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 re- leases of TNF-a and IL-4 by antigen in RBL-2H3 cells. Phyllodulcin and hydrangenol also showed significant in- hibition 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, Ca2 release from intracellular Ca2 stores and ported that compounds 1, 2, and 5 inhibited histamine release influx via Ca2 release-activated Ca2 channels (CRAC). El- from rat peritoneal mast cells stimulated by compound 2 2 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 3 -hydroxy- blank (B): DNP-BSA ( ), test sample ( ) hydrangeaic acid (9) derived from 7 on the degranulations normal (N): DNP-BSA ( ), test sample ( ) 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 2 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-hex- osaminidase 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 (5 107 cells) in 5 ml of Siraganian buffer Hydrangeae Dulcis Folium, and thunberginol G (6) and 3 - 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- (2 105 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 (%) [1 (T N)/(C N)] 100 sity of 2 105 cells per well and were incubated with anti- DNP IgE antibody (0.45 mg/ml) for sensitization of the cells. control (C): DNP-BSA ( ), test sample ( ) Then, the cells were washed twice with Siraganian buffer test (T): DNP-BSA ( ), test sample ( ) normal (N): DNP-BSA ( ), test sample ( ) (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 Ca2 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 (5 104 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. was 10 mg/ml) were added to each well. The ple was calculated by the following equation, and IC50 values inhibition (%) of increase in fluorescence intensity (DRFU) were determined graphically: by the test sample 8 min after stimulation was calculated by the following equation, and IC values were determined inhibition (%) [1 (T B N)/(C N)] 100 50 graphically: control (C): DNP-BSA ( ), test sample ( ) inhibition (%) [1 (T N)/(C N)] 100 test (T): DNP-BSA ( ), test sample ( ) 390 Vol. 30, No. 2
control (C): DNP-BSA ( ), test sample ( ) phenyldihydroisocoumarins [thunberginols C (3), E (4), and test (T): DNP-BSA ( ), test sample ( ) G (6), phyllodulcin (7), and hydrangenol (8)], a benzyli- normal (N): DNP-BSA ( ), test sample ( ) denephthalide [thunberginol F (5)], and a stilbene [3 -hy- Statistics Values are expressed as means S.E.M. One- droxyhydrangeaic acid (9)] on antigen-induced degranulation way analysis of variance followed by Dunnett’s test was used in RBL-2H3 cells were examined. As shown in Table 1, com- for statistical analysis. pounds 1, 2, and 5 substantially inhibited the release of b- hexosaminidase from the cells stimulated by DNP-BSA as an
RESULTS AND DISCUSSION antigen with IC50 values of 17, 5.7, and 19 m M. However, the 3-phenyldihydroisocoumarins (3, 6, 7, 8) except for 4 Effects of the Compounds from Hydrangeae Dulcis (IC50 48 m M) showed weak inhibition of the degranulation Folium on Release of b-Hexosaminidase from RBL-2H3 by 14—52% at 100 m M (Table 1). To confirm that the anti-al- Cells As a marker of the degranulation of RBL-2H3 cells, lergic effects of these active compounds are due to the inhibi- release of b-hexosaminidase into the