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Medicine
Materials Science

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Microwave-assisted FLP-catalyzed hydrogenations

FLP-catalyzed hydrogenations of 15 substrates were compared using microwave irradiation and conventional heating. The direct comparison revealed that a rate acceleration of up to 2.5 was achieved in the presence of microwaves. This heating method is particularly promising for the hydrogenation of nitrogen-containing heterocycles. Acridine, quinines and especially 1-methyl indole were reduced very e ﬃ ciently under mild conditions and only 4 bar hydrogen pressure in high yields.

Introduction
The reduction of organic molecules by frustrated Lewis pairs (FLP) using molecular hydrogen (H 2 ) has emerged to be a powerful tool for organic synthesis. 1 Electron-deficient as well as electron-rich compounds have been successfully hydrogenated 2 and even asymmetric reductions of prochiral substrates were achieved. 3 Usually such reactions are conducted in closed reaction systems e.g. thick-walled vials, pressure tubes including NMR-tubes or steel bomb autoclaves. The appropriate reactor must be selected with regard to the required H 2pressure. The closed reaction systems are typically heated in oil baths or with heating jackets above the boiling point of the solvent. 4 The same specifications hold true for microwavereactors including the advantage of the fast equilibration of the temperature and inverted temperature gradients. 5 Microwave-assisted transition-metal catalyzed hydrogenations and transfer hydrogenations were reported 6 rendering this method attractive for FLP-catalyzed hydrogenations. Interestingly, the application of microwave heating in FLP-catalyzed hydrogenations has not been reported yet.
Herein we report the first microwave-assisted FLP-catalyzed hydrogenation of imines, heterocycles, enamines, silyl enol ethers, nitroolefins, malonates and olefins. This study focuses on the comparison of hydrogenations conducted under conventional and microwave heating. Therefore reported catalyst systems (Lewis acids 1-3 and Lewis bases 4-8, Chart 1) and known substrates susceptible to FLP-catalyzed hydrogenation were investigated.

