Microbial Transformation of 18β-Glycyrrhetinic acid yields: Potent
Inhibitors Urease; In-vitro and In-silco studies
Abstract
Biological structural modification of natural metabolites (products) becomes an important, and
worthy approach to diverse pharmacological properties. The current study focuses on the
structural modification of 18β-glycyrrhetinic acid that was carried out by using Macrophomina
phaseolina and Cunninghamella blakesleeana. Microbial bio-transformation of compound 1
with M. phaseolina yielded metabolites 2-6, while metabolites 7 and 8 were obtained with C.
blakesleeana. Among them, two metabolites 5 and 6 were found to be new metabolites.
Furthermore, all the yielded metabolites were subjected to test their inhibitory potential against
urease, which is a key agent in causing different gastric ulcers and other nephropathies. Among
the metabolites, compound 1 was found to be potent active (IC50 =13.2 ± 0.9 µM), followed by
compounds 7 (IC50 = 22.4 ± 1.13 µM), 4 (IC50 = 23.8 ± 0.64 µM), and 5 (IC50 25.8 ± 0.78 µM).
While compound 2 was found to be moderate active (IC50 = 38.6 ± 2.3 µM). Compounds 3, 6,
and 8 were found inactive. Acetohydroxamic acid (IC50 = 20.3 ± 0.43 µM) was used as a
reference compound in this study.
Keywords
Biotransformation; 18β-glycyrrhetinic acid; Macrophomina phaseolina KUCC 730; Cunninghamella
blakesleeana ATCC 8688A; Urease Inhibition; Gastric Ulcers;
1
Introduction
Plant s' derive secondary metabolites have been one of the common sources of drug
discovery. Due to the number of limitations associated with the plant natural products i.e.
toxicity to normal cells, solubility, and so on, these products are usually modified based on the
structure-activity relationship in delivering candidates to the clinic trial [1,2]. Microorganisms
exist with a high magnitude of biodiversity than plants, and offers exceptional metabolic
adaptability [3]. Therefore, microorganisms with such structural modification capacities will
embark on the biotechnology boom especially in drug development and large-scale production.
18β-Glycyrrhetinic acid/Enoxolone (GA) (1), is a pentacyclic triterpene, is one of the
most important bioactive components of licorice (Glycyrrhiza glabra L., Fabaceae) [4-6]. GA
(1) is well known for its biological activities, such as anti-inflammatory, anticancer, antiviral,
1
and anti-allergic properties [7-9]. Previously, Li, Kun et al. and his co-worker chemical
synthesize a number of derivatives of GA (1) with potent anti-cancer activities [10].
Biotransformation or microbial based transformation is one of the method of choice for
structural modifications of compounds having complex skeleton because reactions are highly
regio- and stereo-selective with environmentally friendly conditions [11]. Biocatalytic
conversions can be achieved by several agents, such as enzymes, animals and plant cell
cultures, and microorganisms. On the other hand, microbes are most effectively used for this
purpose, as they have a cytochrome P450 monooxygenase system that catalyzes the
hydroxylation at various unassailable positions of the complex skeleton [12,13].
Biotransformation on GA (1) has been earlier reported by using various biological
systems, such as bacteria, and fungi [14-17]. The current study is the continuation of our
research on the biotransformation of 18β-glycyrrhetinic acid [15]. This time Macrophomina
phaseolina and Cunninghamella blakesleeana were used for derivatization of compound 1.
Oxidative ring cleavage, Baeyer-villager oxidation, and hydroxylation at various carbons were
observed. Biotransformation of GA acid with these fungi yielded two new 5-6, and five known
2-4, and 7-8 compounds.
Urease (EC 3.5.1.5) is a bi-nickel containing metallic-enzyme that catalyzes the
hydrolytic decomposition of urea into ammonia and carbon dioxide, and thus enables
organisms to use urea as a nitrogen source. Ureases are also had a strict influence on socioeconomic growth and environmental hardals [18-20]. The continuous labor of urease enzyme
in both human and animal cells can cause multiple chronic diseases, such as urolithiasis,
urinary catheter encrustation, hepatic encephalopathy, peptic, and duodenal ulcers.
In view of their diverse functions, urease inhibition is considered as one of the key
factors to avoid such chronic infections. Thereby, specific compounds with high efficiency and
potency could provide an insight into the treatment of such chronic diseases. Thus we designed
the current study to address the current enigma of urease inhibitors.
2
Materials and Methods
2.1
Fungal Strains and Media
A bio-transformational study was done by using strains of M. phaseolina, obtained
from Karachi University Culture Collection (KUCC), Pakistan, and C. blakesleeana (ATCC
2
8688A), which purchased from American Type Culture Collection (ATCC), USA. SDA slants
were used for sufficient growth and kept at 4 ˚C.
