Microbial Transformations of Two Beyerane-Type Diterpenes by Cunninghamella echinulata

Cunninghamella echinulata

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Microbial Transformations of Two Beyerane-Type Diterpenes by

Yu-Qi Gao, Ruoxin Li, Wei-Wei Wang, Shoei-Sheng Lee, and Jin-Ming Gao*

Cite This: J. Agric. Food Chem. 2020, 68, 4624 −4631

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sı Supporting Information

ABSTRACT: Microbial transformations of two tetracyclic beyerane-type diterpenes, ent-16β-oxobeyeran-19-oic acid (1) and its

chemical reduction product, ent-16β-hydroxybeyeran-19-oic acid (2), by the ﬁlamentous fungus Cunninghamella echinulata ATCC

688a yielded eight metabolites (3−10). Incubation of the substrate 2 with C. echinulata aﬀorded three new hydroxylated ones (3−

) along with two known ones (6−7), while incubation of 1 gave three known ones (8−10). The new compounds were

characterized by 1D and 2D NMR as well as HRESIMS analysis, and the stereostructures of 3 and 4 were conﬁrmed by X-ray

crystallography. The bioreactions were involved not only in stereoselective incorporation of hydroxyl groups at inert positions C-7,

9, -12, and -14 of the two beyerane diterpenes but also in glucosidation at C-19 of 2. This is the ﬁrst report on the biotransformation

of the diterpenes by using C. echinulata. All compounds were assayed for their α-glucosidase inhibitory, neurotrophic, anti-

inﬂammatory, and phytotoxic activity, and only in neurotrophic assay compounds, 2 and 9 were found to display nerve growth

factor-mediated neurite-outgrowth promoting eﬀects in PC12 cells; the others were inactive.

KEYWORDS: beyerane diterpenes, biotransformation, Cunninghamella echinulata, biocatalysis, hydroxylation

INTRODUCTION

formation of isosteviol (1) and new biological activities have so

■

0−14

far been limited.

Stevioside, isolated from the leaves of Stevia rebaudiana (family

Asteraceae), is commonly utilized as a non-caloric natural

sugar substitute in many kinds of foods and as food

In order to study the metabolism of the two tetracyclic

beyerane-type diterpenes, isosteviol (ent-16β-oxobeyeran-19-

oic acid, 1) and its chemical reduction product (ent-16β-

hydroxybeyeran-19-oic acid, 2) and to predict their likely

mammalian metabolites, in the current work, biotransforma-

tion of these two diterpenoids by the fungal strain Cunning-

hamella echinulata ATCC 8688a was carried out, leading to the

isolation and identiﬁcation of eight metabolites (3−10)

(Scheme 1) from the fermentation broth. Herein, we deal

with the microbial conversion of 1 and 2 and isolation,

structure elucidation, and biological evaluation of several

metabolites.

supplements in many countries. Stevioside is a complex

diterpenoid glycoside molecule and was hydrolyzed under

alkaline or acidic conditions to generate the stevioside

aglycone, ent-kaurane diterpene steviol or ent-beyerane

diterpene isosteviol (1), respectively. Steviol and isosteviol

have been demonstrated to have many biological properties,

such as anti-inﬂammatory, anti-hypertensive, anti-diabetes,

−3

anticancer, and neuroprotective properties.

Microbial transformations have been proved a useful means

for incorporating hydroxyl groups at non-activated positions in

the diterpenoid or steroid scaﬀold for structural modiﬁca-

MATERIALS AND METHODS

General Experimental Procedures. The speciﬁc rotation was

measured on a JASCO DIP-370 polarimeter. IR spectra were

recorded on a JASCO FT/IR-410 FT-IF spectrometer. 1D- and

■

−7

tions.

Diterpenoids are a diverse group of bioactive

components found in several plant-derived extracts. Structure

modiﬁcations of the diterpenoids to increase their pharma-

ceutical relevance can be eﬃciently performed by the use of

biotransformation processes utilizing microbes or isolated

D-NMR spectra were obtained on a Bruker Avance 400 or Avance

III 500 or 600 Cryo-probe spectrometer (Bruker Instruments, UK),

operating at 400, 500, and 600 MHz ( H) and 100, 125, and 150

MHz ( C) in methanol-d using the signals of the residual solvent

enzymes. In addition, the microbial biotransformation has

many advantages: to surmount the problems present in organic

synthesis reactions; to enhance/reduce the biological activity/

toxicity proﬁles of the drugs; and to mimic mammalian drug

protons (δ_H3.30) and carbons (δ_C49.0) as internal standards.

