Design and synthesis of novel celastrol derivative and its antitumor activity in hepatoma cells and antiangiogenic activity in zebrafish.

Two series of celastrol derivatives were designed and synthesized by modifying carboxylic acid at the 28th position with amino acid, and their intermediates with isobutyrate at the third position. All compounds were evaluated for their antiproliferation activity by four human cancer cell lines (SCG7901, HGC27, HepG2, and Bel7402) and one normal cell LO2. The most promising compound, compound 8, showed superior bioactivity and lower toxicity than others including celastrol. Further underlying tests illustrated that compound 8 induced apoptosis and cell arrest at G2/M and inhibited proliferation and mobility of human hepatoma cells by suppressing the signal transducer and activator of transcription-3 signaling pathway. Besides these, a highly accurate and reproducible high performance liquid chromatography protocol was established to determine celastrol and compound 8 absorption in zebrafish, and results demonstrated that their concentration increased rapidly within 4 hr in a time-dependent manner and the concentration of compound 8 was higher than that of celastrol. In addition, without detection at 12 hr, compound 8 was rapidly metabolized in vivo. These findings are very helpful for the structural modification of celastrol and other bioactive compounds to improve their bioactivity, toxicity, and absorption.

Growth inhibitory effect of KYKZL-1 on Hep G2 cells via inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest.

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G2 growth. We found that KYKZL-1 inhibited the growth of Hep G2 cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE2 and LTB4 production in Hep G2 cells. Accordingly, exogenous addition of PGE2 or LTB4 reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S-G2 checkpoint via the activation of p21(CIP1) protein and down-regulation of cyclin A expression. These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest. Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile.

Inhibition of inflammatory mediators contributes to the anti-inflammatory activity of KYKZL-1 via MAPK and NF-κB pathway.

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the anti-inflammatory activity test focusing on its modulation of inflammatory mediators as well as intracellular MAPK and NF-κB signaling pathways. In acute ear edema model, pretreatment with KYKZL-1 (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice with a higher inhibition than diclofenac. In a three-day TPA-induced inflammation, KYKZL-1 also showed significant anti-inflammatory activity with inhibition ranging between 20% and 64%. In gastric lesion test, KYKZL-1 elicited markedly fewer stomach lesions with a low index of ulcer as compared to diclofenac in rats. In further studies, KYKZL-1 was found to significantly inhibit the production of NO, PGE2, LTB4 in LPS challenged RAW264.7, which is parallel to its attenuation of the expression of iNOS, COX-2, 5-LOX mRNAs or proteins and inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. Taken together, our data indicate that KYKZL-1 comprises dual inhibition of COX and 5-LOX and exerts an obvious anti-inflammatory activity with an enhanced gastric safety profile via simultaneous inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB.

Discovery, synthesis, and evaluation of small-molecule signal transducer and activator of transcription 3 inhibitors.

The signal transducer and activator of transcription 3 (STAT3) oncogene is a promising molecular target and its inhibitors have great potential as anticancer drugs. To identify novel and STAT3-selective inhibitors, a virtual screening based on Specs and Maybridge databases was conducted and a 6,6'-bibenzoxazole type small molecule, compound 3a with a inhibition constant K(i) value of 494.32 nM to STAT3 was explored. Further, a novel series of derivatives originally derived from 3a was synthesized and evaluated through cell-based assays using human breast cancer cell lines, MDA-MB-468 and MCF-7 with or without constitutive expression of STAT3, respectively. In the series, 3a, 3c, 3d and 4e showed a better inhibitory activity with a good selectivity. Among them, 3a and 3c significantly inhibited STAT3 protein level and also displayed binding affinity for STAT3 that detected with flow injection analysis-quartz crystal microbalance (FIA-QCM) analysis system. The results provided a new lead for future design and development of potent STAT3 inhibitors.

Simultaneous quantitation of three major triterpenoid glycosides in Centella asiatica extracts by high performance liquid chromatography with evaporative light scattering detection.

