Three neo-Clerodane Diterpenoids from Tinospora cordifolia Stems and Their Arginase Inhibitory Activities.

Three neo-clerodane diterpenoids, including two new tinocordifoliols A (1) and B (2) and one known tinopanoid R (3), were isolated from the ethyl acetate-soluble fraction of the 70% ethanol extract of Tinospora cordifolia stems. The structures were elucidated by various spectroscopic methods, including one dimensional (1D) and 2D-NMR, high resolution-electrospray ionization (HR-ESI)-MS, and electronic circular dichroism (ECD) data. The T. cordifolia extract and all isolated compounds 1-3 possessed arginase I inhibitory activities. Among them, 3 exhibited moderate competitive inhibition of human arginase I (IC50 = 61.9 µM). Furthermore, docking studies revealed that the presence of a ß-substituted furan in 3 may play a key role in the arginase I inhibitory activities.

New Clerodane Diterpenoids from Tinospora Capillipes with Antibacterial and Anti-Inflammatory Properties.

Four new clerodane diterpenoids, namely tinocapills A-D (1-4), and one known analogue (5) were isolated from the roots of Tinospora capillipes in the present study. The structures of these new compounds, including their absolute configurations, were determined through a combination of detailed spectroscopic analysis and theoretical statistical approaches, including electronic circular dichroism (ECD) analyses and quantum mechanical (QM)-NMR methods. Additionally, the stereostructure of 5 was confirmed via X-ray diffraction analysis. Furthermore, all these isolates were evaluated for their antibacterial and anti-inflammatory activities. Compounds 1, 2 and 5 demonstrated antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with MICs ranging from 4-64 µg/mL, and compounds 3 and 4 exhibited potential anti-inflammatory effects by suppressing LPS-induced TNF-α and NO releases in RAW264.7 cells.

Callicarpnoids A-C, structurally intriguing ent-Clerodane diterpenoid dimers with cytotoxicity against MCF-7 and HCT-116 cell lines from Callicarpa arborea Roxb.

Callicarpnoids A-C (1-3), three new ent-clerodane diterpenoid dimers formed via a [4 + 2] hetero Diels-Alder cycloaddition, appeared as a third example of this type of dimers, were isolated from the stems of Callicarpa arborea Roxb.. Their structures were elucidated by comprehensive spectroscopic analysis, and the absolute configurations were confirmed by single-crystal X-ray diffraction and electronic circular dichroism (ECD) calculations, as well as DP4 + analysis. Cytotoxicity test in two cell lines indicated that compounds 2 and 3 had significant cytotoxic effect against breast cancer cell (MCF-7) and colorectal cancer cell (HCT-116) with IC50 ranging from 5.2 to 7.2 µM, comparable to those of the positive control. Furthermore, the western blot analysis revealed that the protein expression levels of Bax were increased following compounds 2 and 3 treatment, whereas the expression levels of caspase 8, caspase 3, caspase 9 and Bcl2 were decreased in a dose-dependent manner, indicating that compounds 2 and 3 may induce apoptosis via both intrinsic and extrinsic pathways in MCF-7 and HCT-116 cells.

The discovery of potentially active diterpenoids to inhibit the pyroptosis from Callicarpa arborea.

Pyroptosis is a programmed-inflammatory cell death, which leads to release of inflammatory cellular contents and formation of inflammation. Uncontrollable pyroptosis can result in serious immune diseases, such as cytokine release syndrome (CRS), sepsis, disseminated intravascular coagulation (DIC), and acute organ damage, including acute respiratory distress syndrome (ARDS) and acute kidney injury (AKI). Members of the Callicarpa genus are significant raw materials for traditional Chinese medicine, widely used for analgesia, hemostasis, and anti-inflammation. Previously, we have reported some ent-clerodane diterpenoids from Callicarpa arborea, shown potent inhibitory effects against pyroptosis. In this study, we went on investigating this kind of diterpenoids, and yielded 66 ent-clerodane diterpenoids, including 52 new compounds, from Callicarpa arborea. Their structures featured with a 5/6- (1-25) or a 6/6- (26-66)-fused double-ring scaffolds, were elucidated using spectroscopic data, electrostatic circular dichroism (ECD) and X-ray diffraction analyses. Screening for the inhibitory activity against pyroptosis by detecting of IL-1ß secretion in J771A.1 cells, revealed 28 compounds with an IC50 below 10.5 µM. Compound 1 was the most potent with an IC50 of 0.68 µM and inhibited the J774A.1 macrophage pyroptosis by blocking the NLR pyrin domain containing 3 (NLRP3) inflammasome activation. An in vivo study further revealed that compound 1 decreased infiltration of CD11b + F4/80 + macrophages into lung and attenuated the lipopolysaccharide (LPS)-induced lung injury. Taken together, this study indicated the potential of compound 1 as a candidate for pyroptosis-related inflammation treatment, as well as provided the chemical and pharmacological basis for the further development of Callicarpa genus as a herbal medicine.

