Plausible Emergence of Biochemistry in Enceladus Based on Chemobrionics.

Saturn's satellite Enceladus is proposed to have a soda-type subsurface ocean with temperature able to support life and an iron ore-based core. Here, it was demonstrated that ocean chemistry related to Enceladus can support the development of Fe-based hydrothermal vents, one of the places suggested to be the cradle of life. The Fe-based chemical gardens were characterized with Fourier-transform (FT)IR spectroscopy and XRD. The developed chemobrionic structures catalyzed the condensation polymerization of simple organic prebiotic molecules to kerogens. Further, they could passively catalyze the condensation of the prebiotic molecule formamide to larger polymers, suggesting that elementary biochemical precursors could have emerged in Enceladus.

Development and Validation of an HPLC Method for the Analysis of Flowers of Wild-Growing Primula veris from Epirus, Greece.

An HPLC-PDA method was developed for the determination of the flavonoids in the flowers of Primula veris from Epirus, Greece. The aim was to investigate the chemical content of the over-harvested P. veris populations of Epirus and to develop and optimize an extraction protocol to allow fast, exhaustive, and repeatable extraction. Qualitative analysis revealed that the P. veris flowers from Epirus were particularly rich in flavonoids, especially flavonol triglycosides including derivatives of quercetin, isorhamnetin, and kaempferol. A phytochemical investigation of a 70% hydromethanolic extract from the flowers afforded a new flavonoid, namely, isorhamnetin-3-Ο-ß-glucopyranosyl-(1 → 2)-ß-glucopyranosyl-(1 → 6)-ß-glucopyranoside, which is also the main constituent of the flower extracts. Its structure elucidation was carried out by means of 1D and 2D NMR and mass spectrometry analyses. The HPLC-PDA method was developed and validated according to the International Council for Harmonisation guidelines. Since the main flavonol glycoside of the plant is not commercially available, rutin was used as a secondary standard and the response correction factor was determined. Finally, the overall method was validated for precision (% relative standard deviation ranging between 1.58 and 4.85) and accuracy at three concentration levels. The recovery ranged between 93.5 and 102.1% with relative standard deviation values < 5%, within the acceptable limits. The developed assay is fast and simple and will allow for the quality control of the herbal drug.

Inhibitory Effect of Lithospermic Acid on the HIV-1 Nucleocapsid Protein.

The HIV-1 nucleocapsid protein (NC) is a desirable target in antiretroviral therapy due to its high conservation among HIV-1 strains, and to its multiple and crucial roles in the HIV-1 replication cycle. Natural products represent a valuable source of NC inhibitors, with the catechol group being a privileged scaffold in NC inhibition. By coupling molecular modeling with NMR spectroscopy and fluorescence-based assays, we disclosed lithospermic acid, a catechol derivative extracted from Salvia miltiorrhizza, as a potent and chemically stable non-covalent inhibitor of the NC. Being different from other catechol derivative reported so far, lithospermic acid does not undergo spontaneous oxidation in physiological conditions, thus becoming a profitable starting point for the development of efficient NC inhibitors.

A Closed Chemobrionic System as a Biochemical Delivery Platform.

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.

LC-MS- and NMR-Guided Isolation of Monoterpene Dimers from Cultivated Thymus vulgaris Varico 3 Hybrid and Their Antityrosinase Activity.

Targeted isolation based on a combination of NMR and HPLC-PDA-MS of a dichloromethane extract of Thymus vulgaris Varico 3 aerial parts afforded one new p-cymene dimer, 6,3',4'-trihydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl (1: ), together with two known p-cymene derivatives (2: and 3: ), as well as five known compounds, namely, thymol (4: ), oleanolic acid (5: ), ursolic acid (6: ), cirsimaritin (7: ), and xanthomicrol (8: ). The structural elucidation of all compounds was performed by spectroscopic analyses, including 1D and 2D NMR, and HRESIMS experiments. The biphenyls were assayed for their inhibitory activity on tyrosinase. Compounds 2: and 3: showed negligible activity on tyrosinase, while compound 1: effectively inhibited the enzyme with 35% (± 0.3) inhibitory activity, higher than the inhibition of the reference compound kojic acid (18.6 ± 0.02).

