Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting.

Phytoecdysteroids like 20-hydroxyecdysone ("ecdysterone") can exert a mild, non-hormonal anabolic/adaptogenic activity in mammals, and as such, are frequently used in food supplements. Spinach is well-known for its relatively low ecdysteroid content. Cyanotis arachnoidea, a plant native in China, is among the richest sources of phytoecdysteroids, and extracts of this plant are marketed in tons per year amounts via the internet at highly competitive prices. Here we report the investigation of a series of food supplements produced in Germany and claimed to contain spinach extracts. Twelve ecdysteroids including two new compounds were isolated and utilized as marker compounds. A comparative analysis of the products with Cyanotis and spinach extracts provides evidence that they were manufactured from Cyanotis extracts instead of spinach as stated. Based on the chromatographic fingerprints, 20-hydroxyecdysone 2- and 3-acetate are suggested as diagnostic markers for related quality control. This case appears to represent an unusual type of dietary supplement counterfeiting: undeclared extracts from alternative plants would supposedly 'guarantee' product efficacy.

High performance liquid chromatography used for quality control of Achyranthis Radix.

To establish a standard of quality control and to identify reliable Achyranthis Radix, three phytoecdysones including ecdysterone (1), 25R-inokosterone (2) and 25S-inokosterone (3) were determined by quantitative HPLC/UV analysis. Three phytoecdysones were separated with an YMC J'sphere ODS C(18) column (250 mm × 4.6 mm, 4 µm) by isocratic elution using 0.1% formic acid in water and acetonitrile (85:15, v/v%) as the mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 245 nm. The standards were quantified by HPLC/UV from Achyranthes bidentata Blume and Achyranthes japonica Nakai, as well as Cyathula capitata Moq. and Cyathula officinalis Kuan, which are of a different genus but are comparative herbs. The method was successfully used in the analysis of Achyranthis Radix of different geographical origin or genera with relatively simple conditions and procedures, and the assay results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of eighteen A. bidentata Blume samples and ten A. japonica Nakai samples. The results indicate that the established HPLC/UV method is suitable for quantitation and pattern recognition analyses for quality evaluation of Achyranthis Radix.

[Studies on the chemical constituents of Paris bashanensis].

OBJECTIVE: Study the chemical constituents of the rhizomes of Paris bashanensis to search after the alternative resourc for the Chinese medicinal material Rhizoma Paridis. METHODS: The n-BuOH extracts of P. bashanensis was applied to silica gel column and eluted with EtOAc-EtOH,then the gained fractions were further purified by chromatography on Sephadex LH-20 column and PreRP-HPLC to give pure compounds whose structures were elucidated mainly on the basis of analyzing the spectral data of MS,1H-NMR, 13C-NMR,2D-NMR. RESULTS: Five compounds were isolated and identified as P-ecdysone (1), pinnatasterone(2), pennogenin-3-0-alpha-L-rhamnopyranosyl (1-->2)-[alpha-L-arabinofuranosyl ( 1- -4) ] -pf-D-glycopyranoside (3), diosgenin-3-O-alpha-L-rhamnopyranosyl (1-->2) - [ a-L-arabinofuranosyl (1-->4)]-beta-D-glycopyranoside(4), pennogenin-3-O-alpha-L-rhamnopyranosyl (1-->4) -a-L-rhamnopyranosyl (1-4)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glycopyranoside (5). CONCLUSION: Compound 1-5 are obtained from this plant for the first time.

Two minor phytoecdysteroids of the plant Silene viridiflora.

Chemical investigations of Silene viridiflora (L.) yielded a new ecdysteroid, 20-hydroxyecdysone 20,22-monoacetonide-25-acetate (1), and a known ecdysteroid, 2-deoxypolypodine B-3-beta-D-glucoside (2). The elucidation of the chemical structures was established by 1D and 2D NMR experiments.

Furostanol saponins and ecdysones with cytotoxic activity from Helleborus bocconei ssp. intermedius.

