Efficient enzymatic synthesis and antibacterial activity of andrographolide glycoside

Article abstract of DOI:10.1016/j.procbio.2016.02.008

19-O-β-Galactosyl andrographolide, a potential novel antibacterial agent, was synthesized through enzymatic transgalactosylation of andrographolide in co-solvent systems. Organic solvents and their contents have important influences on the regioselective galactosylation of andrographolide catalyzed by β-galactosidase from bovine liver in co-solvent systems. β-Galactosidase showed high activity and stability in 5-15% (v/v) DMSO with 22-52% total molar yields of andrographolide glycosides. The addition of hydrophilic DMSO not only greatly promoted the solubility of the substrate, but also improved the reaction efficiency of the process. β-Galactosidase displayed absolute regioselectivity toward the 19-position of andrographolide. The solubility of andrographolide glycoside in water was 42.1 mg ml^-1, which is about 702 times that of andrographolide. The glycosylated andrographolide showed antibacterial activity against five representative species of food-borne pathogenic bacteria [with minimal inhibitory concentrations (MICs) as low as 8 μg ml^-1], whereas andrographolide exhibited no such activity. These results indicate an enzymatic modification was not only facile and green, but an effective method for the preparation of an andrographolide monoglycoside.

Full text of DOI:10.1016/j.procbio.2016.02.008

Process Biochemistry 51 (2016) 675–680
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Efficient enzymatic synthesis and antibacterial activity of
andrographolide glycoside
Ying Qiao, Yao Huang, Fang Feng, Zhi-Gang Chen∗
Glycomics and Glycan Bioengineering Research Center, College of Food Science &Technology, Nanjing Agricultural University, Nanjing 210095, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
19-O-␤-Galactosyl andrographolide, a potential novel antibacterial agent, was synthesized through
enzymatic transgalactosylation of andrographolide in co-solvent systems. Organic solvents and their
contents have important influences on the regioselective galactosylation of andrographolide catalyzed
by ␤-galactosidase from bovine liver in co-solvent systems. ␤-Galactosidase showed high activity and
stability in 5–15% (v/v) DMSO with 22–52% total molar yields of andrographolide glycosides. The addi-
tion of hydrophilic DMSO not only greatly promoted the solubility of the substrate, but also improved
the reaction efficiency of the process. ␤-Galactosidase displayed absolute regioselectivity toward the
Received 3 December 2015
Received in revised form 28 January 2016
Accepted 12 February 2016
Available online 15 February 2016
Keywords:
Andrographolide
␤
19-position of andrographolide. The solubility of andrographolide glycoside in water was 42.1 mg ml−1
,
which is about 702 times that of andrographolide. The glycosylated andrographolide showed antibacterial
activity against five representative species of food-borne pathogenic bacteria [with minimal inhibitory
concentrations (MICs) as low as 8 ␮g ml−1], whereas andrographolide exhibited no such activity. These
results indicate an enzymatic modification was not only facile and green, but an effective method for the
preparation of an andrographolide monoglycoside.
-Galactosidase
Glycosylation
Regioselectivity
Organic solvent
Antibacterial activity
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Glycosylation has been used to modify the hydrophilicity,
bioactivity and chemical properties of lipophilic natural prod-
Andrographolide (C20H30O5, Scheme 1), a diterpene lactone
[1], is the main and most medicinally active component isolated
from Andrographis paniculata (Burm.f.) Nees, an important herbal
medicine traditionally used to treat different range of diseases
in Asian countries [2–4]. Andrographolide is found in the whole
plant but is most concentrated in the leaves (about 0.5–1.0%,
dry weight) and has multiple pharmacological activities such as
anti-bacterial, anti-inflammatory, antiviral, and anti-allergic activ-
ities [3–5]. However, the solubility of andrographolide in water
was maximal at a concentration of 60 ␮g ml−1. Moreover, andro-
grapholide has a poor solubility in most organic solvents. The low
solubility of andrographolide is disadvantageous not only by lim-
iting its pharmacological use in potential therapeutics but also for
its modification. Andrographolide glycosides are also active com-
ponents from A. paniculata, and have good solubility in water, but
their contents are very low (about 0.01–0.03%, dry weight) [5]. In
fact, andrographolide glycosides derivatives can be achieved by
glycoside modification of andrographolide.
