Inhibitory Effects of Thunberginols A, B, and F on Degranulations and Releases of TNF-A and IL-4 in RBL-2H3 Cells

388 Notes Biol. Pharm. Bull. 30(2) 388—392 (2007) Vol. 30, No. 2 Inhibitory Effects of Thunberginols A, B, and F on Degranulations and Releases of TNF-a and IL-4 in RBL-2H3 Cells a,b a a a b Qilong WANG, Hisashi MATSUDA, Koudai MATSUHIRA, Seikou NAKAMURA, Dan YUAN, and ,a Masayuki YOSHIKAWA* a Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan: and b Shenyang Pharmaceutical University; 103 Wenhua Road, Shenyang, 110016, China. Received October 11, 2006; accepted November 13, 2006; published online November 22, 2006 Thunberginols A, B, and F from the processed leaves of Hydrangea macrophylla var. thunbergii (Hydrangeae Dulcis Folium) substantially inhibited the degranulations by antigen and calcium ionophore A23187, and the releases of TNF-a and IL-4 by antigen in RBL-2H3 cells. Phyllodulcin and hydrangenol also showed significant inhibition for the antigen-induced degranulations, but their effects were weaker than those of thunberginols A, B, and F. Among them, thunberginol B showed the most potent activity. With regard to structural requirements of thunberginols for the activity, the 3,4-double bond was essential for the strong activity and the 6-hydroxyl group and lactone ring enhanced the activity. Thunberginols A, B, and F inhibited increase in intracellular free Ca2؉ levels, which is an essential process for the degranulation and production of cytokines, in RBL-2H3 cells induced by antigen, but not by calcium ionophore A23187. These results suggested that these active compounds inhibited .the degranulation processes both before and after increase in intracellular free Ca2؉ levels Key words thunberginol; degranulation; tumor necrosis factor a (TNF-a); interleukin (IL-4); Hydrangea macrophylla var. thunbergii Basophils as well as mast cells play important roles in lated from Hydrangeae Dulcis Folium, the fermented leaves both immediate and late-phase reactions of type I allergy. of Hydrangea macrophylla SERINGE var. thunbergii MAKINO, Aggregation of FceRI by antigens results in tyrosine phos- as antiallergic constituents.4—7) In the previous study, we re- phorylations, Ca2ϩ release from intracellular Ca2ϩ stores and ported that compounds 1, 2, and 5 inhibited histamine release influx via Ca2ϩ release-activated Ca2ϩ channels (CRAC). El- from rat peritoneal mast cells stimulated by compound 2ϩ 2ϩ 6—8) evation of intracellular free Ca level ([Ca ]i) plays an es- 48/80, calcium ionophore A23187, or antigen, and oral sential role in the degranulation process.1—3) Furthermore, administration of compound 1 inhibited passive cutaneous mast cells and basophils concomitantly synthesize and re- anaphylaxis reactions in rats.9,10) However, their inhibitory ef- lease a variety of cytokines including interleukin (IL)-3, IL- fects on the antigen-induced degranulation and release of cy- 4, granulocyte-macrophage colony-stimulating factor (GM- tokines in basophils have not been reported to date. CSF), tumor necrosis factor a (TNF-a), and these cytokines In our continuing study, we examined the effects of the induce the late-phase reactions, production of IgE, etc.1) isolated compounds [thunberginols A (1), B (2), C (3), E (4), Thunberginols A (1), B (2), and F (5) (Fig. 1) were iso- and F (5), phyllodulcin (7), hydrangenol (8)] from Hy- Fig. 1. Chemical Structures of Compounds 1—9 from Hydrangeae Dulcis Folium ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2007 Pharmaceutical Society of Japan February 2007 389 drangeae Dulicis Folium, thunberginol G (6) and 3Ј-hydroxy- blank (B): DNP-BSA (Ϫ), test sample (ϩ) hydrangeaic acid (9) derived from 7 on the degranulations normal (N): DNP-BSA (Ϫ), test sample (Ϫ) and/or releases of TNF-a and IL-4 via FceRI signaling in rat Tranilast and ketotifen fumarate14) were used as reference basophilic leukemia (RBL-2H3) cells. In addition, effects of compounds. Under these conditions, it was calculated that 2ϩ the active constituents on increase in [Ca ]i were examined 40—70% of b-hexosaminidase was released from the cells to get some information for their mechanism of action. in the control groups by determination of the total b-hexosaminidase activity after sonication of the cell suspension. MATERIALS AND METHODS To clarify that the anti-allergic effects of samples are due to the inhibition of b-hexosaminidase release, but not the false Isolation Thunberginols A (1), B (2), C (3), E (4), and F positive from the inhibition of b-hexosaminidase activity, the (5), phyllodulcin (7), and hydrangenol (8) were isolated from cell suspension (5ϫ107 cells) in 5 ml of Siraganian buffer Hydrangeae Dulcis Folium, and thunberginol G (6) and 3Ј- was sonicated. The solution