Letter pubs.acs.org/OrgLett
Galactomannan from Antrodia cinnamomea Enhances the Phagocytic Activity of Macrophages Namal Perera,†,‡,§,⊥,□ Feng-Ling Yang,†,□ Ching-Ming Chang,† Yueh-Tung Lu,# Shih-Hao Zhan,† Yi-Ting Tsai,∥ Jung-Feng Hsieh,∥ Lan-Hui Li,¶ Kuo-Feng Hua,*,#,△ and Shih-Hsiung Wu*,†,‡ †
Institute of Biological Chemistry and ‡Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan § Department of Chemistry, National Tsing-Hua University, Hsinchu, Taiwan ⊥ Faculty of Applied Sciences, Sabaragamuwa University, Belihuloya 70140, Sri Lanka ∥ Department of Food Science, Fu-Jen Catholic University, Taipei, Taiwan ¶ Department of Laboratory, Kunming Branch, Taipei City Hospital, Taipei, Taiwan # Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan △ Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan S Supporting Information *
ABSTRACT: Galactomannan with an octasaccharide-repeating unit (ACP) was isolated from Taiwan medicinal mushroom Antrodia cinnamomea, and its chemical structure was determined herein. ACP significantly enhanced the phagocytosis and bactericidal activity of J774A.1 murine macrophages against Escherichia coli, with prospects for developing a new immunomodulatory compound or adjuvant in immunotherapy and vaccination.
acetonitrile to remove polar small molecules. Remaining solid was then extracted with cold water and lyophilized to obtain crude polysaccharides (recovery percentage was 8.29 w/w%). After DNase I, RNase, and protease K treatment, to remove nucleic acids and proteins, the partially purified polysaccharide was further separated by size exclusion chromatography to yield the major neutral polysaccharide (ACP) as an off-white amorphous solid (approximately 70 kDa). The detailed extraction procedure is given in Supporting Information (Figure S1). The biological activity of ACP targeting TNF-α and phagocytosis enhancement in murine macrophages were first assessed and further subjected to structural analysis. Monosaccharide composition of ACP was analyzed by acidic hydrolysis with 0.5 M methanolic HCl at 80 °C for 16 h followed by trimethylsilylation [HMDS/TMCS/pyridine, 3:1:9]. The final trimethylsilylated products were analyzed by GC-MS. Sugar composition data revealed the presence of mannose (75%) and galactose (25%) as major components in ACP. Hakamori methylation followed by GC-MS analysis was used to determine
Antrodia cinnamomea (phylum Basidiomycota) is one of the most extensively investigated medicinal fungi in Chinese medicine. Traditionally, it is used as a remedy for liver diseases, abdominal pain, drug intoxication, diarrhea, itchy skin, hypertension, and cancer. Recent scientific studies revealed that this medicinal mushroom’s extracts as well as purified compounds have various biological potential, including anti-inflammatory, antioxidant, cytotoxic, hepatoprotective, and anticancer activities.1 Based on its medicinal efficacy, the total market value of A. cinnamomea products is estimated to be over US$100 million per year.2 Triterpenoids, steroids, benzoquinone derivatives, and polysaccharides have all been identified as important pharmacologically active constituents of A. cinnamomea. Most of the functional secondary metabolites are also identified and wellcharacterized,2 and its crude polysaccharides have shown many biological activities.3 However, the structure-based analysis of Antrodia polysaccharides is limited. For the first time, this study explored the NMR-based complete structural characterization of a polysaccharide from A. cinnamomea, its effects on phagocytosis, and the bactericidal potential of murine macrophages. Commercially available mycelia of A. cinnamomea, which were grown under recommended conditions (25 °C, potato dextrose broth), were lyophilized, pulverized, and washed with aqueous © 2017 American Chemical Society
