Currently, studies on various natural products that aid in wound healing have seen significant growth. Betulin and its derivative, betulinic acid, are triterpenes abundant in the leaves and barks of certain species, such as birch, which has been of particular interest. Clinical and preclinical studies have demonstrated their ability to accelerate wound healing, as well as other properties such as antimicrobial, anti-inflammatory action, and anti-cancer properties, and beneficial effects on HIV, diabetes mellitus, and cardiovascular disorders. However, further research focusing exclusively on betulin and betulinic acid is needed to better understand its therapeutic potential and develop new medications for effective management. These advancements could greatly enhance the treatment of wounds, skin lesions and other diseases, offering more effective and safer therapeutic options to improve the quality of life of the population.

Phytochemical profile of the Central American rainforest endemic Annonaceae species, Annona pittieri and Cymbonopetallum costaricense, were studied in search of novel bioactive compounds. The acetogenin squamocin (1) isolated from A. pittieri showed cytotoxic activity against human acute lymphocytic leukaemia with low activity against healthy blood cells. In addition, other eight compounds were isolated from A. pittieri, including a novel 2-azaanthraquinone alkaloid, 4-methoxybenzo[g]isoquinoline-5,10-dione (2). Furthermore, from C. costaricense three compounds were isolated including a novel 1-azaanthraquinone alkaloid, 6-hydroxy-9-methoxy-cleistopholine (3).

Venoms of medically important scorpions from Buthidae family have been intensively studied, in contrast to non-buthid venoms, for which knowledge is scarce. In this work, we characterized the venom of a Diplocentridae species, Didymocentrus krausi, a small fossorial scorpion that inhabits the Tropical Dry Forest of Central America. D. krausi venom soluble fraction contains proteases with enzymatic activity on gelatin and casein. Mass spectrometry and venomic analysis confirmed the presence of elastase-like, cathepsin-O-like proteases and a neprilysin-like metalloproteinase. We did not detect phospholipase A2, C or D, nor hyaluronidase activity in the venom. By homology-based venom gland transcriptomic analysis, NDBPs, a β-KTx-like peptide, and other putative toxin transcripts were found, which, together with a p-benzoquinone compound present in the venom, could potentially explain its direct hemolytic and cytotoxic effects in several mammalian cell lines. Cytotoxicity of D. krausi venom was higher than the effect of venoms from two buthid scorpion species distributed in Costa Rica, Centruroides edwardsii and Tityus pachyurus. Even though D. krausi venom was not lethal to mice or crickets, when injected in mouse gastrocnemius muscle at high doses it induced pathological effects at 24 h, which include myonecrosis, weak hemorrhage, and inflammatory infiltration. We observed an apparent thrombotic effect in the skin blood vessels, but no in vitro fibrinogenolytic activity was detected. In crickets, D. krausi venom induced toxicity and paralysis in short periods of time.

Ethnopharmacological relevance: Calendula officinalis (pot marigold) flower extracts have a long-lasting tradition in ethnopharmacology. Currently, the European Medicines Agency (EMA) has approved its lipophilic and aqueous alcoholic extracts as traditional medicinal products for the treatment of minor inflammation of the skin and as an aid in the healing of minor wounds.Aim of the study: The purpose of this study was to analyse the molecular mechanism of the wound healing effects of Calendula extracts, which may reflect the phytomedicines currently used in the market.Materials and methods: The effect of three different extracts from Calendula flowers (n-hexanic, ethanolic, aqueous) on the inflammatory phase of wound healing was studied in human immortalized keratinocytes and human dermal fibroblasts. An electrophoretic mobility shift assay on NF-kappa B-DNA binding, qRT-PCR and ELISA experiments were performed. The effect of Calendula extracts on the new tissue formation phase of wound healing was evaluated by studying the migratory properties of these extracts, triterpene mixtures and single compounds in human immortalized keratinocytes using the scratch assay. Finally, the effect of the extracts on the formation of granulation tissue in wound healing was studied using bacterial collagenase isolated from Clostridium histolyticum and the determination of soluble collagen in the supernatant of human dermal fibroblasts.Results: The n-hexanic and the ethanolic extracts from Calendula flowers influence the inflammatory phase by activating the transcription factor NF-kappa B and by increasing the amount of the chemokine IL-8, both at the transcriptional and protein level, in human immortalized keratinocytes. The migration of the keratinocytes during the new tissue formation phase was only marginally influenced in the scratch assay. However, it can be assumed that the granulation tissue was affected, as the ethanolic extract inhibited the activity of collagenase in vitro and enhanced the amount of collagen in the supernatant of human dermal fibroblasts.Conclusions: Our results contribute to a better understanding of the wound healing properties of the traditional medicinal plant Calendula officinalis. However, further studies are necessary to evaluate which of its known constituents are responsible for these effects. Triterpenes seem to play only a marginal role, but carotene and xanthophyll derivatives should garner more attention in future studies.

