extfieldsofstudy: Chemistry Medicine

text:
Single-molecule photochemical reactions of Auger-ionized quantum dots

Photoinduced electron transfer in donor-acceptor systems composed of quantum dots (QDs) and electron donors or acceptors is a subject of considerable recent research interest due to the potential applications of such systems in both solar energy harvesting and degradation of organic pollutants. Herein, we employed single-molecule imaging and spectroscopy techniques for the detection of photochemical reactions between 1,4-diaminobutane (DAB) and CdSe/ZnS single QDs. We investigated the reactions by analyzing photoluminescence (PL) intensity and lifetime of QDs at ensemble and single-molecule levels. While DAB was applied to single QDs tethered on a cover slip or QDs dispersed in a solution, PL intensity of QD continuously decreased with a concomitant increase in the PL lifetime. Interestingly, these changes in the PL properties of QD were predominant under high-intensity photoactivation. We hypothesize that the above changes in the PL properties surface due to the transfer of an electron from DAB to Auger-ionized QD followed by elimination of a proton from DAB and the formation of a QD-DAB adduct. Thus, a continuous decrease in the PL intensity of QDs under high-intensity photoactivation is attributed to continuous photochemical reactions of DAB with single QDs and the formation of QD-(DAB)n adducts. We believe that detection and analysis of such photochemical reactions of single QDs with amines will be of considerable broad interest due to the significant impact of photoinduced electron transfer reactions in energy management and environmental remediation.

C olloidal quantum dots (QDs) show unique optical properties such as broad absorption and narrow emission bands as well as bright, stable, and sizedependent tunable photoluminescence (PL) (1,2). Also, the large surface area of QDs is promising for the conjugation of various organic and biomolecules (3). The unique optical and structural properties formulate QDs to be ideal donors or acceptors of electrons in the construction of donor-acceptor systems for modern photovoltaic devices (4Á14).
Given the significance of QDs in solar energy harvesting, investigation of photoinduced electron transfer in QD-based donor-acceptor systems such as QD-TiO 2 (5Á9), QD-Indium Tin Oxide (ITO) (10), QD-inorganic complexes (11), QD-C 60 (12), and QD-polymer (13)  systems, transfer of an electron from a photoactivated QD to the corresponding acceptor results in the formation of charge-separated states. Solar cells based on donor-acceptor systems are designed by directing the charge carrier recombination through an external electrical circuit. Although photoinduced electron transfer processes in QD-based donor-acceptor systems are greatly investigated, photochemical reactions between organic molecules and photoactivated QDs as well as potentials of such reactions for environmental remediation remain mostly unattended.
Recently, Sharma et al. found that p-phenylenediamine lowers the PL quantum efficiency of CdSe QD by accepting an electron from it (14). On the other hand, n-butylamine is known to passivate surface defects in CdSe QD, suppress non-radiative carrier recombination in the defects, and enhance the PL quantum efficiency of QD (14). Nonetheless, controversies exist in the amineinduced variations of QD's PL quantum efficiency. For example, n-butylamine itself at higher concentrations activates non-radiative carrier recombination in CdSe QD and lowers its PL quantum efficiency (15). To-date electron transfer interactions in the above QD-based donor-acceptor systems are found to be reversible by energy wasting back electron transfer. Although reversible electron transfer interactions of QDs with donors or acceptors of electron are greatly investigated, photochemical reactions between hole-acceptors and QDs remain mostly unattended. Importantly, photochemical reactions of highly photostable QDs with dyes, amines, and other organic pollutants would be of great value for environmental waste management. Here we found that photoactivation of CdSe/ZnS QDs in the presence of 1,4-diaminobutane (DAB) results in a gradual but irreversible decrease in the PL intensity of single QDs. This irreversible decrease in the PL intensity is apparent only under high-intensity photoactivation of QDs, which gives us the notion that Auger-ionized QDs accept electrons from DAB molecules and subsequent elimination of a proton from the QD-DAB ' complex results in the formation of a QD-DAB adduct. Thus, we propose that the gradual but irreversible decrease in the PL intensity of single QDs is due to successive photochemical reactions of Auger-ionized QDs with a large number of DAB molecules and the formation of QD-(DAB) n adducts.

Results and discussion
We have investigated photochemical reactions of DAB with QDs at ensemble and single-molecule levels by recording and analyzing PL spectra of QD solutions and PL intensity trajectories of single QDs both in the presence of DAB and under high-intensity photoactivation. Fig. 1A shows the effect of concentration of DAB on the PL spectra of a QD solution. As the concentration of DAB was increased, PL intensity of QDs decreased. Also, we have examined temporal changes in the PL intensity of QDs under continuous photoactivation. Fig. 1B shows PL spectra of a QD solution in the presence of DAB and under continuous photoactivation. Here, PL intensity of QDs continuously decreased with time but without any changes in the concentration of DAB.
A decrease in the PL intensity of QDs with an increase in the concentration of DAB (Fig. 1A) and a temporal decrease in the PL intensity of QDs under continuous photoactivation ( Fig. 1B) are attributed to photochemical reactions of DAB with QDs and the formation of QD-DAB adducts having lower PL quantum efficiencies than pristine QDs.
To validate the photochemical reaction of DAB with QDs, we have recorded and analyzed the PL intensity trajectories of single QDs in the presence and absence of DAB and under low-(0.22 kW/cm 2 ) and high-(2.2 kW/cm 2 ) intensity photoactivation. Fig. 2 shows PL intensity trajectories of single QDs tethered on a cover slip, immersed in water, and recorded in the presence or absence of DAB. Interestingly, in the presence of DAB and under high-intensity photoactivation, we detected a continuous decrease in the PL intensity of single QDs (Fig. 1BÁF). On the other hand, PL intensities of single QDs under high-intensity photoactivation but without DAB remained intact even after several minutes ( Fig. 2A). The excitation power density (2.2 kW/cm 2 ) applied in these experiments was high enough to activate multiple exciton pairs and induce Auger ionization in QDs, which is apparent from intermittent PL blinking of single QDs. Our hypothesis for the gradual decrease in the PL intensity of single QDs in the presence of DAB is that Auger-ionized QDs react with DAB and form QD-DAB adducts having PL quantum efficiencies lower than that of QDs. The reaction of DAB with QD probably took place by the transfer of an electron from DAB to Auger-ionized QD followed by the elimination of a proton from QD-DAB ' adduct. Steps involved in the photochemical reactions of DAB with photoactivated QD is schematically presented in Fig. 3. Under continuous photoactivation, a large number of DAB molecules can be added onto the surface of QDs. Thus, the continuous decrease in the PL intensity can be attributed to the formation of poorly luminescent QD-(DAB) n adducts.
The above hypothesis was investigated by intermittent photoactivation of single QDs in the presence of DAB and under high excitation intensity (2.2 kW/cm 2 ). Fig. 2C and D shows PL intensity trajectories of single QDs excited at 15 s and 60 s laser 'on'/'off' intervals. We found that the PL intensities of single QDs remained essentially unaffected before and after each laser 'off' period, which indicates that the reaction of DAB with QD should be absolutely photoinduced. In other words, DAB does not react with QDs in the dark. Our hypothesis was further investigated by photoactivating single QDs in the presence of DAB but under low excitation intensity (0.22 kW/cm 2 ), which is insufficient to activate multiple excitons and induce Auger ionization. Under these conditions, we found that PL intensities of single QDs remained essentially intact even after 10 min. The relation between the concentration of DAB and PL intensities of single QDs is shown in Fig. 2B, E, and F. Here, as the concentration of a DAB solution in a single QD sample was increased (10 mM for Fig. 2B, 100 mM for Fig. 2E, and 1 mM for Fig. 2F), the rate at which PL intensities of single QDs decreased was also increased. These results and the gradual decrease in the PL intensity of single QDs support our hypothesis that Auger-ionized QDs react with DAB.
The photochemical reaction between QD and DAB was further investigated by recording and analyzing PL decay profiles of QD solutions. Fig. 4 shows PL decay profiles of a QD solution recorded at 2 min intervals in the presence of DAB and under high-intensity (2.2kW/cm 2 ) photoactivation. Auger ionization of QDs under such high-intensity photoactivation is apparent   Fig. 4A and inset). Interestingly, we detected a gradual increase (from 5.9 ns to 9.8 ns, 66%) in the PL lifetime of QDs with time under photoactivation (Fig. 4A). On the other hand, PL lifetime of a QD solution was increased by only B20% when photoactivated without the addition of DAB (inset of Fig. 4A). The spontaneous increase in the PL lifetime of QDs is associated with an increase in the PL quantum efficiency, which originates from an increase in the contribution of radiative carrier recombination in the surface states (16,17). However, an increase in the PL lifetime of QDs under high-intensity photoactivation in the presence of DAB is associated with a gradual but irreversible decrease in PL intensity. Because amines are known to passivate surface defects in QDs, we attribute the changes in the PL properties of QDs to surface passivation of QDs by photoinduced electron transfer reaction with DAB and the formation of QD-(DAB) n adducts. The contribution of DAB to an increase in the PL lifetime of QDs was further investigated by recording and analyzing PL decays of QDs under lowintensity photoactivation. Fig. 4B shows PL decay profiles of a QD solution recorded at 2 min intervals in the presence of DAB and under low (0.22 kW/cm 2 ) excitation intensity, which is insufficient to activate multi-exciton pairs and induce Auger ionization. Therefore, photochemical reactions of DAB with QD takes place only under high-intensity photoactivation. In other words, the decrease in the PL intensity of QDs with time under photoactivation in the presence of DAB can be attributed to photoinduced electron transfer reactions between Auger-ionized QD and DAB and subsequent formation of poorly luminescent QD-(DAB) n adducts.
In short, single-molecule photochemical reactions of an aliphatic amine on the surface of quantum dots were detected by analyzing photoluminescence intensity trajectories and lifetimes of quantum dots at ensemble and single particle levels. As QDs were photoactivated in the presence of DAB, we found continuous decrease in the photoluminescence intensity of quantum dots and concomitant increase in their photoluminescence lifetime values. These changes in the luminescence properties were apparent only under high-intensity photoactivation of quantum dots, during which Auger ionization is operational. Thus, the changes in the luminescence properties of single quantum dots are attributed to photoinduced electron transfer reaction of amine with Auger-ionized quantum dots and the formation of quantum dot-amine adducts. Such photochemical reactions of organic molecules on the surface of quantum dots and other nanoparticles would be of great value for energy management and environment remediation.

