Enrique Jiménez-Ferrer

Inhibition of angiotensin convertin enzyme (ACE) activity by the anthocyanins delphinidin- and cyanidin-3-O-sambubiosides from Hibiscus sabdariffa

ETHNOPHARMACOLOGICAL RELEVANCE The beverages of Hibiscus sabdariffa calyces are widely used in Mexico as diuretic, for treating gastrointestinal disorders, liver diseases, fever, hypercholesterolemia and hypertension. Different works have demonstrated that Hibiscus sabdariffa extracts reduce blood pressure in humans, and recently, we demonstrated that this effect is due to angiotensin converting enzyme (ACE) inhibitor activity. AIM OF THE STUDY The aim of the current study was to isolate and characterizer the constituents responsible of the ACE activity of the aqueous extract of Hibiscus sabdariffa. MATERIALS AND METHODS Bioassay-guided fractionation of the aqueous extract of dried calyces of Hibiscus sabdariffa using preparative reversed-phase HPLC, and the in vitro ACE Inhibition assay, as biological monitor model, were used for the isolation. The isolated compounds were characterized by spectroscopic methods. RESULTS The anthocyanins delphinidin-3-O-sambubioside (1) and cyanidin-3-O-sambubioside (2) were isolated by bioassay-guided purification. These compounds showed IC(50) values (84.5 and 68.4 microg/mL, respectively), which are similar to those obtained by related flavonoid glycosides. Kinetic determinations suggested that these compounds inhibit the enzyme activity by competing with the substrate for the active site. CONCLUSIONS The competitive ACE inhibitor activity of the anthocyanins 1 and 2 is reported for the first time. This activity is in good agreement with the folk medicinal use of Hibiscus sabdariffa calyces as antihypertensive.

Effect of a Polyphenol-Rich Extract from Aloe vera Gel on Experimentally Induced Insulin Resistance in Mice

Insulin resistance, which precedes type 2 diabetes mellitus (T2DM), is a widespread pathology associated with the metabolic syndrome, myocardial ischemia, and hypertension. Finding an adequate treatment for this pathology is an important goal in medicine. The purpose of the present research was to investigate the effect of an extract from Aloe vera gel containing a high concentration of polyphenols on experimentally induced insulin resistance in mice. A polyphenol-rich Aloe vera extract (350 mg/kg) with known concentrations of aloin (181.7 mg/g) and aloe-emodin (3.6 mg/g) was administered orally for a period of 4 weeks to insulin resistant ICR mice. Pioglitazone (50 mg/kg) and bi-distilled water were used as positive and negative controls respectively. Body weight, food intake, and plasma concentrations of insulin and glucose were measured and insulin tolerance tests were performed. The insulin resistance value was calculated using the homeostasis model assessment for insulin resistance (HOMA-IR) formula. Results showed that the polyphenol-rich extract from Aloe vera was able to decrease significantly both body weight (p < 0.008) and blood glucose levels (p < 0.005) and to protect animals against unfavorable results on HOMA-IR, which was observed in the negative control group. The highest glucose levels during the insulin tolerance curve test were in the negative control group when compared to the Aloe vera extract and pioglitazone treated mice (p < 0.05). In conclusion, Aloe vera gel could be effective for the control of insulin resistance.

Citrus limetta leaves extract antagonizes the hypertensive effect of angiotensin II

ETHNOPHARMACOLOGICAL RELEVANCE Citrus limetta Risso (Rutaceae) is widely used in Mexico for healing purposes, among them as antihypertensive treatment. AIM OF THE STUDY To assess the antihypertensive effect of C. limetta leaves as one of its ethnomedical uses. MATERIALS AND METHODS The acute response of blood pressure to angiotensin II administration was measured in mice. Additionally, the acute oral toxicity profiles were determined. RESULTS The findings of the current investigation showed that different concentrations of the aqueous extract prevented the raise of systolic blood pressure (p< or =0.001 vs. vehicle), diastolic blood pressure (p< or =0.0002 vs. vehicle) and mean blood pressure (p< or =0.0000 vs. vehicle); with a dose dependent effect for diastolic pressures at 125-500mg/kg dosages. The 500 and 1000mg/kg doses inhibited the action of Ang II in similar extent to telmisartan. Toxic signs or deaths were not observed in mice treated at 2000mg/kg of C. limetta extract. CONCLUSIONS All doses of C. limetta aqueous extract, used in this assay, were safe and effective.

In vivo anti-inflammatory and anti-ulcerogenic activities of extracts from wild growing and in vitro plants of Castilleja tenuiflora Benth. (Orobanchaceae).

