Involvement of the Glucocorticoid Receptor in Pro-inflammatory Transcription Factor Inhibition by Daucane Esters from Laserpitium zernyi.

Species of the genus Laserpitium have been used traditionally to treat inflammation and infection. From the herb of Laserpitium zernyi, six new compounds were isolated and their structures elucidated (using IR, NMR, HRMS data) as derivatives of 8-daucene-2,4,10-triol (1, 2, and 4), 7-daucene-2,4,10-triol (3), a lapiferin derivative featuring a C-2 ester moiety (5), and a daucane featuring an exomethylene group at C-8 (6). Also isolated were the rare daucanes vaginatin (7) and laserpitin (8). In a search for selective glucocorticoid receptor (GR) modulators, the compounds were tested for their capacity to inhibit NF-κB and AP-1 pro-inflammatory factors and for a potential competitive effect on a dexamethasone (Dex)-induced GR-driven glucocorticoid response element (GRE) reporter gene. The new 2ß-angeloyloxy-10α-acetoxy-8-daucene-2,4,10-triol (2) significantly inhibited transactivation of both NF-κB and AP-1, while vaginatin (7) was the most active of the compounds tested in blocking AP-1. Both compounds competitively repressed Dex-induced GRE-driven promoter activities, indicative of a potential role for GR. In addition, a decreased potential to inhibit NF-κB was apparent in GR knockout A549 cells. In line with the transcriptional assays, compounds 2 and 7 also significantly lowered CCL-2 chemokine production, albeit to a lesser extent than Dex. The results suggest that daucanes may be interesting candidates in the search for compounds with GR-modulating activities.

Daucane esters from laserwort (Laserpitium latifolium L.) inhibit cytokine and chemokine production in human lung epithelial cells.

BACKGROUND: Laserwort, Laserpitium latifolium L. (Apiaceae), is a European medicinal plant. Its roots and rhizomes were traditionally used as a general tonic and to treat inflammatory and infective diseases. PURPOSE: The anti-inflammatory potential of daucane esters, isolated from underground parts extract of L. latifolium and specific structural features that contribute to their activity were investigated. In addition, we studied their interference with the transactivation capacity of the Glucocorticoid Receptor when added together with a classic glucocorticoid (GC), dexamethasone (DEX). This particular property may be relevant in combination strategies, attempting to circumvent diabetogenic side effects of glucocorticoids upon long-term anti-inflammatory treatments. MATERIALS AND METHODS: Nine L. latifolium daucane esters were isolated and elucidated as derivatives of desoxodehydrolaserpitin, laserpitin and a novel 2ß-esterified laserpitinol analogue. Of all compounds effects on NF-κB- and AP-1-driven pro-inflammatory pathways were assessed using TNF- or PMA-induced reporter gene analysis in A549 cells. Daucanes with a strong and concentration-dependent inhibition of both NF-κB and AP-1, were tested for a potential effect on DEX-stimulated GR-driven Glucocorticoid Response Element (GRE) reporter gene activity. In addition, GRE-driven anti-inflammatory mRNA expression was determined (GILZ and DUSP1). Also anti-inflammatory properties were validated by monitoring effects on CCL-2, IL-6, IL-1ß mRNA expression levels (qPCR) and on CCL-2 chemokine production (ELISA). RESULTS: Daucanes featuring an ester moiety and/or a hydroxy group at positions 2ß, 6α and 10α and especially the novel 2ß-esterified laserpitinol derivative that, in comparison to other isolated compounds, features an additional 9α-hydroxy group, demonstrated suppression of both NF-κB- and AP-1-dependent pro-inflammatory pathways. Remarkably, those entities competitively and concentration-dependently repressed GR-driven GRE-dependent reporter gene activities. The most active compounds inhibited CCL-2 protein excretion and compound 4 downregulated genes coding for IL-1ß and IL-6 induced upon TNF treatment in A549. In absence of TNF, compound 4 upregulated the GRE-mediated anti-inflammatory gene GILZ, but not DUSP1. CONCLUSIONS: Daucane esters are novel anti-inflammatory agents that may, in combination with GCs, potentially improve therapeutic benefit. These results contribute to the ongoing search for novel anti-inflammatory agents as safer alternatives to, or with, GCs.

Laserpitium zernyi Hayek Flower and Herb Extracts: Phenolic Compounds, and Anti-edematous, Antioxidant, and Antimicrobial Activities.

Phenolic compounds and different biological activities of the dry MeOH extracts of the flowers and the herb (aerial parts without flowers) of Laserpitium zernyi Hayek (Apiaceae) were investigated. The total phenolic contents in the extracts were determined spectrophotometrically using Folin-Ciocalteu reagent. In both extracts, apigenin, luteolin, their 7-O-glucosides, and chlorogenic acid were detected by HPLC. Identified phenolics were quantified in both extracts, except luteolin in L. zernyi herb extract. The extracts (p.o.) were tested for anti-edematous activity in a model of carrageenan (i.pl.) induced rat paw edema. Antioxidant activity of the extracts was assessed by FRAP assay and DPPH and • OH radicals scavenging tests. Antimicrobial activity was investigated using broth microdilution test against five Gram-positive and three Gram-negative bacteria, as well as against two strains of Candida albicans. The polyphenol-richer flower extract exerted higher anti-edematous and antioxidant activities. The herb extract exhibited better antimicrobial effect against Micrococcus luteus, Enterococcus faecalis, Bacillus subtilis, and Pseudomonas aeruginosa, while against other tested microorganisms, the activity of both extracts was identical. Demonstrated biological activities of L. zernyi flower and herb extracts represent a good basis for their further investigation as potential new herbal medicinal raw materials.

