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Background/Objectives: Peroxisome proliferator–activated receptor γ (PPARγ) plays a key role in mediating anti-inflammatory and anticancer effects in the tumor microenvironment. Kaurenoic acid (KA), a diterpene compound isolated from Sphagneticola trilobata (L.) Pruski, has been demonstrated to exert anti-inflammatory, anticancer, and antihuman immunodeficiency virus effects. Methods: In this study, we identified KA as a novel activator of PPARγ with potent anti-inflammatory and antitumor effects both in vitro and in vivo. Given the potential of PPARγ regulators in overcoming radioresistance and chemoresistance in cancer therapies, we hypothesized that KA may enhance the efficacy of breast cancer radiotherapy. Results: In a lipopolysaccharide (LPS)-induced mouse inflammation model, KA treatment reduced the levels of pro-inflammatory cytokines, including COX-2, IL-6, IL-1β, and TNFα. In a xenograft mouse mode of breast cancer, KA treatment inhibited tumor growth. Specifically, KA treatment enhanced caspase-3 activity and cytotoxicity against MDA-MB-231 and MCF-7 breast cancer cells. When KA was co-treated with a caspase inhibitor, Z-VAD-FMK, caspase-dependent apoptosis was suppressed in these cells. KA was found to induce the generation of cytosolic calcium ions (Ca2+) and reactive oxygen species (ROS), triggering endoplasmic reticulum (ER) stress via the PERK-ATF4-CHOP axis. Hence, the ER stressor thapsigargin (TG) synergized with KA treatment to enhance apoptosis in these cells, while the loss of the PERK or CHOP function inhibited this phenomenon. KA treatment was shown to induce oxidative stress via the NADPH oxidase 4 (NOX4) and stimulate ROS production. Specifically, NOX4 knockdown (KD) and antioxidant treatment (N-acetyl cysteine or diphenyleneiodonium) suppressed such ER stress–mediated apoptosis by inhibiting KA-enhanced caspase-3 activity, cytotoxicity, and intracellular ROS production in the treated cells. In radioresistant MDA-MB-231R and MCF-7R cells, KA combined with 2 Gy radiation overcame radioresistance by upregulating PPARγ and modulating epithelial–mesenchymal transition (EMT) markers, such as E-cadherin, N-cadherin, and vimentin. In PPARγ KD MDA-MB-231R and MCF-7R cells, this phenomenon was inhibited due to reduced PPARγ and NOX4 expression. Conclusions: In conclusion, these findings demonstrated KA as a novel PPARγ regulator with promising potential to enhance the efficacy of breast cancer radiotherapy. [ABSTRACT FROM AUTHOR]

Acanthopanax gracilistylus is a deciduous plant in the family Araliaceae, which is commonly used in Chinese herbal medicine, as the root bark has functions of nourishing the liver and kidneys, removing dampness and expelling wind, and strengthening the bones and tendons. Kaurenoic acid (KA) is the main effective substance in the root bark of A. gracilistylus with strong anti-inflammatory effects. To elucidate the KA biosynthesis pathway, second-generation (DNA nanoball) and third-generation (Pacific Biosciences) sequencing were performed to analyze the transcriptomes of the A. gracilistylus leaves, roots, and stems. Among the total 505,880 isoforms, 408,954 were annotated by seven major databases. Sixty isoforms with complete open reading frames encoding 11 key enzymes involved in the KA biosynthesis pathway were identified. Correlation analysis between isoform expression and KA content identified a total of eight key genes. Six key enzyme genes involved in KA biosynthesis were validated by real-time quantitative polymerase chain reaction. Based on the sequence analysis, the spatial structure of ent-kaurene oxidase was modeled, which plays roles in the three continuous oxidations steps of KA biosynthesis. This study greatly enriches the transcriptome data of A. gracilistylus and facilitates further analysis of the function and regulation mechanism of key enzymes in the KA biosynthesis pathway. [ABSTRACT FROM AUTHOR]

The EFSA Panel on Food Additive and Flavourings (FAF Panel) provides a scientific opinion on the safety of a new process to produce steviol glycosides by fermentation of simple sugars using a genetically modified strain of Yarrowia lipolytica (named Y. lipolytica VRM). The manufacturing process may result in impurities different from those that may be present in the other steviol glycosides E 960a‐d, therefore the Panel concluded that separate specifications are required for the food additive produced as described in the current application. Viable cells and DNA from the production strain are not present in the final product. The Panel considered that the demonstration of the absence of kaurenoic acid in the proposed food additive, using a method with a limit of detection (LOD) of 0.3 mg/kg, is adequate to dispel the concerns for potential genotoxicity. Given that all steviol glycosides follow the same metabolic pathways, the Panel considered that the current steviol glycosides would fall within the same group of substances. Therefore, the Panel considered that the already existing data on rebaudioside M and structurally related steviol glycosides are sufficient, and a similar metabolic fate and toxicity is expected for the food additive. The results from the bacterial reverse mutation assay and the in vitro micronucleus assay were negative and indicated absence of genotoxicity from the food additive. The existing acceptable daily intake (ADI) of 4 mg/kg body weight (bw) per day, expressed as steviol equivalents, was considered to be applicable to the proposed food additive. The Panel concluded that there is no safety concern for steviol glycosides, predominantly Rebaudioside M, produced by fermentation using Y. lipolytica VRM, to be used as a food additive at the proposed uses and use levels. [ABSTRACT FROM AUTHOR]

