Microbial transformations of racemic 7-hydroxyflavanone using strains of genus
Flavonoids comprise a large group of secondary metabolites derived from phenylalanine. They are commonly found in plants, where their biogenesis takes place under normal physiological conditions (e.g., synthesis of pigments in flowers), but also may be a part of environmental stress response (e.g., synthesis of phytoalexins) [
Practical application of flavonoids in pharmacy or in medicine is often considerably limited due to their low solubility, inefficient transport across biological membranes or low stability. Biotransformation of flavonoid compounds may be a natural method of modification of their structures, aiming at greater structural diversity and better bioaccessibility. It also gives a possibility to trace metabolic transformation of flavonoids [
Enzymatic systems of microorganisms are capable of different reactions, including hydrolysis, carbonyl group reduction, hydroxylation,
Ibrahim and Abul-Hajj described biotransformation of monohydroxyflavones with substituents in the A-ring using the strain of
The substrates that possess several hydroxyl groups in the flavonoid skeleton, like quercetin or fisetin, in the culture of
In this paper we present the study in which we used the filamentous fungi of genera
The course of microbial transformation was monitored by TLC (SiO2, DC Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany). Chromatograms were developed using the following developing systems: hexane : ethyl acetate (7 : 3), dichloromethane : ethyl acetate (1 : 1), toluene : diethyl ether (4 : 1). Column chromatography (SiO2, Kieselgel 60, 230–400 mesh, 40–63
HPLC analyses were performed with a Waters 2690 instrument equipped with Waters 996 photodiode array detector, using ODS 2 column (4.6 × 250 mm, Waters) and a Guard-Pak Inserts
The racemic substrate for biotransformation—7-hydroxyflavanone
C15H12O3; melting point 188–190°C; 1H NMR see Table
1H NMR chemical shifts (
H-2 | 5.43 (dd) | 5.47 (dd) | 5.48 (dd) | 5,15 (dd) | 5.22 (m) | |
H-3 | H-3ax 2.96 (dd) | H-3ax 3.02 (dd) | H-3ax 3.05 (dd) | H-3ax 2.09 (ddd) | H-3ax 3.05 (ddd) | 6.86(s) |
H-4 | 5.03 (dd) | 5.52 (dd) | ||||
H-5 | 7.67 (d) ( | 7.87 (d) ( | 7.87 (d) ( | 7.32 (d) ( | 7.86 (d) ( | |
H-6 | 6.44 (dd) | 6.63 (dd) | 6.62 (dd) | 6.47 (dd) | 7.40 (d) ( | 6.91 (dd) |
H-8 | 6.32 (d) ( | 6.51 (d) ( | 6.51 (d) ( | 6.36 (d) ( | 7.42 (d) ( | 6.97 (d) |
H-2′ | 7.43 (d) ( | 7.48 (d) ( | 7.44 (s) | 7.30–7.43 (m) | 7.44–7.50 (m) | 8.02 (d) ( |
H-3′ | 7.33 (m) | 7.39 (m) | 7.30–7.43 (m) | 7.44–7.50 (m) | 7.53 (m) | |
H-4′ | 7.33 (m) | 7.39 (m) | 7.30–7.43 (m) | 7.44–7.50 (m) | 7.53 (m) | |
H-5′ | 7.33 (m) | 7.39 (m) | 6.24 (d) ( | 7.30–7.43 (m) | 7.44–7.50 (m) | 7.53 (m) |
H-6′ | 7.43 (d) ( | 7.48 (d) ( | 6.50 (d) ( | 7.30–7.43 (m) | 7.44–7.50 (m) | 8.02 (d) ( |
7-OH | 10.80 (s) | |||||
7-OCH3 | 3.86 (s) | 3.84 (s) |
aSolvent CD3OD.
bSolvent CDCl3.
cSolvent DMSO-d6.
13C NMR chemical shifts (
Carbon | Compounds | |||||
1a | 2b | 3b | 4b | 5b | 6c | |
C-2 | 81.1 | 80.2 | 80.0 | 77.0 | 73.8 | 162.8 |
C-3 | 45.2 | 44.3 | 44.2 | 40.3 | 37.8 | 106.6 |
C-4 | 193.1 | 192.2 | 190.8 | 65.6 | 59.5 | 176.4 |
C-5 | 129.9 | 129.7 | 128.8 | 128.3 | 159.7 | 126.5 |
C-6 | 111.9 | 110.1 | 110.3 | 108.8 | 92.0 | 115.1 |
C-7 | 166.9 | 168.0 | 166.2 | 156.3 | 161.5 | 161.9 |
C-8 | 103.9 | 100.9 | 100.9 | 103.0 | 93.5 | 102.6 |
C-9 | 165.4 | 162.2 | 163.6 | 155.4 | 156.5 | 157.5 |
C-10 | 115.1 | 126.2 | 114.7 | 118.7 | 106.3 | 116.1 |
C-1′ | 140.7 | 130.3 | 138.7 | 140.4 | 141.0 | 131.3 |
C-2′ | 127.4 | 127.9 | 115.5 | 126.0 | 126.3 | 126.2 |
C-3′ | 129.7 | 130.1 | 135.2 | 128.7 | 128.8 | 129.1 |
C-4′ | 129.6 | 128.8 | 135.4 | 128.3 | 128.1 | 131.6 |
C-5′ | 129.7 | 130.1 | 115.4 | 128.7 | 128.8 | 129.1 |
C-6′ | 127.4 | 127.9 | 115.9 | 126.0 | 126.3 | 126.2 |
OCH3 | 55.7 | 55.6 |
aSolvent CD3OD.
bSolvent CDCl3.
cSolvent DMSO-d6.
