Study on the absolute configuration and biological activity of rotenoids from the leaves and twigs of Millettia pyrrhocarpa Mattapha, Forest & Hawkins, sp. Nov.

BACKGROUND: M. pyrrhocarpa is a new plant in the Fabaceae: Faboideae family that is found in Thailand. A literature search revealed that the Milletia genus is rich in bioactive compounds possessing a wide range of biological activities. In this study, we aimed to isolate novel bioactive compounds and to study their bioactivities. METHODS: The hexane, ethyl acetate, and methanol extracts from the leaves and twigs of M. pyrrhocarpa were isolated and purified using chromatography techniques. These extracts and pure compounds were tested in vitro for their inhibitory activities against nine strains of bacteria, as well as their anti-HIV-1 virus activity and cytotoxicity against eight cancer cell lines. RESULTS: Three rotenoids, named 6aS, 12aS, 12S-elliptinol (1), 6aS, 12aS, 12S-munduserol (2), dehydromunduserone (3), and crude extracts were evaluated for antibacterial, anti-HIV, and cytotoxic activities. It was found that compounds 1-3 inhibited the growth of nine strains of bacteria, and the best MIC/MBC values were obtained at 3/ > 3 mg/mL. The hexane extract showed anti-HIV-1 RT with the highest %inhibition at 81.27 at 200 mg/mL, while 6aS, 12aS, 12S-elliptinol (1) reduced syncytium formation in 1A2 cells with a maximum EC50 value of 4.48 µM. Furthermore, 6aS, 12aS, 12S-elliptinol (1) showed cytotoxicity against A549 and Hep G2 cells with maximum ED50 values of 2.27 and 3.94 µg/mL. CONCLUSION: This study led to the isolation of constituents with potential for medicinal application, providing compounds (1-3) as lead compounds against nine strains of bacteria. The hexane extract showed the highest %inhibition of HIV-1 virus, Compound 1 showed the best EC50 in reducing syncytium formation in 1A2 cells, and it also showed the best ED50 against human lung adenocarcinoma (A549) and human hepatocellular carcinoma (Hep G2). The isolated compounds from M. pyrrhocarpa offered significant potential for future medicinal application studies.

Dihydrochalcone Derivative Induces Breast Cancer Cell Apoptosis via Intrinsic, Extrinsic, and ER Stress Pathways but Abolishes EGFR/MAPK Pathway.

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4',6'-dihydroxy-2',4-dimethoxy-5'-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2',6'-dihydroxy-4,4'-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3'-dihexyloxacarbocyanine iodide, DiOC6, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

Alkaloids from Stephania venosa as Chemo-Sensitizers in SKOV3 Ovarian Cancer Cells via Akt/NF-κB Signaling.

Crebanine (CN), tetrahydropalmatine (THP), O-methylbulbocapnine (OMBC) and N-methyl tetrahydropalmatine (NMTHP) are isoquinoline derived natural alkaloids isolated from tubers of Stephania venosa. We investigated chemo-sensitizing effects of these alkaloids in ovarian cancer cells and evaluated underlying molecular mechanisms involved in chemo-sensitivity. Detection of cell apoptosis was evaluated by using flow cytometry. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chou-Talalay median effect principle was used to evaluate potential drug interactions. Protein analyses were performed on ovarian carcinoma cells using Western blotting upon treatment with anticancer drug and alkaloids. Aporphine alkaloids, such as CN and OMBC, enhanced cisplatin sensitivity in intrinsic cisplatin resistant SKOV3 cells, but not in cisplatin sensitive A2780 cells. Protoberberine alkaloids, such as THP and NMTHP, had no synergistic effect on cisplatin sensitivity in either cell line. Chemo-sensitizing effects of CN and OMBC in SKOV3 cells were mediated via activating apoptosis-induced cell death through caspase-3, -8 and cleaved poly ADP-ribose polymerase (PARP) and via inhibiting anti-apopotic and survival protein expression, such as Bcl-xL, Baculoviral IAP repeat-containing protein 3 (cIAP-2), survivin and interleukin (IL) -6. Cisplatin stimulated protein kinase B (Akt) and nuclear factor-kappaB (NF-κB) signaling pathways, but not mitogen-activated protein kinase (MAPK), activator protein 1 (AP-1) and signal transducer and activator of transcription 3 (STAT3) in SKOV3 cells. Akt/NF-κB signaling was blocked by CN and OMBC leading to increased sensitization to cisplatin. These findings demonstrate that CN and OMBC sensitizes SKOV3 cells to cisplatin via inhibition of Akt/NF-κB signaling and the down regulation of NF-κB mediated gene products. Our results suggest that alkaloids obtained from S. venosa could be used as chemo-sensitizers in ovarian cancer to sensitize and minimize the dose related toxicity of platinum-based chemotherapeutic drugs.