The media ingredients used for the preparation of the growing culture of M. phaseolina
and C. blakesleeana are the same. For 1 L distilled water, glucose (10 g), peptone (5 g), sodium
chloride (5 g), KH2PO4 (5 g), and glycerol (10.0 mL) were used.
2.2
General experimental details
Substrate 1 (18β-glycyrrhetinic acid, GA) was purchased from Sigma-Aldrich Chemie
GmbH (Germany). Buchi (M-560) machine was used for measuring melting points. TLC cards
(PF254; 20 × 20, 0.25 mm thick) were supplied by Merck (Darmstadt, Germany). Metabolites
were visualized by spraying ceric sulfate on TLC cards. The separation was carried out by
column chromatography on Silica gel (70-230, mesh), acquired from E. Merck. Final
purification of GA metabolites was carried out by using a recycling reverse phase HPLC, fitted
with the JAIGEL ODS-H-80 column. Infrared (IR), Ultraviolet (UV), and optical rotations
experiments were recorded on FT-IR-8900 spectrophotometer, UV-240 spectrophotometer,
Shimadzu (Tokyo, Japan), and Stuart SMP-10 (chloroform/methanol), respectively. The
masses of the resulting metabolite were measured by using a JEOL JMS-600H (JEOL,
Akishima, Japan) mass spectrometer, which was used for both low-resolution (EI-MS) as well
as for high-resolution electron impact mass spectrometry (HREI-MS). For polar compounds,
fast atomic bombardment mass spectroscopic (FAB-MS) measurements were carried out on
JEOL TMS-HX110 (Japan) spectrometer, using glycerol or thioglycerol as a matrix, and KI or
ScI used as an internal standard to determine the exact masses of organic compounds. Onedimensional (1H-NMR), and two-dimensional (13C-NMR, HSQC, HMBC, COSY, and
NOESY) techniques were performed on Bruker Avance-Cryo probe spectrometers, by Bruker,
(Zurich, Switzerland) in CD3OD or DMSO-d6.
2.3
Biotransformation of 18β-Glycyrrhetinic acid (1) with Macrophomina phaseolina
(KUCC 730)
The above-mentioned media (13 L, 500 mL in 1000 mL flasks) was sterilized and
cooled at room temperature, spores of M. phaseolina were added to each flask from a seed
flask. After a few days, when enough growth of fungi was observed, a clear solution of
substrate 1 (2 g) in methanol was dispensed into 26 flasks. This reaction was carried out for 18
days, with continuous shaking at low temperature (26-28 ˚C) on a rotary shaker. The reaction
mixture was also monitored with negative control (A reaction mix. containing media and fungi)
as well as with positive control (A reaction mix. containing media and drug). After completion
3
of the reaction, a suitable solvent mainly ethyl acetate was added in it for quenching the
reaction. The solution was filtered, extracted, and evaporated yielding a brown gummy material
which was then fractionated by column chromatography (4% gradient EtOAc, and 10%
gradient MeOH-DCM). Resulting fraction-1 (at 70% EtOAc) was purified using RP-HPLC
(Mobile phase MeOH/H2O 8/2), and metabolites 2, and 6 were obtained. The fraction-2 (at
10% MeOH) was further purified by using the same solvent system on HPLC, to obtain
metabolite 3-5.
2.3.1 3,4-Seco-4-hydroxy-11-oxo-18β-olean-12-en-3,30-dioic acid 3,4-lactone (2)
White shiny solid (80 mg). m.p.: 266-270 ˚C. [𝛼]25
D : -21.9˚ (c = 0.0012). UV (MeOH):
λmax nm (log ɛ) 248 (3.6). IR (CHCl3); Vmax cm-1: 3578 (OH), 1724 (lactone), 1690 (enone),
1640 (acid). 1H-NMR (DMSO-d6, 400 MHz): Table-2.
13
C-NMR (DMSO-d6, 100 MHz):
Table-2. FAB-MS (+) m/z: 585.3 [M + H]+. HRFAB-MS (mol. formula, calcd value): m/z
585.3281 (C30H45O5, 585.3267).
