Determinación de los principios bioactivos, propiedades farmacológicas y antioxidantes de tres extractos medicinales de Echinacea para su control de calidad y estandarización

Background: The Echinacea genus belongs to the Asteraceae family and is composed of 9 species, of which E. pallida, E. angustifolia and E. purpurea are used as medicinal plants. Sales of medicinal extractsderived from these three Echinacea species reached 21 million dollars in 2002, however, it is important to mention that these extracts are being commercialized without adequate control over the standardization process, defining standardization not only as phytosanitary and agronomic regulation but also including variables that correlate the chemical content with some biological effect, decreasing the therapeutic variability, which is the main problem with herbal treatments. Because of all the above, the principal objective of the present work was to evaluate the chemical, antioxidant, anti-inflammatory, hypoglycemic and antiproliferative capacities of three hydro-alcoholic Echinacea extracts in order to establish the bases for their characterization and the future oftheir standardization. Materials and methods: Three Echinacea extracts were selected. These extracts meet the first step of the standardization process which implies the manufacturing standards described by the United States Department of Agriculture (USDA), where the main requirement is the traceability of the product and the guarantee of origin. The selected extracts were prepared by two different manufacturers using different organs or tissues. Extract A (Echinacea®) was prepared with roots of E. purpurea, extract B (Super Echinacea®) was prepared with roots, leaves, flowers and seeds of E. purpurea and extract C (Echinacea Supreme®) was prepared with roots and aerial parts of E. purpurea and roots of E. angustifolia. The three selected extracts were applied a standardization protocol as proposed by our investigation group and published in 2014, which takes into account three phases subsequent to those included in the post phytosanitary and agronomic production standards established by USDA.These steps were 1) Physical analyses; density and dry matter were assessed. 2) Chemical analyses; some qualitative tests were performed to determine the presence of some functional groups (tannins, saponins, coumarins, anthraquinones and alkaloids), as well as some specific constituents related to functional properties like total phenols, flavonoids, caffeic and chlorogenic acid, sugars and alkylamides. 3) Biological analyses; antioxidant and antiproliferative capacities were measured in vitro and the anti-inflammatory, hypoglycaemic and thermotolerance effects were measured in vivo. Finally, some associations were made between the chemical compounds and the biological measurements. Results: this study showed differences between the three stages of standardization, highlighting some observations that were considered relevant. At first, we observed that both the dry matter and the density were significantly higher in extract C. In the second stage we found that extract A presented higher amounts of total phenols (975.5 mg GAE/gdw) and total flavonoids (6.8 mg QE/ gdw) when expressed in terms of dry weight, a tendency that is modified when the results are expressed as mL of each Echinacea extract. In this case extract C presented higher amounts of phenols and flavonoids compared to the other extracts, which is very important because these types of compounds are dosed with mL of extract. Caffeic acid was only detected in extract C (0.048 ppm) and by analysis of the non-polar fraction of the hydro-alcoholic extracts, 11 alkylamides were determined for extracts A and B and 14 for extract C. In the third stage, extract C showed higher antioxidant capacity when determined by DPPH (19.97 mM TEAC/ gdw; 4.77 mM TEAC/ mL extract) and when determined by ABTS, extract A showed higher antioxidant capacity (70.09 mM TEAC/ gdw), however, when expressed as mL of extract, extract C showed a higher antioxidant capacity (10.45 mM TEAC/ mL extract). Only extract B showed anti-inflammatory activity, extract C showed antiproliferative capacity, extracts A and C showed hyperglycaemic capacity. Discussion: Certain tendencies can be observed that allow us to relate some of the chemical compounds with a biological effect. Extract C showed higher contents of phenols and flavonoids which can be related to higher antioxidant capacity by both DPPH and ABTS. Even when the content of total phenolic compounds was observed to be high, a very low content of caffeic acid was observed, leaving unknown which types of phenolic compounds are present. Extract C also showed a higher number of alkylamides which could be associated to its antiproliferative effect shown only in this extract. Three of these alkylamides were only observed in extract C which is the only one that contains E. angustioflia, which could indicate that they may be used as possible species markers. The anti inflammatory effect observed in extract B could be related to the presence of saponins observed in the qualitative analyses. There is evidence in related literature that these types of compounds are related to anti-inflammatory processes, even though it is important to note that saponins are a very diverse group and for this reason it would be interesting to study them in more detail, starting with the aerial parts of Echinacea which is what extract B is made of and focusing on terpenoids, because within the group of saponins, terpenoids are frequently associated with this kind of pharmacological effect. Conclusions: We observed differences in the physical, chemical and biological parameters showing the need to plan standardization strategies, carry out correlation studies in finer detail that allow us to conclude the proposed association raised in this study, advancethe adequate use of herbal medicine and generate chemical content data related to biological effects. These data need to be added to the current protocols, which are mostly based on ensuring the safety and identity of the product.Antecedentes: El género Echinacea pertenece a la familia Asteraceae y está compuesto de 9 especies de las cuales sólo E. pallida, E. angustifolia