Anti-Inflammatory and Cytotoxic Activities of Clerodane-Type Diterpenes.

The secondary metabolites of clerodane diterpenoids have been found in several plant species from various families and in other organisms. In this review, we included articles on clerodanes and neo-clerodanes with cytotoxic or anti-inflammatory activity from 2015 to February 2023. A search was conducted in the following databases: PubMed, Google Scholar and Science Direct, using the keywords clerodanes or neo-clerodanes with cytotoxicity or anti-inflammatory activity. In this work, we present studies on these diterpenes with anti-inflammatory effects from 18 species belonging to 7 families and those with cytotoxic activity from 25 species belonging to 9 families. These plants are mostly from the Lamiaceae, Salicaceae, Menispermaceae and Euphorbiaceae families. In summary, clerodane diterpenes have activity against different cell cancer lines. Specific antiproliferative mechanisms related to the wide range of clerodanes known today have been described, since many of these compounds have been identified, some of which we barely know their properties. It is very possible that there are even more compounds than those described today, in such a way that makes it an open field to discover. Furthermore, some diterpenes presented in this review have already-known therapeutic targets, and therefore, their potential adverse effects can be predicted in some way.

Metabolism Characterization and Chemical and Plasma Stability of Casearin B and Caseargrewiin F.

Oral preparations of Casearia sylvestris (guacatonga) are used as antacid, analgesic, anti-inflammatory, and antiulcerogenic medicines. The clerodane diterpenes casearin B and caseargrewiin F are major active compounds in vitro and in vivo. The oral bioavailability and metabolism of casearin B and caseargrewiin F were not previously investigated. We aimed to assess the stability of casearin B and caseargrewiin F in physiological conditions and their metabolism in human liver microsomes. The compounds were identified by UHPLC-QTOF-MS/MS and quantified by validated LC-MS methods. The stability of casearin B and caseargrewiin F in physiological conditions was assessed in vitro. Both diterpenes showed a fast degradation (p < 0.05) in simulated gastric fluid. Their metabolism was not mediated by cytochrome P-450 enzymes, but the depletion was inhibited by the esterase inhibitor NaF. Both diterpenes and their dialdehydes showed a octanol/water partition coefficient in the range of 3.6 to 4.0, suggesting high permeability. Metabolism kinetic data were fitted to the Michaelis-Menten profile with KM values of 61.4 and 66.4 µM and Vmax values of 327 and 648 nmol/min/mg of protein for casearin B and caseargrewiin F, respectively. Metabolism parameters in human liver microsomes were extrapolated to predict human hepatic clearance, and suggest that caseargrewiin F and casearin B have a high hepatic extraction ratio. In conclusion, our data suggest that caseargrewiin F and casearin B present low oral bioavailability due to extensive gastric degradation and high hepatic extraction.

Antileishmanial activity of neo-clerodane diterpenes from Croton echioides.

Nowadays, new leishmanicidal drugs are needed and natural products arise as a promising alternative source. Therefore, bioguided fractionation of a hydroethanolic extract from the stem bark of Croton echioides Baill. were conducted based on its antileishmanial activity. Two novel neo-clerodane diterpenoids methyl-15,16-epoxy-3,13(16),14-neo-clerodatrien-17,18-dicarboxylate (1) and dimethyl-3-oxo-15,16-epoxy-13(16),14-neo-clerodadien-17,18-dicarboxylate (2) were isolated, as well as four known compounds (3-6) and lupeol, from the hexane fraction. Their structures were established by NMR analysis. The crude extract, fractions and the compounds (1 and 3-6) were evaluated for their in vitro antileishmanial activity and cytotoxicity against macrophages J774A.1. The selectivity index (SI) were calculated. The most active compound against promastigote forms of L. amazonensis was the clerodane diterpene 4, with IC50 values of 8.3 µM and SI value of 80.9. Our results highlighted stem bark of Croton echioides Baill. as a promising source for the development of a new chemotherapeutic agent to combat leishmaniasis.

A Combined Molecular Docking and Density Functional Theory Nuclear Magnetic Resonance Study of Trans-Dehydrocrotonin Interacting with COVID-19 Main Protease and Severe Acute Respiratory Syndrome Coronavirus 2 3C-Like Protease.

