Biotransformation of ursolic acid by an endophytic fungus from medicinal plant Huperzia serrata

Article abstract of DOI:10.1248/cpb.59.1180

Endophytic fungi were used not only for their producing bioactive products but also for their ability to transform natural compounds. An endophytic fungus, isolated from medicinal plant Huperzia serrata, was identified as Umbelopsis isabellina based on the internal transcribed spacer of ribosomal DNA (rDNA-ITS) region. It was used to transform ursolic acid (1), a pentacyclic triterpene. Incubation of ursolic acid with U. isabellina afforded three products, 3β-hydroxy-urs-11-en-28,13-lactone (2), 3β,7β-dihydroxy-urs-11- en-28,13-lactone (3), 1β,3β-dihydroxy-urs-11-en-28,13-lactone (4). Although product 2 was a known compound, it was first obtained by microbial transformation. Products 3 and 4 were new compounds. The structural elucidation of the three compounds was achieved mainly by the 1D- and 2D-NMR, MS, IR data. The endophytic fungus U. isabellina can hydroxyate the C12-C13 double bond at position 13 of ursolic acid 1 and form a five-member lactone effectively. In the meantime, this fungus can also introduce the hydroxyl group at C-1 or C-7 of ursolic acid 1.

Full text of DOI:10.1248/cpb.59.1180

1180
Note
Chem. Pharm. Bull. 59(9) 1180—1182 (2011)
Vol. 59, No. 9
Biotransformation of Ursolic Acid by an Endophytic Fungus from
Medicinal Plant Huperzia serrata
a
a
a
b
,a
*
Shao-bin FU, Jun-shan YANG, Jin-long CUI, Xu FENG, and Di-an SUN
a Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College;
Beijing 100193, China: and b Navy Medical Research Institute; Shanghai 200433, China.
Received April 17, 2011; accepted May 30, 2011; published online June 3, 2011
Endophytic fungi were used not only for their producing bioactive products but also for their ability to
transform natural compounds. An endophytic fungus, isolated from medicinal plant Huperzia serrata, was iden-
tified as Umbelopsis isabellina based on the internal transcribed spacer of ribosomal DNA (rDNA-ITS) region. It
was used to transform ursolic acid (1), a pentacyclic triterpene. Incubation of ursolic acid with U. isabellina
afforded three products, 3b-hydroxy-urs-11-en-28,13-lactone (2), 3b,7b-dihydroxy-urs-11-en-28,13-lactone (3),
1b,3b-dihydroxy-urs-11-en-28,13-lactone (4). Although product 2 was a known compound, it was first obtained
by microbial transformation. Products 3 and 4 were new compounds. The structural elucidation of the three
compounds was achieved mainly by the 1D- and 2D-NMR, MS, IR data. The endophytic fungus U. isabellina can
hydroxyate the C12–C13 double bond at position 13 of ursolic acid 1 and form a five-member lactone effectively.
In the meantime, this fungus can also introduce the hydroxyl group at C-1 or C-7 of ursolic acid 1.
Key words biotransformation; ursolic acid; endophytic fungus; Umbelopsis isabellina; medicinal plant
Endophytes are bacterial or fungal microorganisms which weight that was two units less than that of the substrate 1.
colonize in the healthy plant tissue inter- and/or intracellu- Seven characteristic methyl group (two doublets and five sin-
larly without causing any apparent symptoms of disease.1) glets in the proton NMR) and proton signals of H-3 weren’t
Endophytes can produce bioactive substances, such as pacli- changed compared to the 1H-NMR spectrum of the substrate
taxel,2,3) podophyllotoxin etc.4,5) Endophytic fungi exten- 1. Two olefinic proton signals appeared at dH 5.94 (1H, br d,
sively metabolized 2-hydroxy-1,4-benzoxazin-3(2H)-one Jꢁ10.8 Hz) and dH 5.51 (1H, dd, Jꢁ4.8, 10.8 Hz). Corre-
(HBOA) and transformed it to less toxic metabolites proba- spondingly, two olefinic carbon signals at dC 134.1 and dC
ble by their oxidase and reductases.6) Thus, endophytes at- 130.0 were shown in its 13C-NMR spectrum. Obviously, the
tracted more and more attention not only for producing many double bond was not consistent with that in the substrate 1.
novel substances but also to transform the natural product.
