The in vitro antiviral property of Azadirachta indica polysaccharides for poliovirus

Ethnopharmacological relevance

Azadirachta indica A. Juss, popularly known as neem, has been extensively used in Ayurvedic medicine by Indian population for over 2000 years. It is used traditionally for the healing of various diseases. Natural products and their derivatives provide an excellent source for new anti-viral drugs.

Aim of the study

The present study aims at evaluating the activity of two polysaccharides (P1 and P2) isolated from the leaves of Azadirachta indica and their chemical sulfated derivatives (P1S and P2S) against poliovirus type 1 (PV-1).

Materials and methods

The cytotoxicity of the compounds was analyzed by MTT and the antiviral effect was determined by plaque reduction assay in different protocols.

Results

The polysaccharides did not show any cytotoxic effects on HEp-2 cells at the highest tested concentration (200 μg/ml) and exhibited significant antiviral activity with inhibitory concentrations (IC₅₀) of 80 μg/ml, 37.5 μg/ml, 77.5 μg/ml, and 12.1 μg/ml for P1, P1S, P2 and P2S, respectively, and the selectivity indexes (SI) ranged from 18 to 131.9. The compounds demonstrated better inhibitory effect when added concomitantly with the virus infection with a dose-dependent curve inhibition. Lesser effect was observed when the compounds were added after viral infection and the least effect at pre-treatment.

Conclusions

We suggested that the polysaccharides obtained from Azadirachta indica act against PV-1 by inhibiting the initial stage of viral replication. Importantly, original polysaccharides showed better virucidal effect than their sulfated derivatives at all tested concentrations. This study provides a scientific basis for the past and present ethnomedical uses of this plant.     

Hyperpigmentant activity of leaves and flowers extracts of Pyrostegia venusta on murine B16F10 melanoma

Ethnopharmacological relevance

Pyrostegia venusta is a native Brazilian plant which has a variety of uses in traditional folk medicine including the treatment of vitiligo. However, its effectiveness on melanogenesis is not yet elucidated.

Aim of the study

This study aimed to investigate the melanogenic activity of hydroalcoholic extracts from the leaves and flowers of P. venusta on murine B16F10 melanoma cells.

Materials and methods

Different concentrations of the hydroalcoholic extracts of flowers and leaves of P. venusta were evaluated in trials of spontaneous melanin content (4 days), and cell viability by the MTT assay in murine B16F10 cells, and in the mushroom tyrosinase activity in vitro.

Results

Both extracts, leaves (0.1; 0.3; 1 and 3 μg/mL) and flowers (0.03 and 0.1 μg/mL) increased the melanin content in a concentration dependent manner after 4 days of incubation on melanoma cells. Leaves extract promoted enhancement of melanogenesis with maximum effect of 33.3 ± 3% (3 μg/mL), and the flower extract increased in 23.4 ± 3% (0.1 μg/mL). The cell viability test using MTT showed that in the same tested concentrations of both extracts no cell death was detected. Actually, either extract was not able to cause any change in the tyrosinase activity. HPLC analysis of P. venusta extracts found 0.09% and 1.08% of allantoin on leaves and flowers extracts, respectively.

Conclusions

The leaves and flowers extracts of P. venusta stimulates B16F10 melanogenesis at very low concentrations. These findings support the folk medicinal use of P. venusta on the treatment of hypopigmentation diseases, such as vitiligo.     

Gut and airways relaxant effects of Carum roxburghianum

Ethnopharmacological relevance

Carum roxburghianum is traditionally used in hyperactive gastrointestinal and respiratory disorders. The present study was carried out to investigate the possible gut and airways relaxant potential of Carum roxburghianum to rationalize its folk uses.

Materials and methods

Crude extract of Carum roxburghianum (Cr.Cr) was studied in in vivo and in vitro techniques.

