Effect of extracts from several sponges of Yucatan Coast on Giardia lamblia and preliminary chemical investigation of the bioactive extract of Haliclona (Reinera) tubifera.

In this study, twenty-four organic extracts from six marine sponge species, collected at shallows of Yucatan, Mexico, were evaluated against Giardia lamblia trophozoites and Vero cells. The dichloromethane and hexane extracts of Haliclona tubifera exhibited the highest antigiardiasic activity (IC50 = 1.00 and 2.11 µg/mL, respectively), as well as high selectivity (SI = 41.8 and > 47.4, respectively), while ethyl acetate and methanol extracts of Cinachyrella alloclada, and methanol extract of Suberites aurantiaca showed moderate activity. Contrastingly, the extracts of Halichondria magniculosa and Oceanapia nodosa were considered non actives. Consequently, the dichloromethane extract of H. tubifera were subject to an exploratory chemical study, isolating cholesterol, two benzaldehyde derivatives, three benzoic acid derivatives, cytosine, and thymine.

Lupane Triterpene Derivatives Improve Antiproliferative Effect on Leukemia Cells through Apoptosis Induction.

Leukemia is one of the most frequent types of cancer. No effective treatment currently exists, driving a search for new compounds. Simple structural modifications were made to novel triterpenes isolated from Phoradendron wattii. Of the three resulting derivatives, 3α-methoxy-24-hydroxylup-20(29)-en-28-oic acid (T1m) caused a decrease in the median inhibitory concentration (IC50) on the K562 cell line. Its mode of action was apparently apoptosis, ROS generation, and loss of mitochondrial membrane potential (MMP). Molecular docking analysis showed T1m to produce lower binding energies than its precursor for the Bcl-2 and EGFR proteins. Small, simple, and viable modifications to triterpenes can improve their activity against leukemia cell lines. T1m is a potentially promising element for future research. Clarifying the targets in its mode of action will improve its applicability.

Apoptotic and Cell Cycle Effects of Triterpenes Isolated from Phoradendron wattii on Leukemia Cell Lines.

Current antineoplastic agents present multiple disadvantages, driving an ongoing search for new and better compounds. Four lupane-type triterpenes, 3α,24-dihydroxylup-20(29)-en-28-oic acid (1), 3α,23-dihydroxy-30-oxo-lup-20(29)-en-28-oic acid (2), 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (3), and 3α,23-dihydroxylup-20(29)-en-28-oic acid (4), previously isolated from Phoradendron wattii, were evaluated on two cell lines of chronic (K562) and acute (HL60) myeloid leukemia. Compounds 1, 2, and 4 decreased cell viability and inhibit proliferation, mainly in K562, and exhibited an apoptotic effect from 24 h of treatment. Of particular interest is compound 2, which caused arrest in active phases (G2/M) of the cell cycle, as shown by in silico study of the CDK1/Cyclin B/Csk2 complex by molecular docking. This compound [3α,23-dihydroxy-30-oxo-lup-20(29)-en-28-oic acid] s a promising candidate for incorporation into cancer treatments and deserves further study.

Microwave-enhanced synthesis of 26-amino-22-oxocholestanes and their cytotoxic activity.

The synthesis of a series of 26-amino-22-oxocholestanes derived from diosgenin was accomplished via the substitution of an iodine atom at C-26 by primary and secondary amines. The reactions were conducted in refluxing acetonitrile and through microwave-assisted heating. The latter shows significant improvements in terms of reaction times going from hours to a few minutes or even seconds for completion. Only one of the selected amines, 4-aminourazole, did not yield the substitution product and the imine formation pathway was investigated instead, achieving the 26-iminourazole-22-oxocholestane. All the final products have been characterized and the cytotoxic activity of three of them has been evaluated in SiHa, MCF-7 and MDA tumor cell lines by the sulforhodamine B assay.

Cytotoxic and antiproliferative activity of thiazole derivatives of Ochraceolide A.

