Screening of anti-cancer compounds from Vaccariae Semen by lung cancer A549 cell fishing and UHPLC-LTQ Orbitrap MS.

Vaccariae Semen, derived from the dried ripe seed of Vaccaria segetalis (Neck.) Garcke, has various therapeutic characteristics in traditional Chinese medicine (TCM), containing promoting blood circulation and unblocking meridians. It exhibits significant anti-cancer activity and is therapeutically utilized to treat and reduce chemotherapy adverse effects in cancer patients, notably those with lung cancer. However, the active ingredients responsible for its anti-lung cancer efficacy remain unknown. In this study, we used A549 cell fishing in conjunction with UHPLC-LTQ Orbitrap MS to screen for anti-lung cancer active components in Vaccariae Semen. The cell counting Kit-8 (CCK-8) assay revealed that the n-butanol extract substantially reduced A549 cell growth. Through the cell fishing assay, we found 14 A549 cell-binding compounds in the n-butanol extract, all of which were identified as triterpenoid saponins. The total saponins of Vaccariae Semen were subsequently purified using macroporous adsorption resin (MAR), and they showed a significant inhibitory effect on the proliferation of A549 lung cancer cells, as well as alterations in cell morphology, apoptosis, and fragmentation. In conclusion, saponins were discovered as the key active components responsible for the anti-lung cancer activity of Vaccariae Semen.

Bixasteroid, a new compound from the fruits of Bixa orellana and its anti-inflammatory activity.

Bixasteroid (1), one new steroid together with five known compounds (2-6), were isolated from the ethyl acetate fraction of ethanol extract of Bixa orellana fruits. All of these known compounds were isolated from the plant for the first time. Their structures were elucidated on the basis of spectroscopic analysis, and the absolute configuration of compound 1 was determined by X-ray crystallographic data analysis as well as by the quantum chemical ECD calculations. All the isolated compounds were tested for their anti-inflammatory activities. Compounds 1 and 2 showed inhibiting NO release activities in LPS-induced RAW 264.7 macrophages with the IC50 values of 4.72 ± 0.28 and 5.48 ± 1.48 µM, respectively.

New phenylpropanoids from the fruits of Xanthium sibiricum and their anti-inflammatory activity.

Seven new phenylpropanoids, including two pairs of enantiomers of 8-O-4'-type neolignans, named (±)-xanthiifructins A-B (1a/1b-2a/2b), a pair of simple phenylpropanoid derivatives (±)-xanthiifructins C (3a/3b), and a racemic phenylpropanoid derivative xanthiifructin D (4), together with four known analogues (5-8) were isolated from the fruits of Xanthium sibiricum. Racemic xanthiifructins A-C were separated on chiral HPLC columns. Their structures were elucidated by comprehensive spectroscopic data analysis and comparison with the literatures. The anti-inflammatory and cytotoxic activities were evaluated for all isolates. Among them, (-)-xanthiifructin C (3b) exhibited potent inhibitory effect against nitric oxide production in lipopolysaccharide-activated RAW 264.7 mouse macrophage cells with an IC50 value of 9.94 ± 0.57 µM. All compounds obviously were inactive for three human tumor cell lines (MCF-7, HepG2, and A549) with IC50 values much more than 10 µM.

One pair of new enantiomeric trinorsesquiterpenes from the aerial parts of Justicia gendarussa.

A pair of new enantiomeric trinorsesquiterpenes, (+)-genpenterpene A (1a) and (-)-genpenterpene A (1b), together with seven known compounds (2-8), were isolated from the aerial parts of Justicia gendarussa Burm.f.. All of these known compounds were isolated from this plant for the first time. Racemic genpenterpene A was separated by chiral HPLC column. Their chemical structures were elucidated based on extensive spectroscopic analysis, single crystal X-ray diffraction, and ECD calculations. (+)-genpenterpene A (1a) exhibited potent inhibitory effect against nitric oxide production in lipopolysaccharide-activated RAW 264.7 mouse macrophage cells with an IC50 value of 9.54 ± 1.02 µM.

New compounds from the aerial parts of Justicia gendarussa Burm.f. and their antioxidant and anti-inflammatory activities.