For the development of drugs that treat SARS-CoV-2, the fastest way is to find potential molecules from drugs already on the market. Unfortunately, there is currently no specific drug or treatment for COVID-19. Among all structural proteins in SARS-CoV, the spike protein is the main antigenic component responsible for inducing host immune responses, neutralizing antibodies, and/or protecting immunity against virus infection. Molecular docking is a technique used to predict whether a molecule will bind to another. It is usually a protein to another or a protein to a binding compound. Natural products are potential binders in several studies involving coronavirus. The structure of the ligand plays a fundamental role in its biological properties. The nuclear magnetic resonance technique is one of the most powerful tools for the structural determination of ligands from the origin of natural products. Nowadays, molecular modeling is an important accessory tool to experimentally got nuclear magnetic resonance data. In the present work, molecular docking studies aimed is to investigate the limiting affinities of trans-dehydrocrotonin molecule and to identify the main amino acid residues that could play a fundamental role in their mechanism of action of the SARS-CoV spike protein. Another aim of this work is all about to evaluate 10 hybrid functionalities, along with three base pairs using computational programs to discover which ones are more reliable with the experimental result the best computational method to study organic compounds. We compared the results between the mean absolute deviation (MAD) and root-mean-square deviation (RMSD) of the molecules, and the smallest number between them was the best result. The positions assumed by the ligands in the active site of the spike glycoprotein allow assuming associations with different local amino acids.

Infraspecific Chemical Variability and Biological Activity of Casearia sylvestris from Different Brazilian Biomes.

Casearia sylvestris is an outstanding representative of the Casearia genus. This representability comes from its distinctive chemical profile and pharmacological properties. This species is widespread from North to South America, occurring in all Brazilian biomes. Based on their morphology, 2 varieties are recognized: C. sylvestris var. sylvestris and C. sylvestris var. lingua. Despite the existence of data about their chemical composition, a deeper understanding of the specialized metabolism correlation and variation in respect to environmental factors and its repercussion over their biological activities was still pending. In this study, an UHPLC-DAD-based metabolomics approach was employed for the investigation of the chemical variation of 12 C. sylvestris populations sampled across 4 Brazilian biomes and ecotones. The correlation between infraspecific chemical variability and the cytotoxic and antioxidant activities was achieved by multivariate data analysis. The analyses showed that C. sylvestris var. lingua prevailed at Cerrado areas, and it was correlated with lower cytotoxic activity and high level of glycosylated flavonoids. Among them, narcissin and isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside showed good correlation with the antioxidant activity. Conversely, C. sylvestris var. sylvestris prevailed at the Atlantic Forest areas, and it was associated with high cytotoxic activity and high content of clerodane diterpenoids. Different casearins showed good correlation (R2 = 0.3 - 0.70) with the cytotoxic activity. These findings highlighted the great complexity among different C. sylvestris populations, their chemical profile, and the related biological activities. Consequently, it can certainly influence the medicinal properties, as well as the quality and efficacy, of C. sylvestris phytomedicines.

Hydroxy-neo-Clerodanes and 5,10-seco-neo-Clerodanes from Salvia decora.

Preliminary analysis of the mass spectrometric (MS) and NMR spectroscopic data of the primary fractions from the biologically active extract of Salvia decora revealed spectra that are characteristic for neo-clerodane-type diterpenoids. MS-guided isolation of the bioactive fractions led to the isolation of three new chemical entities, including two hydroxy-neo-clerodanes (1 and 2) and one acylated 5,10-seco-neo-clerodane (3), along with three known diterpenoids (4-6), ursolic acid (7), and eupatorin (8). The structures of the new compounds were established by analysis of the 1D and 2D NMR and MS data, whereas their absolute configuration was deduced using a combination of experimental and theoretical ECD data and confirmed by X-ray crystallography (1 and 4). Furthermore, compounds 1, 3, 4, and 6-8 were evaluated as hPTP1B1-400 (human protein tyrosine phosphatase) inhibitors, where 7 showed the best activity, with an IC50 value in the lower µM range. Additionally, compound 7 was evaluated as an α-glucosidase inhibitor. The affinity constant of the 7-hPTP1B1-400 complex was determined by quenching fluorescence experiments (ka = 1.3 × 104 M-1), while the stoichiometry ratio (1:1 protein-ligand) was determined by a continuous variation method.

Fragmentation study of clerodane diterpenes from Casearia species by tandem mass spectrometry (quadrupole time-of-flight and ion trap).