A series of 15 novel 1,3-thiazole amide derivatives of the pentacyclic triterpene Ochraceolide A (1) was synthesized, characterized, and evaluated in vitro against three human cancer cell lines (MCF-7, MDA-MB-231 and SiHa) and a normal cell line (Vero). Synthetic derivatives were obtained by acylation of the 2-aminothiazole triterpene 2, previously reported. Remarkably, the 5-nitrofuramide derivative (2o) showed better cytotoxic and antiproliferative activity than compound 2 and the other derivatives against the three cancer cell lines with CC50 and IC50 values of 1.6-12.7 µM. Furthermore, butyramide derivative (2c) was approximately 25 times more selective than 2, as well as 3.4 times more selective than Docetaxel, against SiHa cells in the cytotoxic assay, while the phenyl amide derivatives were inactive against the three cancer cell lines.

Season affects active metabolite composition and cytotoxic effect in Phoradendron wattii methanol extracts.

Plant-derived secondary metabolites are a source of promising bioactive molecules in the search for safer more selective cancer drugs. Mexico's flora is extremely diverse and many species, such as Phoradendron wattii, form part of traditional medicine. Compounds with notable cytotoxic activity have been isolated from P. wattii, but their concentrations may vary seasonally. The aim was to identify any variation in active metabolite concentrations in Phoradendron wattii methanol extracts in response to season. Betulin exhibited the most evident seasonal variations, being most abundant during the midsummer drought. Cytotoxic activity was highest (29 ± 1 µg/mL) in the rainy season methanol extract. Though not the most abundant metabolite in the extracts, 3α,24-dihydroxylup-20(29)-en-28-oic acid is apparently one of the most active among them and is a promising chemotaxonomic biomarker for this species. In summary, secondary metabolite concentrations in P. wattii methanol extracts varied in response to season, and these variations influenced cytotoxic activity.

Cytotoxic activity against breast cancer cells of two species of marine sponges from shallows of Yucatan peninsula.

This study aimed to evaluate the in vitro activity of extracts of two marine sponge species, occurring in the shallows of the Yucatan peninsula coast, on two cancer and one normal mammalian cell lines. Hexane, dichloromethane, ethyl acetate and methanol extracts of Halichondria magniconulosa and Halichondria melanadocia were screened for their cytotoxic activity against hormone-dependent breast cancer (MCF-7) and human cervix cancer (SiHa) cell lines. The ethyl acetate extract of H. magniconulosa exhibited significant cytotoxicity against MCF-7 cells to a CC50 of 0.8 µg/mL, as well as high selectivity (SI = 24.5). On the other hand, SiHa cells were moderately sensitive to the dichloromethane and ethyl acetate extracts of the same species. (CC50 = 34.9 and 31.5 µg/mL, respectively). None of the extracts of H. melanadocia were considered active due their CC50's were ranged from 59.0 to 94.5 µg/mL.

Neuroprotective effect of Mayan medicinal plant extracts against glutamate-induced toxicity.

Neurological disorders are a public health problem worldwide for which there is currently no direct treatment of the cause of the disorder. The goal of this study was to investigate the potential in vitro neuroprotective property of plants used in Mayan traditional medicine. Plant ethanolic extracts were prepared and tested on models in which neuronal damage was induced by glutamate, i.e., a human neuroblastoma cell line (SH-SY5Y) and rat cortical neurons. HPLC profiles from active extracts were also obtained. A total of 51 plant species were identified in the literature as plant species used in Mayan traditional medicine for the treatment of symptoms suggestive of neurological disorders, and we studied 34 of these in our analysis. Six extracts had a neuroprotective effect on SH-SY5Y cells, with the most active extract being that from Schwenckia americana roots (half maximal effective concentration [EC50] 11.3 ± 2.9 µg/mL), and three extracts exhibited a neuroprotective effect in the rat neuron cortical model, with the most active extract being that from Elytraria imbricata aerial parts (EC50 6.8 ± 3.1 µg/mL). These results suggest that the active extracts from such plants have the potential to be a great resource. Future studies should be performed that are more extensive and which isolate the active constituents.

Lupane-Type Triterpenes of Phoradendron vernicosum.

Three new lupane-type triterpenes, 3α,24-dihydroxylup-20(29)-en-28-oic acid (1), 3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (2), and 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (3), together with eight known compounds (4-11) were isolated from a methanol extract of Phoradendron vernicosum aerial parts. The chemical structures of 1-3 were determined on the basis of spectroscopic data interpretation. The isolated compounds were tested against seven human cancer cell lines and two normal cell lines.

Hepatoprotective Effect of Low Doses of Caffeine on CCl4-Induced Liver Damage in Rats.

