Terpenoids from Myrrh and Their Cytotoxic Activity against HeLa Cells.

The oleo-gum resin of Commiphora myrrha (Nees) Engl. has a long history of medicinal use, although many of its constituents are still unknown. In the present investigation, 34 secondary metabolites were isolated from myrrh resin using different chromatographic techniques (silica flash chromatography, CPC, and preparative HPLC) and their structures were elucidated with NMR spectroscopy, HRESIMS, CD spectroscopy, and ECD calculations. Among the isolated substances are seven sesquiterpenes (1-7), one disesquiterpene (8), and two triterpenes (23, 24), which were hitherto unknown, and numerous substances are described here for the first time for C. myrrha or the genus Commiphora. Furthermore, the effects of selected terpenes on cervix cancer cells (HeLa) were studied in an MTT-based in vitro assay. Three triterpenes were observed to be the most toxic with moderate IC50 values of 60.3 (29), 74.5 (33), and 78.9 µM (26). Due to the different activity of the structurally similar triterpenoids, the impact of different structural elements on the cytotoxic effect could be discussed and linked to the presence of a 1,2,3-trihydroxy substructure in the A ring. The influence on TNF-α dependent expression of the intercellular adhesion molecule 1 (ICAM-1) in human microvascular endothelial cells (HMEC-1) was also tested for 4-6, 9-11, 17, 18, 20, and 27 in vitro, but revealed less than 20% ICAM-1 reduction and, therefore, no significant anti-inflammatory activity.

Myrrh and Chamomile Flower Extract Inhibit Mediator Release from IgE-stimulated Mast-Cell-Like RBL-2H3 Cells.

Recent clinical evidence supports the efficacy of a traditional medicinal product (TMP) containing a combination of myrrh (Commiphora myrrha (Nees) Engl.), coffee charcoal (Coffea arabica L.), and chamomile flower dry extract (Matricaria chamomilla L.) in the therapy of diarrhea and inflammatory bowel disease. Mast cells seem to play a key role in the symptom generation of irritable bowel syndrome (IBS). To evaluate the use of the TMP in IBS treatment, the effects of the herbal extracts on the release of mast-cell mediators from stimulated RBL-2H3 cells were investigated. Therefore, degranulation was induced by phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A13187 (CI) or IgE stimulation, and the amounts of released ß-hexosaminidase and histamine were quantified. The extracts showed no effect on the mediator release of PMA- and CI-stimulated RBL-2H3 cells. Myrrh and chamomile were able to reduce the ß-hexosaminidase release of IgE-stimulated cells, while myrrh showed stronger inhibition of the mediator release than chamomile, which reduced only IgE-stimulated histamine release. Thus, these results indicate a mechanistic basis for the use of the herbal combination of myrrh, coffee charcoal, and chamomile flower extract in the symptom-oriented treatment of IBS patients with diarrheal symptoms.

Phytochemical and functional analysis of horseradish (Armoracia rusticana) fermented and non-fermented root extracts.