Experimental procedure
CdSe/ZnS (PL maximum Â600/655 nm) QDs were obtained from Invitrogen Corporation (Japan) and DAB from Sigma Aldrich (Japan). Single-molecule samples were prepared by tethering QDs on cover slips. The procedure for preparation of single-molecule samples is reported elsewhere (9). In short, cover slips were cleaned by dipping in a nascent chlorine solution and silanized using a 0.5% solution of 3-aminopropyltriethoxysilane in acetone. Silanized cover slips were copiously washed with water and acetone, and biotinylated by dipping in a 10 mM aqueous solution of biotin 3-sulfo-Nhydroxysuccinimide ester (Sigma Aldrich, Japan) for 30 min. Biotinylated cover slips were copiously washed with deionized water and successively, CdSe/ZnS QDs were uniformly tethered on the cover slips by placing a 10 pM solution of QD655-streptavidin conjugate on one side of the cover slip. After incubation for 30 min at room temperature, unbound QDs were removed by washing with deionized water. Finally, QDs were dipped in deionized water and recorded single-molecule images, PL intensity trajectories, and PL lifetimes. Single-molecule experiments were carried out in an inverted optical microscope (Olympus IX 70) equipped with a 60) objective lens (Olympus, NA 0.98). Fluorescence signals collected using the objective lens were filtered through a 620 nm long pass filter, magnified using a 2.5 )telescopic lens, and recorded using an image intensifier, chargecoupled device assembly (Hamamatsu Photonics, Japan). For PL lifetime measurements, samples were excited with 400 nm fs laser pulses from an optical parametric amplifier (OPA). The OPA was pumped by 800 nm pulses (200 kHz) from a regenerative amplifier that was seeded by a mode-locked Ti:sapphire laser (76 MHz). Fluorescence decay profiles were recorded using an assembly of a polychromater and a streak-scope (Hamamatsu photonics, Japan).


s2fieldsofstudy: Chemistry Biology

text:
Synthesis of 86 species of 1,5-diaryl-3-oxo-1,4-pentadienes analogs of curcumin can yield a good lead in vivo.

BACKGROUND
Curcumin is known to possess many anti-tumor properties such as inhibition of tumor growth and induction of apotosis. However, limited bioavailability of curcumin prevents its clinical application. A synthesized curcumin analog, 1,5-diaryl-3-oxo-1,4-pentadiene such as GO-Y030, has the improved anti-tumor potential in vitro as well as in mouse model of colorectal carcinogenesis.

RESULTS
These compounds were divided into two groups; one is the higher anti-proliferative group, in which 79.7% of 1,5-diaryl-3-oxo-1,4-pentadienes were clustered. One of the 1,5-diaryl-3-oxo-1,4-pentadiene analogs, GO-Y078 has the most enhanced growth inhibition, and its solubility was improved, compared with curcumin. GO-Y078 inhibits NF-κB transactivation, as well as expression of TP53 and DR5 more effectively than curcumin. In a mouse model, GO-Y078 presented 1.4 fold more survival elongation that was not achieved by curcumin and GO-Y030.

CONCLUSIONS
The 1,5-diaryl-3-oxo-1,4-pentadiene analogs can yield good lead compounds for cancer chemotherapy, to overcome low bioavailability of curcumin.

Background
Naturally derived products are the most valuable source for drugs and their lead compounds. Approximately 74% of anticancer drugs are either natural products or their derivatives [1]. Curcumin, 1,7-bis(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-dione, is a dietary constituent of turmeric. It is well known for its ability to suppress tumor growth [2]. The mechanisms of its tumor suppression have been examined at the molecular level. Curcumin is known to interfere with the transactivation of nuclear factor-B (NF-B) [3], activator protein 1 [4], and β-catenin [5] thus resulting in the negative regulation of various oncogenes such as c-Myc, cyclin D1, Bcl-2, and Bcl-XL. Curcumin arrests the cell cycle at G 0 /G 1 and/or G 2 /M through the upregulation of the cyclin-dependent kinase inhibitors p21 and p27, and the downregulation of Cdc2 and cyclin B1 [6]. Curcumin blocks signaling of growth factors, including human epidermal growth factor receptor-2 [7], plateletderived growth factor [8] and fibroblast growth factor [9]. It also blocks Wnt signalling [10]. Curcumin also has anti-invasive, antimetastastic, and antiangiogenic properties [11,12]. Therefore, curcumin is labeled as a multi-targeted drug. However, the systemic availability of curcumin remains negligible [13,14]. Hydrophobicity, low absorption, and rapid metabolism have been considered as reasons for curcumin's low bioavailability [2]. To address this issue, we synthesized a series of curcumin analogs and screened them. An analog named GO-Y030 was synthesized that possessed 30-to 50-fold enhanced antiproliferative potential against various types of cancers in vitro [15]. The molecular mechanisms of GO-Y030 closely resembled those of curcumin. Oral administration of GO-Y030 prevented the adenoma formation in the familial adenomatous polyposis (FAP) mouse model without any apparent toxicity [16]. It was also demonstrated that the number and the size of β-catenin-positive adenomas were reduced. In this case, bioavailability of GO YO30 was not an issue because it can be directly delivered to the gastrointestinal tract. However, GO-Y030 was hydrophobic and ineffective to the treatment of peritoneal carcinomatosis (PC) of gastric carcinoma in a mouse model. These observations led us to additionally synthesize hyperactive curcumin analogs in order to optimize their pharmacological potential.

Results
Screening of the hyperactive growth inhibitors among the newly synthesized curcumin analogs Eighty eight curcumin analogs, including 69 species of DOPs, curcumin and 1,7-diaryl-1,6-heptadiene-3,5dione, and 17 species of non-DOP analogs were synthesized ( Figure 1). The growth-suppressive potential of these compounds was then examined on cell panels composed of 16 types of cancers. These included gastric cancers (GCIY and SH10TC), colon cancers (HCT116, DLD1 and SW680), lung cancer (A549), pancreatic cancer (PK1), kidney cancer (ACHN), liver cancer (HUH7), ovarian cancer (OVK18), breast cancer (MCF7), skin cancer (A431), bile duct cancer (HuCCT1), thyroid cancer (8505c), melanoma (G361), and prostate cancer (PC3). The IC50 value of each analog was calculated against the individual cell line. These values were analyzed using cluster analysis ( Figure 2). Resulting from the pattern of inhibition, 88 curcuminoids were divided into 2 clusters; one is the high IC 50 group (Group a) composed of 29 analogs, and the other is the low IC 50 group (Group b) composed of 59 analogs. In detail of Group a, 51.7% (15/29) of those are DOPs,  whereas 91.5% (54/59) are DOPs in Group b. From the view point of DOPs, 55 out of 69 (79.7%) belong to Group b. In this study, 64 species out of 69 analogs are classified to acyclic DOPs, and 81.3% species (52/64) of acyclic DOPs belong to Group b. On the other hand, among 17 non-DOPs, 82.4% species belong to Group a. The analogs that exhibited the strongest potential against all of the cell lines were clustered in Group c ( Figure 2). This cluster included GO-Y078, 079, 030, 097, and 098. All of these compounds had at least 10 times higher growth-suppressive potential than curcumin. For example, the value of GO-Y030 reached 76 times, GO-Y078 84 times, and GO-Y079 29 times higher than curcumin against GCIY. In the solution, as each ethoxyethyl group of GO-Y079 and 097 is predicted to be hydrolyzed in an acidic environment, and GO-Y079 and GO-Y097 can convert themselves to 078 and 098, respectively. Essentially, GO-Y078 is nearly identical to 079, and 098 to 097, just as shown in the cluster analysis. For this reason, we excluded GO-Y079 and 097 from further examination. In this analysis, it has been shown A549 and MCF7 were rather resistant to curcumin analogs among them ( Figure 2).

Improvement in solubility of curcumin analogs
The ADME profiles of 100 curcuminoids, including virtual analogous compounds were determined in silico. Concerning solubility of analogs, the predicted values of top 15 are represented in the bar graph ( Figure 3a). Analogs, that bear the improved solubility than curcumin and belong to Group b at the same time, are 8 compounds including GO-Y078 and Y098. The predictive values of them are 1.98 and 2.93 times higher than curcumin, respectively. The highest predictive value is 11.7 mg/L of GO-Y038, which is 21.7 times higher than curcumin. As for GO-Y030, the solubility is 0.48 times lower than curcumin. Then, we practically examined the solubility of curcumin, GO-Y030, 078, and 098 ( Figure 3b). These compounds were first dissolved in ethanol-cremophor EL ® and then diluted in PBS. Concentration of GO-Y078 in ethanol-cremophor EL ® reached the highest level among them at 300 mM. While there were no aggregations seen in GO-Y078 and 098 solutions after 4 serial dilutions, the appearance of aggregation was observed in curcumin and GO-Y030 solutions immediately after the first dilution ( Figure 3c). In fact, GO-Y078 and 098 showed increased solubility, as predicted.

