Qinglin Zhang

UPLC–Q-TOF-MS/MS fingerprinting of Traditional Chinese Formula SiJunZiTang

SiJunZiTang (SJZT), a classic Traditional Chinese Formula (TCMF) consisting of four herbs, Radix Ginseng, Atractylodes macrocephala, Poria cocos and Glycyrrhiza uralensis, has been demonstrated to show protective effects on intestine and stomach injure. The chromatographic quality control is needed. UPLC-Q-TOF-MS is a rapid and efficient technique for analysis of complex sample in combination with UPLC and tandem mass spectrometry (MS/MS). In this paper, UPLC-MS fingerprinting of SJZT was developed. As a result, 66 compounds including ginsenosides, flavonoids, triterpenoid and coumarins were detected, 58 of them were tentatively identified. The major constituents of SJZT were ginsenosides and flavonoids that coming from Radix Ginseng and Glycyrrhiza uralensis.

Mesenchymal stromal cell-conditioned medium prevents radiation-induced small intestine injury in mice.

BACKGROUND AIMS Effective therapy for radiation-induced intestinal injury is currently unavailable. Mesenchymal stromal cells (MSC) are expected to be useful in repairing intestinal damage caused by irradiation. We determined whether the MSC-derived bioactive components could protect radiation-induced small intestine injury in mice. METHODS Human umbilical cord (UC)-derived MSC were isolated, expanded and exposed to hypoxic conditions in vitro. The hypoxia-conditioned medium was ultrafiltrated with a 3-kDa molecular weight cut-off to prepare the high molecular weight fraction (HMWF). The effect of HMWF on the viability of irradiated rat intestinal epithelial cells (IEC-6) was examined by MTT(methyl thiazolyl tetrazolium) assay. HMWF was also delivered to BALB/C male mice by tail intravenous injection immediately after receiving local abdominal irradiation at a selected dose of 10 Gy. Animal body weight, survival and diarrhea were monitored for 30 days. The improvement of mice intestine structure, including epithelium thickness and villus height, was examined by histology. RESULTS HMWF enhanced the viability of irradiated IEC-6 cells in vitro. Repeated infusion of HMWF for 7 days immediately after abdominal irradiation of 10 Gy ((60)Coγ-ray) increased the survival rate, decreased diarrhea occurrence and improved the small intestinal structural integrity of irradiated mice. CONCLUSIONS MSC-derived bioactive components could be a novel therapeutic approach for the treatment of radiation-induced injury.

Structural characterization of polysaccharides from the roots of Urtica fissa

Three polysaccharides were isolated from the roots of Urtica fissa by extraction, ultrafiltration, anion-exchange, and gel-filtration chromatography. The structures were characterized using acetylation, methylation, and spectral methods (GCMS, NMR). All three polysaccharides are mainly composed of d-arabinofuranosyl, d-galactopyranosyl, d-glucopyranosyl residues with different structural characteristics. Polysaccharide A of MW 5.2 × 103 contained a linear chain of 1-linked β-d-glucopyranosyl, 1,6-linked β-d-glucopyranosyl, 1,6-linked α-galactopyranosyl, and 1,5-linked β-arabinofuranosyl moieties. Polysaccharide B of MW 7.7 × 104 possessed a chain consisting of 1,5-linked α-d-arabinofuranosyl, 1,3-linked β-d-mannopyranosyl, 1,6-linked β-d-glucopyranosyl, and 1,6-linked α-d-galactopyranosyl residues, but 4-O of α-d-galactopyranosyl residues were branched by terminal β-d-glucopyranosyl residues. Polysaccharide C of MW 5.3 × 104 composed of a chain of 1,5-linked α-d-arabinofuranosyl, 1,4-linked β-d-galactopyranosyl, 1,5-linked β-d-xylopyranosyl, 1,4-linked β-d-mannopyranosyl, 1-linked β-d-glucopyranosyl residues, and the terminal β-d-glucopyranosyl residues are attached to 3-O positions of 1,6-linked α-d-glucopyranosyl residues.

Microbial transformation of epothilone A by Aspergillus niger AS 3.739.

The microbiological transformation of epothilone A (1) by Aspergillus niger AS 3.739 afforded four main metabolites. Their structures were elucidated by 1H, 13C NMR and HSQC, COSY, HMBC, and NOESY spectra as trans-12,13-hydroxylated epothilone A (2), cis-12,13-hydroxylated epothilone A (3), trans-12,15-epoxidated epothilone A (4), and cis-12,15-epoxidated epothilone A (5). All four compounds were firstly found based on their stereochemistry. These new compounds displayed cytotoxicity against human breast carcinoma cells MCF-7 with IC50 9.88 μg/ml of 2, 2.52 μg/ml of 3, 9.88 μg/ml of 4, and 5.68 μg/ml of 5.

