Bioassay-Guided Isolation and Identification of Cytotoxic Compounds from Melaleuca quinquenervia Fruits.

The fruit extract of Melaleuca quinquenervia yielded a total of 19 compounds, including two novel spiro-biflavonoid enantiomers (1a and 1b) and a chalcone derivative (3). Their structures were determined through spectroscopic analysis. The enantiomers of the racemic mixture of compound 1 were successfully resolved into (+)-1 and (-)-1 using chiral-phase HPLC. Single-crystal X-ray diffraction analysis was also used to confirm the structure of 1. The enantiomeric configurations of 1 and 2 were determined through a comparison of the calculated and experimental electronic circular dichroism spectra. Compounds 2 (melanervin), 14 (methyl betulinate), 15 (3-O-acetylbetulinic acid), and 16 (pyracrenic acid) were found to be highly cytotoxic, with compound 16 showing superior growth inhibition of nonsmall cell lung cancer cells (A549 cells) (IC50 2.8 ± 0.1 µM) compared to cisplatin (IC50 3.3 ± 0.0 µM), a positive control chemotherapeutic drug. Both compound 16 and cisplatin were significantly more cytotoxic toward A549 lung cancer cells compared to nontumorigenic Vero E6 cells.

Altersolanol B, a fungal tetrahydroanthraquinone, inhibits the proliferation of estrogen receptor-expressing (ER+) human breast adenocarcinoma by modulating PI3K/AKT, p38/ERK MAPK and associated signaling pathways.

The present study focused on the apoptosis-inducing effects and cellular signal-modulating properties of altersolanol B (AB), a minor fungal tetrahydroanthraquinone (THAQ) metabolite, in the estrogen receptor positive (ER+) human breast adenocarcinoma cell line, MCF-7. AB demonstrated approximately 4-fold greater antiproliferative activity in ER+ MCF-7 cells (IC50 5.5 µM) compared to the ER-negative (triple-negative) MDA-MB-231 (IC50 21.3 µM). The viability of normal breast fibrocystic epithelial cells, MCF-10A, was unaffected. AB induced intrinsic apoptosis in MCF-7 cells; it triggered the activation of caspase 9 and poly (ADP-ribose) polymerase (PARP), upregulated the expression of pro-apoptotic Bax, and downregulated the expression of anti-apoptotic Bcl-2. AB induced cell cycle arrest at G0/G1, as indicated by the downregulation of key checkpoint proteins operating at the G0/G1 phase of the cell cycle (cyclin D1, CDK4 and CDK2). The observed increase in p21Waf1/Cip1 and p53 expression may facilitate cell cycle arrest, and the subsequent induction of apoptosis. AB lacked significant effects on intracellular ROS levels, while it down-regulated nuclear factor erythroid 2-related factor 2 (Nrf2), and the Nrf2-dependent antioxidant enzyme, heme oxygenase-1. The compound disrupted AKT signaling through the downregulation of phospho-AKT and phospho-FOXO1, and the upregulation of PTEN, a phosphatase and tumor suppressor that negatively regulates the PI3K/AKT pathway. AB also disrupted the phosphorylation of AKT-controlled eukaryotic translation initiation factor, 4E-BP1, and GSK-3ß, both of which are aberrantly regulated in human cancer. The AB-dependent downregulation of NF-κB was corroborated by the inhibition of TNFα-induced NF-κB activity as monitored in a luciferase reporter. The NF-κB inhibitory activity of AB was 3-fold more potent than that of the standard inhibitor, N-p-Tosyl-l-phenylalanine chloromethyl ketone. In addition to reducing the pro-survival effects of NF-кB, the inhibition of AKT phosphorylation by AB may also lead to FOXO1-mediated growth arrest and apoptosis. AB upregulated the expression of phospho-MKK4 and phospho-p38, and downregulated the expression of phospho-MEK1/2 and phospho-ERK1/2 indicating opposing effects on the two important oncogenic signaling cascades that are aberrantly activated in many cancers. AB disrupted both the AKT and ERK1/2 signaling pathways leading to apoptosis in ER+ MCF-7 cells through mitochondria-associated mechanisms coupled with the potent inhibition of NF-кB activation. The clinical limitations of multi-agent combination therapy that targets multiple pathways in cancer may potentially be circumvented by using a single molecule, such as AB, that inhibits both AKT and ERK1/2 signaling. Our preliminary study suggested that the THAQ pharmacophore, with its disrupted conjugated ring system and relative redox inactivity, may possess greater mechanistic advantage against ER+ breast cancer when compared to the fully conjugated ring systems of the anthraquinone that possess intrinsic redox activity and DNA interacting ability. This study supports the continued investigation of THAQs as lead molecules in anticancer drug discovery and development.