Growth suppression and apoptosis induction potencies of GO-Y030, 078, and 098
To examine the growth suppression of 3 DOP analogs, GO-Y030, Y078, and Y098 clustered in Group c, the analysis of cell cycle progression in HCT116 cells was conducted by flow cytometry. Analysis of cell cycle progression indicated the increase of the subG 1 fraction. Those treated with GO-Y030, 078, and 098 at 2 μM were 27.2%, 17.1%, and 19.8%, respectively. These values were 6.0, 3.8, and 4.4 times higher than curcumin at the same concentration (Figure 4a). GO-Y030, 078, and 098 may have a stronger potency to induce apoptosis than curcumin. To further elucidate this observation, caspase cleavage reactions were assessed. At a dose of 2 μM, the amount of cleaved product in GO-Y030, 078, and 098 treatments were 2.12 ± 0.07, 3.47 ± 0.84 and 1.67 ± 0.19 times higher, respectively, than curcumin. GO-Y030, 078, and 098 were also considered to be potent inducers of apoptosis ( Figure 4b).
GO-Y030, 078, and 098 can induce apoptosis through numerous mechanisms just as curcumin Generally, cancer cells have some breakdown in key apoptosis induction mechanisms. These include the inactivation of a cohort of NF-B target genes, as well as inactivation of the TP53 pathway, and extrinsic apoptotic pathways. Hyperactivated NF-B in cancer cells leads to protection from apoptotic cell death via its targets including IAPs. It is well documented that curcumin can inhibit NF-B transactivation [3,4]. The inhibitory effects of NF-B transactivation in 3 DOP analogs, were examined by ELISA. In this assay, NF-B transactivation was estimated by the amount of p50 subunit binding to its DNA consensus sequence. NF-B p50 activates a set of target genes in response to certain inflammatory signals, such as TNFα. In this study, curcumin and DOP analogs decreased TNFα-induced NF-B transactivation at a dose of 2 μM. The relative values of transactivation with curcumin, GO-Y030, 078, and 098 were 0.32 ± 0.02, 0.16 ± 0.05, 0.26 ± 0.07, and 0.40 ± 0.01 compared to the control (= 1.0) (Figure 4c). These DOPs were shown to have comparative potency to curcumin in the inhibition of NF-B transactivation.
The p53 gene is altered in nearly half of human cancers, usually by point mutations, and results in the loss of ability to induce pro-apoptotic genes. Following 24 h of treatment, TP53 induction was assessed in HCT116 cells bearing wild-type p53 as a control. After treatment with a 2 μM dose, the TP53 expression in curcumintreated cells was similar to the control level, while the levels of TP53 in GO-Y030, 078, and 098 were increased 2.32, 1.54, and 1.52 times higher, respectively, than the control. (Figure 4d). Activating certain death receptors (DR), particularly DR4 and DR5, has been shown to selectively kill cancer cells while sparing normal cells [21]. Curcumin activates DR5, but does not activate DR4 [22]. DR5 is regulated by either a p53-dependent or -independent mechanism [23]. DR5 overexpression induces ligand-independent apoptosis [24]. The expression of DR5 was estimated in DLD-1 bearing mutant TP53, by Western blotting. After 24 h of incubation, the expression of DR5 was increased to 3.58, 6.20, and 2.39 times higher than the control, in GO-030, 078, and 098 treated cells, respectively (Figure 4e). It was shown that the apoptosis induction potency, including overexpression of TP53 and DR5 was improved, but NF-B inhibition was not.

Therapeutic effects of DOP analogs in an experimental mouse model
According to the data concerning solubility described above, GO-Y078 and 098 may have improved bioavailability. Initially, the toxicity of these compounds was examined in vitro. We examined the growth suppression of the primary normal liver cells, hNHeps ® with GO-Y030, 078, and 098. They do not affect survival nor have any toxic effects to hNHeps ® at doses as high as 50 μM. Then, we conducted a single IP injection, and determining the maximum tolerable dose (MTD). The MTDs of GO-Y030 and 078 were determined to be 474.5 and 400 mg/kg, respectively. Administration of these doses did not affect the animals for over 1 month. However, the MTD of GO-Y098 was rather toxic at a dose below 204 mg/kg, and this compound was excluded from further examination. The compounds were then applied to the mouse model of PC with GCIY, a gastric cancer notorious for its rapid progression [25]. The PC progresses very rapidly, and increases in ascites fluid and comparative weight gain continues until the animal dies. As a result, mortality occurs within 1 month following tumor inoculation. Experimental treatment was carried out as shown in Figure 5a. The IP administration of curcumin had no effect on the PC model, where the average body weight of untreated group reached to 164.0 ± 20.8% of that before treatment, whereas that treated with curcumin to 150.2 ± 24.0% (P = 0.18). GO-Y030 had a significant antitumor effect on PC progression (p < 0.001, Figure 5b), but no survival benefit was observed (Figure 5c). Similar to that of GO-Y030, GO-Y078 showed a significant suppression of PC progression. At the 17th day after the first treatment, the average body weight of the untreated group was 35.9 ± 2.6 g due to increased ascites fluid accumulation, while that of the treated group was significantly less at 29.1 ± 2.5 g (p = 0.003, Figure 5d). The average body weight before inoculation was 28.4 ± 0.9 g, therefore the ascites fluid accumulation was considered to be completely suppressed by GO-Y078 treatment. Survival time of the untreated group (n = 4) ranged from 21 to 23 days, with a median survival time (MST) of 22 days. Survival time of the GO-Y078 treated group (n = 6) ranged from 25 to 109 days, with a MST of 30.5 days. Treatment with GO-Y078 led to an approximate 40% increase in survival time. Log-rank analysis significantly indicated a survival benefit of GO-Y078 treatment (p = 0.01, Figure 5e). Judging from these results, the bioavailability of GO-Y078 is significantly higher than GO-Y030. In order to examine why the survival was limited in the GO-Y078 treated mice, despite the fact that the ascites fluid accumulation disappeared completely. When necropsies were performed, in nearly all of the GO-Y078-treated mice, residual cancer cells were observed to colonize around the stomach, pancreas, bile duct, and duodenum. Direct invasion to the pancreas, liver, and duodenum was thought to be the causes of death.

Discussion
DOP analogs possess common structural feature, that is an electrophilic α,β-unsaturated ketone, which reacts with nucleophilic groups through the Michael addition. Then, the ketone binds covalently with thiol groups of cysteine residues in different proteins [26]. As thiol group does not exist in DNA, curcuminoids do not attach DNA itself, different from the cytotoxic chemotherapeutic agents. Two independent proteins can be alkylated through two α,β-unsaturated double bonds in DOP that stretch from the center ketone moiety toward both ends [27]. In this alkylation, an association of a cysteine protease, such as caspase, and its target protein may assist in the catalytic reaction of caspase, resulting in target degradation. In our previous study, we observed that DOP analogs increased caspase activity, and a caspase inhibitor inhibited the degradation of βcatenin or KRAS by DOP analogs [15]. Although this contention remains unproven, perhaps this center structure of DOP confering Michael addition is responsible for the multi-targeted nature of curcuminoids. Contribution to enhanced potency by the alteration from a diketone to a monoketone also remains to be elucidated. Shortening the chemical distance by alkylation through conversion from a di-to monoketone structure may result in an enhanced potential. Acyclic DOP analogs have an enhanced cytotoxicity [27]. Substitution in each aromatic ring could improve solubility and the resultant pharmaceutical potency. As the symmetry of the attachment group of each aromatic ring is not necessary, further improvement of solubility may be achievable by various types of substitution in this portion [17]. The hydrophilic nature and total positioning of the attached groups in a compound can reflect the potency of the analogs. In this study, the maximum IC 50 value of DOP analogs reached a level that was 84 times lower than curcumin. The data, that curcuminoids could only affect the malignant growth, lead to the hypothesis that the growth-suppressive potential of these analogs depends on the degradation of oncoproteins that are overexpressed in malignant cells. That might be a reason why curcuminoids are rather safe to normal cells.
Furthermore, it has been shown that some of DOP analogs induce more cell death to cancer cells, compared with curcumin. Apoptosis related proteins, such as TP53 and DR5 were up-regulated with DOP analogs more efficiently than curcumin. However, the precise mechanisms of up-regulation are still unknown [28]. Cancer cells invent numerous ways to inactivate many of the apoptotic pathways. The pluripotent apoptosis induction by curcuminoids could overcome complexity of antiapoptosis in cancer cells. One of DOP analog, GO-Y078 has shown survival benefit in mouse PC model for the first time. That could not be achieved by GO-Y030. Although these two DOPs presented the same level of IC 50 values, the predicted solubility of GO-Y078 was 1.98 mg/L, whereas that of GO-Y030 was 0.26 mg/L. In fact, GO-Y030 was less soluble compared with GO-Y078. The difference of solubility between GO-Y030 and GO-Y078 probably influences the difference in their survival benefits in the PC models. Moreover, additional times of administration of GO-Y078 may lead to further improved survival. Combined treatment with conventional cytotoxic agents, such as 5-FU and taxanes, or a DR5 ligand, for example, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), may synergistically induce better outcomes [22]. Recently, off-target toxicity has become a problem in molecular-targeted medicines which have already been approved [29,30]. Some of the potent DOP analogs may not be as safe as dietary curcumin, shown with GO-Y098, and we should be cautious against that during development.

Conclusions
Synthesis of DOP analogs represented by GO-Y078 can overcome low bioavailability of curcumin, yielding good lead compounds for new cancer chemotherapeutic agents bearing multi-target properties.

Chemicals
Chemical synthesis, physical properties and molecular formulas of the newly synthesized curcumin analogs have been previously described [15,17]. See additional file 1: Physical properties of new curcumin analogs. Curcumin (Sigma-Aldrich) and its analogs were dissolved in DMSO at 10-50 mmol/L as a stock solution. Cremophor EL ® was purchased from Sigma-Aldrich.

Growth-suppression analysis
Growth-suppressive effects of the derivative compounds were measured in different cancer cell lines for 72 h. Cell viability was assayed by quantifying the uptake and digestion of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt according to the manufacturer's instructions (Dojindo Laboratories) by 96-well plate reader, Spectra-Max M2e (Molecular Devices). The percentage cell growth of the control, which was treated with 1% DMSO alone, was calculated and plotted, and the mean growth-inhibitory concentration (IC 50 ) value was then determined.

Cluster analysis
Clustering analysis among analogs was performed with MultiExperiment Viewer software (MeV) from the institute for Genomic Research at that time [18].

Cell Cycle analysis
Cell cycle phase was determined by fluorescence-activated cell sorting analysis. Cells from the HCT116 cell line were inoculated into 6-well plates at a concentration of 5 × 10 5 per well, exposed to the derivative compounds at various concentrations, cultured for 24 h, and collected and sorted using a Cytomics FC500 Flow Cytometry System (Beckman Coulter, Inc.) as previously described [19]. The percentage of each cell fraction corresponding to the subG 1 , G 0 /G 1 , S, and G 2 /M phases was calculated using WinCycle Software (Beckman).