Results and discussion
We initiated our studies with the well-established FLP-catalyzed hydrogenation of N-benzylidene-tertbutyl amine 1c,7 (9) using conventional and microwave heating. A solution of imine 9 in toluene (0.2 M) and 5 mol% B(C 6 F 5 ) 3 was prepared and split into two identical thick walled glass vessels. The vessels were sealed and pressurized with 4 bar of hydrogen. One sample was immersed into a preheated oil bath and the second sample was introduced into a CEM microwave reactor with a power setting to 150 W. Both reactions were heated to 80°C for the indicated time (see Table 1).
Chart 1 Lewis acids and Lewis bases for FLP-mediated hydrogen activation. The hydrogenation product 10 was obtained with both heating methods however in very different yields. The reaction using microwave irradiation gave the product in 60% yield after 10 minutes while using conventional heating furnished only 22% yield. The microwave-assisted reaction went to full conversion with prolongation of the reaction time to 20 minutes while the oil bath heated reaction provided 10 in 47% yield. We attribute this significant rate increase to more efficient heating of the reaction by microwave irradiation.
Encouraged by these results we investigated other substrates bearing polarized double bonds in the microwave-assisted reaction. We selected substrates, which are susceptible to FLPcatalyzed hydrogenation under conventional heating and compared them with yields obtained from microwave experiments.
The standard conditions for the hydrogenations using microwave-assisted and conventional heating were benzene as solvent (0.2 M), 4 bar hydrogen and 150 W power-level in CEM Discover microwave reactor with external IR temperature control or a preheated oil-bath respectively. 12 For best comparability thermal as well as microwave-assisted hydrogenations were conducted under identical conditions using stock-solutions, identical glassware, temperature, hydrogen pressure and reaction time. The reactions were stopped after 10 to 120 minutes and the yields were determined by 1 H NMR with hexamethylbenzene as internal standard. The results are summarized in Table 2. Generally, all substrates underwent FLPcatalyzed hydrogenation under microwave conditions most of them with pronounced rate acceleration. In accord with our observations for imine 9 we found two-fold rate increase for imines 11a and 11b (entries 1 and 2). The imine 11c underwent hydrogenation in comparable yields and diastereoselectivity irrespective of the heating method (entry 3). Particularly, the hydrogenation of nitrogen-containing heterocycles benefits from microwave-assisted heating (entries 4-7). The hydrogenation of acridin (11d), 8-methyl-quinolin (11e) and 2-methyl-quinoline (11f ) proceeded in significantly higher yields (72-99%) compared to the reactions with conventional heating (7-68%). Notably, indol 11g was reduced in 57% yield in 40 minutes at 140°C with only 4 bar of hydrogen pressure under microwave conditions whereas the hydrogenation was not achieved using conventional heating (entry 7). Although yields for the reaction using conventional heating were not reported 2e the decrease of the required pressure by almost 100 bar renders the application of microwave-assisted FLP-hydrogenation of indoles as very promising. We also investigated the core hydrogenation of diphenylamine with 20 mol% catalyst loading (11h). 4b However, even under forcing conditions only traces of the product could be observed irrespective of the heating techniques (entry 8). The electron-rich double bond in enamines 7b and silyl enol ethers 3c, 9,13 were reduced in good to excellent yields in only 10 to 40 minutes (entries 9-11). Again the microwave-assisted reactions provided the products in shorter reaction times compared to reactions using conventional heating. Densely functionalised malonates 10,14 were hydrogenated in quantitative yield in 2 h at 80°C (entry 12) compared to 12 h at room temperature. However, direct comparison of the reactions using microwave and conventional heating after 40 minutes revealed identical reaction rates using the FLP-catalyst 3/7 for transient hydrogen activation. Such electronically modified FLP-catalysts e.g. consisting of less Lewis-acidic boranes B(C 6 F 3 H 2 ) 2 (2) and B(C 6 F 2 H 3 ) 3 (3) or less electron-releasing phosphine 8, display highly reversible H 2 -activation at room temperature or below according to eqn (1). 11 According to an almost ergoneutral reaction the temperature increase shifts the equilibrium to the left side resulting in reduced concentrations of the H 2 -activation products. 7c, 10,11,15 Consequently, substrates e.g. nitroolefins or olefins (entries 13 and 14), which require the application of highly reversible FLP-catalysts are less susceptible to microwave-assisted hydrogenations and conventional heating provides higher yields. The reduction of nitroolefin 11m was achieved in 60% at 50°C, but only 32% yield was obtained if the reaction was conducted at 80°C (entry 13). Similar reactivity was observed for the hydrogenation of the olefin 11n (entry 14). Nevertheless, microwave-assisted FLP-catalyzed hydrogenations are highly valuable for reactions, which require heating leading to a significant rate increase.

Conclusions
In summary we have shown that microwave-assisted heating significantly accelerates FLP-catalyzed hydrogenations rendering this methodology as useful tool in organic synthesis. The yields for the hydrogenation of fifteen common substrates under microwave irradiation and conventional heating were directly compared. Rate increase of 2-2.5 were observed for imines 9, 11a and 11b using only 4 bar of hydrogen pressure. We attribute the reduced reaction times to more efficient heating of the reaction mixture. The hydrogenation of nitrogen-containing heterocycles e.g. acridine (11d), quinolines (11e-f) and 1-methyl indole (11g) proceeds in high yields under mild reaction conditions. Astonishingly, the indole derivative 11g was reduced in 57% yield under only 4 bar hydrogen pressure while the reduction was absent using conventional heating.

