Discovery of inhibitors of protein tyrosine phosphatase 1B contained in a natural products library from Mexican medicinal plants and fungi using a combination of enzymatic and in silico methods*.

This work aimed to discover protein tyrosine phosphatase 1B (PTP1B) inhibitors from a small molecule library of natural products (NPs) derived from selected Mexican medicinal plants and fungi to find new hits for developing antidiabetic drugs. The products showing similar IC50 values to ursolic acid (UA) (positive control, IC50 = 26.5) were considered hits. These compounds were canophyllol (1), 5-O-(ß-D-glucopyranosyl)-7-methoxy-3',4'-dihydroxy-4-phenylcoumarin (2), 3,4-dimethoxy-2,5-phenanthrenediol (3), masticadienonic acid (4), 4',5,6-trihydroxy-3',7-dimethoxyflavone (5), E/Z vermelhotin (6), tajixanthone hydrate (7), quercetin-3-O-(6″-benzoyl)-ß-D-galactoside (8), lichexanthone (9), melianodiol (10), and confusarin (11). According to the double-reciprocal plots, 1 was a non-competitive inhibitor, 3 a mixed-type, and 6 competitive. The chemical space analysis of the hits (IC50 < 100 µM) and compounds possessing activity (IC50 in the range of 100-1,000 µM) with the BIOFACQUIM library indicated that the active molecules are chemically diverse, covering most of the known Mexican NPs' chemical space. Finally, a structure-activity similarity (SAS) map was built using the Tanimoto similarity index and PTP1B absolute inhibitory activity, which allows the identification of seven scaffold hops, namely, compounds 3, 5, 6, 7, 8, 9, and 11. Canophyllol (1), on the other hand, is a true analog of UA since it is an SAR continuous zone of the SAS map.

Echeveria Pallida: Inhibiting Adhesion of Fibroblasts From Pterygium and Neutrophil Extracellular Traps Production.

Context: Pterygium, meaty eyes, is a disease that produces a triangular, conjunctival-epithelial, neovascularized overgrowth covering the cornea, which can cause vision loss. Histological characterization of Pterygium reveals the presence of proliferating fibroblasts (FBs) that remodel the extracellular matrix, with infiltration of immune cells, causing chronic inflammation. The fresh juice of Echeveria pallida E. Walther (Crassulaceae), mechanically extracted from the leaves, can be used to lubricate the eyes and remove Pterygium, even in advanced, degenerative ocular disease. Objective: This study aimed to explore the healing mechanisms of an ethanolic extract of E. pallida on pterygium-derived FBs, lymphocytes, and neutrophils. Design: The research team designed an in-vitro study. Primary cultures of FBs were obtained from fresh, surgical pterygium tissues, and neutrophils and mononuclear cells were purified from the peripheral blood of healthy donors. Intervention: An ethanolic extract of E. pallida was evaluated at 30, 50, 80, 100, 200, and 300 µg/mL-the intervention groups-for viability and proliferation of FBs and lymphocytes. The study included a negative control with no extract, and a positive control, Mitomycin C (MMC), used as a FB proliferation inhibitor and anti-inflammatory. Because some reports have suggested that DMSO at low concentrations can stimulate or inhibit lymphocyte proliferation depending on the cell type, the study also included a DMSO control. Outcome Measures: The measures included an analysis of E. pallida's effects on the proliferation and viability of FBs, the proliferation of human lymphocytes, and human neutrophil extracellular traps (NETs) production. NETs were induced using biochemical and microbiological stimuli-phorbol myristate acetate (PMA), hypochlorous acid (HOCl), Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans-through fluorescence microscopy. Results: The ethanolic extract didn't affect the viability or proliferation of pterygium-derived FBs and human blood lymphocytes, but it showed significant inhibitory activity, from 100 µg/mL, on FB adhesion and the production of NETs. Conclusion: The study found scientific evidence that supports the effects of an extract of the medicinal plant E. pallida in inhibiting the adhesion of FBs derived from human pterygium and NET production.