Kaurenoic acid (KA) is a diterpene extracted from Sphagneticola trilobata (L.) Pruski. KA presents analgesic properties. However, the analgesic activity and mechanisms of action of KA in neuropathic pain have not been investigated so far; thus, we addressed these points in the present study. A mouse model of neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve. Acute (at the 7th-day post-CCI surgery) and prolonged (from 7–14th days post-CCI surgery) KA post-treatment inhibited CCI-induced mechanical hyperalgesia at all evaluated time points, as per the electronic version of von Frey filaments. The underlying mechanism of KA was dependent on activating the NO/cGMP/PKG/ATP-sensitive potassium channel signaling pathway since L-NAME, ODQ, KT5823, and glibenclamide abolished KA analgesia. KA reduced the activation of primary afferent sensory neurons, as observed by a reduction in CCI-triggered colocalization of pNF-κB and NeuN in DRG neurons. KA treatment also increased the expression of neuronal nitric oxide synthase (nNOS) at the protein level as well as the intracellular levels of NO in DRG neurons. Therefore, our results provide evidence that KA inhibits CCI neuropathic pain by activating a neuronal analgesic mechanism that depends on nNOS production of NO to silence the nociceptive signaling that generates analgesia. [ABSTRACT FROM AUTHOR]

Breast cancer is the type of cancer with the highest incidence in women around the world. Noteworthy, the triple-negative subtype affects 20% of the patients while presenting the highest death rate among subtypes. This is due to its aggressive phenotype and the capability of invading other tissues. In general, tumor-associated macrophages (TAM) and other immune cells, are responsible for maintaining a favorable tumor microenvironment for inflammation and metastasis by secreting several mediators such as pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, chemokines like CCL2, and other proteins, as metalloproteinases of matrix (MMP). On the other hand, immunomodulatory agents can interfere in the immune response of TAM and change the disease prognosis. In this work, we prepared nanostructured lipid carriers containing kaurenoic acid (NLC-KA) to evaluate the effect on cytokine production in vitro of bone marrow-derived macrophages (BMDM) and the migratory process of 4 T1 breast cancer cells. NLC-KA prepared from a blend of natural lipids was shown to have approximately 90 nm in diameter with low polydispersity index. To test the effect on cytokine production in vitro in NLC-KA treated BMDM, ELISA assay was performed and pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified. The formulation reduced the secretion of IL-1β and TNF-α cytokines while presenting no hemolytic activity. Noteworthy, an anti-migratory effect in 4 T1 breast cancer cells treated with NLC-KA was observed in scratch assays. Further, MMP9 and CCL2 gene expressions in both BMDM and 4 T1 treated cells confirmed that the mechanism of inhibition of migration is related to the blockade of this pathway by KA. Finally, cell invasion assays confirmed that NLC-KA treatment resulted in less invasiveness of 4 T1 cells than control, and it is independent of CCL2 stimulus or BMDM direct stimulus. Ultimately, NLC-KA was able to regulate the cytokine production in vitro and reduce the migration of 4 T1 breast cancer cells by decreasing MMP9 gene expression. [Display omitted] [ABSTRACT FROM AUTHOR]

Copalic (CA) and kaurenoic (KA) acids are the main diterpenes found in the oleoresin extracted from the copaiba tree (Copaifera sp). This study aimed to characterize the metabolism of CA and KA in rat and human liver microsomes using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The in vitro assays showed deviations from the Michaelian kinetics in the metabolism of CA and KA. Putative metabolites of CA and KA were characterized by LC-MS/MS using electrospray ionization (ESI) with time of flight (LC-ESI-TOF) and ion-trap (LC-ESI-IT) systems and identified as a CA isomer and 16,17-dihydroxy-kaurenoic acid, respectively. CA and KA are subject to extensive metabolism with each passage through the liver with extraction ratios (E) estimated as 0.97 and 0.99, respectively. In conclusion, the kinetic parameters and metabolites described here might support drug development and the traditional use.

Adenostemmoic acid B (AB) is a major compound found in Adenostemma lavenia ; it shows anti-melanogenic, anti-inflammatory, and cytotoxic activities. By modifying the 19th position (carboxy: involved in the avoidance of cytotoxicity) of AB, we succeed to separate these activities. Short-chain alkylation of the carboxy group enhanced anti-melanogenic activity, while long-chain alkylation (hydrophobic) resulted in the suppression of nitric oxide (NO) production and inducible NO synthase (iNOS) expression without anti-melanogenic activity. Re-modification of hydrophilic properties in these long-chain derivatives restored anti-melanogenic activity but did not suppress NO production. Unexpectedly, AB and derivatives with long chains linked by an anhydride bond were new iNOS inhibitors. These results suggest that AB modulates multiple physiological activities by regulating different targets, including iNOS. [Display omitted] • Adenostemmoic acid B is a multipotential ingredient in Adenostemma lavenia. • Adenostemmoic acid B suppresses gene expression of tyrosinase (anti-melanogenesis) and iNOS (anti-inflammatory). • Adenostemmoic acid B and its derivatives with an anhydride bond inhibit iNOS enzyme activity. [ABSTRACT FROM AUTHOR]

This work aims to develop a rapid and reliable method by HPLC-QqQ-MS/MS for identification and quantification of Kaurenoic acid (KA) in extract, tincture and syrup from Mikania glomerata. The developed method allows identifying and quantifying KA in complex samples obtained from extract marketed in pharmaceutical industry, a tincture prepared with the leaves from plant and two syrups purchased in the local market. The quantification was performed using standard addition calibration curve. An accurate method for Kaurenoic acid determination in M. glomerata samples was developed. The concentrations of KA found in M. glomerata were 0.19 mg mL-1 in the commercial extract, 0.93 mg mL-1 in the tincture, 3.00 x 10-3 mg mL-1 in the syrup B and in syrup A was not possible to quantify due to the concentration is below the detection limit. Since most reports describe the quantification of the main active constituent (coumarin) and do not provide relevant information on the quantification of KA in these sample types (syrup and tincture), the developed method represents an excellent alternative for agile and efficient analysis of the KA in complex matrices from simple dilution.

