Synthesis and microbiological evaluation of honokiol derivatives as new antimicrobial agents

Article abstract of DOI:10.1007/s12272-010-2225-7

Honokiol, a major phenolic constituent of Magnolia sp., has various pharmacological activities. To improve the solubility and antibacterial activity of honokiol against E. coli and P. aeruginosa, new honokiol-derivatives (honokiol-acetate, honokiol-succinic acid, honokiol-glycerol, honokiol-glycine, honokiol-glucose and honokiol-mannose) were synthesized and their solubility and antimicrobial activities were investigated. Among the tested compounds, honokiol-glycine showed improved water solubility and antibacterial activities against E. coli and P. aeruginosa when compared to honokiol.

Full text of DOI:10.1007/s12272-010-2225-7

Arch Pharm Res Vol 33, No 1, 61-65, 2010
DOI 10.1007/s12272-010-2225-7
Synthesis and Microbiological Evaluation of Honokiol Derivatives as
New Antimicrobial Agents
Young-Soo Kim*, Ji-Young Lee*, Junho Park, Wangtaek Hwang, Jongsung Lee, and Deokhoon Park
Biospectrum Life Science Institute, SK Ventium 101-701, Gunpo 435-833, Korea
(Received July 2, 2009/Revised October 23, 2009/Accepted November 4, 2009)
Honokiol, a major phenolic constituent of Magnolia sp., has various pharmacological activi-
ties. To improve the solubility and antibacterial activity of honokiol against E. coli and P.
aeruginosa, new honokiol-derivatives (honokiol-acetate, honokiol-succinic acid, honokiol-glyc-
erol, honokiol-glycine, honokiol-glucose and honokiol-mannose) were synthesized and their
solubility and antimicrobial activities were investigated. Among the tested compounds, honok-
iol-glycine showed improved water solubility and antibacterial activities against E. coli and P.
aeruginosa when compared to honokiol.
Key words: Honokiol derivatives, Anti-bacterial, Escherichia coli, Pseudomonas aeruginosa
hydroxy and the 5-allyl groups are responsible for
honokiol-mediated neurite outgrowth-promoting acti-
INTRODUCTION
Honokiol, which is isolated from the stem bark of vity (Esumi et al., 2004). In addition, honokiol has
Magnolia sp., has long been used as traditional medi- strong antibacterial and antifungal activities (Ho et
cine for the treatment of cough, diarrhea, and allergic al., 2001). However, despite these positive attributes
rhinitis in Korea, China and Japan. This compound is of honokiol, it is insoluble in oil and water and exerts
known to possess various biological properties includ- no antibacterial activity against Escherichia coli and
ing anti-oxidative (Ogata et al., 1997), anti-arrhyth- Pseudomonas aeruginosa (Ho et al., 2001). Due to its
mic (Tsai et al., 1999), anti-inflammatory (Liou et al., insolubility, honokiol cannot be used in a broad range
2003), anti-thrombocytic (Teng et al., 1988), anti- of applications including food preservatives, cosmetics
angiogenesis (Bai et al., 2003), anti-tumor (Yang et and medicine.
al., 2002), anxiolytic (Kuribara et al., 1999), and anti-
HIV (human immunodeficiency viruses) activities novel derivatives of honokiol (
(Amblard et al., 2006). In addition, honokiol has been honokiol-succinic acid (
), honokiol-glycerol (
found to exert high antimicrobial activity against kiol-glycine (
), honokiol-glucose ( ) and honokiol-
several types of microorganisms (Park et al., 2004).
mannose ( ) to develop novel substances with
Structurally, honokiol consists of para-allyl-phenol enhanced solubility as well as antibacterial activities
and an ortho-allyl-phenol that are linked together against E. coli and P. aeruginosa
Therefore, in this study, we synthesized a series of
), honokiol-acetate ( ),
), hono-
1
2
3
4
5
6
7
.
through ortho-, para-C-C-coupling. In a structure-
activity relationship, potent activities of honokiol are
attributed to the presence of hydroxyl and allylic
groups on a biphenolic moiety. It is well known that,
while the 3'-allyl group of honokiol plays an important
MATERIALS AND METHODS
Synthesis of honokiol derivatives
Derivative
2
was prepared by acetylation of the 2-
by incubating in acetic
anhydride in pyridine for 18 h at 25oC. Derivative
was prepared by reacting with maleic anhydride
(3,4-dihydrofuran-2,5-dion) in pyridine for 2 days at
50oC. Derivative
was prepared by reacting with
glycerol in pyridine for 8 h at 25oC. The synthesis of
role in cytotoxic activity (Kong et al., 2005), the 4'- and 4'-hydroxyl groups of
1
1
3
*Y.-S. Kim and J.-Y. Lee contributed equally to this work.
Correspondence to: Deokhoon Park, Biospectrum Life Science
Institute, Gunpo 435-833, Korea
Tel: 82-31-436-2090, Fax: 82-31-436-0605
E-mail: pdh@biospectrum.com
1
4
1
5
61