BACKGROUND: The roots of horseradish (Armoracia rusticana) are used for infections of respiratory airway and for urinary tract infections due to isothiocyanates (ITC), enzymatically formed during fermentation of glucosinolates by myrosinase. HYPOTHESIS/PURPOSE: The present study aims to present a comprehensive overview on the phytochemical composition of A. rusticana roots, especially concerning isothiocyanates and respective glucosinolates. The complex flavonoid spectrum of the herbal material is reviewed. Published data on in vitro activity of horseradish extracts and isolated compounds are summarized. These data indicate well-established use of horseradish as an antibacterial remedy against bacterial infections of the airway and urinary tract. STUDY DESIGN: To answer the question if other compounds from A. rusticana beside ITC contribute to the antibacterial activity, non-targeted LC-MS studies were performed with fermented and non-fermented horseradish extracts, and detailed phytochemical profiles were established. RESULTS: Comparative investigations on the antibacterial activity indicated that only ITC-containing extracts and fractions exert antibacterial activity. The huge variety of non-ITC compounds do not significantly contribute to the antibacterial activity, but can be used for analytical characterisation and quality control of the herbal material. Detailed phytochemical analysis additionally revealed a variety of compounds, not described until now for horseradish roots: the flavonol glycosides kaempferol-3-O-ß-d-xylopyranosyl-(1''' â†’ 2'')-ß-d-galactopyranoside, kaempferol-3-O-α-l-rhamnopyranosyl-(1''' â†’ 6'')-ß-d-glucopyranoside, kaempferol-3-O-ß-d-glucopyranoside, Kaempferol-3-O-ß-d-xylopyranosyl-7-O-ß-d-glucopyranoside, Kaempferol-3-O-ß-d-xylopyranosyl-(1'''' â†’ 2''')-ß-d-galactopyranoside-7-O-ß-d-glucopyranoside, the oxo-indole derivative spirobrassinin, the phenylthiazole 2-methylsulfanyl-4-phenyl-4,5-dihydro-1,3-thiazole, a series of lysophophatidylethanolamine and 13 different N-phenylpropenoyl-L-amino acids. CONCLUSION: The antibacterial effects of horseradish are only due to the presence of glucosinolates resp. the corresponding ITC, and the detailed overall composition of horseradish extracts has been reported.

Sesquiterpenes from Myrrh and Their ICAM-1 Inhibitory Activity In Vitro.

By using various chromatographic steps (silica flash, CPC, preparative HPLC), 16 sesquiterpenes could be isolated from an ethanolic extract of myrrh resin. Their chemical structures were elucidated by 1D and 2D NMR spectroscopy and HRESIMS. Among them, six previously unknown compounds (1-6) and another four metabolites previously not described for the genus Commiphora (7, 10, 12, 13) could be identified. Sesquiterpenes 1 and 2 are novel 9,10-seco-eudesmanes and exhibited an unprecedented sesquiterpene carbon skeleton, which is described here for the first time. New compound 3 is an 9,10 seco-guaian and the only peroxide isolated from myrrh so far. Compounds 1, 2, 4, 7-9, 11, 13-16 were tested in an ICAM-1 in vitro assay. Compound 7, as well as the reference compound furanoeudesma-1,3-diene, acted as moderate inhibitors of this adhesion molecule ICAM-1 (IC50: 44.8 and 46.3 µM, respectively). These results give new hints on the activity of sesquiterpenes with regard to ICAM-1 inhibition and possible modes of action of myrrh in anti-inflammatory processes.

Anti-Inflammatory and Barrier-Stabilising Effects of Myrrh, Coffee Charcoal and Chamomile Flower Extract in a Co-Culture Cell Model of the Intestinal Mucosa.

Recent clinical evidence suggests the efficacy of a traditional herbal medicinal product containing myrrh (Commiphora molmol Engl.), coffee charcoal (Coffea arabica L.) and chamomile flower dry extract (Matricaria chamomilla L.) in the therapy of inflammatory bowel diseases (IBD). However, the mechanisms of action in this context have not been entirely elucidated. The present study aimed to evaluate the effects of myrrh, coffee charcoal and chamomile flower extract on the inflammatory cross talk between immune and intestinal epithelial cells together with the resulting intestinal barrier disorders. A complex co-culture cell model consisting of intestinal epithelial cell (IEC) monolayers (Caco-2, HT29-MTX-E12) and macrophages (THP-1) was established for the simultaneous investigation of these two IBD characteristics. The lipopolysaccharide (LPS) activation of the macrophages led to a pro-inflammatory mediator release and thereby an inflammatory stimulation of IECs with chemokine release and reduced barrier function. The effects of the individual plant extracts and a ternary combination on inflammatory mediator release (IL-6, TNF, IL-8, MCP-1, PGE2) was quantified by ELISA. The transepithelial electrical resistance (TEER) of IEC monolayers was measured to evaluate the effects on the barrier function. Budesonide served as a positive control. All three plant extracts exhibited anti-inflammatory properties via the inhibition of the inflammatory mediator release to a varying extent. An intestinal barrier stabilising effect was observed for myrrh and coffee charcoal. Myrrh exerted the most distinct pharmacological activity. Dose reducing and synergistic interactions emerged within the threefold combination. Thus, our results provide a mechanistic basis for the use of the herbal combination of myrrh, coffee charcoal and chamomile flower extract in IBD treatment and underline the potential benefits of the phytotherapeutic multi-component/multi-target approach in this complex pathogenesis.

