Cytotoxic isoprenylated xanthones from Cudrania tricuspidata

Eight new isoprenylated xanthones, cudratricusxanthones A-H (1-8), were isolated from the roots of Cudrania tricuspidata, together with ten known compounds, cudraxanthones H (9) and M (10), xanthone V(1a) (11), toxyloxanthone C (12), macluraxanthone B (13), 1-hydroxy-3, 6, 7-trimethoxyxanthone (14), cycloartocarpesin (15), artocarpesin (16), cudraflavone B (17), and kaempferol (18). Their structures were characterized by spectroscopic methods. Xanthones 5, 7, 10, and 12 showed inhibitory effects on four kinds of human digestive apparatus tumor cell lines (HCT-116, SMMC-7721, SGC-7901, and BGC-823) with IC(50) values of 1.6-11.8 microg/mL. Xanthones 2, 4, and 11 displayed significant cytotoxicity against HCT-116, SMMC-7721, and SGC-7901 (IC(50)=1.3-9.8 microg/mL). Flavonoids 15-17 were almost inactive.     

Isoprenylated xanthones and flavonoids from Cudrania tricuspidata

Further phytochemical investigation on the roots of Cudrania tricuspidata afforded a new isoprenylated xanthone, cudratricusxanthone I (1), two new isoprenylated flavanones, cudraflavanones C and D (2 and 3, resp.), and seven known compounds, 1,7-dihydroxy-3,6-dimethoxyxanthone (4), macluraxanthone C (5), cudraxanthones E, K, and L (6, 7, and 8, resp.), cudraflavanone A (9), and cudraflavone C (10). Their structures were identified by spectroscopic methods. Cudratricusxanthone H (12), macluraxanthone B (13), two xanthones previously isolated from this plant, and 5, showed significant inhibitory effects on four kinds of human digestive apparatus tumor cell lines (HCT-116, SMMC-7721, SGC-7901, and BGC-823) with IC50 values of 2.70-12.66 microM.     

Antioxidant and cytotoxic activities of xanthones from Cudrania tricuspidata

The new catecholic xanthone, 1,3,7-trihydroxy-4-(1,1-dimethyl-2-propenyl)-5,6-(2,2-dimethylchromeno)-xanthone (1), was isolated from the root bark of Cudrania tricuspidata together with seven known xanthones. The structures were fully characterized by analysis of physical and spectral (UV, IR, mass, and NMR) data. Relationships between the structural characteristics of xanthones and their antioxidant activities (DPPH, superoxide, and hydroxyl radical) were studied. Among the range of catecholic xanthones, 6,7-dihydroxyl xanthones (3–8) exhibited a strong scavenging effect on the DPPH radical. When one of the catecholic hydroxyl groups was protected as in compounds 1 and 2, DPPH radical scavenging activity was markedly decreased (IC₅₀ > 200 μM). DPPH activities were consistent with electrochemical response by cyclic voltammetry. Interestingly, compounds (1, 2) which had the weak activities on DPPH, exhibited both potent superoxide and hydroxyl radical scavenging activities. The strong activity on the hydroxyl radical of compounds (1, 2) could be rationalized by their chelating effect with iron (Fe²⁺) due to a redshift of its complex. The catecholic xanthones (3–8), being able to convert quinone methide intermediate, showed potent cytotoxicities against human cancer cell lines (HT-29, HL-60, SK-OV3, AGS, and A549). In particular, compounds 3, 6, and 7 had strong cytotoxic activities against AGS (LD₅₀ < 5 μM). DNA fragmentation patterns induced by catecholic xanthones revealed that tumor cell death was due to apoptosis.     

Structural characteristics of flavanones and flavones from Cudrania tricuspidata for neuraminidase inhibition

The structural characteristics of flavonoids (1–3 and 6–8) from the root of Cudrania tricuspidata required for neuraminidase inhibition were studied and compared with commercially available flavonoids (4, 5, and 9–12). Alkylated flavanones (1–3) display better inhibition than the corresponding parent compound 4. Importantly, flavanone 1 bearing a C-8 hydrated prenyl group showed extremely high inhibition with IC₅₀ of 380 nM. On the other hand, the parent flavone 5 was more effective than alkylated analogues (6–8). Isolated inhibitors (1–3 and 6–8) showed noncompetitive inhibition in kinetic studies. The binding affinity of flavanones (1–4) for neuraminidase in in silico docking experiments correlated well with their IC₅₀ values and noncompetitive inhibition mode.     