Leaf Decoction of Carica papaya Combined with Artesunate Prevents Recrudescence in Plasmodium berghei-Infected Mice.

Malaria treatment and control have become increasingly difficult because of the spread of drug-resistant strains of Plasmodium falciparum and Plasmodium vivax. Thus, there is a continuous need to develop new combination therapies such as artemisinin-based combination therapies (ACTs) to contrast the emergence of resistant Plasmodium strains. Despite ACT has been recommended by the World Health Organization since 2001, its overall deployment in poor endemic areas is very slow, principally due to its high cost. In the malaria endemic areas, plant remedies are still widely used mostly without assurance of their efficacy and/or safety. A variety of widespread herbal drugs or natural products were already reported for their possible plasmodicidal activities, but the studies concerning their activity in combination with artemisinins are very scarce. The antimalarial activity of papaya is mostly anecdotal, and the present study is aimed at investigating the antiplasmodial activity of a decoction obtained by traditional recipe from the mature leaves of Carica papaya. The decoction was analyzed by HPLC-DAD-MS (high performance liquid chromatography coupled with diodoarray detector and mass spectrometry) showing the presence of caffeoyl derivatives and di- and triglycosides of flavonols. The extract was found to be active against P. falciparum 3D7 strains with a synergism in the presence of artemisinin. In vivo activity against the murine malaria model of Plasmodium berghei was disclosed both for the dried extract alone (250, 500, and 750 mg/kg/d) and for its combination with artesunate (250 mg/kg/d papaya plus 10 mg/kg/d artesunate). This combination displayed the greatest antimalarial activity in terms of reduction of parasitemia and prevention of recrudescence in animals recovered from the infection.

A Validated Method for the Quality Control of Andrographis paniculata Preparations.

Andrographis paniculata is a herbal drug of Asian traditional medicine largely employed for the treatment of several diseases. Recently, it has been introduced in Europe for the prophylactic and symptomatic treatment of common cold and as an ingredient of dietary supplements. The active principles are diterpenes with andrographolide as the main representative. In the present study, an analytical protocol was developed for the determination of the main constituents in the herb and preparations of A. paniculata. Three different extraction protocols (methanol extraction using a modified Soxhlet procedure, maceration under ultrasonication, and decoction) were tested. Ultrasonication achieved the highest content of analytes. HPLC conditions were optimized in terms of solvent mixtures, time course, and temperature. A reversed phase C18 column eluted with a gradient system consisting of acetonitrile and acidified water and including an isocratic step at 30 °C was used. The HPLC method was validated for linearity, limits of quantitation and detection, repeatability, precision, and accuracy. The overall method was validated for precision and accuracy over at least three different concentration levels. Relative standard deviation was less than 1.13%, whereas recovery was between 95.50% and 97.19%. The method also proved to be suitable for the determination of a large number of commercial samples and was proposed to the European Pharmacopoeia for the quality control of Andrographidis herba.

Phenols and Polyphenols as Carbonic Anhydrase Inhibitors.

Phenols are among the largest and most widely distributed groups of secondary metabolites within the plant kingdom. They are implicated in multiple and essential physiological functions. In humans they play an important role as microconstituents of the daily diet, their consumption being considered healthy. The physical and chemical properties of phenolic compounds make these molecules versatile ligands, capable of interacting with a wide range of targets, such as the Carbonic Anhydrases (CAs, EC 4.2.1.1). CAs reversibly catalyze the fundamental reaction of CO2 hydration to bicarbonate and protons in all living organisms, being actively involved in the regulation of a plethora of patho/physiological processes. This review will discuss the most recent advances in the search of naturally occurring phenols and their synthetic derivatives that inhibit the CAs and their mechanisms of action at molecular level. Plant extracts or mixtures are not considered in the present review.

Compounds from Sedum caeruleum with antioxidant, anticholinesterase, and antibacterial activities.

