Isolation and Biological Characteristics of a Novel Phage and Its Application to Control Vibrio Parahaemolyticus in Shellfish Meat.

Vibrio parahaemolyticus is a common foodborne pathogenic bacterium. With the overuse of antibiotics, an increasing proportion of drug-resistant strains are emerging, which puts enormous pressure on public health. In this study, a V. parahaemolyticus-specific phage, VP41s3, was isolated. The head length, width, and tail length of the phage were 77.7 nm, 72.2 nm, and 17.5 nm, respectively. It remained active in the temperature range of 30-50°C and pH range of 4-11. The lytic curve of phage VP41s3 showed that the host bacteria did not grow until 11 h under phage treatment at MOI of 1000, indicating that the phage had good bacteriostatic ability. When it was added to shellfish contaminated with V. parahaemolyticus (15°C, 48 h), the number of bacteria in the experimental group was 2.11 log10 CFU/mL lower than that in the control group at 24 h. Furthermore, genomic characterization and phylogenetic analysis indicated that phage VP41s3 was a new member of the Podoviridae family. The genome contained 50 open reading frames (ORFs), in which the ORF19 (thymidine kinase) was an enzyme involved in the pyrimidine salvage pathway, which might lead to the accelerated DNA synthesis efficiency after phage entered into host cells. This study not only contributed to the improvement of phage database and the development of beneficial phage resources but also revealed the potential application of phage VP41s3 in food hygiene and safety.

Caecum Rupture Secondary to Iliac Crest Bone Graft: A Case Report.

Autogenous bone grafting is a common surgical method in orthopaedics. The anterior iliac crest is a common site for harvesting autologous bone grafts. There are many complications after iliac bone harvesting, and pain and discomfort at the donor site are the most common sequelae. However, intestinal rupture after iliac bone harvesting has not been reported. We report a case of caecum rupture in a 58-year-old male after harvesting bone from his iliac crest. After proper surgical repair, the patient was discharged from the ICU and his bowel function recovered. This serious complication of bone harvesting from the iliac crest prompted investigation of the technique of iliac crest harvesting and donor site reconstruction.

Acid-Responsive Aggregation of Gold Nanoparticles for the Photothermal Treatment of Bacterial Infections.

Photothermal therapy (PTT) is considered to be one of the promising methods to combat pathogenic bacteria. However, traditional PTT is prone to generate undesired temperature increase to surrounding normal tissues, which limits the application of PTT. Herein, an acid-responsive PTT system (Au nanoparticles system: AuNPs-S) was constructed based on the photothermal feature of spherical gold nanoparticles (AuNPs) and the low pH of the bacterial infected site. AuNPs-S is composed of two kinds of AuNPs: AuNPs modified with Asp-Asp-Asp-Asp-Asp-Cys (peptide A) were denoted as AuNPs-A; AuNPs modified with 2,3-dimethylmaleic anhydride (DA) grafted Lys-Gly-Gly-Lys-Gly-Gly-Lys-Cys (peptide B) were denoted as AuNPs-B/DA. AuNPs-B/DA with an acid-responsive moiety showed a charge-convertible feature. The negatively charged AuNPs-B/DA became positively charged AuNPs-B at low pH, aggregating with the negatively charged AuNPs-A via an electrostatic interaction, reaching the threshold to the interparticle plasmonic coupling effect among AuNPs, thereby killing bacteria precisely under the irradiation of near-infrared (NIR) light through the elevated temperature at the targeted area. This acid-responsive PTT strategy supplies an excellent mode for combating bacterial infections with no vital damage to normal tissues.

Biological Characterization of Pseudomonas fluorescens Phage Pf17397_F_PD1 and Its Application in Food Preservation.

