Replacement of light by dibutyryl-CAMP and CAMP in betacyanin synthesis

The effect of adenosine 3′,5′-cyclic monophosphate and its N⁶,O^2′- dibutyryl derivative (Bu₂-CAMP) on betacyanin formation in etiolated Amaranthus paniculatus seedlings was investigated. Both substances can replace the action of light in the synthesis of these pigments, the formation of which is controlled by phytochrome. The specificity of this mimicry is underlined by the observations that sodium butyrate does not promote any betacyanin formation and that theophylline enhances the effect of Bu₂-AMP. Puromycin inhibits the induction of betacyanin synthesis by Bu₂-CAMP just as it does the light-induced pigment formation. These findings suggest that phytochrome exerts its controlling role in the synthesis of betacyanins through the agency of CAMP.     

Isolation and purification of silychristin,silydianin and taxifolin in the co-products of the silybin refined process from the silymarin by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of silychristin, silydianin and taxifolin in the co-products of the silybin refined process from the silymarin was successfully established by n-hexane–chloroform–methanol–water (0.5:11:10:6 (0.5 acetic acid), v/v/v/v) as the two-phase solvent system. 146 mg silydianin, 280 mg silychristin and 63 mg taxifolin from 1.463 g co-products sample in one separation were obtained with the purities of 95.1%, 99.3% and 98.2%, respectively, determined by HPLC. The structures of the compounds were identified by means of ESI-MS-MS, TOF-MS, ¹H NMR, ¹³C NMR and 2DNMR-HMBC. Silychristin, silydianin and taxifolin had been separated as standards by HSCCC for the first time. A comparative study between HSCCC and RPLC for separation and isolation of taxifolin, silychristin and silydianin was investigated. The differences between the two preparative chromatographic methods were all discussed. The results demonstrated that HSCCC was a powerful separation tool and could contribute to identifying and quantifying plant ingredients.     

Application of microwave‐assisted extraction coupled with high‐speed counter‐current chromatography for separation and purification of Dehydrocavidine from Corydalis saxicola Bunting

Introduction – Dehydrocavidine is a major component of Corydalis saxicola Bunting with sedative, analgesic, anticonvulsive and antibacterial activities. Conventional methods have disadvantages in extracting, separating and purifying dehydrocavidine from C. saxicola. Hence, an efficient method should be established. Objective – To develop a suitable preparative method in order to isolate dehydrocavidine from a complex C. saxicola extract by preparative HSCCC. Methodology – The methanol extract of C. saxicola was prepared by optimised microwave‐assisted extraction (MAE). The analytical HSCCC was used for the exploration of suitable solvent systems and the preparative HSCCC was used for larger scale separation and purification. Dehydrocavidine was analysed by high‐performance liquid chromatography (HPLC) and further identified by ESI‐MS and 1H NMR. Results – The optimised MAE experimental conditions were as follows: extraction temperature, 60°C; ratio of liquid to solid, 20; extraction time, 15 min; and microwave power, 700 W. In less than 4 h, 42.1 mg of dehydrocavidine (98.9% purity) was obtained from 900 mg crude extract in a one‐step separation, using a two‐phase solvent system composed of chloroform–methanol–0.3 m hydrochloric acid (4 : 0.5 : 2, v/v/v). Conclusion – Microwave‐assisted extraction coupled with high‐speed counter‐current chromatography is a powerful tool for extraction, separation and purification of dehydrocavidine from C. saxicola. Copyright © 2009 John Wiley & Sons, Ltd.     

