Purification of triterpenoid saponins and 25R/25S-inokosterone from Achyranthes bidentata Bl. by high-speed countercurrent chromatography coupled silver nitrate coordination.

An effective method by high-speed countercurrent chromatography coordinated with silver nitrate for the preparative separation of sterones and triterpenoid saponins from Achyranthes bidentata Blume was developed. Methyl tert-butyl ether/n-butanol/acetonitrile/water (4:2:3:8, v/v/v/v) was selected for 20-hydroxyecdysone (compound 1), chikusetsusaponin IVa methyl ester (compound 4), 2'-glycan-11-keto-pigmented saponin V (compound 5), as well as a pair of isomers of 25S-inokosterone (compound 2) and 25R-inokosterone (compound 3), which were further purified by silver nitrate coordinated high-speed countercurrent chromatography. What is more, dichloromethane/methanol/isopropanol/water (6:6:1:4, v/v/v/v) was applied for calenduloside E (compound 6), 3ß-[(O-ß-d-glucuronopyranosyl)-oxy]-oleana-11,13-dien-28-oic acid (compound 7), zingibroside R1 (compound 8) and chikusetsusaponin IVa (compound 9). Adding Ag+ to the solvent system resulted in unique selectivity for 25R/25S isomers of inokosterone, which increased the complexing capability and stability of Ag+ coordinated 25S-inokosterone, as well as the α value between them. These results were further confirmed by the computational calculation of geometry optimization and frontier molecular orbitals assay. Comprehensive mass spectrometry and nuclear magnetic resonance analysis demonstrated the structures of the obtained compounds.

[A new sesquiterpenoid from Lindera aggregata].

The chemical composition of the aqueous part of the extract from Lindera aggregata was studied, which was separated and purified by the macroporous resin column chromatography, MCI medium pressure column chromatography, semi-preparative high-performance liquid phase and other methods. The structures of the compounds were identified according to physical and chemical properties and spectroscopic data. Thirteen compounds were isolated and identified from the aqueous extracts, which were identified as(1S,3R,5R,6R,8S,10S)-epi-lindenanolide H(1), tachioside(2), lindenanolide H(3), leonuriside A(4), 3,4-dihydroxyphenyl ethyl ß-D-glucopyranoside(5), 3,4,5-trimethoxyphenol-1-O-6-α-L-rhamnose-(1→6)-O-ß-D-glucoside(6), 5-hydroxymethylfurfural(7),(+)-lyoniresin-4-yl-ß-D-glucopyranoside(8), lyoniside(9), norboldine(10), norisopordine(11), boldine(12), reticuline(13). Among them, compound 1 was a new one, and compounds 2, 5, 6, 8, 9 were obtained from L. aggregata for the first time. The inflammatory model was induced by lipopolysaccharide(LPS) in the RAW264.7 cells. The results showed that compounds 1, 8, 10 and 12 had significant anti-inflammatory activity.

Efficient discovery of active isolates from Dioscorea spongiosa by the combination of bioassay-guided macroporous resin column chromatography and high-speed counter-current chromatography.

In the present study, twelve compounds from Dioscorea spongiosa were successfully purified by an efficient technique combined bioassay-guided fractionation macroporous resin column chromatography (MRCC) pretreatment and high-speed counter-current chromatography (HSCCC) separation for the first time. Then, D101 MRCC was used to fractionate the crude extract into five parts, which further applied the bioassay-guided fractionation strategy to screen the active fractions of 2 and 4. As for the separation, 200 mg Fr.2 was purified by HSCCC using EtOAc/n-BuOH/H2 O (2:2:3, v/v), leading to annulatomarin (1), dioscoresides C (2), diosniponol C (3), methyl protodioscin (4), pseudoprotodioscin (5), protogracillin (6), as well as 200 mg Fr.4 yielding montroumarin (7), dioscorone A (8), diosniponol D (9), protodioscin (10), gracillin (11), and dioscin (12) using CH2 Cl2 /MeOH/H2 O (3:3:2, v/v) with the purities over 95.0%. Finally, the isolates were assayed for their anti-inflammatory, urico-lowering, and anti-diabetic activities in vitro, which indicated that the steroidal saponins of 5, 6, and 11 showed all these three activities.

Secondary metabolites from Scindapsus officinalis (Roxb.) Schott. with in vitro antidiabetic activities.

One new phenolic cyclobutantetraol ester united chromone glycoside (1), one new amide (2), and three new phenyl ethanol glycosides (3-5) were obtained from the water extract of Scindapsus officinalis (Roxb.) Schott, in which compound 1 was the first reported structure incorporating the phenolic cyclobutantetraol ester and chromone via the glucose phenolic metabolites in nature. Structures of the isolated compounds, including absolute configurations, were elucidated according to the analysis of HRESIMS, NMR, ECD and BLYP/6-31G* geometry optimization calculations of 13C NMR data. All isolates (1-5) were evaluated for the antidiabetic activity by the insulin resistance (IR) model and anti-inflammatory activity against NO production in vitro. Compounds 1-3 showed strong antidiabetic activities, greatly promoting the glucose consumption in the insulin resistance HepG2 cells compared with the model group, however, 1-5 showed weak anti-inflammatory activities.

