Purpose: Inflammatory processes are involved in many diseases. The bark of Cinnamomum verum and its extracts are well known for anti-inflammatory effects, but the underlying active compounds and chemical mechanisms are not yet fully identified. The objective of this study was to elucidate how cinnamon extract, specifically active compounds, and their combinations influence the signaling pathways of inflammation, especially through toll-like receptors TLR2 and TLR4. Methods: Bioassay-guided fractionation was performed for standard ethanolic cinnamon extract using high performance liquid chromatography followed by compound identification in the determined active fractions by high-resolution mass spectrometry and gas chromatography-mass spectrometry. THP-1 monocytes were pre-incubated with cinnamon extract, cinnamon fractions or its compounds and stimulated with lipopolysaccharides (LPS), followed by determination of interleukin 8 (IL-8) secretion, and phosphorylation of protein kinase B (Akt), nuclear factor (NF)-κB inhibitor alpha (IκBα) and p38. Furthermore, testing was performed in stimulated HEK-TLR2 and HEK-TLR4 reporter cells for direct receptor agonistic effects. Results: Among the identified compounds, trans-cinnamaldehyde and p-cymene significantly reduced the LPS-dependent IL-8 secretion in THP-1 monocytes. Synergistic anti-inflammatory effects were observed for combinations of trans-cinnamaldehyde with p-cymene, cinnamyl alcohol or cinnamic acid. Moreover, cinnamon extract as well as trans-cinnamaldehyde and p-cymene mitigated the phosphorylation of Akt and IκBα. Conclusions: Trans-cinnamaldehyde and p-cymene contribute to the strong anti-inflammatory effects of cinnamon extract. Furthermore, our experiments indicate that also synergistic effects among compounds that do not exhibit anti-inflammatory effects themselves might be present to positively influence the beneficial effects of cinnamon bark extract.

The antioxidant activities of Japanese knotweed rhizome bark extracts, prepared with eight different solvents or solvent mixtures (water, methanol, 80% methanol_(aq), acetone, 70% acetone_(aq), ethanol, 70% ethanol_(aq), and 90% ethyl acetate_(aq)), were determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. Low half maximal inhibitory concentration (IC₅₀) values (2.632–3.720 µg mL⁻¹) for all the extracts were in the range of the IC₅₀ value of the known antioxidant ascorbic acid at t₀ (3.115 µg mL⁻¹). Due to the highest extraction yield (~44%), 70% ethanol_(aq) was selected for the preparation of the extract for further investigations. The IC₅₀ value calculated for its antioxidant activity remained stable for at least 14 days, while the IC₅₀ of ascorbic acid increased over time. The stability study showed that the container material was of great importance for the light-protected storage of the ascorbic acid_(aq) solution in a refrigerator. Size exclusion–high-performance liquid chromatography (SEC-HPLC)–UV and reversed phase (RP)-HPLC-UV coupled with multistage mass spectrometry (MSⁿ) were developed for fractionation of the 70% ethanol_(aq) extract and for further compound identification, respectively. In the most potent antioxidant SEC fraction, determined using an on-line post-column SEC-HPLC-DPPH assay, epicatechin, resveratrol malonyl hexoside, and its in-source fragments (resveratrol and resveratrol acetyl hexoside) were tentatively identified by RP-HPLC-MSⁿ. Moreover, epicatechin was additionally confirmed by two orthogonal methods, SEC-HPLC-UV and high-performance thin-layer chromatography (HPTLC) coupled with densitometry. Finally, the latter technique enabled the identification of (−)-epicatechin. (−)-Epicatechin demonstrated potent and stable time-dependent antioxidant activity (IC₅₀ value ~1.5 µg mL⁻¹) for at least 14 days.

The purpose of the study was to investigate the migration of oleamide, a polymer lubricant, and a bioactive compound, from various plastic, marketed containers for food/beverages and medicines into polymer contact liquid. Methanol, food/medicine simulants or real samples were used to extract polymer leachables and extractables. Migrated oleamide into polymer contact liquids was determined by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). The concentration of oleamide in the extracts of medicinal and insulin syringes was 7351 ng mL⁻¹ and 21,984 ng mL⁻¹, respectively. The leachates of intravenous (i.v.) infusion bottle, medicinal and insulin syringes contained 17 ng mL⁻¹, 12 ng mL⁻¹ and 152 ng mL⁻¹, respectively. Oleamide in the extracts of dummies ranged from 30 to 39 ng mL⁻¹, while in the leachates of baby bottles, from 12 to 23 ng mL⁻¹. Leachates of soft drink bottles contained from 6 to 15 ng mL⁻¹ oleamide, milk bottles from 3 to 9 ng mL⁻¹, liquid yogurt bottles 17 ng mL⁻¹ and water bottles from 11 to 18 ng mL⁻¹. Bottled real matrices of oil and milk contained oleamide in the range from 217 to 293 ng mL⁻¹. Moreover, the source of migrated oleamide (e.g., containers, caps, other parts) was identified. Oleamide is listed in the current EU regulations without a specific migration limit. Accordingly, these values are considered of no concern, unless future toxicological studies prove the opposite.

During sample preparation and analysis, samples are coming in contact with different labware materials. By four unrelated analytical (phytochemical and pharmaceutical) case-studies and employing different analytical techniques, we demonstrated the potential misinterpretation of analytical results due to the use of contaminants-leaching labware during sample handling. Oleamide, a common polymer lubricant and a bioactive compound, was identified as a main analytical interference, leaching from different labware items into solvents, recognised as chemically compatible with the tested polymer material. Moreover, anti-inflammatory effect of oleamide at 100 μg mL⁻¹ and considerable pro-inflammatory effect of the plastic syringe extractables (containing oleamide) at the same level were shown in a TLR4-based bioassay. Taking these results into account, together with the fact that oleamide can be a compound of natural origin, we would like to notify the professional public regarding the possible erroneous oleamide-related analytical and bioassay results due to the use of oleamide-leaching labware. Researchers are alerted to double check the real source of oleamide (labware or natural extract), which will prevent further reporting of false results. Analysis of procedural blanks with de-novo developed UHPLC-ESI-MS method is, among some other strategies, proposed for detection of oleamide interference and avoidance of misleading results of certain analyses.