulation by A23187. (−)-β-Conidendrin significantly inhibited A23187-induced degranulation in a dose-dependent manner, and the inhibition rate at 500 μm was 74.0% ± 14.2% (Figure 5a). Even though the significant inhibition of degranulation, intracellular Ca²⁺ concentration was not affected by (−)-β-conidendrin (Figure 5b). This suggested that (−)-β-conidendrin suppressed the degranulation of RBL-2H3 cells without inhibiting Ca²⁺ influx into the cells induced by A23187. This fact also suggested that the action mechanisms of (−)-β-conidendrin on antigen-induced degranulation and A23187-induced one are quite different.

Figure 5.

Effects of (−)-β-conidendrin on A23187-induced degranulation and elevation in intracellular Ca²⁺ concentration in RBL-2H3 cells. (a) After RBL-2H3 cells were treated with (−)-β-conidendrin, degranulation was induced by A23187. (−)-β-Con represents (−)-β-conidendrin. Data are shown as means ± SEM from 3 independent experiments. (b) Relative fluorescence intensity of Fluo 3 reflects intracellular Ca²⁺ concentration. RBL-2H3 cells were treated with (−)-β-conidendrin (500 µm). Open square: cells not treated with (−)-β-conidendrin nor stimulated with A23187; gray square: cells not treated with (−)-β-conidendrin but stimulated with A23187 (control); closed circle: (−)-β-conidendrin. Data are shown as means ± SEM of triplicate wells from a representative experiment from 3 independent experiments. Statistical analysis was performed using a 1-way ANOVA with Dunnett’s post hoc test, **P < .01 and ^****P < .0001 versus the control.

Discussion

Since conidendrin has 3 asymmetric carbon atoms at 7'-, 8-, and 8'-positions, it has 8 stereoisomers (Figure 1). As shown in Table 1, (7'S,8R,8'R)-form and its enantiomer (7'R,8S,8'S)-form did not affect cell viability, suggesting that the relative conformation (7'S*,8R*,8'R*)-form was not cytotoxic. Additionally, (7'R,8R,8'S)-form and its enantiomer (7'S,8S,8'R)-form did not affect cell viability, suggesting that the relative conformation (7'R*,8R*,8'S*)-form had no cytotoxicity. Furthermore, although 8- and 8'-positions are not involved in cytotoxicity even if they are relatively trans or cis, the absolute structure of 7'-position is believed to be important. When 8- and 8'-positions were in the trans form, the (7'R,8R,8'R)-form exhibited cytotoxicity. Conversely, the (8S,8'S)-form exhibited cytotoxicity in the case of the 7'S-form. When 8- and 8'-positions were in the cis form, the (8R,8'S)-form exhibited cytotoxicity in the case of 7'S-form, whereas the (8S,8'R)-form exhibited cytotoxicity in the case of 7'R-form. Particularly, the (7'R,8R,8'R)-form showed the strongest cytotoxicity, suggesting that it was the most cytotoxic form among the 8 stereoisomers of conidendrin. Cytotoxicity tests of conidendrin stereoisomers against RBL-2H3 cells were performed to exclude the possibility that the inhibition of degranulation was due to toxicity to RBL-2H cells. Therefore, the intracellular signaling related to cytotoxicity was not examined in this study. α-Conidendrin has been reported to exhibit antitumor activity by regulating the expression and activity of factors involved in apoptosis and cell cycle (Hafezi et al. 2020; Vo et al. 2021). In addition, arctigenin, a natural lignan compound extracted from the seeds of Arctium lappa, has been reported to induce apoptosis in hepatocellular carcinoma cells via suppression of PI3K/Akt signaling (Jiang et al. 2015). Since we did not examine cytotoxicity at concentrations higher than those used in this study, it is not clear whether all conidendrin stereoisomers exhibit toxicity to RBL-2H3 cells. However, we speculate that the structural differences may have different effects on cell death–inducing factors and intracellular signaling, resulting in different cytotoxicity.

