Effect of Spilanthes acmella hydroethanolic extract activity on tumour cell actin cytoskeleton.

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Numerous natural products have pharmacological activity such that many biologically active compounds have led to the development of cancer chemotherapy drugs. Spilanthes acmella (Asteraceae) is widely cultivated in the State of Pará, Brazil, being employed in folk medicine for its anti-inflammatory, antimicrobial, antioxidant, analgesic, insecticide, and larvicidal properties. However, its cytotoxicity and influence on actin cytoskeleton organisation in tumour cell lines are practically nonexistent. We have verified the cytotoxicity of a hydroethanolic extract of the inflorescence of S. acmella, and examined its effects on the cytoskeleton of tumour cells. Decreasing concentrations of the extract (250, 500 and 1,000 µg/mL) were given to cultures of neoplastic cells (HEp-2). Cytotoxicity was assessed by the MTT test, and the influence on cytoskeleton organisation was examined by fluorescence microscopy. The IC50 of the hydroethanolic extract was 513 µg/mL, confirming the data obtained from the MTT assay that gave high cytotoxicity. The actin cytoskeleton arrangement of HEp2 cells at 500 and 1,000 µg/mL showed depolymerisation of the filaments, causing loss of morphology and consequently compromising cell adhesion.

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Numerous natural products have pharmacological activity such that many biologically active compounds have led to the development of cancer chemotherapy drugs. Spilanthes acmella (Asteraceae) is widely cultivated in the State of Pará, Brazil, being employed in folk medicine for its anti-inflammatory, antimicrobial, antioxidant, analgesic, insecticide, and larvicidal properties. However, its cytotoxicity and influence on actin cytoskeleton organisation in tumour cell lines are practically nonexistent. We have verified the cytotoxicity of a hydroethanolic extract of the inflorescence of S. acmella, and examined its effects on the cytoskeleton of tumour cells. Decreasing concentrations of the extract (250, 500 and 1,000 µg/mL) were given to cultures of neoplastic cells (HEp-2). Cytotoxicity was assessed by the MTT test, and the influence on cytoskeleton organisation was examined by fluorescence microscopy. The IC50 of the hydroethanolic extract was 513 µg/mL, confirming the data obtained from the MTT assay that gave high cytotoxicity. The actin cytoskeleton arrangement of HEp2 cells at 500 and 1,000 µg/mL showed depolymerisation of the filaments, causing loss of morphology and consequently compromising cell adhesion.

Spilanthes acmella (Paracress), a common spice, has been administered as a traditional folk medicine for years to cure toothaches, stammering, and stomatitis. Previous studies have demonstrated its diuretic, antibacterial, and anti-inflammatory activities. However, the active compounds contributing to the anti-inflammatory effect have seldom been addressed. This study isolates the active compound, spilanthol, by a bioactivity-guided approach and indicates significant anti-inflammatory activity on lipopolysaccharide-activated murine macrophage model, RAW 264.7. The anti-inflammatory mechanism of paracress is also investigated. Extracts of S. acmella are obtained by extraction with 85% ethanol, followed by liquid partition against hexane, chloroform, ethyl acetate, and butanol. The ethyl acetate extract exhibits a stronger free radical scavenging capacity than other fractions do, as determined by DPPH and ABTS radical scavenging assays. The chloroform extract significantly inhibits nitric oxide production ( p < 0.01) and is selected for further fractionation to yield the active compound, spilanthol. The diminished levels of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) mRNA and protein expression support the postulation that spilanthol inhibits proinflammatory mediator production at the transcriptional and translational levels. Additionally, the LPS-stimulated IL-1beta, IL-6, and TNF-alpha productions are dose-dependently reduced by spilanthol. The LPS-induced phosphorylation of cytoplasmic inhibitor-kappaB and the nuclear NF-kappaB DNA binding activity are both restrained by spilanthol. Results of this study suggest that spilanthol, isolated from S. acmella, attenuates the LPS-induced inflammatory responses in murine RAW 264.7 macrophages partly due to the inactivation of NF-kappaB, which negatively regulates the production of proinflammatory mediators.

