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Deep networks are vulnerable to adversarial attacks from malicious adversaries. Currently, many adversarial learning algorithms are designed to exploit such vulnerabilities in deep networks. These methods focus on attacking and retraining deep networks with adversarial examples to do either feature manipulation or label manipulation or both. In this paper, we propose a new adversarial learning algorithm for finding adversarial manipulations to deep networks. We formulate adversaries who optimize game-theoretic payoff functions on deep networks doing multi-label classifications. We model the interactions between a classifier and an adversary from a game-theoretic perspective and formulate their strategies into a Stackelberg game associated with a two-player problem. Then we design algorithms to solve for the Nash equilibrium, which is a pair of strategies from which there is no incentive for either the classifier or the adversary to deviate. In designing attack scenarios, the adversary’s objective is to deliberately make small changes to test data such that attacked samples are undetected. Our results illustrate that game-theoretic modelling is significantly effective in securing deep learning models against performance vulnerabilities attached by intelligent adversaries.

In recent years, the advances in data collection and statistical analysis promotes canonical correlation analysis (CCA) available for more advanced research. CCA is the main technique for two-set data dimensionality reduction such that the correlation between the pairwise variables in the common subspace is mutually maximized. Over 80-years of developments, a number of CCA models have been proposed according to different machine learning mechanisms. However, the field lacks an insightful review for the state-of-art developments. This survey targets to provide a well-organized overview for CCA and its extensions. Specifically, we first review the CCA theory from the perspective of both model formation and model optimization. The association between two popular solution methods, i.e., eigen value decomposition (EVD) and singular value decomposition (SVD), are discussed. Following that, we present a taxonomy of current progresses and classify them into seven groups: 1) multi-view CCA, 2) probabilistic CCA, 3) deep CCA, 4) kernel CCA, 5) discriminative CCA, 6) sparse CCA and 7) locality preserving CCA. For each group, we demonstrate two or three representative mathematical models, identifying their strengths and limitations. We summarize the representative applications and numerical results of these seven groups in real-world practices, collecting the data sets and open-sources for implementation. In the end, we provide several promising future research directions that can improve the current state of the art.

The objective of the present study was to investigate the migration characteristics and model of chloride ions in salt storage asphalt mixtures under various working condition through natural leaching test. Three doses of NaCl-based salt storage fillers, two leaching temperatures, and seven freely leaching times were designed to simulate different work environment in this study. Auto ions analyser was utilized to characterize residual chloride ions content in salt storage asphalt mixtures before and after water leaching process, which indirectly stated the movement behaviour of sodium chloride from sample into water. And Fick's second law was used to establish migration model of residual chloride ions content in salt storage asphalt mixtures. The results show that residual chloride ions content and attenuation rate of salt storage asphalt mixtures all decreased with increasing leaching time. Also, high leaching temperature and content of salt storage fillers resulted in accelerating the motion of chloride ions in early leaching stage. The deviation analysis indicated that the established migration model of chloride ions had good accuracy except low usage of salt storage fillers or short water leaching time. The migration model could predict the residual chloride ions content in the salt storage asphalt mixtures under certain work condition.

C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed.

This study investigates the potential drug-excipient interactions of polystyrene sulfonic acid (PSSA) and two weakly basic anticancer drugs, lapatinib (LB) and gefitinib (GB), in amorphous solid dispersions. Based on the strong acidity of the sulfonic acid functional group, PSSA was hypothesized to exhibit specific intermolecular acid-base interactions with both model basic drugs. Ultraviolet (UV) spectroscopy identified red shifts, which correlated well with the color change observed in lapatinib-PSSA solutions. Fourier transform infrared (FTIR) spectra suggest the protonation of the quinazoline nitrogen atom in both model compounds, which agrees well with data from the crystalline ditosylate salt of lapatinib. X-ray photoelectron spectroscopy (XPS) detected increases in binding energy of the basic nitrogen atoms in both lapatinib and gefitinib, strongly indicating protonation of these nitrogen atoms. (15)N solid-state NMR spectroscopy provided direct spectroscopic evidence for protonation of the quinazoline nitrogen atoms in both LB and GB, as well as the secondary amine nitrogen atom in LB and the tertiary amine nitrogen atom in GB. The observed chemical shifts in the LB-PSSA (15)N spectrum also agree very well with the lapatinib ditosylate salt where proton transfer is known. Additionally, the dissolution and physical stability behaviors of both amorphous solid dispersions were examined. PSSA was found to significantly improve the dissolution of LB and GB and effectively inhibit the crystallization of LB and GB under accelerated storage conditions due to the beneficial strong intermolecular acid-base interaction between the sulfonic acid groups and basic nitrogen centers.