2.3.2
3,4-Seco-4-hydroxy-11-oxo-18β-olean-12-en-3,30-dioic acid (3)
White amorphous powder (15 mg). tR: 20 min. m.p.: 289-304 ˚C. [𝛼]25
D : +65.1 (c =
0.0011). UV (MeOH): λmax nm (log ɛ) 248 (3.9). IR (CHCl3); νmaxcm-1: 3410 (OH), 1647
(enone), 1557 (acid). 1H-NMR (CD3OD, 600 MHz): Table-2. 13C-NMR (CD3OD, 150 MHz):
Table-2. FAB-MS (+) m/z: 503.2 [M + H]+. HRFAB-MS (mol. formula, calcd value): m/z
503.3385 (C30H47O6, 585.3373).
2.3.3 3,4-Seco-4,7β-dihydroxy-11-oxo-18β-olean-12-en-3,30-dioic acid (4)
White crystalline solid (15 mg). tR: 18 min. m.p.: 296-310 ˚C. [𝛼]25
D : +59.7 (c = 0.0013).
UV (MeOH): λmax nm (log ɛ) 248 (3.9). IR (CHCl3); νmaxcm-1: 3472 (OH), 1682 (enone), 1653
(acid). 1H-NMR (CD3OD, 400 MHz): Table-2. 13C-NMR (CD3OD, 100 MHz): Table-2. FABMS (+) m/z: 519.2 [M + H] +. HRFAB-MS (mol. formula, calcd value): m/z 519.3333
(C30H47O7, 519.3322).
2.3.4 3,4-Seco-4,15α-dihydroxy-11-oxo-18β-olean-12-en-3,30-dioic acid (5)
White amorphous powder (15 mg). tR: 20 min. m.p.: 258-260 ˚C. [𝛼]25
D : +80.6 (C =
0.0011). UV (MeOH): λmax nm (log ɛ) 243, 248 (3.8). IR (CHCl3); νmaxcm-1: 3457 (OH), 1664
(enone), 1588 (acid). 1H-NMR (CD3OD, 400 MHz): Table-3. 13C-NMR (CD3OD, 100 MHz):
Table-2. FAB-MS (+) m/z: 519.1 [M + H] +. HRFAB-MS (mol. formula, calcd value): m/z
519.3335 (C30H47O7, 519.3322).
4
2.3.5 3,4-Seco-4,15α-dihydroxy-11-oxo-18β-olean-12-en-3,30-dioic acid,3-methyl ester
(6)
White shiny solid (15 mg). tR: 18 min. m.p.: 345-350 ˚C. [𝛼]25
D : +62.7 (c = 0.0011).
UV (MeOH): λmax nm (log ɛ) 248 (3.9). IR (CHCl3); νmaxcm-1: 3446 (OH), 1735 (ester), 1701
(enone), 1650 (acid). 1H-NMR (CD3OD, 400 MHz): Table-3. 13C-NMR (CD3OD, 100 MHz):
Table-2. FAB-MS (+) m/z: 533.4 [M + H] +. HRFAB-MS (mol. formula, calcd value): m/z
533.3498 (C31H49O7, 533.3478).
2.4
Biotransformation of 18β-Glycyrrhetinic acid (1) with Cunninghamella
blakesleeana (ATCC 8688A)
This time C. blakesleeana was used for conversion of the same substrate 1 (2.5 g). It
was dissolved in acetone and distributed among 25 flasks containing sterilized media and fully
grown strains of C. blakesleeana. These flasks were then kept on a rotary shaker for 18 days at
low temperatures. Later on, the reaction was stopped by adding ethyl acetate, and the same
solvent was used for extraction. The organic layers were evaporated to obtain a gummy crude
material which was subjected to column chromatography with 4% acetone in hexanes as
solvent system. An impure fraction of about 300 mg was obtained which was again passed
through a column of silica gel using 0.5% gradient methanol in dichloromethane to obtain two
fractions with small impurity, finally, metabolite 7-8 were purified by recycling HPLC with
methanol and deionized water (70:30) as solvent system.
2.4.1 3β,7β-Dihydroxy-11-oxo-Olean-12-en-30-oic acid (7)
White shiny solid (70 mg). tR: 34 min. m.p.: 320-327 ˚C. [𝛼]25
D : +90 (c = 0.14). UV
(MeOH): λmax nm (log ɛ) 243, 248 nm (4.46). IR (CHCl3); νmaxcm-1: 3393 (OH), 1703 (enone),
1651 (acid). 1H-NMR (DMSO-d6, 400 MHz): Table-3.
13
C-NMR (DMSO-d6, 100 MHz):
Table-2. EI-MS m/z (rel. int., %): 486 [M]+ (22), 468 (34), 303 (100). HREI-MS (mol. formula,
calcd value): m/z 486.3346 (C30H46O5, 486.3345).
