Four New Monoterpenoid Glycosides from the Flower Buds of Magnolia biondii

Four new monoterpenoid glycosides 1–4, named magnoliaterpenoid A–D, were isolated from a 50% aqueous acetone extract of flower buds of Magnolia biondii, along with one known compound, (1′R,3′S,5′R,8′S,2Z,4E)-dihydrophaseic acid 3-O-β-d-glucopyranoside (5). Their structures and relative configuration were identified by extensive spectroscopic analysis (IR, UV, MS, 1D and 2D NMR). The aglycones of these four new compounds possess seven-membered rings systems, which are very rare. A plausible biosynthetic route for the four new compounds was proposed via the biogenetic isoprene rule. Compounds 1, 2, 3, and 4 showed no antimicrobial activity at the concentration range of 1.95–250 µg/mL.


Introduction
The genus Magnolia (family Magnoliaceae) includes about 90 species worldwide, mainly distributed in tropical and temperate regions of Asia and North America to South America. In China, there are about 30 species, mostly distributed in the southern and northern provinces of China [1]. Flos Magnoliae, the flower buds of M. biondii, M. denudata or M. sprengeri, which is unique to China and mostly distributed in the Henan, Hubei, Zhejiang, Anhui, and Shanxi provinces, has been used as a traditional Chinese medicine for more than 1500 years to treat nasal congestion, headaches, sinusitis, and allergic rhinitis [2][3][4]. Its volatile oil has also been processed into all kinds of cosmetics which are widely used in China [4]. It has been reported to possess extensive anti-allergy [5,6], anti-inflammatory [7], anti-angiogenic [8], anti-platelet aggregation [9,10], antimicrobial [11] and antioxidant activities [12,13]. Several types of compounds have been isolated from the plant, including lignans [9], neolignans [9], sesquiterpenoids [14], alkaloids [15,16], and flavonoids [15,16]. In order to further investigate the active components of the herb, a systematic phytochemical study was carried out on the 50% aqueous acetone extract of flower buds of Magnolia biondii. As a result, four new monoterpenoid glycosides1-4, named magnoliaterpenoid A-D (Figure 1), were isolated and structurally identified, along with a known sesquiterpenoid glycoside, (1 1 R,3 1 S,5 1 R,8 1 S,2Z,4E)dihydrophaseic acid 3-O-β-D-glucopyranoside (5). Herein, the isolation and structural elucidation of compounds 1-4 are reported, as well as their antimicrobial activity.

Structure Elucidation of Compounds 1-4
Compound 1, obtained as a colorless gum, was determined to have the molecular formula C16H26O8Cl by HRESIMS (m/z 381.1301 [M + Cl] − , calcd. 381.1310),with four degrees of unsaturation. Its UV spectrum showed an absorption maximum at 217 nm, indicating the presence of an α,β-unsaturated carbonyl moiety. The IR spectrum supported the presence of hydroxyl (3367 cm −1 ), carbonyl (1682 cm −1 ) and double bond (1652 cm −1 ) groups. The 1 H-NMR spectroscopic data of 1 (       and H-6/H-7 displayed the key spin systems. In the HMBC spectrum (Figure 2), the correlation from H-8 to C-5 indicated that the 8-CH 3 was located to C-5; the correlation from H-9 to C-4 indicated that the 9-CH 3 was connected to C-4; the correlation from H-1 1 to C-5 indicated that the β-D-glucose was linked to C-5; the correlations from H-2 and H-7 to C-10 indicated that the carbonyl (C-10) was linked to C-1; the oxygenated quaternary carbon (C-5) was located between C-4 and C-6 from the HMBC correlations from H-4 and H-6 to C-5. The relative configuration of compound 1 was established by the NOESY experiment ( Figure 3) in which the correlation of H-4 and H-8 was observed. Thus, the structure of compound 1 was determined to be as shown in Figure 1, and it was assigned the trivial name magnoliaterpenoid A.