Pentacyclic triterpene mono- and diesters have been isolated from Calendula officinalis flowers. GC-MS, APCI-Exactive Orbitrap HR-MS and NMR allowed to identify the triterpene skeleton in various samples (different triterpene mixtures from Calendula n-hexane extract). NMR provided evidence that triterpene diesters are present in the samples as well. However, the corresponding quasi-molecular ions could not be detected by APCI-Exactive Orbitrap HR-MS. Instability of triterpene diesters and loss of a fatty acid residue, respectively, in the ion-source made their MS detection challenging. Thus, a set of new APCI-QTOF-MS methods (using the TripleTOF 5600+ mass spectrometer) were developed which made it eventually possible to solve this problem and confirm the diester structures by MS via quasi-molecular ion [M+H](+) detection. Direct infusion APCI-QTOF MS experiments in MS/MS high sensitivity scan mode with low collision energy and multi-channel averaging acquisition (MCA) allowed the detection of quasi-molecular ions of triterpene diesters for the first time and unequivocally confirmed the presence of faradiol 3,16-dimyristate and -dipalmitate, as well as the corresponding mixed diesters faradiol 3-myristate,16-palmitate and faradiol 3-palmitate,16-myristate. Preferential loss of the fatty acid in 16-position made it possible to distinguish the mixed diesters by MS/MS spectra. Their chromatographic separations turned out to be challenging due to their bulkiness and extended molecular dimensions. However, separation could be achieved by an uncommon non-aqueous RPLC mode with an in-house synthesized C30 phase. Finally, two (U)HPLC-APCI-QTOF-MS methods with C18- and C30-based non-aqueous RPLC provided suitable, sensitive assays to monitor the presence of monoesters and diesters of various triterpenes (faradiol, maniladiol, arnidiol, arnitriol A and lupane-3β,16β,20-triol esters) in the n-hexane extract of C. officinalis with high mass resolution and good mass accuracy.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits.

Preparations of Deguelia duckeana, known in Brazil as timbó, are used by indigenous people to kill fish. Reinvestigation of its extracts resulted in the isolation and identification of 11 known flavonoids identified as 3,5,4'-trimethoxy-4-prenylstilbene (1), 4-methoxyderricidine (2), lonchocarpine (3), 4-hydroxylonchocarpine (4), 4-methoxylonchocarpine (5), 5-hydroxy-4',7-dimethoxy-6-prenylflavanone (6), 4'-hydroxyisolonchocarpine (7), 4'-methoxyisolonchocarpine (8), 3',4',7-trimethoxyflavone (9), 3',4'-methylenedioxy-7-methoxyflavone (10), and 2,2-dimethyl-chromone-5,4'-hydroxy-5'-methoxyflavone (11). Except for 1, 3, and 4 all of these flavonoids have been described for the first time in D. duckeana and the flavanone 6 for the first time in nature. Compounds 2, 3, 4, 7, 9, and 10 were studied for their potential to induce cell death in neuronal SK-N-SH cells. Only the chalcone 4 and the flavanone 7 significantly induced lactate dehydrogenase (LDH) release, which was accompanied by activation of caspase-3 and impairment of energy homeostasis in the MTT assay and may explain the killing effect on fish. Interestingly, the flavone 10 reduced cell metabolism in the MTT assay without inducing cytotoxicity in the LDH assay. Furthermore, the flavonoids 2, 3, 4, 7, and 10 induced phosphorylation of the AMP-activated protein kinase (AMPK) and the eukaryotic elongation factor 2 (eEF2). The initiation factor eIF4E was dephosphorylated in the presence of these compounds. The initiation factor eIF2alpha was not affected. Further studies are needed to elucidate the importance of the observed effects on protein synthesis and potential therapeutic perspectives.

