Magnolol derivatives as specific and noncytotoxic inhibitors of breast cancer resistance protein (BCRP/ABCG2).

The breast cancer resistance protein (BCRP/ABCG2) transporter mediates the efflux of numerous antineoplastic drugs, playing a central role in multidrug resistance related to cancer. The absence of successful clinical trials using specific ABCG2 inhibitors reveals the urge to identify new compounds to attend this critical demand. In this work, a series of 13 magnolol derivatives was tested as ABCG2 inhibitors. Only two compounds, derivatives 10 and 11, showed partial and complete ABCG2 inhibitory effect, respectively. This inhibition was selective toward ABCG2, since none of the 13 compounds inhibited neither P-glycoprotein nor MRP1. Both inhibitors (10 and 11) were not transported by ABCG2 and demonstrated a low cytotoxic profile even at high concentrations (up to 100 µM). 11 emerged as the most promising compound of the series, considering the ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50), showing a therapeutic ratio (TR) higher than observed for 10 (10.5 versus 1.6, respectively). This derivative showed a substrate-independent and a mixed type of inhibition. The effect of compound 11 on the ABCG2 ATPase activity and thermostability revealed allosteric protein changes. This compound did not affect the expression levels of ABCG2 and increased the binding of the conformational-sensitive antibody 5D3. A docking study showed that 11 did not share the same binding site with ABCG2 substrate mitoxantrone. Finally, 11 could revert the chemoresistance to SN-38 mediated by ABCG2.

Characterization and Interaction with Biomembrane Model of Benzo[k,l]xanthene Lignan Loaded Solid Lipid Nanoparticles.

Benzo[k,l]xanthene lignans are a group of rare natural products belonging to the class of polyphenols with promising biological activities and are studied as potential chemotherapeutic agents. The lipophilic character of a xanthene core makes these molecules difficult to be used in an aqueous medium, limiting their employment in studies for pharmaceutical applications. To overcome this problem, a drug-delivery system which is able to improve the stability and bioavailability of the compound can be used. In this study, a bioactive benzoxanthene lignan (BXL) has been included in SLN. Unloaded and BXL-loaded SLN have been prepared using the Phase Inversion Temperature method and characterized in terms of size, zeta potential, entrapment efficiency and stability. Differential scanning calorimetry was used to evaluate the thermotropic behavior and ability of SLN to act as carriers for BXL. A biomembrane model, represented by multilamellar vesicles, was used to simulate the interaction of the SLN with the cellular membrane. Unloaded and loaded SLN were incubated with the MLV, and their interactions were evaluated through variations in their calorimetric curves. The results obtained suggest that SLN could be used as a delivery system for BXL.

Laccase-mediated synthesis of bioactive natural products and their analogues.

Laccases are a class of multicopper oxidases that catalyse the one-electron oxidation of four equivalents of a reducing substrate, with the concomitant four-electron reduction of dioxygen to water. Typically, they catalyse many anabolic reactions, in which mostly phenolic metabolites were subjected to oxidative coupling. Alternatively, laccases catalyse the degradation or modification of biopolymers like lignin in catabolic processes. In recent years, laccases have proved valuable and green biocatalysts for synthesising compounds with therapeutic value, including antitumor, antibiotic, antimicrobial, and antioxidant agents. Further up to date applications include oxidative depolymerisation of lignin to gain new biomaterials and bioremediation processes of industrial waste. This review summarizes selected examples from the last decade's literature about the laccase-mediated synthesis of biologically active natural products and their analogues; these will include lignans and neolignans, dimeric stilbenoids, biflavonoids, biaryls and other compounds of potential interest for the pharmaceutical industry. In addition, a short section about applications of laccases in natural polymer modification has been included.

Synthesis and in vitro evaluation of chlorogenic acid amides as potential hypoglycemic agents and their synergistic effect with acarbose.