Kuguacin J isolated from bitter melon leaves modulates paclitaxel sensitivity in drug-resistant human ovarian cancer cells.

We previously reported the multidrug resistance-reversing ability of kuguacin J (KJ) in cervical cancer cells via the inhibition of P-glycoprotein (P-gp) function. This study investigated whether KJ could promote cisplatin- and paclitaxel (PTX)-induced cancer cell death in drug-resistance human ovarian cancer cells (SKOV3). Cytotoxicity testing showed that SKOV3 was more resistant to cisplatin and PTX compared to drug-sensitive human ovarian cancer cells (A2780). The cytotoxicity of PTX was significantly increased in SKOV3 cells when co-treated with KJ. We found that enhancement of PTX toxicity in the cells was not related to P-gp inhibition. To elucidate the mechanism by which KJ increases PTX sensitivity, the expression of cell death involving proteins was analyzed by Western blot analysis. The results showed that PTX treatment increased the level of an anti-apoptotic protein, survivin, which may be involved in drug resistance in SKOV3. The co-treatment with PTX and KJ dramatically decreased the level of survivin and markedly induced cleavage of PARP and caspase-3, which are apoptotic-induced molecules. These findings may support the use of KJ as an effective chemosensitizer in combination with conventional chemotherapy to promote PTX sensitization in ovarian cancer patients.

Pinocembrin attenuates gentamicin-induced nephrotoxicity in rats.

Oxidative stress mediated apoptosis of renal tubular cells is a major pathology of gentamicin-induced nephrotoxicity, which is one of the prevailing causes of acute renal failure. Pinocembrin is a major flavonoid found in rhizomes of fingerroot (Boesenbergia pandurata). It has pharmacological and biological activities including antimicrobial, anti-inflammatory, and antioxidant effects. Preclinical studies have suggested that pinocembrin protects rat brain and heart against oxidation and apoptosis induced by ischemia-reperfusion. The aim of the current study was to investigate the mechanisms of renoprotection elicited by pinocembrin in gentamicin-induced nephrotoxicity. Nephrotoxicity was induced in rats by intraperitoneal injection (i.p.) of gentamicin, and pinocembrin was administered via i.p. 30 min before gentamicin treatment for 10 days. Gentamicin-induced nephrotoxicity was indicated by the reduced renal function and renal Oat3 function and expression. Gentamicin treatment also stimulated Nrf2, HO-1, and NQO1, as well as the pro-apoptotic proteins Bax and caspase-3, concomitant with the attenuation of Bcl-XL expression in the renal cortical tissues. Pinocembrin pretreatment improved renal function and renal Oat3 function and reduced oxidative stress and apoptotic conditions. These findings indicate that pinocembrin has a protective effect against gentamicin-induced nephrotoxicity, which may be due in part to its antioxidant and anti-apoptotic effects, subsequently leading to improved renal function.

Enterocarpam-III induces human liver and breast cancer cell apoptosis via mitochondrial and caspase-9 activation.

An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6- tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells. Cell viability was determined by MTT assay to determine IC10, IC20 and IC50 levels, reactive oxygen species (ROS) production with 2',7'-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC6) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the IC50 levels of 26.0±4.45 and 51.3±2.05 µM, respectively. For murine normal fibroblast NIH3T3 cells, the IC50 concentration was 81.3±10.1 µM. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.

Cytotoxic aporphine alkaloids from leaves and twigs of Pseuduvaria trimera (Craib).