Expression analysis
HCT116 cells treated by the analogs were cultured for 24 h, collected and lysed with lysis buffer (500 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, and 1% NP-40). After centrifuging samples, the supernatant fraction was transferred to a fresh tube. Samples were mixed with 5× SDS sample buffer (312.5 mM Tris-HCl pH 6.8, 50% Glycerol, 25% 2-mercaptoethanol, 12.5% SDS, and 0.1% bromophenol blue) before western blotting. Antibodies used for Western blotting were anti-beta-actin monoclonal antibody (A2066, Sigma-Aldrich), anti-DR5 monoclonal antibody (Santa Cruz Biotechnology), and Alexa Fluor 680 (Invitrogen). Membranes were scanned and semiquantification was conducted using the Odyssey detection system (LI-COR). Expression of TP53 was measured using P53 ELISA kit (Promokine) according to the manufacturer's instructions. Samples were collected using the same method as Western blotting. Twenty micrograms of protein were incubated in wells coated with anti-human p53 antibody. Biotin-conjugated antihuman p53 antibody was then added. After incubation, streptavidin conjugated with horse radish peroxidase (HRP) was added. Tetramethyl-benzine, which reacts with HRP, was then added and the absorbance was measured at 450 nm by SpectraMax M2e.
Caspase cleavage reaction HCT116 cells were inoculated at a concentration of 1 × 10 4 per well in a 96-well plate, cultured until cells were semi-confluent, and treated with the analogs for 24 h. Serum from each sample was collected, duplicated, and assessed by ELISA using HRP-conjugated M30 monoclonal antibody (M30-Apoptsense ® ELISA, Peviva) according to the manufacturer's instructions. Anti-M30 monoclonal antibody recognizes a soluble cytokeratin18-Asp396 molecule that becomes exposed after cleavage by caspases (caspase3, 6, 7, and 9) during apoptosis. The absorbance was measured by SpectraMax M2e at 450 nm. These analyses were conducted in triplicate.

NF-B transactivation
NF-B transactivation was measured by ELISA using a NFB p50 Transcription Factor Assay kit (Thermo Scientific) according to the manufacturer's instructions. SW620 cells were treated with the compounds with or without Tumor Necrosis Factorα (TNFα) (10 ng/ml, Sigma) for 6 hours, washed with phosphate buffered saline (PBS), and lysed with provided M-PER ® lysis buffer (Thermo Scientific). Whole-cell extracts containing 20 μg of protein were added with biotinylated-NF-B consensus DNA sequence to a streptavidin-coated 96-well plate. Sample wells were incubated with the monoclonal antibody against NF-B p50, and then incubated with HRP-conjugated secondary antibody. Luminol/enhancer solution and stable peroxide solution were added and NF-B transactivation was detected using an LMax Luminometer (Molecular Devices).
In silico ADME analysis Absorption, distribution, metabolism, excretion (ADME), and solubility, were analyzed in silico by ADMEWORKS/ Predictor (Fujitsu Kyushu Systems Ltd.). This analysis is commercially available and depends on a quantitative structure-activity relationship [20]. Solubility was calculated by the linear multiple regression equation and indicated by the predicted value.

Animal experiment
C57BL/6J mice were obtained from CLEA Japan, Inc. and KSN Slc (nu/nu) mice were obtained from Japan SLC. The injection solution was prepared by first dissolving the analogs with ethanol-cremophor EL ® (1:1) and then diluting the solution 1:8 with PBS. C57BL/6J mice were used for the assessment of toxicity of the C5-curcuminoids. The maximum tolerable dose (MTD) was determined by a single intraperitoneal (IP) administration. Therapeutic experiments were conducted with the IP administration of 155 mg/kg of GO-Y030 (total volume 1.0 mL). IP administration of GO-Y078 was conducted with 133 mg/kg or 266 mg/kg doses. Ethanol-cremophor EL ® alone diluted 1:8 with PBS was injected as a control. The PC model was established by inoculating 5 × 10 6 GCIY cells into the abdominal cavities 6-week-old male KSN/Slc mice, 1 week prior to treatment with the 1,5-diaryl-3-oxo-1,4-pentadiene (DOP) analogs. Pathological examination including hematoxylin-eosin staining was performed as previously described [16]. All animal experiments were performed in accordance with approved institutional animal use guidelines (Tohoku University) based on international guideline.

Statistic analysis
Each experiment was conducted three times, unless specified otherwise. Individual values were compared by Student's t-test. The survival curve was drawn using the Kaplan-Meier method and compared using the log rank test.

Additional material
Additional file 1: Physical properties of new curcumin analogs. The physical properties and molecular formula of the analogs are described in the file.


text:
catena-Poly[[bis(nitrato-κO)cadmium]bis[μ-1,4-bis(pyridin-3-ylmethoxy)benzene-κ2 N:N′]]

In the title compound, [Cd(NO3)2(C18H16N2O2)2]n, the six-coordinated CdII ion is located on an inversion center and has a distorted octahedral environment defined by four N atoms from four 1,4-bis(pyridin-3-ylmethoxy)benzene ligands and two O atoms from two nitrate anions. The ligands link the CdII ions into a ribbon-like structure running along [201]. One O atom of the nitrate anion is disordered over two positions with site-occupancy factors of 0.59 (2) and 0.41 (2).

In the title compound, [Cd(NO 3 ) 2 (C 18 H 16 N 2 O 2 ) 2 ] n , the sixcoordinated Cd II ion is located on an inversion center and has a distorted octahedral environment defined by four N atoms from four 1,4-bis(pyridin-3-ylmethoxy)benzene ligands and two O atoms from two nitrate anions. The ligands link the Cd II ions into a ribbon-like structure running along [201]. One O atom of the nitrate anion is disordered over two positions with site-occupancy factors of 0.59 (2) and 0.41 (2).

Experimental
Crystal data [Cd(NO 3

Comment
The bridging compounds with rigid and flexible pyridyl-containing bidentate or multidentate organic spacers have assembled numerous interesting topology structures by coordination with metals and intermolecular supramolecular interactions. Our group focused attention on study of flexible pyridyl-based aromatic ligands, and obtained some isolated molecules, chain, two-and three-dimensional network structures (Liu et al., 2010a, b). Herein, as a continuing work for pyridyl ligands, we report the synthesis and crystal structure of the title compound, which is a isostructural compound of our previous reports (Liu et al., 2011;Zou et al., 2011).
In the title compound, the Cd II ion lies on an inversion center and is six-coordinated in a distorted octahedral geometry defined by four N atoms of the pyridine derivatives and two O atoms of the nitrate anions ( Fig. 1, Table 1). One O atom of the nitrate anion has a badly thermal ellipsoid, which is split over two positions with site-occupancy factors of 0.59 (2) and 0.41 (2). In the crystal, ribbon-like structures along [2 0 1] are built up by the N-heterocyclic ligands linking the Cd II ions (Fig. 2).

Experimental
The 1,4-bis(pyridin-3-ylmethoxy)benzene ligand was synthesized as the reference method (Liu et al., 2010a). A mixture of 1,4-dihydroxybenzene (1.10 g, 10 mmol), 3-chloromethylpyridine hydrochloride (3.28 g, 20 mmol) and NaOH (1.60 g, 40 mmol) in acetonitrile (50 ml) was refluxed under nitrogen with stirring for 24 h. After cooling to room temperature, the solution was filtered and the residue was washed with acetonitrile for several times. The mixed filtrate was dropped into a 300 ml water solution, giving a powder crude product. A total of 2.51 g (yield 86%) pure product was obtained by recrystallizing from a mixed solution of 10 ml water and 10 ml methanol. The title compound was synthesized by the reaction of 1,4-bis(pyridin-3-ylmethoxy)benzene (0.29 g, 1.0 mmol) and Cd(NO 3 ) 2 .4H 2 O (0.31 g, 1.0 mmol) in a mixed solution of 5 ml water and 5 ml methanol. The mixture was filtered after stirring for about 1 h. The filtate was allowed to stand for 4 d under room temperature to give block-like colorless crystals suitable for X-ray analysis.

Refinement
O5 atom of nitrate is disordered over two positions and the site-occupancy factors were refined to 0.41 (2) for O5 and 0.59 (2) for O5'. The command "isor 0.005 O5 O5" was used to restrain ADP. H atoms bound to C atoms were placed in calculated positions and treated as riding on their parent atoms, with C-H = 0.93 (aromatic) and 0.97 (methylene) Å and with U iso (H) = 1.2U eq (C).


High yield synthesis of carbon nanofibers in an environmental friendly route

An environmental friendly route for the high yield synthesis of carbon nanofibers (CNFs) has been developed. CNFs have been synthesized using nickel formate as catalyst precursor at 680°C using chemical vapor deposition method. Upon pyrolysis this catalyst precursor yields metal catalyst nanoparticles directly. The sodium chloride and potassium chloride was used as catalyst support, it was chosen because of its non-toxic and water soluble nature. The problems such as detrimental effect, environment and even cost has been avoided by using water soluble supports. The structure of the products was characterized by scanning electron microscopy, transmission electron microscopy, Raman spectroscopy and X-ray diffraction method. The purity of as grown products and purified products was determined by thermal analysis. Here, we report the 7,800 and 7,200 wt% yield of CNFs synthesized over NaCl and KCl support. This synthetic route can be used for the large scale synthesis in industries.

