Antiproliferative activity of Artemisia asiatica extract and its constituents on human tumor cell lines.

The extract of Artemisia asiatica herb with antiproliferative activity against four human tumor cell lines (A2780, A431, HeLa, and MCF7) was analyzed by the MTT assay, and bioassay-directed fractionation was carried out in order to identify the compounds responsible for the cytotoxic activity. Guaianolide (1-4), seco-guianolide (5), germacranolide (6) and eudesmanolide sesquiterpenes (7), monoterpenes (8, 9), including the new compound artemisia alcohol glucoside (8), and flavonoids (10-16) were isolated as a result of a multistep chromatographic procedure (CC, CPC, PLC, and gel filtration). The compounds were identified by means of UV, MS, and NMR spectroscopy, including (1)H-and (13)C-NMR, (1)H-(1)H COSY, NOESY, HSQC, and HMBC experiments. The isolated compounds 1-16 were evaluated for their tumor cell growth-inhibitory activities on a panel of four adherent cancer cell lines, and different types of secondary metabolites were found to be responsible for the cytotoxic effects of the extract. Especially cirsilineol (13), 3ß-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide (3), and iso-seco-tanapartholide 3-O-methyl ester (5) exerted marked cytotoxic effects against the investigated cell lines, while jaceosidin (12), 6-methoxytricin (15), artecanin (2), and 5,7,4',5'-tetrahydroxy-6,3'-dimethoxyflavone (14) were moderately active. All the sesquiterpenes and monoterpenes are reported here for the first time from this species, and in the case of artecanin (2), 3α-chloro-4ß,10α-dihydroxy-1ß,2ß-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide (4), ridentin (6), and ridentin B (7), previously unreported NMR spectroscopic data were determined.

Topical analgesic, anti-inflammatory and antioxidant properties of Oxybaphus nyctagineus: phytochemical characterization of active fractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxybaphus nyctagineus (Michx.) Sweet has traditionally been used by several Native American tribes predominantly as a topical anti-inflammatory and analgesic agent. AIM OF THE STUDY: To evaluate the antioxidant, analgesic and anti-inflammatory activity of the extracts prepared from the aerial parts of Oxybaphus nyctagineus and to characterize the major chemical constituents of the bioactive extracts. MATERIALS AND METHODS: Crude polar and apolar extracts (PCE and ACE) of the herb of Oxybaphus nyctagineus were prepared and tested in the models of the CFA-induced hyperalgesia in rat knee and carrageenan-induced paw edema in rat. To identify the active compounds, subfractions were prepared by column chromatography and subjected in vitro assays, such as antioxidant assays (DPPH, peroxynitrite (ONOO-) scavenging), and the LPS-induced IL-1ß release test in human monocytes. Preparative HPLC was employed for the isolation of active substances, while phytochemical analysis was performed by mean of LC-MS/MS and NMR. RESULTS: The topically administered PCE and ACE of Oxybaphus nyctagineus demonstrated a significant analgesic and anti-inflammatory effect in the inflammation animal models. The subfraction A4 of ACE and the subfraction P5 of PCE considerably inhibited the LPS-induced IL-1ß release in human monocytes, while the strongest activity was localized in the subfraction P5 in the antioxidant assays. The HPLC-MS/MS and NMR analysis revealed that 6-methoxyflavonol diglycosides, namely patuletin-3-O-robinobioside (1), 6-methoxykaempferol-3-O-robinobioside (2), spinacetin-3-O-robinobioside (3), and hydroxy-polyenoic fatty acids, namely corchorifatty acid B (4), 9-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9-HOT acid) (5), and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HOD acid) (6) were present in PCE, and in ACE as major compounds. CONCLUSION: The results of this study established a pharmacological evidence for the traditional use of Oxybaphus nyctagineus as an anti-inflammatory agent used topically, and provided data on its phytochemical composition for the first time.

Elemental analysis of Ginkgo biloba leaf samples collected during one vegetation period.

The object of our work was the identification and quantification of inorganic elements in Ginkgo biloba L. leaves (Ginkgonis folium, Ginkgoaceae) by X-ray fluorescence analysis. The plant material was obtained from a 50-years-old female tree at the Comenius University Botanical Garden (Bratislava, Slovakia). Leaves were collected from early May to late September, with the last sample consisting of fallen leaves. The elements analyzed were: phosphorus, sulfur, potassium, calcium, scandium, iron, zinc, yttrium, molybdenum, tellurium, samarium, gadolinium, dysprosium, iridium, thallium and lead. The amounts of the monitored heavy metals were below the limits specified in Ph. Eur. 7 and PhS 1.

