Japanese Knotweed Rhizome Bark Extract Inhibits Live SARS-CoV-2 In Vitro.

Coronavirus disease 2019 (COVID-19), a viral infectious respiratory disease, is caused by highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is responsible for the ongoing COVID-19 pandemic. Since very few drugs are known to be effective against SARS-CoV-2, there is a general need for new therapeutics, including plant-based drugs, for the prophylaxis and treatment of infections. In the current study, the activity of a 70% ethanolic(aq) extract of the rhizome bark of Japanese knotweed, an invasive alien plant species, was tested for the first time against the wild-type SARS-CoV-2 virus using a specific and robust virus neutralization test (VNT) on Vero-E6 cells, which best mimics the mechanism of real virus−host interaction. A statistically significant antiviral effect against SARS-CoV-2 (p-value < 0.05) was observed for the 50.8 µg mL−1 extract solution in cell medium. A suitable extract preparation was described to avoid loss of polyphenols throughout filtration of the extract, which was dissolved in cell medium containing fetal bovine serum (FBS). The significance of the differences between the sums of the test and control groups in the incidence of cytopathic effects (CPE) was determined using the one-way ANOVA test. A dose−response relationship was observed, with the cytotoxic effect occurring at higher concentrations of the extract (≥101.6 µg mL−1). The obtained results suggest possible use of this plant material for the production of various products (e.g., packaging, hygiene products, biodisinfectants, etc.) that would be useful against the spread of and for self-protection against COVID-19.

Antioxidant and Antimicrobial Biofoil Based on Chitosan and Japanese Knotweed (Fallopia japonica, Houtt.) Rhizome Bark Extract.

A 70% ethanol(aq) extract of the rhizome bark of the invasive alien plant species Japanese knotweed (JKRB) with potent (in the range of vitamin C) and stable antioxidant activity was incorporated in 1% w/v into a chitosan biofoil, which was then characterized on a lab-scale. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay confirmed the antioxidant activity of the JKRB biofoil upon contact with the food simulants A, B, C, and D1 (measured half-maximal inhibitory concentrations-IC50) and supported the Folin-Ciocalteu assay result. The migration of the antioxidant marker, (-)-epicatechin, into all food simulants (A, B, C, D1, D2, and E) was quantified using liquid chromatography hyphenated to mass spectrometry (LC-MS). Calculations showed that 1 cm2 of JKRB biofoil provided antioxidant activity to ~0.5 L of liquid food upon 1 h of contact. The JKRB biofoil demonstrated antimicrobial activity against Gram-positive bacteria. The incorporation of JKRB into the chitosan biofoil resulted in improved tensile strength from 0.75 MPa to 1.81 MPa, while elongation decreased to 28%. JKRB biofoil's lower moisture content compared to chitosan biofoil was attributed to the formation of hydrogen bonds between chitosan biofoil and JKRB compounds, further confirmed with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The JKRB biofoil completely degraded in compost in 11 days. The future upscaled production of JKRB biofoil from biowastes for active packaging may support the fights against plastic waste, food waste, and the invasiveness of Japanese knotweed, while greatly contributing to the so-called 'zero-waste' strategy and the reduction in greenhouse gas emissions.

(-)-Epicatechin-An Important Contributor to the Antioxidant Activity of Japanese Knotweed Rhizome Bark Extract as Determined by Antioxidant Activity-Guided Fractionation.

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.

Off-line multidimensional high performance thin-layer chromatography for fractionation of Japanese knotweed rhizome bark extract and isolation of flavan-3-ols, proanthocyanidins and anthraquinones.

A methodology based on off-line multidimensional thin-layer chromatography was developed for isolation of several secondary metabolites from bark of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Successive fractionation steps using PLC silica gel and HPTLC silica gel or HPTLC cellulose plates in combination with various developing solvents enabled isolation of (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, procyanidin B1, procyanidin B2, procyanidin B3, proanthocyanidin B dimer gallate, emodin, emodin-8-O-glucoside and emodin-8-O-malonyl-glucoside. Their identity was confirmed by HPTLC, HPTLC-MSn and for most of them also by 1H NMR and 2D NMR analyses. To the best of our knowledge emodin-8-O-malonyl-glucoside, procyanidins B1 and B2 were for the first time isolated from this plant material. HPTLC and HPTLC-MSn analyses were also performed as support of fractionation/isolation process, leading to first detection of some compounds in bark of Japanese knotweed rhizomes and Japanese knotweed rhizomes in general: procyanidins B1 and B2, methyl derivatives of emodin bianthrone and emodin bianthrone-hexose, resveratrol-malonyl-hexoside and taxifolin derivatives. Characterization of flavan-3-ols and proanthocyanidins was facilitated by post-chromatographic derivatization of the corresponding chromatographic zones with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent.

