Hedyocoronins A and B: two new oleanane saponins from the aerial parts of Hedyotis coronaria

Abstract Two new oleanane saponins, hedyocoronin A (1) and hedyocoronin B (2), were isolated from the aerial parts of Hedyotis coronaria (Kurz) Craib, Rubiaceae, collected at Da Oai district, Lam Dong province in Vietnam. Their chemical structures were elucidated by HR-MS, 1D and 2D-NMR spectra, along with the comparison with those reported in the literature. Compounds 1 and 2 showed weak cytotoxicity against KB and HeLa-S3 cancer cell lines with IC50 values of more than 54 µM. Graphical Abstract


Introduction
The Hedyotis genus (Rubiaceae) are herbaceous plants distributed in tropical areas, comprising about 160 species. Vietnamese traditional medicines, along with Chinese ones, have used several Hedyotis species, for example, H. corymbosa, H. diffusa, H. biflora, H. capitellata, … for treatment of appendicitis, boils, dysentery, hepatitis, snakebites, and tonsillitis, and so on (Vo 1996). In the cancer treatment aspect, H. chrysotricha has been used as a Chinese folk medicine for treatment of cancer and compound (24S)-ergostane-3b,5a,6b-triol, isolated from this species, remarkably reduced the migration of SK-HEP-1 cells by 78.9% at a dose of 30 mM, without any apoptotic effect on this cell line (Ye et al. 2013). Compound asperuloside isolated from H. corymbosa showed inhibitory activities against YMB-1, HL60 and KB cell lines with IC 50 values of 0.7, 11.0 and 104.2 mg/mL, respectively (Artanti et al. 2015). Compounds pomolic acid 3b-acetate from H. pilulifera showed the cytotoxicity against LU-1, HL-60, KB, Hep G2 and SK-Mel2 cancer cell lines with IC 50 values ranging from 51.7 to 78.3 mg/mL . Additionally, H. biflora has been used to treat cancer, especially for digestive system cancer for thousands of years in China. Five cyclotides (hedyotides B5-B9) isolated from leaves and root of H. biflora had strong cytotoxic activities against BxPC3, Capan2, MOH-1 and PANC1 cancer cell lines with IC 50 values from 0.33 to 3.11 mM (Ding et al. 2014). Moreover, according to the review paper on the H. diffusa (Chen et al. 2016), this species was recently noted for its anti-proliferative activity in cancer cells and was the most commonly Chinese herb used for colon cancer and breast cancer patients. The extracts and compounds isolated from H. diffusa were cytotoxically evaluated against some cancer cell lines, for instance anti-colorectal cancer, anti-leukemia, anti-liver cancer, anti-lung cancer, anti-cervical tumor, anti-prostate cancer, anti-multiple myeloma, and other anti-cancer effects. Although H. coronaria was not mentioned to use in traditional for treatment of cancer, this species would hopefully possess the same properties as other species in the same Hedyotis genus. On the chemical constituent aspect, there were several studies on this genus, which reported the presence of steroids, triterpenoids, anthraquinones, alkaloids, flavonoids, iridoids, lignans, glycosides, and so on (Konishi et al. 1998;Lajis and Ahmad 2006;Pham et al. 2010;Ngo et al. 2011;Bui et al. 2012;Luu et al. 2013;Chen et al. 2016;Luo et al. 2016). On the contrary, H. coronaria (the Vietnamese name is ram nui) has not been chemically and biologically studied; therefore, this species was chosen for phytochemical investigation and cytotoxic activity. Our previous study reported the isolation and structural elucidation of four oleanane saponins and two triterpenes from this species collected at Da Oai district, Lam Dong province in Vietnam (Pham et al. 2015). In this paper, further studies on the methanol extract of this plant led to the isolation of two new oleanane saponins, hedycoronin A and hedycoronin B (Figure 1), and the cytotoxicity against two cancer cell lines (KB and HeLa-S3) of these compounds was evaluated as well.

