Cytotoxic Potential of the Monoterpene Isoespintanol against Human Tumor Cell Lines.

Cancer is a disease that encompasses multiple and different malignant conditions and is among the leading causes of death in the world. Therefore, the search for new pharmacotherapeutic options and potential candidates that can be used as treatments or adjuvants to control this disease is urgent. Natural products, especially those obtained from plants, have played an important role as a source of specialized metabolites with recognized pharmacological properties against cancer, therefore, they are an excellent alternative to be used. The objective of this research was to evaluate the action of the monoterpene isoespintanol (ISO) against the human tumor cell lines MDA-MB-231, A549, DU145, A2780, A2780-cis and the non-tumor line MRC-5. Experiments with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fluorescence with propidium iodide (PI), 4',6-diamidino-2-phenylindole dilactate (DAPI) and green plasma revealed the cytotoxicity of ISO against these cells; furthermore, morphological and chromogenic studies revealed the action of ISO on cell morphology and the inhibitory capacity on reproductive viability to form colonies in MDA-MB-231 cells. Likewise, 3D experiments validated the damage in these cells caused by this monoterpene. These results serve as a basis for progress in studies of the mechanisms of action of these compounds and the development of derivatives or synthetic analogues with a better antitumor profile.

Transcriptional Reprogramming of Candida tropicalis in Response to Isoespintanol Treatment.

Candida tropicalis, an opportunistic pathogen, ranks among the primary culprits of invasive candidiasis, a condition notorious for its resistance to conventional antifungal drugs. The urgency to combat these drug-resistant infections has spurred the quest for novel therapeutic compounds, with a particular focus on those of natural origin. In this study, we set out to evaluate the impact of isoespintanol (ISO), a monoterpene derived from Oxandra xylopioides, on the transcriptome of C. tropicalis. Leveraging transcriptomics, our research aimed to unravel the intricate transcriptional changes induced by ISO within this pathogen. Our differential gene expression analysis unveiled 186 differentially expressed genes (DEGs) in response to ISO, with a striking 85% of these genes experiencing upregulation. These findings shed light on the multifaceted nature of ISO's influence on C. tropicalis, spanning a spectrum of physiological, structural, and metabolic adaptations. The upregulated DEGs predominantly pertained to crucial processes, including ergosterol biosynthesis, protein folding, response to DNA damage, cell wall integrity, mitochondrial activity modulation, and cellular responses to organic compounds. Simultaneously, 27 genes were observed to be repressed, affecting functions such as cytoplasmic translation, DNA damage checkpoints, membrane proteins, and metabolic pathways like trans-methylation, trans-sulfuration, and trans-propylamine. These results underscore the complexity of ISO's antifungal mechanism, suggesting that it targets multiple vital pathways within C. tropicalis. Such complexity potentially reduces the likelihood of the pathogen developing rapid resistance to ISO, making it an attractive candidate for further exploration as a therapeutic agent. In conclusion, our study provides a comprehensive overview of the transcriptional responses of C. tropicalis to ISO exposure. The identified molecular targets and pathways offer promising avenues for future research and the development of innovative antifungal therapies to combat infections caused by this pathogenic yeast.

Isoespintanol Antifungal Activity Involves Mitochondrial Dysfunction, Inhibition of Biofilm Formation, and Damage to Cell Wall Integrity in Candida tropicalis.

The growing increase in infections caused by C. tropicalis, associated with its drug resistance and consequent high mortality, especially in immunosuppressed people, today generates a serious global public health problem. In the search for new potential drug candidates that can be used as treatments or adjuvants in the control of infections by these pathogenic yeasts, the objective of this research was to evaluate the action of isoespintanol (ISO) against the formation of fungal biofilms, the mitochondrial membrane potential (ΔΨm), and its effect on the integrity of the cell wall. We report the ability of ISO to inhibit the formation of biofilms by up to 89.35%, in all cases higher than the values expressed by amphotericin B (AFB). Flow cytometric experiments using rhodamine 123 (Rh123) showed the ability of ISO to cause mitochondrial dysfunction in these cells. Likewise, experiments using calcofluor white (CFW) and analyzed by flow cytometry showed the ability of ISO to affect the integrity of the cell wall by stimulating chitin synthesis; these changes in the integrity of the wall were also observed through transmission electron microscopy (TEM). These mechanisms are involved in the antifungal action of this monoterpene.

