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Two immunosuppressive compounds from the mushroom Rubinoboletus ballouii using human peripheral blood mononuclear cells by bioactivity-guided fractionation.



Pistillarin 1-ribofuranosyl-s-triazin-2(1 H)-one Rubinoboletus baltouil bioassay-guided fractionation peripheral blood mononuclear cells (PBMCs)


Rubinoboletus ballouii is an edible mushroom wildly grown in Yunnan province, China. Up till now, little was known about the chemical and biological properties of this mushroom. The aim of this study was to investigate the immunomodulatory effects of the ethanolic extract of Rubinoboletus ballouii and its fractions on human peripheral blood mononuclear cells (PBMCs) using bioactivity-guided fractionation. The crude extract of the fruiting bodies of RB was fractionated by high-speed counter current chromatography (HSCCC). Twelve fractions were obtained and the third fraction (Fraction C) exerted the most potent anti-inflammatory activities in mitogen-activated PBMCs. Further fractionation of fraction C led to the isolation of two single compounds which were elucidated as 1-ribofuranosyl-s-triazin-2 (1 H) -one and pistillarin, respectively. The results showed that both 1-ribofuranosyl-s-triazin-2(1H)-one and pisti I-larin exhibited significant immunosuppressive effects on phytohemagglutinin (PHA)-stimulated human PBMCs by inhibiting [methyl-(3)H]-thymidine uptake and inflammatory cytokines productions such as tumor necrosis factor (TNF)-[alpha], interleukin (IL)-10, interferon (IFN)-[gamma] and IL-1[beta]. Besides, 1-riboluranosyl-s-triazin-2(1H) -one was firstly found in natural resources, and pistillarin was also isolated from the family Boletaceae for the first time. They exhibited great potential in developing as anti-inflammatory reagents.

[c] 2013 Elsevier GmbH. All rights reserved.


Many edible mushrooms exhibited various biological properties including immunomodulatory, anti-inflammatory, anti-tumor, anti-HIV, anti-oxidative activities (El Dine et al., 2008; Jiang et al., 2011; Zhang et al., 2003). For instance, the components isolated from the rhizome of Curcuma longa exerted potent immunostimulatory effects on human peripheral blood mononuclear cells (PBMCs), they stimulated the PBMCs proliferation and increased the releases of IL-2, TNF-[alpha], and IFN--[gamma] (Yue et al., 2010). Fraction AB-BDM-2 from Agaricus blazei Murill could suppress phytohemag-glutinin (PHA)-stimulated PBMCs proliferation, inhibit the release of several cytokines such as IL-2, IL-4 and IFN-[gamma], and arrest the cell cycle progression from the G1 transition to the S phase (Kuo et al., 2002).

Rubinoboletus ballouii P. & D., belonging to the family Boletaceae, was abundantly found in Yunnan province, China. Several compounds were previously isolated from this family, in particular the firstly isolated ones from Tylopilus virens including a new ergostane-type steroidal glycoside, named tylopilosicle (Liu, 2007). Up till now, very little information has been reported on the components and the biological properties of Rubinoboletus ballouii.

Human PBMC represent a heterogeneous population of immune cells including B cells, T cells, monocytes, NK cells and various granulocytes. In a close interplay, these cells orchestrate innate and acquired immune responses that might either be enhanced or reduced by the addition of mushroom compounds to these human PBMC cultures (Jeurink et al., 2008). Hence, our present study was to identify active component (s) from the ethanolic extract of Rubi-nobolerus ballouii with immunomodulatory activities using human PBMC.

High-speed counter current chromatography (HSCCC) is one form of highly efficient preparative counter current chromatography (CCC) technique. Due to limited solid support as stationary phase in this type of chromatography, "tailing", irreversible adsorption from solid support and denaturation of the solute problems are perfectly solved in HSCCC (Ito, 2005). Many successful examples have been reported about employing HSCCC on the separation of various natural and synthetic components, since it was first developed in 1970s (Han et al., 2006; Lopes-Lutz et al., 2011).

As part of the searching for natural immunomodulators from native wild mushrooms, the ethanolic extract of the fruiting bodies of Rubinoboletus ballouii exhibited an ability to suppress the immune response by inhibiting several cytokines such as TNF-[alpha], IL-2, IL-10 and IFN-[gamma]. Herein, the bioassay-guided fractionation, structural determination, and biological properties of an active fraction and two bioactive compounds responsible for the immunosuppressive activity are described in this paper.