The Genus Ageratina (Asteraceae) in America: An Insight into its Chemistry and Pharmacological Potential.

BACKGROUND: Ageratina is an American genus of the tribe Eupatorieae (Asteraceae), comprising about 320 species. In Mexico, some species of this genus are highly valued for their medicinal properties, particularly A. pichinchensis, A. petiolaris, and A. grandifolia. Furthermore, herbal preparations of A. pichinchensis are available for treating several mycoses. AIMS AND OBJECTIVE: The present review is aimed to summarize the chemical and pharmacological properties of 37 species of the Ageratina genus up to April, 2022. METHODS: Data were recorded using online scientific databases, including Scopus, PubMed, Google Scholar, Taylor and Francis Imprints, National Center for Biotechnology Information, Science Direct, JSTOR, and SciFinder. The information was gathered from research articles, relevant books on herbal medicinal plants and the history of medicinal plants from Mexico, theses, reports, and web pages. RESULTS: The specialized metabolites present in the Ageratina genus belong to different chemical classes, including flavonoids, benzyl benzoates, benzofurans, chromenes, and terpenoids. The chromenes, benzofurans, and benzyl benzoates are the metabolites most widespread in the genus. So far, the species more thoroughly investigated is A. adenophora. Ageratina has received little attention from the pharmacological point of view. The studies are limited to 10 species. Biological studies have been conducted on extracts and/or compounds isolated from plants collected mainly from China and Mexico. The results revealed that the extracts and metabolites possess several biological activities, including antiviral, antioxidant, antimicrobial, anti-inflammatory, antinociceptive, antifeedant, larvicidal, acaricidal, antidiabetic, antiprotozoal, and wound-healing properties. In the case of A. pichinchensis, A. petiolaris, and A. grandifolia, the pharmacological studies provided evidence for their use for treating gastrointestinal complaints and diabetes. Furthermore, herbal preparations of A. pichinchensis are now widely used for alleviating onychomycosis. A. adenophora, is the most investigated species, chemically and biologically; however, some hepatotoxicity effect has been recorded. CONCLUSION: This review recapitulates information on the Ageratina genus, highlighting the phytochemistry and biological activities of the species investigated. It is important to point out that the pharmacological potential of this large genus remains largely unexplored.

Protein tyrosine phosphatase 1B inhibitory activity of compounds from Justicia spicigera (Acanthaceae).

An infusion from the aerial parts of Justicia spicigera Schltdl., an herb commonly used to treat diabetes, inhibited the activity of protein tyrosine phosphatase 1B (PTP1B). Two undescribed compounds, 2-N-(p-coumaroyl)-3H-phenoxazin-3-one, and 3″-O-acetyl-kaempferitrin, along with kaempferitrin, kaempferol 7-O-α-L-rhamnopyranoside, perisbivalvine B and 2,5-dimethoxy-p-benzoquinone were isolated from the active extract. Their structures were elucidated by a combination of spectroscopic and spectrometric methods. The isolates were evaluated for their inhibitory activity against PTP1B; the most active compounds were 2-N-(p-coumaroyl)-3H-phenoxazin-3-one, and perisbivalvine B with IC50 values of 159.1 ± 0.02 µM and 106.6 ± 0.01 µM, respectively. However, perisbivalvine B was unstable. Kinetic analysis of 2-N-(p-coumaroyl)-3H-phenoxazin-3-one and 2,5-dimethoxy-p-benzoquinone (obtained in good amounts) indicated that both compounds behaved as parabolic competitive inhibitors and bind to the enzyme forming complexes with 1:1 and 1:2 stoichiometry. Docking of 2-N-(p-coumaroyl)-3H-phenoxazin-3-one and 2,5-dimethoxy-p-benzoquinone to PTP1B1-400 predicted a good affinity of these compounds for PTP1B catalytic site and demonstrated that the binding of a second ligand is sterically possible. The 1:2 complex was also supported by the second docking analysis, which predicted an important contribution of π-stacking interactions to the stability of these 1:2 complexes. Finally, an UHPLC-MS method was developed and validated to quantify the content of kaempferitrin in the infusion of the plant.

