Casearia tomentosa fruit extracts exposed larvicidal activity and morphological alterations in Culex quinquefasciatus and Aedes albopictus under in vitro and semi field conditions.

Mosquitoes are notorious insects that transmit a wide range of infectious diseases, including zika, malaria, chikungunya, filariasis, and dengue. The overuse and incorrect application of synthetic pesticides to control mosquitoes has resulted in resistance development and environmental contamination, both of which have had a negative impact on human health. To address this issue, the larvicidal and pupicidal potential of acetone extract from Casearia tomentosa fruits was investigated. The extract was evaluated in a lab setting against all larval instars and pupa of Culex quinquefasciatus and Aedes albopictus, as well as against third instar larvae in a semi-field condition. Purified compounds through TLC were also tested against 3rd instar larvae of both mosquito and non-target organisms. The FT-IR and GC-MS analyses were used to characterise the extract. Morphological aberration caused by the acetone extract was observed using FESEM. The anal gills and respiratory siphon of both mosquitoes showed significant deformation from their normal state. 100 ppm was found to cause 100% larval mortality at 24 h of exposure in case of Cx. quinquefasciatus and at 72 h of exposure in Ae. albopictus larvae. After 72 h of exposure under in vitro conditions, the extract demonstrated considerable larvicidal activity with LC50 values of 38.33 and 47.56 against 3rd instar larvae of Culex quinquefasciatus and Aedes albopictus, respectively. The acetone extract can be considered as a highly effective mosquito larvicidal agent that is safe for the environment.

Maternal-fetal safety evaluation of an aqueous extract of Casearia sylvestris leaves in rats.

BACKGROUND: This study evaluated the maternal, embryotoxic, and teratogenic effects of the aqueous extract of Casearia sylvestris (AECS), a species listed in the Unique Health System of Brazil, and widely used for treating several conditions, such as diarrhea, wounds, pain, and ulcers. METHODS: Pregnant rats were daily treated orally with 0, 175, 350, or 700 mg/kg/body weight of AECS, from gestational day (GD) 6 to 15 (organogenesis period). On GD 20, the pregnant rats were euthanized, and the litters submitted to an assessment of fetal development. RESULTS: No clinical signs of toxicity were observed in the dams during the treatment. In the embryo-fetal development study, a significant increase in the basal zone height of the placenta was observed in the intermediate dose group. Furthermore, there was a significant increase in the relative anogenital distance measurement of female fetuses in the lowest and intermediate dose groups. Although no visceral abnormalities were observed in the treated-fetuses, skeletal anomalies evidenced by changes in the ossification of the sternum and the presence of supernumerary ribs were found in the intermediate and high dose groups. CONCLUSION: In conclusion, the treatment with AECS during organogenesis at this dose level had detrimental effects on the normal development of fetuses.

Stereochemical assignment of clerodane-type diterpenes from the fruits of Casearia grewiifolia and their ability to inhibit PCSK9 expression.

More than 20 natural products have been reported to modulate PCSK9-mediated cholesterol regulation, and small-molecule-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors continue to be developed and identified. Here, twelve undescribed clerodane-type diterpenes (1-9 and 12-14) and two known compounds were isolated from the chloroform-soluble extract of the dried fruits of Casearia grewiifolia Vent. using a PCSK9 mRNA expression monitoring assay. Among the undescribed compounds, the stereochemistry of two diastereomeric grewiifolins A and B (1 and 2) were extensively elucidated using 2D Nuclear Overhauser Effect Spectroscopy (NOESY) experiments, excitation-sculptured indirect detection experiments (EXSIDE), interproton distance analyses, and computational calculations that included quantum chemical shift calculations combined with DP4+ analysis. All isolates were assessed for their inhibitory activity against PCSK9 and IDOL mRNA expression. Among the compounds tested, compound 3 inhibited PCSK9 and IDOL mRNA expression.

Bioactive diterpenoids isolated from the twigs and leaves of Casearia velutina.

Nine previously undescribed clerodane-type diterpenoids (1-9), named caseabalanspenes A-I, along with six know compounds (10-15), were isolated from the twigs and leaves of Casearia velutina. Spectroscopic data (1D and 2D NMR) analysis permitted the definition of their structures and then determination of the molecular formula of the compound by high resolution mass spectrometry (HR-ESI-MS). It is worth noting that compound 7 contains N- heterocycle. Compounds 1-8 were tested the anti-inflammasome activity, and compound 3 exhibited potent activity and decreased LDH level in a dose-dependent manner, with IC50 values of 2.90 µM.

