Saifullah Khan

Metabolic classification of South American Ilex species by NMR-based metabolomics.

The genus Ilex to which mate (Ilex paraguariensis) belongs, consists of more than 500 species. A wide range of metabolites including saponins and phenylpropanoids has been reported from Ilex species. However, despite the previous works on the Ilex metabolites, the metabolic similarities between species which can be used for chemotaxonomy of the species are not clear yet. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics was applied to the classification of 11 South American Ilex species, namely, Ilex argentina, Ilex brasiliensis, Ilex brevicuspis, Ilex dumosa var. dumosa, I. dumosa var. guaranina, Ilex integerrima, Ilex microdonta, I. paraguariensis var. paraguariensis, Ilex pseudobuxus, Ilex taubertiana, and Ilex theezans. (1)H NMR combined with principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) showed a clear separation between species and resulted in four groups based on metabolomic similarities. The signal congestion of (1)H NMR spectra was overcome by the implementation of two-dimensional (2D)-J-resolved and heteronuclear single quantum coherence (HSQC). From the results obtained by 1D- and 2D-NMR-based metabolomics it was concluded that species included in group A (I. paraguariensis) were metabolically characterized by a higher amount of xanthines, and phenolics including phenylpropanoids and flavonoids; group B (I. dumosa var. dumosa and I. dumosa var. guaranina) with oleanane type saponins; group C (I. brasiliensis, I. integerrima, I. pseudobuxus and I. theezans) with arbutin and dicaffeoylquinic acids, and group D (I. argentina, I. brevicuspis, I. microdonta and I. taubertiana) with the highest level of ursane-type saponins. Clear metabolomic discrimination of Ilex species and varieties in this study makes the chemotaxonomic classification of Ilex species possible.

Biotransformation of oral contraceptive ethynodiol diacetate with microbial and plant cell cultures

BackgroundBiotransformation by using microbial and plant cell cultures has been applied effectively for the production of fine chemicals on large scale. Inspired by the wealth of literature available on the biotransformation of steroids, we decided to investigate the biotransformation of ethynodiol diacetate (1) by using plant and microbial cultures.ResultsThe biotransformation of ethynodiol diacetate (1) with Cunninghamella elegans and plant cell suspension cultures of Ocimum basilicum and Azadirachta indica is being reported here for the first time. Biotransformation of 1 with Cunninghamella elegans yielded three new hydroxylated compounds, characterized as 17α-ethynylestr-4-en-3β,17β-diacetoxy-6α-ol (2), 17α-ethynylestr-4-en-3β,17β-diacetoxy-6β-ol (3), and 17α-ethynylestr-4-en-3β,17β-diacetoxy-10β-ol (4) and a known metabolite, 17α-ethynyl-17β-acetoxyestr-4-en-3-one (5). The biotransformation of 1 with Ocimum basilicum included hydrolysis of the ester group, oxidation of alcohol into ketone, and rearrangement of the hydroxyl group. Thus four major known metabolites were characterized as 17α-ethynyl-17β-acetoxyestr-4-en-3-one (5), 17α-ethynyl-17β-hydroxyestr-4-en-3-one (6), 17α-ethynyl-3 β-hydroxy-17β-acetoxyestr-4-ene (7) and 17α-ethynyl-5α,17β-dihydroxyestr-3-ene (8). Biotransformation of 1 with Azadirachta indica culture yielded compounds 5 and 6. Spectroscopic data of compound 8 is being reported for the first time. Structure of compound 6 was unambiguously deduced through single-crystal x-ray diffraction studies.ConclusionBiotransformation of an oral contraceptive, ethynodiol diacetate (1), by using microbial and plant cell cultures provides an efficient route to the synthesis of a library of new steroids with potential contraceptive properties. These methods can be employed in the production of such compounds with high stereoselectivity.

Biotransformation of (–)-Caryophyllene Oxide by Cell Suspension Culture of Catharanthus roseus

Abstract Catharanthus roseus cell suspension cultures were employed for the biotransformation of (-)-caryophyllene oxide (1), and four metabolites, 15-hydroxycaryophyllene oxide (2), 4β ,5α- dihydroxycaryophyll-8(13)-ene (3), 2β -hydroxycaryophyllene oxide (4), and 2-hydroxy-4,5- epoxycaryophyllan-13-ol (5) were obtained. Metabolites 4 and 5 were found to be new compounds, and their structures were deduced by different spectroscopic techniques.

