Biological properties of aqueous extract and pyranocoumarins obtained from the bark of Brosimum alicastrum tree.

ETHNOPHARMACOLOGICAL RELEVANCE: Brosimum alicastrum is a tree used in Mexican traditional medicine for the treatment of several diseases, including uterine cancer. AIM OF THE STUDY: In this study, the cytotoxic activity of aqueous extract of B. alicastrum bark and isolated compounds xanthyletin (1), luvangetin (2), and 8-hydroxyxanthyletin (3) on three human cancer cell lines was determined. Moreover, the biological effects of 8-hydroxyxanthyletin (3) were investigated. MATERIALS AND METHODS: The aqueous extract was prepared according to the ethnomedical information reported from the bark. The compounds were purified using chromatographic methods and their structures were elucidated by spectroscopic techniques. The antiproliferative effect of aqueous extract and isolates was determined in three human tumor cell lines: HeLa, A2780, and MSTO-211H, and evaluated by trypan blue exclusion assay. The cell cycle and the mitochondrial transmembrane potential (ΔΨ) were measured by flow cytometry, while Reactive Oxygen Species (ROS) levels were determined using 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe. The effect on the relaxation activity, mediated by topoisomerase I and II, was evaluated by electrophoresis, and docking studies were performed using Autodock 4.2 to analyze the interactions. RESULTS: Aqueous extract of B. alicastrum bark showed significant antiproliferative effect on the evaluated cancer cell lines (IC50 = 1.6, 8.5, and 21.4 µg/ml). Four coumarins were identified in the extract and three of them were also evaluated. A2780 cell line exhibited higher sensitivity against pyranocoumarins with IC50 values ranging from 32 to 47 µmol/l. 8-hydroxyxanthyletin (3) exerts an interesting effect on human topoisomerases I and II, by inhibiting the enzymes at concentrations comparable to those obtained in antiproliferative assay. Moreover, 8-hydroxyxanthyletin (3) arrests the cell cycle at G0/G1 phase and induces in A2780 cells a concentration-dependent increase in ROS levels. The results of molecular docking suggest the participation of the hydroxyl group in the interaction between 8-hydroxyxanthyletin (3) and topoisomerase I and II. CONCLUSION: This is the first report that demonstrates the cytotoxic activity of the aqueous extract of B. alicastrum bark, and determines the main metabolites.

Zinc Oxide Nanoparticles Induce an Adverse Effect on Blood Glucose Levels Depending On the Dose and Route of Administration in Healthy and Diabetic Rats.

Different studies in experimental diabetes models suggest that zinc oxide nanoparticles (ZnONPs) are useful as antidiabetic agents. However, this evidence was performed and measured in long-term treatments and with repeated doses of ZnONPs. This work aimed to evaluate the ZnONPs acute effects on glycemia during the next six h after an oral or intraperitoneal administration of the treatment in healthy and diabetic rats. In this study, the streptozotocin-nicotinamide intraperitoneal administration in male Wistar rats were used as a diabetes model. 10 mg/kg ZnONPs did not modify the baseline glucose in any group. Nevertheless, the ZnONPs short-term administration (100 mg/kg) induced a hyperglycemic response in a dose and route-dependent administration in healthy (130 ± 2 and 165 ± 10 mg/dL with oral and intraperitoneal, respectively) and diabetic rats (155 ± 2 and 240 ± 20 mg/dL with oral, and intraperitoneal, respectively). The diabetic rats were 1.5 fold more sensitive to ZnONPs effect by the intraperitoneal route. In conclusion, this study provides new information about the acute response of ZnONPs on fasting glycemia in diabetic and healthy rat models; these data are essential for possible future clinical approaches.

HPLC-DAD method for the detection of five annopurpuricins in root samples of Annona purpurea.

INTRODUCTION: Annona purpurea is a species known in Mexico as "cabeza de negro". In folk medicine A. purpurea root is used to treat patients with kidney diseases and cancer. Our recent studies demonstrated that this species contains five acetogenins named annopurpuricins A-E, which are active against tumoural cell lines in a subnanomolar range. OBJECTIVE: To develop an analytical method using a high-performance liquid chromatography diode array detector (HPLC-DAD) to quantify annopurpuricins A-E in different A. purpurea root samples. METHODOLOGY: To quantify the five annopurpuricins A-E a sample treatment was carried out, which consisted of fractionation by means of cold and hot maceration; using solvents of ascending polarity: hexane, dichloromethane, methanol and water. The resulting extracts were subject to HPLC-DAD analysis. The optimised chromatographic separation on a XBRIDGE C18 column achieved separation of all compounds in around 30 min. RESULTS: The developed method was validated according to ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) validation guide. The developed analytical method was found fast, economic, robust, sensitive, linear and precise. The dichloromethane extract of A. purpurea contains annopurpuricin A in quantities 2- to 25-fold higher than annopurpuricins B-E. This optimised method identified and quantified five annopurpuricins, highly bioactive molecules, in A. purpurea root. CONCLUSIONS: The fingerprint of the dichloromethane extracts of A. purpurea was obtained at 210 nm. The results analysis allowed to quantify annopurpuricins A-E that are present in different collection batches of medium polarity extracts. After data analysis, annopurpuricin A could be establish as the metabolite marker of the root of the species.

Validation of an HPLC-DAD method for the determination of plant phenolics

Abstract A selective, sensitive and precise reversed phase HPLC-DAD method was developed and validated for the simultaneous determination of six phenolic acids in the aqueous extract and their hydrolyzed forms prepared from Solanum elaeagnifolium Cav., Solanaceae, Ampelocissus acapulcensis (Kunth) Planch., Vitaceae, or Brosimum alicastrum Sw., Moraceae. The new method showed good linearity (r > 0.999) in a relatively wide concentration range (0.5-100 mg/l). The limits of detection and quantification for the compounds were in the range of 0.097-0.467 mg/l and 0.097-0.496 mg/l, respectively. The recoveries of compounds were calculated in three different concentrations in the range of 88.07-109.17% and matrix effect was less than 5% for all phenolic acids. Finally, our developed HPLC method is simple, reliable and successfully applied to identify and quantify the phenolic acids in complex aqueous extracts from medicinal species, that can be useful for the analysis of infusions that people consume in folk medicine.