Moreover, there was another new oxygenated carbon at dC
In an attempt to find endophytes that might produce Hu- 90.0 in product 2. The possibility is that the double bond was
perzine A, a natural product isolated from the medicinal transferred from C12–C13 to C11–C12 with C-13 hydroxy-
plant Huperzia serrata that was used for the treatment of lated. The chemical shift of carbon-18 was also affected
Alzheimer’s disease, we collected the medicinal plant Hu- (downshifted from about dC 53.9 to dC 60.8). Deduced from
perzia serrata and isolated over one hundred endophytes. the NMR data, there were two possible structures for this
Unfortunately, we were not able to find any of them that compound. The first one was that the 13-OH and 28-COOH
could produce Huperzine A.
were free. The second one was that there was a lactone for-
Since microorganisms possess multi-enzyme systems mation between 13-OH and 28-COOH. Since its molecular
which have significant regio- and stereo-selectivities, we
hope to use these endophytes to transform some of the triter-
penoids that is usually hard to achieve from chemical modifi-
cation.7)
Ursolic acid was a natural triterpenoid with many bioactiv-
ities, including anti-inflammatory activity,8) anti-bacterial ac-
tivity,9) anti-virus activity.10) Since ursolic acid has limited
active sites for chemical modification, it’s not easy to obtain
large number of its derivatives for effective structure–activity
relationship (SAR) study. In an attempt to modify ursolic
acid, especially to obtain its hydroxylation products, we have
used large number of microorganisms to transform this com-
pound. In this article, we reported the microbial transforma-
tion ursolic acid by an endophytic fungus U. isabellina iso-
lated from medicinal plant H. serrata.
Results and Discussion
Product 2 was obtained as white solid. Its molecular
weight was determined as 454 according to ESI-MS data
Fig. 1. Biotransformation of Ursolic Acid by Endophytic Fungus U. isa-
bellina from H. serrata
(m/z: 455 [MꢀH]ꢀ, 931 [2MꢀNa]ꢀ). It has a molecular
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: diansun@sina.com

September 2011
1181
and H-12 (dH 7.89, 1H, d, Jꢁ10.8 Hz) with C-13 (dC 89.9)
were conspicuous in HMBC spectrum (Fig. 2). The other two
correlations between H-11 with C-8 (DC 42.7) and H-12 with
C-14 (DC 42.9) were also observed from HMBC data. H-27,
H-11 and H-12 were correlated with the hydroxylated C-13
(dC 89.9) in the HMBC spectrum which further confirmed
the above deduction. Since compound 4 also has a molecular
weight of 470 the same as product 3, it was also supposed to
have been formed a five-member lactone between C-28 and
C-13 that was in the same way as product 2 and 3. Based on
Fig. 2. Selected HMBC Correlations of Compounds 3 and 4
weight was 454. It belongs to the second structure. Thus the above evidences, the structure of product 4 was identified
compound 2 was elucidated as 3b-hydroxy-urs-11-en-28,13- as 1b,3b-dihydroxy-urs-11-en-28,13-lactone. It was also a
lactone. It was a known compound obtained from chemical new compound.
modification.11) Since there were no detailed NMR data re-
Many endophytic fungi were isolated from medicinal plant
ported for this compound, we reported its NMR data for fur- H. serrata by other research groups with few of them were
ther reference. used to modify natural products. We investigated the ability
Product 3 was obtained as white power. It had the molecu- of the endophytic filamentous U. isabellina to transform ur-
lar formula of C30H46O4, as evidenced by the HR-ESI-MS solic acid. In all three products, the double bond transferred
data (m/z: 469.3333 [MꢂH]ꢀ, Calcd for C30H45O4, 469.3312). from C12–C13 to C11–C12 with the formation of a lactone
It had one more oxygen atom and two less hydrogen atoms between C-28 and C-13 correspondingly. This strain may
than the substrate 1. It had one more oxygen atom than prod- produce the transferase and esterase that can transfer the
uct 2. In the 1H-NMR spectrum, all seven methyl groups with double bond in this type of structure. The endophytic fungus
the splitting pattern of ursan-type triterpene were appeared in U. isabellina can also introduce hydyoxyl groups into C-1
the high field. There were two olefinic proton signals similar and C-7 positions.
to those of compound 2. From the APT (13C-NMR) and
In conclusion, endophytic fungi were useful resource not
HMQC spectra, two more hydroxyl groups were introduced. only for their bioactivity of their metabolites but also for
One carbon (dC 73.2) was connected with proton dH 4.25 their biotransformation ability of natural products. Endo-
(1H, dd, Jꢁ4.2, 10.8 Hz). The other carbon (dC 90.1) was a phytic fungus U. isabellina transferred the double bond of ur-
quaternary carbon. The correlations of H-27 (dH 1.40) and solic acid 1 effectively, and introduced the hydroxyl groups
two olefinic protons with the carbon (dC 90.1) were observed at saturated carbon in the triterpenoid rings that was hard to
in the HMBC spectra (Fig. 2). It was deduced that the double achieve through chemical methods.
bond was at C-11–C-12. From the molecular weight 470,
product 3 was supposed to have been formed an ester at C-28
Experimental
General Mps were determined by the apparatus that was uncorrected.
