A microbial model of mammalian metabolism: biotransformation of 4,5-dimethoxyl-canthin-6-one using Cunninghamella blakesleeana CGMCC 3.970.

Abstract 1. A filamentous fungus, Cunninghamella blakesleeana CGMCC 3.970, was applied as a microbial system to mimic mammalian metabolism of 4,5-dimethoxyl-canthin-6-one (1). Compound 1 belongs to canthin-6-one type alkaloids, which is a major bioactive constituent of a traditional Chinese medicine (the stems of Picrasma quassioides). 2. After 72 h of incubation in potato dextrose broth, 1 was metabolized to seven metabolites as follows: 4-methoxyl-5-hydroxyl-canthin-6-one (M1), 4-hydroxyl-5-methoxyl-canthin-6-one (M2), canthin-6-one (M3), canthin-6-one N-oxide (M4), 10-hydroxyl-4,5-dimethoxyl-canthin-6-one (M5), 1-methoxycarbonl-β-carboline (M6), and 4-methoxyl-5-O-β-D-glucopyranosyl-canthin-6-one (M7). 3. The structures of metabolites were determined using spectroscopic analyses, chemical methods, and comparison of NMR data with those of known compounds. Among them, M7 was a new compound. 4. The metabolic pathways of 1 were proposed, and the metabolic processes involved phase I (O-demethylation, dehydroxylation, demethoxylation, N-oxidation, hydroxylation, and oxidative ring cleavage) and phase II (glycosylation) reactions. 5. This was the first research on microbial transformation of canthin-6-one alkaloid, which could be a useful microbial model for producing the mammalian phase I and phase II metabolites of canthin-6-one alkaloids. 6. 1, M1−M5, and M7 are canthin-6-one alkaloids, whereas M6 belongs to β-carboline type alkaloids. The strain of Cunninghamella blakesleeana can supply an approach to transform canthin-6-one type alkaloids into β-carboline type alkaloids.


Microorganisms and culture medium
All 24 microorganism strains were presented from College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China. These strains were used for the preliminary screening of the biotransformation of 1 as follows: Absidia coerulea CGMCC 3.3389, Alternaria alternata CGMCC 3.4578, Alternaria longipes CGMCC 3.2875, Aspergillus carbonarius CICC 2087, Aspergillus flavus CGMCC 3.3554, Aspergillus fumigatus  Stock cultures were maintained at 4 C on agar slants containing PDA in an Eyela LTI-700 conventional incubator (Tokyo Rikakikai Co. Ltd, Tokyo, Japan). All initial screening experiments were performed in conical flasks (500 mL) containing 100 mL of PDB, which was sterilized by autoclaving at 121 C for 30 min using a MLS3750 autoclave (Sanyo Electric Co. Ltd, Moriguchi, Japan).

Fermentation procedures
The preliminary screening strains were cultured on slants of PDA at 25 C for days 2À5. Then, the cultures were incubated in the above-described flasks with shaking on a HZQ-8 rotator shaker (HDL Apparatus Co. Ltd, Beijing, China) operating at 220 ppm and 28 C. After 48 h, 5 mg of 1 was added to each flask and maintained for another 72 h under the same conditions. It is worth mentioning that culture controls and substrate controls were carried out to easily discover the positive strains. Culture controls consisted of culture medium, in which microorganisms were grown under identical conditions without 1. Substrate controls consisted of culture medium and the same amount of 1 incubated under the same conditions without microorganisms (Hsu et al., 2002). Scale-up fermentation of 1 followed the above-described procedures except the number of flasks.

Extraction and isolation
The fermented broths were extracted three times with ethyl acetate (EtOAc), and the organic solvent was removed under vacuum to yield a crude extract using an Eyela N-1001 rotary evaporator (Tokyo Rikakikai Co. Ltd, Tokyo, Japan). The crude extract was separated using ODS CC, which was eluted with MeOH-H 2 O to yield fractions. Isolation conditions of fractions were optimized on the Dionex HPLC system, and isolation of fractions was performed on the Shimadzu HPLC system.

Acid hydrolysis
M7 (1.0 mg) was hydrolyzed with 2 N of HCl for 1 h at 90 C. After extracting with EtOAc twice, the H 2 O layer was evaporated in vacuo to furnish a monosaccharide residue. The residue was dissolved in pyridine (1.0 mL) containing Lcysteine methyl ester hydrochloride (1.0 mg) and heated at 60 C. After 1 h, 10 mL of o-tolyl isothiocyanate was added to the reaction mixture and further reacted at 60 C for 1 h. Then, the reaction mixture was directly analyzed by the Dionex HPLC system and detected by an UV detector (at 250 nm).
Analytical HPLC was performed on the Welch XB-C18 column with isocratic elution of CH 3 CN:H 2 O:HCOOH (25:75:0.01, v/v/v) for 40 min at a flow rate of 0.8 mL/min. The standard monosaccharides of D-Glc and L-Glc were subjected to the same method (Chen et al., 2013).

Screening of strains
To search for the strains which can catalyze the biotransformation of 1, 24 strains (including two strains of Cunninghamella) were screened. Analytical TLC of the crude extracts was carried out to screen the positive strains. For visualization of the alkaloidal spots on the TLC plates, modified Dragendorff's reagent was used. The TLC results suggested that only one crude extract of the fermented broths of 1 with C. blakesleeana displayed distinct Dragendorffpositive spots rather than 1 compared with culture controls and substrate controls. Therefore, this strain was selected for scale-up fermentation.
The metabolic pathways of 1 were proposed, and the metabolic processes involved O-demethylation, dehydroxylation, demethoxylation, N-oxidation, hydroxylation, oxidative ring cleavage (Phase I), and glycosylation (Phase II) (Figure 1). Although there is still no report about mammalian metabolism of canthin-6-one alkaloids and there are only few reports on the pharmacokinetics of canthin-6-one alkaloids (Chen et al., 2016;Shi et al., 2015), most of the microbial metabolites of 1 in this research are the same as the expected mammalian metabolites according to the general rules of mammalian metabolism. Mammalian metabolism is mainly represented by ''detoxification'' processes, classified as phase I (functionalization) and phase II (conjugation) reactions. Phase I reactions consist of oxidation (O-dealkylation, Ndealkylation, N-oxidation, hydroxylation, and so on), reduction, and hydrolysis. Phase II reactions are synthetic reactions involving the conjugation of substrates or metabolites with common endogenous substances, including glycosylation, sulfonation, and glutathionylation (Azerad, 1999;Srisilam & Veeresham, 2003). Cunninghamella blakesleeana is a eukaryotic organism that possesses metabolizing enzyme systems similar to those of mammalian systems, and this strain has been proposed as a microbial model of mammalian metabolism in the past (Abourashed et al., 1999;Asha & Vidyavathi, 2009;Piska et al., 2016;Quinn et al., 2015;Xie et al., 2005;Zhang et al., 1996). Therefore, C. blakesleeana could be a potential microbial system for producing the mammalian phase I and phase II metabolites of canthin-6-one alkaloids.
Additionally, metabolite M6, a -carboline type alkaloid, was also produced by biotransformation of 1 in this study. Although the current biotransformation yield is low (0.3%) in this research, this strain of C. blakesleeana may contribute a valuable enzyme or directly serve as an approach to transform canthin-6-one type alkaloids into -carboline type alkaloids if the efficiency of biotransformation is improved by optimization of the fermentation conditions (Gong et al., 2011;  Haldar et al., 2015;Shen et al., 2014). A hypothetical mechanism of oxidative ring cleavage from M1 to M6 is proposed (Figure 3).