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Regioselective ortho-hydroxylations of flavonoids by yeast Sandra Sordon, Anna Madej, Jaros#aw Pop#o#ski, Agnieszka Bartma#ska, Tomasz Tronina, Ewa Brzezowska, Piotr Juszczyk, and Ewa Huszcza J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02210 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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Journal of Agricultural and Food Chemistry

Regioselective ortho-hydroxylations of flavonoids by yeast

Sandra Sordon1*, Anna Madej1, Jarosław Popłoński1, Agnieszka Bartmańska1, Tomasz Tronina1, Ewa Brzezowska1, Piotr Juszczyk2, Ewa Huszcza1

1

) Department of Chemistry, Wrocław University of Environmental and Life Sciences,

Norwida 25, 50-375 Wroclaw, Poland 2

) Department of Biotechnology and Food Microbiology, Wrocław University of

Environmental and Life Sciences, Chełmońskiego 37/41, 51-630 Wrocław, Poland

*) Corresponding author: Sandra Sordon Tel.: 0048713205197, Fax: 00483207744, E-mail addresses: [email protected]

1 Environment ACS Paragon Plus


1

ABSTRACT

2

Natural flavonoids, such as naringenin, hesperetin, chrysin, apigenin, luteolin,

3

quercetin, epicatechin and biochanin A were subjected to microbiological transformations by

4

Rhodotorula glutinis. Yeast was able to regioselective C-8 hydroxylation of hesperetin, luteolin

5

and chrysin. Naringenin was transformed to 8- and 6-hydroxyderivatives. Quercetin,

6

epicatechin and biochanin A did not undergo biotransformation. A metabolic pathway for the

7

degradation of chrysin has been elucidated. The metabolism of chrysin proceeds via an initial

8

C-8 hydroxylation to norwogonin, followed by A-ring cleavage to 4-hydroxy-6-phenyl-2H-

9

pyran-2-one.

10 11

Keywords: flavonoids, biotransformation, yeast, Rhodotorula glutinis, hydroxylation,

12

regioselectivity


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13


INTRODUCTION

14

Flavonoids are a diverse group of ubiquitous plant secondary metabolites. Health

15

benefits associated with them resulted in a significant increase of interest in this class of

16

compounds. Some flavonoids, such as naringenin, naringin, hesperidin or chrysin, are

17

inexpensively available in a large quantities (see supplementary data). They may serve as a

18

viable source of lead compounds for the development of food additives, preservatives,

19

antioxidants and also drug components.1

20

Biotransformation is one of the possible methods for the preparation of various

21

derivatives of flavonoids.2, 3 The use of microorganisms as a biocatalyst is a relevant strategy to

22

obtain high added value natural compounds from both cheap and readily available substrates.3, 4

23

These processes offer more advantages compared to conventional catalysts such as mild

24

reaction conditions, high catalytic activities with high regio- and stereoselectivity.4, 5 Moreover,

25

according to the European Union Law, the products obtained by biotransformation of natural

26

compounds may be considered as natural ones (EU Directive 88/388/EEC).

27

It has been reported that that a protective effect of diet flavonoids against several

28

diseases, among them atherosclerosis6, is connected to their antioxidant properties. It is general

29

considered that a higher number of hydroxyl groups in a flavonoid results in a higher

30

antioxidant activity.7, 8 Briefly, the presence of two hydroxyl groups in an ortho position of ring

31

B, especially the 3´,4´-dihydroxyphenolic structure, is the most important structure for

32

scavenging actions. Also of importance is the presence of hydroxyl groups at positions 5, 6 and

33

7 in the ring A. The ability of flavonoids to chelate metals results from the presence metal

34

complexing domains between the 5-hydroxyl and 4-carbonyl group, the 3-hydroxyl and 4-

35

carbonyl group and between the 3´,4´-hydroxyl groups. Consequently, development of

36

microbial hydroxylation of flavonoids is significant in the synthesis of active antioxidants for

37

biological activity in human.



38

Our previous microbial transformation research indicated the capability of the yeast

39

Rhodotorula marina to efficient hydroxylation of naringenin at C-6 or C-8 position to

40

carthamidin and isocarthamidin, respectively.9,

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greater antioxidant activity than either naringenin and its glycoside naringin.11 These

42

encouraging results prompted us to search for new useful red yeast biocatalysts, and then to

43

undertake studies on preparation of valuable derivatives of natural flavonoids by

44

biotransformation in the selected yeast cultures.

10

These metabolites showed significantly

45 46

MATERIALS AND METHODS

47 48

Compounds

49

Naringenin (1), hesperidin, chrysin (3), biochanin A, (-)-epicatechin, quercetin, ascorbic acid

50

and DMSO-d6 were purchased from Sigma-Aldrich, Sephadex LH-20 from Pharmacia,

51

acetonitrile HPLC grade, silica gel 60 and TLC plates from Merck, peptone from Biocorp

52

(Poland). Other chemicals and solvents were purchased from POCH (Poland).

53 54

Hesperetin (2) was obtained by acidic hydrolysis of hesperidin.

55

A solution of 1.25 g of hesperetin (2) (≥80%), 500 mg of ascorbic (added to prevent oxidation

56

of the products of hydrolysis), 50 mL of 2M hydrochloric acid and 50 mL of methanol was

57

heated under reflux for 10 h. After cooling, Na2CO3 was added to reach the pH about 4 and the

58

reaction mixture was extracted three times with 50 mL of ethyl acetate. The combined fractions

59

were washed with distilled water, dried over anhydrous magnesium sulfate and concentrated.