2.4.2 3β,7β,15α-Trihydroxy-11-oxo-Olean-12-en-30-oic acid (8)
White shiny solid (70 mg). tR: 34 min. m.p.: 320-327 ˚C. [𝛼]25
D : +90 (c = 0.14). UV
(MeOH): λmax nm (log ɛ) 243, 248 nm (4.46). IR (CHCl3); νmaxcm-1: 3393 (OH), 1703 (enone),
1651 (acid). 1H-NMR (DMSO-d6, 400 MHz): Table-3.
13
C-NMR (DMSO-d6, 100 MHz):
Table-2. EI-MS m/z (rel. int., %): 486 [M]+ (22), 468 (34), 303 (100). HREI-MS (mol. formula,
calcd value): m/z 486.3346 (C30H46O5, 486.3345).
3
Biology
5
3.1
Urease inhibition Assay:
The urease inhibition assay in vitro was performed spectrophotometrically. The
reaction mixture is comprised of 25 µL of urease solution (1 U/well) was incubated with 5 µL
of test compound (500 µM) for 15 minutes at 30 °C. Thereafter, 55 µL of urea (substrate) with
100 mM concentration was added, and the plate was again incubated for 15 min at 30 °C. After
incubation, 45 µL of phenol reagents (1 % w/v phenol and 0.005 % w/v sodium nitroprusside),
and 70 µL of alkali reagents (0.5 % w/v sodium hydroxide and 0.1 % sodium hypochlorite)
were added to each well. The plate was again incubated for 50 min at 30 °C. The activity of
urease was monitored with the rate of production of ammonia continuously, following the
method of Weatherburn, [21] and change in absorbance was monitored at 630 nm on an ELISA
plate reader (Spectra Max M2, Molecular Devices, CA, USA). Acetohydroxamic acid was used
as a standard. For kinetic studies, the concentration of test compounds that inhibits the
hydrolysis of substrates (jack bean urease) by 50% (IC50) was determined by monitoring the
urease inhibitory effect of various concentrations of both compounds in the assay. The IC50
(inhibitor concentration that inhibits 50% activity of enzyme) values were then calculated using
the EZ-Fit Enzyme kinetics program (Perrella Scientific Inc., Amherst, MA, USA). Kinetic
Graphs were plotted using the GraFit program (Leather barrow RJ. GraFit Version 4.09.
Staines, UK: Erithacus Software Ltd) [22].
6
4
Results and discussion
Macrophomina phaseolina (KUCC 730) has been used extensively for various
biotransformation activities. In the present study, its potential for oxidative ring cleavage
reactions has been reported for the first time. 18β-Glycyrrhetinic acid (GA) was converted into
3,4-Seco-oleanane-type metabolites, which resulted from the oxidation of 3-oxo intermediates
to seven-membered ring lactone, which were then hydrolyzed to get the resulting metabolites,
and hydroxylated (C-7, C-15) metabolites. Compounds 2-4 were published earlier through the
biotransformation of 18β-glycyrrhetinic acid with Chania antibiotic all spectral data were
compared with reported data [18].
Metabolite 5 was obtained as a white amorphous powder through RP-HPLC, fixed with
column JAIGEL ODS H-80 (MeOH: H2O; 80:20), at a retention time (tR) of 20 minutes. The
molecular formula for metabolite 5 was determined as C30H47O7 by HRFAB-MS ([M + H]+ at
m/z 519.3335 (calcd. 519.3322), with an additional mass of 48 a.m.u. in the substrate. There is
an addition of three oxygen atoms in metabolite 5. The 1H-NMR spectrum of metabolite 5
indicated an additional peak of methane proton as a double doublet at δ 4.22 (J15α,16α = 11.0 Hz,
J15α,16e = 4.5 Hz). The 13C-NMR spectrum of metabolite 5 further indicated the presence of an
additional hydroxyl group through the appearance of a downfield peak at δ 65.5 (C-15).
