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Abstract

In this study, a whole-cell biotransformation by Bacillus amyloliquefaciens Korean Collection for Type Culture 13588 was carried out to acquire naringenin derivatives. High-performance liquid chromatography analysis of the reaction metabolites showed the presence of 3 different products. The structure of the metabolites was confirmed through high-resolution quadrupole-time-of-flight electrospray-ionization mass spectrometry and nuclear magnetic resonance analysis. The result revealed that B. amyloliquefaciens catalyzes naringenin into 3 derivatives, naringenin 7-O-phosphate, naringenin 7-O-glucoside (prunin), and 6''-O-succinylprunin. To our knowledge, this is the first report of the production of 3 different derivatives when naringenin was used as a substrate. Furthermore, our study also revealed that the product naringenin 7-O-phosphate has approximately 45-fold higher water solubility than that of naringenin. Our finding suggests that biotransformation might solve the low bioavailability of flavonoids.

Keywords

biotransformation naringenin derivatives water solubility

Flavonoids are plant secondary metabolites that are important natural products due to their broad spectrum of biological activities. This includes antioxidative, anti-inflammatory, antimutagenic, and anticarcinogenic properties associated with their capacity to alter key cellular enzyme functions.¹ The most abundant flavanone of grapefruits, naringenin, is well known for its hepatoprotective, anti-lipid peroxidation and anticarcinogenic effects.² However, the lower water solubility of this compound results in its low bioavailability and prevents its application in medicinal chemistry.³ The physiochemical, as well as biological, properties of flavonoids are associated with the different functional group attached. In addition, a previous report suggests that modification of flavonoid molecules can result in different biochemical properties to that of the parent compounds.⁴

Several scientific efforts have been carried out in past to improve the bioactivity of flavonoid molecules. Although chemical and physical methods^3,5 can be used for the modification of flavonoid molecules, it is not normally highly regiospecific. In principle, the physiochemical properties of naringenin can be altered by its regiospecific modification, which eventually leads to the development of novel naringenin derivatives. Among several methodologies, microbial biotransformation is an effective process proven in diverse flavonoids.⁶ Hence, microbial biotransformation of flavonoids is of increasing scientific interest.

In this study, Bacillus amyloliquefaciens was used for the whole-cell biotransformation of naringenin. Our finding suggests that B. amyloliquefaciens is a good host for the production of naringenin 7-O-phosphate, naringenin 7-O-glucoside (prunin), and 6''-O-succinylpruin (Figure 1). To our knowledge, this is the first report of the production of 3 naringenin derivatives together using naringenin as a substrate.

Figure 1

Bioconversion of naringenin by Bacillus amyloliquefaceins. Naringenin was converted into naringenin 7-O-phosphate (1), naringenin 7-O-glucoside (prunin) (2), and 6''-O-succinyl pruning (3).

Our previous reports on chemical diversification of genistein and daidzein in whole-cell biotransformation mediated by B. amyloliquefaciens suggest that this probiotic bacterium might be a potential host for the biotransformation of different flavonoid molecules.^7,8 Hence, we used this strain for the biotransformation of naringenin. Bacillus amyloliquefaciens were grown in nutrient broth overnight and the cells were then harvested. The bio-transformation of naringenin was carried out with the cell mass in phosphate buffer supplemented with 2% glycerin at 30°C for 48 hours.

HPLC chromatograms of the reaction mixture showed the presence of 3 new peaks, whereas the cell extract of B. amyloliquefaciens does not show any such metabolites (Figure 2). The total conversion percentage of naringenin was 85%. Naringenin was converted to P1, P2, and P3 with the conversion percentages of 21%, 7%, and 57%, respectively. Furthermore, reaction metabolites were confirmed by high-resolution quadrupole-time-of-flight electrospray-ionization mass spectrometry (HR-QTOF-ESI/MS) analysis. This revealed that Peak 1 at 8.5 minutes has a molecular ion [M+H]⁺ (m/z) of 353.0420, which is equal in mass to naringenin conjugated with a phosphate group (Figure 3a). Peak 2 observed at 9 minutes showed the molecular ion [M+H]⁺ (m/z) of 435.1306, which corresponded to the mass of monosaccharide-conjugated naringenin, and was supported by the neutral loss of the glycan residue (162 Da) (Figure 3b). In addition, Peak 3 with the molecular ion [M+H]⁺ (m/z) of 535.1452 indicated a molecular formula of C₂₅H₂₇O₁₃, which was considered as succinylated naringenin-7-O-glucoside (Figure 3c).