CONTEXT: This is the first study on the phytochemistry, antioxidant, anticholinesterase, and antibacterial activities of Sedum caeruleum L. (Crassulaceae). OBJECTIVE: The objective of this study is to isolate the secondary metabolites and determine the antioxidant, anticholinesterase, and antibacterial activities of S. caeruleum. MATERIALS AND METHODS: Six compounds (1-6) were isolated from the extracts of S. caeruleum and elucidated using UV, 1D-, 2D-NMR, and MS techniques. Antioxidant activity was investigated using DPPH(•), CUPRAC, and ferrous-ions chelating assays. Anticholinesterase activity was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the Ellman method. Antibacterial activity was performed according to disc diffusion and minimum inhibitory concentration (MIC) methods. RESULTS: Isolated compounds were elucidated as ursolic acid (1), daucosterol (2), ß-sitosterol-3-O-ß-D-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6). The butanol extract exhibited highest antioxidant activity in all tests (IC50 value: 28.35 ± 1.22 µg/mL in DPPH assay, IC50 value: 40.83 ± 2.24 µg/L in metal chelating activity, and IC50 value: 23.52 ± 0.44 µg/L in CUPRAC), and the highest BChE inhibitory activity (IC50 value: 36.89 ± 0.15 µg/L). Moreover, the chloroform extract mildly inhibited (MIC value: 80 µg/mL) the growth of all the tested bacterial strains. DISCUSSION AND CONCLUSION: Ursolic acid (1), daucosterol (2), ß-sitosterol-3-O-ß-D-galactopyranoside (3), apigenin (4), apigetrin (5), and apiin (6) were isolated from Sedum caeruleum for the first time. In addition, a correlation was observed between antioxidant and anticholinesterase activities of bioactive ingredients of this plant.

New natural product carbonic anhydrase inhibitors incorporating phenol moieties.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. They represent a typical example of enzyme convergent evolution, as six genetically unrelated families of such enzymes were described so far. The need to find selective CA inhibitors (CAIs) triggered the investigation of natural product libraries, which proved to be a valid source of agents with such an activity, as demonstrated for the phenols, polyamines and coumarins. Herein we report an in vitro inhibition study of human (h) CA isoforms hCAs I, II, IV, VII and XII with a panel of natural polyphenols including flavones, flavonols, flavanones, flavanols, isoflavones and depsides, some of which extracted from Quercus ilex and Salvia miltiorrhiza. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting some important isoforms over the off-target ones hCA I and II.

Effects of Salvia miltiorrhiza on CNS Neuronal Injury and Degeneration: A Plausible Complementary Role of Tanshinones and Depsides.

Salvia miltiorrhiza is a very important herbal drug of traditional Chinese medicine. Bioactive constituents are represented by two main groups of secondary metabolites, the lipophilic diterpenic quinones known as tanshinones and the hydrophilic depsides known as salvianolic acids. S. miltiorrhiza extracts and single constituents have been shown to have positive effects in central nervous system neuronal injury and degeneration in several animal models by various biological mechanisms. Both tanshinones and depsides protect against ß-amyloid-induced toxicity, but their mechanisms are complementary due to their different structure, the lipophilic tanshinones and the hydrophilic depsides. A number of anti-inflammatory mechanisms is also reported for both tanshinones and depsides. Common mechanisms are the effects on cytokines, inducible nitric oxide synthase, and glial fibrillary acidic protein. In addition, depsides are inhibitors of nitric oxide and cyclooxygenase-2, while tanshinones inhibit hypoxia-inducible factor-1α and nuclear factor kappa ß. Both constituents can also modulate the protection of the central nervous system from oxidative stress with different but complementary mechanisms: tanshinones can enhance the activities of superoxide dismutase and glutathione peroxidase, while depsides can decrease reactive oxygen species.Furthermore, neuronal death underlies the symptoms of many human neurological disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, stroke, and amyotrophic lateral sclerosis. Both classes of constituents can enhance the antiapoptotic B-cell leukemia protein-2 family members and decrease the translocation of cytochrome c, and, in addition, depsides decrease caspase-3 and intracellular Ca(2+). Again, both classes of constituents have an activity on vascular endothelial growth factor but it is opposite, whereas tanshinones are inhibitors of acetylcholinesterase.Besides the extensive studies reporting on the biological mechanisms of depsides and tanshinones, pharmacokinetics studies are still very limited and not conclusive, especially for brain distribution. Further research is warranted to address the mechanisms of the multitarget actions of S. miltiorrhiza constituents and to translate this knowledge into clinical practice.