Type 2 Diabetes mellitus is a chronic disease considered one of the most severe global health emergencies. Chlorogenic acid (1) has been shown to delay intestinal glucose absorption by inhibiting the activity of α-glucosidase (α-Glu) and α-amylase (α-Amy). In the present work, eleven chlorogenic acid amides have been synthesized and evaluated for their antioxidant properties (as DPPH and ORAC) and inhibition activity towards the two enzymes and, with the aim to obtain dual-action antidiabetic agents. The two most promising hypoglycemic compounds, bearing a tertiary amine function on an alkyl chain (8) and a benzothiazole scaffold (11), showed IC50 values lower than that of (1) (45.5 µM α-Glu; 105.2 µM α-Amy). Amides 8 and 11 were by far more potent α-Glu inhibitors than the antidiabetic drug acarbose (IC50 = 268.4 µM) and about twice less active toward α-Amy than acarbose (IC50 = 34.4 µM). Kinetics experiments on amides 8 and 11 indicated these compounds as mixed-type inhibitors of α-Glu with K'i values of 13.3 and 6.3 µM, respectively. The amylase inhibition occurred with a competitive mechanism in the presence of 8 (Ki = 79.7 µM) and with a mixed-type mechanism with 11 (Ki = 19.1 µM; K'i = 93.6 µM). Molecular docking analyses supported these results, highlighting the presence of additional binding sites in both enzymes. Fluorescence experiments confirmed the grater affinity of amides 8 and 11 towards the two enzymes respect to (1). Moreover, a significant enhancement in acarbose efficacy was observed when inhibition assays were performed adding acarbose and amide 11. The above outcomes pinpointed the benzothiazole-based amide 11 as a promising candidate for further studies on type 2 diabetes treatment, both alone or combined with acarbose.

Natural Isoflavones and Semisynthetic Derivatives as Pancreatic Lipase Inhibitors.

Obesity, now widespread all over the world, is frequently associated with some chronic diseases. Thus, there is a growing interest in the prevention and treatment of obesity. To date, the only antiobesity drug is orlistat, a natural product-derived pancreatic lipase (PL) inhibitor with some undesired side effects. In the last decades, many natural compounds or derivatives have been evaluated as potential PL inhibitors, and natural polyphenols are among the most promising for possible exploitation as antiobesity agents. However, few studies have been devoted to isoflavones. In this work, we report a study on the PL inhibitory properties of a small library of semisynthetic isoflavone derivatives together with the natural leads daidzein (1), genistein (2), and formononetin (3). In vitro lipase inhibition assay showed that 2 is the most promising PL inhibitor. Among synthetic isoflavones, the hydroxylated and brominated derivatives were more potent than their natural leads. Detailed studies through fluorescence measurements and kinetics of lipase inhibition showed that 2 and the bromoderivatives 10 and 11 have the greatest affinity for PL. Docking studies corroborated these findings highlighting the interactions between isoflavones and the enzyme, confirming that hydroxylation and bromination are useful modifications.

A Rare Natural Benzo[k,l]xanthene as a Turn-Off Fluorescent Sensor for Cu2+ Ion.

Rapid and efficient analyses of copper ions are crucial to providing key information for Cu2+ in living cells because of their biological importance. In this study, we reported one new turn-off fluorescent sensor for Cu2+ with a benzo[k,l]xanthene core, which served as an efficient cation sensor for copper ion over a wide range of other cations (Na+, K+, Ag+, Hg2+, Cd2+, Co2+, Ni2+, Zn2+, Mg2+, and Fe3+) owing to the catechol group in the aromatic core. The sensor showed selectivity for Cu2+ over other ions; the logKß for Cu2+ binding to compound 1 had a value of 13.265. In the presence of Cu2+, sensor 1 provided significant fluorescence decrement; Co2+, and Ni2+ caused a fluorescence decrement when employed at a higher concentration than Cu2+, while Na+, K+, Hg2+, Cd2+, Zn2+, and Mg2+ metal ions produced only minor changes in fluorescence intensity. Fluorescence experiments demonstrate that compound 1 may have an application as a fluorescent probe for detecting Cu2+ with a limit of detection of 0.574 µM.

