Microbial Transformations of 7-Methoxyflavanone

Microbial transformations of racemic 7-methoxyflavanone using strains of the genus Aspergillus (A. niger KB, A. ochraceus 456) and the strain Penicillium chermesinum 113 were described. The strain A. niger KB catalysed carbonyl group reduction, leading to (±)-2,4-cis-7-methoxyflavan-4-ol. Biotransformation with the help of A. ochraceus 456 gave two products: (+)-2,4-trans-7-methoxyflavan-4-ol and 4'-hydroxy-7-methoxyflavone. Transformation by means of P. chermesinum 113 resulted in a dihydrochalcone product, 4,2'-dihydroxy-4'-methoxydihydrochalcone. DPPH scavenging activity test proved that all the biotransformations products have higher antioxidant activity that the substrate.


Introduction
Flavonoids are polyphenolic compounds with diverse chemical structures, which are widely found in plants. Apart from plants, a natural capability to carry out biosynthesis of flavonoid compounds is a feature of some endophytic fungi [1]. Animal and human organisms do non synthesize flavonoids [2,3]. Their specific properties make them useful for pharmaceutical, cosmetics and food industry. Therefore, there is growing interest in chemical synthesis of flavonoids, as well as in their biotechnological production [4][5][6][7][8][9]. The therapeutic potential and low toxicity of flavonoids are accompanied with relatively little information about their metabolic pathways in living organisms. That is why the attention has been directed to biocatalysis [10].

OPEN ACCESS
The microorganisms used in biotransformation of flavonoid compounds have enzymatic systems capable of performing various chemical reactions, including reduction, hydroxylation, O-methylation and hydrolysis [11,12]. Analytical tests carried out during the course of biotransformation allow tracing of the metabolic transformations of flavonoids [13][14][15]. The products are often new compounds, not described in the literature so far and difficult to obtain by chemical synthesis. Moreover, they have often high antioxidant properties [16,17].
The objective of our research was to transform racemic 7-methoxyflavanone into optically pure products with higher antioxidant properties than the starting substrate.
For the obtained 2,4-trans-7-methoxyflavan-4-ol (3) the measured specific optical rotation was [α] 20 546 = +2.57 (c = 0.7, CH 3 OH), and the enantiomeric excess (by HPLC, chiral column) ee = 30%. For the unreacted substrate (1) isolated from the reaction mixture the data was as follows: [α] 20 546 = +6.28 (c = 2.1, CH 3 OH) and ee = 24%. The structure of the independently formed 4'-hydroxy-7-methoxyflavone (4) was established by means of 1 H NMR and 13 C-NMR. Hydroxylation at C 4' in ring B is proved by two doublets integrating each for 2 H at  = 6.95 ppm (J = 8.6 Hz) and  = 7.81 ppm (J = 8.7 Hz), corresponding to H-3', H-5' and H-2', H-6', respectively. In the 13 C-NMR we observe a change in C-4' chemical shift from  = 128.8 ppm for substrate (1) to  = 160.5 ppm for product 4. In the 1 H-NMR spectrum of 7-methoxyflavone (1) the signal of H-2 appears at  = 5.47 ppm as a doublet of doublets (J = 13.3 Hz and J = 2.8 Hz), the signal of H-3 ax at  = 3.04 ppm is a doublet of doublets (J = 16.9 Hz and J = 13.3 Hz) and the signal of H-3 eq at  = 2.84 ppm is also a doublet of doublets (J = 16.9 Hz and J = 2.9 Hz). In the 1 H-NMR of 4'-hydroxy-7-methoxyflavone (4) a one proton singlet is visible at  = 5.69 ppm, attributed to H-3, whereas the signal of H-2 disappears, which confirm the presence of a double bond between C-2 and C-3 in ring C. Additionally, in the 13 C-NMR the signals of C-2 and C-3 were moved from  = 80.2 ppm and  = 44.3 ppm for the substrate (1) to  = 162.8 ppm and  = 104.0 ppm for the dehydrogenation product 4.
In the 1 H-NMR spectrum of product 5 there are two triplets at  = 2.98 ppm and 3.19 ppm, integrating for two protons each, corresponding to H-β and H-α and typical for a dihydrochalcone structure. Opening of the ring C is additionally proved by the presence of the one proton singlet at  = 12.6 ppm, which is attributed to 2'-OH. Substitution at C-4 is confirmed by two dublets at  = 6.77 ppm and  = 7.11 ppm of identical coupling constants (J = 8.5 Hz) and integrating for 2 protons each. The 4-OH hydroxyl proton is visible as a singlet at  = 5.38 ppm.
The IC 50 values (antiradical activity) of the substrate and the biotransformation products were determined spectrophotometrically on the basis of graphs: DPPH radical reduction as a function of concentration of a tested compound [16]. IC 50 means the concentration of an antioxidant (flavonoid) that reduces the initial concentration of DPPH by 50%. The measured IC 50 values are presented in Table 5. Among the products of the microbial transformation of 7-methoxyflavanone the highest antioxidant activity was observed for 4'-hydroxy-7-methoxyflavone (4) (IC 50 = 7.66) and 4,2'-dihydroxy-4'methoxydihydrochalcone (5) (IC 50 = 7.75). This is due to the microbial introduction of a hydroxyl group at 4' position in ring B. The two reduction products: 2,4-cis (2) and 2,4-trans 7-methoxyflavan-4-ol (3) have also higher antioxidant properties than the substrate (1). Comparison of the IC 50 values of 2 and 3 indicates that 2,4-cis-7-methoxyflavan-4-ol (2) is a better antioxidant (IC 50 = 8.20) (Table 5), which shows that stereochemistry of a compound may have an influence on its antioxidant activity. Our earlier research on antioxidant activity of products of biotransformations of flavanone, its monosubstituted derivatives (among them 7-methoxyflavanone) and naringenin allowed us to draw more detailed conclusions concerning the relationship between flavonoid structure and antioxidant activity [16].