Collagenases are the most important group of commercially-produced enzymes. However, even though biological resources are abundant in the sea, very few of these commercially popular enzymes are from marine sources, especially from marine bacteria. We optimized the production of marine collagenases by Pseudoalteromonas sp. SJN2 and investigated the antioxidant activities of the hydrolysates. Media components and culture conditions associated with marine collagenase production by Pseudoalteromonas sp. SJN2 were optimized by statistical methods, namely Plackett–Burman design and response surface methodology (RSM). Furthermore, the marine collagenases produced by Pseudoalteromonas sp. SJN2 were seen to efficiently hydrolyze marine collagens extracted from fish by-products, and remarkable antioxidant capacities of the enzymatic hydrolysates were shown by DPPH radical scavenging and oxygen radical absorbance capacity (ORAC) tests. The final optimized fermentation conditions were as follows: soybean powder, 34.23 g·L−1; culture time, 3.72 d; and temperature, 17.32 °C. Under the optimal fermentation conditions, the experimental collagenase yield obtained was 322.58 ± 9.61 U·mL−1, which was in agreement with the predicted yield of 306.68 U·mL−1. Collagen from Spanish mackerel bone, seabream scale and octopus flesh also showed higher DPPH radical scavenging rates and ORAC values after hydrolysis by the collagenase. This study may have implications for the development and use of marine collagenases. Moreover, seafood waste containing beneficial collagen could be used to produce antioxidant peptides by proteolysis.

A reliable high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for oleanolic acid (OA) determination in rat plasma and liver tissue using glycyrrhetic acid as the internal standard (IS). Plasma and liver homogenate samples were prepared using solid-phase extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The detection was performed by multiple reaction monitoring mode via positive electrospray ionization interface. The calibration curves showed good linearity (R(2) > 0.9997) within the tested concentration ranges. The lower limit of quantification for plasma and liver tissue was ≤0.75 ng/mL. The intra- and inter-day precision and accuracy deviations were within ±15% in plasma and liver tissue. The mean extraction recoveries ranged from 80.8 to 87.0%. In addition, the carryover, matrix effect, stability and robustness involved in the method were also validated. The method was successfully applied to the plasma and hepatic pharmacokinetics of OA after oral administration to rats.

Marine organisms are rich sources of structurally diverse bioactive nitrogenous components. In recent years, numerous bioactive peptides have been identified in a range of marine protein resources, such as antioxidant peptides. Many studies have approved that marine antioxidant peptides have a positive effect on human health and the food industry. Antioxidant activity of peptides can be attributed to free radicals scavenging, inhibition of lipid peroxidation, and metal ion chelating. Moreover, it has also been verified that peptide structure and its amino acid sequence can mainly affect its antioxidant properties. The aim of this review is to summarize kinds of antioxidant peptides from various marine resources. Additionally, the relationship between structure and antioxidant activities of peptides is discussed in this paper. Finally, current technologies used in the preparation, purification, and evaluation of marine-derived antioxidant peptides are also reviewed.

This study investigated the presence of specific drug-excipient interactions in amorphous solid dispersions of lapatinib (LB) and four commonly used pharmaceutical polymers, including Soluplus, polyvinylpyrrolidone vinyl acetate (PVPVA), hydroxypropylmethylcellulose acetate succinate (HPMCAS), and hydroxypropylmethylcellulose phthalate (HPMCP). Based on predicted pKa differences, LB was hypothesized to exhibit a specific ionic interaction with HPMCP, and possibly with HPMCAS, while Soluplus and PVPVA were studied as controls without ionizable functionality. Thermal studies showed a single glass transition (Tg) for each dispersion, in close agreement with predicted values for Soluplus, PVPVA, and HPMCAS systems. However, the Tg values of LB-HPMCP solid dispersions were markedly higher than predicted values, indicating a strong intermolecular interaction between LB and HPMCP. (15)N solid-state NMR provided direct spectroscopic evidence for protonation of LB (i.e., salt formation) within the HPMCP solid dispersions. (1)H T1 and (1)H T1ρ relaxation studies of the dispersions supported the ionic interaction hypothesis, and indicated multiple phases in the cases of excess drug or polymer. In addition, the dissolution and stability behavior of each system was examined. Both acidic polymers, HPMCAS and HPMCP, effectively inhibited the crystallization of LB on accelerated stability, likely owing to beneficial strong intermolecular hydrogen and/or specific ionic bonds with the acidic polymers. Soluplus and PVPVA showed poor physical properties on stability and subsequently poor crystallization inhibition.

