Chemical composition and antiprotozoal activities of Colombian Lippia spp essential oils and their major components

The chemical composition and biological activities of 19 essential oils and seven of their major components were tested against free and intracellular forms of Leishmania chagasi and Trypanosoma cruzi parasites as well as Vero and THP-1 mammalian cell lines. The essential oils were obtained from different species of Lippia, a widely distributed genus of Colombian plants. They were extracted by microwave radiation-assisted hydro-distillation and characterised by GC-FID and GC-MS. The major components were geranial, neral, limonene, nerol, carvacrol, p-cymene, γ-terpinene, carvone and thymol. The essential oil of Lippia alba exhibited the highest activity against T. cruzi epimastigotes and intracellular amastigotes with an IC50 of 5.5 μg/mL and 12.2 μg/mL, respectively. The essential oil of Lippia origanoides had an IC50 of 4.4 μg/mL in L. chagasi promastigotes and exhibited no toxicity in mammalian cells. Thymol (IC50 3.2 ± 0.4 μg/mL) and S-carvone (IC50 6.1 ± 2.2 μg/mL), two of the major components of the active essential oils, were active on intracellular amastigotes of T. cruziinfected Vero cells, with a selective index greater than 10. None of the essential oils or major components tested in this study was active on amastigotes of L. chagasi infected THP-1 cells.

Organic compounds from plant extracts and their effect on soil phosphorus availability

The objective of this work was to evaluate the effect of organic compounds from plant extracts of six species and phosphate fertilization on soil phosphorus availability. Pots of 30 cm height and 5 cm diameter were filled with Typic Hapludox. Each pot constituted a plot of a completely randomized design, in a 7x2 factorial arrangement, with four replicates. Aqueous extracts of black oat (Avena strigosa), radish (Raphanus sativus), corn (Zea mays), millet (Pennisetum glaucum), soybean (Glycine max), sorghum (Sorghum bicolor), and water, as control, were added in each plot, with or without soluble phosphate fertilization. After seven days of incubation, soil samples were taken from soil layers at various depths, and labile, moderately labile and nonlabile P fractions in the soil were analysed. Plant extracts led to an accumulation of inorganic phosphorus in labile and moderately labile fractions, mainly in the soil surface layer (0-5 cm). Radish, with a higher amount of malic acid and higher P content than other species, was the most efficient in increasing soil P availability.

In vitro activity of essential oils of Lippia sidoides and Lippia gracilis and their major chemical components against Thielaviopsis paradoxa, causal agent of stem bleeding in coconut palms

Essential oils of Lippia sidoides, Lippia gracilis and their main chemical components were investigated for in vitro control of Thielaviopsis paradoxa. Mycelial growth and a number of pathogen conidia were inhibited by the essential oil of L. sidoides at all concentrations tested (0.2; 0.5; 1.0; 3.0 µL mL-1). L. sidoides oil contained 42.33% thymol and 4.56% carvacrol, while L. gracilis oil contained 10% thymol and 41.7% carvacrol. Mycelial growth and conidial production of T. paradoxa were completely inhibited by thymol at a 0.3 µL m-1 concentration. The results suggest that thymol could potentially be used for controlling coconut stem bleeding.

Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich.) Briq. and their isolated components in silver catfish (Rhamdia quelen)

