Evaluation of the Anti-Toxoplasma gondii Efficacy, Cytotoxicity, and GC/MS Profile of Pleopeltis crassinervata Active Subfractions.

Pleopeltis crassinervata (Pc) is a fern that, according to ethnobotanical records, is used in Mexican traditional medicine to treat gastrointestinal ailments. Recent reports indicate that the hexane fraction (Hf) obtained from Pc methanolic frond extract affects Toxoplasma gondii tachyzoite viability in vitro; therefore, in the present study, the activity of different Pc hexane subfractions (Hsf) obtained by chromatographic methods was evaluated in the same biological model. Gas chromatography/mass spectrometry (GC/MS) analysis was carried out for hexane subfraction number one (Hsf1), as it showed the highest anti-Toxoplasma activity with a half-maximal inhibitory concentration (IC50) of 23.6 µg/mL, a 50% cytotoxic concentration (CC50) of 398.7 µg/mL in Vero cells, and a selective index (SI) of 16.89. Eighteen compounds were identified by Hsf1 GC/MS analysis, with the majority being fatty acids and terpenes. Hexadecanoic acid, methyl ester was the most commonly found compound (18.05%) followed by olean-13(18)-ene, 2,2,4a,8a,9,12b,14a-octamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,12b,13,14,14a,14b-eicosahydropicene, and 8-octadecenoid acid, methyl ester, which were detected at 16.19%, 12.53%, and 12.99%, respectively. Based on the mechanisms of action reported for these molecules, Hsf1 could exert its anti-Toxoplasma activity mainly on T. gondii lipidomes and membranes.

Antidiabetic and Toxicological Effects of the Tea Infusion of Summer Collection from Annona cherimola Miller Leaves.

Annona cherimola Miller (Ac) is a plant used in Mexican traditional medicine for the treatment of diabetes. In this work, the tea infusion extracts obtained from 1.5 g of leaf powder from Ac collected in May (AcMa), June (AcJun), July (AcJul), and August (AcAu) were evaluated on streptozocin-induced diabetic (STID) mice and for subchronic toxicity in STID and non-diabetic (ND) mice. In addition, extracts were subjected to high-performance liquid chromatography with diode array detection (HPLC-DAD). Results showed that the tea infusion extract of the sample collected in August (AcAu) exhibited the most significant antihyperglycemic activity during all acute assays. The analysis of the extracts (AcMa, AcJu, AcJul, and AcAu) by HPLC-DAD revealed that flavonoid glycosides, rutin, narcissin, and nicotiflorin were the major components. In addition, the sample AcAu contained the best concentration of flavonoids. In the case of subchronic oral toxicity, the AcAu sample did not cause mortality in STID mice, and histopathological analysis revealed significant improvement in the changes associated with diabetes in the liver and kidneys. These findings suggest that the Ac leaves collected in August may be a source of flavonoids such as rutin, with antidiabetic potential. In addition, these findings support the use of Ac to treat diabetes in traditional medicine.

Understanding the Antilymphoma Activity of Annona macroprophyllata Donn and Its Acyclic Terpenoids: In Vivo, In Vitro, and In Silico Studies.

