Bioassay-Guided Fractionation of Erythrostemon yucatanensis (Greenm.) Gagnon & GP Lewis Components with Anti-hemagglutinin Binding Activity against Influenza A/H1N1 Virus.

Erythrostemon yucatanensis (Greenm.) Gagnon & GP Lewis is a legume tree native to and widely distributed in southeast Mexico, where its branches are used in traditional medicine. An in vitro evaluation of the antiviral activity of extracts and fractions from the leaves, stem bark and roots against two strains of the AH1N1 influenza virus was performed, leading to the identification of bioactive compounds in this medicinal plant. In a cytopathic effect reduction assay, the fractions from the leaves and stem bark were the active elements at the co-treatment level. These were further fractionated based on their hemagglutination inhibition activity. The analysis of spectroscopy data identified a combination of phytosterols (ß-sitosterol, stigmasterol and campesterol) in the stem bark active fraction as the main anti-hemagglutinin binding components, while 5-hydroxy-2(2-hydroxy-3,4,5-trimethoxyphenyl)-7-metoxi-4H(chromen-4-ona), which was isolated from the leaf extracts, showed a weak inhibition of viral hemagglutinin. Time of addition experiments demonstrated that the mixture of sterols had a direct effect on viral particle infectivity at the co-treatment level (IC50 = 3.125 µg/mL). This effect was also observed in the virus plaque formation inhibition assay, where the mixture showed 90% inhibition in the first 20 min of co-treatment at the same concentration. Additionally, it was found using qRT-PCR that the NP copy number was reduced by 92.85% after 60 min of co-treatment. These results are the first report of components with anti-hemagglutinin binding activity in the genus Erythrostemon sp.

Anti-inflammatory and anti-hypersensitive effects of the chalcone isocordoin and its semisynthetic derivatives in mice.

Isocordoin (1), a chalcone isolated from different plants, has been found to present a range of interesting biological properties. This study aimed to evaluate the anti-hypersensitive and anti-inflammatory effects of isocordoin (1) and several natural and semisynthetic derivatives (2-10). Initial evaluation of (1), dihydroisocordoin (2) and six semisynthetic derivatives (3-8) in the inhibition of abdominal writhes induced by acetic acid model showed that only isocordoin dimethylether (5) caused more than 70% of inhibition. Further evaluation of 5 for its anti-oedematogenic activity and anti-hypersensitivity effect induced by carrageenan, lipopolysaccharide (LPS), bradykinin (BK), prostaglandin E2 (PGE2), and epinephrine showed that isocordoin dimethylether (5) presented a discrete inhibition of carrageenan- and LPS-induced hypersensitivity, and of carrageenan-induced paw oedema, and that it was able to significantly reduce both the oedema and hypersensitivity induced by BK. Furthermore, when tested in the PGE2 model, 5 interfered only with the paw-oedema, without showing any effect against the paw-hypersensitivity. Evaluation of the natural isocordoin (1), together with the semisynthetic derivatives isocordoin dimethylether (5), isocordoin methylether (9), and dihydroisocordoin methylether (10) in the BK-induced oedema and hypersensitivity showed that the monoalkylated derivatives 10 and 9 had the strongest antinociceptive activity. The results of this investigation indicate that both monoalkylation of the C-4' phenolic hydroxyl group and reduction of the double bond in the α,ß-unsaturated system of the chalcone skeleton favor activity.

Spatio-Temporal Variation of Terpenoids in Wild Plants of Pentalinon andrieuxii.

Pentalinon andrieuxii (Müll.Arg.) B.F.Hansen & Wunderlin (Apocynaceae) is a vine native to the Yucatan peninsula, where it is widely used in Mayan traditional medicine to treat, among other ailments, the wounds caused by cutaneous leishmaniasis. Among the secondary metabolites isolated from P. andrieuxii are the triterpene betulinic acid and the chemically unusual tri-norsesquiterpene urechitol A; however, to date, there is no existing knowledge about the accumulation dynamics of the ubiquitous betulinic acid or the novel urechitol A in the plant. In this article, we report on the accumulation of both secondary metabolites in wild individuals of P. andrieuxii; our results show that while the content of betulinic acid in plant leaves bears no apparent relation to plant ontogeny, the content of urechitol A in root tissue is clearly related to plant development.

Isotopologue profiling of triterpene formation under physiological conditions. Biosynthesis of lupeol-3-(3'-R-hydroxy)-stearate in Pentalinon andrieuxii.

