Biotransformation of α-terpineol by: Alternaria alternata

Article abstract of DOI:10.1039/c9ra08042b

α-Terpineol (1), the main volatile constituent in some traditional Chinese medicines, has been reported to be metabolized to 4R-oleuropeic acid by the larvae of common cutworms. The present study verified that α-terpineol could be converted to 4R-oleurope

Full text of DOI:10.1039/c9ra08042b

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Biotransformation of a-terpineol by Alternaria
alternata†
Cite this: RSC Adv., 2020, 10, 6491
*
Rui-Feng Mei,‡ Ya-Xian Shi,‡ Wei-He Duan, Hao Ding, Xiao-Ran Zhang, Le Cai
*
and Zhong-Tao Ding
a-Terpineol (1), the main volatile constituent in some traditional Chinese medicines, has been reported to
be metabolized to 4R-oleuropeic acid by the larvae of common cutworms. The present study verified that
a-terpineol could be converted to 4R-oleuropeic acid (2) and (1S,2R,4R)-p-menthane-1,2,8-triol (3) by
Alternaria alternata fermentation. Using shortened fermentation times, 7-hydroxy-a-terpineol (2a) was
identified as an oxidative intermediate, which was consistent with the hypothesis put forward by previous
studies. Cytochrome P450 enzymes were also confirmed to catalyze this biotransformation. This is the
first study on the biotransformation of a-terpineol by microbial fermentation.
Received 4th October 2019
Accepted 15th January 2020
DOI: 10.1039/c9ra08042b
rsc.li/rsc-advances
endophyte fungi to conrm its biological oxidation. The results
showed that Alternaria alternata fermentation could facilitate
1 Introduction
the biotransformation of interest. Isolation of the fermentation
broth led to the identication of two oxidative products 4R-
oleuropeic acid (2) and (1S,2R,4R)-p-menthane-1,2,8-triol (3)
(Fig. 1). 7-Hydroxy-a-terpineol (2a), isolated from the fermen-
tation broth by shortening fermentation time, was conrmed to
be an oxidative intermediate. The oxidation of a-terpinol (1) to
compound 2 is consistent with larvae that reported in the
literature.20 The conversion ratio of a-terpineol (1) to 4R-oleur-
opeic acid (2) was determined by high-performance liquid
chromatography (HPLC) and the yield of compound 3 was
determined by gas chromatography (GC). Additionally, cyto-
chrome P450 enzymes were further conrmed to catalyze this
biotransformation. Compared with feeding insects to collect
their excreta, the method of liquid fermentation by fungi to
convert a-terpinol is more potential for mass production. This is
the rst study to conrm the biotransformation of a-terpineol,
Biotransformation is superior to chemical synthesis due to
superior regio- and stereo-selectivities,1 environmentally
friendly procedures,2 and mild reaction conditions.3 Bio-
catalysts play an important role in regio- and stereo-selective
reactions,4,5 including acetylation,6 hydroxylation,7 and epoxi-
dation of steroids.8 Our previous studies have shown that many
transformations have been achieved by microorganisms.9–12
a-Terpineol (1), a cyclic monoterpene alcohol, is widely
present in many traditional Chinese medicines, such as Thymus
vulgaris, Annona squamosa, and Magnolia denudata. a-Terpineol
is also commonly used in industrial syntheses13,14 and certain
pharmaceutical preparations.15–17 The in vivo metabolic path-
ways of a-terpineol were conrmed to be connected to the
digestive system and the P450 enzyme system.18,19 It has been
reported that a-terpineol is oxidized to oleuropeic acid by the
larvae of common cutworms (Spodoptera litura).20 It is reason-
able to speculate that microbes possessing P450 enzyme system
might facilitate above transformation. As a key raw material,
oleuropeic acid (2) can be used to synthesize a variety of active
substances.21–25 Therefore, the investigation of the accumula-
tion of oleuropeic acid (2) by microbial fermentation is impor-
tant and prospective.