Electrospray ionization mass spectra (ESIMS) were recorded on a

Bruker Daltonics Esquire 2000 ESI-Ion Trap Mass Spectrometer or

metabolism. Taking into account that small changes in a

molecular structure may signiﬁcantly aﬀect its bioactivities and

properties, although these two diterpenoids shows many

attractive bioactivities, their poor water solubility limits further

application. In spite of several semisynthesis and structure

modiﬁcations of isosteviol that have been described,

investigations on several diterpenoids derived from biotrans-

Received: January 28, 2020

Revised: March 23, 2020

Accepted: March 25, 2020

Published: March 27, 2020

2020 American Chemical Society

J. Agric. Food Chem. 2020, 68, 4624−4631

624

Journal of Agricultural and Food Chemistry

Article

Scheme 1. Chemo- and Biotransformations of Stevioside and Its Derivatives Isosteviol (1) and ent-16β-Hydroxybeyeran-19-oic

Acid (2)

Bruker Daltonics microTOF II Mass Spectrometer and Thermo

Scientiﬁc Q Exactive HF Orbitrap-FTMS. Column chromatography

control (substrate in organism-free culture medium) and culture

control (organisms in substrate-free culture medium), were incubated

under the same conditions to show the stability of the substrate in the

process of incubation. The results of the transformation were

monitored by TLC. Similarly, a preparative-scale biotransformation

by C. echinulata ATCC 8688a was conducted in 500 mL Erlenmeyer

ﬂasks containing 200 mL of potato-dextrose media, and the

microorganisms were precultured for 2 d.

Bioconversion of 2 by C. echinulata ATCC 8688a was carried out

following the aforementioned procedures of 1. Compound 2 (2.8 g)

was dissolved in acetone (280 mL), and ﬁnal concentrations of 0.1

mg/mL were distributed among the medium.

(

CC) was carried out on 200−300 mesh silica gel (Qingdao Marine

Chemical Ltd., Qingdao, P. R. China), RP-18 (40−63 μM; Merck,

Darmstadt, Germany), and Sephadex LH-20 (20−100 μm;

Amersham Biosciences, Uppsala, Sweden). Thin layer chromatog-

^r^a^p^h^y⁽^T^L^C⁾^aⁿ^a^l^y^sⁱ^s^w^a^s^p^e^r^f^o^r^m^e^d^oⁿ^p^r^e^c^o^a^t^e^d^Sⁱ^g^e^l⁶⁰^G^F²54

(

Qingdao Marine Chemical Ltd.). Developed chromatograms were

visualized by spraying developed plates with 5% H SO in EtOH and

then by heating until maximum development of the spot colors. All

reagents and solvents used were of analytical grade.

Semisynthesis of Compounds 1 and 2. The substrates

isosteviol (1) and ent-16β-hydroxybeyeran-19-oic acid (2) were

Extraction and Isolation. The fermentation cultures of 2 were

pooled and ﬁltered, and the obtained mycelia were ultrasonically

washed three times with EtOAc. The ﬁltrates were extracted thrice

with an equal volume of EtOAc. The combined organic layer was

dried over anhydrous Na SO , ﬁltered, and evaporated in vacua to

0,12

derived from stevioside as described previously

and were

conﬁrmed by NMR and ESIMS data.

Microorganisms and Culture Medium. The microbes

employed in this work included Geotrichum candidum ATCC

34614, Rhizopus stolonifer ATCC 6227a, Rhizopus chinensis CCTCC

give a brown oil. The residue (3.98 g) was submitted to silica gel CC

M201021, Apergillus ochraceus, Aspergillus niger CICC2377, Cunning-

hamella blakesleeana CGMCC 3.970, Cunninghamella elegans ATCC

and eluted with a gradient of CHCl −CH OH (100:0, 98:2, 95:5,

2:8, 90:10, 80:20) to provide fractions A−F. Fraction C was

36112, C. echinulata ATCC 8688a, Penicillium citrinum ATCC 8506,

subjected to CC over silica gel and eluted with CHCl −CH OH

Mucor mucedo CBS228.29, and Mucor wutungchiao CBS 111557. They

were screened for their abilities to convert substrates 1 and 2 in

preliminary work. These microorganisms were maintained on potato-

dextrose agar media slants at 4 °C. Before the conversion experiments,

all organisms were freshly subcultured. Preliminary screening

bioconversion of 1 or 2 by C. echinulata ATCC 8688a was carried

out by a two-stage fermentation procedure in potato-dextrose

medium. The medium was autoclaved at 121 °C for 30 min before

use.

Culture Conditions and Biotransformation Procedures. The

microorganisms were inoculated in 250 mL Erlenmeyer ﬂasks

containing 50 mL of potato-dextrose medium. The ﬂasks were

incubated at 28 °C on a rotary shaker at 180 rpm for 48 h. The

solution of compound 1 in acetone (10 mg/mL) was added to each

ﬂask. Final concentration of 1 in this media was 0.1 mg/mL, and

incubation was continued for 5 d. Both parallel controls, the substrate

(

95:5) to provide four fractions (Fr.C1−4). Fr.C1 (361 mg) was

fractionated by a Sephadex LH-20 column (CH OH), silica gel CC

with CHCl −CH OH (98:2), followed by recrystallization from

CH OH, to aﬀord compound 3 (41 mg). Fr.C3 (202 mg) was

fractionated by a Sephadex LH-20 column with CHCl −CH OH

(

1:1), silica gel CC with CHCl −CH OH (96:4), followed by a RP-

8 column using CH OH−H O (30:70, 40:60, 50:50, 60:40, 30:70,

0:20). Compound 4 (16 mg) from subfraction eluted with

CH OH−H O (50:50) was puriﬁed by Sephadex LH-20 CC

(CH

OH), and 6 (16.6 mg) from subfraction eluted with

Sephadex LH-20 column (CHCl

(CH OH−H O, 94:6) followed by a RP-18 column using CH

O (60:40), furnishing 5 (22 mg). Fractions D and E combined

OH−H

O (60:40). Fr.C4 (213 mg) was fractionated by a

−CH OH, 1:1), a silica gel column

OH−

(295 mg) were chromatographed over a Sephadex LH-20 column

625

J. Agric. Food Chem. 2020, 68, 4624−4631

Journal of Agricultural and Food Chemistry

Article

Table 1. H NMR Spectroscopic Data for Compounds 2−7

no.