Results and Discussion
Molecules 2016, 21, 728 3 of 7 ( Figure 2), the correlation from H-8 to C-5 indicated that the 8-CH3 was located to C-5; the correlation from H-9 to C-4 indicated that the 9-CH3 was connected to C-4; the correlation from H-1′ to C-5 indicated that the β-D-glucose was linked to C-5; the correlations from H-2 and H-7 to C-10 indicated that the carbonyl (C-10) was linked to C-1; the oxygenated quaternary carbon (C-5) was located between C-4 and C-6 from the HMBC correlations from H-4 and H-6 to C-5. The relative configuration of compound 1 was established by the NOESY experiment ( Figure 3) in which the correlation of H-4 and H-8 was observed. Thus, the structure of compound 1 was determined to be as shown in Figure 1, and it was assigned the trivial name magnoliaterpenoid A.   Materials). Comparison of its 13 C-NMR and DEPT data with those of compound 1 indicated that the two compounds possessed a similar skeleton. One of the differences between these two compounds was that the methylene signal at C-7 (δC 26.3) in 1 was replaced by an oxygenated methine at C-7 (δC 71.9) in 2, which was established by the HMBC correlations ( Figure 2) from H-2, H-6 and H-10 to C-7, along with the 1 H-1 H COSY correlations (Figure 2) of H-6/H-7. The other main difference was that the carbonyl at C-10 (δC 170.9) in 1 was replaced by a methyl at C-10 (δC 19.2) in 2, which was determined on the basis of the HMBC correlations ( Figure 2) from H-10 to C-1, C-2 and C-7. The relative configuration of compound 2 was deduced from the NOESY experiment ( Figure 3) in which the correlationof H-4/H-8 and H-7/H-8 was detected. Similarly, the sugar unit of compound 2 was confirmed as β-D-glucose using the same method as for compound 1. Therefore, the structure of 2 was assigned as shown in Figure 1, and it was given the trivial name magnoliaterpenoid B.  Figure 2), the correlation from H-8 to C-5 indicated that the 8-CH3 was located to C-5; the correlation from H-9 to C-4 indicated that the 9-CH3 was connected to C-4; the correlation from H-1′ to C-5 indicated that the β-D-glucose was linked to C-5; the correlations from H-2 and H-7 to C-10 indicated that the carbonyl (C-10) was linked to C-1; the oxygenated quaternary carbon (C-5) was located between C-4 and C-6 from the HMBC correlations from H-4 and H-6 to C-5. The relative configuration of compound 1 was established by the NOESY experiment ( Figure 3) in which the correlation of H-4 and H-8 was observed. Thus, the structure of compound 1 was determined to be as shown in Figure 1, and it was assigned the trivial name magnoliaterpenoid A.   Materials). Comparison of its 13 C-NMR and DEPT data with those of compound 1 indicated that the two compounds possessed a similar skeleton. One of the differences between these two compounds was that the methylene signal at C-7 (δC 26.3) in 1 was replaced by an oxygenated methine at C-7 (δC 71.9) in 2, which was established by the HMBC correlations ( Figure 2) from H-2, H-6 and H-10 to C-7, along with the 1 H-1 H COSY correlations (Figure 2) of H-6/H-7. The other main difference was that the carbonyl at C-10 (δC 170.9) in 1 was replaced by a methyl at C-10 (δC 19.2) in 2, which was determined on the basis of the HMBC correlations ( Figure 2) from H-10 to C-1, C-2 and C-7. The relative configuration of compound 2 was deduced from the NOESY experiment ( Figure 3) in which the correlationof H-4/H-8 and H-7/H-8 was detected. Similarly, the sugar unit of compound 2 was confirmed as β-D-glucose using the same method as for compound 1. Therefore, the structure of 2 was assigned as shown in Figure 1, and it was given the trivial name magnoliaterpenoid B.
Compound 3 was isolated as a colorless gum and had a molecular formula C16H26O7Cl by HRESIMS (m/z 365.1357 [M + Cl] − , calcd. 365.1361), with four degrees of unsaturation. Its UV spectrum showed an absorption maximumat 240 nm, indicating the presence of an α,β-unsaturated carbonyl moiety. The IR spectrum supported the presence of hydroxyl (3364 cm −1 ), carbonyl (1655 cm −1 ) and Compound 2 was isolated as a colorless gum and had a molecular formula C 16 Materials). Comparison of its 13 C-NMR and DEPT data with those of compound 1 indicated that the two compounds possessed a similar skeleton. One of the differences between these two compounds was that the methylene signal at C-7 (δ C 26.3) in 1 was replaced by an oxygenated methine at C-7 (δ C 71.9) in 2, which was established by the HMBC correlations ( Figure 2) from H-2, H-6 and H-10 to C-7, along with the 1 H-1 H COSY correlations (Figure 2) of H-6/H-7. The other main difference was that the carbonyl at C-10 (δ C 170.9) in 1 was replaced by a methyl at C-10 (δ C 19.2) in 2, which was determined on the basis of the HMBC correlations ( Figure 2) from H-10 to C-1, C-2 and C-7. The relative configuration of compound 2 was deduced from the NOESY experiment (Figure 3) in which the correlationof H-4/H-8 and H-7/H-8 was detected. Similarly, the sugar unit of compound 2 was confirmed as β-D-glucose using the same method as for compound 1. Therefore, the structure of 2 was assigned as shown in Figure 1, and it was given the trivial name magnoliaterpenoid B.
Compound 3 was isolated as a colorless gum and had a molecular formula C 16 Supplementary Materials). The 13 C-NMR and DEPT spectroscopic data of 3 (Table 1) were highly similar to those of 2. The only difference between compound 2 and 3 was that the oxygenated methine at C-7 (δ C 71.9) in 2 was replaced by a carbonyl at C-7 (δ C 203.5) in 3, which was established by the HMBC correlations ( Figure 2) from H-2, H-6 and H-10 to C-7. The relative configuration of compound 3 was assigned by the NOESY experiment (Figure 3) in which the correlation of H-4 and H-8 was observed. Similarly, the sugar unit of compound 3 was also confirmed as β-D-glucose using the same method as for compound 1. Therefore, the structure of 3 was established as shown in Figure 1, and the compound was trivially named magnoliaterpenoid C.
Compound 4 was isolated as a colorless gum. Its molecular formula was established as C 16 (Table 1) demonstrated that its structure was closely related to that of compound 2, except that the double bond at C-1 (δ C 138.2) and C-2 (δ C 124.6) in 2 was reduced to a methane at C-1 (δ C 37.9) and a methylene at C-2 (δ C 29.6) respectively in 4, which was established by the HMBC correlations ( Figure 2) from H-10 to C-1 and C-2, along with the 1 H-1 H COSY correlations (Figure 2) of H-1 and H-2. The relative configuration of compound 4was similar to compound 2, which was deduced from the NOESY experiment ( Figure 3) in which the correlation of H-4/H-8, H-7/H-8 and H-1/H-7 was detected. Similarly, the sugar unit of compound 4 was also confirmed as β-D-glucose using the same method as for compound 1. Thus, the structure of 4 was established as shown in Figure 1, and the compound was given the trivial name magnoliaterpenoid D.
The identity of compound 5 was determined by spectroscopic analysis and comparison with literature data [18].

