Wound tissue remodeling by latex exudate of Himatanthus drasticus: A plant species used in Brazilian folk medicine.

This work investigated the healing properties of proteins extracted of latex (HdLP) on excisional wounds. Cell toxicity of HdLP was investigated carried out in murine fibroblasts after incubation with HdLP (12.5-100 µg/ml). The dermal irritability test was performed to evaluate dermal reactions. The wounds were performed and treated with vehicle or HdLP (0.5 %, 1.0 %, and 2.0 %). The macroscopic parameters, histological analysis and measurement of inflammatory markers and mediators were evaluated. HdLP did not exhibit cytotoxicity and did not induce skin irritation. HdLP stimulated the release of IL-1ß at the beginning of the inflammatory phase. This effect probably favored the earlier release of IL-10 by macrophages, during the proliferative phase. The shortening and completeness of healing were characterized by fibroblast proliferation and the presence of newly synthesized collagen fibers. This was accompanied by well-organized re-epithelialization. The involvement of latex proteins in this activity is reported for the first time.

Successful Pre-Clinical Management of Irinotecan-Debilitated Animals: A Protein- Based Accessory Phytomedicine.

BACKGROUND: Calotropis procera is a laticiferous plant (Apocynaceae) found in tropical regions all over the world. The ultrastructural characteristics of laticifers, their restricted distribution among different taxonomic groups, and in some species in each clade, as peptidases from latex, make them very attractive for biological analysis. OBJECTIVE: The study aims to investigate the effects of LP-PII-IAA (laticifer protein (LP) sub-fraction II (PII) of C. procera presenting an iodoacetamide-inhibited cysteine proteinase activity) on irinotecan-induced intestinal mucositis, a serious adverse effect of this medicine for the treatment of cancer. METHODS: LP-PII-IAA is composed of closely related isoforms (90%) of peptidases derived from catalysis and an osmotin protein (5%). Animals receiving co-administration of LP-PII-IAA presented a significant decrease in mortality, absence of diarrhea, histological preservation, and normalization of intestinal functions. RESULTS: Clinical homeostasis was accompanied by a reduction in MPO activity and declined levels of IL-1ß, IL-6 and KC, while the IL-10 level increased in LP-PII-IAA-treated animals. COX-2 and NF-kB immunostaining was reduced and the levels of oxidative markers (GSH, MDA) were normalized in animals that received LP-PII-IAA. CONCLUSION: We suggest that peptidases from the latex of Calotropis procera were instrumental in the suppression of the adverse clinical and physiological effects of irinotecan.

Anti-inflammatory latex proteins of the medicinal plant Calotropis procera: a promising alternative for oral mucositis treatment.

OBJECTIVE AND DESIGN: Oral mucositis (OM) is an intense inflammatory reaction progressing to tissue damage and ulceration. The medicinal uses of Calotropis procera are supported by anti-inflammatory capacity. PII-IAA, a highly homogenous cocktail of laticifer proteins (LP) prepared from the latex of C. procera, with recognized pharmacological properties was tested to treat OM. MATERIALS AND SUBJECTS: Male Golden Sirius hamsters were used in all treatments. TREATMENT: The latex protein samples were injected i.p. (5 mg/Kg) 24 h before mucositis induction (mechanical trauma) and 24 h later. METHODS: Histology, cytokine measurements [ELISA], and macroscopic evaluation [scores] were performed. RESULTS: PII-IAA eliminated OM, accompanied by total disappearance of myeloperoxidase activity and release of IL-1b, as well as reduced TNF-a. Oxidative stress was relieved by PII-IAA treatment, as revealed by MDA and GSH measurements. PII-IAA also reduced the expression of adhesion molecules (ICAM-1) and Iba-1, two important markers of inflammation, indicating modulatory effects. Histological analyses of the cheek epithelium revealed greater deposition of type I collagen fibers in animals given PII-IAA compared with the control group. This performance was only reached when LPPII was treated with iodoacetamide (IAA), an irreversible inhibitor of proteolytic activity of cysteine proteases. The endogenous proteolytic activity of LPPII induced adverse effects in animals. Candidate proteins involved in the phytomodulatory activity are proposed. CONCLUSIONS: Therapy was successful in treating OM with the laticifer protein fraction, containing peptidases and osmotin, from Calotropis procera. The effective candidate from the latex proteins for therapeutic use is PII-IAA.