A high-performance liquid chromatography method with evaporative light scattering detection was established for simultaneous determination of three major triterpenoid glycosides, i.e. asiaticoside, madecassoside and asiaticoside-B, in Centella asiatica extracts. The optimal chromatographic conditions were achieved on a COSMOSIL 5C(18)-MS-II column by constant elution with water (0.01% trifluoroacetic acid, v/v) and acetonitrile (1.0% methyl tert-butyl ether, 0.01% trifluoroacetic acid, v/v) (78:22) as mobile phase at a flow rate of 1.0 mL/min; the column temperature was 30 degrees C. The evaporative light scattering detector was set at an evaporating temperature of 40 degrees C and nitrogen gas pressure of 3.5 bar. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy. All calibration curves showed good linear regression (r(2) > 0.9993) within test ranges. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 1.73-3.06 and 3.89%-4.92%, respectively, and overall recoveries of 97.63-99.39% for the three compounds analyzed. The method developed was successfully applied to quantify the main triterpenoid glycosides in Centella asiatica extracts from different companies.

Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro.

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H(2)O(2) (200 microM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 microM. Furthermore, in the H(2)O(2)-treated group, agarose gel electrophoresis displayed a "DNA ladder". Whereas in the 10 microM crocin-pretreated group, cells remained intact and no "DNA ladder" was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 muM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H(2)O(2), which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).

Alleviation of picryl chloride-induced delayed type hypersensitivity reaction by saponin fraction of Gleditsia sinensis.

The objective of the present study was to investigate the effects of saponin fraction from anomalous fruits of Gleditsia sinensis (SFGS) on picryl chloride-induced delayed type hypersensitivity (PC-DTH) and functions of T lymphocytes and macrophages in mice. SFGS (100, 200 mg/kg), orally administered during either sensitization stage or effector stage, produced remarkable inhibition of PC-DTH. In vitro, SFGS (1, 2, 4 microg/ml) concentration-dependently attenuated concanavalin A (Con A)-elicited mouse splenocyte proliferation and interleukin 2 (IL-2) production. At concentrations of 10 and 20 microg/ml, SFGS inhibited lipopolysaccharide (LPS)-induced production of nitric oxide and interleukin 1beta (IL-1beta) of mouse peritoneal macrophages. The findings indicate that SFGS attenuates PC-DTH in mice, which is probably mediated by preventing proliferation and differentiation of T cells during the sensitization stage and suppressing activation of macrophages during the effector stage.

[Effect of epigallocatechingallate on acute lung injury induced by oleic acid in mice].

AIM: To investigate the effect of epigallocatechingallate (EGCG) on acute lung injury induced by oleic acid in mice and the possible mechanism. METHODS: Acute lung injury was induced by oleic acid in mice. Light microscopy and electron microscopy were used to examine histological changes and lung index as well as wet to dry weight ratio was calculated. Serum TNF-a level was measured by enzyme linked immunosorbent assay (ELISA) and the phosphorylation of p38 MAPK was determined by Western blotting. RESULTS: Pretreatment of EGCG significantly alleviated oleic acid induced lung injury accompanied by reduction of lung index and wet to dry weight ratio, decreased of TNF-a level in serum and inhibition of phosphorylation of p38 MAPK. CONCLUSION: EGCG showed beneficial effect on acute lung injury induced by oleic acid in mice. The ultimate reduction of TNF-alpha in serum caused by inhibition of phosphorylated p38 MAPK is involved in the mechanism of action of EGCG.

[Study of the effect of crocin on rat experimental hyperlipemia and the underlying mechanisms].

OBJECTIVE: To study the effect of crocin on rat experimental hyperlipemia and its mechanisms. METHOD: Hyperlipemia model was established by feeding heavy cholesterol for 2 months and the effect of crocin on blood lipid in experimental hyperlipemia rats was observed. Aortic smooth muscle cells were cultured in different culture media and proliferation was measured by MTT assay. Western blotting was used to detect the effect of crocin on phosphorylation of p38 MAPK. RESULT: Crocin not only decreased greatly the content of cholesterol, triglyceride and density lipoprotein in blood, but also increased the content of high density lipoprotein. In addition, the proliferation of smooth muscle cells and the activation of p38MAPK were inhibited by Crocin. CONCLUSION: Crocin prevents atherosclerosis in hyperlipemia, which may be mediated by the inhibition of both proliferation of smooth muscle cells and activation of p38MAPK.