Vialinins A and B: Novel Bioactive Compounds from - American

Chapter 41

Vialinins A and B: Novel Bioactive Compounds from Thelephora vialis, an Edible Mushroom in China 1

2

2

2

Chun Xie , Hiroyuki Koshino , Yasuaki Esumi , Shunya Takahashi , Jun-ichi Onose , Kunie Yoshikawa , and Naoki Abe Downloaded by MONASH UNIV on April 23, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch041

1

1

1,2

1

Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan Molecular Characterization Team, Advanced D&S Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 2

Thelephora vialis is a mushroom that grows in symbiosis with pine trees found in Yunnan province, China. It is one of the most favorite edible mushrooms due to its special flavor and taste. Several compounds were isolated from the Thelephoraceae family. For example, ganbanjuns B, C, D and Ε were isolated from the fresh fruiting body of Thelephora ganbajun in 2001 (1), and thelephorin A with DPPH radical -scavenging activity was isolated from thefreshfruitingbody of Thelephora vialis in 2002 (2). However, bioactive compounds in Thelephora species have been poorly studied. In the course of our screening for new bioactive compounds from Thelephora vialis, we isolated five pure compounds and an inseparable mixture.

© 2008 American Chemical Society Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Isolation and Purification for Bioactive Compounds Dry fruiting bodies of the Thelephora vialis (420 g) were soaked in 80% aqueous acetone (8.0 L) for 48 hours at room temperature and filtered. The filtrate was evaporated to the aqueous concentrate in vacuo, and then extracted with EtOAc at pH 3.0, to give 36.5 g of the neutral and acidic extract. Ten g of the extract was applied to column chromatography on a Sephadex LH-20 gel filtration by using a mixture of CHC1 and CH OH (6:4), to give two active fractions A and B. Each^active fraction was rechromatographed by using a preparative HPLC column to give five pure compounds (1, 2, 5, 6, and 7) and an inseparable mixture (3:4 = 3:1). Downloaded by MONASH UNIV on April 23, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch041

3

3

Structure Elucidation of Isolated compounds The molecular formula of compound 1 was determined as C H 608 from high-resolution ESI-MS (pos.) data. However, the H - and C-NMR spectra of 1 in acetone-d only exhibited 7 proton and 13 carbon signals. The H-NMR spectrum revealed five aromatic proton signals (8 6.83, 7.03, 7.12, 7.22, and 7.25) and a singlet methylene signal (8 3.36). The C-NMR and HMQC spectral data revealed the existence of a methylene (8 40.6), five aromatic methine groups (8 115.9, 127.7, 129.2, 130.2, and 132.4), and six aromatic and/or ester quaternary carbon (8 123.1, 124.4, 134.7, 141.7, 157.8, and 169.6) signals. Two quaternary carbon signals were distinguish between 134.63 ppm and 134.68 ppm by high-resolution C-NMR method. Compared with the molecular formula of 1, all of the NMR spectra suggested that the structure of 1 was symmetric. The structure was further elucidated through interpretation of HMBC experiments of 1. The HMBC results and proposed structure of 1 were shown in Figure 1. To confirm the proposed structure of 1, tri-p-bromobenzoate was prepared as an unsymmertical derivative for NOE studies. The NOEs of this unsymmertical derivative were fully supported our structure for 1. (Figure 1) Based on all these evidence, the structure of 1 was determined to be 5',6'bis(phenylacetoxy)-l,r:4',r'-terphenyl-2 ,3 ,4,4"-tetraol, a novel compound, which named vialinin A . The 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical-scavenging mechanism of vialinin A (1) was also examined. The structure of a new product between vialinin A and DPPH indicated that vailinin A donated two hydrogen atoms to two molecules of DPPH radical. The molecular formula of compound 2 was determined as C34H24O9 from high-resolution ESI-MS (pos.) spectrum. The *H-NMR spectrum showed 20 protons, including 16 aromatic methine protons and 2 singlet methylene groups at 3.19 ppm and 3.88 ppm. The C-NMR spectrum of 2 observed 34 carbon 34

!

2

13

!

