Unexpected applications of secondary metabolites

Secondary metabolites have been found to have interesting applications over and above their well-known medical uses, e.g., as antimicrobials, etc. These alternative applications include antitumor, cholesterol-lowering, immunosuppressant, antiprotozoal, antihelminth, antiviral and anti-ageing activities. Polyene antibiotics, such as amphotericin B, are of use as antiprion agents, antitumor drugs and against leishmaniasis. Other microbial natural products that show antibiotic activity are used against cancer e.g., doxorubicin, neomycin, β-lactams, bleomycin and rapamycin. Macrolide antibiotics, such as erythromycin, clarithromycin and azithromycin, improve pulmonary function in patients suffering from panbion cholitis. Pigments like prodigiosin and shikonin have antitumor activity, while violacein has anti-ulcer and antitumor activity and also acts as an antiprotozoal agent. Statins, in addition to lowering cholesterol and LDL levels, also decrease elevated C-reactive protein (CRP) levels independent of their cholesterol effects. Immunosppressants have many alternative effects: (i) Cyclosporin is proving useful in treatment of inflammatory disease such as asthma and muscular dystrophy. (ii) Rapamycin is extremely useful in preventing restenosis of stents grafted in balloon angioplasty. (iii) Tacrolimus and ascomycin help in treating inflammatory skin disease such as allergic contact dermatitis and psoriasis. Artemisinin, an antimalarial agent, is also showing antitumor activity. Other natural products, including those from plants (betulinic acid and shikonin), animals (bryostatins) and microbes (squalestatin and sophorolipids) have a multiplicity of potentially useful actions. Unexpected functions of known secondary metabolites are continuously being unraveled, and are fulfilling some of the needs of present day medicine and show great promise for the future.     

Aerobic biodegradation of 2,2'-dithiodibenzoic acid produced from dibenzothiophene metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2'-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2'-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2'-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B12, and it degraded 2,2'-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2'-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2'-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2'-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

Analysis of steroid metabolites produced by theca cells from the adult domestic hen

In a previous study on steroid metabolism by hen ovarian cells we reported on the production of 17-hydroxyprogesterone (17OH), androstenedione (A), testosterone (T), and oestrogens from [3H]progesterone (P) by theca cells. The present study examines further the metabolism of P by theca cells from the preovulatory follicles of the hen. The results show that the major metabolite of P is 20 beta-hydroxy-4-pregnen-3-one (20 beta-DHP), representing up to 40% of the recovered radioactivity. In addition, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-DHP) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta) were identified as metabolites of P, comprising 1 and 3% of the recovered radioactivity, respectively. This is the first evidence that the allylic steroid, 3 alpha-DHP, can be produced by avian ovaries.     

Antimicrobial metabolites produced by an intertidal Acremonium furcatum

In a screening for antimicrobial metabolites, amides of D-allo- and L-isoleucine derivatives were isolated from the culture of a marine strain of Acremonium furcatum. Structural elucidation of these compounds was performed by analysis of spectroscopic data and confirmed by synthesis. All of the compounds, natural and synthetic intermediates, were bioassayed against bacteria and phytopathogenic fungi, with many showing remarkable antifungal activities.     

Potential metabolites of dimethoate produced by bacterial degradation

Microbial degradation of pesticide has been recognized as a potential solution for the disposal of pesticide. Two bacterial strains namely Bacillus licheniformis and Pseudomonas aeruginosa, which were isolated from water and fish intestine, respectively, were allowed to grow in mineral salt solution. The acetonitrile extracts of the bacterial cultures were subjected to thin layer chromatography using two different solvent systems: hexane–chloroform–methanol and cyclohexane–acetone–chloroform. The chromatogram revealed the presence of four metabolites of dimethoate with different R_f values, in the original P. aeruginosa strain using both hexane–chloroform–methanol and cyclohexane–acetone–chloroform solvent systems. Total disappearance of dimethoate spot occurred in the culture of B. licheniformis strain at day 3. Thus, the present study establishes the bacterial degradation of dimethoate and also suggests the role of bacteria in the bioremediation of pesticides.     

