Simultaneous screening and analysis of antiplatelet aggregation active alkaloids from Rhizoma Corydalis.

CONTEXT: The rising problem of atherosclerosis and ischemic heart disease emphasizes the need to look for new antithrombotic components with effective modes of action. Corydalis yanhusuo (Y.H. Chou & Chun C. Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae) (Rhizoma Corydalis) has been used in the traditional medicines for the treatment of cardiovascular disease. OBJECTIVE: The antiplatelet aggregation compounds in Rhizoma Corydalis were screened to validate its traditional medicinal use. MATERIAL AND METHODS: Total alkaloid extract (TAE) of Rhizoma Corydalis was obtained by refluxing 100 g Rhizoma Corydalis powder with 600 mL 70% ethanol, and purified by acidification (20% HCl) and alkalization (5 M NaOH) process. Potential antiplatelet aggregation compounds in TAE were screened by a method involving platelet bio-specific extraction and HPLC-DAD/LC-MS analysis. Further in vitro antiplatelet aggregation activity confirmation of TAE and seven main alkaloids were achieved by turbidimetry method within 3 h after blood collection from rabbit carotid artery, and all the test drugs were at the concentration range of 25-350 µg/mL. Finally, HPLC-DAD was employed for the quantitative determination of seven main components in TAE. RESULTS: Five alkaloids, identified as glaucine, dehydrocorydaline, canadine, tetrahydrocoptisine and corydaline, can be specifically extracted with platelets. The results indicated that all these five alkaloids can inhibit thrombin-induced platelet aggregation in a low dose (IC50 of glaucine, dehydrocorydaline, canadine, tetrahydrocoptisine and corydaline were 49.057, 34.914, 33.547, 84.261 and 54.164 µg/mL, respectively) as compared to TAE (IC50 = 175.426 µg/mL) and aspirin (IC50 = 300.340 µg/mL), while the unbound compounds (palmatine and tetrahydropalmatine) had a very weak antiplatelet effect (IC50 > 200 µg/mL). DISCUSSION AND CONCLUSION: This study is the first reported work for antiplatelet components screening in Rhizoma Corydalis. Seven compounds were detected and identified by HPLC-DAD/LC-MS, of which five platelet-targeted compounds were discovered.

Β-hydroxymyristic acid as a chemical marker to detect endotoxins in dialysis water.

An analytical chemical method has been developed for determination of ß-hydroxymyristic acid (ß-HMA), a component of lipopolysaccharides (LPSs/endotoxins) in dialysis water. In our investigation, the ß-HMA component was used as a chemical marker for endotoxin presence in dialysis water because it is available in the molecular subunit (lipid A) and responsible for toxicity. It is the most abundant saturated fatty acid in that subunit. The developed method is based on fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ). A high-performance liquid chromatographic separation of the ß-HMA derivative was achieved using an octadecyl silica column in gradient elution. A wide dynamic range of ß-HMA was tested and a calibration curve was constructed with accuracy of 90% and variability of less than 10%. The limits of detection and quantification obtained were 2 and 5µM, respectively. The developed method was applied to detect endotoxins in dialysis water by alkaline hydrolysis of LPS using NaOH (0.25M) at 60°C for 2h. After hydrolysis, free acid was detected as its NBD-PZ derivative using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Good recovery rates ranging from 98 to 105% were obtained for ß-HMA in dialysis water.

Optimized pressurized hot water extraction, HPLC/LC-MS characterization, and bioactivity of Tetrapleura tetraptera L. dry fruit polyphenols.