δ_H(J in Hz)

1.70−1.76 m

1.73−1.79 m

1.77−1.81 m

1.69−1.73 m

1.72−1.77 m

1.70 d (9.5)

0.92 dd (13.4, 4.1)

1.31−1.35 m

1.38−1.42 m

1.85−1.90 m

0.86 s

1.29−1.32 m

1.84−1.91 m

0.93−0.99 m

2.11 d (13.2)

1.01−1.04 m

0.84−0.89 m

0.90−0.97 m

1.35−1.38 m

1.88−1.90 m

0.92 dd (13.4, 3.8)

1.32−1.38 (m)

1.36−1.43 m

1.31−1.38 m

.81−1.90 (m)

1.83−1.88 m

1.78−1.90 m

0.99−1.03 (m)

.11, d (13.2)

0.99 td (13.3, 4.1)

0.98−1.04 m

0.99−1.09 m

0.99−1.04 m

1.73−1.79 m

2.12 d (13.3)

2.11 d (13.2)

2.18 d (13.1)

5β

1.06 dd (11.9, 2.1)

1.71−1.76 m

0.98−1.04 m

0.99−1.09 m

1.11 dd (11.5, 3.04)

1.72−1.87 (m)

1.71−1.79 m

1.66−1.73 m

1.67−1.73 m

1.61−1.69 m

1.74−1.84 m

.97−2.0 m

7α

7β

9β

1.48−1.52 m

1.46 td (12.8, 5.8)

1.14−1.18 m

1.45−1.52 m

1.43−1.49 m

1.23−1.31 m

1.49−1.53 m

1.31−1.36 m

1.34−1.38 m

3.22 dd (8.1, 2.8)

1.13−1.15 m

1.55−1.59 m

1.73−1.80 m

1.15−1.19 m

1.75−1.80 m

0.81 dd (7.6, 1.7)

1.84−1.88 m

1.31−1.35 m

0.97−1.03 m

1.09 dd (11.9, 1.9)

0.99−1.09 m

0.98−1.03 m

1.51−1.56 m

1.31−1.35 m

1.29−1.32 m

1.61−1.69 m

1.51−1.59 m

.62−1.70 m

2.01−2.06 m

1.84−1.91 m

1.83−1.85 m

1.64−1.71 m

1.13−1.19 m

1.43−1.49 m

1.57−1.60 m

0.82 dd (11.6, 2.5)

1.13−1.15 m

1.13−1.18 m

1.78 m

1.27 dd (11.0, 2.5)

.75−1.79 m

3.74 br t (1.2)

0.90−1.93 m

1.60−1.64 m

1.83−1.85 m

1.28 dd (11.5, 2.7)

2.99 s

.94 dd (11.6, 3.2)

1.66−1.70 m

1.76−1.79 m

1.66−1.73 m

2.25 br t (11.1)

1.38−1.42 m

1.72−1.77 m

1.64−1.71 m

.83−1.87 m

2.01−2.06 m

1.84−1.91 m

1.83−1.85 m

1.82−1.89 m

6β

7-CH₃

8-CH₃

0-CH₃

′

3.79 dd (10.8, 4.6)

0.89 s

3.77 dd (10.8, 4.2)

3.88 dd (10.9, 4.4)

3.74 dd (7.3, 3.4)

4.11 t (7.4)

0.95 s

1.17 s

0.87 s

3.78 dd (11.4, 4.6)

0.90 s

1.17 s

1.06 s

0.99 s

1.17 s

0.84 s

0.89 s

1.19 s

0.84 s

0.88 s

1.20 s

0.84 s

1.17 s

0.85 s

5.4 d (8.0)

3.30−3.37 m

3.39 d (1.7)

3.34−3.38 m

3.32−3.38 m

3.80 td (3.7, 1.7)

.67 td (4.4, 4.3)

Measured at 500 MHz in methanol-d . Measured at 600 MHz in methanol-d . Measured at 400 MHz in methanol-d . Overlapping.

(

CHCl −CH OH, 1:1) and silica gel CC elutingwith CHCl −

NMR data: see Tables 1 and 2; ESIMS m/z: 359.1 [M + Na] , 335.1

−

CH OH (90:10) to yield 7 (4 mg).

