Isolation of quercetin and mandelic acid from <Emphasis Type="Italic">Aesculus indica</Emphasis> fruit and their biological activities

Background

In this study Aesculus indica fruit was subjected to isolation of phytochemicals. Two antioxidants quercetin and Mandelic acid were isolated in pure state. The free radical scavenging and acetyl choline esterase inhibitory potential of the crude extract and sub fractions were also determined.

Results

The antioxidant capacity of crude extract, fractions and isolated compounds were determined by DPPH and ABTS methods. Folin-Ciocalteu reagent method was used to estimate the total phenolic contents and were found to be 78.34?±?0.96, 44.16?±?1.05, 65.45?±?1.29, 37.85?±?1.44 and 50.23?±?2.431 (mg/g of gallic acid) in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fractions respectively. The flavonoid concentration in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fraction were; 85.30?±?1.20, 53.80?±?1.07, 77.50?±?1.12, 26.30?±?1.35 and 37.78?±?1.25 (mg/g of quercetin) respectively. The chloroform fraction was more potent against enzymes, acetyl choline esterase and butyryl choline esterase (IC₅₀?=?85 and 160 μg/ml respectively). The phenolic compounds in the crude extract and fractions were determined using HPLC standard method. Chlorogenic acid, quercetin, phloroglucinol, rutin, mandelic acid and hydroxy benzoic acid were detected at retention times 6.005, 10.062, 22.623, 30.597, 35.490 and 36.211 in crude extract and different fractions. The ethyl acetate fraction was rich in the targeted compounds and was therefore subjected to column isolation. The HPLC chromatogram of isolated compounds showed single peak at specified retention times which confirms their isolation in pure state. The isolated compounds were then characterized by FTIR and NMR spectrophotometric techniques.

Conclusion

The Aesculus indica fruit extracts showed antioxidant and anticholine esterase inhibitory potentials. Two bioactive compounds were isolated in the pure form ethyl acetate fraction. From results it was concluded that the fruit of this plant could be used to minimize oxidative stress caused by reactive oxygen species.

     

Characterisation of phenolic compounds of the ethyl acetate fraction from Tabernaemontana catharinensis and its potential antidepressant-like effect

This study evaluates the antidepressant-like effect and analysed the qualitative and quantitative 74 phenolic standards of ethyl acetate fraction from Tabernaemontana catharinensis leaves. Acute administration of fraction in mice reduced the immobility time in forced swimming and tail suspension tests confirming its antidepressant-like activity. The anti-immobility effect elicited by this fraction was prevented by the pretreatment of mice with PCPA (100 mg kg^?1), ketanserin (5 mg kg^?1), SCH 23,390 (0.05 mg kg^?1) or yohimbine (1 mg kg^?1). A sub effective dose of the fraction produced a synergistic effect with fluoxetine (5 mg kg^?1). Chromatographic analysis identified 4-hydroxybenzoic and p-coumaric acids in the ethyl acetate fraction from T. catharinensis. Capillary electrophoresis presented 7.34 ± 0.02 mg g^?1 of p-coumaric acid concentration in the fraction. Therefore, it is possible that antidepressant-like effect elicited by ethyl acetate fraction from T. catharinensis be dependent on the p-coumaric acid.     

DOSY NMR applied to analysis of flavonoid glycosides from Bidens sulphurea

2D DOSY ¹H NMR has proved to be a useful technique in the identification of the molecular skeleton of the four major compounds of ethyl acetate extract of aerial parts of Bidens sulphurea (Asteraceae). The combination of this technique with HPLC, mass spectrometry and other NMR techniques enabled the identification of four flavonoid glycosides: quercetin‐3‐O‐β‐D ‐galactopyranoside, quercetin‐3‐O‐β‐D ‐glycopyranoside, quercetin‐3‐O‐α‐L ‐arabinofuranoside and quercetin‐3‐O‐β‐D ‐rhamnopyranoside. Copyright © 2009 John Wiley & Sons, Ltd.     

