Isolation and Structure Characterization of Flavonoids

Flavonoids are one of the most important classes of secondary metabolites from natural products due to their several applications in medicine, foods, diet industries, and so on. Even though a huge number has been reported from natural and synthetic sources, scientists are still interested in flavonoids and derivatives. The biggest challenge for working on secondary metabolites is related to the use of the predicted theoretical method to isolate the expected compound and finally analyse the spectroscopic data to elucidate and fully characterize the structure. This chapter was designed to document useful techniques for isolation and structure characterization of flavonoids. Besides the well-known methods that have been used so far, we would also put together updated information about novel challenge techniques published in recent articles on isolation and characterization of flavonoids. Our data were obtained mainly from academic library and from reported data online by using research links such as Google Scholar, Scopus, SciFinder, Scirus, PubMed, and so on. Our field experience on phytochemistry of isolation and characterization of flavonoids was also used in this chapter

The variation in antimicrobial and antioxidant activities of acetone leaf extracts of 12 Moringa oleifera (Moringaceae) trees enables the selection of trees with additional uses

AbstractBackgroundThe aim of this study was to evaluate the variation in antimicrobial and antioxidant activities of the leaf acetone extracts of 12 Moringa oleifera trees harvested in order to select the best material for clonal propagation.MethodsA two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against a panel of fungal (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans) and bacterial (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) species. The radical scavenging capacity was determined using 2,2 diphenyl-1-picryhydrazyl (DPPH).ResultsThere was a large variation in antimicrobial activities with MICs between 0.04 and 2.50mg/ml against bacteria and from 0.16 to >2.50mg/ml against fungi. For samples harvested in winter: trees L3 and LP2 had significant activity against E. faecalis (MIC 0.08mg/ml) and E. coli (MIC 0.04mg/ml). Trees L5, LP1 and LP6 had weak activity against E. coli (MICs 1.25 and 2.50mg/ml), S. aureus (MIC 1.25mg/ml), and E. faecalis (MIC 2.50mg/ml), while other samples had moderate activity against the four bacteria (MICs 0.16–0.63mg/ml). From samples collected in summer: L5 (MIC 0.08mg/ml), L6 (MIC 0.08mg/ml after 1h incubation), LP1 (MICs 0.08mg/ml), LP2 (MICs 0.08mg/ml after 1h incubation), LP4 (0.08mg/ml) and LP5 (MICs 0.04 and 0.08mg/ml) had significant activity against E. faecalis (L5, L6, LP1, LP2, LP4, and LP5), S. aureus (LP1, and LP5), and E. coli (LP2, and LP5), respectively. Other extracts had weak antibacterial activity with MICs ranging from 0.16 to 0.63mg/ml. Most of the samples harvested in winter had moderate antifungal activity: L1, L2, L3, L4, L5, L6, LP1, LP2, and LP3 had moderate activity against C. albicans (ATCC strains) with MIC of 0.63mg/ml in all cases while L2, L3 and L4 as well as L6, LP1, LP2, LP3, LP5 and LP6 against A. fumigatus (MICs 0.63mg/ml) and C. neoformans (MICs 0.63mg/ml), respectively. Apart from L1 (MIC 0.31mg/ml), L2, L3 and LP6 (MICs 0.63mg/ml in all cases) with moderate activity, all the samples collected during summer had weak activity against A. fumigatus (MICs 1.25–2.50mg/ml). All the extracts had a low radical scavenging activity with the IC50 values ranging from 34.72 to 109.62μg/ml, compared to the reference standard l-ascorbic acid (IC50 2.41μg/ml). This may be related to the extractant used.ConclusionThe large variation in antimicrobial activity and antioxidant activities of 24 acetone leaf extracts of 12 M. oleifera trees may lead to the selection of clonal material to serve as a source of propagation materials. Successful propagation and growth of tree LP with very good activity against E. coli and a high total activity could provide an additional use of this valuable plant species to rural people

Antibacterial and antimycobacterial activity of crude extracts, fractions, and isolated compounds from leaves of sneezewood, Ptaeroxylon obliquum (Rutaceae)

