Chemistry and bioactivity of endemic plant genusschumacheria and vincristine and vinblastine from an endophytic fungus of catharanthus roseus
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Date
2015
Authors
Bandara, C. J.
Journal Title
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Publisher
University of Peradeniya
Abstract
The first part of the thesis describes the chemistry and the bioactivit y of three species of the endemic genus Schumacheria namely, S. castaneifolia, S. angustifolia and S. alnifolia.
Hexane, dichloromethane and methanol extracts of the plant parts of Schumacheria were subjected to, antioxidant assay using DPPH (1,l-diphenyl-2-picrylhydra zine) stable radical, cytotoxic assay using brine shrimps (Artemia salina) and antimicrobial assays against Staphylococcus aureus (NCTC 8532), Escherichia coli (NCTC 10418) and Aspergillus niger. The extracts of S. castaneifolia flowers exhibited high antibacterial, antioxidant and cytotoxic activities. The dichloromethane and methanol extracts of S. alnifolia exhibited anti fungal susceptibility against A. niger. The methanol extracts of S. alnifolia stem-bark and leaves showed antioxidant activity higher than that of a. tocopherol. The total polyphenol content, expressed as the gallic acid equivalent, was determined using Folin-Ciocaltue method. The methanol extracts of S. alnifolia stem -bark and leaf showed the highest polyphenol content closely followed by the methanol extracts of S. castaneifolia flowers.
The extracts of different plant parts of Schumacheria were subjected to several chromatographic fractionations and fifteen compounds were isolated. The structure elucidation and the bioactivity determinations were carried out and found; taraxerol, betulinaldehyde, betulinic acid, β-sitosterol , 3-0 -α -L-arabinosyloleanolic acid and β sitosterol-3-0-β-D-glucopyranoside were present in all three species. The extracts of S. angustifolia and S. alnifolia gave betulin. Betulonic acid, (6β)-Hydroxy-3-oxolup-20(29)- en-28-oic acid, sorbifolin and epicatechin were only found in the extracts of S. castaneifolia and kaempferol , 7-0-methylkaempferol , catechin and gallocatechin were isolated from the extracts of S. angustifolia.
(6β)-Hydroxy-3-oxolup-20(29)-en-28-oic acid exhibited antibacterial activity with minimum inhibitory concentration (MIC) value at 50 ppm against both S. aureus and E. coli . 3 -0-α -L-Arabinosyloleanolic acid showed antibacterial activity (MIC, 75 ppm) and toxicity to brine shrimps (LCso, 7.6 ± 0.6 ppm) .
The HPLC quantification of catechin and epicatechin of the methanol extract of flowers of S. castaneifolia gave 2.3 ± 0.0 and 9.2 ± 0.1 mg , respectively, in 1.0 g of dry flowers. The genus Dillenia was found to be closely related to the genus Schumacheria from the chemotaxonomy data based on the presence of oleanene-type triterpenoids.
The second part of the thesis describes the isolation of vinca alkaloids producing endophytic fun gus, Botryospha eria laricina (CRS1) from Catharanthus roseus and evaluation of factors that affect the production of vinca alkaloids in B. laricina (CRS1). Eight endophytic fungi from the fresh aerial part s of C. roseus were isolated and one of them , CRS 1, produced vinca alka loids. DNA sequencing of CRS1 gave a 100 % match with the GenBank accession number, KC509580.1 , which is related to the Botryosphaeria laricina strain JAS6 .
Growth of the fungus by fermentation using the CZ medium in the presence of the fresh plant extract of C. roseus (7.5 ml) in the dark for 20 days gave catharanthine (3.2 mg), catharanthinic acid (0.1 mg), N-demethylvinblastine (0.4 mg), vinblastine (2.8 mg) and vincristine (2.4 mg ). Growth in the absence of the fresh plant extract of C. roseus (7.5 ml) or with the preheated (80 °C, for 15 min) plant extract (7.5 m l) did not produce vinca alkaloids.
The plant extract was fractionated by dialysis membranes through various molecular weight (MW) cutoff ranges. Vinca alkaloid production was observed with fractions above 20 kDa MW if dialyzed in buffer at 4 "C. The same fraction dialyzed without buffer at room temperature lacked production of vincristine indicating the sensitivity of enzymes in the biosynthetic path way. Growth of B. laricina (CRS1) in the presence of light:dark (12:12 h), fructose (30.0 g 1-¹ ), glucose (30.0 g 1-¹) , Cu 2+ (0. 1 mM) ions or L-tryptophan (0.1 %) and succinic acid ( 1 %), did not promote the production of vinca alkaloids.
In conclusion , the endophytic fungus B. laricina (CRS1) which has the ability to produce catharanthine was found to require some enzymes larger than 20 kDa MW present in the plant extract to enable the production of two vinca alkaloids vinblastine and vincristine.
Description
Keywords
Bioactivity , Chemistry , Schumacheria , Fungus , Catharanthus roseus