The biosynthesis of lupeol-3-(3'R-hydroxy)-stearate (procrim b, 1) was investigated in the Mexican medicinal plant Pentalinon andrieuxii by (13)CO2 pulse-chase experiments. NMR analyses revealed positional enrichments of (13)C2-isotopologues in both the triterpenoid and the hydroxystearate moieties of 1. Five of the six isoprene units reflected a pattern with [1,2-(13)C2]- and [3,5-(13)C2]-isotopologues from the respective C5-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of 1 showed only the [3,5-(13)C2]-connectivity of the original C5-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of (13)C2-isotopologues was indicative of [(13)C2]acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from P. andrieuxii is of the same type as that from Olea europea and Taraxacum officinale, but different from that of Arabidopsis thaliana. The study shows that (13)CO2 pulse-chase experiments are powerful in elucidating, under in vivo conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes.

Phytochemical diversity of the essential oils of Mexican Oregano (Lippia graveolens Kunth) populations along an Edapho-climatic gradient.

Mexican oregano (Lippia graveolens) is an important aromatic plant, mainly used as flavoring and usually harvested from non-cultivated populations. Mexican oregano essential oil showed important variation in the essential-oil yield and composition. The composition of the essential oils extracted by hydrodistillation from 14 wild populations of L. graveolens growing along an edaphoclimatic gradient was evaluated. Characterization of the oils by GC-FID and GC/MS analyses allowed the identification of 70 components, which accounted for 89-99% of the total oil composition. Principal component and hierarchical cluster analyses divided the essential oils into three distinct groups with contrasting oil compositions, viz., two phenolic chemotypes, with either carvacrol (C) or thymol (T) as dominant compounds (contents >75% of the total oil composition), and a non-phenolic chemotype (S) dominated by oxygenated sesquiterpenes. While Chemotype C was associated with semi-arid climate and shallower and rockier soils, Chemotype T was found for plants growing under less arid conditions and in deeper soils. The plants showing Chemotype S were more abundant in subhumid climate. High-oil-yield individuals (>3%) were identified, which additionally presented high percentages of either carvacrol or thymol; these individuals are of interest, as they could be used as parental material for scientific and commercial breeding programs.

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).

Metabolites from the leaf extract of Serjania yucatanensis with trypanocidal activity against Trypanosoma cruzi.

The bioassay-guided phytochemical investigation of the leaf extract of Serjania yucatanensis, a woody climbing plant endemic to the Yucatan peninsula, led to the identification of a mixture of a triterpene [lup-20(29)-en-3-one] and an oxygenated sesquiterpene (ß-caryophyllene oxide), as that responsible for the originally detected trypanocidal activity in the organic crude extract. Results showed that the mixture of lup-20(29)-en-3-one and ß-caryophyllene oxide is active against trypomastigotes of Trypanosoma cruzi (IC(50) =80.3 µg/mL) and inhibits the egress of trypomastigotes from infected Vero cells (when tested at 100 µg/mL) without being cytotoxic.

Diospyros anisandra phytochemical analysis and anti-hemagglutinin-neuraminidase activity on influenza AH1N1pdm09 virus.

Influenza viral proteins Haemagglutinin (HA) and Neuraminidase (NA) are important targets for antiviral design. We analyzed for the first time the anti-HA activity and the NA inhibitory activity of extracts and their fractions from Diospyros anisandra on the influenza AH1N1pdm09 virus. The n-hexane fruit extract exhibited HA inhibitory (HAI) activity, and fraction F3 inhibited the hemagglutination from 12.5 up to 100 µg/ml. Gas chromatography-mass spectrometry analysis (GC-MS) on fraction F3, and the n-hexane fruit extract, identified six compounds that were individually evaluated. Only vitamin E and lupeol showed a slight inhibitory activity on HA at 100 µg/ml. Regarding the NA assays, the presence of fluorescent (coumarin) and antioxidant (α-tocopherol) compounds in the root extract, masked the NA assays when using fluorescence techniques. We concluded that D. anisandra is a promising source of bioactive compounds with diverse properties including anti-HA activity on the influenza AH1N1pdm09 virus.

Flourensia cernua: Hexane Extracts a Very Active Mycobactericidal Fraction from an Inactive Leaf Decoction against Pansensitive and Panresistant Mycobacterium tuberculosis.