Introduction
Since the discovery of carbon nanotubes by Iijima (1991), an intensive research has been carried out on carbon nanofibers (CNFs) and carbon nanotubes (CNTs). These materials have unique mechanical, chemical and electrical properties and hence attracted many researchers. CNTs and CNFs have large number of potential applications which include field emission sources (Milne et al. 2004), hydrogen storage (Dillon et al. 1997), catalyst substrate (Bahome et al. 2005), chemical sensors (Douglas and Alexander 2010), scanning probe tip (Demicheva et al. 2008), electrode material (Jan et al. 2009), nanoelectronics (Brataas 2010) and mechanical reinforcements (Wang et al. 2010). Until now, various synthetic methods have been developed for the production of these nanostructures, including arc discharge (Iijima 1991), laser vaporization (Guo et al. 1995) and chemical vapor deposition (CVD) (Baker et al. 1989;Su et al. 2000). One of the remarkable advantages of CVD method is that it produces cheap CNTs and CNFs with high yields. The choice of the supporting material has been found to be important factor to synthesize these carbon nanostructures by CVD method. The support materials such as silica, alumina or magnesia are widely studied (Nagaraju et al. 2002;Kathyayini et al. 2008;Bris et al. 2010;Seifi 2011). But these supports are soluble in acids and hence acid wash is required to separate these supports from carbon structures after growth. The acid wash causes detrimental effect, environment problems and effects cost efficiency. Therefore, an important approach for the largescale production of high quality CNTs or CNFs is (1) to avoid acids for purification and (2) to reduce the number of purification steps. In this paper, we have developed novel method to synthesize CNFs by CVD method using water soluble substrates. Nickel formate is used as catalyst precursor, as it directly yields metal catalyst nanoparticles on pyrolysis. The main advantage of using nickel formate is that it avoids the lengthy process of reduction and calcination. Sodium chloride and potassium chloride are used as supports, as they are easily soluble in water. The use of acid or base is completely avoided by using water soluble substrates. Here, we report the 7,800 wt% yield of CNFs synthesized over nickel formate as catalyst precursor on NaCl substrate.

Experimental
All the reagents used were AR-grade and purchased from Sigma Aldrich. Nickel formate was prepared by nickel acetate and formic acid at 80°C. The microcrystalline precipitate was obtained on cooling which was filtered, dried at 100°C. The formation of nickel formate was confirmed by X-ray diffraction method (Fig. 1). The supported catalyst was made by dispersing the nickel formate catalyst precursor on substrate. The obtained supported catalysts were denoted as Ni/NaCl and Ni/KCl.
In this study, the synthesis of CNFs was carried out at atmospheric pressure in a fixed bed CVD reactor. The length and diameter of quartz tube were 900 and 20 mm, respectively. The detailed experimental set up is shown in the Fig. 2. 300 mg of catalyst precursor with support was taken in quartz boat. The boat was placed in the centre of the quartz tube after the reactor reaches the 680°C in an argon atmosphere. Acetylene gas was then released in order to grow the CNFs for 15 min. The reactor was cooled in an argon atmosphere and then carbon deposits were collected. The carbon yield is calculated by Eq. 1 where M Total is the total mass of the carbon product and catalyst, and M Cat is the mass of the catalyst.
To purify the sample, the raw material was stirred in warm water to remove the catalyst and support. The sample was finally washed with deionized water and dried in oven.

Results and discussion
In this study, the catalyst precursor with support was introduced after the CVD reactor reaches the 680°C. The pyrolysis of nickel formate in the gas flow system leads to the formation of nickel nanoparticles. Mechanism of formation of nickel nanoparticles from nickel formate is discussed in the literature (Edwards et al. 1997). The time and cost has been reduced in this process because both calcination and reduction is avoided in the preparation of catalyst. The prepared nickel formate precursor was characterized by X-ray diffraction method. Figure 1 show the XRD pattern of prepared nickel formate. They have characteristic peaks A, B, C and D (JCPDS 14-0813). The Table 1 gives summary of catalyst precursor, wt% of metal in support, CVD parameters, yields and morphology of CNFs produced.
The melting point of NaCl is 800°C, so all the reactions were performed below this temperature. A variety of experiments were conducted with argon-acetylene mixture with various volume ratios (20:1, 15:1, 10:1, 5:1, 3:1, 2:1 and 1:1) at reaction temperatures between 600 and 740°C (Figs. 3, 4). The carbon deposit at 680°C was maximum. The metal loading percentage was fixed to 3 wt% in catalyst support in order to avoid sintering effect of catalyst at high temperatures. So all the reaction were performed at 680°C with gas flow of 200 sccm: 20 sccm (Ar:C 2 H 2 ) for 15 min. Figure 5 shows the SEM morphologies of purified CNFs grown on nickel formate/NaCl and nickel formate/KCl. Because of their twisting morphology it is difficult to measure the length of the CNFs from the SEM micrographs, but the length can be estimated to be more than several tens of micrometers. As seen in SEM image, the catalytic particles and support were removed successfully by the purification. Structural details of the products were further investigated by TEM. Figure 6 show the TEM  images of the purified CNFs grown on nickel formate/NaCl and nickel formate/KCl. There is clear evidence form TEM micrograph for the formation of CNFs. The diameters of the CNFs are in the range of 10-50 nm. TGA results in Fig. 7 show the weight loss of CNFs during the oxidation under air atmosphere. The TGA curve in the temperature range of 30-700°C shows a total weight loss of 60 wt% for products grown on nickel formate/NaCl and nickel formate/KCl. There is a little bit of amorphous carbon in our samples because there are negligible mass losses below 400°C in the TGA spectrum. According to curve c, the purity of the purified CNFs increases to 99.0% after purification process.
The representative Raman spectra (Fig. 8) of the samples show the typical features of CNFs. In detail, there exist two strong peaks at 1,592, 1,596 cm -1 and 1,328, 1,334 cm -1 corresponding to the typical Raman peaks of graphitized carbon. The peak at 1,592 and 1,596 cm -1 is attributed to the Raman-active E 2g in-plane vibration mode and is related to the vibration of sp 2 -bonded carbon atoms in a twodimensional hexagonal lattice. The peak at 1,328 and 1,334 cm -1 is associated with vibrations of carbon atoms with dangling bonds in plane terminations of disordered graphite (Eklund et al. 1995). The intensity of D-band peak is stronger than that of G-band peak, which originates from the in-plane defects of the product preventing the layer from extending. Figure 9 shows the XRD pattern of the purified samples. It contains two characteristic peaks at about 26.0°and

Conclusions
In this work, we have developed a simple method to synthesize CNFs in the bulk scale by CVD method. Nickel formate was used as catalyst precursor because it forms nickel nanoparticles on pyrolysis. Sodium chloride and potassium chloride which are easily soluble in water and non-toxic are used as supports. The yield of carbon nanofibers is high on sodium chloride support when compare to potassium chloride support. The uses of acid and toxic materials are completely avoided. By this route we can grow CNFs in large scale by using low cost precursors in an economical and environmental friendly way.


text:
Review of Pharmacological Effects of Antrodia camphorata and Its Bioactive Compounds

Antrodia camphorata is a unique mushroom of Taiwan, which has been used as a traditional medicine for protection of diverse health-related conditions. In an effort to translate this Eastern medicine into Western-accepted therapy, a great deal of work has been carried out on A. camphorata. This review discusses the biological activities of the crude extracts and the main bioactive compounds of A. camphorata. The list of bioactivities of crude extracts is huge, ranging from anti-cancer to vasorelaxation and others. Over 78 compounds consisting of terpenoids, benzenoids, lignans, benzoquinone derivatives, succinic and maleic derivatives, in addition to polysaccharides have been identified. Many of these compounds were evaluated for biological activity. Many activities of crude extracts and pure compounds of A. camphorata against some major diseases of our time, and thus, a current review is of great importance. It is concluded that A. camphorata can be considered as an efficient alternative phytotherapeutic agent or a synergizer in the treatment of cancer and other immune-related diseases. However, clinical trails of human on A. camphorata extracts are limited and those of pure compounds are absent. The next step is to produce some medicines from A. camphorata, however, the production may be hampered by problems related to mass production.

Introduction
About 80% of the world population currently relies on indigenous or traditional medicines for their primary health needs, and most of these therapies involve the use of herbal extracts, often in aqueous solutions [1][2][3]. Antrodia camphorata (Syn. Antrodia cinnamomea) is a fungal parasite on the inner cavity of the endemic species Cinnamomum kanehirae (Bull camphor tree) Hayata (Lauraceae) (Figure 1). The host plant is a large evergreen broad-leaved tree, which only grows in Taiwan, and is distributed over broad-leaved forests at an altitude of 200-2000 m [4]. Cinnamomum kanehirai is a high quality material used to manufacture valuable furniture. The government has recently protected this endemic tree species from forest-denudation since this species in nature is relatively rare [5]. In Taiwan, A. camphorata is called as "Niu-chang-chih" or "Chang-chih" or "Niu-chang-ku" or "Chang-ku" [5]. Locally, it is believed that A. camphorata is a present from heaven for Taiwanese and, is a well-known Chinese folk medicine and claimed "ruby in mushroom" in Taiwan [5]. It grows in the mountain ranges of Taoyuan, Miaoli, Nantou, Kaohsiung, Taitung and Hualien of Taiwan [3]. The trophophase of A. camphorata occurs from June to October [6]. Being a local species, A. camphorata was historically used in Taiwan by the aborigines as a traditional prescription for the discomforts caused by alcohol drinking or exhaustion [5]. Furthermore, the regular consumption is believed to preserve human vitality and promote longevity. The preparations from fruiting bodies have been used for the prevention, or treatment, of numerous diseases including liver diseases, food and drug intoxication, diarrhea, abdominal pain, hypertension, itchy skin and tumorigenic diseases [7,8]. The aim of this contribution is to review the literature covering pharmacological and phytochemical aspects of A. camphorata.