Protective Effect of Monoterpene Isoespintanol in a Rat Model of Prediabetes Induced by Fructose.

A high-fructose diet (HFD) induces murine alterations like those recorded in human prediabetes. Protective effects of isoespintanol (monoterpene isolated from Oxandra cf. xylopioides) on changes induced by HFD were evaluated. Animals were maintained for 21 days with a standard diet (C), 10% fructose (F), and F plus isoespintanol (FI, 10 mg/kg, i.p.). Glycemia, triglyceridemia, total and HDL-cholesterol, and insulin resistance index (IRX) were determined. Intraperitoneal glucose tolerance test (IGTT) was performed. In the liver, we measured glycogen, lipogenic gene expression (SREBP-1c, GPAT, FAS, and CPT1), oxidative stress (GSH and 3'-nitrotyrosine content), inflammation markers (iNOS, TNF-α, and PAI-1 gene expression; iNOS and COX-2 protein levels), p-eNOS, p-Akt, and p-GSK3ß protein levels. Isoespintanol corrected enhanced triglycerides, lipogenic genes, and IRX, and reduced HDL-cholesterol induced by HFD. Increased liver glycogen and inflammatory markers and decreased GSH, p-Akt, and p-GSK3ß measured in F rats were reversed by isoespintanol, and p-eNOS/e-NOS and iNOS/GADPH ratios were normalized. Isoespintanol restored glucose tolerance (IGTT) compared to F rats. These results demonstrate for the first time that isoespintanol prevents endocrine-metabolic alterations induced by HFD in prediabetic rats. These effects could be mediated by Akt/eNOS and Akt/GSK3ß pathways, suggesting its possible use as a therapeutic tool for the prevention of diabetes at early stages of its development (prediabetes).

Antibacterial Screening of Isoespintanol, an Aromatic Monoterpene Isolated from Oxandra xylopioides Diels.

The incidence of nosocomial infections, as well as the high mortality and drug resistance expressed by nosocomial pathogens, especially in immunocompromised patients, poses significant medical challenges. Currently, the efficacy of plant compounds with antimicrobial potential has been reported as a promising alternative therapy to traditional methods. Isoespintanol (ISO) is a monoterpene with high biological activity. Using the broth microdilution method, the antibacterial activity of ISO was examined in 90 clinical isolates, which included 14 different species: (Escherichia coli (38), Pseudomonas aeruginosa (12), Klebsiella pneumoniae (13), Acinetobacter baumannii (3), Proteus mirabilis (7), Staphylococcus epidermidis (3), Staphylococcus aureus (5), Enterococcus faecium (1), Enterococcus faecalis (1), Stenotrophomonas maltophilia (2), Citrobacter koseri (2), Serratia marcescens (1), Aeromonas hydrophila (1), and Providencia rettgeri (1). MIC90 minimum inhibitory concentration values ranged from 694.3 to 916.5 µg/mL and MIC50 values from 154.2 to 457.3 µg/mL. The eradication of mature biofilms in P. aeruginosa after 1 h of exposure to ISO was between 6.6 and 77.4%, being higher in all cases than the percentage of biofilm eradication in cells treated with ciprofloxacin, which was between 4.3 and 67.5%. ISO has antibacterial and antibiofilm potential against nosocomial bacteria and could serve as an adjuvant in the control of these pathogens.

Antifungal potential of isoespintanol extracted from Oxandra xylopioides diels (Annonaceae) against intrahospital isolations of Candida SPP.

