New dihydroxycucurbitacin D's from the Namib desert endemic plant Acanthosicyos horridus (!nara).

Acanthosicyos horridus Welw. ex Hook.f. (!nara) is a leafless, thorny, melon-producing plant endemic to the hyper-arid Namib Desert. The methanol crude extract prepared from the ripe fruits of !nara afforded the known dihydroxycucurbitacin 7ß-hydroxy-23,24-dihydrocucurbitacin D (1), along with four new congeners 7ß,15ß-dihydroxy-23,24-dihydrocucurbitacin D (2), 25-O-ß-glucopyranosyl-7ß-hydroxy-23,24-dihydrocucurbitacin D (3), 25-O-ß-glucopyranosyl-7ß-hydroxy-23,24-dihydroisocucurbitacin D (4) and 25-O-ß-glucopyranosyl-7ß-hydroxy-23,24-dihydro-3-epi-isocucurbitacin D (5). These compounds were isolated through a combination of preparative normal phase thin-layer chromatography (TLC) and semi-preparative reversed phase high performance liquid chromatography (HPLC). Their structures were established by comprehensive analysis of HR-ESI-MS data, 1D and 2D NMR spectroscopic data and by comparison with literature values of similar cucurbitacins. The five isolated compounds exhibited poor cytotoxic activity against the MDA-MB-231 breast cancer cell line. To the best of our knowledge, this is the first report of glycosylated cucurbitacins in Acanthosicyos horridus.

Cucurbitacin IIb from Ibervillea sonorae Induces Apoptosis and Cell Cycle Arrest via STAT3 Inhibition.

BACKGROUND: Cucurbitacin IIb (CIIb) from Ibervillea sonorae has a high capacity to suppress cancer cell proliferation and induce apoptosis. This study investigated the molecular mechanisms related to the antiproliferative and apoptosis induction capacity of CIIb in HeLa cells. MATERIALS AND METHODS: The cell viability and anti-proliferative effect of CIIb were evaluated by using the trypan blue exclusion assay. The effect of CIIb on the mitochondrial membrane potential was determined by flow cytometry using JC-1. The activity of caspase-3 and caspase-9 was evaluated by flow cytometry using commercial kits. The effect of CIIb on the cell cycle was investigated using Fluorescence-Activated Cell Sorting (FACS) analysis. Western blot analysis was used to evaluate both the inhibitory effect of CIIb on the STAT3 signaling pathway and cyclin -B1, and DNA damage by the comet assay. RESULTS: CIIb triggers disruption of the mitochondrial membrane potential (Δψm) and consequently activated the caspases -3 and -9, as a result of the activation of the intrinsic pathway of the apoptosis. Likewise, the CIIbinduced cell cycle was arrested in S and G2/M after 24h of treatment. CIIb also reduced the expression of STAT3 and cyclin -B1. Finally, CIIb produced an antiproliferative effect at 48 and 72 h, inducing DNA damage. CONCLUSION: These results demonstrate CIIb-induced apoptosis and cell cycle arrest in HeLa through the inhibition of STAT3.

Nor-cucurbitacins from the leaves of Mareya micrantha (Benth.) Müll. Arg. (Euphorbiaceae).

Mareya micrantha, an Ivoirian medicinal plant, was investigated for its chemical constituents and antioxidant properties. This study carried out on the hydroethanolic extract of the leaves led to three new nor-cucurbitacins named: 29-nor-1,2,3,4,5,10-dehydro-3,15α,20ß-trihydroxy-16α-acetyl-11,22-dioxo-cucurbita-23-ene 2-O-ß-D-glucopyranoside (1), 29-nor-2ß,20ß,25-trihydroxy-16α-acetyl-3,11,22-trioxo-cucurbita-4,23-diene (2) and 29-nor-2ß,15α,20ß-trihydroxy-16α-acetyl-3,11,22-trioxo-cucurbita-4,23-diene 2-O-ß-D-glucopyranoside (3). The structures were established on the basis of spectral data (NMR, UV, MS and IR). The antioxidant properties evaluated by DPPH and CUPRAC methods gave the best activity with compound 1. The chemotaxonomic significance of the isolation of these compounds in Mareya micrantha, a species belonging to the Euphorbiaceae family, is discussed.

Cucurbitacin IIb, a steroidal triterpene from Ibervillea sonorae induces antiproliferative and apoptotic effects on cervical and lung cancer cells.

