A silica gel orthogonal high-performance liquid chromatography method for the analyses of steviol glycosides: novel tetra-glucopyranosyl steviol.

A silica gel orthogonal method using acetonitrile: water was developed for the analyses of fractions rich in very polar steviol glycosides and resolve regions of co-elution of these compounds in reversed-phase. Additionally, we also used this normal phase analytical method to scale up the purification process of steviol glycosides. Using these approaches, one novel minor tetra-glucopyranosyl diterpene glycosides together with three known compounds were purified from a commercial Stevia rebaudiana leaf extract. Compound 1 was unambiguously elucidated as 13-[(2-O-ß-D-glucopyranosyl-ß-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(6-O-ß-D-glucopyranosyl-ß-D-glucopyranosyl) ester (rebaudioside Y) based on high-performance liquid chromatography retention times, tandem mass spectrometry dissociation pattern and 1D and 2D NMR experiments. Known compounds were isolated in gram quantities and identified as rebaudioside D, E and M.

Tetra-glucopyranosyl Diterpene ent-Kaur-16-en-19-oic Acid and ent-13(S)-Hydroxyatisenoic Acid Derivatives from a Commercial Extract of Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana and its diterpene glycosides are one of the main focuses of food companies interested in developing novel zero calorie sugar substitutes since the recognition of steviol glycosides as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Rebaudioside A, one of the major steviol glycosides of the leaves is more than 200 times sweeter than sucrose. However, its lingering aftertaste makes it less attractive as a table-top sweetener, despite its human health benefits. Herein, we report the purification of two novel tetra-glucopyranosyl diterpene glycosides 1 and 3 (rebaudioside A isomers) from a commercial Stevia rebaudiana leaf extract compounds, their saponification products compounds 2 and 4, together with three known compounds isolated in gram quantities. Compound 1 was determined to be 13-[(2-O-ß-d-glucopyranosyl-6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid-ß-d-glucopyranosy ester (rebaudioside Z), whereas compound 3 was found to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid -ß-d-glucopyranosy ester. Two new tetracyclic derivatives with no sugar at position C-19 were prepared from rebaudiosides 1 and 3 under mild alkaline hydrolysis to afford compounds 2 13-[(2-O-ß-d-glucopyranosyl-6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z1) and 4 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid. Three known compounds were purified in gram quantities and identified as rebaudiosides A (5), H (6) and J (7). Chemical structures were unambiguously elucidated using different approaches, namely HRESIMS, HRESI-MS/MS, and 1D-and 2D-NMR spectroscopic data. Additionally, a high-quality crystal of iso-stevioside was grown in methanol and its structure confirmed by X-ray diffraction.

Development of a high-performance liquid chromatography procedure to identify known and detect novel C-13 oligosaccharide moieties in diterpene glycosides from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae): Structure elucidation of rebaudiosides V and W.

As an aid for structure elucidation of new steviol glycosides, reversed-phase C18 high-performance liquid chromatography method was developed with several previously characterized diterpene glycosides, to identify known and detect novel aglycone-C13 oligosaccharide moieties and indirectly identify C-19 interlinkages. Elution order of several diterpene glycosides and their aglycone-C13 oligosaccharide substituted with different sugar arrangements were also summarized. Comparison of the retention time of a product obtained after alkaline hydrolysis with the aglycone-C-13 portions of known compounds reported herein allowed us to deduce the exact positions of the sugars in the C-13 oligosaccharide portion. The elution position of several steviol glycosides with an ent-kaurene skeleton was helpful to describe an identification key. Two previously uncharacterized diterpene glycosides together with two known compounds were isolated from a commercial Stevia rebaudiana leaf extract. One was found to be 13-[(2-O-ß-d-xylopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) ester (rebaudioside V), whereas the other was determined to be 13-[(2-O-ß-d-xylopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-α-l-rhamnopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) ester (rebaudioside W). Previously reported compounds were isolated in gram quantities and identified as rebaudioside J and rebaudioside H. In addition, a C-19 sugar-free derivative was also prepared from rebaudioside H to afford rebaudioside H1 . Chemical structures were partially determined by the high-performance liquid chromatography method and unambiguously characterized by using one-dimensional and two-dimensional nuclear magnetic resonance experiments.

Rebaudiosides T and U, minor C-19 xylopyranosyl and arabinopyranosyl steviol glycoside derivatives from Stevia rebaudiana (Bertoni) Bertoni.

