Antibacterial activities of chemical constituents from the aerial parts of Hedyotis pilulifera

Abstract Context:Hedyotis pilulifera (Pit.) T.N. Ninh (Rubiaceae) has been used in Vietnamese ethnomedicine; the methanol extract exhibited antibacterial activity in our preliminary screening. Objectives: In this study, compounds from H. pilulifera were isolated and their antibacterial activity in vitro was evaluated. Materials and methods: The aerial parts of H. pilulifera (1.4 kg) were extracted with MeOH, suspended in water and ethyl acetate extract was chromatographed on a silica gel column. The structures of isolated compounds were elucidated by the combination analyses of spectroscopy including 1D-, 2D-NMR, HRMS and in comparison with the reported NMR data in the literature. All isolated compounds were evaluated for inhibitory effect using the microdilution method toward Staphylococcus aureus, Bacillus subtilis and Mycobacterium smegmatis, and MIC values were determined. Results: Twenty compounds were isolated, including five triterpenoids, two steroids, two aromatic compounds, three fatty acids, one quinone derivative, one lignan glycoside, one ceramide and five glycolipids. Among these, oleanolic acid showed significant antibacterial activity against M. smegmatis with the MIC value of 2.5 μg/mL. Remarkably, rotungenic acid showed strong activity against S. aureus, B. subtilis, M. smegmatis with MIC values of 2.5, 2.5 and 1.25 μg/mL, respectively. Rotundic acid exhibited significant antibacterial activity against B. subtilis with the MIC value of 5 μg/mL. To the best of our knowledge, the antibacterial activity of rotungenic acid, stigmast-4-ene-3,6-dione and (2S,3S,4R,2′R)-2-(2′-hydroxytetracosanoylamino) octadecane-1,3,4-triol was reported for the first time. Conclusions: Oleanolic acid, rotungenic acid, and rotundic acid were considered to be useful for developing new antimicrobial therapeutic agents for human.


Introduction
Hedyotis pilulifera (Pit.) T.N. Ninh (Rubiaceae), an herbal plant, is known to be distributed in Laos and Vietnam (Ho 2003;Newman et al. 2007). It has been used as the remedy for abdominal pain and osteoarthritis (Bossi ere et al. 2006). The valuable experience of ethnomedicine should not to be underestimated: for example, according to our studies, the needles of Pinus sylvestris L. (Pinaceae) showed cytotoxic effect against breast cancer cells (Hoai et al. 2015), they have been used against cancer in Estonian ethnomedicine (Sak et al. 2014).
In our previous report, one new iridoid aglycone, 10-O-acetylborreriagenin, and five known iridoidial glycosides were isolated from the aerial parts of H. pilulifera (Hoai et al. 2016). Generally, the chemical composition of genus Hedyotis, including H. pilulifera, has been investigated just to a small extent. In our preliminary unpublished data, the methanol extract of aerial parts of H. pilulifera showed significant antimicrobial activity. Therefore, the current study was conducted to isolate those compounds and to evaluate their antibacterial activity in vitro.

Plant material
The aerial parts of H. pilulifera were collected in the Quang Tri province (17 02'15.2"N 107 03'55.9"E), Vietnam, in August 2014 and were identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST, Vietnam. A voucher specimen (VL01) was deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Vietnam.

Extraction and isolation of constituents
The aerial parts of H. pilulifera (1.4 kg) were extracted by soaking with hot MeOH (3 Â 3 L, 3 h each, 60 C) to yield 38.0 g of a dark solid extract. This extract was suspended in water and successively partitioned with chloroform (CHCl 3 ) and ethyl acetate (EtOAc) to obtain the CHCl 3 (HC, 13.0 g), the EtOAc (HE, 11.0 g) and the water (HW, 14.0 g) extracts after the removal of solvents in vacuo.

Bioassay
Antimicrobial activity was performed using the dilution method to a published procedure (Eloff 1998) with slight modifications.

Results
The chemical investigations led to the isolation and the structural elucidation of 20 known compounds including five triterpenoids, two steroids, two aromatic compounds, three fatty acids, one quinone derivative, one lignan glycoside, one ceramide and five glycolipids.
Compound 1 was obtained as a white amorphous powder.  (Table 1), compound 1 was determined to be oleanolic acid, consistent with reported data (Mahato & Kundu 1994).
Compound 5 was obtained as a white amorphous powder. The 1 H and 13 C NMR chemical shifts of 5 were almost the same as those of 4 (Table 1), suggesting that these two compounds possessed the analogous structures. The only difference is configuration of chiral centre at C-4. Similar to 4, the hydroxyl group was located at C-23 due to the chemical shifts of methyl carbon (d C 12.7) and oxymethylene carbon (d C 67.5) at C-4 (d C 43.3) were similar to those of 23-OH form [d C 67.9 (C-23)/12.8 (C-24)], but quite different from those of 24-OH form [d C 23.5 (C-23)/64.5 (C-24)] (Zhang & Yang 1994). Thus, compound 5 was identified to be rotundic acid (He et al. 2012).
The antibacterial assay of isolated compounds was evaluated against three Gram (þ) bacterial strains, including Staphylococcus aureus, Bacillus subtilis and Mycobacterium smegmatis. As shown in Table 2, compound 4 demonstrated potent inhibition with MIC values ranging from 1.25 to 2.5 lg/mL. The inhibitory effect of 4 against the tested bacterial strains was comparable with that of the positive control, ampicillin. Compounds 1 and 5 exhibited significant antibacterial activity against M. smegmatis, B. subtilis with MIC values of 2.5, and 5 lg/mL, respectively. The other compounds (2, 6, and 15) showed moderate inhibitory effect.

Discussion
To the best of our knowledge, compounds 3-6, 8-9 and 11-20 were firstly isolated from the genus Hedyotis. From our understanding, the antibacterial activity of compounds 4, 6, and 15 was reported for the first time in this study.
The structure-activity relationship of 4 and 5 may be due to the difference in absolute configuration of C-4 under the experimental conditions. The 4S configuration (in 4) was considered to be more active than 4R configuration (in 5). Compounds 1, 4, and 5 represent pentacyclic triterpenoid skeleton which should be responsible for the bioactivity of the methanol extract of this species.

Conclusions
Based on the obtained results, oleanolic acid (1), rotungenic acid (4), and rotundic acid (5) from the aerial parts of H. pilulifera were considered to be useful for developing new antimicrobial therapeutic agents for human.

Acknowledgments
This work was supported from the Japan Society for the Promotion of Science (ID No. P14412). This research was also supported by Ministry of Education and Training, Vietnam (B2015-DHH-126).

Disclosure statement
The authors report no declarations of interests.