ucts [6]. Glycosylation strategy has been established well for
the improvement of therapeutic efficacy by enhancing oral
absorption, selectivity and water-solubility of the parent agents,
etc. [7]. For instance, the water-solubility of the antitumor
drug—geldanamycin was markedly improved upon glycosylation
modification [8]. Despite the many excellent chemical methods
for glycosylation, methods for the direct regioselective glycosy-
lation of andrographolide are limited owing to the presence of
multi active hydroxyl groups in the reactant (Scheme 1). In general,
arduous and tedious protection–deprotection steps and environ-
mentally unfriendly catalysts were involved in traditional chemical
methods. Use of enzymes as the alternative to chemical catalysts
offered many new opportunities for the regioselective glycosy-
lation, because of excellent selectivity, simplicity, mild reaction
conditions and being environmentally benign [9]. Process sim-
plification resulted in an overall reduction in energy use and
waste production because of avoiding protection–deprotection
steps (Fig. 1).
However, our pre-experiments showed that enzymatic glycosy-
lation reaction in buffer was not efficient due to the low solubility
of the andrographolide. Enzymatic glycosylation in hydrophilic
organic solvents provides numerous industrially attractive advan-
∗
Corresponding author. Fax: +86 25 8439 5618
E-mail address: zgchen@njau.edu.cn (Z.-G. Chen).
http://dx.doi.org/10.1016/j.procbio.2016.02.008
1359-5113/© 2016 Elsevier Ltd. All rights reserved.

676
Y. Qiao et al. / Process Biochemistry 51 (2016) 675–680
NO2
15
11
O
O
16
14
HO
14
13
HO
20
O
HO OH
HO
HO
18
O
NO2
12
O
O
1
+
17
OH
2
3
9
10
8
7
HO OH
HO
3
4
5
-Galactosidase
β
HO
HO
6
O
HO
in co-solvent systems
18
O
19
19
OH
Andrographolide
Scheme 1. Enzymatic regioselective galactosylation of andrographolide catalyzed by ␤-galactosidase from bovine liver.
andrographolide
400
300
200
100
0
oNPG
9-O-β-D-galactosyl-
andrographolide
o-nitrophenol
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Fig. 1. HPLC of 19-O-␤-d-galactosyl-andrographolide (1.9 min), oNPG (2.7 min), andrographolide (3.6 min) and o-nitrophenol (4.3 min).
tages, such as the increased solubility of substrate, reversal of
the thermodynamic equilibrium of the hydrolysis reactions, and
elimination of microbial contamination [10,11]. Obviously, the
denaturation of enzymes in hydrophilic media is a major disad-
vantage. The synthetic utility of glycosidase would be considerably
improved if its catalytic activity could be maintained in the pres-
ence of hydrophilic media.
Antimicrobial activities were assessed using five bacteria [Vibrio
parahemolyticus ATCC 17802, Listeria monocytogenes ATCC 19115,
Salmonella enteritidis CMCC 50041, Staphyloccocus aureus CICC
10307, Escherichia coli CICC 10372]. These test microbes were
obtained from the American Type Culture Collection (ATCC), China
Medical Culture Collection Center (CMCC, Beijing) and China Gen-
eral Microbiology Culture Collection Center (CGMCC, Beijing).
Thus, an efficient solvent-resistant glycosidase in the pres-
ence of a certain percentage of hydrophilic organic solvent may
play a pivotal role in a drastic reaction process. To the best of
our knowledge, there have been no reports on the application
of ␤-glycosidase to andrographolide glycoside syntheses. Herein
we demonstrate that the ␤-galactosidase from bovine liver can
be efficiently used for the glycosylation of andrographolide using
o-nitrophenyl-␤-d-galactoside (oNPG) as donor in hydrophilic
organic solvents containing systems (Scheme 1). The activities of
andrographolide glycoside derivatives as new antibacterial agents
are also explored.
2.2. Enzyme activity assay
The enzyme powder was dissolved in phosphate buffer
(100 mM, pH 7.0) at 4 ◦C to form the enzyme solution (10 mg ml−1).
