18ß-glycyrrhetinic acid derivative promotes proliferation, migration and aquaporin-3 expression in human dermal fibroblasts.

Licorice (Glycyrrhiza) species have been widely used as a traditional medicine and a natural sweetener in foods. The 18ß-glycyrrhetinic acid (18ß-GA) is a bioactive compound in licorice that exhibits potential anti-cancer, anti-inflammatory, and anti-microbial activities. Many synthesized derivatives of 18ß-GA have been reported to be cytotoxic and suggested for the treatment of malignant diseases. In this study, we explored the possible pharmacological roles of an 18ß-GA derivative in skin biology using primary human dermal fibroblasts and HaCaT keratinocytes as cell models. We found that this 18ß-GA derivative did not cause cell death, but significantly enhanced the proliferation of dermal fibroblasts and HaCaT keratinocytes. A scratch wound healing assay revealed that the 18ß-GA derivative promoted the migration of fibroblasts. Due to the important role of aquaporin-3 in cell migration and proliferation, we also investigated the expression of aquaporin-3 and found this compound up-regulated the expression of aquaporin-3 in dermal fibroblasts and HaCaT keratinocytes. In dermal fibroblasts, the 18ß-GA derivative induced the phosphorylation of Akt, ERK, and p38. The inhibitor of Akt predominantly suppressed the 18ß-GA derivative-induced expression of aquaporin-3. Collectively, this compound had a positive effect on the proliferation, migration, and aquaporin-3 expression of skin cells, implying its potential role in the treatment of skin diseases characterized by impaired wound healing or dermal defects.

Triterpenoids and an alkamide from Ganoderma tsugae.

Ganoderma tsugae is a medicinal mushroom. In a continual study on the bioactive constituents of this fungus, a new lanostanoid, 3ß-acetoxy-16α-hydroxy-24ξ-methyl-5α-lanosta-8,25-dien-21-oic acid, named tsugaric acid F (1) and a novel palmitamide, N-(3'α,4'ß-dihydroxy-2'ß-(hydroxymethyl)-1'ß-(cyclobutyl)palmitamide (2) were isolated and characterized from the fruit bodies of G. tsugae, and three novel seco-lanostanoids, 3,4-seco-8α,9α-epoxy-5α-lanosta-21-oic acid 3,4 lactone (5), 3,4-seco-5ß-lanosta-7,9(11),4(29)-trien-3,21-dioic acid-3-methyl ester (6), 3,4-seco-5ß-lanosta-7,9(11),4(29)-trien-3,21-dioic acid (7), and a known compound, 3-oxo-5α-lanosta-8-en-21-oic acid (4) were prepared from 3. The structures of new compounds, 1, 2, 5-7 were determined by spectroscopic methods. Compounds 1 and 4 showed inhibitory effects on xanthine oxidase (XO) with an IC50 values of 313.3 ± 80.0 and 43.9 ± 29.9 µM, respectively when 7 exhibited potent inhibitory effect on superoxide anion generation in rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB) with an IC50 values of 1.3 ± 0.2 µM. Compounds 4-7 showed weak cytotoxic activities against PC3 cells. These results indicated that 4 and 7 may be used as cancer chemopreventive agents.

HPLC-fingerprints and antioxidant constituents of Phyla nodiflora.

Phyla nodiflora is a creeping perennial herb, widely distributed in the most tropical and subtropical regions. It has been used as a folk medicine, herbal beverage, or folk cosmetic. For these usages, the development of a chemical quality control method of this plant is necessary. In the present study, ten compounds, namely, 3,7,4',5'-tetrahydroxy-3'-methoxyflavone (1), nodifloretin (2), 4'-hydroxywogonin (3), onopordin (4), cirsiliol (5), 5,7,8,4'-tetrahydroxy-3'-methoxyflavone (6), eupafolin (7), hispidulin (8), larycitrin (9), and ß-sitosterol were isolated from the methanolic extract of the aerial part of P. nodiflora (PNM) and their structures were identified by 1D-NMR comparing their spectra with the literature. The antioxidant activities of these compounds were evaluated by free radical scavenging activity and tyrosinase inhibitory effect in cell-free systems. Compounds 4, 5, and 7 showed strong antioxidant activity. To control the quality of P. nodiflora, a simple and reliable method of high-performance liquid chromatography combined with ultraviolet detector (HPLC-UV) was established for both the fingerprint analysis and the quantitative determination of two selected active compounds, onopordin (4) and eupafolin (7). Statistical analysis of the obtained data demonstrated that our method achieved the desired linearity, precision, and accuracy. The results indicated that the developed method can be used as a quality evaluation method for PNM.