The aim of this study was to evaluate the antifungal activity of isoespintanol (ISO) extracted from Oxandra xylopioides Diels (Annonaceae) against clinical isolates of Candida spp. Isoespintanol was obtained from the petroleum benzine extract of the leaves and was identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). For antifungal activity experiments, the broth microdilution method was used. The results show an inhibitory effect against Candida spp., with minimum inhibitory concentration (MIC) values between 450.4-503.3 µg/mL. Furthermore, the inhibitory effect of ISO against fungal biofilms is highlighted, even in some cases, greater than the effect shown by amphotericin B (AFB) and in others, where AFB showed no effect. Assays with fluorescent staining with acridine orange (AO) and ethidium bromide (EB), transmission electron microscopy (TEM), Evans blue, measurement of extracellular pH and leakage of intracellular material, evidenced damage at the level of fungal membranes and general cell damage, when cells were exposed to ISO, compared to untreated cells. The results of this research, serve as the basis for future studies in the establishment of the mechanisms of antifungal action of ISO, which could serve as an adjunct in the treatment of infections by these yeasts.

Mechanism of Antifungal Action of Monoterpene Isoespintanol against Clinical Isolates of Candida tropicalis.

The growing increase in infections by Candida spp., non-albicans, coupled with expressed drug resistance and high mortality, especially in immunocompromised patients, have made candidemia a great challenge. The efficacy of compounds of plant origin with antifungal potential has recently been reported as an alternative to be used. Our objective was to evaluate the mechanism of the antifungal action of isoespintanol (ISO) against clinical isolates of Candida tropicalis. Microdilution assays revealed fungal growth inhibition, showing minimum inhibitory concentration (MIC) values between 326.6 and 500 µg/mL. The eradication of mature biofilms by ISO was between 20.3 and 25.8% after 1 h of exposure, being in all cases higher than the effect caused by amphotericin B (AFB), with values between 7.2 and 12.4%. Flow cytometry showed changes in the permeability of the plasma membrane, causing loss of intracellular material and osmotic balance; transmission electron microscopy (TEM) confirmed the damage to the integrity of the plasma membrane. Furthermore, ISO induced the production of intracellular reactive oxygen species (iROS). This indicates that the antifungal action of ISO is associated with damage to membrane integrity and the induction of iROS production, causing cell death.

Isoespintanol, a monoterpene isolated from oxandra cf xylopioides, ameliorates the myocardial ischemia-reperfusion injury by AKT/PKCε/eNOS-dependent pathways.

PURPOSE: To determine the actions of isoespintanol (Isoesp) on post-ischemic myocardial and mitochondrial alterations. METHODS: Hearts removed from Wistar rats were perfused by 20 min. After this period, the coronary flow was interrupted by half an hour and re-established during 1 h. In the treated group, Isoesp was administered at the beginning of reperfusion. To assess the participation of ε isoform of protein kinase C (PKCε), protein kinase B (PKB/Akt), and nitric oxide synthase (NOS), hearts were treated with Isoesp plus the respective inhibitors (chelerythrine, wortmannin, and N-nitro-L-arginine methyl ester). Cell death was determined by triphenyl tetrazolium chloride staining technique. Post-ischemic recovery of contractility, oxidative stress, and content of phosphorylated forms of PKCε, Akt, and eNOS were also examined. Mitochondrial state was assessed through the measurement of calcium-mediated response, calcium retention capacity, and mitochondrial potential. RESULTS: Isoesp limited cell death, decreased post-ischemic dysfunction and oxidative stress, improved mitochondrial state, and increased the expression of PKCε, Akt, and eNOS phosphorylated. All these beneficial effects achieved by Isoesp were annulled by the inhibitors. CONCLUSION: These findings suggest that activation of Akt/eNOS and PKCε signaling pathways are involved in the development of Isoesp-induced cardiac and mitochondria tolerance to ischemia-reperfusion.

Participation of NO in the vasodilatory action of isoespintanol / Participación de NO en la acción vasodilatadora del isoespintanol