Despite the global preference for green extraction methods in the recovery of plant bioactives, Tetrapleura tetraptera fruit polyphenols (TTP) are yet to receive considerable attention. For the first time, pressurized hot water extraction (PHWE) of TTP was optimized for total phenol content (TPC) and antioxidant activity (AA) using the Box Behnken design of response surface methodology. Predictor variables were time, temperature, and liquid-to-solid ratio. An optimum solution with a desirability of 0.805 was selected and parameters were 43 min, 220°C, and 60 ml g-1 liquid-to-solid ratio, yielding TPC of 8.92 mg gallic acid equivalent per gram of sample on dry weight basis (GAE g-1  dw-1 ) and AA of 70.35%. Purified, optimized TTP were characterized and quantified using HPLC/LC-MS. PHWE mainly extracted rutin (379.04 µg g-1 ), cyanidin-3-O-glucoside (chloride) (299.55 µg g-1 ), naringenin 7-O-glucoside (240.11 µg g-1 ), p-coumaric acid (177.28 µg g-1 ), isorientin (150.43 µg g-1 ), and gallic acid (118.06 µg g-1 ) whereas cyanidin-3-O-glucoside (chloride) (83.27 µg g-1 ), protocatechuic acid (61.37 µg g-1 ), rutin (28.03 µg g-1 ), and gallic acid (22.62 µg g-1 ) were mainly extracted by hot water extraction, which was a control. PHWE-obtained TTP showed higher cellular antioxidant activity, cytotoxicity in human liver cancer cell lines (HepG2), and antimicrobial property against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis than control. The potential mechanisms underlying the biological activities of some of the major polyphenols extracted were briefly discussed. Considering the wide use of the T. tetraptera (TT) fruit in Africa in foods and medicine, the use of more efficient green extraction methods such as PHWE is recommended. PRACTICAL APPLICATION: This study serves as a baseline for optimizing pressurized hot water extraction, purification, identification, and quantification of Tetrapleura tetraptera polyphenols (TTP) and their biological activities, being the first of its kind. The varied biological effects shown can be exploited further for applications of TTP as nutraceutical agents and preservatives in foods in different forms. Also, the high amounts of gallic acid and other phenolic acids and flavonoids confirmed in this study make TTP good candidates for the development of metal-phenol network nanoparticles to enhance adequate solubility and distribution in food systems in light of the above proposed applications.

Separation of highly hydrophilic nicotinamide metabolites using a COSMOSIL PBr column.

Highly hydrophilic compounds such as nicotinamide metabolites are very difficult to separate via high-performance liquid chromatography (HPLC) using octadecyl (C18) columns. In general, for the separation of hydrophilic compounds, hydrophilic interaction liquid chromatography (HILIC) columns are used instead of reversed phase chromatography using C18 columns. However, HILIC columns generally obey complex separation mechanisms because ionic interactions are involved in the retention process, which hinders the optimization of the separation conditions. Additionally, the resulting peak shapes are disturbed when large amounts of aqueous samples are injected. This study demonstrates that COSMOSIL PBr columns, in which both hydrophobic and dispersive interactions occur, show high retention for various hydrophilic compounds under similar separation conditions as those used with C18 columns. Specifically, using a COSMOSIL PBr column, 11 nicotinamide metabolites could be separated under simpler conditions than those used previously with C18 columns, affording better peak shape for each compound. The applicability of the method was evaluated using a tomato sample, from which the nicotinamide metabolites were successfully separated. The results show that the COSMOSIL PBr column is a good alternative to the C18 column for a good separation of all the peaks, including impurity peaks.

Hallazgo de diclofenaco en un producto fitoterapéutico a base de caléndula comercializado en Colombia / Finding diclofenac in a marigold-based phytotherapeutic product marketed in Colombia

