A new phenylpropanoid glucoside and a chain compound from the roots of Allium tuberosum

Abstract A new phenylpropanoid glucoside tuberosinine D (1) and a chain compound (Z)-11R,12S,13S-trihydroxy-9-octadecenoate (2) were isolated from the roots of Allium tuberosum. The absolute configuration of 1 was established by comparing of experimental and calculated electronic circular dichroism. The absolute configuration of 2 was determined using the modiﬁed Mosher’s method for the first time.


Introduction
Plants of the genus Allium (Liliaceae) contain abundant sulphur compounds (Kim et al. 2016) and steroidal saponins (Jabrane et al. 2011;Timité et al. 2013;Li et al. 2014;Fang et al. 2015). Previous research revealed that the plants of genus Allium have antimicrobial activity and antioxidant activity (Khalid et al. 2014). Chinese chives (Allium tuberosum Rottler), a species from Allium, is a favourite kitchen vegetable in Chinese cuisine. It is a perennial plant whose stems, leaves and inflorescence are edible and has been used as herbal medicine for treatment of abdominal pain, diarrhoea, haematemesis, snakebite and asthma (Hu et al. 2006;Bianchi 2015). Its seeds are officially listed in the Chinese Pharmacopeia for the treatment of impotence and nocturnal emissions (Chinese Pharmacopoeia Committee edition 2010). A. tuberosum roots have proven to be effective in resisting gastric ulcer and treating dyspepsia (Huang et al. 2006). In the course of our phytochemical studies on the roots of A. tuberosum, a new phenylpropanoid glucoside tuberosinine D (1) and a chain compound (Z)-11R,12S,13Strihydroxy-9-octadecenoate (2) (Figure 1) were isolated from the roots of A. tuberosum. The absolute configuration of 1 was established by electronic circular dichroism (eCD) calculation. The absolute configuration of 2 was determined using the modified Mosher's method for the first time. This paper addresses the isolation and structural elucidation of 1 and 2, as well as the establishment of their absolute configurations.
To clarify the absolute configurations of C-7 and C-8, the eCD data of two possible structures for 1 were calculated and compared with experimental spectrum. Two possible 7S,8R or 7R,8S geometries were previously optimised by density functional theory method at the B3LYP/6-31g(d) level (Shuang et al. 2009). excitation energies and rotational strengths were calculated using time-dependent density functional theory at the B3LYP/6-31 g(d,p) level in acetonitrile with PCM model (Liao et al. 2015). The eCD spectrum is simulated from electronic excitation energies and velocity rotational strengths. The results showed that the theoretical eCD data for 7S,8R-isomer (Isomer b) were in good agreement with the experimental spectrum ( Figure S3), and the chiral centres at C-7 and C-8 were determined as S and R, respectively. Thus, the structure of 1 was fully established and named tuberosinine D.
Compound 2 was obtained as an amorphous powder. The molecular formula (C 18 H 34 o 5 ) of 2 was deduced from the pseudo-molecular ion peak at m/z 353.2298 ([M + Na] + ) in the HR-eSI-MS spectrum. The 13 C NMR and HSQC spectra revealed that compound 2 contains 18 carbons, including one methyl group(δ C 14.5), 11 methylenes (δ C : 35.0, 30.2, 26.1, 30.3, 30.3, 30.7, 28.8, 33.7, 26.6, 33.2, 23.8) (all in upfield), five methines (δ C : 130.8, 133.5, 68.0, 78.9, 72.7) (one olefinic and three oxygenated) and one quaternary carbon (δ C : 177.9) (one ketone). The olefinic and carbonyl units accounted for 2° of unsaturation, requiring that 2 is a chain fatty acid compound. The 1 H-1 H CoSY correlations from H-12 (δ H 3.25, dd, J = 4.0, 6.3 Hz, 1H) to H-11 (δ H 4.57, dd, J = 4.0, 7.9 Hz, 1H) and H-13 (δ H 3.57, ddd, J = 2.1, 6.3, 9.1 Hz, 1H) ( Figure  S4) indicated that three methines were connected with each other at the vicinal position of the olefinic bond. eI-MS experiment was used to establish the position of the olefinic moiety. In the eI-MS spectrum of 2 ( Figure S5), positive ions at m/z 199 and 131 implied that the olefinic bond was located between C-9 and C-10. Two fragments of m/z 199 and 131 were obtained by the fragmentation of C-11 and C-12 bond ( Figure S5), which was consistent with the fragmentation style of this kind of compounds in literature (Kato et al. 1986). Basing on the establishment of C (9) =C (10) bond, three oxygenated methines were assigned at C-11, C-12 and C-13. The Z/E configuration of C (9) =C (10) bond was determined based on the chemical shift of C-8. It was reported that the chemical shift of CH 2 attaching to E double bond is about 33 ppm, whereas attaching to Z double bond is about 27 ppm (Mahendran et al. 1979). In our experiment, the chemical shift of C-8, which is adjacent to the C (9) =C (10) bond, appears at 28.1 ppm. Therefore, the configuration of C (9) =C (10) bond was assigned as Z. The planar structure of 2 was thus assigned as (Z)-11,12,13-trihydroxy-9-octadecenoate.
The relative configuration of 2 was assigned by the coupling constants of H-11, H-12 and H-13, as well as Noe correlations ( Figure S6). Thorough studies on the conformation of three vicinal chiral carbons in hunanamycin A by Macmillan and his co-workers (Youcai et al. 2013) have provided an approach of assigning the relative configuration of such moieties on the basis of the coupling constants and Noe correlations. The small coupling constant between H-11 with H-12 (J = 4.0 Hz) and a large coupling constant between H-12 and H-13 (J = 6.3 Hz) in 2 match with the erythro configuration for both 11/12 and 12/13 ( Figure S6). The presence of the Noe correlations from H-10 to H-12/H-13, H-11 to H-13, and the absence of the Noe correlation from H-11 to H-14 further supported above conclusion ( Figure S6). Therefore, there are two possible absolute configurations for 2, either 11R,12S,13S or 11S,12R,13R.
Calculating the eCD and VCD spectra and comparing with experimental data are widely used in the absolute configuration establishment (He et al. 2011). However, for the compounds without obvious ultraviolet absorption, such as 2, eCD calculation does not work. The modified Mosher's method has been used as a routine mean for determining the absolute configuration of organic, natural and synthetic compounds (ohtani et al. 1991;Keebeom et al. 2014;Takahiro et al. 2014). In this experiment, the modified Mosher's method was used for the absolute configuration establishment of 2. In view of the presence of multiple hydroxyl groups in 2, it is very important to improve the selectivity of esterification reaction. The treatment of 2 with (R)-and (S)-MTPA-oH (α-methyloxy-trifluoro-methyl-phenyl-acetic acid) for 5 min yielded the 11,13-di-(R)-MTPA-2 and 11-(S)-MTPA-2, respectively. Notably, oH-12 couldn't be esterified by MPTA, which might be caused by steric hindrance, ensuring the absolute configuration establishment of C-11. The shielding on H-12 depends surely on both 11 and 13-MTPA groups; therefore, the calculation of the 1 H NMR Δδ S-R values for H-12 cannot be used for the assignment of the absolute configuration of C-11. However, 13-MTPA has almost no influence on the chemical shift of H-10 because of their long spatial distance. That is to say, the 1 H NMR Δδ S-R values for H-10 of 11,13-di-(R)-MTPA-2 and 11-(S)-MTPA-2 could be used for the determination of the absolute configuration of C-11. The calculation of the 1 H NMR Δδ S-R values (+0.28) for H-10 of 11,13-di-(R)-MTPA-2 and 11-(S)-MTPA-2 ( Figure S7) finally confirmed an R absolute configuration at C-11. Thus, the structure of 2 was assigned as (Z)-11R,12S,13S-trihydroxy-9-octadecenoate. It is reported that the configurations of some natural analogues of 2 were determined as 11R,12S,13S-and 11S,12S,13S-configurations by partial synthesis (Kato et al. 1986), which was consistent with our study.
It is worth noting that compound 2 could be found in SciFinder. However, in the exported literatures, we didn't find enough evidence for the establishment of the absolute configuration of this compound. This paper assigned the absolute configurations of three chiral carbons in 2 definitely for the first time.