medium was deter- tion on release of b-hexosaminidase, but not the false posi- mined. In the present study, inhibitory effects of two 3- tive due to the inhibition of enzyme activity of b-hex- phenylisocoumarins [thunberginols A (1) and B (2)], five 3- osaminidase, effects of compounds 1, 2, and 5 on the enzyme activity were examined. As a result, compounds 1, 2, and 5 showed only a weak inhibition against enzyme activity of b- Table1. Inhibitory Effects of Compounds 1—9 from Hydrangeae Dulcis Folium on Antigen-Induced Release of b-Hexosaminidase in RBL-2H3 hexosaminidase by 17, 8, and 25% at 100 m M. Cells With regard to the structure–activity relationships, we pre- viously reported some structural requirements of 3- Compounds Conc. (m M) Inhibition (%) IC50 (m M) phenylioscoumarins for degranulation using rat peritoneal mast cells. Briefly, the 3,4-double bond is essential for the Thunberginol A (1)00.0 3.4 3 2.0 2.3 strong activity, and the hydroxyl groups at the 8-, 3 -, and 4 - 10 29.0 1.8** positions of thunberginols, but the 6-hydroxyl group is not 20 59.6 1.2** 17 important, and a lactone moiety enhanced the activity.8) In 30 74.7 1.8** agreement with the previous report, the activities of 1 and 2 60 96.0 1.8** having the 3,4-double bond showed stronger activities than 100 104.8 1.8** Thunberginol B (2)00.0 2.3 those of 3, 4, 6, 7, and 8 lacking the 3,4-double bond [e.g. 1 3 15.8 0.8** (IC50 17 m M) 6 ( 100 m M)] indicating that the 3,4-double 6 52.3 1.2** 5.7 bond is essential for the strong anti-degranulation activity. In 10 81.1 1.7** our previous study about effects on the degranulation,13) 30 101.3 1.0** Thunberginol C (3)00.0 3.0 piceatannol (3,3 ,4 ,5-tetrahydroxystilbene) showed signifi- 10 6.2 1.0 cant inhibition of degranulation in the same bioassay meth- 30 8.2 2.9 100 ods (IC50 24 m M), but the inhibitory effect was weaker than 100 43.6 1.7** that of 2 having lactone moiety. In the present study, 3 -hy- Thunberginol E (4)00.0 1.4 droxyhydrangeaic acid (9) lacked the activity. These results 10 13.9 2.7** 30 32.8 3.1** 48 indicate the importance of lactone moiety for the strong ac- 100 76.8 1.3** Thunberginol F (5)00.0 0.5 Table2. Inhibitory Effects of Compounds 1, 2, 5, 7, and 8 from Hy- 10 23.2 1.3** drangeae Dulcis Folium on Calcium Ionophore A23187-Induced Release of 20 50.4 2.1** b-Hexosaminidase in RBL-2H3 Cells 30 69.4 1.9** 19 60 94.4 1.2** Compounds Conc. (m M) Inhibition (%) IC50 (m M) 100 102.5 0.3** Thunberginol G (6)00.0 1.4 Thunberginol A (1)00.0 9.0 100 13.6 4.1** 100 10 7.8 3.9 Phyllodulcin (7)00.0 4.3 20 45.0 3.0** 22 60 30.0 4.6** 30 70.1 1.1** 100 51.5 2.3** ca. 90 60 95.9 1.6** Hydrangenol (8)00.0 4.1 Thunberginol B (2)00.0 4.8 100 36.3 4.6** 100 3 26.4 3.6** 3 -Hydroxyhydrangeaic 0 0.0 3.5 6 46.3 2.3** 6.2 acid (9) 100 28.6 6.1** 100 10 71.1 3.4** Tranilast14) 0 0.0 1.7 30 100.1 1.0** 30 8.2 1.8 Thunberginol F (5)00.0 3.4 100 22.4 2.5* 282 10 33.0 4.6** 300 56.9 3.4** 20 62.7 2.3** 15 1000 75.0 0.6** 30 81.0 2.7** Ketotifen fumarate14) 0 0.0 1.8 60 98.0 0.6** 30 7.4 1.5 Phyllodulcin (7)00.0 0.8 100 27.6 2.2* 158 100 22.0 6.4** 100 300 80.7 1.8** Hydrangenol (8)00.0 0.8 1000 100.7 1.1** 100 12.2 2.0** 100