Identification of triterpenes from milkweed (Asclepias syriaca).

The chloroform extract of Asclepias syriaca stem were investigated. Three triterpenes were isolated by TLC, VLC, and preparative chromatography, and their structures established by one and two-dimensional NMR spectroscopy. Lupenyl acetate has been isolated for the first time from A. syriaca; this is thefirst representative of a triterpene bearing a lupane skeleton in this species. In addition, alpha-amyrin acetate and alpha-amyrin butyrate were isolated.

Antiinflammatory activities of Hungarian Stachys species and their iridoids.

The antiinflammatory activities of aqueous extracts prepared from the aerial parts of ten Hungarian Stachys species were investigated in vivo in the carrageenan-induced paw oedema test after intraperitoneal and oral administration to rats. Some of the extracts were found to display significant antiphlogistic effects when administered intraperitoneally and orally; in particular, the extracts of S. alpina, S. germanica, S. officinalis and S. recta demonstrated high activity following intraperitoneal administration. At the same dose of 5.0 mg/kg, these extracts exhibited similar or greater potency than that of the positive control diclofenac-Na. The main iridoids present in the investigated extracts, ajugoside, aucubin, acetylharpagide, harpagide and harpagoside, were also assayed in the same test, and high dose-dependent antiphlogistic effects were recorded for aucubin and harpagoside. These results led to the conclusion that most probably iridoids are responsible for the antiinflammatory effect of Stachys species, but other active constituents or their synergism must also be implicated in the antiinflammatory effect.

Antioxidant activity-guided phytochemical investigation of Artemisia gmelinii Webb. ex Stechm.: isolation and spectroscopic challenges of 3,5-O-dicaffeoyl (epi?) quinic acid and its ethyl ester.

Although Artemisia gmelinii Webb. ex Stechm. has long been used in south and south-east Asia to treat many kinds of inflammatory diseases, up until now its bioactivity-coupled phytochemical characterization has not been reported. We identified one fraction of the methanolic extract of A. gmelinii as a hit in our antioxidant screening (DPPH) campaign. In order to identify the active radical scavenger components of the extract, a DPPH-HPLC spiking assay was carried out. Out of six detected known compounds caffeic acid and scopoletin had already been identified in the plant, but four of them, namely chlorogenic acid, 4-O-caffeoylquinic acid, luteolin-7-O-glucoside, and apigenin-7-O-glucoside are first described here. Moreover, the two most active compounds of the mixture, 3,5-O-dicaffeoylquinic acid (7) and its ethyl ester derivative (8) were isolated with preparative HPLC. The spectroscopic identification of 7 and 8 presented a surprising challenge due to literature ambiguities. These questions are discussed in detail.

Bioactivity-guided study of antiproliferative activities of Salvia extracts.

The cytotoxic activities of the n-hexane, chloroform and aqueous methanolic fractions prepared from the methanolic extract of the leaves of 23 Salvia taxa were studied for their cell growth-inhibitory activity against human cervix adenocarcinoma (HeLa), skin carcinoma (A431) and breast adenocarcinoma (MCF7) cells using the MTT assay. The n-hexane fractions of six Salvia taxa (S. hispanica, S. nemorosa, S. nemorosa 1. albiflora, S. pratensis, S. recognita and S. ringens) and the chloroform fraction ofS. officinalis 1. albiflora produced over 50% growth inhibition of the skin carcinoma cell line. None of the tested extracts showed substantial (above 50%) antiproliferative effects against HeLa and MCF7 cells. S. ringens was the most powerful among the studied Salvia species with a 61.8% cell growth inhibitory activity on A431 cells. In the case of S. ringens, other plant parts were also tested for antiproliferative effect, and the highest activities were recorded for the root extract. This was subjected to bioactivity-guided fractionation, which yielded four abietane diterpenes (royleanone, horminone, 7-O-methyl-horminone and 7-acetyl-horminone), one triterpene (erythrodiol-3-acetate) and beta-sitosterol. Horminone, 7-acetyl-horminone and erythrodiol-3-acetate displayed marked concentration-dependent antiproliferative effects, while royleanone and 7-O-methyl-horminone produced weaker activities.

Analysis of the essential oil of Amsonia illustris.