The efficacy of decoction in extracting mycobactericidal compounds from Flourensia cernua (Hojasé) leaves and fractionation with solvents having ascending polarity was compared with that of (i) ethanol extraction by still maceration, extraction with a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration, followed by fractionation with n-hexane, ethyl acetate, and n-butanol; (ii) sequential extraction with n-hexane, ethyl acetate, and n-butanol, by still maceration, using a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration. The in vitro mycobactericidal activity of each preparation was measured against drug-sensitive (SMtb) and drug-resistant (RMtb) Mycobacterium tuberculosis strains. The results of which were expressed as absolute mycobactericidal activity (AMA). These data were normalized to the ΣAMA of the decoction fraction set. Although decoction was inactive, the anti-RMtb normalized ΣAMA (NAMA) of its fractions was comparable with the anti-RMtb NAMA of the still maceration extracts and significantly higher than the anti-SMtb and anti-RMtb NAMAs of every other ethanol extract and serial extract and fraction. Hexane extracted, from decoction, material having 55.17% and 92.62% of antituberculosis activity against SMtb and RMtb, respectively. Although the mycobactericidal activity of decoction is undetectable; its efficacy in extracting F. cernua active metabolites against M. tuberculosis is substantially greater than almost all pharmacognostic methods.

Activity of Semi-Synthetic Mulinanes against MDR, Pre-XDR, and XDR Strains of Mycobacterium tuberculosis.

Tuberculosis causes more than 1.2 million deaths each year. Worldwide, it is the first cause of death by a single infectious agent. The emergence of drug-resistant strains has limited pharmacological treatment of the disease and today, new drugs are urgently needed. Semi-synthetic mulinanes have previously shown important activity against multidrug-resistant (MDR) Mycobacterium tuberculosis. In this investigation, a new set of semi-synthetic mulinanes were synthetized, characterized, and evaluated for their in vitro activity against three drug-resistant clinical isolates of M. tuberculosis: MDR, pre-extensively Drug-Resistant (pre-XDR), and extensively Drug-Resistant (XDR), and against the drug-susceptible laboratory reference strain H37Rv. Derivative 1a showed the best anti-TB activity (minimum inhibitory concentration [MIC] = 5.4 µM) against the susceptible strain and was twice as potent (MIC = 2.7 µM) on the MDR, pre-XDR, and XDR strains and also possessed a bactericidal effect. Derivative 1a was also tested for its anti-TB activity in mice infected with the MDR strain. In this case, 1a produced a significant reduction of pulmonary bacilli loads, six times lower than the control, when tested at 0.2536 mg/Kg. In addition, 1a demonstrated an adjuvant effect by shortening second-line chemotherapy. Finally, the selectivity index of >15.64 shown by 1a when tested on Vero cells makes this derivative an important candidate for future studies in the development of novel antitubercular agents.

Trinorsesquiterpenoids from the root extract of Pentalinon andrieuxii.

Two unusual trinorsesquiterpenoids, urechitols A (1) and B (2), were isolated from the root extract of Pentalinon andrieuxii, a plant used commonly in Yucatecan traditional medicine to treat leishmaniasis. The structures of 1 and 2 were identified by interpretation of their spectroscopic data and chemical correlation reactions. The relative stereochemistry of 1 was confirmed through an X-ray crystallographic study.

Effects of polyphenol removal methods on the in vitro exsheathment inhibitory activity of Lysiloma latisiliquum extracts against Haemonchus contortus larvae.

It has been suggested that the anthelmintic (AH) activity of tannin-rich forages is related to their tannin content. However, reports on tropical fodders such as Lysiloma latisiliquum describe the same AH activity after the addition of tannin-blocking agents, suggesting that the activity either depends on the method of tannin blocking/removal or is due to other type of secondary metabolites. This study compared both the effect of the drying process of the plant material and the effect of different polyphenol removal methods on the AH activity of L. latisiliquum acetone-water extracts when tested against Haemonchus contortus. The results showed that the extraction of oven-dried (OD) leaves of L. latisiliquum yielded a CT-free extract with high AH activity. However, polyphenol-free fractions showed similar or lower AH activity levels as of those original OD extract. HPLC analysis confirmed that common polyphenolic metabolites are not responsible for the AH activity of L. latisiliquum.

Anti-Advanced Glycation End-product and Free Radical Scavenging Activity of Plants from the Yucatecan Flora.