RATIONALE: Clerodane-type diterpenes from Casearia species show important pharmacological activites such as antitumor, antimicrobial and anti-inflamatory. There are several mass spectrometry (MS)-based methods for identification of diterpenes; however, there is still a lack of MS procedures capable of providing characteristic fragmentation pathways for a rapid and unambiguous elucidation of casearin-like compounds. METHODS: Casearin-like compounds were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The fragmentation studies were carried out by tandem mass spectrometry in space (quadrupole time-of-flight (QTOF)) using different collision energies and also by tandem mass spectrometry in time (QIT) by selective isolation of product ions. RESULTS: Casearin-like compounds presented a predominance of sodium- and potassium-cationized precursor ions. Both QIT and QTOF techniques provided sequential neutral losses of esters related to the R1 to R5 substituents linked to the nucleus of the clerodane diterpenes. The fragmentation pathway is initiated with a cleavage of the ester moieties R2 followed by the elimination of the ester groups R3 , both losing neutral carboxylic acids. Using QIT, it was also possible to observe the cleavage of the ester groups R1 or R5 by MS4 experiments. CONCLUSIONS: Through a rational analysis of the fragmentation mechanisms of Casearia diterpenes it was possible to suggest an annotation strategy based on the sequential cleavages of the ester groups related to the R2 , R3 and R5 substituents. These results will assist studies of the dereplication and metabolomics involving casearin-like compounds present in complex extracts of Casearia species.

Diterpenoids with inhibitory activity of nitrite production from Croton floribundus.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton floribundus Spreng. (Euphorbiaceae), popularly known as Capixinguí, stands out due to its widespread use in traditional medicine to treat wounds, syphilis, hemorrhoids, eye diseases and as a purgative. AIM OF THE STUDY: To characterize clerodanes diterpenes from C. floribundus and to evaluate the effects of the fraction and diterpenes (1-5) on inhibition of nitrite production. MATERIALS AND METHODS: The hydroethanolic root extract of C. floribundus was fractionated on a solid phase extraction column to obtain the fraction named Fr80%. From this, five compounds were obtained and characterized. The absolute configuration of compound 1 was determined by a combination of electronic and vibrational circular dichroism spectroscopies. Additionally, compounds 1-5 were evaluated for their inhibitory effects on nitrite production induced by lipopolysaccharide (LPS) in RAW 264 macrophage cell. RESULTS: Five clerodane diterpenoids were characterized, and the absolute stereochemistry of 1 was established as 3R,4R,5R,8R,9R,10S,12S. The IC50 values obtained through inhibition of nitrite production were 28.52 ±â€¯2.21 µM (1), 40.26 ±â€¯2.79 µM (2), 25.47 ±â€¯2.16 µM (3), 35.78 ±â€¯2.93 µM (4) and 40.58 ±â€¯4.78 µM (5). In the tested concentrations, the samples presented low toxicity in macrophages. CONCLUSIONS: Four new diterpenes were characterized from C. floribundus, these being croflorins A-D (1-4) and a known halimane (5). These compounds exhibited inhibitory effect on nitrite production.

Toxicological findings about an anticancer fraction with casearins described by traditional and alternative techniques as support to the Brazilian Unified Health System (SUS).

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts, essential oils and molecules from Casearia sylvestris have popularly shown pharmacological actions against chronic diseases, as anxiety, inflammation, cancer and dyslipidemia. In the context of antitumoral therapy, we investigated in vitro, ex vivo and in vivo toxicological changes induced by a Fraction with Casearins (FC) and its component Casearin X isolated from C. sylvestris on animal and vegetal cells, and upon invertebrates and mammals. MATERIAL AND METHODS: Cytotoxicity was carried out using normal lines and absorbance and flow cytometry techniques, Artemia salina nauplii, Danio rerio embryos and meristematic cells from Allium cepa roots. Acute and 30 days-mice analysis were done by behavioral, hematological and histological investigations and DNA/chromosomal damages detected by alkaline Cometa and micronucleus assays. RESULTS: FC was cytotoxic against lung and fibroblasts cells and caused DNA breaks, loss of integrity and mitochondrial depolarization on ex vivo human leukocytes. It revealed 24 h-LC50 values of 48.8 and 36.7 µg/mL on A. salina nauplii and D. rerio embryos, reduced mitotic index of A. cepa roots, leading to cell cycle arrest at metaphase and anaphase and micronuclei. FC showed i.p. and oral LD50 values of 80.9 and 267.1 mg/kg body weight. Subacute i.p. injections induced loss of weight, swelling of hepatocytes and tubules, tubular and glomerular hemorrhage, microvesicular steatosis, lung inflammatory infiltration, augment of GPT, decrease of albumin, alkaline phosphatase, glucose, erythrocytes, and lymphocytes, and neutrophilia (p > 0.05). FC-treated animals at 10 mg/kg/day i.p. caused micronuclei in bone marrow and DNA strand breaks in peripheral leukocytes. CONCLUSIONS: This research postulated suggestive side effects after use of FC-related drugs, demonstrating FC as antiproliferative and genotoxic on mammal and meristematic cells, including human leukocytes, teratogenicity upon zebrafish embryos, myelosuppression, clastogenicity, and morphological and biochemical markers indicating liver as main target for FC-induced systemic toxicity.