General information
All preparations were carried out in oven-dried glassware under an atmosphere of inert gas (Argon 5.0, Air Liquide) employing both Schlenk line techniques and a Glovebox Systems inert atmosphere glovebox. Microwave assisted reactions were carried out in a CEM Discover microwave system (model 908010) with external IR temperature control in 10 ml reaction vessels equipped with Teflon/rubber crimp caps. [D 6 ]-deuterated benzene and toluene were degassed by 3 freeze-pump-thaw cycles, vacuum transferred from sodium/ benzophenone, dispensed into Straus flasks equipped with Young-type Teflon valve stop-cocks and stored over 4 Å molecular sieves. Hydrogen 6.0 (AirLiquide) was purified through a Johnson Matthey Model HIG 35XL gas purifier. Substrates for hydrogenation reactions were purchased from Sigma Aldrich or abcr and were recrystallized or distilled prior to use if necessary. Reagents for borane synthesis were used as received without further purification. B(C 6 F 2 H 3 ) 3 (3), B(C 6 F 3 H 2 ) 3 (2) were prepared as described earlier. 10 General procedure for microwave assisted hydrogenation reactions In a glovebox, B(C 6 F 5 ) 3 (1) (5-10 mol%), hexamethylbenzene (1.6 mg, 0.01 mmol, 0.1 equiv.) and the corresponding substrate (0.1 mmol, 1.0 equiv.) were dissolved in 0.5 ml of deuterated solvent. The stock solution was divided in three parts, two portions of 0.2 ml were transferred into two 10 ml reaction vessels equipped with magnetic stir bars and were capped with PTFE/butyl rubber crimp caps. The third part of 0.1 ml of the stock solution was used as comparison sample and analysed by 1 H NMR spectroscopy. The reaction vessels were attached to an H 2 /vacuum line via needle, immersed into liquid nitrogen (6 cm), freeze pump thawed for one cycle and charged with H 2 at 77 K. The reactions were carried out in two CEM Discover (model 908010) microwave systems using 150 W power at given times and temperatures. The cooled reaction mixtures were separately analysed by 1 H NMR spectroscopy. Yield as mean value of both measurements. Non-microwave control samples were completely immersed in a preheated oil bath. For substrates requiring an FLP, a suitable base was added matching the concentration of borane as indicated. For the domino hydrosilylation hydrogenation of enones HSiPh 2 Me (0.1 mmol, 1.0 equiv.) was added prior to the addition of a suitable base as described earlier. 9 Reactions of nitroolefins and malonates required the use of B(2,6-F 2 -C 6 H 3 ) 3 (3) or B(2,4,6-F 3 -C 6 H 2 ) 3 (2) (20 mol%). The analytic data of all starting materials and hydrogenation products were reported in the literature. Yields were determined by 1 H NMR spectroscopy using hexamethylbenzene as internal standard.


Antiproliferative Effects of New Dimeric Ellagitannin from Cornus alba in Prostate Cancer Cells Including Apoptosis-Related S-Phase Arrest

Activity-guided isolation of 80% acetone extract of Cornus alba, which is traditionally used as an anti-inflammatory, hemostatic and diuretic in Korea, yielded one novel compound, tentatively designated cornusiin H (13), together with 12 known compounds. The known compounds included four flavonoids (catechin (1), quercetin-3-O-β-d-glucuronide (2), quercetin-3-O-β-d-glucopyranoside (3), kaempferol-3-O-β-d-glucopyranoside (4)) and eight hydrolysable tannins (gallic acid (5), 2,6-di-O-galloyl-hamamelofuranoside (6), 2-galloyl-4-caffeoyl-l-threonic acid (7) 2,3-di-O-galloyl-4-caffeoyl-l-threonic acid (8), 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside (9), cornusiin B (10), cornusiin A (11) and camptothin B (12)). All compounds exhibited potent 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical scavenging activity. Especially, the radical scavenging activities of 6 and 9–13 were higher than that of vitamin C. Compounds 9, 11, 12 and 13 inhibited the production of nitric oxide (NO) in lipopolysaccharide-stimulated RAW264.7 cells to the same degree as NG-Monomethyl-l-arginine (l-NMMA). When the antiproliferative effects of the isolated compounds were assessed in prostate cancer cells, the dimeric ellagitannins (11–13) selectively inhibited LNCaP hormone-dependent prostate cancer cells. Flow cytometry analysis indicated that the dimeric ellagitannins induced apoptosis and S-phase arrest. These results suggest that dimeric ellagitannins from Cornus alba can be developed as functional materials or herbal medicines for prostate tumors such as benign prostate hyperplasia and early-stage prostate cancer.