Based on the findings, we compared the degranulation inhibitory activity among the 4 stereoisomers possessing no cytotoxicity. Significant degranulation inhibitory activity was observed in all stereoisomers; particularly, the (7'R,8R,8'S)-form ((−)-β-conidendrin) showed the highest activity (Figure 2). The activity of α-conidendrin was the same whether it was (−)- or (+)-form, but in β-conidendrin, the (−)-form showed relatively stronger activity. A comparison of (−)-β-conidendrin with other 3 stereoisomers revealed that when the 7'- and 8'-positions were in the R- and S-forms, respectively, the structure with the 8-position in the R-form (7'R,8R,8'S) exhibited stronger activity. Contrastingly, when the 7'-position was in the S-form (7'S,8R,8'S) or the 8'-position was in the R-form (7'R,8R,8'R), it exhibited cytotoxicity. This suggested that conidendrin exhibits stereospecific biological activity in the antiallergic activity by inhibiting degranulation, and (−)-β-conidendrin is the most effective structure. As shown in Figure 3, (−)-, (+)-secoisolariciresinol and (7'R,8R,8'S)-CLar did not exhibit degranulation inhibitory activity, whereas (−)-, (+)-lariciresinol, and (7'R,8S,8'S)-CLar significantly inhibited the degranulation of RBL-2H3 cells at 500 μm without cytotoxicity. Previous studies have reported that (−)-secoisolariciresinol, (+)-lariciresinol, and (+)-isolariciresinol ((7'S,8R,8'R)-CLar) had no degranulation inhibitory activity (Jo et al. 2020; Tao et al. 2023); however, these studies were conducted at lower concentrations than those in our experiments. In the current study, we found that (−)-, (+)-lariciresinol, and (7'R,8S,8'S)-CLar exerted an inhibitory effect on degranulation, whereas the inhibition rate was lower than that of (−)-β-conidendrin. In addition, (−)-β-conidendrin only showed significant activity at 250 μm. Therefore, these results suggested that the bond at the 6- and 7'-positions is important for its activity. A comparison of (−)-β-conidendrin with (7'R,8R,8'S)-CLar revealed that the (7'R,8R,8'S)-form with a butyrolactone structure exhibits degranulation inhibitory activity. By contrast, the inhibition rate of (7'R,8S,8'S)-CLar (26.1% ± 4.0%) was slightly stronger than that of (+)-α-conidendrin (14.6% ± 2.1%), suggesting that when the 8-position is in the S-form, the diol structure is predominant.