62
Y.-S. Kim et al.
was accomplished using the following general peptide
coupling reaction: glycine was reacted with di-tert-
MIC (minimum inhibition concentration) assay
About 104 microbial cells of the pre-cultured micro-
butyl dicarbonate (di-Boc), after which a synthetic bes were inoculated into 3 mL of broth. Subsequently,
Boc-Gly was reacted with , 1,3-diaza-1,3-dicyclohexyl- honokiol and magnolol were then added into the 3 mL
1
propa-1,2-diene (DCC), triethylamine (TEA) and 4- broth containing the microbes and cultured. To deter-
dimethylaminopyridine (DMAP) for 6 h at 40oC. Next, mine the MIC of honokiol derivatives, we employed a
a di-Boc on di-Boc-Gly-
with dry HCl gas for 4 h at room temperature in defined as the lowest concentration that yield no
EtOAc solution. Derivatives and were prepared by microbial cell growth.
processing with glycosylation and deacetylation,
respectively. In the first step, and were coupled
1 was eliminated by bubbling two-fold serial dilution method. The MIC value was
6
7
6
7
MBC (minimum bactericidal concentration)
assay
MBC (minimum bactericidal concentration) was de-
with glucose pentaacetate and mannose pentaacetate
using boron trifluoride diethyl etherate (BF3-Et2O) to
produce synthetic
and -D-tetraacetylmannopyranoside, respectively. Committee for Clinical Laboratory Standards, 1987).
In the second step, synthetic -D-tetraacetylglu- The killing curve assay was performed on the basis of
copyranoside and -D-tetraacetylmannopyranoside a previously published standard protocol (Lorian,
1-β-D-tetraacetylglucopyranoside termined according to NCCLS guidelines (National
1-β
1-β
1-β
were deacetylated by sodium methoxide followed by 1996), and the experiment was performed in dupli-
neutralization by passage through an Amberlite IR- cate. Microbial cells from the logarithmic phase of
120 (H+) ion exchange column to give
1
-
β
-D-glucopyr- growth were collected and they were incubated with
-D-mannopyranoside, respectively. The different concentrations of honokiol derivatives in a 5
structures of the synthesized
-derivatives were then mL total volume of cation-adjusted MHB (105 to 106
assigned based on their H- and 13C-NMR spectral organisms/mL) for 10 min, 30 min, 1 h, or 2 h respec-
anoside and 1-β
1
1
data.
The solubility of
tively. After incubation with honokiol derivatives for
-derivatives were determined by the indicated time, 0.1 mL of the culture (5 mL) was
1
measuring their water or oil solubility. Among them, collected and was mixed with 25 mL molten agar for
while
and
2
,
5
,
6
and
7
showed good water solubility,
3
the preparation of agar pour plates. Since reagents
were diluted at least 250-fold in the plates, the
reagent carryover effect was minimal. In addition, to
obtain the appropriate numbers of Colony Forming
Unit (CFU) in an individual plate (fewer than 150
4
displayed good oil solubility (data not shown).
The standardized filter-paper disc-agar diffu-
sion assay
Antimicrobial activity was evaluated using the colonies/plate) to ensure accurate colony counting, 0.2
standardized filter-paper disc-agar diffusion method, mL of the culture (5 mL) was taken at different time
known as the Kirby-Bauer method. Gram (+) bacteria points, and a series of 10-fold dilutions (10-1 to 10-7)
(Staphylococcus aureus ATCC6538P, Bacillus subtilius was prepared. Then 0.1 mL of the diluted cells was
ATCC6633, Propionibacterium acnes ATCC6919, and used to prepare the plates as described above.
Propionibacterium granulosum ATCC25564), Gram
(-) bacteria (Escherichia coli ATCC8739, and Pseudo-
monas aeruginosa ATCC27853), yeast (Candida albi-
cans ATCC10231, and Filobasidiella neoformans
RESULTS AND DISCUSSION
Derivatives of honokiol,
2, 3, 4, 5, 6, and 7, were
ATCC34144), and fungi (Aspergillus niger ATCC10254 prepared according to the procedure s depicted in Fig.
and Trichophyton mentagrophytes KCTC6077) were 1. The solubility of -derivatives were determined by
employed in these experiments. Approximately 106 measuring their water or oil solubility. Among them,
microbial cells were inoculated into 7 mL agar while and showed good water solubility,
displayed good oil solubility (data not shown).
1
2,
5,
6
7
3
medium (containing 0.8% phytoagar), and then agar and
4
medium containing 106 microbial cells was poured Antimicrobial activity was evaluated using the stand-
onto the agar plate (containing 1.5% phytoagar). After ardized filter-paper disc-agar diffusion method, and by
that, filter-paper discs of uniform size (diameter of 10 MIC/MBC assay. In the disc-agar diffusion assay, with
mm) are impregnated with specified concentrations of the exception of 5, none of the 1-derivatives showed
test materials and then placed on the surface of an any antibacterial or antifungal activities (data not
agar plate that has been seeded with the organism to shown). Specifically, acetate, succinic acid, glycerol, glu-
be tested. Antimicrobial activity was defined by cose, and mannose substituent groups of the hydroxyl
measuring the diameter of the growth inhibition zone. group did not enhance the antibacterial activities