Materials and methods


The mushroom R. ballouii was collected in Yunnan province, China in 2009 and authenticated by Professor YANG Zhuliang, Kunming Institute of Botany, China. A voucher specimen was deposited in the herbarium of the Institute of Chinese Medicine, the Chinese University of Hong Kong, with voucher specimen number 2009-3232. Methanol, ethanol, acetone, n-butanol and formic acid (all of analytical grade) were purchased from TED1A company, Inc., USA. Distilled water was prepared using a Millipore (MA, USA) MILLI-Q SP reagent water system. XIT (2,3-bis-(2-methoxy-4-ni tro-5-sulfopheny1) -2H-tetrazolium-5-carboxanilide) powder and dexamethasone used as positive control were purchased from Sigma-Aldrich (St. Louis, USA).


TBE-1000A preparative high-speed counter current chromatography (Shanghai, Tauto Biotech, China) was employed in this study, which was connected with a BUCHI 615 middle pressure liquid chromatography (MPLC) series (BUCHI Labortechnik AG, Switzerland) contained pump, UV detector and sample collector. Thin layer chromatography (TLC) silica gel G [F.sub.254] plates (20 cm x 20 cm, 05 mm) were purchased from Merck KGaA (Darmstadt, Germany). (1) H, (13) C and 2D NMR spectra were recorded with Tetram-ethylsilane as internal standard. The qualitative analysis of fractions and compounds were performed on a Waters BEH C18 column (2.1 x 100 mm, Ireland) with an Agilent Technologies 1290 system (Agilent, USA).

Preparation of the two-phase solvent system and sample solution

A two-phase solvent system of n-butanol-water (1: 1) was used in our present study. The solvents with the volume ratio of 1: 1 were mixed in a separation funnel and fully equilibrated by hand shaking continuously for at least 15 min at room temperature. The lower and upper phases were separated and degassed by sonication for 20 min before use. The air-dried powder of the fruiting bodies (350g) of R. ballouii was extracted twice with 95% ethanol (10 lx 2). The extracts were combined and centrifuged at 4000 rpm. 20 [degrees] C for 30 min. The supernatant was collected and concentrated at 45 [degrees] C under reduced pressure, and then lyophilized to give a powder crude extract (78.0 g). RBE (6.3g) was redissolved in 80 ml of the two-phase solvent to make the sample solution.

Fractionation and purification by HSCCC and HPLC

The upper phase of the solvent system was filled into the coil column at a flow rate of 50 ml/min using a Buchi 615 MPLC pump. The apparatus was rotated at 500 rpm. After filling with the upper phase, the lower phase was pumped into the column at a flow rate of 8.0 ml/mm. The system temperature was maintained at 25 [degrees] C. The sample solution was injected into the column, after the mobile phase emerged out of the end of the column and hydrodynamic equilibrium was formed in the column. The effluent was collected at 10 min/tube. After separation for 350 min, the system was stopped and all the remaining solution was pushed out of the column using high-pressure gas. Fractions with similar TLC profiles were combined and grouped into twelve major fractions (Fr.A-Fr.L). Fraction C was further fractionated by a preparative C8 column (4.6 x 250 mm, Aglient) eluted with gradient elution ofacetonitrile-distilled water (2%, 20 min), followed by a self-prepared MCI gel column (2.5 x 200 cm) eluted with 30-90% methanol in water and 70% acetone. Two compounds C3-1 (12 mg) and C3-3 (24 mg) were yielded from the elution of 40% methanol and 70% acetone in distilled water.

Analytical reverse-phase ultra performance liquid chromatography (RP-UPLC)

The qualitative analysis of the isolated compounds was performed on a Waters BEH C18 column (2.1 x 100 mm, Ireland) with a Agilent Technologies 1290 system (Agilent, USA). Samples were eluted with a solvent system of A: 0.1% formic acid in [H.sub.2] O, B: Methanol (A: B=8: 2). Flow rate was set at 0.300 ml/min. Column temperature was 40 [degrees] C. The absorbance measurements were performed at 254 nm.

