Detection, Isolation, and 1H NMR Quantitation of the Nitrile Glycoside Sarmentosin from a Bryophyllum pinnatum Hydro-Ethanolic Extract.

Bryophyllum pinnatum (Lam) Pers. (Crassulaceae) is widely used in folk medicine as leaf juice, aqueous, or hydro-ethanolic extracts. It is also listed as a medicinal plant in several countries such as France and Brazil. The main reported constituents are flavone glycosides, especially those with the rare 3-O-α-l-arabinopyranosyl-(1 → 2)-α-l-rhamnopyranoside moiety. Despite several phytochemical screenings indicating the presence of cyanide derivatives or alkaloids, there are no reports of nitrogenous metabolite characterization from this plant species. Nevertheless, the occurrence and the type of such compounds are of particular interest, as they may account for some of the numerous biological activities and ethnomedicinal uses described for B. pinnatum and could be regarded as chemical/taxonomic markers. Consequently, a hydro-ethanolic extract of B. pinnatum was investigated by using UHPLC-HRMS/MS and the nitrile glucoside sarmentosin was detected for the first time within the genus Bryophyllum/Kalanchoe. Considering the wide use of B. pinnatum and its closely related species for health purposes, the target metabolite was isolated by a combination of centrifugal partition chromatography in elution/extrusion mode and MPLC in order to confirm its structure. A linear, selective, precise, fast, and reliable 1H NMR quantitation method was then developed and validated and may become a tool for easy quality assessment of the plant species. The amount of sarmentosin was determined as 2.07% of the examined sample. Sarmentosin was also detected in Kalanchoe laciniata, confirming the occurrence of this compound within the genus.

Antifungal and Antibiofilm Activities of B-Type Oligomeric Procyanidins From Commiphora leptophloeos Used Alone or in Combination With Fluconazole Against Candida spp.

Commiphora leptophloeos (Burseraceae) is a medicinal plant native to Brazil which is popularly used for treating oral and vaginal infections. There has been no scientific evidence pointing to its efficacy in the treatment of these infections. Thus, this study sought to investigate the cytotoxic, antifungal, and antibiofilm activity of C. leptophloeos against Candida spp. and to isolate, identify, and quantify the content of B-type oligomeric procyanidins (BDP) in the extract of C. leptophloeos stem bark. The extract and the n-butanol fraction were obtained by maceration and liquid-liquid partition, respectively. Phytochemical analysis performed by HPLC-PDA/ELSD and FIA-ESI-IT-MS/MS allowed the identification and quantification of BDP in the samples. The application of centrifugal partition chromatography helped isolate BDP, which was identified by 1H NMR and MS analyses. Candida spp. reference strains and clinical isolates (including fluconazole-resistant strains) derived from the blood cultures of candidemic patients and the vaginal secretion of patients with vulvovaginal candidiasis were used for evaluating the antifungal and antibiofilm effects. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were determined by the microdilution technique, and biofilm inhibition was evaluated through crystal violet and XTT assays. The combined action of BDP with fluconazole was determined by the checkerboard method. The extract, the n-butanol fraction, and the BDP exhibited antifungal activity with MIC values ranging from 312.5 to 2500 µg/mL and were found to significantly reduce the biofilm formed in all the Candida strains investigated. BDP showed a fungicidal potential against strains of Candida spp. (especially against fluconazole-resistant strains), with MIC and MFC values ranging from 156.2 to 2500 µg/mL. In addition, the combined application of BDP and fluconazole produced synergistic antifungal effects against resistant Candida spp. (FICI = 0.31-1.5). The cytotoxic properties of the samples evaluated in human erythrocytes through hemolytic test did not show hemolytic activity under active concentrations. The findings of the study show that C. leptophloeos has antifungal and antibiofilm potential but does not cause toxicity in human erythrocytes. Finally, BDP, which was isolated for the first time in C. leptophloeos, was found to exhibit antifungal effect against Candida spp. either when applied alone or in combination with fluconazole.

Gastric Ulcer Healing Property of Bryophyllum pinnatum Leaf Extract in Chronic Model In Vivo and Gastroprotective Activity of Its Major Flavonoid.