Results and discussion
Compound 1 was obtained as colorless powder. Its molecular formula was determined as C 54 H 88 O 22 based on the high-resolution-electrospray ionization mass spectrum (HR-ESI-MS), revealing a sodiated ion peak at m/z 1111.5626 [M þ Na] þ (calcd for C 54 H 88 O 22 þ Na, 1111.5665). The 1 H NMR spectrum exhibited four anomeric proton signals at d H 4.46 (1H, d, 8.0 Hz, H-1 0 ), 4.90 (1H, d, 7.5 Hz, H-1 00 ), 5.28 (1H, d, 7.5 Hz, H-1 000 ) and 5.30 (1H, d, 1.0 Hz, H-1 0000 ) which possessed the HSQC cross-peaks with anomeric carbons at d C 105.6 (C-1 0 ), 102.5 (C-1 00 ), 100.5 (C-1 000 ) and 102.2 (C-1 0000 ), respectively. The COSY correlations demonstrated the connection of these anomeric protons to adjacent oxygenated protons of these four sugar units, as shown in Figure  S1. Among four sugars, the first three ones were assigned as the b-D-glucopyranoses based on the large coupling constants at around 7.5-9.5 Hz of the anomeric protons and other oxygenated protons. Whereas the small coupling constants of 3 J H-1 0000 ,H-2 0000 ¼ 1.0 and J H-2 0000 ,H-3 0000 ¼ 3.5 Hz along with the presence of the typical doublet methyl signal H-6 0000 at 1.27 (d, 6.5 Hz) suggested the fourth one to be an a-L-rhamnopyranose. The HSQC and HMBC confirmed all the proton and carbon signals of the four sugar units. In addition, their proton and carbon chemical shift values were in good agreement with those in the literature (Romussi et al. 1979;Domon and Hostettmann 1984;Dorsaz and Hostettmann 1986;Suter et al. 1986;Thiilborg et al. 1994).
Exception of signals of the four sugar units, the rested proton signals of 1 proved the characteristic of an olean-12-ene type consisting of signals of an olefinic proton at d H 5.24 (1H, dd, 3.5, 3.5 Hz, H-12), an oxygenated proton at d H 3.20 (1H, dd, 12.0, 4.5 Hz, H-3) and seven quaternary methyl groups at the high magnetic field at d ). In addition, it was strongly supported by the 13 C-NMR and HSQC spectra with the presence of the signals at d C 122.9 (-CH=, C-12) and 146.2 (¼C<, C-13) of a double bond at C-12 of olean-12-ene type as usual, an oxygenated methine carbon at d C 92.3 (C-3) and seven methyl carbons in the range from 17 to 34 ppm. These observations suggested that the aglycon of 1 should be the olean-12-ene possessing one oxygenated methine group (C-3) and one carboxyl group (C-28) whose structure fitted nicely to its molecular formula and DEPT NMR data. The position of the CH-O group at C-3 was confirmed by the HMBC correlations of both methyl protons H-23 and H-24 to the carbon at d C 92.3 (C-3). The axial position of the proton H-3 at d H 3.20 (1H, dd, 12.0, 4.5 Hz) was supported by the large coupling constant (Jaa ¼ 12.0 Hz). The HMBC experiment ( Figure S1) also confirmed the positions of seven methyl groups at C-23, C-24, C-25, C-26, C-27, C-29 and C-30 through the correlations of these methyl protons with their nearby carbons, meaning that the carbon C-28 was the carboxyl group in which their chemical shift values showed highly compatible to the one of the 3-O-glycosylolean-12-ene-28-oic acid (Voutquenne et al. 2003).
Compound 2 was obtained as colorless powder. Its molecular formula was assigned as  [d C 106.5, 104.6, 102.9, 95.8], signals of twenty oxygenated carbons from 62 to 91 ppm (one of 3-oxygenated oleanane skeleton and nineteen ones of the four sugars) and eight methyl carbon signals at the high magnetic field (one of L-rhamnose and seven ones of the aglycon). The differences between 1 and 2 were the positions of the four sugar units which were supported by the 2 D-NMR experiments. The HMBC spectrum of 2 with cross-peaks of four anomeric protons H-1 0 , H-1 00 , H-1 000 , H-1 0000 to carbons at d C 90.9 (C-3), 85.0 (C-3 0 ), 178.1 (C-28) and 69.5 (C-6 000 ), respectively, ( Figure S1) suggested the attachment of the three b-D-glucopyranoses at C-3, C-28, and C-6 000 and of a-L-rhamnopyranose at C-3 0 . The b-D-glucopyranose unit at C-28 with the chemical values of the carboxyl carbon (C-28) and the anomeric carbon (C-1 000 ) upfield shifted to d C 178.1 (C-28) and 95.8 (C-1 000 ) instead of d C at around 182 (COOH), and more than 100 ppm (anomeric carbon attaching to another glucose moiety), respectively (Jayasinghe et al. 2002). All the rested of 2D-NMR data confirmed the chemical structure of 2 as b-D-glucopyranosyl-(1!6)-b-D-glucopyranosyl 3-O-[a-L-rhamnopyranosyl-(1!3)-b-D-glucopyranosyl]oleanoate. The new compound 2 was named hedyocoronin B.
Our previous study on this species collected in Lam Dong province, Vietnam (Pham et al. 2015)  had one more L-rhamnose unit at C-2 000 and the position of the third D-glucose unit at C-4 0 instead of at C-3 0 as in 3, whereas 2 differed from 4 by the presence of one more glucose unit at C-6 000 . In general, from Hedyotis coronaria collected in Vietnam, six oleanane saponins in both acid and ester forms were isolated. Among them, three pairs of saponins possessed two sugar units, three sugar units, and four sugar units, respectively. In the Hedyotis genus, beside the four triterpenoid saponins isolated in H. nudicaulis (Konishi et al. 1998), H. coronaria is the second species containing oleanane triterpenoid saponins.
Compounds 1 and 2 were evaluated the cytotoxicity against KB (human epidermoid carcinoma) and HeLa-S3 (human cervix adenocarcinoma) cell lines. The result showed that they had weak cytotoxicity against two tested cancer cell lines with IC 50 more than 54 mM.