Antinociceptive Activity of Compounds from the Aqueous Extract of Melampodium divaricatum.

A decoction prepared from the aerial parts of Melampodium divaricatum showed antinociceptive and antihyperalgesic responses when tested in the formalin model in mice. From the CH2 Cl2 fraction of the decoction, two non-previously reported secondary metabolites, 3-O-ß-D-glucopyranosyl-16α-hydroxy-ent-kaurane (1) and melampodiamide (2) [(2'R*,4'Z)-2'-hydroxy-N-[(2S*,3S*,4R*)-1,3,4-trihydroxyoctadec-2-yl]tetracos-4-enamide] were separated and characterized by spectroscopic, spectrometric, and computational techniques. The flavonoids isoquercitrin and hyperoside, which possessed noted antinociceptive properties, were obtained from the active AcOEt fraction of the decoction. The chemical composition of the essential oil of the plant was also analyzed by gas chromatography-mass spectrometry. The major constituents were (E)-caryophyllene, germacrene D, ß-elemene, δ-elemene, γ-patchoulene, and 7-epi-α-selinene. Headspace solid-phase microextraction analysis detected (E)-caryophyllene as the main volatile compound of the plant.

α-Glucosidase Inhibitors from Ageratina grandifolia.

Fractionation of an aqueous extract from the aerial parts of Ageratina grandifolia yielded a new natural product, namely, 4-hydroxy-3-((S)-1'-angeloyloxy-(R)-2',3'-epoxy-3'-methyl)butylacetophenone (1), along with eight known compounds, including three flavonoids (2-4) and five chromenes (5-9). NMR data interpretation and DFT-calculated chemical shifts combined with DP4+ statistical and J-DP4 probability analyses allowed for the complete characterization of compound 1. The presence of compound 1 in a plant that biosynthesizes 2,2-dimethylchromenes is noteworthy, because an epoxy derivative has long been postulated as the reaction intermediate from the prenylated p-hydroxyacetophenones to cyclic dimethylchromenes. So far, this key intermediate has not been isolated, due to its purported chemical instability. Thus, this is the first report of a potential epoxide intermediate, leading to any of the chromene constituents of this plant. Compounds 1-9 inhibited yeast α-glucosidase with IC50 values ranging from 0.79 to 460 µM (acarbose, IC50 = 278.7 µM). The most active compounds were quercetagetin-7-O-(6-O-caffeoyl-ß-d-glucopyranoside (3) and 6-hydroxykaempferol-7-O-(6-O-caffeoyl-ß-d-glucopyranoside (4). Kinetic analysis of 3 revealed its mixed-type inhibitor nature. Docking studies into the crystallographic structure of yeast α-glucosidase (pdb 3A4A) predicted that 3 and 4 bind at the catalytic site of the enzyme.

Flavonoids and Terpenoids with PTP-1B Inhibitory Properties from the Infusion of Salvia amarissima Ortega.

An infusion prepared from the aerial parts of Salvia amarissima Ortega inhibited the enzyme protein tyrosine phosphatase 1B (PTP-1B) (IC50~88 and 33 µg/mL, respectively). Phytochemical analysis of the infusion yielded amarisolide (1), 5,6,4'-trihydroxy-7,3'-dimethoxyflavone (2), 6-hydroxyluteolin (3), rutin (4), rosmarinic acid (5), isoquercitrin (6), pedalitin (7) and a new neo-clerodane type diterpenoid glucoside, named amarisolide G (8a,b). Compound 8a,b is a new natural product, and 2-6 are reported for the first time for the species. All compounds were tested for their inhibitory activity against PTP-1B; their IC50 values ranged from 62.0 to 514.2 µM. The activity was compared to that of ursolic acid (IC50 = 29.14 µM). The most active compound was pedalitin (7). Docking analysis predicted that compound 7 has higher affinity for the allosteric site of the enzyme. Gas chromatography coupled to mass spectrometry analyses of the essential oils prepared from dried and fresh materials revealed that germacrene D (15) and ß-selinene (16), followed by ß-caryophyllene (13) and spathulenol (17) were their major components. An ultra-high performance liquid chromatography coupled to mass spectrometry method was developed and validated to quantify amarisolide (1) in the ethyl acetate soluble fraction of the infusion of S. amarissima.