Plausible Biogenetic Pathway
Since the aglycones of these four new compounds contained 10 carbon atoms and belonged to the monoterpenoid class of compounds, we propose a plausible biosynthetic route via the biogenetic isoprene rule, as shown in Scheme 1.  Materials). The 13 C-NMR and DEPT spectroscopic data of 3 (Table 1) were highly similar to those of 2. The only difference between compound 2 and 3 was that the oxygenated methine at C-7 (δC 71.9) in 2 was replaced by a carbonyl at C-7 (δC 203.5) in 3, which was established by the HMBC correlations ( Figure 2) from H-2, H-6 and H-10 to C-7. The relative configuration of compound 3 was assigned by the NOESY experiment ( Figure 3) in which the correlation of H-4 and H-8 was observed. Similarly, the sugar unit of compound 3 was also confirmed as β-D-glucose using the same method as for compound 1. Therefore, the structure of 3 was established as shown in Figure 1, and the compound was trivially named magnoliaterpenoid C. Compound 4 was isolated as a colorless gum. Its molecular formula was established as C16H30O7Na by HRESIMS (m/z 357.1878 [M + Na] + , calcd. 357.1883), with two degrees of unsaturation. The IR spectrum displayed the existence of hydroxyl (3362cm −1 ) groups. The 1 H-NMR spectroscopic data of 4 (Table 1) (Table 1) demonstrated that its structure was closely related to that of compound 2, except that the double bond at C-1 (δC 138.2) and C-2 (δC 124.6) in 2 was reduced to a methane at C-1 (δC 37.9) and a methylene at C-2 (δC 29.6) respectively in 4, which was established by the HMBC correlations ( Figure 2) from H-10 to C-1 and C-2, along with the 1 H-1 H COSY correlations (Figure 2) of H-1 and H-2. The relative configuration of compound 4was similar to compound 2, which was deduced from the NOESY experiment ( Figure 3) in which the correlation of H-4/H-8, H-7/H-8 and H-1/H-7 was detected. Similarly, the sugar unit of compound 4 was also confirmed as β-D-glucose using the same method as for compound 1. Thus, the structure of 4 was established as shown in Figure 1, and the compound was given the trivial name magnoliaterpenoid D.
The identity of compound 5 was determined by spectroscopic analysis and comparison with literature data [18].