Several studies have shown the hepatoprotective effect of the consumption of coffee and tea, which is mainly attributed to caffeine. Many experimental studies have demonstrated this effect; however, these studies used high caffeine doses that are not related to human consumption. The aim of this study was to evaluate the hepatoprotective effect of low doses of caffeine on carbon tetrachloride (CCl4)-treated rats. Low doses of caffeine (CAFF) 5 and 10 mg/kg (CAFF5 and CAFF10) were evaluated in chronic liver damage induced by CCl4 (0.75 mL/kg) in rats. CAFF treatment was administered once a day and CCl4 administration was twice weekly for 10 weeks. Liver function tests (biochemical markers) and functional (sleeping time) and histological (hematoxylin-eosin and Masson trichrome stains) parameters were carried out at the end of damage treatment. Daily treatments of CAFF5 and CAFF10 exhibited a hepatoprotective effect supported by a decrease of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AP) serum activities and bilirubin serum levels compared with control and also restored serum albumin levels and liver glutathione (GSH). Moreover, CAFF prevented CCl4-induced prolongation in pentobarbital sleeping time and a decrease of liver fibrosis and cell death. Our results demonstrated that low doses of CAFF exert a hepatoprotective effect against CCl4 -induced liver damage in rats.

Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an âˆ¼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense.

Anti-giardia activity and acute toxicity of a methanol extract of Senna racemosa bark.

ETHNOPHARMACOLOGICAL RELEVANCE: Senna racemosa (Mill.) H.S. Irwin & Barneby (syn. Cassia racemosa Mill.) is a plant used in traditional Mayamedicinal practices to treat diarrhea. A methanol extract of S. racemosa bark has been shown to have in vitro activity against Giardia intestinalis. No studies of its efficacy and toxicity in in vivo models have been done. The present study objective was to analyze the activity of this methanol extract of S. racemosa bark against Giardia intestinalis trophozoites in experimentally infected mice, and evaluate its toxicological effects in rats. MATERIAL AND METHODS: S. racemosa was collected in Merida, Yucatan, Mexico (21°58'N, 89°36'W) in June 2005. The bark methanol extract was obtained and high performance liquid chromatography (HPLC-DAD) was used to generate a constituent profile. In vivo anti-giardia activity was assayed with an experimental model of G. intestinalis infection in neonatal CD-1 mice. Nine doses ranging from 0.25-15mg extract/kg body weight were tested to determine the dose required to kill 50% of the trophozoites (ED50). An acute toxicity assay was run in which one of four single doses (200, 1000, 2000 and3000mg/kg body weight) was orally administered to adult Wistar rats. Animal weight, death rates, toxic effects and behavioral parameters were observed over a 14-d period. They were then euthanized and a necropsy performed. RESULTS: The S. racemosa bark extract inhibited growth of G. intestinalis (ED50=1.14mg/Kg) in neonatal CD-1 mice. No toxic or lethal effects were observed even at the highest dosage (3000mg/Kg), and neither were signs of toxicity observed in internal organs. The active compounds chrysophanol and physcion were present in the extract at a 1.76 ratio. CONCLUSIONS: The results strongly support traditional use of S. racemosa bark for treatment of diarrhea caused by Giardia intestinalis infection.

Mercury levels assessment and its relationship with oxidative stress biomarkers in children from three localities in Yucatan, Mexico.

Mercury (Hg) is a global pollutant that is released into the environment from geologic and anthropogenic sources. Once it enters an organism, it generates several toxicity mechanisms and oxidative stress has been proposed as the main one. Metal susceptibility is greater in children, which is a result of their physiology and behavior. In Yucatan, Mexico, burning of unregulated garbage dumps and household trash, ingestion of top marine predators, and pottery manufacturing are among the conditions that could promote Hg exposure. However, for Yucatan, there are no published studies that report Hg levels and associated oxidative stress status in children. Therefore, this study aimed to assess Hg levels in blood and urine and oxidative stress biomarkers levels in a sample of 107 healthy children from three localities in Yucatan, Mexico, as well as investigate the relationship between these parameters. Hg was detected in 11 (10.28%) of blood samples and 38 (35.51%) of urine samples collected from the participating children. Fourteen subjects showed Hg above recommended levels. The oxidative stress biomarkers were slightly elevated in comparison with other studies and were statistically different between the sampling sites. No linear correlation between Hg levels and oxidative stress biomarkers was found. Nevertheless, exploratory univariate and multivariate analysis showed non-linear relations among the measured variables. Globally, the study provides, for the first time, information regarding Hg levels and their relationship with oxidative stress biomarkers in a juvenile population from Mexico's southeast (Yucatan) region. In agreement with worldwide concern about Hg, this study should stimulate studies on metal monitoring in humans (especially children) among scientists working in Mexico, the establishment of polices for its regulation, and the reduction of human health risks.