Background: accumulating evidence suggests that natural compounds and specifically monoterpenes exert a vasodilator action. Objetive: to investigate the vascular effects of isoespintanol (2-isopropil-3,6-dimetoxi-5-metilfenol, ISO) monoterpene isolated from the leaves of Oxandra cf xylopioides. Methods: thoracic aortic rings isolated from Wistar rats were contracted with KCl 80 mM and then relaxed by exposure to Ca2+-free solution in absence and in presence of ISO 0.6 mg/mL. The force/tissue ratio (F/W) and the time to obtain 50% of relaxation (T-50) were used to assess the maximal contractile response and the relaxation, respectively. To examine the participation of NO additional experiments were performed under inhibition of nitric oxide synthase with L-NAME (L-NG-Nitroarginine methyl ester). Results: ISO significantly decreased the F/W ratio (257 ± 19 vs. 360 ± 18) and did not change T-50. In presence of L-NAME the effects of ISO on contractile response was abolished. Conclusions: these results demonstrate that ISO exerts a vasodilator effect through NO-dependent pathways and suggest that an inhibition of calcium influx could be the involved mechanism

Antecedentes: la evidencia acumulada sugiere que los compuestos naturales, especialmente monoterpenos, ejercen una acción vasodilatadora. Objetivo: investigar los efectos vasculares del monoterpeno isoespintanol (2-isopropil-3,6-dimetoxi-5-metilfenol, ISO) aislado de hojas de Oxandra cf xylopioides. Métodos: anillos de aorta torácica aislados de ratas Wistar fueron contraídas con cloruro de potasio 80 mM y luego relajadas por exposición a una solución libre de Ca2+ en ausencia y presencia de isoespintanol 0,6 µg/mL. El radio fuerza/tejido (F/T) y el tiempo para obtener 50% de relajación (T50) se usaron para lograr la máxima respuesta contráctil y de relajación, respectivamente. Para evaluar la participación del óxido nítrico, se realizaron experimentos adicionales bajo la inhibición de la óxido nítrico sintetasa con L-NAME (L-NG-éster metílico de nitroarginina). Resultados: Isoespintanol disminuyó el radio F/W significativamente (257 ± 19 vs. 360 ± 18) y no cambió T-50. En presencia de L-NAME, los efectos del isoespintanol en la respuesta contráctil fueron suprimidos. Conclusiones: Estos resultados demuestran que el isoespintanol ejerce un efecto vasodilatador a través de vías NO dependientes y sugiere que la inhibición de la entrada de calcio puede ser el mecanismo involucrado.

Intestinal, urinary and uterine antispasmodic effects of isoespintanol, metabolite from Oxandra xylopioides leaves.

BACKGROUND: Isoespintanol is a monoterpene isolated from the leaves of Oxandra xylopioides Diels. (Annonaceae) with antioxidant and antiinflammatory effects. It was of interest to know whether it has antispasmodic effects such as other known drugs, phloroglucinol and trimethoxybenzene, used in therapeutics for treating biliary, urinary and uterine spasms. PURPOSE: To assess whether isoespintanol possesses antispasmodic effects on intestine, uterus and bladder. STUDY DESIGN: A preclinical study was performed in which isoespintanol, phloroglucinol and trimethoxybenzene were evaluated with concentration-contractile response curves (CRC) of carbachol in isolated rat intestine and bladder, and with CRC of serotonin (5-HT) in rat uterus. Moreover, it was assessed whether isoespintanol interferes with Ca2+ influx by making CRC of Ca2+ in high-K+ medium in intestine and bladder. RESULTS: Isoespintanol non-competitively inhibited the CRC of carbachol with affinity constant (pK) of 4.78 ±â€¯0.09 in intestine and 4.60 ±â€¯0.09 in bladder. Phloroglucinol and trimethoxybenzene were also non-competitive antagonists, but isoespintanol was 8 times more potent than trimethoxybenzene and similarly potent than phloroglucinol in intestine. In bladder, isoespintanol resulted 8 times more potent than trimethoxybenzene. The maximal inhibition of contraction followed the order of isoespintanol > trimethoxybenzene > phloroglucinol in intestine, and isoespintanol > trimethoxybenzene in bladder. Moreover, isoespintanol also completely and non-competitively inhibited the CRC of Ca2+, with a pK of 5.1 ±â€¯0.1 in intestine, and 4.32 ±â€¯0.07 in bladder. In uterus isoespintanol reduced, completely and non-competitively, the contraction produced by 5-HT with pK of 5.05 ±â€¯0.07. CONCLUSION: Results demonstrate that isoespintanol is a very good intestinal, urinary and uterine antispasmodic, with higher potency than the other drugs used in therapeutics. The mechanism of action of isoespintanol is the interference with Ca2+ influx, at a difference of trimethoxybenzene and phloroglucinol.