Molecular Docking and Molecular Dynamics Simulation Studies of Triterpenes from Vernonia patula with the Cannabinoid Type 1 Receptor.

A molecular docking approach was employed to evaluate the binding affinity of six triterpenes, namely epifriedelanol, friedelin, α-amyrin, α-amyrin acetate, ß-amyrin acetate, and bauerenyl acetate, towards the cannabinoid type 1 receptor (CB1). Molecular docking studies showed that friedelin, α-amyrin, and epifriedelanol had the strongest binding affinity towards CB1. Molecular dynamics simulation studies revealed that friedelin and α-amyrin engaged in stable non-bonding interactions by binding to a pocket close to the active site on the surface of the CB1 target protein. The studied triterpenes showed a good capacity to penetrate the blood-brain barrier. These results help to provide some evidence to justify, at least in part, the previously reported antinociceptive and sedative properties of Vernonia patula.

Siamese Crocodile White Blood Cell Extract Inhibits Cell Proliferation and Promotes Autophagy in Multiple Cancer Cell Lines.

Cancer represents one of the most significant threats to human health on a global scale. Hence, the development of effective cancer prevention strategies, as well as the discovery of novel therapeutic agents against cancer, is urgently required. In light of this challenge, this research aimed to evaluate the effects of several potent bioactive peptides and proteins contained in crocodile white blood cell extract (cWBC) against LU-1, LNCaP, PC-3, MCF-7, and CaCo-2 cancer cell lines. The results demonstrate that 25, 50, 100, and 200 µg/ml cWBC exhibits a strong cytotoxic effect against all investigated cell lines (IC50 70.34-101.0 µg/ml), while showing no signs of cytotoxicity towards noncancerous Vero and HaCaT cells. Specifically, cWBC treatment caused a significant reduction in the cancerous cells' colony forming ability. A remarkable suppression of cancerous cell migration was observed after treatment with cWBC, indicating potent antimetastatic properties. The mechanism involved in the cancer cell cytotoxicity of cWBC may be related to apoptosis induction, as evidenced by typical apoptotic morphology features. Moreover, certain cWBC concentrations induced significant overproduction of ROS and significantly inhibited the S-G2/M transition in the cancer cell. The molecular mechanisms of cWBC in apoptosis induction were to decrease Bcl-2 and XIAP expression levels and increase the expression levels of caspase-3, caspase-8, and p53. These led to a decrease in the expression level of the cell cycle-associated gene cyclin-B1 and the arrest of cell population growth. Consequently, these findings demonstrate the prospect of the use of cWBC for cancer therapy.

Solid-Phase Synthesis and Antibacterial Activity of Cyclohexapeptide Wollamide B Analogs.