Microorganisms
The wild strain A. niger KB was obtained from the collection of the Department of Biotechnology and Food Microbiology of Wrocław University of Environmental and Life Sciences (Poland). The microorganism was maintained on potato slants (sterilized piece of potato) at 5 °C.
The wild strains A. ochraceus 456 and P. chermesinum 113 were obtained from the collection of the Department of Chemistry of Wrocław University of Environmental and Life Sciences (Poland). The microorganisms were maintained on agar slants at 5 °C.

Screening Procedure
Cultivation media consisted of 3% glucose (The Industrial and Trading Enterprise "Stanlab" Co. Ltd., Lublin, Poland) and 1% peptobac (BTL sp. z o.o., Warszawa, Poland) in water. The microorganisms were transferred from the slants to 500 mL Erlenmayer flasks, each containing 200 mL of the medium. Preincubation was performed at 25 °C for 24-48 h. Then portions of 1 mL of the culture solution were transferred to inoculate 500 mL flasks, each containing 200 mL of the medium. After cultivation at 25 °C for 24 h on a rotary shaker, 10 mg of a substrate, dissolved in 0.5 mL of THF, was added to the grown culture. Control cultivation with no substrate was also performed. After 1, 3, 6 and 9 days of incubation under the above conditions, portions of 5 mL of the transformation mixture were withdrawn and extracted with ethyl acetate (3 × 3 mL). The extracts were dried over MgSO 4 (5 min), concentrated in vacuo and analyzed by TLC. Quantitative analyses of the mixtures were performed by means of HPLC. Calibration curves for quantitative analyses were prepared using isolated and purified biotransformation products as standards.

Preparative Biotransformation
Portions of 1 mL of the preincubation culture solution were used to inoculate three 2000 mL flasks, each containing 500 mL of the cultivation medium. The cultures were incubated at 25 °C for 48 h on a rotary shaker. Then 50 mg of the substrate dissolved in 2.5 mL of THF was added to each flask (100 mg of the substrate per 1 L of the cultivation mixture). After 9 or 10 days of incubation the mixtures were extracted with ethyl acetate (3 × 200 mL), dried (MgSO 4 ) and concentrated in vacuo. The transformation products were separated by column chromatography. Pure products were identified by means of spectral analyses (TLC, 1 H-NMR, 13 C-NMR, IR).

Measurement of Antioxidant Properties of the Substrate and the Products
A methanolic solution of DPPH (1,1-diphenyl-2-picryl-hydrazyl) with an absorbance of about 1.00, was mixed with a proper amount of a tested flavonoid 1-5. After 20 min, disappearance of absorbance at 520 nm was measured. The initial concentration of DPPH was determined by means of calibration curve. The IC 50 value (antiradical activity) was determined graphically-DPPH radical reduction (expressed in %) as a function of concentration of the tested compound. IC 50 means concentration of the antioxidant that reduces the initial concentration of DPPH by half.

Conclusions
The study on A. niger KB described in this article and in our previous papers prove that this strain is a good catalyst for carbonyl group reduction in flavanone and its monosubstituted derivatives [19,20]. The strain P. chermesinum 113 performs mainly reactions of hydroxylation in ring B, which are often accompanied with the ether bond cleavage in ring C, leading to dihydrochalcone structure [15].
All the products of biotransformations of 7-methoxyflavanone have higher antioxidant properties than the substrate.