In the present study, the biotransformations of a-terpineol
were screened in liquid fermentation using nine plant
Functional Molecules Analysis and Biotransformation Key Laboratory of Universities in
Yunnan Province, Key Laboratory of Medicinal Chemistry for Natural Resource
Ministry of Education, School of Chemical Science and Technology, Yunnan
University, Kunming 650091, P. R. China. E-mail: ztding@ynu.edu.cn; caile@ynu.
edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra08042b
‡ The rst two authors contributed equally to this work.
Fig. 1 The biotransformation of 1 by Alternaria alternate.
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and this study could serve as a reference for further studies on
the internal metabolism of this compound.
2.7 Screening of biotransformation
PDA slant culture medium was inoculated with the aforemen-
tioned fungi and kept in a constant temperature incubator at
28 ꢂC for 7 days. Then, the mature slants were added to conical
asks (150 mL) that contained 20 mL of potato dextrose broth
(PDB: 1 L of water, 200 g of potato starch, and 20 g of dextrose)
seed culture medium and placed on a rotary shaker shaking at
120 rpm at 28 ꢂC for 3 days. And then 150 mL of PDB was added
to a 500 mL conical ask to serve as the fermentation culture
medium. Aer the fermentation culture medium was sterilized
in an autoclave at 121 ꢂC and 1.06 kg cmꢀ2 for 30 min, the seed
cultures and 1 mL (5 mL mLꢀ1) of substrate were added and the
medium was incubated at room temperature for 7 days. Then,
the PDB before and aer fermentation was immersed in EtOAc
(200 mL) and extracted with an ultrasonic cleaner three times
(30 min each), extracts were analyzed with thin layer chroma-
tography (TLC).
2 Experimental
2.1 Chemicals
a-Terpineol (compound 1, CAS: 10482-56-1) was purchased
from Aladdin Industrial Corporation. Silica gel (200–300 mesh)
for column chromatography was purchased from Qingdao
Marine Chemical Corporation, Qingdao, China. Acetonitrile
and methanol (Hipure Chem, China) were of HPLC grade. 1-
Aminobenzotriazole (ABT, 98%), piperonyl butoxide (PBO,
90%), and n-tetradecane (98%) were obtained from J&K Scien-
tic Ltd. (Beijing, China). CDCl3 (99.9 atom% D, containing
0.03% TMS) was purchased from ARMAR Chemicals (Ningbo,
China) and MeOD (99.9 atom% D, contains 0.03% TMS) was
purchased from Sigma-Aldrich (Shanghai, China). The water
used in this study was puried using a water purication system
(Chengdu, China) and had a resistivity $18.25 MU cmꢀ1. Other
reagents were of analytical grade and were purchased from
Tianjin Fuyu Fine Chemical Co. Ltd. (Tianjin, China).
2.8 Biotransformation process investigation
A. alternata was grown in shaking cultures at 28 ꢂC for 3 days in
150 mL conical asks that contained 50 mL of sterile PDB. The
a-terpineol (10.0 mL each) dissolved in MeOH (0.5 mL) was
evenly distributed between two asks containing 50 mL of the
previously mentioned sterile PDB and strains that had been
cultured for 3 days. The a-terpineol mixtures were incubated for
a further 1, 5, 7, or 9 days each. The broth was extracted with
EtOAc (200 mL) three times to afford the extract. HPLC was used
to determine the content of compound 2a and 2 in samples at
different times.
2.2 Nuclear magnetic resonance (NMR) spectroscopy
The NMR spectra were obtained on a Bruker Avance 400 MHz
spectrometer (400.00 MHz, 1H; 100.00 MHz, 13C) using tetra-
methylsilane (TMS) as the internal reference.
2.3 Polarimeter
Optical rotation was measured with a Jasco P-1020 digital
polarimeter (Jasco, Tokyo, Japan). A Shimadzu UV-vis 2550.