In Vitro Evidence for the Use of Astragali Radix Extracts as Adjuvant against Oxaliplatin-Induced Neurotoxicity.

The repeated exposure to the anticancer drug oxaliplatin induces a disabling, painful neuropathy. The current pharmacological treatments are unsatisfactory and unable to modify the complex nervous damage induced by the platin derivative. Recently, we described a system of cellular measures of oxidative stress as a method for studying features of oxaliplatin neurotoxicity and screening new compounds able to reduce oxaliplatin-induced neuropathy. Based on this experimental design, the protective properties of Astragali radix were studied comparing aqueous and two hydroalcoholic root extracts. Aqueous and the 20 % hydroalcoholic (20 % water) extract were prepared from plant material, while the 50 % hydroalcoholic (50 % water) extract was a commercial one. All of the extracts were characterized in terms of drug extract ratio and content of typical isoflavonoids, Astragaloside IV, and related saponins. Furthermore, the molecular weight of the polysaccharide fraction was evaluated by light scattering analysis. Oxaliplatin increased the superoxide anion production both in the neuronal-derived cell line SH-SY5Y and in primary cultures of rat cortical astrocytes. Aqueous and the 50 % hydroalcoholic extract (50 µg/mL) showed significant antioxidant effects. In astrocytes, aqueous and the 50 % hydroalcoholic extract showed protective effects against oxaliplatin-induced lipid peroxidation (malonyl dialdehyde levels), protein (carbonylated proteins), and DNA oxidation (8-OH-2-dG levels). The 50 % hydroalcoholic extract was the most active in preventing the activation of the apoptotic enzyme caspase-3 and it was the only able to stimulate astrocyte viability. None of the tested extracts interfered with the toxicity elicited by oxaliplatin in the human colon adenocarcinoma cell line HT-29. The pharmacological profile of Astragali radix extracts, in particular, the aqueous and 50 % hydroalcoholic extracts, makes these natural products candidates as therapeutic adjuvant agents against oxaliplatin neurotoxicity.

Antioxidant, anti-inflammatory potential and chemical constituents of Origanum dubium Boiss., growing wild in Cyprus.

Origanum dubium Boiss. is a flavouring herb widely used in Cyprus. In this study, both lipophilic and polar extracts of the aerial parts of O. dubium were investigated for their chemical contents and their antioxidant potential. Overall, 20 constituents were isolated and identified, belonging mainly to three significant classes of compounds: terpenes, phenolic derivatives, such as hydroquinone glycosides and flavonoids and alicyclic derivatives. None of them was previously reported as constituent of O. dubium The inhibitory potencies of all total extracts and the isolated compounds on lipid peroxidation and their interaction with 1,1-diphenyl-picrylhydrazyl (DPPH) activity is discussed. The polar extract showed strong interaction with DPPH stable radical and significant inhibition of lipoxygenase and lipid peroxidation.

Kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus.

The protein coded by the open-reading-frame 3a of SARS coronavirus has been demonstrated to form a cation-selective channel that may become expressed in the infected cell. The activity of the channel is involved in the mechanism of virus release. Drugs that inhibit the ion channel can, therefore, inhibit virus release, and they could be a source for development of novel therapeutic antiviral agents. Various drugs found in Chinese herbs that are well known as anticancer agents also have an antiviral potency. Here we tested the flavonols kaempferol, kaempferol glycosides, and acylated kaempferol glucoside derivatives with respect to their potency to block the 3a channel. We used the Xenopus oocyte with a heterologously expressed 3a protein as a model system to test the efficacy of the flavonols. Some of these drugs turned out to be potent inhibitors of the 3a channel. The most effective one was the glycoside juglanin (carrying an arabinose residue) with an IC50 value of 2.3 µM for inhibition of the 3a-mediated current. Kaempferol derivatives with rhamnose residue also seem to be quite effective. We suggest that viral ion channels, in general, may be a good target for the development of antiviral agents, and that, in particular, kaempferol glycosides are good candidates for 3a channel proteins of coronaviruses.

