Histochemistry and immunolocalisation of glucokinin in antidiabetic plants used in traditional Mexican medicine.

Mexico is a megadiverse country that has 3,600 to 4,000 species of medicinal plants, of which approximately 800 are used to treat conditions related to diabetes mellitus (DM). DM is a chronic degenerative disease of energy metabolism that exists as two types: type 1 (DM1) and type 2 (DM2). DM is considered a public health problem that affects 7% of the Mexican population older than 20 years. DM is clinically controlled with hypoglycaemic drugs, alpha-glucosidase inhibitors, insulin secretion stimulants or the direct application of insulin. The hypoglycaemic effectiveness of specific molecules has been determined only for some medicinal plants in Mexico used to treat DM2. The presence of molecules called glucokinins, wich are similar to animal insulin molecules, has been reported in some plant species; glucokinins act as both growth factors and regulators of glucose metabolism in plants. Therefore, we hypothesized that the hypoglycaemic effectiveness of some of the popularly used species for the control of DM could be due to the presence of glucokinin, as reported for Bauhinia variegata. The goal of this work was to use histochemistry to detect, the accumulation of protein that is immunocytochemically compatible with glucokinin in slide sections of hypoglycaemic species used as remedies for DM2. The top fourteen most used medicinal plants in Mexico were selected for study via microscopic sections. Proteins were histochemically detected using naphthol blue black and Johansen's quadruple stain, and the immunocytochemical correspondence of the proteins with glucokinin was investigated using an insulin antibody. All species studied reacted positively to proteins and glucokinin in the same structures. The presence of glucokinin in these structures and the corresponding hypoglycaemic effects are discussed in the contex of the actions of other compounds.

Sexual and vegetative propagation of the medicinal Mexican species Phyllonoma laticuspis (Phyllonomaceae) / Propagación sexual y vegetativa de la especie medicinal mexicana Phyllonoma laticuspis (Phyllonomaceae)

Abstract:Phyllonoma laticuspis leaves are used in Carrizal de Bravo, Guerrero, Mexico, to heal skin lesions such as injuries and smallpox sequelae and to treat diabetes mellitus type 2, and organic extracts of these leaves have been reported to exert antibacterial effects. High demand of P. laticuspis as a medicinal plant has decreased its natural populations, and propagation of the species has not yet been reported. Therefore, the purpose of this study was to assess the vegetative propagation of the species through cutting and air layering, as well as its sexual propagation in a preserved population. For this, concentrations of 1 000, 4 000 and 6 000 ppm of a commercial root enhancer, with indole butyric acid (IBA), and a control treatment without IBA, were applied to the cuttings and air layers. Germination was evaluated under light and dark conditions using lots of freshly collected seeds and lots of seeds that had been stored for three months at 4 °C or 24 ± 2 °C. All experiments were performed in a completely randomized design. The cuttings did not develop roots in any concentration, whereas 100 % of the air layers rooted, displaying vigorous roots in the presence of 4 000 ppm IBA, after four month of treatment application. Regarding germination, more than 60 % of the freshly collected seeds germinated, whereas less than 20 % of the seeds stored at 4 °C, and close to 50 % of the seeds stored at 24 ± 2 °C germinated under light and dark conditions. No significant differences were observed between light and dark conditions, so they were categorized as indifferent photoblastic seeds. The observed moisture content of 13.5 % and germination behaviour as the response to cold storage, suggest that the resultant seed quality was intermediate. P. laticuspis propagation for short-term foliage production can be carried out in air layers, in populations with a high density of adult plants as a source of plant material and for the restoration of disturbed areas, in the same locality. On the other hand, large-scale seedling production, medium-term foliage production and preservation of species variability can be achieved using seeds. Rev. Biol. Trop. 65 (1): 9-19. Epub 2017 March 01.