In order to explore the application prospects of phages for controlling bacterial contamination, a lytic phage Pf17397_F_PD1 (Later abbreviated as PD1) was isolated from fish guts using Pseudomonas fluorescens ATCC 17397 as the host bacterium. The phage displayed short latency (18 min), long lysis period (212 min), and high lysis volume (1.47 × 102 PFU/each cell). It displayed wide temperature (30-70°C) and pH (4-11) tolerance. Genomic comparison revealed a maximum sequence identity of 48.65% between phage PD1 and other identified phages, indicating that PD1 was a new phage. The phage PD1 significantly inhibited the growth of P. fluorescens in milk and grass carp at 4°C and 25°C. Compared to the negative control, bacterial levels in milk stored at 25°C for 48 h were reduced by 2.71 log CFU/mL and 2.84 log CFU/mL at the multiplicity of infection (MOI) of 100 and 1,000, respectively. In contrast, when grass carp were stored at 25°C for 24 h, the bacterial load was reduced by 1.28 log CFU/g and 2.64 log CFU/g compared to the control (MOI of 100 and 1,000). When the phage was applied for preservation of grass carp blocks, total volatile salt nitrogen (TVB-N) values of phage-treated samples increased by 6.8 mg/100 g and 7.5 mg/100 g at MOI of 100 and 1,000, respectively, after 7 days of storage, which was significantly lower than that of the control group (15.83 mg/100 g). This study showed that phage PD1 was a good natural biological antimicrobial agent against P. fluorescens ATCC 17397.

Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

Inhibitory Effect of Two Closely Related Phages on Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a foodborne pathogenic bacterium commonly found in seafood. The emergence of drug-resistant strains poses a threat to human public health and economic development. Therefore, there are increasing needs to develop new technologies in controlling multidrug-resistant V. parahaemolyticus strains and to evaluate their practical efficiency in seafood or mariculture. In this study, we screened two genetically related V. parahaemolyticus phages, F23s2 and H256D1, which belonged to the siphoviridae family and podoviridae family, respectively. They showed 97.13% and 96.13% identity with Vibrio phage vB_Vpap_MGD1, respectively. Both phages were stable at pH 4-11 and displayed temperature tolerance (<70°C). Meanwhile they showed a broad host spectrum for multidrug-resistant V. parahaemolyticus, and Phage F23s2 lysed 16 of all 23 V. parahaemolyticus strains, while phage H256D1 lysed 10 strains. Phage F23s2 and H256D1 had a good inhibitory effect on V. parahaemolyticus in shrimp meat. Compared with the negative group, the bacterial amount of experimental group with phage F23s2 decreased by 1.60 log colony-forming unit (CFU)/mL at 12 h. For phage H256D1, the bacterial concentration of shrimp meat contaminated with V. parahaemolyticus H256 increased to 5.65 log CFU/mL at 72 h, while the concentration of the experimental group in presence of phage H256D1 was 3.58 log CFU/mL. All live clams infected with V. parahaemolyticus died after 96 h in the absence of phage, whereas clams with phage F23s2 and H256D1 still had a survival rate of 12% and 4%, respectively. Understanding the gene function and biology of phages facilitates its application for control of V. parahaemolyticus contamination worldwide.

Bacteriostatic effects of phage F23s1 and its endolysin on Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a common foodborne pathogenic bacterium and drug-resistant strains are now widespread. Phages led by drug-resistant V. parahaemolyticus strains are promising means to decrease the pressure on public health. We isolated a V. parahaemolyticus-specific bacteriophage F23s1 that was active at wide ranges of temperature (30-60°C) and pH (4-10). Phage F23s1 exhibited a specific host range; in that, only 13 of the 23 V. parahaemolyticus strains were lysed. F23s1 effectively inhibited the growth of V. parahaemolyticus strain F23 in shrimp at 25°C within 12 h at a multiplicity of infection of 1000. We sequenced the genome of phage F23s1 which comprised a 76,648-bp DNA with 105 open reading frames (ORFs) and identified an endolysin gene ORF52 that was then cloned and successfully expressed in Escherichia coli. The recombinant ORF52 protein significantly decreased OD600 nm of V. parahaemolyticus F23 from 0.978 to 0.249 when used at 20 µmol/L within 60 min. The endolysin also showed lytic activity against a panel of 23 drug-resistant V. parahaemolyticus and 12 Salmonella strains with a higher lytic ability for V. parahaemolyticus. The phage F23s1 and its endolysin will be useful for preventing and controlling V. parahaemolyticus in food safety.