Acidic and hepatic derivatives of bioactive clerodane diterpenes casearins J and O.

Clerodane diterpenes from Casearia sylvestris are antiulcerogenic and anti-inflammatory. The finding that they may undergo acid degradation or hepatic metabolization led to an investigation of their degradation products. Purified clerodane diterpenes (casearins J and O) were subjected to in vitro assays to simulate their oral administration. Resulting derivatives were identified using chromatographic and spectrometric techniques. Nitric oxide synthesis by LPS-stimulated macrophages was assayed to verify whether structural modifications alter the anti-inflammatory activity of diterpenes. Nine compounds (1-9) were identified after acid degradation remaining 5.05% of casearin J. Besides the remaining casearin O (13.1%), eight compounds (10-17) were identified. The dialdehydes from each casearin were the major constituents. S9 rat liver treatment of casearins J and O generated two compounds identical to some of those produced by acid degradation, which remained 36.8% and 36.5% intact, respectively. Both casearins and its derivatives were not cytotoxicity at concentrations lower than 0.312 µg/mL (0.555 µM for casearin J and 0.516 µM for casearin O) and did not inhibit the nitric oxide production in this concentration. Thus, the structural modifications conducted did not alter the activity of casearins and the anti-inflammatory pathway of diterpenes probably is not involved on nitric oxide modulation.

Clerodane Diterpenes from the Marine Sponge Raspailia bouryesnaultae Collected in South Brazil.

The marine sponge Raspailia bouryesnaultae, collected in South Brazil, was selected for detailed investigation considering the results of a screening that pointed to an in vitro antiproliferative effect against non-small cells of human lung cancer (A549) and anti-herpes activity against Herpes Simplex virus type 1 (KOS and 29R strains) of ethanolic extracts. The fractionation and chemical investigation of the sponge's hexanic fraction led to the isolation and structural elucidation of six clerodane diterpenes. The main component was identified as the already-reported raspailol (1), isolated from a sponge of the same genus collected in New Zealand. The structure of a new diterpene (2) with a rearranged skeleton was established by high-resolution mass spectrometry (HRMS) and 1D and 2D Nuclear magnetic resonance spectroscopy (NMR) experiments, and named here as raspadiene. Furthermore, four diterpenes were elucidated as isomers of clerodane diterpenes previously obtained from plants, namely kerlinic acid (3), kerlinic acid methyl ester (4), annonene (5), and 6-hydroxyannonene (6). They differ in their stereochemistry, since these diterpenes are characterized by a trans ring fusion at the decalin moiety and the relative configuration of the two methyl groups at C-8 and C-9 in a cis relationship (type trans/cis). The Raspailia diterpenes have a cis ring fusion at the decalin moiety, and the two methyl groups at C-8 and C-9 are in a trans relationship (type cis/trans). The isolated compounds were evaluated for their potential antiproliferative effects on human cancer cell line A549, and it was observed that the diterpenes bearing a hydroxyl group at C-6 exhibited moderate cytotoxic activity, with 50% inhibitory concentration (IC50) values lower than 25 µM. The evaluation of the potential anti-herpes activity against Herpes Simplex Virus type 1 (HSV-1, KOS and 29R strains) showed that the more promising results were observed for the new compound 2, since it inhibited HSV-1 (KOS and 29R strains) replication by 83% and 74%, respectively.

Characterization of Casearin X Metabolism by Rat and Human Liver Microsomes.

