2016 Volume 64 Issue 2 Pages 96-103
Mast cells and basophils play important roles in both immediate- and late-phase reactions of type 1 allergy. Histamine, which is released from mast cells and basophils stimulated by an antigen or degranulation inducers, is usually determined as a degranulation marker in experiments on immediate allergic reactions in vitro. β-Hexosaminidase is also stored in secretory granules of the cells and is released concomitantly with histamine when the cells are immunologically activated, and recently this enzyme activity in the medium has been used as a marker of the degranulation. In this paper, we review our studies on the search for degranulation inhibitors, such as flavonoids, stilbenes, and curcuminoids, from medicinal plants using rat basophilic leukemia (RBL-2H3) cells.
Recently, an increase in the number of patients with allergies such as pollinosis and asthma in Japan has necessitated the search for more effective, safe antiallergic agents, although excellent antiallergic agents have been developed. Mast cells and basophils play important roles in both immediate- and late-phase reactions of type 1 allergy. The aggregation of high-affinity Fcε receptor I (FcεRI) by antigens results in tyrosine phosphorylations, Ca2+ release from intracellular Ca2+ stores and influx via Ca2+ release-activated Ca2+ channels. The elevation of intracellular free Ca2+ ([Ca2+]i) level plays an essential role in the degranulation process.1–4)
Histamine, which is released from mast cells and basophils stimulated by an immunoglobulin E (IgE)/antigen or a degranulation inducer, is usually determined as a degranulation marker of immediate allergic reactions in in vitro experiments. β-Hexosaminidase is also stored in secretory granules of the cells, and is released concomitantly with histamine when the cells are immunologically activated. Therefore, this enzyme activity is used as a marker of degranulation of cells, and this assay has been used for the evaluation of antiallergic compounds as an alternative to passive cutaneous anaphylaxis (PCA) reactions in laboratory animals.5) In the course of our search for bioactive constituents from medicinal plants, we have isolated various degranulation inhibitors from medicinal plants using rat basophilic leukemia (RBL-2H3) cells. For example, flavonoids from various medicinal plants including the aerial parts of Cissus sicyoides6–8); stilbenes, stilbene dimers, and phenanthrenes from the rhizomes of Rheum undulatum, the tuber of Gymnadenia conopsea, and the whole plant of Cyperus longus9–11); diarylheptanoids including curcuminoides from the barks of Myrica rubra and Acer nikoense and the rhizomes of Curcuma zedoaria and C. comosa12–15); 3-phenyl-isocoumarins from the processed leaves of Hydrangea macrophylla var. thunbergii16,17); phenylpropanoids from the rhizomes of Alpinia galanga18); β-carboline-type alkaloids from the roots of Stellaria dichotoma19); sesquiterpenes from the rhizomes of Hedychium coronarium and the fruits of Alpinia oxyphylla20,21); meroterpenes from the seeds of Psoralea corylifolia22); and acylated oleanane-type triterpene saponin from Camellia sinensis and C. assamica23–25) were reported.
In this review, we focus on flavonoids, stilbenes, and curcuminoids from several medicinal plants with antiallergic activity in in vitro experiments, their structural requirements for antiallergic activity, and some mechanisms of action.
Flavonoids, which are widely distributed in the plant kingdom, are recognized to have various biological activities including antiallergic actions, and various in vitro and in vivo studies of the antiallergic effects of flavonoids were reported. However, their structure–activity relationships were not discussed satisfactorily because of insufficient numbers of examples in vitro and/or technical limitations in vivo.