Oleamide, a Bioactive Compound, Unwittingly Introduced into the Human Body through Some Plastic Food/Beverages and Medicine Containers.

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.

Determination of d-Cycloserine Impurities in Pharmaceutical Dosage Forms: Comparison of the International Pharmacopoeia HPLC-UV Method and the DOSY NMR Method.

d-cycloserine is a broad-spectrum antibiotic that is currently being used as a secondary choice in the treatment of tuberculosis. In recent years, it has become more popular, due to its effect on the nervous system. In this current study, we provide evidence that The International Pharmacopoeia HPLC-UV method for d-cycloserine impurity profiling is not repeatable due to the variable response of cycloserine dimer, one of d-cycloserine impurities. Therefore, we introduced the DOSY (diffusion ordered spectroscopy) NMR (nuclear magnetic resonance) technique to determine the levels of d-cycloserine impurities in pharmaceutical dosage forms. The DOSY NMR technique allowed separation of d-cycloserine, its degradation products, and key process impurities in concentrations below pharmacopoeial specification limits. The proposed DOSY NMR method allowed accurate identification and quantification of the cycloserine dimer, which was not possible through the use of the pharmacopoeial HPLC method. The current method has the potential for practical use in analytical laboratories of the pharmaceutical industry.

Interference of oleamide with analytical and bioassay results.

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.

A Novel Testing Approach for Oxidative Degradation Dependent Incompatibility of Amine Moiety Containing Drugs with PEGs in Solid-State.

Reactive impurities originating from excipients can cause drug stability issues, even at trace amounts. When produced during final dosage form storage, they are especially hard to control, and often, factors inducing their formation remain unidentified. Oxidative degradation dependent formation of formaldehyde and formic acid is responsible for N-methylation and N-formylation of amine-moiety-containing drug substances. A very popular combination of polyethylene glycols and iron oxides, used in more than two-thirds of FDA-approved tablet formulation drugs in 2018, was found to be responsible for increased concentrations of N-methyl impurity in the case of paroxetine hydrochloride. We propose a novel testing approach for early identification of potentially problematic combinations of excipients and drug substances. The polyethylene glycol 6000 degradation mechanism and kinetics in the presence of iron oxides is studied. The generality of the proposed stress test setup in view of the susceptibility of amine-moiety-containing drug substances to N-methylation and N-formylation is evaluated.

Anti-inflammatory effects of cinnamon extract and identification of active compounds influencing the TLR2 and TLR4 signaling pathways.

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.

Analysis of triterpenoids and phytosterols in vegetables by thin-layer chromatography coupled to tandem mass spectrometry.

Three TLC methods were used for an initial screening of some common plant triterpenoids and phytosterols in cuticular wax extracts of different vegetables (zucchini, eggplant, tomato, red pepper, mangold, spinach, lettuce, white-colored radicchio di Castelfranco, raddichio Leonardo, white cabbage, red cabbage and savoy cabbage). The preliminary experiments showed that the studied vegetables are potential sources of triterpenoids and phytosterols. To identify the compounds present in the extracts with high certainty, the first TLC-MS(2) method was developed for the analysis of eight triterpenoids (lupeol, α-amyrin, ß-amyrin, cycloartenol, cycloartenol acetate, lupeol acetate, lupenone and friedelin) and two phytosterols (ß-sitosterol and stigmasterol). This method takes the advantages of: (1) a satisfactory separation of the target compounds; (2) their differentiation according to the band colors; and (3) the potential of their discrimination by the acquired first-order mass (MS) and product ion (MS(2)) spectra. Since the closely eluting compounds have complex and similar MS(2) spectra, distinguishing between them was possible by the proposed characteristic ions. Using a custom-built mass spectral library, the head to tail MS(2) spectra comparison of sample test solution zones and standard aided the compound identification. In addition to the molecular mass information, the developed atmospheric pressure chemical ionization method (APCI) in positive ion mode provided structural information, regarding the presence of functional group in the molecule. This approach resulted in many positively assigned compounds in the investigated vegetable waxes, from which more than a half are reported for the first time.