Developmental and Environmental Effects on Sesquiterpene Lactones in Cultivated Arnica montana L.

The amount of sesquiterpene lactones and the lactone profile of Arnica montana L. in flowering and seed formation stages in vitro and in vivo propagated from seeds of German, Ukrainian, and Austrian origin and grown in two experimental fields were studied. It was found that in vitro propagated 2-year plants in full flowering stage accumulated higher amount of lactones in comparison to in vivo propagated 3-year plants and to the seed formation stage, respectively. Helenalins predominated in in vivo propagated 2-year or in vitro propagated 3-year plants. 2-Methylbutyrate (2MeBu) was the principal ester in the samples with prevalence of helenalins, while isobutyrate (iBu) was the major one in the samples with predominance of 11,13-dihydrohelenalins. The results revealed that the environmental conditions on Vitosha Mt. are more suitable for cultivation of A. montana giving higher content of lactones.

Chemometrics-based Approach in Analysis of Arnicae flos.

INTRODUCTION: Arnica montana flowers have a long history as herbal medicines for external use on injuries and rheumatic complaints. OBJECTIVE: To investigate Arnicae flos of cultivated accessions from Bulgaria, Poland, Germany, Finland, and Pharmacy store for phenolic derivatives and sesquiterpene lactones (STLs). MATERIALS AND METHODS: Samples of Arnica from nine origins were prepared by ultrasound-assisted extraction with 80% methanol for phenolic compounds analysis. Subsequent reverse-phase high-performance liquid chromatography (HPLC) separation of the analytes was performed using gradient elution and ultraviolet detection at 280 and 310 nm (phenolic acids), and 360 nm (flavonoids). Total STLs were determined in chloroform extracts by solid-phase extraction-HPLC at 225 nm. The HPLC generated chromatographic data were analyzed using principal component analysis (PCA) and hierarchical clustering (HC). RESULTS: The highest total amount of phenolic acids was found in the sample from Botanical Garden at Joensuu University, Finland (2.36 mg/g dw). Astragalin, isoquercitrin, and isorhamnetin 3-glucoside were the main flavonol glycosides being present up to 3.37 mg/g (astragalin). Three well-defined clusters were distinguished by PCA and HC. Cluster C1 comprised of the German and Finnish accessions characterized by the highest content of flavonols. Cluster C2 included the Bulgarian and Polish samples presenting a low content of flavonoids. Cluster C3 consisted only of one sample from a pharmacy store. CONCLUSION: A validated HPLC method for simultaneous determination of phenolic acids, flavonoid glycosides, and aglycones in A. montana flowers was developed. The PCA loading plot showed that quercetin, kaempferol, and isorhamnetin can be used to distinguish different Arnica accessions. SUMMARY: A principal component analysis (PCA) on 13 phenolic compounds and total amount of sesquiterpene lactones in Arnicae flos collection tended to cluster the studied 9 accessions into three main groups. The profiles obtained demonstrated that the samples from Germany and Finland are characterized by greater amounts of phenolic derivatives than the Bulgarian and Polish ones. The PCA loading plot showed that quercetin, kaemferol and isorhamnetin can be used to distinguish different arnica accessions.

Influence of nutrient medium composition on in vitro growth, polyphenolic content and antioxidant activity of Alchemilla mollis.

Alchemilla mollis (Buser) Rothm. (Rosaceae) is a high-mountain medicinal plant growing in the Balkan Peninsula, with only one population in Bulgaria. Alchemilla plants (Lady's mantle) are commonly used in traditional medicine for treatment of many gynecological diseases. The commercial drugs "Herba Alchemillae" induce a rapid regeneration of skin epithelium and have styptic and anti-inflammatory actions. Because of the high content of phenolic compounds (tannins and flavonoids) and the ecological plasticity of the species, field cultivation or in vitro biomass production of A. mollis are possible alternatives to its collection from nature. Four MS based nutrient media differing in the concentration of the minerals and supplemented with alpha-naphthaleneacetic acid (NAA) and benzylaminopurine (BAP) were tested in order to examine their influence on the shoot multiplication effectiveness and the antioxidant activity of A. mollis, and also the possible relation between these parameters under the conditions of in vitro culture. The cultures grown for two months on these media differed significantly in their multiplication rates (p < 0.001), as well as in their morphological features--height, leaf color and root development. Methanol extracts of in vitro cultivated and ex vitro adapted and acclimated on Vitosha Mt. (1500 m a. s. l.) plants were analyzed for tannin and flavonoid content and for free radical scavenging activity. The contents of flavonoids and tannins in the in vitro cultures of A. mollis cultivated on the four tested media differed significantly (p < 0.05). The highest flavonoid content was found in the shoots cultivated on the control MS medium, as well as in the ex vitro adapted plants. The antioxidant activity of the in vitro cultures correlated positively with the concentrations of the PGRs in the respective media, and the ex vitro adapted plants had the highest antioxidant activity (IC50 13.1 +/- 1.9 microg/mL) commensurable with that of the commercial antioxidant butylated hydroxytoluene (BHT) used as a positive control, with an IC50 of 12.65 microg/mL.