Two furostanol saponins helleboroside A (1) and helleboroside B (2) were isolated from the methanol extract of Helleborus bocconei Ten. subsp. intermedius (Guss.) Greuter and Burdet, along with the furospirostanol saponin 4 and two ecdysones: ecdysterone (5) and polypodyne B (6). Compound 2 was enzymatically hydrolysed to give product 3. The biological activity of all compounds was tested against rat C6 glioma cells showing a significant cytotoxicity for compounds 3, 4 and 6.

Simultaneous determination of three phytoecdysteroids in the roots of four medicinal plants from the genus Asparagus by HPLC.

INTRODUCTION: The genus Asparagus is known to contain phytoecdysteroids that have been shown to exhibit many beneficial pharmacological properties such as improving lipid metabolism, modulating immunological responses, etc. Currently, knowledge about the contents of phytoecdysteroids in the roots of Asparagus species is limited and HPLC methods for their analyses are unsatisfactory. OBJECTIVE: To develop an HPLC method for the simultaneous determination of three phytoecdysteroids, 20-hydroxyecdysone, ecdysone and ajugasterone C, in the roots of four Asparagus species. METHODOLOGY: Reference standards of phytoecdysteroids were isolated from the roots of Asparagus filicinus by open column chromatography. HPLC analysis was performed on an Alltima C(18) column with gradient elution using aqueous 0.2% formic acid solution containing 0.2% isopropanol and acetonitrile. RESULTS: All calibration curves showed good linear correlation coefficients (r(2) > 0.9994) within the tested ranges. Limits of detection (S/N = 3) and quantification (S/N = 10) for the three analytes were less than 2.7 and 9.9 ng, respectively. Intra- and inter-day RSDs of retention times and peak areas were less than 2.61%. The recoveries were between 93.2 and 107.5%, and the RSDs were less than 3.83% for the root samples of A. filicinus. CONCLUSION: The HPLC method established is appropriate for the efficient quantitative and qualitative analyses of important phytoecdysteroids in Asparagus species. This study showed that A. filicinus is rich in phytoecdysteroids, especially 20-hydroxyecdysone. However the three studied phytoecdysteroids were not detected in A. cochinchinensis, A. officinalis and A. setaceus.

Ecdysteroids act as inhibitors of calf skin collagenase and oxidative stress.

Three new phytoecdysteroids have been isolated from the seeds of Chenopodium quinoa and structurally identified as 20,26-dihydroxy, 28-methyl ecdysone, 20,26-dihydroxy, 24(28)-dehydro ecdysone, and 20-hydroxyecdysone 22-glycolate using serial chromatographic and spectroscopic methods. Both previously reported compounds and newly identified phytoecdysteroids were evaluated for their inhibitory effect on calf skin collagenase, as this enzyme is involved in aging skin diseases. Their effectiveness on scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, as well as in chelating the iron metal ions was also investigated. All isolated compounds demonstrated a higher potency to inhibit this matrix metalloproteinase and to chelate the iron ion, both with respect to the number of carbonyl groups bearing their carbon skeleton, suggesting that their mechanism of action involves their ability to coordinate both ions (either the zinc ion of the catalytic domain of collagenase or the iron ion), acting as an electron donor. The DPPH-scavenging ability was hydroxyl dependent. Satisfactory results obtained from the enzyme in vitro experiment were further supported by the gel electrophoresis. These results indicate that ecdysteroids might be considered as potent chemical agents to prevent or delay both collagenase-related skin damages and oxidative stress.

A new phytoecdysteroid from Ajuga taiwanensis.

A new phytoecdysteroid, namely ajugalide-E (1), together with twenty five known compounds were isolated from Ajuga taiwanensis collected from Tainan, Taiwan. Their structures were determined by spectral analysis including high resolution one- and two-dimensional NMR spectroscopy.

Phytoecdysteroids and glycoceramides from Eriophyton wallchii.