Synthesis, DNA/RNA-interaction and biological activity of benzo[k,l]xanthene lignans.

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants - presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring - influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.

Mass Spectrometry and 1H-NMR Study of Schinopsis lorentzii (Quebracho) Tannins as a Source of Hypoglycemic and Antioxidant Principles.

The ethyl acetate extract of the commercial tannin Tan'Activ QS-SOL (from Schinopsis lorentzii wood), employed for the production of red wine, was subjected to chromatography on Sephadex LH-20, providing nine fractions (A-1-A-9), which were estimated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase and α-amylase inhibition). All the fractions were analyzed by means of HPLC/ESI-MS/MS and 1H-NMR to identify the principal active constituents. Fractions A-1 and A-3 showed the highest antioxidant activity and gallic acid (1), pyrogallol (3), eriodictyol (6), catechin (12), and taxifolin (30) were identified as the major constituents. The highest α-glucosidase and α-amylase inhibitory activity was observed in fractions A-7-A-9 containing condensed (9', 15, 18, 19, 23, and 27) hydrolysable tannins (13 and 32) as well as esters of quinic acid with different units of gallic acid (5, 11, 11', 14, and 22). This last class of gallic acid esters are here reported for the first time as α-glucosidase and α-amylase inhibitors.

Special Issue: From Natural Polyphenols to Synthetic Bioactive Analogues.

In recent years, phenolic compounds from plant sources, commonly referred to as 'plant polyphenols', have been the subject of an impressive number of research studies, to a large extent focused on the healthy properties attributed to diet polyphenols, including antioxidant, anti-inflammatory, antineoplastic, antidiabetic, neuroprotective, and other biological activities [...].

Substantial Equivalence of a Transgenic Lemon Fruit Showing Postharvest Fungal Pathogens Resistance.

The development of genetically modified (GM) crops speeds up the obtainment of novel varieties with improved agronomic characteristics. However, the risk evaluation of the use of GMs is mandatory before their release in the market. In this paper, an untargeted and comprehensive nuclear magnetic resonance-based metabolomic study was carried out on the peel and flesh of a transgenic lemon clone (E23) expressing the chit42 gene and exhibiting an increased tolerance to some pathogenic fungi and on its wild type. Results highlighted a substantial equivalence of the metabolomics profile of the transgenic clone compared to the wild type. In addition, an enhanced response of the E23 clone toward fungal pathogens affecting the postharvest management in lemon was evidenced. These results confirm the potential of genetic engineering for the punctual modification of specific agronomic traits without altering the whole pattern of metabolites and open new perspectives for a more sustainable and effective management of specific postharvest diseases in citrus.

Synthesis of Bisphenol Neolignans Inspired by Honokiol as Antiproliferative Agents.

Honokiol (2) is a natural bisphenol neolignan showing a variety of biological properties, including antitumor activity. Some studies pointed out 2 as a potential anticancer agent in view of its antiproliferative and pro-apoptotic activity towards tumor cells. As a further contribution to these studies, we report here the synthesis of a small library of bisphenol neolignans inspired by honokiol and the evaluation of their antiproliferative activity. The natural lead was hence subjected to simple chemical modifications to obtain the derivatives 3-9; further neolignans (12a-c, 13a-c, 14a-c, and 15a) were synthesized employing the Suzuki-Miyaura reaction, thus obtaining bisphenols with a substitution pattern different from honokiol. These compounds and the natural lead were subjected to antiproliferative assay towards HCT-116, HT-29, and PC3 tumor cell lines. Six of the neolignans show GI50 values lower than those of 2 towards all cell lines. Compounds 14a, 14c, and 15a are the most effective antiproliferative agents, with GI50 in the range of 3.6-19.1 µM, in some cases it is lower than those of the anticancer drug 5-fluorouracil. Flow cytometry experiments performed on these neolignans showed that the inhibition of proliferation is mainly due to an apoptotic process. These results indicate that the structural modification of honokiol may open the way to obtaining antitumor neolignans more potent than the natural lead.