Neo-clerodane Diterpenoids with Hypoglycemic Effects in Vivo from the Aerial Parts of Salvia hispanica L.

A new neo-clerodane diterpenoid, salvihispin H (1), and six known ones (2-7) were identified from the aerial parts of Salvia hispanica L. The structure and absolute configuration of 1 were elucidated by extensive analysis of spectroscopic (1 H, 13 C, and 2D NMR, and HR-ESI-MS) and single-crystal X-ray diffraction data. The anti-diabetic effects of salvihispin H (1) and salvifaricin (2) were evaluated in diabetic db/db mice. The data showed that 1 and 2 could significantly reduce fasting blood glucose level and improve insulin resistance, and compound 1 exerted glucose-lowering effect more quickly than metformin. In addition, 1 and 2 could also reduce serum TG level in db/db mice. These results demonstrated that compounds 1 and 2 could be considered as potent candidates for the therapy of type 2 diabetes mellitus (T2DM).

Clerodane Diterpenoids from Callicarpa hypoleucophylla and Their Anti-Inflammatory Activity.

Plants of the genus Callicarpa are known to possess several medicinal effects. The constituents of the Taiwan endemic plant Callicarpa hypoleucophylla have never been studied. Therefore, C. hypoleucophylla was selected for our phytochemical investigation. Two new clerodane-type diterpenoids, named callihypolins A (1) and B (2), along with seven known compounds were isolated from the leaves and twigs of the Lamiaceae plant C. hypoleucophylla and then characterized. The structures of compounds 1 and 2 were elucidated by spectroscopic data analysis, specifically, two-dimension nuclear magnetic resonance (NMR). The anti-inflammatory activity of compounds 1-9 based on the suppression of superoxide anion generation and elastase release was evaluated. Among the isolates, compounds 2-4 showed anti-inflammatory activity (9.52-32.48% inhibition at the concentration 10 µm) by suppressing superoxide anion generation and elastase release. Our findings not only expand the description of the structural diversity of the compounds present in plants of the genus Callicarpa but also highlight the possibility of developing anti-inflammatory agents from Callicarpa endemic species.

A (-)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum.

Salvia divinorum (Lamiaceae) is an annual herb used by indigenous cultures of Mexico for medicinal and ritual purposes. The biosynthesis of salvinorin A, its major bioactive neo-clerodane diterpenoid, remains virtually unknown. This investigation aimed to identify the enzyme that catalyzes the first reaction of salvinorin A biosynthesis, the formation of (-)-kolavenyl diphosphate [(-)-KPP], which is subsequently dephosphorylated to afford (-)-kolavenol. Peltate glandular trichomes were identified as the major and perhaps exclusive site of salvinorin accumulation in S. divinorum. The trichome-specific transcriptome was used to identify candidate diterpene synthases (diTPSs). In vitro and in planta characterization of a class II diTPS designated as SdKPS confirmed its activity as (-)-KPP synthase and its involvement in salvinorin A biosynthesis. Mutation of a phenylalanine into histidine in the active site of SdKPS completely converts the product from (-)-KPP into ent-copalyl diphosphate. Structural elements were identified that mediate the natural formation of the neo-clerodane backbone by this enzyme and suggest how SdKPS and other diTPSs may have evolved from ent-copalyl diphosphate synthase.

In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes.

1. The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes. 2. Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway. 3. Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH. 4. Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by ß-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite. 5. This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.

New cytotoxic neo-clerodane diterpenoids from Scutellaria strigillosa.

Three new neo-clerodane diterpenoids, named scutestrigillosins A-C (1-3), were isolated from the whole plant of Scutellaria strigillosa. Their chemical structures including absolute configurations were established on the basis of detailed physical data analyses. In vitro, the isolated three new compounds exhibited significant cytotoxic activities against four tumor cell lines (HONE-1, P-388, MCF7 and HT29), and gave IC50 values in the range 3.5-7.7µM.

New neo-clerodane diterpenoids from Scutellaria strigillosa with cytotoxic activities.

Two new neo-clerodane diterpenoids, named scutestrigillosins D and E (1 and 2), were isolated from the whole plant Scutellaria strigillosa. Their structures were established on the basis of detailed physical data analyses. In vitro, two new compounds exhibited cytotoxic activities against four tumor cell lines (HONE-1, P-388, MCF7, and HT29), and gave IC50 values in the range of 3.4-8.9 µΜ.