Chemical components of Hyssopus cuspidatus Boriss.: isolation and identification,characterization by HPLC-DAD-ESI-HRMS/MS,antioxidant activity and antimicrobial activity

Abstract

Eighteen compounds were isolated from the ethyl acetate fraction of the aerial part of Hyssopus cuspidatus. Their structures were established by analysis of mass and NMR data, as well as comparison with previous published data in the literatures. Among them, ten compounds were found from the Hyssopus genus for the first time, and one compound was isolated from H. cuspidatus for the first time. HPLC-DAD-ESI-HRMS/MS investigations was applied to further obtain the phenolic profiling of the ethyl acetate fraction, and nine derivatives of caffeic acid and ferulic or isoferulic acid were identified. Antioxidant activity against DPPH free radical and antibacterial activity against Candida albicans, Escherichia coli and Staphylococcus aureus were evaluated. The ethyl acetate fraction exhibited weak antioxidant activity and moderate antibacterial activity. The isolated compounds showed weak to potent antioxidant and antibacterial activity. To the best of our knowledge, this is the first report on the antioxidant and antibacterial activity of H. cuspidatus.     

Bioassay Guided Isolation and Identification of a Cytotoxic Compound from Azadirachta indica Leaves

This study aimed to investigate the structural features of the isolated flavonol glycoside, which might behave as a cytotoxic compound. The hexane, chloroform, ethyl acetate, and aqueous fractions of an 80% methanol solution of Neem (Azadirachta indica) (Family: Meliaceae) leaves were subjected to a cytotoxicity bioassay against brine shrimp, Artemia salina. The ethyl acetate fraction exhibited the highest cytotoxic effect, supported by the lowest lethal concentration, a LC₅₀ value of 1.35±0.40 ppm. A compound, Quercetin 3‐O‐β‐D‐glucopyranoside, was isolated from the most toxic fraction of the ethyl acetate via preparative liquid chromatography and then identified via ultraviolet‐visible (UV‐Vis), infrared (IR), mass spectrum (MS) and nuclear magnetic resonance (NMR) analyses. The compound was further confirmed by physical state, color, solubility, and melting point determination. The cytotoxic results suggest that the leaf ethyl acetate fraction consists of toxic compounds, which point towards the isolation of Quercetin 3‐O‐β‐D‐glucopyranoside.     

A methoxylated quercetin glycoside harnesses HCC tumor progression in a TP53/miR-15/miR-16 dependent manner

Abstract

This study focused on studying the impact of flavonoids isolated from Cleome droserifolia on HCC cell lines and to further unveil their possible impact on TP53 and its downstream tumor suppressor miRNAs. Three flavonol glycosides were isolated from C. droserifolia namely, Isorhamnetin-3-O-β-D-glucoside (1), Quercetin-3`-methoxy-3-O-(4``-acetylrhamnoside)-7-O-α-rhamnoside (2), and Kaempferol-4`-methoxy-3,7-O-dirhamnoside (3). They showed a concentration and time dependent reduction in cellular viability and anchorage-independent growth of HCC cells. Moreover, they exhibited a decrease in the migrating capacity of HepG2 cells in a pattern similar to positive control cells. (2) Showed the most potent effects in halting HCC tumorigenic activity (IC₅₀=36?±?1.70?µM) and a repression of the cellular proliferation rate of HepG2 cells. Restoration of TP53 and its downstream tumor suppressor miRNAs; miR-15a, miR-16, miR-34a by (2) was observed. Moreover, attenuation of (2) mediated actions was shown upon using anti-miR-15a and anti-miR-16. To conclude, this study crystallizes a novel role of C. droserifolia in harnessing HCC progression in-vitro with a possible contribution of TP53/miR-15a/miR-16.     

Flavonoids from Plinia cauliflora (Mart.) Kausel (Myrtaceae) with antifungal activity

Plinia cauliflora is a plant species with edible fruits but its chemical composition is not totally known yet although former studies showed its potential as antifungal agent. This work aimed the chemical analysis of the leaves, the activity against fungi species and evaluated cytotoxicity. Extract was obtained with 70% ethanol. An ethyl acetate fraction was obtained and glycosylated quercetin and myricetin were isolated. Samples were tested against Candida species, dermatophytes and entomopathogenic fungi. Cytotoxicity was evaluated against fibroblast cells. Extract showed good activity against C. albicans (minimum inhibitory concentration at 156 μg/mL), C. parapsilosis (78 μg/mL), C. krusei (19 μg/mL), Trichophyton rubrum (78 μg/mL) and Microsporum canis (156 μg/mL). Isolated compounds were more active against C. krusei and T. rubrum. The extract, which was the more active sample, demonstrated low cytotoxic effect and encourage more studies against rising non-albicans species and dermatophyte T. rubrum.     

Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity

This investigation was undertaken to identify the major secondary metabolite, produced by two Trichoderma harzianum strains (T-4 and T-5) with their antifungal activity against phytopathogenic fungi using poison food technique. The ethyl acetate extract was subjected to column chromatography using n-hexane, ethyl acetate and methanol gradually. Chromatographic separation of ethyl acetate extract of T. harzianum (T-4) resulted in the isolation and identification of palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2), 6-pentyl-2H-pyran-2-one (3), 2(5H)-furanone (4), stigmasterol (5) and β-sitosterol (6), while T. harzianum (T-5) gave palmitic acid (1), 1-hydroxy-3-methylanthraquinone (7), δ-decanolactone (8), 6-pentyl-2H-pyran-2-one (3), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11) as major metabolites. Among compounds screened for antifungal activity, compound 10 was found to be most active (EC₅₀ 35.9–50.2 μg mL^? 1). In conclusion, the present investigation provided significant information about antifungal activity and compounds isolated from two different strains of T. harzianum obtained from two different Himalayan locations.     

Chemical components of Hyssopus seravshanicus: antioxidant activity,activations of melanogenesis and tyrosinase,and quantitative determination by UPLC-DAD

Hyssopus seravshanicus (Dubj.) Pazij has been used as traditional herb and food due to its wide biological properties. Seventeen known compounds were isolated from the ethyl acetate fraction. Their structures were identified by spectroscopic data and comparison with published data. Among them, 14 ones were identified from H. seravshanicus for the first time. DPPH and ABTS radical scavenging activities for crude ethanol extract (CEE), ethyl acetate fraction (EAF), butanol fraction (BF) and compounds 1, 3, 8, 10, 11 and 13 were performed. CEE, compounds 1, 3, 11 and 13 exhibited potent antioxidant activities. Compound 1 was found to increase the melanin content and tyrosinase activity of B16 melanoma cells. Moreover, the quantitative estimation of compound 1 in the ethyl acetate fraction was carried out by UPLC-DAD and the method was validated. This is the first report on the isolation and bioactivity research on the non-valotile components of H. seravshanicus.     

Separation of caffeoylquinic acids and flavonoids from Asteris souliei by high‐performance counter‐current chromatography and their anti‐inflammatory activity in vitro

Eleven compounds were successfully separated from Asteris souliei by using a two‐step high‐performance counter‐current chromatography method. The first step involved a reversed phase isocratic counter‐current chromatography separation using hexane/ethyl acetate/methanol/water (1:0.8:1:1 v/v/v/v), which produced three fractions, the first two of which were mixtures. The second step used step‐gradient reversed‐phase counter‐current chromatography with hexane/butanol/ethyl acetate/methanol/water (1:0.5:3.5:1:4 v/v/v/v/v) initially followed by hexane/ethyl acetate/methanol/water (1:2:1:2 v/v/v/v) to separate Fraction 1 into seven compounds; and hexane/ethyl acetate/methanol/water (1:1:1:1.2 v/v/v/v) to separate Fraction 2 into three further compounds. The chemical structures of the separated compounds were identified by ESI‐MS and NMR spectroscopy (¹H and ¹³C). Baicalin ( 5 ), eriodictyol ( 7 ), apigenin‐7‐glycoside ( 8 ), quercetin ( 9 ), luteolin ( 10 ), and apigenin ( 11 ) showed obvious inhibitory effects on lipopolysaccharide‐induced nitric oxide production in RAW264.7 cells at a concentration of 10 μg/mL.     