Herein we report the antibacterial structure-activity relationships of cyclic hexapeptide wollamide analogs derived from solid-phase library synthesis. Wollamide B, a cyclic hexapeptide natural product, has been previously found to have activity against Mycobacterium bovis. To further evaluate its antimycobacterial/antibacterial potential, 27 peptides including wollamides A/B, and desotamide B, were synthesized and subsequently tested against a panel of clinically significant bacterial pathogens. Biological evaluation revealed that the cyclic scaffold, amide functionality in position I, tryptophan residue in position V, and the original stereochemistry pattern of the core scaffold were key for antituberculosis and/or antibacterial activity. In addition, against M. tuberculosis and Gram-positive bacteria, residues in position II and/or VI greatly impacted antibacterial activity and selectivity. Wollamides A (3) and B (2) along with their corresponding II (l-Leu) analog 10 retained the most promising antituberculosis activity, with the lowest minimum inhibitory concentration (MIC) against virulent M. tuberculosis H37Rv (MIC = 1.56 µg/mL), as well as desirable selectivity indices (>100). Importantly, the antimicrobial activities of wollamides A and B do not result from disruption of the bacterial membrane, warranting further investigation into their mechanism of action.

Solid-phase synthesis of cyclic hexapeptides wollamides A, B and desotamide B.

Solid-phase synthesis of antibacterial cyclohexapeptides including wollamides A, B and desotamide B has been developed. Briefly, the protected linear hexapeptides were assembled on 2-chlorotrityl chloride resin using standard Fmoc chemistry and diisopropylcarbodiimide/hydroxybenzotriazole coupling reagents, cleaved off-resin with hexafluoroisopropanol/dichloromethane to keep side-chain protecting groups intact, and cyclized in solution. Final global removal of all protecting groups using a cocktail of trifluoroacetic acid/triisopropylsilane/dichloromethane afforded the desired cyclic hexapeptides, which were characterized by 1H, 13C NMR, and HRMS. Subsequent investigation of macrocyclization parameters such as terminal residues, coupling reagents, and cyclization concentration revealed the optimized conditions for the synthesis of this class of cyclic hexapeptides.

Bioactive Constituents from an Endophytic Fungus, Penicillium polonicum NFW9, Associated with Taxus fauna.

BACKGROUND: Endophytic fungi are being recognized as vital and untapped sources of a variety of structurally novel and unique bioactive secondary metabolites in the field of natural products drug discovery. Herein, this study reports the isolation and characterization of secondary metabolites from an endophytic fungus Penicillium polonicum (NFW9) associated with Taxus fuana. METHOD: The extracts of the endophytic fungus cultured on potato dextrose agar were purified using several chromatographic techniques. Biological evaluation was performed based on their abilities to inhibit tumor necrosis factor-alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) and cytotoxicity assays. RESULTS: Bioactivity-directed fractionation of the ethyl acetate extract of a fermentation culture of an endophytic fungus, Penicillium polonicum led to the isolation of a dimeric anthraquinone, (R)- 1,1',3,3',5,5'-hexahydroxy-7,7'-dimethyl[2,2'-bianthracene]-9,9',10,10'-tetraone (1), a steroidal furanoid (-)-wortmannolone (2), along with three other compounds (3-4). Moreover, this is the first report on the isolation of compound 1 from an endophytic fungus. All purified metabolites were characterized by NMR and MS data analyses. The stereo structure of compound 1 was determined by the measurement of specific optical rotation and CD spectrum. The relative stereochemistry of 2 was confirmed by single-crystal X-ray diffraction. Compounds 2-3 showed inhibitory activities in the TNF-α-induced NF-κB assay with IC50 values in the range of 0.47-2.11 µM. Compounds 1, 4 and 5 showed moderate inhibition against NF-κB and cancer cell lines. CONCLUSION: The endophytic fungus Penicillium polonicum of Taxus fuana is capable of producing biologically active natural compounds. Our results provide a scientific rationale for further chemical investigations into endophyte-producing natural products, drug discovery and development.

Litsea Species as Potential Antiviral Plant Sources.

