Isolation Of Secondary Metabolites Research Articles

Secondary metabolites isolation in natural products chemistry: Comparison of two semipreparative chromatographic techniques (high pressure liquid chromatography and high performance thin-layer chromatography)

Chemical investigations on secondary metabolites in natural products chemistry require efficient isolation techniques for characterization purpose as well as for the evaluation of their biological properties. In the case of phytochemical studies, the performance of the techniques is critical (resolution and yield) since the products generally present a narrow range of polarity and physicochemical properties. Several techniques are currently available, but HPLC (preparative and semipreparative) is the most widely used. To compare the performance of semipreparative HPLC and HPTLC for the isolation of secondary metabolites in different types of extracts, we have chosen carvone from spearmint essential oil (Mentha spicata L.), resveratrol from Fallopia multiflora (Thunb.) Haraldson, and rosmarinic acid from rosemary (Rosmarinus officinalis L.) extracts. The comparison was based on the chromatographic separation, the purity and quantity of isolated compounds, the solvent consumption, the duration and the cost of the isolation operations. The results showed that semipreparative HPTLC can in some case offer some advantages over conventional semipreparative HPLC.

Nitrogen losses due to nitrification: plant based remedial prospects

Nitrification of nitrogen fertilizers results into loss of nitrogen nutrient from soil along with environmental hazards, like, global warming, contamination of ground water and so on. To tackle the problem, many synthetic inhibitors of nitrification have been formulated but till now none of them has proved to be attractive option due to their cost and non-ecofriendly nature. A shift towards natural nitrification inhibitors would be beneficial, economically viable and environmentally safer option. Also, it is a matter of exhaustive research and appropriate testing before one can come out with infallible system with holistic integrated approach. To increase the potential of natural nitrification inhibitors, some neglected aspects, like, effect of genotypic variations, broader plant exploration for nitrification inhibition capacity, identification and isolation of secondary metabolites which are responsible for nitrification inhibition activity, manipulation of germplasm for superior nitrification inhibition performance require incessant persistent scientific input for transfer of information to the farmers.

Di- and sesquiterpenoids from Cystoseira genus: structure, intra-molecular transformations and biological activity.

Natural products have been the single most productive source of leads for the development of drugs, because of the great variety of their chemical structures. Previous chemical investigation of members of the genus Cystoseira resulted in the discovery of various bioactive secondary metabolites. The secondary metabolites isolated and characterized are very interesting, both from the biological activity and structural complexity points of view, which make this genus an attractive target for further investigations. The present review covers the research progress on natural products isolated from this genus since January 1995 until now, concerning the isolation and structural elucidation of the secondary metabolites from Cystoseira species. In this contribution significant biological properties are briefly discussed. Simultaneously, we gradually construct an intra-molecular pathway that logically interrelates the isolated compounds.

ChemInform Abstract: When the Nine‐Membered Enediynes Play Hide and Seek

AbstractReview: strategy to develop masked enediyne analogues; 36 refs.

Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity

The compound 3-nitropropionic acid is a potent neurotoxic agent in animals and well-known as a potent inhibitor of Mycobacterium tuberculosis. In this research, we were able to extract this compound from the endophytic fungus, Phomopsis longicolla (FJ62759), isolated from Trichilia elegans A. JUSS ssp. elegans. The aim of this study was the isolation of secondary metabolites produced by P. longicolla, the chemical identification of these compounds and evaluation of their antimicrobial and insecticidal activity. To accomplish these goals, the fungus was cultured in BD broth for 25 days without agitation at 28 °C, and then the broth was separated from the mycelium. The supernatant was partitioned with dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and butanol (BuOH) solvents resulting in 3 extracts. However, only the EtOAc extract was used for fractionation and chemical identification because it had the greatest mass. After common chromatographic procedures, the fractions were analyzed by nuclear magnetic resonance to elucidate the chemical components. This procedure resulted in the identification of 3-nitropropionic acid in the D fraction. Evaluation of the insecticidal and antimicrobial activity of this compound has been accomplished, and the results indicate good inhibition of the citrus pathogen Guignardia citricarpa and cocoa pathogen Moniliophthora perniciosa and slight inhibition of the human bacterial pathogens Micrococcus luteus, Salmonella typhi and slight inhibition of phytopathogenic bacteria Xanthomonas axonopodis pv. phaseoli. The evaluation of insecticide activity did not show mortality of the Diatraea saccharalis larvae by the metabolite 3-nitropropionic acid in the D fraction. The results suggest that P. longicolla is a bioactive metabolic producing endophytic fungus with biotechnological properties.