Annona macroprophyllata Donn (A. macroprophyllata) is used in traditional Mexican medicine for the treatment of cancer, diabetes, inflammation, and pain. In this work, we evaluated the antitumor activity of three acyclic terpenoids obtained from A. macroprophyllata to assess their potential as antilymphoma agents. We identified the terpenoids farnesyl acetate (FA), phytol (PT) and geranylgeraniol (Gg) using gas chromatography-mass spectroscopy (GC-MS) and spectroscopic (1H, and 13C NMR) methods applied to petroleum ether extract of leaves from A. macroprophyllata (PEAm). We investigated antitumor potential in Balb/c mice inoculated with U-937 cells by assessing brine shrimp lethality (BSL), and cytotoxic activity in these cells. In addition, to assess the potential toxicity of PEAm, FA, PT and Gg in humans, we tested their acute oral toxicity in mice. Our results showed that the three terpenoids exhibited considerable antilymphoma and cytotoxic activity. In terms of lethality, we determined a median lethal dose (LD50) for thirteen isolated products of PEAm. Gg, PT and AF all exhibited a higher lethality with values of 1.41 ± 0.42, 3.03 ± 0.33 and 5.82 ± 0.58 µg mL-1, respectively. To assess cytotoxic activity against U-937 cells, we calculated the mean cytotoxic concentration (CC50) and found that FA and PT were closer in respect to the control drug methotrexate (MTX, 0.243 ± 0.007 µM). In terms of antilymphoma activity, we found that FA, PT and Gg considerably inhibited lymph node growth, with median effective doses (ED50) of 5.89 ± 0.39, 6.71 ± 0.31 and 7.22 ± 0.51 mg kg-1 in females and 5.09 ± 0.66, 5.83 ± 0.50 and 6.98 ± 0.57mg kg -1 in males, respectively. Regarding acute oral toxicity, we classified all three terpenoids as category IV, indicating a high safety margin for human administration. Finally, in a molecular docking study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, we found binding of terpenoids to some amino acids of the catalytic site, suggesting an effect upon activity with a resulting decrease in the synthesis of intermediates involved in the prenylation of proteins involved in cancer progression. Our findings suggest that the acyclic terpenoids FA, PT, and Gg may serve as scaffolds for the development of new treatments for non-Hodgkin's lymphoma.

Antitumor Potential of Annona muricata Linn. An Edible and Medicinal Plant in Mexico: In Vitro, In Vivo, and Toxicological Studies.

Annona muricata (Am) is a plant used in traditional Mexican medicine to treat cancer. In this study, ethanol extracts of Am collected in Acapulco and Tecpan from Guerrero state were evaluated orally on Balb/c mice inoculated with 4T1 cells, for cytotoxic activity (CA) on 4T1 cells, in brine shrimp lethality assay (BSLA), and for acute oral toxicity in mice. In addition, ethanol extracts were subjected to high-performance liquid chromatography (HPLC) with diode array detection. Results showed that the extracts collected in December in Acapulco (AcDe) and Tecpan (TeDe) exhibited the most significant antitumor and cytotoxic activity. In the BSLA, the most important effect was observed in the extracts from Acapulco and Tecpan collected in June (AcJu) and August (TeAg), respectively. The samples from Acapulco (AcJu, and AcAg) and Tecpan (TeJu and TeAg) showed the highest toxicity. The analysis of the extracts, AcDe and TeDe, by HPLC revealed that flavonoids, rutin, narcissin, and nicotinflorin were the major components. These findings suggest that extracts from Am collected in Acapulco and Tecpan in the month of December may be an important source to obtain flavonoid glycosides with anticancer potential specifically against breast cancer. This also supports the use of Am to treat cancer in Mexican traditional medicine.

Antihyperglycemic and Antilipidemic Properties of a Tea Infusion of the Leaves from Annona cherimola Miller on Streptozocin-Induced Type 2 Diabetic Mice.

The antihyperglycemic and antilipidemic effects of the tea infusion extracts of leaves from Annona cherimola Miller (IELAc-0.5, IELAc-1.5, and IELAc-3.0) were evaluated on normoglycemic (NG) and streptozocin-induced diabetic (STID) mice. In the acute test, IELAc-1.5 at 300 mg/kg bodyweight (bw) exhibited antihyperglycemic activity on STID mice since the first hour of treatment. Then, its antidiabetic potential was analyzed in a subchronic evaluation. IELAc-1.5 was able to reduce the blood glucose level, glycated hemoglobin (HbA1c), cholesterol (CHO), and triglycerides (TG); high-density lipoprotein (HDL) showed an increase at the end of treatment. IELAc-1.5 did not modify the urine profile at the end of the evaluation, and neither toxicity nor macroscopic organ damage were observed in acute and subchronic assays. In addition, a major flavonol glycoside present in the tea infusion extracts was identified using high-performance liquid chromatography with diode array detection (HPLC-DAD). The analysis of the tea infusion extracts by HPLC revealed that rutin was the major component. This study supports the use of tea infusions from Annona cherimola for the treatment of diabetes and suggests that rutin could be responsible, at least in part, for their antidiabetic properties.

Antihyperglycemic and Lipid Profile Effects of Salvia amarissima Ortega on Streptozocin-Induced Type 2 Diabetic Mice.