The root and leaf essential oils, present in trace amounts in Amsonia illustris Woods. (Apocynaceae), were isolated by steam distillation and their chemical constituents identified by GC-FID and GC-MS. More than 80% of the thirty volatile compounds in the leaf oil were identified, the major constituents being mainly sesquiterpenes like a-humulene (14.5%), beta-caryophyllene (12.4%) and guaiol (11.6%). The volatile ingredients of the root oil were pinocampheol, methyl salicylate, (2E,4E)- decadienal, eugenol and trans-isoeugenol.

Betaine yields from marine algal species utilized in the preparation of seaweed extracts used in agriculture.

Ascophyllum nodosum, and to a lesser extent, Laminaria digitata, L. hyperborea and Fucus serratus, are marine algal species utilized in the commercial production of seaweed extracts used in agriculture. Betaines have been shown to be important constituents of these extracts, but there appears to have been no study made on whether there are variations in the betaine contents of these species based on either the place or date of collection. Samples of each of the four species were collected from widely separated areas at different times of the year. Also, in the case of A. nodosum, approximately monthly collections were made from one location. The betaines detected in the various collections of the same species showed little variation, although in the case ofA. nodosum, glycinebetaine was found as a minor constituent in some samples, but was not detected in others. Trigonelline was found in all the tested samples of the two Laminaria species; this is, to our knowledge, the first record of this betaine in marine algae. With the exception of trigonelline in the Laminaria species, the betaine yields from the various samples of L. digitata, L. hyperborea and F. serratus showed little variation, regardless of either the place or date of collection. The trigonelline contents of the Laminaria species collected at one location (Finavarra, Ireland), in particular of L. hyperborea, was substantially greater than those from the other places of collection. In the case of A. nodosum, the betaine yields from samples collected at one site (Dale, Pembrokeshire, UK) were significantly higher than those from the other places of collection, which were very similar to each other. There was no clear indication of seasonal variation in betaine yields from A. nodosum.

Comparative study of the antioxidant activities of eleven Salvia species.

The lipid peroxidation-inhibiting activities of aqueous methanolic extracts of eleven Salvia species (Fam. Lamiaceae) were evaluated in an enzyme-independent biological system. The total polyphenol contents and the amounts of the most abundant phenoloids of the genus, caffeic and rosmarinic acids, were also determined. The EC50 values of the extracts displayed substantial differences. All of the investigated species except S. jurisicii (EC50 191.2 microg/mL) exhibited higher activities than that of ascorbic acid (EC50 123.8 microg/mL), the reference compound. Among the studied species, S. scabiosifolia (EC50 5.4 microg/mL) demonstrated the highest effect, followed in sequence by S. dumetorum, S. transsylvanica, S. officinalis l albiflora, S. nemorosa l albiflora and S. recognita (EC50 6.5 - 10.2 microg/mL). The close correlation was confirmed between the antioxidant activities and the total phenol contents of the extracts. For caffeic and rosmarinic acids, the correlation was much weaker, indicating the important role of other polyphenols in the antioxidant activity.

Antiproliferative activity of Hungarian Asteraceae species against human cancer cell lines. Part II.

The antiproliferative activities of aqueous and organic extracts prepared from 26 Hungarian species of the tribes Cynereae and Lactuceae (Asteraceae) were tested in vitro against HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma) and MCF7 (breast epithelial adenocarcinoma) cells by using the MTT assay. Of the tested 200 extracts of different plant parts obtained with n-hexane, chloroform, 50% methanol and water, 16 extracts displayed noteworthy cell growth inhibitory activity (>50% inhibition at a concentration of 10 microg/mL). The IC50 values of these extracts were determined, and their direct cytotoxic effects were measured. High differences between the antiproliferative and cytotoxic activities, demonstrating a real cell proliferation inhibitory activity rather than direct killing effects, were found for some Centaurea, Cirsium, Cichorium, Lactuca, Onopordum and Scorsonera extracts.

Antiproliferative activity of Hungarian Asteraceae species against human cancer cell lines. Part I.