Results

Cr.Cr exhibited protective effect against castor oil-induced diarrhea in mice at 100–1000 mg/kg. In rabbit jejunum preparations, Cr.Cr (0.03–3.0 mg/mL) caused relaxation of spontaneous and K⁺ (80 mM)-induced contractions at similar concentrations, like papaverine. Pretreatment of tissues with Cr.Cr (0.1–1.0 mg/mL) shifted Ca⁺⁺ concentration–response curves (CRCs) to right, like verapamil. Cr.Cr (0.03 and 0.1 mg/mL) caused leftward shift of isoprenaline-induced inhibitory CRCs, similar to papaverine. In isolated guinea-pig ileum, Cr.Cr (0.01 and 0.03 mg/mL) produced rightward parallel shift of acetylcholine-curves, like atropine. Cr.Cr (1.0–30 mg/kg) caused suppression of carbachol (CCh, 100 μg/kg)-induced increase in inspiratory pressure of anaesthetized rats. In guinea-pig trachea, Cr.Cr (0.03–1.0 mg/mL) relaxed CCh and high K⁺-induced contractions, shifted isoprenaline-induced inhibitory CRCs to left at 0.1 and 0.3 mg/mL and CCh-curves parallel to right (0.01 and 0.03 mg/mL). Cr.Cr did not cause any mortality of mice up to 10 g/kg dose.

Conclusion

These results indicate that Carum roxburghianum possess combination of antidiarrheal, antispasmodic and bronchodilatory effects, which provides pharmacological basis to its traditional use in the disorders of gut and airways hyperactivity, like diarrhea, colic and asthma.     

Screening of the topical anti-inflammatory activity of the bark of Acacia cornigera Willdenow, Byrsonima crassifolia Kunth, Sweetia panamensis Yakovlev and the leaves of Sphagneticola trilobata Hitchcock

Ethnopharmacological relevance

An investigation of topical anti-inflammatory activity was undertaken on plants used in Central America traditional medicine.

Aim of study

Four herbal drugs used in the folk medicine of Central America to treat inflammatory skin affections (Acacia cornigera bark, Byrsonima crassifolia bark, Sphagneticola trilobata leaves and Sweetia panamensis bark) were evaluated for their topical anti-inflammatory activity.

Materials and methods

Petroleum ether, chloroform and methanol extracts were obtained for herbal medicines and then extracts were tested on Croton oil-induced ear dermatitis model in mice.

Results

Almost all the extracts reduced the Croton oil-induced ear dermatitis in mice and the chloroform ones showed the highest activity, with ID₅₀ (dose giving 50% oedema inhibition) values ranging from 112 μg/cm² (Byrsonima crassifolia) to 183 μg/cm² (Sphagneticola trilobata). As reference, ID₅₀ of the non-steroidal anti-inflammatory drug indomethacin was 93 μg/cm².

Conclusions

Lipophilic extracts from these species can be regarded as potential sources of anti-inflammatory principles.     

Sesquiterpene lactones from Gynoxys verrucosa and their anti-MRSA activity

Ethnopharmacological relevance

Because of its virulence and antibiotic resistance, Staphylococcus aureus is a more formidable pathogen now than at any time since the pre-antibiotic era. In an effort to identify and develop novel antimicrobial agents with activity against this pathogen, we have examined Gynoxys verrucosa Wedd (Asteraceae), an herb used in traditional medicine in southern Ecuador for the treatment and healing of wounds.

Materials and methods

The sesquiterpene lactones leucodine (1) and dehydroleucodine (2) were extracted and purified from the aerial parts of Gynoxys verrucosa, and their structure was elucidated by spectroscopic methods and single-crystal X-ray analysis. The in vitro anti-microbial activity of Gynoxys verrucosa extracts and its purified constituents was determined against six clinical isolates including Staphylococcus aureus and Staphylococcus epidermidis strains with different drug-resistance profiles, using the microtiter broth method.

Results

Compound 1 has very low activity, while compound 2 has moderate activity with MIC₅₀s between 49 and 195 μg/mL. The extract of Gynoxys verrucosa has weak activity with MIC₅₀s between 908 and 3290 μg/mL.

Conclusions

We are reporting the full assignment of the ¹H NMR and ¹³C NMR of both compounds, and the crystal structure of compound 2, for the first time. Moreover, the fact that compound 2 has antimicrobial activity and compound 1 does not, demonstrates that the exocyclic conjugated methylene in the lactone ring is essential for the antimicrobial activity of these sesquiterpene lactones. However, the weak activity observed for the plant extracts, does not explain the use of Gynoxys verrucosa in traditional medicine for the treatment of wounds and skin infections.     