2.1. Structural Requirements of Flavonoids for the Antigen-Induced Release of β-Hexosaminidase from RBL-2H3 CellsTo clarify the structural requirements of flavonoids for antiallergic activity, we reported the inhibitory effects of more than 50 flavonoids on the release of β-hexosaminidase induced by dinitrophenylated bovine serum albumin (DNP-BSA) as an antigen from RBL-2H3 cells sensitized with anti-DNP IgE, which is a convenient screening test for the immediate phase of type I antiallergic reactions.6) Figure 1 shows representative flavones and flavonols and their IC50 values. Among them, luteolin (5, IC50=3.0 µM), fisetin (8, 3.0 µM), and diosmetin (2.1 µM) were found to show potent inhibitory activity without direct inhibition of the enzyme activity of β-hexosaminidase, and the results suggested the following structural requirements of flavonoids: 1) The C-2/C-3 double bond of flavones and flavonols is essential for the activity, 2) the glycoside moiety markedly reduced the activity, 3) as the hydroxy groups at the C-5, C-6, C-7, C-3′, and C-4′ positions increased in number, the inhibitory activities became more potent, 4) the flavonols with a pyrogallol-type moiety (the 3′,4′,5′-trihydroxy group) at the B ring showed less activity than those with a p-phenol-type moiety (the 4′-hydroxy group) or catechol-type moiety (the 3′,4′-dihydroxy group) at the B ring, 5) Flavones tended to show stronger activity than flavonols, 6) Methylation of flavonols at the C-3 position reduced the activity, although several exceptions were observed6) (Fig. 2). However, isoflavones and flavan-3-ols showed less activity (>100 µM).
a) Data taken from ref. 6.
In type 1 allergy, IgE antibodies bind to FcεRI on the surface of mast cells and basophils. After sensitization with IgE antibodies, exposure to the same allergen cross-links the bound IgE on sensitized cells, resulting in degranulation and the secretion of physiologically active mediators such as histamine, leukotrienes (LTs), and prostaglandins (PGs) that cause the inflammation of the surrounding tissues. Type 1 allergy is further classified into immediate- and late-phase reactions. The immediate-phase reaction occurs within minutes after the exposure to an allergen and is caused by the release of vasoactive amines and lipid mediators. The late-phase reaction occurs 2–4 h after the exposure and is mediated by the release of cytokines such as interleukins (ILs)-3, 4, 5, and 6; tumor necrosis factor α (TNF-α); and granulocyte-macrophage colony-stimulating factor (GM-CSF). The late-phase reaction is caused by release of mediators such as major basic protein (MBP), eosinophil cationic protein (ECP), and LTC4 from eosinophils and are dependent on the activity of Th2 cells.1–4)
Several flavones [apigenin (4), luteolin (5)], flavonols [kaempferol (7), quercetin (9)] inhibit the antigen-induced release of TNF-α and IL-4 in sensitized RBL-2H3 cells6) (Table 1). In addition, a chalcone (isoliquiritigenin) also inhibits degranulation and the release of TNF-α and IL-4.7,8) These findings suggest that these active flavonoids could be effective for the treatment of late-phase as well as immediate-phase reactions.
| IC50 (µM) | ||
|---|---|---|
| TNF-α | IL-4 | |
| Apigenin (4) | 5.3 | 3.6 |
| Luteolin (5) | 5.8 | 3.7 |
| Diosmetin | 5.3 | 3.0 |
| Fisetin (8) | 4.2 | 3.8 |
| Quercetin (9) | 5.6 | 7.5 |
TNF-α and IL-4 levels in the medium were determined 4 h after the stimulation. a) Data taken from ref. 6.
