Edward J. Kennelly

Anthocyanin antioxidants from edible fruits

Abstract The edible fruits of 12 plants were extracted in methanol and subjected to solvent–solvent partitioning to yield three fractions, hexane, ethyl acetate, and aqueous. The semi-purified aqueous fractions were separated over Diaion HP-20SS resin to remove sugars and ascorbic acid. These fractions were then screened for antioxidant activity using the 1,1-diphenyl-2-picrylhydrazyl assay. Nine of the semi-purified fractions exhibited high antioxidant capacity. Cyanidin-3- O -β-glucopyranoside, an anthocyanin antioxidant, was identified from semi-purified aqueous fractions of the tropical fruit star apple ( Chrysophyllum cainito ), Surinam cherry ( Eugenia uniflora ), and jaboticaba ( Myrciaria cauliflora ). Delphinidin-3- O -β-glucopyranoside was identified from E. uniflora .

White and green teas (Camellia sinensis var. sinensis): variation in phenolic, methylxanthine, and antioxidant profiles.

Recent investigations have associated white teas with anti-carcinogenic, immune-boosting, and antioxidative properties that may impact human health in a manner comparable to green teas. An in-depth chemical analysis of white tea types was conducted to quantify polyphenols and antioxidant potential of 8 commercially available white teas, and compare them to green tea. Extraction and HPLC protocols were optimized and validated for the quantification of 9 phenolic and 3 methylxanthine compounds to examine inter- and intra-variation in white and green tea types and subtypes. A sampling strategy was devised to assess various subtypes procured from different commercial sources. Variation in antioxidant activity and total phenolic content (TPC) of both tea types was further assessed by the 1-1-diphenyl-2-picrylhydrazyl (DPPH) and Folin-Ciocalteau (F-C) assays, respectively. Total catechin content (TCC) for white teas ranged widely from 14.40 to 369.60 mg/g of dry plant material for water extracts and 47.16 to 163.94 mg/g for methanol extracts. TCC for green teas also ranged more than 10-fold, from 21.38 to 228.20 mg/g of dry plant material for water extracts and 32.23 to 141.24 mg/g for methanol extracts. These findings indicate that statements suggesting a hierarchical order of catechin content among tea types are inconclusive and should be made with attention to a sampling strategy that specifies the tea subtype and its source. Certain white teas have comparable quantities of total catechins to some green teas, but lesser antioxidant capacity, suggesting that white teas have fewer non-catechin antioxidants present. Practical Application: In this investigation white and green teas were extracted in ways that mimic common tea preparation practices, and their chemical profiles were determined using validated analytical chemistry methods. The results suggest certain green and white tea types have comparable levels of catechins with potential health promoting qualities. Specifically, the polyphenolic content of green teas was found to be similar to certain white tea varieties, which makes the latter tea type a potential substitute for people interested in consuming polyphenols for health reasons. Moreover, this study is among the first to demonstrate the effect subtype sampling, source of procurement, cultivation, and processing practices have on the final white tea product, as such analysis has previously been mostly carried out on green teas.

Growth inhibitory activity of extracts and purified components of black cohosh on human breast cancer cells.

The purpose of this study was to determine whether black cohosh contains constituents that inhibit the growth of human breast cancer cells, and therefore might eventually be useful in the prevention or treatment of breast cancer. Black cohosh rhizomes were extracted with methanol/water and fractionated by solvent–solvent partitioning to yield three fractions: hexane, ethyl acetate and water. The ethyl acetate fraction displayed the highest potency in two cell-based assays, growth inhibition and cell cycle analysis. This fraction inhibited growth of both the ER+ MCF7 and ER−MDA-MB-453 human breast cancer cell lines with IC50 values of about 20 and 10 µg/ml, respectively. It also induced cell cycle arrest at G1 when tested at 30 µg/ml and at G2/M at 60 µg/ml in MCF7 cells. This suggests that the extract contains a mixture of components with the more active (or more abundant) causing G1 arrest and the less active causing G2/M arrest. We then examined specific components in this extract. The triterpene glycoside fraction obtained by polyamide column chromatography, and the specific triterpene glycosides actein, 23-epi-26-deoxyactein and cimiracemoside A, inhibited growth of the MCF7 human breast cancer cells and induced cell cycle arrest at G1. The most potent compound, actein, decreased the level of cyclin D1, cdk4 and the hyperphosphorylated form of the pRb protein and increased the level of p21cip1 in MCF7 cells, changes that may contribute to the arrest in G1. Further studies are in progress to identify the mechanisms by which actein and related compounds present in black cohosh inhibit growth of human breast cancer cells.