Rosmarinic Acid Production from Origanum dictamnus L. Root Liquid Cultures In Vitro.

In the present work Origanum dictamnus L. was studied as a suitable in vitro adventitious root culture system for the production of important bioactive molecules, such as rosmarinic acid (RA). Callus culture was initiated from leaf, petiole and root explants on solid MS medium supplemented with either 5 µM NAA + 5 µM kinetin (ODK3) or 5 µM NAA + 0.5 µM kinetin (ODK4). New roots formed from leaf, petiole and root calluses were aseptically transferred into Erlenmeyer flasks containing 100 mL liquid medium and shaken at 120 rpm in the dark. The liquid medium used was the MS supplemented either with 35 µM IBA + 2.5 µM kinetin (ODY1) or 5 µM NAA + 0.5 µM kinetin (ODY2). Biomass production parameters, RA content (%) and yield index (YI) were recorded for each treatment explant type, medium composition and incubation period. Results showed, in every case, the production of RA in vitro. Between the two liquid media (ODY1, ODY2) and the different culture periods, the ODY1 medium and the longest 200-day-culture period were more effective for RA and biomass production, regardless of the initial explant type used. The combination of ODK4-ODY1 resulted in higher RA (5.1% and 4.7%), fresh biomass production (19.0 g and 11.6 g), mean YI (93.7 mg and 51.4 mg) and YI per explant (3.75 mg and 2.06 mg) for roots derived from leaf calluses and root calluses, respectively. However, the solid ODK3 (200 days)-liquid ODY1 (40 days) transition treatment was more beneficial for roots derived from petiole calluses leading to an 18.8-fold increase in fresh biomass growth rate. RA accumulation and YIs were also significantly influenced by explant type, with the highest value produced from root petiole calluses (6.6% RA dry weight, 115.3 mg mean YI and 4.61 mg YI per explant) after 240 days.

Extensive analysis of the cultivated medicinal herbal drug Origanum dictamnus L. and antimicrobial activity of its constituents.

Phytochemical investigations of the methanol extract from Origanum dictamnus L. (Lamiaceae) resulted in the isolation of forty compounds belonging to the classes of terpenes, resorcinol derivatives, flavonoids, depsides, neolignans and jasmonates. Chromatographic isolations were targeted by using two analytical platforms, NMR and HPLC-PDA-MS. In parallel, HPLC-PDA-MS of individual fractions enabled the unambiguous identification of additionally eight components. In total 48 constituents were isolated/identified. Among the isolated constituents are four undescribed compounds, one resorcinol derivative, one monoterpene, one diterpene and one acylated flavonoid glycoside. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR, and HPLC-ESI-MS and HRMS experiments. Representative compounds were tested for their antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. I4-II7-dicarvacrol was the most potent constituent.

HPLC- and NMR-Based Chemical Profiling, Wound-Healing Potential, Anti-Inflammatory and Antibacterial Activities of Satureja pilosa (Lamiaceae), a Neglected Medicinal-Aromatic Herb.