TiO2-based metal oxide affinity chromatography (MOAC) nanomaterials show high potential in phosphoproteome mass-spectrometric (MS) analysis. However, a drawback of TiO2 nanomaterials is poor water solubility, which greatly reduces the enrichment efficiency of phosphopeptides and eventually limits their use in phosphoproteome MS analysis. In this work, a hydrophilic TiO2 hybrid material (denoted as NH2@TiO2) is successfully designed with 1,6-hexanediamine modified on the surface of TiO2 nanoparticles and applied as a biofunctional adsorbent for selective enrichment of phosphopeptides. The novel TiO2 hybrid material with high hydrophilicity and biocompatibility is characterized using scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and infrared (IR) spectroscopy, and its performance in selective enrichment of phosphopeptides is evaluated with the standard protein digests, human serum and the tryptic digests of nonfat milk.

In order to prepare antioxidant peptide through hydrolyzing low-value protein resources with bacterial extracellular proteases and to discover novel proteases, crude extracellular protease from Pseudoalteromonas sp. SHK1-2 was obtained through fermentation which was used to hydrolyze collagen extracted from Cirrhinus molitorella skin. Small peptide fraction was isolated from hydrolysate by ultrafiltration and Sephadex LH-20 size exclusion chromatography and showed 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity (35.6%±7%), oxygen radical absorbance capacity and inhibition of DNA oxidation damage. The molecule weight was 776.2 Da, and amino acid sequence was Thr-Ala-Gly-His-Pro- Gly-Thr-His through liquid chromatography mass spectrum. Our findings suggest that peptide obtained from low-value protein of fish waste by hydrolysis with bacterial protease has antioxidant activity.为了以细菌胞外蛋白酶酶解低值蛋白资源制备抗氧化活性肽以及挖掘新型蛋白酶，采用液体发酵培养的方法对假交替单胞菌Pseudoalteromonas sp. SHK1-2 进行发酵产酶，获得胞外蛋白酶粗酶液用于酶解热水法提取的鲮鱼胶原蛋白，通过超滤、sephadex LH-20 分子筛层析获得具有DPPH 自由基清除能力 (35.6%±7%)、氧自由基清除能力 (Oxygen radical absorbance capacity，ORAC) 及DNA 氧化损伤抑制活性的小分子肽，液相色谱质谱联用鉴定该活性肽分子量为776.2 Da，预测氨基酸序列为Thr-Ala-Gly-His-Pro-Gly-Thr-His。ORAC检测验证了人工合成的多肽同样具有抗氧化活性。研究表明细菌胞外蛋白酶在低值资源高值化中具有一定的应用前景，对于新型蛋白酶及其应用的挖掘具有一定的参考意义。.

Background SiNiSan (SNS) is a traditional Chinese medicine (TCM) prescription that has been widely used in the clinical treatment of irritable bowel syndrome (IBS). However, the underlying active substances and molecular mechanisms remain obscure. Purpose A bioinformatics/topology based strategy was proposed for identification of the drug targets, therapeutic agents and molecular mechanisms of SiNiSan against irritable bowel syndrome. Materials and methods In this work, a bioinformatics/network topology based strategy was employed by integrating ADME filtering, text mining, bioinformatics, network topology, Venn analysis and molecular docking to uncover systematically the multicomponent synergy mechanisms. In vivo experimental validation was executed in a Visceral Hypersensitivity (VHS) rat model. Results 76 protein targets and 109 active components of SNS were identified. Bioinformatics analysis revealed that 116 disease pathways associated with IBS therapy could be classified into the 19 statistically enriched functional sub-groups. The multi-functional co-synergism of SNS against IBS were predicted, including inflammatory reaction regulation, oxidative-stress depression regulation and hormone and immune regulation. The multi-component synergetic effects were also revealed on the herbal combination of SNS. The hub-bottleneck genes of the protein networks including PTGS2, CALM2, NOS2, SLC6A3 and MAOB, MAOA, CREB1 could become potential drug targets and Paeoniflorin, Naringin, Glycyrrhizic acid may be candidate agents. Experimental results showed that the potential mechanisms of SiNiSan treatment involved in the suppression of activation of Dopaminergic synapse and Amphetamine addiction signaling pathways, which are congruent with the prediction by the systematic approach. Conclusion The integrative investigation based on bioinformatics/network topology strategy may elaborate the multicomponent synergy mechanisms of SNS against IBS and provide the way out to develop new combination medicines for IBS.