Cytotoxicity and antibacterial activity of Lindera strychnifolia essential oils and extracts

Aim of the study

Lindera strychnifolia (LS) is widely used in traditional Chinese medicine. In the present study, we investigated cytotoxicity and antibacterial activity of essential oils and various fractions of ethanol extract of LS to explore the active components of LS and their pharmacological effects.

Materials and methods

The in vitro cytotoxicities of essential oils and various solvent fractions of LS on three human cancer cell lines (A549, HeLa and Hep G2) and a non-cancerous cell line (HUVEC) were examined using a modified MTT assay. And by using agar disc diffusion and broth microdilution methods, the antibacterial activity of these samples was evaluated against 10 bacteria including 5 clinically isolated strains. The compositions of the essential oils from the leaves and roots of LS were also analyzed by GC and GC–MS.

Results

The leaf oil showed the strongest cytotoxicity on the cancer cell lines tested with the IC₅₀ values ranged from 22 to 24 μg/ml after 24 h of treatment. The most sensitive microbial strain to all the samples was Staphylococcus aureus ATCC 25923.

Conclusions

Our results showed that the essential oils of LS exhibited greater cytotoxicity and antibacterial activity than the solvent fractions of ethanol extract of LS.     

Involvement of the L-arginine-nitric oxide pathway in the antinociception caused by fruits of Prosopis strombulifera (Lam.) Benth

Ethnopharmacological relevance

Prosopis strombulifera (Lam.) Benth. is a rhizomatous shrub that grows in the north and central zone of Argentina. In folk medicine, the fruits of this plant have been used as an astringent, anti-inflammatory and odontalgic agent and anti-diarrheic.

Aim of the study

To investigate the antinociceptive effect of ethanol (EE), chloroform (CE) and ethyl acetate (EtOAcE) extracts of Prosopis strombulifera fruits and the involvement of the l-arginine-nitric oxide pathway in this effect.

Materials and methods

The antinociceptive effects of the EE, CE and EtOAcE of Prosopis strombulifera fruits were evaluated in vivo using the formalin-induced pain test in mice with aspirin and morphine as reference antinociceptive compounds. The participation of the l-arginine-nitric oxide pathway in the antinociceptive effect was investigated in the same animal model using l-arginine as a nitric oxide (NO) precursor. The in vitro inhibitory effect of the extracts on LPS-induced nitric oxide production and iNOS expression was investigated in a J774A.1 macrophage-derived cell line.

Results

CE (300 mg/kg), in contrast to EE and EtOAcE, caused significant inhibition (p < 0.05) of the in vivo nociceptive response. Moreover, CE (100–1000 mg/kg, p.o.) produced a dose-dependent inhibition of the neurogenic and the inflammatory phases of the formalin test with inhibition values (at 600 mg/kg) of 42 ± 7% and 62 ± 7%, respectively. CE inhibition was more potent in the inflammatory phase, with an ID₅₀ of 400.1 (252.2–634.8) mg/kg. The antinociception caused by CE (600 mg/kg, p.o.) was significantly attenuated (p < 0.05) by i.p. treatment of mice with l-arginine (600 mg/kg). In addition, CE (100 μg/mL) produced significant in vitro inhibition (p < 0.001) of LPS-induced NO production, which was not observed with EE and EtOAcE at the same concentration. The inhibition of NO production by CE (10–100 μg/mL) was dose-dependent, with an IC₅₀ of 39.8 (34.4–46.1) μg/mL, and CE significantly inhibited LPS-induced iNOS expression in J774A.1 cells.

Conclusions

This study supports, in part, the ethnomedical use of Prosopis strombulifera fruits by showing that its CE produces moderate antinociception in vivo. The findings also provide scientific information for understanding the molecular mechanism involved in the analgesic effect of this plant.     

In vitro antiprotozoal and cytotoxic activity of 33 ethonopharmacologically selected medicinal plants from Democratic Republic of Congo

Ethnopharmacological relevance

The antiprotozoal and cytotoxic activity of the aqueous extracts from 33 medicinal plants, used by traditional healers for the treatment of various parasitic diseases and collected after an ethnopharmacological inventory conducted in the Bolongo area, Bandundu province in DR Congo, was evaluated.