Abstract Background Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli. Results A genome-engineered E. coli strain, which coexpressed 5′ untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5′-UTR–engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP+ ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5′-UTR–engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation. Conclusions 5′-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids—including steviol glycosides and carotenoids—in microorganisms.

A known kaurenoic acid (1), together with a new kaurane derivative named 3α-isobutyryloxykaurenoic acid (2), was isolated from the roots of Lasianthaea aurea, a plant endemic to Mexico. The compounds were characterized using their physical and spectroscopic data. In order to verify the structure and absolute configuration of 2, the derived methyl ester 3, obtained after diazomethane treatment, was subjected to single-crystal X-ray diffraction analysis, whose Flack and Hooft parameter calculations evidenced that the new natural product is an ent-kaurane derivative. [ABSTRACT FROM AUTHOR]

Breast cancer continues to be the major cause of cancer death among women. The high genetic heterogeneity and chemoresistant phenotypes of breast tumors require the search for new compounds to expand the options for the treatment of this disease. Kaurenoic acid (KA) is an ent‐kaurane type‐diterpene with cytotoxic and genotoxic effects against gastric and cervical cancer cells. In the present study, we report the antiproliferative effects of KA in the breast tumoral cell line MCF‐7 and the normal breast counterpart MCF‐10 A. The long‐term cytotoxic effects of KA were observed by the reduction of survival fraction at 50 and 100 μM. At these concentrations, the treatment with KA increased the extension of DNA damage followed by G1 cell cycle phase accumulation and induction of apoptosis in both cell lines. Taken together, the results contained herein support the promising antiproliferative effects of KA. [ABSTRACT FROM AUTHOR]

Background: The roots of Aralia continentalis have been used in traditional Korean medicine to treat pain, lumbago, lameness, and rheumatism. Objective: Simultaneous determination of two diterpenoids; continentalic acid (CA) and kaurenoic acid, in the water extract of A. continentalis using the high-performance liquid chromatography (HPLC) was established for quality control, as well as evaluation of their wound-healing activity. Materials and Methods: The separation was conducted on YMC hydrosphere C18 column with isocratic elution of 65% acetonitrile. Furthermore, the isolated diterpenoids were screened for their wound-healing activity using keratinocytes (HaCaT cells). Results: The calibration curves were linear over the established range with R2 > 0.9999. The intraday and interday RSDs for each compound were 0.13%–0.89% and 0.14%–0.73%, respectively. The limits of detection and limits of quantification for the two tested diterpenoids were 0.0912, 0.0018 μg/μL, and 0.2764, 0.0056 μg/μL, respectively. In addition, the CA-treated groups showed accelerated wound closure compared to the control group. Conclusion: The HPLC method could be accomplished to the quality control and stable experiment for the preparations consisted of two diterpenoids. Compared to the control group, the CA-treated group showed that wound closure was accelerated, indicating its potential in promoting migration of skin cells which is the most important step of wound closure. [ABSTRACT FROM AUTHOR]