Optical rotations were measured using a Perkin-Elmer 341 apparatus at
589 nm and 20 °C. The IR spectra were measured on a FTIR-8400S infrared
spectrometer with KBr pellets. NMR spectra were acquired with Bruker
with C-13 and thus lost one molecule water similar to prod-
uct 2. The carbon at dC 73.2 was assigned to C-7 based on its
correlation with H-26 (dH 1.56) in the HMBC spectrum. The
b-orientation of 7-OH was deduced from the coupling con-
DRX-600 spectrometer operating at 600 MHz (for proton NMR) and 150
1
MHz (for carbon NMR) using TMS as internal standard. UV spectra were
stants of H-7 (dH 4.25, 1H, dd, Jꢁ4.2, 10.8 Hz) in H-NMR
recorded on Shimadzu UV2550 spectrometer. High resolution mass (HR-EI-
spectrum with H-7 at axial (a) position. The carbon with dC
61.2 was attributed to C-18 because of its correlation with H-
29 (dH 1.05, 3H, d, Jꢁ6.6 Hz) in HMBC spectrum (Fig. 2).
MS) spectra employed a monoisotopic mass spectrometer. High-resolution
electrospray ionization mass spectra (HR-ESI-MS) and ESI-MS were ob-
tained with a Thermo LTQ Orbitrap XL mass spectrometer. Sterilization was
Thus, the structure of compound 3 was elucidated as 3b,7b- carried out in an YX-2800 autoclave. Aseptic operations were carried out in
SW-CJ-ID laminar flow cabinet. Incubations were on a HZQ-X100 constant
dihydroxy-urs-11-en-28,13-lactone. It was a new compound.
temperature shake incubator. Column chromatography was carried out on
silica gel (100—200, 200—300, 300—400 mesh, Qingdao Oceanic Chemi-
cals, China). TLC was performed on 0.25 mm thick silica prepared plates
also from Qingdao Oceanic Chemicals, China. Spot visualizations were
made by spraying with H2SO4/95% EtOH (9 : 1, v/v) followed by heating.
Substrate, Microorganism, and Culture Medium. Substrate The
substrate UA (with purity ꢃ98%) was purchased from Changsha Staherb
Compound 4 was obtained as white amorphous power. Its
molecular formula was determined as C30H46O4 according to
the HR-EI-MS data (m/z: 470.3365 [Mꢀ], Calcd for
1
C30H46O4, 470.3396). Compared to product 2, the H-NMR
spectrum of 4 exhibited a new hydroxylated proton signals at
dH 3.85 (1H, dd, Jꢁ4.2, 10.8 Hz) except for H-3 (dH 3.61,
Natural Ingredients Co., Ltd., Changsha, China. Its structure was character-
1H, dd, Jꢁ5.6, 12.6 Hz). Correspondingly, the 13C-NMR
ized by comparison its 1H- and 13C-NMR data with those reported in the lit-
erature.12)
spectrum of 4 showed a new oxygenated carbon signals at dC
Microorganism Endophytes were isolated from healthy medicinal plant
80.2. In the HMQC spectrum, the carbon at dC 80.2 was cor-
H. serrata collected from Guizhou, China with a method described by Stro-
related with the proton at dH 3.85. The correlation between
bel et al. with slightly modification.13) Leaves and stems of the plant were
H-25 (dH 1.29) with the carbon at dC 80.2 in the HMBC
washed under running water 2 h, and rinsed 5 times with sterile distilled
spectrum (Fig. 2) clearly showed that it had a C-1 hydroxyla-
tion. The hydroxyl group at C-1 was -orientation was based
water. The leaves were sterilized with 75% EtOH (v/v) for 1 min and 0.1%
mercuric chloride (v/v) for 5 min. The stems sere sterilized with 0.1% mer-
curic chloride (v/v) for 8 min. Finally, the samples were rinsed six times
again in sterile water and were cut into small sections using a sterile knife.