60

After purification by column chromatography on silica gel 60 using mixture chloroform :

61

methanol (10 : 1, v/v) as eluent, 307.0 mg of hesperetin (2) was obtained with purity greater

62

than 95% (according to HPLC).1H NMR (600 MHz, DMSO-d6) δ (ppm): 2.70 (dd, 1H, J =


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17.1, 3.1 Hz, H-3ax), 3.18 (dd, 1H, J = 17.1, 12.3 Hz, H-3eq), 3.77 (s, 3H, C4´-O-CH3), 5.42

64

(dd, 1H, J = 12.3, 3.1 Hz, H-2), 5.88 (d, 1H, J = 2.1 Hz, H-6), 5.89 (d,1H, J = 2.1 Hz, H-8),

65

6.87 (dd, 1H, J = 8.3, 2.1 Hz, H-6´), 6.92 (d, 1H, J = 2.0 Hz, H-2´), 6.93 (d, 1H, J = 8.3 Hz, H-

66

5´), 8.29 (s, 1H, 3´-OH), 9.12 (s, 1H, 7-OH), 12.12 (s, 1H, 5-OH).

67

DMSO-d6) δ (ppm): 42.1 (C-3), 55.8 (C4´-O-CH3), 78.3 (C-2), 95.1 (C-8), 95.9 (C-6), 101.9

68

(C-10), 112.1 (C-5´), 114.1 (C-2´), 117.8 (C-6´), 131.2 (C-1´), 146.5 (C-3´), 148.0 (C-4´),

69

162.9 (C-9), 163.6 (C-5), 166.7 (C-7), 196.3 (C-4).

13

C NMR (150 MHz,

70 71

Apigenin (4) was obtined from naringenin (1) by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone

72

(DDQ) - oxidation of C2-C3 bond according to the method described by Kim et al.12 1H NMR

73

(600 MHz, DMSO-d6) δ (ppm): 6.19 (m, 1H, H-8), 6.48 (m, 1H, H-6), 6.77 (s, 1H, H-3), 6.92

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(m, 2H, H-3´,5´), 7.92 (m, 2H, H-2´, 6´), 10.35 (s, 1H, 4´-OH), 10.82 (s, 1H, 7-OH), 12.96 (s,

75

1H, 5-OH).

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103.7 (C-10), 116.0 (C-3´,5´), 121.2 (C-1´), 128.5 (C-2´,6´), 157.3 (C-5), 161.2 (C-4´), 161.5

77

(C-9), 163.7 (C-2), 164.1 (C-7), 181.8 (C-4).

13

C NMR (150 MHz, DMSO-d6) δ (ppm): 94.0 (C-6), 98.8 (C-8), 102.9 (C-3),

78 79

Luteolin (5) was isolated from artichoke (Cynara scolymus L.) leaf dry extract. Flavonoid

80

glycosides included in the dry extract of artichoke were hydrolyzed. For this purpose a mixture

81

of 9 g of the extract, 200 mg of ascorbic (added to prevent oxidation of the products of

82

hydrolysis), 240 mL of 2M hydrochloric acid and 240 mL of methanol was heated under reflux

83

for 6 h. After cooling, Na2CO3 was added to reach the pH about 4 and the reaction mixture was

84

extracted three times with 250 mL of ethyl acetate. The combined fractions were washed with

85

distilled water, dried over anhydrous magnesium sulfate and concentrated to give 2.75 g of

86

extract. After purification by column chromatography on Sephadex LH-20 using methanol as

87

eluent, 86.2 mg of luteolin (5) was obtained with purity greater than 98% (according to HPLC).



88

1

89

Hz, H-6), 6.68 (s, 1H, H-3), 6.88 (d, 1H, J = 8.2 Hz, H-5´), 7.39 (s, 1H, H-2´), 7.41 (d, 1H, J =

90

9.6 Hz, 6´-H), 9.49 (s, 1H, 4´-OH), 9.99 (s, 1H, 3´-OH), 10.84 (s, 1H, 7-OH), 12.98 (s, 1H, 5-

91

OH). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 93.9 (C-8), 98.9 (C-6), 102.9 (C-3), 103.8 (C-

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10), 113.4 (C-2´), 116.1 (C-5´), 119.1 (C-6´), 121.5 (C-1´), 145.8 (C-3´), 149.8 (C-4´), 157.4

93

(C-9), 161.5 (C-5), 164.0 (C-2), 164.2 (C-7), 181.7 (C-4).

H NMR (600 MHz, DMSO-d6) δ (ppm): 6.18 (d, 1H, J = 1.80 Hz, H-8), 6.44 (d, 1H, J = 1.8

94 95

Analytic procedures

96

TLC was carried out on silica gel 60 F254 (0.2 mm thick) plates. Compounds were detected by

97

spraying the plates with 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4] in 10% H2SO4. HPLC was

98

performed on a Waters 2695 Alliance instrument with a photodiode array detector Waters 2996

99

(detection at 290 and 370 nm wavelength) equipped with the analytical HPLC column Agilent

100

ZORBAX Eclipse XDB 5 µm (46 × 250 mm), which was eluted at 0.8 mL min. A linear

101

gradient was composed of (A) acetonitrile and (B) 0.1% acetic acid in water. After injection

102

solvent A increased from 25% to 38% over 10 min and held at 38% for 18 min, then further

103

decreased from 38% to 25% over 2 min, and finally held 25% for 6 min.