From ethyl acetate-methanol extracts of leaves and twigs of Pseuduvaria trimera a new aporphine alkaloid; 8-hydroxy-1,4,5-trimethoxy-7-oxoaporphine or 8-hydroxyartabonatine C (1) was isolated, together with the known 1,2,3-trimethoxy-4,5-dioxo-6a,7-dehydroaporphine (ouregidione, 2). Their structures were elucidated by a combination of spectral methods; mainly 2D NMR; IR and MS. Compounds 1 and 2 exhibited cytotoxic activity with IC50 values of 26.36±5.18 µM and 12.88±2.49 µM, respectively, for human hepatocellular carcinoma HepG2 cells, and 64.75±4.45 and 67.06±3.5 µM, respectively, for human breast cancer MDA-MB231 cells. Both compounds displayed anti-cancer activity but less than that of doxorubicin; a conventional chemotherapeutic drug, the IC50 levels of which were 2.21±1.72 and 1.83±0.09 µM for HepG2 and MDA-MB231 cells, respectively.

Sawtehtetronenin from Goniothalamus sawtehii and its cytotoxicity.

A new acetogenin has been isolated from the ethyl acetate extract of leaves and twigs of G. sawtehii (Annonaceae). The structure of compound 1 was identified as sawtehtetronenin on the basis of spectral evidence (UV, IR, MS and 1H, and 13C NMR) and by comparison with related compounds. Sawtehtetronenin was found to be cytotoxic to human hepatocellular carcinoma HepG2 and breast cancer MDA-MB231 cells with IC50 values of 79.3 ± 11.9 µM and 108.1 ± 1.5 µM, respectively. Compound 1 was less toxic to both cell lines when compared with camptothecin, a chemotherapeutic drug.

Dihydrochalcones with antiinflammatory activity from leaves and twigs of Cyathostemma argenteum.

A new dihydrochalcone derivative, 4',6'-dihydroxy-2',4-dimethoxy-5'-(2''-hydroxybenzyl)dihydrochalcone (1) and one known dihydrochalcone, 4',6'-dihydroxy-2',4- dimethoxydihydrochalcone (2) were isolated from leaves and twigs of Cyathostemma argenteum. Their structures were established by spectral methods, mainly 2D NMR spectroscopic techniques, which involved combined applications of DEPT, COSY, HMQC and HMBC. The molecular structure of 1 was also confirmed by single crystal X-ray diffraction. The test compounds 1 and 2 displayed significant inhibitory activity at a dose of 1 mg/ear on edema formation at all determination times, with similar intensity as phenylbutazone.

Antiproliferative effect of alkaloids via cell cycle arrest from Pseuduvaria rugosa.

CONTEXT: Pseuduvaria rugosa (Blume) Merr. (Annonacaea) grows widely in the south and southeast regions of Thailand. Preliminary screening for biological activities revealed that crude hexane, ethyl acetate, and acetone extracts from mixtures of leaves and twigs of P. rugosa showed cytotoxicity. OBJECTIVE: Chemical constituents and their antiproliferative activity in K562, U937, and HL-60 human leukemic cell lines from P. rugosa were performed for the first time. MATERIALS AND METHODS: The isolated compounds were obtained from chromatographic separation. The structures were established by spectroscopic techniques including IR, UV, NMR together with 2D NMR (HMBC, COSY, and NOE) and MS. The K562, U937, and HL-60 cell lines were treated with isolated aporphine alkaloids (0-100 µg/mL) and cell viability was measured with the MTT assay. Cell cycle analysis was performed using propidium iodide (PI) based staining methods. RESULTS: Two known aporphine alkaloids, 1,2,3-trimethoxy-5-oxonoraporphine (1) and ouregidione (2) were isolated. Treatment of the cells with compounds 1 and 2 at a concentration of 100 µg/mL for 72 h reduced the viability of K562, U937, and HL-60 cell lines to 63 and 64, 38 and 66, and 49 and 64%, respectively. In addition, compounds 1 and 2, at a concentration of 100 µg/mL, exposed to U937 and HL-60 cell lines showed cell cycle arrest. The U937 cell line treated with compounds 1 and 2 increased significantly the proportion of the cell in S phase, whereas the HL-60 cell line-induced G2/M and G1 phase, respectively. DISCUSSION AND CONCLUSION: The results showed that 1,2,3-trimethoxy-5-oxonoraporphine and ouregidione-induced cytotoxicity with HL-60, U937, and K562 cells where 1,2,3-trimethoxy-5-oxonoraporphine was more active than ouregidione.