C-glucosidic ellagitannins and galloylated glucoses as potential functional food ingredients with anti-diabetic properties: a study of α-glucosidase and α-amylase inhibition.

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, which can be counteracted by inhibition of α-glucosidase and α-amylase, both involved in the carbohydrate metabolism. Fourteen C-glucosidic ellagitannins and three galloylated glucoses were studied as potential α-glucosidase and α-amylase inhibitors. Most of the compounds were found to be moderate inhibitors of α-amylase, but potent inhibitors of α-glucosidase, showing low-micromolar IC50 values, far lower than that of the antidiabetic drug acarbose. This selectivity can be an advantage for their possible application as functional food ingredients with anti-diabetic properties because strong α-amylase inhibition generally causes undesired side effects. The best inhibitors were selected for further studies. Intrinsic fluorescence measurements confirmed their high affinity towards α-glucosidase, highlighting a static quenching mechanism. Circular dichroism measurements and kinetics of inhibition indicated that the most active C-glucosidic ellagitannin roburin D (RobD) is a competitive inhibitor, whereas α-pentagalloylglucose (α-PGG) acts as a mixed-type inhibitor.

Bioinspired benzoxanthene lignans as a new class of antimycotic agents: synthesis and Candida spp. growth inhibition.

In this work we synthetized the bioinspired benzoxanthene lignans (BXLs) 3, 14-22, and the phenazine derivative 23 as potential antimycotic agents. MICs and MFCs against Candida strains were determined. In a preliminary screening, compounds 3, 15, 20, 21, 22 were substantially inactive. Compounds 14 and 17 showed antifungal activity, being able to inhibit the growth of the majority of Candida strains with MIC values in the range 4.6-19.2 µM (14) and 26.0-104.3 µM (17); for three strains, the MICs were lower than those obtained using the antimycotic drug fluconazole. The three BXLs 18, 19 and 23 showed some MIC values lower than that of fluconazole; 18 was also active against two non-albicans Candida strains resistant to fluconazole. Phenazine 23, although active only against one strain (MIC = 1.3 µM), was one order of magnitude more potent than fluconazole. All the BXLs were fungicidal.

Novel benzoxanthene lignans that favorably modulate lipid mediator biosynthesis: A promising pharmacological strategy for anti-inflammatory therapy.

Lipid mediators (LM) encompass pro-inflammatory prostaglandins (PG) and leukotrienes (LT) but also specialized pro-resolving mediators (SPM) which display pivotal bioactivities in health and disease. Pharmacological intervention with inflammatory disorders such as osteoarthritis and rheumatoid arthritis commonly employs anti-inflammatory drugs that can suppress PG and LT formation, which however, possess limited effectiveness and side effects. Here, we report on the discovery and characterization of the two novel benzoxanthene lignans 1 and 2 that modulate select LM biosynthetic enzymes enabling the switch from pro-inflammatory LT to SPM biosynthesis as potential pharmacological strategy to intervene with inflammation. In cell-free assays, compound 1 and 2 inhibit microsomal prostaglandin E2 synthase-1 and leukotriene C4 synthase (IC50 ∼ 0.6-3.4 µM) and potently interfere with 5-lipoxygenase (5-LOX), the key enzyme in LT biosynthesis (IC50 = 0.04 and 0.09 µM). In human neutrophils, monocytes and M1 and M2 macrophages, compound 1 and 2 efficiently suppress LT biosynthesis (IC50 < 1 µM), accompanied by elevation of 15-LOX-derived LM including SPM. In zymosan-induced murine peritonitis, compound 1 and 2 ameliorated self-limited inflammation along with suppression of early LT formation and elevation of subsequent SPM biosynthesis in vivo. Together, these novel benzoxanthene lignans promote the LM class switch from pro-inflammatory towards pro-resolving LM to terminate inflammation, suggesting their suitability as novel leads for pharmacotherapy of arthritis and related inflammatory disorders.