Salvia amarissima Ortega was evaluated to determinate its antihyperglycemic and lipid profile properties. Petroleum ether extract of fresh aerial parts of S. amarissima (PEfAPSa) and a secondary fraction (F6Sa) were evaluated to determine their antihyperglycemic activity in streptozo-cin-induced diabetic (STID) mice, in oral tolerance tests of sucrose, starch, and glucose (OSTT, OStTT, and OGTT, respectively), in terms of glycated hemoglobin (HbA1c), triglycerides (TG), and high-density lipoprotein (HDL). In acute assays at doses of 50 mg/kg body weight (b.w.), PEfAPSa and F6Sa showed a reduction in hyperglycemia in STID mice, at the first and fifth hour after of treatment, respectively, and were comparable with acarbose. In the sub-chronic test, PEfAPSa and F6Sa showed a reduction of glycemia since the first week, and the effect was greater than that of the acarbose control group. In relation to HbA1c, the treatments prevented the increase in HbA1c. In the case of TG and HDL, PEfAPSa and F6Sa showed a reduction in TG and an HDL increase from the second week. OSTT and OStTT showed that PEfAPSa and F6Sa significantly lowered the postprandial peak at 1 h after loading but only in sucrose or starch such as acarbose. The results suggest that S. amarissima activity may be mediated by the inhibition of disaccharide hydrolysis, which may be associated with an α-glucosidase inhibitory effect.

Antihyperglycemic Effects of Annona diversifolia Safford and Its Acyclic Terpenoids: α-Glucosidase and Selective SGLT1 Inhibitiors.

Annona diversifolia Safford and two acyclic terpenoids were evaluated to determine their antihyperglycemic activity as potential α-glucosidase and selective SGLT-1 inhibitiors. Ethanolic extract (EEAd), chloroformic (CHCl3Fr), ethyl acetate (EtOAcFr), aqueous residual (AcRFr), secondary 5 (Fr5) fractions, farnesal (1), and farnesol (2) were evaluated on normoglycemic and streptozocin-induced diabetic mice. EEAd, CHCl3Fr, Fr5, (1) and (2) showed antihyperglycemic activity. The potential as α-glucosidase inhibitors of products was evaluated with oral sucrose and lactose tolerance (OSTT and OLTT, respectively) and intestinal sucrose hydrolysis (ISH) tests; the potential as SGLT-1 inhibitors was evaluated using oral glucose tolerance (OGTT), intestinal glucose absorption (IGA), and urinary glucose excretion (UGE) tests. In OSTT and OLTT, all treatments showed significant activity at two and four hours. In ISH, half maximal effective concentrations (CE50) of 565, 662 and 590 µg/mL, 682 and 802 µM were calculated, respectively. In OGTT, all treatments showed significant activity at two hours. In IGA, CE50 values of 1059, 783 and 539 µg/mL, 1211 and 327 µM were calculated, respectively. In UGE Fr5, (1) and (2) showed significant reduction of the glucose excreted compared with canagliflozin. These results suggest that the antihyperglycemic activity is mediated by α-glucosidase and selective SGLT-1 inhibition.

Antioxidant Properties and Protective Effects of Some Species of the Annonaceae, Lamiaceae, and Geraniaceae Families against Neuronal Damage Induced by Excitotoxicity and Cerebral Ischemia.

This study aimed to compare the antioxidant activities of extracts obtained from three plant families and evaluate their therapeutic effect on strokes. Ethanol extracts were obtained from either the leaf or the aerial parts of plants of the families Annonaceae (Annona cherimola, A. diversifolia, A. muricata, A. purpurea, and A. reticulata), Lamiaceae (Salvia amaríssima and S. polystachya), and Geraniaceae (Geranium niveum and G. mexicanum). Extracts were analyzed in terms of hydroxyl radical (OH•), peroxyl radical (ROO•), and superoxide anion (O2•-). The efficiency of the extracts to prevent neuronal death induced by excitotoxicity was tested with the tetrazolium assay, the O2•- scavenging capacity was evaluated with the dihydroethidium dye, and the protective effect of the extracts with the highest antioxidant activity was tested on a stroke experimental model. The extracts' IC50 values (µg/mL) of scavenging varied from 98.9 to 155.04, 4.5 to 102.4, and 20.2 to 118.97 for OH•, ROO•, and O2•-, respectively. In the excitotoxicity model, Annonaceae extracts were highly cytotoxic while Lamiaceae and Geraniaceae reduced intracellular O2•- production and protect neurons against oxidative stress. Salvia polystachya reduced cerebral damage, as well as improved survival and behavior after ischemia. Our results encouraged the use of plant extracts as natural antioxidants to minimize neuronal injury following stroke.