However, the 13C-NMR spectrum also showed one of the two carboxyl carbons at δ 170.3, and
a downfield signal for tertiary carbinol carbon (C-4) at δ 76.1. This indicated the opening of the
ring between C-3, and C-4. The position of newly incorporated hydroxyl moiety was deduced
from HMBC correlations. H-15 (δ 4.22, dd) showed correlations with C-16 (δ 36.4), and C-28
(δ 22.8), and indicated hydroxylation at C-15. The hydroxyl moiety at C-15 was further
supported by COSY correlations of H2 -16 (δ 1.72, m; 1.46, m) with H-15 (δ 4.22, dd). The
position of the other hydroxyl group at C-4 was deduced from HMBC correlations of H-23 (δ
1.26, s), H-24 (δ 1.29, s), and H-5 (δ 1.42, m) with C-4 (δ 76.1). Similarly, H2-1 (δ 2.40, m;
1.93, m), and H2-2 (δ 2.60, m; 2.40, m) showed HMBC correlations with carboxyl C-3 (δ 170.3)
and indicated a ring cleavage. The stereochemistry of -OH group at C-15 was assigned as αoriented based on NOESY correlations of β-oriented H-15 with β-oriented H-26 (δ 1.38, s),
and H-28 (δ 0.75, s). Based on 1H-NMR, 13 C-NMR, IR, and mass spectrometric analyses,
metabolite 5 was deduced as a new compound 3,4-Seco-4,15α-dihydroxy-11-oxo-18β-Olean12-en-3,30-dioic acid (5). Metabolite 6 was isolated as a white shiny solid with the help of
recycling RP-HPLC, fixed with column JAIGEL ODS H-80 (MeOH: H2O; 80:20), and
retention time (tR) of 18 minutes. Its molecular composition was determined as C31H49O7 by
HRFAB-MS ([M + H]+ at m/z 533.3498, calcd. 533.3478), with an increment of 62 a.m.u. This
7
suggested the incorporation of three oxygen atoms and a methyl group. The 1H-NMR spectrum
of compound 6 was found to be different from substrate 1 in many ways. First, an additional
methyl signal appeared at δ 3.59 as a single, which was not present in the substrate. Secondly,
a methine proton appeared as a double doublet at δ 4.22 (J15α,16α = 12.0 Hz, J15α,16e = 4.0 Hz).
The 13C-NMR spectrum further indicated the presence of an additional methoxy carbon at δ
51.9 (C-31) and suggested the insertion of a methoxy group. The incorporation of a hydroxyl
group at C-15 was observed through the downfield signal at δ 65.3 (C-15). The position of the
newly introduced methoxy group was deduced based on the HMBC correlation of H-31 (3.59,
s) with C-3. Additionally, H2-1 (δ 2.55, m; 2.18, m), and H2-2 (δ 2.66, m; 2.34, m) showed
HMBC correlations with C-3 (δ 176.9), which indicated the incorporation of a methoxy group
at C-3. The relative configuration of -OH group at C-15 was assigned as α on the basis of
NOESY correlations of geminal β-oriented H-15 with β-oriented, H-26 (δ 1.37, s), and H-28
(δ 0.76, s). Based on spectroscopic data, metabolite 6 was deduced as a new compound, 3,4seco-4,15α-dihydroxy-11-oxo-18β-Olean-12-en-3,30-dioic acid, 3-methyl ester (6).
4.1
Biological Evaluation and Structure-Activity Relationship:
18β-Glycyrrhetinic acid (1), and its metabolites 2–8 were evaluated for their urease
inhibitory assay. The addition of different substituted groups on the icosahydropicene skeleton
were carefully studied to elucidate the best possible structure-activity relationship. Compound
1 (substrate) showed a potent activity with an (IC50 =13.2 ± 0.9 µM), as compared to the
reference compound, acetohydroxamic acid (IC50 = 20.3 ± 0.43 µM). The activity might be due
to the icosahydropicene skeleton and up to some extent due to the hydroxal groups and ketone
groups present on the icosahydropicene skeleton. The π-electron of the icosahydropicene
skeleton might forms the hydrophobic bonds with the side chains of the amino acids and with
the bi-nickel center of the urease enzyme. Such π-electron-metal interactions are very well
known for their role in the enzyme-ligand interactions
While comparing compounds 7 (IC50 = 22.4 ± 1.13 µM), 4 (IC50 = 23.8 ± 0.64 µM) and
5 (IC50 = 25.8 ± 0.78 µM) with compound 1, a slight decrease in their IC50 values were
8
observed. Such variation in the activity might be due to the modification in their structure by
the addition of different functional groups at different positions of the icosahydropicene
skeleton. Compound 7 where hydroxal were substituted at C-7, similarly, in compound 4 where
a phenyl ring is replaced with the propanoic acid at C-10, hydroxal group and methyl groups
at C-4 the compound showed further decrease in the activity in comparison to compound 7,
while in compound 5 where a hydroxal group is placed at C-15 the compound activity further
increases as compared to compound 4. An interesting correlation was observed from the pattern
of the activities of these compounds that the addition of Hydroxal groups at different position
significantly vary the compound activity. Whereas, compound 2 was found to be weak active
with (IC50 = 38.6 ± 2.3 µM) in comparison to compound 1. While compounds 3, 6, and 8 were
found to be inactive.