Casearia Essential Oil: An Updated Review on the Chemistry and Pharmacological Activities.

Casearia species are found in the America, Africa, Asia, and Australia and present pharmacological activities, besides their traditional uses. Here, we reviewed the chemical composition, content, pharmacological activities, and toxicity of the essential oils (EOs) from Casearia species. The EO physical parameters and leaf botanical characteristics were also described. The bioactivities of the EOs from the leaves and their components include cytotoxicity, anti-inflammatory, antiulcer, antimicrobial, antidiabetic, antioxidant, antifungal, and antiviral activities. The main components associated with these activities are the α-zingiberene, (E)-caryophyllene, germacrene D, bicyclogermacrene, spathulenol, α-humulene, ß-acoradiene, and δ-cadinene. Data on the toxicity of these EOs are scarce in the literature. Casearia sylvestris Sw. is the most studied species, presenting more significant pharmacological potential. The chemical variability of EOs components was also investigated for this species. Caseria EOs have relevant pharmacological potential and must be further investigated and exploited.

16,17-dinor-abietane diterpenoids from Casearia kurzii.

Eleven undescribed 16,17-dinor-abietane diterpenoids, caseazins A-K (1-11), and ten known diterpenoids (12-21) were isolated from the twigs and leaves of Casearia kurzii (Flacourtiaceae). Caseazins A-K were the first abietane -type dinorditerpenoids to have been isolated from the plant of Casearia kurzii. Their chemical structures were elucidated using a combination of 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configurations of 5 and 10 were established by electronic circular dichroism calculations. Moreover, compounds 2, 3, 13, 14, and 18 exhibited anti-inflammatory activity with IC50 values of 0.17, 0.36, 6.55, 1.30, and 4.53 µM, respectively. IL-1ß and caspase-1 analyses suggested that compound 14 inhibited NLRP3 inflammasome activation and blocked macrophage pyroptosis.

Bioactive Clerodane Diterpenoids from the Leaves of Casearia coriacea Vent.

Casearia coriacea Vent., an endemic plant from the Mascarene Islands, was investigated following its antiplasmodial potentialities highlighted during a previous screening. Three clerodane diterpene compounds were isolated and identified as being responsible for the antiplasmodial activity of the leaves of the plant: caseamembrin T (1), corybulosin I (2), and isocaseamembrin E (3), which exhibited half maximal inhibitory concentrations (IC50) of 0.25 to 0.51 µg/mL. These compounds were tested on two other parasites, Leishmania mexicana mexicana and Trypanosoma brucei brucei, to identify possible selectivity in one of them. Although these products possess both antileishmanial and antitrypanosomal properties, they displayed selectivity for the malaria parasite, with a selectivity index between 6 and 12 regarding antitrypanosomal activity and between 25 and 100 regarding antileishmanial activity. These compounds were tested on three cell lines, breast cancer cells MDA-MB-231, pulmonary adenocarcinoma cells A549, and pancreatic carcinoma cells PANC-1, to evaluate their selectivity towards Plasmodium. This has not enabled us to establish selectivity for Plasmodium, but has revealed the promising activity of compounds 1-3 (IC50 < 2 µg/mL), particularly against pancreatic carcinoma cells (IC50 < 1 µg/mL). The toxicity of the main compound, caseamembrin T (1), was then evaluated on zebrafish embryos to extend our cytotoxicity study to normal, non-cancerous cells. This highlighted the non-negligible toxicity of caseamembrin T (1).

Caseatardies A-K, eleven undescribed clerodane diterpenoids isolated from Casearia tardieuae and their anti-inflammatory activity.

A phytochemical investigation to obtain bioactive substances as lead compounds or agents for anti-inflammatory led to the obtainment of eleven previously undescribed clerodane diterpenoids, named caseatardies A-K (1-11), and four known clerodane diterpenoids (12-15) from the twigs and leaves of Casearia tardieuae. The structural elucidation of these clerodane diterpenoids was based on 1D and 2D-NMR spectroscopy (COSY, HSQC, HMBC and ROESY) as well as high resolution mass spectrometry (HR-ESI-MS). The relative configurations were defined by ROESY correlations. The anti-inflammatory activity of all the isolated compounds was screened and compound 15 decreased LDH level in a dose-dependent manner, showing IC50 value of 2.89 µM.