This study evaluated the sedative and anesthetic effects of the essential oils (EO) of Hyptis mutabilis (Rich.) Briq. and their isolated components on silver catfish (Rhamdia quelen). Quantitative chemical differences between the EOs obtained from leaves and inflorescences were verified, and a new chemotype rich in globulol was described. Although there were no significant differences in the time of induction for sedation and anesthesia between the EOs, only the leaf EO at 344 mg/L anesthetized all fish without side effects. Fractionation of the leaf EO was carried out by column chromatography. The isolated compounds [(+)-1-terpinen-4-ol and (-)-globulol] showed different activity from that detected for the leaf EO in proportional concentrations and similar sedation to a eugenol control at 10 mg/L. However, fish exposed to 1-terpinen-4-ol (3 and 10 mg/L) did not remain sedated for 30 min. Anesthesia was obtained with 83-190 mg/L globulol, but animals showed loss of mucus during induction and mortality at these concentrations. Synergism of the depressor effects was detected with the association of globulol and benzodiazepine (BDZ), compared with either drug alone. Fish exposed to BDZ or globulol+BDZ association showed faster recovery from anesthesia in water containing flumazenil, but the same did not occur with globulol. In conclusion, the use of globulol in aquaculture procedures should be considered only at sedative concentrations of 10 and 20 mg/L, and its mechanism of action seems not to involve the GABAA-BDZ system.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Evaluation of biological activities of Physalis peruviana ethanol extracts and expression of Bcl-2 genes in HeLa cells

Physalis species are used in folk medicine for phytotherapeutic properties. The extracts of medicinal plants are known to possess cytotoxic and chemopreventative compounds. In this study we investigated antibacterial, antioxidant, DNA damage preventative properties of Physalis peruviana (golden berry) on leaf and shoot ethanol extracts and their effects on cytotoxicity of HeLa cells and expression of apoptotic pathway genes. Among the tested bacteria for antibacterial activity, maximum inhibition zone was determined in Lactococcus lactis. The phenolic content was found higher in leaf extracts than shoot extracts. The antioxidant activity showed the highest TEAC values of the leaf (2 mg/mL) and the shoot (0.5 mg/mL) extracts as 0.291±0.04 and 0.192±0.015, respectively. In DNA damage prevention assay both leaf and shoot extracts, especially 30 and 20 µg/mL concentrations, exhibited significant protection against DNA damage-induced by hydroxyl radical generated by Fenton reaction. Our results suggest that leaf and shoot extracts possess cytotoxic effect on HeLa cells when applied as 100 µg/mL concentration. Also mRNA expression analysis showed the alteration of antiapoptotic genes, so the results suggest that P. peruviana ethanol extracts induce apoptotic cell death and should be investigated for identification of active compounds and their mechanisms of action.

Microbicidal effect of medicinal plant extracts (Psidium guajava Linn. and Carica papaya Linn.) upon bacteria isolated from fish muscle and known to induce diarrhea in children

Out of the twenty-four samples of shrimp and fish muscle used for this study, twelve were collected near a large marine sewer for waste disposal, 3 km off the coast of Fortaleza (Brazil) and used for the isolation of E. coli. Other twelve were collected at the Mucuripe fresh fish market (Fortaleza, Brazil) and used for the isolation of Staphylococcus aureus. Ethanol, water and acetone-diluted extracts of guava and papaya leaf sprouts were tested on the bacteria in order to verify their microbicidal potential. The E. coli strains used in the trials were rated LT positive. The papaya leaf extracts (Carica papaya Linn) showed no microbicidal activity while the guava sprout extracts (Psidium guajava Linn) displayed halos exceeding 13 mm for both species, an effect considered to be inhibitory by the method employed. Guava sprout extracts by 50% diluted ethanol most effectively inhibited E. coli (EPEC), while those in 50% acetone were less effective. It may be concluded that guava sprout extracts constitute a feasible treatment option for diarrhea caused by E. coli or by S. aureus-produced toxins, due to their quick curative action, easy availability in tropical countries and low cost to the consumer.