Plasmid pUDK-HGF encoding human hepatocyte growth factor gene attenuates gentamicin-induced kidney injury in rats

The clinical application of gentamicin has been limited by its nephrotoxicity, which is characterized by kidney injury, interstitial fibrosis and progressive renal impairment. In this paper, we examine effects of plasmid pUDK-HGF which encodes the human hepatocyte growth factor (HGF) gene on gentamicin-induced renal injury in rats. The kidney injury was intentionally induced by injecting gentamicin intraperitoneally. On the third day after last gentamicin treatment, pUDK-HGF was injected into the left kidney tissue only once via a sterile back incision. At day 30 after gentamicin treatment, RI, Scr, BUN, 24 h-UTP and apoptotic cell death were determined. Tubulointerstitial injury and the renal interstitial vessel regeneration were evaluated by histological scoring. pUDK-HGF treatment significantly improved the renal function with decreasing RI, Scr and BUN. 24 h-UTP also presented ameliorating trend compared to the control group with kidney injury. pUDK-HGF treatment significantly decreased the score of tubulointerstitial injury and enhanced angiogenesis, also prevented kidney cells from apoptosis. The tubulointerstitial injury was significantly reduced in the pUDK-HGF injected left kidney and right kidney also showed some improvements. Our results showed that pUDK-HGF may become a novel therapeutic agent for kidney injury and renal fibrosis.

Structure of a polysaccharide from Urtica fissa determined by NMR spectra

A polysaccharide, isolated and purified from the aqueous extract of nettle plant Urtica fissa, was found to consist of d-glucose and d-arabinose. Molecular weight was determined to be Mn 4140. The NMR experiments (1H, 13C, 1H–1H COSY, TOCSY, HSQC, NOESY, and HMBC) revealed the structure as the following repeating unit:

Local intramuscular injection of a plasmid encoding human proenkepahlin attenuates incision pain in rats.

We investigated the antinociceptive effect of local intramuscular injection of a plasmid encoding human proenkephalin (pVAX1-hPPE) on postoperative pain in rats. Male Sprague-Dawley rats with incision-induced pain were intramuscularly injected into injured plantaris muscle with empty vector (pVAX1) or pVAX1-hPPE, respectively. Paw mechanical threshold and thermal latency in the 200μg pVAX1-hPPE treated rats were significantly higher at 6h and on 1day, and lasted until day 7 after intramuscular administration, respectively. The analgesic effects were reversed by methylnaltrexone, suggesting that the antinociceptive effect of pVAX1-hPPE was mediated through peripheral opioid receptor pathway. In contrast, incisional or pVAX1-treated rats did not significantly affect pain thresholds. These results demonstrated that single intramuscular injection of pVAX1-hPPE attenuated incision-induced pain in rats, and it is worthy of further study as a potential gene therapy for postoperative pain.

Nonviral vector plasmid DNA encoding human proenkephalin gene attenuates inflammatory and neuropathic pain-related behaviors in mice

Inflammatory pain and neuropathic pain are major clinical health issues that represent considerable social and economic burden worldwide. In the present study, we investigated the anti-nociceptive efficacy of delivery of human proenkephalin gene by a plasmid DNA vector (pVAX1-PENK) on complete Freunds adjuvant (CFA) induced inflammatory pain and spared nerve injury (SNI) induced neuropathic pain in mice. Mice were intramuscularly or intrathecally administered pVAX1 or pVAX1-PENK, respectively. Pain thresholds in the pVAX1-PENK treated mice were significantly higher at day 3, then reached a peak at day 7 and lasted until day 28 after gene transfer, and the analgesic effect of pVAX1-PENK was blocked with naloxone hydrochloride. In contrast, pVAX1 treated mice did not significantly improve pain thresholds. These results indicate that peripheral or spinal delivery of a plasmid encoding human proenkephalin gene provides a potential therapeutic strategy for inflammatory pain and neuropathic pain.

Chemical constituents from Bletilla striata and their NO production suppression in RAW 264.7 macrophage cells

Abstract A novel glucoside bletilloside A (1) was isolated from the tubers of Bletilla striata, together with seven known compounds (2–8). Their structures were determined on the basis of extensive spectroscopic analyses. All compounds were evaluated for the inhibition on NO production effects in RAW 264.7 macrophage cells, while militarine (4) and dactylorhin A (5) exhibited moderate inhibitory effects.

Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia

ABSTRACT The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.