104

NMR spectra (1H NMR,

105

(HSQC, HMBC)) were recorded on a MHz DRX Bruker Avance™ 600 (600 MHz) instrument

106

in DMSO-d6. OD 600 nm analysis were performed using a Spectrofotometer Cintra 303, GBC,

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Positive-ion HR-ESIMS spectra were measured on a Bruker micrOTOF-Q spectrometer.

13

C NMR, DEPT 135°, 1H-1H NMR (COSY), and 1H-13C NMR

108 109

Microorganisms

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Microorganisms used in this work were purchased from Institute of Biology and Botany,

111

Wrocław Medical University (indexed AM) and Department of Chemistry, Wrocław

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University of Environmental and Life Sciences (indexed KCh). The red yeast cultures used for


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preliminary study were as follows: Rhodotorula glutinis AM242, Rhodotorula glutinis

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KCh735, Rhodotorula rubra AM4 and Rhodotorula rubra AM82.

115 116

Biotransformation procedure

117

The yeast were cultivated on rotary shakers (130 rpm, 6.5 amplitude) at 28 °C in 100-

118

mL Erlenmeyer flasks with 30 mL of the medium in the preliminary screening experiments and

119

in 300-mL Erlenmeyer flasks containing 100 mL of the medium in the preparative scale

120

fermentation. The growth medium for biotransformation processes (Sabouraud medium)

121

contained 3% glucose and 1% peptone. The cultures on agar slants were used to obtain the

122

inoculation culture, and then 2-day inoculation culture were transferred to the main cultures

123

media (1 mL to the 30 mL and 3 mL to the 100 mL). After three days of growth the substrates

124

were added.

125

In the screening studies, 5 mg of naringenin (1) dissolved in 0.5 mL of acetone was

126

added to the culture. Experiments were performed in triplicate. Biotransformations were

127

performed for 48 hours, acidified using 1M HCl to pH about 5. Then each of the reaction

128

mixture was extracted with ethyl acetate (2 x 10 mL).

129

In the preparative biotransformations, a total of 80 mg of each substrate dissolved in 4 mL of

130

methanol (for substrates 1, 2, 5) and DMSO (for substrates 3, 4) or was equally distributed

131

among four flasks with 3-day fungal cultures of OD600 = 22.40 ± 0.99 (1 mL each) to give a

132

final concentration of 200 mg / L. Reactions were carried out for 1 to 6 days, acidified using

133

1M HCl to pH about 5, and then each of the reaction mixture was extracted with ethyl acetate

134

(3 x 40 mL).

135 136

The progress of biotransformations was monitored by TLC and HPLC. Substrate controls consisted of substrate and a sterile growth medium incubated without microorganism.



137 138

Preparative transformation of chrysin (3) leading to its degradation was carried out under similar conditions except that tetrahydrofuran was used to dissolve the substrate.

139 140

Products isolation and analysis

141

Extracts were dried with anhydrous magnesium sulphate, filtered and evaporated to

142

dryness. The resulting materials were redissolved in methanol and analyzed by TLC and

143

HPLC. The combined organic phases were evaporated and column chromatographed over

144

Sephadex LH-20 gel using chloroform/methanol (4:1 v/v) as eluent for 1, 3, 4 and 5, and

145

chloroform/methanol (10:1 v/v) for 2. Quantitative analysis of the ethyl acetate extract of the

146

biotransformation mixture was performed by means of HPLC, using calibration curves for the

147

substrate and for all the isolated products.

148 149

The structures of the obtained compounds were confirmed by NMR spectra analysis. Spectroscopic data of the products 6-12 are with agreement with literature data.10, 11, 13-19

150 151

carthamidin (5, 6, 7, 4´- tetrahydroxyflavanone, 6)

152

1

153

J = 17.1, 12.5 Hz, H-3ax), 5.36 (dd, 1H, J = 12.5, 2.9 Hz, H-2), 5.94 (s, 1H, H-8), 6.79 (d, 2H, J

154

= 8.5 Hz, H-3´, 5´), 7.31 (d, 2H, J = 8.5 Hz, H-2´,6´), 8.11 (s, 1H, 7-OH), 9.57 (s, 1H, 4´-OH),

155

10.42 (s, 1H, 6-OH), 11.98 (s, 1H, 5-OH). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 42.3 (C-

156

3), 78.5 (C-2), 94.7 (C-8), 101.7 (C-10), 115.2 (C-3´, 5´), 126.3 (C-6), 128.3 (C-2´, 6´), 129.2

157

(C-1´), 150.2 (C-9), 155.3 (C-5), 155.8 (C-7), 157.7 (C-4´), 196.7 (C-4).

H NMR (600 MHz, DMSO-d6) δ (ppm): 2.65 (dd, 1H, J = 17.1, 2.9 Hz, H-3eq), 3.22 (dd, 1H,

158 159

isocarthamidin (5, 7, 8, 4´- tetrahydroxyflavanone, 7)

160

1

161

J = 17.1, 12.3 Hz, H-3ax), 5.42 (dd, 1H, J = 12.3, 3.0 Hz, H-2), 5.93 (s, 1H, H-6), 6.79 (d, 2H, J

H NMR (600 MHz, DMSO-d6) δ (ppm): 2.71 (dd, 1H, J = 17.1, 3.0 Hz, H-3eq), 3.22 (dd, 1H,


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= 8.5 Hz, H-3´, 5´), 7.35 (d, 2H, J = 8.5 Hz, H-2´, 6´), 8.11 (s, 1H, 7-OH), 9.57 (s, 1H, 4´-OH);

163

10.42 (s, 1H, 8-OH), 11.75 (s, 1H, 5-OH). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 42.3 (C-

164

3), 78.5 (C-2), 95.4 (C-6), 101.7 (C-10), 115.1 (C-3´,5´), 125.6 (C-8), 128.4 (C-2´, 6´), 129.2

165

(C-1´), 149.4 (C-9), 156.5 (C-5), 155.8 (C-7), 157.7 (C-4´), 196.6 (C-4).