Signs of malnutrition were observed in mice fed raw pejibaye (Bactris gasipaes) meal as a substitute of corn in the feed. No problems appeared when the animals were fed the same mixtures previously autoclaved. An aqueous extract of pejibaye meal inhibited the caseinolytic action of porcine pancreatine. The inhibiting effect disappeared when boiling the extract for one hour upon dialysis. ApparentIy a relatively low molecular weight pro tease inhibitor is present in raw pejibaye meal.

Recently we described an unusual way of activating a cryptic gene cluster when we explored the origin of the bald phenotype of Streptomyces calvus. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a new natural products. In this study we report on the expression of bldA in several Streptomyces strains that have been described as "poorly sporulating" strains. In seven out of 15 cases, HPLC profiling revealed the production of new compounds, and in two cases the overproduction of known compounds. Two compounds were isolated and their structures were determined.

Calendula officinalis L. (marigold) flower extracts are recommended for the treatment of minor inflammation of the skin and wounds [1]. Triterpenes from the 20-taraxastene type, especially faradiol seem to be crucial for the anti-inflammatory potential [2, 3]. Besides monols triterpene diols and triols are present in the form of their C3 lauric, myristic and palmitic acid monoesters. Here we report on the isolation of a mixture of novel faradiol fatty acid diesters (20-taraxastene 3β,16β-diester). 1D and 2D NMR analysis revealed the triterpene skeleton and the esterification position. However, only after UHPLC-QTOF-MS analysis the fatty acids could be identified and assigned to the respective triterpene. This method allowed identification of intact [M+H+]+ molecular ion peaks of faradioldimyristate (863.7354 m/z), faradioldipalmitate (919.8302 m/z) and faradiolmyristate, palmitate (891.7633 m/z). Moreover, this technique is also suitable for the separation and identification of the arnitriol A 3- and the lupane-3β,16β,20-triol 3-fatty acid monoesters which is here reported for Calendula officinalis L. flowers for the first time.

The anthocyanins, coumarins, and casearins are interesting compounds due to their chemical and biological properties. In the present study, the in vitro effect of 4 ́-O-methyl-gallocatechin (an anthocyanin), columbianatin (a coumarin), and casearin G on the mitogenic response and phagocytic activity from whole blood cells, was evaluated. In addition, the cytotoxic effect of these compounds on the cell lines B16/BL6 (murine melanoma) and COS-7 (kidney fibroblasts transfected with SV40 virus) was measured by a colorimetric assay (MTS/PMS, IC50: inhibitory concentration) and the sulphorhodamine B assay (GI50: growth inhibition, TGI: total growth inhibition, LC50: lethal concentration). The results show that 4 ́-O-methyl-gallocatechin and columbianatin reduced lymphoproliferation. Columbianatin reduced both the phagocytic index and the percentage of phagocytic monocytes/macrophages. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against the tumor cells, B16/BL6 (IC50 = 30.8 μM; GI50 = 12.4 μM; LC50 = 34.7 μM) and COS-7 (IC50 = 137.3 μM; GI50 = 3.8 μM; LC50 = 29.9 μM). In conclusion, 4 ́-O-methyl-gallocatechin and columbianatin showed immunosuppressive properties in vitro while casearin G was the most cytotoxic.

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 μM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.

Deguelia duckeana A.M.G. Azevedo (Fabaceae) is known as timbó and used by indigenous people for killing fish. Recently the phytochemical analysis showed flavonoids [1]. Reinvestigation of D. duckeana afforded further flavonoids identified by means of MS and 1H and 13C NMR as 3,5,4'-trimethoxy-4-prenylstilbene (1), 4-methoxyderricidine (2), lonchocarpin (3), 4-hydroxylonchocarpin (4), 4-methoxylonchocarpin (5), 5-hydroxy-4',7-dimethoxy-6-prenylflavanone (6), 4'-hydroxyisolonchocarpin (7), 5-hydroxy-4'-methoxyisolonchocarpin (8), 3',4',7-trimethoxyflavone (9), 3',4'-methylenedioxy-7-methoxyflavone (10), 2”,2”-dimethypyrano-5,4'-dihydroxy-5'-methoxyflavone (11). Except of 1, 3, and 4 all these flavonoids have been described for the first time in D. duckeana.