Three new compounds (1-3), together with six known compounds (4-9), were isolated from the ethyl acetate fraction of ethanol extract of the aerial parts of Justicia gendarussa Burm.f. All of these known compounds were isolated from the plant for the first time. Their structures including absolute configurations were elucidated on the basis of HR-ESI-MS, NMR spectroscopic analyses, single crystal X-ray diffraction and comparison with the literatures. Compounds 1-9 were tested for their antioxidant and anti-inflammatory activities. Compounds 5 and 8 showed the antioxidant activities with IC50 values of 28.61 ± 1.56 and 22.55 ± 1.38 µM, respectively. Compounds 2 and 3 showed anti-inflammatory activity in LPS-induced RAW 264.7 macrophages with the IC50 values of 20.95 ± 1.11 and 16.50 ± 1.04 µM, respectively.

[Chemical constituents from Xanthii Fructus].

This study was carried out to investigate the chemical constituents from Xanthii Fructus(the fruits of Xanthium sibiricum). The compounds were separated and purified by silica gel column chromatography, Sephadex LH-20 and ODS chromatography and semi-preparative HPLC. Base on HR-ESI-MS, NMR and other spectral data, their structures were identified. The anti-inflammatory activity of the isolated compounds was evaluated by lipopolysaccharide(LPS)-induced macrophage RAW264.7 as a screening model. A total of twenty-one compounds were isolated from the EtOAc fraction of 95% ethanol extract and identified as uracil(1), thymine(2), uridine(3), indole-3-carbaldehyde(4), indole-3-carboxylic acid(5), 2'-O-methyluridine(6), guanosine(7), 2,4(1H,3H)-quinazolinedione(8), 3-hydroxy-3-(2-hydroxyethyl)indolin-2-one(9), nicotinamide(10), N-acetyl-L-phenylalaninol(11), heliolactam(12), terresoxazine(13), caudatin(14), qingyangshengenin(15), caudatin-3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside(16), caudatin-3-O-ß-D-cymaropyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside(17), caudatin-3-O-α-L-cymaropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-α-L-cymaropyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranoside(18), qinyangshengenin-3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranoside(19), qinyangshengenin-3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-digitoxopyranoside(20), rostratamine-3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside(21). Compounds 5-21 are obtained from genus Xanthium for the first time. Compounds 12 and 13 indirectly exhibited anti-inflammatory activity by suppressing LPS-induced NO production in RAW264.7 cells with IC_(50) values of(15.45±0.56) and(20.14±0.78) µmol·L~(-1), respectively.

Bioactive sulfur-containing compounds from Xanthium sibiricum, including a revision of the structure of xanthiazinone.

Seven previously undescribed sulfur-containing compounds, (+)- and (-)-xanthiazinone A, (+)- and (-)-xanthiazinone B, (+)- and (-)-xanthiazinone C and xanthiazinone D, and four known thiazinedione derivatives, together with three thiophene derivatives were isolated from the fruits of Xanthium sibiricum. Racemic xanthiazinones A-C were separated by chiral HPLC columns. Their chemical structures were elucidated based on extensive spectroscopic analyses, ECD calculations, and single crystal X-ray diffractions. The X-ray crystallographic analyses for xanthiazinones A-C represent the first example described for the structure elucidation of the thiazinedione with the five-membered lactone ring attached via an oxygen atom. Accordingly, the previously proposed structure for xanthiazinone was revised. The anti-inflammatory and cytotoxic activities were evaluated for all the isolated compounds. (+)-xanthiazinone B and 2-hydroxy-xanthiazone exhibited potent inhibitory effects against nitric oxide production in lipopolysaccharide-activated RAW 264.7 mouse macrophage cells with IC50 values of 8.75 and 10.90 µM, respectively. All compounds obviously were inactive for three human tumor cell lines (HepG2, MCF-7, and A549) with IC50 values more than 10 µM.

Responses of Labile Organic Nitrogen Fractions and Enzyme Activities in eroded Mollisols After 8-year Manure Amendment.