Anti-atherosclerotic and anti-inflammatory activities of catecholic xanthones and flavonoids isolated from Cudrania tricuspidata

The catecholic xanthones and flavonoids 1-13 were isolated from the root bark of Cudrania tricuspidata. Compounds 1 and 3-8 exhibited significant antioxidant activity against low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance (TBARS) assay. Among them, prenylated flavonoids 10-12 showed an inhibitory effect on the NO production and iNOS expression in RAW264.7 cells. Also, compounds 1, 2, 5, 7, 9, and 11 preferentially inhibited hACAT-2 than hACAT-1, whereas compounds 3, 4, 6, and 8 showed a similar specificity against hACAT-1 and -2. However, flavonoids 10, 12, and 13 dominantly inhibited hACAT-2, not hACAT-1.     

Xanthones from Cudrania tricuspidata displaying potent alpha-glucosidase inhibition

We have proven that xanthones 1-8 isolated from the root of C. tricuspidata possess highly potent alphaalpha-glucosidase inhibition properties. Compound 1 was identified as a new isoprenylated tetrahydroxy xanthone, 1,3,6,7-tetrahydroxy-2-(3-methylbut-2-enyl)-8-(2-methylbut-3-en-2-yl)-9H-xanthen-9-one (1). These are the first natural xanthones documented to exhibit such inhibition. The IC(50) values of compounds 1-8 inhibiting alpha-glucosidase activity were determined to be up to 16.2microM. Mechanistic analysis showed the xanthones 1-8 exhibited full mixed inhibition.     

Xanthones from the stems of Cudrania tricuspidata and their inhibitory effects on pancreatic lipase and fat accumulation

Nine new xanthones, cudracuspixanthones I - Q (12–14, 25, 32–36), and 30 known xanthones (1–11, 15–24, 26–31, 37–39) were isolated from the stems of Cudrania tricuspidata (Moraceae). The structures of isolated compounds were established by using 1D and 2D NMR in combination with HR-TOF-MS. Xanthones from the stems of C. tricuspidata exerted pancreatic lipase inhibitory activity. In addition, cudracuspixanthone P (35), a new xanthone, reduced the fat accumulation in liver cells stimulated with fatty acids. Therefore, these compounds might be beneficial in the treatment of metabolic diseases.     

Selective ABTS radical-scavenging activity of prenylated flavonoids from Cudrania tricuspidata

The antioxidative properties of five prenylated flavonoids, including new flavanone (2), from the root bark of Cudrania tricuspidata were examined against the ABTS, DPPH, and hydroxyl radicals. In most of the assays to determine their antioxidative properties, the ABTS activity was strongly correlated with DPPH because both methods are responsible for the same chemical property of hydrogen- or electron-donation to the antioxidant. On the other hand, the prenylated flavonoids (1-5) acted differently with both methods; namely, all the prenylated flavonoids strongly scavenged the ABTS radical (IC(50) < 10 microM), while they were inactive against the DPPH radical (IC(50) > 300 microM). Even though isolated 5,7,2',4',-tetrahydroxy-6,5'-diprenylflavanone (3) showed weak reducing power (746 mV) by cyclic voltammetry when compared to quercetin (394 mV), both had similar ABTS activity (IC(50) < 5 microM).     

Antimicrobial activity of isoprenoid-substituted xanthones from Cudrania cochinchinensis against vancomycin-resistant enterococci.

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against vancomycin-resistant enterococci (VRE). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against five VRE strains (VanA, VanB and VanC) (MICs = 1.56 microg/ml). Four xanthones, 1,3,7-trihydroxy-2-prenylxanthone, gerontoxanthone I, alvaxanthone and isoalvaxanthone, showed weaker antibacterial activity against these VREs (MICs = 3.13-6.25 microg/ml). .     

Water-Soluble Constituents of Cudrania tricuspidata （Carr.） Bur.

In order to find new structural and biologically active compounds, the constituents of the bark of Cudrania tricuspidata （Carr.） Bur. were investigated and a new 6-p-hydroxybenzyltaxifolin glucoside, named tricusposide （compound 1）, together with 16 known compounds, was isolated by solvent partition, macroporous adsorption resin AB-8, silica gel, Sephadex LH-20 chromatography. Using spectroscopic methods, the structures of the compounds were elucidated as 6-p-hydroxybenzyl taxifolin-7-O-β-D-glucoside （compound 1）, dihydroquerctin-7-O-β-D-glucoside （compound 2）, dihydrokaempferol-3-O-β-D-glucoside （compound 3）, dihydroquercetin （compound 4）, peonoside （compound 5）, sphaerobioside （compound 6）, quercimeritrin （compound 7）, genistein （compound 8）, aromadendrin （compound 9）, kaempferol （compound 10）, genistin （compound 11）, 3,4-dihydroxystyryl alcohol （compound 12）, sucrose （compound 13）, 1,3,5,6-tetrahydroxyxanthone （compound 14）, gericudranin E （compound 15）, gericudranin C （compound 16）, and orobol （compound 17）. Compounds 2-6, 8, 9, 12-14, and 17 were isolated from this genus for the first time.     