Identification of kazinol Q, a natural product from Formosan plants, as an inhibitor of DNA methyltransferase.

DNA methylation plays a pivotal role in the epigenetic regulation of the transcription of a number of cancer-related genes, thereby representing an important target for cancer prevention and treatment. In our search for DNA methyltransferase (DNMT) inhibitors from Formosan plants, by screening against a library consisting of 12 structurally distinct natural products, we identified kazinol Q {4-[6-(1,1-dimethyl-allyl)-7-hydroxy-chroman-2-yl]-3,6-bis-(3-methyl-but-2-enyl)-benzene-1,2-diol} as an inhibitor of recombinant DNMT1 with IC50 of 7 µM. The effect of kazinol Q on DNMT inhibition was validated by its ability to reactivate the expression of a DNA methylation-silenced gene, E-cadherin, in MDA-MB-231 breast cancer cells. Moreover, kazinol Q suppressed the proliferation of MCF-7 breast and LNCaP prostate cancer cells, in part, through apoptosis induction. The role of DNMT1 inhibition in mediating kazinol Q's antiproliferative effect was supported by the protective effect of ectopic expression of DNMT1 on kazinol Q-induced cell death. Molecular modeling analysis suggests that kazinol Q inhibited DNMT activity by competing with cytosine binding, a mechanism similar to that described for (-)-epigallocatechin-3-gallate (EGCG). Relative to EGCG, kazinol Q exhibits several desirable features for drug development, including chemical stability and increased hydrophobicity, and might have therapeutic relevance to cancer treatment.

Xanthine oxidase inhibitory lanostanoids from Ganoderma tsugae.

Two new lanostanoids, 3α-acetoxy-22-oxo-5α-lanosta-8,24-dien-21-oic acid, named tsugaric acid D (1) and 16α-hydroxy-3-oxo-5α-lanosta-6,8,24(24(1))-trien-21-oic acid, named tsugaric acid E (2) were isolated from the fruit bodies of Ganoderma tsugae. The structures 1 and 2 were determined by spectroscopic methods. Compound 1 and known compounds 3 and 6 exhibited significant inhibitory effects on xanthine oxidase (XO) activity with an IC50 values of 90.2±24.2, 116.1±3.0, and 181.9±5.8 µM, respectively. Known compound 5 was able to protect human keratinocytes against damage induced by UVB light, which showed 5 could protect keratinocytes from photodamage. The 1 and 5 µM 1 combined with 5 µM cisplatin, respectively, enhanced the cytotoxicity induced by cisplatin. It suggested that 1 and 5 µM 1 combined with low dose of cisplatin may enhance the therapeutic efficacy of cisplatin and reduce side effect and cisplatin resistant.

Cytotoxic and antioxidant constituents from Garcinia subelliptica.

Two new triterpenoids, garcinielliptones Q (1) and S (3), and a new phloroglucinol, garcinielliptone R (2), were isolated from the seed of Garcinia subelliptica. Their structures were established by analysis of their spectroscopic data. Phloroglucinol, garcinielliptone FC (4) from this plant exhibited a significant increase of antiproliferative effect, while 4 combined with cisplatin significantly caused decrease of cell inhibition induced by cisplatin in NTUB1. Exposure of NTUB1 cells to 4 cotreated with cisplatin for significantly decreased the amount of reactive oxygen species (ROS) than that of the total amount generated by 4 and cisplatin. These results suggested that 4 could protect the cisplatin toxicity through reduction of ROS in NTUB1. Phloroglucinols, garcinielliptones, A (5) and F (7), and garsubelline A (6), from this plant, revealed ABTS radical cation scavenging activity and 5 displayed an inhibitory effect on xanthine oxidase. These finding showed that 5-7 may be used as antioxidants.