Chemical profiling of secondary metabolites from Himatanthus drasticus (Mart.) Plumel latex with inhibitory action against the enzymes α-amylase and α-glucosidase: In vitro and in silico assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Himatanthus drasticus is an important medicinal plant whose latex is traditionally used in Northeast Brazil to treat various diseases, including diabetes. The use of α-amylase and α-glucosidase inhibitors can be an effective strategy to modulate levels of postprandial hyperglycemia via control of starch metabolism. AIMS OF THE STUDY: This study aimed to verify if H. drasticus latex has inhibitory activity against enzymes linked to type 2 diabetes, besides chemically characterizing the metabolites responsible for such activities. In addition, in silico analysis was performed to support the traditional claim of possible antidiabetic activity of this latex. MATERIALS AND METHODS: Latex from H. drasticus stems was sequentially partitioned with n-hexane (FHDH), CHCl3 (FHDC) and EtOH (FHDHA). Wash extraction of the FHDHA fraction was performed to obtain the other extract fractions. The FHDHA was submitted to chromatography in a SPE C18 cartridge using gradient elution with MeOH/H2O to produce five fractions: FHDHA1, FHDHA2, FHDHA3, FHDHA4 and FHDHA5. The FHDHA1 was subjected to semi-preparative reverse phase HPLC. Lineweaver-Burk plots were used to investigate the kinetic parameters of α-amylase and α-glucosidase inhibitory activity. The interactions between plumieride and porcine pancreatic α-amylase and α-glucosidase were analyzed through an in silico molecular docking study. RESULTS: Phytochemical identification of compounds present in the FHDHA fraction of H. drasticus latex was possible by 1H, 13C NMR analysis and mass spectrometry, and the results were compared with the literature. The identified compounds were α-ethyl glucoside, protocatechuic acid, 3-O-caffeoylquinic acid, 15-demethylplumieride acid, 5-O-caffeoylquinic acid, caffeic acid, vanillic acid, plumieride, and catechin. The inhibition results of the fractions tested against α-amylase and α-glucosidase showed inhibitory activities dependent on the increase of fractions and compound concentrations. The IC50 results obtained from FHDHA, FHDHA1 and plumieride fractions against α-amylase were 36.46, 72.61, 33.87 µg/mL respectively. The IC50 of plumieride was the closest to that of acarbose (22.52 µg/mL), a result similar to that obtained for α-glucosidase. The type of inhibition was competitive for both enzymes. CONCLUSIONS: There was strong inhibition of α-amylase and α-glucosidase by FHDHA, FHDHA1 and plumieride, suggesting that these enzymes slow glucose absorption.

A phytomodulatory hydrogel with enhanced healing effects.

The healing performance of a hydrogel composed of hemicelluloses extracted from seeds of Caesalpinia pulcherrima (Fabaceae) and mixed with phytomodulatory proteins obtained from the latex of Calotropis procera was characterized on excisional wounds. The hydrogel did not induce dermal irritability. When topically used on excisional wounds, the hydrogel enhanced healing by wound contraction. Histology and the measurement of inflammatory mediators (myeloperoxidase, interleukin-1ß, and interleukin-6) suggested that the inflammatory phase of the healing process was intensified, stimulating fibroplasia and neovascularization (proliferative phase) and tissue remodeling by increasing new collagen fiber deposition. In addition, reduction on levels of malondialdehyde in the groups that the hydrogel was applied suggested that the oxidative stress was reduced. The hydrogel performed better than the reference drug used, as revealed by the extended thickness of the remodeled epithelium.

Proteins from latex of Calotropis procera prevent septic shock due to lethal infection by Salmonella enterica serovar Typhimurium.

AIM OF THE STUDY: The latex of Calotropis procera has been used in traditional medicine to treat different inflammatory diseases. The anti-inflammatory activity of latex proteins (LP) has been well documented using different inflammatory models. In this work the anti-inflammatory protein fraction was evaluated in a true inflammatory process by inducing a lethal experimental infection in the murine model caused by Salmonella enterica Subsp. enterica serovar Typhimurium. MATERIALS AND METHODS: Experimental Swiss mice were given 0.2 ml of LP (30 or 60 mg/kg) by the intraperitoneal route 24 h before or after lethal challenge (0.2 ml) containing 10(6) CFU/ml of Salmonella Typhimurium using the same route of administration. RESULTS: All the control animals succumbed to infection within 6 days. When given before bacterial inoculums LP prevented the death of mice, which remained in observation until day 28. Even, LP-treated animals exhibited only discrete signs of infection which disappeared latter. LP fraction was also protective when given orally or by subcutaneous route. Histopathological examination revealed that necrosis and inflammatory infiltrates were similar in both the experimental and control groups on days 1 and 5 after infection. LP activity did not clear Salmonella Typhimurium, which was still present in the spleen at approximately 10(4) cells/g of organ 28 days after challenge. However, no bacteria were detected in the liver at this stage. LP did not inhibit bacterial growth in culture medium at all. In the early stages of infection bacteria population was similar in organs and in the peritoneal fluid but drastically reduced in blood. Titration of TNF-alpha in serum revealed no differences between experimental and control groups on days 1 and 5 days after infection while IL-12 was only discretely diminished in serum of experimental animals on day 5. Moreover, cultured macrophages treated with LP and stimulated by LPS released significantly less IL-1beta. CONCLUSIONS: LP-treated mice did not succumb to septic shock when submitted to a lethal infection. LP did not exhibit in vitro bactericidal activity. It is thought that protection of LP-treated mice against Salmonella Typhimurium possibly involves down-regulation of pro-inflammatory cytokines (other than TNF-alpha). LP inhibited IL-1beta release in cultured macrophages and discretely reduced IL-12 in serum of animals given LP. Results reported here support the folk use of latex to treat skin infections by topic application.

Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model.

AIM OF THE STUDY: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. It comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). MATERIAL AND METHODS: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII) and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. RESULTS: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. CONCLUSIONS: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production.

Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: involvement of cytokines and nitric oxide.

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125-1000 microg/cavity), and at 2-96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 microg/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-alpha, IL-1beta and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 microg/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1beta, TNF-alpha and CINC-1.

Antitumor properties of a sulfated polysaccharide from the red seaweed Champia feldmannii (Diaz-Pifferer).

In recent years, much attention has been focused on polysaccharides isolated from natural sources. The aim of this study was to investigate the in vitro and in vivo antitumor properties of a sulfated polysaccharide isolated from the seaweed C. feldmannii (Cf-PLS). Hematological, biochemical and histopathological analyses were performed in order to evaluate the toxicological aspects related to Cf-PLS treatment. Its effects on the immunological system were also investigated. The Cf-PLS did not show any significant in vitro cytotoxicity at the experimental exposure levels that were used, but showed in vivo antitumor effect. The inhibition rates of sarcoma 180 tumor development were 48.62 and 48.16% at the doses of 10 and 25 mg kg(-1), respectively. In addition, Cf-PLS was also able to increase the response elicited by 5-fluorouracil (5-FU) from 48.66 to 68.32%. The histopathological analysis of liver and kidney showed that both organs were moderately affected by Cf-PLS-treatment. Neither enzymatic activity of alanine aminotransferase nor urea or creatinine levels were significantly altered. In hematological analysis, leucopeny was observed after 5-FU treatment, but this effect was prevented when the treatment was associated with the Cf-PLS. It was also demonstrated that Cf-PLS acts as an immunomodulatory agent, raising the production of specific antibodies, and increasing the production of OVA-specific antibodies. It also induced a discreet hyperplasia of lymphoid folicules of the white pulp in the spleen of treated mice. In conclusion, Cf-PLS has some interesting anticancer activity that could be associated with its immunostimulating properties.

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

In this study, we have addressed the role of H(2)S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H(2)S synthesis inhibitors, dl-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H(2)S donors, NaHS or Lawesson's reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB(4). Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K(ATP)(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K(ATP)(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H(2)S augments neutrophil adhesion and locomotion, by a mechanism dependent on K(ATP)(+) channels.

In vivo growth inhibition of sarcoma 180 by piperlonguminine, an alkaloid amide from the Piper species.

Many authors have already emphasized that phytochemicals from spices have biological applications. Piperlonguminine is a known alkaloid amide from peppers, including Piper divaricatum. The aim of this study was to investigate the in vitro and in vivo antitumor effects of piperlonguminine in experimental models. In order to evaluate the toxicological aspects related to piperlonguminine treatment, hematological, biochemical, histopathological and morphological analyses of treated animals were performed. Piperlonguminine did not show any significant in vitro cytotoxic effect at experimental exposure levels, but showed an in vivo antitumor effect. After 7 days of treatment, the inhibition rates were 38.71% and 40.68% at doses of 25 mg kg(-1) and 50 mg kg(-1), respectively. The histopathological analysis suggests that the liver and kidney were only weakly affected by piperlonguminine treatment. Neither the enzymatic activity of transaminases (AST and ALT) nor the urea levels were significantly altered. In the hematological analysis, all parameters analysed remained constant after piperlonguminine treatment. In conclusion, these data reinforce the anticancer potential of spice components.

Latex proteins from the plant Calotropis procera are partially digested upon in vitro enzymatic action and are not immunologically detected in fecal material.

Soluble proteins from the latex of Calotropis procera (LP) were investigated in vitro and in vivo for digestibility as the latex has previously been shown to produce considerable toxic effects on animals. The latex is also an important biologically active compound that displays antiinflammatory and antidiarrhea properties. The proteins were digested by the action of trypsin, pepsin or chemotrypsin as revealed by gel filtration and SDS-PAGE analysis. Furthermore, the full LP digestion was easily achieved by protease treatment. Rabbit polyclonal antibodies raised against LP failed to detect cross-reactive molecules in fecal material of experimental rats following 35 consecutive days of LP consumption in water. Similar patterns of electrophoresis were observed for the negligible amounts of protein observed in the fecal extracts of control and test animals. No death or toxic effects were observed among animals. Taken together these results suggest that harmful and toxic effects on animals of the latex from C. procera are present in its rubber and low molecular weight fractions rather than its protein content.