Background: Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli. Results: A genome-engineered E. coli strain, which coexpressed 5′ untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5′-UTR–engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP+ ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5′-UTR–engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation. Conclusions: 5′-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids—including steviol glycosides and carotenoids—in microorganisms. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana Bertoni has been promoted for having sweet leaves as well as pharmaceutical and industrial properties. The sweet taste of Stevia leaves is due to the presence of steviol glycosides (a group of diterpene glycosides) found in a small number of plants. In the biosynthetic pathway of steviol glycosides (SGs), 15 enzymes that express the genes are associated with these enzymes under the influence of the elicitors. Due to the individuality of the stevia and few studies on the biosynthesis pathway of SGs, this paper attempted to investigate the effects of some of the elicitors, including methyl jasmonate (MeJA), salicylic acid (SA), auxins (Aux), cytokinins (CKs), gibberellins (GAs) and its inhibitors including paclobutrazol (BPZ) and chloroquate (CCC)), on the responsible genes for the biosynthesis of SGs. Some of these elicitors, including MeJA, SA and GA have great potential in increasing secondary metabolites. Moreover, the biosynthetic pathway of GAs and SGs are shared till ent-kaurenoic acid (ent-KA) biosynthesis, which raises the question of whether this hormone and its inhibitors are effective in the SGs biosynthesis. Type 2 diabetes and obesity are two major risk factors for cardiovascular disease that are caused by over-consumption of sugar. Since artificial sweeteners have been shown to be harmful to health, natural extracts including steviol glycosides (SGs) are now considered the best substitute for sugar. We present new knowledge on the effect of elicitors on SGs biosynthesis, and show that an increase in plant yield can also increase these secondary metabolites. [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Copaiba oleoresin has been used in folk medicine in the treatment of bronchitis, syphilis, skin diseases and ulcers due to its anti-inflammatory and antiseptic activities, but there is no information about major compounds oral absorption to support the traditional use. Aim of study Considering the potential of copalic (CA) and kaurenoic acid (KA) – major biological activity (in vitro) diterpenes found in the oleoresin, this study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 cells model as predictive test for oral drug absorption. Materials and methods Chemical stability at pH 1.2 and 7.4 and plasma stability were evaluated to mimic physiological conditions of the gastrointestinal tract. The intestinal permeability of CA and KA was evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. Results CA and KA were rapidly degraded at pH 1.2 (0.2 M Clark-Lubs buffer). At pH 7.4 (0.1 M phosphate buffer), CA was stable for up to 24 h and KA for up to 6 h. In human plasma, CA and KA can be considered stable for 24 h and 12 h at 37 °C, respectively. Caco-2 cells were considered viable when incubated with CA or KA in the range of 3.9–250 μM for 24 h. CA and KA exhibited moderate apparent permeability (P app) of 4.67 (±0.08) × 10−6 cm/s and 4.66 (±0.04) × 10−6 cm/s, respectively. Simultaneous incubation with verapamil showed that P-glycoprotein does not play a relevant role on CA and KA oral absorption, with P app of 4.48 (±0.26) × 10−6 cm/s and 5.37 (±0.72) × 10−6 cm/s observed for CA and KA, respectively. Conclusion The oral absorption of both CA and KA is driven by mainly passive permeability, is not limited by p-glycoprotein, but enteric-coated dosage forms should be used to avoid chemical instability in the gastric pH. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance The oleoresin of Brazilian Copaifera reticulata is a traditional remedy used for the treatment of skin and urinary tract infections, respiratory diseases, rheumatism, ulcer and tumours; thus, playing an important role in the primary health care of the indigenous population. Aim As most previous pharmacological tests used the crude oleoresin and only a few studies so far dealt with enriched fractions or pure chemically defined compounds, the aim of this study was to systematically evaluate the antimicrobial and cytotoxic properties of the Copaifera reticulata oleoresin and to assign traditional uses to specific secondary metabolites. Materials and methods The oleoresin, as well as its neutral and acidic fractions were tested for their activity against six cancer cell lines, two clinically relevant bacterial strains, and two dermatophytes. Both fractions were analysed by GC-MS and UHPLC-ELSD, respectively. The antibacterial acidic phase was further fractionated by preparative chromatography to purify and characterize the compounds responsible for the observed pharmacological effect. Results Whereas no cytotoxic activity was detected, the crude oleoresin and its acidic fraction showed antibacterial activity against gram-positive bacteria Enterococcus faecium (IC 50 values 4.2 and 4.8 µg/mL, respectively) and methicillin-resistant Staphylococcus aureus (MRSA, IC 50 values 5.3 and 7.2 µg/mL, respectively). Purification of the acidic fraction of the C. reticulata oleoresin yielded two dicarboxylic diterpene acids and the four main diterpene acids, comprising three different diterpene scaffolds. Interestingly, the activity was not restricted to a particular diterpene-type but rather depended on the compounds' lipophilicity, with the most active constituent showing IC 50 values of 1.6 (E. faecium) and 2.5 µg/mL (MRSA), respectively. Furthermore, ent -polyalthic acid, the major diterpenoid, was significantly active against dermatophytes with IC 50 values of 6.8 µg/mL (Trichophyton rubrum) and 4.3 µg/mL against (T. mentagrophytes). Conclusion The present study proved the antimicrobial effects of the C. reticulata oleoresin and its diterpenoid constituents, confirming its wide use in folk medicine for the treatment of skin and urinary tract infections. The inhibitory activity of copaiba diterpenoids against dermatophytic fungi as well as the gram-positive bacteria E. faecium and MRSA is being reported for the first time, providing potential lead structures for the treatment of these clinically relevant bacterial strains. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Aristolochia odoratissima L. is employed for the treatment of pain and as an antidote against the poison of venomous animals in traditional medicine. However, reports have not been found, to our knowledge, about the evaluation of the antinociceptive activity of extracts nor about the presence of compounds associated with this activity. Thus, the purpose of this work was to evaluate the antinociceptive activity of extracts and compounds isolated from the stems of Artistolochia odoratissima L. The extracts were obtained with solvents of increasing polarity and the compounds were isolated and characterized by column chromatography, HPLC, and NMR. The antinociceptive activity was carried out by the formalin test in mice. Ethyl acetate (AoEA) and methanolic (AoM) extracts decreased the paw licking in both phases of the formalin test. The isolated compounds (kaurenoic acid and hinokinin) from AoEA showed the highest antinociceptive activity in both phases of the formalin test. These results confirmed the analgesic effect of this specie described in traditional medicine and provided a base for a novel analgesic agent. They also allowed an approach for the development of standardized plant extracts with isolated metabolites.

We report herein the synthesis of six diterpene derivatives, three of which are new, generated through known organic chemistry reactions that allowed structural modification of the existing natural products kaurenoic acid (1) and copalic acid (2). The new compounds were fully characterized using high resolution mass spectrometry, infrared spectroscopy, 1H- and 13C-NMR experiments. We also report the evaluation of the anti-tuberculosis potential for all compounds, which showed some promising results for Micobacterium tuberculosis inhibition. Moreover, the toxicity for each of the most active compounds was also assessed.