Biotransformation of perfumery terpenoids, (−)-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

BackgroundBiotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture.ResultsBiotransformation of (−)-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2), 6β-hydroxyambrox (3), 1α-hydroxy-3oxoambrox (4), 1α,3β-dihydroxyambrox (5), 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6), 3-oxoambrox (7), 2α-hydroxyambrox (8), 3β-hydroxysclareolide (9), and 2α,3β-dihydroxyambrox (10). Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies.ConclusionNine oxygenated metabolites of (−)-ambrox (1) were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner.

Efficient Plant Genomic and Viral DNA Isolation from Mature Leaf Midrib of Banana (Musa spp)

The genomic DNA isolation from mature leaf midrib is a tough job, because of the abundance of polysaccharides and secondary metabolites, which interferes with DNA isolation as well as polymerase chain reaction (PCR) studies. The leaf midrib of 3rd leaf from 3-moths old, ex-vitro developing banana [AAA, Dwarf Cavendish-Basrai (Sindhri banana)] plants (healthy and BBTV infected) was grinded in liquid N2. Exact 0.3 g of leaf midrib powder was washed with washing buffer (100 mM Tris-Cl, 5 mM EDTA, 0.35 M sorbitol, 1% 2-mercaptoethanol) then homogenized in 0.8 ml of three different pre-heated (60°C) DNA isolation buffers. Supernatant was extracted through phenol: chloroform:isoamyl alcohol (25:24, v/v), chloroform: isoamyl alcohol (24:1, v/v) and finally with chloroform (100%) one by one. Maximum yields were ranged from 49.33 and 27.73 μg mg −1 DNA with impurities 5.67 and 5.87 μg mg−1 through buffer I, while 45.77 and 25.53 μg mg−1 DNA with 6.13 and 6.16 μg mg−1 impurities through buffer III from healthy and infected plants respectively. Best one RAPD was observed in all the DNA samples isolated with different buffers, while viral amplification was good in DNA isolated with buffer I and II, when 10 (RAPD) and 25 ng DNA (C1 gene) was used as a template in a reaction of 25 μl. Meanwhile, buffer II is limited for viral DNA isolation while buffer I (1M Tris-Cl, 5M NaCl, 2 % cTAB, 50mM EDTA, 1 % PVP, 0.2 % 2-mercaptoethanol) has dual capacity for plant and virus DNA isolation. This described protocol is economic in terms of times, labor and cost.

Chenisterol, a New Antimicrobial Steroid from Chenopodium badachschanicum

Chenisterol (1), a new steroid, has been isolated from the EtOAc soluble subfraction of Chenopodium badachschanicum Tzvelev, along with ergosterol (2), stigmasterol (3), fucosterol (4), morin (5), sinapic acid (6), and thymol (7), reported for the first time from this species. Their structures were elucidated with the help of spectroscopic techniques including 2D NMR. Compound 1 showed significant antimicrobial activity against three Gram-positive and one Gram-negative bacterial strains.

Biotransformation of Dehydroepiandrosterone by Cell Suspension Culture of Codiaeum variegatum

Dehydroepiandrosterone (1) was incubated with a cell suspension culture of Codiaeum variegatum for biotransformational studies. After 10 days of incubation, four metabolites, 5α-androstane-3,17-dione (2), 5β-androstane-3,17-dione (3), androst-4-ene-3,17-dione (4), and 17β-hydroxyandrost-4-en-3-one (5), were obtained. The structures of these biotransformed products were elucidated by spectroscopic methods. Biotransformation using a cell suspension culture of Codiaeum variegatum provides an effective and convenient method to oxidize the 3β-hydroxy group in 1.

Potential of Azadirachta indica cell suspension culture to produce biologically active metabolites of dehydroepiandrosterone

Dehydroepiandrosterone (1) was investigated for biotransformation studies using the plant cell suspension culture of Azadirachta indica A. Juss. for the first time, yielding metabolites 2–6: 5α,3,17-androstanedione (2), 5-androstene-3β,17β-diol (3), 3β-hydroxyandrostan-17-one (4), 3β,11α-dihydroxy-5-androsten-17-one (5), and 3β,7α-dihydroxy-5-androsten-17-one (6), whose structures were solved through modern spectroscopic methods. All five compounds 2–6 have not been reported obtained by this way before. This is a new method to biosynthesize compounds 2–6 employing cultured cells of A. indica. Metabolites 2, 3, and 6 are important biologically active compounds, whereas 4 is a precursor for the production of the 7-hydroxylated compound having antiglucocorticoid and neuroprotective effects.