Identification of Anti- Helicobacter pylori Compounds From Usnea undulata

Abstract

In this study, we evaluated 3 extracts (n-hexane, acetone, and methanol) of Usnea undulata Stirton (Usneaaceae) and 5 isolated compounds [methyl orsellinate (1), methyl β-orsellinate (2), 7-hydroxyl-5-methoxy-6-methylphtalide (3), usnic acid (UA) (4), and salazinic acid (5)] for their inhibitory activity against Helicobacter 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, besides UA 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.

Clarithromycin-resistant Helicobacter pylori is ranked in the high priority pathogen group by the World Health Organization, rendering the current antibiotics ineffective, thus, screening for potential antimicrobial compounds is an urgent demand. ¹ Lichens are stable, consistent, and identifiable mutualistic associations between algae and/or cyanobacteria (the photobiont) and fungi (the mycobiont). Lichens synthesize a wide range of phenolic compounds that can be principally classified as depsides, depsidones, dibenzofurans, and pulvinic acid derivatives with a great variety of effects such as antibiotic, antimycobacterial, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative, and cytotoxic activities. ² Species Usnea undulata is a fruticose lichen that grows on tree. Very little has been published on the secondary metabolites and pharmacological activity of U. undulata. Except for usnic acid (UA, from U. undulata) that showed high antibacterial activities, ³ there is no report on anti-H. pylori activity of the other metabolites.

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/disk, the n-hexane (H) extract produced the biggest clear zone (d = 14 mm) (Figure 1(a), 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 to 23 mm at 240 to 2000 µg/disk, U. undulata extracts were significantly more potent against H. pylori. ^{4 -6} Notably, 2 reference drugs at 50 µg/disk only yielded 8-mm diameter zones.

Table 1

Anti-H. pylori Activity of Extracts and Isolated Compounds From Usnea undulata.

Extracts/compounds	Methanol a	Acetone a	n-Hexane a	1 b	2 b	3 b	5 b	MTZ	AMX
Diameter (mm)	7	9	14	22	27	-	-	8	8
MIC (µg/mL)			80		25

AMX, amoxicillin; MTZ, metronidazole.

^a100 µg/disk.

^b50 µg/disk.

Figure 1

Inhibitory effect of Usnea undulata extracts (a) and compounds 1 to 3 and 5 (b). Total extracts: A, acetone; M, methanol; H, n-hexane; R, reference drug.

The known isolated compounds (1-5) were identified by comparison of their physical and spectral data with the literature values as methyl orsellinate (1), ⁷ methyl β-orsellinate (2) ⁷ 7-hydroxyl-5-methoxy-6-methylphtalide (3), ⁸ (R)-(+)-UA (4), ^9,10 and salazinic acid (5). ³ Previously, a few phytochemical studies were performed on the Australian, Indian, and South African species ³ ; here, the constituents of the Vietnamese species were reported for the first time. Among the isolated compounds, 3 was newly found in U. undulata.

As shown in Figure 1(b), compound 2 had the strongest anti-H. pylori 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 that yielded IZD between 13 and 14 mm at 1 mg, compounds 1 and 2 showed potential application in drug development. ¹¹ 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. 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 high performance liquid chromatography (HPLC) in n-hexane extract, it gave a suitable explanation for the highest activity of such extract. Minimum inhibitory concentrations (MICs) of this extract and compound 2 (Table 1) were similar or lower than those in the strong-moderate activity category against 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

Nuclear magnetic resonance, Bruker DMX 300 and 500 spectrometers; melting points, melting point meter M5000 Krüss; optical rotation, Perkin Elmer 341 polarimeter; column chromatography, normal phase silica gel (40-63 µm, Kieselgel 60, Merck 7667). All other routine chemicals used were of analytical grade.

HPLC Quantification of UA in the n-Hexane Extract

The HPLC analysis was carried out on Shimadzu LC20A system with autosampler, GL ODS C18 column (150 × 4.6 mm, 5 µm) and UV detector, at 28°C with solvent methanol: phosphate buffer (75:25) pH 7.4 during 10 minutes. The detection wavelength was 245 nm and the injection volume was 10 µL with a flow rate of 1 mL/min. (+)-UA 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 area under curve corresponding to UA (R _t = 5.03 minutes) was evaluated on the basis of calibration curve y = 35 977x + 20 0961 and R² = 0.9994.

Extraction and Isolation

Air-dried crushed thallus of the lichen U. undulata (150 g) was 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 extracts were stored at 4°C for further work. n-Hexane extract was dissolved in diethyl ether and stored at 4°C for 24 hours 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 Culture

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, United States). Positive colonies were visible after 5 days with diameter from 0.5 to 1 mm.

Anti-Helicobacter pylori Activity Evaluation

Agar diffusion method was performed as per Mitscher et al with slight modifications. ¹⁶ Prior to the test, 20 µL of extract (200 mg/mL in dimethyl sulfoxide) were added to the paper disks (d = 6 mm) and air dried for 3 hours. Helicobacter pylori colonies were resuspended into a solution with McFarland turbidity of 2 (OD₆₂₅ = 0.451, approximately 6 × 10⁸ cells) and streaked on the plates. After that, the disks 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

Supplemental Material

Supplementary material - Supplemental material for Identification of Anti-Helicobacter pylori Compounds From Usnea undulata

Supplemental material, Supplementary material, for Identification of Anti-Helicobacter pylori Compounds From Usnea undulata by Trung Do, Trang T.H. Nguyen, Thai N. Ha, Nguyen T.H. Nhu, Nguyen Van Lam, Nguyen T.T. Tram, and Yen Pham in Natural Product Communications