Figure 2

High-performance liquid chromatogram of biotransformation reaction. Crude biotransformation reaction (a) contains 3 new metabolites (P₁, P₂, and P₃) when compared to standard naringenin (peak S) (b) and negative control (c).

Figure 3

Liquid chromatography mass spectrometry and UV spectra of different naringenin derivatives. (a) (i) ESI/MS; (ii) λ_max of peak 1; (b) (i) ESI/MS; (ii) λ_max of peak 2; and (c) (i) ESI/MS and (ii) λ_max of peak 3.

For further structural analysis, the metabolites were purified by preparative HPLC and their structures were determined by NMR spectroscopy. The result of the NMR analysis of Peak 1 showed a ³¹P NMR spectrum with one signal at −6.5 ppm supporting the phosphorylation of naringenin, while both ¹H and ¹³C NMR spectra of Peak 1 were similar to those of naringenin used as the substrate. A specific rotation in MeOH was a similar negative value ([α]²⁵ _D −38, c 0.004) as naringenin ([α]²⁵ _D −25 in MeOH), which indicated the same absolute configuration of both compounds. Furthermore, the position of the phosphate group was identified to be attached to the 7-OH by the disappearance of a signal corresponding to the 7-OH in ¹H NMR spectrum of naringenin. The phosphorylation at 7-OH was also clarified by carbonphosphorous coupling to C-6, C-7, and C-8 as 6 Hz (⁴ J _CP) at 100.5 ppm, 5 Hz (³ J _CP) at 160.1 ppm, and 5 Hz (⁴ J _CP) at 99.6 ppm in the ¹³C NMR spectrum (Figures S1-S3). Further HSQC and HMBC analysis further supported the structure of Peak 1 as naringenin 7-O-phosphate (Figures S4 and S5).

According to the combined NMR and MS data, the compound corresponding to Peak 2 was a glucoside of naringenin. The ¹H NMR spectrum of Peak 2 (Figure S6) indicated the presence of β-D-glucopyranose, which was conjugated at C-7 via the O-glycosidic linkage. Its structure was identified from comparisons of its ¹H NMR and UV data with those in the literature.⁹ In comparison with the ¹H NMR spectrum of 2, the ¹H NMR of compound 3 showed resonances for 4 methylene protons (2.44 and 2.33 ppm) with typical chemical shifts of methylene groups adjacent to a carbonyl group, suggesting a succinyl functionality (Figure S7). Based on no detection of the 6''-OH signal in the ¹H NMR spectrum, Peak 3 was elucidated as naringenin-6''-succinyl-7-O-glucose (6''-O-succinyl prunin). A recent paper reported the production of 3 through a similar biotransformation approach.¹⁰ Although some of the carbon signals could only be observed from HSQC and HMBC data, the NMR spectra were almost identical to those reported,¹⁰ which supported the structure of 3 as 6''-O-succinyl prunin (Figures S8-S10).

When 20 mg of naringenin was used as a substrate in 10 mL of reaction mixture with 2 OD₆₀₀ cell density, the yields of naringenin 7-O-phosphate, prunin and 6''-O-succinyl prunin were 4 , 1.2 and 11.2 mg, respectively.

Bacillus species, including B. amyloliquefaciens, are attractive industrial microorganisms due to their versatile functionality as well as their biological safety, enough to be recognized as GRAS (generally regarded as safe). This study demonstrated the capability of B. amyloliquefaciens to convert naringenin into 3 different derivatives with different physiochemical properties. A previous report revealed that only 15% of ingested naringenin was absorbed by the human body while conjugation with different functional groups can greatly enhance the physiochemical properties of the compounds,¹¹ which eventually play a role in the absorption process. Thus, the bioavailability of such compounds might be improved by bioconversion.

Water solubility of small molecules has a significant role in the pharmacological properties^3,12 and effective bioactive compounds are often abandoned during the drug development process due to poor physiochemical characteristics.¹³ Among the 3 derivatives, naringenin 7-O-glucoside (prunin) is less absorbed by the human body as compared to its aglycone form naringenin.¹⁴ Compared to naringenin, 6''-O-succinyl prunin has approximately 102 times higher water solubility with slightly improved antibacterial properties.¹⁰ However, no biological as well as physiochemical properties of naringenin 7-O-phosphate have been reported. To calculate the solubility of naringenin 7-O-phosphate on the basis of partition coefficient of organic solvent and water, we compared the peak areas of the corresponding compound by HPLC. Our result showed that solubility was increased by 45-fold after conjugation with phosphate at 7-OH position of naringenin.. Increased water solubility of naringenin 7-O-phosphate was expected and this compound may also have different biological properties to that of naringenin.¹⁰

New flavonoid derivatives, obtained by biotransformation, are associated with many advantages such as different physiochemical properties.^15,16 The results of this study may help in the development of naringenin derivatives, which might be candidates for future pharmaceutical applications. Further research focusing on the biological activity and application of these naringenin derivatives is going on in our laboratory.

