Yuzhong Zhang

Amino Acid Derivatives of Ligustrazine-Oleanolic Acid as New Cytotoxic Agents

A series of novel ligustrazine-oleanolic acid (TOA) derivatives were designed, and synthesized by conjugating amino acids to the 3-hydroxy group of TOA by ester bonds. Their cytotoxicity was evaluated on four cancer cell lines (HepG2, HT-29, Hela and BGC-823) by standard MTT assays. The ClogP values were calculated by means of computer simulation, and logP values of both 3β-glycine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6a) and TOA were determined using a shake flask-ultraviolet spectrophotometry method. It was found that 6a and the 3β-l-lysine ester-6g not only displayed good cytotoxicity (IC50 < 3.5 μM) but also possessed better hydrophilicity than TOA. Moreover, 6a (IC50 = 4.884 μM) had lower nephrotoxicity than both 6g (IC50 = 2.310 μM) and cisplatin (CDDP, IC50 = 3.691 μM) on MDCK cells. Combining Giemsa and DAPI staining, it was further verified that 6a could induce HepG2 apoptosis via nuclei fragmentation and had lower nephrotoxicity. In addition, the structure-activity relationships of these derivatives are briefly discussed.

A New Ligustrazine Derivative-Selective Cytotoxicity by Suppression of NF-κB/p65 and COX-2 Expression on Human Hepatoma Cells. Part 3.

A new anticancer ligustrazine derivative, 3β-hydroxyolea-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methylester (T-OA, C38H58O3N2), was previously reported. It was synthesized via conjugating hepatoprotective and anticancer ingredients of traditional Chinese medicine. We found that T-OA exerted its anticancer activity by preventing the expression of nuclear transcription factor NF-κB/p65 and COX-2 in S180 mice. However, the selective cytotoxicity of T-OA on various kinds of cell lines has not been studied sufficiently. In the present study, compared with Cisplatin, T-OA was more toxic to human hepatoma cell line Bel-7402 (IC50 = 6.36 ± 1.56 µM) than other three cancer cell lines (HeLa, HT-29, BGC-823), and no toxicity was observed toward Madin–Darby canine kidney cell line MDCK (IC50 > 150 µM). The morphological changes of Bel-7402 cells demonstrated that T-OA had an apoptosis-inducing effect which had been substantiated using 4ʹ,6-diamidino-2-phenylindole (DAPI) staining, acridine orange (AO)/ethidium bromide (EB) staining, flow cytometry and mitochondrial membrane potential assay. Combining the immumohistochemical staining, we found T-OA could prevent the expression of NF-κB/p65 and COX-2 in Bel-7402 cells. Both of the proteins have been known to play roles in apoptosis and are mainly located in the nuclei. Moreover subcellular localization was performed to reveal that T-OA exerts in nuclei of Bel-7402 cells. The result was in accordance with the effects of down-regulating the expression of NF-κB/p65 and COX-2.

An overview on structural modifications of ligustrazine and biological evaluation of its synthetic derivatives

Ligustrazine, a natural product from Rhizoma Chuanxiong containing a pyrazine ring, has been widely used in China for treatment of heart diseases. Because of its remarkable effects but rapid metabolism, studies on structural modification of ligustrazine have attracted much attention. Ligustrazine synthetic derivatives reported in recent decades can be divided into ethers, esters, amides and piperazines, olefins, and others. These derivatives are mainly derived from four primary intermediates. The lack of a comprehensive and recent review on this topic prompted us to gather more new information. This paper is a compilation of almost all of the 304 synthetic ligustrazine derivatives we encountered; the information included is expected to be of benefit to further studies of ligustrazine.

A Novel Ligustrazine Derivative T-VA Prevents Neurotoxicity in Differentiated PC12 Cells and Protects the Brain against Ischemia Injury in MCAO Rats

Broad-spectrum drugs appear to be more promising for the treatment of acute ischemic stroke. In our previous work, a new ligustrazine derivative (3,5,6-trimethylpyrazin-2-yl) methyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (T-VA) showed neuroprotective effect on injured PC12 cells (EC50 = 4.249 µM). In the current study, we show that this beneficial effect was due to the modulation of nuclear transcription factor-κB/p65 (NF-κB/p65) and cyclooxygenase-2 (COX-2) expressions. We also show that T-VA exhibited neuroprotective effect in a rat model of ischemic stroke with concomitant improvement of motor functions. We propose that the protective effect observed in vivo is owing to increased vascular endothelial growth factor (VEGF) expression, decreased oxidative stress, and up-regulation of Ca2+–Mg2+ ATP enzyme activity. Altogether, our results warrant further studies on the utility of T-VA for the potential treatment of ischemic brain injuries, such as stroke.