CO2 improves the anaerobic biodegradation intensity and selectivity of heterocyclic hydrocarbons in heavy oil.

Heterocyclic hydrocarbons pollution generated by oil spills and oilfield wastewater discharges threatens the ecological environment and human health. Here we described a strategy that combines the greenhouse gas CO2 reduction with microbial remediation. In the presence of nitrate, CO2 can improve the biodegradation efficiency of the resins and asphaltenes in heavy oil, particularly the biodegradation selectivity of the polar heterocyclic compounds by the newly isolated Klebsiella michiganensis. This strain encoded 80 genes for the xenobiotic biodegradation and metabolism, and can efficiently utilize CO2 when degrading heavy oil. The total abundance of resins and asphaltenes decreased significantly with CO2, from 40.816% to 26.909%, to 28.873% with O2, and to 36.985% with N2. The transcripts per million (TPM) value of accA gene was 57.81 under CO2 condition, while respectively 8.86 and 21.23 under O2 and N2 conditions. Under CO2 condition, the total relative percentage of N1-type heterocyclic compounds was selectively decreased from 32.25% to 22.78%, resulting in the heavy oil viscosity decreased by 46.29%. These results demonstrated a novel anaerobic degradation mechanism that CO2 can promote the anaerobic biodegradation of heterocyclic hydrocarbons in heavy oil, which provides a promising biotreatment technology for the oil-contaminated water.

[Effects of short-segment pedicle screw internal fixation surgery combined with hyperbaric oxygen treatment for acute spinal injury on the morphology and function of the spine].

OBJECTIVE: To explore effect of short-segment pedicle screw internal fixation combined with hyperbaric oxygen in treating acute spinal fractures and its influence on recovery of spinal nerve function. METHODS: A total of 96 patients with acute spinal fracture admitted from February 2017 to March 2020 were divided into combined group and control group, with 48 cases in each group. Both groups were treated with short-segment pedicle screw internal fixation. The combined group was given hyperbaric oxygen after surgery. The operation time, surgical blood loss, incision length and other general operation conditions between two groups were recorded. The differences in spinal morphology and function, Ameraican Spinal Injury Assiciation(ASIA) neurological function grade, serum inflammatory factors and ability of daily living activities were observed before and after surgery. RESULTS: There was no significant difference in operation time, surgical blood loss, and incision length between combined group and control group(P>0.05). There were no significant differences in anterior height ratio and Cobb angle between two groups before surgery, 1 week and 6 months after surgery(P>0.05). The height ratio of anterior margin of the injured spine was significantly improved in both groups at 1 week and 6 months after surgery compared with preoperative period (P<0.05), and Cobb angle was significantly reduced in both groups compared with preoperative period (P<0.05). There was no statistically significant difference in serum interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α(TNF-α) levels between two groups at 1 d after surgery(P>0.05);the serum IL-6, IL-8, and TNF-α levels of combined group were lower than those of control group at 1 week after surgery (P<0.05). At 6 months after surgery, ASIA neurological function grade of combined group was C grade in 2 cases, D grade in 23 cases, E grade in 22 cases. In control group, 7 cases was grade C, 26 cases was grade D, 13 cases was grade E, and the difference between two groups was statistically significant(P<0.05). The Barthel score of combined group was higher than that of control group at 1 month and 3 months after surgery, and the difference was statistically significant (P<0.05);at 6 months after surgery, there was no significant difference in Barthel score between two groups(P>0.05). CONCLUSION: Short-segment pedicle screw internal fixation combined with hyperbaric oxygen for the treatment of acute spinal fractures is beneficial to the recovery of spinal nerve function after surgery, and has a certain effect on the early improvement of the patients' activities of daily living.

Chemical constituents from the bulbs of Lilium davidii var. unicolor and anti-insomnia effect.

In this work, twenty-five water-soluble constituents were isolated from the bulbs of Lilium davidii var. unicolor, including two new compounds termed liliumtides A and B (1-2), and seventeen known compounds (3-4, 6-13, 15, 17, 21-25) isolated from the genus of Lilium for the first time. Their structures were established using high-resolution electrospray ionization mass spectroscopy and 1D and 2D nuclear magnetic resonance data. To confirm furtherly the absolute configuration of liliumtide A, and to accumulate enough sample to study the anti-insomnia effect, a total synthesis for liliumtide A was achieved by four steps. The pentobarbital-induced sleeping time test showed that compared with the blank control group, the liliumtide A decreased sleep latency and significantly increased the sleep time. These results suggested that liliumtide A could be investigated as a natural anti-insomnia lead compound in the pharmaceutical and food industries.