In FcεRI signal transduction, the nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1), which is composed of c-fos and c-jun, induce the transcription of target genes of cytokines by binding at gene promoters and enhancers.1–4,26) Several flavonoids such as luteolin (5) and quercetin (9) were reported to inhibit IgE-mediated Ca2+ influx and the activation of protein kinase C, extracellular signal-regulated kinases (ERKs) and c-jun NH2-terminal kinase (JNK), but not the activation of p38 mitogen-activated protein kinase (p38 MAPK), and then inhibit the release of LTs and PGD2 and expression of GM-CSF mRNA in cultured human mast cells.27) Hirano et al. also reported that luteolin (5) inhibited phosphorylation of c-jun and DNA-binding activity of AP-1 in a human basophilic cell line (KU812) induced by A23187 and 12-myristate 13-acetate (PMA).28)
In our study, kaempferol (7) and quercetin (9), which showed significant inhibition of the release of TNF-α and IL-4 in RBL-2H3 cells, markedly inhibited phosphorylation of ERK1/2 and JNK/SAPK, but obvious inhibition of the phosphorylation of p38 MAPK was not observed, consistent with a previous report.8)
Shichijo et al. reported that flavonoids inhibited the activation of spleen tyrosine kinase (Syk) which plays a pivotal role in FcεRI-mediated degranulation of mast cells.29) In addition, luteolin (5) and quercetin (9) were reported to inhibit calcium ionophore A2318-induced histamine release in human cultured mast cells,27) and the calmodulin pathway, at least myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin, and positively regulate Ca2+-induced degranulation in RBL-2H3 cells.30) Ludowyke et al. reported that Ca2+-induced degranulation correlated with the phosphorylation of myosin light chain by protein kinase C,31) whereas kaempferol (7) was reported to inhibit MLCK of bovine aorta.32) The FcεRI signaling pathway is partly shown in Fig. 3.1–4,26,30,31,33)
In our study using commercially available kinases (Z′-LYTE assay kit, Invitrogen, Carlsbad, CA, U.S.A.), 7 and 9 inhibited the enzyme activity of Syk and MLCK2 partly correlated with their effects on the degranulation of RBL-2H3 cells. Furthermore, 7 and 9 inhibited the phosphorylation of the linker for activation of T cells (LAT) which is a target protein of Syk, and myosin regulatory light chain (MRLC, myosin light chain 2) which is a target protein of MLCK.30) These findings suggest that the possible target molecules of antiallergic activity in RBL-2H3 are Syk and MLCK, although further studies are need to clarify the mechanism of action (Fig. 3).
CaM: calmodulin, DAG: diacylglycerol, ERK: extracellular signal-regulated kinase, Gads: grb2-related adaptor downstream of src, Grb2: growth factor receptor binding protein 2, IP3: inositol-1,4,5-trisphosphate, JNK: Jun amino-terminal kinase, LAT: linker for activation of T cells, Lyn: a Src family tyrosine–protein kinase, MLCK: myosin-light chain kinase, MRLC: myosin regulatory light chain, p38: p38 mitogen-activated protein kinase, PKC: protein kinase C, PLCγ: phospholipase Cγ, Ras: a small G-protein, SLP76: SH2-containing leukocyte-specific protein of 76 kDa, Shc: (Src homology 2 domain containing) transforming protein, Sos: son of sevenless, Syk: spleen tyrosine kinase, Vav1: vav 1 guanine nucleotide exchange factor.
The rhizomes of R. undulatum (Polygonaceae) have been used as a remedy for blood stagnation syndrome as well as a purgative agent in Japanese, Korean, and Chinese traditional medicines. This rhubarb has been considered to have a less purgative effect but to be more effective in treating blood stagnation syndrome than other rhubarbs34) and contains stilbenes such as rhaponticin, piceatannol 3′-O-β-D-glucopyranoside, rhapontigenin, piceatannnol as the principal constituents.35,36) Stilbenes have been reported to have antibacterial, anti-inflammatory, anti-tumor, antimalarial, and anti-aging effects.37–41) We previously reported the antioxidant activity of stilbenes isolated from this herb and their inhibitory effects on nitric oxide production from macrophages stimulated by a lipopolysaccharide (LPS).35,36) In addition, the effects of the principal stilbene constituents on antigen-induced histamine release from rat peritoneal mast cells, 48-h homologous PCA in rats (type I allergic model), and sheep blood cell-induced delayed-type hypersensitivity in mice (type IV allergic model) were also reported.42) However, the structure–activity relationships of stilbenes for antiallergic activity were not studied in sufficient detail.
