Purification of thonningianins A and B and four further derivatives from Thonningia sanguinea by one- and two-dimensional centrifugal partition chromatography.

Thonningia sanguinea is a parasitic herb widely used in traditional African medicine. Dihydrochalcone glucosides (unsubstituted, substituted with hexahydroxydiphenoyl or galloyl moieties) are the main constituents in the subaerial parts of this plant. In the present study, purification of the six major compounds from a methanol extract of the plant's subaerial parts was achieved by centrifugal partition chromatography. A first dimension centrifugal partition chromatography separation with the solvent system methyl tert-butyl ether/1,2-dimethoxyethane/water (1:2:1) in the ascending mode enabled the isolation of the two major bioactive compounds thonningianin A and B from 350 mg of methanol extract within only 16 min with respectable yields (25.7 and 21.1 mg), purities (87.1 and 85%), and recoveries (71.2 and 70.4%). Using a multiple heart-cutting strategy, the remaining four major dihydrochalcone glucosides of the extract were further separated in a second dimension centrifugal partition chromatography with the solvent system ethyl acetate/1,2-dimethoxyethane/water (2:1:1) in the descending mode with high purities (88.9-98.8%).

Development and Validation of a UHPLC-DAD Method for the Quantitative Analysis of Major Dihydrochalcone Glucosides from Thonningia sanguinea VAHL.

Thonnigia sanguinea is a plant widely used in traditional African medicine against a variety of diseases. The obligate parasite is growing throughout tropical African forests and utilizes a large variety of hosts. Dihydrochalcone glucoside derivatives isolated from the subaerial parts of this plant were identified as potential antidiabetic lead compounds. In this study, an ultrahigh-performance liquid chromatographic method coupled with a photodiode array detector was developed for the quantitation of six major dihydrochalcone derivatives. The analytes were baseline separated in complex samples within 14 minutes on a Phenomenex Luna Omega 1.6 µm C18 column using a mobile phase consisting of water and acetonitrile (each + 0.01% trifluoroacetic acid) in gradient elution. Method validation confirmed the selectivity, linearity (R2 ≥ 0.9992), precision (inter-day ≤ 1.98%, intraday ≤ 2.00%), and accuracy (recovery rates of 97.4 - 106.3% for all analytes). At 280 nm, the LODs and LOQs were found to be lower than 1.42 and 4.30 µg/mL, respectively. Eight plant batches from the northern Angolan province of Uíge (collected in the wild or bought on markets) were extracted with methanol using an ultrasound-assisted extraction protocol and subsequently analyzed with the validated method. Results indicated high contents of dihydrochalcone glucosides in all eight samples. Most notably, the two bioactive constituents thonningianin A and B were present in fairly large amounts (2.42 - 5.35 w%).

Dihydrochalcone Glucosides from the Subaerial Parts of Thonningia sanguinea and Their in Vitro PTP1B Inhibitory Activities.

Six new and four known dihydrochalcone glucoside derivatives (1-10), the phenylpropanoid coniferin (11), and the lignans (+)-pinoresinol (12) and lariciresinol (13) were isolated from the subaerial plant parts of Thonningia sanguinea in the course of a screening campaign for new antidiabetic lead compounds. The structures of the new substances were elucidated by HRESIMS, NMR, GC-MS, and ECD data evaluation. 2'- O-(3-Galloyl-4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)-3-hydroxyphloretin (4), 2'- O-(4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)phloretin (5), 2'- O-(3- O-galloyl-4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)phloretin (6), and thonningianin B (9) showed moderate protein tyrosine phosphatase-1B inhibition in an enzyme assay (IC50 values ranging from 19 to 25 µM), whereas thonningianin A (10) was identified as a more potent inhibitor (IC50 = 4.4 µM). The observed activity differences could be explained by molecular docking experiments. The activity of 10 could further be confirmed in HEPG2 liver carcinoma cells, where the compound was able to increase the level of phosphorylated insulin receptors in a concentration-dependent manner.

Medicinal plants of northern Angola and their anti-inflammatory properties.

ETHNOPHARMACOLOGICAL RELEVANCE: 32 plants, from which 30 are used in local traditional medicine - identified by interviews with the resident population - in the province of Uíge in northern Angola for the treatment of inflammation related disorders, were screened on different anti-inflammatory parameters. Three extracts were selected for a detailed ethnobotanical, pharmacological and phytochemical investigation based on their in vitro activity. AIM OF THE STUDY: We aimed to assess the in vitro anti-inflammatory activity of these plants and highlight the active principles of the three most promising candidates. MATERIALS AND METHODS: Plant material was collected in northern Angola during eight field trips from 2013 to 2015 based on data documented in 61 interviews with 142 local informants. 36 methanol (MeOH) extracts were prepared and tested at different concentrations (100, 50, 10µg/mL) to evaluate their inhibition on cyclooxygenase (COX)-2 expression and on nitric oxide (NO) release in lipopolysaccharide (LPS)-stimulated J774A.1 macrophages. Five selected extracts were additionally tested at the lower concentrations of 5, 2.5, and 1.25µg/mL and for their potential on inhibition of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) release. The major compounds of three of those five extracts were either identified by HPLC coupled with (tandem) mass spectrometry and comparison with data from literature or isolated from the respective extracts and confirmed by NMR experiments (one and two dimensional). RESULTS: 30 plant species with in total 161 citations were mentioned by the informants to have anti-inflammatory properties. The predominantly used plant part is the leaf (39%), followed by underground organs like roots and rhizomes (25%), bark (18%) as well as fruits and seeds with 15%. With 47%, decoction is the most frequent preparation form. A large number of the MeOH extracts showed promising activities in our preliminary screening for the inhibition of COX-2 expression and NO release. Five extracts with high activities in both assays showed also concentration dependent inhibition at lower concentrations and a decreased release of two additional pro-inflammatory mediators (IL-6 and TNF-α) vs. LPS. Three leaf extracts where chosen for a detailed investigation, which lead to the identification of several constituents: verbascoside and isoverbascoside (Acanthus montanus), geraniin, chebulagic acid and a large flavonoid fraction (Alchornea cordifolia) as well as the four flavonoids astilbin, isovitexin, isoorientin and swertisin (Chaetocarpus africanus). Their implication in the observed biological activity was proved by comparison with published data of these compounds in identical or similar pharmacological models. CONCLUSIONS: The indigenous use of these plants against inflammation related ailments could be - at least partly - verified by our in vitro models for many of the investigated extracts. Acanthus montanus and Alchornea cordifolia particularly stood out with their high activity in all four performed assays, which was in accordance with pharmacological studies of their major constituents in literature. In addition, this study was the first phytochemical investigation of Chaetocarpus africanus and first description of the occurrence of the ellagitannins geraniin and chebulagic acid in Alchornea cordifolia. These results support the traditional use and should encourage further investigations of medicinal plants of northern Angola.