Cytotoxic Acetogenins from the Roots of Annona purpurea.

Annona purpurea, known in Mexico as "cabeza de negro" or "ilama", belongs to the Annonaceae family. Its roots are employed in folk medicine in several regions of Mexico. Taking that information into account, a chemical and biological analysis of the components present in the roots of this species was proposed. Our results demonstrated that the dichloromethane (DCM) extract was exclusively constituted by a mixture of five new acetogenins named annopurpuricins A-E (1-5). These compounds have an aliphatic chain of 37 carbons with a terminal α,ß unsaturated γ-lactone. Compounds 1 and 2 belong to the adjacent bis-THF (tetrahydrofuran) α-monohydroxylated type, while compounds 3 and 4 belong to the adjacent bis-THF α,α'-dihydroxylated type; only compound 5 possesses a bis-epoxide system. Complete structure analysis was carried out by spectroscopy and chemical methods. All compounds were evaluated for their antiproliferative activity on three human tumor cell lines (MSTO-211H, HeLa and HepG2). Compounds 1-4 inhibited significantly the growth of HeLa and HepG2 cells, showing GI50 values in the low/subnanomolar range, while 5 was completely ineffective under the tested conditions. The investigation of the mechanism of action responsible for cytotoxicity revealed for the most interesting compound 1 the ability to block the complex I activity on isolated rat liver mitochondria (RLM).

Synthesis, anti-inflammatory activity and modeling studies of cycloartane-type terpenes derivatives isolated from Parthenium argentatum.

The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema model in mice determined the anti-inflammatory activities in vivo of argentatins A, B and D, the main cycloartenol-type triterpenes present in Parthenium argentatum. Our results showed that argentatin B (ED50=1.5×10(-4)mmol/ear) and argentatin A (ED50=2.8×10(-4)mmol/ear) were more potent anti-inflammatory agents than indomethacin (ED50=4.5×10(-4)mmol/ear), the reference drug. Based on these findings, we decided to evaluate 13 derivatives of argentatins A and B. All the derivatives showed anti-inflammatory activity in the TPA-induced edema model in mice. The most active compound was 25-nor-cycloart-3, 16-dione-17-en-24-oic acid, obtained from argentatin A (ED50=1.4×10(-4)mmol/ear). Argentatin B was assayed as inhibitor of COX-2 activity one of the key enzymes involved in the TPA assay. The results showed that argentatin B at 15µM doses inhibited 77% COX-2 activity. Docking studies suggest that argentatin B interacts with Arg 120, a key residue for COX-2 activity.

Synthesis and comparative molecular field analysis (CoMFA) of argentatin B derivatives as growth inhibitors of human cancer cell lines.

Synthesis, characterization, anticancer activity, and comparative molecular field analysis (CoMFA) of 14 argentatin B (1) analogs are described. The effect of argentatin B derivatives on the growth of K562 (leukemia), PC-3 (prostate), U251 (CNS), and HCT-15 (colon) human cancer cell lines was determined using the sulforhodamine B test. The most active compound in this series, 2-formyl-(16beta,24R)-16,24-epoxy-25-hydroxycycloart-1-en-3-one (12), was about 35-50 times more potent than argentatin B (1). Structures were built using the X-ray crystallography of six derivatives for 3D modeling with Sybyl6.9. CoMFA of Log (1/IC50) in K562 cell line gave q2 = 0.507, r2 = 0.907, and three components. The standard deviation CoMFA contours indicate that increased activity is associated with a bulky group at C-2, a C1-C2 double bond, and low electronic density at C-25. Experimental Log P values for argentatin B and one derivative were 1-2 Log units more hydrophilic than the calculated CLog P values.

Evaluation of the cytotoxicity, cytostaticity and genotoxicity of argentatins A and B from Parthenium argentatum (Gray).

Argentatins A and B are abundant triterpenes present in Parthenium argentatum. Both compounds have shown cytotoxic properties on K562, MCF-7, PC-3, HCT-15 and U251 human cancer cell lines. Furthermore the cytotoxic, cytostatic and genotoxic effects of the argentatins on proliferating lymphocytes were evaluated using cytokinesis-block micronucleus test. Argentatin A had no cytostatic properties, but it was cytotoxic for proliferating lymphocytes at a concentration of 25 microM (P < 0.005). On the other hand, argentatin B showed significant cytostatic effects (P < 0.001) at concentrations of 5 to 25 microM and it did not show cytotoxic effects at the same concentrations. Neither argentatin showed genotoxic effects in terms of micronucleus frequency in human lymphocytes. According to these results the argentatins are not able to cause injury on DNA by clastogenic or aneugenic mechanisms.

Synthesis of argentatin A derivatives as growth inhibitors of human cancer cell lines in vitro.

The syntheses of nine argentatin A analogs are described. These compounds were assessed for their ability to inhibit growth in vitro in four human cancer cell lines. Our results showed that the presence of either a double bond at C-1/C-2, or a bromine atom or formyl moiety at C-2 as well as the presence of an isoxazol ring in argentatin A enhanced its potency in all cell lines tested. In addition, an X-ray study of (16S,17R,20S,24R)-3-oxime-20,24-epoxy-16,25-dihydroxy-cycloartan-3-one led to the determination of the correct stereochemistry of argentatin A.