y E. purpurease emplean como plantas medicinales. Las ventas de los extractos medicinales en donde se emplean estas tres especies ascendierona 21 millones de dólares en el 2002.Sin embargo, es importante aclarar que estos extractos son comercializados sin tener un control adecuado en el proceso de estandarización, definiendo estandarización no solo como la regulación fitosanitaria y agronómica sino incluyendo además variables que correlacionen el contenido químico con algún efecto biológico, disminuyendo con ello la variabilidad terapéutica la cual es el principal problema de los tratamientos herbales.Es por ello que en el presente trabajo se contempló comoobjetivo principal evaluar el contenido químico, capacidad antioxidante, antiinflamatoria, hipoglucemiante y antiproliferativa de tres extractos hidroalcohólicos de Echinacea, con el fin de establecer las bases para su caracterización y a futuro de su estandarización. Materiales y Métodos: Se seleccionaron tres extractos de Echinaceaque cumplen con la primera etapa de estandarización que corresponde a las normas de manufactura descritas por United States Department of Agriculture(USDA), en las que el principal requisito es la trazabilidad del producto y la garantía de origen.Los extractos seleccionados con esta condiciónfueron preparados por dos casas comerciales diferentes empleando distintos órganos o tejidos:extracto A (Echinacea®)el cual fue elaborado con raíces de Echinacea purpurea; extracto B (Super Echinacea®) elaborado con raíces, hojas, flores y semillas de E. purpurea y el extracto C (Echinacea Supreme®) elaborado con raíces y partes aéreas de E. purpurea y raíces de E. angustifolia. A los tres extractos seleccionados en el estudio,se lesaplicó un protocolo de estandarización propuesto por el grupo de investigación y publicado en el 2014,que contempla tres etapasadicionales a las incluidas en las normas fitosanitarias y de producción agronómicas establecidas por USDA.Estas etapasfueron: 1) Análisis físico:Se determinó la densidad y cantidad de materia seca. 2) Análisis químico: Se realizaron ensayos cualitativos para determinar la presencia de algunos grupos fitoquímicos(taninos, saponinas, cumarinas, antraquinonas y alcaloides), así como la determinación de algunos constituyentes específicos relacionados con las propiedades funcionales como son fenólicos totales, flavonoides totales, ácido cafeico, ácido clorogénico,azúcares yalquilamidas. 3) Análisis biológico: Se determinó la capacidad antioxidante yantiproliferativain vitro, así como el efecto antiinflamatorio, hipoglucemiante y de termotoleranciain vivo. Finalmente, se asoció la presencia de los compuestos químicos con algunos efectos biológicos.Resultados:El estudio mostródiferencias en los tres parámetros de estandarización.Seobservó que tanto la cantidad de materia secacomo la densidad fueron significativamente mayoresen el extracto C. En la segunda etapa se encontró queel extracto A presentóla mayor cantidad de compuestos fenólicos totales (975.5 mg EAG/gps) y flavonoides totales (6.8 mg EQ/gps), en base seca, tendencia que se modifica al analizarlo por mL de extracto hidroalcohólico, en queel extracto C mostró los valores más altos comparado con los otros dos extractos estudiados, lo cual es de suma importancia ya que este tipo de compuestos se dosifican por mL de extracto.La presencia del ácido cafeico sólo se encontró en el extracto C (0.048 ppm).Del análisis de la fracción no polar del extracto hidroalcohólico seencontraron11 alquilamidas para los extractos A y B, y 14 para el extracto C. En relación a la tercera etapa, la capacidad antioxidante fue mayor en el extracto C cuando se determinó por el método de DPPH (19.97 mM/ gps; 4.77 mM/ mL extracto)y cuando se determinó por ABTS, la capacidad antioxidantefue mayor en el extracto A (70.09 mM/ gps);sin embargo, la tendencia cambiócuando se expresó por mL de extracto, en que se observóla mayor capacidad antioxidanteen el extracto C (10.45 mM/mL extracto).Se observó también efecto antiinflamatorio para el extracto B, efecto antiproliferativo para el extracto C y efecto hipoglucemiante para los extractos A y C. Discusión. Se puedenobservar ciertas tendencias que nos permiten asociar algunos componentes químicos con los efectos biológicos, tales como mayores cantidades de compuestos fenólicos totales y flavonoides totales para el extracto Cexpresado por mL de extracto,esto se puede asociar con lo observado en la determinación de capacidad antioxidantes por los métodos deDPPH y ABTSen los que el extracto C mostró la mayor capacidad antioxidante.Aunque la cantidad de fenoles totales fue abundante, la cantidad de ácido cafeico fue muy baja, quedando la incógnita de qué tipo de compuestos polifenólicos están presentes en el extracto. Por otra parte, en el extracto C se encontró elmayor número dealquilamidas, lo cual podría asociarse al efecto antiproliferativo observado en este extracto.Tres de estas alquilamidas sólo se observaron en el extracto C que es el único que contiene E. angustifolia lo que podría indicar que pueden ser utilizadas como marcadores de especie. El efecto antiinflamatorio que se observó para el extracto B se podría correlacionar con la presencia de saponinas existiendo evidencia en la literatura de compuestos de este tipo asociados a procesos antiinflamatorios, aunque es importante mencionar que las saponinas son un grupo muy diverso lo cual sería interesante evaluar este grupo de compuestos de manera específica iniciando por parte aéreas donde observó la mayor cantidad y centrándonos en terpenoides que dentro de las saponinas son los que se asocian frecuentemente a este tipo de efecto farmacológico. Conclusiones. Se observaron diferencias en los parámetros físicos, químicos y biológicos en los tres extractos evaluados, mostrando la necesidad de plantear estrategias de estandarización llevando a cabo estudios que establezcan una correlación de una manera más fina y que permitan concluir la propuesta de asociación planteada en este estudio, avanzando con ello en el uso adecuado de los medicamentos herbales generando datos de composición química y efecto biológico, datos que se deben sumar a los protocolos actuales que se basan en su mayoría en garantizar la inocuidad e identidad del producto

