Biocatalytic Synthesis of the Anti-diabetes Agent-corosolic Acid by Whole Cells of Microorganisms

Article abstract of DOI:10.1002/jccs.201500035

Diabetes is one of the most prevalent and costly global diseases. For diabetes, frequent insulin treatment and synthetic drugs are very expensive and may cause unwanted side effects. Corosolic acid (CA), a natural product, was reported to be efficient in the treatment of diabetes, meanwhile without induction of anti-insulin antibodies and obesity. The preparation of CA attracted many researchers in the world. This study investigated the biocatalytic synthesis method of CA from ursolic acid by Streptomyces griseus subsp. griseus 4.18. LC-MS analysis demonstrated that 5 day, 125 μg/mL substrate, pH=9 and 10% strain concentration were the appropriate conditions. It is estimated that biocatalysis will contribute to the development of green and sustainable synthetic processes with less time-consuming and more environmentally friendly.

Full text of DOI:10.1002/jccs.201500035

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Biocatalytic Synthesis of the Anti-diabetes Agent-corosolic Acid by Whole Cells of
Microorganisms
Shaobin Fu,a* Qingfeng Meng,b Shu-an Long,a Yun Zhang,a Di-an Sunc and Gang Weid
aDepartment of Pharmacy, Zunyi Medical University, Zunyi 563000, China
bDepartment of Public Health, Zunyi Medical University, Zunyi 563000, China
cChinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
dMaterials Science and Engineering, Commonwealth Scientific and Industrial Research Organization (CSIRO), 2070,
Australia
(Received: Jan. 28, 2015; Accepted: Aug. 11, 2015; Published Online: Sept. 7, 2015; DOI: 10.1002/jccs.201500035)
Diabetes is one of the most prevalent and costly global diseases. For diabetes, frequent insulin treatment and
synthetic drugs are very expensive and may cause unwanted side effects. Corosolic acid (CA), a natural
product, was reported to be efficient in the treatment of diabetes, meanwhile without induction of anti-insu-
lin antibodies and obesity. The preparation of CAattracted many researchers in the world. This study investi-
gated the biocatalytic synthesis method of CAfrom ursolic acid by Streptomyces griseus subsp. griseus 4.18.
LC–MS analysis demonstrated that 5 day, 125 mg/mL substrate, pH = 9 and 10% strain concentration were
the appropriate conditions. It is estimated that biocatalysis will contribute to the development of green and
sustainable synthetic processes with less time-consuming and more environmentally friendly.
Keywords: Biocatalysis; Synthesis; Anti-diabetes; Corosolic acid; Microbes.
INTRODUCTION
Pentacyclic triterpene acids are one group of promis-
be prepared by a chemical procedure, a green synthetic
method would be desirable. The method of preparation of
CA through plant cell culture techniques was developed
with the low productivity and instability which prevented
the method from being applied to relevant industry.10 It is
necessary to look for a new method for the preparation of
CA. As more and more attentions were paid to health and
environmental protection, the sustainable and green chemi-
cal synthesis is much more popular.11,12 Biocatalysis is con-
sidered more efficient and less side-reactions. The essence
of biocatalysis is the reaction of enzymes. Due to their spe-
cific three-dimensional structure, enzymes may distinguish
between functional groups and other groups in different re-
gions of the substrate molecule.13 As a consequence,
biocatalysis is of chemoselectivity, regioselectivity, enanti-
oselectivity.
ing natural product which distributed in many species of
plants. Corosolic acid (CA), a pentacyclic triterpenoid
acid, also named 2a-hydroxyursolic acid, is found in a va-
riety of plant species. Extensive research has revealed sev-
eral important pharmacological activities of CA, such as
anti-cancer,1,2 anti-inflammatory activities,3,4 cardiopro-
tective effect.5 This compound, known as ‘plant insulin’,
has attracted interests worldwide owing to its remarkable
ability of anti-diabetes for application of weight-loss man-
agement and blood sugar balance on animal experiments
and clinical trials.6-8 Both the pure compound corosolic
acid and the herbal product are considered as one of the
good sources for a new drug or a lead to make a new drug to
treat diabetes. KFDA (Korean FDA) has approved blood
glucose lowering effects of banaba alcohol extract as func-
tion II grade with a restricted dose of 50-100 mg/day
corosolic acid.9 Extraction from medicinal plants is the
main preparation method to obtain CA. However, CA was
low in plant- based resource in nature.
Herein, dozens of strains were screened to synthesis
of CA by biotransformation and S. griseus subsp. griseus
CGMCC 4.18 can catalyze the natural product with similar
structure and low price-ursolic acid (UA) into CA. Optimi-
zation experiments were also performed.
Additionallly, CA and its isomers usually coexist in
the same plant which also brings difficulty in separation.
Besides, most of plant-originated useful materials have
complicated structures having been synthesized through
various stages of biosynthesis pathways. Though CA can
RESULTS AND DISCUSSION
Optimization of reaction conditions
Effect of media on transformation
In two media, the PDA medium was fond to con-
* Corresponding author. Tel: +86-0851-28609461; Fax: +86-0851-28609493; Email: fushb@126.com
J. Chin. Chem. Soc. 2015, 62, 871-874
© 2015 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
871