Preparation of lymphocytes from Human Peripheral Blood Mononuclear Cells (PBMCs)

Peripheral blood buffy coat from Hong Kong Red Cross Blood Transfusion Service was used (Yue et al., 2010; Yue et al., 2012). Buffy coat was diluted with PBS. After centrifugation, the resulting cell pellet was resuspended in RPMI1640. The cell suspension was then layered onto an equal volume of Ficoll-Paque (TM) Plus gradient solution (Amersham Pharmacia Biotech, Uppsala, Sweden) in a 15-ml centrifuge tube and centrifuged at 800 xg for 20 minutes. PBMC were collected at the interface between the two layers and then washed twice with RPM! 1640 medium. The cells were resuspended in RPM! medium supplemented with 10% (v/v) fetal bovine serum, 100 IU/ml penicillin, and 100 [micro]g/ml streptomycin for subsequent experiments.

Evaluation of immlinomodulatoiy effects

Ten thousand cells were seeded in 100 pi of medium per well of 96-well flat-bottom culture plates (Iwaki, Japan). Cells were exposed to different concentrations of the fractions, co-incubated with phytohemagglutinin (PHA, 10 [micro]g/ml). After incubation at 37 [degrees] C and in 5% C [O.sub.2] atmosphere for 24 hours, supernatant was collected for cytokine assay. Cells were further incubated for 48 hours, XTT assay and [methyl-(3)H]-thymidine uptake assay were then conducted (Yue et al., 2012). For [methyl-(3)-H]-thymidine uptake assay, [methyl-(3)H]-thymidine (0.5 [micro] Ci/well) was added into each well and incubated for 6 hours. The cells were harvested on glass fiber filters by cell harvester and the radioactivity in the filters was determined by Packard TopCount NXTT [TB] Microplate Scintillation and Luminescence Counter (PerkinElmer Inc., MA, USA).

Determination of human cytokines IL-1[beta], IL-2, IL-10, IFN- [gamma] and TNF-[alpha] production

The concentrations of IL-1[beta], IL-2, IL-10, IFN--[gamma] and TNF-[alpha] from the collected supernatants were quantified by ELISA (The BD OptEIA [TM] ELISA, BD Bioscience PharMingen (CA, USA)) and the assay procedures were carried out as described in the kit manual recommended.

CD marker expression from human PBMC

The isolated human PBMC (3 x [10.sup.6]/m1) were treated with various concentrations (50-100 [micro] g/ml) of fraction C and PHA (10 [micro] g/ml) in 24-well plates for 24 hours. The cells were harvested and blocked with 1% human serum in PBS for 20 min at room temperature. Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD4 and CD14, phycoerythrin (PE)-conjugated CDS and mouse IgG1 isotype were added into the tubes, then the tubes were incubated at 4 [degrees] C for 30 min in the dark. The cells were washed with PBS supplement with 0.05% sodium azide and 1% bovine serum albumn for three times before subjecting to flow cytometry for analysis. 10,000 viable cells were gated and analyzed for the expressions of CD4, CD8 and CD14 by flow cytometry (Becton Dickinson FACSCanto II, USA).

Cell cycle analysis of the active fraction on human PBMC

The isolated human PBMC (2 x [10.sup.6]/m1) were treated with various concentrations (50-100 [micro] g/ml) of fraction C and the cells were co-incubated with PHA (10 [micro] g/ml) in a 24-well plate for 24 hours. The cells were then collected in flow tubes and fixed in 70% (v/v) ethanol at 4 [degrees] C overnight. Before flow analysis, cells were resuspended in PBS with propidium iodide (PI, 20 [micro] g/ml) and RNase A (10 [micro] g/ml) at room temperature for 40 min in the dark. Ten thousand cells per sample were collected by flow cytometry. Modfit LT (Verity Software House, ME, USA) was employed for analysis of cell cycle and apoptotic peak modeling.

Statistical analyses Data were expressed as the mean [+ or -] the standard error of the mean (SEM). Statistical analyses and significance, as measured by one-way analysis of variance (ANOVA) were performed by Repeated Measures ANOVA, Tukey test using GraphPad PRISM software version 5.0 (GraphPad Software, San Diego, CA, USA). In all comparison, p <0.05 was considered statistically significant.

Results and discussion

Fractionation by HSCCC

In our screening, the 95% ethanol extract of R. ballouii with yield over 20% (w/w) exerted immunosuppressive effects on PHA-stimulated human PBMC in terms of cytokines production of TNF-[alpha], IL-10, IL-2 and IFN-[gamma], (data not shown). To identify the active components in RB, the ethanolic extract of RB was fractionated by HSCCC using the two solvent system of n-butanol and distilled water with the ratio of 1: 1. Fig. 1A showed that twelve fractions named fraction A--fraction L were produced from 28 collected tubes from HSCCC, based on the similar TLC profiles. The 1000 ml solvent remained in the HSCCC column after the separation was pushed out by high-pressure gas to make the last fraction L. All the fractions were dried using rotary evaporator in vacuum and weighed. Over 75% of the ethanolic extract of RB was water-soluble components in the first two fractions. The non-polar part was around 10% in the last fraction. 6.06g fractions were collected from 6.30g of loaded sample, giving the sample recovery of 96.2%.