Each value represents the mean S.E.M. (n 4). Significantly different from the con- Each value represents the mean S.E.M. (n 4). Significantly different from the con- trol, ∗ p 0.05, ∗∗ p 0.01. trol, ∗ p 0.05, ∗∗ p 0.01. February 2007 391
Fig. 2. Effects of Compounds 1, 2, and 5 on the Changes of Fluorescence-Intensity in Fluo-3 Loaded RBL-2H3 Cells Each value represents the mean with S.E.M. (n 4). Significantly different from the control, ∗ p 0.05, ∗∗ p 0.01. tivity. Table3. Inhibitory Effects of Compounds 1, 2, 5, 7, and 8 from Hy- On the other hand, different from the previous report,8) the drangeae Dulcis Folium on Antigen-Induced Releases of TNF-a and IL-4 in activities of 2, 3, and 4 having the 6-hydroxyl groups were RBL-2H3 Cells stronger than those of 1, 7, and 8 lacking the 6-hydroxyl TNF-a IL-4 Conc. group [2 (5.7 m M) 1 (17 m M), 4 (48 m M) 7 (ca. 90 m M), 3 Compounds (m M) (inhibition at 100 m M: 43.6%) 8 (36.3%)] and methylation Inhibition (%) IC50 (m M) Inhibition (%) IC50 (m M) of the 4 -hydroxyl group tended to enhance the activity [7 Thunberginol A (1)0 0.0 8.3 0.0 3.9 (inhibition at 100 m M: 51.5%) 6 (13.6%)]. 10 19.9 7.1 4.0 2.2 In addition, compounds 1, 2, and 5 were reported to show 30 59.5 10.8** 23 8.0 1.1** 43 inhibitory activity for degranulation induced by calcium 60 86.2 13.9** 65.3 1.7** ionophore A23187 in mouse peritoneal mast cells.8) In agree- 100 — 99.9 0.1** ment with the previous report, compounds 1, 2, and 5 inhib- Thunberginol B (2)0 0.0 1.4 0.0 1.0 6 25.5 1.8 — ited the release of b-hexosaminidase, but not 7 and 8, in 10 44.2 2.8** 11 16.3 2.7** 19 RBL-2H3 cells. 20 78.4 1.6** 50.3 1.2** Effects of Thunberginols A (1), B (2), and F (5) on 30 96.1 5.4** 78.6 0.9** 2 60 — 99.5 0.0 Change in [Ca ]i In RBL-2H3 cells, tyrosin kinase Syk recruited by aggregated FceRI phosphorylates phospholipase Thunberginol F (5)0 0.0 15.4 0.0 3.9 30 38.3 10.2** 41 — 100 Cg, which leads to the generation of inositol 1,4,5-triphos- 60 63.5 4.7** 10.1 3.5** 2 phate (IP3). IP3 causes the release of Ca from intracellular 100 85.0 3.3** 36.8 2.4** Ca2 stores and activates Ca2 influx via CRAC channels to Phyllodulcin (7)00.0 11.8 0.0 1.0 replenish the depleted Ca2 stores. The Ca2 influx is essen- 100 18.6 10.3 100 26.0 0.9** 100 Hydrangenol (8)00.0 4.6 0.0 0.6 tial for the degranulation and cytokine production in RBL- 1—3) 100 24.8 23.4 100 11.6 1.2 100 2H3 cells. Calcium ionophores are compounds that en- Luteolin14) 0 0.0 3.1 0.0 2.1 2 hance Ca influx into the cell by increasing membrane per- 3 25.0 3.0** ca. 5.8 41.6 1.3** ca. 3.7 2 2 10 89.0 1.3** 89.4 1.0** meability of Ca . The increase in [Ca ]i is followed by the movement of granules to plasma membrane followed by the 30 101.3 1.2** 99.2 0.2** degranulation of mast cells or basophils and activates the for- Each value represents the mean S.E.M. (n 4). Significantly different from the con- mation of inflammatory mediators such as prostaglandins trol, ∗ p 0.05, ∗∗ p 0.01. and leukotrienes.1) Therefore, we examined effects of thun- 2 berginols A (1), B (2), and F (5) on increase in [Ca ]i in- tenuated by 2. These findings suggested that compounds 1, 2, duced by antigen and/or calcium ionophore A23187 in RBL- and 5 inhibited the degranulation processes both before and 2 