was then centrifuged, and the su- hydroxyhydrangeaic acid (9) were derived from 7 as de- pernatant was diluted with the incubation buffer and adjusted scribed in our previous reports.4—7,11) to the equal enzyme activity of the degranulation tested Reagents Eagle’s minimum essential medium (MEM) above. The enzyme solution (45 ml) and test sample solution and anti-DNP IgE (Monoclonal Anti-DNP) were purchased (5 ml) were transferred into a 96-well microplate and enzyme from Sigma; fetal calf serum (FCS) was from Gibco; the activity was examined as described above. ELISA kits for determination of TNF-a (TNF-a, rat) and IL- Inhibitory Effects on Antigen-Induced Releases of 4 (IL-4, rat) were from Amersham Pharmacia Biotech; Cal- TNF-a and IL-4 from RBL-2H3 Cells RBL-2H3 cells cium Kit-Fluo 3TM from Dojindo Laboratories; the dinitro- (2ϫ105 cells/well) were sensitized with anti-DNP IgE as de- phenylated bovine serum albumin (DNP-BSA) was prepared scribed above. The cells were washed twice with 500 ml of as described previously,9,12) and other chemicals were from MEM containing 10% FCS, pencillin (100 units/ml), and Wako Pure Chemical Industries. 24-Well multiplates and 96- streptomycin (100 mg/ml), and exchanged with 320 ml of the well microplates were from Sumitomo Bakelite; 96-well fresh medium. Then, 40 ml of test sample solution and 40 ml black microplates were from Nunc. of antigen (DNP-BSA, final conc. 10 mg/ml) were added to Inhibitory Effects on the Release of b-Hexosaminidase each well and incubated at 37 °C for 4 h. The supernatant from RBL-2H3 Cells and on Enzyme Activity of b-Hex- (50 ml) was transferred into a 96-well ELISA plate and TNF- osaminidase As a marker of the degranulation of RBL- a and IL-4 concentrations were determined using commer- 2H3 cells, release of b-hexosaminidase into the medium was cial kits (Amersham). The test samples were dissolved in determined as described previously.13,14) Briefly, RBL-2H3 DMSO, and the solution was added to MEM (final DMSO cells [Cell No. JCRB0023, obtained from Health Science Re- conc. was 0.1%). The inhibition (%) of the releases of TNF- search Resources Bank (Osaka, Japan)] in MEM containing a and IL-4 by the test sample was calculated by the follow- 10% FCS and penicillin (100 units/ml) and streptomycin ing equation, and IC50 values were determined graphically: (100 g/ml) were seeded into 24-well multiplates at the den- m inhibition (%)ϭ[1Ϫ(TϪN)/(CϪN)]ϫ100 sity of 2ϫ105 cells per well and were incubated with anti- DNP IgE antibody (0.45 mg/ml) for sensitization of the cells. control (C): DNP-BSA (ϩ), test sample (Ϫ) Then, the cells were washed twice with Siraganian buffer test (T): DNP-BSA (ϩ), test sample (ϩ) normal (N): DNP-BSA (Ϫ), test sample (Ϫ) (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 25 mM PIPES, and 14) 40 mM NaOH, pH 7.2) supplemented with 5.6 mM glucose, Luteolin was used as a reference compound. 2؉ 1mM CaCl2, and 0.1% bovine serum albumin (BSA) and in- Inhibitory Effects on [Ca ]i Change Stimulated by cubated in 160 ml of buffer for 10 min at 37 °C. Then, cells Antigen The Ca2ϩ responses in RBL-2H3 were assessed were added with 20 ml of test sample solution, and were stim- with Calcium Kit-Fluo 3TM (Dojindo Laboratories). RBL- ulated with 20 ml of DNP-BSA (final conc. 10 mg/ml) as an 2H3 cells (5ϫ104 cells/well) in 96-well black micro plate antigen or calcium ionophores A23187 (final conc. 0.3 m M) were sensitized with anti-DNP IgE antibody (0.45 mg/ml) for for 10 min. The reaction was stopped by cooling in an ice 24 h. The cells were washed twice with PBS buffer and incu- bath for 10 min. The supernatant (50 ml) was transferred into bated with 4.5 m M Fluo 3-AM at 37 °C for 1 h. The Fluo 3- a 96-well microplate and incubated with 50 ml of substrate AM loading cells were washed twice with PBS buffer and in- (1 mM p-nitrophenyl-N-acetyl-b-D-glucosaminide) in 0.1 M cubated with 80 ml recording medium (Calcium Kit-Fluo citrate buffer (pH 4.5) at 37 °C for 1 h. The reaction was 3TM). Then, 10 ml of test sample solution (0.1% DMSO) was stopped by adding 200 ml of stop solution (0.1 M Na2CO3/ added to each well and incubated in FLUOstar OPTIMA NaHCO3, pH 10.0). The absorbance was measured with a (BMG Labtechnologies, Offenburg, Germany) at 37 °C, and microplate reader at 405 nm. The test sample was dissolved the fluorescence of the solution was monitored for 2 min be- in dimethylsulfoxide (DMSO), and the solution was added to fore stimulation and 8 min after stimulation (ex. 485 nm, ex. incubation buffer (final DMSO conc. was 0.1%). The inhibi- 520 nm). After 5th determination, 10 ml of antigen (DNP- tion (%) of the release of b-hexosaminidase by the test sam- BSA, final conc.

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