Two new compounds, 28-epi-cyasterone and eriophytonoide, along with 11 known compounds, cyasterone, ajuforrestins A and B, 20-hydroxyecdysone, polypodin B, ajugalactone, 8-O-acetylharpagid, apigenin, N-[2hydroxy-(nonadecanoyl-tricosanoyl)]-4-hydroxy-trans-8-sphingenine, beta-sitosterol, and daucosterol, were isolated from the aqueous ethanolic extract of the whole herb of Eriophyton wallchii Benth. The structures of 28-epi-cyasterone and eriophytonoide were elucidated as (22R,24S,25S,28S)-5beta-stigmast-7-en-26-oic acid, 2beta,3beta,14,20,22,28-hexa hydroxy-6-oxo-gamma-lactone, and 1-O-beta-d-glucopyranosyl-(2S,3S,4R,8E)-2-[(2R)-2-hydroxyhexadecanoylamino]-1,3,4-octadecanetriol-8-ene, respectively, on the basis of spectral and chemical evidence.

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.

Benzoxazinoids and iridoid glucosides from four Lamium species.

A new class of compounds for the plant family Lamiaceae, benzoxazinoids, was found in Lamium galeobdolon. From the aerial parts of the species were isolated the new 2-O-beta-D-glucopyranosyl-6-hydroxy-2H-1,4-benzoxazin-3(4H)-one (6-hydroxy blepharin) together with four known benzoxazinoids, DHBOA-Glc, blepharin, DIBOA, DIBOA-Glc, as well as harpagide, 8-O-acetyl-harpagide and salidroside. Eight known iridoid glucosides, 24-epi-pterosterone and verbascoside were isolated from Lamium amplexicaule, L. purpureum and L. garganicum. The iridoids, 5-deoxylamiol and sesamoside, as well as the phytoecdysone, 24-epi-pterosterone, were found for the first time for the genus Lamium. The phytochemical data are discussed from a systematic and evolutionary point of view.

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.

[Chemical constituents of Cyanotis arachnoidea].

AIM: To investigate the chemical constituents of Cyanotis arachnoidea. METHODS: By using chromatographic methods for separation and combination with spectral analysis, their chemical structures were determined. RESULTS: Six compounds were identified as ajugasterone C-20, 22-acetonide (1), 20-hydroxyecdysone-20, 22-acetonide (2), 22-oxo-ajugasterone C (3), 22-oxo-20-hydroxyecdysone (4), beta-sitosterol (5), daucosterol (6). CONCLUSION: Compound 3 is a new compound, 4 was a new natural compound.

Phytoecdysteroids effects on mammalians, isolation and analysis.

Ecdysteroids are known insect moulting hormones, regulating the insects' metamorphosis. At the same time, ecdysteroids reveal beneficial effects on humans and animals alike. Medicinal plants have been subjected to an intensive research, addressing the presence of ecdysteroids. The possible utilization of medicinal plant deals with their use as raw materials for health preparations and also for the isolation of new phytoecdysteroids. Research on the plant ecdysteroids involves two basic lines. Isolation of major compounds and scout their physiological effects; and isolation of minor ecdysteroids to find new compounds. This review summarizes the recent efforts in the ecdysteroid research including their indication as health improvement preparations, chromatography of ecdysteroids and certain methods for identification of new ecdysteroids.

Ecdysteroids from a Zoanthus sp.

A new ecdysteroid, zoanthusterone, has been isolated from a marine zoanthid, Zoanthus sp. Ten known ecdysteroids, ponasterone A, 20-hydroxyecdysone 2-acetate, viticosterone E, integristerone A 25-acetate, 2-deoxy-20-hydroxyecdysone, ecdysone, ajugasterone C, dacryhainansterone, inokosterone, and 20-hydroxyecdysone, have also been isolated. This is the first report of ecdysteroids in a Zoanthus species.

3-Deoxy-1beta,20-dihydroxyecdysone from the leaves of Diploclisia glaucescens.