Metabolite Profiling of Jaboticaba (Myrciaria cauliflora) and Other Dark-Colored Fruit Juices

Many dark-colored fruit juices, rich in anthocyanins, are thought to be important for human health. Joboticaba (Myrciaria cauliflora) fruits, native to Brazil, have phenolics including anthocyanins and are processed into juice and other products. The phenolic constituents in the fruits of jaboticaba were studied by high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. Twenty-two compounds were identified or tentatively determined by detailed analysis of their mass spectral fragmentation patterns; 11 compounds including 7 gallotannins, 2 ellagic acid derivatives, syringin, and its glucoside were detected for the first time in the fruit. The compositional differences among the fruit extracts and their commercial products were also compared by principal component analysis; two anthocyanins, delphinidin 3-O-glucoside and cyanidin-3-O-glucoside, as well as two depsides, jaboticabin and 2-O-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxyphenylacetic acid, present in the fruit extracts were not detected unexpectedly in commercial jaboticaba juice or jam. Therefore, the stability of anthocyanins in jaboticaba fresh fruits and products has been compared directly with that of other dark-colored fruit products made from blueberry and Concord grape, and the same trend of decreasing amounts of anthocyanins was observed in all tested products. The antioxidant activities (DPPH(•) and ABTS(•+)) of jaboticaba fresh fruit extract and commercial samples were also compared. Principal component analysis proved to be a useful way to discern changes between fresh and processed fruits. Jaboticaba is a promising fruit with antioxidant capacity similar to those of other so-called superfruits; however, during processing the levels of some of anthocyanins and other polyphenols decrease significantly, and therefore the capacity of these products to affect human health may vary significantly from that of the fresh fruit.

Saponins from the traditional medicinal plant Momordica charantia stimulate insulin secretion in vitro.

The antidiabetic activity of Momordica charantia (L.), Cucurbitaceae, a widely-used treatment for diabetes in a number of traditional medicine systems, was investigated in vitro. Antidiabetic activity has been reported for certain saponins isolated from M. charantia. In this study insulin secretion was measured in MIN6 β-cells incubated with an ethanol extract, saponin-rich fraction, and five purified saponins and cucurbitane triterpenoids from M. charantia, 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (1), momordicine I (2), momordicine II (3), 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-β-glucopyranoside (4), and kuguaglycoside G (5). Treatments were compared to incubation with high glucose (27 mM) and the insulin secretagogue, glipizide (50 μM). At 125 μg/ml, an LC-ToF-MS characterized saponin-rich fraction stimulated insulin secretion significantly more than the DMSO vehicle, p=0.02. At concentrations 10 and 25 μg/ml, compounds 3 and 5 also significantly stimulated insulin secretion as compared to the vehicle, p≤0.007, and p=0.002, respectively. This is the first report of a saponin-rich fraction, and isolated compounds from M. charantia, stimulating insulin secretion in an in vitro, static incubation assay.

A new liquid chromatography-mass spectrometry-based strategy to integrate chemistry, morphology, and evolution of eggplant (Solanum) species.

This study presents a strategy based on repeatable reversed-phase LC-TOF-MS methods and statistical tools, including untargeted PCA and targeted PLS/OPLS-DA models, to analyze 31 accessions representing 24 species in the eggplant genus Solanum (Solanaceae), including eight species whose metabolic profiles were studied for the first time. Sixty-two Solanum metabolites were identified after detailed analysis of UV absorbance spectra, mass spectral fragmentation patterns, NMR spectra, and/or co-injection experiments with authentic standards. Among these were two new 5-O-caffeoylquinic acid derivatives that were identified by analyzing their MS/MS fragmentation. Based on these results, a Solanum metabolic database (SMD) and a detailed biosynthetic pathway of Solanum metabolites were created. Results of analyses identified seven marker metabolites that distinguish four Solanum sections, and revealed species-specific chemical patterns. Combining LC-MS data with multivariate statistical analysis was proven effective in studying the metabolic network within the large genus Solanum, allowing for integration of complicated chemistry, morphology, and evolutionary relationships.

Growth inhibition of colon cancer cells by polyisoprenylated benzophenones is associated with induction of the endoplasmic reticulum response