ANTIPROTOZOAL ACTIVITY OF EXTRACTS OF ELAEODENDRON TRICHOTOMUM (CELASTRACEAE).

BACKGROUND: Chagas disease, amebiasis, giardiasis and trichomoniasis represent a serious health problem in Latin America. The drugs employed to treat these illnesses produce important side effects and resistant strains have appeared. The present study was aimed to evaluate the antiprotozoal activity of leaves, stem bark and root bark of Elaeodendron trichotomum, a celastraceus, that is used in Mexico as an anti-infective in febrile-type diseases. MATERIALS AND METHODS: Dichloromethane and methanol extracts of leaves, bark and roots of Elaeodendron trichotomum were tested against Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Trypanosoma cruzi. A quantitative HPLC analysis of pristimerin and tingenone was performed. RESULTS: The dichloromethane extract of roots was active against E. histolytica, G. lamblia, T. vaginalis, and T. cruzi, at IC50's of 0.80, 0.44, 0.46, and 2.68 µg/mL, respectively. The HPLC analysis revealed the presence of tingenone (3.84%) and pristimerin (0.14%). CONCLUSIONS: The dichloromethane extract of the roots bark showed significant activity against all screened protozoa.

Isolation and Identification of Cytotoxic Compounds from Aeschynomene fascicularis, a Mayan Medicinal Plant.

The plant Aeschynomene fascicularis (Fabaceae) has been used in Mayan traditional medicine in the Yucatan peninsula. However, the compounds present in the plant responsible for its curative properties have not yet been investigated. Aeschynomene fascicularis root bark was extracted with 100% methanol to obtain a crude extract. The methanol extract was partitioned successively with solvents with increasing polarity to obtain the corresponding hexane (Hx), dichloromethane (DCM) and ethyl acetate fractions (EtOAc), as well as a residual water-alcoholic fraction. These fractions were tested for their cytotoxic activities using an MTT assay against Hep-2 cancer cell lines. The Hx fraction led to the isolation of spinochalcone C (1), spinochalcone A (2), isocordoin (3) and secundiflorol G (4). Their structures were identified based on spectroscopic evidence and chemical properties. All compounds were subjected to cytotoxicity and antiproliferative assays against a panel of seven cell lines, including one normal-type cell line. Spinochalcone A (2) exhibited cytotoxic activity against DU-145 cell line and antiproliferative activity against the KB cell line. Secundiflorol G (4) showed strong cytotoxic activity towards KB and Hep-2 cell lines. In addition, isocordoin (3) showed moderate activity on KB, Hep-2 and DU-145 cell lines. The active Compounds 2, 3 and 4 are potential therapeutic entities against cancer.

In vitro and in vivo trypanocidal activity of native plants from the Yucatan Peninsula.

Ethanol extracts of Senna villosa, Serjania yucatanensis, Byrsonima bucidaefolia, and Bourreria pulchra were evaluated for their in vitro activity against epimastigotes and trypomastigotes of Trypanosoma cruzi. Results showed that the leaf extracts of S. yucatanensis and B. pulchra were the most active against epimastigotes (IC(100) = 100 µg/mL) and trypomastigotes of T. cruzi (95% or more reduction in the number of parasites at 100 and 50 µg/mL). However, only the leaf extract of S. yucatanensis showed significant trypanocidal activity when tested in vivo, reducing 75% of the parasitemia in infected mice at 100 mg/kg. This same extract inhibited the egress of trypomastigotes from infected cells and proved not to be cytotoxic (IC(50) = 318.8 ± 2.3 µg/mL).

Long-lasting resistance to haloperidol-induced catalepsy in male rats chronically treated with caffeine.