Chemical investigation of methanol extract of the leaves of Diploclisia glaucescens of the family Menispermaceae furnished a new ecdysteroid, 3-deoxy-1beta,20-dihydroxyecdysone. The structure of the new ecdysteroid was established on detailed analysis of spectral data. The 3-deoxy ecdysteroid showed 40% potency of 20-hydroxyecdysone in the spiracle index assay using the fourth instar larvae of the silkworm Bombyx mori.

Limnantheoside C (20-hydroxyecdysone 3-O-beta-D-glucopyranosyl-[1-->3]-beta-D-xylopyranoside), a phytoecdysteroid from seeds of Limnanthes alba (limnanthaceae).

A new ecdysteroid glycoside, limnantheoside C (20-hydroxyecdysone 3-O-beta-D-glucopyranosyl-[-->3]-beta-D-xylopyranoside [1]), together with limnantheoside A (20-hydroxyecdysone 3-O-beta-D-xylopyranoside [2]) and 20-hydroxyecdysone (3) have been isolated by bioassay/RIA-directed HPLC analyses of a methanol extract of the seedmeal of Limnanthes alba Hartw. ex Benth. The structure of the novel ecdysteroid glycoside (1) was determined unambiguously by UV, LSIMS and a combination of 1D- and 2D-NMR experiments. These three compounds are isolated from Limnanthes alba for the first time.

A new phytoecdysone from the roots of Rhaponticum uniflorum.

A new ecdysteroid named rhapontisterone R1 (1) together with two known phytoecdysones, rhapontisterone (2) and ecdysterone (3) were isolated from the roots of Rhaponticum uniflorum (L.) DC. The new compound was shown to be 2beta,3beta,11alpha,14alpha,20xi,22xi-hexahydroxy-stigma-7,24(28)-dien-6-oxo-28,25-carbolactone. The structure has been determined primarily on the basis of physico-chemical properties and spectral analysis.

1 alpha,20R-dihydroxyecdysone from Axyris amaranthoides.

Bioassay/RIA-directed phytochemical examination of the seeds of Axyris amaranthoides afforded a new ecdysteroid: 1 alpha,20R-dihydroxyecdysone [1-epi-integristerone A], together with 20-hydroxyecdysone and polypodine B. The structure of 1 alpha,20R-dihydroxyecdysone was determined unequivocally by UV, LSIMS, and a combination of 1D and 2D NMR techniques.

Evidence that Y-organs of the crab Cancer antennarius secrete 3-dehydroecdysone.

Y-organs are paired glands in crustaceans that secrete a class of steroid hormones (ecdysteroids) that regulate growth, molting and development. The glandular secretion has been assumed to be solely the ecdysteroid, ecdysone, a polyhydroxylated derivative of cholesterol. We previously reported that Y-organs of a crab (Cancer antennarius) additionally secreted an ecdysteroid that is less polar than ecdysone. Evidence is presented here that the other secretion product is 3-dehydroecdysone (3-dhE). The compound co-chromatographed with authentic 3-dhE in both normal-phase, and reversed-phase, high-performance liquid chromatography. Mass spectrometry of the ecdysteroid gave results consistent with its identity as 3-dhE. The putative 3-dhE was radiolabeled by injecting crabs with [3H]cholesterol and then incubating the Y-organs. The putative [3H]3-dhE secretion was then subjected to chemical reduction. The reaction yielded labeled products that co-chromatographed with authentic ecdysone and 3-epiecdysone. Results of other experiments gave the following results: (1) Putative 3-dhE was not altered (chromatographic criteria) by incubations with snail hydrolases. (2) Putative [3H]3-dhE, added to incubations of Y-organ halves or homogenates, was not significantly converted to ecdysone; also, no conversion was evident after incubation in medium alone in which the hemolymph serum supplement was raised to 50% of the volume. (3) [3H]Ecdysone was not converted to putative 3-dhE in vitro by Y-organ halves or homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)