Characteristics of Antimicrobial-Resistant Vibrio parahaemolyticus Strains and Identification of Related Antimicrobial Resistance Gene Mutations.

Multidrug-resistant (MDR) Vibrio parahaemolyticus strains have become a great threat to public health. The purpose of this study was to investigate differences in biological characteristics and antimicrobial resistance gene (ARG) mutations of V. parahaemolyticus that displayed different levels of antimicrobial resistance. The susceptibility of 74 V. parahaemolyticus strains to 9 common antimicrobials was investigated, of which 88% were resistant to 3-4 antimicrobials and 3% to 5-7 antimicrobials. Interestingly, only 9% were resistant to 1-2 antimicrobials. The MDR strains possessed longer growth lag time than the non-MDR strains and displayed weaker swimming abilities. Whole genome sequencing was performed on strains VP41, VP44, 460, and 469 that were resistant to two to three classes of antimicrobials. ARGs were identified and compared with that of reference strain ATCC17802, and some important mutations were deduced. The Val189Ile mutation emerged in qnr gene of a single strain. Besides, the nonsynonymous mutations existed in four ARGs in different strains, including CatB (Pro165Ser, Gly208Asp), VmeA (Ile313Thr), VmeC (Glu329Ala), and VmeD (Asn205Ser). These results linked resistance gene mutations to enhance resistance in V. parahaemolyticus strains and provide a reference for more effective monitoring and prevention of V. parahaemolyticus infections.

Gastric Acid Powered Nanomotors Release Antibiotics for In Vivo Treatment of Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) infection has ≈75% probability of causing gastric cancer, so it is considered to be the strongest single risk factor for gastric malignancies. However, the harsh gastric acid environment has created obstacles to medical treatment. This work reports a nanomotor with a bottle-shaped container that can be loaded with small molecules of clarithromycin, nano calcium peroxide (CaO2 ), and Pt nanoparticles (Pt NPs) by ultrasound. Nanomotors can quickly consume gastric acid through the chemical reaction of CaO2 to temporarily neutralize gastric acid. The product hydrogen peroxide (H2 O2 ) is catalytically decomposed into a large amount of oxygen (O2 ) by Pt NPs. The local concentration gradient of O2 bubbles causes it to be expelled from the nanobottles through a narrow opening, and then push the nanobottles forward to provide maximum release and prodrug efficacy. Experiments in animal models show that 15 mg nanomotors can safely and quickly neutralize gastric acid in the stomach and simultaneously release prodrugs to achieve good therapeutic effects without causing acute toxicity. H. pylori burden in mice was 2.6 orders of magnitude lower than that in the control group. The stomach returns to normal pH within 1 d after administration.

Photothermally triggered nitric oxide nanogenerator targeting type IV pili for precise therapy of bacterial infections.

Nitric oxide (NO) is an important biological messenger involved in the treatment of bacterial infections, but its controlled and targeted release in bacterial infections remains a major challenge. Herein, an intelligent NO nanogenerator triggered by near-infrared (NIR) light is constructed for targeted treatment of P. aeruginosa bacterial infection. Since maleimide can recognize and attach to the pilus of T4P of P. aeruginosa, we adopt this strategy to achieve the accurate release of therapeutic drugs at the infection site, i.e., after maleimide targets Gram-negative bacteria, the SNP@MOF@Au-Mal nanogenerator will release NO and generate ROS in situ from the inorganic photosensitizer gold nanoparticles under NIR irradiation to achieve synergistic antibacterial effect. In vivo experiments proved that the bacterial burden on the wound was reduced by 97.7%. Additionally, the nanogenerator was shown to promote the secretion of growth factors, which play a key role in regulating inflammation and inducing angiogenesis. This strategy has the advantage of generating a high concentration of NO in situ to promote the transfer of more NO and its derivatives (N2O3, ONOO-) to bacteria, thereby significantly improving the antibacterial effect. The multifunctional antibacterial platform has been demonstrated as a good carrier for gas therapy because of its simple and efficient gas release performance, indicating its great potential for the treatment of drug-resistant bacterial infections.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Correction to Fabrication of Bis-Quaternary Ammonium Salt as an Efficient Bactericidal Weapon Against Escherichia coli and Staphylococcus aureus.

