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.

[Epidemiological Characteristics and Drug Resistance of Microbial Pathogens of Patients With Silicosis Combined With Pulmonary Infection].

Objective: To investigate the epidemiological characteristics of patients with silicosis combined with pulmonary infection in recent years, to study the distribution and the drug susceptibility of fungal and bacterial pathogens in their sputum samples, and to provide references for the prevention and treatment of silicosis and the appropriate drug use. Methods: The clinical data and drug sensitivity test results of patients with silicosis combined with pulmonary infection diagnosed at the Department of Occupational Diseases, West China Fourth Hospital, Sichuan University were retrospectively analyzed. Results: A total of 318 patients with silicosis combined with pulmonary infection who received treatment between January 2017 and December 2020 were enrolled. All the patients had positive microorganism test results. All participants were male. Their median age at the time of onset was 51.00 years and the median time of exposure to silica dust at work was 12.40 years. They worked mostly in construction, non-ferrous metal mining, and coal mining. The main types of work they did were pneumatic drilling, coal digging, and mining. The positive detection rates for the first, second and third phases of silicosis were 27.54%, 28.32%, and 32.97%, respectively. A total of 341 strains of fungal and bacterial pathogens were isolated, of which, 54.1% were fungi, including 114 strains (35.8%) of Candida albicans, and 53.1% were bacteria, including 168 strains (52.8%) of gram-negative bacteria, most of which being Klebsiella pneumoniae (30.2%). There was only 1 strain (0.3%) of gram-positive bacteria, namely Staphylococcus hemolyticus. Gram-negative bacilli were most resistant to ampicillin and highly sensitive to penicillin G and ofloxacin. Conclusion: Among patients with silicosis combined with pulmonary infection, the incidence of pulmonary infection increases along with the progress of silicosis. Microorganism analysis reveals high detection rates for fungi and the bacteria detected are predominantly gram-negative bacteria. The overall prospect for drug resistance rate was not optimistic.

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.

Study on the Salivary Microbial Alteration of Men With Head and Neck Cancer and Its Relationship With Symptoms in Southwest China.

This study explored the association between oral microbes and head and neck cancer (HNC) as well as symptoms related to patients with HNC before surgical treatment. Fifty-six patients with HNC and 64 matched healthy controls were recruited from West China hospital in Southwest China. The demographic, clinical, and symptom data were collected. Salivary samples were collected to determine the microbial characteristics using 16S rRNA gene sequencing. Patients with HNC presented increased Capnocytophaga abundances. The oral microbial markers as Capnocytophaga (area under the curve=0.81) achieved a high classification power between the HNC patients and healthy controls. Moreover, using Capnocytophaga in conjunction with symptom of voice/speech difficulty achieved an overall predicting accuracy of 92.5% comparing with using Capnocytophaga alone (79.2% accuracy) in distinguishing the HNC patients from healthy controls. Salivary microbial profiles and HNC symptoms may be potential biomarkers for HNC screening.

[Identification of the Strain Which Highly Produces Protease and ß-D-glucosidase Isolated from Shuidouchi Produced in Sichuan and Evaluating Its Ability of Producing Protease].

OBJECTIVE: To select and identify the bacterium which highly produces protease and ß-D-glucosidase from 72 strains of Shuidouchi from Sichuan, and to provide evidence for further research on its nutritional value and fermentation strain exploiting. METHODS: Casein degradation test and pNPG chemical test were applied respectively to detect the capacity to produce protease and ß-D-glucosidase of each strain. Characteristics of morphology, biochemistry, 16S rRNA and MALDI-TOF-MS were used to identify the fermentation strain, which genetic stability, curves of growth and enzyme producing were also obtained. RESULTS: The strain with the highest enzyme activity of ß-D-glucosidase (0.084 U/L) among the top 10 strains for producing protease was selected as the fermentation strain and was identified as Bacillus subtilis, which curves of growth and enzyme producing conformed as well. The result of genetic stability showed that capacity of enzyme producing was stable until the 10th generation. CONCLUSIONS: The fermentation strain which highly produced protease and ß-D-glucosidase was selected from 72 strains of shuidouchi from Sichuan and was identified as Bacillus subtilis.

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.

Molecular characterization of enterovirus 71 sibling strains for thermal adaption in Vero cells with adaptive laboratory evolution.