Synthesis of Rosmarinic Acid Amides as Antioxidative and Hypoglycemic Agents.

Type 2 diabetes mellitus (T2DM) is an important metabolic disorder for which there is an urgent need for new antidiabetic drugs. α-Glucosidase inhibition is an established protocol for T2DM therapy. Because hyperglycemia causes oxidative tissue damage, the development of agents with both α-glucosidase inhibition and antioxidant activity from natural or natural-derived polyphenols such derivatives of rosmarinic acid (RA) represents an attractive therapeutic option. We report a study on amides 1-10 derived from RA and their evaluation for yeast α-glucosidase inhibition and antioxidant activity (DPPH and ORAC tests). All amides showed higher inhibitory activity than that of RA, were by far more potent than the antidiabetic drug acarbose, and proved to be effective antioxidants. A molecular docking study displayed significant binding interactions of RA amides with the active site of α-glucosidase. This in silico optimization study led to the design and synthesis of amides 9 (IC50 = 42.3 µM) and 10 (IC50 = 35.2 µM), showing the most potent α-glucosidase inhibition and good antioxidative properties. A kinetic study showed that 10 acts as a mixed type inhibitor.

Chemical Proteomics-Guided Identification of a Novel Biological Target of the Bioactive Neolignan Magnolol.

Understanding the recognition process between bioactive natural products and their specific cellular receptors is of key importance in the drug discovery process. In this outline, some potential targets of Magnolol, a natural bioactive compound, have been identified by proteomic approaches. Among them, Importin-ß1 has been considered as the most relevant one. A direct binding between Magnolol and this nuclear chaperone has been confirmed by DARTS and molecular docking, while its influence on Importin-ß1 translocation has been evaluated by in vitro assays.

A mass spectrometry and 1H NMR study of hypoglycemic and antioxidant principles from a Castanea sativa tannin employed in oenology.

The ethanol extract of the commercial tannin Tan'Activ C, (from Castanea sativa wood), employed in oenology, was subjected to chromatography on XAD-16 affording fractions X1-X5, evaluated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase inhibition). Fraction X3 showed GAE, radical scavenging activity, and α-glucosidase inhibition higher than those of the Castanea sativa extract, and was subjected to chromatography on Sephadex LH-20 to obtain fractions S1-S7, analyzed by HPLC/ESI-MS/MS and 1H NMR to identify the main active constituents. In fractions with higher antioxidant activity, gallic acid (4), grandinin (5), valoneic acid dilactone (9), 1,2,3-tri-O-galloyl-ß-glucose (14), 3,4,6-tri-O-galloyl-ß-glucose (15) and galloyl derivative of valoneic acid dilactone (21) were identified as the major constituents. The highest hypoglycemic activity was found in fractions S6 and especially S7; the major constituents of these fractions are valoneic acid dilactone (9), three tetragalloyl glucose isomers (16-18) and 1,2,3,4,6-penta-O-galloyl-ß-glucose (23), previously reported as α-glucosidase inhibitors.

Identification by Inverse Virtual Screening of magnolol-based scaffold as new tankyrase-2 inhibitors.

The natural product magnolol (1) and a selection of its bioinspired derivatives 2-5, were investigated by Inverse Virtual Screening in order to identify putative biological targets from a panel of 308 proteins involved in cancer processes. By this in silico analysis we selected tankyrase-2 (TNKS2), casein kinase 2 (CK2) and bromodomain 9 (Brd9) as potential targets for experimental evaluations. The Surface Plasmon Resonance assay revealed that 3-5 present a good affinity for tankyrase-2, and, in particular, 3 showed an antiproliferative activity on A549 cells higher than the well-known tankyrase-2 inhibitor XAV939 used as reference compound.