In our study, the methanolic extract also inhibited the release of β-hexsominidase induced by the antigen in the sensitized RBL-2H3 cells. Therefore, the effects of principal stilbenes isolated from this herb and their related compounds on the antigen-induced degranulation in the RBL-2H3 cells were examined.9)
3.1. Structural Requirements of Stilbenes for the Antigen-Induced Release of β-Hexosaminidase from RBL-2H3 CellsAmong the compounds tested, resveratrol (20, IC50=17 µM), piceatannol (21, 24 µM) desoxyrhapontigenin (22, 18 µM), trimethylresveratrol (23, 5.1 µM), rhapontigenin (29, 11 µM), isorhapontigenin (30, 12 µM), 3,3′,4′-trimethylpiceatannol (31, 16 µM), tetramethylpiceatannol (32, 13 µM), and 3,5,4′-trimethylpiceatannol (33, 2.1 µM) exhibited potent activity, and their activities were stronger than those of two antiallergic agents, tranilast and ketotifen fumarate. trans-Stilbene (19, >100 µM), which lacks oxygen functions on the aromatic rings, was inactive. Our results suggested that stilbenes substituted with methoxy groups at the C-3, C-5, and C-4′ positions exhibited higher activity than those with hydroxy groups. The substitution of glycoside moiety on the ring markedly decreased the activity (Fig. 4).
a) Data taken from ref. 9.
Our results suggested that the positions of the methoxy groups of stilbenes were important for their antiallergic activity. Briefly, 33 (2.1 µM) with methoxy groups at the C-3, C-5, and C-4′ positions exhibited activity ca. 8-fold higher than that of 31 (16 µM) with methoxy groups at the C-3, C-3′, and C-4′ positions. In addition, the three compounds 23, 32, and 33, which have three methoxy groups at the C-3, C-5, and C-4′ positions but have a different group substituted at the C-3′ position, also showed different IC50 values (Fig. 5).
Inamori et al. reported that the inhibitory effect of piceatannol (21) was greater than that of the α,β-dihydro derivative (25) on antigen-induced histamine release in rat peritoneal mast cells.43) Consistent with that report, our results showed that the activities of resveratrol (20, 17 µM) and 21 (24 µM) were greater than those of dihydroresveratrol (24, >100 µM) and 25 (81 µM), respectively. Thus, the C-α/C-β double bond is essential for higher activity.
Cheong et al. also reported the inhibitory effects of hydroxystilbenes on the release of β-hexosaminidase,44) and they came to two main conclusions; 1) The antiallergic effect decreased in proportion to the number of methoxy groups, and hydroxy substituents on the benzene rings might be important for the activity, 2) glycoside substitution of stilbenes decreased the activity. Our results supported the previous report and suggested some additional or revised structural requirements: 1) The oxygen functions (–OCH3, –OH) are essential and their positions on aromatic rings were important for the activity, especially that of methoxy groups, which differed from the previous report,44) 2) the glycoside moiety markedly decreased the activity, 3) The C-α/C-β double bond increased the activity, 4) the substitution group at the C-3′ position in trimethylresveratrol was preferably OH>H>OCH3 for the activity.
Furthermore, 20, 21, 23, and 33 inhibited the release of β-hexosaminidase by ionomycin, and their IC50 values (IC50=23, 31, 8.5, 2.9 µM, respectively) were similar to those by the antigen (17, 24, 5.1, 2.1 µM, respectively) in RBL-2H3 cells.
Our previous study on the effects of stilbenes against nitric oxide production in LPS-activated macrophages indicated structural requirements similar to those for the antiallergic activity, except that the C-α/C-β double bond of stilbenes did not affect nitric oxide production,36) whereas the C-α/C-β double bond is essential for the antiallergic activity.