166 167

8-hydroxyhesperetin (3´,5,7,8-tetrahydroxy-4´-metoxyflavanone, 8)

168

1

169

J = 11.8 Hz, 17.1 Hz, H-3ax), 3.77 (s, OCH3), 5.41 (1H, dd, J = 11.8, 3.1 Hz, H-2), 5.92 (1H, s,

170

H-6), 6.91 (1H, d, J = 1.8 Hz, H-2´), 6.94 (1H, dd, J =12.9, 1.8 Hz, H-6´), 6.95 (1H, d, J = 12.1

171

Hz, H-5´). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 42.4 (C-3), 55.8 (OCH3), 78.3 (C-2), 95.5

172

(C-6), 101.8 (C-10), 112.0 (C-2´), 114.3 (C-5´), 117.8 (C-6´), 125.7 (C-8), 131.5 (C-1´), 146.5

173

(C-3´), 147.9 (C-4´), 149.3 (C-9), 155.8 (C-7), 156.5 (C-5), 196.5 (C-4).

H NMR (600 MHz, DMSO-d6) δ (ppm): 2.72 (1H, dd, J = 17.1, 3.1 Hz, H-3eq); 3.15 (1H, dd,

174 175

norwogonin (5,7,8-trihydroxyflavone, 9)

176

1

177

5´), 8.16 (2H, m, H-2´,6´), 12.26 (1H, s, 5-OH). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 98.8

178

(C-6), 103.6 (C-10), 104.7 (C-3), 125.2 (C-8), 126.6 (C-2´, 6´), 129.1 (C-3´, 5´), 130.9 (C-1´),

179

132.0 (C-4´), 145.6 (C-9), 153.1 (C-5), 153.8 (C-7), 163.0 (C-2), 182.3 (C-4).

H NMR (600 MHz, DMSO-d6) δ: 6.30 (1H, s, H-6), 6.94 (1H, s, H-3), 7.59 (3H, m, H-3´, 4´,

180 181

4-hydroxy-6-phenyl-2H-pyran-2-one (10)

182

1

183

3´, 4´, 5´), 7.81-7.87 (2H, m, H- 2´, 6´). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 89.7 (C-3),

184

98.5 (C-5) 125.6 (C-2´, 6´), 129.3 (C-3´, 5´), 131.1 (C-1´), 131.2 (C-4´), 160.25 (C-4), 163.5

185

(C-6), 170.8 (C-2). HRESI-MS [M+H]+ (calculated/found) (m/z 189.0546/189.0545).

H NMR (600 MHz, DMSO-d6) δ: 5.40 (1H, m, H-3), 6.75 (1H, m, H-5), 7.49-7.54 (3H, m, H-

186



187

8-hydroxyapigenin (4´,5,7,8-tetrahydroxyflavone, 11)

188

1

189

8.01 (2H, m, H-2´,6´), 8.75 (1H, s, 8-OH), 10.33 (1H, s, 4´-OH), 10.46 (1H, s, 7-OH), 12.38

190

(1H, s, 5-OH).

191

10), 115.9 (C-3´, 5´), 121.4 (C-1´), 125.0 (C-8), 128.6 (C-2´, 6´), 145.5 (C-9), 153.0 (C-5),

192

153.4 (C-7), 161.1 (C-4´), 163.5 (C-2), 182.1 (C-4).

H NMR (600 MHz, DMSO-d6) δ: 6.26 (1H, s, H-6), 6.74 (1H, s, H-3), 6.93 (2H, m, H-3´, 5´),

13

C NMR (150 MHz, DMSO-d6) δ (ppm): 98.6 (C-6), 102.3 (C-3), 103.3 (C-

193 194

hypoletin (3´,4´,5,7,8-pentahydroxyflavone, 12)

195

1

196

8.2 Hz, H-5´), 7.49 (2H, d, J = 12.6 Hz, H-2´, 6´); 13C NMR (150 MHz, DMSO-d6) δ (ppm):

197

98.6 (C-6), 102.4 (C-3), 103.4 (C-10), 113.7 (C- 2´), 116.0 (C-5´), 119.2 (C-6´), 121.8 (C-1´),

198

125.1 (C-8), 145.6 (C-3´), 145.7 (C-5), 149.7 (C-4´), 153.0 (C-7), 153.3 (C-9), 163.8 (C-2),

199

182.1 (C-4).