2H3 cells using calcium probe Fluo 3-AM. after increase in [Ca ]i. As shown in Fig. 2, thunberginols A (1), B (2), and F (5) Effects of Thunberginols A (1), B (2), and F (5) on concentration-dependently inhibited the increase in intensity Antigen-Induced Releases of TNF-a and IL-4 in RBL- of fluorescence induced by the antigen. This finding indicates 2H3 Cells The antigen-induced mast cells and basophils that compounds 1, 2, and 5 inhibited antigen-induced in- produce and release cytokines, which alter the local microen- 2 crease in [Ca ]i. The IC50 values of 1 and 2 were 26 and vironment and lead to the recruitment of inflammatory cells 1) 9.6 m M respectively 8 min after the stimulation, but the in- such as neutrophils and eosinophils eventually. The in- hibitory effect of 5 was weaker than those of 1 and 2. On the hibitory effects on releases of TNF-a and IL-4 were exam- 2 other hand, A23187-induced increase in [Ca ]i could not at- ined by ELISA method. In pre-examination about releases of 392 Vol. 30, No. 2 them in the medium, the releases in the medium reached to ed., W. H. Freeman and Company, New York, 2002. the maximum level 2—4 h after stimulation (data not 2) Matsubara M., Masaki S., Ohmori K., Karasawa A., Hasegawa K., Biochem. Pharmacol., 67, 1315—1326 (2004). shown). As shown in Table 3, thunberginols A (1), B (2), and 3) Fischer M. J., Paulussen J. J., De Mol N. J., Jassen L. H., Biochem. F (5) substantially inhibited releases of TNF-a and/or IL-4 in Pharmacol., 55, 1255—1262 (1998). the culture medium 4 h after the stimulation. Among them, 2 4) Yoshikawa M., Uchida E., Chatani N., Murakami N., Yamahara J., showed the strongest activity. Chem. Pharm. Bull., 40, 3121—3123 (1992). In conclusion, the 3-phenylisocoumarins [thunberginols A 5) Yoshikawa M., Uchida E., Chatani N., Kobayashi H., Naitoh Y., Okuno Y., Matsuda H., Yamahara J., Murakami N., Chem. Pharm. (1) and B (2)] and a benzylidenephthalide [thunberginol F Bull., 40, 3352—3354 (1992). (5)] from the processed leaves of Hydrangea macrophylla 6) Yoshikawa M., Harada E., Naitoh Y., Inoue K., Matsuda H., Shimoda var. thunbergii (Hydrangeae Dulcis Folium) substantially in- H., Yamahara J., Murakami N., Chem. Pharm. Bull., 42, 2225—2230 hibited the degranulation by antigen and calcium ionophore (1994). A23187, and the releases of TNF-a and IL-4 by antigen in 7) Yoshikawa M., Matsuda H., Shimoda H., Shimada H., Harada E., Naitoh Y., Miki A., Yamahara J., Murakami N., Chem. Pharm. Bull., RBL-2H3 cells. With regard to structural requirements of the 44, 1440—1447 (1996). 3-phenylisocoumarins for the activity, the 3,4-double bond 8) Matsuda H., Shimoda H., Yoshikawa M., Bioorg. Med. Chem., 7, was essential for the strong activity and the 6-hydroxyl group 1445—1450 (1999). and lactone ring enhanced the activity. The active compounds 9) Yamahara J., Matsuda H., Shimoda H., Wariishi N., Yagi N., Mura- 1, 2, and 5 inhibited increase in [Ca2 ] in RBL-2H3 cells in- kami N., Yoshikawa M., Folia Pharmacol. Jpn., 105, 365—379 i (1995). duced by antigen, but not by calcium ionophore A23187. 10) Matsuda H., Shimoda H., Yamahara J., Yoshikawa M., Biol. Pharm. Bull., 22, 870—872 (1999). 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