First report of acaricidal efficacy from plumbagin on larvae of Rhipicephalus microplus and Rhipicephalus sanguineus resistant to conventional acaricides.

The problem of resistance to acaricides in ticks such as Rhipicephalus microplus and R. sanguineus has motivated the search for control alternatives, such as the use of extracts and secondary metabolites from plants. Plumbagin is a natural product present in plants such as Plumbago zeylanica L., Diospyros kaki, and D. anisandra, of which acaricidal activity has been reported. Therefore, the objective of this study was to evaluate in vitro the acaricidal efficacy of plumbagin on larvae of R. microplus and R. sanguineus resistant to conventional acaricides. Larvae from engorged female ticks, collected from naturally infested dairy cattle and domiciled dogs, in Yucatan, Mexico, were used. The larval packet test and the larval immersion test were performed to detect acaricide susceptibility. Both tick populations were detected as resistant to cypermethrin and amitraz. Then, the modified larval immersion test was used and plumbagin was evaluated at concentrations of 1%, 0.5%, 0.25%, and 0.125% (%w/v), obtaining a mortality of 100% in the four concentrations for both tick species. Subsequently, lower doses of plumbagin were evaluated at concentrations of 0.0625%, 0.03125%, 0.015625% and 0.0078125%, obtaining mortalities of 100 to 36.26% for R. microplus and 100%-5.33% for R. sanguineus. Using Probit analysis, lethal concentrations at 50% (LC50), 99% (LC99) and confidence intervals at 95% (CI95%) were calculated. R. microplus showed a LC50 of 0.011% (CI95%: 0.010-0.011) and LC99 of 0.019% (CI95%: 0.018-0.022). R. sanguineus presented a LC50 of 0.017% (CI95%: 0.015-0.018) and CL99 of 0.031% (CI95%: 0.027-0.036). It was concluded that plumbagin has high acaricidal efficacy against larvae of R. microplus and R. sanguineus resistant to amitraz and cypermethrin. R. microplus larvae were significantly more susceptible to LC50 and LC99 compared to R. sanguineus. This is the first report on the acaricidal efficacy of plumbagin on larvae of R. microplus and R. sanguineus resistant to conventional acaricides.

Plumbagin: A Promising In Vivo Antiparasitic Candidate against Schistosoma mansoni and In Silico Pharmacokinetic Properties (ADMET).

Schistosomiasis, a potentially fatal chronic disease whose etiological agents are blood trematode worms of the genus Schistosoma spp., is one of the most prevalent and debilitating neglected diseases. The treatment of schistosomiasis depends exclusively on praziquantel (PZQ), a drug that has been used since the 1970s and that already has reports of reduced therapeutic efficacy, related with the development of Schistosoma-resistant or -tolerant strains. Therefore, the search for new therapeutic alternatives is an urgent need. Plumbagin (PLUM), a naphthoquinone isolated from the roots of plants of the genus Plumbago, has aroused interest in research due to its antiparasitic properties against protozoa and helminths. Here, we evaluated the in vivo schistosomicidal potential of PLUM against Schistosoma mansoni and the in silico pharmacokinetic parameters. ADMET parameters and oral bioavailability were evaluated using the PkCSM and SwissADME platforms, respectively. The study was carried out with five groups of infected mice and divided as follows: an untreated control group, a control group treated with PZQ, and three groups treated orally with 8, 16, or 32 mg/kg of PLUM. After treatment, the Kato-Katz technique was performed to evaluate a quantity of eggs in the feces (EPG). The animals were euthanized for worm recovery, intestine samples were collected to evaluate the oviposition pattern, the load of eggs was determined on the hepatic and intestinal tissues and for the histopathological and histomorphometric evaluation of tissue and hepatic granulomas. PLUM reduced EPG by 65.27, 70.52, and 82.49%, reduced the total worm load by 46.7, 55.25, and 72.4%, and the female worm load by 44.01, 52.76, and 71.16%, for doses of 8, 16, and 32 mg/kg, respectively. PLUM also significantly reduced the number of immature eggs and increased the number of dead eggs in the oogram. A reduction of 36.11, 46.46, and 64.14% in eggs in the hepatic tissue, and 57.22, 65.18, and 80.5% in the intestinal tissue were also observed at doses of 8, 16, and 32 mg/kg, respectively. At all doses, PLUM demonstrated an effect on the histopathological and histomorphometric parameters of the hepatic granuloma, with a reduction of 41.11, 48.47, and 70.55% in the numerical density of the granulomas and 49.56, 57.63, and 71.21% in the volume, respectively. PLUM presented itself as a promising in vivo antiparasitic candidate against S. mansoni, acting not only on parasitological parameters but also on hepatic granuloma. Furthermore, in silico, PLUM showed good predictive pharmacokinetic profiles by ADMET.

