Triterpenoid Saponin and Lignan Glycosides from the Traditional Medicine Elaeagnus angustifolia Flowers and Their Cytotoxic Activities

A new triterpenoid saponin, named terpengustifol A (1), and two new lignan glucosides, phengustifols A and B (2 and 3), were isolated from the flowers of Elaeagnus angustifolia. Their structures were determined by the extensive analysis of the spectroscopic data (including NMR and HRMS) and ECD calculations. Compound 1 possesses an unusual monoterpene (Z)-6-hydroxy-2,6-dimethylocta-2,7-dienoyl unit at C-21. Compounds 2 and 3 are a pair of diastereoisomers, while their aglycones are a pair of enantiomers. Compounds 1 and 2 exhibited moderate cytotoxic activities against A375 cell lines with IC50 values at 12.1 and 15.6 μM, respectively. This is firstly reported the triterpenoid saponin and lignans isolated from the Elaeagnus angustifolia flowers.


Introduction
Elaeagnus angustifolia L., commonly called oleaster or Russian olive, is a medicinal plant belonging to the Elaeagnacea family. It is widely distributed from the northern regions of Asia to the Himalayas and Europe [1]. E. angustifolia is mainly planted in various provinces of Northwest of China, for wind breaks and sand fixation [2]. The flowers of E. angustifolia were traditionally used to treat asthma and tetanus in Iran and China [3][4][5]. In Chinese Uygur medicine, E. angustifolia flowers were considered as dry and hot materials, which is mainly used to treat brain disease, thoracalgia and asthma in the clinic. According to the specialists in traditional medicine, E. angustifolia flowers are a herbal medication which can stimulate sexuality, especially in young girls and women [6]. Zeinalzadeh et al. reported a randomized clinical trial to compare the effect of E. angustifolia flower capsule and sildenafil citrate tablet on sexual interest/arousal disorder [7]. Previous phytochemical investigations of E. angustifolia flowers focused on the essential oil and the total flavonoids content, and few studies were conducted on the isolation and identification of compounds present in the flowers [8][9][10]. Our group previously reported the identification of a novel macrocyclic flavonoid glycoside from the flowers of this plant [11]. In continuation of this program, a new triterpenoid saponin (1), and two new lignan glucosides (2 and 3) (Figure 1 and Supplementary Materials), were further isolated and identified from E. angustifolia flowers. Herein, the isolation, structural elucidation, and cytotoxic activities of the compounds, were presented.

Results and Discussion
Compound 1 was isolated as white powder; its molecular formula was identified as C 69 9), and two ester carbonyls (δ C 176.3 and 169.3). The above-mentioned data was typical for triterpenoid saponin, and the olefinic proton signal at δ H 5.30 (1H, t, J = 3.3 Hz, H-12) implied that it is an oleanolic-type saponin [12].
Acid hydrolysis of compound 1 yielded xylose, glucose and rhamnose, their configurations were identified as d-configuration for xylose and glucose, and l-configuration for rhamnose by the method gave in the experiment part. Analysis of HSQC, HMQC-TOCSY, and 1 H-1 H COSY spectrum clarified the proton and carbon signals of five sugar units (a xylose, three glucoses and a rhamnose) as shown in Table 1. Aside from the signals for the sugar units, 40 other carbon signals were observed in the 13 C-NMR spectrum. Further analysis of 2D NMR data revealed the presence of an aglycone with 3,21-dihydroxyolean-12-en-28-oic acid skeleton. There are 10 carbon signals remaining except the five sugar units and 3,21-dihydroxyolean-12-en-28-oic acid skeleton.   Figure 2a) from H 3 -9 to C-1 (δ C 169.3), C-2 (δ C 129.0) and C-3 (δ C 144.3); from H 3 -10 to C-5 (δ C 41.7), C-6 (δ C 73.7) and C-7 (δ C 146.1), indicated the presence of a 6-hydroxy-2,6-dimethyl-octa-2,7-dienoyl monoterpene moiety. Thus, the three units (sugars, triterpenoid and monoterpene) of 1 were determined, and their connections are the next thing to solve. The HMBC correlation from H-1 to C-3 (δ C 90.0) connected the xylose to C-3. A rhamnose and glucose were linked to positions 2 and 3 of xylose, respectively, by the HMBC correlations ( Figure 2a) from H-1 to C-2 (δ C 75.5) and from H-1 to C-3 (δ C 83.2). The HMBC correlation from H-1 to C-2 (δ C 83.9) assigned another glucose to position 2 of the glucose connected to xylose. The last glucose was connected to C-28 on the basis of the HMBC correlation from H-1 to C-28 (δ C 176.3). Compared the CH-21 (δ H 4.81, δ C 77.0) chemical shift of 1 to that of known compound machaerinic acid [13], obvious down-field shift indicated the monoterpene moiety was attached to C-21, although the HMBC correlation from H-21 to C-1 was not observed. The NOESY correlations (Figure 2b): from H-3 to H-5, from H-5 to H-9, from H-9 to H 3 -27, from H 3 -27 to H-16α, from H-16α to H-21, and from H-21 to H 3 -29, indicated they were in the same face. Correspondingly, the NOESY correlations from H 3 -24 to H 3 -25, from H 3 -25 to H 3 -26, and from H-18 to H 3 -30, revealed they took β-orientation. The Z-geometry of ∆ 2 was determined by the NOESY correlation between H-3 and H 3 -9 . Therefore, the structure of compound 1 was elucidated as shown in Figure 1, and it was given the trivial name terpengustifol A. Compounds 2 and 3 were obtained originally as a mixture as they were showed as one peak in the HPLC chromatography using a reverse phase C 18 column. The 1 H-NMR spectrum of the mixture cannot distinguish they are two compounds, while there are some subtle differences of several carbon resonances in the 13 C-NMR spectrum. Chiral chromatography analysis of the mixture showed that there were two peaks with a ratio around 1:1. Thus, compounds 2 and 3 were separated by chiral chromatography. The molecular formula of compounds 2 and 3 were established as C 26 (Table 2)  Further analysis of 2D NMR data ( 1 H-1 H COSY, HSQC, HMBC) of 2 established the planar structure. A hexose moiety (C-1 to C-6 ) and two subunits (C-7 to C-9 and C-7 to C-9 ) (drawn with bold bond in Figure 3) were established based on the 1 H-1 H COSY correlations. The three fragments were connected to the other functional groups by the HMBC correlations ( Figure 3). The linkage between C-7 and C-1 was determined by the HMBC correlations from H-7 to C-1, C-2 and C-6, and the linkage between C-8 and C-4 via ether bond by the HMBC correlations from H-8 to C-4 . The fragment C-7 to C-9 was linked to C-1 by the HMBC correlation from H-7 to C-2 and C-6 .  The HMBC correlation from H-1 to C-4 indicated the hexose moiety was connected to C-4. Thus, the planar structure of 2 was elucidated as depicted.
Compound 3 had the same planar structure as 2 based on analysis of the NMR data. Both compounds 2 and 3 produced d-glucose after acid hydrolysis, indicating that the difference between them was the stereochemistry of C-7 and C-8. The large coupling constant between H-7 and H-8 (J = 6.1 Hz) of compounds 2 and 3 indicated they had the same 7,8-threo configuration [14]. Therefore, compounds 2 and 3 are a pair of diastereoisomers, making their aglycones a pair of enantiomers. An ECD calculation was applied to elucidate the absolute configurations of C-7 and C-8. Due to the excessive number of chiral centers of glucose, a model compound 4 (Figure 4a) was selected to simplify the calculation. As shown in the Figure 4b, the calculated ECD of (7R,8R)-4 showed a similar Cotton effect as compound 3, which indicated that compound 3 had a 7R,8R-configuration. Correspondingly, compound 2 possesses a 7S,8S configuration. Thus, the structures of compounds 2 and 3 were determined as shown in Figure 1, and they were given the trivial names phengustifol A and B, respectively. There are many reports about the cytotoxicity of triterpenoid saponins and lignan glucosides against various cell lines [15][16][17][18], thus, the three new compounds obtained in the current study were tested for cytotoxic activity against the A375 human melanoma cell line using the CCK8 method [19]. The results revealed that compounds 1 and 2 exhibited moderate cytotoxic activities, with IC 50 values at 12.1 and 15.6 µM, respectively, while compound 3 showed weak cytotoxicity with an IC 50 value of 62.8 µM. The IC 50 value for the positive control cabazitaxel was 0.11 µM.