A Series of New Ligustrazine-Triterpenes Derivatives as Anti-Tumor Agents: Design, Synthesis, and Biological Evaluation

A series of novel ligustrazine-triterpenes derivatives was designed, synthesized and screened for their cytotoxicity against five cancer cell lines (Bel-7402, HepG2, HT-29, Hela, and MCF-7) and Madin-Darby canine kidney (MDCK). Current study suggested that most of the ligustrazine-triterpenes conjunctions showed better cytotoxicity than the starting materials. In particular, compound 4a exhibited better cytotoxic activity (IC50 < 5.23 μM) against Bel-7402, HT-29, MCF-7, Hela, and HepG2 than the standard anticancer drug cisplatin (DDP). The cytotoxicity selectivity detection revealed that 4a exhibited low cytotoxicity (IC50 > 20 μM) towards MDCK cells. A combination of fluorescence staining observation and flow cytometric analysis indicated that 4a could induce HepG2 cell apoptosis. Further studies suggested that 4a-induced apoptosis is mediated through depolarization of the mitochondrial membrane potential and increase of intracellular free Ca2+ concentration. In addition, the structure-activity relationships of these derivatives were briefly discussed.

New synthesis method for sultone derivatives: synthesis, crystal structure and biological evaluation of S-CA.

There has been no remarkable progress in the synthesis of sultones in recent years. To facilitate more detailed studies of this functional group, we found a new method to synthesize the sulfonic acid lactone derivatives and finish its ring-closing reaction. A new sultone derivative, (E)-ethyl 4-oxo-6-styryl-3,4-dihydro-1,2-oxathiine-5-carboxylate 2,2-dioxide (S-CA), was synthesized and structurally identified by 1H-NMR, 13C-NMR, HMQC and X-ray single crystal diffraction analysis. The new rapid synthesis extended the method of ring-closing reaction of sulfonic acid lactone derivatives. The angiogenesis activities of S-CA were evaluated by the chick chorioallantoic membrane (CAM) model. It could selectively suppress small angiogenesis in CAM, without influencing either middle and large angiogenesis. In addition, anticancer efficacy of S-CA was evaluated in vivo using a murine sarcoma S180 model. Reduction of the tumor weight and tumor HE staining regions demonstrated that S-CA (10 mg/kg, intraperitoneal injection) had potent inhibition effects and a 44.71% inhibitory rate in S180 mice. Moreover, an acute toxicity test showed that the LD50 value of S-CA via intraperitoneal injection was 25.624 mg/kg.

Synthesis and biological evaluation of T-OA analogues as the cytotoxic agents

The lead compound T-OA, 3β-hydroxyolean-12-en-28-oic acid-(3,5,6-trimethylpyrazine-2-yl) methyl ester, which exhibited promising anticancer effects in vitro and in vivo, has previously been reported. According to the structural features, a series of novel T-OA analogues were synthesized via amino condensation reaction. These analogues’ cytotoxic activities were evaluated on five cancer cell lines (Bel-7402, HepG-2, HT-29, Hela, BGC-823) and hepatic stellate cell line (HSC-6). Among the candidates, compounds 8 and 16 showed promising effects; 3β-hydroxy-lup-20(29)-ene-28-oic acid-(3,5,6-trimethylpyrazin-2-yl) methyl amine (16) even possessed superior bioactivities to positive drugs (cisplatin and ursolic acid), which was worthy of further study. In addition, structure–activity relationships and Clog P values of T-OA analogues were briefly discussed.