Received: May 15, 2017 Published: June 13, 2017 3486
DOI: 10.1021/acs.orglett.7b01468 Org. Lett. 2017, 19, 3486−3489
Letter
Organic Letters
signal beyond 103 ppm led to the conclusion that all sugar residues are in pyranoside form. Inverted signals at 61 ppm (A, B, C, and F), 65 ppm (G and H), and 66 ppm (D and E) of the 13CDEPT spectrum clearly showed the methylene protons (C-6 positions) of each sugar residue (Figure S5). The relatively downfield 1H and 13C chemical shift values of the sixth position of D, E, G, and H (δ = 3.6−4.0 and 65−66 ppm), second positions of A, C, and D (δ = 3.99−4.10 and 78 ppm), and third position of residue F (δ = 3.94 and 77 ppm) indicated substitutions at O-6, O-2, and O-3, respectively. Linkages between sugar residues were analyzed using NOESY (800 MHz; mixing time 400 ms) and HMBC (500 MHz) spectra. In the NOESY spectrum (Figures 3 and S11), the inter-
the glycosyl linkages of ACP. Results revealed the presence of 3,4,6-tri-O-methyl-D-mannose, 2,4,6-tri-O-methyl-D-mannose, 3,4-di-O-methyl-D-mannose, and 2,3,4-tri-O-methyl-D-galactose, indicating that the repeating unit of ACP mainly consisted of (1→2)-linked mannose, (1→3)-linked mannose, (1→2,6)linked mannose, and (1→6)-linked galactose residues (Figure S2 and Table S1). NMR spectra of ACP in D2O were recorded on Bruker Avance 500 and 800 spectrometers (equipped with a cryoprobe) at 305 K. The proton NMR (500 MHz) spectrum (Figures 1 and S3) of
Figure 1. 1H NMR spectrum (500 MHz) of ACP (D2O, 305 K); anomeric signals are annotated.
ACP contained eight major signals (at δ 5.27, 5.13, 5.10, 5.07, 5.04, 5.03, 4.99, and 4.98 ppm) in the anomeric region as well as a number of overlapping signals in the ring proton region. The presence of eight anomeric signals indicated the octasaccharide nature of the repeating unit. The anomeric signals were designated as A, B, C, D, E, F, G, and H, whereas ring protons linked to each anomeric signal were completely assigned using 1D-TOCSY, 2D-TOCSY (mixing time 80−100 ms), and COSY experiments (Table S2). Furthermore, coupling constants smaller than 3 Hz (JH−H < 3 Hz) indicated the presence of α-configured sugar residues. The H-2 signals of the first six residues (A−F, α-mannose) and H-2 signals of G and H (α-galactose) appeared as a reasonably separated set of peaks around 3.6−4 ppm, which are in agreement with previous reports.4 Chemical shift values of the corresponding 13C anomeric signals of A, B, C, D, E, F, G, and H were, respectively, assigned as 100.5, 102.3, 98.0, 97.8, 102.2, 102.0, 97.8, and 97.8 ppm using 13C NMR, HSQC, and HSQCTOCSY spectral data (Figures 2 and S4−S10). The absence of
Figure 3. Part of the NOESY spectrum (800 MHz) of ACP (D2O, 305 K); correlations are given as H/H.
residual connectivities were observed between AH-1/DH-2, BH1/FH-3, CH-1/HH-6 and 6′, DH-1/GH-6 and 6′, EH-1/AH-2, FH-1/CH-2, GH-1/EH-6 and 6′, and HH-1/DH-6 and 6′, indicating the presence of (1→2), (1→3), (1→6), and (1→2,6) linkages. The HMBC spectrum, which showed AH-1/DC-2, BH-1/FC-3, CH-1/HC-6, DH-1/GC-6, EH-1/AC-2, FH-1/ CC-2, GH-1/EC-6, and HH-1/DC-6 cross peaks (Figures 4 and S12), firmly supported the inter-residual connectivities revealed by NOESY experiment. Considering the above spectral evidence, A and C were assigned as (1→2)-α-D-mannopyranosyl, whereas D was assigned as (1→2,6)-α-D-mannopyranosyl residues.
Figure 2. 13C NMR spectrum (500 MHz) of ACP (D2O, 305 K); most relevant signals are annotated.