Soil erosion will cause a degradation in soil nitrogen supplying capacity (SNSC) and manure amendment is an effective way to restored eroded soils. Both labile fractions of soil organic N (SON) and N transformation enzymes are indicators for SNSC, but the effect of manure amendments on labile SON fractions and the relationship between labile SON fractions and enzyme activities remains unclear. In this study, five degrees of erosion were simulated in Mollisols (removal of 0, 5, 10, 20 and 30 cm of topsoil) to analyse the changes in labile SON fractions and nitrogen transformation enzyme activities after 8-year manure amendment. We found that soil total N (TN), labile SON fractions and enzyme activities all increased after manure amendments. The largest labile SON fraction was particle organic nitrogen (POM-N) and the second was light fraction organic nitrogen (LFOM-N), which accounted >60% for TN in total. Correlation analysis showed that both urease and protease activities were significantly correlated with POM-N, LFOM-N, microbial biomass N and dissolvable organic N, indicating that both urease and protease activities can be used to predict labile SON pools and enzyme activities worked similarly in indicating SNSC with labile SON fractions. Altogether, 8-year manure amendment could recover SNSC of lightly eroded Mollisols to natural levels, i.e. erosion depths at 5 cm and 10 cm; however, it is not able to recover SNSC in Mollisols suffering severe erosion.

[Clinical Trials for Treatment of Stroke Patients with Dysphagia by Vitalstim Electroacupuncture Combined with Swallowing Rehabilitation Training].

OBJECTIVE: To observe the clinical effect of vitalstim electroacupuncture (EA) combined with swallowing rehabilitation training in the treatment of stroke patients with dysphagia. METHODS: A total of 80 stroke patients with dysphagia were randomized into treatment and control groups (n=40 in each group). Patients of the control group were treated by regular medication for anti-platelet aggregation and anti-coagulation, lipid-lowering, neuroprotection, blood glucose control and blood pressure control, etc. and swallowing function rehabilitation training, and those of the treatment group treated by EA stimulation of Fengchi (GB 20), Jinjin (EX-HN 12) and Yuye (EX-HN 13) with a Vitalstim Electrostimulator and manual acupuncture stimulation of Lianquan (CV 23), Tiantu (CV 22) in combination with regular medication plus swallowing function training as those mentioned in the control group. The EA and manual acupuncture stimulation treatment was conducted once daily, 6 times a week and 4 weeks altogether. The therapeutic effect was assessed by using Kubota swallowing ability test (6 levels), dysphagia subscale (0-6 scores) of the neurological deficit degrees, videofluorography (VFG) assessment (markedly effective, effective and invalid, for evaluating the function and symmetry state of the swallowing movements), and the MOS Item Short Form Health Survey (SF-36, 8 minor items of two major aspects in physiological function, mental health, emotional function, social function and overall health) for assessing the patients' daily-life quality. RESULTS: After the treatment, the dysphagia score of the treatment group was signi-ficantly lower than that of the control group (P<0.05). VFG outcomes showed that, of the two 40 patients in the control and treatment groups, 16 and 23 experienced a marked improvement, 20 and 15 were effective, 4 and 2 were ineffective, with the markedly effective rate being 40.0% and 57.5%, respectively. The daily-life quality scores for physiological function, mental health, emotional function, social function and overall health were all notably increased after the treatment in both groups, particularly in the treatment group (P<0.05). The therapeutic effects of the treatment group were remarkably superior to those of the control group in improving dysphagia (showed by dysphagia score and VFG outcomes) and life quality. CONCLUSIONS: EA treatment combined with swallowing function rehabilitation training is effective in improving swallowing ability and daily-life quality in stroke patients with dysphagia.

["Xingnao Kaiqiao" Acupuncture(Acupuncture Technique for Restoring Consciousness) Combined with Hyperbaric Oxygen in Patients with Hypoxic Ischemic Encephalopathy: Clinical Observation and Its Influence on Blood Rheology].

OBJECTIVE: To observe the effect of "Xingnao Kaiqiao" acupuncture(acupuncture technique for restoring consciousness) combined with hyperbaric oxygen in patients with hypoxic ischemic encephalopathy and assess its influence on blood rheology. METHODS: Forty patients with hypoxic ischemic encephalopathy were randomly divided into treatment group and control group with 20 cases in each group. The treatment group received "Xingnao Kaiqiao" acupuncture combined with hyperbaric oxygen, and the control group received hyperbaric oxygen only. The two groups of patients were treated once per day for 6 consecutive days, which consisted of one course of treatment, and the total treatment lasted for four courses. Before and after treatment, the patients underwent the mini mental state examination (MMSE) to evaluate cognitive function and the barthel index score for activities of daily living. The changes of whole blood viscosity, plasma viscosity, and erythrocyte aggregation index were determined. RESULTS: After treatment, both the treatment and control groups showed improvements in cognitive function，activities of daily living, and blood rheology compared with those before treatment (P<0.05). However, these improvements in the treatment group were significantly greater than those in the control group (P<0.05). CONCLUSIONS: Application of "Xingnao Kaiqiao" acupuncture combined with hyperbaric oxygen in patients with hypoxic ischemic encephalopathy can result in an integrated curative effect, a significant improvement in the blood rheology indexes, and promote the recovery of brain cells.

