Flavonoids from Piper Species as Promising Antiprotozoal Agents against Giardia intestinalis. Structure-Activity Relationship and Drug-Likeness Studies.

Diarrhea diseases caused by the intestinal protozoan parasite Giardia intestinalis are a major global health burden. Moreover, there is an ongoing need for novel anti-Giardia drugs due to drawbacks with currently available treatments. This paper reports on the isolation and structural elucidation of six new flavonoids (1-6), along with twenty-three known ones (7-29) from the Piper species. Their structures were established by spectroscopic and spectrometric techniques. Flavonoids were tested for in vitro antiprotozoal activity against Giardia intestinalis trophozoites. In addition, structure-activity relationship (SAR) and in silico ADME studies were performed to understand the pharmacophore and pharmacokinetic properties of these natural compounds. Eight flavonoids from this series exhibited remarkable activity in the micromolar range. Moreover, compound 4 was identified as having a 40-fold greater antiparasitic effect (IC50 61.0 nM) than the clinical reference drug, metronidazole (IC50 2.5 µM). This antiprotozoal potency was coupled with an excellent selectivity index (SI 233) on murine macrophages and in silico drug-likeness. SAR studies revealed that the substitution patterns, type of functional group, and flavonoid skeleton played an essential role in the activity. These findings highlight flavonoid 4 as a promising candidate to develop new drugs for the treatment of Giardia infections.

Trichilones A-E: New Limonoids from Trichilia adolfi.

In addition to the trichilianones A-D recently reported from Trichilia adolfi, a continuing investigation of the chemical constituents of the ethanol extract of the bark of this medicinal plant yielded the five new limonoids 1-5. They are characterized by having four fused rings and are new examples of prieurianin-type limonoids, having a ε-lactone which in 4 and 5 is α, ß- unsaturated. The structures of the isolated metabolites were determined by high field NMR spectroscopy and HR mass spectrometry. The new metabolites were shown to have the ε-lactone fused with a tetrahydrofuran ring which is connected to an oxidized hexane ring joined with a cyclo-pentanone having a 3-furanyl substituent. As the crude extract possesses antileishmanial activity, the compounds were assayed for cytotoxic and antiparasitic activities in vitro in murine macrophage cells (raw 264.7 cells) and in Leishmania amazoniensis as well as L. braziliensis promastigotes. Metabolites 1-3 and 5 showed moderate cytotoxicity (between 30-94 µg/mL) but are not responsible for the antileishmanial effect of the extract.

Trichilianones A-D, Novel Cyclopropane-Type Limonoids from Trichilia adolfi.

The fractionation of an ethanol extract of the bark of Trichilia adolfi yielded four novel limonoids (trichilinones A-D, 1-4), with five fused rings and related to the hortiolide-type limonoids. Starting with an ε-lactone, which is α,ß-unsaturated in trichilinones A and D (1 and 4), attached to a tetrahydrofuran ring that is connected to an unusual bicyclo [5.1.0] hexane system, joined with a cyclopentanone with a 3-furanyl substituent [(2-oxo)-furan-(5H)-3-yl in trichilinone D (4)], the four compounds isolated display a new 7/5/3/5/5 limonoid ring system. Their structures were established based on extensive analysis of NMR spectroscopic data. As the crude extract possessed anti-leishmanial properties, the compounds were assayed for cytotoxic and anti-parasitic activities in vitro in murine macrophages cells (Raw 264.7) and leishmania promastigotes (L. amazoniensis and L. braziliensis), respectively. The compounds showed moderate cytotoxicity (approximately 70 µg/mL), but are not responsible for the leishmanicidal effect of the extract.

(E)-Piplartine Isolated from Piper pseudoarboreum, a Lead Compound against Leishmaniasis.

The current therapies of leishmaniasis, the second most widespread neglected tropical disease, have limited effectiveness and toxic side effects. In this regard, natural products play an important role in overcoming the current need for new leishmanicidal agents. The present study reports a bioassay-guided fractionation of the ethanolic extract of leaves of Piper pseudoarboreum against four species of Leishmania spp. promastigote forms, which afforded six known alkamides (1-6). Their structures were established on the basis of spectroscopic and spectrometric analysis. Compounds 2 and 3 were identified as the most promising ones, displaying higher potency against Leishmania spp. promastigotes (IC50 values ranging from 1.6 to 3.8 µM) and amastigotes of L. amazonensis (IC50 values ranging from 8.2 to 9.1 µM) than the reference drug, miltefosine. The efficacy of (E)-piplartine (3) against L. amazonensis infection in an in vivo model for cutaneous leishmaniasis was evidenced by a significant reduction of the lesion size footpad and spleen parasite burden, similar to those of glucantime used as the reference drug. This study reinforces the therapeutic potential of (E)-piplartine as a promising lead compound against neglected infectious diseases caused by Leishmania parasites.