For our research we used a wild strain of
The wild strains
Cultivation media consisted of 3% glucose (The Industrial and Trading Enterprise “Stanlab” Co. Ltd., Poland) and 1% peptobac (BTL sp. z o.o., 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 hours 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 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 MgSO4 (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.
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 hours 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 3, 6, or 9 days of incubation the mixtures were extracted with ethyl acetate (3 × 200 mL), dried (MgSO4), and concentrated in vacuo. The transformation products were separated by column chromatography. Pure products were identified by means of spectral analyses (TLC, 1H NMR, 13C NMR, IR).
Physical and spectral data of the products obtained are presented next.
C16H14O3; melting point 89-90°C; 24% yield; purity 98% (HPLC);
C16H14O5; oily liquid; 19% yield; purity 98% (HPLC);
C15H14O3; oily liquid; 74% yield; purity 98% (HPLC);
C15H14O4; melting point 275–277°C; 12% yield; purity 98% (HPLC);
C15H10O3; melting point 246–248°C; 98% yield; purity 99% (HPLC); 1H NMR see Table
A methanolic solution of DPPH (1,1-diphenyl-2-picryl-hydrazyl) of absorbance of about 1.00 was mixed with a proper amount of a tested flavonoid. After 20 min, disappearance of absorbance at 520 nm was measured. The initial concentration of DPPH was determined by means of calibration curve. The IC50 value (antiradical activity) was determined on the basis of graphs—DPPH radical reduction (expressed in %) as a function of concentration of the tested compound. IC50 means concentration of the antioxidant that reduces the initial concentration of DPPH by half.
At first screening tests on racemic 7-hydroxyflavanone
A six-day transformation of 7-hydroxyflavanone
The structures of newly formed products: 7-methoxyflavanone (
In the 1H NMR of 3′,4′-dihydroxy-7-methoxyflavanone (
Microbial transformation of 7-hydroxyflavanone (
X-ray analysis of 2,4-
Selected 1H NMR data for 2,4-
2,4- | 5.17 | 5.08 | 2.13 | 2.51 | 13.06 | 11.62 | 1.83 | 10.59 | 6.27 |
2,4- | 5.15 | 5.03 | 2.09 | 2.49 | 13.20 | 11.30 | 1.80 | 9.90 | 6.20 |
2,4- | 5.22 | 5.52 | 3.05 | 2.89 | 16.90 | 8.40 | 16.70 | 13.30 | 2.90 |
The other product of biotransformation of 7-hydroxyflavanone (
Hydroxylation at C-5 was confirmed in the 1H NMR spectrum of
Transformation of 7-hydroxyflavanone (
In the 1H NMR of substrate
Comparison of UV spectra of 7-hydroxyflavanone (
UV absorption of 7-hydroxyflavanone
Compound | 1st band | 2nd band | 3rd band | |||
log | log | log | ||||
7-Hydroxyflavanone ( | 237 | 4.15 | 275 | 4.18 | 310 | 3.95 |
7-Methoxyflavanone ( | 235 | 4.18 | 273 | 4.19 | 310 | 3.89 |
3′,4′-Dihydroxy-7-methoxyflavanone ( | 238 | 4.25 | 272 | 4.39 | 335 | 4.01 |
2,4- | 220 | 3.95 | 282 | 4.20 | — | — |
2,4- | 237 | 4.08 | 284 | 4.46 | — | — |
7-Hydroxyflavone ( | 254 | 4.32 | 302 | 4.34 | — | — |
For product
For substrate
The IC50 values of the 7-hydroxyflavanone
Substrate | Product | IC50* (±SD) [ |
---|---|---|
7-Hydroxyflavanone ( | 9.44 (±0.06) | |
3′,4′-Dihydroxy-7-methoxyflavanone ( | 6.70 (±0.07) | |
2,4- | 7.07 (±0.05) | |
7-Hydroxyflavone ( | 8.80 (±0.05) | |
2,4- | 9.17 (±0.03) | |
7-Methoxyflavanone ( | 9.50 (±0.07) |
*Mean values of IC50 calculated as an average of at least three measurements.
Microbial transformations of 7-hydroxyflavanone (
the strain of the strain of
The strain
A nontypical reaction of hydroxylation at C-5 along with carbonyl group reduction was observed for the strain
The highest yields of products were observed for 7-hydroxyflavan-4-ol (
Microbial transformation of flavonoids may be used as a natural method leading to the derivatives of higher antioxidant properties than the initial substrates.
The authors report no conflict of interests. The authors alone are responsible for the content and writing of the paper.
Publication/Project “Biotransformations for pharmaceutical and cosmetics industry” no. POIG.01.03.01-00-158/09 was part-financed by the European Union within the European Regional Development Fund.