Efficient quantification of the phenolic profiles of Zanthoxylum bungeanum leaves and correlation between chromatographic fingerprint and antioxidant activity

Sixteen subsequent fractions were prepared from the ethyl acetate fraction of Zanthoxylum bungeanum leaves after bio-guided chromatographic separation. The HPLC profiles and antioxidant activity of the various fractions indicated that the content of eight phenolic compounds (chlorogenic acid, epicatechin, rutin, hyperoside, trifolin, quercitrin, afzelin and quercetin) and antioxidant activity vary significantly, and high concentrations of a combination of eight phenolic compounds would result in an increase of the antioxidant activity. These results suggested that the eight compounds could be used as chemical markers for quality assessment of Z. bungeanum leaves. Correlation between chromatographic fingerprint and antioxidant activity of the fractions showed that quercitrin and hyperoside play crucial roles in the antioxidant activity, and they can be seen as the milestone for quality control. The findings also suggested that five obtained fractions (E-3-3, E-2-4, E-7, E-5 and E-4) could become useful supplements for functional food ingredients and health-related products.     

Preferential solvation of indomethacin and naproxen in ethyl acetate + ethanol mixtures according to the IKBI method

The preferential solvation parameters of indomethacin and naproxen in ethyl acetate + ethanol mixtures are derived from their thermodynamic properties by using the inverse Kirkwood–Buff integrals method. It is found that both drugs are sensitive to solvation effects, so the preferential solvation parameter, δx_EA,D, is negative in ethanol-rich and ethyl acetate-rich mixtures but positive in compositions from 0.36 to 0.71 in mole fraction of ethyl acetate. It is conjecturable that in ethanol-rich mixtures, the acidic interaction of ethanol on basic sites of the analgesics plays a relevant role in the solvation. The more solvation by ethyl acetate in mixtures of similar co-solvent compositions could be due to polarity effects. Finally, the slight preference of these compounds for ethanol in ethyl acetate-rich mixtures could be explained as the common participation of basic sites in both solvents and the acidic site of ethanol. Nevertheless, the specific solute–solvent interactions remain unclear.     

Preparative purification of plasmin activity stimulating phenolic derivatives from Gastrodia elata using centrifugal partition chromatography

Gastrodia rhizome, a dried and steamed tuber of Gastrodia elata Blume (Orchidaceae), has been traditionally used in Korea, China and Japan for the treatment of neurological and nervous disorders such as headaches, dizziness, vertigo and convulsive illnesses. The ethyl acetate and water extracts of G. elata stimulated plasmin activity. The active ethyl acetate fraction was subjected to centrifugal partition chromatography (CPC) with a two‐phase solvent system, composed of n‐hexane–ethyl acetate–methanol–water (3:7:4:6, v/v) followed by semi‐preparative HPLC purification to separate active compounds and the water fraction was purified by Diaion HP‐20 resin and semi‐preparative HPLC. In ethyl acetate extract, 4‐hydroxybenzyl alcohol (1), 4‐hydroxybenzoic acid (2), 4‐hydroxybenzaldehyde (3), 4‐ethoxymethylphenol (4), 4,4′‐oxybis(methylene)diphenol (5) and 4,4′‐methylenediphenol (6) were obtained with high purities. Parishin (7) and parishin B (8) were isolated from water extract. Among isolated compounds, 4‐hydroxybenzyl alcohol (1), 4‐hydroxybenzaldehyde (3) and 4‐ethoxymethylphenol (4) significantly stimulated plasmin activity. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro inhibition of protein glycation and advanced glycation end products formation by hydroethanolic extract and two fractions of Simaba trichilioides roots

Abstract

Long-term hyperglycemia maintenance is responsible for increased protein glycation and formation of advanced glycation end products (AGEs), both are associated with the onset of diabetes mellitus complications. Efforts have been made to discover new agents having antiglycation potential. The aim of this study was to investigate the effects of the hydroethanolic extract and the ethyl acetate and methanolic fractions of Simaba trichilioides roots on the formation of AGEs. In an in vitro model system of protein glycation, incubations with hydroethanolic extract, ethyl acetate or methanolic fractions of S. trichilioides decreased the fluorescent AGEs, and markers of tyrosine and tryptophan oxidation. Protein crosslinking was reduced in the presence of the ethyl acetate fraction of S. trichilioides. Simaba trichilioides roots seem to be a promising source of compounds having ability to prevent glycoxidation changes, with potential applications in complementary therapies for management of diabetic complications.     