Cilnidipine (CN) is a novel dihydropyridine calcium antagonist that is practically insoluble in aqueous media and exhibits a low oral bioavailability or limited clinical efficacy.This study investigated the effects of three commercial and chemically diverse polymers - PVP, PVP/VA and Soluplus - on crystallization tendency and in vitro dissolution profiles of CN in order to determine an optimum carrier for composing the preferred solid dispersion (SD) of CN.All these co-evaporated systems were characterized up to 3 months by thermoanalytical (DSC), crystallographic (POM, PXRD), microscopic (SEM) and spectroscopic (FTIR) techniques.The results showed that the polymers could be sorted by their effects of inhibiting CN crystallization in the ascending order: Soluplus, PVP/VA, PVP. The sequence was in accordance with that of the strength of drug-polymer hydrogen bonds revealed by FTIR spectra. It could be ascribed to relative hydrogen-bonding acceptor strengths of N-vinylpyrrolidone moiety in the polymer molecules. On the other hand, all the SDs showed enhanced dissolution profiles compared to pure CN alone. On their effects of enhancing CN dissolution, the polymers could be sorted in the descending order: Soluplus, PVP, PVP/VA.It implied that the dissolution behavior of CN could bear a close relationship to both hydration capacity and hydrogen-bonding interaction tendency of moieties of the polymers. It might suggest an optimal formulation for CN comprising both PVP and Soluplus.

In this study, a reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated for simultaneous quantification of seven flavonoids including nobiletin, tangeretin, naringin, naringenin, hesperidin, neohesperidin, and hesperetin in Pericarpium Citri reticulatae (PCR). Ultrasonic-assisted extraction (UAE) conditions were optimized using response surface methodology (RSM) to obtain maximum extractive contents of the seven flavonoids. A Box–Behnken design (BBD) was employed to study the main effects and interactions of independent variables. Following UAE, chromatographic separation was accomplished on a C18 column with a linear gradient elution. Full validation of the assay was implemented. It was found that this method had good linearity and specificity. Limit of detection (LOD) and limit of quantification (LOQ) for the tested flavonoids were 0.17–0.49and 0.75–1.75 μg/mL, respectively. Precisions (relative standard deviation, RSD) for intra- and interday were better than 4.54 %. Reproducibility was less than 4.91 %. Analyzed samples were stable for at least 48 h. Analysis had a good robustness (RSD

Extracted salmon skin collagen was hydrolysed with the free or immobilized extracellular protease of Vibrio sp. SQS2-3. The hydrolysate exhibited anti-freezing activity (>3 kDa) and antioxidant activity (

Pseudoalteromonas sp. CSN423, a marine strain, can express a major protease designated as E423 and it was secreted into the supernatant. To improve the protease E423 yield, Pseudoalteromonas sp. CSN423 was subjected to mutagenesis using UV irradiation. Mutant strain with 5.1-fold higher protease yield was isolated and named as Pseudoalteromonas sp. CSN423-M. Three protease bands were detected by zymography with casein as substrate, and results of mass spectrometry (MS) showed that two lower molecular weight protein bands were the same protease but with different mature forms. The entire protease operon was sequenced and no mutation was found. Mutant strain-associated changes of expression levels of protease synthesis and secretion-related genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Mutant strain had higher expression of e423 than wild-type strain. Such result was consistent with protease activity profiles. Moreover, the mutant strain had higher transcriptional levels of citrate synthase (cs), α-ketoglutarate decarboxylase (kgd), cytochrome c oxidase subunit I (coxI), tolC, hlyD (membrane protein), luxR3, luxO, and luxT (transcriptional regulator). However, hexokinase (hk), pyruvate dehydrogenase E1 (pd-e1), epsD (membrane protein), and luxR1 remained unchanged, and luxR2 decreased sharply in the mutant. These results suggested that the redox pathway was promoted in the mutant strain, and LuxR family transcriptional regulators in Pseudoalteromonas spp. may play some role in regulating protease expression. Meanwhile, the secretion of extracellular protease was closely related to ABC transport system. These results may shed some light on the molecular mechanism underlying higher yield of protease E423 from Pseudoalteromonas sp. CSN423-M.