The cut segments were placed on Petri dishes containing potato dextrose
agar and were incubated at 28 °C in darkness. The Petri dishes with the last
sterile water as the control incubated under the same conditions. Pure cul-
b
on the coupling constants and splitting pattern of H-1 (dH
3.85,1H, dd, Jꢁ4.2, 10.8 Hz). Similar to compounds 2 and 3,
it also had the double bond transferred from C12–
C13 to C11–C12 and C-13 was hydroxylated. The correla-
tions between both H-11 (dH 5.65, 1H, dd, Jꢁ2.4, 10.8 Hz) tures were then transformed to potato dextrose agar (PDA) plates. The puri-

1182
Vol. 59, No. 9
Table 1. 1H- (600 MHz) and 13C-NMR (150 MHz) Data of Compounds 3
and 4 (Pyridine-d5)
fied fungi were stored in the PDA slant at 4 °C.
Culture Media The preliminary screening experiment was carried out
in liquid PDA medium (consisting of 200 g potatoes boiling for 20 min; dex-
trose, 20 g; peptone, 10 g; distilled H2O, 1000 ml).
3
4
No.
Identification of Endophytic Fungus The fungus was identified by se-
quencing the internal transcribed region of ribosome deoxyribonucleic acid
(rDNA-ITS). The universal primers ITS1 OF (5ꢄ-AACTCGGCCATTTA-
GAGGAAGT-3ꢄ) and ITS4 OF (5ꢄ-TCCTCCGCTTATTGATATGC-3ꢄ) were
used to amplify the ITS regions from the DNA extract.14) The polymerase
chain reaction (PCR) reaction was performed with the following cycles: (1)
94 °C for 3 min; (2) 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for
1 min, and (3) 72 °C for 10 min.
dH
dC
dH
dC
1
2
3
4
5
1.86 (o), 0.96 (m)
1.92 (o)
3.49 (m)
38.8 3.85 (dd, 4.2, 10.8)
28.7 2.35 (o), 2.39 (o)
78.2 3.61 (dd, 5.6, 12.6)
80.2
39.7
75.9
0.1
—
39.7
—
1.05 (m)
2.08 (o)
4.25 (dd, 4.2, 10.8)
53.3 0.84 (o)
30.0 1.67 (o), 1.71 (o)
73.2 1.13 (o), 1.44 (o)
43.8
53.4 2.49 (o)
37.1
130.3 5.65 (dd, 2.4, 10.8) 128.2
133.3 7.89 (d, 10.8)
90.1
48.0
53.0
18.3
31.9
42.7
54.9
43.2
6
7
8
9
10
11
12
13
14
Fermentation of Compound and Extraction of Metabolites. Prelimi-
nary Screening The preliminary screenings were carried out in 100 ml Er-
lenmeyer flasks containing 40 ml of liquid PDA medium. After incubation at
28 °C and 160 rpm in the shaker for 2 d, the substrate 0.5 mg (dissolved in
ethanol) were added into the flaks for another 10 d and harvested. Substrate
controls with the substrate but without fungi and culture controls with the
fungi but without substrate were incubated at the same conditions described
as above. The cultures were extracted with ethyl acetate for 40 min at ultra-
sonic condition. The extracts were evaporated under reduced pressure on a
rotary evaporator (50 °C) and then checked by TLC plates. The controls with
the substrate but without fungi and the controls with the fungi but without
substrate were incubated at the same conditions described as above.
Preparative Fermentation The preparative fermentation was carried
out according to standard two-stage fermentation. The seed cultures were
grown in 250 ml flasks each containing 100 ml of medium. 2% of the seed
cultures were added fifteen 1000 ml flask that contains 400 ml liquid PDA
media and several grains of glass beads which can increase the oxygen.
After 3 d, 800 mg substrate ursolic acid (suspended in ethanol : DMSOꢁ1 : 1)
were distributed evenly in fifteen flasks. The cultures were further incubated
for 10 d and then harvested. Mycelia were filtered and were ultrasonic ex-
tracted with ethyl acetate 40 min (3 times). The aqueous layer was extracted
with the equal volumes of the same solvent for 3 times. Both extracts were
combined and evaporated under reduced pressure on a rotary evaporator
(50 °C) and 490 mg crude extracts were obtained.