Bioactive Plant Compounds in Coffee Charcoal (Coffeae carbo) Extract Inhibit Cytokine Release from Activated Human THP-1 Macrophages.

The herbal preparation coffee charcoal is produced by over-roasting and milling green dried Coffea arabica L. seeds, and has a long-standing tradition in the treatment of inflammatory and gastrointestinal disorders. Its therapeutic properties are commonly attributed to adsorptive and astringent effects. This insufficiently explains its mode of action, especially when used in the treatment of inflammatory diseases in lower dosages. Our investigations aimed to identify bioactive secondary plant metabolites affecting cytokine-signaling. Thus, a phytochemical analysis of coffee charcoal extract was conducted using HPLC and LC/MS. Trigonelline, neochlorogenic acid, chlorogenic acid, caffeine, cryptochlorogenic acid, feruloylquinic acid isomers, and a caffeoylquinolacton were identified in the extract. Subsequently, the effects of coffee charcoal extract, chlorogenic acid isomers, their metabolite caffeic acid, caffeine, and trigonelline on cytokine (TNF, IL-6, MCP-1) release from LPS-challenged human THP-1 macrophages were examined to evaluate anti-inflammatory activity. Coffee charcoal showed concentration-dependent mild-to-medium inhibitory effects. The chlorogenic acid isomers and caffeic acid inhibited the TNF release, with cryptochlorogenic acid exerting the most distinct effects, as well as decreasing the release of IL-6 and MCP-1. In addition, scanning electron microscopic images provided an impression of the particle constitution, indicating a larger particle size and less structured surface of coffee charcoal in comparison to activated charcoal. In conclusion, our findings underline that beyond adsorptive effects, coffee charcoal exhibits pharmacological properties, which derive from a spectrum of secondary plant metabolites and support the therapeutic use in inflammatory diseases. Chlorogenic acids, particularly cryptochlorogenic acid, appear as pivotal bioactive compounds.

An aeroplysinin-1 specific nitrile hydratase isolated from the marine sponge Aplysina cavernicola.

A nitrile hydratase (NHase) that specifically accepts the nitrile aeroplysinin-1 (1) as a substrate and converts it into the dienone amide verongiaquinol (7) was isolated, partially purified and characterized from the Mediterranean sponge Aplysina cavernicola; although it is currently not known whether the enzyme is of sponge origin or produced by its symbiotic microorganisms. The formation of aeroplysinin-1 and of the corresponding dienone amide is part of the chemical defence system of A. cavernicola. The latter two compounds that show strong antibiotic activity originate from brominated isoxazoline alkaloids that are thought to protect the sponges from invasion of bacterial pathogens. The sponge was shown to contain at least two NHases as two excised protein bands from a non denaturating Blue Native gel showed nitrile hydratase activity, which was not observed for control samples. The enzymes were shown to be manganese dependent, although cobalt and nickel ions were also able to recover the activity of the nitrile hydratases. The temperature and pH optimum of the studied enzymes were found at 41 °C and pH 7.8. The enzymes showed high substrate specificity towards the physiological substrate aeroplysinin-1 (1) since none of the substrate analogues that were prepared either by partial or by total synthesis were converted in an in vitro assay. Moreover de-novo sequencing by mass spectrometry was employed to obtain information about the primary structure of the studied NHases, which did not reveal any homology to known NHases.