Biotransformation of the Antimelanoma Agent Betulinic Acid by Bacillus megaterium ATCC 13368

Abstract

Microbial transformation of the antimelanoma agent betulinic acid was studied. The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound. Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11␣-hydroxy-lup-20(29)-en-28-oic acid, 1␤-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3␤,7␤,15␣-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses. In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).Betulinic acid (compound 1, Fig. 1), 3␤-hydroxy-lup-20(29)-en-28-oic acid, is a pentacyclic lupane type of triterpene that is widely distributed in the plant kingdom (8,19). Betulinic acid has been shown to exhibit a variety of biological activities, including inhibition of human immunodeficiency virus (HIV) replication in H9 lymphocyte cells (11), blockage of HIV type 1 entry into cells (20), and inhibition of DNA polymerase ␤ (18). Synthetic derivatives of betulinic acid have also been investigated as specific inhibitors of HIV type 1 (11-13). In addition, betulinic acid has been reported to be a melanomaspecific cytotoxic agent in both in vitro cell culture and in vivo studies (24). The antitumor activity of this compound is mediated by the induction of apoptosis, as demonstrated by a variety of cellular responses (24). Due to its high level of antitumor activity and lack of toxicity, betulinic acid is an attractive and promising new lead compound for use against human melanoma and is currently undergoing preclinical development for treatment or prevention of malignant melanoma.An essential part of the preclinical development of a drug is elucidation of its mammalian metabolism. Since there have been no reports on the mammalian metabolism of betulinic acid, a prospective approach was used to study its metabolism by utilizing microorganisms as in vitro model systems. Fungi, bacteria, and yeasts have been utilized successfully as in vitro models to mimic and predict the metabolic fate of drugs and other xenobiotics in mammalian systems (7,9,15,16,28). Studies have shown that many of the mammalian phase I and phase II biotransformation reactions, including hydroxylation, N oxidation, O and N dealkylation, dehydrogenation, and glucuronide and sulfate conjugation reactions, occur in microbial systems as well (27). The biochemical bases for this parallelism in metabolic reactions lie in the similarity between mammalian and microbial enzyme systems, such as cytochrome P450 monooxygenases and copper oxidases. These studies, which have been extensively reviewed in the literature, highlighted the potential value of microbial transformations as a powerful tool for mimicking and predicting mammalian biotransformations ...

An efficient process for the transformation of betulin to betulinic acid by a strain of Bacillus megaterium

“…For getting crystals of betulinic acid, the elute was mixed twice the volume of elute with the mixture of ethyl acetate and methanol (9:1) and is kept at 4 ± 1°C overnight. This crystallized sample was further analyzed for biophysical characterization of betulinic acid by NMR and ESI-MS (Carpenter et al 1980;Chatterjee et al 2000).…”

“…The column was packed with silica gel and saturated with ethyl acetate before loading the sample. Gradient mobile phase consisting of ethyl acetate and hexane with 7:3 ratios was used for elution of betulinic acid (Chatterjee et al 2000). The eluent was tested by HPLC at 1.0 ml/min flow rate and with 210 nm wavelength at room temperature (Liu et al 2011;Feng et al 2013).…”

“…However, there are not enough literatures available on microbial transformations of betulin. Reports suggest that betulin can be transformed 3-5 metabolites (Chatterjee et al 2000;Feng et al 2013). The bio-chemical modifications at positions C-28 of the parent structure of betulin can produce betulinic acid (Fig.…”

Microbial Biotransformation of Gentiopicroside by the Endophytic Fungus Penicillium crustosum 2T01Y01

“…The medium was sterilized at 121°C and 18 lb/in 2 for 20 min. Fermentations were carried out according to a standard two-stage protocol (23). Endophytic fungus stock inoculums were first prepared by suspending the fungus from one agar slant in 1 ml of sterile distilled water.…”

Production of a highly potent epoxide through the microbial metabolism of 3β-acetoxyurs-11-en-13β,28-olide by Aspergillus niger culture

“…A number of publications are available regarding the biotransformation of triterpenes. In one experiment, using Cunninghamella species, betulinic acid was transformed into its glycosylated product (Chatterjee et al 1999), similarly in another report, different hydroxylated products of betulinic acid were produced using Bacillus megaterium (Chatterjee et al 2000).…”