Skin wound healing is a well organized process to restore tissue integrity and can be divided into three overlapping phases; inflammation, new tissue formation and remodelling. Complications in the wound healing process are a high physiological and psychological interface for patients and a tremendous socio- economic problem [1]. Calendula officinalis L. (marigold) flower extracts are recommended for the treatment of wounds by the Pharm. Eur., the ESCOP, the WHO and the EMA. Despite some studies for the wound healing activity the active components are still a matter of debate. In addition to polysaccharides, carotenoids and triterpene saponins, free and esterified triterpenes are major constituents of Calendula officinalis L. flowers with proven anti-inflammatory activity in the Croton oil ear test [2]. Previously Fronza et al. observed remarkable effects for the myristic and palmitic acid ester of faradiol, the major triterpene in Calendula officinalis L. flowers, in the scratch assay using 3T3-mouse fibroblasts [3]. Here we report on phytochemical studies of the hexane extract which afforded the isolation of 6 triterpene alcohols from the psi-taraxene, taraxene, oleanene, lupene and dammarane type, of which 3 are described for Calendula officinalis L. flowers for the first time. Some of the isolated triterpenes were studied for their effect on the migration and proliferation of keratinocytes in the scratch assay and first structure-activity-relationships are drawn. Studies are in progress to gain further insights in the complex wound healing mechanism of preparations from Calendula officinalis L. flowers.

Four new flavonol glycosides were isolated from the leaves of Brugmansia suaveolens: kaempferol 3-O-β-D-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside (1), kaempferol 3-O-β-D-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside-7-O-į-D-gluco-pyranoside (2), kaempferol 3-O-β-D-[6'''-O-(E-caffeoyl)]-glucopyranosyl-(1'''→2'')-O-α-l-arabinopyranoside-7-O-β-D-glucopyranoside (3), and kaempferol 3-O-β-D-[2'''-O-(E-caffeoyl)]-glucopyranosyl-(1'''→2'')-O-α-l-arabinopyranoside-7-O-β-D-glucopyranoside (4). The structure elucidation was performed by MS, 1D and 2D NMR analyses.

De las raices de Croton hirtus (Euphorbiaceae), recolectadas en Paraiso de Cartago (Costa Rica) se aislaron, a parte de unos pocos compuestos conocidos, 20 compuestos que no habían sido publicados antes, el bisnordolabradano 1, los dolabradanes 2 y 3, los kauranes 4-7, cyclopropakauranos 8 y 9, los hirtusanos 10-15, los ésteres de germacreno 16-18, los elemanos 19 y 20 y un compuesto C-25, 21, el cual es formado presumiblemente por una reacción de Diels-Alder entre el compuesto 18 y un monoterpeno. Todas las estructuras fueron elucidadas con la utilización de técnicas de RMN de una y dos dimensiones. Los patrones de fraccionamiento de los compuestos nuevos también se registran. Las separaciones fueron llevadas a cabo con técnicas cromatográficas modernasThe roots of Croton hirtus (Euphorbiaceae) collected in Paraiso, Cartago (Costa Rica) afforded, in addition to the few known metabolites, 20 new compounds: the bis-nor dolabradane 1, the dolabradanes 2 and 3, the kauranes 4-7, the cyclopropakauranes 8 and 9, the hirtusanes 10-15, the germacradiene esters 16-18 and the C-25 compound 21, presumably formed by a Diels-Alder reaction between compound 18 and a monoterpene. All structures were elucidated using high field 1D and 2D NMR techniques. MS fragmentation patterns are here reported. The absolute configurations of 4 and 9 were elucidated by using circular dichroism. The separation was performed with modern chromatographic technics