Anti-Hyperglycemic Activity of Major Compounds from Calea ternifolia.

Demethylisoencecalin (1) and caleins A (4) and C (5) (3.16-31.6 mg/kg, p.o.), the major components from an infusion of Calea ternifolia controlled postprandial glucose levels during an oral sucrose tolerance test (OSTT, 3 g/kg) in normal and nicotinamide/streptozotocin (NA/STZ, 40/100 mg/kg) hyperglicemic mice. The effects were comparable to those of acarbose (5 mg/kg). During the isolation of 1, 4, and 5, four additional metabolites not previously reported for the plant, were obtained, namely 6-acetyl-5-hydroxy-2-methyl-2-hydroxymethyl-2H-chromene (3), herniarin (6), scoparone (7), and 4',7-dimethylapigenin (8). In addition, the structure of calein C (5) was confirmed by X-ray analysis. Pharmacological evaluation of the essential oil of the species (31.6-316.2 mg/kg, p.o.) provoked also an important decrement of blood glucose levels during an OSTT. Gas chromatography coupled with mass spectrometry (GC-MS) analysis of the headspace solid phase microextraction (HS-SPME)-adsorbed compounds and active essential oil obtained by hydrodistillation revealed that chromene 1 was the major component (19.92%); sesquiterpenes represented the highest percentage of the essential oil content (55.67%) and included curcumene (7.10%), spathulenol (12.95%) and caryophyllene oxide (13.0%). A suitable High Performance Liquid Chromatography (HPLC) method for quantifying chromenes 1 and 6-hydroxyacetyl-5-hydroxy-2,2-dimethyl-2H-chromene (2) was developed and validated according to standard protocols.

Spasmolytic Action of Preparations and Compounds from Hofmeisteria schaffneri.

Hofmeisteria schaffneri is used in Mexican folk medicine for treating painful gastric complaints. Therefore, in this paper the smooth muscle relaxant effect of the essential oil, and an infusion of the whole plant were evaluated using the gastrointestinal transit test in mice. The results revealed that both preparations at 316 mg/kg inhibited gastrointestinal transit by 47.5 and 52.1%, respectively. The common component of the infusion and essential oil was 8.9 -epoxy-10-acetoxythymol angelate (2), which inhibited the gastrointestinal transit by 53.4% at a dose of 31.6 mg/kg. An HPLC-UV method was developed and validated to quantify 2. The chromatographic conditions were: A LiChrospher® 100 RP-18 column (250 x 4 mm i.d., 5µm) with a mobile phase composed of CH3CN-H2O, in a gradient run at a flow rate of 0.6 mL/min, using a wavelength of 215 nm. The method was linear, precise, accurate, and showed excellent recovery. According to the results, compound 2 can be used as a marker for the quality control procedures of the crude drug of H. schaffneri.

α-Glucosidase Inhibitors from Vauquelinia corymbosa.

The α-glucosidase inhibitory activity of an aqueous extract and compounds from the aerial parts of V. corymbosa was demonstrated with yeast and rat small intestinal α-glucosidases. The aqueous extract inhibited yeast α-glucosidase with a half maximal inhibitory concentration (IC50) of 28.6 µg/mL. Bioassay-guided fractionation of the extract led to the isolation of several compounds, including one cyanogenic glycoside [prunasin (1)], five flavonoids [(-)-epi-catechin (2), hyperoside (3), isoquercetin (4), quercitrin (5) and quercetin-3-O-(6''-benzoyl)-ß-galactoside (6)] and two simple aromatic compounds [picein (7) and methylarbutin (8)]. The most active compound was 6 with IC50 values of 30 µM in the case of yeast α-glucosidase, and 437 µM in the case of the mammalian enzyme. According to the kinetic analyses performed with rat and yeast enzymes, this compound behaved as mixed-type inhibitor; the calculated inhibition constants (Ki) were 212 and 50 µM, respectively. Molecular docking analyses with yeast and mammalian α-glucosidases revealed that compound 6 bind differently to these enzymes. Altogether, the results of this work suggest that preparations of V. corymbosa might delay glucose absorption in vivo.