ETHNOPHARMACOLOGICAL RELEVANCE Castilleja tenuiflora Benth. (Orobanchaceae) is a perennial shrub used since the 16(th) century in Mexican traditional medicine for the treatment of a number of health disorders including inflammation, stomach pain and tumors. The aim of the present study was to evaluate the cytotoxic, anti-inflammatory and anti-ulcerogenic activities of ethyl acetate (EaE), methanol (ME) and aqueous extracts (AE) of Castilleja tenuiflora wild grown (CtW) and in vitro plants (CtIv). MATERIAL AND METHOD Phytochemical analysis of the phenylethanoid glycoside (PhG) and iridoid glycoside (IG) components was carried out by chromatographic methods. In vitro cytotoxic activity of the extracts was evaluated in the following four carcinoma cell lines: colon (HF-6), breast (MCF-7), prostate (PC-3), and nasopharyngeal (KB). The topical anti-inflammatory activity was evaluated in mouse ear edema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Anti-ulcerogenic activity was evaluated in rats using an absolute ethanol-induced acute gastric ulcer model. RESULTS The main compounds in the extracts were isoverbascoside, verbascoside and aucubin and their concentration depended both on the solvent used and on the plant material origin. None of the extracts showed cytotoxicity against the tested cell lines. In contrast, CtWEaE, CtWAE and CtIvEaE (1.6 mg/ear) showed moderate anti-inflammatory activity similar to dexamethasone (1 mg/ear) with a 38.2, 39.3 and 49.1% decrease of inflammation, respectively. CtWEaE and CtIvEaE (100 mg/kg) showed high anti-ulcerogenic activity with 88.3 and 83.1% inhibition, respectively, compared to famotidine (20 mg/kg, 32.8% inhibition). CONCLUSION Castilleja tenuiflora extracts provided significant gastric protection in an acute ulcer induction model and topical anti-inflammatory activity in a mouse ear edema model. These activities are related to verbascoside and may explain the traditional use of Castilleja tenuiflora in the treatment of anti-inflammatory and gastrointestinal disorders. Cultured Castilleja tenuiflora plants (in vitro) exhibited pharmacological activities and also have the potential to produce bioactive compounds.

Anti-inflammatory Activity of Hautriwaic Acid Isolated from Dodonaea viscosa Leaves

The aim of this study was to identify an anti-inflammatory compound from D. viscosa leaves. The structure of this bioactive substance was elucidated by IR and NMR studies, which indicated that this natural product corresponds to hautriwaic acid (HA). This diterpene exhibited good anti-inflammatory activity in 12-O-tetradecanoylphorbol 13-acetate (TPA) mice ear edema models by applications at doses of 0.25, 0.5 and 1.0 mg/ear (60.2, 70.2 and 87.1% inhibition, respectively); additionally Dodonaea viscosa dichloro-methane extract (DvDE) displays a 97.8% anti-inflammatory effect at 3 mg/kg. Multiple applications of DvDE at doses of 100 mg/kg on TPA mice ear edema inhibited the edema-associated inflammation by 71.8%, while HA at doses of 15 mg/kg, reduced edema to 64% and indomethacin 40%.

Anti-Inflammatory Activity of Different Agave Plants and the Compound Cantalasaponin-1

Species of the agave genus, such as Agave tequilana, Agave angustifolia and Agave americana are used in Mexican traditional medicine to treat inflammation-associated conditions. These plants’ leaves contain saponin compounds which show anti-inflammatory properties in different models. The goal of this investigation was to evaluate the anti-inflammatory capacity of these plants, identify which is the most active, and isolate the active compound by a bio-directed fractionation using the ear edema induced in mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) technique. A dose of 6 mg/ear of acetone extract from the three agave species induced anti-inflammatory effects, however, the one from A. americana proved to be the most active. Different fractions of this species showed biological activity. Finally the F5 fraction at 2.0 mg/ear induced an inhibition of 85.6%. We identified one compound in this fraction as (25R)-5α-spirostan-3β,6α,23α-triol-3,6-di-O-β-D-glucopyranoside (cantalasaponin-1) through 1H- and 13C-NMR spectral analysis and two dimensional experiments like DEPT NMR, COSY, HSQC and HMBC. This steroidal glycoside showed a dose dependent effect of up to 90% of ear edema inhibition at the highest dose of 1.5 mg/ear.

Interaction of the natural anxiolytic Galphimine-B with serotonergic drugs on dorsal hippocampus in rats.