[neo-Clerodane diterpenoids from Scutellaria galericulata].

By means of preparative HPTLC and column chromatography over silica gel and Sephadex LH-20, fourteen diterpenoids were isolated and purified from the whole plants of Scutellaria galericulata. Based on the physico-chemical properties and spectral data, their structures were elucidated and identified as：scutebarbatine D(1), scutolide A(2), scutolide K(3), scutebata J(4), scutebata I(5), 6-O-acetylscutehenanine A(6), barbatin C(7), scutolide E(8), barbatine C(9), scutebarbatine Y(10), scutebarbatine B(11), scutestrigillosin A(12), scutebata O(13), scutolide B(14). Compounds 1-14 were isolated from S. galericulata for the first time.

New clerodane diterpenoids from the twigs and leaves of Croton euryphyllus.

Three new nor-clerodane diterpenoids, crotoeurins A-C (1-3), together with four known ones were isolated from the twigs and leaves of Croton euryphyllus. Among them, crotoeurin A (1) is a new nor-clerodane diterpenoid dimer with a unique cyclobutane ring via a [2+2] cycloaddition. Their structures were elucidated by spectroscopic analyses and the stereochemistry of 1 was confirmed by single-crystal X-ray diffraction analysis. Compounds 1-3 exhibited neurite outgrowth-promoting activity on NGF-mediated PC12 cells at concentration of 10µM.

Rapid Bioassay-Guided Isolation of Antibacterial Clerodane Type Diterpenoid from Dodonaea viscosa (L.) Jaeq.

Plant extracts are complex matrices and, although crude extracts are widely in use, purified compounds are pivotal in drug discovery. This study describes the application of automated preparative-HPLC combined with a rapid off-line bacterial bioassay, using reduction of a tetrazolium salt as an indicator of bacterial metabolism. This approach enabled the identification of fractions from Dodonaea viscosa that were active against Staphylococcus aureus and Escherichia coli, which, ultimately, resulted in the identification of a clerodane type diterpenoid, 6ß-hydroxy-15,16-epoxy-5ß, 8ß, 9ß, 10α-cleroda-3, 13(16), 14-trien-18-oic acid, showing bacteriostatic activity (minimum inhibitory concentration (MIC) = 64-128 µg/mL) against test bacteria. To the best of our knowledge, this is the first report on antibacterial activity of this metabolite from D. viscosa.

A new diterpenoid from the leaves and twigs of Croton lachnocarpus Benth.

A new clerodane diterpenoid, crotolanin A (1), along with three known clerodane diterpenoids, crotoeurin B (2), teucvidin (3) and teucvin (4), was isolated from the ethanol extract of the leaves and twigs of Croton lachnocarpus Benth. Their structures were identified by extensive NMR spectroscopic and HRESIMS analyses. The dopaminergic neuroprotective activity of compounds 1-4 was tested by using transgenic Caenorhabditis elegans pathological model. Compound 2 alleviated dopaminergic neuron degeneration of worms induced by 6-hydroxydopamine (6-OHDA) that represented a potential therapy for Parkinson's disease (PD).

Two new clerodane diterpenoids and a new pyran-2-one derivative with anti-neuroinflammatory activities from Croton crassifolius.

Two new clerodane diterpenoids (1 and 2), a new pyran-2-one derivative (3), along with five known compounds (4‒8), were isolated from Croton crassifolius. Notably, crassifolin X (1) is a novel clerodane diterpenoid, characterized with a peculiar δ-lactone core being formed between C-1 and C-4. Their structures, including absolute configurations, were established on the basis of spectroscopic methods (UV, IR, HRESIMS and NMR), and circular dichroism experiments. In addition, all compounds were evaluated for their anti-neuroinflammatory activities based on the expression of TNF-α and IL-6 levels on LPS-induced BV2 cells, and compounds 1‒3 and 5 showed potential anti-neuroinflammatory activity.

Phytochemical Screening and Isolation of New Ent-Clerodane Diterpenoids from Croton guatemalensis Lotsy.