Antimicrobial activity and chemical composition of the essential oil of Hofmeisteria schaffneri.

OBJECTIVES: The aims of this study were to establish the antimicrobial potential of Hofmeisteria schaffneri essential oil and its chemical composition. METHODS: The essential oils of Hofmeisteria schaffneri harvested at flowering (batches I and IV) and non-flowering (batches II and III) seasons were prepared by hydrodistillation and analysed by GC and GC-MS. The aqueous and organic (CH(2) Cl(2) -MeOH 1 : 1) extracts were prepared by using infusion and maceration techniques, respectively. The in-vitro antimicrobial activity of the preparations and compounds against Candida albicans and some bacteria (Gram-negative and Gram-positive) was assessed using the broth dilution method in 96-microplate wells. KEY FINDINGS: Forty-four compounds, representing ∼90% of the total constituents, were identified in the essential oil of Hofmeisteria schaffneri collected in flowering (batches I and IV) and non-flowering (batches II and III) seasons. In all cases, several thymol analogues were the major components of the oils (∼65%); some small differences in the relative proportions of these constituents were observed. The infusion exhibited an antibacterial activity against Staphylococcus aureus and Bacillus subtilis, with a MIC value of 64 µg/ml in each case. The essential oil batches were active against Staphylococcus aureus, with MIC ranging from 48 to 192 µg/ml. They were, however, inactive against Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi (MIC > 1024 µg/ml). On the other hand, the infusion of the plant as well as the oil from batch I displayed anti-Candida albicans activity, with MIC of 128 and 192 µg/ml, respectively. Finally, the organic extract did not displayed significant activity against the tested microorganisms (MIC ≥ 1024 µg/ml). Some of the compounds isolated from the plant were also tested. Compounds 8 and 38, which were present in the essential oils, displayed the best antibacterial effect against Gram-positive bacteria (MIC ranging between 32 and 64 µg/ml). Compounds 6 (present in the infusion) and 10 (present in all preparations) showed higher activity against the yeast (MIC = 128 µg/ml) than the remaining compounds, with MIC values ranging from 256 to 512 µg/ml. CONCLUSIONS: The composition and antimicrobial activity of the oils changed slightly from flowering to non-flowering seasons. The results of the present investigation provide in-vitro scientific support for the use of the plant against skin infections in Mexican folk medicine.

Antinociceptive effect of extracts and compounds from Hofmeisteria schaffneri.