Plants used for the treatment of diarrhoea from Mexican flora with amoebicidal and giadicidal activity, and their phytochemical constituents.

ETHNOPHARMACOLOGICAL RELEVANCE: Flora of the Mexican region is represented by approximately 30,000 vascular plant species, many of which are used as traditional medicines based on knowledge compiled and refined since ancient times (e.g. Cruz-Badiano and Florentino Codexes). The traditional use of plants as medicines, including the treatment of infectious diseases such as diarrhoea, is generally practiced in communities that are geographically isolated or in human settlements where health services are scarce. AIM OF THE STUDY: The aim of this review is to evaluate current research advances in the ethnopharmacology and phytochemistry of Mexican medicinal plants exhibiting antiprotozoal activity, used to treat diarrhoea, and to identify the gaps in this research area for future studies. METHODS: The literature study and compilation of information relied on books and scientific journals from leading electronic databases including Scopus, Springer, SciFinder, ISI Web of Science, PubMed, and Google Scholar; the topics searched for were antiprotozoal activity, followed by Entamoeba histolytica or Giardia lamblia. Ethnopharmacological data were obtained from books regarding medicinal plants and their uses in Mexico as well as from the government databases of "Comisiòn Nacional para el Conocimiento y Uso de la Biodiversidad (CONABIO)" as "NaturaLista" and "EncicloVida". RESULTS: A total of 80 plant species used in the Mexican traditional medicine for the treatment of diarrhoea have been evaluated as potential sources of antiprotozoal agents against E. histolytica or G. lamblia. From these samples, 150 compounds have been isolated and screened in vitro, including specialized metabolites such as flavonoids and terpenoids as well as semisynthesized derivatives. However, in vivo pharmacological studies on these substances are limited. Additional pharmacological and molecular studies of the most active compounds have also been summarized. CONCLUSION: Research performed in the past 25 years on specialized metabolites derived from plants with antiprotozoal activity has yielded relevant findings whose results provide evidence-based support for the use of these plants in the traditional medicine of Mexico to treat diarrhoea. Toxicological and clinical trials of standardized extracts and bioactive compounds are proposed as priority future works in this research area. In addition, in vivo assays are required, of more extracts and/or pure compounds. The optimization of the pharmacological properties of the bioactive specialized metabolites through semisynthetic derivatives and computational methods could aid in developing new antiprotozoal phytomedicines and novel drugs for the treatment of these types of infections. Furthermore, elucidation of the mechanism of action of these bioactive compounds through pharmacological and molecular studies are also necessary.

Antiprotozoal, Antibacterial and Antidiarrheal Properties from the Flowers of Chiranthodendron pentadactylon and Isolated Flavonoids.