Polyisoprenylated benzophenones derived from Garcinia xanthochymus have cytotoxic activity in vitro and antitumor activity in rodent models, but the mechanism is unknown. The purpose of our study was to examine in parallel molecular pathways that are targeted by 3 Garcinia‐derived benzophenones‐xanthochymol (X), guttiferone E (GE) and guttiferone H (GH), in 3 human colon cancer cell lines, HCT116, HT29 and SW480. The IC50 concentrations were determined and the cells were then treated with X, GE or GH at their respective IC50 or IC50x2 concentrations. Effects on the cell cycle, mitochondrial membrane potential and apoptosis were assessed by flow cytometry and caspase activation. Changes in gene expression were assessed with Illumina 24 K gene arrays. We found that X, GE and GH induced loss of mitochondrial membrane potential and G1 arrest at their IC50 concentrations and induced caspase activation at IC50 × 2 concentrations. An analysis of the changes in gene expression revealed that with all 3 compounds and all 3 cell lines there was a marked increase in expression of several genes, including XBP1, ATF4 and DDIT3/CHOP, which are components of the endoplasmic reticulum stress response. The DDIT4/REDD1 gene, an inhibitor of the mTOR survival pathway, was also up‐regulated. Therefore, X, GE and GH appear to inhibit the growth of human colon cancer cells, at least in part, by activating the endoplasmic reticulum stress response and inhibiting the mTOR cell survival pathway. These combined effects may contribute to the anticancer activity of these novel compounds.

Analysis of thirteen populations of Black Cohosh for formononetin

Black cohosh (Actaea racemosa L. syn. Cimicifuga racemosa (L.) Nutt.), a North American perennial plant, is a promising natural alternative to hormone replacement therapy for treating menopausal symptoms, but the mechanism of action is not understood. The clinical actions of this plant have been attributed to the isoflavonone formononetin since 1985, when its presence was reported in a black cohosh extract. Others have since looked for formononetin, but have not detected it. We looked for formononetin in extracts of black cohosh roots and rhizomes collected in thirteen locations in the eastern United States, including Maryland, New Jersey, New York, North Carolina, Pennsylvania, Virginia, and Tennessee. The rhizome samples were extracted using 80% methanol, and the extracts were partially purified using solid-phase extraction to concentrate any isoflavonoids that might be present. We tested for formononetin in these partially purified samples using thin-layer chromatography and high-performance liquid chromatography with a photodiode array detector and a mass spectrometer. Formononetin was not detected in any of the thirteen plant populations examined. Remifemin, a German product now on the United States market, and CimiPure, a commercially available black cohosh rhizome extract, were also analyzed. We did not detect formononetin, or ononin (formononetin-7-glucoside), in any sample tested by the above-mentioned chemical analyses. Therefore, the clinically observed estrogen-like actions of black cohosh, such as reduction of hot flashes, are likely due to a compound, or combination of compounds, other than formononetin.

Immunostimulatory activities of polysaccharide extract isolated from Curcuma longa

Several curcuminoids and sesquiterpenoids isolated from Curcuma longa (CL) have been shown to have many pharmacological activities. In the present study, the immunomodulatory activities of the polar fractions of CL hot water extracts were investigated using human peripheral blood mononuclear cells (PBMC). Our results showed that the high polarity fraction of the hot water extract exhibited stimulatory effects on PBMC proliferation as shown in [methyl-(3)H]-thymidine incorporation assay. In an attempt to isolate the active components responsible for the activities, further partition with ethyl acetate, n-butanol and ethanol, progressively were performed. The cytokine productions (TGF-beta, TNF-alpha, GM-CSF, IL-1alpha, IL-5, IL-6, IL-8, IL-10, IL-13, etc.) have been modulated by a polysaccharide-enriched fraction as shown in ELISA and cytokine protein array. The proportion of CD14 positive stained PBMC was increased by such fraction. The composition of monosaccharide of the active fraction has been determined by GC-MS and gel permeation chromatography. The immunostimulatory effects of C. longa polysaccharides on PBMC were shown for the first time. The findings revealed the potential use of C. longa crude extract (containing curcuminoids and polysaccharides) as an adjuvant supplement for cancer patients, whose immune activities were suppressed during chemotherapies.

Anthocyanins and antioxidant capacities of six Chilean berries by HPLC-HR-ESI-ToF-MS

The HPLC profiles of six fruits endemic of the VIII region of Chile were investigated using high resolution mass analysis (HR-ToF-ESI-MS). The anthocyanin fingerprints generated for the fruits were compared and the antioxidant capacities measured by the scavenging of the DPPH radical, the ferric reducing antioxidant power (FRAP), the superoxide anion scavenging activity assay (SA), and correlated with the inhibition of lipid peroxidation in human erythrocytes (LP) and total content of phenolics, flavonoids and anthocyanins measured by spectroscopic methods. Several anthocyanins were identified, including 3-O-glycosides derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin. Three phenolic acids (feruloyl-quinic acid, chlorogenic acid, and neochlorogenic acid) and five flavonols (hyperoside, isoquercitrin, quercetin, rutin, myricetin and isorhamnetin) were also identified. Calafate fruits showed the highest antioxidant activity. However, the highest LP activity was found for Chilean blueberries (>95%) followed by calafate fruits (91.27%) and luma (83.4%).