2.9 Purication of metabolites
The extracts of strains that had been fermented for 5 days were
subjected to a Sephadex LH-20 column (CHCl3–MeOH (1 : 1))
and silica gel CC (PE–EA, (15 : 1, 7 : 1, and 4 : 1)) to obtain
compound 2a (7.8 mg). In addition, extracts of strains that had
been fermented for 9 days were subjected to a Sephadex LH-20
column (CHCl3–MeOH (1 : 1)) and silica gel CC (PE–EA, (4 : 1))
to obtain compound 2 (14.5 mg), or the same extract was sub-
jected to silica gel CC (PE–EA (2 : 1)) and a Sephadex LH-20
column (MeOH) to yield compound 3 (12.3 mg).
2.4 HPLC
An Agilent Technologies 1200 Series HPLC equipped with an
Agilent dual pump, a diode array detector (DAD), an Agilent
ZORBAX SB-C18 (4.6 ꢁ 250 mm, 5 mm) and an Agilent Guard
Column Hardware Kit was used for all HLPC analyses.
2.5 GC
An Agilent Technologies 7890B GC System with a ame ioni-
zation detector (FID), an Agilent 19091J-413 HP-5 (30 m ꢁ 320
mm ꢁ 0.25 mm) was used for all GC analyses.
2.10 Spectroscopic data
7-Hydroxy-a-terpineol (2a). White oil; [a]2D5 ꢀ25.1 (c 0.020,
CHCl3); EIMS m/z 170 M+; for 1H and 13C NMR spectral data, see
Table 1.
2.6 Microorganisms and culture medium
A. alternata, Fusarium solani, Fusarium avenaceum, Aspergillus
tubingensis, Fusarium oxysporum, Myrothecium inundatum,
Aspergillus sydowii, Penicillium virgatum and Aspergillus versi-
color were obtained from the Yunnan Institute of Microbiology,
Yunnan Province, China. A. tubingensis, A. sydowii, P. virgatum
and A. versicolor were separated from the root of Panax noto-
ginseng. F. oxysporum, M. inundatum, A. alternata, F. solani, F.
avenaceum were separated from the tuber of Gastrodia elata.
Culture and biotransformation experiments used fungi grown
4R-Oleuropeic acid (2). White amorphous powder;
1
[a]2D5 ꢀ38.9 (c 0.020, MeOH); EIMS m/z 184 M+; for H and 13C
NMR spectral data, see Table 1.
(1S,2R,4R)-p-Menthane-1,2,8-triol (3). Amorphous powder;
1
[a]D25 +16.3 (c 0.020, CHCl3); EIMS m/z 188 M+; for H and 13C
NMR spectral data, see Table 1.
2.11 Determining reaction yield by HPLC and GC analysis
in potato dextrose agar (PDA, 1 L of water, 200 g of potato, 20 g Standards and samples were dissolved in MeOH (1.000 mL) and
of dextrose and 15 g of agar) slant culture medium.
ltered through a 0.45 mm lter prior to injection into the HPLC
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Table 1 The NMR spectroscopic data for 2a, 3 in CDCl3 and 2 in MeOD
2a
2
3
dH
dH
Position
dC
dH mult. (J in Hz)
dC
mult. (J in Hz)
dC
mult. (J in Hz)
1
2
3
137.6 s
122.6 d
23.7 t
131.7 s
140.8 d
24.6 t
71.0 s
74.1 d
30.0 t
5.69 (m, 1H)
2.05 (m, 1H)
1.24 (m, 1H)
1.54 (m,1H)
1.55 (m, 1H)
1.36 (m, 1H)
1.76 (m, 1H)
1.52 (m, 1H)
4.01 (t, 2H, 12.0)
6.98 (t, 1H, 2.4)
2.02 (m, 1H)
1.22 (m, 1H)
1.53 (m, 1H)
2.48 (br d, 1H, 16.0)
2.08 (m, 1H)
3.61 (brs, 1H)
1.79 (m, 2H)
4
5
45.2 d
26.5 t
45.6 d
26.4 t
41.1 d
22.3 t
1.63 (tt, 1H, 12.0, 6.0)
1.54 (t, 1H, 8.0)
1.34 (td, 1H, 8.0, 4.0)
1.75 (t, 1H, 2.0)
1.50 (t, 1H 3.6)
6
26.7 t
28.5 t
2.34 (br d, 1H, 16.0)
2.01 (m, 1H)
33.7 t
7
8
9
10
67.2 t
72.9 s
27.5 q
26.7 q
171.1 s
72.9 s
26.4 q
27.0 q
27.8 q
72.8 s
26.7 q
27.6 q
1.22 (s, 3H)
1.19 (s, 3H)
1.24 (s, 3H)
1.17 (s, 3H)
1.17 (s, 3H)
1.18 (s, 3H)
1.14 (s, 3H)
and GC. Different concentrations of compound 2 and 3 methanol terpineol was oxidized to 4R-oleuropeic acid by the enteric
solution were injected into HPLC or GC and integrate the peak microorganism in the larvae of common cutworms. Therefore,
area to draw a standard working curve. Each sample was analyzed in the present study, the biotransformation of a-terpineol was
in triplicate. The contents of products were determined accord- screened in liquid fermentation using nine plant pathogenic
ing to the corresponding calibration curves; further conversions fungi to conrm its biological oxidation process. TLC was used
were calculated on the basis of these contents.
