INTRODUCTION

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia. There are many and diverse therapeutic strategies in the management of type 2 diabetes. Predominantly herbal drugs have been widely used globally for diabetic treatment over thousands of years due to their traditional acceptability and lesser side effects. Therefore, screening of -amylase and -glucosidase inhibitors in medicinal plants has received much attention.1

(Taylor) Hale is a foliose lichen; belonging to the genus one of the largest genera of the Parmeliaceae family.2 Previous studies of this lichen reported that its methanolic extract had various pharmacological activities such as antimicrobial, antioxidant, and insecticidal activities.3 However, metabolites possessing antidiabetic activity unexplored well as no chemical data of this lichen are reported. Therefore, to search for new classes of -glucosidase inhibitors, we nine compounds' isolation and structural elucidation from the chloroform extract of the lichen (Taylor) Hale and the -glucosidase inhibitory activity of these compounds.

MATERIALS AND METHODS

RESULTS

By using efficient separation techniques, the chemical investigation on the chloroform extract of the lichen P. cristiferum led to the isolation of nine compounds. Their chemical structures were elucidated by 1D and 2D NMR as well as the HR-ESI-MS. They were four phenolic compounds, orcinol (1), orsellinic acid (2), atranol (3), methyl -orsellinate (4), three depsides, atranorin (5), diffractaic acid (6), lecanoric acid (7), and two depsidones, bailesidone (8), and tinctorinone (9). Their H and C-NMR data of compounds 1-4 were presented in Table 1, Table 3; of compounds 5-9 were presented in Table 2, Table 3; and the following characteristics.

DISCUSSION

The structures of nine isolated compounds were elucidated based on modern physicochemical methods such as HR-MS and 1D and 2D NMR compared their data with those in the literature. The compounds possessing a similar framework were structurally discussed.

Compound 1 was isolated as colorless needles. Its molecular formula was determined as CHO through its molecular ion peak at 125.0601 [M+H] in the HR-ESI-MS spectrum. The H-NMR data of 1 gave one methyl group, three aromatic methine protons, and two hydroxyl protons (Table 1). The C-NMR spectrum showed five signals, including one methyl group, three aromatic methines, and three quaternary aromatic carbons (Table 3). The good compatibility of its NMR and MS data with those of orcinol in the literature6, 7proposed that compound 1 was orcinol.

Compound 2 was obtained as colorless needles. The similarity in the NMR data (Table 1, Table 3) just with the replacement of one aromatic proton in the 1 by a carboxyl group in 2 suggested that it was orselinic acid.6, 7 This was supported by the pseudomolecular ion peak at 169.0491 [M+H] in the HR-ESI-MS spectrum which fitted well with the molecular formula of CHO. Therefore, 2 was orselinic acid.

Compound 3 was quickly identified as atranol because it possessed similar NMR data (Table 1, Table 3) with those in the literature.6, 8The position of the aldehyde group was clarified by HMBC cross-peaks of its proton( 10.23) to C-3 ( 109.3) and C-2, 4 ( 163.2) (Figure 2). Its molecular formula CHO was determined through the pseudomolecular ion peak at 153.0536 [M+H].

The comparison NMR data of 1 and 4 showed that the latter possessed one more methoxycarbonyl ( 3.71, 173.5, 52.1, COO-CH) and one methyl group ( 1.82, 8.1, C-9). The positions of these two groups were evidenced by HMBC cross-peaks of the methoxy protons at 3.71 (7-OCH) with the carboxyl carbon at 173.5 (C-7), and of the methyl protons ( 1.82) with adjacent aromatic carbons at 164.1 (C-2), 109.5 (C-3) and 161.0 (C-4). Based on the compatibility of the NMR and HR-ESI-MS data of 4 with the literature,94 was methyl -orsellinate.