Satureja pilosa Velen. (Lamiaceae) is a perennial and melliferous aromatic-medicinal subshrub which is range-restricted in adjacent parts of Greece and Bulgaria and locally in Italy, known in Northern Greece as wild oregano ("agriorigani") and traditionally collected from the wild for culinary purposes. Since the ethnopharmacological data and modern biological activities of Satureja spp. suggest promising applications in skin conditions, the present study aimed to investigate the hitherto unknown phenolic content of cultivated S. pilosa and its potential biological activities, focusing mainly on wound-healing and anti-inflammatory effects. An HPLC-PDA-MS-targeted phytochemical investigation, along with NMR, allowed for the isolation and characterization of the main constituents, resulting in 18 compounds. Representative extracts and purified compounds were tested for wound-healing activity on NIH/3T3 fibroblasts. The butanol extract exhibited a significantly higher cell migration rate (73.4%) compared to aqueous (50.6%) and methanolic (49.6%) ones, enhancing the cell migration more rapidly at both concentration levels, whilst rosmarinic acid was the most potent among the isolated compounds, with a migration rate of 64.0% at the concentration level of 10-5 mg/mL, followed by 3,4-dihydrophenyllactic acid (54.7%). Moreover, potential effects on endothelial activation processes were explored, including the leukocyte-endothelial cell interaction during inflammatory processes and the migratory capacity during angiogenic actions, since these processes are commonly associated with skin diseases. Finally, extracts and purified compounds demonstrated weak antibacterial potential against two important pathogens (Staphylococcus aureus and Pseudomonas aeruginosa), suggesting that further investigation is warrented.

Sesquiterpene lactones from Centaurea zuccariniana and their antimicrobial activity.

Twenty-nine compounds were isolated from the aerial parts of the Greek plant C. zuccariniana DC. The structures of the isolated compounds were established by means of NMR- ((1) H,(1) H-COSY, (1) H,(13) C-HSQC, HMBC, NOESY, and ROESY) and mass-spectral analyses. These compounds comprise 13 sesquiterpene lactones, 14 flavonoids, two lignans, and one simple lactone. Among the isolated sesquiterpene lactones, three are new, namely one heliangolide, (1E,4Z)-15-hydroxy-8α-O-(4'-acetoxy-3'-hydroxy-2'-methylidenebutanoyl)-6ßH,7αH-germacra-1,4,11(13)-trien-6,12-olide; and two eudesmanolides, 8α-(4',5'-diacetoxyangeloyl)sonchucarpolide and one unusual eudesmanolide with an oxygenated bridge linking C(1) and C(4), named zuccarinin. The main sesquiterpene lactones were malacitenolide, cnicin, and 4'-O-acetylcnicin. These results are in agreement with those obtained from the previously studied Greek Centaurea sp. belonging to the section Acrolophus (Cass.) DC.; this finding could be of chemotaxonomic significance for the genus Centaurea. The in vitro antimicrobial activities of the isolated new sesquiterpene lactones were against eight bacteria and eight fungal species. A 96-well microbioassay procedure for fast and easy evaluation of antibacterial and antifungal activities was applied to compare these compounds with commercial antibiotic and fungicide standards, and with previously isolated analogous sesquiterpene lactones tested by the same bioassay. All of the compounds tested showed moderate antibacterial, but significant antifungal activities; the present results corroborate with previous data, indicating that these types of compounds exhibit low or moderate antibacterial, but potent antifungal activities. The unusual eudesmanolide zuccarinin proved to be the most potent among the present tested sesquiterpene lactones, as well as among all previously tested eudesmanolides isolated from Greek Centaurea sp.

A Validated Method for the Determination of Carnosic Acid and Carnosol in the Fresh Foliage of Salvia rosmarinus and Salvia officinalis from Greece

In the framework of a project aiming at identifying genotypes of Greek rosemary and sage producing high amounts of carnosic acid, an HPLC-PDA method was developed for the determination of the main antioxidant in the fresh leaves. To this end, an effective and repeatable extraction process of the labile diterpene was developed to ensure a good extraction yield. A fast RP-HPLC protocol was developed and optimized to allow for a short and reliable analysis of the unstable target constituent. The HPLC-PDA method was validated for precision and accuracy according to ICH guidelines. Finally, the overall method was validated for precision and accuracy at three concentration levels. The precision was acceptable with % RSD values ranging between 1.42 and 4.35. The recovery ranged between 85.1% and 104.6% with RSD values < 5%, within the acceptable limits. The developed assay was fast and simple and allowed for the fast and accurate determination of carnosic acid and carnosol in the fresh herbs. The methodology was applied to the quantitative analysis of several cultivated samples of S. rosmarinus and S. officinalis, and some of them were revealed to be promising starting materials for the development of Greek genotypes rich in carnosic acid.