BACKGROUND: Chiranthodendron pentadactylon Larreat. (Sterculiaceae) is a Mexican plant used in traditional medicine for the treatment of heart disease symptoms and infectious diarrhea. OBJECTIVE: To evaluate in vitro antiprotozoal and antibacterial activities and in vivo antidiarrheal activity from the flowers of C. pentadactylon using the extract, fractions, and major isolated flavonoids. MATERIALS AND METHODS: Bioassay-guided fractionation of the methanol extract of C. pentadactylon (MECP) led to the isolation of five flavonoids, tiliroside, astragalin, isoquercitrin, (+)-catechin, and (-)-epicatechin. Antimicrobial activities were tested on two protozoa (Entamoeba histolytica and Giardia lamblia) and nine bacterial enteropathogens (two Escherichia coli strains, two Shigella sonnei strains, two Shigella flexneri strains, two Salmonella sp. strains, and Vibrio cholerae) isolated from feces of children with acute diarrhea or dysentery and resistant to chloramphenicol. Also, antidiarrheal activity was tested on cholera toxin-induced diarrhea in male Balb-c mice. RESULTS: Epicatechin was the most potent antiamoebic and antigiardial compound with IC50 values of 1.9 µg/mL for E. histolytica and 1.6 µg/mL for G. lamblia; tiliroside showed moderate antiprotozoal activity against both protozoan. In contrast, in the antibacterial activity, tiliroside was the most potent compound on all microorganisms with minimum inhibitory concentration values less than 0.7 mg/mL. In the case of cholera toxin-induced diarrhea, epicatechin was the most potent flavonoid with IC50 of 14.7 mg/kg. CONCLUSION: Epicatechin and tiliroside were the flavonoids responsible for antimicrobial andantidiarrheal activities of C. pentadactylon. Its antiprotozoal, antibacterial, and antidiarrheal properties are in good agreement with the traditional medicinal use of C. pentadactylon for the treatment of infectious diarrhea. SUMMARY: Epicatechin was the most potent antiamoebic and antigiardial compound with IC50 values of 1.9 µg/mL for E. histolytica and 1.6 µg/mL for G. lamblia.Tiliroside showed antibacterial activity against all microorganisms tested with MIC values less than 0.7 mg/mL.Epicatechin was the most potent flavonoid on cholera toxin-induced diarrhea with IC50 of 14.7 mg/kg. Abbreviations used: MECP: Methanol extract of C. pentadactylon.

Antiprotozoal Activities of Tiliroside and other Compounds from Sphaeralcea angustifolia (Cav.) G. Don.

BACKGROUND: Sphaeralcea angustifolia (Malvaceae) is extensively used in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. OBJECTIVE: The current study was to validate the traditional use of S. angustifolia for the treatment of diarrhea and dysentery on biological grounds using in vitro antiprotozoal activity and computational experiments. MATERIALS AND METHODS: The ethanol extract, subsequent fractions, flavonoids, phenolic acids, and a sterol were evaluated on Entamoeba histolytica and Giardia lamblia trophozoites. Moreover, molecular docking studies on tiliroside were performed; it was tested for its affinity against pyruvate:ferredoxin oxidoreductase (PFOR) and fructose-1,6-bisphosphate aldolase (G/FBPA), two glycolytic enzymes of anaerobic protozoa. RESULTS: Bioassay-guided fractionation of extract of the aerial parts of S. angustifolia gives tiliroside and apigenin, caffeic acid, protocatechuic acid, and ß-sitosterol. The in vitro antiprotozoal assay showed that tiliroside was the most potent antiprotozoal compound on both protozoa with 50% inhibitory concentration values of 17.5 µg/mL for E. histolytica and 17.4 µg/mL for G. lamblia. Molecular docking studies using tiliroside showed its probable antiprotozoal mechanism with PFOR and G/FBPA. In both cases, tiliroside showed high affinity and inhibition constant theoretic for PFOR (lowest free binding energy from -9.92 kcal/mol and 53.57 µM, respectively) and G/FBPA (free binding energy from -7.17 kcal/mol and 55.5 µM, respectively), like to metronidazole, revealing its potential binding mode at molecular level. CONCLUSION: The results suggest that tiliroside seems to be a potential antiprotozoal compound responsible for antiamoebic and antigiardial activities of S. angustifolia. Its in vitro antiprotozoal activities are in good agreement with the traditional medicinal use of S. angustifolia in gastrointestinal disorders such as diarrhea and dysentery. SUMMARY: Bioassay-guided fractionation of extract of the aerial parts of S. angustifolia gives: tiliroside and apigenin, caffeic acid, protocatechuic acid) and ß-sitosterol. The in vitro antiprotozoal assay showed that tiliroside was the most potent antiprotozoal compound on both protozoa with IC50 values of 17.5 mg/mL for E. histolytica and 17.4 µg/mL for G. lamblia. Molecular docking studies using tiliroside showed its probable antiprotozoal mechanism with PFOR and G/FBPA. In both cases tiliroside showed high affinity and inhibition constant theoretic for PFOR (lowest free binding energy from -9.92 kcal/mol and 53.57 mM, respectively) and G/FBPA (free binding energy from -7.17 kcal/mol, respectively and 55.5 µM), like to metronidazole, revealing its potential binding mode at molecular level. The results suggest that tiliroside seems to be a potential antiprotozoal compound responsible for antiamoebic and antigiardial activities of Sphaeralcea angustifolia. Abbreviations Used: PFOR: Pyruvate:ferredoxin oxidoreductase; G/FBPA: Fructose 1,6 bisphosphate aldolase.