EFFECTS OF SINGLE, BINARY AND TERTIARY COMBINATIONS WITH Jatropha gossypifolia AND OTHER PLANT-DERIVED MOLLUSCICIDES ON REPRODUCTION AND SURVIVAL OF THE SNAIL Lymnaea acuminata

The effect of sub-lethal doses (40% and 80% of LC50/24h) of plant derived molluscicides of singly, binary (1:1) and tertiary (1:1:1) combinations of the Rutin, Ellagic acid, Betulin and taraxerol with J. gossypifolia latex, leaf and stem bark powder extracts and their active component on the reproduction of freshwater snail Lymnaea acuminata have been studied. It was observed that the J. gossypifolia latex, stem bark, individual leaf and their combinations with other plant derived active molluscicidal components caused a significant reduction in fecundity, hatchability and survival of young snails. It is believed that sub-lethal exposure of these molluscicides on snail reproduction is a complex process involving more than one factor in reducing the reproductive capacity.

Antitumor and Antiviral Activity of Colombian Medicinal Plant Extracts

Extracts of nine species of plants traditionally used in Colombia for the treatment of a variety of diseases were tested in vitro for their potential antitumor (cytotoxicity) and antiherpetic activity. MTT (Tetrazolium blue) and Neutral Red colorimetric assays were used to evaluate the reduction of viability of cell cultures in presence and absence of the extracts. MTT was also used to evaluate the effects of the extracts on the lytic activity of herpes simplex virus type 2 (HSV-2). The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration of the viral effect (EC50) for each extract were calculated by linear regression analysis. Extracts from Annona muricata, A. cherimolia and Rollinia membranacea, known for their cytotoxicity were used as positive controls. Likewise, acyclovir and heparin were used as positive controls of antiherpetic activity. Methanolic extract from Annona sp. on HEp-2 cells presented a CC50 value at 72 hr of 49.6x103mg/ml. Neither of the other extracts examined showed a significant cytotoxicity. The aqueous extract from Beta vulgaris, the ethanol extract from Callisia grasilis and the methanol extract Annona sp. showed some antiherpetic activity with acceptable therapeutic indexes (the ratio of CC50 to EC50). These species are good candidates for further activity-monitored fractionation to identify active principles.

Cytotoxic and Antiviral Activities of Colombian Medicinal Plant Extracts of the Euphorbia genus

Forty-seven plant extracts of 10 species of the genus Euphorbia (Euphorbiaceae) used by Colombian traditional healers for the treatment of ulcers, cancers, tumors, warts, and other diseases, were tested in vitro for their potential antitumour (antiproliferative and cytotoxic) and antiherpetic activity. To evaluate the capacity of the extracts to inhibit the lytic activity of herpes simplex virus type 2 (HSV-2) and the reduction of viability of infected or uninfected cell cultures, the end-point titration technique (EPTT) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay were used, respectively. The therapeutic index of the positive extracts for the antiviral activity was determined by calculating the ratio CC50 (50% cytotoxic concentration) over IC50 (50% inhibitory concentration of the viral effect). Five of the 47 extracts (11%) representing 3 out of 10 Euphorbia species (30%) exhibited antiherpetic action; the highest activity was found in the leaf/stem water-methanol extracts from E. cotinifolia and E. tirucalli. The therapeutic indexes of these two plant species were > 7.1; these extracts exhibited no cytotoxicity. Six extracts (13%) representing 4 plant species (40%) showed cytotoxic activity. The highest cytotoxicity was found in the dichloromethane extract obtained from E. cotinifolia leaves and the CC50 values for the most susceptible cell lines, HEp-2 and CHO, were 35.1 and 18.1 µg/ml, respectively.

Screening and fractionation of plant extracts with antiproliferative activity on human peripheral blood mononuclear cells

Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA) stimulated proliferation of human peripheral blood mononuclear cells (PBMC). The proliferation was evaluated by the amount of [³H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 µg/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the ¹H and13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation.