Enzyme solution (20 ␮l) was added to 0.6 ml phosphate buffer
(100 mM, pH 7.0) containing oNPG (50 mM). The reaction was con-
ducted for 30 min at 45 ◦C, and then stopped by adding 5.38 ml 1 M
Na2CO3. The released o-nitrophenol was assayed at 420 nm. One
unit of glycosidase activity was defined as the amount of enzyme
required to catalyze the release of 1 ␮mol o-nitrophenol per minute
under the conditions given. The specific activity of the enzyme was
5 U ml−1
.
2. Materials and methods
2.1. Chemical and biological materials
2.3. Solubility determination
The ␤-galactosidase from bovine liver (≥0.15 U mg−1 protein)
was obtained from Sigm–Aldrich. Andrographolide (purity >99%)
was purchased from Nanjing TCM Institute of Materia Medica,
Nanjing, China. o-Nitrophenyl-␤-d-galactoside (oNPG) was from
Guangzhou Genebase Bioscience Co., Ltd., China. Dimethyl sulfox-
ide (DMSO), tetrahydrofuran (THF), pyridine and acetone (all HPLC
grade) were purchased from Sinopharm (Shanghai, China). All other
chemicals were obtained from commercial sources and of the high-
est purity available.
Andrographolide or andrographolide glycoside was mixed with
0.5 ml of solvent in an Eppendorf tube at 45 ◦C. An ultrasonic cleaner
(Type NP-B-400-15; Newpower Co., Ltd., Kunshan, China) was used
to maximize the solubility of each compound. After 1 h sonication
and centrifugation at 10,000 × g for 20 min to remove insoluble
material, the sample was diluted and filtered through a 0.45 ␮m
membrane, and used for HPLC analysis to determine the sample
solution concentration.

Y. Qiao et al. / Process Biochemistry 51 (2016) 675–680
677
Table 1
Effects of solvents on the galactosylation of andrographolide catalyzed by ␤-galactosidase.a
b
Reaction media
log P [13]
Dielectric constant [14]
Solubility (mg ml−1
)
Reaction time (h)
Glycosylation (%)c
d
PBS (pH 6.5)
DMSO
Acetone
Ethanol
Acetonitrile
THF
Pyridine
Butanol
10% (v/v) DMSO–PBS
10% (v/v) acetone–PBS
10% (v/v) ethanol–PBS
10% (v/v) acetonitrile–PBS
10% (v/v) THF–PBS
10% (v/v) pyridine–PBS
10% (v/v) butanol–PBS
–
80.2 (20 ◦C)
6.1 0.2 × 10−2
60
60
60
60
60
60
60
60
48
48
48
48
48
48
48
<1
−1.30
−0.23
−0.24
−0.34
0.49
0.71
0.88
–
47.2 (20 ◦C)
25.5 1.5
10.5 0.4
7.4 0.6
<5
<5
5.0 0.7
<5
21.0 (20 ◦C)
5.9 0.4
20.1 0.8
15.2 0.6
18.5 0.9
10.8 0.8
2.1 0.3
–
–
–
–
–
–
–
24.5 (25 ◦C)
37.5 (21 ◦C)
7.5 (22 ◦C)
13.3 (20 ◦C)
7.8 (19 ◦C)
<5
–
–
–
–
–
–
–
40.0 2.3
18.4 1.1
13.5 0.9
14.6 1.6
13.2 0.8
10.3 0.5
5.2 0.8
–
–
–
–
–
–
a
Reaction conditions: andrographolide 0.1 mmol, o-NPG 0.6 mmol, NaH2PO4–Na2HPO4 buffer (100 mmol l−1, pH 6.5)–organic solvent (ꢀ = 10%), 0.75 U ␤-galactosidase,
45 ◦C, 200 rpm, total volume 5 ml.
b
The solubility of andrographolide in each solvent was determined by HPLC analysis of saturated solutions at 45 ◦C.
Glycosylation efficient was calculated on the bases of remaining acceptor.
No data or not determined.
c
d
2.4. General procedure for enzymatic glycosylation of
andrographolide
2.6. The stability of ˇ-galactosidase in DMSO-containing systems
The ␤-galactosidase (final concentration 0.5 U ml−1) was added
to various systems in universal bottles and incubated at 45 ◦C with
shaking at 200 rpm. Every 3 h, incubated ␤-galactosidase was sam-
pled, and the remaining activity was measured as described above.