Litsea verticillata Hance (Lauraceae), a Chinese medicine used to treat swelling caused by injury or by snake bites, was the first plant identified by our National Institutes of Health (NIH)-funded International Cooperative Biodiversity Group (ICBG) project to exhibit anti-HIV activities. From this plant, we discovered a class of 8 novel litseane compounds, prototypic sesquiterpenes, all of which demonstrated anti-HIV activities. In subsequent studies, 26 additional compounds of different structural types were identified. During our continuing investigation of this plant species, we identified two new litseanes, litseaverticillols L and M, and a new sesquiterpene butenolide, litseasesquibutenolide. Litseaverticillols L and M were found to inhibit HIV-1 replication, with an IC[Formula: see text] value of 49.6[Formula: see text][Formula: see text]M. To further determine the antiviral properties of this plant, several relatively abundant isolates, including a litseane compound, two eudesmane sesquiterpenes and three lignans, were evaluated against an additional 21 viral targets. Lignans 8 and 9 were shown to be active against the Epstein-Barr Virus (EBV), with EC[Formula: see text] values of 22.0[Formula: see text][Formula: see text]M ([Formula: see text]) and 16.2[Formula: see text][Formula: see text]M ([Formula: see text]), respectively. Since many antiviral compounds have been discovered in L. verticillata, we further prepared 38 plant extracts made from the different plant parts of 9 additional Litsea species. These extracts were evaluated for their anti-HIV and cytotoxic activities, and four of the extracts, which ranged across three different species, displayed 97-100% inhibitory effects against HIV replication without showing cytotoxicity to a panel of human cell lines at a concentration of 20 µg/mL.

The Role of Endophytic Fungi in the Anticancer Activity of Morinda citrifolia Linn. (Noni).

We hypothesize that the fungal endophytes of noni may possibly play a role in its overall pharmacological repertoire, especially since the perceived efficacy of the fruit in ethnomedicinal use is associated with the fermented juice. The foremost goal of this study is to explore the role of endophyte-derived secondary metabolites in the purported anticancer properties of noni. To that end, culturable endophytic fungi resident within the healthy leaves and fruit of the plant were isolated and identified by molecular sequence analysis of the 5.8S gene and internal transcribed spacers (ITS). Purified organisms were subjected to in vitro fermentation in malt extract broth for 8 weeks under anaerobic conditions at room temperature (25°C), in order to simulate the conditions under which traditional fermented noni juice is prepared. The cytotoxic potential of organic extracts derived from the fermented broths of individual endophytes was then tested against three major cancers that afflict humans. Twelve distinct endophytic fungal species were obtained from the leaves and 3 from the fruit. Three of the leaf endophytes inhibited the growth of human carcinoma cell lines LU-1 (lung), PC-3 (prostate), and MCF-7 (breast) with IC50 values of ≤10 µg/mL.

Synthesis and structure-activity relationships of lansine analogues as antileishmanial agents.

Clear and rational thinking: A series of rationally designed, lansine-derived carbazoles was synthesized and evaluated for activity against promastigotes and amastigotes of Leishmania donovani, the causative agent of leishmaniasis. Some structural modifications gave rise to compounds with enhanced activity and selectivity over lansine, allowing structure-activity relationships to be elucidated and providing a foundation for the further development of this pharmacophore.

An ethnobotanical survey of medicinal plants of Laos toward the discovery of bioactive compounds as potential candidates for pharmaceutical development.

CONTEXT: An ethnobotany-based approach in the selection of raw plant materials to study was implemented. OBJECTIVE: To acquire raw plant materials using ethnobotanical field interviews as starting point to discover new bioactive compounds from medicinal plants of the Lao People's Democratic Republic. METHODS: Using semi-structured field interviews with healers in the Lao PDR, plant samples were collected, extracted, and bio-assayed to detect bioactivity against cancer, HIV/AIDS, TB, malaria. Plant species demonstrating activity were recollected and the extracts subjected to a bioassay-guided isolation protocol to isolate and identify the active compounds. RESULTS: Field interviews with 118 healers in 15 of 17 provinces of Lao PDR yielded 753 collections (573 species) with 955 plant samples. Of these 955, 50 extracts demonstrated activity in the anticancer, 10 in the anti-HIV, 30 in the anti-TB, and 52 in the antimalarial assay. Recollection of actives followed by bioassay-guided isolation processes yielded a series of new and known in vitro-active anticancer and antimalarial compounds from 5 species. DISCUSSION: Laos has a rich biodiversity, harboring an estimated 8000-11,000 species of plants. In a country highly dependent on traditional medicine for its primary health care, this rich plant diversity serves as a major source of their medication. CONCLUSIONS: Ethnobotanical survey has demonstrated the richness of plant-based traditional medicine of Lao PDR, taxonomically and therapeutically. Biological assays of extracts of half of the 955 samples followed by in-depth studies of a number of actives have yielded a series of new bioactive compounds against the diseases of cancer and malaria.