Gastric ulcer is a common disease that develops complications such as hemorrhages and perforations when not properly treated. Extended use of drugs in the treatment of this pathology can provoke many adverse effects. Therefore, finding medicinal plants with gastroprotective and mucosal healing properties has gained increasing interest. Bryophyllum pinnatum (Crassulaceae), popularly known in Brazil as "saião" or "coirama," has been used to treat inflammatory disorders. It is rich in flavonoids, and quercetin 3-O-α-L-arabinopyranosyl-(1→2)-O-α-L-rhamnopyranoside-Bp1 is its major compound. In this study, we aimed to investigate ulcer healing properties of B. pinnatum against an acetic acid-induced chronic ulcer model and the gastroprotective activity of Bp1 against gastric lesions induced by ethanol and indomethacin. Ultrafast liquid chromatography was used to quantify the main compounds (mg/g of the extract)-quercetin 3-O-α-L-arabinopyranosyl-(1→2)-O-α-L-rhamnopyranoside (33.12 ± 0.056), kaempferol 3-O-α-L-arabinopyranosyl-(1→2)-O-α-L-rhamnopyranoside (3.98 ± 0.049), and quercetin 3-O-α-L-rhamnopyranoside (4.26 ± 0.022) and showed good linearity, specificity, selectivity, precision, robustness, and accuracy. In vivo studies showed that treatment with the extract at 250 and 500 mg/kg stimulated the healing process in the gastric mucosa with significant ulceration index reduction, followed by improvement in the antioxidant defense system [increased glutathione (GSH) levels, decreased superoxide dismutase upregulation, and malondialdehyde (MDA) levels]. Moreover, the extract decreased interleukin-1ß and tumor necrosis factor-a levels and myeloperoxidase (MPO) activity, increased interleukin 10 levels, showed a cytoprotective effect in histological analyzes and also downregulated the expression of cyclooxygenase-2 and NF-κB (p65). The pretreatment with Bp1 at a dose of 5 mg/kg reduced gastric lesions in the ethanol and indomethacin models, increased GSH, and decreased MDA levels. In addition, the pretreatment decreased MPO activity, interleukin-1ß and tumor necrosis factor-α levels, while also showing a cytoprotective effect in histological analyzes. Our study suggests that treatment with B. pinnatum extract showed a higher inhibition percentage than pretreatment with the Bp1. This might in turn suggest that Bp1 has gastroprotective activity, but other compounds can act synergistically, potentiating its effect. We conclude that B. pinnatum leaf extract could be a new source of raw material rich in phenolic compounds to be applied in food or medicine.

Anti-Inflammatory and Chemopreventive Effects of Bryophyllum pinnatum (Lamarck) Leaf Extract in Experimental Colitis Models in Rodents.

Inflammatory bowel diseases, mainly ulcerative colitis and Crohn's disease are characterized by chronic inflammation in the intestine. Currently several therapeutic strategies available to treat inflammatory bowel diseases. Though, most treatments can be associated with serious adverse effects what justifies the search for new treatments. In this sense, we highlight the interest in herbal products rich in bioactive compounds which immunomodulatory and antioxidant properties as is the case of Bryophyllum pinnatum (Crassulaceae). This plant is used in traditional medicine in Brazil for treating inflammatory diseases. We hypothesized that hydroethanolic B. pinnatum leaf extract has intestinal anti-inflammatory effects on two experimental colitis models: 2.4-dinitrobenzene sulfonic acid (DNBS) in rats, and dextran sulfate sodium (DSS) in mice. Ultra-fast liquid chromatography method used for the quantification of the main compounds indicated good linearity, specificity, selectivity, precision, robustness and accuracy. The major flavonoids (mg/g of the extract) quantified were: quercetin 3-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside (35.56 ± 0.086 mg/g), kaempferol 3-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside (4.66 ± 0.076 mg/g) and quercetin-3-O-rhamnopyranoside (4.56 ± 0.026 mg/g). The results obtained in the DNBS and DSS models indicate that extract has both chemopreventive and anti-inflammatory effects, observing a significant reduction in the disease activity index score, and less macroscopic and microscopic damage. The extract promoted downregulation of Toll-like receptor and kappa B p65 nuclear factor gene expression, leading to a reduction in pro-inflammatory and oxidative mediators, chemokines, and cell adhesion molecules. This immunomodulatory property was proposed that one of the possible action mechanisms of extract. An improvement in intestinal damage was also associated with a reduction in oxidative stress and infiltration of leukocytes, as evidenced by the reduction in malonaldialdehyde and myeloperoxidase activity and increase in total glutathione in the colonic tissue. Moreover, the extract improved the cytoarchitecture of the colonic tissue and the integrity of the intestinal epithelial barrier by restoring the expression of the proteins associated with mucosa protection. In view of the beneficial effects showed by the B. pinnatum leaf extract in preclinical rodent models of colitis there is the potential to conduct some future clinical studies to ensure safe and effective development of a phytotherapeutic treatment for human inflammatory bowel diseases.

