Reversed-Phase HPLC Analysis of Steviol Glycosides Isolated from Stevia rebaudiana Bertoni

High Performance Liquid Chromatography (HPLC) experiments have been performed on nine steviol glycosides namely rebaudioside A, steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A isolated from the leaves of Stevia rebaudiana using Reversed-Phase (RP) column. Using RP-HPLC method, the individual retention times for nine naturally occurring ent-kaurane diterpene glycosides of S. rebaudiana reported in JECFA have been determined at four different temperatures: 20℃, 40℃, 60℃, and 79℃. Also, calculated the relative retention times of the eight steviol glycosides steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A against the major steviol glycoside rebaudioside A. HPLC results suggested that temperatures 40℃ and 60℃ would be ideal conditions for better separation of steviol glycosides.

Antimicrobial activity of kojic acid from endophytic fungus Colletotrichum gloeosporioides isolated from Sonneratia apetala,a mangrove plant of the Sundarbans

Objective: To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate(EtOAc) extract of endophytic fungus Colietotrichum gloeosporioides(C.gloeosporioides) isolated from Sonneratia apetala. Methods: Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Grampositive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay. Results: The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8 S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites.Kojic acid(1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1 D, 2 D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms.Whilst the minimum inhibitory concentration(MIC) values from the EtOAc extract ranged between 2.4×10^(-4)mg/mL and 2.5 mg/mL, those of kojic acid(1) were between 0.125 mg/mL and1 mg/mL. The EtOAc extract and kojic acid(1) were most active against Pseudomonas aeruginosa(MIC = 2.4×10^(-4). mg/mL) and Micrococcus luteus(MIC = 0.125 mg/mL), respectively. Conclusions:The results revealed that the endophytic fungus C. gloeosporioides could be a good source of commercially important kojic acid, which exhibited antimicrobial properties.

Chemical Composition and Antioxidant Activity of <i>Bunchosia glandulifera</i>Fruit at Different Ripening Stages

Bunchosia glandulifera belongs to the family Malpighiaceae and is popularly known as “caferana” in the city of Santo Antonio da Patrulha. The fruits have a high economic value and their inclusion in the diet has health benefits. They are natural sources of antioxidants because of the presence of bioactive compounds. However, their chemical composition can be influenced by the maturation process. This study aims to evaluate the changes in the bioactive antioxidant compounds in the pulp and seeds of B. glandulifera fruit during different stages of ripening. The fruits, were analyzed for caffeine, carotenoids, flavonoids, vitamin C, and total phenolic content as well as for antioxidant activity, at four different stages of ripening. An increase in bioactive compounds was observed from the second maturation stage to the ripened fruit, and the ripened fruit had the highest bioactive compound content. Phenolic compound and carotenoid levels were found to be highest in the pulp. Carotenoid content showed a positive correlation with the antioxidant activity. Colorimetric analysis also showed a high correlation between the levels of carotenoids and color change during the ripening stages.

Human sperm motility stimulating activity of a sulfono glycolipid isolated from Sri Lankan marine red alga Gelidiella acerosa

Aim: To evaluate the sperm motility stimulating activity of a sulfono glycolipid (S-ACT-1) isolated from Gelidiellaacerosa, a Sfi Lankan marine red algae. Methods: S-ACT-I, a white amorphous powder was separated from morepolar fractions of the hexane soluble of 1:1 CH_2Cl_2/MeOH extract and subjected to ~1H, ^(13)C NMR and IR Spectroscopyafter reverse phase HPLC for identification. Effects of S-ACT-1 on human sperm motility was assessed in vitro at 10,100 and 1000μg/mL concentrations at 37℃ for 0, 5, 15, 30 and 60 min. Results: S-ACT-1 was identified as aglycolipid sulfate. The lower dose increased the sperm motility slightly, whilst the medium dose significantly increasedthe motility (P < 0.05) from 5 min of incubation reaching a peak at 15 min and the stimulant effect was sustainedthroughout the experimental period. Furthermore, the medium dose rendered 80% of the immotile viable sperm motile.In contrast, the highest dose impaired the sperm motility. The sperm stimulating activity of S-ACT-1 was dose-depen-dent and had a bell-shaped dose response curve for all the 5 incubation periods. Conclusion: S-ACT-1 of Gelidiellaacerosa is a Sulfono glycolipid. S-ACT-1 has a potent sperm motility stimulating activity in vitro and has the potentialto be developed into a sperm stimulant. (Asian J Androl 2001 Mar; 3: 27-31)

A New Penta <i>β</i>-D-Glucopyranosyl Diterpene from <i>Stevia rebaudiana</i>

A new steviol diterpene glycoside, 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-{2-O-[6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl} ester (1) has been isolated from the commercial extract of the leaves of Stevia rebaudi-ana Bertoni. Structure of the new compound has been established on the basis of extensive NMR spectroscopy (1H &13C, TOCSY, HMQC, and HMBC) and High Resolution (HR) mass spectroscopic data as well as enzymatic and acid hydrolysis studies.

