Supercritical CO2 pretreatment and subcritical water extraction of sulfated flavonoids from Flaveria bidentis leaves

FALLETTI P.; BARRERA VAZQUEZ F.; SANTOS P.H.; GONÇALVES RODRIGUES L.G; LANZA M.; MARTINI, R.E.; COMINI LR .

Cordoba

Conferencia; VI IBEROAMERICAN CONFERENCE ON SUPERCRITICAL FLUIDS (PROSCHIBA); 2023

VI IBEROAMERICAN CONFERENCE ON SUPERCRITICAL FLUIDS (PROSCHIBA)

Flaveria bidentis is an annual weed native to South America and has spread rapidly, posing serious threats to crop and native biodiversity in different countries. This species is capable of synthesizing two quercetin derivatives with the highest degree of sulfation known: quercetin 3,7,3',4'-tetrasulfate (QTS) and quercetin 3-acetyl-7,3',4'-trisulfate (ATS), which present important antithrombotic pharmacological activities and inhibitors of the aldose reductase enzyme. These sulfated flavonoids (SF) are not commercially available and they are obtained exclusively from the plant kingdom. Thus, large volume of biomass generated by F. bidentis during its weeding can be exploited in order to obtain these bioactive compounds. However, they are generally extracted through conventional methods that present low yields and extraction efficiencies and for this reason, alternative extraction methods should be explored.In this sense, the extraction of natural compounds with subcritical water (SWE) implies the use of water as extraction solvent, at temperature between 100-374°C and pressure high enough to maintain it in a liquid state. SWE is considered a green technology because it is ecological, economical and safe. On the other hand, supercritical CO2 (SC-CO2) has been used to carry out sample treatments prior to the extraction of the active principles, to weaken the cell walls of the plant matrix and increase its recovery.Thus, in this work we propose the design, development and optimization of the pretreatment of F. bidentis leaves with SC-CO2 (P-SC-CO2), and its subsequent SWE extraction, with the aim to increases the extraction yield and efficiency of total SF (QTS+ATS), in comparison with the extractive method in the absence of pretreatment (SWE). For this purpose, the SWE was performed under optimal conditions previously determined (Falletti et al., 2023b).During each P-SC-CO2 assay, 3 g of leaves (420 - < 1190 μm) were pretreated at 50°C. The CO2 was removed by depressurization and the pretreated plant material was reserved for later extraction. To optimize and evaluate the effects of time (15 and 30 min), pressure (15 and 30 MPa) and depressurization speed (0.2 and 2 Kg CO2/h), on the SWE performance of total SF (mg of total SF/ 100 g of leaves), a Response Surface Methodology (RSM) was applied based on a 23 factorial experimental design of two replicates. Data analysis was performed using ANOVA, significance with p value < 0.05, suitability of the model through the coefficient of determination R2, and maximization of the desirability function to determine the optimal conditions. All pretreated samples were subjected to SWE under optimal conditions (2.86 ml/min, 101.2 °C, 420 -< 1190 μm, 100 bar, 35 min, continuous flow) (Falletti et al., 2023b). The extracts were purified in total SF and finally quantified by high performance liquid chromatography (HPLC). Additionally, the effect of P-SC-CO2 on plant material morphology was studied by scanning electron microscopy (SEM) (10 kV, 500X and 1000X, gold coating). In the SEM images it can be seen that the leaves subjected to P-SC-CO2 show significant fragmentation due to the high speed of depressurization used. Regarding the RSM, the ANOVA showed a first-order polynomial equation, with R2=0.99 and a statistically significant (p