Two new alkaloid metabolites produced by endophytic fungus Stagonosporopsis oculihominis isolated from Dendrobium huoshanense

Two new alkaloids (1–2), together with six known compounds (3–8), were isolated from cultures of the endophytic fungus Stagonosporopsis oculihominis. Their structures were elucidated through extensive spectroscopic methods including 2D NMR and HRMS analyses. The absolute configurations of 1 was determined by the comparison of experimental and theoretical electronic circular dichroism spectra. The new compounds were investigated for their cytotoxicity against five human cancer cell lines.     

Patterns of metabolites produced from the fluoroquinolone enrofloxacin by basidiomycetes indigenous to agricultural sites

Supernatants of mycelial cultures of seven basidiomycetous fungi indigenous to agricultural sites were evaluated for metabolites generated from the veterinary fluoroquinolone enrofloxacin (EFL) by employing high–performance liquid chromatography/high–resolution electrospray ionization mass spectrometry. From exact masses, molecular formulae were derived, and the most probable chemical structures were deduced. Patterns of major metabolites were surprisingly similar but differed greatly from that provided by Gloeophyllum striatum due to the absence of monohydroxylated EFL congeners and a greater variety of metabolites with a modified piperazine moiety. The structures of three metabolites were elucidated by ¹H–nuclear magnetic resonance spectroscopy. Of 61 compounds detected, 48 were new, while 13 were known from a pattern of 87 EFL metabolites identified for G. striatum. Ethylpiperazine moieties carrying oxido, hydroxy, oxo, and acetoxy groups, or showing partial degradation, were linked to the unmodified, oxidatively decarboxylated, or multiply hydroxylated core of EFL and to isatin– and anthranilic acid–type EFL congeners. Cleavage of the fluoro–aromatic bond was observed for two, ¹⁴CO₂ formation for six species. Metabolites with a hydroxylated aromatic part implied subsequent ring cleavage to be brought about by the formation of potentially four oxidizable ortho–aminophenol– and one catechol–type intermediates. EFL degradation appears to be a common activity among basidiomycetes.     

Bioactive metabolites produced by Chaetomium globosum,an endophytic fungus isolated from Ginkgo biloba

A novel cytotoxic chlorinated azaphilone derivative named chaetomugilin D (1), together with three known metabolites, chaetomugilin A (2), chaetoglobosins A (3) and C (4), has been isolated by a bioassay-guided fractionation from the EtOAc extract of the cultures of Chaetomium globosum, an endophytic fungus found in the leaves of Ginkgo biloba. Structure of 1 was established by analyses of spectroscopic methods, including 2D-NMR experiments (COSY, NOESY, HMQC, and HMBC). Compounds 1–4 displayed significant growth inhibitory activity against the brine shrimp (Artemia salina) and Mucor miehei.     

Bioactive secondary metabolites produced by microorganisms associated with plants

In the past few decades groups of scientists have focused their study on relatively new microorganisms called endophytes. By definition these microorganisms, mostly fungi and bacteria, colonise the intercellular spaces of the plant tissues. The mutual relationship between endophytic microorganisms and their host plants, taxanomy and ecology of endophytes are being studied. Some of these microorganisms produce bioactive secondary metabolites that may be involved in a host-endophyte relationship. Recently, many endophytic bioactive metabolites, known as well as new substances, possesing a wide variety of biological activities as antibiotic, antitumor, antiinflammatory, antioxidant, etc. have been identified. The microorganisms such as endophytes may be very interesting for biotechnological production of bioactive substances as medicinally important agents. Therefore the aim of this review is to briefly characterize endophytes and summarize the structuraly different bioactive secondary metabolites produced by endophytic microorganisms as well as microbial sources of these metabolites and their host plants.     