Antimicrobial activity of hydrophobic xanthones from Cudrania cochinchinensis against Bacillus subtilis and methicillin-resistant Staphylococcus aureus

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against B. subtilis (MIC = 1.56 microg/ml). Four xanthones, gerontoxanthone I, toxyloxanthone C, cudraxanthone S, and 1,3,7-trihydroxy-2-prenylxanthone, showed weak antibacterial activity against the bacterium (MICs = 3.13-6.25 microg/ml). These compounds also exhibited similar MIC values against methicillin-sensitive S. aureus, MRSAs, and Micrococcus luteus.     

Isolation, purification, and immunomodulatory activity in vitro of three polysaccharides from roots of Cudrania tricuspidata

Three water-soluble polysaccharides (CTPS-1A, CTPS-2B, and CTPS-3A) were obtained from roots of Cudrania tricuspidata (Carr.) Bur. in this study. The homogeneity of polysaccharides was determined, and the average molecular weight, ultraviolet, infrared, monosaccharide composition, and methylation analyses were carried out. Immunomodulatory activity assays in vitro showed that the three polysaccharides could directly stimulate the proliferation of mouse splenocytes alone or combining with concanavalin A or lipopolysaccharide. Furthermore, their stimulating activities were higher than that of the widely clinically used lentinan at optimal concentrations. CTPS-1A and CTPS-2B also enhanced the pinocytic activity of mouse peritoneal macrophages.     

Molecular Characterization and Phylogenetic Analysis of Two Novel Regio-specific Flavonoid Prenyltransferases from Morus alba and Cudrania tricuspidata

Prenylated flavonoids are attractive specialized metabolites with a wide range of biological activities and are distributed in several plant families. The prenylation catalyzed by prenyltransferases represents a Friedel-Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylated flavonoids and contributes to the structural diversity and biological activities of these compounds. To date, all identified plant flavonoid prenyltransferases (FPTs) have been identified in Leguminosae. In the present study two new FPTs, Morus alba isoliquiritigenin 3′-dimethylallyltransferase (MaIDT) and Cudrania tricuspidata isoliquiritigenin 3′-dimethylallyltransferase (CtIDT), were identified from moraceous plants M. alba and C. tricuspidata, respectively. MaIDT and CtIDT shared low levels of homology with the leguminous FPTs. MaIDT and CtIDT are predicted to be membrane-bound proteins with predicted transit peptides, seven transmembrane regions, and conserved functional domains that are similar to other homogentisate prenyltransferases. Recombinant MaIDT and CtIDT were able to regioselectively introduce dimethylallyl diphosphate into the A ring of three flavonoids with different skeleton types (chalcones, isoflavones, and flavones). Phylogenetic analysis revealed that MaIDT and CtIDT are distantly related to their homologs in Leguminosae, which suggests that FPTs in Moraceae and Leguminosae might have evolved independently. MaIDT and CtIDT represent the first two non-Leguminosae FPTs to be identified in plants and could thus lead to the identification of additional evolutionarily varied FPTs in other non-Leguminosae plants and could elucidate the biosyntheses of prenylated flavonoids in various plants. Furthermore, MaIDT and CtIDT might be used for regiospecific prenylation of flavonoids to produce bioactive compounds for potential therapeutic applications due to their high efficiency and catalytic promiscuity.     

Optimization of pancreatic lipase inhibition by Cudrania tricuspidata fruits using response surface methodology

The fruits of Cudrania tricuspidata (Carr.) Bur. (Moraceae) significantly inhibited pancreatic lipase, which plays a key role in fat absorption. Optimization of extraction conditions with minimum pancreatic lipase activity and maximum yield was determined using response surface methodology with three-level-three-factor Box–Behnken design (BBD). Regression analysis showed a good fit of the experimental data and the optimal condition was obtained as ethanol concentration, 74.5%; temperature 61.9 °C and extraction time, 13.5 h. The pancreatic lipase activity and extraction yield under optimal conditions were found to be 65.5% and 54.0%, respectively, which were well matched with the predicted value of 65.8% and 47.1%. Further fractionation of C. tricuspidata extract resulted in the isolation of compound 1, which was identified as 5,7,4′-trihydroxy-6,8-diprenylisoflavone. It inhibited pancreatic lipase activity with IC₅₀ value of 65.0 μM. HPLC analysis suggested positive correlation between pancreatic lipase inhibition and 5,7,4′-trihydroxy-6,8-diprenylisoflavone of C. tricuspidata fruits.     