ResumenLas hojas de Phyllonoma laticuspis se utilizan en Carrizal de Bravo, Gro. México, para curar lesiones de la piel como heridas, secuelas de viruela y afecciones de la diabetes mellitus tipo 2. Los extractos orgánicos de hojas, mostraron efectos antibacterianos. La gran demanda de P. laticuspis como planta medicinal, ha disminuido sus poblaciones naturales, y no existen reportes de la propagación de la especie. Por lo tanto, el propósito del presente trabajo fue evaluar la propagación vegetativa de la especie a través de estacas y acodos, y su propagación sexual en una población conservada. En la propagación por estacas y acodos se aplicaron concentraciones de 1 000, 4 000 y 6 000 ppm de un enraizador comercial con ácido indolbutírico (AIB), y un tratamiento control sin AIB. La germinación se evaluó en luz y oscuridad mediante el uso de lotes de semillas recién recolectadas y lotes de semillas almacenadas por tres meses a 4 °C y a 24 ± 2 ºC. Todos los experimentos se realizaron en un diseño completamente al azar. Las estacas no desarrollaron raíces en ninguna de las concentraciones, mientras que el 100 % de los acodos enraizaron, produciendo raíces vigorosas con la concentración de 4 000 ppm de AIB, a los cuatro meses de la aplicación. Con respecto a la germinación bajo condiciones de luz y oscuridad, las semillas recién recolectadas germinaron más del 60 %, mientras que las semillas almacenadas a 4 ºC menos del 20 % y las almacenadas a 24 ± 2 ºC cerca del 50 %. No hubieron diferencias significativas entre la germinación en luz y oscuridad, por lo que se consideraron como semillas fotoblásticas indiferentes. El contenido de humedad de 13.5 % y el comportamiento de la germinación en respuesta al almacenamiento en frío, sugieren la cualidad de semillas de tipo intermedias. La propagación de P. laticuspis para la producción de follaje a corto tiempo, puede hacerse por acodos en poblaciones con alta densidad de plantas adultas como fuente de material vegetal y para la restauración de áreas alteradas en la misma localidad. Mientras que la producción de follaje a mediano plazo y la conservación de la variabilidad de la especie, se puede lograr con la obtención masiva de plántulas a través de semillas.

A Histochemical Technique for the Detection of Annonaceous Acetogenins.

Annonaceous acetogenins (ACGs) are molecules with carbon numbers C35-C37, usually with tetrahydrofuran and tetrahydropyran rings and one terminal γ-lactone (usually α,ß-unsaturated), in a large aliphatic chain that is varyingly hydroxylated, acetoxylated or ketonized. ACGs have ecological functions as insecticides and are pharmacologically promising due to their cytotoxic and antitumoral properties. They are found in the seeds, leaves, roots, flowers and fruits of annonaceous plants and can be detected during isolation via thin-layer chromatography using Kedde's reagent, which reacts with the unsaturated lactone. This chapter describes the location in situ of ACGs in fresh sections of annonaceous seeds using Kedde's reagent.The acetogenins are located in the idioblasts, in the endosperm and in the embryonic axis during differentiation. This method can aid in the detection of ACGs with a terminal unsaturated γ-lactone in organs and tissues.

Sexual and vegetative propagation of the medicinal Mexican species Phyllonoma laticuspis (Phyllonomaceae).

Phyllonoma laticuspis leaves are used in Carrizal de Bravo, Guerrero, Mexico, to heal skin lesions such as injuries and smallpox sequelae and to treat diabetes mellitus type 2, and organic extracts of these leaves have been reported to exert antibacterial effects. High demand of P. laticuspis as a medicinal plant has decreased its natural populations, and propagation of the species has not yet been reported. Therefore, the purpose of this study was to assess the vegetative propagation of the species through cutting and air layering, as well as its sexual propagation in a preserved population. For this, concentrations of 1 000, 4 000 and 6 000 ppm of a commercial root enhancer, with indole butyric acid (IBA), and a control treatment without IBA, were applied to the cuttings and air layers. Germination was evaluated under light and dark conditions using lots of freshly collected seeds and lots of seeds that had been stored for three months at 4 °C or 24 ± 2 °C. All experiments were performed in a completely randomized design. The cuttings did not develop roots in any concentration, whereas 100 % of the air layers rooted, displaying vigorous roots in the presence of 4 000 ppm IBA, after four month of treatment application. Regarding germination, more than 60 % of the freshly collected seeds germinated, whereas less than 20 % of the seeds stored at 4 °C, and close to 50 % of the seeds stored at 24 ± 2 °C germinated under light and dark conditions. No significant differences were observed between light and dark conditions, so they were categorized as indifferent photoblastic seeds. The observed moisture content of 13.5 % and germination behaviour as the response to cold storage, suggest that the resultant seed quality was intermediate. P. laticuspis propagation for short-term foliage production can be carried out in air layers, in populations with a high density of adult plants as a source of plant material and for the restoration of disturbed areas, in the same locality. On the other hand, large-scale seedling production, medium-term foliage production and preservation of species variability can be achieved using seeds.

