Two new steroidal saponins from the roots of Cordyline fruticosa (L.) A. Chev

Abstract Two new steroidal saponins, 26-O-β-D-glucopyranosyl-22α-methoxy-5α-furost-25(27)-ene-1β,3β,26-triol 1-O-β-D-glucopyranoside (1), and 26-O-β-D-glucopyranosyl-22α-methoxy-furosta-5,25(27)-diene-1β,3β,26-triol 1-O-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranoside (2) were isolated and elucidated from the roots of Cordyline fruticosa (L.) A. Chev. Their structures were established by interpretation of spectroscopic data (1 D and 2 D NMR) and mass spectrometry (HR-ESI-MS). Graphical Abstract


Introduction
The genus Cordyline consists of 27 species distributed in tropical region (Mimaki et al. 1998).Some species of the genus have been used in the traditional medicine to treat diseases (Calixto et al. 1990;Mimaki et al. 1997;Mimaki et al. 1998;Lense 2012;Manicketh et al. 2022).Previous investigations on these plants revealed the presence of steroidal saponins, some of those substances possess interesting biological properties, mainly in cytotoxic and antimicrobial activities (Jewers et al. 1974;Blunden et al. 1984;Yang et al. 1989;Yang et al. 1990;Yokosuka et al. 2012;Fouedjou et al. 2014;Ponou et al. 2019;Nguyen et al. 2021).
Cordyline fruticosa (L.) A. Chev. is an ornamental plant which could be found in Viet Nam.Previous study on the leaves of C. fruticosa (L.) A. Chev.reported the presence of cholestane glycosides with cytotoxic activity (Yokosuka et al. 2012).As our interest of searching for new compounds from the Vietnamese plants, we reported herein the isolation and structural determination of two new steroidal saponins from the roots of C. fruticosa (L.) A. Chev.The structural determination of isolated compounds was carried out by a combination of spectroscopic analysis (1 D and 2 D NMR) and mass spectrometry (HR-ESI-MS), together with comparison their values in literature data.

Results and discussion
The aqueous ethanolic crude extract of the roots of C. fruticosa (L.) A. Chev.was isolated and purified by various chromatographic methods running on normal phased silica gel resulting new compounds 1 and 2. The structural determination of isolated compounds was carried out by spectroscopic analysis ( 1 H, 13 C, COSY, TOCSY, HSQC, HMBC, ROESY) and mass spectroscopy (HR-ESI-MS in positive mode).The sugar moieties of these compounds were determined by 2 D NMR analyses including HSQC, HMBC, COSY, TOCSY and ROESY as two glucopyranosyl for 1, and one fucopyranosyl, one rhamnopyranosyl, one glucopyranosyl for 2. The absolute configurations of sugar moieties were further identified to be D for glucose (Glc) and fucose (Fuc), and L for rhamnose (Rha).The relatively large values from 7.6 to 8.2 Hz revealed a b anomeric orientation for Fuc and Glc.The relatively large J C-1, H-1 value of Rha from 166 Hz indicated an a-equatorial pyranoid form for this anomeric proton.
Compound 1 was isolated as a white amorphous powder with a molecular formular of C 40 H 66 O 15 , proved by the HR-ESI-MS (m/z 809.4334 [M þ Na] þ ).The 1 H and 13 C NMR spectroscopic of the aglycone moiety of 1 was in good agreement with the signals of a furostanol saponin as described in the literature data (Mimaki et al. 1998;Nguyen et al. 2021; Table S1).The 1 H NMR spectrum of the aglycone moiety of 1 displayed signal of two tertiary methyl groups at d H 0.82 (s, H-18), 1.01 (s, H-19), a secondary methyl group at d H 1.08 (d, J ¼ 6.4 Hz, H-21), and two exomethylene protons at d H 5.07 and 5.37 (each 1H, br s, H-27).Moreover, in the 1 H NMR spectrum, the signal of another tertiary methyl group at d H 3.33 (s) revealed the presence of a methoxy group at C-22 position of the aglycone.The 13 C NMR spectrum displayed signals of a hemiacetal function at d C 120.4, three secondary alcoholic functions at d C 80.9 (C-1), 65.7 (C-3), and 81.0 (C-16), and one primary alcoholic function at d C 71.6 (C-26), suggested a 22a-methoxy-5a-furost-25(27)-ene type steroid.The COSY spectrum showed the signal of an oxygen bearing a methine proton at d H 3.99 (dd, J ¼ 11.1, 4.7 Hz, H-1) correlated to the d H 2.02 (H-2ax) and d H 2.87 (H-2eq), and the HMBC correlation between d H 0.82 (s, H-19) and d C 81.2 (C-1) revealed the location of a hydroxyl group at the C-1 position at the b-orientation and a glycosidic linkage at the C-1 position of the aglycone.Furthermore, tracing out the correlations in the ROESY spectrum led to an identification of a-orientation for the H-1 and H-5 of the aglycone, proved by the cross-peaks at d H 3.99 (H-1)/d H 2.25 (H-5), d H 2.02 (H-2ax)/d H 0.82 (s, H-19), and d H 2.87 (H-2eq)/d H 2.25 (H-5).Thus, the aglycone of 1 was elucidated as 22a-methoxy-5a-furost-25(27)ene-1b,3b,26-triol.The HSQC spectrum of sugar moiety of 1 displayed two cross-peaks at d H /d C 5.02 (d, J ¼ 7.6 Hz)/99.8, and 4.91 (d, J ¼ 8.2 Hz)/104.7,indicating the presence of two sugar units, Glc I and Glc II (Table S2).The linkage of Glc I to the C-1 position of the aglycone was confirmed by the HMBC correlation at d H /d C 5.02 (d, J ¼ 7.6 Hz, Glc I H-1)/80.9 (C-1), and by the ROESY correlation at d H /d H 5.02 (d, J ¼ 7.6 Hz, Glc I H-1)/3.99 (dd, J ¼ 11.1, 4.7 Hz, H-1).The Glc II was proved to be linked at C-26 of the aglycone by the HMBC correlation at Compound 2 was isolated as a white amorphous powder with a molecular formular of C 46 H 74 O 18 , proved by the HR-ESI-MS (m/z 937.4948 The NMR signals of the aglycon of 2 were in good with those of 1, except for the presence of a double bond at C-5 position of the aglycone in 2 (Table S1).Thus, the structure of 2 was elucidated as 22a-methoxy-furosta-5,25(27)-diene-1b,3b,26-triol.The HSQC spectrum of sugar moiety of 2 showed three anomeric signals with cross-peaks at d H /d C 4.78 (d, J ¼ 7.6 Hz)/99.8,4.90 (d, J ¼ 7.6 Hz)/103.4,and 6.35 (br s)/101.2,indicating the presence of three sugar units including one Fuc, one Glc, and one Rha, respectively (Table S2).The linkage of Fuc unit to the C-1 position of the aglycone was confirmed by the HMBC correlation at d H /d C 4.78 (d, J ¼ 7.6 Hz, Fuc H-1)/80.7 (C-1), and by the ROESY correlation at d H /d H 4.78 (d, J ¼ 7.6 Hz, Fuc H-1)/4.43 (dd, J ¼ 11.1, 3.5 Hz, H-1).The Rha unit was attached to the C-2 position of the Fuc unit proved by the HMBC correlation at d H /d C 6.35 (br s, Rha H-1)/74.2 (Fuc C-2), and by the ROESY correlation at d H /d H 6.35 (br s, Rha H-1)/4.50 (dd, J ¼ 9.4, 7.6 Hz, Fuc H-2).The Glc unit was proved to be linked at C-26 position of the aglycone by the HMBC correlation at d H /d C 4.90 (d, J ¼ 7.6 Hz, Glc H-1)/71.8 (C-26), and by the ROESY correlation at d H /d H 4.90 (d, J ¼ 7.6 Hz, Glc H-1)/4.62 (br d, J ¼ 12.3 Hz, H-26).Therefore, the structure of 2 was characterized as 26-O-b-D-glucopyranosyl-22a-methoxy-furosta-5,25(27)-diene-1b,3b,26-triol 1-O-a-L-rhamnopyranosyl-(1!2)-b-D-fucopyranoside (Figure S18).