OCCURRENCE OF LOROXANTHIN,LOROXANTHIN DECENOATE,AND LOROXANTHIN DODECENOATE IN TETRASELMIS SPECIES (PRASINOPHYCEAE,CHLOROPHYTA)1

The pigment composition of six species of Tetraselmis (Prasinophyceae) was analyzed using improved HPLC methods. All pigment extracts showed three peaks corresponding to unknown carotenoids. The isolated pigments were analyzed using UV–Vis spectroscopy, electrospray ionization–mass spectrometry (ESI–MS), and when carotenoid esters were suspected, gas chromatography–mass spectrometry (GC–MS) of the methyl ester and dimethyloxazoline derivative of the corresponding fatty acid. The new pigments were determined to be loroxanthin, loroxanthin 19‐(2‐decenoate), and loroxanthin 19‐(2‐dodecenoate); this is the first time these pigments have been described in the genus Tetraselmis. Moreover, this is the first report of esterification of 2‐decenoic acid to loroxanthin. The relative contents of these pigments depended on the light regime, with the lowest proportions measured at the highest photon flux density assayed. The implications of the identification of these pigments in the genus Tetraselmis for the pigment types previously described in the class Prasinophyceae are discussed.     

Carotenoid Production by a Novel Isolate of <Emphasis Type="Italic">Microbacterium paraoxydans</Emphasis>

This study reports extraction and characterization of carotenoid pigments from Microbacterium paraoxydans, a non-photosynthetic bacterium, cultivated in Luria–Bertani (LB) medium. The isolate was identified to be moderately halo- and osmo-tolerant capable of withstanding high (~ 6%) salt and sugar (30% w/v sucrose, 20% w/v glucose) concentrations after a brief period of adaptation. The pigments were characterized using a combination of UV–Vis spectral analysis with the λ_max at 407, 436 and 466 nm and ESI–MS with an m/z value at 536.44. The absorption profile of the pigments and their nature was influenced by carbon, nitrogen source and presence of salt in the growth medium. Highest level of pigment (~ 16 g kg dry wt cells^?1) was produced in NH₄Cl supplemented LB medium. The pigment displayed free radical scavenging, anticancer activity, characteristic of the plant carotenoids. Based on the accumulation of pigments under different conditions, a biochemical pathway for synthesis of neurosporene was proposed.     

Determination of botulinum toxins after peptic sample pre-treatment by multidimensional nanoscale liquid chromatography and nano-electrospray ion-trap mass spectrometry

Botulinum neurotoxin types A to G are produced from different strains of Clostridium botulinum. The complex neurotoxins belong to the most toxic substances known and cause botulism both in humans and animals. Botulinum toxin complexes are produced with molecular weights of 300, 500 and 900 kDa. These large protein complexes contain beside the toxic zinc protease of 150 kDa, additional neurotoxin associated proteins, which are responsible for the extreme pH and protease stability. In this study we present for the first time a rugged detection method of botulinum toxins at femtomole levels in complex culture media after peptic sample pre-treatment and 2D-nano-LC–ESI–MS–MS-technique. In contrast to other studies, we used progenitor toxins directly from culture supernatant of C. botulinum strains A, B, E and F without further purification, to simulate complex, protein-containing sample conditions. We were able to demonstrate, that peptic pre-treatment is a great challenge in reducing ubiquitous proteins as well as proteins from suspicious samples. The study also found that multidimensional chromatography leads to significant better peptide differentiation and identification in protein loaded matrices than one dimensional nano-LC–ESI–MS.     

Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii

Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline beta-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas-liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of alpha-Rhap-(1 --> 3)-alpha-Rhap-(1 --> 3)-alpha-Manp-(1 --> 2)-Man-ol substituted at O-6 of mannitol with an alpha-Glcp-(1 --> 4)-beta-Galp group. Species containing one and two additional alpha-Glcp-(1 --> 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by approximately 75%, whereas fraction 3 inhibited by approximately 55%.     

盐地碱蓬甜菜红素苷的鉴定及环境因素对其积累的影响

以C3盐生植物盐地碱蓬（Suaeda salsa）为材料研究了体内红色素的理化性质、随发育时期的变化及光照、温度、盐分对红色素积累的影响。结果表明：该红色素溶于水而不溶于有机溶剂,光抑制其积累,在酸性条件下稳定,为红色,在碱性条件下不稳定,为黄色,最大吸收峰为538nm。根据这些特性,初步确定该类红色素为甜菜红素苷（betacyanin）。黑暗、低温、高盐环境有利于盐地碱蓬甜菜红素苷的积累,其中萌发期黑暗是诱导甜菜红素苷积累的重要因子。     

Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy

Four betacyanin pigments were analysed by LC NMR and subjected to extensive NMR characterisation after isolation. Previously, low pH values were applied for NMR investigations of betalains resulting in rapid degradation of the purified substances thus preventing extensive NMR studies. Consequently, up to now only one single (13)C NMR spectrum of a betalain pigment, namely that of neobetanin (=14,15-dehydrobetanin), was available. Because of its sufficient stability under highly acidic conditions otherwise detrimental for betacyanins, this pigment remained an exemption. Since betalains are most stable in the pH range of 5-7, a new solvent system has been developed allowing improved data acquisition through improved pigment stability at near neutral pH. Thus, not only (1)H, but for the first time also partial (13)C data of betanin, isobetanin, phyllocactin and hylocerenin isolated from red-purple pitaya [Hylocereus polyrhizus (Weber) Britton & Rose, Cactaceae] could be indirectly obtained by gHSQC- and gHMQC-NMR experiments.     

Morphological and phytochemical aspects of three alien Opuntia species on Euganean Hills in North-Eastern Italy

In this study, Opuntia stricta, O. engelmannii, and O. humifusa, naturalized alien units in the Euganean Hills (North-Eastern Italy), are investigated. Different morphological and physiological features both in cladodes collected in summer and winter and in fruits arisen from two blooms are reported. The morphological observations showed a different organization in epidermis/hypodermis of cladodes collected in winter and in summer, probably correlated to the individual plant reaction to a stressful factor as low temperature. Scanning electron microscopy observations revealed no differences in the deposition of waxes and stomata distribution in the epidermis of cladodes. The betacyanin analysis showed that cladodes produce these pigments only in the winter season. In all the species, the higher content of betacyanins was reached in the fruits from the second bloom, with O. stricta being the most rich. The amount of betacyanins is comparable to some commercial red beets used as a common source of betacyanins as natural food color.     

Preparative isolation and purification of four flavonoids from the petals of nelumbo nucifera by high‐speed counter‐current chromatography

Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high‐speed counter‐current chromatography (HSCCC). Methodology – Following an initial clean‐up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC‐PAD, ESI‐MS, ¹H‐NMR and ¹³C‐NMR. Results – The separation was performed using a two‐phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow‐rate of 1.0 mL/min in the head‐to‐tail elution mode. Ultimately, 5.0 mg syringetin‐3‐O‐β‐d‐glucoside, 6.5 mg quercetin‐3‐O‐β‐d‐glucoside, 12.8 mg isorhamnetin‐3‐O‐β‐d‐glucoside and 32.5 mg kaempferol‐3‐O‐β‐d‐glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.     

Correlation of pigment production with mycelium morphology in extractive fermentation of Monascus anka GIM 3.592

Extractive fermentation with nonionic surfactants is a potential method for producing Monascus pigments. In this study, the correlation between mycelium morphology and pigment production was investigated in extractive fermentation of Monascus anka GIM 3.592. The results demonstrated that pigment biosynthesis was associated with mycelial morphology and the accumulation of granular inclusions in cells. The physiological status in terms of hyphal and pellet diameters exhibited an excellent correlation with pigment accumulation, especially the yield of extracellular pigment in extractive fermentation (r > 0.85, p < 0.05). Nonionic surfactants could reduce pigment yield by influencing the morphology of hyphae and mycelium pellets. High yields of both intracellular and extracellular pigments could be achieved by controlling variations in hyphal diameters in two-stage extractive fermentation. Moreover, continuous extractive fermentation led to stable pigment production, with a relatively high productivity of total pigments reaching 72.3 AU/day. This study proposed a simple method for monitoring pigment biosynthesis in extractive fermentation using mycelium morphology as an indicating factor.     