Figure 4. Part of the HMBC spectrum (500 MHz) of ACP (D2O, 305 K); correlations are given as C/H. 3487
DOI: 10.1021/acs.orglett.7b01468 Org. Lett. 2017, 19, 3486−3489
Letter
Organic Letters Similarly, E and F were, respectively, assigned as (1→6)-α-Dmannopyranosyl and (1→3)-α-D-mannopyranosyl residues. In addition, G and H were identified as (1→6)-α-D-galactopyranosyl moieties. None of the ring protons of residue B showed inter-residual connectivity with other anomeric signals, so it was considered as a terminal nonreducing 1-α-D-mannopyranosyl unit. Based on the NMR and GC-MS results, we determined the putative structure of ACP to be {→6)-D-Manp-(α1→2)-DManp-(α1→2)[D -Manp-(α1→3)- D-Manp-(α1→2)-D -Manp(α1→6)- D-Galp-(α1→6)]-D -Manp-(α1→6)- D-Galp-(α1→}. Using this deduced structure of ACP, mannose and galactose contents were theoretically calculated as 75 and 25%, respectively. These values corroborate the experimental sugar composition data given by GC-MS analysis. Besides being a major dietary component providing energy for cellular function, polysaccharides have gained attention recently for their therapeutic potential as immunomodulators. Immunostimulants can nonspecifically enhance the innate immunity of the host by stimulating immune cells to produce cytokines, modifying a specific antigen-based response, and enhancing defense mechanisms such as phagocytosis. Phagocytic ability can be characterized by the increased phagocytosis and bactericidal activity of macrophages. Immunostimulatory potential of ACP was investigated by measuring its cytokine induction activity in J774A.1 macrophages, and it was found that ACP induced TNF-α production in a dose-dependent manner (Figure 5A). ACP was further investigated for its enhancing effects on phagocytosis and the bactericidal potential of J774A.1 macrophages. After treatment with ACP (100 μg/mL), cells were infected with E. coli for 1 h. The number of engulfed bacteria was determined 1 and 18 h after infection. As shown in Figure 5B,C, ACP significantly increased the number of engulfed bacteria (2553 ± 356 CFU/mL) compared to the untreated cells (1426 ± 245 CFU/mL). Moreover, the reduced number of bacteria after 24 h incubation indicates the enhanced bactericidal activity of ACP-pretreated cells compared to that of the untreated cells. In the treated cells, 1.5 times more bacteria were killed compared to the untreated cells (Figure 5D), revealing the potency of ACP on phagocytosis enhancement and bactericidal activity of macrophages. There was a considerable increase of TNF-α after 1 h infection and a dramatic decrease after 18 h in the ACP-treated cell culture medium (Figure S13). These results demonstrated that ACP pretreatment activated the macrophages to produce immune mediators, which might enhance immune responses against the bacteria in the early stage but attenuate the risk of severe inflammation in the latter stages of infection. The nutritional and therapeutic value of mannan polysaccharides has been investigated in many in vitro and in vivo studies.5 Mannose-containing polysaccharides are major structural components in the fungal cell wall. Additionally, a number of fungal species (Morchella esculenta, Aspergillus spp., Cordyceps sinensis, and yeast spp.) contain biologically active galactomannans.6 A previous report of an antiangiogenic nonadecasaccharide repeating unit structure of fucosylated (1→6) mannogalactan (400 kDa) from hot water extract of A. cinnamomea7 was the first study of the detailed structure of the polysaccharide component of A. cinnamomea. The antiangiogenic activity might be due to the high ratio of α-1,6-branched galactose and partial fucose containing terminals in the structure. In contrast, ACP in the present study was extracted by cold water. The molecular weight distributed around 70 kDa, and a higher percentage of mannose was found in ACP. There are various
Figure 5. ACP induces TNF-α production and increases the phagocytosis activity of macrophages. J774A.1 macrophages were incubated for 24 h with or without ACP. The TNF-α levels in the supernatants were measured by ELISA (A). J774A.1 macrophages were incubated for 18 h with or without ACP (100 μg/mL). After being washed, cell cultures were infected with or without E. coli for 1 and 18 h; the cells were lysed, and the engulfed live E. coli was determined by CFU assay (B,C). The number of killed bacteria was calculated by 1 h CFU − 18 h CFU (D). Data are expressed as the means ± SD of three separate experiments; ***indicates a significant difference at the level of p < 0.001 compared to the untreated control group (A) or infection-alone group (B,D).