Taxonomical Description
Ku is a Chinese common name meaning mushroom; chih means a famous Ganoderma-like fungus. Antrodia camphorata was first published and identified as new ganoderma species, Ganoderma camphoratum, by Zang and Su in 1990 [9]. However, according to fruiting-body morphology and cultural characteristics, this fungus has been proposed the name as A. camphorata [5,10]. In 2004, a phylogenetic analysis based on sequence data derived from large ribosomal subunit sequences of ribosomal RNA genes indicated that A. camphorata is distantly related to other species in Antrodia and, consequently, the fungus was transferred to the new genus Taiwanofungus [11]. However, using polymorphism analysis of internal transcribed spacer regions of the ribosomal RNA gene, A. camphorata was reconsidered as an Antrodia species [12]. The current taxonomic position of A. camphorata is as follows [13]: Fungi, Basidiomycota, Homobasidiomycetes, Aphyllophorales, Polyporaceae. Clearly, however, the nomenclature and exact taxonomy (genus and species) of A. camphorata is still the subject of debate and needs further research. In this article, we have chosen the name as A. camphorata to describe this unique Formosan fungus. The fruiting bodies of A. camphorata assume different plate-like, bell-like, hoof-like or tower-like shapes. They are flat on the surface of wood at the beginning of growth. Then the brim of the front edge rises to roll into plate-shaped or stalactites. The top surfaces of A. camphorata are lustrous, brown to dark brown in color, with unobvious wrinkles, flat and blunt edges. The bottom sides are orange red or partially yellow with ostioles all over [12]. In addition, A. camphorata exhales strong smell of sassafras (camphor aroma), becomes pale yellowish brown when sun-dried and has a strong bitter taste. The red to light cinnamon fruiting bodies of A. camphorata are bitter and have a mild camphor scent like the host woods [5]. The mycelia isolated from the fruiting bodies of A. camphorata form orange red and orange brown to light cinnamon-colored colonies [5]. The hyphae of A. camphorata possess generative hyphae 2-3.5 μm with clamp connections, and hyaline to light brown skeletal hyphae up to 4.5 μm wide with weakly amyloid. Basidia, 12-14 × 3.0-5.0 μm, is clavate and 4-sterigmate with a basal clamp. Basidiospores, 3.5-5.0 × 1.5-2 μm, are cylindrical, hyaline, smooth and sometimes slightly bent [14].

Ethnomedicine
Antrodia camphorata has long been used in traditional medicines of Taiwan for the treatment of twisted tendons and muscle damage, terrified mental state, influenza, cold, headache, fever and many internally affiliated diseases [14]. In 1773, a traditional Chinese medical doctor Wu-Sha found that Taiwan aborigines have often chewed the fruiting bodies and/or decoction of A. camphorata for the discomfort caused by excess alcohol or exhaustion because of lifestyle [14].

Chemical Constituents
A total of 78 compounds have been identified and structurally elucidated. Predominant in fruiting bodies are generally terpenoids in a large number (39 compounds) [17][18][19][20][21][22][23][24][25], though there are a few publications on the constituents of the solid-state cultivated mycelium and, mycelium from submerged cultivations [26][27][28][29][30][31][32][33][34][35][36]. A large number of triterpenoid compounds (31 structures) with similar or even the same structures were described within the last few years. A common feature of these structures is ergostane or lanostane skeleton. Due to the high amount of 63% of terpenoids in the fruiting bodies of A. camphorata, this group of natural compounds has been in the focus of many phytochemical studies. Interestingly, no distinct terpenoid glycosides have ever been isolated from this species; in contrast to polysaccharides that have been elucidated. Furthermore, several other constituents were described from A. camphorata comprising benzenoids, lignans, benzoquinones and maleic/succinic acid derivatives, in addition to polysaccharides. Finally, sterols, nucleotides and fatty acids were detected in this species [37][38][39][40]. Typical structures and their activities of isolated constituents from A. camphorata are depicted in Figure 2 and Table 1, respectively.

Pharmacological Effects of Crude Extracts
The scientific world's particular interest in A. camphorata and its curative properties originated from the realm of traditional medicine. Ethnic medicine has come to be an irreplaceable source of knowledge of medicinal mushrooms and their curative qualities, as well as creating clues for scientific research, which usually confirms the legitimacy of their usage [41,42]. This part of review will deal with the pharmacological effects of crude extracts of the A. camphorata in different models of in vivo and in vitro studies.

Anti-Cancer Activities.
Both the fruiting bodies and mycelium of A. camphorata have potent anti-proliferative activity against various cancers in vitro and in vivo. It was indicated that there were multiple potent mechanisms underlying the anti-cancerous effects of A. camphorata. The crude CHCl 3 /MeOH extract from fruiting bodies of A. camphorata exhibited significant cytotoxic activity with an IC 50 value of 4.1 μg ml −1 against P-388 murine leukemia cells [18]. The ethylacetate extract from fruiting bodies of A. camphorata (EAC) exhibited apoptotic effects in two human liver cancer cell lines, Hep G2 and PLC/PRF/5 in a dosedependent manner [43]. In addition, EAC also initiated mitochondrial apoptotic pathway through regulation of Bcell lymphoma (Bcl)-2 family proteins expression, release of cytochrome c, and activation of caspase-9 both in Hep G2 and PLC/PRF/5 cells [43]. Furthermore, EAC also inhibited the cell survival signaling by enhancing the amount of Iκ-α in cytoplasm and reducing the level and activity of nuclear factor (NF)-κB in the nucleus, and subsequently attenuated the expression of Bcl-X L in Hep G2 and PLC/PRF/5 cells [43]. Treatment with EAC also caused another human liver cancer cell line Hep 3B to undergo apoptotic cell death by way of calcium-calpain-mitochondria signaling pathway [44]. Another study reported that EAC could inhibit the invasiveness and metastasis of liver cancer cell line PLC/PRF/5 cells through the inhibition of angiogenesis [45]. We previously reported that the CHCl 3 extract from fruiting bodies of A. camphorata (FBAC) showed cytotoxic activity with an IC 50 value of 22, 150, 65 and 95 μg ml −1 , against cancer cell lines Jurkat, Hep G2, Colon 205 and MCF 7, respectively. Furthermore, MeOH extract from FBAC also cytotoxic (IC 50 = 40 μg ml −1 ) to Jurkat cells [46]. Antrodia camphorata solid-state cultured mycelium (AC-SS, 1 μg ml −1 ) showed adjuvant anti-proliferative effects with cisplatin (10 μM) or mitomycin (10 μM) in hepatoma cell lines C3A and PLC/PRF/5 cells (in vitro) and, on xenografted cells in tumor implanted nude mice (in vivo). Furthermore, AC-SS showed its adjuvant effects through the inhibition of MDR gene expressions and the pathway of COX-2dependent inhibition of AKT phosphorylation [47]. In terms of the very recent literature, Lu et al. [48] noted that ethanol extract from wild fruiting bodies of A. camphorata (EEAC) dose-dependently induced human premyelocytic leukemia HL 60 cells apoptosis via histone hypoacetylation, upregulation of histone deacetyltransferase 1 (HDAC 1), and downregulation of histone acetyltransferase activities including GCN 5, CBP and PCAF. Furthermore, combined treatment with 100 nM of trichostatin A (histone deacetylase inhibitor) and 100 μg ml −1 EEAC caused synergistic inhibition of cell growth and increase of apoptotic induction through the upregulation of DR5 and NF-κB activation [48].
There are relatively fewer studies on extracts from the solid-state or submerged cultivated mycelium or culture filtrates. Aqueous extract from submerged cultivation mycelium (SCM) of A. camphorata exhibited significant cytotoxicity against HL-60 cells but not against cultured human endothelial cells [49]. The SCM resulted dose (25-150 μg ml −1 ) and time-dependent apoptosis, as shown by loss of cell viability, chromatin condensation and internucleosomal DNA fragmentation in HL-60 cells [50]. Furthermore, apoptosis in these cells was accompanied by the release of cytochrome c, activation of caspase-3, specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), and also with a reduction in the levels of Bcl-2 [50]. In an another study, the ethanolic extract (0.2-2%, v/v) from solid-state cultivated mycelia of A. camphorata showed potent antiproliferation effect in human non-small cell lung carcinoma A549 cells but not primary human fetal lung fibroblast MRC-5 cells [51]. In addition, this extract triggered the apoptosis in the A549 cells by downregulated human galectin-1, human eukaryotic translation initiation factor 5A, human Rho GDP dissociation inhibitor α, human calcium-dependent protease small subunit and human annexin V [51]. To continue, the effects of A. camphorata on cancer cells was investigated,  No.

78.
77. methanol extract of SCM exhibited the cytotoxicity in Hep G2 (wild-type p53) and Hep 3B (delete p53) cells with IC 50 values of 49.5 and 62.7 μg ml −1 , respectively, after 48 h of incubation. Cell-cycle analysis revealed that the above SCM extract treatment induced apoptosis on Hep G2 via G0/G1 cell-cycle arrest followed by the apoptosis through activation of the caspase-3 and -8 cascades [52]. Furthermore, these authors also reported that the mechanism of MEM-mediated apoptosis in Hep G2 cells through the Fas/Fas ligand (FasL) death receptor pathway [53]. In parallel, Chen et al. [54] noted that the ethanolic extract from SCM has anti-proliferation against Hep G2 and Hep G3 cells with 54.2 and 82.9 μg ml −1 , respectively. On the other hand, Yang et al. [55] reported that fermented culture broth of A. camphorata (FCBAC) exhibits dose (25-150 μg ml −1 ) and time-dependent anti-proliferative effect by induction of apoptosis in breast cancer cell line MCF-7. In addition, this apoptic effect is associated with cytochrome c translocation, caspase-3 activation, PARP degradation and dysregulation of Bcl-2 and Bax in MCF-7 cells [55]. These authors also reported that FCBAC has the dose (40-240 μg ml −1 ) and time-dependent apoptotic effect in estrogen-nonresponsive human breast cancer cell line MDA-MB-231 with a similar kind of mechanism as mentioned above [56]. In addition, FCBAC treatment also inhibited the cyclooxygenase (COX)-2 protein expression and prostaglandin E 2 (PGE 2 ) production in MDA-MB-231 cells [56]. Furthermore, FCBAC treatment induced cell-cycle arrest and apoptosis in MDA-MB-231 both in vitro and in vivo [57]. The A. camphorata crude extract (ACCE) at 50 μg ml −1 acts as an anti-metastatic agent, by anti-proliferative through induces G2/M cell-cycle arrest followed by suppress the active form of matrix metalloproteinase (MMP)-9 in bladder cancer cell T24 cells [58]. In addition, ACCE (100 μg ml −1 ) showed significant antiproliferation effect in transitional cell carcinomas (TCC) cell lines RT4, TSGH-8301 and T24 [59]. In RT4 cells, 100 μg ml −1 of ACCE showed the p53-independent over expression of p21 followed by downregulation of pRb. On the contrary, treatment with ACCE at 50 μg ml −1 resulted in downregulations of Cdc2 and Cyclin B1 in the cell lines TSGH-8301 and T24 [59]. In another study, the ACCE extract at 150 μg ml −1 concentration showed anticancer effect in androgen responsive prostate cancer cell line LNCaP through pathway Akt → p53 → p21 → CDK4/cyclin D1 → G1/S-phase arrest → apoptosis [60]. In addition, ACCE also inhibited the androgen independent prostate cancer cell line PC-3 through G2/M-phase arrest mediated through pathway p21 → cyclin B1/Cdc2 with limited degree of apoptosis [60]. Recently, Lu et al. [61] noted that submerged cultivated A. camphorata extract prevents serum-deprived PC-12 cell apoptosis through a PKA-dependent pathway and by suppression of JNK and p38 activities. Ho et al. [62] reported that crude extract of A. camphorata (AC) at concentrations of 5-50 μg ml −1 did not affect tumor cells PC-3 viability, but at 100-200 μg ml −1 decreased viability and induced apoptosis in a concentration-dependent manner. In addition, 25-200 μg ml −1 did not alter basal [Ca 2+ ] i , however at 25 μg ml −1 decreased the [Ca 2+ ] i induced by ATP, bradykinin, histamine and thapsigargin [62]. The mycelia powder of A. camphorata (MAC) at 25-50 μg ml −1 , did not affect the cell viability in MG63 human osteosarcoma cells, however, at 100-200 μg ml −1 decreased viability and induced Evidence-Based Complementary and Alternative Medicine 7 apoptosis via inhibition of ERK MAPK phosphorylation [63].
In summary, extracts of A. camphorata inhibited markedly intracellular signaling and invasive behavior of cancer cells. This complexity can also bring significant advantages. For example, certain components in the natural products can reduce the cytotoxicity of the whole product (and vice versa). Also, the interaction between different biologically active components can be responsible for their effects in vivo. Different compounds can modulate unrelated signaling and therefore, can possess synergistic effects [73]. However, the molecular mechanism(s) has not been fully elucidated. Further studies are needed to explore the benefits and safety to cancer patients.

