LC-MS/MS Evaluation of Pyrrolizidine Alkaloids Profile in Relation to Safety of Comfrey Roots and Leaves from Polish Sources.

Comfrey (Symphytum officinale L.) has a long tradition of use in the treatment of musculoskeletal disorders. However, due to hepatotoxic pyrrolizidine alkaloids (PAs), the EMA restricts the use of comfrey root (CR) to external use only and for short periods of time. Recent studies indicate a low permeability of PAs across the skin, calling into question the safety of topical application of products containing comfrey preparations. The aim of our work was to develop and validate an HPLC method enabling the separation of isomeric PAs from comfrey and, on this basis, to assess the potential toxicity of CR and comfrey leaf (CL) obtained from various Polish sources. The qualitative and quantitative analysis of PAs via HPLC-MS/MS was performed in MRM mode. The results obtained confirmed a lower content of PAs in CL than in CR and showed a wide variation in the composition of PAs in CR, with a much more stable profile of PAs in CL. Factor analysis confirmed that CRs and CLs differ in PA content, which is influenced by the growth conditions and geographical origin. The determined concentrations of PAs prove that in some CRs available on the Polish herbal market, the content of PAs may exceed the daily dose considered safe.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

CONTEXT: Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. OBJECTIVE: The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. MATERIALS AND METHODS: Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5-120 µg/mL) and compounds (1-50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. RESULTS: The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 µg/mg) and flavone C-glycosides (820.18 ± 0.05 µg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. CONCLUSIONS: The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

TLC-densitometric analysis of allantoin in Symphytum officinale L. roots.

A TLC-densitometric method for determination of allantoin in Symphytum officinale root was developed. Densitometric quantification of allantoin was carried out on TLC Si60 plates with butanol-50 % methanol/formic acid, 66.5:33.2:0.3 (V/V/V) as developing solvent, at a wavelength of 190 nm. The method was preliminarily validated in terms of specificity, linearity, precision, limit of detection, limit of quantification, recovery and robustness. The results of TLC quantification were compared with HPLC analysis carried out on a HILIC Luna NH2 100A column, with mobile phase consisting of acetonitrile/water 80:20 (V/V) and UV detection at 190 and 210 nm. Allantoin content was determined in two herbal products and it varied from 0.94 to 2.09 %, depending on the producer, and was in agreement with literature reports.