Materials and methods

Decoctions were prepared, lyophilized and evaluated for in vitro antiprotozoal activity against Trypanosoma b. brucei, Trypanosoma cruzi, Leishmania infantum, and the chloroquine- and pyrimethamine-resistant K1 strain of Plasmodium falciparum. Cytotoxicity against MRC-5 cells was included to assess selectivity of activity.

Results

Most of the tested extracts exhibited pronounced (IC₅₀ ≤ 5 μg/ml) or good (5 < IC₅₀ ≤ 10 μg/ml) antiprotozoal activity against one or more of the selected protozoa. A total of 19 plant extracts inhibited Trypanosoma b. brucei, especially the extract from Isolona hexaloba stem bark (IC₅₀ = 1.95 μg/ml, SI = 16.5); 8 plant extracts were active against Trypanosoma cruzi, the extracts from Enanatia chlorantha stem bark and Quassia africana root bark being the most active with IC₅₀ values of 1.87 and 1.88 μg/ml, respectively (SI = 3.0 and 3.3, respectively); 8 plant extracts showed activity against Leishmania infantum, with extracts from Napoleona vogelii stem bark and Quassia africana root bark as the most active with IC₅₀ values of 5.66 and 5.04 μg/ml (SI = 11.3 and 1.2). Finally, 9 plant extracts inhibited Plasmodium falciparum K1 with the extracts from Quassia africana (root bark and stem bark) being the most active ones with IC₅₀ values of 0.46 and 1.27 μg/ml (SI = 13.7 and 13.6). Extracts from Enantia chlorantha stem bark, Piptadeniastrum africanum stem bark and Quassia africana root bark were cytotoxic for MRC-5 cells (CC₅₀ < 10 μg/ml).

Conclusions

These results can partly support and justify the traditional use of some of these plant species for the treatment of parasitic diseases.     

Aegiceras corniculatum extract suppresses initial and late phases of inflammation in rat paw and attenuates the production of eicosanoids in rat neutrophils and human platelets

Aim of the study

The present study is designed to explore the anti-inflammatory potential of Aegiceras corniculatum Linn. Blanco stems extracts and their mechanism of action against various pro-inflammatory mediators and to validate its traditional use against inflammatory diseases.

Materials and methods

Rat paw edema and peritonitis models were employed for in vivo studies. For in vitro studies human platelets and rat neutrophils were stimulated with Ca²⁺-ionophore A23187 leading to the production of various pro-inflammatory metabolites, i.e., 12-HTT, 12-HETE and LTB₄ and 5-HETE which were quantified by HPLC.

Results

The highly polar methanol extract (100 mg/kg) caused ∼90% reduction in the carrageenan- and prostaglandin E2-induced paw edema in rats. It also caused the inhibition of cycloxygenase-1 metabolite, 12-HHT (IC₅₀ 41.1 ± 1.5 μg/ml) with a concomitant rise in 12-lipoxygenase metabolite, 12-HETE in A23187 stimulated human platelets. Conversely, the non-polar hexane extract attenuated (IC₅₀ 0.36 ± 0.12 μg/ml) 12-HETE formation with a parallel rise in 12-HHT, thereby displaying a selectivity towards 12-lipoxygenase. Non-polar hexane extract also antagonized the production of 5-lipoxygenase metabolites, i.e., leukotriene B₄ and 5-HETE in the rat neutrophils. Furthermore, ethyl acetate extract inhibited both COX and 5-LOX with a marked decline in the production of 12-HHT (IC₅₀ 0.08 ± 0.002 μg/ml) and LTB₄ (IC₅₀ 0.86 ± 0.03 μg/ml), respectively. The anti-inflammatory effect of hexane and ethyl acetate extracts was also reflected by the diminution of carrageenan-induced cell infiltration in rat peritoneum. Additionally, plant extracts caused ∼60% suppression in dextran-induced paw edema implying that they also ameliorate histamine and serotonin release.

Conclusion

Hexane, ethyl acetate and methanol extracts derived from Aegiceras corniculatum possess significant anti-inflammatory activity via multiple mechanisms and validate their traditional use against inflammation-related diseases.     