Anti-Inflammatory/Immunomodulatory
Effects. Compounds that are capable of interacting with the immune system to up regulate or down regulate specific aspects of the host response can be classified as immunomodulators or biologic response modifiers [74][75][76]. In peripheral human neutrophils, extracts from SCM of A. camphorata displayed anti-inflammatory effects by inhibiting reactive oxygen species (ROS) production with an IC 50 ranging from 2-20 μg ml −1 [77]. The aqueous extract from SCM dosedependently (25-100 μg ml −1 ) inhibited the lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor (TNF-α), interleukin (IL)-1β and PGE 2 production, and inducible nitric oxide synthase (iNOS) and COX-2 protein expression via NF-κB pathway, in macrophages [78]. These results are in parallel to our previous report that CHCl 3 (3-25 μg ml −1 ) and MeOH (6-50 μg ml −1 ) extracts from fruiting bodies of A. camphorata significantly inhibited the enhanced production NO through reducing iNOS expression and, TNF-α and IL-12 productions from macrophages [46]. Liu et al. [79] reported that the methanol extract (50 μg ml −1 ) from wild fruiting bodies have more potency than water extracts on the anti-inflammatory activity through inhibiting iNOS, COX-2 and TNF-α expression in mouse microglia cell line EOC13. 31. In addition, the extracts from solidstate culture were similar to wild-fruiting body in antiinflammatory activity, but liquid-state fermentation was less effective [79]. To continue, a hot water extracts (fraction MII from mycelium, fractions EII and EIII from culture filtrate) of submerged cultured A. camphorata show dose-dependent (5-60 μg ml −1 ) induction of TNF-α and IL-6 in peripheral blood culture. Furthermore, these fractions at 20 μg ml −1 also showed marked activity in enhancing phagocytosis in human polymorphonuclear neutrophils (PMN), in addition to CD11b upregulation, and monocytes [80]. According to Chang et al. [72] in vivo data, hexane extract (100, 200 and 400 mg kg −1 ) from SCM of A. camphorata has protection from nephritis by suppression of urine protein and serum blood urea nitrogen levels and decreased the thickness of the kidney glomerular basement membrane in SLE-prone NZB/W F1 mice [72].

Anti-Hepatitis B Virus Replication.
It was reported that extracts from the mycelium of A. camphorata have in vivo anti-hepatitis B virus (HBV) activity in a dose-dependent manner without cytotoxicity [81]. The ethanol extract displayed anti-HBV effects on both wild-type and lamivudine-resistant HBV mutants [71]. The ability of A. camphorata to inhibit the replication of HBV in vivo and in vitro may be one additional reason for considering this fungus as a potential therapeutic for HBV infection.

Anti-Oxidant Activities.
Accumulating data have shown that A. camphorata is a potent direct free radical scavenger [82][83][84][85]. The stable free radical 1,1-diphenyl-2picrylhydrazyl (DPPH) is scavenged by the extracts of A. camphorata [83,84]. Aqueous extracts of A. camphorata inhibited nonenzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC 50 value of 3.1 mg ml −1 [83]. It has been reported that, compared to other A. camphorata extracts, the fermented culture broth of A. camphorata (FCBA) and aqueous extracts of the mycelia from A. camphorata (AEMA) harvested from submerged cultures are the most potent inhibitors of lipid peroxidation, possessing marked free-radical-scavenging activity [82]. The aqueous extract from SCM of A. camphorata is possessing anti-oxidant property with respect to oxidative modification of human low-density lipoproteins (LDL) in a timeand concentration-dependent manner [86]. A recent study reported that FCBA and AEMA at 25-100 μg ml −1 and 50-200 μg ml −1 , respectively, possess antioxidant properties in human umbilical vein endothelial cell (EC) culture system [87]. In addition, both FCBA and AEMA treatment significantly inhibited apoptotic cell death in the ECs, as evidenced by reduced DNA fragmentation, cytochrome c release, caspase-3 activation and dysregulation of Bcl-2 and Bax [87]. Shu and Lung [85] observed the antioxidant activity (lipid peroxidation, scavenging effect on DPPH radical, hydroxyl free radicals, superoxide anion, reducing power activity and chelating effect on ferrous ions) of methanolic extracts from mycelia and filtrates of A. camphorata at two different concentrations (0.2 and 0.6 mg ml −1 ). To continue, 2.5 mg ml −1 of methanolic extract irradiated with 20 kGy γ-rays showed potent anti-oxidant property by scavenging abilities of 92.3-103% on DPPH radicals [88].

Hepatoprotective Activity.
Ao et al. [89] reviewed the potential of A. camphorata in treating liver diseases, which provided the major biologically active constituents and their effect or mode of action. The fruiting bodies and mycelium of A. camphorata are shown to have protective activity against liver hepatitis and fatty liver induced by acute hepatotoxicity of alcohol [90]. The methanolic extract from wild and solid-state cultures of A. camphorata exhibited angiotensinconverting enzyme (ACE) inhibitory activities in spontaneously hypertensive rats [91]. The dry matter of submerged cultivation (DMC) filtrate and aqueous extracts from fruiting bodies have been reported to possess hepatoprotective activity against liver damage induced by CCl 4 [83,92]. Both of the extracts reduce glutathione (GSH)-dependent enzymes such as glutathione peroxidase, glutathione reductase and glutathione S-transferase. Histopathological evaluation of the rat liver revealed that the DMC reduced the incidence of liver lesions, including neutrophil infiltration,     hydropic swelling and necrosis induced by CCl 4 [92]. A new formulation comprising the filtrate of A. camphorata and extracts from Astragalus membranaceus, Salvia miltiorrhiza and Lycimm chinense found to have significant inhibitory activity against the elevated ALT level in CCl 4 -treated animals to an extent that was even better than when the filtrate was used alone [93].
In conclusion, the reported data showed that A. camphorata may exert its hepatoprotective effects, though different mechanisms such as scavenging free radicals responsible for cell damage, enhancing the enzymes responsible for antioxidant activity, inhibiting the inflammatory mediators and/or induction of the regeneration of the liver cells. Previously reported data revealed that A. camphorata is a potent free radical scavenger [82][83][84][85]. It is therefore possible that hepatoprotective action of A. camphorata is partially due to its antioxidant activity. The antioxidant activities of the filtrate and mycelium extracts were correlated with the presence of total polyphenols, crude triterpenoids and the protein/polysaccharide ratio of the crude polysaccharides [82]. Aqueous extracts of A. camphorata inhibited nonenzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC 50 value of about 3.1 mg ml −1 [83].
These results suggest that A. camphorata exerts effective protection against chemical-induced hepatic injury in vivo by free radical scavenging activities.

Prevention of Liver Fibrosis.
Using CCl 4 -treated rats as an experimental model, it is found that the filtrate of fermented mycelium from A. camphorata has the preventive and curative properties of liver fibrosis [94]. Post treatment with mycelium to CCl 4 -administered rats clearly accelerated the reversal of fibrosis and lowered the elevated mRNA levels of hepatic collagen I, transforming growth factor (TGF)-β1 and tissue inhibitors of matrix metalloproteinase (TIMP)-1. Furthermore, it is confirmed that hepatic lipid peroxidation is increased during fibrogenesis where hepatic malondialdehyde (MDA) and hydroxyproline (HP) contents in curative groups were remarkably restored [94]. Another study reported that fermented mycelium is effective in reversing liver fibrosis induced by dimethylnitrosamine (DMN), while the lowered activities of antioxidative enzymes (SOD, catalase and GSH-Px) in the liver were not restored [95]. Therefore, more in vivo studies and randomized controlled clinical studies should be performed to further elucidate the mechanisms of action of A. camphorata.

Neuroprotective Effect.
It is reported that mycelium extract from submerged cultivation of A. camphorata prevents serum-deprived PC-12 cell apoptosis through PKAdependent pathway and by suppression of JNK and p38 activities [61,96].

Antihypertensive
Effect. The extracts of wild and solidstate cultures A. camphorata were obtained by sequential extraction with cold water (CWS), methanol (MS) and hot water (HWS), respectively. Among these three, only extract MS (10 mg kg −1 BW) showed potent antihypertensive effects in spontaneously hypertensive rats by decreased systolic blood pressure and diastolic blood pressure, however these effects were absent in Wistar Kyoto rats [97]. These results might have a scope to develop A. camphorata to be a healthy (or functional) food to regulate blood pressure.

