Original Study Abstracts
1. Aim or purpose
A new type of crystals of betulinic acid + vitamin C in isopropyl alcohol/ methanol / ethanol monosolvate (BA_VitC IPA/MeOH /EtOH) was obtained in a hydrothermal experiment, with superior antitumoral activity on various cell lines.
2. Materials and methods
Materials
All reagents used in the assay procedures were of analytical standard and were purchased from Sigma-Aldrich, Germany and Chemical Company SA, Iasi, Romania.
Methods
• Synthesis of (BA_VC IPA/EtOH/MeOH) cocrystals from isopropyl alcohol / methanol/ ethanol was done by using BA, vit.C and the solvents. The mixture was heated closed to the boiling temperature of the solvents, then cool slowly to room temperature protected from light. The formation of the first crystals was after 10 days
• Physico chemical properties: The morphology of the samples were determined by Scanning electron microscopy (SEM) and the thermal response of each product was performed via differential scanning calorimetry (DSC). Each sample was measured in the temperature interval of 25-350°C at a heating rate of 10°C/min under constant argon flow. X-ray powder diffraction and FT-IR spectra were realized in order to determine the structure of the new synthesized crystals.
• Antioxidant activity of the compound was evaluated by DPPH radical scavenging assay. A solution of radical DPPH was used as a standard antioxidant stock solution and a solution of ascorbic acid (0.167 mmol•L-1) in ethanol 96% (v/v) was used as etalon. Samples were analyzed by using a Uvi Line 9400 Spectrophotometer from SI Analytics at 516 nm for 20 minutes.
• Determination of cell viability was performed on several cancerous cell lines (MCF7, MDA-MB-231, HeLa, A375, B164A5 and B16F0) and non-tumorigenic human cells (HaCat) in order to verify the cytotoxic effects of the BA-VC IPA / EtOH / MeOH. The cells were stimulated with different concentrations (3, 10 and 30 μM) and incubated at three intervals of time. The antiproliferative activities of the tested samples were determined by MTT and Alamar blue assay and the absorbance measured using a xMark™ Microplate Spectrophotometer (Biorad) at 570 nm and 600 nm (reference) wavelengths.
3. Results
SEM images show morphological changes between the pure ingredients and their co-crystallization product and we can indicate the formation of a new phase. Different solvent molecules BA: Vit C obtained in MeOH / EtOH// IPA result in the dissimilarities among patterns were needle-shaped or plate-shaped.
XRPD spectra of cocrystals differ significantly from the ones recorded for pure ingredients, that suggest the formation of cocrystals instead of physical mixtures.
Differential scanning calorimetry shows a single broad peak corresponding to the water loss below 100 C for all types of cocrystals, in EtOH, MeOH and IPA. In all casses we can notice the disappearance of the pure substances’ characteristic peaks .
4. Conclusions
Physico chemical analysis (optical, SEM, DSC, XRPD, FTIR) antioxidant and in vitro activity of the new compounds obtained gave us the information that were formed new compounds with improved characteristics and superior antitumoral activity when compared with the components.

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