Anti-proliferative effects offractions on human PBMCs

All fractions were screened for the proliferative effects on PHA-stimulated human PBMCs using XTT and [methyl-(3)H]-thymidine uptake assays. The concentrations of tested fractions were evaluated at 100[micro]g/ml. Fig. 1B indicated that all fractions showed no cytotoxicity against mononuclear cells using XIT assay. Among all fractions, fraction C showed significant anti-proliferative effects on PHA-stimulated PBMCs even when the concentration was at 1001 [micro] g/ml. The cell proliferative effects of PHA-stimulated human PBMCs of the crude extract were not affected until the concentration was as high as 400 [micro] g/ml (Fig. 1C). Therefore, the immunomodulatory effects of fraction C were further evaluated in human PBMC.

Immunomodulatory activities offraction C in human PBMC

The cultured supernatants were collected after treatment for 24 hours, and tested for the concentrations of cytokines by ELISA. Fig. 2A-2D showed that the productions of TNF-[alpha], IFN--[gamma], 1L-2 and IL-10 were significantly decreased by fraction Cat 100 [micro] g/ml.

Effects of fraction C on the expression of cell surface marker CD4, CD8 and CD14, and cell cycle arrest in human PBMC.

Fig. 2E showed that the ratio of CD4 (+)/CD8 (+) lymphocytes was unaffected when treating with fraction C for 24 hours. In the meanwhile, the percentage of CD14 (+) lymphocytes in the total lymphocytes population was also not changed (Fig. 2F).Taken together, fraction C did not exert selective effects on different subsets of lymphocytes. Besides, [G.sub.2]/M phase of the cell cycle was arrested after treatment with 50 [micro] g/ml of fraction C for 24 hours (Fig. 2G).

Identification of bioactive components

Two compounds 1-ribofuranosyl-s-triazin-2(1H)-one (12 mg) and pistillarin (24 mg) were isolated from the elution of 40% methanol and 70% acetone in distilled water. The structures were elucidated by use of NMR and MS analysis. The physical and chemical data were in agreement with those described in the literature (Capon et al., 2007; Steglich, 1984) (Fig. 3).

Effects of pistillarin and 1-ribofuranosyl-s-triazin-2(1H)-one on human PBMC

Eight blood samples were evaluated in three assays: XTT, [methy1-(3)H]-thymidine uptake and ELISA (Yue et al., 2012). Dexamethasone, an anti-inflammatory and immunosuppressant, was used as a positive control. The collected supernatants were subjected to test for the cytokine concentrations using ELISA kit after co-incubation with various concentrations of 1-ribofuranosyl-s-triazin-2(1H)-one or pistillarin for 24 hours. Fig. 4 showed that in PHA-stimulated PBMC, RT (8-100 [micro]M) could significantly increase the viable cell number, whilst P (8-100 [micro]M) and dexamethasone (Dex, 1 [micro]M) did not show any effects on the viable cell numbers. RT and P could significantly inhibit [methyl-(3)H]-thymidine uptake at 20-100 [micro]M. The releases of TNF-[alpha], IL-1[beta] and IL-10 in the supernatant were significantly decreased over 50% when exposed with 100 [micro]M of 1-ribofuranosyl-s-triazin-2(1H)-one or pistillarin. In the meanwhile the concentrations of IFN-[gamma], cytokines expressed by T lymphocytes were also significantly decreased for about 50% when treating with 8-10011M of 1-ribofuranosyl-s-triazin-2(1H)-one or 20-100 [micro]M of pistillarin. The productions of TNF-[alpha], IL-10, IFN-[gamma] and IL-1[beta] in the cell free supernatants were all down-regulated in a dose-dependent manner. The potencies of the inhibitory activities of pistillarin and 1-ribofuranosyl-s-triazin-2(1H)-one on human PBMC were comparable to curcuminoids and turmerones that we isolated from the rhizome of Curcuma longa (Yue et al., 2010).