Antimalarial activity of Aspilia pruliseta, a medicinal plant from Uganda.

Aspilia pruliseta Schweinf. (Asteraceae) is a medicinal plant indigenous to Uganda and the neighboring countries of East Africa. It has been used extensively by the rural population for the treatment of fevers and malaria. During the antimalarial evaluation of this plant, four nontoxic diterpenes were isolated that possessed moderate activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2) clones of Plasmodium falciparum, with IC(50) values ranging from 14 to 23 µM. These moderately active compounds included the previously undescribed diterpene, ENT-15 ß-senecioyloxy-16,17-epoxy-kauran-18-oic acid that demonstrated an IC(50) value of 23.4 µM against clone D6, but was devoid of activity against clone W2. Four additional diterpenes were obtained from the aerial parts of A. pruliseta, but these known compounds were essentially inactive. The moderate activities of select diterpenes of A. pruliseta could account collectively for the historical and enduring use of this plant in traditional African medicine.

Bioactive sulfated sesterterpene alkaloids and sesterterpene sulfates from the marine sponge Fasciospongia sp.

Two new sulfated sesterterpene alkaloids, 19-oxofasciospongine A (3) and fasciospongine C (4), and a new sesterterpene sulfate, 25-hydroxyhalisulfate 9 (5), along with two known sesterterpene sulfates, halisulfates 7 (6) and 9 (7), were isolated from an organic extract of the marine sponge Fasciospongia sp. The structures of the new compounds were established on the basis of one- and two-dimensional NMR spectroscopic studies as well as by HRESIMS analysis. Compounds 1-7 exhibited inhibitory activity against Streptomyces 85E in the hyphae-formation inhibition assay. Compounds 1, 2, and 4-7 were not cytotoxic when tested at 20 microg/mL with MCF-7, LNCaP, and LU cell lines. Only compound 3 demonstrated a moderate cytotoxic effect on the MCF-7 (IC(50) 13.4 microM), LNCaP (IC(50) 21.8 microM), and LU-1 cells (IC(50) 5.0 microM), respectively.

A first new antimalarial pregnane glycoside from Gongronema napalense.

As a part of the UIC-based ICBG project in Laos, plants were collected based on ethnomedical interviews and evaluated for antimalarial activity. A CHCl3 extract from the vine of Gongronema napalense (Wall.) Decne. (Asclepiadaceae) showed promising anti-malarial activity while exhibiting low levels of cytotoxicity and was thus followed up with further fractionation and biological evaluation. Bioassay-guided fractionation led to the isolation of a new steroidal glycoside, gongroneside A, which showed antimalarial activity in vitro with an IC50 value of 1.60 and 1.39 µM against the Plasmodium falciparum D6 and W2 clones, respectively.

"Studies on biodiversity of Vietnam and Laos" 1998-2005: examining the impact.

The impact of the University of Illinois at Chicago-based Vietnam-Laos International Cooperative Biodiversity Group (ICBG) Program "Studies on Biodiversity of Vietnam and Laos", which has been in operation for the period of 1998-2005, touches on five major areas of endeavor: (a) biodiversity inventory and conservation; (b) studies on medicinal plants; (c) drug discovery and development; (d) economic development; and (e) issues on intellectual property rights and benefit sharing in natural products drug discovery and development. Highlights are presented and the significance is discussed.

Apoptotic anticancer effect of alvaradoin E isolated from Alvaradoa haitiensis.