Apoptotic activity of isoespintanol derivatives in human polymorphonuclear cells / Actividad apoptótica de derivados semisintéticos del isopentanol en células polimorfonucleares humanas

Background: Inflammation is a complex physiopathologic response to different stimuli. Recently, some pharmacological strategies have been proposed that could be used for resolution of inflammation by enhancing apoptosis of inflammatory cells. Objectives: To study in vitro apoptotic activity of isoespintanol [ISO] and of two semi-synthetic derivatives, bromide isoespintanol [BrI] and demethylated isoespintanol [DMI], in human polymorphonuclear (PMN) cells. Methods: PMN were exposed to the different concentrations of ISO, BrI and DMI for 30 min in phosphate-buffered saline pH 7.4 containing 1 mg/mL glucose, 0.4 mM Mg2+, and 1.20 mM Ca2+. Viability was assessed by dimethylthiazol diphenyl tetrazolium bromide (MTT). To distinguish between the two modes of cell death, apoptosis and necrosis, we examined differences in morphological and biochemical changes of cells stained with annexin V- FITC (An) and/or propidium iodide (PI) using two different assays based on flow cytometry Results: The MTT assay revealed the ability of cells to reduce MTT salt to formazan. In the presence of BrI and DMI a significant concentration-dependent decrease of cell viability was observed. The annexin V- FITC binding assay showed a high proportion of apoptotic cells for those treated with BrI (An+/ PI- : 62.3 ± 8.2% vs. 2.1 ± 0.5% of control, P<0.05). The population of PMN treated with DMI produced the highest percentage (An+/IP+: 43.4 ± 5.2 % vs. 0.4 ± 0.3 % of control, P<0.05) of necrotic cells. Apoptotic nuclei were analyzed by PI staining. The cell population in the sub G0/G1 region represents cells with hypodiploidal DNA, an indicator of apoptosis. When cells were incubated with 50 and 100 µM of BrI, the cell population in the sub G0/G1 region increased, suggesting a dose-dependent increase in the population of apoptotic cells. The presence of the pan-inhibitor of caspases (Z-VAD-fmk) showed a significant reduction in cell population in the sub G0/G1 region, indicating less degradation of DNA. Conclusions: Bromide isoespintanol [BrI] induces an apoptotic process in PMN, mediated ­at least in part­ by activation of caspases, although this compound may probably act through other caspase-independent mechanisms as well.

Antecedentes: La inflamación es una respuesta fisiopatológica compleja generada por diferentes estímulos. Recientemente, se han propuesto nuevas estrategias farmacológicas que podrían ser utilizadas para conducir a la resolución de la inflamación mediante el aumento de la apoptosis de células inflamatorias. Objetivo: Estudiar la actividad apoptótica in vitro del isopentanol [ISO] y dos derivados semisintéticos ­bromuro de isoespintanol [BrI] e isoespintanol desmetilado [DMI] ­ en células polimorfonucleares humanas (PMN). Métodos: Las PMN fueron expuestas a diferentes concentraciones de los compuestos durante 30 min en una disolución salina tamponada con fosfato (pH 7,4). La viabilidad celular se evaluó utilizando el ensayo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difenil tetrazolio (MTT). Para distinguir entre los dos modos de muerte celular, la apoptosis y la necrosis, se examinaron las diferencias en los cambios morfológicos y bioquímicos de las células teñidas con anexina V (An) y/o yoduro de propidio (PI) usando dos técnicas de citometría de flujo. Resultados: Mediante el ensayo con MTT, se demostró que los compuestos BrI y DMI disminuyeron significativamente y de manera concentración-dependiente la viabilidad celular. El ensayo de unión con la anexina V-FITC mostró una alta proporción de células apoptóticas en las células tratadas con BrI (An+/PI- : 62,3 ± 8,2% versus 2,1 ± 0,5% del control, P<0,05). El análisis de núcleos apoptóticos se llevó a cabo a través de tinción con PI. La población de células en la región sub G0/G1 representa células con ADN hipodiploidal, que es un indicador de apoptosis. Cuando las células se incubaron con BrI, la población de células en la región sub G0/G1 aumentó, confirmando su mecanismo citotóxico. En presencia de un inhibidor de caspasas (Z-VAD-FMK), se observó una reducción significativa en la población celular en la región sub G0/G1, indicando una menor degradación del ADN. Conclusiones: El bromuro de isoespintanol [BrI], induce un proceso apoptótico en PMN que está mediado ­al menos en parte­ por la activación de las caspasas, aunque este compuesto probablemente podría actuar también a través de otros mecanismos independientes de las caspasas.

