Physico-Chemical Characterization of High Dilutions of Symphytum officinale and Therapeutic Effect in Hepatic and Osteosarcoma Cells

Background and Aim: Symphytum officinale (SO) root and leaf extracts are used as a remedy for bone fracture healing, anti-inflammation, and also prescribed clinically in high dilution as a homeopathic medicine. These extracts have lycopsamine, rosmarinic acid, symphytine and coumaric acid as major pyrrolizidine alkaloids. The objective of this study was to characterize SO mother tincture (SOMT), 6c, 30c and 200c, and to study biological responses with hepatic epithelial WRL-68 and osteosarcoma Saos-2 cells.

Methods: Physico-chemical characterization of SO were studied with high resolution liquid chromatogram mass spectrometer (HRLCMS), nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy, scanning electron microscopy and transmission electron microscopy (TEM). In-vitro drug efficacy was studied in WRL-68 and Saos-2 cells using a confocal microscope, MTT cell viability assay and flow cytometer techniques with commercially available SOMT, 6c, 30c and 200c.

Results: Lycopsamine mass peak of 300.47 Da, symphytine 381.075 Da, symviridine 382.45 Da and other pyrrolizidine fragments were detected using HRLCMS for SOMT, 6c, and 30c. Chemical shifts of 58.49ppm and 18.47ppm in 13C NMR confirm the presence of carbonyl, alkyl, amines, alkanes and alcohol functional groups. Silicates and micro-elements (K, Na, Mg, P, S) were detected in SO samples using high resolution TEM. Hepatotoxicity and anti-proliferative effects of SOMT were observed in WRL-68 and Saos-2 cells, whereas cell proliferation and hormesis were confirmed by MTT assay when treated with 6c to 200c for 48hrs. Morphology of Saos-2 and WRL-68 cells showed cytoskeletal disorientation with intact plasma membrane and nucleus with SOMT, 6c, 30c after 24hrs in confocal micrographs.

Conclusions: SO retained lycopsamine, symviridine and other pyrrolizidine alkaloids in high dilution up to 30c potency. This corresponds to silica encapsulation of starting material as shown in TEM micrographs. Significant proliferation of Saos-2 cells treated with SOMT to 200c confirms SO efficacy for bone healing.

Keywords: Symphytum officinale, HRLCMS, high dilution, lycopsamine, TEM