BACKGROUND: Formation and accumulation of advanced glycation end-products (AGE) is recognized as a major pathogenic process in diabetic complications, atherosclerosis and cardiovascular diseases. In addition, reactive oxygen species and free radicals have also been reported to participate in AGE formation and in cell damage. Natural products with antioxidant and antiAGE activity have great therapeutic potential in the treatment of diabetes, hypertension and related complications. Objective: to test ethanolic extracts and aqueous-traditional preparations of plants used to treat diabetes, hypertension and obesity in Yucatecan traditional medicine for their anti-AGE and free radical scavenging activities. MATERIALS AND METHODS: ethanolic extracts of leaves, stems and roots of nine medicinal plants, together with their traditional preparations, were prepared and tested for their anti-AGE and antioxidant activities using the inhibition of advanced glycation end products and DPPH radical scavenging assays, respectively. RESULTS: the root extract of C. fistula (IC50= 0.1 mg/mL) and the leaf extract of P. auritum (IC50= 0.35 mg/mL) presented significant activity against vesperlysine and pentosidine-like AGE. Although none of the aqueous traditional preparations showed significant activity in the anti-AGE assay, both the traditional preparations and the ethanolic extracts of E. tinifolia, M. zapota, O. campechianum and P. auritum showed significant activity in the DPPH reduction assay. CONCLUSIONS: the results suggest that the metabolites responsible for the detected radical-scavenging activity are different to those involved in inhibiting AGE formation; however, the extracts with antioxidant activity may contain other metabolites which are able to prevent AGE formation through a different mechanism. SUMMARY: Ethanolic extracts from nine plants used to treat diabetes, hypertension and obesity in Yucatecan traditional medicine were tested for their anti-AGE and free radical scavenging activities.Significant activity against vesperlysine and pentosidine-like AGE was detected in the root extract of Cassia fistula and the leaf extract of Piper auritum.Traditional preparations and the ethanolic extracts of Ehretia tinifolia, Manilkara zapota, Ocimum campechianum and Piper auritum showed significant activity in the DPPH reduction assay.Results suggest that the metabolites responsible for the detected radical-scavenging activity are different to those involved in inhibiting AGE formation. Abbreviations Used: AGE: Advanced glycation end-product; DPPH: 2,2-Diphenyl-1-picrylhydrazyl; DM: Diabetes mellitus; ROS: Reactive oxygen species; BSA: Bovine serum albumin; EtOH: Ethanol; EtOAc: Ethyl acetate; ANOVA: Analysis of variance; BA: Brosimum alicastrum; BS: Bunchosia swartziana; CF: Cassia fistula; CN: Cocos nucifera; ET: Ehretia tinifolia; MZ: Manilkara zapota; OC: Ocimum campechianum; PA: Piper auritum; RM: Rhizophora mangle; L: Leaves; S: Stems; R: Roots; T: traditional preparation; I: Inflorescences; W: Water.

Chemometrics-enhanced high performance liquid chromatography-ultraviolet detection of bioactive metabolites from phytochemically unknown plants.

This work describes the use of Colubrina greggii as a model to investigate the use of chemometric analysis combined with data from a leishmanicidal bioassay, using Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures (O-PLS), to detect biologically active natural products in crude extracts from plants having little or no phytochemical information. A first analysis of the HPLC-UV profiles of the extract and its semi-purified fractions using both Principal Component Analysis (PCA) and Orthogonal Partial Least Squares (O-PLS) indicated that the components at tR 48.2, 48.7, 51.8min correlated with the variation in bioactivity. However, a further O-PLS analysis of the HPLC-UV profiles of fractions obtained through a final semi-preparative HPLC purification showed two components at tR 48.7 and 49.5min which correlated with the variation of the bioactivity in a high performance predictive model, with high determination coefficient, high correlation coefficient values (R(2) and Q(2)=0.99) and a low root mean square error (RMSE=0.018). This study demonstrates that the association of chemometric analysis with bioassay results can be an excellent strategy for the detection and isolation of bioactive metabolites from phytochemically unknown plant crude extracts.

Natural zinniol derivatives from Alternaria tagetica. Isolation, synthesis, and structure-activity correlation.

Two novel phytotoxins, 8-zinniol methyl ether (5) and 8-zinniol acetate (6), in addition to 6-(3',3'-dimethylallyloxy)-4-methoxy-5-methylphthalide (2), 5-(3',3'-dimethylallyloxy)-7-methoxy-6-methylphthalide (3), and the novel metabolites 8-zinniol 2-(phenyl)ethyl ether (4) and 7-zinniol acetate (7) have been identified as natural zinniol derivatives from the organic crude extract of Alternaria tagetica culture filtrates. Using zinniol as the starting material, phytotoxin 5 was synthesized, together with a number of synthetic intermediates (8-13). Both natural and synthetic zinniol derivatives were evaluated in the leaf-spot bioassay against marigold leaves (Tagetes erecta).

Bioactive metabolites from Cnidoscolus souzae and Acmella pilosa.