Two diterpene glycosides were isolated from a commercial Stevia rebaudiana leaf extract. One was found to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-ß-d-xylopyranosyl-3-O-ß-d-glucopyranosyl- ß-d-glucopyranosyl) ester (rebaudioside T), whereas the other was determined to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(6-O-α-l-arabinopyranosyl-ß-d-glucopyranosyl) ester (rebaudioside U). In addition, five C-19 sugar free derivatives were prepared and identified as follows: 13-[(2-O-α-l-rhamnopyranosyl-ß-d-glucopyranosyl)]oxy]kaur-16-en-19-oic acid (dulcoside A1); 13-[(2-O-ß-d-xylopyranosy-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]kaur-16-en-19-oic acid; 13-[(2-O-ß-d-xylopyranosyl-ß-d-glucopyranosyl-)oxy]kaur-16-en-19-oic acid; 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-xylopyranosyl-)oxy]kaur-16-en-19-oic acid (rebaudioside R1) and 13-[(2-O-6-deoxy-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]kaur-16-en-19-oic acid, respectively. Chemical structures were determined by NMR experiments. HPLC analyses were also useful to differentiate different steviol-C13 sugar substituent patterns by elution position.

Rebaudiosides R and S, Minor Diterpene Glycosides from the Leaves of Stevia rebaudiana.

Two new diterpene glycosides have been isolated from a commercial extract of the leaves of Stevia rebaudiana. Compound 1 was shown to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-xylopyranosyl)oxy]ent-kaur-16-en-19-oic acid ß-d-glucopyranosyl ester (rebaudioside R), while compound 2 was determined to be 13-[(2-O-α-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 2-O-α-l-rhamnopyranosyl-ß-d-glucopyranosyl ester (rebaudioside S). Six additional known compounds were identified, dulcoside B, 13-[(2-O-ß-d-xylopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid ß-d-glucopyranosyl ester, eugenol diglucoside, rebaudioside G, 13-[(2-O-6-deoxy-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid ß-d-glucopyranosyl ester, and rebaudioside D (3), respectively. The structures of 1 and 2 were determined based on comprehensive 1D and 2D NMR (COSY, HSQC, and HMBC) studies. A high-quality crystal of compound 3 allowed confirmation of its structure by X-ray diffraction.

HPLC Plasma Assay of a Novel Anti-MRSA Compound, Kaempferol-3-O-Alpha-L-(2",3"-di-p-coumaroyl)rhamnoside, from Sycamore Leaves.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious pathogen that is resistant to current antibiotic therapy. Thus, there is an urgent need for novel antimicrobial agents that can effectively combat these new strains of drug-resistant "superbugs". Recently, fractionation of an extract from Platanus occidentalis (American sycamore) leaves produced an active kaempferol molecule, 3-O-alpha-L-(2",3"-di-p-coumaroyl)rhamnoside (KCR), in four isomeric forms; all four isomers exhibit potent anti-MRSA activity. In order to further the preclinical development of KCR as a new antibiotic class, we developed and validated a simple analytical method for assaying KCR plasma concentration. Because KCR will be developed as a new drug, although comprising four stereoisomers, the analytical method was devised to assay the total amount of all four isomers. In the present work, both a plasma processing procedure and an HPLC method have been developed and validated. Mouse plasma containing KCR was first treated with ethanol and then centrifuged. The supernatant was dried, suspended in ethanol, centrifuged, and the supernatant was injected into an HPLC system comprising a Waters C18, a mobile phase composing methanol, acetonitrile, and trifluoroacetic acid and monitored at 313 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 0.27 microg/mL, and high accuracy. In summary, this method allows a rapid analysis of KCR in the plasma samples for pharmacokinetics studies.

Gastroprotective activity of the hydroethanolic extract and isolated compounds from the leaves of Solanum cernuum Vell.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum cernuum Vell. (Solanaceae) is a Brazilian medicinal plant, traditionally known as "panaceia". Its folk name is probably due to its wide range of applications in traditional medicine including the treatment of ulcers. AIM OF THE STUDY: To evaluate the gastroprotective activities of the hydroethanolic extract (ESC) of S. cernuum and its major isolated compounds using in vivo gastric ulcer models. MATERIAL AND METHODS: The ESC extract was obtained by maceration followed by percolation of the dried and powdered leaves of S. cernuum in ethanol:water (7:3). The major compounds in the extract were isolated by applying various preparative chromatographic techniques. The gastroprotective activity was evaluated in mice using different gastric ulcer-induced models. The anti-Helicobacter pylori activity was performed using the agar-well diffusion and broth microdilution methods. RESULTS: The ESC extract showed gastroprotective effects in the assay of acute gastric ulcer-induced by HCl/EtOH, nonsteroidal anti-inflammatory drug, and acetic acid-induced chronic ulcer protocols. The results also demonstrated that the gastroprotection induced by ESC extract is related to the activity of nitric oxide and endogenous sulfhydryls, which are important gastroprotective factors. The ESC extract and the alkaloid cernumidine did not show activity against H. pylori in the concentrations tested. CONCLUSIONS: The present study showed that the crude extract of S. cernuum possessed gastroprotective activity which corroborating the traditional use of this plant for the treatment of gastric ulcers. The isolated flavonoids, quercitrin and afzelin as well as the phenylpropanoid, isoferulic acid are suggested to be the compounds responsible for the gastroprotective activity of S. cernuum extract.