Local anti-inflammatory activity: Topical formulation containing Kalanchoe brasiliensis and Kalanchoe pinnata leaf aqueous extract.

Kalanchoe brasiliensis and Kalanchoe pinnata are used interchangeably in traditional medicine in the treatment of wound healing. In this context, the objective of the present study was to evaluate the local anti-inflammatory activity of a topical formulation containing aqueous extract of both species. The in vivo model used was ear edema induced by croton oil and paw edema induced by carrageenan. The Swiss mice treatments use formulations containing aqueous extract at different concentrations (1.25%, 2.5%, and 5%) or dexamethasone (1 mg/g), all administered topically and immediately after edema induction. The treatment with formulations containing aqueous extract of both species reduced ear and paw edema, besides that, the decrease in edema was evidenced by reduction of myeloperoxidase activity, IL-1ß, and TNF-α levels and increase IL-10 levels. In conclusion, the two species showed local anti-inflammatory activity; however K. brasiliensis showed a better result in both edematogenic models since it had activity in the lowest concentration.

Gastroprotective and Antioxidant Activity of Kalanchoe brasiliensis and Kalanchoe pinnata Leaf Juices against Indomethacin and Ethanol-Induced Gastric Lesions in Rats.

Kalanchoe brasiliensis and Kalanchoe pinnata are used interchangeably in traditional medicine for treating peptic ulcers and inflammatory problems. In this context, this study aims to characterize the chemical constituents and evaluate the gastroprotective activity of the leaf juices of the two species in acute gastric lesions models. Thin Layer Chromatography (TLC) and Ultra High Performance Liquid Chromatography coupled to Mass Spectrometer (UHPLC-MS) were performed for chemical characterization. Wistar rats were pre-treated orally with leaf juices (125, 250 and 500 mg/kg) or ranitidine (50 mg/kg). The peaks observed in the chromatogram of K. brasiliensis showed similar mass spectra to flavonoid glycosides derived from patuletin and eupafolin, while K. pinnata showed mass spectra similar to compounds derived from quercetin, patuletin, eupafolin and kaempferol. K. brasiliensis at all doses and K. pinnata at doses of 250 mg/kg and 500 mg/kg significantly reduced the lesions in the ethanol induction model. In the indomethacin induction model, both species showed significant results at doses of 250 and 500 mg/kg. Also, the pre-treatment with leaf juices increased the antioxidant defense system, glutathione (GSH), whereas malondialdehyde (MDA), myeloperoxidase (MPO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels were significantly decreased. Treatment with leaf juices led to the upregulation of zone occludes-1 (ZO-1) and the downregulation of inducible nitric oxide synthase (iNOS) and factor nuclear-κβ transcription (NF-κB-p65), while also showing a cytoprotective effect and maintaining mucus production. These findings show that the leaf juices of the two species showed gastroprotective effects on ethanol and gastric indomethacin injury which were a consequence of gastric inflammation suppression, antioxidant activity and the maintenance of cytoprotective defenses and mucosal structure architecture.

In Vivo and In Vitro Toxicity Evaluation of Hydroethanolic Extract of Kalanchoe brasiliensis (Crassulaceae) Leaves.

The species Kalanchoe brasiliensis, known as "Saião," has anti-inflammatory, antimicrobial, and antihistamine activities. It also has the quercetin and kaempferol flavonoids, which exert their therapeutic activities. With extensive popular use besides the defined therapeutical properties, the study of possible side effects is indispensable. The objective of this study is to evaluate the toxicity in vitro and in vivo from the hydroethanolic extract of the leaves of K. brasiliensis. The action of the extract (concentrations from 0.1 to 1000 uL/100 uL) in normal and tumor cells was evaluated using the MTT method. Acute toxicity and subchronic toxicity were evaluated in mice with doses of 250 to 1000 mg/kg orally, following recognized protocols. The in vitro results indicated cytotoxic activity for 3T3 cell line (normal) and 786-0 (kidney carcinoma), showing the activity to be concentration-dependent, reaching 92.23% cell inhibition. In vivo, the extract showed no significant toxicity; only liver changes related to acute toxicity and some signs of liver damage, combining biochemical and histological data. In general, the extract showed low or no toxicity, introducing itself as safe for use with promising therapeutic potential.

