Quantitative Chemical Composition, Anti-Oxidant Activity, and Inhibition of TNF Release by THP-1 Cells Induced by Extracts of Echinodorus macrophyllus and Echinodorus grandiflorus.

This study investigated the similarities between Echinodorus macrophyllus and Echinodorus grandiflorus, plant species that are traditionally used in Brazil to treat rheumatism and arthritis, whose anti-inflammatory effects are supported by scientific evidence. The contents of cis- and trans-aconitic acid, homoorientin, chicoric acid, swertisin, caffeoyl-feruloyl-tartaric acid, and di-feruloyl-tartaric acid were quantified by UPLC-DAD in various hydroethanolic extracts from the leaves, whereas their anti-oxidant activity and their effect on TNF release by LPS-stimulated THP-1 cells were assessed to evaluate potential anti-inflammatory effects. The 50% and 70% ethanol extracts showed higher concentrations of the analyzed markers in two commercial samples and a cultivated specimen of E. macrophyllus, as well as in a commercial lot of E. grandiflorus. However, distinguishing between the species based on marker concentrations was not feasible. The 50% and 70% ethanol extracts also exhibited higher biological activity, yet they did not allow differentiation between the species, indicating similar chemical composition and biological effects. Principal component analysis highlighted comparable chemical composition and biological activity among the commercial samples of E. macrophyllus, while successfully distinguishing the cultivated specimen from the commercial lots. In summary, no differences were observed between the two species in terms of the evaluated chemical markers and biological activities.

Definition of chemical markers for Hancornia speciosa Gomes by chemometric analysis based on the chemical composition of extracts, their vasorelaxant effect and α-glucosidase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa Gomes (Apocynaceae) is a tree found in the Brazilian savannah, traditionally used to treat several diseases, including diabetes and hypertension. The anti-hypertensive activity of H. speciosa leaves (HSL) has been demonstrated in different models and is credited to the vasodilator effect and ACE (angiotensin-converting enzyme) inhibition. The hypoglycemic effect of HSL has been also reported. AIM OF THE STUDY: To establish correlations between the biological activities elicited by H. speciosa extracts and the contents of their major compounds, aiming to define chemical markers related to the potential antihypertensive and antidiabetic effects of the species. Additionally, it aimed to isolate and characterize the chemical structure of a marker related to the α-glucosidase inhibitory effect. MATERIALS AND METHODS: Extracts of a single batch of H. speciosa leaves were prepared by extraction with distinct solvents (ethanol/water in different proportions; methanol/ethyl acetate), employing percolation or static maceration as extraction techniques, at different time intervals. The contents of chlorogenic acid, rutin and FlavHS (a tri-O-glycoside of quercetin) were quantified by a developed and validated HPLC-PDA method. Bornesitol was determined by HPLC-PDA after derivatization with tosyl chloride, whereas total flavonoids were measured spectrophotometrically. Identification of other constituents in the extracts was performed by UPLC-DAD-ESI-MS/MS analysis. The vasorelaxant activity was assayed in rat aortic rings precontracted with phenylephrine, and α-glucosidase inhibition was tested in vitro. Principal component analysis (PCA) was employed to evaluate the contribution of each marker to the biological responses. Isolation of compound 1 was carried out by column chromatography and structure characterization was accomplished by NMR and UPLC-DAD-ESI-MS/MS analyses. RESULTS: The contents of the chemical markers (mean ± s.d. % w/w) varied significantly among the extracts, including total flavonoids (2.68 ± 0.14 to 5.28 ± 0.29), bornesitol (5.11 ± 0.26 to 7.75 ± 0.78), rutin (1.46 ± 0.06 to 1.97 ± 0.02), FlavHS (0.72 ± 0.05 to 0.94 ± 0.14) and chlorogenic acid (0.67 ± 0.09 to 0.91 ± 0.02). All extracts elicited vasorelaxant effect (pIC50 between 4.97 ± 0.22 to 6.48 ± 0.10) and α-glucosidase inhibition (pIC50 between 3.49 ± 0.21 to 4.03 ± 0.10). PCA disclosed positive correlations between the vasorelaxant effect and the contents of chlorogenic acid, rutin, total flavonoids, and FlavHS, whereas a negative correlation was found with bornesitol concentration. No significant correlation between α-glucosidase inhibition and the contents of the above-mentioned compounds was found. On the other hand, PCA carried out with the areas of the ten major peaks from the chromatograms disclosed positive correlations between a peak ascribed to co-eluted triterpenes and α-glucosidase inhibition. A triterpene was isolated and identified as 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol. CONCLUSION: According to PCA results, the vasorelaxant activity of H. speciosa extracts is related to flavonoids and chlorogenic acid, whereas the α-glucosidase inhibition is associated with lipophilic compounds, including esters of lupeol like 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol, described for the first time for the species. These compounds can be selected as chemical markers for the quality control of H. speciosa plant drug and derived extracts.