Compound 5 was preliminarily proposed as a depside via the comparison of its NMR data (Table 2, Table 3) with corresponding depsides in the literature. A close search showed that 5 was composed of the combination of 3 and 4 moieties. This observation was well supported by its HMBC pattern (Figure 1) and HR-ESI-MS spectrum (negative mode) with a pseudomolecular ion peak at 373.0881 [M-H] (calcd. for CHO-H, 373.0923). Thus, the structure of 5was assigned as atranorin.8

Compound 6 was obtained as a white amorphous powder. A careful search of the NMR data (Table 2, Table 3) showed that 6 was also a depside and composed of two moieties of -orsellinic acid (the carboxylic acid of 4). This was proved by the HR-ESI-MS spectrum (positive mode) with a pseudomolecular ion peak at 375.1437 [M+H] (calcd. for CHO+H, 375.1443), by HMBC experiment (Figure 1), and by the similar NMR data with those of diffractaic acid.10, 11 Finally, the chemical structure of 6 was determined to be diffractaic acid.

Compound 7 was isolated as colorless needles. In the same above argument, the similarity in the spectral data (Table 2, Table 3) suggested that 7 was composed of two moieties of orsellinic acid (2). The HR-MS spectrum ( 317.0672 [M-H], calcd. for CHO-H, 317.0661) and good accordance in the NMR data12(Table 1, Table 2) proposed 7 as lecanoric acid.

Compound 9 was isolated as a white amorphous powder. The comparison NMR data of 9 and 8 (Table 2, Table 3) showed that they were depsidones with a lactone ring, with some differences. HMBC experiment of 9 confirmed that its lactone moiety possessed one acetal carbon ( 7.10, 102.4) and one ethoxy side-chain [ 3.97, 66.2 (O-CH-CH),and 1.34 (t, 7.0 Hz), 15.6 (O-CH-CH]. In addition, its A ring contained a further hydroxymethylene [ 4.96 (, 11.5, 8.5 Hz), 4.79 (, 11.5, 8.5 Hz), 53.0, C-9]. The comparison of these NMR data with those of tinctorinone, a depsidone in the lichen showed the compatibility. The specific optical rotation data of 9 was zero, thus it was a racemic mixture of tinctorinone.

The inhibitory assay against -glucosidase for isolated compounds was evaluated. The results (Table 4) indicated that these compounds (1-9) were inactive.

CONCLUSION

From the chloroform extract of the lichen collected at Paksong district, Champasack province, Laos, using various chromatographic methods, nine phenolic compounds were isolated. They were orcinol (1), orsellinic acid (2), atranol (3),methyl -orsellinate (4), atranorin (5), diffractaic acid (6), lecanoric acid (7), a racemic mixture of bailesidone (8), and a racemic mixture of tinctorinone (9). Except for compounds 2, 4, and 7,14 the remaining six compounds were reported for the first time in this species. All nine compounds were evaluated against -glucosidase enzyme but none of them were active. Further research on the remaining extracts is continued.

ABBREVIATIONS

HR-ESI-MS: High resolution- Electrospray ionization-Mass spectrometry

H-NMR: Proton Nuclear Magnetic Resonance

C-NMR: Carbon-13 Nuclear Magnetic Resonance

HMBC: Heteronuclear Multiple Bond Correlation

: singlet

: doublet

: doublet of doublets

: triplet

: multiplet

COMPETING INTEREST

The authors declare no competing financial interest.

AUTHORS’ CONTRIBUTION

Huynh B.L.C, Ngo T.T.D contributed in conducting experiments, acquisition of data, interpretation of data. Nguyen T.N.V, Nguyen M.A.N, Tran T.B interpreted NMR and MS data as well as searched the bibliography. Nguyen T.L.T, Bui T. T did the biological assay. Nguyen K.P.P. gave the final correction for the manuscript

Corresponding author: Nguyen Thi Ngoc Van, University of Science, National University-Ho Chi Minh City, Street, , , . Email: ntnvan101096@gmail.com

ACKNOWLEDGMENT

We would like to thank Dr. Vo Thi Phi Giao for the lichen authentication.