Aqueous and organic extracts of 25 selected species from four tribes of Hungarian Asteraceae were screened in vitro for antiproliferative activity against HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma) and MCF7 (breast epithelial adenocarcinoma) cells, using the MTT assay. Twenty five of the 228 tested extracts from different parts of the species of Astereae (6), Inuleae (3), Heliantheae (5) and Anthemideae (11) demonstrated a substantial antiproliferative effect (at least 50% inhibition of cell proliferation) at 10 microg/mL against one or more of the cell lines. Complete dose-response curves were generated and IC(50) values were calculated for these active extracts, and their direct cytotoxic effects were determined. In summary, 11 of the tested 25 plants were found to be active and 4 of them (Anthemis ruthenica, Erigeron canadensis, Erigeron annuus and Inula ensifolia) had not been studied previously for either active compounds or anticancer properties.

[Chemical diversity of the biological active ingredients of salvia officinalis and some closely related species]. / A Salvia officinalis és néháiny rokon faj biológiailag aktiv anyagainak kémiai diverzitása.

Comparative studies on the volatile and non-volatile fractions of 6 species. i.e. Salvia officinalis, S. tomentosa, S. fruticosa, S. candelabrum, S. ringens, S. lavandulifolia of the Section Salvia (Lamiaceae) have been carried out. Both fractions provide the chemical pattern matches to the chemotaxonomic character of Subfamily Nepetoideae in Erdtmanr two subfamiliar system. S. lavandulifolia had the highest essential oil content, followed by S. fruticosa, S. tomentosa, S. officinalis and S. candelabrum. S. ringens contains volatile oil only in traces. The neurotoxin thujone content was the highest in the S. officinalis oils and in that of S. fruticosa. No thujone was detected in S. lavandulifolia. The other species, e.g.: S. tomentosa contain this compound only in moderate concentrations (less than 10%). Among the non-volatile fractions of the plant ingredients the triterpene ursolic and oleanolic acids had the highest concentration in the leaves. Despite some rare cases, ursolic acid dominates the tritepene fraction. Rosmarinic and caffeic acids were measured in similar concentrations, in all species. As the case of S. officinalis shows, these compounds vary significantly in all organs during the vegetation period. Caffeic acid is also ubiquitous in the genus Salvia but as our data suggest it occurs in an order of magnitude lower concentration than rosmarinic acid. The isolation of phenylethanolid martynoside, though obtained in a rather small concentration, is of great chemotaxonomic significance, as this is the first phenylethanolid type glycoside isolated not only from the Salvia genus but also from the entire Subfamily Nepetoideae. As pheylethanolids are rather common and accumulate in significant concentrations in plants of the Subfamily Lamioideae, our opinion that the chemical differences between the two subfamilies are less qualititative than quantitative, is confirmed. This holds true of other chemical markers like monoterpenes, ursolic and oleanolic acids, caffeic acid and now phenylethanolids. Diterpenes i.e. 7-methyl carnosoate, rosmanol 7- methylether, sageon from S. officinalis, 7alpha-acetoyroyleanone, 7alpha-hydroxyroyleanone, royleanone, 6,7-dehydroroyleanone from S. tomentosa and candesalvoquinone, candelabroquinone, 12-O-methylcandesalvone, candesalvone B methyl ester and candelabrone have been isolated from Salvia candelabrum. All of the compounds belong to the abietane type of diterpenoids and have pronounced antioxidant effect.

Diterpenoids and flavonoids from the fruits of Vitex agnus-castus and antioxidant activity of the fruit extracts and their constituents.

From the n-hexane fraction of the fruits of Vitex agnus-castus, two labdane-type diterpenes, vitetrifolin B and C, were isolated by means of multiple chromatographic separations, together with the previously identified rotundifuran, vitexilactone and the sesquiterpene spathulenol. From the EtOAc fraction, eupatorin was identified for the first time, besides the known casticin, penduletin, vitexin and orientin. The n-hexane, EtOAc and MeOH-H(2)O fractions of the MeOH extract of Agni-casti fructus were subjected to in vitro antioxidant assays. The EtOAc extract displayed a significant concentration-dependent effect when tested by 1,1-diphenyl-2-picrylhydrasyl (DPPH) free radical assay (IC(50) = 68 microg/mL) and against the autooxidation of a standard rat brain homogenate (IC(50) = 14 microg/mL). The MeOH-H(2)O fraction was less active with 3643 microg/mL (DPPH test) and IC(50) = 125 microg/mL (rat brain homogenate), while the n-hexane phase proved to be inactive. The main flavonoid constituents of the EtOAc extract, casticin, vitexin and orientin were assayed for antioxidant activity and found that only casticin possesses a marked lipid peroxidation inhibitory effect (IC(50) = 0.049 mm) compared with that of the positive control ascorbic acid (IC(50) = 0.703 mm).