[M−H] ; HRESIMS m/z: 335.2232 [M − H] (calcd for C H O ,

20 31

The procedures were similar to those described above. From 100

mg of 1, a crude extract was obtained, which was separated by silica

gel CC eluting with a gradient of petroleum ether−acetone. Eluting

with petroleum ether−acetone (9:1) led to the recovery of substrate 1

335.2228). All NMR data were in agreement with the literature

data.

ent-16β-Hydroxybeyeran-19-α-D-glucopyranosyl ester (7): white

solid powder (MeOH); [α]_D−40 (c 0.3, MeOH); IR (KBr): ν

3420 (OH), 1725 (ester), 1073 cm ; C and H NMR data: see

Tables 1 and 2; ESIMS m/z: 505.2 [M + Na] , 184.9 [Glu + Na] ,

481.0 [M − H] , 319.0 [M − Glu] ; HRESIMS m/z: 481.2809 [M −

H] (calcd for C H O , 481.2805). All NMR data were in

max

−

(

50 mg). Further elution with petroleum ether−acetone (8:2) gave

metabolites 8 (16 mg), 9 (3 mg), and 10 (3 mg).

−

ent-9α,16β-Dihydroxybeyeran-19-oic acid (3): white needle crystal

−

(

MeOH−EtOAc); [α]_D−45 (c 0.3, MeOH); IR (KBr): ν 3476,

max

26 41

−

384 (OH), 2932, 2874, 1695 (COOH), 1455 cm ; C and H

accordance with the literature data.

NMR data: see Tables 1 and 2; ESIMS m/z: 359.2 [M + Na] , 335.0

ent-9α-Hydroxy-16-ketobeyeran-19-oic acid (8): white needle

crystal (MeOH); IR (KBr) ν_m_a_x: 3504, 3448 (OH), 2995, 2922,

2867, 1719 (CO), 1689 (COOH), 1451, 1254, 1142 cm ; C NMR

(125, acetone-d ) data: see Table 2; ESIMS m/z: 357.1 [M + Na] ,

335.1 [M + H] , 317.1 [M − OH] . All NMR data were concordant

−

[

M − H] ; HRESIMS m/z: 335.2232 [M − H] (calcd for

−

1 13

C H O , 335.2228).

ent-12α,16β-Dihydroxybeyeran-19-oic acid (4): white needle

−

crystal (MeOH); [α]_D−71 (c 0.3, MeOH); IR (KBr): ν 3491

(

max

−

OH), 2946, 2845, 1702 (COOH), 1245 cm ; C and H NMR

with the literature data.

data: see Tables 1 and 2; ESIMS m/z: 359.1 [M + Na] , 335.1 [M −

X-ray Crystallographic Data for Compounds 3 and 4. The

−

H] ; HRESIMS m/z: 335.2238 [M − H] (calcd for C H O ,

crystals of 4 were obtained from MeOH−H O (3:1) and those of 3

35.2228).

from MeOH. The method used for structure analysis and the detailed

crystallographic data are described in the Supporting Information.

Crystal data for 3: C H O , M = 368.50, colorless crystals,

ent-7β,16β-Dihydroxybeyeran-19-oic acid (5): white needle crystal

(

MeOH); [α]_D²³−60 (c 0.3, MeOH); IR (KBr): ν 3413 (OH),

max

21 36

−1

937, 2869, 1701 (COOH), 1455, 1193, 1069 cm ; C and H

orthorhombic, a = 8.0964(9) Å, b = 14.0282(13) Å, c = 17.5127(17)

Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1989.1(3) Å , space group

P2 2 2 , λ(Mo Kα) = 0.71073 Å, T = 298(2) K, Z = 4, μ = 0.086

mm , 10062 reﬂections collected, 2024 independent reﬂections (R

= 0.0338). Final R indices (I > 2σ(I)): R = 0.0366, wR = 0.0806.

Final R indices (all data): R = 0.0561, wR = 0.0949. Goodness of ﬁt

1 2

on F : 1.078. Flack parameter: −3.1(18). Crystallographic data for 3

NMR (150, 600 MHz, CD OD) data: see Tables 1 and 2; ESIMS m/

−

z: 359.2 [M + Na] , m/z: 335.1 [M − H] ; HRESIMS m/z: 335.2231

−

−1

[

M − H] (calcd for C H O , 335.2228).

int

ent-14β,16β-Dihydroxybeyeran-19-oic acid (6): white needle

1 2

crystal (MeOH); [α]_D−54 (c 0.3, MeOH); IR (KBr): ν 3452

(

max

−

OH), 2951, 2926, 2844, 1697 (COOH), 1460 cm ; C and H

626

J. Agric. Food Chem. 2020, 68, 4624−4631

Journal of Agricultural and Food Chemistry

Article

Table 2. ¹³C NMR Spectroscopic Data for Compounds 2−8

no.