Active stilbenes (20, 21, 23, 33) with degranulation inhibitory activity on the release of TNF-α and IL-4 in RBL-2H3 cells were also investigated, and 33 exhibited potent activity against TNF-α and IL-4 release (IC50=10, 39 µM, respectively), whereas 20 exhibited the most potent activity against IL-4 with an IC50 value of 32 µM. Trimethylresveratrol (23), which is active against the release of β-hexosaminidase (IC50=5.1 µM), exhibited only moderate or less activity against the release of TNF-α and IL-4 (64, >100 µM, respectively). These results indicate that the mechanism of action of 23 on degranulation is somewhat different from that on the release of TNF-α and IL-4. Piceatannol (21) exhibited moderate activity against both TNF-α and IL-4, with IC50 values of 30 and 72 µM, respectively.9)
Regarding the stilbene-related compounds, 3-phenyl-isocoumarins [e.g. thunberginol B (5,6,3′,4′-tetrahydroxy 3-phenyl-isocoumarin)] from the process leaves of Hydrangea macrophylla var. thunbergii also showed the inhibitory activity with an IC50 value of 5.7 µM, and the 3,4-dihydro derivarives also showed weaker activity. In addition, the gene expression of several cytokines and c-fos in RBL-2H3 cells treated with thunberginol B was similar to that of a flavone, luteolin (5).16,17) Stilbene derivatives and phenanthrenes from the tubers of G. conopsea also showed moderate activity.10)
3.2. Possible Target Molecules of Piceatannol (21)Piceatannol (21) was reported to be a selective inhibitor of Syk in RBL-2H3, and the inhibition of Syk has been considered to be an inhibitory mechanism of degranulation by 21.45,46) However, our results, in which 21 inhibited a calcium ionophore ionomycin-stimulated degranulations, suggested that other mechanisms such as the inhibition of Ca2+ influx or degranulation mechanisms after Ca2+ influx as well as the inhibition of Syk were involved in the inhibitory activity of 21.9)
The rhizomes of C. zedoaria have been used as antiinflammatory and antiflatulance agents as well as a spice and fragrance in Asia. Pharmacological studies of this herb resulted in reports on the antiinflammatory effects of the extract and sesquiterpenes were reported.47–50) In our previous studies, we reported the hepatoprotective, vasodilative, and nitric oxide production–inhibitory activities of various sesquiterpenes and curcuminoids isolated from the rhizomes of C. zedoaria cultivated in China.51–56)
In our study, the 80% aqueous acetone extract of the dried rhizomes of C. zedoaria cultivated in Thailand (Thai zedoary)57) was found to inhibit the release of β-hexosaminidase induced by the antigen in sensitized RBL-2H3 cells and ear PCA reactions in mice.14) Four curcuminoids and two bisabolane-type sesquiterpenes were isolated from the active fraction. Therefore, the effects of curcuminoids and their derivatives on the release of β-hexosaminidase induced by the antigen in sensitized RBL-2H3 cells were examined.
4.1. Structural Requirements of Curcuminoids for the Antigen-Induced Release of β-Hexosaminidase from RBL-2H3 CellsCurcumin (34), monomethylcurcumin (35), and bisdemethoxycurcumin (38) exhibited potent inhibitory activities with IC50 values of 5.3, 15, and 11 µM, respectively. Other compounds showed moderate or weak activity14) (Fig. 6). Yano et al. reported that the extract of the rhizomes of Curcuma longa and several curcuminoids isolated from the active fraction inhibited histamine release induced by compound 48/80, concanavalin A, and/or calcium ionophore A23187 in rat peritoneal mast cells.58) However, the structure–activity relationships of antiallergic activity of curcuminoids have not been sufficiently studied.
a) Data taken from ref. 14.
Regarding the structural requirements of curcuminoids for antiallergic activity, when the conjugated olefins at the C-1 to C-7 positions and the C-3′ and C-3″ methoxy groups decreased in number, the activities decreased. This indicates the importance of the conjugated olefins and methoxy groups of curcuminoids. However, the conjugated olefins seem to be more important than the methoxy groups for the activity. Compound 38 was reported to show stronger activity than 34 against compound 48/80-induced histamine release in rat peritoneal mast cells.58) However, in our study, the activity of 34 was greater than that of 38 against antigen-IgE mediated degranulation in RBL-2H3 cells.