Phytochemical screening of an ethanol-water extract (EWE) from the bark of Croton guatemalensis led to the isolation and identification of eight compounds, among them: five ent-clerodane diterpenoids [junceic acid (1), 6(s)-acetoxy-15,16-diepoxy-ent-cleroda-3,13(16),14-trien-20-oic acid (crotoguatenoic acid A) (2), 6(s)-hydroxyoxy-15,16-diepoxy-ent-cleroda-3,13(16),14-trien-20-oic acid (crotoguatenoic acid B) (3), formosin F (4), bartsiifolic acid (5)], and three flavonoids [rutin (6), epicatechin (7), and quercetin (8)]. Of these, 2 and 3 are reported here for the first time. Structures were established through conventional spectroscopy methods and their absolute configurations were determined by optical rotation and comparison of experimental electronic circular dichroism (ECD) and theoretical calculated ECD spectra. A suitable high performance liquid chromatography (HPLC) method for quantifying rutin (6) was developed and validated according to standard protocols. Affinity-directed fractionation was used to identify possible in vitro active compounds on α-glucosidases from Saccharomyces cerevisiae. HPLC-ESI-MS was used to identify the inhibitors as free ligands after being released from the enzymatic complex by denaturing acidic conditions. The affinity studies led to the identification of ent-clerodane diterpenoids as active compounds. In silico analysis allowed us to determine the best conformational rearrangement for the α-glucosidase inhibitors.

Bioactive terpenoids from Croton laui.

Two new highly-oxygenated neo-clerodane diterpenoids, 3S-acetoxyl-mollotucin D dilactone ester (1) and 6S-crotoeurin C (2), and a new lupane-type triterpene, 16ß-hydroxyl-3ß-O-trans-coumaroyl-betulin (6), as well as three known analogues (3-5) were obtained from the leaves of Croton laui. The structures of the new compounds were determined by extensive spectroscopic methods, and their absolute configurations were determined by combination of single-crystal X-ray diffraction analysis, electronic circular dichroism (ECD) spectra, and literature data. Compounds 2 and 3 exhibited inhibitory activities of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages with IC50 values of 1.2 and 1.6 µM, respectively. Additionally, compound 6 exhibited activity against Col205 and HepG2 cell lines with IC50 values of 12.9 and 17.7 µM, respectively.

A pair of new neo-clerodane diterpenoid epimers from the roots of Croton crassifolius and their anti-inflammatory.

A pair of new neo-clerodane diterpenoid epimers, 3S-methoxyl-teucvin (1) and 3R-methoxyl-teucvin (2), were isolated from the Roots of Croton crassifolius. Their structures were completely established on the basis of spectroscopic methods, and the absolute configurations were determined by analysis of electronic circular dichroism (ECD) spectroscopy and X-ray diffraction analysis. Compounds 1 and 2 exhibited anti-inflammatory activities with IC50 values of 0.82 and 0.54 µM, respectively, while the IC50 value of dexamethasone as a positive control was found to be 0.14 µM.

Anti-proliferative activity-guided isolation of clerodermic acid from Salvia nemorosa L.: Geno/cytotoxicity and hypoxia-mediated mechanism of action.

The adaptation of solid tumors to the low oxygen/nutrient environment is mediated by the pivotal transcription role of hypoxia-inducible factor-1 (HIF-1). Thus, the HIF-1 and its subunits have been considered to be hopeful anti-cancer targets. Various natural compounds were reported to persuade cell cytotoxicity through targeting and downregulation of the HIF-1. The genus Salvia is a rich source of bioactive terpenoids which show promising anti-cancer activities. Here, the identification of natural anti-proliferative compound targeting the HIF-1α expression was reported. A bioassay-guided isolation was employed for the discovery of natural anti-proliferative compounds from Salvia extracts using MTT assay against A549 cells. In this direction, clerodermic acid (CDA) as a potent cytotoxic compound was purified from Salvia nemorosa and identified using 1D and 2D NMR analysis. Results indicated that CDA has anti-proliferation activity (IC50 value of 35 µg/mL) which was confirmed by genotoxicity and apoptosis detection analyses. The quantitative qPCR analysis showed that the expression level of HIF-1 alpha was strongly inhibited in the hypoxic cells treated with CDA compared to the untreated cells tolerated hypoxia. Findings exhibited that S. nemorosa and clerodermic acid have significant potential for reducing HIF-1α expression and could be considered for further studies for cancer therapy.

Sulfated neo-clerodane diterpenoids and triterpenoid saponins from Sheareria nana S. Moore.

Three novel neo-clerodane diterpenoids Sheareria A-C (1-3) together with three known triterpenoid saponins were isolated from the whole herb of Sheareria nana S. Moore. Their structures were established by spectroscopic and chemical method. This is the first natural sulfated neo-clerodane diterpenoids. This is the first report of all these compounds from this plant. These neo-clerodane diterpenoids and triterpenoid saponins from S. nana S. Moore may be considered as chemotaxonomic markers for the genus. The compounds isolated were evaluated for their cytotoxic effects against three cancer cell lines, the test substances demonstrated selectivity toward the cancer cells. To date, this is the first report on the phytochemical and biological activity of secondary metabolites from S. nana S. Moore.