H NMR (600 MHz, DMSO-d6) δ (ppm): 6.26 (1H, s, H-6), 6.63 (1H, s, H-3), 6.89 (1H, d, J =

200 201

RESULTS AND DISCUSSION

202

Screening test carried out on four red yeasts cultured on Sabouraud medium with

203

naringenin (1) as a model substrate lead to selection of Rhodotorula glutinis KCh735 strain,

204

which was capable for transformation of 1. The oleaginous species of Rhodotorula glutinis can

205

be potentially used for biodiesel production and also other important products, such as

206

carotenoids.20, 21

207

Our studies carried out on a larger scale with the use of four natural flavonoids, having

208

flavanone (1, 2), flavone (3, 4, 5), flavonol (quercetin), flavane (epicatechin) and isoflavone

209

(biochanin A) skeletons, have demonstrated the ability of this organism to perform

210

hydroxylation reaction of the first two flavonoid groups (Fig. 1). Hydroxylation reaction is the

211

capacity rarely observed in yeast. Preparative-scale biotransformations afforded seven


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212

metabolites (6-12). The structures of the products were identified and confirmed by comparison

213

of the spectroscopic data with those reported in literature.10, 11, 13-19

214

Naringenin (1) after 24 h was converted to carthamidin (6) and isocarthamidin (7) in a

215

ratio of 1 : 19, respectively (according to HPLC). The starting material was not observed. The

216

35.8 mg of mixture of 5 and 6 was isolated with the yield of 42.3%. These metabolites were

217

investigated by Miyake et al. by using of free radical-scavening system of 1,1-diphenyl-2-

218

picrylhydrazyl (DPPH) and the methyl linoleate oxidative system.11 In both of these assays

219

they exhibited higher antioxidant activity than the naringenin (1). The activity of the

220

isocarthamidin (7) was stronger that of carthamidin (6) and nearly equal to the α-tocopherol.

221

The both products of naringenin (1) biotransformation were also recognized and patented as a

222

antidiabetic agents.22 Recent study on the screening of new tyrosinase inhibitors resulted in the

223

identification of isocarthamidin (5,7,8,4´-tetrahydroxyflavanone, 7), and other orto-

224

hydroxylated flavonoids (6,7,4´-trihydroxyisoflavone, 7,8,4´-trihydroxyisoflavone and 5,7,8,4´-

225

tetrahydroxyisoflavone) as a potent tyrosinase inhibitors.23-25 C-6 and C-7 hydroxyl groups in

226

the A-ring strongly affect both the inhibitory strength and the inhibitory mode of the flavonoids

227

on mushroom tyrosinase. Inhibiting the tyrosinase activity has been the subject of many studies

228

because this enzyme is responsible among others for the undesirable hyperpigmentation in skin

229

and browning in fruits.26 In light of these information the yeast Rhodotorula glutinis which lead

230

regioselective hydroxylation in these key positions flavonoids may be a very useful tool for

231

obtaining natural inhibitors of tyrosinase.

232

The content of 6 and 7 in plants is very low27, therefore from a pharmaceutical point of view

233

the preparation of these valuable compounds in an efficient biotransformation of much cheaper

234

substrate (see supplementary data) is very promising. Biohydroxylation of 1 at position 6 or 8

235

was earlier described for filamentous fungi Aspergillus saitoi11, Aspergillus niger28 and for

236

yeast Rhodotorula marina.9,10 In the process using the yeast R. marina substrate conversion and



237

the ratio of products in the biotransformation mixture were comparable to results obtained for

238

the fungus A. niger (the maximum concentration of isocarthamidin was 21.7 mg/L and

239

carthamidin 21.5 mg/L, with the initial concentration naringenin equal to 60 mg/L), however 5-

240

fold more of the substrate can be introduced to the yeast culture (300 mg /L).

241

The results of biotransformations described in this article, concerning another species of

242

red yeast - Rhodotorula glutinis, indicate high similarity in metabolizing of 1 for both

243

organisms. The application of R. glutinis gave acceptable ratio of more valuable isocarthamidin

244

(7) to carthamidin (6) (1:19), but the most important advantage of the newly selected

245

biocatalyst is its ability to regioselective ortho-hydroxylation of other natural flavonoids. The

246

tested yeast was capable of converting flavonoids that belong to the flavanones and flavones

247

groups, but did not metabolize substrates having flavonol, flavane and isoflavone skeletons.

248

Biotransformations of hesperetin (2), chrysin (3), apigenin (4) and luteolin (5) are described

249

below.

250

The incubation of hesperetin (2) with Rhodotorula glutinis for 72 hours gave 8-

251

hydroxyhesperetin (8) (14.3 mg, 17.0%). The antioxidant activity of this compound

252

investigated by Miyake et al. is high, its ability to scavenge DPPH radicals is similar to α-

253

tocopferol, at a concentration of 10 µM was 63%.11

254

8-Hydroxyhesperetin (8) was previously obtained by biotransformation of hesperetin

255

(2) in the cultures of filamentous fungi Aspergillus saitoi11 and Mucor ramannianus ATCC

256

2628.14 Both of these transformations occurred with the low yield (3.5%, 0.03%) and - in the

257

case of Aspergillus saitoi - in a long process lasting 20 days. Our new method for preparing 8-

258

hydroksyhesperetin (8) by using the yeast Rhodotorula glutinis shortens the time of

259

transformation to three days and allows to obtain the product with the nearly five times higher

260

yield.


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261

Preparative scale 6-day fermentation of chrysin (3) yielded one metabolite -

262

norwogonin (9) (26.2 mg, 30.8%), which is the one of the active components in the root of

263

Scutellaria baicalensis. According to literature, 9 is more effective than wogonin (5,7-

264

dihydroxy-8-methoxy flavone) in induction of apoptosis in leukemia HL-60 cells.32 It is also

265

one of the most potent antimicrobial natural compound against multidrug-resistant bacteria

266

Acinetobacter baumannii.33 Thus, the above described experiment may serve after optimizing

267

as a method for obtaining valuable norwogonin (9) from chrysin (3) - a cheap source, in a one-

268

step simple biotransformation process.