Biological effects of polyphenol-rich extract and fractions from an oenological oak-derived tannin on in vitro swine sperm capacitation and fertilizing ability.

Although excessive ROS levels induce sperm damage, sperm capacitation is an oxidative event that requires low amounts of ROS. As the antioxidant activity of the ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R®) and its four fractions (FA, FB, FC, FD) has been recently reported, the present study was set up to investigate the biological effects of TRE and its fractions in an in vitro model of sperm capacitation and fertilization. Boar sperm capacitation or gamete coincubation were performed in presence of TRE or its fractions (0, 1, 10, 100 µg/ml). TRE at the concentration of 10 µg/ml (TRE10) stimulated sperm capacitation, as it increased (p < .001) the percentage of spermatozoa with tyrosine-phosphorylated protein positivity in the tail principal piece (B pattern) (67.0 ±â€¯10.6 vs. 48.6 ±â€¯9.0, mean ±â€¯SD for TRE10 vs. Ctr respectively). Moreover T10 significantly (p < .001) increased oocyte fertilization rate (91.9 ±â€¯4.0 vs. 69.0 ±â€¯14.8, TRE10 vs. Ctr respectively). An opposite effect of TRE at the concentration of 100 µg/ml (TRE100) on both sperm capacitation (B pattern cell percentage 33.3 ±â€¯29.2) and fertilizing ability (fertilization rate 4.9 ±â€¯8.3), associated with a higher sperm viability (66.9 ±â€¯9.3 vs. 35.4 ±â€¯10.8, TRE100 vs. Ctr respectively) (p < .001), was recorded. The potency of the TRE fractions seems to be highest in FB followed by FC, faint in FD and nearly absent in FA. Our results show that TRE and its fractions, in a different extent, exert a powerful biological effect in finely modulating capacitation and sperm fertilizing ability.

Resveratrol, piceatannol and analogs inhibit activation of both wild-type and T877A mutant androgen receptor.

Prostate cancer growth and progression are mainly dependent on androgens and many current prostate cancer treatment options target the synthesis or function of androgens. We have previously reported that resveratrol and synthetic analogs of resveratrol with a higher bioavailability inhibit the synthesis of androgens in human adrenocortical H295R cells. Now we have studied the antiandrogenic properties of resveratrol, piceatannol and analogs in two different prostate cell lines; LNCaP and RWPE. LNCaP carry a T877A mutation in the androgen receptor while RWPE has a wild-type androgen receptor. We found that resveratrol, piceatannol and all studied analogs were able to inhibit a dihydrotestosterone-induced activation of the androgen receptor, showing that they act as antiandrogens. In LNCaP cells, all studied compounds were able to statistically significantly decrease the androgenic signaling in concentrations ≥1µM and the synthetic analogs trimethylresveratrol (RSVTM) and tetramethylpiceatannol (PICTM) were the most potent compounds. RWPE cells were not as responsive to the studied compounds as the LNCaP cells. A statistically significant decrease in the androgenic signaling was observed at concentrations ≤5µM for most compounds and RSVTM was found to be the most potent compound. Further, we studied the effects of resveratrol, piceatannol and analogs on the levels of prostate-specific antigen (PSA) in LNCaP cells and found that all studied compounds decreased the level of PSA and that the synthetic analogs diacetylresveratrol (RSVDA), triacetylresveratrol (RSVTA) and RSVTM were the most potent compounds, decreasing the PSA level by approx. 50% at concentrations ≥10µM. In a cell-free receptor binding assay we were unable to show binding of resveratrol or analogs to the ligand binding domain of the androgen receptor, indicating that the observed effects are mediated via other mechanisms than direct ligand competition. We conclude that the resveratrol, piceatannol and analogs are highly interesting for chemoprevention of prostate cancer, since they have a high potency both as inhibitors of androgen synthesis and androgen receptor activation.