Composition and antimicrobial activity of the essential oils of Laserpitium latifolium L. and L. ochridanum Micevski (Apiaceae).

The chemical composition and antimicrobial activity of essential oils of Laserpitium latifolium and L. ochridanum were investigated. The essential oils were isolated by steam distillation and characterized by GC-FID and GC/MS analyses. All essential oils were distinguished by high contents of monoterpenes, and α-pinene was the most abundant compound in the essential oils of L. latifolium underground parts and fruits (contents of 44.4 and 44.0%, resp.). The fruit essential oil was also rich in sabinene (26.8%). Regarding the L. ochridanum essential oils, the main constituents were limonene in the fruit oil (57.7%) and sabinene in the herb oil (25.9%). The antimicrobial activity of these essential oils as well as that of L. ochridanum underground parts, whose composition was reported previously, was tested by the broth-microdilution method against four Gram-positive and three Gram-negative bacteria and two Candida albicans strains. Except the L. latifolium underground-parts essential oil, the other investigated oils showed a high antimicrobial potential against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, or Candida albicans (minimal inhibitory concentrations of 13.0-73.0 µg/ml), comparable to or even higher than that of thymol, which was used as reference compound.

Extracts of three Laserpitium L. species and their principal components laserpitine and sesquiterpene lactones inhibit microbial growth and biofilm formation by oral Candida isolates.

Antimicrobial properties of extracts of underground parts of three Laserpitium L. (Apiaceae) species, namely Laserpitium latifolium L., Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski, were investigated. The investigated species are widely used as functional foods, as spices and for preparations in traditional medicine for treating complaints connected with infection and inflammation. Furthermore, antimicrobial and antibiofilm effects of laserpitine, the most abundant compound in the chloroform extract of L. latifolium, and guaianolide sesquiterpene lactones, such as, isomontanolide, montanolide and tarolide, principal components of the extracts of L. zernyi and L. ochridanum were assessed. The antimicrobial activity was tested using the microdilution method against five pathogenic bacteria and five fungi, as well as in the microplate biofilm assay on two Candida clinical isolates (C. albicans and C. krusei). Among the extracts, L. latifolium showed the most prominent activity. Isolated metabolites exerted higher effects against fungal than against bacterial strains, isomontanolide being the most active. Interestingly, all constituents showed higher potential on inhibition of biofilm formation than fluconazole, a reference compound. Tested metabolites may be good novel agents with high antifungal and antibacterial potential that might find practical applications in food industry as food preservatives in order to retard the growth of food spoiling microbes, but only after detailed safety assessments.

Antinociceptive and anti-edematous activities of the essential oils of two Balkan endemic Laserpitium species.

In this paper antinociceptive and anti-edematous effects are examined of the essential oils of the underground parts of two Balkan endemic Laserpitium species (Apiaceae), L. zernyi and L. ochridanum. Furthermore, the essential oil of the underground parts of L. ochridanum is chemically characterised by GC and GC-MS. Antinociceptive and anti-edematous effects were measured in a rat model of localized inflammation, induced by carrageenan, using apparatus for the modified paw-pressure test, and plethysmometer, respectively. The effects of both Laserpitium essential oils were measured after oral gavage administration to male Wistar rats in doses of 25, 50 and 100 mg/kg. The main constituents of L. ochridanum essential oil were: alpha-pinene (33.2%), alpha-bisabolol (10.3%) and chamazulene (14.9%). The essential oil of L. zernyi was previously shown to be rich in alpha-pinene (31.6%) and alpha-bisabolol (30.9%). Both examined essential oils produced a significant dose-dependent antinociceptive effect. The corresponding ED50 +/- SEM in producing antinociception were 45.9 +/- 4.9 mg/kg and 42.4 +/- 2.1 mg/kg for L. zernyi and L. ochridanum oil, respectively. Both essential oils also significantly reduced paw edema in a dose-dependent manner. The estimated ED50 +/- SEM values for the anti-edematous effect were 36.3 +/- 4.5 mg/kg for L. zernyi oil and 45.1 +/- 11.3 mg/kg for L. ochridanum oil. These results suggest that the essential oils of both investigated Laserpitium species may be effective against pain and edema present in various inflammatory conditions.

Antihyperalgesic and antiedematous activities of bisabolol-oxides-rich matricaria oil in a rat model of inflammation.