Polyphenol-rich extract and fractions of Terminalia phaeocarpa Eichler possess hypoglycemic effect, reduce the release of cytokines, and inhibit lipase, α-glucosidase, and α-amilase enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: species of Terminalia (Combretaceae) are used to treat diabetes and metabolic disorders in Asia, Africa, and America. Terminalia phaeocarpa Eichler is an endemic tree from Brazil, popularly known as capitão. This species is closely related to Terminalia argentea Mart., also vulgarly known as capitão, a native but not endemic tree. Due to their phenotype similarity, these species might eventually prove inseparable and they are indistinctly used by locals to treat diabetes, among other diseases. The potential antidiabetic effect of T. argentea has been previously reported, whereas the biological effects and chemical composition of T. phaeocarpa have never been addressed so far. AIM OF THE STUDY: investigate the hypoglycaemic effect of an ethanol extract (EE) of T. phaeocarpa leaves and its ethyl acetate (FrEtOAc) and hydromethanolic (FrMEOH) fractions, in addition to their activity on the release of pro-inflammatory mediators and inhibition of lipase, α-amylase, and α-glucosidase enzymes. Additionally, it aimed to characterize the chemical composition of the extract and fractions, seeking to identify the compounds related to the biological activities. MATERIALS AND METHODS: The effect on the release of TNF-α, IL-1ß, and CCL-2 was evaluated in LPS-stimulated THP-1 cells (ATCC TIB-202). The inhibition of lipase, α-amylase, and α-glucosidase was tested in vitro, whereas the hypoglycemic effect was assayed in the oral starch tolerance test. The chemical composition was investigated by extensive UHPLC-DAD-ESI-MS/MS analyses. RESULTS: The extract and derived fractions reduced TNF-α (EE pIC50 = 4.58 ± 0.01; FrEtOAc pIC50 = 4.69 ± 0.01; FrMeOH pIC50 = 4.54 ± 0.02) and IL-1ß (EE pIC50 = 4.86 ± 0.02; FrEtOAc pIC50 = 4.86 ± 0.02; FrMeOH pIC50 = 4.75 ± 0.01) release by LPS-stimulated THP-1 cells in a concentration-dependent manner, whereas the inhibitory effect on CCL-2 release did not reach a clear linear relationship for the tested concentrations. The extract and fractions also inhibited in vitro the activity of lipase (EE pIC50 = 3.97 ± 0.12; FrEtOAc pIC50 = 3.87 ± 0.04; FrMeOH pIC50 = 3.67 ± 0.14), α-amylase (EE pIC50 = 4.46 ± 0.27; FrEtOAc pIC50 = 5.47 ± 0.27; FrMeOH pIC50 = 4.26 ± 0.22), and α-glucosidase (EE pIC50 = 5.46 ± 0.05; FrEtOAc pIC50 = 5.79 ± 0.11; FrMeOH pIC50 = 5.74 ± 0.05). The pIC50 values of the test samples were lower than those obtained with orlistat (7.59 ± 0.08) and acarbose (6.04 ± 0.37 and 7.63 ± 0.04) employed as the positive controls respectively in the lipase, α-amylase, and α-glucosidase assays. When assayed in the oral starch tolerance test, the extract and fractions also reduced animal glycaemia. UHPLC-DAD-ESI-MS/MS analyses of the extract and fractions led to the identification of 38 phenolic compounds, mainly phenolic acids, ellagitannins and flavonoids, among others, all of them first-time described for the species. CONCLUSION: Based on our findings, T. phaeocarpa has hypoglycaemic activity and polyphenols are the probable bioactive compounds, which support the ethnomedical use of the species.

Forced degradation of l-(+)-bornesitol, a bioactive marker of Hancornia speciosa: Development and validation of stability indicating UHPLC-MS method and effect of degraded products on ACE inhibition.

The antihypertensive activity of the medicinal plant Hancornia speciosa has been previously demonstrated by us, being the activity ascribed to polyphenols and cyclitols like l-(+)-bornesitol. We herein evaluated the stability of the bioactive marker bornesitol submitted to forced degradation conditions. Bornesitol employed in the study was isolated from H. speciosa leaves. An UHPLC-ESI-MS/MS method was developed to investigate bornesitol stability based on MRM (Multiple Reaction Monitoring) acquisition mode and negative ionization mode, employing both specific (m/z 193 → 161 Da) and confirmatory (m/z 193 → 175 Da) transitions. A gradient elution of 0.1% formic acid in water and acetonitrile was performed on a HILIC column. The method was validated and showed adequate linearity (r2 > 0.99), selectivity, specificity, accuracy, and precision (RSD < 2.9%). The method was robust for deliberate variations on dessolvation temperature, but not for changes in the flow rate and dessolvation gas. The results from the stability studies allowed us to classify bornesitol as labile for acidic and alkaline hydrolysis, but as very stable for oxidative and neutral hydrolysis exposure. Bornesitol was categorized as practically stable under photolysis degradation, whereas a considerable reduction on its contents was induced by metal ions and thermolysis exposure. Degraded samples from neutral hydrolysis and thermolysis were assayed in vitro for ACE inhibition and showed a substantial decrease in biological activity as compared to intact bornesitol. myo-Inositol was identified as the major degradation products in both matrices. This is the first report on bornesitol stability under different stress conditions and the obtained data are relevant for the development and quality control of standardized products from H. speciosa leaves.