A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Saracura-Mirá, a Proposed Brazilian Amazonian Adaptogen from Ampelozizyphus amazonicus.

The Amazon Forest is known all over the world for its diversity and exuberance, and for sheltering several indigenous groups and other traditional communities. There, as well as in several other countries, in traditional medical systems, weakness, fatigue and debility are seen as limiting health conditions where medicinal plants are often used in a non-specific way to improve body functions. This review brings together literature data on Ampelozizyphus amazonicus, commonly known in Brazil as "saracura-mirá" and/or "cerveja de índio", as an Amazonian adaptogen, including some contributions from the authors based on their ethnographic and laboratory experiences. Topics such as botany, chemistry, ethnopharmacological and pharmacological aspects that support the adaptogen character of this plant, as well as cultivation, market status and supply chain aspects are discussed, and the gaps to establish "saracura-mirá" as an ingredient for the pharmaceutical purposes identified. The revised data presented good scientific evidence supporting the use of this Amazonian plant as a new adaptogen. Literature data also reveal that a detailed survey on natural populations of this plant is needed, as well as agronomical studies that could furnish A. amazonicus bark as a raw material. Another important issue is the lack of developed quality control methods to assure its quality assessment.

Ethnopharmacological Evaluation of Breu Essential Oils from Protium Species Administered by Inhalation.

BACKGROUND: Breu is an aromatic oleoresin which has been used by Amazonian traditional communities as a remedy for headaches and migraines by burning and inhaling the smoke produced during its combustion. This study evaluated the antinociceptive and sedative activities of formulations containing breu essential oils administered by inhalation. METHODS: Five different formulations (A-E) containing breu essential oils were evaluated for their sedative and antinociceptive activities in mice. They were delivered for 20 minutes using an inhalation chamber coupled with a nebulizer and the air inside was collected by static headspace and analyzed by GC-FID. RESULTS: All nebulized formulations had similar chemical compositions and major compounds as the original essential oils. None of them resulted in significant increase in response time during the hot plate test. In the formalin test, Formulation E showed a significant inhibition of licking responses in the early (46.8%) and late (60.2%) phases. Formulation B was effective (36.9%) in the first phase and Formulation D (37.9%) in the second. None of the formulations presented sedative effects. CONCLUSION: Breu essential oils, when inhaled, may present antinociceptive and anti-inflammatory properties without sedation. Additionally, nebulization proved to be an efficient method for administration of formulations containing these essential oils.

Spray-dried extract from the Amazonian adaptogenic plant Ampelozizyphus amazonicus Ducke (Saracura-mirá): Chemical composition and immunomodulatory properties.

Ampelozizyphus amazonicus Ducke is a medicinal plant used in the Amazon region to prepare a drink with tonic, immunomodulatory and adaptogenic properties. Due to the growing interest in dietary supplements with these properties and, to provide a new functional ingredient, barks from A. amazonicus were extracted. The extract was spray dried without drying adjuvants, resulting in a powder (SARF), which was characterized by its physico-chemical properties and proximate, mineral and saponin contents. The SARF saponins were characterized by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry (HPLC-HRMSn) analysis. The SARF particles tended to have a spherical shape and a unimodal size distribution. The particles also had good rehydration characteristics and high saponin content (33%). The effect of SARF on antibody production was investigated, and we found that SARF increased the basal levels of anti-ovalbumin, anti-LPS and anti-dextran IgM antibodies, and the anti-dextran IgG antibodies in unimmunized mice. No increase in antibody titers was observed after SARF treatment in immunized mice. These results suggest that SARF could be an interesting new functional ingredient for food applications or pharmaceutical products.

Gradient x Isocratic Elution CCC on the Isolation of Verbascoside and Other Phenylethanoids: Influence of the Complexity of the Matrix.

Verbascoside is a phenylethanoid glycoside widely distributed in nature, especially among the order Lamiales, occurring in numerous plants that are constituents of folk medicine preparations. This natural compound, previously isolated by our group from the ethyl acetate extract of Lantana trifolia using the gradient approach in countercurrent chromatography, was now isolated from the butanol extract of the same plant and from Lippia alba f. intermedia (Verbenaceae) using countercurrent chromatography in either gradient or isocratic elution modes. The ethyl acetate extract of L. alba, rich in phenylethanoids and flavonoids, was fractionated using countercurrent chromatography in the step-gradient elution approach. The four-step solvent system was composed of n-hexane-ethyl acetate-n-butanol-water (4 : 10 : X : 10), where X = 1 (solvent system A), 3 (solvent system B), 5 (solvent system C), and 7 (solvent system D), and allowed for the isolation of verbascoside along with other phenylethanoids and flavonoids from both plants. Verbascoside and 2'-O-ß-apiosylverbascoside were further isolated from the n-butanol extract of L. trifolia using the solvent system ethyl acetate-n-butanol-water 10 : 2 : 10 on an isocratic run. The difference in the complexity of the two plant extracts demanded different purification steps, which included a second high-speed countercurrent chromatography purification using the isocratic elution mode.

Immunobiologic and antiinflammatory properties of a bark extract from Ampelozizyphus amazonicus Ducke.

Ampelozizyphus amazonicus is used in the treatment and prevention of malaria. The effect of an aqueous extract from this plant (SART) on the immune response was investigated by measuring immunoglobulin production induced by immunization with the antigen TNP-Ficoll in Plasmodium chabaudi-infected mice. SART treatment increased antigen-specific IgM and IgG levels in TNP-Ficoll-immunized mice. The B cell response during malarial infection was also modified by SART. There was an increase in total serum IgM and IgG and a decrease in the percentage of splenic plasma cells (CD138+ cells) in P. chabaudi-infected, SART-treated animals. SART (1, 3 or 10 mg/kg, p.o.) and the reference drug dexamethasone (5 mg/kg) were also tested in carrageenan-induced leukocyte migration to the subcutaneous air pouch (SAP). All SART doses significantly reduced leukocyte migration into the SAP. The protein concentration resulting from extravasation into the peritoneum was also significantly reduced. Our data indicate that SART possesses immunomodulatory properties, inducing an in vivo modification of the B lymphocyte response and anti-inflammatory properties, which are partly due to a reduction in cell migration and are most likely due to an inhibition of the production of inflammatory mediators. Preliminary HPLC-ESI-MS/MS analysis of SART shows a complex saponin profile with deprotonated molecule [M-H](-) ions in the range of m/z 800-1000.

Chemical composition and antimycobacterial activity of the essential oil from Anemia tomentosa var. anthriscifolia.

The essential oil from Anemia tomentosa var. anthriscifolia showed in vitro activity against Mycobacterium tuberculosis (MIC 100 microg/ml) and therefore was characterized by gas chromatography (GC) and by gas chromatography coupled with mass spectrometry (GC-MS). The major constituents of this essential oil were triquinane sesquiterpenes: silphiperfol-6-ene (14.7%), (-)-epi-presilphiperfolan-1-ol (30.6%), presilphiperfol-7-ene (3.9%), cameroonan-7 alpha-ol (4.4%), prenopsan-8-ol (1.9%) and presilphiperfolan-8-ol (8.3%), suggesting the existence of different chemotypes for this species. The essential oil was fractionated by column chromatography and its major constituent and fractions were assayed against Mycobacterium tuberculosis and M. smegmatis. (-)-epi-Presilphiperfolan-1-ol exhibited an MIC of 120 microg/ml against M. tuberculosis H37Rv.