Gastroprotective and gastric healing effects of the aqueous extract of Casearia sylvestris in rodents: Ultrasound, histological and biochemical analyzes.

ETHNOPHARMACOLOGICAL IMPORTANCE: Casearia sylvestris Sw. (Salicaceae) is a native plant from the Americas, where it is also known as "guaçatonga" or "erva-de-bugre." Although its leaves have been commonly used to treat inflammation and gastrointestinal disorders in South America, the antiulcer effects of an aqueous extract from this medicinal plant, similar to popular use, have not to be investigated yet. AIM OF THE STUDY: This study evaluated the hypothesis that the aqueous extract a of C. sylvestris (AEC) prevents the gastric ulcers and accelerates the healing of ulcers already installed, by assessing ultrasound imaging, histological and biochemical analyses. MATERIALS AND METHODS: Rats (females) were treated with AEC (3, 30 or 300 mg/kg) prior to the ethanol or piroxicam-induced gastric ulcers. The healing effect of AEC (300 mg/kg) was examined in 80% acetic acid-induced ulcer in rats, whereas the quality of healing was evaluated in recurrent 10% acetic acid-induced ulcer in mice with recurrence induced by interleukin 1ß. To assess the responses of the lesions, in addition to the classical methods used to analyze gastroprotection (ex vivo), we also measured the gastric wall thickness (in vivo) using ultrasonography. After euthanasia, the extent of ulcer was determined and the levels of reduced glutathione (GSH), lipid hydroperoxides (LOOH), nitrate, and the activities of myeloperoxidase (MPO), N-acetyl-ß-D-glycosaminidase (NAG), superoxide dismutase (SOD), and glutathione S-transferase (GST) were measured. The antisecretory activity of AEC was also examined based on pylorus ligated rats. Furthermore, gastric tissue samples were analyzed histologically, and phytochemical analyses of the C. sylvestris extract were parallelly performed. RESULTS: The AEC (30 or 300 mg/kg) prevented ulcers in the ethanol- and piroxicam-induced acute. Moreover, the AEC at a dose of 300 mg/kg also accelerated the gastric healing of acetic acid-induced ulcer in rats by 48% and the ultrasonography records shown a decrease in the wall thickness and the extent of edema of ulcerous lesions promoted by the extract. The gastric healing effect of AEC was also accompanied by reduced MPO and NAG activities at acetic acid-induced ulcer in rats; as well as was by the reduction in the nitrate and LOOH levels, the increase in mucin and SOD activity, and by a partial recovery of GSH levels. The AEC (300 mg/kg) minimized the ulcer recurrence in mice exposed to IL-1ß, but the extract administration did not change pH or peptic activity of gastric juice in pylorus ligated rats. CONCLUSION: The results of this study provide convincing evidence for the therapeutic efficacy of C. sylvestris with respect to gastroprotection and indicate that ultrasound examination would be a potentially promising approach for evaluating gastroprotective effects in vivo. Collectively, our findings indicate that the gastric the gastroprotective and healing effects of aqueous extract C. sylvestris involve a reduction in acid secretion, promotion of the antioxidant system, reductions in the migration of neutrophils and mast cells, with a consequent lower inflammatory response, and the preservation of mucin.

Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators.

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABAA receptors with an α1ß2γ2 subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC50 values of 0.5, 4.6, and 1.4 µM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABAA receptors of the α1ß2γ2S and α1ß2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC50 (α1ß2γ2S) = 43.6 µM; Emax = 809% and EC50 (α1ß2) = 57.6 µM; Emax = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

α-zingiberene, a sesquiterpene from essential oil from leaves of Casearia sylvestris, suppresses inflammatory angiogenesis and stimulates collagen deposition in subcutaneous implants in mice.

α-zingiberene is a phytochemical of the sesquiterpenes class, the major constituent of the essential oil from the leaves of Casearia sylvestris, a plant widely used in traditional medicine for the treatment of inflammatory diseases, tumours, and bacterial infections. In the present study, we evaluated the effects of daily administration of α-zingiberene (0.01, 0.1 and 1 µg diluted in 10 µl of 0.5% DMSO) on the inflammatory, angiogenic, and fibrogenic components, induced by subcutaneous sponge implants in an animal model. Treatment with sesquiterpene resulted in a reduction in macrophage activation, as well as in mean blood vessels and in the activity of metalloproteinases 2 and 9. Furthermore, it resulted in an increase in collagen deposition near the implants. These results show the therapeutic potential of α-zingiberene in the treatment of pathologies, in which processes such as inflammation and angiogenesis are exacerbated, or even for the treatment of chronic wounds.