Plausible Biogenetic Pathway
Since the aglycones of these four new compounds contained 10 carbon atoms and belonged to the monoterpenoid class of compounds, we propose a plausible biosynthetic route via the biogenetic isoprene rule, as shown in Scheme 1.

Acid Hydrolysis and Sugar Analysis
Compounds 1-4 (1.0 mg, respectively) were refluxed with 8% HCl (2 mL) for 3 h. After the reaction mixture was extracted with EtOAc (2 mLˆ3), the aqueous layer was dried under vacuum. Then the residue was dissolved in pyridine (0.3 mL) containing L-cysteine methyl ester hydrochloride (1.5 mg) and heated at 60˝C for 1 h. A 0.3 mL solution of phenyl isothiocyanate (1.5 mg) in pyridine was added to the mixture, which was heated at 60˝C for 1 h. The reaction mixture was directly analyzed by gas chromatography (GC). The D-configuration of glucose was confirmed by comparing the retention time with a standard sample [t R (min): D-glucose (8.5)].

Antimicrobial Assay
The antimicrobial activity of compounds 1-4 was evaluated against Gram-positive bacteria (Staphylococcus aureus ATCC25923), Gram-negative bacteria (Escherichia coli ATCC35150 and Proteus vulgaris ATCC33420) and fungi (Aspergillusniger ATCC6257 and Candida albicans ATCC90029) by a microdilution titre technique [19]. Kanamycin and fluconazole was used as positive controls. All tests were performed in triplicate.

Conclusions
In conclusion, although these four new compounds did not show antimicrobial activity at the tested concentration range of 1.95-250 µg/mL, the aglycones of these four new compounds possess seven-membered rings system which are very rare and a plausible biogenetic pathway of 1-4 was proposed via the biogenetic isoprene rule. This kind of skeleton was also isolated from the Magnoliaceae for the first time.

Conflicts of Interest:
The authors declare that there are no conflicts of interest.