269

In the described biotransformation it was also observed the presence of trace amounts of

270

the second product, which interestingly was the main product in the analogous process, in

271

which chrysin (3) was added to the culture as a solution in tetrahydrofuran. Transformation of

272

chrysin (3) added to the yeast culture in tetrahydrofuran for 48 hours led to 4-hydroxy-6-

273

phenyl-2H-pyran-2-one (10) (30.5 mg, 50.2%). The product had the molecular formula

274

C11H8O3 as indicated by HR-ESIMS of the [M+M]+ ion peak at m/z 189.0545 (calc. 189.0546).

275

The

276

signals what suggested degradation of substrate. Signals of B-ring had nearly the same

277

chemical shifts as recorded for chrysin (3). Three signals at the lower field belongs to C-ring

278

carbons: C-2 (170.8 ppm), C-6 (163.5 ppm) and C-4 (160.3 ppm). Further evidence came from

279

the 1H-NMR spectrum which showed only four signals from seven protons. The absence of two

280

singlets at 12.8 ppm (C5-OH), 10.92 ppm (C7-OH) and two doublets at 6.22 ppm (H-6) and

281

6.52 ppm (H-8), presented in the spectrum of the substrate, confirmed degradation of the A-

282

ring of chrysin (3). Correlations observed in the HMBC spectrum permitted the assignment of

283

the H-3 and H-5 protons of lactone product (Fig. 2). The aromatic A-ring proton appeared as a

284

multiplet at 6.75 ppm resonated with four carbons: at 89.7 ppm (C-3), 131.1 ppm (C-1´), 160.3

285

ppm (C-4) and 170.8 ppm (C-2), what suggested that it is bonded to the carbon atom C-5. In

13

C NMR spectrum differed from that of starting compound (3) in the reduction of four



286

turn the other A-ring proton appeared as a multiplet at 5.40 ppm resonated with three carbons:

287

at 98.5 ppm (C-5), 163.5 ppm (C-6) and 170.8 ppm (C-2). No correlation of this proton with

288

the C-1´ carbon atom indicates the considerable spatial distance between mentioned atoms and

289

unambiguously assigns it to the carbon C-3.

290

The chemical synthesis of 4-hydroxy-6-phenyl-2H-pyran-2-one (10) has been

291

described.18 No information is found on the direct use of the product 9, but it is considered that

292

compounds containing in their molecule the structure of 4-hydroxy-6-phenyl-2H-pyran-2-one

293

can be HIV protease inhibitors so that they can show antiviral activity.31 The simple one-pot

294

process for preparing 4-hydroxy-6-phenyl-2H-pyran-2-one (10) by biotransformation can be an

295

alternative to the mentioned three step chemical method.

296

Degradation of chrysin (3) in the culture of Rhodotorula glutinis, involving the break-

297

up of the A-ring, prompted us to investigate its course. The presence of the lactone moiety in

298

the product 10 allow to assume that an intermediate of this pathway have a hydroxyl group and

299

the C-8 position of flavone skeleton. This assumption is also consistent with the demonstrated

300

ability of yeast selected by us to ortho-hydroxylation of the A ring of other flavones and

301

flavanones. The OH groups makes the flavonoids capable of autooxidation under prolonged

302

aerobic environments, which becomes more rapid in alkaline conditions. The course of the A-

303

ring cleavage by Rhodotorula glutinis proceeds via an initial C-8 hydroxylation of chrysin (3)

304

to norwogonin (9), oxidative ring-fission followed by a hydrolase-type reaction to yield 4-

305

hydroxy-6-phenyl-2H-pyran-2-one (10).

306

Flavonoids were generally decomposed via C-ring fission.34,35 To the best our

307

knowledge degradation of flavonoids via A-ring cleavage was described only for bacteria from

308

the genus Pseudomonas. Pseudomonas sp. grown on (+)-catechin, oxidized dihydrogossypetin

309

(3´,4´,5,7,8-pentahydroxyflavanonol) by cleaving the A-ring to form 5-(3,4-dihydroxyphenyl)-

310

4-hydroxy-3-oxovalero-8-lactone and oxaloacetic acid.36 If the lactone - type intermediates


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311

have a C-3 hydroxyl group at an unsaturated site, they can be substrates for a second

312

dioxygenase

313

Hydroxyflavanones, such as taxifolin, were not oxidized.

and

underwent

further

degradation

by

Pseudomonas

putida.37

3-

314

Chrysin (4) biotransformations described above indicate a substantial influence of the

315

solvent in which the substrate is administered, on the rate of its metabolism. Addition of

316

terahydrofuran to the well - grown yeast culture accelerates chrysin (4) degradation.

317

8-Hydroxyapigenin (11) was the only product of the 6-day biotransformation of

318

apigenin (4). The amount of 10.9 mg of this compound was isolated with the yield of 12.9%.

319

Apigenin (4) is widely used as nutritional supplement. The rich source of 4 are among others

320

parsley and celery. 8-Hydroxyapigenin (11) significantly inhibited lung virus proliferation

321

when administered intranasally or orally to mice without being toxic against mice.38

322

Biotransformation of luteolin (3) was carried out for 36 hours and hypoletin (12) was

323

the only product isolated (21.3 mg, 25.3%). Hypoletin (12) is present in plants in small

324

quantities, and exhibits anti-inflammatory29 and antidiabetic activities.30

325

To sum up, Rhodotorula glutinis KCh735 was found to be the only one of tested red

326

yeast strains, which showed the ability of naringenin biotransformation in the preliminary

327

studies. This selected strain is capable of regioselective ortho-hydroxylation of the A-ring of

328

flavonoids, that is unique in the phylum of yeasts. Described biotransformations of natural

329

flavones and flavanones using this biocatalyst are the method that allows to specifically convert

330

cheap natural bioactive molecules into rare and pharmacologically improved metabolites.

