Monoterpenoid Glycosides from the Leaves of Ligustrum robustum and Their Bioactivities

The leaves of Ligustrum robustum have been applied as Ku-Ding-Cha, a functional tea to clear heat, remove toxins, and treat obesity and diabetes, in Southwest China. The phytochemical research on the leaves of L. robustum led to the isolation and identification of eight new monoterpenoid glycosides (1–8) and three known monoterpenoid glycosides (9–11). Compounds 1–11 were tested for the inhibitory activities on fatty acid synthase (FAS), α-glucosidase, α-amylase, and the antioxidant effects. Compound 2 showed stronger FAS inhibitory activity (IC50: 2.36 ± 0.10 μM) than the positive control orlistat (IC50: 4.46 ± 0.13 μM), while compounds 1, 2, 5 and 11 displayed more potent ABTS radical scavenging activity (IC50: 6.91 ± 0.10~9.41 ± 0.22 μM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM). This study provided a theoretical basis for the leaves of L. robustum as a functional tea to treat obesity.


Introduction
Ku-Ding-Cha has been used widely as a functional tea to clear heat, remove toxins, and treat obesity, diabetes and so on, in Southwest China for a long time [1,2]. It was produced from the leaves of more than 30 plants from 13 genera in 12 families, in which the most common categories were from the genus Ligustrum (Oleaceae) and the genus Ilex (Aquifoliaceae) [3]. Ligustrum robustum (Roxb.) Blume, distributed widely in Southwest China, India, Burma, Vietnam and Cambodia, has been consumed as Ku-Ding-Cha in Southwest China, especially in Guizhou Province [4]. L. robustum has been classified as a food by the Chinese Ministry of Health since 2011 [5]. In the past two decades, the phytochemical studies on L. robustum led to the isolation and identification of monoterpenoid glycosides, phenylethanoid glycosides, iridoid glycosides, flavonoid glycosides and triterpenoids [1, [6][7][8][9][10][11]. The biological research on L. robustum reported the anti-obesity activity of the total glycosides and the aqueous extract [2,5], the antioxidative, anti-inflammatory and hepato-protective effects of the aqueous extract [4], and the antioxidant effect of some constituents [1,10]. In our previous study on L. robustum [12], some antioxidative and α-glucosidase inhibitory components, which might be a part of anti-diabetic ingredients of L. robustum [13][14][15][16], were discovered. However, to the best of our knowledge, the exact anti-obesity ingredients of L. robustum and their mechanisms are still unclear so far.

General Experimental Procedure
First, 1D and 2D NMR spectra were measured on a Bruker Ascend TM 400 NMR spectrometer (Bruker, Germany) ( 1 H at 400 MHz, 13 C at 100 MHz) or an Agilent 600/54 Premium Compact NMR spectrometer (Agilent, Santa Clara, CA, USA) ( 1 H at 600 MHz, 13 C at 150 MHz) with CD 3 OD as the solvent at 25 • C. Chemical shifts are expressed in δ (ppm) with tetramethylsilane (TMS) as the internal standard, and coupling constants (J) are reported in Hz. High-resolution electrospray ionization mass spectroscopy (HRESIMS) was carried out on a Waters Q-TOF Premier mass spectrometer (Waters, Milford, MA, USA). The IR absorption spectrum was measured with a PerkinElmer Spectrum Two FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). UV spectrum was recorded using a UV2700 spectrophotometer (Shimadzu, Kyoto, Japan). Optical rotation value was analyzed with an AUTOPOL VI automatic polarimeter (Rudolph, Hackettstown, NJ, USA).

Plant Material
The leaves of L. robustum were collected from Yibin City, Sichuan Province, China, in April 2017, and identified by Professor Guo-Min Liu (Kudingcha Research Institute, Hainan University, Haikou, 570228, China). A voucher specimen (No. 201704lsh) was deposited in West China School of Pharmacy, Sichuan University, China.

Extraction and Isolation
The fresh leaves of L. robustum were stirred and dried at 120 • C for 50 min and then powdered. The dried raw powder (7.0 kg) was extracted under reflux with 70% ethanol (28 L × 1) in a multi-function extractor for 2 h. The ethanol extract was filtrated and concentrated in vacuo to obtain a dark brown paste (2.2 kg). The paste was dissolved in 95% ethanol (3 L), and then, the distilled water (3 L) was added to precipitate the chlorophyll. After filtration, the filtrate was concentrated in vacuo to gain a brown residue (1.0 kg). The residue was chromatographed on 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.

Determination of Bioactivities
The inhibitory activities on FAS, α-glucosidase and α-amylase, and the DPPH and ABTS radical scavenging effects of compounds 1-11 were evaluated according to the methods described in the literature [12,18,20], while orlistat, acarbose and L-(+)-ascorbic acid were used as the positive controls, respectively (S1).

Statistical Analyses
Statistical analyses were carried out on GraphPad Prism 5.01. All samples were measured in triplicate. The IC 50 (the final concentration of sample needed to inhibit 50% of enzyme activity or scavenge 50% of free radical) was obtained by plotting the inhibition or scavenging percentage of each sample against its concentration. The results are reported as mean ± standard deviation (SD). Differences of means between groups were analyzed by one-way analysis of variance (ANOVA) on statistical package SPSS 13.0. The differences between groups were believed to be significant when p < 0.05.