Honokiol Derivatives as a New Antimicrobial Agent
63
Fig. 1. General synthetic scheme of honokiol derivatives
against E. coli and P. aeruginosa. When the disc stronger antibacterial activities against E.coli. and P.
diffusion assay was conducted using several other aeruginosa than . Specifically, although showed no
bacteria and fungi, no antibacterial or antifungal antibacterial or bactericidal activities, the MICs of
activities were observed, which indicates that acetate, against E.coli. and P. aeruginosa were found to be 100
succinic acid, glycerol, glucose, and mannose substi- g/mL and 200 g/mL, respectively. Moreover, the
tuents of attenuate antibacterial and antifungal MBCs of against E.coli. and P. aeruginosa were also
activities rather than enhancing them. In the case of 100 g/mL and 200 g/mL, respectively. Foodborne
, improved antibacterial activities against E. coli and illness resulting from the consumption of food con-
1
1
5
µ
µ
1
5
µ
µ
5
P. aeruginosa were observed when compared to taminated with pathogenic bacteria has been of vital
honokiol. In addition, similar to the hydroxyl group of concern to public health. Among the reported out-
1
(Ho et al., 2001), glycine showed a comparable breaks in the United States from 1993-1997 for which
contribution to antibacterial and antifungal activities the etiology was determined, bacterial pathogens
against the previously mentioned bacteria and fungi. caused the largest percentage of outbreaks (75%) and
Improved solubility was also obtained by substitution the largest percentage of individual cases (86%) (Olsen
of the hydroxyl group with glycine. Therefore,
5 dis- et al., 2000). Bacterial contamination of unwashed
played high water solubility. Furthermore, additional raw food, leaking packages, hands, and surfaces may
studies conducted using the MIC (minimal inhibitory directly contaminate other stored foods or attach to
concentration) and MBC (minimal bactericidal con- and persist on the internal surface of refrigerators,
centration) assays revealed that
lower anti-microbial and bactericidal activities when ation during subsequent food preparation activities
compared to (Table I). However, displayed much (Michaels et al., 2001). Of these, E. coli and P. aeru-
5 showed slightly thereby posing risks for indirect longer-term contamin-
1
5