Vegetable moisturizing raw material from "Caatinga" Brazilian biome: safety and efficacy evaluations of O/W cosmetic emulsions containing Kalanchoe brasiliensis extract

The purpose of this study was to evaluate the safety of Kalanchoe brasiliensis extract, followed by the development of an oil in water emulsion containing the K. brasiliensis leaves extract and evaluating its clinical moisturizing efficacy. The formulations containing sodium acrylates/ Beheneth-25 methacrylate Crosspolymer (and) hydrogenated polydecene (and) lauryl glucoside and 0.5% of extract were prepared. The extract was considered as non-irritating through skin irritant tests. The stability testing was carried out in different conditions for 90 days. The skin hydration was measured by capacitance measurement and transepidermal water loss using biophysical techniques. The results indicate that the formulation containing 0.5% of extract increased the hydration of the stratum corneum up to 5 h after application on the forearm. The transepidermal water loss was reduced when compared to the untreated area and placebo area. Therefore, we can conclude that the increased skin hydration and protection of barrier function can be attributed to the K. brasiliensis extract. This research presents a new raw material from the Brazilian Caatinga biome and shows its possible application in the development of cosmetic products.

Correction: Inhibitory Effects of Hydroethanolic Leaf Extracts of Kalanchoe brasiliensis and Kalanchoe pinnata (Crassulaceae) against Local Effects Induced by Bothrops jararaca Snake Venom.

[This corrects the article DOI: 10.1371/journal.pone.0168658.].

Inhibitory Effects of Hydroethanolic Leaf Extracts of Kalanchoe brasiliensis and Kalanchoe pinnata (Crassulaceae) against Local Effects Induced by Bothrops jararaca Snake Venom.

The species Kalanchoe brasiliensis and Kalanchoe pinnata, both known popularly as "Saião," are used interchangeably in traditional medicine for their antiophidic properties. Studies evaluating the anti-venom activity of these species are scarce. This study aims to characterize the chemical constituents and evaluate the inhibitory effects of hydroethanolic leaf extracts of K. brasiliensis and K. pinnata against local effects induced by Bothrops jararaca snake venom. Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography coupled with Diode Array Detection and Electrospray Mass Spectrometry (HPLC-DAD-MS/MS) were performed for characterization of chemical markers of the extracts from these species. For antiophidic activity evaluation, B. jararaca venom-induced paw edema and skin hemorrhage in mice were evaluated. In both models, hydroethanolic extracts (125-500 mg/kg) were administered intraperitoneally in different protocols. Inhibition of phospholipase enzymatic activity of B. jararaca was evaluated. The HPLC-DAD-MS/MS chromatographic profile of extracts showed some particularities in the chemical profile of the two species. K. brasileinsis exhibited major peaks that have UV spectra similar to flavonoid glycosides derived from patuletin and eupafolin, while K. pinnata showed UV spectra similar to flavonoids glycosides derived from quercetin and kaempferol. Both extracts significantly reduced the hemorrhagic activity of B. jararaca venom in pre-treatment protocol, reaching about 40% of inhibition, while only K. pinnata was active in post-treatment protocol (about 30% of inhibition). In the antiedematogenic activity, only K. pinnata was active, inhibiting about 66% and 30% in pre and post-treatment protocols, respectively. Both extracts inhibited phospholipase activity; however, K. pinnata was more active. In conclusion, the results indicate the potential antiophidic activity of Kalanchoe species against local effects induced by B. jararaca snake venom, suggesting their potential use as a new source of bioactive molecules against bothropic venom.

Use of Opuntia ficus-indica (L. ) Mill extracts from Brazilian Caatinga as an alternative of natural moisturizer in cosmetic formulations

ABSTRACT The aim of this work was the obtainment of Opuntia fícus-indica (L.) Mill extract for the development of cosmetic formulations and in vivo evaluation of its moisturizing effects. The formulations were tested for preliminary and accelerated stability. Organoleptic characteristics, pH values and rheological behavior were assessed. The evaluation of moisturizing efficacy of the emulsions formulated with 3.0% of Polyacrylamide (and) C13-14 Isoparaffin (and) Laureth-7 containing 1.0 and 3.0% of O. ficus-indica hydroglycolic extract (EHG001) was performed using the capacitance method (Corneometer(r)) and the transepidermal water loss - TEWL evaluation (Tewameter(r)). The emulsions formulated were stable, exhibiting pseudoplastic and thixotropic behavior. The results of evaluation of moisturizing efficacy showed increased skin hydration after five hours by mainly increasing the skin barrier effect. The formulations containing 1.0 and 3.0% of EHG001 enhanced the skin barrier effect by reducing TEWL up to four hours after application. The results observed suggest that O. ficus-indica hydroglycolic extract may act through a humectant and occlusion mechanism.