The stabilities of ␤-galactosidase are expressed as the remaining
activity compared with the initial activity in a buffer system (taken
as 100%).
In a typical experiment, ␤-galactosidase (final concentration
0.15 U ml−1) was added to the reaction mixture (5 ml) containing
0.1 mmol andrographolide, 0.6 mmol oNPG and phosphate buffer
(100 mM, pH 6.5) in 15% (v/v) DMSO. The reaction was conducted
at 45 ◦C with shaking at 200 rpm. Aliquots were withdrawn at
intervals, held at 100 ◦C for 5 min to denature the enzyme, and
then diluted by 10 times with water/methanol (30/70, v/v) prior
to HPLC analysis. A control reaction was performed in which no
enzyme was added to the above procedure; no chemical glycosyla-
tion of andrographolide was detectable in the absence of enzyme.
All experiments were performed in triplicate, and the results are
reported as the mean standard deviation.
2.7. Structure determination of andrographolide glycoside
The product was separated and purified through flash column
chromatography using ethyl acetate/methanol (6/3, v/v) as the elu-
ent. The product structure was determined by 13C NMR and 1H NMR
(Bruker AVANCE AV-500 NMR spectrometer, Germany). CD3OD
was used as a solvent.
2.5. HPLC analysis
19-O-ˇ-d-Galactosyl-andrographolide 1H NMR (ı): H1 (1.85, m,
1H; 1.29, m,1H), H2 (1.78, m, 2H), H3 (3.39, t, J = 8.1, 1H), H5 (1.31,
m, 1H), H6 (1.87, m, 1H; 1.38, m, 1H), H7 (2.43, m, 1H; 2.03, m, 1H),
H9 (1.93, m, 1H), H11 (2.60, m, 2H), H12 (6.85, td, J = 6.8, 1.7 Hz,
1H), H14 (5.01, d, J = 6.1 Hz, 1H), H15 (4.16, dd, J = 10.2, 2.2 Hz, 1H;
The reaction mixture was analyzed by RP-HPLC on a SinoChrom
ODS-BP column (4.6 mm × 150 mm, 5 ␮m, Dalian Elite Analytical
Instruments Co., Ltd., China) using a Hitachi L-7110 pump and
a Hitachi L-7420 UV detector (Hitachi Co., Ltd., Tokyo, Japan) at
225 nm. The mobile phase was a mixture of water and methanol
(30/70, v/v) at a flow rate of 1.0 ml min−1. The retention times
for 19-O-␤-d-galactosyl-andrographolide, oNPG, andrographolide,
and o-nitrophenol were 1.9, 2.7, 3.6 and 4.3 min (Fig. 1), respec-
tively. All data are averages of experiments performed at least in
duplicate, and no more than 2% deviation was observed.
100
5% DMSO
10% DMSO
15% DMSO
20% DMSO
80
60
40
20
0
Table 2
Effect of DMSO contents on enzymatic galactosylation of andrographolide.a
Medium
pH
Maximal yield (%)
19-Regioselectivity (%)b
PBS
6.50
6.52
6.60
6.75
6.85
6.92
<1
>99
>99
>99
>99
>99
>99
1%(v/v)DMSO–PBS
5%(v/v)DMSO–PBS
10%(v/v)DMSO–PBS
15%(v/v)DMSO–PBS
20%(v/v)DMSO–PBS
5.0 0.2
22.3 1.4
40.5 1.8
51.5 2.4
42.8 2.1
a
Reaction conditions: andrographolide 0.1 mmol, o-NPG 0. 6 m mol,
NaH2PO4–Na2HPO4 buffer (100 mmol l−1
, pH 6.5)–organic solvent, 0.75 U ␤-
0
10
20
30
40
50
60
galactosidase from bovine liver, 45 ◦C, 200 rpm, reaction time 48 h, total volume
5 ml.
Tmie (h)
b
Regioselectivity was defined as the ratio of the HPLC peak area corresponding
Fig. 2. The deactivation profile of ␤-galactosidase across a range of time in different
media.
to the indicated product to that of all the products formed.