Antifungal activity of extracellular metabolites produced by Sporothrix flocculosa

The antifungal properties of extracellular compounds produced by the epiphytic fungus Sporothrix flocculosa were bioassayed against phytopathogenie fungi on the basis of inhibition of spore germination, and mycelial growth and induction of cellular leakage. Following incubation in stationary culture, S. flocculosa released antifungal metabolites into the culture medium which were extractable with méthylene chloride. When separated by thin layer chromatography, extracted metabolites yielded a compound(s) at R_f0.65 which inhibited development of Cladosporium cucumerinum and several other phytopathogenic fungi. Treatment of Botrytis cinerea and Fusarium oxysporum f.sp. radicis‐lycopersici (FORL) with the same compound(s) greatly reduced spore germination and biomass growth of both fungi. Additionally, both B. cinerea and FORL leaked electrolytes and proteins when grown in presence of the metabolites. Observations under electron microscopy revealed that FORL reacted to the presence of S. flocculosa metabolites by retraction of the plasmalemma and rapid disintegration of the cytoplasm. These reactions were similar to the ones induced by conidia of S. flocculosa when applied on powdery mildew fungi. These results provide strong evidence of the production of antifungal compounds in vivo and of their role in the antagonistic properties of S. flocculosa.     

Low molecular weight metabolites produced by various Penicillium species

Low-molecular weight volatile metabolites produced by Penicillium farinosum, P. citrinum, P. camemberti and P. chrysogenum were investigated. During first 40 days of cultivation the fungi produced mainly C-8 compounds, and later mainly 2-hexenal was synthesized. Addition of 0.1% linoleic acid significantly stimulated the secretion of volatile metabolites. P. citrinum and P. farenosum produced large quantities of geosmin.     

Secondary metabolites produced by an endophytic fungus Cordyceps ninchukispora from the seeds of Beilschmiedia erythrophloia Hayata

Four new amide derivatives, designated as cordycepiamides A–D (1–4), together with 14 known compounds (5–18), were isolated from the EtOAc-soluble fraction of the 95% EtOH extract of long-grain rice fermented with the endophytic fungus C. ninchukispora BCRC 31900, derived from the seeds of medicinal plant Beilschmiedia erythrophloia Hayata. Their structures were elucidated by means of spectroscopic and mass-spectrometric analyses, particularly 1D and 2D NMR spectroscopy as well as HRESIMS. All known isolates except 11, were isolated for the first time from this species. The antiinflammatory activities of selected isolated 10 compounds (1, 2, 4–6, 9–12, and 14) were evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. Compound 3→4 was shown to have modest anti-inflammatory effects through inhibition of NO production in LPS-stimulated RAW264.7 cells.     

Characterization of antifungal metabolites produced by Lactobacillus plantarum and Lactobacillus coryniformis isolated from rice rinsed water

Molecular Biology Reports - A recent spike in demand for chemical preservative free food has derived the scientific community to develop natural ways of food preservation. Therefore,...     

Further secondary metabolites produced by the fungus Pyricularia grisea isolated from buffelgrass (Cenchrus ciliaris)

The fungal pathogen Pyricularia grisea has been studied to evaluate its production of phytotoxins for the biocontrol of the buffelgrass (Cenchrus ciliaris L.) weed. A first investigation allowed to isolate several new and known phytotoxic metabolites. However, the further investigation on the organic extract obtained from the fungus liquid culture showed the presence of other metabolites possibly contributing to its phytotoxicity. Thus, four known metabolites were isolated and identified by spectroscopic (nuclear magnetic resonance [NMR] and high-resolution electrospray ionization mass spectrometry [HRESIMS]) methods as dihydropyriculol ( 1 ), epi-dihydropyriculol ( 2 ), 3-methoxy-6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin ( 3 ), and (R)-mevalonolactone ( 4 ). The absolute configuration of 1 – 3 was determined for the first time by a computational analysis of their electronic circular dichroism (ECD) spectra. When the isolated compounds were bioassayed at a concentration of 5 × 10^–3 M in a buffelgrass coleoptile and radicle elongation test no toxicity was detected. On the contrary, compounds 1 and 3 showed a significant stimulating effect of radical elongation. Furthermore, the difference in growth stimulation between 1 and its epimer 2 highlights the tight relationship between absolute configuration and biological activity of these fungal metabolites.     