Xanthine oxidase inhibitory triterpenoid and phloroglucinol from guttiferaceous plants inhibit growth and induced apoptosis in human NTUB1 cells through a ROS-dependent mechanism.

A known triterpenoid, ß-amyrin (1), and a known and a new phloroglucinol, cohulupone (2) and garcinielliptone P (3), were isolated from the pericarp and heartwood and seed of Garcinia subelliptica, respectively. A new xanthonolignoid, hyperielliptone HF (4), was isolated from the heartwood of Hypericum geminiflorum. The new compounds were established by analysis of their spectroscopic data. Compounds 1-3 showed an inhibitory effect on xanthine oxidase (XO). Treatment of NTUB1, a human bladder cancer cell, with 1 or 1 cotreated with cisplatin for 24 h resulted in a decreased viability of cells. Exposure of NTUB1 to 1 or 1 cotreated with cisplatin for 24 h significantly increased the level of production of reactive oxygen species (ROS). Flow cytometric analysis indicated that treatment of NTUB1 with 1 or 1 cotreated with cisplatin led to the cell cycle arrest, accompanied by an increase in the extent of apoptotic cell death in 1 or 1 combined with cisplatin-treated NTUB1 after 24 h. These data suggested that the presentation of cell cycle arrest and apoptosis in 1 or 1 combined with cisplatin-treated NTUB1 for 24 h was mediated through an increased amount of ROS in cells exposed to 1 or 1 cotreated with cisplatin.

New diterpenoids and cytotoxic and anti-inflammatory diterpenoids from Amentotaxus formosana.

Two new abietane diterpenoids, ramentoxide (1) and ramentoxidone (2) and a new icetexane diterpenoid, amentonone (3) were isolated from the barks of Amentotaxus formosana. The structures of 1-3 were determined by spectroscopic methods. Known compounds brevitaxin (4), and (+)-ferruginol (5) and ent-kaur-16-en-15-one (6) isolated from this plant revealed potent cytotoxic activity against human breast adenocarcinoma cells, MCF-7 cells with an IC(50) value of 0.08 ± 0.05 µg/mL, and significant anti-inflammatory activities, respectively.

Terpenoids induce cell cycle arrest and apoptosis from the stems of Celastrus kusanoi associated with reactive oxygen species.

Bioguided fractionation of the CHCl(3) extracts obtained from Celastrus kusanoi stems led to isolation of two new terpenoids, 3beta-hydroxy-11,14-oxo-abieta-8,12-diene (1) and 3beta-trans-(3,4-dihydroxycinnamoyloxy)-11alpha-methoxy-12-ursene (2), and four known compounds characterized by spectroscopic methods. Compounds 1 and 2 and known triterpenoid erythrodiol (3) exhibited cytotoxic activity against bladder cancer cells (NTUB1) with IC(50) values of 58.2 +/- 2.3, 160.1 +/- 60.9, and 18.3 +/- 0.5 microM, respectively. Exposure of NTUB1 to 3 (5 and 10 microM) for 24 h significantly increased the level of production of reactive oxygen species (ROS). Flow cytometric analysis showed that treatment of NTUB1 with 3 led to the cell cycle arrest at G0/G1 accompanied by an increase in the extent of apoptotic cell death after 24 h. These data suggest that the presentation of G1 phase arrest and apoptosis in 3-treated NTUB1 for 24 h was mediated through an increased amount of ROS in cells exposed to 3.

Involvement of caspases and apoptosis-inducing factor in bufotalin-induced apoptosis of Hep 3B cells.