linkages, including α-1,2-, 1,3-, and 1,2,6-linkages, in mannopyranosyl residues. The minor part is α-1,6-galactose. Therefore, different extraction methods might bring different kinds of fungal polysaccharides and different medicinal functions. In summary, the current study is the first to report the occurrence of galactomannan (ACP) in A. cinnamomea and provide a detailed structural analysis. Furthermore, its potential to enhance phagocytosis and the bactericidal activity of murine macrophages was demonstrated. The current findings will enable investigation into the detailed mechanism of ACP-induced immunostimulation and phagocytosis. Based on the structure and biological activity, ACP is a strong candidate for the development of new carbohydrate-based nutraceutical supplements and adjuvants in immunotherapy for many kinds of diseases.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01468. 3488
DOI: 10.1021/acs.orglett.7b01468 Org. Lett. 2017, 19, 3486−3489
Letter
Organic Letters
■
Detailed extraction process, bioassay, NMR spectra, and spectroscopic data tables (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Tel.: +886-(03) 931-7626. Fax: +886-(03) 931-1526. *E-mail:
[email protected]. Tel.: +886-(02) 2785-5696. Fax: +886-(02) 2653-9142. ORCID
Namal Perera: 0000-0003-3576-6148 Author Contributions □
N.P. and F.-L.Y. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research was financially supported by Ministry of Science and Technology (MOST), Taiwan (MOST 105-2628-B-197001-MY3), and the Taiwan International Graduate Program (TIGP), Academia Sinica. We gratefully acknowledge NMR and Biophysics cofacility of Institute of Biological Chemistry for technical support, and Dr. Y.K. Rao for manuscript proofreading.
■
REFERENCES
(1) (a) Lu, M. C.; El-Shazly, M.; Wu, T. Y.; Du, Y. C.; Chang, T. T.; Chen, C. F.; Hsu, Y. M.; Lai, K. H.; Chiu, C. P.; Chang, F. R.; Wu, Y. C. Pharmacol. Ther. 2013, 139, 124−156. (b) Yue, P. Y.; Wong, Y. Y.; Chan, T. Y. K.; Law, C. K. M.; Tsoi, Y. K.; Leung, K. S. Y. Int. J. Med. Mushrooms 2012, 14, 241−256. (c) Geethangili, M.; Tzeng, Y. M. eCAM. 2011, 2011, 212641. (d) Cheng, J. J.; Huang, N. K.; Chang, T. T.; Wang, D. L.; Lu, M. K. Life Sci. 2005, 76, 3029−3042. (2) Wang, H. C.; Chu, F. H.; Chien, S. C.; Liao, J. W.; Hsieh, H. W.; Li, W. H.; Lin, C. C.; Shaw, J. F.; Kuo, Y. H.; Wang, S. Y. J. Agric. Food Chem. 2013, 61, 8556−8564. (3) (a) Lin, C. C.; Pan, I. H.; Li, Y. R.; Pan, Y. G.; Lin, M. K.; Lu, Y. H.; Wu, H. C.; Chu, C. L. PLoS One 2015, 10, e0116191. (b) Chen, C. C.; Liu, Y. W.; Ker, Y. B.; Wu, Y. Y.; Lai, E. Y.; Chyau, C. C.; Hseu, T. H.; Peng, R. Y. J. Agric. Food Chem. 2007, 55, 5007−5012. (4) (a) Cheng, H. N.; Neiss, T. G. Polym. Rev. 2012, 52, 81−114. (b) Bubb, W. A. Concepts Magn. Reson. 2003, 19A, 1−19. (5) (a) Meng, X.; Liang, H.; Luo, L. Carbohydr. Res. 2016, 424, 30−41. (b) Ramberg, J. E.; Nelson, E. D.; Sinnott, R. A. Nutr. J. 2010, 9, 54. (6) (a) Duncan, C. J. G.; Pugh, N.; Pasco, D. S.; Ross, S. A. J. Agric. Food Chem. 2002, 50, 5683−5685. (b) Bardalaye, P. C.; Nordin, J. H. J. Biol. Chem. 1977, 252, 2584−2591. (c) Miyazaki, T.; Oikawa, N.; Yamada, H. Chem. Pharm. Bull. 1977, 25, 3324−3328. (d) Ikuta, K.; Shibata, N.; Hanehiko, H.; Kobayashi, H.; Suzuki, S.; Okawa, Y. FEBS Lett. 1977, 414, 338−342. (e) Gorin, P. A. J.; Spencer, J. F. T. Can. J. Chem. 1968, 46, 2299−2304. (7) Cheng, J. J.; Lu, M. K.; Lin, C. Y.; Chang, C. C. Carbohydr. Polym. 2011, 83, 545−553.
3489
DOI: 10.1021/acs.orglett.7b01468 Org. Lett. 2017, 19, 3486−3489