Introduction
Prostate tumors can be categorized as benign prostate hyperplasia (BPH), which is an overgrowth of the prostate caused by extensive androgen-dependent tissue remodeling [1], and prostate cancer (PCa), which is a malignant tumor caused by oncogenic mutations, aberrant signaling or inflammatory conditions [2]. The proliferation of prostate tissue which could make urination difficult is an important problem for BPH [3]. The α 1 -Adrenergic agonists and 5α-reductase inhibitors are typically used to treat BPH, with the goals of improving urination and decreasing prostate growth. However, long-term issues with these medications include adverse effects, such as low blood pressure and sexual dysfunction. Hormonal therapy is used for early-stage prostate cancer. Frequently, the cancer becomes hormone-refractory. Being a typical chronic disease in middle-aged and old men, prostate tumors are likely to increase with age. BPH has been described in 62% of European middle-aged men [4]. PCa, the most common cancer in Western men, occurred mostly between 54 and 75 years of age [2]. The incidence of prostate tumors is rapidly increasing in Korea. A recent Korean survey reported that the prevalence of BPH was about 40% in men over 65 years of age [5], with the incidence of PCa increasing by an average of 12.1% annually over the past decade, ranking as the fifth most prevalent cancer in men [6]. Also, on the worldwide scale, PCa, as second in the estimated new cases and fifth in the estimated deaths in males, is one of the most prevalent cancers [7].
The Cornus species of plants is also used as an East Asian folk remedy for urinary health. C. officinalis hips are a renowned natural tonic in aging men [10], and the stems of C. walteri are used as a diuretic [11,12]. Various phytochemicals including lignans, iridoids, terpenoids, flavonoids and tannins have been more recently isolated from these species [13][14][15], and Cornus extracts reportedly possess antioxidative, anti-inflammatory and anti-cancer biological activities [16]. Cornus alba (CA), also known as red-barked or Siberian dogwood, is native to Siberia, northern China and Korea [16]. The stems and leaves of CA have been used as antiphlogistic, hemostatic and diuretic treatments in Korea. [17] This ethnopharmacologic usage comes despite limited knowledge of chemistry and biological activity of this plant.
The present study was undertaken to provide clarity on its phytochemicals and biological activities. We tried to isolate active constituents and evaluated their biological activities with the goal of developing natural prostate tumor medications.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activity
Reducing oxidative stress is related to the anti-cancer and anti-inflammatory effect. [28] To assess the antioxidative activity of the compounds isolated from CA, DPPH radical scavenging activity was investigated. All compounds showed radical scavenging activity in a dose-dependent manner (data The positive cotton effect at the short wavelength region ([θ] 224 8.81) in the CD spectrum indicated that the absolute configuration of the valoneoyl group was S [26]. The downfield-shifted H-4 R (δ H 5.11, 5.12) as well as the separately observed H-6a R (δ H 5. 27-5.32) and H-6b (δ H 3.76-3.96), which were correlated with acylated anomeric H-1 R (δ H 6.18, 6.18) in TOCSY, suggested that the valoneoyl group should be connected at OH-4/OH-6 of the right glucose core [27]. ( Figure S4) In the left glucose core, the downfield-shifted H-4 L (δ H 5.50, 5.57) as well as the adjoined H-6a L (δ H 4.47, 4.51) and H-6b L (δ H 4.23, 4.30), which correlated with the non-acylated H-1 L (δ H 4.74, 5.42) in TOCSY, ( Figure S4) and no observation of any characteristic chemical shifts of the HHDP group at δ 6.40-6.60 indicated that the two galloyl groups should connect with the OH-4 and OH-6 of the left glucose core, respectively. ( Figure S3) The upfield-shifted H-1 of the β-anomer (δ 4.74 (0.7H, J = 7.8 Hz, H-1 L of β-β form) compared with the α-anomer in the left-side glucose core (δ 5.42 (1H, d, J = 3.0 Hz, H-1 L of α-β form)) indicated that the valoneoyl group at OH-2 of the left-side glucose core induced an anisotropic shielding effect in H-1 L . ( Figure S1) In addition, the downfield-shifted Val H A at δ H 6.68 and 6.69, due to absence of the shielding effect by the galloyl group at OH-3 in the right glucose core, indicated that the left glucose core is connected at the bottom of the valoneoyl group. The ellagitannin composed with an unacylated anomeric center and substituted by two galloyl groups at OH-4 and OH-6 in the left glucose core was tentatively designated cornusiin H.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activity
Reducing oxidative stress is related to the anti-cancer and anti-inflammatory effect. [28] To assess the antioxidative activity of the compounds isolated from CA, DPPH radical scavenging activity was investigated. All compounds showed radical scavenging activity in a dose-dependent manner (data not shown). Especially compounds 6 and 9-13, which are hydrolysable tannins, possessed more potent DPPH free radical scavenging activities than the vitamin C positive control, while the scavenging activities of simple gallic acids (5) and galloyl caffeoyl threonic acids (7,8) were moderate ( Table 1). The hydrolysable tannins (6 and 9-13) had more than two galloyl groups known to eliminate free radicals, and their efficiency depended on the number of galloyl groups (6 < 9 < 10, 11 < 12, 13). Flavonols are well-known antioxidative compounds. Especially, the hydroxyl group at C-5, the ketone at C-4 and B-ring phenolic hydroxyl groups are crucial as electron acceptors. In this case, the radical scavenging activities of compounds 2 and 3, which have a 3 1 ,4 1 -dihydroxylphenyl B-ring, were more potent than compound 4, which possesses a 4 1 -hydroxylphenyl B-ring. a Values are presented as the mean˘SD (n = 3); b Positive controls. Vit.C: L-ascorbic acid; L-NMMA: N G -monomethyl-L-arginine.