to monitor the transformation process during fermentation.
A gradient elution system consisting of solvents A (0.1% The results showed that A. alternata fermentation could facili-
HAc) and B (acetonitrile) was used for the HPLC analysis. The tate the biotransformation of interest.
gradient program was as follows: 0–10 min, 20–70% solvent B;
10–40 min, 70–80% solvent B; 40–45 min, 80–100% solvent B;
and 45–46 min, 100–20% solvent B and then held for 10 min.
3.2 Identication of compounds
All 1H and 13C NMR spectral data of the known compounds 2a, 2
The detecting wavelength of the diode array detector was set to
and 3 agreed with spectral data reported in the literature.20,28,29
The absolute conguration of compound 3 was determined
based on the ROESY spectrum. As mentioned above, chiral
center at C-4 remained unchanged during the fermentation
process. The a-orientation of OH-2 was assigned on the basis of
NOE correlations between H-4b [dH 1.63 (tt, J ¼ 12.0, 6.0 Hz)]
and H-2 [dH 3.61 (brs, 1H)]. The NOE correlation (Fig. 2) between
CH3-7 [dH 1.22 (s, 3H)] and H-2 indicated that they were oriented
in the same direction. Thus, the chiral centers in 3 were
assigned as 1S, 2R and 4R. Compound 3 was chemically
synthesized by a previously reported method,30 and this is the
rst report of its synthesis via microbial fermentation.
225 nm. The ow rate was 1.0 mL minꢀ1, the column temper-
ꢂ
ature was set at 25 C, and the injection volume was 10 mL.
Argon was used in the mobile phase of GC analysis, argon at
a ow rate of 1 mL minꢀ1 was used as a carrier gas. The ovꢀe1n
temperature was programmed from 200 to 250 ꢂC at 4 ꢂC min
.
The injector and detector temperatures were 270 and 280 ꢂC,
respectively.
2.12 Effects of the inhibitors ABT and PBO and of the
inducer n-tetradecane
The inhibitors ABT and PBO (40 mg mLꢀ1, nal concentration)
and the inducer n-tetradecane (2%, v/v, nal concentration)
were added to the spore suspension aer it had been incubated
for 24 h. Aer an additional 48 h of culturing, 1 mL of the
substrate (10 mL mLꢀ1) was added. The spore suspension
without the inhibitors and inducer was used as the positive
control. The concentrations of the product and reactant were
determined by HPLC and GC analysis aer 6 h, 24 h, 48 h, 72 h
and 96 h. Each sample was repeated in triplicate.
3.3 A. alternata fermentation of a-terpineol
Fermentation of a-terpineol by A. alternata for seven days
allowed the isolation of two oxidative products, 4R-oleuropeic
acid (2) and (1S,2R,4R)-p-menthane-1,2,8-triol (3). Aer
reducing fermentation time to 5 days, we isolated 7-hydroxy-a-
terpineol (2a) as a key oxidative intermediate, which conrmed
the oxidation process of a-terpineol (1) to oleuropeic acid (2).