Effects of Pasteurization on Antihyperglycemic and Chemical Parameter of Xoconostle (Stenocereus stellatus) Juice

Background and Objective: The antihyperglycemic effect is associated with the pre-hispanic fruit xoconostle or tunillo (Stenocereus stellatus, Pfeiffer and Riccobono). This fruit includes in various varieties, distinguished by color. Xoconostle fruits are highly perishable. Therefore, the aim of this study was to assess antihyperglycemic effects of xoconostle juice before (fresh) and after pasteurization. The study focused on the white and red varieties of xoconostle. Material and Methods: In this study, the method involved collecting juice from xoconostle fruits, followed by pasteurization. Chemical, physical and microbial parameters were assessed for the juice and the ability to decrease capillary glucose levels (antihyperglycemic effect) was assessed in male Wistar rats. Results and Conclusion: Pasteurization process led to decreases in total phenolic content of the red variety of xoconostle fruit, while the white variety showed increases in malic acid content. Despite these changes, fresh and pasteurized juices of the two varieties showed lower blood glucose levels, compared to the control group. Red variety demonstrated a stronger antihyperglycemic effect. In conclusion, pasteurization did not affect pharmacological effects of xoconostle juice, making it a viable preservation method without compromising the antihyperglycemic charac-teristics. Results of this research suggest a conservation method which preserve the antihyperglycemic effects while extending its shelf life. Conflict of interest: The authors declare no conflict of interest

Micropropagation of Buddleja cordata and the content of verbascoside and total phenols with antioxidant activity of the regenerated plantlets

Buddleja cordata is a medicinal plant distributed in Mexican territory that is characterized for producing phenolic compounds possessing antioxidant activity. It was evaluated the type of morphogenetic responses induced by plant growth regulators. Furthermore, the content of verbascoside and total phenols was determined, as well as the antioxidant activity in regenerated plantlets. The greatest shoot proliferation (29.2 shoots per explant) was achieved in the stem-nodes that were grown in a half-strength Murashige and Skoog medium containing 4.44 µM N6-benzyladenine. The greatest shoot heights of 4.3 and 4.7 cm (statistically not different) were obtained with gibberellic acid at 4.34 and 8.67 µM, respectively. The highest percentage of rooting (89.4%) occurred with 2.45 µM indole-3-butyric acid with 20.3 roots per shoot and an average root length of 4.4 cm. Of the rooted shoots, 91.7% were able to survive after 30 days of acclimatization. The verbascoside (1.0 mg g-1) and total phenols (24.8 mg of gallic acid equivalents g-1) content was related to antioxidant activity of the regenerated plantlets. The micropropagation of B. cordata might represent an alternative about massive production and depict the basis of the establishment of commercial crops and genetic studies

Micropropagación de Buddleja cordata y el contenido de verbascósido y fenoles totales con actividad antioxidante de las plántulas regeneradas