AIM OF THE STUDY Galphimine-B (G-B) is a nor-seco triterpene with an anxiolytic-like effect obtained from the plant species Galphimia glauca Cav. By means of a double blind clinical trial, it has been demonstrated that the extract from this plant, standardized in G-B content, possesses therapeutic effectiveness in patients with generalized anxiety. The mechanism of action of this compound remains unknown to date, but it has already demonstrated a non interaction with the γ-aminobutyric acid (GABA)ergic system. For this reason, the objective of this work was to evaluate the pharmacological interaction between G-B with the 5-hydroxytryptamine 1A (5HT(1A)) and 5-hydroxytryptamine 2A (5HT(2A)) serotonergic receptors on CA1 neurons of hippocampus. MATERIALS AND METHODS Electrophysiological records were performed as the frequency of discharge of in vivo CA1 cells from dorsal hippocampus in rats. RESULTS G-B was able to increase the frequency of discharge of neurons of the CA1 cells with some characteristics that support an interaction with the serotonergic system in this zone. It was demonstrated that this triterpene modulates the induced response of 5HT(1A) receptors, in an allosteric manner. CONCLUSION This effect demonstrated an interaction between G-B and the serotonergic system in dorsal hippocampus and evidenced that the mechanism of action of this compound could involve a complex series of actions on different neurotransmitter systems related with the anxiety disorder.

Hypoglycemic and Hypotensive Activity of a Root Extract of Smilax aristolochiifolia, Standardized on N-trans-Feruloyl-Tyramine

The metabolic syndrome (MS) is a condition consisting of various metabolic abnormalities that are risk factors for developing kidney failure, cardiovascular, vascular and cerebrovascular diseases, among others. The prevalence of this syndrome shows a marked increase. The aim of this study was to investigate the pharmacological effect of Smilax aristolochiifolia root on some components of MS and obtain some of the active principle using chromatographic techniques. The compound isolated was N-trans-feruloyl tyramine NTF (1), and its structure was determined by spectroscopic and spectrometric analyses. The whole extract and the standardized fractions were able to control the weight gain around 30%; the fraction rich in NTF was able to decrease the hypertriglyceridemia by 60%. The insulin resistance decreased by approximately 40%; the same happened with blood pressure, since the values of systolic and diastolic pressure fell on average 31% and 37% respectively, to levels comparable to normal value. The treatment also had an immunomodulatory effect on the low-grade inflammation associated with obesity, since it significantly decreased the relative production of pro-inflammatory cytokines regarding anti-inflammatory cytokines, both kidney and adipose tissue. Therefore it can be concluded that the extract and fractions of Smilax aristolochiifolia root with NTF are useful to counteract some symptoms of MS in animal models.

MICROPROPAGATION OF LEPIDIUM VIRGINICUM (BRASSICACEAE), A PLANT WITH ANTIPROTOZOAL ACTIVITY

SummaryMicropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA∶KN (0.57∶13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC50 antiprotozoal value between 141.90 and 268.53 μg ml−1.

Interactions of a standardized flavonoid fraction from Tilia americana with Serotoninergic drugs in elevated plus maze

ETHNOPHARMACOLOGICAL RELEVANCE Tilia americana var. mexicana (Schltdl) Hardin (Tiliaceae) aerial parts (bracts and flowers) are used in the traditional Mexican medicine to treat nervous disorders, as sedative and to treat insomnia. A fraction of this species called FC1 (organic fraction from this plant) was proposed, described as anxiolytic and characterized by the presence of flavonoids. In the present work, this fraction was standardized, and its interaction with different serotonergic drugs was tested. We used the elevated plus maze model as anxiety test and the open field test so as to observe a possible effect on mice׳s motor behavior. MATERIAL AND METHODOLOGY HPLC technique was used to quantify the flavonoids contained in a fraction called F1C. Different doses of F1C were administered to ICR mice (12.5, 25, 37.5 and 50mg/kg, oral pathway) then they were exposed to elevated plus maze or open field test. After, each dose of F1C fraction was co-administered with different drugs, in order to evaluate the animal׳s behavior: DOI agonist (2.0mg/kg) and KET antagonist (0.03mg/kg) of 5-HT2A receptors; 8-OH-DPAT (0.1mg/kg) selective agonist and WAY100635 (0.5mg/kg) antagonist of 5HT1 receptors. RESULTS The HPLC quantitative analysis revealed the F1C composition (mg/g of extract): tiliroside (28.56), glucoside of quercetin (16.25), quercitrin (7.96), rutin (3.93), Kaempferol (2.83). The Emax for F1C curve was 80.6% for time to open arms with an ED50 of 15.09 mg/kg. The combination of F1C with DOI gives a significant increase of the F1C anxiolytic effect (Emax=111% and ED50=13.51 mg/kg), while KET blocks it completely (Emax=12.25% and ED50=2.4 mg/kg). The administration of F1C with 8-OH-DPAT does not generate significant changes on the time to open arms, although it does induce a decrement in F1C potency (Emax=83.3% and ED50=33.3mg/kg). When F1C and WAY-100365 are combined, the anxiolytic activity of the fraction decreases (Emax=33.3% and ED50=102.10mg/kg). CONCLUSIONS The medicinal use attributed to Tilia americana for their effect on central nervous system, could be in part in the flavonoid fraction (F1C) with anxiolytic activity which is dose dependent, and has the ability to interact with the serotonergic system. It is necessary to advance in the study of the mechanism of action, using other techniques such in vitro analysis.