Mineral Analysis, In Vitro Evaluation of Alpha-Amylase, Alpha-Glucosidase, and Beta-Galactosidase Inhibition, and Antibacterial Activities of Juglans regia L. Bark Extracts.

In traditional medicine, various parts of the plant Juglans regia L. are used to treat several pathological conditions including diabetes and infectious and periodontal diseases. This includes the bark of Juglans regia. The present study is aimed at evaluating for the first time the mineral composition, investigating the antidiabetic and antibacterial properties of Moroccan J. regia bark, and finally determining the correlations between the chemical composition of the tested extracts and their biological activities. The mineral composition was determined using inductively coupled plasma atomic emission spectroscopy. Then, nine extracts were prepared by different methods and modalities of extractions and investigated for their antidiabetic activities, via tests of inhibition of alpha-amylase, alpha-glucosidase, and beta-galactosidase enzymes, and for their antibacterial activities against six strains involved in infectious diseases and periodontology. Finally, the correlation between the chemical compositions of the different extracts prepared and their antidiabetic and antibacterial potencies was determined by Principal Component Analysis (PCA). J. regia is an important source of mineral elements, mainly Fe (19849.8), K (3487.8), Mg (2631.03), and P (691.02) mg/kg plant material. All the extracts of J. regia possess antidiabetic activity, and in particular, the macerated acetone extract gave the highest inhibitory activity against alpha-amylase (IC50 value of 5445.33 ± 82.58 µg/mL), alpha-glucosidase (IC50 value of 323.7 ± 1.71 µg/mL), and beta-galactosidase (IC50 value of 811.2 ± 8.32 µg/mL). For the results of antibacterial activity, the macerated acetone extract at the concentration of 80 mg/mL was found to be the most active by inducing inhibition diameters of 12, 17, 18, 11, 14.5, and 16 mm against Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Listeria innocua, respectively. PCA allowed us to deduce that the extracts richer in polyphenols, in particular, the two acetone and ethanol macerates, have a better antidiabetic activity against alpha-glucosidase as well as a better antibacterial activity. The results of the present study revealed that the aqueous and organic macerate extracts showed a better antidiabetic activity and justified the use of J. regia bark as an antibacterial and antiseptic agent in traditional Moroccan medicine in the treatment of dental affections.

Isolation from Stevia rebaudiana of DMDP acetic acid, a novel iminosugar amino acid: synthesis and glycosidase inhibition profile of glycine and ß-alanine pyrrolidine amino acids.

DMDP acetic acid [N-carboxymethyl-2,5-dideoxy-2,5-imino-D-mannitol] 5 from Stevia rebaudiana is the first isolated natural amino acid derived from iminosugars bearing an N-alkyl acid side chain; it is clear from GCMS studies that such derivatives with acetic and propionic acids are common in a broad range of plants including mulberry, Baphia, and English bluebells, but that they are very difficult to purify. Reaction of unprotected pyrrolidine iminosugars with aqueous glyoxal gives the corresponding N-acetic acids in very high yield; Michael addition of both pyrrolidine and piperidine iminosugars and that of polyhydroxylated prolines to tert-butyl acrylate give the corresponding N-propionic acids in which the amino group of ß-alanine is incorporated into the heterocyclic ring. These easy syntheses allow the identification of this new class of amino acid in plant extracts and provide pure samples for biological evaluation. DMDP N-acetic and propionic acids are potent α-galactosidase inhibitors in contrast to potent ß-galactosidase inhibition by DMDP.

Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes.

Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1ß and IL-6, senescence associated-ß-galactosidase (SA-ß-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1ß) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-ß-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes.

Small molecule inhibits activity of scavenger receptor A: Lead identification and preliminary studies.

Scavenger receptor A (SRA) has been implicated in the processes of tumor invasion and acts as an immunosuppressor during therapeutic cancer vaccination. Pharmacological inhibition of SRA function thus holds a great potential to improve treatment outcome of cancer therapy. Macromolecular natural product sennoside B was recently shown to block SRA function. Here we report the identification and characterization of a small molecule SRA inhibitor rhein. Rhein, a deconstructed analog of sennoside B, reversed the suppressive activity of SRA in dendritic cell-primed T cell activation, indicated by transcription activation of il2 gene and production of IL-2. Rhein also inhibited SRA ligand polyinosinic:polycytidylic acid (poly(I:C)) induced activation of transcriptional factors, including interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1). Additionally, this newly identified lead compound was docked into the homology models of the SRA cysteine rich domain to gain insights into its interaction with the receptor. It was then found that rhein can favorably interact with SRA cysteine rich domain. Collectively, rhein, being the first identified small molecule inhibitors for SRA, warrants further structure-activity relationship studies, which may lead to development of novel pharmacological intervention for cancer therapy.

Green tea extract and its major constituent epigallocatechin-3-gallate inhibit growth and halitosis-related properties of Solobacterium moorei.