Chemical studies on Ibervillea sonorae (S. Watson) Greene root led to isolation and chemical characterization of diverse cucurbitacin triterpenoid compounds such as kinoin A, B, C, and their glucosides. In previous studies, we demonstrated that kinoin A inhibits the cell proliferation on diverse cell line and induce apoptosis in HeLa cells. Therefore, the study of the isolated compounds from the extracts continued to be necessary. The objective of the present work was to isolate and chemically characterize the active compounds of the methanolic extract of the roots of I. sonorae and to evaluate their antiproliferative activity and induction of apoptosis. By chromatographic column separation and using NMR spectroscopy experiments, cucurbitacin IIb (CIIb), known as 23,24-dihydrocucurbitacin F or hemslecin B, was isolated and identified for the first time as a chemical constituent of the crude methanolic extract of this plant. The antiproliferative activity of CIIb was evaluated by MTT assay, and the apoptosis induction capacity was monitored by annexin V-FITC/propidium iodide using flow cytometry. CIIb showed a pronounced effect on the proliferation of HeLa and A549 tumor cells, with IC50 of 7.3 and 7.8 µM, respectively, but was less effective against L929 non-cancerous murine cell line. Apoptosis induction capacity of CIIb on HeLa and A549 was monitored by annexin V-FITC/propidium iodide using flow cytometry. Exposure of HeLa and A549 with CIIb (8 µM) for 24 h increased 56.9 and 52.3% respectively of the total apoptosis compared to the negative control (p < 0.005). CIIb, isolated for the first time from I. sonorae, showed antiproliferative activity against HeLa and A549 cell lines by inducing cell death by apoptosis.

Efficient extraction of cucurbitacins from Diplocyclos palmatus (L.) C. Jeffrey: Optimization using response surface methodology, extraction methods and study of some important bioactivities.

Diplocyclos palmatus (L.) C. Jeffrey is an important medicinal plant used in several reproductive medicines. It serves as a wide source of tetracyclic triterpens called cucurbitacins. Response surface methodology (RSM) with Box-Behnken design (BBD) was studied to optimize the production of cucurbitacins. RSM put forth the ideal conditions such as 1:30 SS ratio (g/mL), 80 rpm (mixing extraction speed), 150 µm mean particle size, 30 min extraction time and 50 °C using chloroform in continuous shaking extraction (CSE) and showed the highest cucurbitacin I (CUI) content (2.345 ± 0.1686 mg/g DW). Similarly, the highest yield of cucurbitacin B (CUB) (1.584 ± 0.15 mg/g DW) was recorded at ideal conditions (1:40 g/mL SS ratio and 60 min time and others similar to CUI). Among the tested extraction methods, the highest CUI, CUB, and CUI + B yield (1.437 ± 0.03, 0.782 ± 0.10, 2.17 ± 0.35 mg/g DW, respectively) as well as promising DPPH radical scavenging activity (25.06 ± 0.1 µgAAE/g DW) were recorded from the SBAE (steam bath assisted extraction). In addition, MAE and UAE revealed the highest inhibition of α-amylase (68.68%) and α-glucosidase (56.27%) enzymes, respectively. Fruit extracts showed potent anticancer activity against breast (MCF-7) and colon (HT-29) cancer cell lines (LC50 - 44.27 and 46.88 µg/mL, respectively). Our study proved that SS ratio, particle size and temperature were the most positively influencing variables and served to be the most efficient for the highest recovery of CUI and CUB. Based on the present study, the fruits of D. palmatus were revealed as a potent antioxidant, anti-diabetic and anticancer bio-resource that could be explored further to develop novel drug to manage diabetes, cancer and oxidative stress related disorders.

Cucurbitacins extracted from Cucumis melo L. (CuEC) exert a hypotensive effect via regulating vascular tone.