Antihyperglycemic Activity of the Leaves from Annona cherimola Miller and Rutin on Alloxan-induced Diabetic Rats.

BACKGROUND: Annona cherimola, known as "chirimoya" has been reported in Mexican traditional medicine for the treatment of diabetes. OBJECTIVE: The aims of the present study were to validate and assess the traditional use of A. cherimola as an antidiabetic agent. MATERIALS AND METHODS: The ethanol extract from A. cherimola (300 mg/kg, EEAc), subsequent fractions (100 mg/kg), and rutin (30 mg/kg) were studied on alloxan-induced type 2 diabetic (AITD) and normoglycemic rats. In addition, oral glucose tolerance test (OGTT) and oral sucrose tolerance test (OSTT) were performed in normoglycemic rats. Molecular docking technique was used to conduct the computational study. RESULTS: Bioassay-guided fractionation of EEAc afforded as major antihyperglycemic compound, rutin. EEAc attenuated postprandial hyperglycemia in acute test using AITD rats (331.5 mg/dL) carrying the glycemic levels to 149.2 mg/dL. Rutin after 2 h, attenuated postprandial hyperglycemia in an acute assay using AITD rats such as EEAc, with maximum effect (150.0 mg/dL) being seen at 4 h. The antihyperglycemic activities of EEAc and rutin were comparable with acarbose (151.3 mg/dL). In the subchronic assay on AITD rats, the EEAc and rutin showed a reduction of the blood glucose levels since the 1st week of treatment, reaching levels similar to normoglycemic state (116.9 mg/kg) that stayed constant for the rest of the assay. OGTT and OSTT showed that EEAc and rutin significantly lowered blood glucose levels in normoglycemic rats at 2 h after a glucose or sucrose load such as acarbose. Computational molecular docking showed that rutin interacted with four amino acids residues in the enzyme α-glucosidase. CONCLUSION: The results suggest that rutin an α-glucosidase inhibitor was responsible in part of the antihyperglycemic activity of A. cherimola. Its in vivo antihyperglycemic activity is in good agreement with the traditional use of A. cherimola for the treatment of diabetes. SUMMARY: The ethanol extract from Annona cherimola (300 mg/kg, EEAc), subsequent fractions (100 mg/kg) and rutin (30 mg/kg) were studied on alloxan-induced type 2 diabetic (AITD) and normoglycemic rats. The results suggest that rutin; an α-glucosidase inhibitor was responsible in part of the antihyperglycemic activity of A. cherimola. Its in vivo antihyperglycemic activity is in good agreement with the traditional use of A. cherimola for the treatment of diabetes. Abbreviations Used: EEAc: The ethanol extract from Annona cherimola, AITD: Alloxan-induced type 2 diabetic rats, OGTT: Oral glucose tolerance test, OSTT: Oral sucrose tolerance test, DM: Diabetes mellitus.

Antiprotozoal Constituents from Annona cherimola Miller, a Plant Used in Mexican Traditional Medicine for the Treatment of Diarrhea and Dysentery.

BACKGROUND: Annona cherimola Miller (Annonaceae) is a medicinal plant frequently recommended in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. OBJECTIVE: This work was undertaken to obtain information that support the traditional use of A. cherimola, on pharmacological basis using in vitro and computational experiments. MATERIAL AND METHODS: Bioassay-guided fractionation of the ethanol extract of the leaves of A. cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin, and rutin. RESULTS: The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 µg/mL for Entamoeba histolytica and 8.7 µg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different than metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase (PFOR). CONCLUSION: Considering that PFOR is a target of metronidazole; kaempferol may be a lead compound for the development of novel antiprotozoal agent. Also, these findings give support to the use of A. cherimola in the traditional medicine from México for the treatment of diarrhea and dysentery. SUMMARY: Bioassay-guided fractionation of the ethanol extract of the leaves of Annona cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin and rutin. The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 µg/mL for Entamoeba histolytica and 8.7 µg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different that metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase. Abbreviations used: PFOR:Pyruvate:ferredoxin oxidoreductase, G: lamblia: Giardia lamblia, E: histolytica: Entamoeba histolytica.