An unprecedented chlorine-containing piperamide from Piper pseudoarboreum as potential leishmanicidal agent.

A phytochemical investigation of the ethanolic extract of leaves from Piper pseudoarboreum led to the isolation of 3-chlorosintenpyridone 1, an unprecedented chlorinated piperamide, together with the known compounds 2-12. Their structures were established based on 1D and 2D (COSY, ROESY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The proposed biosynthetic pathway of compound 1 is discussed. Compounds 1-12 were tested in vitro for their leishmanicidal potential against promastigote stages of Leishmania amazonensis, L braziliensis, L. guyanensis and L. infantum. Two compounds from this series, the alkamide 1 (IC50 3.4-5.2 µM) and the fatty acid 9 (IC50 18.7-29.6 µM) displayed higher or similar potency to Miltefosine, used as the reference drug.

Leishmanicidal and cytotoxic activity from plants used in Tacana traditional medicine (Bolivia).

ETNOPHARMACOLOGICAL RELEVANCE: Thirty-eight Tacana medicinal plant species used to treat skin problems, including leishmania ulcers, skin infections, inflammation and wound healing, were collected in the community of Buena Vista, Bolivia, with the Tacana people. Twenty two species are documented for the first time as medicinal plants for this ethnic group living in the northern area of the Department of La Paz. AIM OF THE STUDY: To evaluate the leishmanicidal effect (IC50) and cytotoxicity (LD50) of the selected plants. To carry out bioguided studies on the active extracts. To assess the potential of Bolivian plant biodiversity associated with traditional knowledge in the discovery of alternative sources to fight leishmaniasis. MATERIALS AND METHODS: Seventy three ethanol extracts were prepared from 38 species by maceration and were evaluated in vitro against promastigotes of Leishmania amazonensis and L. braziliensis. Active extracts (IC50 ≤ 50 µg/mL) were fractionated by chromatography on Silica gel column and the fractions were assessed against the two Leishmania strains. The most active fractions and the crude extracts were evaluated against reference strains of L. amazonensis, L. braziliensis, L. aethiopica, two native strains (L. Lainsoni and L. braziliensis) and for cytotoxicity against HeLa cells. The chromatographic profile of the active fractions was obtained by reverse phase chromatography using HPLC. RESULTS: From the 73 extracts, 39 extracts (53.4%) were inactive and 34 showed activity. Thirteen species were sselected for bioguided studies. The crude extracts and their 36 fractions were evaluated against two Leishmania strains. The most active fraction were tested in a panel of five leishmania strains and for cytotoxicity. The Selective Index (SI = LD50/IC50) was calculated, and were generally low. Retention time and UV spectra were recorded for the active fractions by HPLC-DAD using a reverse phase column. Profiles were very different from each other, showing the presence of different compounds. CONCLUSION: Bolivian traditional knowledge from the Tacanba was useful to identify plants with effect on Leishmania promastigotes. Chromatographic bioguided studies showed stronger leishmanicidal and cytotoxic activity for the medium polar fraction. HPLC analysis showed different chromatographic profiles of the active fractions.

Flavonoids from Piper delineatum modulate quorum-sensing-regulated phenotypes in Vibrio harveyi.

Quorum sensing (QS), or bacterial cell-to-cell communication, is a key process for bacterial colonization of substrata through biofilm formation, infections, and production of virulence factors. In an ongoing investigation of bioactive secondary metabolites from Piper species, four new flavonoids (1-4), along with five known ones (5-9) were isolated from the leaves of Piper delineatum. Their stereostructures were established by spectroscopic and spectrometric methods, including 1D and 2D NMR experiments, and comparison with data reported in the literature. The compounds were screened for their ability to interfere with QS signaling in the bacterial model Vibrio harveyi. Four compounds from this series (2, 3, 6, and 7) exhibited remarkable activity in the micromolar range, being compounds 3 and 7 particularly attractive since they did not affect bacterial growth. The results suggest that these flavonoids disrupt QS-mediated bioluminescence by interaction with elements downstream LuxO in the QS circuit of V. harveyi, and also, they exhibited a strong dose-dependent inhibition of biofilm formation. The present findings shed light on the QS inhibition mechanisms of flavonoids, underlining their potential applications.