Highlights • First UPLC-MS/MS report for determination of kaurenoic acid in plasma. • This method was successfully applied to a pharmacokinetic study of kaurenoic acid after oral administration in rats. • Compared to the previous HPLC-PDA method, our method provided a sensitive and rapid assay. • Extensive evaluation of pseudo MRM of kaurenoic acid in plasma. Abstract Kaurenoic acid (KA), a kaurane diterpene found in several medicinal plants, is an active ingredient with potential anti-inflammatory, anticonvulsant, antibacterial and antitumor activities. In this work, an ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was firstly developed and validated to quantify kaurenoic acid in rat plasma. Rhein was chosen as the internal standard (IS) and the plasma was processed with one-step acetonitrile protein precipitation; the chromatographic separation was achieved on a HSS T3 (2.1 × 50 mm, 1.8 μm) column with the mobile phase consisting of acetonitrile and water containing 0.1% formic acid via gradient elution. An electrospray ionization source was applied and operated in the negative ion and multiple reaction monitoring (MRM) modes. Kaurenoic acid and IS were quantified using the transitions of m/z 301.2→301.2 (pseudo MRM) and m/z 283.2 → 238.9, respectively. The calibration curves were linear over the range of 5∼ 100 ng/mL (R2 = 0.990). The lower limit of quantification (LLOQ) was 5 ng/mL. The intra- and inter- day precision (RSD) ranged from 3.0% to 11.4%. The matrix effect and extraction recovery were within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of kaurenoic acid in rats after oral administration at three dosages. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Quantitation of the five major and three minor diterpene acids in Copaifera reticulata oleoresin. • Solid phase extraction procedure for the quantitative separation of the diterpene acids from the oil. • Alternative strategy for the determination of diterpene acids using a quantitative HSQC approach. Abstract In this study, we present the quantitation of eight diterpene acids in the oleoresin of Copaifera reticulata Ducke by UHPLC-ELSD and quantitative HSQC (heteronuclear single quantum correlation spectroscopy). UHPLC was performed using reversed phase material and external calibration and showed RSD values of ≤ 3% (repeatability) and ≤ 4% (precision), and mean recovery rates of 91.2–104.8%. LOQs were determined with 10 and 20 μg/mL, and LODs with 4 and 8 μg/mL, respectively. For the qHSQC method, calibration curves of eight different NMR cross-peaks (furylic, endo- and exocyclic methine signals, exocyclic methylene and methyl signals) were established and normalized with dimethyl terephthalate, which served as internal standard. This approach allowed the direct quantification of four major and one minor diterpene, whereas simple calculation procedures led to the contents of the remaining minor compounds. Comparison with the results of the UHPLC assay showed good agreement for seven of the eight diterpene acids. In terms of precision, the qHSQC method was advantageous for the quantification of the three main compounds, whereas UHPLC-ELSD was superior in the determination of the minor components. In contrast to previous reports, kolavenic acid was identified as a major diterpene acid in the oleoresin of Copaifera reticulata , with amounts of 4.0 ± 0.3%. [ABSTRACT FROM AUTHOR]

Kaurenoic acid (KA) is a kaurane diterpene found in several medicinal plants that displays biological activities, such as anti-inflammatory, smooth muscle relaxant and hypotensive response. However, there are no pharmacokinetic data available about this molecule. The purpose of the study was to determine the pharmacokinetic profile and the oral bioavailability of KA in rats. Wistar rats submitted to jugular vein cannulation received 50 mg/kg of KA by intravenous or oral route. The implanted cannula allowed intravenous administration and serial blood collection along 10 h. Analytical quantification was performed by reversed phase HPLC-UV and mobile phase composed by acetonitrile:acidified water (70:30  v /v). The validated analytical method showed precision, accuracy, robustness, reliability and linearity between 0.75 and 100 μg/mL. KA administered intravenously showed a linear and two-compartment kinetic behavior at the tested dose. The following pharmacokinetic parameters were determined: C max  = 22.17 ± 1.65 mg/L; V = 14.53 ± 1.47 L/kg; CL = 17.67 ± 1.50 mL/min/kg; AUC 0-∞  = 2859.65 ± 278.42 mg·min/L, K = 0.073 ± 0.005 h −1 and t 1/2β  = 9.52 ± 0.61 h. Oral treatment did not provide detectable plasma levels of KA, avoiding the determination of its bioavailability. [ABSTRACT FROM AUTHOR]

Activity, mechanisms of action, and toxicity of natural compounds have been investigated in a context in which knowledge on which pathway is activated remains crucial to understand the action mechanism of these bioactive substances when treating an infected host. Herein, we showed an ability of copaiba oil and kaurenoic acid to eliminate Trypanosoma cruzi forms by infected macrophages through other mechanisms in addition to nitric oxide, reactive oxygen species, iron metabolism, and antioxidant defense. Both compounds induced an anti-inflammatory response with an increase in IL-10 and TGF-β as well as a decrease in IL-12 production. Despite being able to modulate the immune response in host cells, the antimicrobial activity of copaiba oil and kaurenoic acid seems to be a direct action of the compounds on the parasites, causing their death. [ABSTRACT FROM AUTHOR]

BACKGROUND Parasitic weeds are widespread and cause significant losses in important crops. Their germination requires the detection of crop-derived molecules such as strigolactones. Strigolactone mimics are germination-inducing molecules with the potential to apply a suicidal germination strategy for seed bank control of parasitic weeds. RESULTS The D-ring, which is instrumental in the germination process of seeds of parasitic weeds, was attached to gibberellin (GA3) and kaurenoic acid as the scaffold. It was shown that indeed strigolactone mimics prepared from GA3 and kaurenoic acid are active as stimulants when a D-ring is present; some of the mimics are as active as GR24. CONCLUSIONS The starting molecules were plant hormones that had previous growth-regulating activity in other organisms and the products showed enhanced activity towards parasitic weeds. The information generated may contribute to a better understanding of the germination biochemistry of the weed species used. Further research is required in this area but it is clear that the results are promising. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