Experimental

Chemical and Media Preparation

Naringenin was purchased from Selleck chemical (Houston, TX, USA). Acetonitrile and methanol were of HPLC analytical grade. Bacteriological peptone and beef extract were obtained from Difco (Baltimore, MD, USA). Bacillus . amyloliquefaciens Korean Collection for Type Culture (KCTC) 13588 was purchased from KCTC. Sodium phosphate monobasic anhydrous and sodium phosphate dibasic anhydrous were of analytical grade. Phosphate buffer was prepared by maintaining the pH 7.2 after addition of 2% glycerin.

Preparation of Biotransformation Reaction Mixture

A single colony of B. amyloliquefaciens KCTC 13588 was cultured in 3 mL of nutrient broth for 12 hours at 30°C and 200 rpm and scaled up to 80 mL. The culture broth was centrifuged at 6000 rpm for 15 minutes at 4°C and the supernatant was discarded. The cell mass was then washed with phosphate buffer. The reaction was carried using naringenin (2 mg/mL) as a substrate in 10 mL phosphate buffer with 2% glycerin supplement. In this way, the prepared reaction mixture was incubated at 30°C for 48 hours at 200 rpm.

Analysis and Identification of Products

After 48 hours of reaction, the whole-cell biotransformation reaction was extracted with 2 mL of acetone and incubated at room temperature for 2 hours. The cell mass was then separated from the supernatant by centrifugation at 5000 rpm for 5 minutes, and the liquid content was evaporated and dissolved in 80% methanol. For the analysis of the crude sample, a Shimadzu SpectroMonitor 3200 digital UV/Vis detector equipped with a Shim-pack GIS 0.5 µm ODS C18 column (250 × 4.6, 4.0 µm id) was used. The mobile phase was water with 0.1% TFA (solvent A) and acetonitrile (solvent B) under a gradient system. Throughout the gradient system, solvent B was increased from 10% to 70% in 12 minutes and continuously increased to 100% in 16 minutes, which was maintained up to 20 minutes. Afterward, solvent B contraction was reduced to 10% in the next 5 minutes and hold for 5 minutes. Furthermore, purification of metabolites was carried out using preparative scale HPLC using the same program with a Shim-pack GIS 0.5 µm ODS C18 column (250 × 10, 0.5 µm id).

For the determination of molecular mass of different peaks, HR-QTOF ESI/MS analysis was applied in positive ion mode using an ACQUITY (UPLC, Waters Corp., Milford, MA, USA), coupled with an SYNAPT G2-Si column (Waters Corp.). Obtained mass data were analyzed by massLynx version 4.1. For structure elucidation, purified sample was dissolved in DMSO-d ₆ and NMR was performed with the Aglient 400 MHz WB NMR.

Naringenin 7-O-Phosphate (1)

[α]²⁵ _D: −38 (c 0.004 MeOH).

¹H NMR: (400 MHz, DMSO-d ₆): δ 11.98 (1H, s, 5-OH), 9.65 (1H, s(br), 4'-OH), 7.33 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.80 (2H, d, J = 8.5 Hz, H-3', H-4'), 6.32 (2H, s, H-6, H-8) , 5.51 (1H, dd, J = 13.0, 2.8 Hz, H-2), 3.36 (1H, dd, J = 17.0, 13.0 Hz, H-3a), 2.74 (1H, dd, J = 17.0, 3.0 Hz, H-3b);

¹³C NMR: δ 198.07 (C-4), 162.95 (C-5 or C-9), 162.85 (C-5 or C-9), 160.06^* (C-7), 158.24 (C-4'), 128.95 (C-1'), 128.78 (C-2', C-6'), 115.61 (C-3', C-5'), 104.75 (C-10), 100.50^* (C-6), 99.61^* (C-8), 79.09 (C-2), 42.57 (C-3); ^*Observed as doublet due to C-P coupling (see text for details).

³¹P NMR: δ −6.5 ppm.

HRMS: calculated for C₁₅H₁₄O₈P [M+H]⁺ 353.0421; observed 353.0420.