Inhibition of Nitric Oxide (NO) Production
It is reported that there is a strong correlation between the inflammation and the pre-cancerous lesion, [29] so the anti-inflammatory effect could also partly reflect the anti-cancer effect. To evaluate the anti-inflammatory activities of the compounds isolated from CA, their inhibition of NO production in LPS-stimulated RAW264.7 cells was investigated as described previously [30]. While the flavonoids (1-4) and mono-or di-galloyl substituted tannins (5-8) had no effect, the hydrolysable tannins (9-13) dramatically inhibited NO production to almost the same degree as N G -monomethyl-L-arginine (L-NMMA), except for 10, which has a substituted lactonized valoneoyl group (Table 1). These findings indicated that the lactonized valoneoyl moiety may not be helpful for the intracellular effects of hydrolysable tannins.

Antiproliferative Activity on Prostate Cancer Cells
Besides the indirect anti-cancer effect, which includes anti-oxidative and anti-inflammatory activities, the antiproliferative activity on cancer cells could more directly affect the anti-cancer activity. To evaluate the antitumor effects of the compounds isolated from CA, the antiproliferative effect of each compound was determined using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay on androgen-dependent LNCaP and androgen-insensitive DU145 cell lines. The antiproliferative activity of 50 µM of each compound was screened in LNCaP and DU145 cells and RWPE-1 normal prostate epithelial cells together with other tannin-related compounds previously isolated from other plant sources. The compounds were divided into five groups as follows: (1) condensed tannins: 1; (2) flavononol glycosides: 2-4; (3) gallotannins: 5-9; (4) monomeric ellagitannins: 10; and (5) dimeric ellagitannins: 11-13. All dimeric ellagitannins showed selective antiproliferative effects on LNCaP cells. These compounds decreased the cell viability of LNCaP four to five times more than that of RWPE-1. This finding suggested that the HHDP group on ellagitannins might increase the antiproliferative effects of androgen-dependent LNCaP. Interestingly, compound 10, which possesses a lactonized valoneoyl group at OH-2, was decreased. Thus, the rigid valoneoyl group at OH-2 and the flexible galloyl group at OH-2 and OH-3 may be essential for selective antiproliferation. The antiproliferative effects of compounds 9-13 were assessed at various concentrations using LNCaP and DU145 cells. Especially, dimeric ellagitannins (11)(12)(13) inhibited the proliferation of LNCaP cells seven times more potently than DU145 cells, which means dimeric ellagitannins could inhibit the proliferation of hormone-dependent PCa cells more powerfully than hormone-insensitive PCa cells (Table 2), and decreased the cell viability of LNCaP cells more effectively than monomeric ellagitannins did ( Figure 2). All dimeric ellagitannins showed selective antiproliferative effects on LNCaP cells. These compounds decreased the cell viability of LNCaP four to five times more than that of RWPE-1. This finding suggested that the HHDP group on ellagitannins might increase the antiproliferative effects of androgen-dependent LNCaP. Interestingly, compound 10, which possesses a lactonized valoneoyl group at OH-2, was decreased. Thus, the rigid valoneoyl group at OH-2 and the flexible galloyl group at OH-2 and OH-3 may be essential for selective antiproliferation. The antiproliferative effects of compounds 9-13 were assessed at various concentrations using LNCaP and DU145 cells. Especially, dimeric ellagitannins (11)(12)(13) inhibited the proliferation of LNCaP cells seven times more potently than DU145 cells, which means dimeric ellagitannins could inhibit the proliferation of hormone-dependent PCa cells more powerfully than hormone-insensitive PCa cells (Table 2), and decreased the cell viability of LNCaP cells more effectively than monomeric ellagitannins did ( Figure 2).