Ptaeroxylon obliquum (Thunb.) Radlk. (Rutaceae) is traditionally used to treat human and animal diseases in South Africa. In this study, the activity of leaf extracts, fractions, and isolated compounds was determined against nonpathogenic mycobacterial species and nosocomial bacterial pathogens. An acetone leaf extract was partitioned by liquid-liquid fractionation, and obliquumol, a mixture of lupeol and β-amyrin, and eranthin were isolated. Antimicrobial activity was determined using a serial microdilution assay against Mycobacterium smegmatis (American Type Culture Collection [ATCC] 1441), M. bovis (BCG P1172), M. aurum (NCTC 10437), M. fortuitum (ATCC 6841), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 25922), and Escherichia coli (ATCC 27853). The n-hexane fraction had minimal inhibitory concentration (MIC) values as low as 20 and 40 µg/mL against M. fortuitum and S. aureus, respectively. The chloroform fraction also had promising activity with an MIC value of 80 µg/mL against both P. aeruginosa and M. fortuitum. Obliquumol had excellent activity (MIC 8 µg/mL) against M. fortuitum. Fractionation of the crude extract potentiated the antimicrobial activity of the nonpolar fractions. The isolated compound, obliquumol, had good antimicrobial and excellent antimycobacterial activities. The antimicrobial activity provides some scientific rationale for the use of P. obliquum against infectious diseases and related symptoms. This is the first report on the antibacterial activity of obliquumol.https://journals.sagepub.com/home/npxpm2020Paraclinical Science

The potential role of GLUT4 transporters and insulin receptors in the hypoglycaemic activity of Ficus lutea acetone leaf extract

BACKGROUND: Some Ficus species have been used in traditional African medicine in the treatment of diabetes. The antidiabetic potential of certain species has been confirmed in vivo but the mechanism of activity remains uncertain. The aim was to investigate the hypoglycaemic potential of ten Ficus species focussing on glucose uptake, insulin secretion and the possible mechanism of hypoglycaemic activity. METHODS: The dried and ground leaves of ten Ficus species were extracted with acetone. The dried acetone extract was reconstituted with DMSO to a concentration of 100 mg/ml which was then serially diluted and used to assay for glucose uptake in muscle, fat and liver cells, and insulin secretion in pancreatic cells. RESULTS: Only the F. lutea extract was able to modulate glucose metabolism. In comparison to insulin in the primary muscle cells, the glucose uptake ability of the extract was 33% as effective. In the hepatoma cell line, the extract was as effective as metformin in decreasing extracellular glucose concentration by approximately 20%. In the pancreatic insulin secretory assay, the extract was 4 times greater in its secretory activity than commercial glibenclamide. With F. lutea extract significantly increasing glucose uptake in the primary muscle cells, primary fat cells, C2C12 muscle and H-4-II-E liver cells, the extract may act by increasing the activity of cell surface glucose transporters. When the 3T3-L1 pre-adipocytes were compared to the primary muscle, primary fat and C2C12 cells, the differences in the former’s ability to transport glucose into the cell may be due to the absence of the GLUT4 transporter, which on activation via the insulin receptor decreases extracellular glucose concentrations. Because the pre-adipocytes failed to show any active increase in glucose uptake, the present effect has to be linked to the absence of the GLUT4 transporter. CONCLUSION: Only F. lutea possessed substantial in vitro activity related to glucose metabolism. Based on the effect produced in the various cell types, F. lutea also appears to be a partial agonist/antagonist of the insulin cell membrane receptor. While the clinical effectiveness of F. lutea is not known, this plant species does possess the ability to modify glucose metabolism

Some Strychnos spinosa (Loganiaceae) leaf extracts and fractions have good antimicrobial activities and low cytotoxicities

BACKGROUND : Strychnos spinosa Lam. is a deciduous tree used in traditional medicine to treat infectious diseases. This study is designed to determine the antimicrobial, antioxidant and cytotoxic activities of extracts and fractions from leaves of S. spinosa. METHODS : Extracts were obtained by maceration with acetone, methanol and dichloromethane/methanol (1/1) while fractions were prepared by liquid-liquid fractionation of the acetone extract. A broth serial microdilution method with tetrazolium violet as growth indicator was used to determine the minimum inhibitory concentration (MIC) against fungi, Gram-positive and Gram-negative bacteria. The antioxidant activity was determined using free-radical-scavenging assays, and the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay was used to determine cytotoxicity. RESULTS : Four extracts and five fractions had good to weak antimicrobial activity with MICs ranging from 0.04 to >1.25 mg/ml against both fungi and bacteria. The chloroform and ethyl acetate fractions had an MIC of 0.08 mg/ml against Aspergillus fumigatus. The n-butanol fraction had an MIC of 0.04 mg/ml against Cryptococcus neoformans. The hexane and chloroform fractions had an MIC of 0.08 mg/ml against Staphylococcus aureus. The antioxidant activities were much lower than that of the positive controls. Except for the alkaloid extract, all the extracts and fractions had free-radical-scavenging activity (IC50 ranging from 33.66 to 314.30 μg/ml). The cytotoxicity on Vero cells was reasonable to low with LC50 values ranging between 30.56 and 689.39 μg/ml. CONCLUSION : The acetone extract and the chloroform fraction had the highest antibacterial activity. By solvent-solvent fractionation it was possible to increase the activity against A. fumigatus and to decrease the cytotoxicity leading to a potentially useful product to protect animals against aspergillosis. Our results therefore support the use of S. spinosa leaves in traditional medicine to treat infectious diseases.A.I.I is grateful to the Nigerian Tertiary Education Training Fund (TETFund) through Ahmadu Bello University Academic Unit for supporting the work in this paper. M.D.A and J.P.D received Postdoctoral Fellowships from the University of Pretoria to work in the Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science.http://www.biomedcentral.com/bmccom/plementalternmedam201