331

Further optimization of fermentation conditions may provide an efficient route for massive

332

production of this valuable substances.

333 334 335



336

SUPPORTING INFORMATION

337

The Supporting Information is available free of charge on the ACS Publications website at

338

DOI:

339 340

Detailed spectroscopic and structural information of biotransformation substrates and products,

341

the approximate prices list of same used and obtained flavonoids (PDF).


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REFERENCES 1. Chahar, M. K.; Sharma, N.; Dobhal, M. P.; Joshi, Y. C. Flavonoids: A versatile source of anticancer drugs. Pharmacogn Rev. 2011, 5, 1-12. 2. Das, S.; Rosazza, J. P. Microbial and enzymatic transformations of flavonoids. J. Nat. Prod. 2006, 69, 499-508. 3. Wang A.; Zhang F.; Huang L.; Yin X.; Li H.; Wang Q.; Zeng Z.; Xie T. New progress in biocatalysis and biotransformation of flavonoids, J. Med. Plants Res. 2010, 4, 847856. 4. Cao, H.; Chen, X.; Jassbi, A. R.; Xiao J. Microbial biotransformation of bioactive flavonoids. Biotechnol. Adv. 2014, 33, 214-223. 5. Bommarius, A. S.; Riebel - Bommarius, B. R. Biocatalysis: Fundamentals and Applications. Wiley-VCH Verlag, Weinheim, 2004. 6. Siasos, G.; Tousoulis, D.; Tsigkou, V.; Kokkou, E.; Oikonomou, E.; Vavuranakis, M.; Basdra, E. K.; Papavassiliou, A. G.; Stefanadis, C. Flavonoids in atherosclerosis: an overview of their mechanisms of action. Curr. Med. Chem. 2013, 20, 2641-2660. 7. Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20, 933956. 8. Burda, S.; Oleszek, W. Antioxidant and antiradical activities of flavonoids. J. Agric. Food Chem. 2001, 49, 2774–2779. 9. Madej, A.; Popłoński, J.; Huszcza, E. Transformation of naringenin by Rhodotorula marina. Przemysł Chemiczny, 2012, 91, 856-859. 10. Madej, A.; Popłoński, J.; Huszcza, E. Improved oxidation of naringenin to carthamidin and isocarthamidin by Rhodotorula marina. Appl. Biochem. Biotechnol. 2014, 173, 6773.



11. Miyake, Y.; Minato, K.; Fukumoto, S.; Yamamoto, K.; Oya-Ito, T.; Kawakishi, S.; Osawa, T. New potent antioxidative hydroxyflavanones produced with Aspergillus saitoi from flavanone glycoside in citrus fruit. Biosci. Biotechnol. Biochem. 2003, 67, 1443-1450. 12. Kim, J.; Park, K.-S.; Lee, C.; Chong, Y. Synthesis of a complete series of O-methyl analogues of naringenin and apigenin. Bull. Korean Chem. Soc. 2007, 28, 2527-2530. 13. Obara, H.; Onodera, J.; Kurihara, Y.; Yamamoto, F. Synthesis of 2´,3´,4,4´,6´pentahydroxychalcone, an aglycone of carthamin, and its isomerization into 4´,5,6,7and 4´,5,7,8-tetrahydroxyflavanone, carthamidin and isocarthamidin. Bull. Chem. Soc. Jpn. 1978, 51, 3627-3630. 14. Herath, W.; Khan, I. A. Microbial metabolism. Part 13. Metabolites of hesperetin. Bioorg. Med. Chem. Lett. 2011, 21, 5784-5786. 15. Bilia, A.R.; Ciampi, L.; Mendez, J.; Morelli, I. Phytochemical investigation of Licania genus. Flavonoids from Licania pyrifolia. Pharm. Acta Helv. 1996, 71, 199-204. 16. Gurung, S.K.; Pyo Kim, H.; Park, H. Inhibition of prostaglandin E2 production by synthetic wogonin analogs. Arch. Pharm. Res. 2009, 32, 1503-1508. 17. Tomimori, T.; Miyaichi, Y.; Imoto, Y.; Kizu, H.; Tanabe, Y. Studies on the constituents of Scutellaria species. II. On the flavonoid constituents of the root of Scutellaria baicalensis Georgi (2). Yakugaku Zasshi. 1983, 103, 607-611. 18. Schmidt, D.; Conrad, J.; Klaiber, I.; Beifuss, U. Synthesis of the bis-potassium salts of 5-hydroxy-3-oxopent-4-enoic acids and their use for the efficient preparation of 4hydroxy-2H-pyran-2-ones and other heterocycles. Chem. Commun. 2006, 4732–4734. 19. Teles, Y. C.; Horta, C. C.; de Fátima Agra, M.; Siheri, W.; Boyd, M.; Igoli, J. O.; Gray, A. I.; de Fátima Vanderlei de Souza, M. New sulphated flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae). Molecules 2015, 20, 20161-20172.