Plant material
Cordyline fruticosa (L.) A. Chev.was collected in the natural site in Thai Nguyen city, Viet Nam in 2021 (21 34'14.3"N, 105 48'47.0"E)under code number NDH202101.The voucher specimen was stored in the Department of Biology, Thai Nguyen University of Education, Thai Nguyen, Viet Nam.

Acid hydrolysis and GC analysis
The sugar moieties and those absolute configurations of isolated compounds were determined as previously described (Nguyen et al. 2019).Each compound (2 mg) was hydrolysed with 2 N aq.CF 3 COOH (5 mL) for 3 h at 95 C.After extraction with CH 2 Cl 2 (3 Â 5 mL), the aqueous layer was repeatedly evaporated to dryness with MeOH until neutral, and then analysed by TLC over silica gel (CHCl 3 /MeOH/H 2 O 8:5:1) by comparison with authentic samples.Furthermore, the sugars residue was dissolved in anhydrous pyridine (100 lL), and L-cysteine methyl ester hydrochloride (0.06 mol/L) was added.The mixture was stirred at 60 C for 1 h, then 150 lL of HMDS-TMCS (hexamethyldisilazane/trimethylchlorosilane 3:1) was added, and the mixture was stirred at 60 C for another 30 min.The precipitate was centrifuged off, and the supernatant was concentrated under a N 2 stream.The residue was partitioned between n-hexane and H 2 O (0.1 mL each), and the hexane layer (1 lL) was analysed by GC.The absolute configurations were determined by comparing the retention times with thiazolidine derivatives prepared in a similar way from standard sugars (Sigma-Aldrich).The retention times of the sugars were found to be t R ¼ 11.07 min (L-Rha), 13.82 min (D-Fuc), 17.51 min (D-Glc).

Conclusion
Phytochemical study on the roots of C. fruticosa (L.) A. Chev.lead to the isolation and elucidation of two new steroidal saponins.Previous investigation on species of Cordyline genus revealed the presence of steroidal saponins, so this study has a significant benefit to complete the chemotaxonomic data about saponins from genus Cordyline and thus Asparagaceae family.The biological test of these compounds should be further carried out in the next study.