Targeting Tyrosinase: Development and Structural Insights of Novel Inhibitors Bearing Arylpiperidine and Arylpiperazine Fragments.

The inhibition of tyrosinase (Ty, EC 1.14.18.1) represents an efficient strategy of decreasing melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies on two different tyrosinases, that from Bacillus megaterium (TyBm) and that from a mushroom (TyM), has contributed to increasing our knowledge about their structural information and translating that information to the most druggable human Ty (TyH) isozyme. In particular, we designed and synthesized a series of 1-(4-fluorobenzyl)piperazine and 1-(4-fluorobenzyl)piperidine derivatives showing inhibitory activities on TyM at micromolar ranges and more potency than that of the reference compound, kojic acid. The crystal structures of TyBm with inhibitor 3 (IC50 value of 25.11 µM) and 16 (IC50 value of 5.25 µM) were solved, confirming the binding poses hypothesized by in silico studies and revealing the main molecular determinants for the binding recognition of the inhibitors.

Anti-angiogenic activity and phytochemical screening of fruit fractions from Vitex agnus castus.

Although the antitumour activity of Vitex agnus castus fruits has been already addressed, no work has yet assessed their anti-angiogenic potential. To this purpose, several extractive fractions of such fruits were tested on zebrafish embrios by EAP assay, so that only the bioactive fractions could be subsequently tested on the chick chorioallantoic membrane by CAM assay. Bioactive fractions were also phytochemically screened to identify those bioactive compounds responsible for anti-angiogenic activity. A marked inhibition of vessel formation was detected only in zebrafish embryos treated with chloroform or ethyl acetate fractions. Considering CAM assay, chloroform fraction induced a strong reduction of microvasculature and haemoglobin content; while lower anti-angiogenic effects of the ethyl acetate fraction were determined. Phytochemical analyses confirmed the presence of several bioactive anti-angiogenic compounds. Overall, obtained preliminary results highlighted a potential anti-angiogenic activity of V. agnus castus fruits.

Chemical exploration of 4-(4-fluorobenzyl)piperidine fragment for the development of new tyrosinase inhibitors.

Tyrosinase is involved in the production of melanin through the hydroxylation of monophenols to o-diphenols. The role of this enzyme was extensively studied in order to identify new therapeutics preventing skin pigmentation and melanoma. In this work we initially identified the 3-(4-benzylpiperidin-1-yl)-1-(1H-indol-3-yl)propan-1-one (1a) as promising mushroom tyrosinase inhibitor (IC50 = 252 µM). Then, several chemical modifications were performed and new analogues related to compound 1a were synthesized. Biochemical assays demonstrated that several obtained compounds proved to be effective inhibitors showing IC50 values lower both than "lead compound" 1a and reference inhibitor kojic acid, as a well-known tyrosinase inhibitor. The inhibition kinetics analyzed by Lineweaver-Burk plots revealed that compounds 2 a-c and 10b act as non-competitive inhibitors while the most active inhibitor 2d (IC50 = 7.56 µM) is a mixed-type inhibitor. Furthermore, experimental and computational structural studies were performed in order to clarify the binding mode of the derivative 2d.

Chemical Composition of Juniperus phoenicea and J. drupacea Essential Oils and their Biological Effects in the Choriallantoic Membrane (CAM) Assay.

The aim of the present study was the chemical analysis of the essential oils from Juniperus phoenicea and J. drupacea female cones and evaluation of their biological effects. Fresh samples, collected in Greece, were subjected separately to hydrodistillation and the oils obtained analyzed by GC-FID and GC-MS. The oils Were assessed using the CAM (chorioallantoic membrane) assay to evaluate their anti-angiogenic potential and the lack of irritant effects in topical application. GC analysis showed that mainly quantitative differences among the samples were observed: limonene was the most abundant compound in J. drupacea (27.0%) compared with J. phoenicea oil (1.6%); the content of a-pinene was high in both essential oils (J. phoenicea 22.1%, J. drpacea 26.1%) followed by germacrene D (J. phoenicea 7.4%, J. drpacea 7.1%, respectively). Nevertheless, qualitative differences were also detected as the diterpene 4-epi-abietal was present in a considerable amount (13.2%) in J. phoenicea essential oil, but was not detected in J. drupacea oil. In the CAM assay, only J. phoenicea. essential oil evidenced a rather weak anti-angiogenic activity compared with the standard retinoic acid, but no irritant effect was observed for either essential oil suggesting their safety for topical application.

Searching for indole derivatives as potential mushroom tyrosinase inhibitors.

Tyrosinase is a copper-containing enzyme widely distributed in nature, involved in the biosynthesis of melanin whose role is to protect the skin from ultraviolet damage. A great interest has been shown on the melanin involvement in malignant melanoma and other carcinogenetic processes. These phenomena have encouraged the research of tyrosinase inhibitors useful in therapeutic field as well as in foods and cosmetics to prevent browning. The idea was to screen our "in house" database to select suitable lead compounds for the discovery of potential drug-inhibiting enzyme. The obtained biological results demonstrated that compounds containing 4-fluorobenzyl moiety at N - 1 position of indole system showed the best activity. In addition, the role of the portion linked to the carbonyl group at C - 3 was discussed. A Lineweaver-Burk kinetic analysis of the most active indoles, CHI 1043 and derivative 4, showed a mixed-type inhibition in the presence of L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate.