In response to pathogens, activated macrophages are triggered to synthesize and release several pro-inflammation cytokines including TNF--[alpha] and IL-1[beta] (Gordon, 2003). These cytokines are believed to serve as inducers of a local inflammatory response, which help to promote the activation of innate cells such as vascular endothelium cells and the release of other pro-inflammatory cytokines to trigger the related adaptive immune responses (Beutler, 1995; Packard and Libby, 2008; Zganiacz et al., 2004). In the present study, both pistillarin and 1-ribofuranosyl-s-triazin-2(1H)-one exhibited immunosuppressive effects on PHA-stimulated human PBMC by inhibiting the [methyl-(3)H]-thymidine uptake and decreasing the production of TNF-[alpha], IL-10, IFN-[gamma] and IL-1[beta]. This is the first report on the isolation and immunomodulatory characterization of pistillarin and 1-ribofuranosyl-s-triazin-2(1H)-one from the family Boletaceae.

Pistillarin, as a rare cate cholate siderophore, was found in several macro fungi and marine species such as Penicillium bilaii when grown in relatively high iron environments (Capon et al., 2007; Steglich, 1984). Functionally, little was known about pistillarin. Pistillari n has been shown to possess protective activities against DNA damage through iron chelation and free radical-scavenging activity (In-Kyoung Lee, 2011). To our best of knowledge, this is the first study on the immunsuppressive effects of pistillarin on human immune cells.

1-Ribofuranosyl-s-triazin-2(1H)-one which was elucidated based on the COSY and HMBC spectra, contained a rare six-membered aromatic ring alternated with three carbon atoms and three nitrogen atoms. The structure was similar to 5-azacyticline (azacytidine), a well-known chemical analogue of cytidine. Azacytidine was used in the treatment of myelodysplastic syndrome. It had been found that low close of azacytidine could inhibit DNA methyltransferase in human myeloid leukemic HL-60 cells (Momparler et al., 1985). As a ribonucleoside, azacytidine would mainly incoporate into RNA. This would lead to the dissembly of polyribosomes (Lin and Glazer, 1981), incomplete methylation (Almstedt et al., 2010) and acceptor function of transfer RNA (Daskalakis et al., 2002). As an analogue of azacytidine, 1-ribofuranosyl-s-triazin-2(1H)-one might have similar actions in the cells.


In vitro bioassay-guided fractionation of the edible mushroom Rubinoboletus ballouii suggesting that pistillarin and 1-ribofuranosyl-s-triazin-2(1H)-one might be the active components which possess immunosuppressive and anti-inflammatory effects by attenuating the syntheses of a panel of inflammatory cytokines: TNF-[alpha], IL-10, IFN-[gamma] and IL-2 as well as [methyl-(3)H]-thymidine uptake in human PBMCs. Further studies of these isolated compounds include the underlying anti-inflammatory mechanisms and in vivo activities might contribute to the development of novel agents from edible mushroom in the treatment of inflammatory disorders such as asthma and other autoimmune diseases.


This project was financially supported by C. C. Wu Cultural & Education Foundation Fund and The Open Fund (P2009-KF03) of the State Key Laboratory of Phytochemistry and Plant Resources in West China.

* Corresponding author at: E205, Science Centre East Block, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China. Tel.: +852 39436873; fax: +852 26035248.

** Corresponding author at: Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201. China. Tel.: +86 871 65216327; fax: +86871 65216327.

E-mail addresses: (J.-K. Liu), (K.-P. Fung).

0944-7113/$--see front matter [c] 2013 Elsevier GmbH. All rights reserved.


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Long-Fei Li (a), (b), Ben Chung-Lap Chan (a), (b), Grace Gar-Lee Yue (a), (b), Clara Bik-San Lau (a), (b), Quan-Bin Han (d), Ping-Chung Leung (a), (b), Ji-Kai Liu (e), (**), Kwok-Pui Fung (a), (b), (c), (*)

(a) Institute of Chinese Medicine, The Chinese University of Hong Kong, Shoatin, N. T., Hong Kong SAR, China

(b) State Key Laboratory of Phytochemistry and Plant Resources in West China (CUHK), The Chinese University of Hong Kong Shatin, N. T., Hong Kong SAR, China

(c) School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N. T, Hong Kong SAR, China

(d) School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong SAR, China

(e) State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
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Author:Li, Long-Fei; Chan, Ben Chung-Lap; Yue, Grace Gar-Lee Yue; Lau, Clara Bik-San; Han, Quan-Bin; Leung,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Oct 15, 2013
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