Chronic caffeine consumption has been inversely associated with the risk of developing Parkinson's disease. Here we assessed whether chronic caffeine treatment increases the resistance of male Wistar rats to haloperidol (1mg/kg, s.c.)-induced catalepsy, measured in the bar test at 15 min intervals during 3h. Caffeine (5mg/kg/day) was delivered for 6 months via drinking water. Control rats received only tap water. Treatments began when animals were 3-4 months old. In order to unveil long-lasting catalepsy refractoriness not attributable to the presence of caffeine in the brains of rats, they were evaluated from day 18 to day 27 after caffeine withdrawal, a time that is far in excess for the full excretion of a caffeine dose in this species. The average cataleptic immobility measured in caffeine-treated rats (n=23) was 1148+/-140 s, a value 34+/-8% lower than that recorded in control animals (n=20), whose mean immobility was 1736+/-137 s (P=0.0026, t-test). The percentage of catalepsy reduction measured in caffeine-treated rats evaluated on days 18-20 after caffeine discontinuation (-32+/-13%, n=12, P<0.05) was comparable to the catalepsy decrease recorded in those animals tested on days 21-27 (-36+/-10%, n=11, P<0.02), a finding compatible with the notion that the effect was indeed mediated by enduring changes of brain functioning and not by the physical presence of caffeine or its metabolites. Caffeine-treated rats also had higher catalepsy latency scores compared with control rats (P<0.01, U-test). The present findings show that chronic consumption of caffeine produces perdurable resistance to catalepsy induced by dopamine receptor blockade, possibly through enhancement of dopamine transmission, giving further support to the epidemiological results indicating that prolonged caffeine consumption affords neuroprotection against Parkinson's disease.

Antiprotozoal activity of Senna racemosa.

Methanol extracts of leaves, roots and bark of Senna racemosa (Mill.) H.S. Irwin & Barneby (syn. Cassia racemosa Mill.) were tested for antiprotozooal activity against Giardia intestinalis and Entamoeba histolytica. All of the tested extracts showed good activity against both protozoa species. Extracts from stem bark and leaves were most active, with an IC(50) of 2.10 microg/mL for Giardia intestinalis and 3.87 microg/mL for Entamoeba histolytica. Of the previously isolated compounds from Senna racemosa, the piperidine alkaloid cassine had greater activity against Giardia intestinalis with an IC(50) of 3.28 microg/mL and chrysophanol, a 1,8-dihydroxy-anthraquinone, was the most active agent against Entamoeba histolytica, with an IC(50) of 6.21 microg/mL.

Antigiardial activity of triterpenoids from root bark of Hippocratea excelsa.

Two new triterpenoids, 21 beta-hydroxyolean-12-en-3-one (1) and a seco-dinor derivative of pristimerine named dzununcanone (2), were isolated from the root bark of Hippocratea excelsa. Their structures were assigned on the basis of spectroscopic evidence, mainly 1H and 13C 1D and 2D NMR including DEPT, COSY, ROESY, HSQC, and HMBC experiments, as well as EIMS and HREIMS. The known 21alpha-hydroxy-3-oxofriedelane (3), a compound new to the species, and the known methide quinones pristimerine (4) tingenone (5), and xuxuarine Ebeta (7) were also isolated. The antiprotozoal activities were determined against Giardia intestinalis. Pristimerine and tingenone were the most active antigiardial compounds, with IC50 values of 0.11 and 0.74 microM, respectively, compared with metronidazole, the current drug of choice (IC50 1.23 microM).

A novel automated rat catalepsy bar test system based on a RISC microcontroller.

Catalepsy tests performed in rodents treated with drugs that interfere with dopaminergic transmission have been widely used for the screening of drugs with therapeutic potential in the treatment of Parkinson's disease. The basic method for measuring catalepsy intensity is the "standard" bar test. We present here an easy to use microcontroller-based automatic system for recording bar test experiments. The design is simple, compact, and has a low cost. Recording intervals and total experimental time can be programmed within a wide range of values. The resulting catalepsy times are stored, and up to five simultaneous experiments can be recorded. A standard personal computer interface is included. The automated system also permits the elimination of human error associated with factors such as fatigue, distraction, and data transcription, occurring during manual recording. Furthermore, a uniform criterion for timing the cataleptic condition can be achieved. Correlation values between the results obtained with the automated system and those reported by two independent observers ranged between 0.88 and 0.99 (P<0.0001; three treatments, nine animals, 144 catalepsy time measurements).