Influence of cultivation conditions, season of collection and extraction method on the content of antileishmanial flavonoids from Kalanchoe pinnata.

Ethnopharmacological Relevance: Leaves from Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) are popularly used for healing wounds. Its antileishmanial properties are established in experimental animals, and its active flavonoid components have been identified.
Aim of the Study: In this study, we attempted to standardize the extract from K. pinnata leaves by evaluating the influence of season of harvest, sunlight exposure and method of extraction on antileishmanial flavonoids content.
Materials and Methods: HPLC-DAD-MS was used to identify and quantify the active antileishmanial flavonoids in different extracts. ANOVA test for analyses of variance followed by the Tukey test of multiple comparisons were used in the statistical analysis. The antileishmanial potential was assessed by the activation of nitric oxide production by murine macrophage using the Griess method.
Results: We demonstrated that active flavonoids were significantly more abundant when the leaves were collected in the summer, and that aqueous extraction at 50°C allowed the highest flavonoid extraction. The benefit of sunlight exposure was confirmed in plants cultivated under direct sunlight when compared with those that grown under shade. Under sunny conditions the yield of the most active antileishmanial favonoid quercitrin was increased by 7-fold. All aqueous extracts tested were capable to enhance the macrophage nitric oxide production. However, hot aqueous extract from leaves collected in summer exhibited the higher activity, in agreement with HPLC-DAD-MS analysis tendency. In addition, with the aim of reducing the individual chemical variations of the plant constituents and optimizing the production of the active extract, it was obtained in vitro monoclonal KP specimens that were easily adapted to field conditions and were able to produce antileishmanial flavonoids.
Conclusion: Our study reports the better conditions of cultivation, harvest and extraction protocol for obtaining a K. pinnata extract exhibiting the highest antileishmanial activity. Additionally, we propose the flavonoids quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside and quercitrin, as satisfactory chemical markers for standardization purposes.
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