—
—
2.06 (o)
—
5.75 (dd, 1.2, 10.2)
6.05 (br d, 10.8)
—
—
139.4
89.9
42.9
26.4
23.6
45.6
60.7
38.6
40.7
31.4
32.3
28.8
16.1
15.5
20.2
16.7
179.9
18.1
19.5
—
—
—
15 1.25 (o)
30.5 1.81 (o), 1.19 (o)
1.41 (o), 2.22 (td, 4.8, 12.6) 23.8 2.15 (o),1.38 (o)
— 45.6
1.71 (d, 12.0)
16
17
18
—
61.2 1.65 (d, 12.6)
39.6 1.81 (m)
40.8 0.75 (o)
31.4 1.43 (o)
32.4 1.85 (o), 1.90 (o)
29.9 1.25 (o)
16.3 1.10 (s)
15.5 1.29 (o)
15.5 1.36 (s)
17.0 1.29 (o)
19 1.83 (o)
20 0.78 (o)
21 1.42 (o)
22 1.88 (o)
23 1.25 (s)
24 1.04 (o)
25 0.98 (s)
26 1.56 (s)
27 1.40 (o)
28
—
180.0
18.3 0.99 (d, 6.0)
18.4 0.86 (d, 6.0)
—
29
30
1.05 (d, 6.6)
0.88 (o)
Isolation of the Metabolites The crude residue (490 mg) was first puri-
fied by column chromatography on silica gel (200—300 mesh, 21 g) eluted
with a stepwise petroleum ether/acetone from 100 : 1 to 1 : 1. Two fractions
were obtained: fraction A (101 mg), B (60 mg). Fraction A was further puri-
fied by column chromatography on silica gel (300—400 mesh, 7 g) eluted
with a stepwise petroleum ether/acetone from 10 : 1 to 8 : 1 to afford metabo-
lite 2 (5 mg), and eluted with a stepwise dichloromethane /methanol from
90 : 1 to 60 : 1 to afford metabolite 3 (4 mg). Fraction B was further purified
by column chromatography on silica gel (100—200 mesh, 12 g) eluted with
a stepwise trichloromethane/methanol from 3 : 1 to 1 : 1 to afford metabolite
4 (7 mg).
Chemical shifts: ppm; coupling constants: Hz; Mult, multiplicity: s, singlet; d,
doublet; t, triplet; o, overlap. Assignments were based on 1H-NMR, 13C-NMR, APT,
HMQC and HMBC experiments.
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Product 2: White solid, mp 236—237 °C, ESI-MS: m/z: 455 [MꢀH]ꢀ
,
1
931 [2MꢀNa]ꢀ, H-NMR (C5D5N): 0.77 (3H, s), 0.89 (3H, s), 0.97—0.98
(6H, o), 1.04 (3H, (3H, s), 0.92 (3H, s), 1.14 (3H, s), 3.20 (m), 5.51 (1H, dd,
Jꢁ10.8, 4.8 Hz, 11-H), 5.94 (1H, d, Jꢁ10.8 Hz, 12-H); 13C-NMR (C5D5N):
38.5 (C-1), 23.5 (C-2), 78.3 (C-3), 39.9 (C-4), 55.4 (C-5), 18.3 (C-6), 31.9
(C-7), 42.4 (C-8), 53.7 (C-9), 37.0 (C-10), 130.0 (C-11), 134.1 (C-12), 89.8
(C-13), 42.6 (C-14), 28.2 (C-15), 26.2 (C-16), 45.5 (C-17), 60.8 (C-18), 38.9
(C-19), 40.7 (C-20), 21.3 (C-21), 32.3 (C-22), 28.7 (C-23), 16.2 (C-24), 16.5
(C-25), 19.5 (C-26), 18.6 (C-27), 179.8 (C-28), 18.5 (C-29), 19.7 (C-30).
Product 3: White solid, mp 269—270 °C, [a]D20 ꢀ5.3° (cꢁ0.75, MeOH),
UV lmax (MeOH): 203 nm; IR (KBr) cmꢂ1: 3446, 3421, 2951, 2925, 2869,
2856, 1759, 1457, 1387, 1219. HR-ESI-MS: m/z: 469.3333 [MꢀH]ꢀ,
1
(Calcd for C30H45O4, 469.3312). H-NMR (500 MHz, pyridine-d5) and 13C-
NMR (150 MHz, pyridine-d5) see Table 1.
Product 4: White solid, mp 274—277 °C, [a]D20 ꢀ70.6° (cꢁ0.11, MeOH),
UV lmax (MeOH): 205 nm; IR (KBr) cmꢂ1: 3546, 3437, 2969, 2951, 2933,
2868, 1757, 1467, 1387, 1360, 1220. HR-EI-MS: m/z: 470.3365 [M]ꢀ,
1
(Calcd for C30H46O4, 470.3396). H-NMR (500 MHz, pyridine-d5) and 13C-
NMR (150 MHz, pyridine-d5) see Table 1.
Acknowledgements This work is financially supported by the National
Nature Science Foundation of China (Project No. 20772158) and the New
Drug Development Project of China (Project No. 2009ZX09301-003).
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