678
Y. Qiao et al. / Process Biochemistry 51 (2016) 675–680
Table 3
Antibacterial activity of andrographolide and its derivatives.
b
MIC in broth (␮g ml−1
)
Solubilitya (mg ml−1
)
Compound
S1
>128c
S2
>128
32
0.5
S3
S4
S5
Andrographolide
6.0 × 10−2
42.1
>128
16
2
>128
8
nd
2
>128
16
1
19-O-␤-Galactosylandrographolide
8
1
4
Gentamicin sulfated
nde
Benzylpenicillin sodiumd
nd
2
nd
nd
a
The solubility of andrographolide and 19-O-␤-galactosyl andrographolide was determined by HPLC analysis of saturated solutions at 45 ◦C.
The calculated average MIC values are presented. S1: Escherichia coli CICC 10372; S2: Staphyloccous aureuCICC 10307; S3: Vibrio parahemolyticusATCC 17802; S4: Listeria
b
monocytogenes ATCC 19115; S5: Salmonella enteritidis CMCC 50041.
c
MIC > 128 ␮g ml−1 was considered to be inactive.
Gentamicin sulfate and benzylpenicillin sodium were used as positive controls.
nd: not determined.
d
e
4.46, dd, J = 10.2, 6.1 Hz, 1H), H17 (4.67, s, 1H; 4.88, s, 1H), H18
(1.22, s, 3H), H19 (4.15, d, J = 11.0 Hz, 1H; 4.91, d, J = 11.0 Hz, 1H),
H20 (0.75, s, 3H); H2ꢀ + H3ꢀ + H4ꢀ + H5ꢀ + H6ꢀ (3.62–3.93, m, 6H), H1ꢀ
(4.51, d, J = 7.5 Hz, 1H). 13C NMR (ı): C1 (38.2), C2 (29.0), C3 (80.9),
C4 (40.6), C5 (56.4), C6 (25.2), C7 (38.9), C8 (148.8), C9 (57.4), C10
(39.9), C11 (25.7), C12 (149.4), C13 (129.8), C14 (66.7), C (76.1), C16
(172.6), C17 (109.2), C18 (23.4), C19 (67.6), C20 (15.5); C6ꢀ (60.7),
C4ꢀ (68.3), C2ꢀ (70.3), C3ꢀ (72.5), C5ꢀ (74.9), C1ꢀ (102.7).
Enzyme-catalyzed reactions in non-aqueous media are affected
by the nature of the solvent [10,11]. In general, polar solvents
may be employed to overcome the solubility problem. However,
these solvents usually strip the essential water off of the enzymes,
thereby inactivating the biocatalyst [15]. In our system, the reac-
tion media had an obvious effect on enzymatic glycosylation of
andrographolide (Table 1). As shown in Table 1, ␤-galactosidase
was unstable and inactivated substantially in organic solvents.
Glycosylation efficiency was disappointingly low because of the
␤-galactosidase deactivation. The detrimental impacts of organic
solvents on the enzyme may be relieved by decreasing their con-
tents. When organic solvent except DMSO is used as cosolvent,
enzymatic glycosylation efficiency in 10% (v/v) organic solvent–PBS
(pH 6.5) systems was not significantly improved. But, very interest-
ingly, 40% glycosylation rate occurred when DMSO was used as the
cosolvent. The catalytic performance of ␤-galactosidase correlated
well with log P and the dielectric constants of organic solvents in co-
solvent systems, among which the enzyme displayed the highest
activity in DMSO-containing system.
2.8. Antibacterial activity test
The minimum inhibitory concentrations (MICs) of andro-
grapholide and its derivatives were determined using a liquid
dilution method performed in 96 well micro-trays [12].
3. Results and discussion
3.1. Effect of organic solvents on enzymatic galactosylation of
andrographolide
To better understand ␤-galactosidase-mediated glycosylation
reactions conducted in the DMSO–PBS and to further optimize the
reaction, the reactions were carried out in the DMSO co-solvent
system as a function of the DMSO content and reaction time.
The solubility of andrographolide in water was very low. Rel-
atively high concentrations of andrographolide were achieved in
hydrophilic organic solvents (Table 1). The andrographolide con-
centration in DMSO was over 418 times greater than that in PBS (pH
6.5). To demonstrate an application of glycosidase, we examined
the performance of ␤-galactosidase in hydrophilic solvents such as
DMSO, acetone, THF and pyridine, which have been employed to
overcome the solubility of andrographolide. Initially, galactose and
lactose were tested as galactosyl donors. Unfortunately, no galac-
tosylated derivative was formed. So the active glycosyl donor oNPG
was used in this work.