[This corrects the article DOI: 10.1021/acsomega.8b01265.].

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Endogenous stimulus-powered antibiotic release from nanoreactors for a combination therapy of bacterial infections.

The use of an endogenous stimulus instead of external trigger has an advantage for targeted and controlled release in drug delivery. Here, we report on cascade nanoreactors for bacterial toxin-triggered antibiotic release by wrapping calcium peroxide (CaO2) and antibiotic in a eutectic mixture of two fatty acids and a liposome coating. When encountering pathogenic bacteria in vivo these nanoreactors capture the toxins, without compromising their structural integrity, and the toxins form pores. Water enters the nanoreactors through the pores to react with CaO2 and produce hydrogen peroxide which decomposes to oxygen and drives antibiotic release. The bound toxins reduce the toxicity and also stimulate the body's immune response. This works to improve the therapeutic effect in bacterially infected mice. This strategy provides a Domino Effect approach for treating infections caused by bacteria that secrete pore-forming toxins.

One Stone with Two Birds: Functional Gold Nanostar for Targeted Combination Therapy of Drug-Resistant Staphylococcus aureus Infection.

The development of new antibacterial agents to deal with the emergence and spread of antibiotic resistance in Gram-positive bacterial pathogens has become an increasing problem. Here, a new strategy is developed for the effective targeting and killing of Gram-positive bacteria based on vancomycin (Van)-modified gold nanostars (AuNSs). Our work has demonstrated that the Van-modified AuNSs (AuNSs@Van) can not only selectively recognize methicillin-resistant Staphylococcus aureus (MRSA) but also kill MRSA under near-infrared laser irradiation in vitro. Additionally, AuNSs@Van shows satisfactory biocompatibility and antibacterial activity in treating bacterial infection in vivo. The attractive trait of AuNSs@Van is attributed to the physical effect of its antibacterial activity, with less potential for resistance development. The aforementioned advantages indicate the potential of AuNSs@Van as a photothermal antibacterial agent for effectively combating Gram-positive bacteria in the field of health care.

Synergistic antibacterial effects of curcumin modified silver nanoparticles through ROS-mediated pathways.

Due to their remarkable antibacterial properties, silver nanoparticles (Ag NPs) and curcumin (CCM) have been widely used in the antimicrobial field. In our study, we have fabricated the uniform and stable silver/curcumin composite nanoparticles by a facile ultrasound treatment process and the synergistic antibacterial activity were evaluated. The curcumin not only played a role of reducing agent but also acted as a capping agent. The antibacterial effects of silver/curcumin (cAgNPs) were studied by measuring the growth curve and surface plate assay based on the E. coli and B. subtilis, which showed concentration dependent bacteriostatic and bactericidal effects of cAgNPs. The presence of CCM enhance the binding of Ag to bacterial membrane and Ag+ release in comparison to that without CCM, so that creating a temporary and local high Ag+ concentration near the surface of the bacterium, meanwhile, generation of more reactive oxygen species, lead to membrane damage, bacterial lipases and induce leakage of intracellular contents followed by bacterial death that lead to growth inhibition of the bacteria. The antibacterial effects were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the effect which were further found to decrease by introducing antioxidant N-acetyl-l-cysteine (NAC) act as a reactive oxygen species (ROS) scavenging agent. These initial data suggest that cAgNPs have a highly antibacterial efficient and might have potential to be developed as an effective antimicrobial nanomaterial.

Nitrogen-Doped Carbon Quantum Dots for Preventing Biofilm Formation and Eradicating Drug-Resistant Bacteria Infection.