Secondary metabolites produced by callus cultures of various Ruta species

Callus cultures were established from hypocotyl explants of R. bracteosa, R. chalepensis and R. macrophylla. Calli were maintained for more than three years on MS-medium supplemented with 1 mg l^-1 of each 2,4-D and kinetin. Acridone and furoquinoline alkaloids and coumarins have been isolated from four week old calli grown on a hormone containing and hormone-free medium. A new chlorinated acridone alkaloid has been detected.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog [6]     

Non-estrogenic metabolites of diethylstilbestrol produced by prostaglandin synthase mediated metabolism

Incubation of trans-diethylstilbestrol (E-DES) with prostaglandin synthase (PGS) in vitro leads to the formation of the metabolites cis, cis-dienestrol (Z,Z-DIES) and cis-diethylstilbestrol (Z-DES) which have considerably decreased estrogenic activity compared to their parent compound. Incubations of (14C)-E-DES with PGS in the presence of arachidonic acid (AA) predominantly catalyze formation of the oxidative metabolite Z,Z-DIES, accompanied by the formation of protein bound radioactivity. Inhibition of peroxidative metabolism through addition of indomethacin or absence of AA favors isomerization of E-DES to Z-DES without concomitant formation of protein bound radioactivity. Isomerization is inhibited by phenidone (1-phenyl-3-pyrazolidone). Since PGS activity is present in uterine tissue, these pathways may play a role in the metabolism of DES in its target tissue.     

毛筒壳科真菌次级代谢产物生物活性的评价

毛筒壳科Tubeufiaceae真菌具有产新结构、新活性次级代谢产物的潜力,目前对该科真菌次级代谢产物的研究较少。为了寻找具有生物活性的新化合物,有必要对毛筒壳科真菌次级代谢产物及其活性进行系统深入的研究。本文采用平板对峙法、生长速率法和MTT法,分别测定已分离得到的19株该科真菌活体菌株抑菌活性、发酵物抑菌活性以及发酵物粗提物对不同人体肿瘤细胞株增殖的抑制作用。通过平板对峙法,试验共筛选获得13株活性菌株,其中,红棕毛筒腔菌菌株Tubeufia rubra PF02-2对7种植物病原真菌有明显的抑菌效果,抑制率均高于60%且抑菌谱广。采用生长速率法,发现红棕毛筒腔菌菌株PF02-2经液体发酵后,发酵液对其中4种植物病原真菌仍有一定的抑制作用,且菌丝体部分的乙酸乙酯提取物对马铃薯早疫病病菌Alternaria solani（ZYB）的抑制效果最好。通过MTT法,发现发酵物粗提物对3种肿瘤细胞均具有不同程度的细胞毒活性,其中在300μg/mL时,剑叶莎毛筒腔菌菌株Tubeufia machaerinae ML03-2发酵液部分的乙酸乙酯提取物对人宫颈癌细胞株HeLa和人前列腺癌细胞株PC-3的抑制率（%）分别达到了98.92±0.15和97.86±0.18,在400μg/mL时,对人肝癌细胞株HEPG2的抑制率（%）达到了98.88±0.04;在500μg/mL时,明孢新旋卷孢菌菌株Neohelicosporium hyalosporum ML05-1菌丝体部分的乙酸乙酯提取物对人宫颈癌细胞株HeLa细胞的抑制率（%）为98.32±0.02,在600μg/mL时,对人肝癌细胞株HEPG2的抑制率（%）达到了97.62±0.20,在300μg/mL时,对人前列腺癌细胞株PC-3的抑制率（%）达到了98.91±0.02。该研究结果为开发利用毛筒壳科真菌提供了科学依据。