Aristolactam-type alkaloids from Orophea enterocarpa and their cytotoxicities.

A new aristolactam, named enterocarpam-III (10-amino-2,3,4,6-tetramethoxy phenanthrene-1-carboxylic acid lactam, 1) together with the known alkaloid stigmalactam (2), were isolated from Orophea enterocarpa. Their structures were elucidated on the basis of interpretation of their spectroscopic data. Compounds 1 and 2 exhibited significant cytotoxicities against human colon adenocarcinoma (HCT15) cell line with IC(50) values of 1.68 and 1.32 µM, respectively.

Polyoxypregnane glycoside from Dregea volubilis extract inhibits IL-1ß-induced expression of matrix metalloproteinase via activation of NF-κB in human chondrocytes.

Interleukin-1ß (IL-1ß) induces the expression of matrix metalloproteinases (MMPs) implicated in cartilage and joint degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). Polyoxypregnane glycoside (PPG), active compound was identified from Dregea volubilis extract by chemical analysis, shown to exert chondroprotective effects in cartilage explant models. However, no studies have been undertaken for the molecular investigation of whether PPG constituents protect the human articular chondrocyte (HAC). In the present studies, HAC was co-treated with IL-1ß and PPG. The expression of MMPs, type II collagen, phosphorylation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathway were determined by Western immunoblotting. PPG (6.25-25 µM) decreased the IL-1ß-induced HA release from chondrocyte to culture medium. The mode of action of PPG was likely mediated through inhibiting expression of MMP-1, -3 and -13 in the medium, which was associated with the inhibition of mRNA expression. PPG had no effect on IL-1ß-induced phosphorylation of MAPK pathway. Conversely, PPG decreased phosphorylation of IκB kinase and IκBα degradation. Taken together, these results indicate that PPG may inhibit cartilage degradation in OA and may also be used as nutritional supplement for maintaining joint integrity and function.

New clerodane diterpenoid from the bulbils of Dioscorea bulbifera.

A new clerodane diterpenoid has been isolated from the acetone extract of bulbils of Dioscorea bulbifera. The structure of compound 1 was established as 15,16-epoxy-6alpha-O-acety1-8beta-hydroxy- 9-nor-clero-13(16),14-diene-17,12;18,2-diolide on the basis of comprehensive spectroscopic techniques.

4'-Hydroxycinnamaldehyde from Alpinia galanga (Linn.) induces human leukemic cell apoptosis via mitochondrial and endoplasmic reticulum stress pathways.

Rhizomes of Alpinia galanga (Linn.) or 'Kha' in Thai are used in food and as folk medicine in South and Southeast Asia. The aims of this study were to identify the mechanism of cell death of human leukemic HL-60 and U937 cells induced by 4'-hydroxycinnamaldehyde (4'-HCA) isolated from A. galanga. 4'-HCA was cytotoxic to both cell lines in a dose-dependent manner (p<0.05) as demonstrated by MTT assay. Apoptosis induced by 4'-HCA was demonstrated by a variety of methods: visualization of propidium iodide (PI)-stained cells under fluorescence microscope, detection of subdiploid cells by PI-staining and flow cytometry, and assay of active caspase-3 using a specific fluorogenic substrate. 4'-HCA-treated cells (10 and 50 µg/ml for 4 h) showed significant increase in reactive oxygen species production and decreased mitochondrial transmembrane potential as detected by dichlorohydrofluorescein diacetate and 3,3'-dihexyloxacarbocyanine iodide respectively, together with flow cytometry. The apoptotic death involved cytochrome c release, increase in Bax level and concomitant decreases in levels of Bcl-2 and Bcl-xL (using Western blotting), and elevation in cytosolic and mitochondrial Ca²âº contents (using compartment-specific fluorescent Ca2+ dyes). These results indicate that 4'-HCA induces apoptosis of human leukemic cell through a combination of mitochondrial and ER stress pathways.