Naringenin 7-O-Glucoside (2, Prunin)

¹H NMR: (400 MHz, DMSO-d ₆): δ 12.06 (1H, d, J = 2.4 Hz, 5-OH), 9.62 (1H, s, OH-4'), 7.33 (2H, d, J = 8.6 Hz, H-2', H-6'), 6.80 (2H, d, J = 8.6 Hz, H-3', H-5'), 6.15 (1H, m, H-8), 6.13 (1H, t, J = 2.4 Hz, H-6), 5.50 (1H, dt, J = 12.9, 2.9 Hz, H-2), 5.37 (1H, d, J = 4.9 Hz, OH-2''), 5.12 (1H, d, J = 4.7 Hz, OH-3''), 5.05 (1H, d, J = 5.1 Hz, OH-4''), 4.97 (1H, dd, J = 9.8, 7.5 Hz, H-1''), 4.57 (1H, td, J = 5.7, 3.0 Hz, OH-6''), 3.65 (1H, dd, J = 11.4, 4.9 Hz, H-6''a), 3.44 (1H, m, H-6''b), 3.31–3.40 (2H, m, H-5'', H-3a), 3.26 (1H, m, H-3''), 3.21 (1H, m, H-2''), 3.12 (1H, m, H-4''), 2.73 (1H, dt, J = 17.1, 2.8 Hz, H-3b).⁹

HRMS: calculated for C₂₁H₂₃O₁₀ [M+H]⁺ 435.1286; observed 435.1306.

6''-O-Succinyl prunin (3)

¹H NMR: (400 MHz, DMSO-d ₆): δ 12.17(1H, s(br), COOH), 12.02 (1H, d, J = 2.2 Hz, 5-OH), 9.17 (1H, s, OH-4'), 7.32 (2H, d, J = 8.6 Hz, H-2', H-6'), 6.79 (2H, d, J = 8.6 Hz, H-3', H-5'), 6.17 (1H, m, H-8), 6.11 (dd, J = 3.2 and 2.3 Hz, H-6), 5.50 (1H, m, H-2), 5.47 (1H, d, J = 4.9 Hz, OH), 5.33 (1H, dd, J = 5.4, 0.9 Hz, OH), 5.24 (1H, d, J = 4.6 Hz, OH), 4.99 (1H, t, J = 7.8 Hz, H-1’’), 4.32 (1H, m, H-6’’a), 3.99 (1H, ddd, J = 12.3, 7.5, 5.2 Hz, H-6''b), 3.65 (1H, ddd, J = 9.8, 7.6, 2.0 Hz, H-5''), 3.35-3.40 (2H, m, H-3a, H-5''), 3.20-3.30 (2H, m, H-2'', H-3''), 3.13 (1H, m, H-4''), 2.71 (1H, dd, J = 17.2, 3.0 Hz, H-3b), 2.44 (2H, m, H-2'''), 2.33 (2H, m, H-3''').

¹³C NMR^*: 197.9 (C-4), 173.9 (C-4'''), 172.5 (C-1'''), 165.5 (C-7 and C-9), 163.3 (C-5), 158.3 (C-4'), 129.0 (C-1'), 128.9 (C-2' and C-6'), 115.6 (C-3' and C-5'), 103.8 (C-10), 99.6 (C-1''), 97.0 (C-6), 95.9 (C-8), 79.1 (C-2), 74.3 (C-5''), 70.3 (C-4''), 29.0 (C-2''' and C-3'''). ^*13C chemical shifts were assigned by HSQC and HMBC analyses.

HRMS: calculated for C₂₅H₂₇O₁₃ [M+H]⁺ 535.1446; observed 535.1452.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1C1B5018228) to Prof. Seung-Young Kim. The research was also financially supported by the Ministry of Trade, Industry and Energy (MOTIE), Korea under “Regional Specialized Industry Development Program” (Grant No: P0004692) to S.-Y. Kim and by the Ministry of Oceans and Fisheries, Korea under the “Development of Overseas Marine Bioresource and a System for their Utilization” fund (Grant No. 20170488) to J.-S. Park.

Supplemental Material

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Supplementary Material

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Biotransformation of Naringenin by Bacillus amyloliquefaciens Into Three Naringenin Derivatives

Abstract

Keywords

Experimental

Chemical and Media Preparation

Preparation of Biotransformation Reaction Mixture

Analysis and Identification of Products

Naringenin 7-O-Phosphate (1)

Naringenin 7-O-Glucoside (2, Prunin)

6''-O-Succinyl prunin (3)

Footnotes

Declaration of Conflicting Interests

Funding

Supplemental Material

References

Supplementary Material