Effect of methionine on production of naphthoquinones in Impatiens balsamina root cultures and detection of some secondary metabolites

Context: Lawsone, lawsone methyl ether and 3,3′-methylelnebislawsone are the main active compounds of Impatiens balsamina L. (Balsaminaceae). These compounds possess various pharmacological activities that have been shown to assist with the treatment of skin diseases.Objective: This work focused on increased naphthoquinone production in I. basamina root cultures using methionine feeding.Materials and methods: I. balsamina root cultures were maintained in liquid Gamborg’s B5 medium supplemented with 0.1 mg/L α-naphthalene acetic acid, 0.1 mg/L kinetin, 1.0 mg/L 6-benzyladenine and 20 g/L sucrose. The effect of methionine concentration (50, 100, 300, 500 and 1000 mg/L) on naphthoquinone production of I. basamina root cultures was determined. Isolation of secondary metabolites from I. balsamina root cultures was also carried out.Results and discussion: Feeding of 300 mg/L methionine to the root cultures at the beginning of the growth cycle increased the production of 3,3′-methylelnebislawsone almost two-fold (0.63 mg/g dry weight, compared to the control group 0.32 mg/g dry weight). Optimization of the feeding conditions showed that adding 500 mg/L methionine to a 21-day old root cultures increased production of lawsone methyl ether and 3,3′-methylenebislawsone up to 2.6- and 3.1-fold higher, respectively, compared to the controls. In addition, various pharmacologically interesting secondary metabolites were isolated from I. balsamina root cultures, such as a flavonoid, luteolin, a naphthoquinone, 2,3-dihydroxy-1,4-naphthoquinone, and a triterpenoid, echinocystic acid. This is the first report of the occurrence of these compounds in this plant.

Isolation of secondary metabolites from a marine derived Streptomyces sp. 9CM17 and investigation of their antimicrobial activities

Isolation of secondary metabolites from a marine derived Streptomyces sp. 9CM17 and investigation of their antimicrobial activities

When the nine-membered enediynes play hide and seek

The lack of stability of the 9-membered enediynes not associated with an apoprotein may explain the low number of isolated natural compounds containing this core. To overcome such a problem, particular attention should be paid during the process of extraction and isolation of secondary metabolites, especially from microorganisms such as actinomycetes in order to identify the non-cycloaromatized derivatives.

English

Tridax procumbens is a common weed plant belonging to the family Asteraceae . The leaf juice has antiseptic, insecticidal and parasiticidal properties. The present work is based on developing a protocol for the callus induction in Tridax procumbens from various explants like leaf, internodes and shoot apical buds. The sterilized explants were inoculated in MS media containing various combination of auxins such as 2, 4, dichlorophenoxy acetic acid (2, 4-D) and naphthalene acetic acid (NAA) and cytokinins such as kinetin and 6 benzyl amino purine (BAP). Leaf and apical bud explants showed early and profuse callus induction whereas internodal explants showed comparatively delayed but profuse callus induction. Leaf and apical bud explants showed maximum response in terms of callus by using MS media with the combination 2, 4-D 0.5mg/lit and BAP 0.5mg/lit which was followed by 2, 4-D 0.5mg/lit and KIN 0.5mg/lit, 2, 4 -D 0.5mg/lit and BAP+KIN 0.5 mg/lit, NAA 0.5mg/lit and BAP 0.5mg/lit, NAA 2mg/lit and BAP 0.5mg/lit respectively. Whereas internodal explants showed maximum callus induction by using a hormonal concentration of 2mg/lit 2,4, D and 0.5mg/lit BAP. In vitro generated callus can be used as a source for the isolation of secondary metabolites from Tridax plant.