Next, we analyzed the action mechanism of (−)-β-conidendrin, which is the (7'R,8R,8'S)-form that had the strongest degranulation inhibitory activity. In the immediate phase of degranulation, antigen-specific IgE is secreted from antibody-secreting cells that underwent class switching to IgE upon antigen presentation (Turner and Kinet 1999). IgE is then bound to FcεRI on basophils and mast cells. Subsequently, the cells encounter the re-entered antigen, and cross-linking between the IgE on the cell surface and the antigen initiates a series of degranulation processes, including activation of intracellular signal molecules, influx of Ca²⁺ into the cells, increase in the amount of intracellular reactive oxygen species, and translocation of granules along with reorganized microtubules (Turner and Kinet 1999; Metcalfe, Peavy and Gilfillan 2009). After cross-linking between antibodies and antigens, activation of signal molecules depleted Ca²⁺ in the endoplasmic reticulum and induced subsequent influx of extracellular Ca²⁺ from calcium channels. As shown in Figure 4a, the increase in intracellular Ca²⁺ concentration was delayed in (−)-β-conidendrin-treated cells than in the control cells. At 10 s after stimulation, the intracellular Ca²⁺ concentration in the control cells was 1.3-fold higher than before stimulation, whereas in (−)-β-conidendrin-treated cells, it was still the same level as blank cells at 10 s, and gradually started to increase after 20 s of stimulation. Additionally, the control cells reached the maximum level at 80 s and then became constant, whereas (−)-β-conidendrin-treated cells reached the maximum level at 50 s. These results suggested that (−)-β-conidendrin induces the delay of Ca²⁺ influx into cells and suppresses the continuous increase in intracellular Ca²⁺ concentration. Ca²⁺ influx into the cells is triggered by Ca²⁺ depletion in the endoplasmic reticulum, which is induced via phosphorylation of tyrosine kinases (Metcalfe, Peavy and Gilfillan 2009). (−)-β-Conidendrin did not affect Lyn phosphorylation (Figure 4b), suggesting that it does not influence the earliest events in the degranulation response that occur on the cell surface, such as the antigen-antibody cross-linking or the activation of FcεRI. To examine the effect of (−)-β-conidendrin on antigen-antibody cross-linking, DNP-HSA was immobilized on the microplate wells and incubated with (−)-β-conidendrin, followed by DNP-IgE. After washing the plate, an enzyme-labeled secondary antibody against IgE was added to the wells, and the enzyme activity was measured. This assay indicated that (−)-β-conidendrin does not affect antigen-antibody cross-linking (data not shown). In contrast, (−)-β-conidendrin significantly inhibited the phosphorylation of Syk and Akt, which are downstream of Lyn and PI3K (Figure 4). These results suggested that (−)-β-conidendrin either interferes with signal transduction from phosphorylated Lyn and PI3K to downstream signaling molecules, or directly inhibits the phosphorylation of Syk and Akt. Alternatively, (−)-β-conidendrin may indirectly affect the activation of signaling molecules through a receptor with which it interacts. Although research on the biological activity of β-conidendrin is limited, the antiallergic activity and mechanisms of action of natural lignans such as arctigenin and saucerneol F have been reported (Lu, Son and Chang 2012; Kee and Hong 2017). However, these studies have not clarified the detailed mechanisms by which they downregulate the activation of intracellular signaling molecules. To fully understand the mechanism of (−)-β-conidendrin in suppressing degranulation, it is necessary to clarify whether (−)-β-conidendrin directly targets the intracellular signaling molecules and whether the conidendrin-interacting receptor is present inside or on the cell surface of mast cells and basophils. Furthermore, (−)-β-conidendrin suppressed A23187-induced degranulation, but no change was observed in intracellular Ca²⁺ concentration (Figure 5). (−)-β-Conidendrin may also affect events including the increase in intracellular reactive oxygen species, movement of granules due to microtubule formation, and fusion of granule membrane with cell membrane that occur after Ca²⁺ influx.

In conclusion, among the 8 stereoisomers of conidendrin, a metabolite of plant lignans, (−)-β-conidendrin was found to exhibit stereospecific antiallergic activity. Additionally, (−)-β-conidendrin was suggested to inhibit Ca²⁺ influx into cells by downregulating the Syk/PLCγ and PI3K/Akt signaling pathways, thereby suppressing degranulation. Our findings suggest that conidendrin may be useful as an antiallergic plant ingredient that helps prevent and alleviate allergic diseases. Our study also has large-scale implications for promoting the mass production of plants and fiber-rich foods, including whole-grain products and oilseeds, such as flaxseed and sesame seed, which are known to have a high content of lignans.

Data availability

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

Author contribution

M.I., K.N., and T.S. devised and designed research. Experiment was conducted by M.I. and I.M. Organic synthesis was conducted by S.Y. The manuscript was written by M.I. All authors reviewed and approved the manuscript.

Funding

This work was supported in part by a Grants-in-Aid for Scientific Research (KAKENHI grant number 21K05439) from the Japan Society for the Promotion of Science.

Disclosure statement

No potential conflict of interest was reported by the authors.

Acknowledgments

This study was supported by the Division of Genetic Research Support in the Advanced Research Support Center (ADRES), Ehime University.