Vasorelaxation Effect. The SCM extract of strain B85
shown to have concentration-dependent vasorelaxation with maximal relaxation of 40.34 ± 7.53% through an endothelium-dependent mechanism, whereas strains 35 398, 35 396 and B71 had mild effects in isolated rat aortic rings [98]. In conclusion, preclinical and clinical studies are necessary for the validation of this natural product in the prevention and/or therapy of above mentioned applications. Also, the effects of isolated compounds require to be tested further as discussed subsequently.

Bioactivities of Isolated Compounds
6.1. Terpenoids. The bitter components of A. camphorata are triterpenoids and have known pharmacological activities (Table 1). Triterpenes are considered to be potential anticancer agents due to activity against growing tumors, they have direct cytotoxicity against tumor cells rather than to normal cells. Cultivated mycelium has been reported to contain similar compounds with wild fruiting bodies [8]. Biological study revealed that zhankuic acids A (10) and C (16) exhibited cytotoxic activity against P-388 murine leukemia cells with an IC 50 value of 1.8 and 5.4 μg ml −1 , respectively [18]. However, the molecular mechanism(s) responsible for the inhibitory effects have not been fully elucidated. We reported that the isolates (10, 16, 20, 25-27, 29 and 32) from fruiting bodies of A. camphorata showed inhibitory effects on Spodoptera frugiperda Sf 9 insect cells where zhankuic acids A (10) and C (16) and methyl antcinate B (20) being most potent [64]. In continuation of our studies on the activities of pure compounds from fruiting bodies of A. camphorata, these eight compounds together with antcins A (9) and C (11) were examined for their cytotoxic data against various cancer cell types. The three zhankuic acids, 10, 16 and 20 displayed the tumor-specific cytotoxicity with an IC 50 range from 22.3 to 75.0 μM against the colon, breast, liver and lung cancer cell lines [65]. One of the most potent triterpene was methyl antcinate B (20). Furthermore, compounds 10, 16 and 20 demonstrated to induce apoptosis in HT-29 cells, as confirmed by sub-G1 cell-cycle arrest as well as DNA fragmentation. Furthermore, the expression of poly-(ADP-ribose) polymerase cleavage, Bcl-2 and procaspase-3 were also suppressed, in addition to their synergistic cytotoxic effect (4 μM each) in HT-29 cells [65]. Our previous results state that the chloroform extract of A. camphorata demonstrated inhibitory activity on colon cancer cells (see previously). Analysis suggested that the active principles in vivo were triterpenoids. These results indicate that the triterpenoids fraction of A. camphorata may be a useful ingredient in the treatment of colon cancer. To continue, our results also reveal that compounds 9, 18 and 20 displayed potential anti-Helicobacter pylori activity and its associated inflammation in human gastric epithelial AGS cells, by inhibition of adhesion and invasion, NF-κB activation and the subsequent release of IL-8 in AGS cells [99].
Scientific investigations concerning the inhibition of anti-HBV activity by polysaccharides from fruiting bodies and cultured mycelia of A. camphorata were reported in 2002 [38]. Polysaccharides from strain B86 at a dosage 50 μg ml −1 exhibited the highest anti-hepatitis B surface antigen effect, which was higher than that of α-interferon at a concentration of 1000 U/ml [38]. It is interesting to note that the anti-HBV activity has not been reported for polysaccharides from any other mushroom. Thus, further studies on the relationship between specific polysaccharide fraction and their biological activities are required. Recently, extensive studies on the immunomodulatory and anti-tumor effects polysaccharides from different sources have been reported [100]. For example, a partially purified polysaccharide inhibited the proliferation of human leukemic U937 cells via activation of human mononuclear cells [101]. In addition, these in vitro anti-tumor activity was substantiated by the in vivo study in sarcoma 180-bearing mice where the intraperitoneal and oral administration of 100 and 200 mg kg −1 significantly suppressed the tumor growth with the inhibition rate of 69.1% and 58.8%, respectively [101]. Polysaccharides isolated from A. cinnamomea reported to have anti-angiogenic activities in endothelial cells, by dose-dependent inhibition of cyclin D1 expression through vascular endothelial growth factor receptor signal pathway [102]. To continue, Han et al. [103] reported that a neutral polysaccharide named ACN2a from the hot water extract of the mycelium of A. camphorata to have in vivo hepatoprotective activity in mouse model of liver injury that was induced by Propionibacterium acnes-LPS [103]. Another study reported that the polysaccharide fractions (from SCM), AC-1, AC-2, AC-3, AC-4 and AC-5 belonged to the category of glycoprotein with mean molecular mass in the range of 394-940 kDa showed antiinflammatory activity in macrophages [40]. At a concentration of 1 μm, polysaccharides AC-1 and AC-2 showed DPPH radical scavenging activity by 74.5 and 50.5%, respectively. In addition, AC-2 dose-dependently (50-200 μg ml −1 ) inhibit the LPS-induced NO production and iNOS protein expression in macrophages [40]. Polysaccharides from SCM possess immunomodulatory activity by modulating the proinflammatory cytokines [104], through inducing Th1-type cytokines such as IFN-γ and TNF-α in a time-dependent manner but not of Th2 cytokines [70]. A recent study reported that 3-6 weeks oral administration with 2.5 mg of polysaccharides derived from A. camphorata (AC-PS) modulate the expression of Th1 cytokines in splenocytes as well as the type1 differentiation of T and B lymphocytes, in addition to reduce the infection rate of Schistosoma mansoni in mice [105]. Furthermore, water-soluble polysaccharides (200 μg ml −1 ) from the fermented filtrate and mycelia of A. cinnamomea significantly reduced the oxidative DNA damage and ROS induced by hydrogen peroxide in Chang liver cells [106]. A recent study for the first time reported the sulfated polysaccharides (SPSs) from submerged cultivation medium of A. cinnamomea. These SPSs dose-dependently inhibited in vitro Matrigel tube formation in an angiogenesis model, in addition to their prevention of serum-deprived apoptosis in neuronal-like PC-12 cells [107].

Summary and Outlook
This review summarized important areas of investigation being performed on A. camphorata with particular emphasis on crude extracts and isolated compounds. Some correlation between the ethnomedical employment and the pharmacological activities has been duly observed in the present review. Antrodia camphorata extracts from its fruiting bodies, Evidence-Based Complementary and Alternative Medicine 13 mycelium and cultivation filtrate showed multiple cancer preventive and anti-inflammatory activities. In addition, these extracts provide a variety of anti-cancer and antiinflammatory active secondary metabolites and polysaccharides. Of particular promise, due to their potent cytotoxic activity against a number of cancer cell lines, are the triterpenoids with ketonic functional groups. In fact, these triterpenoids, which have also been found in a small number of other mushrooms, are currently under active investigation as potential therapeutic leads [109]. Because the antioxidant action is also a means of lowering chronic anti-inflammatory action, A. camphorata hold potential in functional food approaches aimed at normalizing metabolic syndrome.
In the search for active compounds from A. camphorata, the majority of research has been performed on extracts from the fruiting bodies and mycelium and, there have been fewer studies on extracts from the submerged cultivated medium. Further studies would be desirable to isolate useful new secondary metabolites by varying cultivation conditions. The pharmacological studies so far have mostly been performed in vitro and in vivo with animals. Therefore, clinical studies are needed in order to confirm traditional wisdom in the light of a rational phytotherapy. Nevertheless, the former reports could be considered as providing leads for more scientific research. The biological activities of the pure compound administrated or consumed alone were found to be lower than those obtained from the original mixture of active ingredients present in natural medicines including A. camphorata. Thus, the combined, synergistic effects of a mixture of active components that are present in A. camphorata on biological activities need to be thoroughly assessed. Finally, though we recently developed a cyclodextrin-modified capillary electrophoresis method for the separation and analysis of achiral and chiral triterpenoids from fruiting bodies of A. camphorata [110], there however, is a need to establish suitable quality parameters and analytical methods to determine active compounds.


text:
Ethyl 2-diethylamino-4-oxo-3,5-diphenyl-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidine-7-carboxylate

In the title compound, C25H26N4O3, the two fused pyrrolo[3,2-d]pyrimidine rings form a dihedral angle of 3.7 (2)°. The two substituent phenyl rings are twisted with respect to the pyrrole and pyrimidine rings, making dihedral angles of 57.2 (2) and 69.0 (2)°, respectively. The ethyl and ethoxy groups are disordered over two positions; the site occupancies are 0.53 (1) and 0.47 (1) for ethyl, and 0.63 (1) and 0.37 (1) for ethoxy. The crystal packing features C—H⋯O hydrogen bonds.

In the title compound, C 25 H 26 N 4 O 3 , the two fused pyrrolo[3,2d]pyrimidine rings form a dihedral angle of 3.7 (2) . The two substituent phenyl rings are twisted with respect to the pyrrole and pyrimidine rings, making dihedral angles of 57.2 (2) and 69.0 (2) , respectively. The ethyl and ethoxy groups are disordered over two positions; the site occupancies are 0.53 (1) and 0.47 (1) for ethyl, and 0.63 (1) and 0.37 (1) for ethoxy. The crystal packing features C-HÁ Á ÁO hydrogen bonds.

Experimental
The title compound was obtained in good yield via aza-Wittig reaction. Crystals suitable for single-crystal X-ray diffraction were obtained by recrystallization from a mixture of ethanol and dichloromethane (1:1 v/v) at room temperature.

Refinement
All H atoms were located in difference maps and treated as riding atoms with C-H = 0.93 Å, U iso =1.2U eq (C) for Csp 2 , C-H = 0.97 Å, U iso = 1.2U eq (C) for CH 2 , C-H = 0.96 Å, U iso = 1.5U eq (C) for CH 3 . N-H = 0.86 Å, U iso = 1.2U eq (N) for NH. Fig. 1. The molecular structure of the title compound showing the atom-labeling scheme with displacement ellipsoids drawn at the 30% probability level.

Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.
Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.