In addition, mechanism-based studies of these compounds revealed the type of
inhibition and their Ki values. Kinetic study of the compound 1 inferred that the compound
inhibits urease enzyme in a competitive-manner, in competitive inhibition Km changes while
Vmax remains constant. On the other hand, compounds 7 and 5 showed a mixed-type of
inhibition where an increase occurred in both Km and Vmax values. Whereas, compound 4 was
found to be a non-competitive inhibitor, where Km remains constant and Vmax varied. The Ki
values and type of inhibition are tabulated in Table-1.
Table-1: The IC50 and Ki values of active compounds.
Compounds code
Ki values µM
IC50 values µM
Type of Inhibition
1
21.3 ± 0.32
13.2 ± 0.9
Competitive inhibitor
7
25.6 ± 1.62
22.4 ± 1.13
Mixed type inhibitor
4
28.2 ± 1.21
23.8 ± 0.64
Non-competitive inhibitor
5
32.8 ± 0.82
25.8 ± 0.78
Mixed type inhibitor
Standard*
17.2 ± 0.21
20.3 ± 0.43
Competitive inhibitor
*Acetohydroxamic acid was used as positive control
9
V
0.8
25.0000
0.6
20
20.0000
A
B
15.0000
Slope = Km/Vmax
0.4
0.2
1/V
G
0
-0.2
10
10.0000
5.0000
0
0.0000
-10
-0.4
-20
-0.6
-0.8
-0.08
-3
-2
-1
0
1
2
0
0.04
0.08
Inhibitor
1/S
2.0000
0.2
1.0000
C
0.1
1/V
-0.04
3
0.5000
0.2500
0
-0.1
-0.2
-20
-10
0
10
20
Inhibitor
Figure-1: Urease inhibition by compound 1, (A) Line-weaver Burk plot of reciprocal rate of reaction 1/v against
reciprocal of substrate 1/s (urea) in the absence (▲) and in the presence of 5 µM (∆), 10 µM (■), 15 µM (□), 20
µM (●) and 25 µM (○) of compound 1. (B) Secondary replot of Line-weaver Burk plot is between slopes of each
line on Line-weaver Burk vs different concentrations of compound 1, and (C) Dixon plot reciprocal of rate of
reaction vs different concentrations of compound 1.
10
0.4
Slope = Km/Vmax
A
0.2
1/V
25.0000
0.1
20.0000
0
15.0000
0.05
Km/Vmax
B
10.0000
0
5.0000
0.0000
-0.05
-0.2
-0.1
-0.4
-2
-1
0
1
-20
2
-10
0
10
20
Inhibitor
1/S
0.4
2.0000
1.0000
1/V
0.2
C
0.5000
0.2500
0
-0.2
-0.4
-20
-10
0
10
20
Inhibitor
Figure-2: Urease inhibition by compound 7, (A) Line-weaver Burk plot of reciprocal rate of reaction 1/v against
reciprocal of substrate 1/s (urea) in the absence (▲) and in the presence of 10 µM (■), 15 µM (□), 20 µM (●), and
25 µM (○) of compound 7. (B) Secondary replot of Line-weaver Burk plot is between slopes of each line on Lineweaver Burk vs different concentrations of compound 7, and (C) Dixon plot reciprocal of rate of reaction vs
different concentrations of compound 7.
11
0.4
1
16.0000
Slope = Km/Vmax
A
0.5
1/V
(
20.0000
0
-0.5
B
0.2
12.0000
8.0000
0
4.0000
0.0000
-0.2
-1
-0.4
-2
-1
0
1/S
1
2
-20
-10
0
10
20
Inhibitor
1.5
2.0000
1
1.0000
C
0.5000
0.5
1/V
0.2500
0
-0.5
-1
-1.5
-20
-10
0
10
20
Inhibitor
Figure-4: Urease inhibition by compound 4, (A) Line-weaver Burk plot of reciprocal rate of reaction 1/v against
reciprocal of substrate 1/s (urea) in the absence (▲) and in the presence of 10 µM (∆), 15 µM (■), 20 µM (□), 25
µM (●) and 30 µM (○) of compound 4. (B) Secondary replot of Line-weaver Burk plot is between slopes of each
line on the Line-weaver Burk vs different concentrations of compound 4, and (C) Dixon plot reciprocal of rate of
reaction vs different concentrations of compound 4.