Extraction and Isolation
Air-dried ground powder of E. angustifolia flowers (1.0 kg) was sequentially extracted with petroleum ether (8 L × 3) and methanol (10 L × 3) by maceration at room temperature (7 days each time) to afford a crude methanol extract. The crude methanol extract was suspended in distilled water and then extracted successively with petroleum ether (PE), ethyl acetate and n-butanol. The n-butanol fraction (69.

Acid Hydrolysis of Compounds 1-3 and Sugar Analysis
Each compound (1.0 mg) was added to 1 mL HCl (1 N) and refluxed for 2 h. The solution was evaporated under a stream of N 2 after cooling. Mixture of the residue, anhydrous pyridine solution (0.1 mL) and l-cysteine methyl ester hydrochloride (0.06 N) was heated at 60 • C for half an hour. Removing the solvent, the residue was partitioned between water and cyclohexane. The cyclohexane layer was dried and dissolved in 200 µL acetone for GC analysis.

Cytotoxicity Assay
The Cell Counting Kit 8 (CCK-8) assay was used to evaluate the cytotoxicity of the compounds against A375 cells. The cells were cultured in DMEM with 10% FBS in a humidified incubator with 5% CO 2 at 37 • C for 24 h. The test compounds and positive control were added to the cultures and incubated for another 24 h. After that, the supernatant was removed, 10 µL of CCK8 (Dojindo, Kumamoto, Japan) reagent was added per well and incubated at 37 • C for 2 h. The samples were then transferred to a microplate reader to measure the optical density at a wavelength of 450 nm. Cabazitaxel was used as positive control.

Conclusions
In the present work, three new compounds 1-3, including a triterpenoid saponin and two lignan glucosides, were isolated and identified from Elaeagnus angustifolia flowers. Compound 1 possesses an unusual (Z)-6-hydroxy-2,6-dimethylocta-2,7-dienoyl monoterpene unit at C-21. Compounds 2 and 3 are a pair of diastereoisomers, while their aglycones are a pair of enantiomers. The bioassay indicated that all of the compounds showed somehow cytotoxic activities against A375, compounds 1 and 2 exhibited moderate activities. Interestingly, compound 2 showed stronger cytotoxic activities than that of 3 due to the different absolute configurations of their aglycones.
Supplementary Materials: The following are available online: 1 H, 13 C, 2D NMR, and HRMS spectra for compounds 1-3.
Author Contributions: T.Y. suggested the idea of the investigations and designed the experiments; J.H. and X.C. performed the isolation and identification of new compounds; W.L. and H.C. contributed to the evaluation of cytotoxicity; T.Y. prepared the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the National Natural Science Foundation of China (U1703331, 21502222), and Jiangxi "Shuangqian" Program (JXSQ2018101008).