Enterovirus 71 is the main pathogen that causes severe and fatal hand-foot-mouth-disease (HFMD) cases. As the enterovirus virus mutation has implications for pathogenesis, vaccine development, antiviral therapy, and epidemiological disease management of the virus. In this study, we investigated the variations of enterovirus 71 in thermal adaption, using the method of adaptive laboratory evolution. The sibling virus strains were isolated from a 2-year-old severe case of HFMD (#100) and her symptomless close contact (#101). Both strains were cultured in Vero cells by serial passage of 36 generations at the temperatures of 28.0 °C, 33.0 °C and 39.5 °C to construct adaptive lineages. According to the comparative analysis of phenotypes between adapted strains and parental strains, differences in growth rate were observed in the sibling lineages and a larger plaque was found mainly in the hot adapted strains for lineage #101. Two sets of adaptive strains from six time points (parental, 12th 17th, 31st, 35th passage and endpoint) were sequenced and analyzed by both Sanger sequencing and Next Generation Sequencing. Several variations in most coding genes and one reverse mutation in 5'UTR was observed, along with the identity of 99.8% for complete genome for both lineages. Notably, thermal specific non-synonymous mutations were found in the gene of VP1\VP3\3A\2C\3C. Moreover, the concurrent mutations A292G, A434G and A355C/T of sibling lineages in VP1 showed quantificational trace with distinguishing patterns for different temperatures, which were suspected to be the thermo-sensitive mutation hotspots. These results highlight the possible rules of thermal adaption in enterovirus 71, produce a novel picture of genome evolution of the virus, and shed light on viral variation and evolution.

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.

Gut bacteria alteration in obese people and its relationship with gene polymorphism.

AIM: To investigate the differences in cultivable gut bacteria and peroxisome proliferator-activated receptor γ2 (PPAR-γ2) gene Pro12Ala variation in obese and normal-weight Chinese people. METHODS: Using culture methods, the amounts of Escherichia coli, Enterococci, Bacteroides, Lactobacilli, Bifidobacteria and Clostridium perfringens (C. perfringens) in the feces of 52 obese participants [body mass index (BMI): ≥ 28 kg/m(2)] and 52 participants of normal-weight (BMI: 18.5-24 kg/m(2)) were obtained. Study participants completed comprehensive questionnaires and underwent clinical laboratory tests. The polymerase chain reaction-restriction fragment length polymorphism (PCR-PFLP) assay was used to analyze PPAR-γ2 gene Pro12Ala variation. RESULTS: The obese group exhibited a lower amount of C. perfringens (6.54 ± 0.65 vs 6.94 ± 0.57, P = 0.001) and Bacteroides (9.81 ± 0.58 vs 10.06 ± 0.39, P = 0.012) than their normal-weight counterparts. No major differences were observed in Pro12Ala genotype distribution between the two groups; however, obese individuals with a Pro/Ala genotype had a significantly lower level of Bacteroides (9.45 ± 0.62 vs 9.93 ± 0.51, P = 0.027) than those with a Pro/Pro genotype. In addition, the obese group demonstrated a higher stool frequency (U = 975, P < 0.001) and a looser stool (U = 1062, P = 0.015) than the normal-weight group. CONCLUSION: Our results indicated interactions among cultivable gut flora, host genetic factors and obese phenotype and this might be helpful for obesity prevention.

[Study on polymorphism of UCP2 gene in Chengdu simple obesity and normal-weight people and a preliminary investigation of its relationship with gut bacteria].

OBJECTIVE: To study on the polymorphism of UCP2 gene in Chengdu simple obesity and normal-weight people and to initially investigate the relationship between UCP2 Ala55Val variation and gut bacteria. METHODS: PCR-PFLP was applied to determine the genotypes of Ala55Val variant in the UCP2 gene of 86 Chengdu people (the simple obesity group, 43 subjects; the normal-weight group, 43 subjects). And six kinds of gut bacteria among different genotypes in different groups were analyzed. RESULTS: Both the simple obesity and the normal-weight group had the Ala55Val variants of Ala/Ala, Val/Val and Ala/Val in the UCP2 gene, and the Ala55Val genotype distributions between the two groups was significantly different (chi2=11.97, P< 0.05). The allelic mutation frequency in the simple obesity group was higher than that of the normal-weight group (chi2=10.06, P<0.05). However, no significant difference was observed in the population of six gut bacteria among different genotypes in different groups (P>0.05). CONCLUSION: The UCP2 gene mutation might be a risk factor of obesity in Chengdu area. However, this gene mutation may not be an impact factor on the alternation of gut bacteria.