As an effective medicine for jaundice in traditional Chinese medicine, Cucumis melo L. has been widely used in China. However, its effect on vascular function is still unclear. In this study, we extracted the compounds of Cucumis melo L., and the major ingredients were identified as cucurbitacins (CuEC, cucurbitacins extracted from Cucumis melo L.), especially cucurbitacin B. We replicated the toxicity in mice by intraperitoneal injection of a high dose of CuEC (2 mg/kg) and demonstrated that the cause of death was CuEC-induced impairment of the endothelial barrier and, thus, increased vascular permeability via decreasing VE-cadherin conjunction. The administration of low doses of CuEC (1 mg/kg) led to a decline in systolic blood pressure (SBP) without causing toxicity in mice. More importantly, CuEC dramatically suppressed angiotensin II (Ang II)-induced SBP increase. Further studies demonstrated that CuEC facilitated acetylcholine-mediated vasodilation in mesenteric arteries of mice. In vitro studies showed that CuEC induced vasodilation in a dose-dependent manner in mesenteric arteries of both mice and rats. Pretreatment with CuEC inhibited phenylephrine-mediated vasoconstriction. In summary, a moderate dose of CuEC reduced SBP by improving blood vessel tension. Therefore, our study provides new experimental evidence for developing new antihypertensive drugs.

Isolation of anticancer constituents from Cucumis prophetarum var. prophetarum through bioassay-guided fractionation.

BACKGROUND: Cucumis prophetarum var. prophetarum is used in Saudi folk medicine for treating liver disorders and grows widely between Abha and Khamis Mushait City, Saudi Arabia. METHODS: Bioassay-guided fractionation and purification were used to isolate the main active constituents of Cucumis prophetarum var. prophetarum fruits. These compounds were structurally elucidated using NMR spectroscopy, mass spectral analyses and x-ray crystallography. All fractions, sub-fractions and pure compounds were screened for their anticancer activity against six cancer cell lines. RESULTS: The greatest cytotoxic activity was found to be in the ethyl acetate fraction, resulting in the isolation of five cucurbitacin compounds [E, B, D, F-25 acetate and Hexanorcucurbitacin D]. Among the cucurbitacins that were isolated and tested cucurbitacin B and E showed potent cytotoxicity activities against all six human cancer cell lines. CONCLUSION: Human breast cancer cell lines were found to be the most sensitive to cucurbitacins. Preliminary structure activity relationship (SAR) for cytotoxic activity of Cucurbitacins against human breast cancer cell line MDA-MB-231 has been reported.

Two new cucurbitane-type triterpenoid saponins isolated from ethyl acetate extract of Citrullus colocynthis fruit.

Two new cucurbitacins I (1 and 2), together with eight known compounds (3-10), were isolated from the ethyl acetate extract of the fruit of Citrullus colocynthis. Compounds 3, 5-9 were isolated from C. colocynthis for the first time. The structures of new compounds were determined primarily from IR, HR-MS, 1D-, and 2D-NMR analysis.

The antigluconeogenic activity of cucurbitacins from Momordica charantia.

Five new cucurbitacins, kuguacins II-VI (1-5), along with five known analogues (6-10), were obtained from the fruit of Momordica charantia. Structures of the new compounds were elucidated as 5ß,19-epoxycucurbit-23-en-7-on-3ß,25-diol (1), 5ß,19-epoxycucurbit-7,23-dion-3ß,25-diol (2), 5ß,19-epoxycucurbit-6-en-19,23-dion-3ß,25-diol (3), 5ß,19-epoxy-23,24,25,26,27-pentanorcucurbit-6-en-7,19-dion-3ß,22-diol (4), and cucurbit-5-en-7,23-dion-3ß,19,25-triol (5) by extensive spectroscopic and single-crystal X-ray diffraction analyses. Some cucurbitane compounds from this species were screened for their potential antidiabetic properties in terms of antigluconeogenic activity. As a result, compounds 1, 10, 11, and 12 (at 25-100 µM) showed concentration-dependent inhibition on glucose production from liver cells. In addition, compounds 11 and 12 (at 100 µM) showed around 20-30 % inhibition on PEPCK activity.

Potential activity of fevicordin-A from Phaleria macrocarpa (Scheff) Boerl. seeds as estrogen receptor antagonist based on cytotoxicity and molecular modelling studies.