<i>Seco</i>-limonoid 11<i>α</i>,19<i>β</i>-dihydroxy-7-acetoxy-7- deoxoichangin promotes the resolution of <i>Leishmania panamensis</i>infection

The high morbidity generated by the infection caused by parasites of the genus Leishmania, make of this infection into one of the vector-borne infectious diseases most relevant worldwide, which added to the fact that the drugs used for its treatment are far from be optimal and considering that prophylactic approaches (such as the development of a vaccine) still seems far from being achieved, make of the search for new therapeutic alternatives for safe and effective treatment of this disease one of the most accurate approaches to the control of this disease. In this study we evaluated the antileishmanial and immunomodu- latory activity of the compound 11α,19β-dihydroxy- 7-acetoxy-7-deoxoichangin (a seco-limonid molecule) through: 1) evaluation of its cytotoxicity over promastigotes and axenic amastigotes of L. (V) panamensis, 2) determination of its ability to induce the control of in vitro infection, using infected murine cells (J774.2) and human dendritic cells (hDCs), 3) quantifying the levels of pro-inflammatory cytokines, (iv) evaluating the expression of cell markers associated with hDCs maturation, and (v) determinating the production of nitric oxide free radicals (NO). In this regard, this seco-limonoid exhibited an antileishmanial activity represented in the reduction of in vitro infection in J774.2 cells and hDCs, with a EC50 of 7.9 μM (4.48 μg/mL) and 25.5 μM (14.39 μg/mL), respectively, and additionally, we observed an increase on the production of IL-12p70, TNF-α and NO, as also, in the number of hDCs HLA-DR-positive in treated infected hDCs. These findings suggest that anti-lei- shmanial activity of this compound could be associated with the potential “reactivation” of phagocytic cell that is “paralyzed” by the infection, generating an immune phenotype associated with protection.

The Contribution of dα-Tocopherol and dγ-Tocopherol to the Antioxidant Capacity of Several Edible Plant Oils

Many oils from plants are important components of our food chain and maintaining their oxidative stability (OS) is economically and nutritionally important. OS is dependent in part on antioxidant capacity (AC) arising from the electron donating ability of endogenous compounds in the oils. Attention has focused on the contribution to AC of phenolic compounds in oils as many have bioactivities in vitro. However, the relevance of such phenolics to healthy nutrition remains unclear. In contrast, many plant-derived oils also contain tocopherol homologs, which as vitamin E are dietary-essential, lipid-soluble antioxidants. We have determined the AC of twelve “off the shelf” edible oils by assessing their ability to quench galvinoxyl, a stable free radical species. The stochiometric reactivity of vitamin E with galvinoxyl indicates that the combined dα-tocopherol and dγ-tocopherol homologs contribute between 20% (olive) and 85% (soya) to AC depending on type of oil. Tocopherols are important contributors to the AC of several vegetable oils. Breeding and photo mixotrophic programmes to enhance content in edible oil crops of this important vitamin may have a positive impact not only on oil stability during storage but also in contributing to provision of optimum dietary intakes for health.

Sunitinib Reduces Acute Myeloid Leukemia Clonogenic Cells in Vitro and Has Potent Inhibitory Effect on Sorted AML ALDH+ Cells

Sunitinib is an orally administered, multi-target tyrosine kinase inhibitor that has been approved by the FDA for the treatment of renal cell carcinoma and imatinib resistant gastro-intestinal tumors. Anti-leukemic activity of sunitinib has been examined in early clinical trials with limited success. However, recent trials on acute myeloid leukemia (AML) patients carrying FLT3 mutations have shown promising results. Effects of sunitinib on leukemic clonogenic cells and potential leukemic stem cells have not been examined so far. We analyzed the anti-proliferative and apoptotic properties of sunitinib on AML-derived cell lines. We also tested the effect of sunitinib on AML patient derived clonogenic cells (AML-CFC), as well as flow-sorted potential leukemic progenitors. Peripheral blood or bone marrow samples were obtained from newly diagnosed AML patients and flow sorted for CD34+ CD133+ or ALDH+ cells. Umbilical cord blood derived CD34+ cells were used as normal controls. Sunitinib induced growth arrest and apoptosis in AML derived cell lines. In addition, 7 μM sunitinib induced 75% reduction of AML-CFC as compared to DMSO treated control (±6.79%;n = 4). In contrast, 7 μM sunitinib treatment of umbilical cord blood derived normal CD34+ cells showed 29% reduction in AML-CFC (±6.77%;n = 5). Treatment of ALDH+ cells sorted from 2 AML cases and CD34+ CD133+ cells from one patient showed reduction of AML-CFC on treatment with sunitinib. Our study highlighted a potent anti-proliferative and proapoptotic effect of sunitinib on AML cell lines, AML patient derived clonogenic cells and potential leukemic stem cells.