From the dried flower heads of Matricaria recutita L., essential oil was isolated by hydrodistillation, and in the obtained blue oil, α-bisabolol oxide A (21.5%), α-bisabolol oxide B (25.5%) and (Z)-spiroether (cis-en-yn-spiroether) (10.3%) were identified as the main compounds, by gas chromatography (GC) and GC-mass spectrometry analyses. The antihyperalgesic effects of this oil were examined in a rat model of inflammation induced by carrageenan, through a modified 'paw-pressure' test. Antiedematous effects were examined in a rat model of inflammation induced by carrageenan, dextran and histamine, through plethysmometry. Matricaria oil (25, 50 and 100 mg/kg, p.o.) exhibited a significant dose-dependent reduction of hyperalgesia and edema induced by carrageenan in both prophylactic and therapeutic treatment schemes. It was more efficacious in the prophylactic treatment scheme, and the corresponding median effective dose (ED50 ) ± standard error of the mean (SEM) values were 49.8 ± 6.0 and 42.4 ± 0.2 mg/kg for antihyperalgesic and antiedematous effects, respectively. Prophylactic treatments with matricaria oil (25, 50 and 100 mg/kg, p.o.) caused a significant dose-dependent antiedematous effect in dextran-induced edema with lower efficacy than in the carrageenan model. In a dose of 100 mg/kg, p.o., matricaria oil caused a slight reduction of histamine-induced edema. These results suggest that bisabolol-oxide-rich matricaria oil may be effective against pain and edema present in various inflammatory conditions, which supports matricaria traditional uses.

Mycotherapy of cancer: an update on cytotoxic and antitumor activities of mushrooms, bioactive principles and molecular mechanisms of their action.

Mycotherapy is defined as the study of the use of extracts and compounds obtained from mushrooms as medicines or health-promoting agents. The present review updates the recent findings on anticancer/antitumor agents derived from mushroom extracts and their metabolites. The increasing number of studies in the past few years revealed mushroom extracts as potent antitumor agents. Also, numerous studies were conducted on bioactive compounds isolated from mushrooms reporting the heteropolysaccharides, ß-glucans, α-glucans, proteins, complexes of polysaccharides with proteins, fatty acids, nucleoside antagonists, terpenoids, sesquiterpenes, lanostanoids, sterols and phenolic acids as promising antitumor agents. Also, molecular mechanisms of cytotoxicity against different cancer cell lines are discussed in this review. Findings with Antrodia camphorata and Ganoderma lucidium extracts and isolated compounds are presented, as being the most deeply studied previously.

Sesquiterpene lactones from the extracts of two Balkan endemic Laserpitium species and their cytotoxic activity.

Chloroform extracts of the underground parts of two Balkan endemic Laserpitium species, Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski, were chemically investigated. Five unknown guaianolides from the class of slovanolides, of which four were additionally 2ß-esterified, as well as two lactones, previously identified in other Laserpitium species, were isolated from the L. ochridanum extract. From the L. zernyi extract one slovanolide derivative was isolated for the first time in the genus Laserpitium. In addition, the phenylpropanoid latifolone and six known sesquiterpene lactones, characterised as derivatives of slovanolide and silerolide, were isolated from the extracts of both species. The cytotoxic activities of the total extracts and the isolated compounds were tested using MTT and SRB assays on the two human breast cancer cell lines, MCF 7/6 and MCF 7/AZ. The extracts exerted cytotoxic activities with the IC(50) values ranging 65.21-348.25 µg/mL. The L. ochridanum extract was most potent in the MTT test with IC(50) values of 65.21 and 66.09 µg/mL in the MCF 7/AZ and MCF 7/6 cell lines, respectively. The highest cytotoxic activity exerted 2ß,8α-di-angeloyloxy-10ß-hydroxy-6αH-guaian-3,(7-11)-dien-12,6-olide, a slovanolide derivative with an additional double bond in lactone ring, on highly invasive MCF 7/6 cell line, with IC(50) value 0.7 µM in both assays tested. Generally, guaianolides with a higher number of ester moieties at the positions 2ß, 8α, 10ß or 11α exhibited IC(50) values in the micromolar range, while eudesmanolides and guaianolides with a lower number of esters did not induce significant cytotoxicity.

Essential oil from the underground parts of Laserpitium zernyi: potential source of alpha-bisabolol and its antimicrobial activity.

The composition and antimicrobial activity of the essential oil from the underground parts (roots and rhizomes) of Laserpitium zernyi Hayek (Apiaceae) were investigated. The pale blue essential oil, obtained by hydrodistillation, was analyzed by GC and GC-MS. Forty-three compounds were identified (94.3% of total oil). The main constituent, beside alpha-pinene (31.6%) was alpha-bisabolol (30.9%), so this oil may be a novel potential natural source of this sesquiterpene alcohol. The antimicrobial activity was tested using the microdilution method against Gram (+) bacteria (Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Enterococcus faecalis), Gram (-) bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli), and two strains of yeast (Candida albicans). L. zernyi oil showed significant antibacterial activity against S. epidermidis, S. aureus and M. luteus, but lower activities against the other tested strains.