We report the bifunctional activity of the native ent-kaurene oxidase from Montanoa tomentosa (MtKO) and its N-terminal modified version (LMtKO) for producing both isokaurenoic acid and kaurenoic acid in Saccharomyces cerevisiae. The K of MtKO showed more affinity for ent-kaurene (80.5 µM) than for isokaurene (96.4 µM). Interestingly, LMtKO exhibited an increase of the affinity for isokaurene (79.6 µM) but simultaneously showed an enhancement in the V for both substrates (32.6-38.9 μmol mg h). Biotransformation assays using isokaurene and yeasts containing LMtKO, resulted in 70% more production of isokaurenoic acid, when compared with the yields from yeasts expressing MtKO. Likewise, biotransformation assays using geranylgeraniol and double transformed cells of S. cerevisiae containing an optimized version the ent-kaurene synthase from Phaeosphaeria sp. L487 (optKS) and the LMtKO, produced ~25% more kaurenoic acid than the yeasts containing optKS and MtKO. The isokaurenoic acid synthesized by transgenic yeasts was tested for its anti-acetylcholinesterase and antimicrobial properties. Isokaurenoic acid generated a non-competitive inhibition on acetylcholinesterase, decreasing the V from 0.0249 to 0.0104 mM min but not affecting the K (0.714 mM). The same diterpene showed antifungal activity against Fusarium oxysporum, Aspergillus niger and Phytophtora infestans with a minimum inhibitory concentration of 15.3, 18.3 and 19.2 µg mL, respectively. [ABSTRACT FROM AUTHOR]

Background: Kaurenoic acid (ent-kaur-16-en-19-oic acid: KA) is a key constituent found in the roots of Aralia continentalis Kitagawa (Araliaceae) that has been used for treating rheumatism in traditional Asian medicine.Hypothesis: Although KA was reported to suppress inflammation by activating Nrf2, the anti-inflammatory function of KA is less characterized. Given the complex nature of the inflammatory response and the critical role of TGF-β in resolving inflammation, we hypothesized that KA suppresses inflammatory response by activating TGF-β signaling.Methods: Murine macrophage RAW 264.7, human lung epithelial cell MRC-5, and a TGFβRII defective cell HCT116 were treated with various amounts of KA. KA was also administered to mouse lung via intratracheal (i.t.) route. Phosphorylated Smad2 and Smad3 were analyzed by western blot. TGFβ-dependent gene expression was determined by immunoblotting of α-SMA and luciferase assay.Results: KA induced the phosphorylation of Smad2 and Smad3, key activator molecules in TGF-β signaling. EW7197, an inhibitor for activin receptor-like kinase 5/TGF-β receptor I (TGFβR1) suppressed KA-mediated phosphorylation of Smad2. Similarly, KA failed to phosphorylate Smad2 in HCT116, suggesting that KA acts through the prototypic TGFβR. KA treatment increased the transcriptional activity driven by a Smad-binding element in a luciferase reporter assay and induced the α-smooth muscle actin (α-SMA). Similarly, i.t. KA induced the phosphorylation of Smad2 and increased the expression ofα-SMA in mouse lungs.Conclusion: KA activated TGF-β signaling, suggesting that TGFβ signaling is associated with KA suppressing inflammation. [ABSTRACT FROM AUTHOR]

The kaurenoic acid-type diterpenoids in Acanthopanacis Cortex have been reported to be the major active components. However, the diterpenoids are present as position isomers that exacerbate the challenges in obtaining standards compounds. Little work has been done on the quantitative analysis of the diterpenoids in the herb. In the present study, two diterpenoid isomers ent -16 β H,17-isovalerate-kauran-19-oic acid ( 1 ) and ent -16 β H,17-methyl butanoate-kauran-19-oic acid ( 2 ) with high purity were separated by analytical HPLC, followed by recrystallization in acetone. Furthermore, an HPLC-ELSD method was developed and validated for simultaneous determination of 1 and 2 in 9 batches of Acanthopanacis Cortex samples. The HPLC separation and quantification was achieved in 40 min using an Agela Promosil C 18 column eluted with a gradient of water and acetonitrile. The calibration curves showed good linearity ( r 2 ≥ 0.999 9) within the test ranges. The LOD ranged from 0.407 2 to 0.518 0 μg and LOQ ranged from 1.018 0 to 1.295 0 μg. The precisions (%RSD) were within 1.47% for the two isomers. The recovery of the assay was in the range of 98.78%–99.11% with RSD values less than 2.76%. It is the first time to establish a quantitative HPLC method for the analysis of the bioactive kaurenoic acid isomers in the herb. [ABSTRACT FROM AUTHOR]

A rapid ultra-performance convergence chromatography method was developed for the quantitative determination of bioactive compounds in Aralia continentalis as quality control markers. Quantitative analysis indicated the presence of two major bioactive compounds: diterpenoid acids continentalic acid and kaurenoic acid. Using a Torus 1-aminoanthracene column, continentalic acid and kaurenoic acid were separated in less than 8 min. The method was validated with respect to precision, accuracy, and linearity according to the International Conference on Harmonization guidelines. The optimized method exhibited a good linear correlation ( r2 > 0.996), excellent precision (RSD < 1.0%), and acceptable recoveries (99.97-100.26%). Limits of detection for continentalic acid and kaurenoic acid were 0.068 and 0.097 μg/mL, respectively, while their corresponding limits of quantitation were 0.207 and 0.295 μg/mL. The system performance of ultra-performance convergence chromatography was compared with that of conventional high-performance liquid chromatography with respect to analysis time and efficiency. The proposed method was found to be reliable and convenient for the quantitative analysis of continentalic acid and kaurenoic acid in A. continentalis from South Korea and A. pubescens from China. This study is expected to serve as a guideline for the quality control of Aralia continentalis. [ABSTRACT FROM AUTHOR]