Spondias tuberosa (Anacardiaceae) leaves: profiling phenolic compounds by HPLC-DAD and LC-MS/MS and in vivo anti-inflammatory activity.

Spondias tuberosa is a medicinal plant used by several local communities in northeast Brazil to treat infections, digestive disorders and inflammatory conditions. The study aimed to identify and quantify the major phenolic in hydroethanolic extract of leaves from S. tuberosa and to evaluate its anti-inflammatory potential. The chemical profile of extract was analyzed by HPLC-DAD and HPLC-MS. The in vivo anti-inflammatory activity was investigated in carrageenan-induced hind paw edema and peritonitis models in mice. Identified and quantified through HPLC-DAD or HPLC-MS analyses of S. tuberosa extract were the following compounds: chlorogenic acid, caffeic acid, rutin and isoquercitrin. The inflammatory response to carrageenan was significantly reduced in both models by S. tuberosa extract. In hind paw edema, the edematogenic response was reduced by up to 63.6% and the myeloperoxidase activity was completely inhibited. In the peritonitis model, the total cell migration into the peritoneal cavity was reduced by up to 65%. The results obtained give evidence of the anti-inflammatory action of S. tuberosa and suggest the potential therapeutic benefit of this plant on inflammatory conditions. The chlorogenic acid, caffeic acid, rutin and isoquercitrin identified and quantified in S. tuberosa leaves enable us to suggest that these compounds could be used as chemical markers for quality control of derivative products from this species. Copyright © 2016 John Wiley & Sons, Ltd.

Neutralizing effects of Mimosa tenuiflora extracts against inflammation caused by Tityus serrulatus scorpion venom.

Scorpion bite represents a significant and serious public health problem in certain regions of Brazil, as well as in other parts of the world. Inflammatory mediators are thought to be involved in the systemic and local immune response induced by Tityus serrulatus scorpion envenomation. The aim of this study was to evaluate the effect of extracts of Mimosa tenuiflora on model envenomation. In mice, the envenomation model is induced by Tityus serrulatus venom. Previous treatment of mice with fractions from M. tenuiflora was able to suppress the cell migration to the peritoneal cavity. The treatment of mice with M. tenuiflora extracts also decreased the levels of IL-6, IL-12, and IL-1ß. We concluded that the administration of the extract and fractions resulted in a reduction in cell migration and showed a reduction in the level of proinflammatory cytokines. This study demonstrates, for the first time, the anti-inflammatory effect of aqueous extract from the Mimosa tenuiflora plant on T. serrulatus venom.

Antioxidant and Antiproliferative Activities of Leaf Extracts from Plukenetia volubilis Linneo (Euphorbiaceae).

Plukenetia volubilis Linneo, or Sacha inca, is an oleaginous plant from the Euphorbiaceae family. The aim of this work was to perform a chemical and biological analysis of different leaf extracts from P. volubilis such as aqueous extract (AEL), methanol (MEL), ethanol (EEL), chloroform (CEL), and hexane (HEL). Thin layer chromatography analysis revealed the presence of phenolic compounds, steroids, and/or terpenoídes. Furthermore, the antioxidant activities were analyzed by in vitro assays and their effects on cell lineages by in vivo assays. The Total Antioxidant Capacity (TCA) was expressed as equivalent ascorbic acid (EEA/g) and it was observed that the extracts showed values ranging from 59.31 to 97.76 EAA/g. Furthermore, the DPPH assay values ranged from 62.8% to 88.3%. The cell viability assay showed that the extracts were able to reduce viability from cancer cells such as HeLa and A549 cells. The extracts MEL and HEL (250 µg/mL) were able to reduce the proliferation of HeLa cells up to 54.3% and 48.5%, respectively. The flow cytometer results showed that these extracts induce cell death via the apoptosis pathway. On the other hand, the extracts HEL and AEL were able to induce cell proliferation of normal fibroblast 3T3 cells.