3.2. Effect of DMSO contents on enzymatic galactosylation of
andrographolide
The solubility of the substrate was greatly improved in DMSO
compared to buffer. DMSO was found to be freely miscible with
buffer in all proportions tested. Besides, pH values of reaction
media are not significantly affected by DMSO addition (1–25%, v/v)
(Table 2). The effect of the concentration of DMSO on the galac-
tosylation reaction was then examined to determine the optimal
concentration in the co-solvent mixture.
A higher galactosylation of andrographolide was achieved when
DMSO (15%, v/v) was added to phosphate buffer than in phosphate
buffer alone (Table 2). This result occurred because the addition
of DMSO led to an improvement in the solubility of the andro-
grapholide, enhancing the mass transfer of the substrates and
products to and from the active site of the enzyme, thus result-
ing in enhanced galactosylation activity. However, with too much
DMSO present in the reaction system (>15%, v/v), the galactosy-
lation activity of ␤-galactosidase dropped sharply, which may be
attributed to more serious inactivation of the enzyme by DMSO
(Fig. 2).
60
50
40
30
20
10
0
3
6
9
12 15 18 21 24 27 30 33 36 39
Time (h)
3.3. Stability of ˇ-galactosidase in DMSO-containing systems
Fig. 3. Time course of ␤-galactosidase-catalyzed glycosylation of andrographolide.
The reaction conditions were as follows: andrographolide 0.1 mmol, o-NPG
From both a practical and a theoretical viewpoint, it was impor-
tant to understand the influence of the DMSO-containing system
on the thermal stability of the enzyme. A comparative study was
0.6 mmol, NaH2PO4–Na2HPO4 buffer (100 mmol l−1
,
pH 6.5)–organic solvent
(ꢀ = 15%), 0.75 U ␤-galactosidase from bovine liver, 45 ◦C, 200 rpm, total volume 5 ml.

Y. Qiao et al. / Process Biochemistry 51 (2016) 675–680
679
thus performed on ␤-galactosidase stability by incubating it in the
5–20% (v/v) DMSO–PBS at 45 ◦C and a range of incubation time from
1 to 60 h, followed by measurement of its residual activity (Fig. 2).
After incubation in 5%, 10% and 15% (v/v) DMSO–PBS for 24 h
at 45 ◦C, ␤-galactosidase retained approximately 65%, 53% and 45%
of its original activity, respectively; however, when incubated in
20% (v/v) DMSO-PBS under the same condition, ␤-galactosidase
retained only 15% of its original activity. The better stability of ␤-
galactosidase in ≤15% (v/v) DMSO solvent system suggests that
␤-galactosidase can potentially be used as an industrial catacyst
for enzymatic glycosylation in water-miscible organic solvents.
The reusability of biocatalysts is one of the essential factors for
cost reduction. For practical application, ␤-galactosidase should
be immobilized (e.g. on a macroporous acrylic resin) and recycled.
Upon completion of the reaction, the reaction mixture can be fil-
tered or centrifugated to acquire the immobilized enzyme. Then
the immobilized ␤-galactosidase is used in the next batch reaction
composed of new substrates, highlighting the cost-effectiveness of
the enzyme.
for new antibacterial agents [20]. Natural products are known to be
a rich source of antimicrobial compounds with novel mechanisms
and chemical structures that may have the potential to provide new
and effective therapies against bacteria resistant to drugs currently
in use [21]. Andrographolide and its glycoside derivative (19-O-␤-
galactosyl andrographolide) were evaluated for their antibacterial
action against five representative species of food-borne pathogenic
bacteria that are the most common in food or most dangerous to
human health [20]. In vitro antibacterial activity was assessed by
measuring minimum inhibitory concentration (MIC) values via the
liquid dilution method performed in 96 well plates [12]. Andro-
grapholide did not demonstrate measurable antibacterial activity,
but the glycosylated andrographolide derivatives exhibited rela-
tively broad antibacterial spectra against most of the strains of
food-borne pathogenic bacteria that were tested (Table 3). The
most powerful activities were against the L. monocytogenes (G+)
and E. coli (G−) (MIC = 8 ␮g ml−1 in both cases), which are com-
parable to the activities of the established antibiotics gentamicin
and benzylpenicillin against these strains (Table 3). These results
highlight the critical role of the glycoside moiety on the lactone-
ring in determining the antibacterial activity of andrographolide
derivatives.
