Chemical Constituents from the Leaves of Ligustrum robustum and Their Bioactivities

The leaves of Ligustrum robustum have been consumed as Ku-Ding-Cha for clearing heat and removing toxins, and they have been used as a folk medicine for curing hypertension, diabetes, and obesity in China. The phytochemical research on the leaves of L. robustum led to the isolation and identification of two new hexenol glycosides, two new butenol glycosides, and five new sugar esters, named ligurobustosides X (1a), X1 (1b), Y (2a), and Y1 (2b) and ligurobustates A (3a), B (3b), C (4b), D (5a), and E (5b), along with seven known compounds (4a and 6–10). Compounds 1–10 were tested for their inhibitory effects on fatty acid synthase (FAS), α-glucosidase, and α-amylase, as well as their antioxidant activities. Compound 2 showed strong FAS inhibitory activity (IC50 4.10 ± 0.12 μM) close to that of the positive control orlistat (IC50 4.46 ± 0.13 μM); compounds 7 and 9 revealed moderate α-glucosidase inhibitory activities; compounds 1–10 showed moderate α-amylase inhibitory activities; and compounds 1 and 10 displayed stronger 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ammonium salt (ABTS) radical scavenging effects (IC50 3.41 ± 0.08~5.65 ± 0.19 μM) than the positive control l-(+)-ascorbic acid (IC50 10.06 ± 0.19 μM). This study provides a theoretical foundation for the leaves of L. robustum as a functional tea to prevent diabetes and its complications.


Introduction
Diabetes, which affects nearly 10.5% of the population worldwide, is a chronic metabolic disease characterized by hyperglycemia caused by insulin resistance, a deficiency in insulin secretion, or both [1]. Its complications, including diabetic neuropathy, nephropathy, and cardiovascular diseases, lead to serious morbidity and mortality [1]. Current drugs, such as insulin, metformin, sulfonylureas, and acarbose, can control hyperglycemia, but their effect on preventing the complications of diabetes is not ideal. Therefore, it is significant to search for new resources for the prevention of diabetes and its complications.
Studies have revealed that long-term obesity might trigger specific metabolic disorders, such as cardiovascular diseases, insulin resistance, and diabetes [2,3]; fatty acid synthase (FAS), which catalyzes the synthesis of saturated long-chain fatty acids, is a potential target to prevent obesity [4]; carbohydrate digestive enzymes, such as α-glucosidase and α-amylase, play a crucial role in promoting hyperglycemia by releasing monosaccharides in the course of digestion [5]; and the contribution of reactive oxygen species generated by oxidative stress induced by chronic hyperglycemia has been linked to the onset and progression of diabetes and its complications [6]. Thus, natural products with inhibitory activities on FAS, α-glucosidase, and α-amylase as well as an antioxidant effect might be a new resource to prevent diabetes and its complications.

The Bioactivities of Compounds 1-10
Compounds 1-10 isolated from L. robustum were tested for their inhibitory activities on FAS, α-glucosidase, and α-amylase as well as their antioxidant effects. The results of the bioactivity assays are listed in Table 5.

Plant Material
The fresh leaves of L. robustum were gathered from Yibin City, Sichuan Province, China, in April 2017, and confirmed by Guo-Min Liu (Kudingcha Research Institute, Hainan University, Haikou, China). A voucher sample (No. 201704lsh) was saved at the West China School of Pharmacy, Sichuan University, Chengdu, China.

Extraction and Isolation
The fresh leaves of L. robustum were turned and heated at 120 • C for 50 min and then crushed. The crushed leaves (7.0 kg) were extracted with 70% ethanol (28 L × 1) under reflux in a multifunction extractor for 2 h [4]. The ethanol extract was filtered and condensed in vacuo to acquire a paste (2.2 kg). The paste was dissolved with 3 L 95% ethanol, and then 3 L of purified water was added to deposit the chlorophyll. After percolation, the filtrate was concentrated in vacuo to obtain a residue (1.

Statistical Analyses
The statistical analyses were executed using GraphPad Prism 5.01. Every sample was tested in triplicate. The IC 50 value of a compound (the ultimate concentration of a compound needed to inhibit 50% of the enzyme activity or clear away 50% of the free radicals) was obtained by plotting the inhibition or scavenging percentage of every sample of the compound against its concentration. The results are expressed as the mean ± standard deviation (SD). The difference of the means between groups was analyzed by oneway analysis of variance (ANOVA) using the statistical package SPSS 25.0. The difference between groups was considered to be significant when p < 0.05.

Conclusions
In summary, nine novel compounds, including two hexenol glycosides (1a and 1b), two butenol glycosides (2a and 2b), and five sugar esters (3a, 3b, 4b, 5a, and 5b), together with seven known compounds (4a and 6-10), were isolated from the leaves of L. robustum and identified with spectroscopic methods (i.e., 1 H, 13 C NMR, 1 H-1 H COSY, HSQC, HMBC, and HRESIMS) and a chemical method. The biological assays showed that the FAS inhibitory activity of compound 2 (IC 50 4.10 ± 0.12 µM) was as strong as the positive control orlistat (IC 50 4.46 ± 0.13 µM); the α-glucosidase inhibitory activities of compounds 7 and 9 and the α-amylase inhibitory activities of compounds 1-10 were moderate; the DPPH radical scavenging effects of compound 6 (IC 50 46.66 ± 1.58 µM) were weaker than L-(+)-ascorbic acid (IC 50 13.66 ± 0.13 µM); the ABTS radical scavenging effects of compounds 1 and 10 (IC 50 3.41 ± 0.08~5.65 ± 0.19 µM) were more potent than the positive control L-(+)-ascorbic acid (IC 50 10.06 ± 0.19 µM), while the ABTS radical scavenging effects of compounds 3, 4, 7, and 9 (IC 50 8.78 ± 0.09~12.04 ± 0.08 µM) were as strong as L-(+)-ascorbic acid. Based on this work and previous studies [4,15,16], phenylethanoid, phenylmethanoid, monoterpenoid, hexenol, and butenol glycosides, together with sugar esters, are considered as the main active constituents of L. robustum for the prevention of diabetes and its complications. This study provides a theoretical foundation for the leaves of L. robustum as a functional tea to prevent diabetes and its complications. It is well known, however, that the effect of a compound in vitro is not necessarily equal to its actual effect in vivo. Therefore, further study should be performed to evaluate the activity of the isolates in vivo in the future.  Figure S5); Section S1: Determination of bioactivities; Section S2: 1 H NMR and 13 C NMR data of 4a and 6-10.