Regio-selective prenylation of flavonoids by plant cell suspension cultures of Cudrania tricuspidata and Morus alba

The cell suspension cultures of Cudrania tricuspidata could regio-selectively prenylate chrysin (1) at C-8, while the cell suspension cultures of Morus alba could regio-selectively prenylate genistein (2), sophoricoside (3) and diosmetin (4) at C-6. Eight products (5–12) were isolated, and five of them (8–12) were new compounds. Additionally, the bioconversion of 1–4 using microsomes of the cell cultures was performed, and the results showed that the bioconversion patterns were identical to those using cell cultures. These investigations would provide an approach to the selective prenylation and structural diversification of flavonoids.     

Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension

A pharmacological inhibition of nitric oxide synthase (NOS) in rats for 4-6 weeks produces renal vasoconstriction, renal dysfunction, and severe hypertension. The present study was aimed at investigating whether Cudrania tricuspidata (C. tricuspidata) water extract ameliorates N(G)-Nitro-L-arginine methylester (L-NAME)-induced hypertension. Treatment of L-NAME (60 mg/L drinking water, 4 weeks) causes a sustained increase in systolic blood pressure (SBP). The concentration of plasma NO metabolites and NO/cGMP productions in the vascular tissues of the L-NAME-treated group were significantly reduced as compared with those in the control. C. tricuspidata water extract blocked increase of SBP in the L-NAME-treated group and restored SBP to normal level. Futhermore, C. tricuspidata water extract was able to preserve the vascular NO/cGMP production and plasma NO metabolites concentration. However, there are no changes in the expression of ecNOS and iNOS of thoracic aorta among the rats of control, L-NAME-treated group, and L-NAME and C. tricuspidata water extract co-treated group. The urinary sodium level, urine volume, and creatinine clearance were significantly higher in rats co-treated with C. tricuspidata water extract and L-NAME than in L-NAME-treated group. Taken together, these results suggest that C tricuspidata water extract prevents the increase of SBP in the L-NAME-induced hypertension that may have been caused by enhanced generation of vascular NO/cGMP.     

Cudrania tricuspidata Stem Extract Induces Apoptosis via the Extrinsic Pathway in SiHa Cervical Cancer Cells

The focus of this study is the anti-cancer effects of Cudrania tricuspidata stem (CTS) extract on cervical cancer cells. The effect of CTS on cell viability was investigated in HPV-positive cervical cancer cells and HaCaT human normal keratinocytes. CTS showed significant dose-dependent cytotoxic effects in cervical cancer cells. However, there was no cytotoxic effect of CTS on HaCaT keratinocytes at concentrations of 0.125–0.5 mg/mL. Based on this cytotoxic effect, we demonstrated that CTS induced apoptosis by down-regulating the E6 and E7 viral oncogenes. Apoptosis was detected by DAPI staining, annexin V-FITC/PI staining, cell cycle analysis, western blotting, RT-PCR, and JC-1 staining in SiHa cervical cancer cells. The mRNA expression levels of extrinsic pathway molecules such as Fas, death receptor 5 (DR5), and TNF-related apoptosis-inducing ligand (TRAIL) were increased by CTS. Furthermore, CTS treatment activated caspase-3/caspase-8 and cleavage of poly (ADP-ribose) polymerase (PARP). However, the mitochondrial membrane potential and expression levels of intrinsic pathway molecules such as Bcl-2, Bcl-xL, Bax, and cytochrome C were not modulated by CTS. Taken together, these results indicate that CTS induced apoptosis by activating the extrinsic pathway, but not the intrinsic pathway, in SiHa cervical cancer cells. These results suggest that CTS can be used as a modulating agent in cervical cancer.     

Bacterial neuraminidase inhibitory effects of prenylated isoflavones from roots of Flemingia philippinensis

Bacterial neuraminidase (NA) is one of the key enzymes involved in pathogenesis of inflammation during infection. The organic extract of the roots of Flemingia philippinensis showed high bacterial NA inhibitory activity with an IC₅₀ of around 5 μg/mL. Activity-guided separation of the methanol extract yielded nine prenylated isoflavones together with the novel species isoflavone (2) which was given the name flemingsin. Isolated prenylated isoflavones (1–9) were evaluated for NA inhibition and their IC₅₀ values were determined to range between 0.30 and 56.8 μM. The most potent inhibitor 4 (IC₅₀ = 300 nM, K_i = 130 nM) features a catechol motif in the B-ring and a furan in the A-ring. Structure–activity analysis also showed a 4-hydroxyl group within the B-ring was essential for NA inhibitory activity, because isoflavone (9) having protected 4-hydroxyl group was much less potent than its hydroxylated counterpart. All neuraminidase compounds screened were found to be reversible noncompetitive inhibitors. Furthermore, the most active NA inhibitors (1–9) were proven to be present in the native roots in high quantities by HPLC and LC-DAD-ESI/MS.