Fevicordin-A (FevA) isolated from Phaleria macrocarpa (Scheff) Boerl. seeds was evaluated for its potential anticancer activity by in vitro and in silico approaches. Cytotoxicity studies indicated that FevA was selective against cell lines of human breast adenocarcinoma (MCF-7) with an IC50 value of 6.4 µM. At 11.2 µM, FevA resulted in 76.8% cell death of T-47D human breast cancer cell lines. Critical pharmacophore features amongst human Estrogen Receptor-α (hERα) antagonists were conserved in FevA with regard to a hypothesis that they could make notable contributions to its pharmacological activity. The binding stability as well as the dynamic behavior of FevA towards the hERα receptor in agonist and antagonist binding sites were probed using molecular dynamics (MD) simulation approach. Analysis of MD simulation suggested that the tail of FevA was accountable for the repulsion of the C-terminal of Helix-11 (H11) in both agonist and antagonist receptor forms. The flexibility of loop-534 indicated the ability to disrupt the hydrogen bond zipper network between H3 and H11 in hERα. In addition, MM/GBSA calculation from the molecular dynamic simulations also revealed a stronger binding affinity of FevA in antagonistic action as compared to that of agonistic action. Collectively, both the experimental and computational results indicated that FevA has potential as a candidate for an anticancer agent, which is worth promoting for further preclinical evaluation.

Cytotoxic cucurbitane triterpenoids isolated from the rhizomes of Hemsleya amabilis.

Two new cucurbitane triterpenoids, 7ß-hydroxycucurbitacin F-25-O-acetate (1) and 2ß,3ß,20(S),26,27-pentahydroxy-16α,23(S)-epoxycucurbita-5,24-dien-11-one (2) along with eleven known cucurbitane triterpenoids (3-13, resp.) were isolated from the rhizomes of Hemsleya amabilis Diels. The chemical structures of the new isolated compounds were elucidated unambiguously by spectroscopic data analysis. The cytotoxic activities of the isolated cucurbitane triterpenoids were evaluated against the HeLa human cancer cell lines. Hemslecin A (5), the main ingredient of H. amabilis, exhibited the significant cytotoxicity with IC50 value of 0.389 µM.

Anti-proliferative effect of 23,24-dihydrocucurbitacin F on human prostate cancer cells through induction of actin aggregation and cofilin-actin rod formation.

PURPOSE: The cucurbitacins are a class of triterpenoid molecules that possess cytotoxic characteristics for plant defense against herbivore feeding. 23,24-dihydrocucurbitacin F (DHCF), a derivative of the cucurbitacin family, has been isolated as an active component from the root of Hemsleya amabilis (Cucurbitaceae), an ancient Chinese remedy for bacillary dysentery, gastroenteritis, and cancers. While the toxicity of other cucurbitacins has been explored in several cancers, little data exist on the effect of DHCF on human cancers, including prostate cancer (PCa). In this study, we explore the level and mechanisms of DHCF toxicity on human PCa cell lines. METHODS: Human PCa DU145, PC3, and LNCaP cells were treated with graded doses of DHCF in vitro, and anti-proliferative, cytotoxic, and proteomic effects were determined using MTS assay, cell cycle analysis, immunofluorescent staining, and western blotting. RESULTS: DHCF inhibited cell growth and induced cell cycle arrest at G(2)/M phase, formation of binucleated cells, and increased levels of apoptosis in all PCa cell lines tested. G-actin depletion, actin aggregation, and rod-like actin fibers, with little effect on microtubule structure, were observed after DHCF treatment. Actin aggregation and cofilin-actin rod formation were highly correlated with rapid and persistent dephosphorylation of cofilin-1 (cofilin). DHCF treatment resulted in upregulation of p21(Cip1) and downregulation of cyclin A in all three PCa cell lines. CONCLUSIONS: The anti-proliferative activity of DHCF on human PCa cells may be brought about by inducing actin aggregation and cofilin-actin rod formation, leading to cell cycle arrest, cytokinesis failure, and apoptosis.

In vitro and in vivo study of cucurbitacins-type triterpene glucoside from Citrullus colocynthis growing in Saudi Arabia against hepatocellular carcinoma.

Chromatographic investigation of fruits obtained from Citrullus colocynthis, growing in Saudi Arabia, led to isolation of two compounds; Cucurbitacin E glucoside (Cu E, 1), and Cucurbitacin I glucoside (Cu I, 2). The chemical structures of 1 and 2, were elucidated by spectroscopic analyses include; 1D ((1)H and (13)C) and 2D (COSY, HMQC and HMBC) NMR and ESI-MS spectroscopy. The in vitro cytotoxic activity against hepatoma cell line (HepG2) and mice-bearing tumor of Ehrlich's ascites carcinoma (EAC) of the compounds were estimated. Both compounds had potent inhibitory activity on HepG2 with IC(50) 3.5 and 2.8 nmol/mL, respectively. In addition to these activities, the in vivo study employing EAC, showed the capability of both compounds to prolong the survival time, life span and normalize the biochemical parameters of the infected mice with EAC.