Casearin X (CAS X) is the major clerodane diterpene isolated from the leaves of Casearia sylvestris and has been extensively studied due to its powerful cytotoxic activity at low concentrations. Promising results for in vivo antitumor action have also been described when CAS X was administered intraperitoneally in mice. Conversely, loss of activity was observed when orally administered. Since the advancement of natural products as drug candidates requires satisfactory bioavailability for their pharmacological effect, this work aimed to characterize the CAS X metabolism by employing an in vitro microsomal model for the prediction of preclinical pharmacokinetic data. Rat and human liver microsomes were used to assess species differences. A high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for the quantification of CAS X in microsomes was developed and validated according to European Medicines Agency guidelines. CAS X was demonstrated to be a substrate for carboxylesterases via hydrolysis reaction, with a Michaelis-Menten kinetic profile. The enzyme kinetic parameters were determined, and the intrinsic clearance was 1.7-fold higher in humans than in rats. The hepatic clearance was estimated by in vitro-in vivo extrapolation, resulting in more than 90% of the hepatic blood flow for both species. A qualitative study was also carried out for the metabolite identification by mass spectrometry and indicated the formation of the inactive metabolite CAS X dialdehyde. These findings demonstrate that CAS X is susceptible to first-pass metabolism and is a substrate for specific carboxylesterases expressed in liver, which may contribute to a reduction in antitumor activity when administered by the oral route.

New diterpenes from Salvia pseudorosmarinus and their activity as inhibitors of monoacylglycerol lipase (MAGL).

As a part of our ongoing research program on compounds from higher plants with lactate dehydrogenase (LDH) and monoacylglycerol lipase (MAGL) inhibitory activities, three new neoclerodane diterpene 12-deacetylsplendidin C (1), pseudorosmaricin (2), and 2-dehydroxysalvileucanthsin A (3) along with six known compounds were isolated from Salvia pseudorosmarinus aerial part extracts. Their structures were determined by spectroscopic and spectrometric techniques including 1D- and 2D NMR, and MS analyses. The isolated diterpenes were assayed for their inhibitory activity on LDH5 and MAGL, two enzymes covering key roles in the peculiar energetic metabolism of malignant tumours. All the assayed diterpenes showed negligible activity on LDH5, whereas the known jewenol A (4) displayed a moderate inhibition activity on MAGL, showing an IC50 value of 46.8µM and it proved to be a reversible MAGL inhibitor. Docking and molecular dynamic simulation studies where thus performed to evaluate the binding mode of 4 within MAGL.

Gastroprotective Activity of Parastrephia quadrangularis (Meyen), Cabrera from the Atacama Desert.

Forty-three metabolites including several methoxylated flavonoids, tremetones, and ent-clerodane diterpenes were accurately identified for the first time in the ethanolic extract of P. quadrangularis by means of hyphenated UHPLC-quadrupole Orbitrap mass spectrometry, and seven isolated compounds were tested regarding gastroprotective activity using the HCl/EtOH-induced lesion model in mice. A new tremetone (compound 6) is reported based on spectroscopic evidence. The isolated clerodanes and tremetones showed gastroprotective activity in a mouse model, evidenced by compound 7 (p-coumaroyloxytremetone), which showed the highest gastroprotective activity (76%), which was higher than the control drug lansoprazole (72%). Our findings revealed that several constituents of this plant have gastroprotective activity, and particularly, p-coumaroyloxytremetone could be considered as a lead molecule to explore new gastroprotective agents. This plant is a rich source of biologically active tremetones and terpenoids which can support the ethnobotanical use of the plant.

Antinociceptive effect of neo-clerodane diterpenes obtained from Baccharis flabellata.

We report here for the first time antinociceptive effects of extracts from Baccharis flabellata. Two extracts in this analysis, one obtained in summer and the other during winter time. Our results indicate that both extract show strong antinociceptive effects, being the extracts obtained during the summer significantly more active. Our results suggest that this activity is mainly due to the presence of the diene-acid clerodane ent-15,16-epoxy-19-hydroxy-1,3,13(16),14-clerodatetraen-18-oic acid (DAC) and its dimer called DACD. Employing naloxone as an antagonist of opioid receptors, we demonstrated that both compounds act on opioid receptors, being the antinociceptive effect of DACD stronger than DAC. Thus, the antinociceptive activity of DACD was almost two times stronger than DAC (44.8 over 24.6 s in the hot-plate test) after one hour of treatments. In order to better understand the mechanism of action at molecular level of these compounds, we conducted a molecular modeling study analyzing the molecular interactions of DAC and DACD complexes with the κ-ORs. Our results suggest interactions for both DAC and DACD with Gln115, Val118, Tyr119, Asn122 and Tyr313 stabilizing their complexes; however, these interactions are significantly stronger for DACD with respect to DAC. This finding could explain why DACD have a higher affinity for the κ-ORs. These results are in agreement with the obtained antinociceptive effect. In addition, our results indicate that these neoclerodanes would have a mechanism of action similar to that of salvinorin A; such information can be very useful for the design of new inhibitors of κ-ORs.