Si-Ni-San (SNS) is a widely used traditional Chinese medicine formula (TCMF) in treating various diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, a liquid chromatography coupled with diode array detection and triple-quadrupole spectrometry (LC-DAD-MS/MS) method was developed for detection and identification of SNS metabolites in rat plasma and urine at a normal clinical dosage. Accurate structural elucidation was performed using MS/MS, UV data and n-octanol/water partition coefficient. Based on the proposed strategy, 36 absorbed compounds and 29 metabolites in plasma and 33 metabolites in urine were detected by a highly sensitive MRM method. Our results indicated that phase II reactions (e.g., methylation, glucuronidation and sulfation) were the main metabolic pathways of gallic acid and flavanones, while phase I reactions (e.g., hydroxylation) were the major metabolic reaction for triterpenoid saponins. The metabolite profile analysis of SNS provided a comprehensive understanding of the in vivo metabolic fates of constituents in SNS. Moreover, the results in this work demonstrated the present strategy based on the combination of chromatographic, spectrophotometric, mass-spectrometric, and software prediction to detect and identify metabolites was effective and reliable. And such a strategy may also be extended to investigate the metabolism of other TCMF.

A rapid ultra-high-performance liquid chromatographic-tandem mass spectrometric (UHPLC-MS-MS) method has been developed for rapid screening and quantitative analysis of sulfonate derivatives (SDs) in commercial white peony root. Separation was performed on an Agilent Zorbax Eclipse Plus-C18 column by gradient elution with acetonitrile-0.1% (v/v) formic acid as the mobile phase. In-source fragmentation was used to generate the characteristic fragment ion at m/z 259 and to screen for nine SDs. Detection of these SDs was further performed in multiple reaction monitoring (MRM) mode to improve sensitivity and to quantify the two SDs paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate. The method was validated for specificity, linearity, limits of detection and quantification, precision, accuracy, and matrix effects. Nine commercial white peony root samples were examined by use of this method, which revealed great variety in the paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate content.

The binary systems of capsaicin (CP) and β-cyclodextrin (βCD) or hydroxypropyl-β-cyclodextrin (HPβCD) were investigated in an attempt to improve formulations of this pesticide. UV spectral shift methods indicated guest–host complex formation between CP and the two cyclodextrins (CDs). Phase solubility analysis showed Bs type diagrams with βCD, AL type with HPβCD indicating the formation of an inclusion complex at 1:1 stoichiometric ratio in solution state. Solubility profiles indicated a 50-fold enhancement of CP solubility could be achieved in the presence of 60 mM HPβCD with respect to CP alone. Solid co-evaporated systems (CES) with 1:0.5–1:5 molar ratios of CP/CDs were physicochemically characterized, revealing that the true inclusion complexes could be formed in the solid CP/βCD systems with 1:5 molar ratio and in the solid CP/HPβCD systems with the molar ratios more than 1:3, respectively. In contrast, crystalline drug was detectable in all other systems. Compared with corresponding physical mixtures (PMs), the CES exhibited significant enhancement with regard to CP dissolution and the protection from CP degradation under the accelerated conditions. It was also revealed that complexation of CP with HPβCD had a pronounced improved effect on the pesticide formulations and greatly reduced the amount of CP adsorbed in the soil. These results demonstrate that HPβCD may be a preferred excipient, enabling more efficient and intelligent use of CP/CDs inclusion complexes in the development of pesticide formulations.