Evaluation of the wound healing property of Boesenbergia longiflora rhizomes

Ethnopharmacological relevance

The rhizomes of Boesenbergia longiflora (Wall.) Kuntze (Zingiberaceae) have been traditionally used for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess by decoction with alcohol.

Aim of the study

The rhizomes of Boesenbergia longiflora were carried out to investigate for anti-inflammatory and wound healing activities in order to support the traditional use.

Material and methods

The ethanolic extract of Boesenbergia longiflora and its fractions were tested using relevant in vitro anti-inflammatory and wound healing assays. For the in vitro studies, murine macrophage RAW264.7 cells and mouse fibroblast L929 cells were assessed for anti-inflammatory and fibroblast stimulatory activities, respectively. In vivo anti-inflammatory activity was determined by carrageenan-induced rat paw edema model as well as acute toxicity estimated by the up-and-down method in mice.

Results

The present study has demonstrated that the ethanolic extract of Boesenbergia longiflora rhizomes possesses a potent anti-inflammatory and wound healing activities. Among the isolated fractions, the CHCl₃ fraction showed potent anti-inflammatory effect through nitric oxide inhibitory activity (IC₅₀=5.5 μg/ml) and reduction of carrageenan-induced rat paw edema (ED₅₀=222.7 mg/kg), whereas this fraction exhibited wound healing property via fibroblast migration on both day 1 (77.3%) and day 2 (100%) as well as enhanced collagen production (187.5 μg/ml) at concentration of 3 μg/ml, compared to that of the controls, 39.4% for fibroblast and 60.8 μg/ml for collagen, respectively. The anti-inflammatory mechanism of the CHCl₃ fraction is found to suppress the iNOS and COX-2 mRNA expression.

Conclusion

The scientific investigation of wound healing activity of Boesenbergia longiflora rhizomes support the Thai traditional uses for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess. The EtOH extract and CHCl₃ fraction exert potential wound healing property through NO inhibition, anti-oxidant effect and stimulation of fibroblast migration and collagen production. The phytochemical screening revealed that the CHCl₃ fraction of Boesenbergia longiflora rhizomes contains diarylheptanoids, flavonoids and terpenes. The isolation of the compounds responsible for the wound healing effect is now in progress.     

Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts

Pharmacological relevance

Presentation of the scratch assay as a convenient and inexpensive in vitro tool to gain first insights in the wound healing potential of plant extracts and natural compounds.

Aim of the study

The present study deals with the optimization of the scratch assay which can be used as an in vitro model for quantification of fibroblast migration to and proliferation into the wounded area. It is suitable for the first evaluation of the wound re-epithelialization potential of crude herbal extracts, isolated compounds and pharmaceutical preparations. As a proof of concept three preparations from traditional medicinal plants were investigated.

Materials and methods

Swiss 3T3 albino mouse fibroblasts were used in monolayers and platelet derived growth factor as positive control. Hexane and ethanolic extracts from Calendula officinalis and Matricaria recutita, Hypericum oil as well as the triterpenoids faradiol myristate and palmitate were studied. To differentiate between proliferation and migration antimitotic mitomycin C was added.

Results

Both extracts of Calendula officinalis stimulated proliferation and migration of fibroblasts at low concentrations, e.g. 10 μg/ml enhanced cell numbers by 64.35% and 70.53%, respectively. Inhibition of proliferation showed that this effect is mainly due to stimulation of migration. Faradiol myristate and palmitate gave comparable stimulation rates at an almost 50 μg/ml concentration, indicating that they contribute partially, but not most significantly to the wound healing effects of Calendula preparations. Extracts from Matricaria recutita were only moderately active. Hypericum oil was cytotoxic at concentrations higher than 0.5 μg/ml.

Conclusions

The scratch assay in the present form can be used as a promising scientific approach and platform to differentiate between plant extracts known for their wound healing and their anti-inflammatory properties.     

Evaluation of mechanism for antihypertensive action of Clerodendrum colebrookianum Walp., used by folklore healers in north-east India

Ethnopharmacological relevance

The present investigation was aimed to justify the pharmacological basis in traditional use of Clerodendrum colebrookianum as antihypertensive agent in north-east India.