Antibacterial Activities of Metabolites from Platanus occidentalis (American sycamore) against Fish Pathogenic Bacteria.

One approach to the management of common fish diseases in aquaculture is the use of antibiotic-laden feed. However, there are public concerns about the use of antibiotics in agriculture and the potential development of antibiotic resistant bacteria. Therefore, the discovery of other environmentally safe natural compounds as alternatives to antibiotics would benefit the aquaculture industries. Four natural compounds, commonly called platanosides, [kaempferol 3-O-α-L-(2″,3″-di-E-p-coumaroyl)rhamnoside (1), kaempferol 3-O-α-L-(2″-E-p-coumaroyl-3″-Z-p-coumaroyl)rhamnoside (2), kaempferol 3-O-α-L-(2″-Z-p-coumaroyl-3″-E-p-coumaroyl)rhamnoside (3), and kaempferol 3-O-α-L-(2″,3″-di-Z-p-coumaroyl)rhamnoside (4)] isolated from the leaves of the American sycamore (Platanus occidentalis) tree were evaluated using a rapid bioassay for their antibacterial activities against common fish pathogenic bacteria including Flavobacterium columnare, Edwardsiella ictaluri, Aeromonas hydrophila, and Streptococcus iniae. The four isomers and a mixture of all four isomers were strongly antibacterial against isolates of F. columnare and S. iniae. Against F. columnare ALM-00-173, 3 and 4 showed the strongest antibacterial activities, with 24-h 50% inhibition concentration (IC50) values of 2.13 ± 0.11 and 2.62 ± 0.23 mg/L, respectively. Against S. iniae LA94-426, 4 had the strongest antibacterial activity, with 24-h IC50 of 1.87 ± 0.23 mg/L. Neither a mixture of the isomers nor any of the individual isomers were antibacterial against isolates of E. ictaluri and A. hydrophila at the test concentrations used in the study. Several of the isomers appear promising for the potential management of columnaris disease and streptococcosis in fish.

Minor diterpene glycosides from the leaves of Stevia rebaudiana.

Two new diterpene glycosides in addition to five known glycosides have been isolated from a commercial extract of the leaves of Stevia rebaudiana. Compound 1 (rebaudioside KA) was shown to be 13-[(O-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 2-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl ester and compound 2, 12-α-[(2-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid ß-d-glucopyranosyl ester. Five additional known compounds were identified, rebaudioside E, rebaudioside M, rebaudioside N, rebaudioside O, and stevioside, respectively. Enzymatic hydrolysis of stevioside afforded the known ent-kaurane aglycone 13-hydroxy-ent-kaur-16-en-19-oic acid (steviol) (3). The isolated metabolite 1 possesses the ent-kaurane aglycone steviol (3), while compound 2 represents the first example of the isomeric diterpene 12-α-hydroxy-ent-kaur-16-en-19-oic acid existing as a glycoside in S. rebaudiana. The structures of the isolated metabolites 1 and 2 were determined based on comprehensive 1D- and 2D-NMR (COSY, HSQC, and HMBC) studies. A high-quality crystal of compound 3 has formed, which allowed the acquisition of X-ray diffraction data that confirmed its structure. The structural similarities between the new metabolites and the commercially available stevioside sweeteners suggest the newly isolated metabolites should be examined for their organoleptic properties. Accordingly rebaudiosides E, M, N, O, and KA have been isolated in greater than gram quantities.

Preparative chromatography and natural products discovery.

Methodology is reviewed which significantly enhances the economics and efficiency of larger scale preparative normal phase chromatography. Maintenance of hydration of the silica media and regeneration and re-equilibration of the column after each separation is demonstrated to allow repeated use of the column without loss of performance or requirement of repacking.

Stevioside methanol tetra-solvate.

Stevioside is a naturally occurring diterpenoid glycoside in Stevia rebaudiana Bertoni. The title compound, C38H60O18·4CH3OH, crystallized as its methanol tetrasolvate. Stevioside consists of an aglycone steviol (a tetra-cyclic diterpene in which the four-fused-ring system consists of three six-membered rings and one five-membered ring) and a sugar part (three glucose units). A weak intra-molecular O-H⋯O hydrogen bond occurs. In the crystal, the methanol mol-ecules participate in a two-dimensional hydrogen-bonded network parallel to b axis with the sugars and together they form a hydrophilic tunnel which encloses the lipophilic part of the molecule.