2019

NPC Natural Product Communications Vol. 14

No. 0
Identification of Potential Anti-Helicobacter pylori Compounds from 1-2
Usnea undulata Extracts
Do Thi Thanh Trunga, Nguyen Huyen Trang b, Thai Ngan Hac, Nguyen Thi Huynh Nhuc,
Nguyen Van Lamc, Nguyen Thi Thu Tramc* and Pham Bao Yena*
a
The Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai,
Hanoi, Vietnam
b
Laboratory Center, Hanoi University of Public Health, 1A Duc Thang street, Hanoi, Vietnam
c
Can Tho University of Medicine and Pharmacy, 179 Nguyen Van Cu, Ninh Kieu, Can Tho, Vietnam
*
These authors contributed equally to this work.

yenpb@vnu.edu.vn

Received: March 14th, 2019; Accepted: XX, 2019

In this study, we evaluated three extracts (n-hexane, acetone and methanol) of Usnea undulata Stirton (Usneaaceae) and five isolated compounds (methyl
orsellinate (1), methyl β-orsellinate (2), 7-hydroxyl-5-methoxy-6-methylphtalide (3), usnic acid (4), and salazinic acid (5)) for their inhibitory activity against
H. pylori. The results showed that the n-hexane extract exhibited the strongest activity with the largest inhibition zone (d=14 mm). For the isolated metabolites,
beside usnic acid that has been reported as a potent anti-H. pylori agent, compounds 1 and 2 showed higher inhibition compared to the reference drugs. Our
results demonstrated that U. undulata could be a promising source of potential anti-H. pylori metabolites.

Keywords: gastric disease, Helicobacter pylori, natural compounds, Usnea undulata.

Clarithromycin-resistant Helicobacter pylori is ranked in the High undulata extracts were significantly more potent against H. pylori
priority group by the World Health Organization, rendering current [4-6]. Notably, two reference drugs at 50 µg/disc only yielded 8-
antibiotics ineffective, thus, screening for potential antimicrobial mm diameter zones.
compounds is an urgent demand [1]. Lichens are stable, consistent
and identifiable mutualistic associations between algae and/or Table 1: Anti-H. pylori activity of extracts and isolated compounds from U. undulata.
cyanobacteria (the photobiont) and fungi (the mycobiont). Lichens Extracts/ Methanol a Acetonea n- 1b 2b 3b 5b MTZb AMXb
Compounds Hexanea
synthesize a wide range of phenolic compounds that can be Diameter (mm) 7 9 14 22 27 - - 8 8
principally classified as depsides, depsidones, dibenzofurans and MIC (µg/ml) 80 25
a
pulvinic acid derivatives with a great variety of effects such as 100 µg/disc; b 50 µg/disc; MTZ metronidazole; AMX Amoxicilin.
antibiotic, antimycobacterial, antiviral, anti-inflammatory,
analgesic, antipyretic, antiproliferative and cytotoxic activities [2]. The known isolated compounds (1–5) were identified by
Species Usnea undulata is a fruticose lichen that grows on tree. comparison of their physical and spectral data with literature values
Very little has been published on the secondary metabolites and as methyl orsellinate (1) [7], methyl β-orsellinate (2) [8], 7-
pharmacological activity of U. undulata. Except for usnic acid hydroxyl-5-methoxy-6-methylphtalide (3) [9], (R)-(+)-usnic acid (4)
(from U. undulata) that showed high antibacterial activities [3], [10,11] and salazinic acid (5) [3]. Previously, a few phytochemical
there is no report on anti-H. pylori activity of the other metabolites. studies have been performed on the Australian, Indian and South
African species [3], here the constituents of Vietnamese species
were reported for the first time. Amongst the isolated compounds, 3
was newly found in U. undulata.

As shown in Fig. 1B, compound 2 had the strongest anti-H. pylori

(A) (B)
Figure 1: Inhibitory effect of U. undulata extracts (A) and compounds 1-3,5 (B). Total
extracts, H – n-hexane, A –acetone, M – methanol, R – reference drug.
In this study, we obtained total extracts using different solvents,
then, isolated 5 compounds from U. undulata, and characterized
their anti-H. pylori activity. At 100 µg/disc, the n-hexane (H)
extract produced the biggest clear zone (d = 14 mm) (Fig. 1A, Table
1). At 2 mg, the acetone extract produced a halo with d = 33 mm,
smaller than that obtained with the n-hexane extract at 0.5 mg (37
mm). Compared to many other studies with inhibition zone
diameters (IZD) ranging from 8-23 mm at 240-2000 µg/disc, and U.
effect (IZD = 27 mm) while 3 and 5 did not produce visible clear
zones (Table 1). Compared to a previous study on gallic acid and
catechin at 1 mg and yielded IZD between 13-14 mm, compounds 1
and 2 showed potential application in drug development [9].
Interestingly, both have been shown to possess antimicrobial
activity against different bacterial and fungal species; yet, this study
reported the inhibitory effect on H. pylori for the first time. Usnic
acid (UA) has previously been reported to possess high activity
against H. pylori strains [12]. Compared to the same species, the
Vietnamese U. undulata contained a substantially higher UA
amount [3]. With a large ratio (44.92%) determined by HPLC in n-
hexane extract, it gave a suitable explanation for the highest activity
of such extract. MICs of this extract and compound 2 (Table 1)
were similar or lower than those in the strong-moderate activity
2 Natural Product Communications Vol. 14 (0) 2019 Author A et al.