Induction of Cell Cycle Arrest and Apoptosis
The apoptosis effect of PCa is helpful to assay the anti-cancer effect. To observe cell cycle distribution and apoptosis by the ellagitannins (9-13) obtained from CA, flow cytometry analysis was performed. Compounds 11-13 which effectively are dimeric ellagitannins induced apoptosis within 48 h, and the potency of dimeric ellagitannins was two to three times better than that of monomeric ellagitannins (Figure 3). Compound 11 elevated the subG1 phase of both LNCaP and DU145 cells in a dose-dependent manner, and the potency for LNCaP cells was 10 times greater than that of DU145 cells (Figure 3). Compound 11 applied at a low concentration also increased the S-phase, while the cells in G0/G1 and G2/M were decreased (Figure 3). These findings suggested that dimeric ellagitannins may induce S-phase arrest and apoptosis.

Induction of Cell Cycle Arrest and Apoptosis
The apoptosis effect of PCa is helpful to assay the anti-cancer effect. To observe cell cycle distribution and apoptosis by the ellagitannins (9-13) obtained from CA, flow cytometry analysis was performed. Compounds 11-13 which effectively are dimeric ellagitannins induced apoptosis within 48 h, and the potency of dimeric ellagitannins was two to three times better than that of monomeric ellagitannins (Figure 3). Compound 11 elevated the subG1 phase of both LNCaP and DU145 cells in a dose-dependent manner, and the potency for LNCaP cells was 10 times greater than that of DU145 cells (Figure 3). Compound 11 applied at a low concentration also increased the S-phase, while the cells in G0/G1 and G2/M were decreased (Figure 3). These findings suggested that dimeric ellagitannins may induce S-phase arrest and apoptosis.

Extraction and Isolation
Several extractions of CA (5.7 kg) using 80% acetone at room temperature followed by removal of the acetone under vacuum yielded 463 g of extract. The extract was dissolved in water and filtered through Celite 545 (Duksan Pure Chemical, Ansan, Korea). Then 356 g of water-soluble fraction was obtained together with 89 g of water-insoluble residue; 243 g of the water-soluble fraction applied to a Sephadex LH-20 column (15ˆ100 cm) equilibrated with water. The column was eluted with a water-methanol gradient system and washed in 60% acetone, which yielded 14 fractions.
Cornusiin H (13) The product was an amorphous brown powder. Structural data are as follows:

Measurement of DPPH Radical Scavenging Activity
Antioxidant activity was determined on the basis of the scavenging activity of the stable DPPH free radical (Sigma-Aldrich, St. Louis, MO, USA). Each 20 µL of sample in absolute ethanol was added to 180 µL of 0.1 mM DPPH in absolute ethanol. After mixing gently and standing for 30 min, the optical density was measured at 540 nm using an ELISA reader (TECAN, Salzburg, Austria). The free radical scavenging activity was calculated as inhibition rate (%) = [1´(sample O.D./control O.D.)]ˆ100; IC 50 was the concentration that could scavenge 50% DPPH free radical. L-ascorbic acid was used as positive control.