In Vitro Schistosomicidal Activities of the Leaf Extracts from Casearia sylvestris Varieties.

Schistosomiasis, a tropical disease caused by flatworms, may affect the liver, spleen, bladder, and intestine. Casearia sylvestris Swartz, a medicinal plant, displays antiprotozoal, antimicrobial, antifungal, and antiulcer activities. We have evaluated the in vitro schistosomicidal activity of two C. sylvestris varieties against Schistosoma mansoni adult worms at concentrations between 12.5 and 200 µg/mL. At 100 and 200 µg/mL, the ethanolic C. sylvestris var. sylvestris leaf extract enriched in casearin-like diterpenes eliminated 100 % of the parasites after incubation for 72 h and 48 h, respectively, whilst the same extract at 200 µg/mL eliminated 96 %, 100 %, and 100 % of the parasites after incubation for 24, 48, and 72 h, respectively. On the other hand, the hydroalcoholic C. sylvestris var. lingua leaf extract at 200 µg/mL eliminated 60.4 and 66.7 % of the parasites after incubation for 48 and 72 h, respectively. The presence of casearin-like diterpenes and glycosylated flavonoids was confirmed based on chromatographic techniques and mass spectrometry data.

Salicinoyl Quinic Acids and Their Prostaglandin E2 Production Inhibitory Activities from the Fruits of Casearia grewiifolia.

Phytochemical investigation on the dried fruits of Casearia grewiifolia led to the identification of 10 new salicinoyl quinic acid derivatives (1-10), a new benzoyl quinic acid (11), and two known compounds (12 and 13). The structures of the new compounds were elucidated by interpreting 1D and 2D NMR spectroscopic data including HMBC and EXSIDE along with a chemical method for sugar unit analysis. All isolates were evaluated for their inhibitory activities against prostaglandin E2 (PGE2) production in ultraviolet B (UVB)-irradiated HaCat keratinocytes. Of the isolates tested, compounds 6 and 12 were found to inhibit PGE2 production with IC50 values of 20.5 and 28.8 µM, respectively.

Casearia sylvestris Essential Oil Degradation Products Generated by Leaf Processing.

Casearia sylvestris is an endemic tree of the Latin America that the essential oil (EO) has anti-inflammatory and gastroprotective actions. This study evaluates the chemical composition of the EO from the volatile fractions of in natura, fresh, and dried C. sylvestris var. sylvestris and var. lingua leaves. For both varieties, the dried leaves presented higher EO yield as compared to fresh leaves. The major EO chemical components were (E)-caryophyllene, α-humulene, germacrene D, bicyclogermacrene, spathulenol, caryophyllene oxide, and humulene epoxide II. In both varieties, the content of sesquiterpene hydrocarbons decreased and oxygenated sesquiterpenes increased on going from in natura to fresh and dried leaves, which indicated that leaf drying and hydrodistillation modified the volatile composition. The results also suggested that bicyclogermacrene and (E)-caryophyllene were oxidized during processing, to generate spathulenol and caryophyllene oxide, respectively. C. sylvestris varieties and in natura, fresh, and dried leaves differed in terms of the chemical composition of volatiles, which could affect the EO biological activities.

Identification of flavonoid-3-O-glycosides from leaves of Casearia arborea (Salicaceae) by UHPLC-DAD-ESI-HRMS/MS combined with molecular networking and NMR.