BACKGROUND: Solobacterium moorei is a volatile sulfide compound (VSC)-producing Gram-positive anaerobic bacterium that has been associated with halitosis. The aim of this study was to investigate the effects of green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) on growth and several halitosis-related properties of S. moorei. METHODS: A microplate dilution assay was used to determine the antibacterial activity of green tea extract and EGCG against S. moorei. Their effects on bacterial cell membrane integrity were investigated by transmission electron microscopy and a fluorescence-based permeability assay. Biofilm formation was quantified by crystal violet staining. Adhesion of FITC-labeled S. moorei to oral epithelial cells was monitored by fluorometry. The modulation of ß-galactosidase gene expression in S. moorei was evaluated by quantitative RT-PCR. RESULTS: The green tea extract as well as EGCG inhibited the growth of S. moorei, with MIC values of 500 and 250 µg/ml, respectively. Transmission electron microscopy analysis and a permeabilization assay brought evidence that the bacterial cell membrane was the target of green tea polyphenols. Regarding the effects of green tea polyphenols on the S. moorei colonization properties, it was found that biofilm formation on EGCG-treated surfaces was significantly affected, and that green tea extract and EGCG can cause the eradication of pre-formed S. moorei biofilms. Moreover, both the green tea extract and EGCG were found to reduce the adherence of S. moorei to oral epithelial cells. The ß-galactosidase activity of S. moorei, which plays a key role in VSC production, was dose-dependently inhibited by green tea polyphenols. In addition, EGCG at ½ MIC significantly decreased the ß-galactosidase gene expression. CONCLUSION: Our study brought evidence to support that green tea polyphenols possess a number of properties that may contribute to reduce S. moorei-related halitosis. Therefore, these natural compounds may be of interest to be used to supplement oral healthcare products.

Quercetin-3-O-ß-D-glucuronide isolated from Polygonum aviculare inhibits cellular senescence in human primary cells.

Cellular senescence is known to contribute to tissue aging, a variety of age-related diseases, tissue regeneration, and cancer. Therefore, aging intervention might be useful for prevention of aging as well as age-related disease. In this study, we investigated compounds from Polygonum aviculare to determine if they inhibited cellular senescence in human primary cells, human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs). Ten compounds from P. aviculare were purified and their inhibitory effects on adriamycin-induced cellular senescence were measured by observing senescence-associated ß-galactosidase (SA-ß-gal) activity and reactive oxygen species. Among them, compound 9 (quercetin-3-O-ß-D-glucuronide) showed inhibitory effects against cellular senescence in HDFs and HUVECs treated with adriamycin. Additionally, compound 9 rescued replicative senescence in HDFs and HUVECs. These data imply that compound 9 represses cellular senescence in human primary cells and might be useful for the development of dietary supplements or cosmetics that ameliorate tissue aging or aging-associated diseases.

Estimation of resveratrol content in peanut pericarp and its relation to the in vitro inhibitory activity on carbohydrate metabolizing enzymes.

The aim of this work was the estimation of resveratrol content in two successive extracts (EtOAc and MeOH) of peanuts (Arachis hypogaea L.) pericarp of Egypt, by TLC and HPLC methods. Results showed the presence of 3.0 and 0.5 microg/mL resveratrol in EtOAc and MeOH extracts respectively. The in vitro carbohydrate hydrolyzing enzyme inhibition activity showed higher percentage of inhibition of alpha-amylase, alpha-glucosidase and beta-galactosidase with EtOAc (4.32, 5.93 and 13.7%) than with MeOH extract (3.9, 4.9 and 14.1%) but lower than the standard resveratrol concentration (5.18, 5.94 and 13.26%) and the reference acarbose (5.88, 5.9 and 13.0%). It could be concluded that the content of resveratrol in peanut pericarp is related to the percentage of inhibition activity of carbohydrate hydrolyzing enzymes. These results strongly reflect the benefit of using peanut pericarp, the waste product, as a natural antidiabetic agent.

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy.

Deficiencies of lysosomal ß-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant ß-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal ß-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable.

Phytochemical screening of Nepeta cataria extracts and their in vitro inhibitory effects on free radicals and carbohydrate-metabolising enzymes.

This research was performed to investigate in vitro the biological activities of successive as well as 70% ethanol extracts of Nepeta cataria on some biochemical parameters including oxidative markers and carbohydrate-hydrolysing enzyme activities (α-amylase, ß-galactosidase and α-glucosidase). Powdered N. cataria and its successive extracts were screened for their phytochemical constituents. Tests for tannins, carbohydrates, glycosides and flavonoids were positive in ethanolic extract, but those for steroids and terpenoids were positive in petroleum ether and chloroform extracts. Also, different extracts were chromatographically investigated. The results obtained demonstrated that different successive extracts of N. cataria exhibited an inhibitory effect on oxidative stress indices and carbohydrate-hydrolysing enzymes. It is observed that 70% ethanol, petroleum ether and chloroform extracts showed, respectively, the most potent inhibitory activities, while ethyl acetate and ethanol successive extracts appeared with moderate or low reducing activities.

High-resolution dose-response screening using droplet-based microfluidics.

A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC(50) values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC(50) of 27 ± 0.83 µM.

An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries.