General procedure
Melting point was determined on a XRC-1 melting point apparatus and was uncorrected. optical rotation was measured with a Jasco P-1020 digital polarimeter. A Nicolet Magna-IR 550 spectrometer was used for scanning IR spectroscopy with KBr pellets. NMR spectra were acquired with either a Bruker AM-400 spectrometer (400 MHz for 1 H NMR and 100 MHz for 13 C NMR) or a Bruker DRX-600 (600 MHz for 1 H NMR and 150 MHz for 13 C NMR) spectrometer. eSI-MS analysis was recorded with Agilent g3250AA and Auto Spec Premier P776 spectrometers. Silica gel (200-300 mesh or 300-400 mesh) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography.

Plant material
The roots of A. tuberosum were collected from guandu District, Kunming City in Yunnan Province of China, in June 2011, and identified by professor Shu-gang Lu from School of life Sciences, Yunnan university. The voucher specimen (2011-JCg-1) has been deposited in the school of Chemical Science and Technology of Yunnan university.

Conclusion
The chemical investigation of the roots of A. tuberosum resulted in the isolation of a new phenylpropanoid glucoside tuberosinine D (1) and a chain compound (Z)-11R,12S,13Strihydroxy-9-octadecenoate (2). The absolute configuration of 1 was established by eCD calculation. The absolute configuration of 2 was determined using the modified Mosher's method for the first time. Compounds 1 and 2 showed no obvious antibacterial activity against Bacillus subtilis, Escherichia coli and Candida albicans.

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

Funding
This work was financially supported by a Natural Science Foundation of China (grant number 81460648).