Materials and Methods

The aqueous extract (AECc), its aqueous, n-butanol (nBFCc), Ethyl-acetate (EtFCc) and Chloroform fractions of C. colebrookianum leaves were evaluated for antihypertensive potential by using fructose-induced hypertension model in rats and in isolated frog heart. The ex-vivo muscarinic action in isolated rat ileum, in-vitro assay for Rho-kinase (ROCK –II), phosphodiesterase-5 (PDE-5) and angiotension converting enzyme (ACE) were also carried out to establish the mechanism of action of samples. The total phenolic and flavonoied contents in test samples were estimated to establish phyto–pharmacological relationship.

Results

The 100 μg/mL test samples were showed calcium antagonism in rat ileum and at 50 μg/mL and 75 μg/mL doses exhibited ROCK-II and PDE-5 inhibition respectively where, EtFCc was caused maximum 68.62% (ROCK-II) and 52.28% (PDE-5) inhibition, but none of the sample was exhibit effect in ACE at 100 μg/mL. The test samples also showed negative inotropic and chronotropic effect on isolated frog heart and significant (P<0.001) reduction in systolic blood pressure and heart rate in hypertensive rats compared to control. The total phenolic content maximum 80 μg gallic acid equivalents in nBFCc and flavonoids content maximum 69.57 μg Quercetin equivalent in AECc were estimated.

Conclusions

These observations established the traditional claim and thus C. colebrookianum could be a potent antihypertensive agent for use in future. The antihypertensive effect mediated by cholinergic action and following ROCK – II, PDE-5 inhibition of C. colebrookianum.     

Quercus cerris extracts limit Staphylococcus aureus biofilm formation

Ethnopharmacological relevance

Quercus cerris L., Fagaceae has been used in traditional Mediterranean medicine for numerous purposes, including anti-infective therapies for diarrhea and wound care.

Aim of the study

To evaluate the anti-staphylococcal activity of fractions of ethanolic extracts of Quercus cerris leaf and stem/fruit samples in models for biofilm and growth inhibition.

Materials and methods

Ethanolic extracts of Quercus cerris leaves and stems/fruits were prepared, resuspended in water and fractioned by successively partitioning with hexane, ethyl acetate and butanol. The ability of the fractions to inhibit Staphylococcus aureus biofilm formation was tested using static crystal violet staining methods and confocal laser scanning microscopy. Growth studies were conducted to determine if the diminished capacity to form a biofilm was related to growth inhibition.

Results

The butanol extracts of both the leaf and stem/fruit samples were the most active, and at a dose of 200 μg/ml, the capacity to form a biofilm was limited to a level equivalent to that of the sarA mutant controls. Further examination of the impact of these fractions on Staphylococcus aureus growth revealed that biofilm inhibition by the leaf butanol fraction was due to its bacteriostatic activity. The stem/fruit butanol fraction, however, showed a limited impact on growth, thus demonstrating that biofilm inhibition in this case is not related to the bacteriostatic activity of the extract.

Conclusion

Our evaluation of a medicinal plant used in Mediterranean ethnotherapies for infectious disease has demonstrated significant activity in the inhibition of staphylococcal biofilm formation with a mechanism unrelated to staphylococcal growth inhibition. These results contribute towards validation of this botanical remedy and form the groundwork for future studies in the search for novel biofilm inhibiting drugs.     

Antiplasmodial activity of the andiroba (Carapa guianensis Aubl., Meliaceae) oil and its limonoid-rich fraction

Ethnopharmacological relevance

From seeds of Carapa guianensis the Amazon native people extracts the andiroba oil, which is traditionally used as febrifuge, anti-malarial, insecticidal and repellant. The non-saponifiable fraction separated from the oil is rich in limonoids, which assigns its pharmacological effects.

Materials and methods

The andiroba oil and its limonoid-rich fraction were submitted to in vitro antiplasmodial bioassay using W₂ and Dd₂ strains of Plasmodium falciparum. The acute toxicity of andiroba oil was evaluated. The limonoid-rich fraction was subjected to fractionation and identified its major constituents.