Tong-Xie-Yao-Fang (TXYF), a famous traditional Chinese medicine formula, has efficient effects on treatment of the diarrhea-predominant irritable bowel syndrome (D-IBS), a disease with high incidence worldwide. However, the active principles for this complex formula have not been fully explored so far. In this paper, high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) was applied for the qualitative and quantitative analysis of major chemical constituents in TXYF. Two monoterpene glycosides, one chromone and five polymethoxylated flavones were tentatively characterized based on the retention times, UV spectra and MS data. Fifteen compounds were unambiguously identified by comparison with reference standards. Constituents such as lactone and steroidal, which could not be found by single HPLC method due to the low content in the formula, were identified in this paper. Seven compounds (gallic acid, prim-O-β-D-glucosylcimifugin, paeoniflorin, cimifugin, naringin, hesperidin and 4'-O-β-D-glucosyl-5-O-methylvisamminol) were quantified by HPLC-DAD using a C18 column and gradient elution with acetonitrile and water-0.1% formic acid. The method exhibited intra- and inter-day precision of less than 2.35% and 3.14%, respectively. The LODs and the LOQs for the analytes were less than 0.47 and 1.82 μg ml(-1), respectively. The overall recoveries ranged from 96.82% to 102.47%, with the R.S.D. ranging from 1.17% to 3.94%. These results demonstrated that our present method was effective and reliable for comprehensive quality evaluation of TXYF. Meanwhile, the study might provide the chemical evidence for revealing the material basis of its therapeutic effects.

For CD-based formulations, it is important to directly monitor the complexation status of the drug present in final dosage form pharmaceuticals. In this work, Raman spectroscopy was explored for the detection and identification of the Nimodipine/hydroxypropyl-β-cyclodextrin (NMD/HPβCD) complexation present in the tablet. The evident, consistent Raman spectral changes in the shift, height ratio and area ratio for the characteristic bands of NMD molecule were featured in the NMD/HPβCD complex, and employed to distinguish between complexed and uncomplexed NMD in tablets. A number of practical issues for the Raman measurements performed on the tablets were considered and addressed. The Raman approach and dissolution test were applied to different tablets prepared experimentally at variable granulations. The results demonstrated that the Raman approach can serve as a promising methodology for tablet identification on the complexation. Wet granulation facilitated the process-induced transformations in the complexation. The adequate ethanol in the granulating fluid appeared optimized for the complexation of the two components. The spectral characteristics for dissociation in the tablets were in full accordance with the observations of their diminished initial dissolution parameters. It implied the possibility that the tablet dissolution can be predicted from the Raman interpretation

Seven samples of seeds from the genera Mucuna and Stizolobium (Leguminosae) were collected throughout southern China. HPLC analysis revealed l-dopa concentrations ranging from 3.9 to 6.2%. The seeds of the most commercially available species, S. pruriens var. utilis, collected biweekly during the fruit period, were also assayed using the same method, and their l-dopa concentrations were found to vary between 4.2 to 10.6%. Other nitrogenous compounds, which are significant in chemotaxonomy between the two plant types or have potential influence on analysis of l-dopa in the seeds were identified as stizolamine 3,4-dihydro-5-hydroxymethyl-4-methyl-3-oxopyrazinyl-guanidine), l-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, and its derivatives.

There is a growing concern for the sensitive quantification of multiple components using advanced data acquisition method in herbal medicines (HMs). An improved and rugged UPLC-MS/MS method has been developed and validated for sensitive and rapid determination of multiply analytes from Tong-Xie-Yao-Fang (TXYF) decoction in three biological matrices (plasma/brain tissue/urine) using geniposide and formononetin as internal standards. After solid-phase extraction, chromatographic separation was performed on a C18 column using gradient elution. Quantifier and qualifier transitions were monitored using novel Triggered Dynamic multiple reaction monitoring (TdMRM) in the positive ionization mode. A significant peak symmetry and sensitivity improvement in the TdMRM mode was achieved as compared to conventional MRM. The reproducibility (RSD%) was ≤7.9% by applying TdMRM transition while the values were 6.8-20.6% for MRM. Excellent linear calibration curves were obtained under TdMRM transitions over the tested concentration ranges. Intra- and inter-day precisions (RSD%) were ≤14.2% and accuracies (RE%) ranged from -9.6% to 10.6%. The validation data of specificity, carryover, recovery, matrix effect and stability were within the required limits. The method was effectively applied to simultaneously detect and quantify 1 lactone, 2 monoterpene glucosides, 1 alkaloid, 5 flavonoids and 2 chromones in plasma, brain tissue and urine after oral administration of TXYF decoction. In conclusion, this new and reliable method is beneficial for quantification and confirmation assays of multiply components in complex biological samples.

