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.

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.

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.

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.

Zanthoxylum bungeanum seed oil inhibits RANKL-induced osteoclastogenesis by suppressing ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum bungeanum Maxim (ZBM), a traditional Chinese medicine, is traditionally used for osteoporosis treatment recorded in ancient Chinese medicine work Benjingshuzheng and reported to have the anti-bone loss activity in recent studies. However, the anti-osteoporotic activities of the seed of ZBM have not been elucidated yet. Our previous study found that Zanthoxylum bungeanum Maxim seed oil (ZBSO) was rich in polyunsaturated fatty acids (PUFAs), which were reported to prevent bone loss. Thus, we propose a hypothesis that ZBSO could be a potential natural resource for anti-bone loss. AIM OF THE STUDY: To investigate whether ZBSO could prevent bone loss by targeting osteoclastogenesis and investigate the potential mechanisms in receptor-activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. MATERIALS AND METHODS: RAW264.7 cells were treated with RANKL in the presence or absence of ZBSO. The effect of ZBSO on osteoclast differentiation and bone resorption activity of RAW264.7 cells were evaluated by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, and bone resorption assay. Differentially expression genes (DEGs) and relevant pathways of different cell groups were obtained from RNA sequencing and protein-protein interaction (PPI) network analysis followed by KEGG pathway enrichment analysis. The effect of ZBSO on the RANKL-induced cell cycle change was analyzed by flow cytometry assay, and the expression of genes and proteins related to the selected pathways was further verified by RT-qPCR and western blot analysis. RESULTS: The inhibitory effects of ZBSO on osteoclast differentiation and bone resorption activity in a dose-dependent manner were demonstrated by TRAP staining, F-actin ring staining, and bone resorption assay in RANKL-induced RAW264.7 cells. Osteoclast differentiation and cell cycle pathways were the most enriched pathways based on DEGs enrichment analysis among different cell groups. The reversion effect of ZBSO on the RANKL-induced RAW264.7 cell cycle arrest at the G1 phase was observed by flow cytometry assay. Western blot results showed that ZBSO markedly decreased RANKL-induced activation of ERK, as well as the phosphorylation of c-JUN and NFATc1 expression, and subsequently suppressed osteoclast-specific genes, such as Ctsk, Trap, and Dc-stamp. CONCLUSIONS: ZBSO exhibited an inhibitory effect on osteoclastogenesis via suppressing the ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest induced by RANKL, suggesting that ZBSO may serve as a promising agent for anti-bone loss.

In Vitro and In Vivo Evaluation of Antitumor Activity of Ligustrum robustum, A Chinese Herbal Tea.

OBJECTIVE: To examine the effect of the aqueous extract of Ligustrum robustum on tumor growth in vitro and in vivo and explore the possible molecular mechanisms. METHODS: In in vitro study, cell viabilities of human cervical carcinoma cells (HeLa), human breast cancer cells (MCF-7), human prostate cancer cells (PC-3), human hepatoma cells (7721) and human colon carcinoma cells (SW480) were evaluated with cell counting kit-8. For L. robustum-treated Hela cells, early or late apoptosis were evaluated by annexin V/PI staining. Mitochondrial membrane potential was measured by staining cells with JC-1. Apoptosis was monitored by nuclear morphology based on chromatin condensation and fragmentation by 4',6-diamidino-2-phenylinole (DAPI) staining. Caspase-3 and -8 activity levels were measured by a colorimetric assay. In vivo, to evaluate the possible mechanism of L. robustum-mediated antitumor effect, nude mouse xenograft study was also conducted. RESULTS: In in vitro study, L. robustum was found to be toxic to HeLa, MCF-7, PC-3, 7721, SW480, with an half maximal inhibitory concentration value of 2-5 mg/mL (P<0.05). Moreover, externalization of phosphatidylserine, loss of mitochondrial membrane potential, DNA fragmentation and activation of caspase-3 and -8 were detected in L. robustum-treated Hela cells. Using a nude mouse model bearing Hela xenografts, we found that L. robustum reduced tumor volume and tumor weight (P<0.05), but had no effect on body weight and histological damage of important organs. Intraperitoneal injection of L. robustum caused a significant reduction in serum aspartate transaminase and alanine transaminase levels (P<0.05). Furthermore, cleaved caspase-3-positive and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells were observed in L. robustum-treated tumor tissues. CONCLUSIONS: L. robustum inhibits tumor cell growth both in vitro and in vivo by inducing apoptosis in a caspase-dependent way without apparent hepatic toxicity and histological damage, which may offer partial scientific support for the ethnopharmacological claims of L. robustum as a herbal tea for its antitumor activity.

The antiosteoporosis effect of icariin in ovariectomized rats: a systematic review and meta-analysis.

Herba Epimedii (Yinyanghuo or Horny Goat Weed) is a traditional Chinese medicine widely used in treating osteoporosis. As the major active component, icariin is intensively investigated in the prevention and treatment of osteoporosis in ovariectomized rats. However, misleading conclusions can be generated in animal studies with various experimental designs. Therefore, a systematic review and meta-analysis was performed to evaluate the efficacy of icariin against osteoporosis in ovariectomized rats. PubMed, EMBASE and Chinese National Knowledge Infrastructure databases were searched to identify studies of icariin in ovariectomized rats. Two independent authors selected and reviewed the publications. Data were pooled using a DerSimonian and Laird random-effects model. The results demonstrated that ovariectomized rats treated with icariin had significantly higher bone mineral density (femur and lumbar spine) and lower bone turnover markers (serum alkaline phosphatase and osteocalcin) compared with the ovariectomized control group. For bone histomorphometric parameters, the percentages of trabecular area and trabecular thickness were significantly higher while the trabecular separation was significantly lower in the ovariectomized rats treated with icariin. Based on these results, the presnet study suggested that icariin might possess substantial antiosteoporosis effect in ovariectomized rats. Safety studies and large randomized clinical trials are needed to further support possible clinical applications of icariin in postmenopausal women with osteoporosis.

Effects of Ligustrum robustum on gut microbes and obesity in rats.

AIM: To investigate the anti-obesity and antibacterial effects of Ligustrum robustum (L. robustum) in vivo and in vitro and its possible mechanisms. METHODS: The effects of L. robustum aqueous extract (LR) on various gut bacteria in vitro were evaluated. The effects of LR on high-fat diet-fed (HFD) rats in vivo were also assessed. Culture methods, quantitative polymerase chain reaction, and terminal-restriction fragment length polymorphism were used to analyze the effects of LR on gut bacteria. Biochemical tests were also performed to detect the changes in obesity-related indicators after LR treatment. RESULTS: LR treatment lowered adipose weight and decreased Lee's index, blood glucose, total cholesterol, and lipid in the tested groups relative to control (P < 0.05). To determine the reasons for these changes, we assessed the potential bacteriostatic and bactericidal effects of LR on specific bacterial species in vitro. LR affected the richness, diversity, and evenness of gut bacteria, increased fecal Lactobacillus, and decreased Enterococci in HFD rats (P < 0.05). CONCLUSION: L. robustum may be a safe and effective food for weight loss and obesity control, and the effects of L. robustum might be mediated by the regulation of gut bacteria.