Monoterpenoid Glycosides from the Leaves of Ligustrum robustum and Their Bioactivities (II)

Ligustrum robustum has been not only used as a heat-clearing and detoxicating functional tea (Ku-Ding-Cha) but also consumed as a hypotensive, anti-diabetic, and weight-reducing folk medicine. From the leaves of L. robustum, ten new monoterpenoid glycosides named ligurobustosides T10 (1a), T11 (1b), T12 (2a), T13 (2b), T14 (3a), T15 (3b), F1 (4b), T16 (5a), T17 (5b), and E1 (6b), together with five known ones (4a, 6a, 7, 8a, 8b), were separated and identified using the spectroscopic method and chemical method in this research. The results of biological tests exhibited that the fatty acid synthase (FAS) inhibitory action of compound 5 (IC50: 4.38 ± 0.11 μM) was as strong as orlistat (IC50: 4.46 ± 0.13 μM), a positive control; the α-glucosidase inhibitory actions of compounds 1–4 and 7–8, and the α-amylase inhibitory actions of compounds 1–8 were medium; the ABTS radical scavenging capacities of compounds 1–3 and 5–8 (IC50: 6.27 ± 0.23 ~ 8.59 ± 0.09 μM) were stronger than l-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM) served as a positive control. This research offered a theoretical foundation for the leaves of L. robustum to prevent diabetes and its complications.


Introduction
Diabetes, which affects about 10.5% of the population in the world, is a chronic metabolic disease with the characteristic of hyperglycemia, and its complications, such as diabetic nephropathy, neuropathy, and cardiovascular diseases, result in high morbidity and mortality [1].Present anti-diabetic agents, including insulin, metformin, α-glucosidase inhibitors acarbose and N-substituted iminosugar C-glycoside [2], can deal with hyperglycemia, whereas their function to prevent the complications of diabetes is not ideal.Recent research [3] indicated that antioxidant natural ingredients with inhibitory actions on fatty acid synthase (FAS), α-glucosidase, and α-amylase might be a novel resource for preventing diabetes and its complications.

The Bioactivities of Compounds 1-8
The bioactivities of compounds 1-8 isolated from the leaves of L. robustum, including the inhibitory actions on FAS, α-glucosidase and α-amylase, and the antioxidant capacities, were measured.The results of the biological tests are listed in Table 3.As exhibited in Table 3, the FAS inhibitory action of compound 5 (IC 50 : 4.38 ± 0.11 µM) was as strong as orlistat (IC 50 : 4.46 ± 0.13 µM) applied as a positive control, whereas the FAS inhibitory actions of compounds 4, 6, and 8 (IC 50 : 6.78 ± 0.18~24.68± 0.27 µM) were weaker than orlistat; the α-glucosidase inhibitory actions of compounds 1-4 and 7-8 were medium and weaker than acarbose, a positive control; the α-amylase inhibitory actions of compounds 1-8 were medium and weaker than the positive control acarbose.Although the DPPH radical scavenging capacities of compounds 1-8 were not observed, the ABTS radical scavenging capacities of compounds 1-3 and 5-8 (IC 50 : 6.27 ± 0.23~8.59± 0.09 µM) were stronger than L-(+)-ascorbic acid (IC 50 : 10.06 ± 0.19 µM), which was used as a positive control.Because antioxidant compositions with inhibitory actions on FAS, α-glucosidase, and α-amylase might be a novel resource for preventing diabetes and its complications [3], strong antioxidant compounds 1-8, with strong or medium FAS, α-glucosidase, and α-amylase inhibitory actions, might be partial functional ingredients of L. robustum for preventing diabetes and its complications.

General Experimental Procedure
HRESIMS was measured using a Waters Q-TOF Premier mass spectrometer (Waters, Milford, MA, USA).The IR spectrum was recorded on a PerkinElmer Spectrum Two FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). 1 H NMR, 13 C NMR, 1 H-1 H COSY, HSQC, HMBC, and NOEDS experiments were carried out on an Agilent 600/54 Premium Compact NMR spectrometer (Agilent, Santa Clara, CA, USA) or a Bruker Ascend TM 400 NMR spectrometer (Bruker, Mannheim, Germany) with CD 3 OD as solvent at 25 • C, while tetramethylsilane (TMS) was used as an internal standard.Chemical shifts are expressed in δ (ppm), and coupling constants (J) are reported in Hz.The UV spectrum was performed with a UV2700 spectrophotometer (Shimadzu, Kyoto, Japan).The optical rotation value was examined on an AUTOPOL VI automatic polarimeter (Rudolph, Hackettstown, NJ, USA).

Plant Material
The fresh leaves of L. robustum collected from Yibin City, China, were affirmed by Doctor Guo-Min Liu, Hainan University.A voucher sample (No. 201704lsh) of the leaves of L. robustum was preserved at the West China School of Pharmacy, Sichuan University.

Extraction and Separation
The fresh leaves of L. robustum were turned and heated at 115-120 • C for about 1 h, then powdered.The powdered leaves (7.0 kg) were extracted under reflux in a multi-function extractor with 70% ethanol (28 L × 1) for 2 h [15].After filtration, the ethanol extract was condensed in vacuo to give a brownish-black paste (2.2 kg).The paste was dissolved with 95% ethanol (3 L), and then distilled water (3 L) was added to deposit the chlorophyll.After percolation, the filtrate was condensed in vacuo to acquire a brown residue (1.0 kg).The residue was chromatographed using a silica gel column, which was eluted with CH 2 Cl 2 -MeOH (10:0-0:10) to afford Fr.II (145 g) and three other fractions.Fr.II was chromatographed repeatedly on silica gel column (CH

Acid Hydrolysis of Compounds 1-6
Compounds 1-6 (2 mg, each) were hydrolyzed with 1 M H 2 SO 4 aqueous solution at 92-95 • C for 6 h, respectively, and then neutralized with Ba(OH) 2 solution.After filtration, the solvent of the hydrolyzed solution was volatilized.The monosaccharides in the concentrated solution were identified by TLC with L-rhamnose and D-glucose references, which were developed with EtOAc-MeOH-HOAc-H 2 O (8:1:1:0.7)[3].The R f values of L-rhamnose and D-glucose were 0.73 and 0.43, respectively.

Statistical Analyses
GraphPad Prism 5.01 was applied in statistical analyses.All compounds were measured in triplicate, and the results are reported as mean ± SD.The difference in means was determined by ANOVA with the statistical package SPSS 25.0.The significant difference between groups was confirmed at p < 0.05.
a Coupling constants (J values in Hz) are reported in parentheses.b At 600 MHz.c At 400 MHz.

Table 2 .
13C NMR data of compounds 1-6 from the leaves of L. robustum in CD 3 OD.
a At 150 MHz.b At 100 MHz.

Table 3 .
Results of the biological tests of compounds 1-8 from the leaves of L. robustum a .Result is reported as the mean ± SD (n = 3).The means noted with different letters have significant differences (ANOVA, α = 0.05).b IC 50 : the eventual concentration of the tested compound needed to inhibit 50% of enzyme activity or clear away 50% of free radicals.
a c NA: no activity.d Positive control.