Synthesis and attioxidant activity of two isoespintanol derivatives / Síntesis y ativiidad antioxidante de dos derivados de isoespintaoll / Síntese eatividaade antioxidante dos derivados do isoespintanol

The antioxidant activity of isoespintanol (1) hemisynthetic analogues, 4-bromo-2-isopropyl-3,6-dimethoxy-5-methylphe-nol (2) and 3-isopropyl-6-methylbenzene-1,2,4-triol (3), was evaluated using ABTS, DPPH and FRAP assays. Partial rationalization of the results is provided in terms of quantum chemical calculations of bond dissociation enthalpy (BDE) and ionization potential (IP).

La actividad antioxidante de los análogos hemisintéticos del isoespintanol (1), 4-bromo-2-isopropil-3,6-dimetoxi-5-metil-fenol (2) y 3-isopropil-6-metilbenceno-1,2,4-triol (3), se evaluó empleando los ensayos ABTS, DPPH y FRAP. La racionalización de los resultados es provista de forma parcial en términos de cálculos cuánticos de entalpia de disociación de enlace (BDE) y potencial de ionización (IP).

A atividade antioxidante dos análogos hemi-sintéticos de isoespintanol (1), 4-bromo-2-isopropil-3,6-dimetoxi-Smetilfenol (2) e 3-isopropil-6metilbenze-no- 1,2,4-triol (3 foi avahada através das técnicas de ABTS, DPPH e FRAP A racionalizagáo dos resultados está prevista, em parte, em termos de cálculos quánticos de entalpia de dissociagáo da ligagáo (BDE) e potencial de ionizagáo (IP).

Determinación d la actividad antioxidante en u modelo de peroxidación lipídicca de mantequilla inhibida por el isoespintanol / Anioxidant activvity determinationin a lipidic peroxidation modell of butter inhibited by isoespintanol

En este trabajo se evalúa la actividad antioxidante (Φ), definida como la pendiente de la regresión simple lineal de f o /f i contra la concentración µM, en un modelo de inhibición de la peroxidación lipídica de mantequilla a 60ºC, donde fi es el efecto de cada concentración del isoespintanol y fo es el control. Se encuentra que la formación de hidroperóxidos (valor de peróxido PV) y las especies mas oxidadas reactivas al ácido tiobarbiturico (TBARS) fueron retardadas de manera similar por el isoespintanol (ΦTBARS=9.46x10-5 ± 1.33x10-5 y ΦPV = 9.41x10-5 ± 1.62x10-5). De igual manera, el butilhidroxi tolueno y el isoespintanol inhiben el proceso oxidativo de mantequilla en un 28.0 y 23.3 % a concentraciones de 2380.9 y 3809.5 µM respectivamente.

The antioxidant activity (Φ) is defined as the slope of the simple linear regression of f0/f i against the concentration µM, in an inhibition model of lipid peroxidation of butter at 60° C, where f i is the concentration effect of isoespintanol and f0 is the control. It is found that the formation of: hydroperoxides (peroxide value of PV) and the most oxidized species reactive to thiobarbituric acid (TBARS), were similarly delayed by isoespintanol (ΦTBARS = 9.46x10-5 ± 1.33x10-5 and ΦPV = 9.41x10-5 ± 1.62x10-5). Likewise, the butilhidroxy toluene and isoespintanol inhibit the oxidative process of butter in a 28.0 and 23.3% at concentrations of 2380.9 and 3809.5 µ M respectively.