The variation in antimicrobial and antioxidant activities of acetone leaf extracts of 12 Moringa oleifera (Moringaceae) trees enables the selection of trees with additional uses

BACKGROUND : The aim of this study was to evaluate the variation in antimicrobial and antioxidant activities of the leaf acetone extracts of 12 Moringa oleifera trees harvested in order to select the best material for clonal propagation. METHODS : A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against a panel of fungal (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans) and bacterial (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) species. The radical scavenging capacity was determined using 2,2 diphenyl-1-picryhydrazyl (DPPH). RESULTS : There was a large variation in antimicrobial activities with MICs between 0.04 and 2.50 mg/ml against bacteria and from 0.16 to N2.50 mg/ml against fungi. For samples harvested in winter: trees L3 and LP2 had significant activity against E. faecalis (MIC 0.08 mg/ml) and E. coli (MIC 0.04 mg/ml). Trees L5, LP1 and LP6 had weak activity against E. coli (MICs 1.25 and 2.50 mg/ml), S. aureus (MIC 1.25 mg/ml), and E. faecalis (MIC 2.50 mg/ml), while other samples had moderate activity against the four bacteria (MICs 0.16–0.63 mg/ml). From samples collected in summer: L5 (MIC 0.08 mg/ml), L6 (MIC 0.08 mg/ml after 1 h incubation), LP1 (MICs 0.08 mg/ml), LP2 (MICs 0.08 mg/ml after 1 h incubation), LP4 (0.08 mg/ml) and LP5 (MICs 0.04 and 0.08 mg/ml) had significant activity against E. faecalis (L5, L6, LP1, LP2, LP4, and LP5), S. aureus (LP1, and LP5), and E. coli (LP2, and LP5), respectively. Other extracts had weak antibacterial activity with MICs ranging from 0.16 to 0.63 mg/ml. Most of the samples harvested in winter had moderate antifungal activity: L1, L2, L3, L4, L5, L6, LP1, LP2, and LP3 had moderate activity against C. albicans (ATCC strains) with MIC of 0.63 mg/ml in all cases while L2, L3 and L4 as well as L6, LP1, LP2, LP3, LP5 and LP6 against A. fumigatus (MICs 0.63 mg/ml) and C. neoformans (MICs 0.63 mg/ml), respectively. Apart from L1 (MIC 0.31 mg/ml), L2, L3 and LP6 (MICs 0.63 mg/ml in all cases) with moderate activity, all the samples collected during summer had weak activity against A. fumigatus (MICs 1.25–2.50 mg/ml). All the extracts had a low radical scavenging activity with the IC50 values ranging from 34.72 to 109.62 μg/ml, compared to the reference standard L-ascorbic acid (IC50 2.41 μg/ml). This may be related to the extractant used. CONCLUSION : The large variation in antimicrobial activity and antioxidant activities of 24 acetone leaf extracts of 12 M. oleifera trees may lead to the selection of clonal material to serve as a source of propagation materials. Successful propagation and growth of tree LP with very good activity against E. coli and a high total activity could provide an additional use of this valuable plant species to rural people.The Limpopo Department of Agriculture (LDA), the National Research Foundation and MDA received a Postdoctoral Fellowship from the University of Pretoria. Open Access funded by SAAB.http://www.elsevier.com/locate/sajbhb2017Paraclinical Science

Clerodendrumic acid, a new Triterpenoid from Clerodendrum glabrum (Verbanaceae), and antimicrobial activities of fractions and constituents