The goal of our study was to evaluate the effect of kaurenoic acid, obtained from copaiba oil resin, in gastric cancer (GC) and a normal mucosa of stomach (MNP01) cell lines. The compound was tested at concentrations of 2.5, 5, 10, 30 and 60 μg/mL. Comet and micronucleus assays were used to access its potential genotoxicity in vitro . Moreover, we evaluated the effect of kaurenoic acid in cell cycle progression and in the transcription of genes involved in the control of the cell cycle: MYC , CCND1 , BCL2 , CASP3 , ATM , CHK2 and TP53 . Kaurenoic acid induced an increase on cell DNA damage or micronucleus frequencies on GC cell lines in a dose-dependent manner. The GC and MNP01 cell lines entering DNA synthesis and mitosis decreased significantly with kaurenoic acid treatment, and had an increased growth phase compared with non-treated cells. The treatment induced apoptosis (or necrosis) even at a concentration of 2.5 μg/mL in relation to non-treated cells. GC cell lines presented reduced MYC , CCND1 , BCL2 and CASP3 transcription while ATM , CHK2 and TP53 increased in transcription in relation to non-treated cells, especially at a concentration above 10 μg/mL. The gene transcription in the MNP01 (non-treated non-cancer cell line) was designated as a calibrator for all the GC cell lines. In conclusion, our results showed that kaurenoic acid obtained from Copaifera induces DNA damage and increases the micronuclei frequency in a dose-dependent manner in GC cells, with a significant genotoxicity observed above the concentration of 5 μg/mL. Moreover, this compound seems to be able to induce cell cycle arrest and apoptosis in GC cells. [ABSTRACT FROM AUTHOR]

The diterpene kaurenoic acid (KA) has vasorelaxant, antimicrobial, anti-tumoural and anti-leishmanial effects. Semi-synthetic derivatives were obtained to achieve more satisfactory responses. The assessment of genotoxicity is part of the toxicological evaluation of therapeutic compound candidates. The present study investigated the cytotoxicity and genotoxicity of KA and its semi-synthetic derivatives methoxy kaurenoic acid (MKA) and kaurenol (KRN) using the CHO-K1 cell line. The cytotoxicity evaluation demonstrated that treatments with 200 and 400 μM KA reduced cellular proliferation to 36.5 and 4.43%, respectively, and that 100 and 200 μM KA reduced the survival fraction (SF) to 48.1 and 5.5%, respectively. MKA and KRN at concentrations of 400 μM reduced proliferation to 81 and 86.8%, respectively, while 100 and 200 μM KRN reduced the SF to 50%, and 200 μM MKA reduced the SF to 74%. No genotoxicity was observed for KA or MKA. However, 100 μM KRN increased the DNA damage index, as detected by comet assay, although a micronucleus assay did not confirm these data. The results demonstrated that KA and its semi-synthetic derivative MKA were not genotoxic when tested at noncytotoxic concentrations, but KRN was genotoxic at the highest concentration that was tested, as demonstrated by the comet assay. [ABSTRACT FROM AUTHOR]

The naturally occurring diterpenoids beyerenoic ( 1 ) and kaurenoic acids ( 2 ) extracted from Perymenium buphthalmoides DC., were treated with NaOH and reacted with ZnCl 2 to afford two μ 4 -Oxo tetrazinc clusters ( μ 4 -oxo)-hexakis( μ 2 -beyerenate)-tetra-zinc(II) ( 5 ) and ( μ 4 -oxo)-hexakis( μ 2 -kaurenate)-tetra-zinc(II) ( 6 ). Both species were unequivocally characterized and their structures in the solid state determined by single crystal X-ray diffraction techniques. [ABSTRACT FROM AUTHOR]

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

A series of new thiosemicarbazones derived from natural diterpene kaurenoic acid were synthesized and tested against the epimastigote forms of Trypanosoma cruzi to evaluate their antitrypanosomal potential. Seven of the synthesized thiosemicarbazones were more active than kaurenoic acid with IC50 values between 2-24.0 mM. The o-nitro-benzaldehyde-thiosemicarbazone derivative was the most active compound with IC50 of 2.0 mM. The results show that the structural modifications accomplished enhanced the antitrypanosomal activity of these compounds. Besides, the thiocyanate, thiosemicarbazide and the p- methyl, p-methoxy, p-dimethylamine, m-nitro and o-chlorobenzaldehyde-thiosemicarbazone derivatives displayed lower toxicity for LLMCK2 cells than kaurenoic acid, exhibing an IC50 of 59.5 mM.

The objective of this study was to characterize pharmacokinetics (PKs) of kaurenoic acid (KAU) after administration of the clinical usual dose of Araliae Continentalis Radix extract powder to Korean subjects for the first time and evaluate the mechanism of its absorption in vitro. A simple, sensitive, and selective analytical method was developed for the detection of KAU in human plasma. Concentrations of KAU were quantified by ultra-performance liquid chromatography tandem mass spectrometry after simple liquid⁻liquid extraction. This pharmacokinetic model of KAU was best described by a two-compartment model with first-order absorption. To identify efflux transporters involved in the absorption of KAU, a Caco-2 monolayer model was used. Estimated PK parameters were: systemic clearance, 23.89 L/h; inter-compartmental clearance, 15.55 L/h; rate constant for absorption, 1.72 h−1; volume of distribution of the central compartment, 24.44 L; and volume of distribution of the peripheral compartment, 64.05 L. Results from Caco-2 bidirectional transport study suggested that KAU was a potential substrate of efflux transporters. In summary, PKs of KAU were successfully characterized after administration of a usual dose of Araliae continentalis Radix extract powder in human with the newly developed bioanalytical method and the mechanism of absorption of KAU was identified clearly.