Article
Fu et al.
tribute to the growth of mycelia, but with no transforma-
tion product CA. Therefore, The rest experiments were
performed on GYM media.
most bacteria and actinomycetes are suitable for the growth
of pH in the neutral and alkaline environment. In this study,
strain S. griseus subsp. griseus CGMCC 4.18 grew slowly
and CA was difficult to be detected at pH 6. UA was con-
verted into CA with a higher conversion rate in pH 7 and 9,
while strain in the pH 8 media was polluted which was dealt
with no statistically significance (Fig. 3).
Effect of substrate concentration on transformation
Substrates (S1 to S5) of 31.25 mg/mL, 62.5 mg/mL,
125 mg/mL, 250 mg/mL, 500 mg/mL were investigated in
the experiment. All the five concentrations were harvested
on the 5 day. Apparently, 125 mg/mL UA was optimum for
CAproduction; above this concentration, production of UA
decreased (Fig. 1).
Effect of strain concentration on transformation
The strain concentration also affected the catalytic
activity. According to Fig. 4, when the initial concentration
of was 10%, the transformation rate reached the peak. The
conversion rate decreases above or below this concentra-
tion.
Time-course experiment
Every flask was harvested ever day from the second
day to the eighth day with the same substrate amount. The
results demonstrated that the obtained CA reached a maxi-
mum at 5 days culture time (Fig. 2). The proruction of CA
reached the most yield 959.4 mg/mL.
Separation and identification of transformation
product CA
The crude extract 830 mg was subjected to column
chromatography on silica gel (300–400 mesh, 20 g) with a
stepwise elution with petroleum ether/acetone/acetic acid
from 100:2:0.1 to acetone. The fractions were purified by
Effect of pH on transformation
Different pH values from 6 to 9 were investigated. All
the flasks were harvested on the same time. In commen,
Fig. 1. Effect of substrate concentration on transfor-
mation.
Fig. 3. Effect of pH on transformation.
Fig. 2. Time-course experiment.
Fig. 4. Effect of strain concentration on transforma-
tion.
872
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
One-step Synthesis of Anti-diabetes Agent-corosolic Acid from Ursolic Acid by Biotransformation
column chromatography on silica gel eluted with trichloro-
methane/THF (9:1 to 1:1) and sephadex LH-20 trichloro-
methane/methanol (1:1). CA (4 mg) were obtained after
recrystallization. The structure of CA was confirmed by
NMR (Table 1), MS (ESI-MS: m/z: [M-H]-: 471,) and ref-
erence.14
(300-400 mesh, Qingdao Oceanic Chemicals, China) and Se-
phadex LH-20. 10% H2SO4 in 95% ethanol spray reagent was
used for TLC plated followed by heating. General solvents and re-
agents were purchased from Beijing Chemical Industry Com-
pany, Beijing, China. The microbe was purchased from China
General Microbiological Culture Collection center (CGMCC).
Substrate and Microorganisms
Table 1. 13C NMR data and characteristic 1H-NMR of CA
The substrate ursolic acid (with purity > 98%) was pur-
chased from Changsha Staherb Natural Ingredients Co., Ltd in
China and strain S. griseus subsp. griseus CGMCC 4.18 was pur-
chased from the Institute of Microbiology, Chinese Academy of
Sciences (AS), Beijing, China.
No
13C-NMR