category against anti-H. pylori [13]. Importantly, similar to UA, a
potent anti-H. pylori agent with liver toxicity [14], compound 2
could be toxic, as indicated by the brine shrimp lethality assay [15].
Hence, the synthetic derivative approach is recommended to obtain
analogs of high activity and lower toxicity.
Experimental
Lichen material: Usnea undulata Stirton (Usneaaceae) was
collected in Lam Dong province, Vietnam in December 2017. The
identification was conducted by Dr. Buaruang (Department of
Biology, Faculty of Science, Ramkhamhaeng University, Thailand).
A voucher specimen (No Usnea-1217) was deposited at the
Department of Chemistry, Faculty of Science, Can Tho University
of Medicine and Pharmacy, Can Tho City, Vietnam.
General experimental procedures: NMR, Bruker DMX 300 and
500 spectrometers; Melting points, Melting Point Meter M5000
Krüss; Optical rotation, Perkin Elmer 341 polarimeter; CC, normal
phase silica gel (40-63 µm, Kieselgel 60, Merck 7667). All other
routine chemicals used were of analytical grade.
HPLC quantification of usnic acid in the n-hexane extract: The
HPLC analysis was carried out on Shimadzu LC20A system with
autosampler, GL ODS C18 column (150 x 4.6 mm, 5 µm) and UV
detector, at 28°C with solvent methanol : phosphate buffer (75:25)
pH 7.4 during 10 min. The detection wavelength was 245 nm and
the injection volume was 10 µl with a flow rate of 1 mL/ min. (+)
Usnic acid standard (purity > 98%) was purchased from Sigma-
Aldrich. The n-hexane extract was dissolved in acetone to give a
final concentration at 1 mg/mL. The AUC corresponding to usnic
acid (Rt = 5.03 min) was evaluated on the basis of calibration curve
y = 35977x + 200961, R2 = 0.9994.
Extraction and isolation: Air-dried crushed thallus of the lichen U.
undulata (150 g) were successively and exhaustively extracted by a
hot Soxhlet process with 1 L of each solvent (n-hexane, acetone and
methanol). Solvent removal under reduced pressure gave n-hexane
extract (7.31 g, yield 4.87%), acetone extract (9.15 g, yield 6.10%)
and methanol extract (5.72 g, yield 3.81%), respectively. All the
extracts were stored at 4°C for further work. n-hexane extract was
dissolved in diethyl ether and stored at 4°C for 24 h to precipitate.
After filtration, the precipitate was recrystallized in chloroform to
give compound 4 (1.24 g). The acetone extract was subjected to a
silica gel column and eluted with n-hexane: ethyl acetate (90:10) to
yield compound 1 (55.4 mg), compound 2 (28.7 mg), compound 3
(16.2 mg) and compound 5 (42.0 mg).
Helicobacter pylori strains: Culture plates containing 5% sheep
blood and selective antibiotics were incubated at 37°C under
microaerophilic conditions created by the GasPak™ EZ Pouch
Systems (Becton Dickinson, USA). Positive colonies were visible
after 5 days with diameter from 0.5-1 mm.
Anti-H. pylori activity evaluation: Agar diffusion method was
performed as per Mitscher et al. (1972) with slight modifications
[16]. Prior to the test, 20 μL of extract (200 mg/ml in DMSO) were
added to the paper discs (d=6 mm) and air dried for 3 hours. H.
pylori colonies were resuspended into a solution with McFarland
turbidity of 2 (OD625 = 0.451, approximately 6x108 cells) and
streaked on the surface. After that, the paper discs were placed on
the surface and the plates were incubated under microaerophilic
conditions at 37°C. Antibacterial zone results could be observed
after 5 days. MIC determination was adapted from Yee et al. [17].
Supplementary data: Supplementary data are included as
Supporting information.
Acknowledgments - This study received funding from National
Foundation for Science and Technology Development
(NAFOSTED) with the code: 106-NN.02-2013.55.

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