A reduction in temperature induces bioactive red pigment production in a psychrotolerant Penicillium sp. GEU_37 isolated from Himalayan soil

Filamentous fungi are being globally explored for the production of industrially important bioactive compounds including pigments. In the present study, a cold and pH tolerant fungus strain Penicillium sp (GEU_37), isolated from the soil of Indian Himalaya, is characterized for the production of natural pigments as influenced by varying temperature conditions. The fungal strain produces a higher sporulation, exudation, and red diffusible pigment in Potato Dextrose (PD) at 15 °C as compared to 25 °C. In PD broth, a yellow pigment was observed at 25 °C. While measuring the effect of temperature and pH on red pigment production by GEU_37, 15 °C and pH 5, respectively, were observed to be the optimum conditions. Similarly, the effect of exogenous carbon and nitrogen sources and mineral salts on pigment production by GEU_37 was assessed in PD broth. However, no significant enhancement in pigmentation was observed. Chloroform extracted pigment was separated using thin layer chromatography (TLC) and column chromatography. The two separated fractions i.e., fractions I and II with Rf values 0.82 and 0.73, exhibited maximum light absorption, λ_max, at 360 nm and 510 nm, respectively. Characterization of pigments using GC–MS showed the presence of the compounds such as phenol, 2,4-bis (1,1-dimethylethyl) and eicosene from fraction I and derivatives of coumarine, friedooleanan, and stigmasterole in fraction II. However, LC-MS analysis detected the presence of derivatives of compound carotenoids from fraction II as well as derivative of chromenone and hydroxyquinoline as major compounds from both the fractions along with other numerous important bioactive compounds. The production of such bioactive pigments under low temperature conditions suggest their strategic role in ecological resilience by the fungal strain and may have biotechnological applications.     

Isolation of three new ent-labdane diterpenes from Dodonaea viscosa Jacquin (Sapindaceae): Preliminary evaluation of antiherpes activity

Investigation of the crude ethanolic extract obtained from the aerial parts of Dodonaea viscosa led to the isolation of three new ent-labdane diterpenes (1–3). The structures were established on the basis of their ESI–MS, UV, IR and NMR spectral data. The crude extract, fractions and compounds were evaluated for in vitro antiviral activity against Herpes Simplex Virus type 1 (HSV-1). The crude extract showed promising antiherpes activity expressed by a selectivity index of 6.2.     

Minor betalains in fruits of Hylocereus species

Betacyanins in peel and flesh of fruits of different Hylocereus species were identified by means of GC/MS, electrospray MS/MS, HPLC as well as (1)H and (13)C NMR techniques. As hitherto unknown pigments: betanidin 5-O-(2'-O-beta-D-apiofuranosyl)-beta-D-glucopyranoside, betanidin 5-O-(4'-O-malonyl)-beta-D-glucopyranoside and betanidin 5-O-[(5'-O-E-sinapoyl)-2'-O-beta-D-apiofuranosyl]-beta-D-glucopyranoside were elucidated. The sinapoyl moiety attachment position in the structure of betacyanins was established for the first time. The peel contained a more complex pattern of betacyanins with apiofuranosyl moiety. Other recently identified pigments were also present in the samples and their (1)H or (13)C NMR spectra were recorded. In the case of phyllocactin and its 4'-isomer the migration of the malonyl group was noticed.     

Chlorosome antenna complexes from green photosynthetic bacteria

Chlorosomes are the distinguishing light-harvesting antenna complexes that are found in green photosynthetic bacteria. They contain bacteriochlorophyll (BChl) c, d, e in natural organisms, and recently through mutation, BChl f, as their principal light-harvesting pigments. In chlorosomes, these pigments self-assemble into large supramolecular structures that are enclosed inside a lipid monolayer to form an ellipsoid. The pigment assembly is dictated mostly by pigment–pigment interactions as opposed to protein–pigment interactions. On the bottom face of the chlorosome, the CsmA protein aggregates into a paracrystalline baseplate with BChl a, and serves as the interface to the next energy acceptor in the system. The exceptional light-harvesting ability at very low light conditions of chlorosomes has made them an attractive subject of study for both basic and applied science. This review, incorporating recent advancements, considers several important aspects of chlorosomes: pigment biosynthesis, organization of pigments and proteins, spectroscopic properties, and applications to bio-hybrid and bio-inspired devices.     