Synthesis of a disulfide cross-linked polygalacturonic acid hydrogel for biomedical applications.

Polygalacturonic acid (PGA) hydrogel cross-linked via disulfide bonds was synthesized using a thiol oxidation reaction. PGA was grafted with cysteine to yield thiolated PGA (denoted PGAcys). Per gram, PGA-conjugated cysteine was 725 ± 77 µmol, and the degree of modification was 16.24 %. A PGAcys hydrogel film was fabricated under physiological conditions, with gel content 91.6 % and water content 43.3 %. The PGAcys hydrogel was used as a drug carrier for rosmarinic acid (RA) (denoted PGAcys/RA) and to prevent postsurgical adhesion. The in vitro dynamic release behavior of RA from the PGAcys hydrogel was analyzed. The profiles showed that 80 % of the total RA was released from the hydrogel within 15 min, followed by zero-order kinetic release. Animal implant studies showed that PGAcys and PGAcys/RA hydrogel films reduced adhesion incidence by over 90 %, significantly higher than did Hyaluronate/Carboxymethylcellulose (analogous Seprafilm™) (42 %). The PGAcys/RA hydrogel film also reduced the early inflammatory reaction.

Flavonoids with iNOS inhibitory activity from Pogonatherum crinitum.

Pogonatherum crinitum has long been used as a folk remedy for the treatment of many inflammatory diseases in Taiwan, and till now there is still no report concerning its active principles as well as their pharmacological studies. That prompted us to investigate the bioactive constituents of Pogonatherum crinitum. Two novel chemical entities, luteolin 6-C-beta-boivinopyranoside (1) and 6-trans-(2''-O-alpha-rhamnopyranosyl)ethenyl-5,7,3',4'-tetrahydroxyflavone (2), along with luteolin (3), kaempferol (4), luteolin 6-C-beta-fucopyranoside (5), kaempferol 3-O-alpha-L-rhamnopyranoside (6), luteolin 6-C-beta-glucopyranoside (7), rutin (8) and kaempferol 3-O-rutinoside (9) were isolated from this plant, and identified by spectroscopic analysis. The effect of these compounds on the inhibition of NO production in LPS-activated macrophages was further evaluated. All these compounds inhibited NO production in activated RAW 264.7 cells to various degrees without affecting the cellular viability. Among the compounds examined, both compounds 1 and 2 suppressed LPS-induced NO production, with E(max) values of 99.51+/-0.23% and 92.41+/-3.22%, respectively. The most potent compounds, 3 and 4, inhibited NO production with IC(50) values of 10.41+/-0.02 microM and 10.61+/-0.44 microM, respectively. These effects were attributed to suppression of mRNA expression of inducible NO synthase (iNOS). Our results clearly demonstrated that these naturally occurring iNOS inhibitors may be beneficial to the treatment of inflammatory diseases associated with overproduction of NO, which provides an explanation, at least a part, for the anti-inflammatory property of Pogonatherum crinitum.

Potential of a simple solid-phase extraction method coupled to analytical and bioanalytical methods for an improved determination of microcystins in algal samples.

Artemia assays and protein phosphatase assays are commonly used for the screening of microcystins (MCs) in algal samples instead of the standard mouse toxicity assay. However, it has been shown that their results are often biased because of the matrix effects. To eliminate the possible interferences in the algal matrices, a new solid-phase extraction (SPE) method using silica gel as a sorbent was developed and evaluated. Results show that this SPE method could not only reduce the toxicity of the Microcystis samples towards brine shrimp by 50-80% but also eliminate 90-100% of the endogenous phosphatase activity from Spirulina and Chlorella samples, thus improving the determination of microcystins in algal samples using either of the two bioanalytical methods. The application of this SPE method as an off-line cleanup for high-performance liquid chromatography (HPLC) with UV detection is also described in this study. After SPE, the HPLC chromatograms of Microcystis samples have clear baselines that have no interferences with the analyte peaks.