In vivo photoinduced [4 + 2] dimerization of a neo-clerodane diterpene in Baccharis flabellata. ROS and RNS scavenging abilities.

Secondary metabolites play a major role in the adaptation of plants to the environment. Furan neo-clerodane diterpenes are characteristic secondary metabolites in Baccharis flabellata Hook. & Arn. var. flabellata. One of the main compounds is the diene ent-15,16-epoxy-19-hydroxy-1,3,13(16),14-clerodatetraen-18-oic acid (DAC). In this work a new dimeric compound (DACD) has been isolated and identified by NMR and MS techniques. The presence of other minor dimers was also observed in the same plant methanolic extracts. Assuming that they may be the products of [4 + 2] condensation of two monomeric moieties, the formation of adducts by photochemical dimerization was checked by inducing the in vitro [4 + 2] cycloaddition of DAC. Moreover, the DAC and DACD accumulation rates in aerial parts of B. flabellata specimens were analyzed monthly during a complete phenological cycle. The accumulation of monomer depends on the plant phonological stage; meanwhile the dimer proportion arises in detriment of the monomer as the solar UV radiation increases. Since plants exposed to strong UV intensities produce radical species, the scavenger properties of these compounds toward reactive nitrogen species (RNS), and reactive oxygen species (ROS), were analyzed. Albeit DAC and DACD show significant superoxide radical scavenger activities, the monomer proved to be more effective than the dimer toward ROS, while DACD was an excellent RNS scavenger.

Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway.

This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 µM), or norbinaltorphimine (norBIN, 1 µM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

Chemical variability between different organs of the medicinal plant Casearia sylvestris

The phytochemical profile of essential oils and extracts from Casearia sylvestris leaves, flowers and fruits have been investigated here. Leaf and flower extracts were prepared by sonication and analyzed by thin-layer chromatography and high-performance liquid chromatography. The phenolic content was determined by ultraviolet spectrophotometry. Leaves, flowers, and fruits essential oils were extracted by hydrodistillation. The highest extracts yields were 20.3 % (leaves) and 23.4 % (flowers) with ethanol 70 %. Essential oil extraction yields were 0.3 % (leaves) and 0.1 % (flowers and fruits). Bicyclogermacrene was the major component in all essential oil. Thin-layer chromatography suggests a chemical profile similar for leaves and flowers. The leaves and flowers phenolic content were similar (14.0 and 15.0 %, respectively). Chromatography analyses indicated the predominance of casearin clerodane diterpenes in leaves (λmax 232-235), whereas in flowers, diterpenes with a different standard diene in side-chain C13(16) and C14 (λmax 223-229). The different phytochemical profile of C. sylvestris flowers as compared to the leaves could be explored by the search for new bioactive components. This is the first report on the fruit and flower C. sylvestris essential oil composition. These data could be used as quality control of herbal medicine derived from C. sylvestris leaves.(AU)

Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway

This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 μM), or norbinaltorphimine (norBIN, 1 μM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

neo-Clerodane Diterpenoids from Salvia polystachya Stimulate the Expression of Extracellular Matrix Components in Human Dermal Fibroblasts.

Eleven neo-clerodane diterpenoids (1-11) including the new analogues 1, 2, and 10, and 3',5,6,7-tetrahydroxy-4'-methoxyflavone (12) were isolated from the aerial parts of Salvia polystachya. Polystachyne G (1) and 15-epi-polystachyne G (2) were isolated as an epimeric mixture, containing a 5-hydroxyfuran-2(5H)-one unit in the side chain at C-12 of the neo-clerodane framework. Polystachyne H (10) contains a 1(10),2-diene moiety and a tertiary C-4 hydroxy group. The structures of these compounds were established by analysis of their NMR spectroscopic and MS spectrometric data. The absolute configurations of compounds 3, 4, and 10 were determined through single-crystal X-ray diffraction analysis. The antibacterial, antifungal, and phytotoxic activities of the diterpenoids were determined. In addition, the stimulatory effect of the expression of extracellular matrix components of nine of the isolates (1-8 and 11) was assayed. Compounds 1-4, 8, and 11 increased the expression of the genes codifying for type I, type III, and type V collagens and for elastin.