3.4. Time course of andrographolide glycosylation
To gain a deeper insight into the enzymatic process, the
time courses of the synthesis of O-galactosylated andrographolide
derivative was investigated. The time course of ␤-galactosidase-
catalyzed glycosylation of andrographolide under the optimum
conditions is depicted in Fig. 3. The reaction proceeded at a high
rate during the first 12 h. A substantial deceleration of the reac-
tion was observed when the reaction time extended beyond 18 h.
Several issues might account for the decrease in enzyme activity
over time, including increasing inactivation by the solvent and the
o-nitrophenyl byproduct of oNPG hydrolysis or product. After 36 h,
about 50% yield of the desired product was achieved.
Analysis of the product structures indicated that, within the
range examined, reaction media and reaction time had little effect
on regioselectivity; only the hydroxyl groups attached to the C-
19 of andrographolide participated in glycoside bond formation
(detailed NMR information shown in Section 2.7).
␤-Galactosidase, also known as a lactase, is considered to be
one of the most widely used industrial enzymes [16,17]. Recently,
in view of the potential of ␤-galactosidase transgalactosylation,
a new field of development for galactose containing compounds
is being extensively studied due to their promising role in the
food industry, cosmetics and medicine [16–19]. Despite the fact
that ␤-galactosidase is abundant in nature, the most common
enzyme sources are microorganisms, and the synthesis of galacto-
sides almost entirely microbial enzymes have been used [16–18].
In present study, the ␤-galactosidase of animal origin, produced
by bovine liver, was used to synthesize andrographolide gly-
coside. As mentioned above, ␤-galactosidase displayed a high
efficiency, stability and regioselectivity. These results suggest that
␤-galactosidase can potentially be used as an industrial catalyst for
enzymatic glycosylation.
Further work is warranted to determine whether the 19-
glycosylated derivatives of andrographolide reported here have a
novel mechanism of antibacterial action and hence would be attrac-
tive lead compounds for development of a valuable class of new
antibacterial agents. The results also indicate that enzymatic mod-
ification is a good method for the preparation of andrographolide
derivatives with new properties for new applications.
4. Conclusions
The synthesis of andrographolide glucoside derivatives can be
successfully conducted with excellent regioselectivity and mod-
erate yield by means of ␤-galactosidase-catalyzed glycosylation
in DMSO-containing systems. The solubilities and antibacterial
activities of andrographolide glycoside were higher than those
of andrographolide. Enzymatic modification of andrographolide
offers simplicity, excellent selectivity and environmentally benign
processes. If further scale-up is possible, the reaction may be attrac-
tive for industrial application.
Acknowledgements
This work was partly supported by Qing Lan Project, PAPD, NSFC
and the Scientific Research Foundation for the Returned Overseas
Chinese Scholars (State Education Minisry).
References
[1] S. Pramanick, S. Banerjee, B. Achari, B. Das, A.K. Sen, S. Mukhopadhyay, A.
Neuman, T. Prangé, Andropanolide and isoandrographolide, minor
diterpenoids from Andrographis paniculata: structure and X-ray
crystallographic analysis, J. Nat. Prod. 69 (2006) 403–405.
[2] N. Pholphana, N. Rangkadilok, S. Thongnest, S. Ruchirawat, M. Ruchirawat, J.
Satayavivad, Determination and variation of three active diterpenoids in
Andrographis paniculata (Burm.f.) Nees, Phytochem. Anal. 15 (2004) 365–371.
[3] W.W. Chao, B.F. Lin, Isolation and identification of bioactive compounds in
Andrographis paniculata (Chuanxinlian), Chin. Med. 5 (2010) 17–32.
[4] J. Wang, W. Yang, G. Wang, P. Tang, Y. Sai, Determination of six components of
Andrographis paniculata extract and one major metabolite of andrographolide
in rat plasma by liquid chromatography–tandem mass spectrometry, J.
Chromatogr. B (951–952) (2014) 78–88.