Results

Andiroba oil and its limonoid-rich fraction inhibited the growth of W₂ clone in 100%, between 24 and 72 h, at concentrations of 8.2 μg/mL and 3.1 μg/mL, respectively. Under the same conditions, the parasitaemia of Dd₂ clone provoked by the andiroba oil showed inhibition of 31% (IC₅₀ >82 μg/mL) with a time-dependent relationship of 24 h and inhibition of 88% (IC₅₀ 8.4 μg/mL) after 72 h, while for the limonoid-rich fraction the inhibition of Dd₂ clone was 56% (IC₅₀ 2.8 μg/mL) at 24 h and 82% (IC₅₀ 0.4 μg/mL) after 72 h. Andiroba oil in acute toxicity test with a fixed dose (LD₅₀ >2000 mg/kg) was not toxic The limonoids identified in the oil were gedunin, 6α-acetoxygedunin, 7-deacetoxy-7-oxogedunin, 7-deacetylgedunin, 1,2-dihydro-3β-hydroxy-7-deacetoxy-7-oxogedunin and andirobin. Gedunin and derivatives has been reputed as anti-malarials.

Conclusion

The results support the traditional use of andiroba oil as antiplasmodial, which additionally proved not to be toxic in bioassays conducted with mice.     

Identification of rosmarinic acid as the major active constituent in Cordia americana

Ethnopharmacological relevance

Preparation from leaves of Cordia americana have been widely used in traditional medicine in South Brazil to treat wounds and various inflammations.

Aim of the study

The objective of this work was to identify the effective compounds in the ethanolic extract prepared from the leaves of Cordia americana, which is used in traditional South Brazilian medicine as anti-inflammatory and wound healing remedy.

Materials and methods

Isolation and structure elucidation techniques were performed in order to identify the compounds of Cordia americana and HPLC analysis was used for the quantification. The major constituent and the ethanolic extract were investigated for inhibition of 5-lipoxygenase, p38α MAPK, TNFα release and NF-κB as well as in the fibroblast scratch assay.

Results

Rosmarinic acid (1) was identified as the major compound with an amount of 8.44% in the ethanolic extract of the leaves of Cordia americana. The ethanolic extract as well as (1) exhibited the highest inhibitory effects on 5-lipoxygenase (IC₅₀ = 0.69 and 0.97 μg/mL, resp., IC₅₀ of BWA4C as reference: 0.3 μM) and p38α (IC₅₀ = 3.25 and 1.16 μg/mL, resp., IC₅₀ of SB203580 as reference: 0.046 μM) and moderate inhibitory effects on TNFα release. Slight effects were observed in the fibroblast scratch assay.

Conclusions

This study increases our knowledge on the effective compound in Cordia americana and supports its use in traditional medicine. We demonstrated for the first time pharmacological effects of Cordia americana and we provide evidences for a crucial role of rosmarinic acid as the major key player.     

Effect of Brazilian copaiba oils on Leishmania amazonensis

Ethnopharmacological relevance

Copaiba oil has been used in folk medicine since the 19th century. The use of copaiba oils to treat leishmaniasis is cited in several ethnopharmacological studies. Nevertheless, the potential antileishmania of copaiba oils had not been studied.

Aim of the study

Eight different kinds of Brazilian copaiba oils were screened for antileishmanial activity.

Materials and methods

The antiproliferative effect of copaiba oil on promastigote and amastigote axenic were determined. To determine the survival index peritoneal macrophage were infected with promastigotes of Leishmania amazonensis and treated with copaiba oil. The cytotoxic effect of copaiba oil was assessed on macrophage strain J774G8 by assay of sulforhodamine B.

Results

Copaiba oils showed variable levels of activity against promastigote forms with IC₅₀ values in the range between 5 and 22 μg/mL. The most active oil was that from Copaifera reticulata (collected in Pará State, Brazil) with IC₅₀ values of 5, 15, and 20 μg/mL for promastigote, axenic amastigote and intracellular amastigote forms, respectively. Amphotericin B showed IC₅₀ of 0.058 and 0.231 μg/mL against promastigote and amastigote forms, respectively. Cytotoxicity assay showed that this copaiba oil obtained from Copaifera reticulata showed low cytotoxicity against J774G8 macrophages.

Conclusion

Copaiba oils showed significant activity against the parasite Leishmania amazonensis.     