Euphorbia dioeca Kunth as a novel source for alpha-glucosidase inhibitors / Euphorbia dioeca Kunth como una nueva fuente de inhibidores de alfa-glucosidasa

Euphorbia dioeca Kunth belongs to the Wanderer’s herb complex that is traditionally used for skin diseases and recently as antidiabetic. The methanol and aqueous extracts were evaluated for their in vitro alpha-glucosidase inhibitory activity and an oral starch tolerance test. These extracts showed an IC50 of 0.55 and 0.85 mg/mL, respectively. In diabetic Long Evans rats, the methanol and aqueous extracts reduced significantly the postprandial hyperglycemia peak in 15.2 percent and 12.8 percent, respectively. The alpha-glucosidase inhibitory activity is related with the presence of glycosides, phenolic compounds and flavonoids. Additionally, the safety parameters of both extracts were assessed by means of an acute toxicity test, being classified as innocuous. The traditional use of E. dioeca to control type 2 diabetes was confirmed, being an important source of alfa-glucosidase inhibitors.

Euphorbia dioeca Kunth, pertenece al complejo de plantas medicinales denominado Hierba de la Golondrina; el cual se utiliza para diversos padecimientos, destacando su uso como antidiabético. En dicho marco, se evaluó tanto la actividad inhibitoria de alfa-glucosidasa in vitro, como su desempeño en una prueba de tolerancia a una carga de almidón postprandial. Los extractos inhibieron la actividad de la alfa-glucosidasa con una CI50 de 0.55 y 0.85 mg/mL, respectivamente. Los extractos metanólico y acuoso disminuyeron significativamente el pico hiperglucémico postprandial en un 15.2 por ciento y un 12.8 por ciento, respectivamente, cuando se evaluó en ratas diabéticas. La actividad inhibitoria de alfa-glucosidasa, reflejada en ambas pruebas, está relacionada con la presencia de glicósidos, compuestos fenólicos y flavonoides. De manera adicional, ambos extractos fueron evaluados en una prueba de toxicidad aguda, siendo clasificados como inocuos. Se corroboró el uso tradicional de E. dioeca para el control de la diabetes tipo 2, siendo una importante fuente de compuestos inhibidores de alfa-glucosidasa.

Chemical composition, potential toxicity, and quality control procedures of the crude drug of Cyrtopodium macrobulbon.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyrtopodium macrobulbon ("cañaveral") has been long used in Mexican traditional medicine for the treatment of painful urinary ailments ("mal de orin") in men. This study was conducted (i) to establish the potential acute toxicity and the antinociceptive activity of some preparations of Cyrtopodium macrobulbon, in order to demonstrate its preclinical efficacy for treating symptoms of "mal de orin"; and (ii) to determine the chemical composition and quality control parameters of this medicinal orchid. MATERIALS AND METHODS: The antinociceptive effect was assessed using the acetic acid-induced writhing and the hot-plate tests. Investigation of the acute toxicity was accomplished by the Lorke method. The organic extract (OE) was subjected to conventional phytochemical study using chromatographic conventional procedures. The volatile components profile of the species was accomplished via GC-MS analysis of HS-SPME-adsorbed compounds. Furthermore, an HPLC method to quantify ephemeranthol B (10) was developed and validated according to the International Conference on Harmonization Guidelines. Microscopic anatomy studies were performed using light and scanning electron microscopies. Finally, a potential distribution map was generated using the MaxEnt modeling method. RESULTS: AE and OE were not toxic to mice since the LD50 was higher than 5000 mg/kg. OE was only active in the acetic acid-induced writhing assay at the doses of 100 and 316 mg/kg. Conventional phytochemical analysis of OE led to the isolation and characterization of n-hexacosyl-trans-p-coumarate (1), n-octacosyl-trans-p-coumarate (2), n-triacontyl-trans-p-coumarate (3), 4-methoxy-benzyl alcohol (4), 4-hydroxybenzaldehyde (5), 1,5,7-trimethoxy-9,10-dihydrophenanthrene-2,6-diol (6), confusarin (7), gigantol (8), batatasin III (9), and ephemeranthol B (10). The major volatile components identified by HS-SPME analysis were 6,10,14-trimethyl-2-pentadecanone, eucalyptol (11), and isobornyl formate. An HPLC analytical method for the quantification of compound 10 in the plant was developed and fully validated for selectivity, accuracy, and precision. The microscopic studies revealed that the epidermal tissue displayed a layer of enlarged, crenate and cell thin-walled cells with a thickened cuticle; these cells are described for first time for this species. The potential distribution map generated revealed that this species is widespread in Mexico from Sinaloa to Merida states. CONCLUSIONS: The results of the pharmacological studies tend to support the traditional use of Cyrtopodium macrobulbon for "mal de orin"; the presence of compounds 8, 9, and 11 with known antinociceptive activity might be related with the pharmacological effect demonstrated. The HPLC and microscopic analyses developed in this work will be valuable tools for quality control purposes for this plant.