Synthesis and protective effect of new ligustrazine derivatives against CoCl2-induced neurotoxicity in differentiated PC12 cells. Part 2

Ligustrazine-benzoic acid derivatives which exhibited promising neuroprotective activities have previously been reported. To further improve the neuroprotective effect of ligustrazine, a series of new ligustrazine derivatives was synthesized and evaluated for their protective effect against cobalt chloride-induced neurotoxicity in differentiated PC12 cells. Most compounds exhibited higher activity than ligustrazine, of which, we found compound (E)-(3,5,6-trimethylpyrazin-2-yl)methyl 3-(2,3,4-trimethoxyphenyl)acrylate (9) displayed the highest protective effect on the PC12 cells damaged by CoCl2 (EC50 = 0.719 μM). Structure–activity relationships are briefly discussed.

A New Oleanolic Acid Derivative against CCl4-Induced Hepatic Fibrosis in Rats

A novel hepatoprotective oleanolic acid derivative, 3-oxours-oleana-9(11), 12-dien-28-oic acid (Oxy-Di-OA), has been reported. In previous studies, we found that Oxy-Di-OA presented the anti-HBV (Hepatitis B Virus) activity (IC50 = 3.13 µg/mL). Remarkably, it is superior to lamivudine in the inhibition of the rebound of the viral replication rate. Furthermore, Oxy-Di-OA showed good performance of anti-HBV activity in vivo. Some studies showed that liver fibrosis may affiliate with HBV gene mutations. In addition, the anti-hepatic fibrosis activity of Oxy-Di-OA has not been studied. Therefore, we evaluated the protective effect of Oxy-Di-OA against carbon tetrachloride (CCl4)-induced liver injury in rats. Daily intraperitoneally administration of Oxy-Di-OA prevented the development of CCl4-induced liver fibrosis, which was evidenced by histological study and immunohistochemical analysis. The entire experimental protocol lasted nine weeks. Oxy-Di-OA significantly suppressed the increases of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (p < 0.05). Furthermore, Oxy-Di-OA could prevent expression of transforming growth factor β1 (TGF-β1). It is worth noting that the high-dose group Oxy-Di-OA is superior to bifendate in elevating hepatic function. Compared to the model group, Oxy-Di-OA in the high-dose group and low-dose group can significantly reduce the liver and spleen indices (p < 0.05). The acute toxicity test showed that LD50 and a 95% confidence interval (CIs) value of Oxy-Di-OA were 714.83 mg/kg and 639.73–798.73 mg/kg via intraperitoneal injection in mice, respectively. The LD50 value of Oxy-Di-OA exceeded 2000 mg/kg via gavage in mice. In addition, a simple and rapid high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed and validated to study the pharmacokinetic characteristics of the compound. After single-dose oral administration, time to reach peak concentration of Oxy-Di-OA (Cmax = 8.18 ± 0.66 μg/mL) was 10 ± 2.19 h; the elimination half-life and area under the concentration-time curve from t = 0 to the last time of Oxy-Di-OA was 2.19 h and 90.21 μg·h/mL, respectively.

Compositions, Formation Mechanism, and Neuroprotective Effect of Compound Precipitation from the Traditional Chinese Prescription Huang-Lian-Jie-Du-Tang

Compounds in the form of precipitation (CFP) are universally formed during the decocting of Chinese prescriptions, such as Huang-Lian-Jie-Du-Tang (HLJDT). The formation rate of HLJDT CFP even reached 2.63% ± 0.20%. The identification by liquid chromatography mass spectrometry (LC-MSn) proved that the main chemical substances of HLJDT CFP are baicalin and berberine, which is coincident with the theory that the CFP might derive from interaction between acidic and basic compounds. To investigate the formation mechanism of HLJDT CFP, baicalin and berberine were selected to synthesize a simulated precipitation and then the baicalin–berberine complex was obtained. Results indicated that the melting point of the complex interposed between baicalin and berberine, and the UV absorption, was different from the mother material. In addition, 1H-NMR integral and high-resolution mass spectroscopy (HR-MS) can validate that the binding ratio was 1:1. Compared with baicalin, the chemical shifts of H and C on glucuronide had undergone significant changes by 1H-, 13C-NMR, which proved that electron transfer occurred between the carboxylic proton and the lone pair of electrons on the N atom. Both HLJDT CFP and the baicalin–berberine complex showed protective effects against cobalt chloride-induced neurotoxicity in differentiated PC12 cells. It is a novel idea, studying the material foundation of CFP in Chinese prescriptions.