The fermentation itself process was also investigated. As shown
in Fig. 3, no absorption peak was detected at the beginning
(Fig. 3a), when the fermentation time increased, compound 2a
3 Results and discussion
3.1 Screening of microorganism
Oxidation reactions caused by microbes are commonly reported gradually appeared, reached a peak at 5 days (Fig. 3b), then
in the literature;26,27 thus it is reasonable to propose that a- decreased quickly (Fig. 3c) and nally disappeared (Fig. 3d). In
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Fig. 2 ROESY spectrum and Key NOE (
) correlations of 3.
addition, the content of compound 2 gradually increased and conditions, the production of compound 2 was very slow and we
achieved its highest level aer 9 days of fermentation (Fig. 3d), did not detect compound 2 by HPLC in these cases. In contrast,
corresponding to a maximum conversion ratio of 44.9% ꢃ this transformation could be signicantly induced by 2% (v/v) n-
2.4%. This biotransformation facilitates the possibility of mass tetradecane (Fig. 4c). In the early stage of the fermentation,
production of 4R-oleuropeic acid (2). The isolation of 3 indi- compound 2a was produced much more quickly than it was in
cated that A. alternata fermentation could facilitate cis-dihy-
droxylation of the C(1)–C(2) double bond in a-terpineol, which is
the same as the reported in the literature.31–33 Compound 3
could not be detected by FID until four days fermentation. The
yield of compound 3 aer 9 days of fermentation was 9.6% ꢃ
0.8%.
3.4 Effects of the inhibitors ABT and PBO and the inducer n-
tetradecane
Cytochrome P450 monooxygenases catalyze the oxidation of
a wide variety of endogenous and exogenous organic substrates
typically by inserting an oxygen atom from atmospheric dioxy-
gen into a carbon–hydrogen bond to produce the corresponding
alcohol34,35 or carboxylic acid.36 In this study, compound 2 was
proposed to be derived from a-terpineol via cytochrome P450
oxidative catalysis.
To determine the enzyme system involved in this biotrans-
formation, special inhibition and induction experiments were
performed. PBO (piperonyl butoxide, which contains a methyl-
enedioxyphenyl (MDP) or benzodioxole moiety) is used to esti-
mate the inhibition and induction of reactions mediated by
cytochrome P450.37,38 ABT is a triazole P450-14DM inhibitor that
is widely used as an inactivator of many P450 isozymes.39 n-
Tetradecane is an effective inducer of cytochrome P450
Fig. 3 Detection of the biotransformation process of compound 2a to
2 by HPLC. Figure (a–d) shows the fermentation time: 1, 5, 7, 9 days. a-
Terpineol was first oxidized to compound 2a then decreased quickly
enzymes.40 As shown in Fig. 4a and b, ABT and PBO exhibited
a signicant inhibitory effect on the conversion of a-terpineol to
compound 2a at a concentration of 40 mg mLꢀ1. Under such and finally oxidized to 2.
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4 Conclusions
In summary, the present study veried that a-terpineol could be
converted to 4R-oleuropeic acid (2) and a cis-dihydroxyl deriva-
tive (3) by A. alternata. 7-Hydroxy-a-terpineol (2a) was shown to
be an intermediate in the biotransformation of a-terpineol to 2.
Cytochrome P450 enzymes were conrmed to catalyse this
biotransformation. This is the rst study to clarify the
biotransformation of a-terpineol, and this study could serve as
a reference for further investigations on the internal metabo-
lism of this compound.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was nancially supported by a grant from the Natural
Science Foundation of China (No. 81960760), Program for
Changjiang Scholars and Innovative Research Team in Univer-
sity (IRT_17R94), the project of Yunling Scholars of Yunnan
province, a Program for Excellent Young Talents, and the grant
from Yunnan University’s Research Innovation Fund for Grad-
uate Students (No. 2018184).
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