Antiparasitic Activity of Plumbago auriculata Extracts and Its Naphthoquinone Plumbagin against Trypanosoma cruzi.

Chagas disease (CD) caused by the protozoan Trypanosoma cruzi affects more than six million people worldwide. Treatment is restricted to benznidazole (Bz) and nifurtimox (Nf) that display low activity in the later chronic stage besides triggering toxic events that result in treatment abandonment. Therefore, new therapeutic options are necessary. In this scenario, natural products emerge as promising alternatives to treat CD. In the family Plumbaginaceae, Plumbago sp. exhibits a broad spectrum of biological and pharmacological activities. Thus, our main objective was to evaluate, in vitro and in silico, the biological effect of crude extracts of root and of aerial parts of P. auriculata, as well as its naphthoquinone Plumbagin (Pb) against T. cruzi. The phenotypic assays revealed potent activity of the root extract against different forms (trypomastigote and intracellular forms) and strains (Y and Tulahuen), with a compound concentration that reduced 50% of the number of the parasite (EC50) values ranging from 1.9 to 3.9 µg/mL. In silico analysis showed that Pb is predicted to have good oral absorption and permeability in Caco2 cells, besides excellent probability of absorption by human intestinal cells, without toxic or mutagenic potential effects, not being predicted as a substrate or inhibitor of P-glycoprotein. Pb was as potent as Bz against intracellular forms and displayed a superior trypanosomicidal effect (about 10-fold) in bloodstream forms (EC50 = 0.8 µM) as compared to the reference drug (8.5 µM). The cellular targets of Pb on T. cruzi were evaluated using electron microscopy assays and the findings on bloodstream trypomastigotes showed several cellular insults related to the autophagic process. Regarding toxicity in mammalian cells, the root extracts and the naphthoquinone present a moderate toxic profile on fibroblasts and cardiac cell lines. Then, aiming to reduce host toxicity, the root extract and Pb were tested in combination with Bz, and the data showed additive profiles with the sum of the fractional inhibitory concentration indexes (ΣFICIs) being 1.45 and 0.87, respectively. Thus, our work reveals the promising antiparasitic activity of Plumbago auriculata crude extracts and its purified naphthoquinone Plumbagin against different forms and strains of Trypanosoma cruzi in vitro.

Anthelmintic Activity of Extracts and Active Compounds From Diospyros anisandra on Ancylostoma caninum, Haemonchus placei and Cyathostomins.