ETHNOPHARMACOLOGICAL RELEVANCE: Hofmeisteria schaffneri (Asteraceae) is a medicinal plant widely commercialized in the most important Markets of Mexico City for the treatment of gastro-intestinal complaints and skin afflictions. AIM OF THE STUDY: The main goals of this study were to establish the potential acute toxicity and the antinociceptive activity in animal models of several preparations and compounds from Hofmeisteria schaffneri. MATERIALS AND METHODS: The aqueous and organic extracts as well as the essential oil of Hofmeisteria schaffneri were prepared by infusion, maceration and hydrodistillation, respectively. Investigation of the acute toxicity was accomplished by the Lorke method. The antinociceptive effect was assessed using the writhing and the hot plate tests. Natural compounds were isolated by standard phytochemical procedures. In addition, a few thymol esters were prepared by chemical synthesis. The stability of natural and synthetic esters was qualitatively analyzed by measuring their susceptibility to hydrolysis by pig liver estearase and mouse plasma at 37 degrees C. RESULTS: The LD(50) for each preparation tested was higher than 5000 mg/kg revealing that they were not toxic to mice after exposure for short space of time. On the other hand, the extracts showed significant antinociceptive effect when tested in the hot plate model. The most active natural product as antinociceptive agent was hofmeisterin III (1) which also was the most stable in the stability study. Its pharmacological effect seems to be partially mediated by an opioid mechanism since naloxone inhibits its action. Using compound 1 as a lead molecule, several synthetic thymol esters were prepared and only compounds 13, 15 and 17 were antinoceptive at the dose of 1 mg/kg. CONCLUSIONS: The present investigation provided evidence of the efficacy of several preparations of Hofmeisteria schaffneri as antinociceptive agents. The most active preparation was the essential oil which contained large amount of hofmeisterin III (1) and other thymol derivatives. Some novel synthetic analogs of hofmeisterin III with antinociceptive properties were discovered. The nature of the ester chain of these analogs did not have a clear impact on the antinociceptive activity. The phyto-preparations analyzed in this study were not toxic to mice according to the Lorke's test; therefore considering their long term use of the plant they might be secure for human consumption.

Hypoglycemic activity of extracts and compounds from the leaves of Hintonia standleyana and H. latiflora: potential alternatives to the use of the stem bark of these species.

The CH(2)Cl(2)-MeOH (1:1) extract of the leaves of Hintonia standleyana and H. latiflora caused significant decrease in blood glucose levels in both normal and streptozotozin (STZ)-induced diabetic rats when compared with vehicle-treated groups (p < 0.05). These extracts were not toxic to mice according to the Lorke criteria. From the hypoglycemic extract of H. standleyana, two new 4-phenylcoumarins, namely, 6''-O-acetyl-5-O-beta-d-galactopyranosyl-7,4'-dihydroxy-4-phenylcoumarin (1) and 6''-O-acetyl-5-O-beta-d-galactopyranosyl-7,3',4'-trihydroxy-4-phenylcoumarin (2), were obtained. The analogous extract of H. latiflora yielded the new 5-O-[beta-d-xylopyranosyl-(1-->6)-beta-d-glucopyranosyl]-7,4'-dimethoxy-4-phenylcoumarin (3) along with several known compounds, including ursolic acid and desoxycordifolinic acid. Phenylcoumarins 1 and 2 showed hypoglycemic activity. HPLC profiles of the leaf extracts of both plants revealed the presence of known hypoglycemic phenylcoumarins as well as chlorogenic acid. The overall results have indicated that the leaves of H. standleyana and H. latiflora possess similar antidiabetic potential to their stem bark. Therefore, the leaves from these species could represent an alternative to the use of their stem bark, which, in turn, would contribute to the conservation of these Mexican medicinal plants.

Phytotoxins from Hofmeisteria schaffneri: isolation and synthesis of 2'-(2' '-hydroxy-4' '-methylphenyl)-2'-oxoethyl acetate1.

Activity-directed fractionation of a CH(2)Cl(2)-MeOH (1:1) extract of Hofmeisteria schaffneri led to the isolation of a new phytotoxin characterized as 2'-(2' '-hydroxy-4' '-methylphenyl)-2'-oxoethyl acetate and designated the trivial name of hofmeisterin (1). In addition, the known compounds beta-carotene, euparin, and 3',4',4a',9a'-tetrahydro-6,7'-dimethylspiro[benzofuran-3(2H),2'-pyrano[2,3-b]benzofuran]-2,4a'-diol (2) were obtained. The identification of the isolates was accomplished by spectroscopic methods. The structure of 1 was unequivocally confirmed by synthesis. The methyl derivative 1a was also synthesized following the same strategy. Compounds 1 and 2 inhibited radicle growth of Amaranthus hypochondriacus (IC(50) = 3.2 x 10(-4) and 1.2 x 10(-5) M, respectively) and significantly inhibited activation of the calmodulin (CaM)-dependent enzyme cAMP phosphodiesterase (PDE) with IC(50) values of 4.4 and 4.22 microM, respectively.