12
0.3
45.0000
0.06
0.2
35.0000
A
0.1
1/V
Slope = Km/Vmax
0.04
0
-0.1
B
30.0000
0.02
25.0000
0
0.0000
-0.02
(Column 7)
-0.04
-0.2
-0.06
-3
-2
-1
0
1
2
3
-30
1/S
-20
-10
0
10
20
Inhibitor
0.4
2.0000
C
0.2
1.0000
1/V
0.5000
0.2500
0
-0.2
-0.4
-40
-20
0
20
40
Inhibitor
Figure-5: Urease inhibition by compound 5, (A) Line-weaver Burk plot of reciprocal rate of reaction 1/v against
reciprocal of substrate 1/s (urea) in the absence (∆), and the presence of 15 µM (■), 20 µM (□), 25 µM (●), and
30 µM (○) of compound 5. (B) Secondary replot of Line-weaver Burk plot is between slopes of each line on Lineweaver Burk vs different concentrations of compound 5, and (C) Dixon plot reciprocal of rate of reaction vs
different concentrations of compound 5.
4
Conclusion
A total of seven 18β-glycyrrhetinic acid (1) derivatives were synthesized via
biotransformation with M. phaseolina and C. blakesleeana. Two of them, metabolites 5 and 6
were found to be new. Hydroxylations at C-7, and C-15, and oxidative ring cleavage between
C-3, and C-4 (Bayer-Villiger-type oxidation), and esterification were observed. In vitro urease
inhibition is an important approach for the treatment of diseases caused by urease producing
bacteria. The resulted metabolites were evaluated against urease enzyme. Some of the
transformed metabolites have shown potent activity against the urease enzyme. This study will
help to open new ways in the development of new anti-urease agents.
13
30
Table-2: 1H- and 13C- NMR Data (J in Hz) for metabolites 1–4, chemical shifts are in ppm.
1
Carbon
2
δC
1
38.4
2
26.9
3
4
5
6
76.5
38.7
54.0
17.0
7
32.0
8
9
10
11
12
13
14
15
42.8
61.1
36.6
198.9
127.2
169.5
44.8
26.0
16
25.7
17
18
19
31.4
48.0
40.5
20
21
43.0
30.3
22
37.4
23
24
25
26
27
28
29
30
28.1
15.9
18.3
16.1
22.9
28.3
27.7
177.5
δH
2.56, m;
0.95,m
1.52, overlap;
1.39, overlap
3.00, m
0.69, overlap
1. 50, overlap;
1.35, overlap
1.62, m;
1.32, overlap
2.31,s
5.38, s
1.73, m;
1.13, m
2.06, m;
0.95, m
2.04, m
1.69, m;
1.66, m
1.77, overlap;
1.35, overlap
1.25, m;
1.31, overlap
0.89, s
0.67, s
1.01, s
1.02, s
1.35, s
0.74, s
1.08, s
-
δC
3
δH
39.0
31.9
174.5
84.9
51.9
21.6
30.9
43.0
60.7
39.7
198.0
127.3
170.0
44.7
26.0
25.7
31.5
47.9
40.7
43.1
30.3
37.4
31.2
26.0
17.7
17.5
22.7
28.4
27.8
δC
2.30, m;
1.59, m
2.62, m;
2.42, m
1.80, overlap
1.49, m
32.7
1.71, overlap;
1.32, overlap
2.62, s
5.44, s
1.75, m;
1.15, m
2.05, m;
0.95, m
2.10, m
1.69, m;
1.68, m
1.77, m;
1.35, m
1.34, m ;
1.27, m
1.39, s
1.32, s
1.24, s
1.05, s
1.36, s
0.75, s
1.08, s
-
33.2
4
δH
δC
δH
2.93, m;
2.47, m
2.64, m;
2.34, m
1.40, overlap
1. 85, m;
1.25, overlap
1.71, overlap;
1.40, overlap
2.85, s
5.59, s
1.57, overlap
32.1
2.12, m;
1.02, m
2.19, m
27.8
45.0
32.2
39.0
2.48, m;
2.13, m
1.40, overap
2.38, overlap;
1.93, m
2.60, m;
2.35, m
2.21, m
167, m;
1.60, m
3.97, dd
(J7α,6α = 11.2, J7α,6e = 4.4)
2.77, s
5.63, s
2.09, overlap;
1.67, overlap
2.09, overlap;
0.98, m
1.53, m
1.89, m;
1.72, m
1.37, overlap
39.0
1.39, m
32.8
28.4
19.0
20.3
23.4
29.2
28.8
180.8
1.28, s
1.27, s
1.16, s
1.38, s
1.44, s
0.83, s
1.16, s
-
32.7
28.4
12.3
20.0
23.4
29.2
28.9
181.8
1.29, s
1.27, s
1.12, s
1.38, s
1.59, s
0.82, s
1.29, s
-
36.4
171.9
76.0
52.6
27.5
54.8
54.8
52.5
202.3
129.3
171.9
47.0
22.5
27.3
33.0
49.8
42.3
45.1
31.5
36.1
171.9
75.5
50.7
33.3
72.4
42.5
55.3
46.6
201.7
129.5
171.5
51.8
31.2
33.0
49.8
42.7
Table-2: 1H- and 13C- NMR Data (J in Hz) for metabolites 5–8, chemical shifts are in ppm.