Composition and antimicrobial activity of Achillea distans essential oil.

The essential oil of Achillea distans W. et K. flower heads was analyzed by GC and GC-MS. Altogether 43 components in concentrations more than 0.1% were identified representing 93.5% of the oil composition. The main constituents were 1,8-cineole (16.8%), trans-thujone (9.8%), sabinene (8.2%), borneol (7.5%), beta-pinene (6.5%), and camphor (5.8%). The oil showed moderate activity against Staphylococcus aureus and Candida albicans, and weak activity against Salmonella typhimurium, Proteus vulgaris, and Escherichia coli.

Flavonoid constituents and free radical scavenging activity of Alchemilla mollis.

Antioxidant capacity of the methanolic extract of Alchemilla mollis was measured by its ability to scavenge the DPPH radical. The EtOAc fraction obtained after partition of the total extract was found to be the most active radical scavenger (IC50 9.8 +/- 1.8 microg/mL) and was subjected to fractionation by Sephadex LH-20 CC. Further purification by RP-18 CC led to the isolation of eight flavonoid glycosides: cis- and trans-tiliroside (1 and 2), rhodiolgin (3), hyperoside (4), isoquercitrin (5), miquelianin (6), sinocrassoside D2 (7), and gossypetin-3-O-beta-D-galactopyranosyl-7-O-alpha-L-rhamnopyranoside (8). It was found that 8 is a new compound and its antioxidant activity is also reported. Identification of the isolated compounds was carried out by spectroscopic and spectrometric analysis (1D and 2D NMR, UV and MS).

Essential oil composition of Achillea clusiana from Bulgaria.

The essential oil compositon of Achillea clusiana Tausch from Bulgaria has been studied by GC and GC/MS. Fifty-four components were registered, representing 92.5% of the oil. The oil was characterized by the presence of oxygenated mono- and sesquiterpenoids. The main components were beta-thujone (17.2%), 1,8-cineole (11.2%), camphor (11.1%) and alpha-thujone (7.8%). Farnesol (3.1%), nerolidol (2.7%) and oxygenated nerolidol derivatives (cabreuva oxides A-D, isohumbertiols A-D, bejarol and 7-hydroxy-6,7-dihydro-5,6E-dehydronerolidol) were the main sesquiterpenoids in the oil.

Chemotypes in Achillea collina based on sesquiterpene lactone profile.

The lactone profile of six origins of Achillea collina growing in Bulgaria was studied and significant variability was observed. The reasons for the differences in the lactone composition are discussed. Twenty-five components in total were isolated and identified, while the presence of ten lactones was proved by intensive TLC analysis in comparison with reference compounds. The structures of the components 17, 20, 25-30 were established by spectroscopic methods. The structure of 7, a cyclization product of 6, was also discussed. The anti-inflammatory activity of some extracts, fractions and individual compounds was tested in vitro by determining the inhibitory effects on induced human neutrophils.

Sesquiterpene lactones from Achillea collina J. Becker ex Reichenb.

The flower heads of Achillea collina afforded 31 individual sesquiterpene lactones, among which nine guaianolides (1, 16, 20, 21, 23, 27-30), a germacranolide (18) and a dimeric guaianolide (31) were found to be new natural products. The lactones 2-4, 12, 14, 15, 17, 19 and 25 were isolated for the first time from the investigated species. Their structures were established by spectral methods.

Terpenoids from Anthemis austriaca Jacq.

The aerial parts of Anthemis austriaca Jacq. afforded five new sesquiterpene lactones (two of which are dimeric guaianolides) and three new guaiane type sesquiterpene acids In addition, seven known terpenoids were also found in the studied species. Their structures were elucidated by spectral methods.