Compound 34 with the C-4′ hydroxy group exhibited activity ca. 3-fold more potent than 35 with the C-4′ methoxy group. Moreover, 36 with both the C-4′ and C-4″ methoxy groups exhibited less activity. These findings suggested that at least one p-hydroxy group of the aromatic rings of 34 was essential to exhibit the activity, and we concluded that the conjugated olefins at the C-1 to C-7 positions and the C-4′ and C-4″ hydroxy groups of curcuminoids are essential for the activity, whereas the C-3′ and C-3″ methoxy groups only enhanced the activity14) (Fig. 7).
Calcium ionophores are compounds that enhance Ca2+ influx into the cell by increasing the membrane permeability of Ca2+. The increase in [Ca2+]i is followed by the degranulation of mast cells or basophils and activates the formation of inflammatory mediators such as PGs and LTs.1–3) Yano et al. reported that curcumin (34) inhibited histamine release induced by concanavalin A, compound 48/80, and A23187 in rat peritoneal mast cells, and they concluded that 34 potently suppressed the histamine release, probably through the inhibition of the degranulation process following a rise in [Ca2+]i levels.58) Consistent with that report, 34 and 38 inhibited A23187 and ionomycin-induced degranulation of RBL-2H3 cells, and their IC50 values (7.2 and 13 µM for A23187, 6.0 and 15 µM for ionomycin) were nearly the same as those of the antigen (5.3 and 11 µM).14)
Curcumin (34) was previously reported to inhibit the production of TNF-α and IL-1 in a human macrophage cell line induced by LPS and to inhibit IL-12 production in mouse splenic macrophages induced by LPS,59–61) but the effects of 34 on the antigen-induced production of cytokines in basophils was not investigated. In our study, 34 inhibited the release of TNF-α and IL-4 with IC50 values of 20 and 18 µM respectively, while the values of 38 were 38 and 34 µM.14)
4.2. Possible Molecular Targets of Curcumin (34)Regarding the mechanism of action of curcuminoids, Lee et al. reported that 34 inhibited the activity of Syk which plays a pivotal role in FcεRI-mediated degranulation of mast cells.62) However, a previous study58) and our results, showing that 34 inhibited calcium ionophore A23187-stimulated degranulation, suggested that other mechanisms such as degranulation mechanisms after Ca2+ influx as well as the inhibition of Syk were involved, as for flavonoids and stilbenes.
Curcumin (34) has poor bioavailability and is metabolized to less active reduction metabolites such as 40 and 43 and their glucuronides.63) However, Li et al. reported that the oral administration of 34 significantly attenuated IgE/antigen-induced passive systemic anaphylaxis in mice, as determined by serum LTC4, PGD2, and histamine levels.64)
Several antiallergic agents clinically used, such as tranilast and ketotifen fumarate, show weak inhibitions of the degranulation in rat peritoneal mast cells and RBL-2H3 cells in vitro (IC50>100 µM), contrary to our expectations. We found many degranulation inhibitors with a variety of chemical structures from medicinal plants using RBL-2H3 cells. Recently, many researchers have also reported various active constituents using similar methods, and these active compounds may be promising resources for developing new antiallergic agents. However, most active constituents found in in vitro examinations have not yet been examined in animal experiments (in vivo), except for several flavonoids, stilbenes, and curcuminoids. Their effectiveness should be confirmed in in vivo experiments. In addition, since the antiallergic constituents such as flavonoids are contained in foods as well as medicinal plants, the relation between eating habits and allergic conditions of patients should be investigated.
This research was supported by the 21st COE Program, Academic Frontier Project, and a Grant-in-Aid for Scientific Research from the Ministry of Education Culture, Sports, Science and Technology of Japan.
The authors declare no conflict of interest.