Bufotalin is one of the bufadienolides isolated from Formosan Ch'an Su, which is made of the skin and parotid glands of toads. Ingestion of toad venom results in severe morbidity and high mortality. Although Ch'an Su is clinically toxic, it has been used as an important traditional Chinese medicine for heart failure and pains. In this study, bufotalin-induced apoptosis in human hepatocellular carcinoma Hep 3B cells was investigated. The results indicate that externalization of phosphatidylserine, accumulation of sub-G(1) cells, fragmentation of DNA, and formation of apoptotic bodies were observed in bufotalin-treated Hep 3B cells. The signaling pathway might be via the activation of caspase-8, increase in mitochondrial tBid, disruption of mitochondrial membrane potential, and translocation of apoptosis-inducing factor (AIF). Active caspase-8 might activate caspase-9 and caspase-3 leading to the cleavage of nuclear PARP. Presence of AIF and cleaved PARP in the nuclei might lead to DNA fragmentation. Caspase-8 inhibitor (Z-IETD) or wide-ranging caspase inhibitor (Z-VAD) significantly suppressed the bufotalin-induced apoptosis, while the anti-Fas neutralization antibody had no effect. These data suggest that bufotalin-induced apoptosis in Hep 3B cells might involve caspases and AIF.

Phloroglucinols with antioxidant activity and xanthonolignoids from the heartwood of Hypericum geminiflorum.

A new phloroglucinol, hyperielliptone HA (1/1a), a new spirophloroglucinol possessing an unprecedented skeleton, hyperielliptone HB (2/2a), and two new xanthonolignoids, hyperielliptones HC (3) and HD (4), were isolated from the heartwood of Hypericum geminiflorum. Compounds 1/1a and 2/2a were obtained as tautomeric pairs. The structures and relative configurations of these compounds were elucidated by spectroscopic methods. In biological testing, compound 2/ 2a revealed significant inhibition of oxidative DNA damage and an inhibitory effect on xanthine oxidase.

Phloroglucinols with prooxidant activity from Garcinia subelliptica.

A new phloroglucinol, garcinielliptone HF ( 1), possessing an unprecedented skeleton, and the tautomeric pair of garcinielliptone FC ( 2/ 2a) were isolated from the heartwood and pericarp of Garcinia subelliptica, respectively. Their structures, including relative configurations, were elucidated by means of spectroscopic methods. The ability of compounds 1 and 2/ 2a to induce DNA-cleavage activity was examined using supercoiled plasmid pBR322 DNA. In the presence of Cu(II), compounds 1 and 2/ 2a caused significant breakage of pBR322 DNA. The involvement of H2O2 and O2 (*-), and H2O2, O2 (*-), and OH (*) in 1- and 2/ 2a-mediated scission, respectively, was established by inhibition or no protection of DNA breakage by various oxygen radical scavengers. Thus, in the presence Cu(II), 1 and 2/ 2a may show a prooxidant effect on DNA and induce cell death.

Flavonoids with DNA strand-scission activity from Rhus javanica var. roxburghiana.

The flavonoids isolated from the stems of Rhus javanica var. roxburghiana, taxifolin (1), fisetin (2), fustin (3), 3,7,4'-trihydroxyflavanone (4) and 3,7,4'-trihydroxyflavone (5) caused breakage of supercoiled plasmid pBR322 DNA in the presence of Cu(II). Cu(I) was shown to be an essential intermediate by using the Cu(I)-specific sequestering reagent neocuproine. The Cu(II)-mediated DNA scissions induced by 1, 2, 3 and 5 were inhibited by the addition of catalase and exhibited DNA strand break by the addition of KI and superoxide dimutase (SOD), while in the Cu(II)-mediated DNA scissions induced by 4 was inhibited by the addition of KI, SOD, and catalase. It is concluded that 1, 2, 3, and 5 can induce H2O2 and superoxide anion, while 4 can induce OH* and H2O2 and subsequent oxidative damage of DNA in the presence of Cu(II).

Potent suppressive effects of 3-O-methylquercetin 5,7,3',4'-O-tetraacetate on ovalbumin-induced airway hyperresponsiveness.