1H-NMR
1
48.4
69.0
84.2
40.2
56.3
19.2
33.9
40.4
48.4
38.8
24.1
126.0
139.7
42.9
29.0
25.3
48.5
53.9
39.9
39.8
31.5
37.8
29.7
18.1
17.4
17.9
24.3
180.2
17.9
21.8
2
4.11 (1H, td. J = 4.2, 10.2 Hz)
3.42 (1H, d, J = 9.6 Hz)
3
4
Biocatalytic synthesis of CA from UA
5
Effect of media on transformation: The preliminary
screening experiment was carried out in two media. One is PDA
medium consisting of 200 g potatoes (boiling 20 min; dextrose,
20 g; peptone, 10 g; distilled H2O, 1000 mL). The other medium is
GYM medium consisting of yeast extract 4.0 g, malt extract 10.0
g, glucose 4.0 g, pH 7.3. The preliminary screening experiment of
S. griseus subsp. griseus CGMCC 4.18 was carried out in PDA
medium and GYM medium. Fermentations were carried out ac-
cording to standard two-stage fermentation.15
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.49 (t-like, 12-H)
Effect of substrate concentration on transformation: As
previously report, the concentration of substrate played an impor-
tant role in biotransformation.16 Different concentrations of UA
(from 1.25 mg to 20 mg per flask) were added in the transforma-
tion broth and the yield of CA was evaluated.
2.65 (1H, d, J = 11.4 Hz)
Time-course experiment: The biotransformation process
was performed by a procedure similar to normal transformation
examinations in 7 flasks. Reaction was terminated in only one
flask every day.
1.33 (3H, s)
1.23 (3H, s)
1.07 (3H, s)
1.09 (3H, s)
1.29 (3H, s)
Effect of pH on transformation: Different pH values from
6 to 9 were investigated. All the flasks were harvested on the same
time.
1.01 (3H, d, J = 6.6 Hz)
0.97 (3H, d, J = 6.6 Hz)
1H-NMR (600 MHz) and 13C-NMR (150 MHz) Data of CA
Effect of strain concentration on transformation: Five
strain concentrations 2.5%, 5%, 10%, 17.5%, 27.5% (1 mL, 2 mL,
4 mL, 7 mL seed broth in 40 mL media) were tested on the trans-
formation. The incubation and other conditions were same as the
pH.
(Pyridine-d5).
EXPERIMENTAL
General experimental procedure: NMR spectra were re-
corded on a Bruker DRX-600 spectrometer operating at 600 MHz
Separation and identification of transformation product
CA: The preparative scale biotransformation from UA to CA by
S. griseus subsp. griseus CGMCC 4.18 was similar to the prelimi-
nary screening experiment. 600 mg substrate was added in total.
The target compound was obtained after column chromatography
on silica gel, sephadex LH-20, recrystallization from 830 mg
crude extract. The product was separated by column chromato-
graphy and sephadex LH-20. The sructure was identified by
(for H) and 150 MHz (for 13C) in pyridine-d5. LC-MS (AB
1
SCIEX) was carried out on C-18 column with methanol - 0.2% FA
water = 95:5 and with the condition of CUR: 40.00, CAD; Me-
dium; IS: -4500.00; TEM: 550.00; GS1: 55.00; GS2: 55.00; DP:
-140.00; EP: -10.00; CE: -40.00; CXP: -15.00. TLC experiments
were performed on GF254 plates (Qingdao Oceanic Chemicals,
China). Column chromatography was carried out on silica gel
J. Chin. Chem. Soc. 2015, 62, 871-874
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Fu et al.
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21462057), Program of Ministry of Education “Chunhui
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