The present study aimed to evaluate the anthelmintic activity of leaf and bark extracts of Diospyros anisandra collected during different seasons and their major constituents on eggs of Ancylostoma caninum, Haemonchus placei, and cyathostomins. Specifically, the eclosion inhibition of the methanolic extracts of the leaves and bark of D. anisandra collected during the dry and rainy seasons (600-37.5 µg/ml) were evaluated in addition to the fractions, sub-fractions (300-37.5 µg/ml) and active major constituents (150-2.3 µg/ml). The rainy season bark extract had the highest percentage of eclosion inhibition (PEI) against the evaluated nematodes (≥ 90% at 75 µg/ml) along with high ovicidal activity (90.0 to 93.4% at 75 µg/ml). The purification of the rainy season bark extract showed that its biological activity came from the non-polar n-hexane fraction (≥ 93% at 75 µg/ml). The bioguided fractionation pointed to sub-fraction 5 as having the highest anthelmintic activity against the three evaluated genera of nematodes (PEI ≥ 93% at 37.5 µg/ml). Gas chromatography and mass spectrometry revealed that the major constituent in sub-fraction 5 was plumbagin. Upon evaluation, plumbagin was confirmed to be responsible for the anthelmintic activity of D. anisandra, with a PEI ≥ 90% at 2.3 µg/ml on the three evaluated nematodes. Additionally, the compounds betulin and lupeol in the bark of D. anisandra were evaluated but presented low anthelmintic activity (PEI ≤ 5.3% at 2.3 µg/ml). In conclusion, the rainy season bark extract of D. anisandra exerts a high ovicidal activity against the eggs of the three studied nematodes. Plumbagin is the active compound responsible for this activity and represents a potential alternative for the control of different genera of gastrointestinal nematodes given the current scenario of anthelmintic resistance.

Actividad biológica de Plumbago scandens L. sobre cepas multidrogoresistente de Mycobacterium tuberculosis / Biological activity of Plumbago scandens L. against multidrug-resistance strains of Mycobacterium tuberculosis

Tuberculosis control is a high priority for the Ministry of Health from Peru. In the present work the inhibitory effect of both metanolic (MeOH) and ethyl acetate (AcOEt) crude extracts and the minimum inhibitory concentration (MIC) of both wild plants and in vitro plantlets of Plumbago scandens L. -Plumbaginaceae- against multidrug-resistance (MDR) strains of Mycobacterium tuberculosis were determined. The plant material was constituted by roots and seeds, collected in the Motupe (Lambayeque) area. The in vitro plantlets were obtained from seedlings and micropropagated by shoot tips and nodal segments in Murashige and Skoog (MS) culture medium. The microbiological material consisting of one control strain and two strains of Mycobacterium tuberculosis resistant to isoniazid (INH) and rifampicin (RIF) was cultivated using the Lowenstein–Jensen culture medium. The MIC values varied from 0,65 to 1,3 mg/mL. Bacterial strains showed more sensitivite to the AcOEt crude extract. The GC analysis of the plant material showed the presence of the naphtoquinone plumbagin and other aromatic compounds. In conclusion, the MeOH y AcOEt crude extracts from roots of the wild plants and MeOH crude extract of in vitro plantlets of P. scandens showed a strong inhibitory activity against MDR strains of M. tuberculosis.

El control de la tuberculosis es un objetivo de alta prioridad para el Ministerio de Salud del Perú. En el presente trabajo se determinó el efecto inhibitorio de los extractos crudos metanólico (MeOH) y de acetato de etilo (AcOEt), expresados como concentración mínima inhibitoria (CMI) de plantas silvestres y plántulas in vitro de Plumbago scandens L. (Plumbaginaceae) sobre cepas multidrogoresistente (MDR) de Mycobacterium tuberculosis. El material vegetal estuvo constituido por raíces de plantas silvestres, en tanto que las plántulas in vitro fueron obtenidas de semillas y micropropagadas en medio de cultivo Murashige y Skoog (MS). El material microbiológico, constituido por una cepa control y dos cepas resistentes a isoniacida (INH) y rifampicina (RIF), fue cultivado en medio de cultivo Lowenstein-Jensen. Los valores CMI variaron entre 0,65 y 1,3 mg/mL, mostrándose más sensibles las cepas bacterianas frente al extracto crudo de AcOEt de plantas silvestres. El análisis cromatografía de gases (GC) determinó la presencia de la naftoquinona plumbagina y otros compuestos aromáticos. En conclusión, los extractos crudos de MeOH y AcOEt de raíces de plantas silvestres y extracto crudo de MeOH de plántulas in vitro de P. scandens ejercieron una fuerte acción inhibitoria sobre cepas MDR de M. tuberculosis.