We investigated the suppressive effects of 3-O-methylquercetin 5,7,3',4'- O-tetraacetate (QMTA), a more-potent phosphodiesterase (PDE)3/4 inhibitor than quercetin 3-O-methyl ether (3-MQ), which has been reported to have the potential for treating asthma, against ovalbumin (OVA)-induced airway hyperresponsiveness (AHR). The IC50 value of QMTA for PDE3 was significantly less than that for PDE4. According to the Lineweaver-Burk analysis, QMTA (1-10 microM) competitively inhibited PDE3 and PDE4 activities. The Ki values were 0.9+/-0.3 (n=5) and 3.9+/-0.5 (n=5) microM, respectively, which significantly differed from each other, suggesting that QMTA has higher affinity for PDE3 than for PDE4. QMTA (3-10 microM) concentration-dependently relaxed the baseline level, and significantly inhibited cumulative OVA (10-100 microg/mL)-induced contractions in isolated sensitized guinea pig trachealis suggesting that QMTA has bronchodilator and inhibiting effects on mast cell degranulation. After the secondary challenge, the AHR was measured in unrestrained OVA-sensitized mice, with nebulized methacholine (MCh, 6.25-50 mg/mL), by barometric plethysmography using a whole-body plethysmograph. In the present results, QMTA (3-10 micromol/kg, I. P.) dose-dependently attenuated the enhanced pause (Penh) value induced by MCh (25-50 mg/mL). QMTA (3-10 micromol/kg, I. P.) also significantly inhibited total inflammatory cells, macrophages, neutrophils, lymphocytes, and eosinophils in BALF after determination of Penh values. It also significantly suppressed the release of interleukin (IL)-2, IL-4, IL-5, IFN-gamma, and TNF-alpha, with the exception that 3 micromol/kg QMTA did not suppress the releases of IL-5. QMTA even at 1 micromol/kg significantly inhibited eosinophils, IL-2, and TNF-alpha. In conclusion, our results strongly suggest that QMTA has greater potential than 3-MQ for the treatment of asthma.

Cytotoxic and apoptotic effects of prenylflavonoid artonin B in human acute lymphoblastic leukemia cells.

AIM: To investigate the anticancer effects and molecular mechanism of artonin B on the human acute lymphoblastic leukemia CCRF-CEM cells compared with other prenylflavonoid compounds. METHODS: The effects of four prenylflavonoids on the growth of CCRF-CEM and HaCa cells were studied by 3-(4,5)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Apoptosis were detected through Hoechst 33258 staining. The effect of artonin B on the cell cycle of CCRF-CEM cells were studied by propidium iodide method. The change in mitochondrial membrane potential was detected by rohdamine 123 staining. The cytochrome c release and caspase 3 activity were checked by immunoassay kits, respectively. The expression of Bcl-2 family proteins was detected by Western blot. RESULTS: Our data revealed that artonin B strongly induced human CCRF-CEM leukemia cell death in a dose- and time-dependent manner by MTT assay, but not on normal epithelia cells (HaCa cells). Artonin B-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by Hoechst 33258 staining. The induction of human CCRF-CEM leukemia cancer cell death was caused by an induction of apoptosis through mitochondrial membrane potential change, cytochrome c release, sub-G1 proportion increase, downregulation of Bcl-2 expression, upregulation of Bax and Bak expression and activation of caspase 3 pathways. CONCLUSION: These results clearly demonstrated that artonin B is able to inhibit proliferation by induction of hypoploid cells and cell apoptosis. Moreover, the anticancer effects of artonin B were related to mitochondrial pathway and caspase 3 activation in human CCRF-CEM leukemia cells.

Antiplatelet prenylflavonoids from Artocarpus communis.