6

H

13

H

C

C

C

13

,

,

3)

13

Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Downloaded by MONASH UNIV on April 23, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch041

Figure 1. Summary ofHMBC and NOE results for vaialinins A (I) and B (2) and tri-p-bromobenzoate (V).


468 signals, included characteristic signals of two ester (5 171.1 and 170.9) and two sp methylene (5 41.5 and 41.0) carbon signals. Further structural information was obtained by HMBC and NOESY experiments (Figure 1). On the basis of the spectral analyses, the structure of 2 was determined to be 3-(4-hydroxyphenyl) dibenzofuran-1, 2, 4, 7, 8-pentaol 1, 2-O-diphenylacetate. This compound was also a novel compound, designated vialinin B (4). The *H and C-NMR data including 2D NMR data for the other compounds gave enough evidence to identify compound 3 and 4 as an inseparable mixture of ganbajunins D and E, 5 as atromentin, 6 as ganbajunin B and 7 as cycloleucomelone. Those compounds were divided into two groups: (1) terpheny group (1, 3, 4 and 5) and (2) dibenzofuran group (2, 6 and 7) based on their skeleton. (Figure 2) C

2

C


13

Antioxidant Activity The antioxidative activities of those compounds were examined by using the DPPH free radical-scavenging system. Except atromentin and cycloleucomelone, they all showed strong DPPH radical- scavenging activities compared to BHT. The results were shown in Table 1.

Table I. DPPH-Radical Scavenging Activities Compounds

EC (uM)

Vialinin A (1) Vialinin B (2) Ganbajunins D (3)/E (4) Atromentin (5) Ganbajunin B (6) Cycloleucomelone (7) BHT

24.0 10.0 24/6 not detected 10.4 not detected 56.7

50

Anti-allergic Activity In addition to our search for other bioactivities of these compounds, we focused on anti-allergic activity. To clarify the anti-allergic activity of isolated compounds, the inhibitory effects on the release of (^-hexosaminidase, as a marker of degranulation of rat basophilic leukemia-2H3 cells (RBL-2H3), and the inhibitory effects on the production of an inflammatory cytokine TNF-oc were examined.


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OTTO HO

r^N

OH

O

OH

O

OH

vialinin A (1) Downloaded by MONASH UNIV on April 23, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch041

ganbajunin D (3)

ay atromentin (5) ganbajunin E (4)

ganbajunin B (6) HO

H

\P o^o

cycloleucomelone (7) Figure 2. Structures of bioactive compounds isolatedfrom Thelephora vialis.


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Effects on p Hexosaminidase Release and TNF-ot Production Induced by Antigen in RBL-2H3 Cells


5

RBL-2H3 cells were dispensed in 24-well plate at a concentration of 2xl0 cells/well, and then incubated with anti-dinitrophenol (DNP) IgE for 16 h at 37°C for sensitization of the cells. The supernatants were discarded, and the cells were washed 2 times with DMEM containing 2% (v/v) fetal calf serum. Then, 400 pL of testing compounds were added to the cells and they were incubated for 15 min at 37°C. Finally, 100 pL of DNP-bovine serum albumin (BSA) (50 pg/mL) was added, and the cells were incubated for 3 h at 37°C. For measuring the effects on P-hexosaminidase release, the supernatant was withdrawn from each well, to 50 pL of supernatant, 200 mL of substrate solution [1.3 mg of /?-nitrophenyl-2-acetamide-2-deoxy-P-glucopyranoside per mL of 0.1 M sodium citrate buffer (pH 4.5)] was added, and the mixture was incubated for 60 min at 37°C. The reaction was terminated by addition of 500 pL of 0.2 M glycine (adjusted to pH 10.0 with 1 N NaOH), and absorbance at 405 nm was measured. To quantify the enzyme activity remaining in the cells, they were then treated with 500 pL of 0.2% (v/v) Triton X-100, and the extract was analyzed as described above. For measuring the produced TNF-oc from the cells, the supernatant was collectedfromeach well and absolute amount of these mediators was determined using rat TNF-ot ELISA system (Biosource). A clinical immunosuppressant FK506 (Prograf®) was used as a positive control.