Alpinia galanga extracts downregulate interleukin-1ß-induced matrix metalloproteinases expression in human synovial fibroblasts.

Alpinia galanga has been used as alternative medicine for anti-rheumatic activities. However, the precise action of the extract on arthritic diseases is not yet fully understood. In this study, we investigated the effects of A. galanga extracts on the expression of genes involved in catabolic activities in an interleukin-1ß (IL-1ß)-induced human synovial fibroblast as an inflammatory model. Confluent primary human synovial fibroblasts were treated for 24 h with A. galanga hexane extracts in the presence of recombinant human IL-1ß. MMPs in the culture medium were monitored by gelatin zymography. Total RNA was isolated from the cell lysate and analyzed via semi-quantitative RT-PCR. After treatment with A. galanga extracts, MMP-2 activity in the culture medium was significantly reduced. In addition, MMP-1, MMP-3, MMP-13, and Cox-2 expression were downregulated. These data suggest that the decrease of gene expression and production of MMPs in synovial fibroblasts against inflammatory stimuli could be due to the effects of the A. galanga extracts. Therefore, A. galanga extracts might be a promising therapeutic agent for arthritis.

Ginger extract inhibits human telomerase reverse transcriptase and c-Myc expression in A549 lung cancer cells.

The rhizome of ginger (Zingiber officinale Roscoe) has been reputed to have many curative properties in traditional medicine, and recent publications have also shown that many agents in ginger possess anticancer properties. Here we show that the ethyl acetate fraction of ginger extract can inhibit the expression of the two prominent molecular targets of cancer, the human telomerase reverse transcriptase (hTERT) and c-Myc, in A549 lung cancer cells in a time- and concentration-dependent manner. The treated cells exhibited diminished telomerase activity because of reduced protein production rather than direct inhibition of telomerase. The reduction of hTERT expression coincided with the reduction of c-Myc expression, which is one of the hTERT transcription factors; thus, the reduction in hTERT expression might be due in part to the decrease of c-Myc. As both telomerase inhibition and Myc inhibition are cancer-specific targets for cancer therapy, ginger extract might prove to be beneficial as a complementary agent in cancer prevention and maintenance therapy.

A new azafluorenone from the roots of Polyalthia cerasoides and its biological activity.

Chromatographic separation of the ethyl acetate extract of roots of Polyalthia cerasoides has led to the isolation of the new compound, 6,8-dihydroxy-7-methoxy-1-methyl-azafluorenone. This compound exhibited potent cytotoxic activities with IC50 values in the range of 2.64-3.58 microg x mL(-1) for A549, GLC4 and GLC4/Adr cells, but was not recognized by ABCC1/MRP1 protein. The compound also showed very strong inhibition of M. tuberculosis using a broth microdilution method, with an MIC value of 0.78 microg x mL(-1), which was equal to that of ofloxacin, one of the four antibiotic drugs used as a positive control.

The effects of p-hydroxycinnamaldehyde from Alpinia galanga extracts on human chondrocytes.

Osteoarthritis (OA) is the most common form of arthritis and affects millions of people worldwide. Patients have traditionally been treated with non-steroidal anti-inflammatory drugs (NSAIDs), but these are associated with significant side effects. Purification of the acetone extract of Alpinia galanga afforded p-hydroxycinnamaldehyde, as identified by nuclear magnetic resonance and mass spectrometry analyses. By exploiting the cartilage explant culture, p-hydroxycinnamaldehyde suppressed loss of uronic acid, resulting in release of hyaluronan (HA), sulfated glycosaminoglycans (s-GAGs) and matrix metalloproteinases (MMPs). p-Hydroxycinnamaldehyde and interleukin-1beta (IL-1beta), when incubated in primary human chondrocytes, also reduced release of HA, s-GAG and MMP-2. The results demonstrated: (a) that expression levels of the catabolic genes MMP-3 and MMP-13 were suppressed and (b) mRNA expression levels of anabolic genes of collagen II, SOX9 and aggrecan were increased. This study shows that p-hydroxycinnaldehyde from A. galanga Linn. is a potential therapeutic agent for treatment of OA.