Droplet-based microfluidic systems allow biological and chemical reactions to be performed on a drastically decreased scale. However, interfacing the outside world with such systems and generating high numbers of microdroplets of distinct chemical composition remain challenging. We describe here an automated system in which arrays of chemically distinct plugs are generated from microtiter plates. Each array can be split into multiple small-volume copies, thus allowing several screens of the same library. The system is fully compatible with further on-chip manipulation(s) and allows monitoring of individual plugs over time (e.g. for recording reaction kinetics). Hence the technology eliminates several bottlenecks of current droplet-based microfluidic systems and should open the way for (bio-)chemical and cell-based screens.

Evaluation of antiglycosidase and anticholinesterase activities of Boehmeria nivea.

In this era, major community worldwide is suffering from diabetes type II, cancer and neurodegenerative disorders. To overcome these diseases, in the screening of Korean medicinal plants, we studied the whole plant of Boehmeria nivea (B. nivea). The methanolic leaf, stem and root extracts of B. nivea and their respective n-hexane, methylene chloride (CH(2)Cl(2)), ethyl acetate (EtOAc), n-butanol (BuOH) and aqueous fractions were investigated for their total phenolic content (TPC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme inhibition activities. Profound TPC and DPPH free radical scavenging activities were observed in the EtOAc and BuOH fractions of root, where the BuOH fraction showed high-pitched alpha-glucosidase inhibition and the EtOAc layer showed the maximum beta-glucosidase inhibition. Furthermore, the leaf extract demonstrated the highest beta-galactosidase inhibitory activity, but no alpha-galactosidase inhibition was seen in any of the plant parts. Notable BChE and moderate AChE inhibitory activity was found in whole plant. It can be suggested that whole plant of B. nivea provides a strong biochemical rationale as one of the good choices for the treatment of diabetes type II, cancer and neurodegenerative diseases (AD, etc).

Differential binding with ERá and ERâ of the phytoestrogen-rich plant Pueraria mirifica

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERá and hERâ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the â-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the â-galactosidase activity (EC50) of the tuber extracts in relation to 17â-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERá and hERâ, respectively, with a higher relative estrogenic potency with hERâ than with hERá. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17â-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.

Differential binding with ERalpha and ERbeta of the phytoestrogen-rich plant Pueraria mirifica.

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERalpha and hERbeta and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the beta-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the beta-galactosidase activity (EC(50)) of the tuber extracts in relation to 17beta-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERalpha and hERbeta, respectively, with a higher relative estrogenic potency with hERbeta than with hERalpha. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17beta-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.

Beneficial effects of substrate reduction therapy in a mouse model of GM1 gangliosidosis.

GM1 gangliosidosis is an inherited neurodegenerative disorder caused by lysosomal beta-galactosidase deficiency, resulting in the storage of GM1 and GA1, primarily in the central nervous system. This disease typically afflicts infants and young children and there is currently no effective therapy. Substrate reduction therapy (SRT) could be of potential benefit. The imino sugars N-butyldeoxynojirimycin (NB-DNJ, miglustat, Zavesca) and N-butyldeoxygalactonojirimycin (NB-DGJ) used for SRT inhibit glucosylceramide synthase (GlcCerS) that catalyses the first committed step in glycosphingolipid biosynthesis. We have compared the efficacy and tolerability of NB-DNJ and NB-DGJ in the beta-galactosidase knockout mouse. NB-DGJ was better tolerated than NB-DNJ, due to intrinsic gastrointestinal tract dysfunction that was exacerbated by NB-DNJ. However, functional improvement was greatest with NB-DNJ treatment which may potentially be caused by novel anti-inflammatory properties of NB-DNJ.

Iminosugars from Baphia nitida Lodd.

Chromatographic separation of the 50% aqueous EtOH extract of the leaves of the African medicinal tree Baphia nitida resulted in isolation of 10 iminosugars. The plant contained 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) as a major alkaloid. The structure of a new alkaloid was also elucidated by spectroscopic methods as the 1-O-beta-D-fructofuranoside of DMDP, and this plant produced 3-O-beta-D-glucopyranosyl-DMDP as well. DMDP is a potent inhibitor of beta-glucosidase and beta-galactosidase, whereas the other two derivatives lowered inhibition toward both of these enzymes and improved inhibitory activities toward rice alpha-glucosidase and rat intestinal maltase.

Isoflavonoids with antiestrogenic activity from Millettia pachycarpa.

Three new isoflavonoids, named millewanins G (1) and H (2) and furowanin B (3), were isolated from the leaves of Millettia pachycarpa. Their structures were elucidated on the basis of spectroscopic analyses. The antiestrogenic activity in the yeast two-hybrid assay of these isoflavonoids was examined and shown to be comparable with that of 4-hydroxytamoxifen.

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases.

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.

Synthesis and enzymatic evaluation of five-membered iminocyclitols and a pseudodisaccharide.

Described here are the synthesis of five-membered iminocyclitols with galacto-configuration and a pseudodisaccharide, and their inhibitory activities against beta-galactosyltransferase, beta-galactosidase and alpha-mannosidase.