Characterization of the molluscicidal activity of Bauhinia variegata and Mimusops elengi plant extracts against the fasciola vector lymnaea acuminata

The molluscicidal activity of Bauhinia variegata leaf and Mimusops elengi bark was studied against vector snail Lymnaea acuminata. The toxicity of both plants was time and concentration-dependent. Among organic extracts, ethanol extracts of both plants were more toxic. Toxicity of B. variegata leaf ethanolic extract (96h LC50- 14.4 mg/L) was more pronounced than M. elengi bark ethanolic extract (96h LC50-15.0 mg/L). The 24h LC50 of column purified fraction of B. variegata and M. elengi bark were 20.3 mg/L and 18.3 mg/L, respectively. Saponin and quercetin were characterized and identified as active molluscicidal component. Co-migration of saponin (Rf 0.48) and quercetin (Rf 0.52) with column purified bark of M. elengi and leaf of B. variegata on thin layer chromatography demonstrate same Rf value i.e. 0.48 and 0.52, respectively. The present study clearly indicates the possibility of using M. elengi and/or B. variegata as potent molluscicide.

CHARACTERIZATION OF MOLLUSCICIDAL COMPONENT OF Moringa oleifera LEAF AND Momordica charantia FRUITS AND THEIR MODES OF ACTION IN SNAIL Lymnaea acuminata

SUMMARY The molluscicidal activity of the leaf powder of Moringa oleifera and lyophilized fruit powder of Momordica charantia against the snail Lymnaea acuminata was time and concentration dependent. M. oleifera leaf powder (96 h LC50: 197.59 ppm) was more toxic than M. charantia lyophilized fruit powder (96 h LC50: 318.29 ppm). The ethanolic extracts of M. oleifera leaf powder and Momordica charantia lyophilized fruit powder were more toxic than other organic solvent extracts. The 96 h LC50 of the column purified fraction of M. oleifera leaf powder was 22.52 ppm, while that of M. charantia lyophilized fruit powder was 6.21 ppm. Column, thin layer and high performance liquid chromatography analysis show that the active molluscicidal components in M. oleifera leaf powder and lyophilized fruit of M. charantia are benzylamine (96 h LC50: 2.3 ppm) and momordicine (96 h LC50: 1.2 ppm), respectively. Benzylamine and momordicine significantly inhibited, in vivo and in vitro, the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activities in the nervous tissues of L. acuminata. Inhibition of AChE, ACP and ALP activity in the nervous tissues of L. acuminata by benzylamine and momordicine may be responsible for the molluscicidal activity of M. oleifera and M. charantia fruits, respectively.

EVALUATION OF ANTIMICROBIAL AND CYTOTOXIC ACTIVITIES OF PLANT EXTRACTS FROM SOUTHERN MINAS GERAIS CERRADO

The antimicrobial activity of plant hidroethanolic extracts on bacteria Gram positive, Gram negative, yeasts, Mycobacterium tuberculosis H37 and Mycobacterium bovis was evaluated by using the technique of Agar diffusion and microdilution in broth. Among the extracts evaluated by Agar diffusion, the extract of Bidens pilosa leaf presented the most expressive average of haloes of growth inhibition to the microorganisms, followed by the extract of B. pilosa flower, of Eugenia pyriformis' leaf and seed, of Plinia cauliflora leaf which statistically presented the same average of haloes inhibitory formation on bacteria Gram positive, Gram negative and yeasts. The extracts of Heliconia rostrata did not present activity. Mycobacterium tuberculosis H37 and Mycobacterium bovis(BCG) appeared resistant to all the extracts. The susceptibility profile of Candida albicans and Saccharomyces cerevisiae fungi were compared to one another and to the Gram positive Bacillus subtilis, Enterococcus faecalis and the Gram negative Salmonella typhimurium bacteria (p > 0.05). The evaluation of cytotoxicity was carried out on C6-36 larvae cells of the Aedes albopictus mosquito. The extracts of stem and flower of Heliconia rostrata, leaf and stem of Plinia cauliflora, seed of Anonna crassiflora and stem, flower and root of B. pilosa did not present toxicity in the analyzed concentrations. The highest rates of selectivity appeared in the extracts of stem of A. crassiflora and flower of B. pilosa to Staphylococcus aureus, presenting potential for future studies about a new drug development.