Nephroprotective and antioxidant activities of ethyl acetate fraction of Euphorbia geniculata Ortega family Euphorbiaceae

In continuation of our work on genus Euphorbia, the phytochemical investigation of ethyl acetate fraction of the aerial parts of Euphorbia geniculata Ortega (Eg) family Euphorbiaceae revealed the isolation and identification of eight polyphenolic compounds; gallic acid (1), ellagic acid (2), quercetin-3-O-rutinoside) (rutin) (3), quercetin-3-O-glucopyranoside-7-O-rhamnoside (4), quercetin-3-O-rhamnoside (5), quercetin-3-O-β-D- glucopyranoside (6), and quercetin-3-O-β-D-arabinoside (7) and quercetin (8) using spectrophotometric and physicochemical analysis. Quantitative estimation of total flavonoids and phenolics contents were carried out for total methanolic extract. Biological activities of ethyl acetate fraction including nephroprotective and antioxidant activities were evaluated for the first time. The nephroprotective potential of Eg was evaluated in male rats with thioacetamide induced kidney injury. Antioxidant activity was evaluated using DPPH method.Results; Quantitative estimation of total phenolics and flavonoids content in the total methanolic extract of Eg revealed high phenolics content (285.4 mg GAE /gm extract) in comparison with flavonoids content (36.9 mg RE /gm extract). Isolation and characterization of eight polyphenolic compounds. The nephroprotictive activity was studied using thioacetamide as nephrotoxicant agent resulted in marked nephrotoxicity. While pretreatment of rats with ethyl acetate fraction of Eg significantly attenuated the nephrotoxicity through alteration of kidney biomarkers, improving the redox status of the tissue and so brought the serum biochemical parameters nearly toward the normal levels. The study revealed significant antioxidant activity of Eg in comparison with ascorbic acid. Conclusion; The results suggested that E. geniculata Ortega ethyl acetate fraction could be used in future therapy as nephroprotective and antioxidant drugs of natural source.     

Phytochemical and Biological Characterization of Tephrosia nubica Boiss. Growing in Saudi Arabia

The chloroform (TNC), ethyl acetate (TNE) and n-butanol (TNB) fractions of Tephrosia nubica Bioss. growing in Saudi Arabia were investigated for the first time using UPLC-ESI-MS/MS in two ionization modes. The analysis revealed the tentative identification of 107 compounds. Moreover, the therapeutic potential of T. nubica fractions was determined by in vitro evaluation of their cytotoxic, antioxidant, and anti-obesity activities using MTT assay, DPPH radical scavenging activity and pancreatic lipase inhibitory assay, respectively. The results showed that TNE, TNB, TNC fractions revealed weak antioxidant activity with SC₅₀ 139.9 ± 0.8, 144.9 ± 1.5, 148.9 ± 1.3 µg/mL, respectively compared to ascorbic acid 14.2 ± 0.5 µg/ml. Moreover TNE, TNC fractions showed more significant cytotoxic activity against HepG-2 with IC₅₀ 82.1 ± 3.1, 101 ± 2.8 µg/mL and MCF-7 with IC₅₀ 114 ± 3.2, 124 ± 3.9 µg/mL respectively. The TNB fraction showed weak cytotoxic activity against both cell lines compared to the other fractions. Ultimately, TNE fraction showed a remarkable anti-obesity activity with IC₅₀ 62.4 ± 1.5 µg/mL compared to chloroform fraction with IC₅₀ 535.6 ± 2.1 µg/mL and n-butanol fraction which did not show any activity. In conclusion, these findings represent the first insights into the phytochemical constituents and pharmacological properties of T. nubica. The ethyl acetate fraction of T. nubica might be a promising source of functional constituents with antioxidant, cytotoxic and anti-obesity potentials. It might be a natural alternative therapy and nutritional strategy, for obesity treatment without dangerous side effects. Isolation of the bioactive compounds from the ethyl acetate fraction of T. nubica and evaluating their biological activities are recommended.     