δ_Ctype

41.3 t

20.1 t

39.2 t

44.6 s

58.4 d

23.0 t

43.0 t

43.1 s

57.4 d

39.4 s

21.4 t

34.9 t

43.2 s

56.6 t

43.6 t

81.0 d

25.4 q

29.6 q

181.7 s

13.9 q

38.9 t

20.1 t

37.9 t

44.8 s

49.6 d

22.9 t

33.0 t

47.8 s

78.9 s

44.5 s

26.8 t

31.3 t

42.8 s

49.3 t

45.8 t

80.2 d

25.2 q

29.8 q

182.0 s

17.4 q

41.0 t

20.0 t

39.2 t

44.7 s

58.4 d

23.0 t

42.6 t

43.6 s

52.3 d

38.9 s

29.7 t

72.3 d

47.6 s

49.7 t

42.8 t

81.4 d

22.2 q

29.6 q

181.8 s

13.9 q

40.9 t

20.0 t

39.0 t

44.4 s

56.6 d

32.0

76.6 d

49.1 s

55.4 d

39.4 s

21.1 t

34.7 t

42.5 s

51.1 t

33.7 t

80.9 d

25.4 q

29.5 q

181.5 s

14.0 q

41.4 t

20.1 t

39.1 t

44.6 s

58.0 d

22.3 t

37.4 t

47.6 s

56.9 d

39.4 s

21.0 t

34.3 t

47.7 s

79.6 d

41.4 t

92.9 d

20.2 q

29.6 q

181.7 s

14.2 q

41.1 t

20.0 t

39.0 t

45.1 s

58.9 d

22.8 t

43.0 t

43.1 s

57.4 d

39.4 s

21.4 t

35.0 t

43.2 s

56.6 t

43.4 t

81.1 d

25.4 q

29.3 q

178.2 s

14.3 q

95.6 d

74.0 d

78.7 d

71.1 d

78.6 d

32.4 t

20.2 t

38.6 t

41.1 s

48.8 d

22.6 t

37.1 t

48.6 s

76.7 s

44.1 s

26.8 t

33.8 t

45.1 s

48.3 t

50.6 t

220.7 s

19.7 q

29.5 q

179.3 s

17.0 q

1′

2′

3′

4′

5′

6′

62.4 t

Measured at 125 MHz in methanol-d . Measured at 150 MHz in methanol-d . Measured at 100 MHz in methanol-d . Measured at 125 MHz in

acetone-d₆.

have been deposited with the Cambridge Crystallographic Data

Center as supplementary publication number CCDC-773007.

Crystal data for 4: C H O , M = 368.50, colorless needles,

without treatment were used as a negative control. Cells treated with

nerve growth factor (NGF; 20 ng/mL) were employed as a positive

control. One concentration experiment was repeated in three wells.

After further 72 h incubation, neurite outgrowth in PC-12 cells was

observed under an inverted microscope, and ﬁve images were chosen

randomly under a microscope for each well. At least 100 cells in each

of ﬁve randomly separated ﬁelds were scored, and the cells with

neurites equal to or greater than the length of one cell body were

positive for neurite outgrowth and expressed as a percentage of the

total cell number in ﬁve ﬁelds.

orthorhombic, a = 6.7541(8) Å, b = 13.6991(17) Å, c = 21.781(3) Å;

α = 90.00°, β = 90.00°, γ = 90.00°, V = 2015.3(4) Å , space group

P2 2 2 , λ(Mo Kα) = 0.71073 Å, T = 296.15(2) K, Z = 4, μ = 0.085

1 1

−

mm , 10,615 reﬂections collected, 3824 independent reﬂections (R

int

0.0194). Final R indices (I > 2σ(I)): R = 0.0418, wR = 0.1184.

1 2

Final R indices (all data): R = 0.0471, wR = 0.1238. Goodness of ﬁt

on F : 1.065. Flack parameter: −0.4(4). Crystallographic data for 4

have been deposited with the Cambridge Crystallographic Data

Center as supplementary publication number CCDC-804761.

α-Glucosidase Inhibitory Assay. The α-glucosidase inhibition

NO Inhibition Assay. The cell viability and inhibition of NO

production in BV-2 cells were assayed as reported previously.

Phytotoxicity Assay. Phytotoxic activity was tested by the

17,18

23,24

test of the compounds was carried out as reported previously.

Brieﬂy, Saccharomyces cerevisiae α-glucosidase was supplied by Sigma

EC 3.2.1.20). A solution of the enzyme in 200 μL of 10 mM

previously described method.

(

RESULTS AND DISCUSSION

phosphate buﬀer (pH 6.80) was incubated with 12 μL of the

compound tested in dimethylsulfoxide at 37 °C for 5 min. After the

addition of 36 μL of 4-nitrophenyl α-D-glucopyranoside, the reaction

was maintained at 37 °C for 40 min. The released 4-nitrophenol

amount was examined at 400 nm. The test was carried out with ﬁve

diﬀerent concentrations around the IC₅₀values. In each set of

experiments, the bioassay was conducted in triplicate. The IC values

■

Initial screening biotransformation of 11 organisms indicated

that the fungal strain C. echinulata ATCC 8688a was able to

transform substrate 1 or 2 into several metabolites. As a result,

they were selected for detailed studies of the scale-up

conversion of 2. Preparative incubation of the substrates, 1

and 2, with C. echinulata resulted in the isolation of three new

metabolites, 3−5, as well as ﬁve known ones, 6−10 (Scheme

were calculated as described previously.

Neurite-Outgrowth Assay. The cell viability assay was

performed to examine the cytotoxicity of the tested compounds in

PC-12 cells (Shanghai Institutes for Biological Sciences, Shanghai)

1). Assignments of all of the H and C NMR data for the new

metabolites (Tables 1 and 2) described here were done from

9,20

according to the MTT assay.

