Validation of a New HPLC-DAD Method to Quantify 3-Deoxyanthocyanidins Based on Carajurin in Medicinal Plants and for Chemical Ecology Studies.

Anthocyanidins are found in nature mainly as pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin derivatives. These compounds are found free or as glycoside derivatives which are responsible for the color (red, blue, and violet) of some foods and are responsible for attracting seed dispersers. They are grouped into 3-hydroxyanthocyanidins, 3-deoxyanthocyanidins (3D-anth), and O-methylated anthocyanidins. A new method was developed and validated to quantify 3D-anth in plant-rich extracts. To test the new method, Arrabidaea chica Verlot was selected as it is widely used in folk medicine, and it is rich in 3D-anth. The new method was developed by HPLC-DAD and expressed 3D-anth as carajurin content. Carajurin was chosen as the reference standard due to its role as a biological marker for the antileishmanial activity for A. chica. The selected method used a silica-based phenyl column, a mobile phase composed of potassium dihydrogen phosphate buffer, acetonitrile, and methanol, in a gradient elution mode and detection at 480 nm. The method reliability was confirmed by verifying selectivity, linearity, precision, recovery, and robustness. This method contributes to quality control and development of a possible active pharmaceutical ingredient from A. chica as well as it can be used to evaluate 3D-anth in plant extracts with chemical ecology interest.

Carajurin Induces Apoptosis in Leishmania amazonensis Promastigotes through Reactive Oxygen Species Production and Mitochondrial Dysfunction.

Carajurin is the main constituent of Arrabidaea chica species with reported anti-Leishmania activity. However, its mechanism of action has not been described. This study investigated the mechanisms of action of carajurin against promastigote forms of Leishmania amazonensis. Carajurin was effective against promastigotes with IC50 of 7.96 ± 1.23 µg.mL-1 (26.4 µM), and the cytotoxic concentration for peritoneal macrophages was 258.2 ± 1.20 µg.mL-1 (856.9 µM) after 24 h of treatment. Ultrastructural evaluation highlighted pronounced swelling of the kinetoplast with loss of electron-density in L. amazonensis promastigotes induced by carajurin treatment. It was observed that carajurin leads to a decrease in the mitochondrial membrane potential (p = 0.0286), an increase in reactive oxygen species production (p = 0.0286), and cell death by late apoptosis (p = 0.0095) in parasites. Pretreatment with the antioxidant NAC prevented ROS production and significantly reduced carajurin-induced cell death. The electrochemical and density functional theory (DFT) data contributed to support the molecular mechanism of action of carajurin associated with the ROS generation, for which it is possible to observe a correlation between the LUMO energy and the electroactivity of carajurin in the presence of molecular oxygen. All these results suggest that carajurin targets the mitochondria in L. amazonensis. In addition, when assessed for its drug-likeness, carajurin follows Lipinski''s rule of five, and the Ghose, Veber, Egan, and Muegge criteria.

Carajurin: a anthocyanidin from Arrabidaea chica as a potential biological marker of antileishmanial activity.

Leishmaniasis is a group of neglected tropical diseases whose treatment with antimonials bears limitations and has changed little in over 80 years. Medicinal plants have been evaluated as a therapeutic alternative for leishmaniasis. Arrabidaea chica is popularly used as a wound healing and antiparasitic agent, especially as leishmanicidal agent. This study examined the leishmanicidal activity of a crude extract (ACCE), an anthocyanidin-rich fraction (ACAF), and three isolated anthocyanidins from A. chica: carajurin, 3'-hydroxy-carajurone, and carajurone. We evaluated the antileishmanial activity against promastigote and intracellular amastigote forms of Leishmania amazonensis and determined cytotoxicity in BALB/c peritoneal macrophages, as well as nitrite quantification, using the Griess method. Molecular docking was carried out to evaluate interactions of carajurin at the nitric oxide synthase enzyme. All compounds were active against promastigotes after 72 h, with IC50 values of 101.5 ± 0.06 µg/mL for ACCE and 4.976 ± 1.09 µg/mL for ACAF. Anthocyanidins carajurin, 3'-hydroxy-carajurone, and carajurone had IC50 values of 3.66 ± 1.16, 22.70 ± 1.20, and 28.28 ± 0.07 µg/mL, respectively. The cytotoxicity assay after 72 h showed results ranging from 9.640 to 66.74 µg/mL for anthocyanidins. ACAF and carajurin showed selectivity against intracellular amastigote forms (SI> 10), with low cytotoxicity within 24 h, a statistically significant reduction in all infection parameters, and induced nitrite production. Molecular docking studies were developed to understand a possible mechanism of activation of the nitric oxide synthase enzyme, which leads to an increase in the production of nitric oxide observed in the other experiments reported. These results encourage us to suggest carajurin as a biological marker of A. chica.