Reaction between silver trifluoroacetate and quercetin in low-polarity organic media

CF₃COOAg-Qr-P systems, where Qr is quercetin and P is an organic solvent, have been studied by spectroscopic methods. The reaction between silver trifluoroacetate and quercetin has been shown to produce colloid solutions, whose destruction terminates with the precipitation of a silver phase. The kinetic characteristics of the reaction between silver trifluoroacetate and quercetin in ethyl acetate have been determined.     

Phytochemical Profiling and Evaluation of the Hepatoprotective Effect of Cuscuta Campestris by High-Performance Liquid Chromatography with Diode Array Detection

Plant-derived antioxidant compounds have the potential to prevent cell damage caused by free radicals. As a holoparasitic plant, Cuscuta campestris Yuncker is being valorized for treatment of liver injury and cancer prevention in traditional medicine. The main purpose of this present study is to elucidate the antioxidant- and anticancer-associated contents of C. campestris by spectroscopic and chromatographic methods. Diethyl ether, ethyl acetate, methanol, n-butanol, and water were used as extraction solvents to reach a wide range of secondary metabolites synthesized by this plant. Antioxidant potentials of these extracts were characterized by (2,2-diphenyl-1-picrylhydrazyl)-free radical scavenging activity. Their anticancer activities were evaluated on SNU-398 hepatocellular carcinoma cells and controlled on the normal adult human dermal fibroblasts (hDFs) cells. Their phenolic compounds were analyzed by high-performance liquid chromatography with diode array detector to illuminate the responsible anticancer agent(s). The ethyl acetate extract revealed the most significant antioxidant effect. Methanol and ethyl acetate extracts were found to be cytotoxic on the SNU-398 cell lines with CC₅₀ values of 18.7 and 19.6?µg?mL^?1, respectively, whereas these extracts were not cytotoxic to the adult hDFs cells. Due to their abundance in the extracts, isorhamnetin, kaempferol, and quercetin may have caused this anticancer activity. Methanol extract has the highest concentration of phenolic compounds. Based on chromatographic analyses, we propose that these anticancer effects were positively correlated with plant phenolic compounds. The results showed that this plant is worth further study for its therapeutic uses.     

Enrichment and isolation of barbigerone from Millettia pachycarpa Benth. using high‐speed counter‐current chromatography and preparative HPLC

Enrichment of the anti‐tumor compound barbigerone along with a rotenoid derivative from Millettia pachycarpa Benth. was performed by a two‐step high‐speed counter‐current chromatography (HSCCC) separation process. In the first step, 155.8 mg of target fraction (Fra6) was obtained from 400 mg ethyl acetate extract of M. pachycarpa Benth. with an increase in barbigerone from 5.1 to 13% via HSCCC using a solvent system of n‐hexane–ethyl acetate–methanol–water (5:4:5:3, v/v) under normal phase head to tail elution. HSCCC was repeated to eliminate the major contaminant in this initial fraction 6. After a separation time of 65 min, 22.1 mg barbigerone of 87.7% purity was obtained from Fra6 with the ternary solvent system of n‐hexane–methanol–water (2:2:1, v/v) under normal phase elution. Finally, preparative HPLC was employed for the further isolation of barbigerone and the rotenoid derivative. The structures were confirmed by ESI‐MS, ¹H NMR and ¹³C NMR.     

Pulse radiolysis study on the mechanisms of reactions of CCl3OO. radical with quercetin, rutin and epigallocatechin gallate

The mechanisms of reactions between CC1₃OO^? radical and quercetin, rutin and epigallocatechin gallate (EGCG) have been studied using pulse radiolytic technique. It is suggested that the electron transfer reaction is the main reaction between CC1₃OO^? radical and rutin, EGCG, but there are two main pathways for the reaction of CC1₃OO^? radical with quercetin, one is the electron transfer reaction, the other is addition reaction. The reaction rate constants were determined. It is proved that quercetin and rutin are better CC1₃OO^? radical scavengers than EGCG.