Isolation, structure elucidation, and biological evaluation of 16,23-epoxycucurbitacin constituents from Eleaocarpus chinensis.

Eight new 16,23-epoxycucurbitacin derivatives, designated as elaeocarpucins A-H (1-8), and five known cucurbitacins (9-13) were isolated from the chloroform-soluble partitions of separate methanol extracts of the fruits and stem bark of Elaeocarpus chinensis collected in Vietnam. Isolation work was facilitated using a LC/MS dereplication procedure, and bioassay-guided fractionation was monitored using HT-29 human cancer cells. The structures of compounds 1-8 were determined on the basis of spectroscopic data interpretation, with the absolute configurations of isomers 1 and 2 established by the Mosher ester method. Compounds 1-13 were evaluated in vitro against the HT-29 cell line and using a mitochondrial transmembrane potential assay. Elaeocarpucin C (3), produced by partial synthesis from 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one (13), was found to be inactive when evaluated in an in vivo hollow fiber assay using three different cancer cell types (dose range 0.5-10 mg/kg/day, i.p.).

New cytotoxic cucurbitacins from Wilbrandia ebracteata Cogn.

Chemical investigation of the roots of Wilbrandia ebracteata Cogn. (Cucurbitaceae) led to the isolation of two new (1- 2) and four known (3- 6) cucurbitacins. Their structures were elucidated by NMR and MS and compared with related compounds. The in vitro cytotoxicity of isolated compounds was evaluated against RD, KB, HCT-8, and A549 cell lines showing strong activity.

Actin-aggregating cucurbitacins from Physocarpus capitatus.

Bioassay-guided fractionation of Physocarpus capitatus yielded two new cucurbitacins (3 and 4) along with the known cucurbitacin F (1) and dihydrocucurbitacin F (2). Preliminary mechanism of action studies indicate that the cucurbitacins cause actin aggregates and inhibit cell division.

Trinorcucurbitane and cucurbitane triterpenoids from the roots of Momordica charantia.

Five cucurbitacins, kuguacins A-E (1-5), together with three known analogues, 3beta,7beta,25-trihydroxycucurbita-5,(23E)-diene-19-al (6), 3beta,25-dihydroxy-5beta,19-epoxycucurbita-6,(23E)-diene (7), and momordicine I (8), were isolated from roots of Momordica charantia. Structures of 1-5 were elucidated by NMR and MS spectroscopic analysis. Among them, compounds 3-5 possess an unprecedented 25,26,27-trinorcucurbitane backbone. Compounds 3 and 5 showed moderate anti-HIV-1 activity with EC(50) values of 8.45 and 25.62 microg/ml, and exerted minimal cytotoxicity against C8166 cells (IC(50)>200 microg/ml), with a selectivity index more than 23.68 and 7.81, respectively.

[Determination of the cucurbitacins from Cucubita pepo cv dayangua by HPLC].

OBJECTIVE: To establish an HPLC method for the simultaneous determination of four bioactive cucurbitacins in Cucubita pepo cv Dayangua. METHODS: The HPLC chromatography was carried out with a lineal gradient programming and detection wavelength at 215 nm. Kromasil C8 column (150 mm x 4.6 mm ID, 5 microm)was used. The mobile phase was acetonitrile-water (containing 2.0% HAc) and the flow rate was 1.0 ml/min. RESULTS: The linear range of 23, 24-dihydrocucurbitacin F was 0.28-5.6 microg/ml (r = 0.9978), 23, 24-dihydrocucurbitacin D 0.39-7.8 microg/ml (r = 0.9986), cucurbitacin B 0.304-6.08 microg/ml (r = 0.9983), cucurbitacin E 2. 52 -50. 4 microg/ml (r = 0.9998). The method was accurate with variation less than 5% and recovery more than 95%. CONCLUSION: The method is successfully applied to determination of the four cucurbitacins from Cucubita pepo cv dayangua.

Plant anticancer agents, XXXIV. Cucurbitacins from Elaeocarpus dolichostylus.

Elaeocarpus dolichostylus has afforded the new cucurbitacin derivative, hexanorcucurbitacin F (3), whose structure was determined by spectroscopic and chemical correlation with curcurbitacin F (1). Cucurbitacin F (1) and 23,24-dihydrocucurbitacin F (2) were also isolated in this study.