Morphological analysis and

DEPT, H− H COSY, HSQC, HMBC, and NOESY

correlations. The known compounds were determined to be

ent-14β,16β-dihydroxybeyeran-19-oic acid (6), ent-16β-hy-

droxybeyeran-19-β-D-glucopyranosyl ester (7), ent-9α-hy-

droxy-16-ketobeyeran-19-oic acid (8), ent-7α-hydroxy-16-

ketobeyeran-19-oic acid (9), and ent-7β-hydroxy-16-ketobeyer-

quantiﬁcation of neurite-bearing cells were carried out by using a

phase-contrast microscope according to previous reported proce-

dures. Brieﬂy, PC-12 cells were seeded on poly-L-lysine-coated 24-

well plates (2 × 10 cells/mL) in DMEM containing 10% HS, 5%

FBS, 100 μg/mL streptomycin, and 100 IU/mL penicillin at 37 °C

under 5% CO and a humidiﬁed atmosphere of 95% air for 24 h. Cells

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Journal of Agricultural and Food Chemistry

Article

an-19-oic acid (10) by comparison of the NMR and ESIMS

10−12,16

data with the literature.

Metabolite 3 showed the molecular formula of C H O , as

−

deduced from its HRESIMS m/z: 335.2232 [M − H] and C

NMR data, suggesting that 3 contained one more oxygen atom

than 2. The IR spectrum showed absorptions typical of

−

carbonyl (1695 cm ) and hydroxyl groups (3476, 3384

−

cm ), revealing that a hydroxylation had taken place and that

the carboxylic groups were not aﬀected. The H NMR

spectrum of 3 (Table 1) displayed three methyl signals

characteristic of H -17 (δ 0.90), H -20 (δ 1.06), and H -18

(

δ_H1.17). The C NMR (DEPT) spectra of 3 (Table 2)

displayed the presence of 20 carbon signals including three

tertiary methyls, nine methylenes, two methines, and six

quaternary carbons. In comparison with the H and C NMR

spectra of 2 and 3, one CH signal at δ 57.4 and δ 0.97−1.03

in 2 was replaced by one hydroxyl-bearing carbon signal at δ_C

8.9 in 3, suggesting the new OH group to be located at C-9.

Furthermore, in the C NMR spectrum, the resonances of C-

, C-9, C-10, and C-11 shifted signiﬁcantly downﬁeld from δ_C

3.1 to 47.8, from δ 57.4 to 78.9, from δ 39.3 to 44.5, and

from δ 21.4 to 26.9, respectively, and the resonances of C-1,

C-7, and C-14 shifted upﬁeld from δ 41.3 to 38.9, from δ_C

3.0 to 33.0, and from δ_C56.5 to 49.3, respectively.

Consequently, the tertiary hydroxyl group was located at C-

. This deduction was conﬁrmed by the HMBC correlations of

H -1, H -11, H -12, H -14, and H -20 with C-9 (Figure 1).

Furthermore, the structure of 3 was conﬁrmed by X-ray

crystallographic data (Figure 2). Therefore, 3 was assigned as

ent-9α,16β-dihydroxybeyeran-19-oic acid.

Metabolite 4 showed the molecular formula of C H O , as

−

deduced from its HRESIMS m/z: 335.2238 [M − H] and C

NMR spectra, indicating one more oxygen atom than 2. The

IR spectrum showed absorptions typical of carbonyl (1702

Figure 1. Selected HMBC, NOESY, and COSY correlations for 3−5.

−

−1

cm ) and hydroxyl groups (3491 cm ), indicating that a

hydroxylation had taken place and that the carboxyl group was

not aﬀected. The H NMR spectrum of 3 (Table 1) displayed

characteristic signals of three methyls H -20 (δ 0.84), H -17

(

δ_H0.99), and H -18 (δ_H1.17). The C NMR (DEPT)

spectra of 4 (Table 2) displayed the presence of 20 carbon

signals including three tertiary methyls, eight methylenes, four

methines, and ﬁve quaternary carbons. Compared to the H

NMR and DEPT spectra of 2 (Tables 1 and 2), the

disappearance of one CH signal at δ 34.9 and δ 1.13−

.19 (m) and 1.75−1.79 (m) in 2, which was replaced by one

hydroxyl-bearing methine signal at δ 72.3 and δ 3.74 (br. t, J

Figure 2. ORTEP drawing of the X-ray structures of 3.

1.2 Hz) in 4, suggesting the additional OH group to be

attached at C-12. Furthermore, the resonances of C-11 and C-

3 shifted downﬁeld from δ 21.4 to 29.7 and from δ 43.2 to

C C

7.5, respectively, and the resonances of C-9, C-14, and C-17

shifted upﬁeld from δ 57.4 to 52.3, from δ 56.6 to 49.7, and

from δ 25.4 to 22.2, respectively. As a result, the hydroxyl

group was located at C-12. This deduction was also conﬁrmed

by the COSY correlations of H-12 (δ 3.74) with the H -11

(

δ 1.64−1.71 and 1.84−1.91) (Figure 1). Irradiation of the

signal of α-orientated H-16 (δ_H3.88) led to a NOE

enhancement of a small broad triplet signal at H-12 (δ_H

.74, 100%), thereby indicating the 12-OH group to be in

the axial (β) position. Finally, the structure of 4 was conﬁrmed

by X-ray crystallographic analysis (Figure 3). Thus, 4 was

established as ent-12α,16β-dihydroxybeyeran-19-oic acid.