Polar and antioxidant fraction of Plumbago europaea L., a spontaneous plant of Sardinia

Secondary metabolite isolation and analysis of Plumbago europaea L. (Plumbaginaceae) is the aim of this work, which is included in a project of our groups on spontaneous and endemic Sardinian species. Plumbago europaea is the only specie of the genera that grows wild in Italy. Metabolites from leaves and roots of this specie were compared. Moreover, at present, literature on the phytochemical characterisation of P. europaea is poor. Plumbagin was isolated in both of the samples analysed, confirming its nature in the Plumbaginaceae family as chemotaxonomic marker of the Plumbaginae tribe. In this work, hydroplumbagin-4-O-glucoside and myricetin-3-O-rhamnoside are isolated for the first time in the genus.

Rapid method for extracting the antibiotic mutacin 1140 from complex fermentation medium yeast extract

The peptide antibiotic mutacin 1140 belongs to the group of antibiotics called lantibiotics. They are ribosomally synthesized and undergo extensive enzymatic modifications before being excreted into the culture medium. By using reverse-phase-high-performance liquid chromatography (RP-HPLC) and a semiquantitative bacteriocin bioassay to track mutacin 1140, an efficient ammonium sulfate (AS) precipitation method has been developed for removing mutacin 1140 from a complex medium containing 5% yeast extract. This method minimizes the amounts of fermentation by-products and media components that make downstream purification processes more difficult and economically infeasible. The method may be adaptable for the initial purification step of other lantibiotics. A threefold decrease in the precipitation of the medium components found in yeast extract, at pH 8.0 vs. pH 2.0, may have broad utility for the isolation of secondary metabolites produced in this complex medium. The average yield of mutacin 1140 from the fermentation medium was determined as 66%.

Isolation of secondary metabolites from Hortia oreadica (Rutaceae) leaves through high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system (hexane–ethanol–acetonitrile–water 10:8:1:1, v/v) was applied to examine the leaves of Hortia oreadica, which afforded the known limonoid guyanin ( 1), the alkaloids rutaecarpin ( 2) and dictamnine ( 6), the dihydrocinnamic acid derivatives methyl 5,7-dimethoxy-2,2-dimethyl-2 H-1-benzopyran-6-propanoate ( 3), 5,8-dimethoxy-2,2-dimethyl-2 H-1-benzopyran-6-propanoic acid ( 4), together with the new E-3,4-dimethoxy-α(3-hydroxy-4-carbomethoxyphenyl)cinnamic acid ( 5). The recovery of compounds 1–6 was determined by comparison with LC-atmospheric pressure chemical ionization MS/MS data: 66.2%, 93.1%, 102.5%, 101.2%, 99.0% and 84.9%, respectively. Compound 3 showed IC 50 of 23.6 μM against Plasmodium falciparum and 15.6 μM against Trypanosoma brucei rhodesienses and was not toxic to KB cells (IC 50 > 100 μM).

Nocardamin Production by Streptomyces avermitilis

Desferrioxamine B (desferal®) is the only therapeutic agent for chronic iron overload and acute iron intoxication. Therefore, new drug candidates are required for more effective treatments. Recent genome analyses of microorganisms have enabled us to identify cryptic gene clusters of secondary metabolism. In a genomic analysis of Streptomyces avermitilis, we found the putative biosynthetic gene cluster for nocardamin and desferrioxamine derivatives, the production of which by S. avermitilis had been unknown. To determine the synthesis of nocardamin and discover new derivatives in S. avermitilis, we performed a comprehensive analysis and isolation of secondary metabolites of S. avermitilis. We obtained nocardamin and its related compounds, including a derivative that initially was isolated as a microbial product. The biosynthetic pathway of nocardamin and the substrate specificity of its biosynthesis enzymes were proposed based on the structures of the isolated nocardamin derivatives. The production of nocardamin by S. avermitilis was completely suppressed by the addition of more than 5 μM ferric ions. An iron-dependent regulatory protein (IdeR)-binding motif was located upstream of the sidABCD operon, using the profile hidden Markov model method. To determine the regulation of nocardamin synthesis by IdeR in S. avermitilis, we constructed ideR deletion mutants. The production of nocardamin by the deletion mutants was not suppressed by ferric ions (up to 100 μM).