Buddleja cordata is a medicinal plant distributed in Mexican territory that is characterized for producing phenolic compounds possessing antioxidant activity. It was evaluated the type of morphogenetic responses induced by plant growth regulators. Furthermore, the content of verbascoside and total phenols was determined, as well as the antioxidant activity in regenerated plantlets. The greatest shoot proliferation (29.2 shoots per explant) was achieved in the stem-nodes that were grown in a half-strength Murashige and Skoog medium containing 4.44 µM N6-benzyladenine. The greatest shoot heights of 4.3 and 4.7 cm (statistically not different) were obtained with gibberellic acid at 4.34 and 8.67 µM, respectively. The highest percentage of rooting (89.4%) occurred with 2.45 µM indole-3-butyric acid with 20.3 roots per shoot and an average root length of 4.4 cm. Of the rooted shoots, 91.7% were able to survive after 30 days of acclimatization. The verbascoside (1.0 mg g-1) and total phenols (24.8 mg of gallic acid equivalents g-1) content was related to antioxidant activity of the regenerated plantlets. The micropropagation of B. cordata might represent an alternative about massive production and depict the basis of the establishment of commercial crops and genetic studies

Pharmacological and phytochemical potential study of plants collected in Amecameca, State of Mexico, Mexico

62-67<span style="font-size:11.0pt;font-family: " times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" mangal;mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:="" hi"="" lang="EN-US">This study involved several plants collected from February to May of 2014 in Amecameca, State of Mexico, Mexico. The collected species were Castilleja tenuiflora Benth., Lupinus campestris Schltdl. & Cam., Salvia gesneriiflora Lindl. & Paxton, Senecio barba-johannis DC., Salvia hispanica L., Stevia monardifolia Kunth, Senecios alignus DC., Zephyranthes verecunda Herb., Asclepias notha W.D. Stevens, Cestrum roseum Kunth, Bouvardia ternifolia (Cav.) Schltdl., Phaseolus coccineus L. The plant material underwent to a qualitative assessment of the present main chemical groups and some secondary metabolites such as caffeic and chlorogenic acid, total phenols and flavonoids. Also, the antioxidant capacity of the collected species was evaluated using the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) method. Our results indicated that the plant commonly known as ‘chamiso’ and taxonomically identified as Stevia monardifolia showed promising antioxidant properties (109±11.0 mMEAA). These results correlate with the high content of caffeic and chlorogenic acid (6.1 and 553 ppm, respectively), phenolic compounds (967±36 mEGA/gdw) and total flavonoids (136± 10 mEQ/gdw), which makes this plant an interesting candidate for more specific pharmacological studies.</span

Studies on phytochemical, antioxidant, anti-inflammatory, hypoglycaemic and antiproliferative activities of Echinacea purpurea and Echinacea angustifolia extracts

Context: Echinacea (Asteraceae) is used because of its pharmacological properties. However, there are few studies that integrate phytochemical analyses with pharmacological effects. Objective: Evaluate the chemical profile and biological activity of hydroalcoholic Echinacea extracts. Materials and methods: Density, dry matter, phenols (Folin–Ciocalteu method), flavonoids (AlCl3 method), alkylamides (GC-MS analysis), antioxidant capacity (DPPH and ABTS methods), antiproliferative effect (SRB assay), anti-inflammatory effect (paw oedema assay, 11 days/Wistar rats; 0.4 mL/kg) and hypoglycaemic effect (33 days/Wistar rats; 0.4 mL/kg) were determined in three Echinacea extracts which were labelled as A, B and C (A, roots of Echinacea purpurea L. Moench; B, roots, leaves, flowers and seeds of Echinacea purpurea; C, aerial parts and roots of Echinacea purpurea and roots of Echinacea angustifolia DC). Results: Extract C showed higher density (0.97 g/mL), dry matter (0.23 g/mL), phenols (137.5 ± 2.3 mEAG/mL), flavonoids (0.62 ± 0.02 mEQ/mL), and caffeic acid (0.048 mg/L) compared to A and B. A, B presented 11 alkylamides, whereas C presented those 11 and three more. B decreased the oedema (40%) on day 2 similar to indomethacin. A and C showed hypoglycaemic activity similar to glibenclamide. Antiproliferative effect was only detected for C (IC50 270 μg/mL; 8171 μg/mL; 9338 μg/mL in HeLa, MCF-7, HCT-15, respectively). Discussion and conclusion: The difference in the chemical and pharmacological properties among extracts highlights the need to consider strategies and policies for standardization of commercial herbal extracts in order to guarantee the safety and identity of this type of products