Context: The aerial parts ofSphagneticola trilobata(L.) Pruski (Asteraceae) are popularly used to treat topical inflammation, but have not been fully investigated. Objective: To identify polar compounds inS. trilobataextracts and develop a new topical phytomedicine based on the kaurenoic acid (KA) content while monitoring and demonstrating its topical anti-inflammatory activity. Materials and methods: Ethanol spray-dried extract ofS. trilobatawas analysed by LC-MS while the KA content from semisolid was analysed by LC-UV. The extent of ear edema induced by applying 20 μL of croton oil (2.5%), arachidonic acid (AA; 2 mg/ear) and decanoylphorbol-13-acetate (TPA; 2.5 mg/ear) in mice was used to evaluate the biological activity of the semisolids, which were applied 30 min before the phlogistic agents. Results: Eight phenylpropanoids and four oleanane-type triterpenoid saponins were identified, majority of them reported for the first time in this species, in addition to KA. The semisolid containing 1.0% of dried extract reduced the ear edema induced by croton oil [77.2 ± 4.5%; ID50 = 0.49 (0.28–0.87%)], TPA (81.5 ± 2.4%) and AA (39.1 ± 6.9%), with decreasing effect at higher KA concentrations. This was accompanied by neutrophil migration inhibition as investigated by biochemical and histological assays. Discussion and conclusion: The anti-inflammatory effects were (at least in part) due to the interference in protein kinase C (PKC) activation, AA-cascade products and neutrophil migration inhibition, demonstrating the efficacy of the folk topical usage of this plant. The results support the development of a novel topical anti-inflammatory phytomedicine properly standardized to treat inflammatory dermatological diseases. [ABSTRACT FROM AUTHOR]

Kaurenoic acid ( ent- kaur-16-en-19-oic acid: KA) is a key constituent found in the roots of Aralia continentalis Kitagawa (Araliaceae), a remedy to treat patients with inflammatory diseases in traditional Asian medicine. Since KA activates Nrf2, a key anti-inflammatory factor, at the cellular level, we explored a possible therapeutic usage of KA against neutrophilic inflammatory lung disease such as acute lung injury (ALI). Intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) to C57BL/6 mice induced lung inflammation as in ALI. 2 h after i.p. LPS, intratracheal (i.t.) delivery of KA (0.3, 3, or 30 μg/kg body weight) improved lung structure and significantly suppressed neutrophil infiltrations to mouse lungs, with concomitant reduction of myeloperoxidase activity and of the expression of pro-inflammatory cytokine genes. While activating Nrf2 and expressing Nrf2-dependent genes in mouse lungs, KA did not significantly suppress neutrophil lung inflammation in Nrf2 KO mice. In a mouse model of sepsis, a major cause of ALI, single i.t. KA (3 μg/kg) 2 h after the onset of sepsis significantly decreased the mortality of mice. Together, these results suggest that KA has a therapeutic potential against inflammatory lung disease, the effect of which is associated with Nrf2 activation. [ABSTRACT FROM AUTHOR]

Kaurenoic acid, a kauranic diterpenoid, presents in vitro activity against trypomastigote forms of Trypanosoma cruzi, showing, however, lytic activity on blood erythrocytes, as a side effect. The syntheses of twelve new derivatives of kaurenoic acid, four amides, four amines (and three hydroclorides) and four oximes, was carried out aiming at the improvement of the therapeutic activity and without the side effect. Among the derivatives prepared, one compound showed enhanced trypanocidal activity in vitro towards Trypanosoma cruzi trypomastigote erythrocytic forms, when compared to kaurenoic acid, but continued to show discrete lytic activity on erythrocytes; another compound showed a level of activity similar to that of kaurenoic acid, but without lysis.

Kaurenoic acid is a diterpene isolated from copaiba oleo resin. Based on the reported moderate cytotoxic activity of kaurenoic acid, a series of amides and diols derivatives of kaurenoic acid were synthesized and their abilities to inhibit the growth of MCF-7 cells were evaluated. Among the derivatives, compound 2f was the most active with an IC of 7.84 µM. [ABSTRACT FROM AUTHOR]

Background: Kaurenoic acid (KA), a diterpene extracted from copaíba oil-resin, is known to have potent antioxidant and anti-inflammatory properties. l-Arginine (LA) is an amino acid and a nitrogenous precursor for the synthesis of nitric oxide (NO). NO paper in wound healing has already been well documented. The aim of this study was to investigate the protective effects of LA and KA against ischemia reperfusion injury in a randomized skin flap model in rats. Methods: A modified McFarlane flap model measuring 2.5 wide × 8 cm long was established in 36 anesthetized rats and evaluated within 3 groups: group control, group l-arginine, and group kaurenoic acid. Each group was subdivided into two subgroups ( T1 and T2, n = 6 each). Samples were collected 24 h ( T1)/48 h ( T2) postoperatively for oxidative stress (glutathione), as non-protein thiols, malondialdehyde (MDA), NO, inflammation [myeloperoxidase (MPO)], and cytokines TNF-α and IL-1β assays. Results: KA promoted a significant decrease of TNF-α and IL-1 expression and MPO activity at T1/ T2 time points. NSGH levels increased significantly in KA-treated rats, while MDA levels decreased significantly in the same rats. Arginine promoted a significant decrease in MDA levels at the T1 time point and a significant increase in non-protein thiols concentrations at T1/ T2 time points. NO concentration also decreased at the T1 time point. Conclusions: KA may attenuate the oxidative stress and the inflammation, thereby reducing tissue damage induced by ischemia/reperfusion in rats subjected to dorsal skin flaps. No Level Assigned: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors . [ABSTRACT FROM AUTHOR]

We report herein the synthesis of six diterpene derivatives, three of which are new, generated through known organic chemistry reactions that allowed structural modification of the existing natural products kaurenoic acid (1) and copalic acid (2). The new compounds were fully characterized using high resolution mass spectrometry, infrared spectroscopy, ¹H- and 13C-NMR experiments. We also report the evaluation of the anti-tuberculosis potential for all compounds, which showed some promising results for Micobacterium tuberculosis inhibition. Moreover, the toxicity for each of the most active compounds was also assessed. [ABSTRACT FROM AUTHOR]