Antiamoebic and Antigiardial Activity of Clerodane Diterpenes from Mexican Salvia Species Used for the Treatment of Diarrhea.

Terpenoids from Salvia species have been identified to possess biological properties as antiprotozoal agents. Here, we evaluated the antiamoebic and antigiardial activities of 14 known clerodane and modified clerodane-type diterpenes isolated from five Mexican Salvia species against Entamoeba histolytica and Giardia lamblia, and analyzed the effects of the functionalities in decalin ring or in the whole clerodane framework to visualize the structural requirements necessary to produce an antiprotozoal activity. Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 µM for E. histolytica and of 28.2 µM in the case of G. lamblia. In this context it may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea.

Evaluation of the diuretic activity in two Mexican medicinal species: Selaginella nothohybrida and Selaginella lepidophylla and its effects with ciclooxigenases inhibitors.

ETHNOPHARMACOLOGICAL RELEVANCE: Doradilla is a plant that has a long history in the Mexican traditional system of medicine for gall and renal stones, diuresis, stomach and liver inflammation among other diseases. Major components isolated from these plants include biflavonoids as amentoflavone (1), robustaflavone (2) and (S)-2,3-dihydrorobustaflavone (3) and the carbohydrate trehalose (4). The aim of this study was to evaluate the diuretic effect of the decoction of Selaginella nothohybrida Valdespino and Selaginella lepidophylla (Hook & Grev) Spring (Selaginellaceae), and compounds 1-4. We also explored the probable mode of action comparing the effects when using nonspecific and specific COX׳s inhibitors. MATERIALS AND METHODS: Three biflavonoids (1-3) were isolated from the ethyl acetate extraction of the aqueous decoction and the carbohydrate trehalose (4) from the aqueous phase. The structures of all compounds were elucidated by spectroscopic methods and comparisons were made against published data. The diuretic activity was assessed in mice by oral administration of the decoctions in doses of 1000 and 2000mg/kg and biflavonoids 1-3 and trehalose (4) in a dose range of 10mg/kg using furosemide as a standard drug. Inhibitors of COX׳s such as acetyl salicylic acid, sodium naproxen, indomethacin and Celebrex were also assayed to analyze the involvement of renal prostaglandins in diuresis. Water excretion rate, pH, density, conductivity, and contents of Na(+) and K(+) were measured in the urine of mice. RESULTS: Decoction of Selaginella lepidophylla showed lower effect in the urine output at doses of 1000 and 2000mg/kg, while decoction of Selaginella nothohybrida produced an increase at 2000mg/kg (P<0.05). Urinary electrolytes excretion was also affected by this last extract and pure compounds: decoction diminished urinary excretion of sodium and potassium ions, so as compounds 1 and 4; compounds 2 and 3 observed just a natriuretic effect. Pretreated mice with COX׳s inhibitors and then with test compounds 1, 2, 4 and decoction showed inhibition of diuresis in all cases exception for treatment with trehalose (4); natriuretic effect was observed in all cases except for biflavonoid robustaflavone (2) which behaved as the reference compound furosemide. Selaginella nothohybrida decoction behaved similarly to COX-2 inhibitor Celebrex (8), inhibiting diuresis. CONCLUSIONS: Selaginella nothohybrida presents a moderate diuretic effect, which appears to be in partly mediated by the presence of biflavonoids and trehalose. Renal prostaglandins may be involved in the mechanism of diuresis. The present results provide a quantitative basis explaining the traditional folk medicine use of Selaginella nothohybrida as a diuretic agent by Mexican population.

Effect of the sesquiterpene lactone incomptine A in the energy metabolism of Entamoeba histolytica.

Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism.

Comparison of antioxidant activity of hydroethanolic fresh and aged garlic extracts and their effects on cerebral ischemia.