Cell Culture
RAW 246.7, LNCaP and DU145 cells were purchased from the Korean Cell Line Bank. These cells were grown at 37˝C in a humidified atmosphere (5% CO 2 ) in DMEM or RPMI (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) and 100 IU/mL penicillin G (Gibco BRL, Grand Island, NY, USA).

Viability Assay
Approximately 10 5 /well of RAW264.7 or DU145 cells were seeded in wells of a 24-well plate and incubated for 4 h in 5% CO 2 at 37˝C. LNCaP cells were seeded at a density of 10 4 cells/well and were incubated for 24 h. The medium was replaced with phosphate buffered saline (PBS) containing 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and incubated for 4 h. The supernatant was removed and the MTT-formazan was dissolved in 200 µL dimethylsulfoxide. The extent of the reduction of MTT to formazan within the cells was measured at 540 nm with microplate reader (TECAN, Salzburg, Austria). The cell viability was calculated as sample O.D./blank O.D.ˆ100 (%).

Measurement of Inhibition of Nitric Oxide (NO) Production
RAW 264.7 cells were cultured in wells of a 24-well plate and incubated for 4 h at 37˝C in a humidified atmosphere of 5% CO 2 . The cells were treated with i1 µg/mL lipopolysaccharide (LPS; Sigma-Aldrich) and incubated for 24 h. The NO content was determined by the Griess assay. Griess reagent (100 µL of 0.1% naphthylethylenediamine and 1% sulfanilamide in 5% H 3 PO 4 solution; Sigma-Aldrich) was added to 100 µL of each supernatant. NO was then measured at 540 nm with a microplate reader (TECAN). NO was quantified using a sodium nitrite standard curve as previously described [30].

Flow Cytometry Analysis of Cell Cycle Arrest
The cells were harvested and washed once in PBS. The cell pellet was re-suspended in 400 µL PBS containing 2% FBS and fixed in 1.2 mL 70% ethanol for 24h at´20˝C. Cells were washed twice in PBS and treated with 0.1% Triton X-100 and ribonuclease A (100 µg/mL) in PBS for 20 min. Propidium iodide (PI; 25 µg/mL) was added and flow cytometry was carried out using a BD-LSR II flow cell cytometer (BD, San Jose, CA, USA) using Cell Quest software.

Flow Cytometry Quantification of Apoptosis
Cells were harvested and washed once in PBS and once in annexin V binding buffer. The cells were resuspended in binding buffer and stained with annexin V-fluorescein isothiocynate (FITC) and PI for 15 min in the dark at 4˝C. The fluorescence was analyzed by flow cytometry as described above. The percentages of necrotic cells, early and late apoptotic cells, and viable cells were compared.

Conclusions
A new dimeric ellagitannin, designated cornusiin H (13), was isolated from CA together with 12 known phenolic compounds. Dimeric ellagitannins (11)(12)(13) showed potent DPPH radical scavenging activity and inhibited NO production in LPS-stimulated RAW 264.7 cells. Also, they selectively inhibited proliferation of LNCaP hormone-dependent prostate tumor cells. The rigid 4,6-HHDP group and flexible 2,3-O-digalloyl were essential for the antiproliferative effects and dimerization via the valoneoyl group's increased activity. The dimeric ellagitannins efficiently induced apoptosis as well as S-phase arrest. Thus, the dimeric ellagitannins or ellagitannin-rich fraction from CA might be developed as functional material to alleviate prostate tumors (BPH or early-stage PCa) through future clinical study.