Four flavonoids, dihydroartomunoxanthone (1), artomunoisoxanthone (2), cyclocomunomethonol (3) and artomunoflavanone (4), together with three known compounds, artochamins B (5), D and artocommunol CC (6) were isolated from the cortex of the roots of Artocarpus communis. The structures of 1-4 were determined by spectroscopic methods. The antiplatelet effects of the flavonoids, 1-3, 5 and 6 on human platelet-rich plasma (PRP) were evaluated. Of the compounds tested in human PRP, compounds 1, 5 and 6 showed significant inhibition of secondary aggregation induced by adrenaline. It is concluded that the antiplatelet effect of 1, 5 and 6 is mainly owing to an inhibitory effect on thromboxane formation.

Mechanisms of suppression of nitric oxide production by 3-O-methylquercetin in RAW 264.7 cells.

Rhamnus nakaharai Hayata (Rhamnaceae) is used as a folk medicine in Taiwan for treating constipation, inflammation, tumors, and asthma. 3-O-Methylquercetin (3-MQ), a main constituent of the plant, has been reported to have potential for use in the treatment of asthma. The mechanisms of anti-inflammation of 3-MQ are still unclear. Nitric oxide (NO) production induced by lipopolysaccharide (LPS) through iNOS expression in RAW 264.7 cells, a mouse macrophage cell line, may reflect the degree of inflammation and may provide a measure for assessing the effect of drugs on the inflammatory process. Therefore, we were interested in investigating the mechanisms of suppression of NO production by 3-MQ in RAW 264.7 cells. 3-MQ (1-10 microM) concentration-dependently inhibited LPS (100 ng/mL)-induced NO production in RAW 264.7 cells. The IC(50) value was calculated to be 4.23 microM. 3-MQ (1-10 microM) significantly and concentration-dependently inhibited LPS (100 ng/mL)-induced iNOS protein and mRNA expressions in cells. The IC(50) values were calculated to be 4.36 and 6.53 microM, respectively. There was no significant difference among these three IC(50) values of 3-MQ. In conclusion, 3-MQ may exert its anti-inflammatory effect through the inhibition of iNOS DNA transcription.

Cytotoxic prenylflavonoids from Artocarpus elasticus.

Five new prenylated flavonoids, artelastoheterol (1), artelasticinol (2), cycloartelastoxanthone (3), artelastoxanthone (4), and cycloartelastoxanthendiol (5), along with five known compounds, were isolated from the root bark of Artocarpus elasticus. The structures of 1-5 were elucidated by spectroscopic methods and through comparison with data reported in the literature. The previously known compound artonol A (6) exhibited cytotoxic activity against the A549 human cancer cell line, with an ED50 value of 1.1 microg/mL.

Constituents of the pericarp of Garcinia subelliptica.

A new benzophenone, garcinielliptone FA (1), and a new benzoylphloroglucinol, garcinielliptone FB (2), along with six known compounds, were isolated from the pericarp of Garcinia subelliptica. The structures and relative configurations of 1 and 2 were elucidated by spectroscopic methods and supported by computer-generated molecular modeling. Compound 2 exhibited cytotoxic activity against several human cancer cell lines.

New phenylpropane and anti-inflammatory diterpene derivatives from Amentotaxus formosana.

One new diterpene, 8(14),15-sandaracopimaradiene-2alpha,3beta,18-triol (1), two new phenylpropane derivatives, i.e., (E)-methyl 2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoate (2) and (E)-2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoic acid (3), and two known diterpenes, ent-8(14),15-sandaracopimaradiene-2alpha,18-diol (4) and 8(14),15-sandaracopimaradiene-2alpha,18,19-triol (5), were isolated from the heartwoods and barks of Amentotaxus formosana, respectively. The anti-inflammatory activity of the diterpenes 1, 4, and 5 was assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, macrophages, and microglial cells. Compounds 1, 4, and 5 showed significant concentration-dependent inhibitory effects on the release of beta-glucuronidase from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC50 values of 5.5 +/- 1.8, 8.4 +/- 2.9 and 19.2 +/- 3.3 microM, respectively. Compounds 1 and 5 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB and phorbol 12-myristate 13-acetate (PMA) with IC50 values of 12.6 +/- 1.2 and 9.4 +/- 1.7, and 10.7 +/- 3.3 and 12.9 +/- 0.9 microM, respectively.