Phytochemical screening of Artemisia arborescens L. by means of advanced chromatographic techniques for identification of health-promoting compounds.

Artemisia arborescens, also known as tree wormwood, is a typical species of the Mediterranean flora. It has been used in folk medicine for its antispasmodic, anti-pyretic, anti-inflammatory, and abortifacient properties. In the current study, the application of multidimensional comprehensive gas chromatography (GC×GC), allowed to obtain a detailed fingerprint of the essential oil from A. arborescens aerial parts, highlighting an abundant presence of chamazulene followed by camphor, ß-thujone, myrcene, and α-pinene. Moreover, flavonoids in the dichloromethane extract were analyzed by means of liquid chromatography with photodiode array and atmospheric pressure chemical ionization-mass spectrometry detections (HPLC-PDA and HPLC-APCI-MS). Six polymethoxyflavones were identified and three of them, including chrysosplenetin, eupatin, and cirsilineol, were described in this species for the first time. The anti-angiogenic activity was investigated in the dichloromethane extract by two in vivo models, chick chorioallantoic membrane (CAM) and zebrafish embryos. Results showed that this extract produced a strong reduction on vessel formation, both on zebrafish (57% of inhibition, 0.1 mg/mL) and chick chorioallantoic membrane (58% of inhibition, 0.8 mg/mL). The high separation power and sensitivity of the analytical methodology applied confirmed the safety of A. arborescens essential oil for human consumption, due to the very low level of the psychotrope α-thujone determined. Moreover, the knowledge of the flavonoidic profile holds a great significance for the use of A. arborescens as a valuable source of anti-angiogenic compounds that might contribute to the valorization of the phytotherapeutic potential of this plant.

Anti-angiogenic activity of Entada africana root.

Entada africana roots are used in African traditional medicine for various diseases including inflammation. This application may be mediated through anti-angiogenic effects. Thus, in this study the anti-angiogenic activity of E. africana root extracts (n-hexane, chloroform, chloroform/methanol and methanol) was preliminarily evaluated by the quantitative determination of endogenous alkaline phosphatase in zebrafish embryos. A bioactivity-guided fractionation of chloroform/methanol extract yielded apigenin and robinetin as the main constituents from the most active fractions. In addition, a marked reduction on capillary formation was evidenced in chick chorioallantoic membrane after treatment with the active fractions or isolated compounds. Results obtained in this study suggest that the anti-angiogenic effects of E. africana root may account for its use in inflammatory diseases and other related pathological conditions.

Betula pendula Roth leaves: gastroprotective effects of an HPLC-fingerprinted methanolic extract.

In this study, a methanolic extract of Betula pendula leaves (BLE) was investigated for its gastroprotective effects against 90% ethanol-induced ulcer in rats. Oral pretreatment of rats with BLE (100, 200 and 400 mg kg(- 1)) significantly reduced the incidence of gastric lesions induced by ethanol administration as compared with misoprostol (0.50 mg kg(- 1)). Furthermore, BLE inhibited the increase in malondialdehyde (MDA) and prevented depletion of total sulhydryl and non-protein sulhydryl groups in rat stomach homogenate when compared with ethanol group. With regard to the effect of lipid peroxidation in vitro, BLE showed the ability to reduce methyl linoleate autoxidation. Chemical characterisation of the main biologically active constituents of BLE was also achieved by means of high-performance liquid chromatography with photodiode array and mass spectrometry detection, showing the presence of myricetin-3-O-galactoside, quercetin glycosides, kaempferol glycosides.

Bioassay-guided isolation of proanthocyanidins with antiangiogenic activities.

The proangiogenic members of the vascular endothelial growth factor (VEGF) family and related receptors play a central role in the modulation of pathological angiogenesis. In order to identify plant compounds able to interfere in the VEGFs/VEGFR-1 (Flt-1) recognition by VEGF family members, the extracts of the aerial parts of Campsiandra guayanensis and Feretia apodanthera were screened by a competitive ELISA-based assay. By using this bioassay-oriented approach five proanthocyanindins, including the new natural compounds (2S)-4',5,7-trihydroxyflavan-(4ß→8)-afzelechin (1) and (2S)-4',5,7-trihydroxyflavan-(4ß→8)-epiafzelechin (2) and the known geranin B (3), proanthocyanidin A2 (4), and proanthocyanidin A1 (5), were isolated. The study of the antiangiogenic activities of compounds 1-5 using ELISA and SPR assays showed compound 1 as being the most active. The antiangiogenic activity of 1 was also confirmed in vivo by the chicken chorioallantoic membrane assay. Our results indicated 1 as a new antiangiogenic compound inhibiting the interaction between VEGF-A or PlGF and their receptor VEGRF-1.