There is a growing need both clinically and experimentally to improve the determination of the blood levels of multiple chemical constituents in herbal medicines. The conventional multiple reaction monitoring (cMRM), however, is not well suited for multi-component determination and could not provide qualitative information for identity confirmation. Here we apply a dynamic triggered MRM (DtMRM) algorithm for the quantification of 20 constituents in an herbal prescription Bu-Zhong-Yi-Qi-Tang (BZYQT) in rat plasma. Dynamic MRM (DMRM) dramatically reduced the number of concurrent MRM transitions that are monitored during each MS scan. This advantage has been enhanced with the addition of triggered MRM (tMRM) for simultaneous confirmation, which maximizes the dwell time in the primary MRM quantitation phase, and also acquires sufficient MRM data to create a composite product ion spectrum. By allowing optimized collision energy for each product ion and maximizing dwell times, tMRM is significantly more sensitive and reliable than conventional product ion scanning. The DtMRM approach provides much higher sensitivity and reproducibility than cMRM.

Based on digital image analysis techniques and inhibitor-substrate immersing zymography, intrinsic properties of each active component in the enzymatic system secreted by marine bacteria were studied. This method provides an easy way to characterize the proteases in situ, which can be further verified by Mass spectrometry. Compared to the Folin phenol method, a traditional method used to determine proteases activities, the inhibitor-substrate immersing zymography method coupled with digital image analysis used in this study could determine caseinolytic activity and measure gelatinolytic activity at the same time. The effect on activities of extracellular proteases by inhibitor (phenylmethylsulfonyl fluoride or 1, 10-Phenanthroline) can be quantified by gray value changes of the corresponding band after electrophoretic separation. Because of its high throughput, great sensitivity, and convenient operation, inhibitor-substrate immersing zymography can be used to demonstrate the natural diversity of protein hydrolases and multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of proteases secreted by marine bacteria.

We evaluated the ability of hydroxypropyl-beta-cyclodextrin (HPbetaCD) to influence the percutaneous absorption of capsaicin (CP) through isolated rat skin. Phase solubility analysis and phase distribution studies suggested the potential of HPbetaCD as a solubilizer and permeation enhancer for CP. In vitro permeation studies showed the trend that, the penetration flux (J(s)) of CP increased with the increasing concentration of HPbetaCD from 0 to 2.20% (w/v), and then decreased dramatically when the concentration of HPbetaCD kept on increasing up to 15% (w/v). 2.20% (w/v) of HPbetaCD provided both just adequate solubilization and preferred J(s) for the permeation of CP (0.075%, w/v). Similar change patterns of the permeation parameters were also observed in the hydrogels, but the J(s) of CP was reduced significantly along with the increasing concentration of Carbopol U21. Histological analysis showed an invasive action of HPbetaCD on the stratum corneum (SC) of rat skin, which could only reduce the lag time (T(L)) but could not increase the J(s) of CP. On the other hand, the complexation of HPbetaCD with CP could attenuate this invasive action. It is inferred that excess of HPbetaCD could not only disturb the percutaneous absorption of CP but also disrupt the structure of SC.

Chewing gums containing catechins were prepared by applying a novel solid dispersion and hot‐melt fluid bed coating method. The effect of varying levels of Eudragit® coating to the granules on the prolonged release of catechins from chewing gum was investigated. In vivo release profiles of catechins from the formulations were investigated to determine its release kinetics. Dissolution data were used as the primary basis for comparison. The equations with zero order, first‐order, or square root of time release kinetics were used to fit data. The results showed that the releases of catechins from chewing gum were well extended with the growing coating level of Eudragit® to the granules. The statistical analysis using nonlinear regression software suggested that the Higuchi equation describes the data better than others. The relatively high degree of fit using the Higuchi equation implies that the kinetic process is involved in the release of drug from the dosage form. A possible release mechanism based on this profile is presented in this report. This approach suggested the potential of using chewing gum as a promising controlled‐release delivery system of drugs. The effect of polyvinyl acetates with different crosslinkings on the release profiles of catechins was also studied. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:293–299, 2004