Transcriptomic Analysis, Motility and Biofilm Formation Characteristics of Salmonella typhimurium Exposed to Benzyl Isothiocyanate Treatment.

Salmonella typhimurium (S. typhimurium) is a common foodborne pathogen that not only causes diseases and contaminates food, but also causes considerable economic losses. Therefore, it is necessary to find effective and feasible methods to control S. typhimurium. In this study, changes in S. typhimurium after treatment with benzyl isothiocyanate (BITC) were detected by transcriptomics to explore the antibacterial effect of BITC at subinhibitory concentration. The results showed that, in contrast to the control group (SC), the BITC-treated group (SQ_BITC) had 197 differentially expressed genes (DEGs), of which 115 were downregulated and 82 were upregulated. We screened out eight significantly downregulated virulence-related genes and verified gene expression by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). We also selected motility and biofilm formation to observe the effects of BITC on the other virulence related factors of S. typhimurium. The results showed that both swimming and swarming were significantly inhibited. BITC also had a significant inhibitory effect on biofilm formation, and showed an effect on bacterial morphology. These results will be helpful for understanding the mechanism of the antibacterial action of BITC against S. typhimurium and other foodborne pathogens.

Lycium Barbarum Polysaccharides Alleviates Oxidative Damage Induced by H2O2 Through Down-Regulating MicroRNA-194 in PC-12 and SH-SY5Y Cells.

BACKGROUND/AIMS: Currently, scientists attempt to improve outcome of spinal cord injury (SCI) via reducing secondary injury during SCI. Oxidative stress is critical for pathophysiology of secondary damage, thus we mainly focused on the anti-oxidant effects of Lycium barbarum polysaccharides (LBPs) on PC-12 and SH-SY5Y cells as well as the underlying mechanisms. METHODS: Oxidative stress was induced by H2O2 stimulation. Effects of LBPs on cell viability, apoptosis, and expression of proteins associated with apoptosis and autophagy in H2O2-induced cells were assessed by CCK-8 assay, flow cytometry assay and Western blot analysis, respectively. Then, expression of miR-194 was determined by qRT-PCR. Expression of miR-194 was dysregulated, and whether LBPs affected H2O2-treated cells through modulating miR-194 was verified. The expression of key kinases in the PI3K/AKT pathway and the intracellular levels of ROS and NO were testified by Western blot analysis and flow cytometry with fluorescent probes. RESULTS: H2O2-induced decrease of cell viability and increases of apoptosis and autophagy in PC-12 cells were mitigated by LBPs treatment. Next, we found that miR-194 expression was both down-regulated by LBPs treatment in PC-12 and SH-SY5Y cells. More experiments consolidated that influence of LBPs on H2O2-treated cells was reversed by miR-194 overexpression while was augmented by miR-194 inhibition. LBPs elevated the phosphorylated levels of PI3K and AKT and reduced levels of ROS and NO through miR-194. CONCLUSION: LBPs alleviated H2O2-induced decrease of cell viability, and increase of apoptosis and autophagy through down-regulating miR-194. Moreover, LBPs activated the PI3K/AKT pathway and reduced oxidative stress through miR-194.

Effectiveness of meibomian gland massage combined with topical levofloxacin against ocular surface flora in patients before penetrating ocular surgery.

OBJECTIVE: To investigate the bacterial profile in the conjunctiva and meibomian glands in patients before penetrating ocular surgeries, and to compare the anti-bacterial efficacy of 0.5% levofloxacin and its combination with meibomian gland massage. DESIGN: Hospital-based, case-control study. PARTICIPANTS: Two hundred and twenty-six eyes from 226 patients with non-infective ocular diseases and scheduled for penetrating ocular surgeries. METHODS: Tested eyes were administered topical 0.5% levofloxacin (4 times daily) for 2 days. Among them, 91 eyes received meibomian gland massage before levofloxacin application. Samples from the conjunctival sac and meibomian glands were collected for aerobic and anaerobic cultures. MAIN OUTCOME MEASURES: Culture-positivity and bacterial strains. RESULTS: Before treatment, aerobes and anaerobes were cultured from 38.5% and 11.0% of the conjunctival samples respectively, compared with 38.5% and 8.8% in the meibomian secretions respectively. Staphylococcus epidermidis and Propionibacterium acnes were the commonest isolated aerobe and anaerobe. Two-day application of levofloxacin reduced aerobic growth to 29.6% in the conjunctiva and 19.3% in the meibomian glands. It had no effect on the anaerobes in these regions (13.3% in the conjunctiva and 10.4% in the meibomian glands). Combined levofloxacin with meibomian gland massage further reduced aerobic growth to 19.8% in the conjunctiva and 11.0% in the meibomian glands. It also drastically decreased anaerobic growth in the meibomian glands (1.1%). CONCLUSIONS: Meibomian glands carrying considerable bacteria should be considered as a potential source of contamination in ocular surgery. Meibomian gland massage shows additional anti-bacterial effects to topical levofloxacin and could be recommended as a complementary preoperative prophylaxis.