One new triterpenoid, (3β,11α,19β)-3-(butanoyloxy)-11-hydroxytaraxast-20(30)-ene-23,28-dioic acid (clerodendrumic acid; 1) was isolated from the hexane extract of the leaves of Clerodendrum glabrum var. glabrum along with heptadecanoic acid (2). The structure of the new compound was elucidated by interpretation of its NMR (1D and 2D), MS, and IR data. Combined fractions C and D from the column chromatography of the hexane extract exhibited significant antifungal activities (average MIC of 0.10 mg/ml) against Candida albicans and Cryptococcus neoformans. C. albicans was relatively resistant to clerodendrumic acid (1; MIC 125 μg/ml) and was resistant to heptadecanoic acid (2; MIC 188 μg/ml). Both compounds had low antibacterial activities against two Gram-positive and two Gram-negative bacteria with average MIC values of 157 and 172 μg/ml, respectively. Compounds 1 and 2 were relatively nontoxic against monkey kidney Vero cells in vitro with IC50 values of 202.6 and 108.4 μg/ml, respectively.National Research Foundation (NRF) and the Netherlands Universities Foundation for International Cooperation (NUFFIC).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1522-2675hb201

Antimicrobial activity and cytotoxicity of the ethanol extract, fractions and eight compounds isolated from Eriosema robustum (Fabaceae)

BACKGROUND: The aim of this study was to evaluate the antimicrobial activity and the cytotoxicity of the ethanol crude extract, fractions and isolated compounds from the twigs of Eriosema robustum, a plant used for the treatment of coughs and skin diseases. METHODS: Column chromatographic and spectroscopic techniques were used to isolate and identify eight compounds, robusflavones A (1) and B (2), orostachyscerebroside A (3), stigmasterol (4), 1-O-heptatriacontanoyl glycerol (5), eicosanoic acid (6), 3-O-β-D-glucopyranoside of sitosterol (7) and 6-prenylpinocembrin (8), from E. robustum. A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against fungi and bacteria, and the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay was used to evaluate the cytotoxicity. RESULTS: Fraction B had significant antimicrobial activity against Aspergillus fumigatus and Cryptoccocus neoformans (MIC 0.08 mg/ml), whilst the crude extract and fraction A had moderate activity against A. fumigatus and Candida albicans (MIC 0.16 mg/ml). Fraction A however had excellent activity against Staphylococcus aureus (MIC 0.02 mg/ml), Enterococcus faecalis and Escherichia coli (MIC 0.04 mg/ml). The crude extract had significant activity against S. aureus, E. faecalis and E. coli. Fraction B had good activity against E. faecalis and E. coli (MIC 0.08 mg/ml). All the isolated compounds had a relatively weak antimicrobial activity. An MIC of 65 μg/ml was obtained with robusflavones A (1) and B (2) against C. albicans and A. fumigatus, orostachyscerebroside A (3) against A. fumigatus, and robusflavone B (2) against C. neoformans. Compound 8 had the best activity against bacteria (average MIC 55 μg/ml). The 3 fractions and isolated compounds had LC50 values between 13.20 to > 100 μg/ml against Vero cells yielding selectivity indices between 0.01 and 1.58. CONCLUSION: The isolated compounds generally had a much lower activity than expected based on the activity of the fractions from which they were isolated. This may be the result of synergism between different compounds in the complex extracts or fractions. The results support the traditional use of E. robustum to treat infections. The crude extract had a good activity and low preparation cost, and may be useful in topical applications to combat microbial infections.The authors are grateful to the University of Pretoria for the Post-doctoral Fellowship awarded to MD Awouafack to work at the Faculty of Veterinary Science, Department of Paraclinical Sciences, Phytomedicine Programme, and the National Research Foundation and Medical Research Council for research funding.http://www.biomedcentral.com/1472-6882/13/289am2014mn201

Anticandidal activity of cell extracts from 13 probiotic Lactobacillus strains and characterisation of lactic acid and a novel fatty acid derivative from one strain

This study investigated the anti-Candida activity of methanol extracts from freeze-dried probiotic cells and the isolation of some constituents in the extracts. The MIC values of the probiotic methanol cell extracts against Candida albicans ranged between 1.25 and 5 mg/ml after 48 h of incubation. However, Lactococcus latics subsp. lactis strain X and Lactobacillus casei strain B extracts had an MIC of 10 mg/ml after 48 h of incubation. The extracts had fungistatic rather than fungicidal activity. These extracts had a much higher antifungal activity than antifungal compounds isolated from the growth medium by many other authors. This indicates that probiotics may also release antifungal compounds in their cells that could contribute to a therapeutic effect. Lactic acid (1) and 6-O-(α-D-glucopyranosyl)-1,6-di-O-pentadecanoyl-α-D-glucopyranose a novel fatty acid derivative (2) were isolated from methanol probiotic extracts and the structure of these compounds were elucidated using NMR (1 and 2D) and mass spectrometry (MS).Dr. M.D. Awouafack received a Postdoctoral Fellowship from the University of Pretoria to work at the Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science. The Medical Research Council provided funding.http://www.elsevier.com/locate/foodchem2015-12-31hb201