According to Indian Systems of Medicine, Spilanthes acmella (L.) Murr. (Family - Asteraceae), is considered effective in the treatment of sexual deficiencies especially due to ageing. In the present study, characterization of ethanolic extracts of the Spilanthes acmella flower and its effect on general mating pattern, penile erection and serum hormone levels of normal male Wistar albino rats were investigated and compared with sildenafil citrate. In vitro nitric oxide release was also investigated in human corpus cavernosum cell line. As N-alkylamides are a promising group, their profiling was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS(1) and MS(2) fragmentation data were used for identification purposes. For assessment of sexual behavior, animals were divided into five groups of eight male rats. The extracts (50, 100 and 150mg/kgbodyweight/day) and sildenafil citrate (5mg/kgbodyweight/day) (positive control) were administered orally for 28 days. The behavioral and sexual parameters were observed at days 0, 15, 28 and after a lapse of 7 and 14 days of discontinuance of drug treatment. Five N-isobutylamides, one 2-methylbutylamide and one 2-phenylethylamide were identified. The orally administered extract had a dose dependent positive effect on mounting frequency, intromission frequency and ejaculation frequency and the most significant effects (p<0.05) were observed at 150mg/kg treatment, even after a lapse of 7 and 14 days of discontinuance of drug treatment. A dose dependent effect was also observed on the FSH, LH and testosterone serum levels. With 150mg/kg of ethanolic extract the values for FSH, LH and testosterone were 3.10±0.25mlU/ml, 6.87±0.18mlU/ml and 3.72±0.12ng/ml, respectively. In vitro nitric oxide release was 21.7±2.9μM, which was significantly higher compared to the control group (p<0.01). Sildenafil citrate exhibited also a significant effect on NO release, but no effect on hormone levels of rats was observed. The aphrodisiac potential of an ethanolic Spilanthes acmella extract was demonstrated in vitro and in vivo. N-Alkylamides might attribute to the improved sexual potential. Study lends support to the traditional utilization of S. acmella as a sexual stimulating agent.

Natural products could play an important role in the challenge to discover new anti-malarial drugs. In a previous study, Dicoma tomentosa (Asteraceae) was selected for its promising anti-plasmodial activity after a preliminary screening of several plants traditionally used in Burkina Faso to treat malaria. The aim of the present study was to further investigate the anti-plasmodial properties of this plant and to isolate the active anti-plasmodial compounds.
Eight crude extracts obtained from D. tomentosa whole plant were tested in vitro against two Plasmodium falciparum strains (3D7 and W2) using the p-LDH assay (colorimetric method). The Peters' four-days suppressive test model (Plasmodium berghei-infected mice) was used to evaluate the in vivo anti-plasmodial activity. An in vitro bioguided fractionation was undertaken on a dichloromethane extract, using preparative HPLC and TLC techniques. The identity of the pure compound was assessed using UV, MS and NMR spectroscopic analysis. In vitro cytotoxicity against WI38 human fibroblasts (WST-1 assay) and haemolytic activity were also evaluated for extracts and pure compounds in order to check selectivity.
The best in vitro anti-plasmodial results were obtained with the dichloromethane, diethylether, ethylacetate and methanol extracts, which exhibited a high activity (IC50 ≤ 5 μg/ml). Hot water and hydroethanolic extracts also showed a good activity (IC50 ≤ 15 μg/ml), which confirmed the traditional use and the promising anti-malarial potential of the plant. The activity was also confirmed in vivo for all tested extracts. However, most of the active extracts also exhibited cytotoxic activity, but no extract was found to display any haemolytic activity. The bioguided fractionation process allowed to isolate and identify a sesquiterpene lactone (urospermal A-15-O-acetate) as the major anti-plasmodial compound of the plant (IC50 < 1 μg/ml against both 3D7 and W2 strains). This was also found to be the main cytotoxic compound (SI = 3.3). While this melampolide has already been described in the plant, this paper is the first report on the biological properties of this compound.
The present study highlighted the very promising anti-plasmodial activity of D. tomentosa and enabled to identify its main active compound, urospermal A-15-O-acetate. The high anti-plasmodial activity of this compound merits further study about its anti-plasmodial mechanism of action. The active extracts of D. tomentosa, as well as urospermal A 15-O-acetate, displayed only a moderate selectivity, and further studies are needed to assess the safety of the use of the plant by the local population.

Spilanthes acmella (Family: Asteraceae) commonly known as "toothache plant" is known to possess strong insecticidal and larvicidal properties. Experiments have been conducted to isolate and characterise the biolarvicidal compounds from the flower head extract of micropropagated S. acmella plants employing various tools like FT-IR, TLC, CC, NMR. FT-IR spectroscopy of the crude hexane extract sample revealed the presence of amide (secondary metabolite) as functional group in S. acmella flower heads. The crude extract was separated into 85 fractions (100 ml each) through silica gel column chromatography using hexane-ethyl acetate mobile phase. All fractions were tested for their larvicidal activity against late III/early IV instar Anopheles stephensi larvae and fraction showing maximum bioefficacy against aforesaid larvae was further resolved into three separate bands on Preparative TLC plate, the respective R (f) values being (a) 0.18, (b) 0.23 and (c) 0.27. Based on Proton NMR spectrum of the eluted compounds and their comparison with published results, three different compounds were identified: N-isobutyl-2,6,8-decatrienamide (compound 1), undeca-2E,7Z,9E-trienoic acid isobutylamide (compound 2) from band a and (2E)-N-(2-methylbutyl)-2-undecene-8,10-diynamide (compound 3) from band b. The amount of the compounds obtained were 338 mg (compounds 1 and 2) and 188.4 mg (compound 3), respectively. This is the first report of biolarvicidal compounds isolation and characterisation from micropropagated S. acmella plants.

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