The balm of the Norway spruce (Picea abies) is a well-known traditional herbal medicine used to cure wounds. Even though clinical trials have confirmed its empirical use, the active constituents, their mode of action, and the exact composition of this natural product are still unknown. In this study, the balm was subjected to fractionated extraction and further purified employing flash chromatography, HPLC-PDA-ELSD, preparative and analytical TLC. Hydroxycinnamic acids (1–3), the lignan pinoresinol (4), four hydroxylated derivatives of dehydroabietic acid (DHAA) (5 – 8), and dehydroabietic acid (9) were isolated. Their structures were elucidated by LC-MS, 1D- and 2D-NMR. Four extracts, two commercially available resin acids–pimaric acid (10) and isopimaric acid (11)–and the isolated compounds were tested for increased re-epithelialization of cell-free areas in a human adult low calcium high temperature keratinocytes monolayer. Lysophosphatidic acid (10 µM) served as positive control and ranged between 100% and 150% rise in cell-covered area related to the vehicle control. Two extracts containing carboxylic acids and non-acidic apolar constituents, respectively, boosted wound closure by 47% and 36% at 10 and 3 µg/mL, respectively. Pinoresinol, DHAA, three of its hydroxylated derivatives, and pimaric and isopimaric acid as well as defined combinations of the hydroxylated DHAA derivatives led to a significantly enhanced wound closure by up to 90% at concentrations between 1 and 10 µM. Overall, lignans and diterpene resin acids, main constituents of Norway spruce balm, are able to increase migration or proliferation of keratinocytes in vitro. The presented data link the phytochemistry of this natural wound healing agent with boosted re-epithelialization.

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In a cytopathic effect inhibition assay, a standardized Rhodiola rosea root and rhizome extract, also known as roseroot extract (SHR-5), exerted distinct anti-influenza A virus activity against HK/68 (H3N2) (IC50 of 2.8 µg/mL) without being cytotoxic. For fast and efficient isolation and identification of the extractʼs bioactive constituents, a high-performance countercurrent chromatographic separation method was developed. It resulted in a three-stage gradient elution program using a mobile phase solvent system composed of ethyl acetate/n-butanol/water (1 : 4 : 5 → 2 : 3 : 5 → 3 : 2 : 5) in the reversed-phase mode. The elaborated high-performance countercurrent chromatographic method allowed for fractionation of the complex roseroot extract in a single chromatographic step in a way that only one additional orthogonal isolation/purification step per fraction yielded 12 isolated constituents. They cover a broad polarity range and belong to different structural classes, namely, the phenylethanoid tyrosol and its glucoside salidroside, the cinnamyl alcohol glycosides rosavin, rosarin, and rosin as well as gallic acid, the cyanogenic glucoside lotaustralin, the monoterpene glucosides rosiridin and kenposide A, and the flavonoids tricin, tricin-5-O-β-D-glucopyranoside, and rhodiosin. The most promising anti-influenza activities were determined for rhodiosin, tricin, and tricin-5-O-β-D-glucopyranoside with IC50 values of 7.9, 13, and 15 µM, respectively. The herein established high-performance countercurrent chromatographic protocol enables fast and scalable access to major as well as minor roseroot constituents. This is of particular relevance for extract standardization, quality control, and further in-depth pharmacological investigations of the metabolites of this popular traditional herbal remedy.

Mulberry Diels−Alder-type adducts (MDAAs) derived from the white mulberry tree were discovered recently as dual inhibitors of influenza viruses and pneumococci. For the development of a natural product based remedy for respiratory infections, the aim was to (i) identify the most prolific natural source of MDAAs, (ii) develop a protocol to maximize the content of MDAAs in Morus alba extracts, (iii) unravel constituents with the highest anti-infective potential within multicomponent mixtures, and (iv) select and characterize a hit extract as a candidate for further studies. Validated quantitative UPLC-PDA analysis of seven MDAAs (1−7) revealed the root bark as the best starting material and pressurized liquid extraction (PLE) as the optimum technique for extraction. Extracts enriched in MDAAs of a total content above 20% exerted a potent dual anti-influenza virus and antipneumococcal activity. For a detailed analysis of the most bioactive chemical features and molecules within the extracts, 1 H NMR-based heterocovariance analysis (HetCA) was used. According to the multivariate statistical analysis procedure conducted, MDAAs exclusively accounted for the in vitro anti-influenza viral effect. The anti-infective profile of one hit extract (MA60) investigated showed a good tolerance by lung cells (A549, Calu-3) and pronounced in vitro activities against influenza viruses, S. pneumoniae, S. aureus, and inflammation.