Antioxidant properties and protective effect of aged garlic extract (AGE) and of 20% hydroethanolic fresh extracts from garlic clove (GCE) and skin (GSE) on cerebral ischemia were evaluated by administering extracts at the beginning of reperfusion in a rat model of stroke. All three extracts scavenged superoxide anion, peroxynitrite anion, and peroxyl radicals, but with different efficiencies; furthermore, GCE and GSE scavenged hydroxyl radicals and GSE scavenged singlet oxygen. These extracts significantly prevented reduction of neuronal nuclear antigen in the infarcted area, although no improvement in neurological function was observed. Importantly, GCE and GSE contained S-allylcystein, a compound associated with AGE's neuroprotective effect against damage induced by cerebral ischemia. Extracts decreased mRNA expression of NR1- and NR2B-NMDA-receptor subunits and prevented ischemia-induced reduction in mitochondrial potential and in ATP synthesis. These results indicate that antioxidants present in garlic extracts may regulate ROS concentrations during ischemia, favour pro-survival pathways, and attenuate mitochondrial dysfunction.

Incomptines C and D, two heliangolides from Decachaeta incompta and their antiprotozoal activity.

Two new heliangolides, incomptines C (3) and D (4), were isolated from the leaves of Decachaeta incompta. The structures were established by spectroscopic methods and confirmed by X-ray crystallography. The antiprotozoal activity of incomptines C and D was evaluated. Additionally, the chromatographic profile of the leaves and roots extracts were compared to identify incomptines A-D (1-4) in each extract.

Anti-diarrheal activity of (-)-epicatechin from Chiranthodendron pentadactylon Larreat: experimental and computational studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Chiranthodendron pentadactylon Larreat is frequently used in Mexican traditional medicine as well as in Guatemalan for several medicinal purposes, including their use in the control of diarrhea. AIM OF THE STUDY: This work was undertaken to obtain additional information that support the traditional use of Chiranthodendron pentadactylon Larreat, on pharmacological basis using the major antisecretory isolated compound from computational, in vitro and in vivo experiments. MATERIALS AND METHODS: (-)-Epicatechin was isolated from ethyl acetate fraction of the plant crude extract. In vivo toxin (Vibrio cholera or Escherichia coli)-induced intestinal secretion in rat jejunal loops models and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on Vibrio cholera toxin were used in experimental studies while the molecular docking technique was used to conduct computational study. RESULTS: The antisecretory activity of epicatechin was tested against Vibrio cholera and Escherichia coli toxins at oral dose 10 mg/kg in the rat model. It exhibited the most potent activity on Vibrio cholera toxin (56.9% of inhibition). In the case of Escherichia coli toxin its effect was moderate (24.1% of inhibition). SDS-PAGE analysis revealed that both (-)-epicatechin and Chiranthodendron pentadactylon extract interacted with the Vibrio cholera toxin at concentration from 80 µg/mL and 300 µg/mL, respectively. Computational molecular docking showed that epicatechin interacted with four amino acid residues (Asn 103, Phe 31, Phe 223 and The 78) in the catalytic site of Vibrio cholera toxin, revealing its potential binding mode at molecular level. CONCLUSION: The results derived from computational, in vitro and in vivo experiments on Vibrio cholera and Escherichia coli toxins confirm the potential of epicatechin as a new antisecretory compound and give additional scientific support to anecdotal use of Chiranthodendron pentadactylon Larreat in Mexican traditional medicine to treat gastrointestinal disorders such as diarrhea.

Evaluation of the antiprotozoal activity of neo-clerodane type diterpenes from Salvia polystachya against Entamoeba histolytica and Giardia lamblia.

Chia (Salvia polystachya Ort., Lamiaceae) is frequently used in Mexican traditional medicine to treat dysentery. In this study the main neo-clerodane diterpenes (polystachynes A, B and D, as well as linearolactone) were isolated from the aerial parts of chia, and their antiprotozoal activities toward Entamoeba histolytica and Giardia lamblia trophozoites were evaluated in vitro. Linearolactone was the most potent antiamoebic and antigiardial compound with IC(50) values of 22.9 microM for E. histolytica and 28.2 microM for G. lamblia. Polystachynes A, B and D, showed moderate antiprotozoal activity against both protozoans with IC(50) values ranging from 117.0 to 160.6 microM for E. histolytica and from 107.5 to 134.7 microM for G. lamblia. These data suggest that linearolactone may play an important role in the antidiarrhoeal activity of S. polystachya.