[5] Z. Cao, The Synthesis and Biolological Research of Andropanolide Derivatives
(in Chinese with English Abstract) Master Thesis, University of Henan, China,
2007, pp. 1–20.
[6] I. Gill, R. Valivety, Enzymatic glycosylation in plasticized glass fhases: a novel
and efficient route to O-glycosides, Angew Chem. Int. Ed. 39 (2000)
3804–3808.
3.5. Solubility and antibacterial activities of andrographolide
glycoside
The solubility of andrographolide glycoside in water was dra-
matically higher than that of andrographolide (Table 3). The
apparent solubility of andrographolide glycoside in water was
42.1 mg ml−1, which is about 702 times that of andrographolide.
This suggests that the attachment of a galactosyl residue to andro-
grapholide greatly enhanced the aqueous solubility.
Food-borne illnesses usually result from food contaminated
with pathogenic bacteria. Spread of food-borne pathogenic bacteria
is now a critical problem in public health and there is an urgent need

680
Y. Qiao et al. / Process Biochemistry 51 (2016) 675–680
´
[7] F.J. Li, H. Maag, T. Alfredson, Prodrugs of nucleoside analogues for improved
[16] M. Carevic´, M. Corovic´, M. Mihailovc´, K. Banjanac, A. Milisavljevic´, D.
oral absorption and tissue targeting, J. Pharm. Sci. 97 (2008) 1109–1134.
[8] C.Z. Wu, J.H. Jang, M. Woo, J.S. Ahn, J.S. Kim, Y.S. Hong, Enzymatic
glycosylation of non-benzoquinone geldanamycin analogs via Bacillus
UDP-glycosyltransferase, Appl. Environ. Microb. 78 (2012) 7680–7686.
[9] N. Li, T.J. Smith, M.H. Zong, Biocatalytic transformation of nucleoside
derivatives, Biotechnol. Adv. 28 (2010) 348–366.
[10] A.M. Klibanov, Improving enzymes by using them in organic solvents, Nature
409 (2001) 241–246.
[11] K.M. Koeller, C.H. Wong, Enzymes for chemical synthesis, Nature 409 (2001)
232–240.
Velicˇkovic´, D. Bezbradica, Galacto-oligosaccharide synthesis using chemically
modified ␤-galactosidase from Aspergillus oryzae immobilised onto
macroporous amino resin, Int. Dairy J. 54 (2016) 50–57.
[17] M. Carevic´, D. Velicˇkovic´, M. Stojanovic´, N. Milosavic´, H. Rogniaux, D. Ropartz,
D. Bezbradica, Insight in the regioselective enzymatic transgalactosylation of
salicin catalyzed by ␤-galactosidase from Aspergillus oryzae, Process Biochem.
50 (2015) 782–788.
[18] S.E. Lee, H.Y. Lee, K.H. Jung, Production of chlorphenesin galactoside by whole
cells of ␤-galactosidase-containing Escherichia coli, J. Microbiol. Biotechnol. 23
(2013) 826–832.
[12] Clinical and Laboratory Standards Institute, Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically approved
standards, seventh ed., Clinical and Laboratory Standards Institute, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087–1898, USA, 2006.
[13] C. Reichardt, Solvatochromic dyes as solvent polarity indicators, Chem. Rev.
94 (1994) 2319–2358.
[19] L.Q. Yan, N. Li, M.H. Zong, First enzymatic galactosylation of acyclic nucleoside
drugs by ␤-galactosidase: synthesis of water-soluble ␤-d-galactosidic
prodrugs, Biotechnol. Bioproc. E 19 (2014) 586–591.
[20] J.A. Haagsma, S. Polinder, C.E. Stein, A.H. Havelaar, Systematic review of
foodborne burden of disease studies: quality assessment of data and
methodology, Int. J. Food Microbiol. 166 (2013) 34–47.
[14] J.A. Dean, Lange’s Handbook of Chemistry, 15th ed., McGraw-Hill, New York,
1998, pp. 105.
[21] E.D. Brown, G.D. Wright, New targets and screening approaches in
antimicrobial drug discovery, Chem. Rev. 105 (2005) 759–774.
[15] A.M. Klibanov, Why are enzymes less active in organic solvents than in
water? Trends Biotechnol. 15 (1997) 97–101.