Phenylethanoid and Phenylmethanoid Glycosides from the Leaves of Ligustrum robustum and Their Bioactivities

The phytochemical study on the leaves of Ligustrum robustum, which have been used as Ku-Ding-Cha, led to the isolation and identification of three new phenylethanoid glycosides and three new phenylmethanoid glycosides, named ligurobustosides R1 (1b), R2–3 (2), R4 (3), S1 (4b), S2 (5), and S3 (6), and five reported phenylethanoid glycosides (7–11). In the bioactivity test, (Z)-osmanthuside B6 (11) displayed strong fatty acid synthase (FAS) inhibitory activity (IC50: 4.55 ± 0.35 μM) as the positive control orlistat (IC50: 4.46 ± 0.13 μM), while ligurobustosides R4 (3) and S2 (5), ligupurpuroside B (7), cis-ligupurpuroside B (8), ligurobustoside N (9), osmanthuside D (10), and (Z)-osmanthuside B6 (11) showed stronger ABTS radical scavenging activity (IC50: 2.68 ± 0.05~4.86 ± 0.06 μM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM). This research provided a theoretical basis for the leaves of L. robustum as a tea with function in treating obesity and diabetes.


General Experimental Procedure
Optical rotation value was determined with an AUTOPOL VI automatic polarimeter (Rudolph, Hackettstown, NJ, USA). The UV spectrum was measured on a UV2700 spectrophotometer (Shimadzu, Kyoto, Japan). IR absorption spectrum was carried out with a PerkinElmer Spectrum Two FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). NMR spectra were recorded using an Agilent 600/54 Premium Compact NMR spectrometer (Agilent, Santa Clara, CA, USA) ( 1 H at 600 MHz, 13 C at 150 MHz) or a Bruker Ascend TM 400 NMR spectrometer (Bruker, Germany) ( 1 H at 400 MHz, 13 C at 100 MHz) with CD3OD

General Experimental Procedure
Optical rotation value was determined with an AUTOPOL VI automatic polarimeter (Rudolph, Hackettstown, NJ, USA). The UV spectrum was measured on a UV2700 spectrophotometer (Shimadzu, Kyoto, Japan). IR absorption spectrum was carried out with a PerkinElmer Spectrum Two FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). NMR spectra were recorded using an Agilent 600/54 Premium Compact NMR spectrometer (Agilent, Santa Clara, CA, USA) ( 1 H at 600 MHz, 13 C at 150 MHz) or a Bruker Ascend TM 400 NMR spectrometer (Bruker, Germany) ( 1 H at 400 MHz, 13 C at 100 MHz) with CD 3 OD (compound 3: CD 3 OD + DMSO-d 6 ) as the solvent at 25 • C. Chemical shifts are reported in δ (ppm) with tetramethylsilane (TMS) as the internal standard, while coupling constants (J) are expressed in Hz. High-resolution electrospray ionization mass spectroscopy (HRESIMS) was measured on a Waters Q-TOF Premier mass spectrometer (Waters, Milford, MA, USA).

Plant Material
The leaves of L. robustum were harvested in April 2017 from Yibin City, Sichuan Province, China, and authenticated by Professor Guo-Min Liu (Kudingcha Research Institute, Hainan University, China). A voucher specimen (No. 201704lsh) was conserved at West China School of Pharmacy, Sichuan University, China.

Extraction and Isolation
The fresh leaves of L. robustum were agitated and baked at 120 • C for 50 min and then smashed. The raw powder (7.0 kg) was extracted with 70% ethanol (28 L × 1) under reflux in a multi-function extractor for 2 h [13]. The ethanol extract was percolated and condensed in vacuo to gain a paste (2.2 kg). The paste was dissolved in 3 L 95% ethanol, and then 3 L purified water was infunded to sediment the chlorophyll. After percolation, the filtrate was condensed in vacuo to obtain a residue (1.0 kg). The residue was separated on a silica gel column, eluting with CH 2 Cl 2 -MeOH (10:0-0:10), to yield Fr. I (84 g), Fr. II (145 g), Fr. III (93 g), and Fr. IV (70 g). Fr. II was isolated repeatedly by CC on silica gel, eluting with     Table 3; 13 C NMR (CD 3 OD, 100 MHz) data, see Table 4; HRESIMS m/z 585.1943 [M + Na] + (calculated for C 28 H 34 NaO 12 , 585.1948).

Enzymatic Hydrolysis of Compounds 2
Compound 2 (20 mg) and cellulase (30 mg) were added to 12 mL HOAc-NaOAc buffer solution (pH 5.0) and kept at 37 • C for 6 h. The hydrolyzed product was extracted with EtOAc and purified on a silica gel column (eluting with EtOAc) to afford (R)- (

Statistical Analyses
Statistical analyses were performed on GraphPad Prism 5.01. All samples were determined in triplicate. The IC 50 (the ultimate concentration of sample needed to inhibit 50% of enzyme activity or clear away 50% of free radicals) was acquired by plotting the inhibition or scavenging percentage of every sample against its concentration. The results are recorded as mean ± standard deviation (SD). Differences of means between several groups were analyzed by one-way analysis of variance (ANOVA) on the statistical package SPSS 25.0. The differences between groups were deemed to be significant when p < 0.05.