The development of novel antimicrobial agents is a top priority in the fight against drug-resistant bacteria. Here, we synthesized a green nanoantibiotic, nitrogen-doped carbon quantum dots (N-CQDs) from bis-quaternary ammonium salt (BQAS) as carbon and nitrogen sources. The as-obtained N-CQDs possess high antibacterial activity (>99%) against both methicillin-resistant Staphylococcus aureus (MRSA) and Ampicillin-resistant Escherichia coli bacteria in vitro than some known clinical antibiotics (vancomycin and gentamicin). The N-CQDs can kill MRSA pathogens without inducing resistance, prevent biofilm formation and eliminate established biofilm and persister cells. The treatment of N-CQDs can significantly reduce the amount of bacteria on the infected tissue and accelerate wound healing. The N-CQDs are positively charged, thus enabling them to interact with bacterial cell membrane through electrostatic interaction, leading to severe damage and an increased permeability of the cell membrane, which further promotes the penetration of N-CQDs into the membrane and induces the degradation of DNA by N-CQDs generated reactive oxygen species. The N-CQDs also play a role in obstructing the intracellular metabolic pathways of MRSA. The overall data demonstrate the green nanoantibiotic as an excellent eradicator of biofilm and persister cells as well as a promising antibacterial candidate for treating infections induced by drug-resistant bacteria.

Fabrication of Bis-Quaternary Ammonium Salt as an Efficient Bactericidal Weapon Against Escherichia coli and Staphylococcus aureus.

Combating bacterial pathogens has become a global concern, especially the emergence of drug-resistant bacteria have made conventional antibiotics lose their efficiency. This grim situation suggests the necessity to explore novel antibacterial agents with favorable safety and strong antibacterial activity. Here, we took the advantage of quaternary ammonium compounds and synthesized a long-chain high-molecular organic bis-quaternary ammonium salt (BQAS) with a broad-spectrum bactericidal activity through a facile one-pot reaction. The bactericidal effect of BQAS was evaluated by two bacterial human pathogens: Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), which are the major cause of diarrheal infections in children and adults. Our experimental results indicate that the bactericidal activity of BQAS is linked to the strong contact between the positively charged quaternary ammonium groups and the bacterial cells, thus leading to a temporary and locally high concentration of reactive oxygen species, which subsequently triggers oxidative stress and membrane damage in the bacteria. This mechanism was further confirmed by several assays, such as the membrane permeabilization assay, fluorescent-based cell live/dead test, scanning electron microscopy, transmission electron microscopy, together with the lactate dehydrogenase release assay, which all indicated that BQAS induced damage to the cytoplasmic membrane and the leakage of intracellular fluid containing essential molecules. The excellent bactericidal activity of BQAS suggests its great application potential as a promising candidate against the rapid emergence of drug-resistant bacterial pathogens.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

Simultaneous determination of matrine, sophoridine and oxymatrine in Sophora flavescens Ait. by high performance liquid chromatography.

Following a detailed study, a reversed-phase high performance liquid chromatographic method (HPLC) has been developed and validated for analysis of three bioactive alkaloids, matrine, sophoridine and oxymatrine, in Sophora flavescens Ait. HPLC separation of the alkaloids was performed on a Kromasil C(18) column and detected by ultraviolet absorbance at 208 nm. The column temperature was maintained at 40 degrees C. A mobile phase composed of 0.01 mol/L KH(2)PO(4) buffer-methanol-triethylamine in the ratios 94:6:0.01 (v/v) was found to be the most suitable for this separation at a fl ow-rate of 1.0 mL/min and enabled the baseline separation of the three analytes free from interferences with isocratic elution. The analysis time was 24 min per injection. The calibration was linear in the range of 0.2-120.0 micro g/mL for matrine, 0.2-115.2 micro g/mL for sophoridine and 0.2-110.4 micro g/mL for oxymatrine, respectively. For assaying Sophora Flavescens Ait. samples, the relative standard deviations were 2.0% for matrine, 2.8% for sophoridine and 1.8% for oxymatrine analysis. The average recoveries of matrine, sophoridine and oxymatrine were 93.9, 95.3 and 93.5% for the Sophora flavescens Ait. samples, respectively. The method has been successfully applied to the simultaneous determination of matrine, sophoridine and oxymatrine in Sophora Flavescens Ait. samples collected in different habitats.