64
Y.-S. Kim et al.
Table I. MIC (Minimum inhibition concentration) and MBC/MFC (Minimum bactericidal/Fungicidal concentration) of
honokiol ( ) and honokiol-glycine (
1
5)
MIC (
µ
g/mL)
MBC (µg/mL)
Organism
1
5
Antibiotics
1
5
Gram (+) bacteria
S. aureus
B. subtilius
P. acnes
P. granulosum
Gram (-) bacteria
E. coli
P. aeruginosa
Yeast
C. albicans
F. neoformans
Fungi
A. niger
T. mentagrophytes
13.31
18.26
14.13
18.26
145.54
128.69
111.38
122.77
0.37b
<11.74b
<0.18b
<0.18b
126.63
116.78
116.78
116.78
145.54
128.69
145.54
145.54
a
-
100.00
200.00
183.50b
N.T.d
-
-
100.00
200.00
-
26.63
16.66
191.07
111.38
0.92c
0.23c
153.27
113.32
182.15
122.77
53.27
13.32
113.84
111.38
1.85c
0.46c
106.54
113.32
113.84
122.77
aNo activity, bErythromycin, cAmphotericin B, dNot tested.
(2004).
ginosa accounted for the largest number of outbreaks,
Ho, K-Y., Tsai, C-C., Chen, C-P., Huang, J-S., and Lin, C-C.,
Antimicrobial activity of honokiol and magnolol isolated
from Magnolia officinalis. Phytother. Res., 15, 139-141
cases, and deaths. Overall, these enhanced antibac-
terial activities of
5 against E.coli. and P. aeruginosa
indicate that it may have a broad range of applica-
tions.
(2001).
Kong, Z-L., Tzeng, S-C., Liu, Y-C., Cytotoxic neolignans: an
SAR study. Bioorg. Med. Chem. Lett., 15, 163-166 (2005).
Kuribara, H., Kishi, E., and Hattori, N., Application of the
elevated plus-maze test in mice for evaluation of the con-
tent of honokiol in water extracts of magnolia. Phytother.
Res., 13, 593-596 (1999).
Liou, K. T., Shen, Y. C., and Chen, C. F., The anti-inflamma-
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inhibition of reactive oxygen species production. Eur. J.
Pharmacol., 475, 19-27 (2003).
In conclusion, in this study, we demonstrated that
5,
a derivative of , exhibited increased anti-microbial
1
activity against E.coli. and P. aeruginosa, which are
major foodborne pathogenic microorganisms. Further-
more,
solubility when compared to
that may be broadly used in areas such as the
5
was found to have greatly improved water
1. These results suggest
5
cosmetic, food, and pharmaceutical industries.
ACKNOWLEDGEMENTS
Lorian, V., Antibiotics in laboratory medicine. The Williams
& Wilkins Co., Baltimore, (1996).
This work was supported by a grant from the
Ministry of Commerce, Industry, and Energy, Republic
of Korea (IH-9-12-10018068).
Michaels, B., Ayers, T., Gangar, V., Inactivation of refri-
gerator biofilm bacteria for application in the food service
environment. Food Ser. Technol., 1, 169-179 (2001).
Ogata, M., Hoshi, M., Shimotohno, K., Ureno, S., and Endo,
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Olsen, S. J., MacKinnon, L. C., Goulding, J. S., and Slutsker,
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