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20. Bhosale, P.; Gadre, R. V. Production of β-carotene by a Rhodotorula glutinis mutant in sea water medium. Biores. Technol. 2001, 76, 53-55. 21. Xue, F.; Miao, J.; Zhang, X.; Tan, T. A new strategy for lipid production by mix cultivation of Spirulina platensis and Rhodotorula glutinis. Appl. Biochem. Biotechnol. 2010, 160, 498-503. 22. Satoru, K.; Tsumoru, W.; Yoshiharu, I.; Atsuko E. Antidiabetic agent, JP 2008133192 A, 2007. 23. Chang, T. S.; Ding, H. Y.; Lin, H. C. Identifying 6,7,4´-trihydroxyisoflavone as a potent tyrosinase inhibitor. Biosci. Biotechnol. Biochem. 2005, 69, 1999-2001. 24. Chang, T. S. Two potent suicide substrates of mushroom tyrosinase: 7,8,4´trihydroxyisoflavone and 5,7,8,4´-tetrahydroxyisoflavone. J. Agric. Food Chem. 2007, 55, 2010-2015. 25. Chang, T. S.; Lin, M. Y.; Lin, H. J. Identifying 8-hydroxynaringenin as a suicide substrate of mushroom tyrosinase. J. Cosmet. Sci. 2010, 61, 205-210. 26. Rescigno, A.; Sollai, F.; Pisu, B.; Rinaldi, A.; Sanjust, E. Tyrosinase inhibition: General and applied aspects, J. Enzyme Inhib. Med. Chem. 2003, 17, 207-218. 27. Werawattanachai, N.; Kaewamatawong, R. Chemical constituents from Parinari anamense. Biochem. Syst. Ecol. 2010, 38, 836-838. 28. Xu, J.; Yang, L.; Wang, Z. T.; Zhao, S. J.; Hu, Z. B. An efficient way from naringenin to carthamidine and isocarthamidine by Aspergillus niger. World J. Microbiol. Biotechnol. 2012, 28, 1803-1806. 29. Ferrándiz, M. L.; Alcaraz, M. J. Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids. Agents Actions. 1991, 32, 283-288.



30. Kato, A.; Nasu, N.; Takebayashi, K.; Adachi, I.; Minami, Y.; Sanae, F.; Asano, N.; Watson, A. A.; Nash, R. J. Structure-activity relationships of flavonoids as potential inhibitors of glycogen phosphorylase. J. Agric Food Chem. 2008. 56, 4469-4473. 31. Prasad, V. J. V. N.; Tummino, P. J.; Ferguson, D.; Saunders, J.; Roest, S. V.; McQuade, T. J.; Heldsinger, A.; Reyner, E. L.; Stewart, B. H.; Guttendorf, R. J.; Para, K. S.; Lunney, E. A.; Gracheck, S. J.; Domagala, J. M. Nonpeptidic HIV protease inhibitors: 4-hydroxy-pyran-2-one inhibitors with functional tethers to P1 phenyl ring to reach S3 pocket of the enzyme. Biochem. Biophys. Res. Commun. 1996, 221, 815-820. 32. Chow, J. M.; Huang, G. C.; Shen, S. C.; Wu, C. Y.; Lin, C. W.; Chen, Y. C. Differential apoptotic effect of wogonin and nor-wogonin via stimulation of ROS production in human leukemia cells. J. Cell Biochem. 2008, 103, 1394-404. 33. Miyasaki, Y.; Rabenstein, J. D.; Rhea, J., Crouch, M. L.; Mocek, U. M.; Kittell, P. E.; Morgan, M. A.; Nichols, W. S.; Van Benschoten, M. M.; Hardy, W. D.; Liu, G. Y. Isolation and characterization of antimicrobial compounds in plant extracts against multidrug-resistant Acinetobacter baumannii. PLoS One, 2013, 8, e61594. 34. Rao, J. R.; Cooper, J. E. Rhizobia catabolize nod gene-inducing flavonoids via C-ring fission mechanisms. J. Bacteriol. 1994, 176, 5409-5413. 35. Winter, J.; Moore, L. H.; Dowell, V. R.; Bokkenheuser, V. D. C-ring cleavage of flavonoids by intestinal bacteria. Appl. Environ. Microbiol. 1989, 55, 1203-1208. 36. Jeffrey, A. M.; Jerina, D. M.; Self, R.; Evans, W. C. The bacterial degradation of flavonoids. Oxidative fission of the A-ring of dihydrogossypetin by a Pseudomonas sp. Biochem. J. 1972, 130, 383-390. 37. Schultz, E.; Engle, F. E.; Wood, J. M. New oxygenases in the degradation of flavones and flavanones by Pseudomonas putida. Biochem. 1974, 13, 1768-1776.


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38. Nagai, T.; Miyaichi, Y.; Tomimori, T.; Suzuki, Y.; Yamada, H. In vivo anti-influenza virus activity of plant flavonoids possessing inhibitory activity for influenza virus sialidase. Antiviral Res. 1992, 19, 207-217.

Funding Information The Project was supported by Wroclaw Centre of Biotechnology, The Leading National Research Centre Programme (KNOW) for years 2014-2018.



FIGURE CAPTIONS Figure 1. Transformation of naringenin (1), hesperetin (2), chrysin (3), apigenin (4) and luteolin (5) by Rhodotorula glutinis. Figure 2. Key HMBC correlations for compound 10. Figure 3. Putative pathway of chrysin (3) degradation via A-ring cleavage conducted by Rhodotorula glutinis.


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Figure 1



Figure 2


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Figure 3




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Regioselective ortho-Hydroxylations of Flavonoids ... - ACS Publications

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