Carbon
5
δC
1
33.0
6
δH
2.40, m; 1.93, m
δC
30.7
7
δH
2.55, m; 2.18, m
δC
38.1
8
δH
2.52, overlap;
δC
38.2
0.84, m
δH
2.55, m;
0.88, overlap
2
36.7
2.62, m; 2.40, m
36.2
2.66, m; 2.34, m
28.4
1.56, m; 1.33, m
26.7
1.64, m; 1.50, m
3
170.3
-
176.9
-
76.4
3.00, m
76.3
3.01, m
14
4
76.1
-
75.7
-
38.4
-
38.4
-
5
52.3
1.42, m
52.7
1.34, m
50.7
0.70, m
50.5
0.72, overlap
6
22.6
1.57, overlap
22.1
1.58, m; 1.48, m
29.5
2.02, m; 1.52, m
26.9
1.43, m; 1.40, m
7
33.3
1.70, m;
33.1
1.74, m; 1.43,
70.7
3.89, dd (J7α,6α =
70.3
3.89, dd (J7a,6a =
overlap
1.41, overlap
10.4, J7α,6e = 4.8)
10.8, J7a,6e = 4.8 )
8
47.2
-
46.8
-
42.9
-
42.9
-
9
54.0
2.83, s
54.1
2.77, s
61.3
2.24, s
61.3
2.21, s
10
46.1
-
42.0
-
36.6
-
36.7
-
11
202.1
-
201.8
-
198.6
-
198.2
-
12
129.3
5.63, s
129.0
5.65, s
127.3
5.41, s
127.8
5.48, s
13
170.3
-
170.3
-
169.7
-
169.9
-
14
47.0
-
47.0
-
44.2
-
49.4
-
15
65.5
4.22, dd (J15α,16α =
65.3
4.22, dd (J15α,16α =
26.1
2.03, m; 0.89,
65.5
4.09, dd (J15α,16α =
11.0, J15α,16e = 4.5 )
16
36.4
1.72, m; 1.46, m
12.0, J15α,16e = 4.0 )
36.1
1.74, m; 1.48,
overlap
11.6, J15α,16e = 5.2 )
26.9
1.49, m; 1.38, m
34.8
2.00, m; 1.18, m
overlap
17
37.9
-
37.7
-
31.4
-
31.5
-
18
51.8
2.41, m
51.6
2.43, m
48.7
2.06, m
49.1
2.03, m
19
42.5
1.91, m; 1.66, m
42.5
1.91, m; 1.69, m
40.8
1.68, m; 1.65, m
40.4
1.69, m; 1.62, m
20
45.2
-
45.2
-
43.0
-
50.7
-
21
31.8
1.96, m; 1.36,
31.7
1.95, m; 1.37,
30.3
1.76, m; 1.30, m
30.3
1.77, overlap; 1.34,
overlap
overlap
overlap
22
32.6
2.10, m; 1.13, m
32.8
1.12, m; 2.01, m
37.4
1.26, m ; 1.22, m
37.1
1.36, m; 1.22, m
23
32.9
1.29, s
32.7
1.27, s
28.0
0.89, s
27.8
0.90, s
24
28.3
1.26, s
28.2
1.26, s
15.9
0.66, s
16.0
0.67, s
25
19.1
1.17, s
18.9
1.17, s
16.0
1.00, s
15.8
1.00, s
26
20.3
1.38, s
20.1
1.37, s
12.2
0.98, s
12.5
1.00, s
27
24.5
1.51, s
24.2
1.50, s
23.0
1.37, s
17.9
1.32, s
28
22.8
0.75, s
22.6
0.76, s
28.3
0.74, s
28.9
0.76, s
29
29.0
1.15, s
28.8
1.14, s
27.8
1.07, s
28.0
1.08, s
30
182.3
-
182.4
-
177.7
-
177.5
-
51.8
3.59, s
31
15
Figure-1: Biotransformation of 18β-glycyrrhetinic acid (1) with M. phaseolina.
16
Figure-2: Biotransformation of 18β-glycyrrhetinic acid (1) with C. blakesleean
Figure-3: Key HMBC, COSY, and NOESY correlations in metabolite 5.
Figure-4: Key HMBC, COSY, and NOESY correlations in metabolite 6.
17
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