General experimental procedures
The NMR experiments using residual solvent signal CD 3 OD as an internal reference (d H 3.31 and d C 49.0) were measured on a Bruker Avance 500 III spectrometer, at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR. HR-ESI-MS were recorded on a Bruker MicroOTOF-Q II mass spectrometer. Thin layer chromatography was carried out on precoated Kieselgel 60 F 254 or silica gel 60 RP-18 F 254 S (Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution, followed by heating. Gravity column chromatography was performed with silica gel 60 (0.040-0.063 mm, Himedia) or Li Chroprep RP-18 (0.040-0.063 mm, Merck).

Plant material
The aerial parts of H. coronaria (Kurz) Craib. were collected at Da Oai district (11.453862N, 107.514610E), Lam Dong province, Vietnam in December 2013. The scientific name of the plant was authenticated by the deceased botanist Phan Duc Binh, vice-editor of the Vietnam Medicine and Health Semi-monthly. A voucher specimen (N o US-C018) was deposited in the herbarium of the Department of Organic Chemistry, Faculty of Chemistry, University of Science, Vietnam National University -Ho Chi Minh City.

Extraction and isolation
The air-dried powder (1.7 kg) was macerated with methanol (7 L Â 3) at room temperature, and after filtration, the methanolic solution was evaporated at reduced pressure to yield a residue of 120 g. This crude residue was applied to silica gel solid phase extraction and then successively eluted with increasing polar solvents to give corresponding extracts, including petroleum ether (10.1 g), chloroform (6.8 g), ethyl acetate (26.2 g) and methanol extracts (56.3 g).

Biological assays
The cytotoxic activities against KB and HeLa-S3 cell lines were determined as previous reports ).

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.