A new ursane and a new oleanane triterpene acids from the whole plant of Spermacoce latifolia

A phytochemical study on the whole plant of Spermacoce latifolia led to the isolation of a new ursane triterpene acid, 3β,6β,23-trihydroxy-urs-12,20(30)-dien-28-oic acid (1), and a new oleanane triterpene acid, 3β,6β,29-trihydroxy-olean-12-en-28-oic acid (2), together with seven known triterpenic acids (3–9). Their structures were established on the basis of detailed spectroscopic analysis, including one- and two-dimensional NMR, ESI–MS and HR–ESI–MS techniques. All the compounds were isolated from S. latifolia for the first time. Compounds 3, 5, 6 and 9 were found to show in vitro growth inhibitory activity against three assayed human cancer cell lines MCF-7, NCI-H460 and HepG-2 with IC₅₀ values from 9.53 to 80.20 mM. Compounds 1 and 3–9 were further revealed to show significantly in vitro α-glucosidase inhibitory activity with IC₅₀ values from 0.009 to 0.422 mM, most of which were more potent than the reference compound acarbose (IC₅₀ 0.409 mM).     

过氧化氢参与了黑暗诱导的盐地碱蓬叶片甜菜红素积累

该文比较研究了黑暗和光照条件下C₃盐生植物盐地碱蓬(Suaeda salsa)叶片甜菜红素积累和H₂O₂含量及其抗氧化酶活性的关系,实验分析了甜菜红素体外抗氧化性能,以期揭示诱导盐地碱蓬甜菜红素积累的可能机制以及甜菜红素积累的生理生态意义。结果表明:暗期处理和营养液中加入一定浓度的H₂O₂都明显促进盐地碱蓬叶片H₂O₂含量、甜菜红素的含量、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活性,而且叶片中 H₂O₂含量与甜菜红含量、SOD和CAT活性具有正相关性;盐地碱蓬甜菜红素体外清除羟自由基的能力明显强于维生素C,而清除超氧阴离子能力低于维生素C。这些结果表明:黑暗作为一种环境胁迫因子诱导盐地碱蓬叶片甜菜红素的积累可能是由自由基介导的,甜菜红素的积累可能与提高植物的抗氧化能力有关。     

Betalains of Celosia argentea

The betalains of yellow, orange and red inflorescences of common cockscomb (Celosia argentea var. cristata) were compared and proved to be qualitatively identical to those of feathered amaranth (Celosia argentea var. plumosa). In addition to the known compounds amaranthin and betalamic acid, the structures of three yellow pigments were elucidated to be immonium conjugates of betalamic acid with dopamine, 3-methoxytyramine and (S)-tryptophan by various spectroscopic techniques and comparison to synthesized reference compounds; the latter two are new to plants. Among the betacyanins occurring in yellow inflorescences in trace amounts, the presence of 2-descarboxy-betanidin, a dopamine-derived betacyanin, has been ascertained. The detection of high dopamine concentration may be of toxicological relevance in use of yellow inflorescences as a vegetable and in traditional Chinese medicine, common uses for the red inflorescences of common cockscomb.     

Betalain producing cell cultures ofBeta vulgaris L. var. bikores monogerm (red beet)

Summary The betalains are a class of natural pigments comprising the yellow betaxanthins and the violet betacyanins. Callus lines developed fromBeta vulgaris, L. var. bikores monogerm exhibited cell colors ranging from white/green (nonpigmented) through yellow, orange, red, and violet and were representative of all betalain pigments found in the whole plant. The betalains have gained particular interest from the food industry as potential natural alternatives to synthetic food colorants in use today. Red beet extracts (E162), which contain significant amounts of the betacyanins, are currently used in products such as yogurts and ice creams. We describe here the characteristics of culture growth and betalain production for cell suspensions derived from the orange (predominantly betaxanthin-producing) and violet (betacyanin producing) callus lines. The major factors affecting betalain biosynthesis in both cultured and whole plant tissues are reviewed. Presented in the Session-in-Depth Batch Production and Fermentation at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–29, 1991.