Metabolite 5 showed the molecular formula of C H O , as

Figure 3. ORTEP drawing of the X-ray structures of 4.

−

deduced from its HRESIMS m/z: 335.2231 [M − H] .

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Journal of Agricultural and Food Chemistry

J. Agric. Food Chem. 2020, 68, 4624−4631

Article

Figure 4. Eﬀect of compounds 1−10 on the NGF-induced neurite outgrowth in PC12 cells. (Top) Morphological change of PC12 cells treated

with 2 and 9 at 20 μM. The cell morphological characteristics were observed under a light microscope. (Bottom) Percentage of neurite outgrowth

of PC12 cells after treatment with compounds 1−10 at 20 μM in the presence of NGF for 72 h.

Examination of DEPT and HSQC spectra of 5 indicated that a

Mortierella isabellina, Actinoplanes sp., Mucor recurvatus,

monohydroxylation had occurred. Comparison of H and

Cunninghamella bainieri, Bacillus megaterium, A. niger,

Gibberella fujikuroi, and Glomerella cingulata and Mortierella

These species have the capacity of functionalizing

1 or 2 at the C-7 or C-9 position. This is a common

NMR spectra of 2 and 5 revealed that the substitution of an

additional hydroxyl group in 5 might be at C-7. This coincided

25,26

elongate.

with the C NMR signals observed for downﬁeld shifts of C-6

Δδ +9.0) and C-8 (Δδ +6.0) and upﬁeld shifts of C-5 (Δδ

(

occurrence for other microorganisms with beyerane-type

,6,16

−

1.8), C-9 (Δδ −2.0), C-14 (Δδ −5.5), and C-15 (Δδ −9.9),

scaﬀolds.

respectively, in comparison with those of 2 (Table 2). In the

In this study, C. echinulata was accountable for the most

common biohydroxylation reactions that took place in the

microbial conversions, and several hydroxylated ent-beyerane

congeners (3−8) were prepared regio- and stereoselectively by

hydroxylation at 9β-, 12β-, 14α-, 7α- and 7α-, 7β-, 9β-positions

on the ent-beyerane skeleton by using this strain. Interestingly,

it has been reported that some xenobiotics are known to

HMBC spectrum, H-7 at δ 3.22 exhibited cross-peaks with C-

(δ 32.0), C-9 (δ 55.4), C-14 (δ 51.1), and C-15 (δ 33.7)

C C C C

(

Figure 1). Therefore, the hydroxylation occurred at C-7 (δ_C

6.6). In the H NMR spectrum, the α-conﬁguration of the

OH group at C-7 was deduced from the double-doublet

multiplicity of the C-7 signal (1H, dd, J = 8.1, 2.8 Hz) in 5.

12,27

Furthermore, the relative stereochemistry of OH-7 was also

generate conjugates with glucose in mammalian systems.

conﬁrmed by a NOESY experiment (Figure 1), in which H-7

Selected microbes, especially fungi, have been employed

successfully as in vitro models to predict and imitate the

metabolic fate of drugs and other xenobiotics in mammalian

(

δ 3.22) showed obvious correlations with H-5 (δ 1.01), H-

(δ 1.14), and H-14 (δ 0.81). Thus, the structure of 5 was

H H

28−30

established to be ent-7β,16β-dihydroxybeyeran-19-oic acid.

Metabolite 7 has a molecular formula of C H O , as

systems.

Compound 7, a conjugated fungal metabolite of

2, had been obtained previously through glucosidation by the

−

determined by HRESIMS at m/z: 481.2809 [M − H] . Six

additional resonances in the ¹³C NMR spectra of 7 (Table 2)

revealed a hexose moiety in the structure. The hexose moiety

was veriﬁed to be characteristic of ß-D-glucose by the NMR

spectra. The coupling constant of the anomeric proton at δ_H

bacterium B. megaterium. However, the glycoside formation

by C. echinulata has been ﬁrst reported for the tetracyclic

beyerane diterpene motifs.

We next tested the neurite-outgrowth promoting, α-

glucosidase and NO production inhibiting, and phytotoxic

eﬀects of the transformation products. Results showed that

only compounds 2 and 9 were found to promote neurite-

outgrowth eﬀects in the presence of NGF in PC-12 cells, the

others did not display any activity (Figure 4). The prevalence

of neurodegenerative diseases (ND), such as Alzheimer’s

disease, will continue to increase steadily. Despite the

advancement of treatment, the management of these disorders

remains highly ineﬀective. Therefore, it is essential to discover

new biologically active natural products and analogues thereof

to mitigate ND. Over the past decades, neurotrophic factors,

for example, NGF, have attracted great interest because of their

.4 ppm (d, J = 8.0 Hz, H-1′) of the glucose residue in 7

disclosed the ß-conﬁguration of the glucosidic linkage.

Comparison of the ¹³C NMR spectra of compounds 2 and 7

suggested that 7 was a glucopyranosyl ester of 2 at C-19 due to

the upﬁeld shift of C-19 from 181.7 to 178.2 ppm (Table 2).