INTRODUCTION: Casearia is an essential source of cytotoxic highly oxidised clerodane diterpenes, in addition to phenolics, flavonoids, and glycoside derivatives. Here we identify flavonoid-3-O-glycoside derivatives in the ethyl acetate (EtOAc) fraction of the methanolic extract from leaves C. arborea leaves. OBJECTIVE: To characterise the EtOAc phase from the methanolic extract of C. arborea leaves using ultra-high-performance liquid chromatography diode array detector high-resolution tandem mass spectrometry (UHPLC-DAD-HRMS/MS) and molecular networking-based dereplication. Methodology We identified compounds not annotated in the GNPS platform by co-injection of standards in HPLC-DAD or by isolation and characterisation of the metabolites using nuclear magnetic resonance (NMR) spectroscopy. A workflow on the GNPS platform aided the organisation of spectral data and dereplication by annotations. We subjected the EtOAc phase to HPLC-DAD analysis using standard compound co-injection to corroborate the GNPS annotations. We isolated unidentified compounds with semi-preparative HPLC-DAD for structural identification using NMR. RESULTS: We annotated a molecular family of flavonoid-3-O-glycosides in the molecular networking created using the GNPS platform. These included avicularin, cacticin, isoquercitrin, quercitrin, rutin, and a quercetin-3-O-pentoside cluster. We confirmed the annotations with standard compounds using HPLC-DAD co-injection analysis, besides identifying quercetin-3-O-robinobioside and kaempferol. We isolated three flavonoid-3-O-pentosides and characterised them using one- and two-dimensional NMR; we identified them as reynoutrin, guaijaverin, and avicularin. CONCLUSION: This work describes the isolation of kaempferol and nine known flavonoid-3-O-glycosides from the polar fraction of the methanolic extract (EtOAc) from C. arborea leaves using molecular networking to guide the chromatographic procedures. We identified eight compounds for the first time in Casearia that amplify and reinforce the genus' chemotaxonomy with the presence of glycosylated flavonoids.

Macroscopic, biochemical and hystological evaluation of topical anti-inflammatory activity of Casearia sylvestris (Flacourtiaceae) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Inflammatory skin diseases presents high prevalence and lack of alternatives that can be used for self-care by the population. Casearia sylvestris is a plant used topically in different communities in Brazil, to treat wounds or promote cutaneous healing. To evaluate the topical anti-inflammatory activity for the crude hydroalcoholic extract of Casearia sylvestris (HCE-CS) in the models of single or multiple administration of chroton oil to induce ear edema in mice. MATERIALS AND METHODS: Experimental study using male Swiss mice (25-35g) kept under constant conditions in the Laboratory of Experimental Neuroscience (LaNEx)-UNISUL. Edema was induced in both models, respectively, by the single or multiple application of croton oil (CO, 2.5%, in 20 µl) on the external surface of the ear. The different groups of animals (n = 8) received different treatments: vehicle, dexamethasone (DEXA) or different doses of HCE-CS. Edema was evaluated macroscopically for 6 h (early edema) or 8 days (late edema) after the first application of the CO and immediately after the animals were submitted to euthanasia for the collection of the samples (treated ears). For early edema, the tissue was biochemically evaluated for myeloperoxidase activity (MPO) and levels of nitrite/nitrate. In the late edema model, the ears were histologically evaluated for general morphometry, degranulated and non-degranulated mast cells, as well as acanthosis. RESULTS: Topic treatment with HCE-CS significantly reduced the early and late edema, as well as MPO activity and tissue levels of nitrite/nitrate. Finally, in the late edema model there was a lower density of degranulated mast cells in relation to the vehicle treated group and decreased thickness of the epidermis (acanthosis). CONCLUSION: These results suggest a possible benefit of topical treatment with HCE-CS in inflammatory conditions of the skin.

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

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

Dolabellane and Clerodane Diterpenoids from the Twigs and Leaves of Casearia kurzii.

A pair of enantiomeric 15-nordolabellane diterpenoids, (-)- and (+)-caseadolabellols A (1a and 1b), three dolabellane diterpenoids, caseadolabellols B-D (2-4), two dolastane diterpenoids, caseadolastols A and B (5 and 6), 10 clerodane diterpenoids, caseakurzins A-J (7-16), and nine known diterpenoids (17-25) were isolated from the twigs and leaves of Casearia kurzii. The structures of the new compounds were established on the basis of extensive spectroscopic data, and those of compounds 1a, 1b, and 2 were verified by single-crystal X-ray crystallographic analysis. The enantiomers 1a and 1b were separated by chiral-phase HPLC. The absolute configurations were determined by experimental and calculated ECD data, the modified Mosher's method, or literature comparison. Compounds 1a and 5 showed significant quinone reductase-inducing activity in Hepa 1c1c7 cells, while 1b showed moderate activity. Molecular docking studies showed that 1a had greater binding affinity with Nrf2 protein (5FNQ) than 1b. The cytotoxic activity of compounds 1a, 1b, 2-12, 15, and 16 was evaluated, among which compounds 8 and 16 exhibited significant inhibitory activity against the A549 cell line. Compounds 8 and 16 induced the A549 cells to arrest at G2/M and S phases, respectively, and both compounds induced apoptosis in A549 cells.

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

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