The leaves of Zuelania guidonia yielded eight new clerodane diterpenes, namely, zuelaguidins A-H (1-8), and the known clerodane diterpene esculentin A (9). Some of these structures contained a 3,6-dihydro-1,2-dioxin moiety. The new compounds were isolated and identified using 1D- and 2D-NMR experiments. All compounds were evaluated for cytotoxicity against the CCRF-CEM (human acute lymphocytic leukemia), CEM-ADR5000 (human acute lymphocytic leukemia resistant to doxorubicin), and MIA-PaCa-2 (human pancreatic carcinoma) cell lines as well as for their selectivity against peripheral blood mononuclear cells from healthy human subjects. Zuelaguidins B, C, and E were the most potent compounds against the CCRF-CEM cell line, with IC50 values ranging from 1.6 to 2.5 mu M.

BACKGROUND:The rainforest is an important source of natural compounds with therapeutic properties. Although there are many anti-inflammatory and antineoplastic drugs available to the clinician, there is an ongoing need for new therapeutic drugs with fewer serious adverse effects.AIM:To evaluate the in vitro cytotoxic effects of lupeol and casearin G on tumor cells, on phagocytic activity and nitric oxide (NO) production by blood mononuclear cells.MATERIAL AND METHODS:The cytotoxic effect of these compounds on cell lines MCF-7 (human breast adenocarcinoma) and PC-3 (human prostate cancer) was measured by a colorimetric assay (MTS/PMS) and the sulphorhodamine B assay. Peripheral blood mononuclear cells were obtained from eight healthy volunteers. The effect of these compounds on nitric oxide (NO) production was measured using the Griess reaction. Their effect on phagocytic activity of PBMC was also evaluated.RESULTS:Lupeol (≥ 2 mM) resulted in a reduction of both the phagocytic index and the percentage of phagocytic monocytes and macrophages. Treatment of monocytes/macrophages with lupeol (72 µM) and casearin G (4 µM) reduced the production of NO. Neither lupeol (< 969 µM) nor casearin G (< 55 µM) had cytotoxic effects on PBMC. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against tumor cells, PC-3 (IC50 = 12.5 µM; GI50 = 13.3 µM; LC50 = 51.9 µM) and MCF-7 (IC50 = 112.8 µM; GI50 = 11.8 µM; LC50 = 49.4 µM), as well as a hemolytic effect (≥ 182 µM).CONCLUSIONS:These observations indicate that lupeol and casearin G might be useful compounds in the preparation of anti-inflammatory drugs, whereas casearin G might be useful in the elaboration of antitumor drugs.

The rainforest is an important source of natural compounds with therapeutic properties. Although there are many anti-inflammatory and antineoplastic drugs available to the clinician, there is an ongoing need for new therapeutic drugs with fewer serious adverse effects.To evaluate the in vitro cytotoxic effects of lupeol and casearin G on tumor cells, on phagocytic activity and nitric oxide (NO) production by blood mononuclear cells.The cytotoxic effect of these compounds on cell lines MCF-7 (human breast adenocarcinoma) and PC-3 (human prostate cancer) was measured by a colorimetric assay (MTS/PMS) and the sulphorhodamine B assay. Peripheral blood mononuclear cells were obtained from eight healthy volunteers. The effect of these compounds on nitric oxide (NO) production was measured using the Griess reaction. Their effect on phagocytic activity of PBMC was also evaluated.Lupeol (≥ 2 mM) resulted in a reduction of both the phagocytic index and the percentage of phagocytic monocytes and macrophages. Treatment of monocytes/macrophages with lupeol (72 µM) and casearin G (4 µM) reduced the production of NO. Neither lupeol (< 969 µM) nor casearin G (< 55 µM) had cytotoxic effects on PBMC. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against tumor cells, PC-3 (IC50 = 12.5 µM; GI50 = 13.3 µM; LC50 = 51.9 µM) and MCF-7 (IC50 = 112.8 µM; GI50 = 11.8 µM; LC50 = 49.4 µM), as well as a hemolytic effect (≥ 182 µM).These observations indicate that lupeol and casearin G might be useful compounds in the preparation of anti-inflammatory drugs, whereas casearin G might be useful in the elaboration of antitumor drugs.