Based on the above evidence and literature data, the

structure of 7 was characterized as shown. This is the ﬁrst

report to obtain the glucosidation of 2 by C. echinulata.

An array of microbial transformations of the two beyerane-

type diterpenoids, especially isosteviol (1), involved oxidation

reactions by diﬀerent species of microorganisms, such as

629

J. Agric. Food Chem. 2020, 68, 4624−4631

Journal of Agricultural and Food Chemistry

(2) Wolwer-Rieck, U. The leaves of Stevia rebaudiana (Bertoni),

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In summary, we demonstrated for the ﬁrst time that eight

oxygenated metabolites were obtained and characterized from

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hydroxybeyeran-19-oic acid (2) with C. echinulata. The

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ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jafc.0c00592.

■

9) Li, X.-J.; Shi, X. W.; Shuai, Q.; Gao, J. M.; Zhang, A. L. Bioactive

012, 75, 90−98.

Experimental details of assays and 1D NMR, 2D NMR,

and HRESIMS spectra for 3−5 (PDF )

X-ray crystallographic data for 3 (CIF)

metabolites from biotransformation of paeonol by the white-rot

basidiomycete Coriolus versicolor . Nat. Prod. Commun. 2011, 6, 1129−

1130.

X-ray crystallographic data for 4 (CIF)

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AUTHOR INFORMATION

Corresponding Author

■

(

581−1587.

Jin-Ming Gao − Shaanxi Key Laboratory of Natural Products

11) Hsu, F.-L.; Hou, C.-C.; Yang, L.-M.; Cheng, J.-T.; Chi, T.-C.;

Chemical Biology, College of Chemistry & Pharmacy,

Liu, P.-C.; Lin, S.-J. Microbial transformations of isosteviol. J. Nat.

Northwest A&F University, Yangling 712100, Shaanxi, People’s

Republic of China; orcid.org/0000-0003-4801-6514 ;

Phone: 86-29-87092335; Email: jinminggao@nwsuaf.edu.cn

Prod. 2002, 65, 273−277.

(12) Yang, L. M.; Hsu, F. L.; Cheng, J. T.; Chang, C. H.; Liu, P. C.;

Lin, S. J. Hydroxylation and glucosidation of ent -16 β -hydroxybeyeran-

19-oic acid by Bacillus megaterium and Aspergillus niger . Planta Med.

Authors

2004, 70, 359−363.

Yu-Qi Gao − Shaanxi Key Laboratory of Natural Products &

Chemical Biology, College of Chemistry & Pharmacy,

Northwest A&F University, Yangling 712100, Shaanxi, People’s

Republic of China

Ruoxin Li − Shaanxi Key Laboratory of Natural Products &

Chemical Biology, College of Chemistry & Pharmacy,

Northwest A&F University, Yangling 712100, Shaanxi, People’s

Republic of China

(13) Chou, B.-H.; Yang, L.-M.; Chang, S.-F.; Hsu, F.-L.; Lo, C.-H.;

Liaw, J.-H.; Liu, P.-C.; Lin, S.-J. Microbial transformation of isosteviol

lactone and evaluation of the transformation products on androgen

response element. J. Nat. Prod. 2008, 71, 602−607.

(14) Wonganan, O.; Tocharus, C.; Puedsing, C.; Homvisasevongsa,

S.; Sukcharoen, O.; Suksamrarn, A. Potent vasorelaxant analogs from

chemical modification and biotransformation of isosteviol. Eur. J. Med.

Chem. 2013, 62, 771−776.

15) Ali, M. S.; Hanson, J. R.; de Oliveira, B. H. The

Wei-Wei Wang − Shaanxi Key Laboratory of Natural Products

biotransformation of some ent-beyeran-19-oic acids by Gibberella

Chemical Biology, College of Chemistry & Pharmacy,

fujikuroi . Phytochemistry 1992, 31, 507−510.

Northwest A&F University, Yangling 712100, Shaanxi, People’s

Republic of China

(16) de Oliveira, B. H.; Strapasson, R. A. Biotransformation of

isosteviol by Fusarium verticilloides . Phytochemistry 1996, 43, 393−

17) Wei, J.; Zhang, X.-Y.; Deng, S.; Cao, L.; Xue, Q.-H.; Gao, J.-M.

95.

Shoei-Sheng Lee − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 10051, Taiwan, ROC

α -Glucosidase inhibitors and phytotoxins from Streptomyces

xanthophaeus. Nat. Prod. Res. 2017, 31, 2062−2066.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jafc.0c00592

(18) Yin, H.; Dan, W.-J.; Fan, B.-Y.; Guo, C.; Wu, K.; Li, D.; Xian,

K.-F.; Pescitelli, G.; Gao, J.-M. Anti-inflammatory and α -Glucosidase

Inhibitory Activities of Labdane and Norlabdane Diterpenoids from

the Rhizomes of Amomum villosum. J. Nat. Prod. 2019, 82, 2963−

Funding

This research was ﬁnancially supported by the Fundamental

Research Funds for the Central Universities (2452019185).

Notes

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of 1-O-acetylbritannilactone analogues from Inula britannica and in

vitro evaluation of their anticancer potential. Med. Chem. Commun.

The authors declare no competing ﬁnancial interest.

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