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In this study, an integrated in silico−in vitro approach was employed to discover natural products (NPs) active against SARS-CoV-2. The two SARS-CoV-2 viral proteases, i.e., main protease (M pro ) and papain-like protease (PL pro ), were selected as targets for the in silico study. Virtual hits were obtained by docking more than 140,000 NPs and NP derivatives available in-house and from commercial sources, and 38 virtual hits were experimentally validated in vitro using two enzyme-based assays. Five inhibited the enzyme activity of SARS-CoV-2 M pro by more than 60% at a concentration of 20 μM, and four of them with high potency (IC 50 < 10 μM). These hit compounds were further evaluated for their antiviral activity against SARS-CoV-2 in Calu-3 cells. The results from the cell-based assay revealed three mulberry Diels−Alder-type adducts (MDAAs) from Morus alba with pronounced anti-SARS-CoV-2 activities. Sanggenons C (12), O (13), and G (15) showed IC 50 values of 4.6, 8.0, and 7.6 μM and selectivity index values of 5.1, 3.1 and 6.5, respectively. The docking poses of MDAAs in SARS-CoV-2 M pro proposed a butterfly-shaped binding conformation, which was supported by the results of saturation transfer difference NMR experiments and competitive 1 H relaxation dispersion NMR spectroscopy.

Introduction Preparations from the Rhodiola rosea are experiencing an increase in popularity: extracts of dried roots and rhizomes are used as adaptogen to treat stress, fatigue, and weakness. To meet high pharmaceutical standards, fast and reliable methods to assess phytochemical variations in respect of quality control are needed. Objective The aim of this study was to extract and quantify seven characteristic secondary metabolites of R. rosea, namely p‐tyrosol (1), rosin (2), rosiridin (3), salidroside (4), rosarin (5), rosavin (6), and tricin‐5‐O‐β‐d‐glucopyranoside (7) in 24 herbal drugs and seven commercial preparations using a newly established supercritical fluid workflow. Methods The developed protocol allowed for an exhaustive extraction of compounds 1–7 using 60% carbon dioxide (CO2) and 40% methanol. The constituents were analysed on an ultra‐high‐performance supercritical fluid chromatography (UHPSFC) instrument using a charged surface hybrid fluoro‐phenyl (CSH FP) column (3.0 mm × 100 mm, 1.7 μm; mobile phase: CO2 and methanol). Results The seven compounds were separated in a remarkably short time (< 3.5 minutes). For their quantitation, good results in terms of selectivity, linearity (R2 ≥ 0.99), precision (intraday ≤ 3.03%, interday ≤ 5.17%) and accuracy (recovery rates 96.6–102.4%) were achieved using selected ion recording on a Quadrupole Dalton (QDa) mass detector. Conclusion The quantitative analysis of the investigated herbal drugs showed a highly differing metabolite pattern which was also observed in the investigated commercial products. None of the commercial dietary products met the declared content of rosavins and salidroside. The developed and validated protocol offers a novel and reliable method to assess the quantitative composition of Rhodiola herbal drugs and preparations.

The importance of hops (the flowers of Humulus lupulus) as food and an herbal remedy is reflected by a large number of analytical methods published. However, supercritical fluid chromatography, a highly efficient, rapid, and “green” separation technique, has not been considered for hops samples so far. This prompted us to establish the first supercritical fluid chromatography-based protocol for the separation, identification, and quantitation of five prenylated constituents of hops. Hulupinic acid (1), a prominent oxidation product of hop acids, three flavanones, i.e., 8-prenylnaringenin (2), 6-prenylnaringenin (3), and isoxanthohumol (4), as well as the chalcone xanthohumol (5) could be baseline separated in less than 5 minutes using a Viridis BEH 2-EP column (3.0 × 100 mm; 1.7 µm particle size) and a mobile phase consisting of CO2 and isopropanol. Good results regarding selectivity, accuracy (recovery rates: 85.0 – 113.1%), precision (intra-day ≤ 2.1%, inter-day ≤ 3.5%), and linearity (R2 ≥ 0.99) were obtained for both photodiode array and mass detection. The lowest detection limit at 220 nm was at 0.1 µg/mL (1, 3, and 4), with mass detection even at 0.001 µg/mL (4). As an application example of the validated method, the five hops constituents were quantified in three dietary supplements, one herbal medicinal product, and two batches of hop flowers (Lupuli flos). In most samples analyzed, the major component was 5 (0.01 – 1.02%), whereas the major component in Lupuli flos samples was compound 1 (0.12 – 0.21%). This protocol offers a fast and environmentally friendly alternative to liquid chromatography for the quality control of hops.