Two anthracenone C-glycosides, alvaradoins E and F, isolated from the leaves of Alvaradoa haitiensis Urb. (Simaroubaceae), were found to have potent inhibitory activities with cultured cancer cells. Using the in vivo hollow fiber model, these compounds demonstrated significant growth inhibition at the i.p. site when tested with KB, LNCaP, and Col2 cells. To determine if these anthracenone C-glycosides mediated anticancer activity through an apoptotic pathway, a series of assays were performed with the 10S isomeric compound, alvaradoin E. With a DAPI assay, treatment of LNCaP cells with alvaradoin E at concentrations of 0.4, 2, 10, or 50 microM for 24 or 48 h showed chromatin condensation, a morphological characteristic of apoptosis. Mitochondrial membrane potential, analyzed with a DiOC6 uptake assay, showed that treatment of LNCaP cells with 0.07, 0.14, 0.28, 0.56, 0.86, and 1.12 microM alvaradoin E for 12 h caused dose-dependent membrane depolarization, another indication of early apoptosis. Also, with an annexin V-FITC assay system, treatment of HL-60 cells with 0.07 microM alvaradoin E for 24 h increased annexin V-FITC binding from 3 to 25.9% (8.6-fold). Finally, with the TUNEL assay system, treatment of HL-60 cells with 1.12 microM alvaradoin E for 32 h increased FITC-dUTP binding from 1.2 to 12.1% (10-fold). These data suggest alvaradoin E is an effective anticancer agent that induces apoptosis. Additional studies to establish clinical utility should be of interest.

New 3-O-acyl betulinic acids from Strychnos vanprukii Craib.

Three new betulinic acid derivatives, 3beta-O-trans-feruloylbetulinic acid (1), 3beta-O-cis-feruloylbetulinic acid (2), and 3beta-O-cis-coumaroylbetulinic acid (4), along with two known triterpenes, 3beta-O-trans-coumaroylbetulinic acid (3) and ursolic acid (6) were isolated from the leaves and twigs of Strychnos vanprukiiCraib. All isolates showed moderate anti-HIV activity with IC50 values ranging from 3 to 7 microg/mL (5 to 15 microM) in an indicator cell line for HIV infectivity. The structures of the new isolates were elucidated by spectroscopic techniques including 1D and 2D NMR spectroscopy. In addition, the structure of 1 was confirmed by X-ray crystallography.

Cytotoxic flavone analogues of vitexicarpin, a constituent of the leaves of Vitex negundo.

Bioassay-guided fractionation of the chloroform-soluble extract of the leaves of Vitex negundo led to the isolation of the known flavone vitexicarpin (1), which exhibited broad cytotoxicity in a human cancer cell line panel. In an attempt to increase the cytotoxic potency of 1, a series of acylation reactions was performed on this compound to obtain its methylated (2), acetylated (3), and six new acylated (4-9) derivatives. Compound 9, the previously unreported 5,3'-dihexanoyloxy-3,6,7,4'-tetramethoxyflavone, showed comparative cytotoxic potency to compound 1 and was selected for further evaluation. However, this compound was found to be inactive when evaluated in the in vivo hollow fiber assay with Lu1, KB, and LNCaP cells at the highest dose (40 mg/kg/body weight) tested, and in the in vivo P-388 leukemia model (135 mg/kg), using the ip administration route.

Pseudoirroratin A, a new cytotoxic ent-kaurene diterpene from Isodon pseudo-irrorata.

A new ent-kaurene diterpene, pseudoirroratin A (1), and a known diterpene, pseurata A, were isolated from Isodon pseudo-irrorata. Compound 1 showed significant cytotoxicity against the Lu1, SW626, LNCaP, KB, and HOS cancer cell lines with IC(50) values of 0.26 (0.75), 0.20 (0.57), 0.90 (2.59), 0.90 (2.59), and 0.50 (1.44) microg/mL (microM), respectively. The structure of 1 was elucidated by spectroscopic means including 1D and 2D NMR techniques.