Resumen Introducción: la consulta de un particular que trajo un producto fitoterapéutico a base de caléndula cuyo consumo le causó fuertes reacciones adversas, originó esta investigación sobre la composición de este producto. Objetivo: caracterizar la composición química de muestras de lotes diferentes de un producto comercial denominado fitoterapéutico a base de caléndula (Calendula officinalis) (PFC) comercializado en Colombia. Metodología: se analizaron tabletas de ocho cajas del PFC de cuatro lotes diferentes de producción (2017 y 2018). Se llevó a cabo el análisis de espacio de cabeza (HS) de tabletas por microextracción en fase sólida (SPME), con una fibra PDMS/DVB (65 µm), expuesta al HS de la muestra durante 30 min a 50 °C. Las fracciones volátiles se analizaron por cromatografía de gases acoplada a espectrometría de masas (GC/MS). Los extractos de tabletas obtenidos con mezcla de metanol:agua (1:1, v/v) se analizaron por cromatografía líquida (LC) de alta (HPLC) y ultra-alta eficiencia (UHPLC), con detectores de arreglo de diodos (DAD) y espectrometría de masas de alta resolución (HRMS), respectivamente; la cuantificación de diclofenaco se hizo por calibración con patrón externo y por adición de estándar. Los espectros de masas de baja y alta resolución y patrones de fragmentación de las sustancias detectadas se estudiaron, usando GC/HRMS y LC/HRMS-Orbitrap. Resultados: en tabletas analizadas por HSSPME, se encontraron monoterpenoides y sesquiterpenoides de origen vegetal, ftalatos, residuos de solventes (2-cloroetanol, etilenglicol) y sustancias químicas intermediarias en la síntesis de diclofenaco (2,6-dicloroanilina y 2,6-cloro-N-fenil-bencenamina). En los cromatogramas, obtenidos por GC/MS de los extractos de tabletas obtenidos con diclorometano, se detectaron diclofenaco, sus impurezas A, B y C, los ésteres de diclofenaco y algunas otras impurezas. Diclofenaco en cantidad ca. 40 mg (7-8%) se cuantificó por HPLC en tabletas (> 70 analizadas) escogidas al azar de ocho cajas del PFC, adquirido en el mercado local de Bucaramanga (Colombia). Conclusión: en cada tableta analizada se determinaron alrededor de 40 mg del compuesto sintético diclofenaco (sustancia no declarada en la etiqueta del producto) y en ninguna se detectaron ésteres de los triterpenoides oleanano o faradiol, constituyentes del extracto de caléndula que poseen actividad antiinflamatoria; se encontraron algunos flavonoides comunes a muchas plantas, en cantidades mil veces menores que la de diclofenaco.

Abstract Introduction: The consultation of a person who brought a marigold-based phytotherapeutic product whose consumption caused strong adverse reactions, originated this investigation of the composition of this product. Objective: to characterize the chemical composition of samples of different lots of a commercial product called calendula-based phytotherapeutic product (Calendula officinalis) (PFC) commercialized in Colombia. Methodology: Tablets of eight packs of the phytotherapeutic product from four different production batches (2017 and 2018) were analyzed. Headspace analysis (HS) of tablets by solid phase microextraction (SPME) was carried out with a PDMS/ DVB fiber (65 µm), exposed to the HS of the sample for 30 min at 50 °C. Volatile fractions were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Tablet extracts obtained with methanol:water mixture (1:1, v / v) were analyzed by liquid chromatography (LC) of high (HPLC) and ultra-high performance (UHPLC) with diode array (DAD) and high-resolution mass spectrometric (HRMS) detectors, respectively; diclofenac was quantified by external calibration and standard addition. Low- and high-resolution mass spectra (MS, HRMS) and fragmentation patterns of detected substances were studied, using GC/HRTOF-MS and LC/HRMS-Orbitrap. Results: in tablets analyzed by HS-SPME, monoterpenoids and sesquiterpenoids of plant origin, phthalates, solvent residues (2-chloroethanol, ethylene glycol) and intermediary chemicals in diclofenac synthesis (2,6-dichloroaniline and 2,6- chloro-N-phenyl-benzenamine) were found. In the chromatograms (GC/MS) of the extracts of tablets obtained with organic solvent (dichloromethane), diclofenac, its impurities A, B and C, diclofenac esters, and some other compounds were detected; diclofenac quantification by HPLC found amounts of ca. 40 mg (7 - 8%) in tablets (> 70 analyzed) chosen at random from eight packs of the calendula-based phytotherapeutic product, purchased in the local market in Bucaramanga (Colombia). Conclusion: each analyzed tablet contained around 40 mg of the synthetic compound diclofenac (substance not declared in the product's label) and no tablet contained detectable amounts of esters of the triterpenoids oleanane or faradiol, which are calendula extract constituents that possess antiinflammatory activity; a few flavonoids that are common to many plants were found in amounts a thousand times smaller than that of diclofenac.