Antiadhesion as a functional concept for protection against uropathogenic Escherichia coli: In vitro studies with traditionally used plants with antiadhesive activity against uropathognic Escherichia coli

Ethnopharmacological relevance

Investigation of medicinal plant extracts traditionally used against uncomplicated urinary tract infections (UTI) and identification of antiadhesive effects under in vitro conditions against binding of uropathogenic Escherichia coli (UPEC) on bladder cell surface.

Materials and methods

Literature search on traditionally used medicinal plants for UTI was performed by online data bases and standard herbal monographs. For further identification shortlisting was done by intensive evaluation of results by plausibility and phytochemical aspects. Plant material with documented antibacterial effects was not considered for further investigations. Direct cytotoxicity of EtOH–water (1:1; v/v) extracts of the shortlisted plants was investigated against UPEC strain 2980 and bladder cell line T24. Inhibition of UPEC adhesion to T24 cells was monitored either after pretreatment of bacteria or eukaryotic cells by flow cytometry.

Results

Literature search on traditionally used medicinal plants for UTI resulted in 275 plant species, from which 20 were shortlisted by a validated selection process for experimental testing. While direct cytotoxicity of the extracts (1–2000 μg/mL) against UPEC and T24 cells was excluded significant antiadhesive effects were monitored for five plant extracts. Two of them, prepared from the rhizome of Agropyron repensL. and the stigmata of Zea maysL. decreased bacterial adhesion (IC₂₅ 630 μg/mL, IC₅₀ 1040 μg/mL, resp.) by interacting with bacterial outer membrane proteins, which was shown by pretreatment of UPEC. Preparations of three plant extracts from the leaves of Betula spp. (according to European pharmacopoeia 7.0), Orthosiphon stamineusBENTH. and Urtica spp. showed antiadhesive effects by interacting with T24 cells (IC₅₀ 415, 1330 μg/mL, resp. IC₂₅ 580 μg/mL). Combination of two extracts, one interacting with the bacterial surface (Zea maysL., Agropyron repensL.) and one with the eukaryotic target (Orthosiphon stamineusBENTH.) revealed synergistic effects, as shown by strongly decreased IC₅₀ values (131 μg/mL, 511 μg/mL, resp.).

Conclusions

Different plant extracts, traditionally used for UTI, exhibit antiadhesive effects against UPEC under in vitro conditions. Molecular targets can be different, either on the bacterial or on the host cell surface. Combination of these medicinal plants with different targets, as observed often in phytotherapy, results in synergistic effects.     

Effect of an alkaloidal fraction of Tabernaemontana elegans (Stapf.) on selected micro-organisms

Ethnopharmacological relevance

Bacterial infections remain a significant threat to human health. Due to the emergence of widespread antibiotic resistance, development of novel antibiotics is required in order to ensure that effective treatment remains available. There are several reports on the ethnomedical use of Tabernaemontana elegans pertaining to antibacterial activity.

Aim of the study

The aim of this study was to isolate and identify the fraction responsible for the antimicrobial activity in Tabernaemontana elegans (Stapf.) root extracts.

Materials and methods

The active fraction was characterized by thin layer chromatography (TLC) and gas chromatography–mass spectrometry (GC–MS). Antibacterial activity was determined using the broth micro-dilution assay and antimycobacterial activity using the BACTEC radiometric assay. Cytotoxicity of the crude extract and fractions was assessed against primary cell cultures; lymphocytes and fibroblasts; as well as a hepatocarcinoma (HepG2) and macrophage (THP-1) cell line using the Neutral Red uptake and MTT assays.

Results

The crude root extracts were found to contain a high concentration of alkaloids (1.2%, w/w). GC–MS analysis identified the indole alkaloids, voacangine and dregamine, as major components. Antibacterial activity was limited to the Gram-positive bacteria and Mycobacterium species, with MIC values in the range of 64–256 μg/ml. When combined with antibiotics, additive antibacterial effects were observed. Marked cytotoxicity to all cell lines tested was evident in the MTT and Neutral Red uptake assays, with IC₅₀ values <9.81 μg/ml.

Conclusions

This study confirms the antibacterial activity of Tabernaemontana elegans and supports its potential for being investigated further for the development of a novel antibacterial compound.