The bioassay-guided purification of the ethanol extracts of Acmella pilosa and Cnidoscolus souzae, two plants of the native flora of the Yucatan Peninsula used in traditional medicine to treat inflammation and pain, resulted in the identification of rosmarinic acid (1) and caffeic acid (2) as the bioactive metabolites from A. pilosa, and of 7-deoxynimbidiol (4) as the major bioactive metabolite from C. souzae. Metabolites 1, 2, and 4 proved to be responsible for the antioxidant activity originally detected in the corresponding organic crude extracts; 7-deoxynimbidiol (4) showed good analgesic and anti-inflammatory activities, inhibiting the pain induced by PGE2 and reducing the edema induced by carrageenan, respectively.

Synergistic Effect of Lupenone and Caryophyllene Oxide against Trypanosoma cruzi.

The in vitro trypanocidal activity of a 1 : 4 mixture of lupenone and caryophyllene oxide confirmed a synergistic effect of the terpenoids against epimastigotes forms of T. cruzi (IC50 = 10.4 µ g/mL, FIC = 0.46). In addition, testing of the terpenoid mixture for its capacity to reduce the number of amastigote nests in cardiac tissue and skeletal muscle of infected mice showed a reduction of more than 80% at a dose level of 20.8 mg·kg(-1)·day(-1).

Antihypertensive and vasorelaxant effects of dihydrospinochalcone-A isolated from Lonchocarpus xuul Lundell by NO production: computational and ex vivo approaches.

Current work was conducted to evaluate the vasorelaxant effect of dihydrospinochalcone-A (1) and isocordoin (2), compounds type chalcone isolated from Lonchocarpus xuul, an endemic tree of the Yucatan Peninsula, Mexico. Compounds 1 and 2 were found to induce significant relaxant effect in a concentration-dependent manner on aortic rat rings pre-contracted with noradrenaline (NA, 0.1 µM). Compound 1 was the most active and its effect was endothelium-dependent (Emax=79.67% and EC50=21.46 µM with endothelium and Emax=23.58% and EC50=91.8 µM without endothelium, respectively). The functional mechanism of action for 1 was elucidated. Pre-incubation with L-NAME (unspecific nitric oxide synthase inhibitor), indomethacin (unspecific COX inhibitor), ODQ (soluble guanylyl cyclase inhibitor), atropine (cholinergic receptor antagonist), TEA (unspecific potassium channel blocker) reduced relaxations induced by 1. Oral administration of 50 mg/kg of compound 1 exhibited significant decrease in diastolic and systolic blood pressure in SHR rats. The heart rate was not modified. Compound 1 was docked with a crystal structure of eNOS. Dihydrospinochalcone-A showed calculated affinity with eNOS in the C1 binding pockets, near the catalytic site; Trp449, Trp447 and His373 through aromatic and π-π interactions, also His463 and Arg367 are the residues that make hydrogen bonds with the carbonyl and hydroxyl groups. In conclusion, dihydrospinochalcone-A induces a significant antihypertensive effect due to its direct vasorelaxant action on rat aorta rings, through NO/sCG/PKG pathway and potassium channel opening.

Bioactive dicaffeoylquinic acid derivatives from the root extract of Calea urticifolia

ABSTRACT Calea urticifolia (Mill.) DC., Asteraceae, is a native plant of the Yucatan Peninsula used in traditional medicine to treat inflammation and pain. The bioassay-guided purification of the ethanol root extract allowed the isolation of the main bioactive metabolites, which were identified as an inseparable mixture of thymol (1) and 3-methyl-4-isopropylphenol (2), together with 3,4-O-dicaffeoylquinic acid methyl ester (3), 3,4-O-dicaffeoyl-epi-quinic acid methyl ester (4), 3,5-O-dicaffeoyl-epi-quinic acid methyl ester (5) and 3,5-O-dicaffeoylquinic acid (6). The results showed that the analgesic activity detected in the root extract of C. urticifolia could be attributed mainly to the mixture of 1 and 2 and to the novel 3,4-O-dicaffeoyl-epi-quinic acid methyl ester (4). Alternatively, the similarity on the antiinflammatory and antioxidant activities of the dicaffeoylquinic acid derivatives 3-5 suggests that the former might be related to their ability as radical scavengers.

Dihydrospinochalcone-a and epi-flemistrictin-B, natural isocordoin derivatives from the root extract of Lonchocarpus xuul.

Purification of the root extract of Lonchocarpus xuul resulted in the isolation and identification of dihydrospinochalcone A (1) and epi-flemistrictin B (2) as additional natural isocordoin derivatives. Identification was based on the analysis of their spectroscopic data and by comparing these with those of previously reported metabolites.