[Investigation of the diterpene alkaloids of Aconitum species native to the Carpathian Basin]. / Kárpat-medencében honos Aconitum fajok diterpénalkaloidjainak vizsgálata.

Aconitum species accumulate highly toxic diterpene alkaloids, which have attracted considerable interest because of their complex structure, interesting chemistry and noteworthy physiological effects. The authors carried out phytochemical analysis of two Aconitum species native to the Carpathian Basin, aiming the isolation of diterpene alkaloids. The multistep chromatographic purification of the extracts of A. vulparia whole plant and A. toxicum root resulted in the isolation of 16 alkaloids. Structure elucidation by means of spectroscopic investigations revealed the presence of 3 (acovulparine, vulparine, acotoxinine) new and 13 already known compounds (anthranoyllycoctonine, delectinine, delcosine, finetiadine, lycoctonine, N-methyl-N-deethyllycocotine, septentriodine, aconitine, aconosine, dolaconine, neoline, songorine, songo-ramine). Alkaloids isolated from A. vulparia are C19 diterpene alkaloids possessing aconitane skeleton, whilst from A. toxicum two C20 diterpene alkaloids with napelline and five diterpene alkaloids with aconitane skeleton were identified. The pharmacological testing of the majority of the isolated compounds has not been reported in the literature yet. Since some of the diterpene alkaloids are structurally very similar to compounds which were active in different in vitro or in vivo experiments, the pharmacological testing of the alkaloids reported in this article seems to be reasonable. The investigation of the compounds may help to reveal structure-activity relationships as well.

Two new ecdysteroids from Serratula wolffii.

11alpha-Hydroxypoststerone (1) and herkesterone (2), two new natural ecdysteroids, were isolated from the herb Serratula wolffii. The former compound is the first 11-hydroxylated C21 ecdysteroid, while the latter is a new ecdysteroid with a 7,9(11)-dien-6-one chromophore. Their structures were determined using a combination of spectroscopic techniques.

A new ecdysteroid with unique 9beta-OH and four other ecdysteroids from Silene italica ssp. nemoralis.

A new natural ecdysteroid, 9beta,20-dihydroxyecdysone (1) and four related compounds 5alpha-20-hydroxyecdysone (2), 5alpha-2-deoxy-integristerone A (3), integristerone A (4) and 22-deoxy-integristerone A (5) were isolated from the herb of Silene italica ssp. nemoralis. Compound 1 is the C-9 epimer of the known 9alpha,20-dihydroxyecdysone (6) and represents a peculiar steroid skeleton. The structures of the compounds were elucidated by 1D and 2D NMR, IR and MS spectroscopy.

Preparative scale purification of shidasterone, 2-deoxy-polypodine b and 9a,20-dihydroxyecdysone from Silene italica ssp. nemoralis.

A suitable combination of preparative scale separation methods results in effective clean-up of the ecdysteroids of Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The isolation of minor ecdysteroids from the partially purified extract is based on the use of both droplet counter-current chromatography and low-pressure reversed-phase liquid chromatography. The purification is completed by preparative thin-layer chromatography and preparative high-performance liquid chromatography to obtain the minor ecdysteroids, such as 2-deoxy-20-hydroxyecdysone, shidasterone, 2-deoxy-polypodine B, makisterone C, and 9alpha,20-dihydroxyecdysone.

New diterpenoids from the aerial parts of Salvia candelabrum.

The new diterpenoids, candesalvoquinone, 12- O-methylcandesalvone B, candesalvone B methyl ester with the 3,4-secoabietane skeleton, and candelabroquinone with the abietane skeleton were isolated from the aerial parts of Salvia candelabrum, together with the known compound candelabrone. The structures were established by mass spectroscopy and advanced two-dimensional NMR methods, including 1H-NMR, JMOD, 1H- 1H COSY, HSQC, HMBC and NOESY experiments.

9alpha,20-Dihydroxyecdysone, a new natural ecdysteroid from Silene italica ssp. nemoralis.

A new natural compound, 9alpha,20-dihydroxyecdysone (1), and two known related compounds, 20-hydroxyecdysone and ecdysone, were isolated from the herb Silene italica ssp. nemoralis. Compound 1 is the first C-9 hydroxylated ecdysteroid with a cis-fused A/B ring junction to have been isolated from a plant source, and its structure was determined using a combination of spectroscopic techniques.