Chemical investigation of Finlaysonia obovata : part I – a rare triterpene acid showing antibacterial activity against fish pathogens

The antibacterial screening of extracts of the leaves of Finlaysonia obovata with hexane, chloroform and alcohol was carried out against fresh water fish pathogenic bacteria viz., Micrococcus Sp. (multidrug resistant strain), Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio alginolyticus, Staphylococcus aureus, Escherichia Coli, Edwardsiella tarda by disc-assay method. The hexane and chloroform extracts were found active against four and five pathogens, respectively. The highly active chloroform extract was taken up for fractionations, further screening and isolation of secondary metabolites by chromatographic techniques. The triterpene acid Urs-3β-hydroxy-12-en-27-oic acid (3), which is very rarely found from plant sources is isolated first time from this plant along with known compounds; Lupeol acetate (1) and β-Sitosterol (2). This article presents for the first time all the spectral data for Urs-3β-hydroxy-12-en-27-oic acid (3), which showed moderate activity against four pathogens. This is the first report of antibacterial activity of a triterpene against fish pathogens.

The isolation of secondary metabolites and in vitro potent anti-cancer activity of clerodermic acid from Enicosanthum membranifolium

Four compounds were isolated from Enicosanthum membranifolium. The structures of the compounds were confirmed by spectroscopic data. Their structures were determined as N- trans-feruloyltyramine, R-(−)-mellein, clerodermic acid, and salicifoline chloride as a quaternary alkaloid compound. The structures of R-(−)-mellein and salicifoline chloride were confirmed by using X-ray diffraction. Clerodermic acid was shown to induce potent apoptosis against human leukemia HL60 cells.

A Comparison of Cell Wall Disruption Techniques for the Isolation of Intracellular Metabolites from Pleurotus and Lepista sp.

Different techniques were compared for their effectiveness in the disruption of the rigid cell walls of Basidiomycetes. Grinding under liquid nitrogen, stirred glass bead milling and enzymatic cell lysis were applied to the mycelia of Pleurotus sapidus and Lepista irina grown submerged. Each of the disruption procedures was evaluated by testing the quantity and quality of released intracellular metabolites: DNA, RNA, enzymes, and secondary metabolites. The most suitable method for nucleic acid isolation was grinding under liquid nitrogen, while bead mill homogenization was the superior technique for isolation of active enzymes. A new effective method is proposed for isolation of secondary metabolites with the aid of bead milling of fungal mycelia.

Isolation of secondary metabolites from roots ofPlumbago auriculata Lam. by countercurrent chromatography

Centrifugal liquid-liquid partition chromatography presents significant advantages for the separation and purification of plant metabolites owing to the short operational time of the process and the elimination of possible irreversible adsorption of compounds. The crude chloroform extract from roots of Plumbago auriculata was analysed by countercurrent chromatography using hexane:ethyl acetate:methanol:water (40:10:10:2, v/v) as solvent system. The isolation of the naphthoquinones plumbagin and epi-isoshinanolone, the steroids sitosterol and 3-O-glucosylsitosterol, plumbagic and palmitic acids was easily achieved. Naphthoquinones are typical components of Plumbago species and they show interesting biological activities.

Isolation of secondary metabolites of Aspergillus ochraceus by HPLC

A method is described for the isolation and purification of ochratoxin A, ochratoxin B, ochratoxin ß mellein, 4-hydroxymellein and penicillic acid produced byAspergillus ochraceus in a synthetic liquid medium. Ochratoxin α, which was not found in the culture medium, was obtained by acid hydrolysis of ochratoxin A. A high pressure liquid Chromatograph equipped with Lichrosorb 100 and Lichrosorb RP-18 columns and UV and/or Refractive Index detectors was used.