Table II. Inhibitory Effects on ^-Hexosaminidase Release and TNF-a Production from RBL-2H3 Cells Inhibition: IC (nM) 50

P-hexosaminidase release Compounds

antigen

Vialinin A (1) 500 Vialinin B (2) 500 Ganhajunins D (3)/E (4) > 10,000 Atromentin (5) >10,000 Ganbajunin B (6) 10,000 Cycloleucumelone (7) 10,000 FK506 0.03

DTBHQ-PMA > 10,000 > 10,000 > 10,000 > 10,000 10,000 > 10,000 0.03

TNF-a production antigen DTBHQ-PMA 0.09 0.02 > 10,000 > 10,000 5,000 3,500 0.25

5,000 5,000 9,000 10,000 > 10,000 > 10,000 0.25


471 As shown in Table II, in terphenyl group, vialinin A showed a strong inhibitory activity on p-hexosaminidase release with an IC value of 500 nM, while ganbajunins D and E mixture, and atromentin showed no remarkable activities. Meanwhile, in the dibenzofuran group, vialinin B exhibited the most potent activity with an IC value of 500 nM. In the experiment of the inhibitory effects on TNF-a production of each compound induced by antigen, vialinins A and B were potently inhibit its production. In this assay, the IC values of vialinins A and B were 0.09 nM and 0.02 nM, respectively, suggesting that vialinins were stronger inhibitors than FK506 (IC = 0.25 nM), clinical immunosuppressant, and were approximately 2xl0 -fold more effective than the related compounds. (Table II). 50

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50

50


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Effects on (3-Hexosaminidase Release and TNF-a Production Induced by DTBHQ and PMA in RBL-2H3 Cells To investigate the detailed mechanisms of degranulation and cytokine production in mast cells, an inhibitor of the endoplasmic reticulum Ca -ATPase [2,5-di(/-butyl)-l,4-hydroquinone, DTBHQ] and a protein kinase C (PKC) activating agent [12-O-teradcanoyl-phorbol-13-acetate, TPA] were used for PKC-calcium activation. The inhibitory effects on P-hexosaminidase release and TNF-a production induced by DTBHQ and PMA in RBL-2H3 cells were evaluated similar as described above. RBL-2H3 cells were dispensed in a 24-well plate at a concentration of 2xl0 cells/well, after adding the testing compounds, instead of antigen-antibody reaction, DTBHQ and PMA were added to each well for sensitization of the cells. And then, the supernatant was collected from each well and inhibitory effects were determined. As shown in Table II, in the conditions, FK506 was maintained with its strong inhibitory activities against P-hexosaminidase release and TNFa production. In contrast, vialinins A and B have lost their potent inhibitory activities. 2+

5

Conclusion In the present study, vialinins A and B potently inhibited the phexosaminidase release and TNF-a production in RBL-2H3 cells by the antigen-antibody reaction. These activities, however, were dramatically decreased in the case of stimulating with PKC-calcium system. The results strongly suggested that the inhibitory events of vialinins A and B may happen


472 before the increase of calcium ion concentration and the activation of PKC. We deduced that anti-allergic activities of vialinins A and B were highly depended on their chemical structures from our SAR study. Further investigation on the inhibitory mechanisms of vialinins is in progress.

Acknowledgement


This work was supported by the Granting-Aid for Pursuing the Sophistication of Education Research in Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References 1. Hu, L.; Gao, J.-M.; Liu, J.-K. Helv. Chim. Act. 2001, 84, 33420-3349. 2. Tsukamoto, S.; Abdulgafor, D. M . ; Abe, T.; Hirota, H.; Othta, T. Tetrahedron 2002, 58, 1103-1105. 3. Xie, C.; Koshino, H.; Esumi, Y.; Takahashi, S.; Yoshikawa, K.; Abe, N . Biosci. Biotechnol. Biochem. 2005, 69, 2326-2332. 4. Xie, C.; Koshino, H.; Esumi, Y.; Onose, J.; Yoshikawa, K.; Abe, N. Bioorg. Med. Chemi. Lett. 2006, 16, 5424-5426.


Vialinins A and B: Novel Bioactive Compounds from - American

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