Journal of Pharmaceutical Analysis

Recent advances and perspectives of nucleic acid detection for coronavirus

Minzhe Shen, Ying Zhou, Jiawei Ye, Abdu Ahmed Abdullah AL-maskri, Yu Kang, Su Zeng, Sheng Cai

2020, 10(2): 97-101.

Abstract(1232) PDF(18)

Abstract:
The recent pneumonia outbreak caused by a novel coronavirus (SARS-CoV-2) is posing a great threat to global public health. Therefore, rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments, saving people's lives and preventing epidemics. It is important to establish a quick standard diagnostic test for the detection of the infectious disease (COVID-19) to prevent subsequent secondary spread. Polymerase chain reaction (PCR) is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity. Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion. A variety of improved or new approaches also have been developed. This review summarizes the currently available detection methods for coronavirus nucleic acid. It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.

Molecular immune pathogenesis and diagnosis of COVID-19

Xiaowei Li, Manman Geng, Yizhao Peng, Liesu Meng, Shemin Lu

2020, 10(2): 102-108.

Abstract(1084) PDF(20)

Abstract:
Coronavirus disease 2019 (COVID-19) is a kind of viral pneumonia which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of SARS-CoV-2 has been marked as the third introduction of a highly pathogenic coronavirus into the human population after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coro-navirus (MERS-CoV) in the twenty-first century. In this minireview, we provide a brief introduction of the general features of SARS-CoV-2 and discuss current knowledge of molecular immune pathogenesis, diagnosis and treatment of COVID-19 on the base of the present understanding of SARS-CoV and MERS-CoV infections, which may be helpful in offering novel insights and potential therapeutic targets for combating the SARS-CoV-2 infection.

Hollow fiber-based liquid phase microextraction followed by analytical instrumental techniques for quantitative analysis of heavy metal ions and pharmaceuticals

Wajid Ali Khan, Muhammad Balal Arain, Yadollah Yamini, Nasrullah Shah, Tasneem Gul Kazi, Stig Pedersen-Bjergaard, Mohammad Tajik

2020, 10(2): 109-122.

Abstract(194) PDF(8)

Abstract:
Hollow-fiber liquid-phase microextraction (HF-LPME) and electromembrane extraction (EME) are miniaturized extraction techniques, and have been coupled with various analytical instruments for trace analysis of heavy metals, drugs and other organic compounds, in recent years. HF-LPME and EME provide high selectivity, efficient sample cleanup and enrichment, and reduce the consumption of organic sol-vents to a few micro-liters per sample. HF-LPME and EME are compatible with different analytical in-struments for chromatography, electrophoresis, atomic spectroscopy, mass spectrometry, and electrochemical detection. HF-LPME and EME have gained significant popularity during the recent years. This review focuses on hollow fiber based techniques (especially HF-LPME and EME) of heavy metals and pharmaceuticals (published 2017 to May 2019), and their combinations with atomic spectroscopy, UV-VIS spectrophotometry, high performance liquid chromatography, gas chromatography, capillary elec-trophoresis, and voltammetry.

Quantitative computed tomography analysis for stratifying the severity of Coronavirus Disease 2019

Cong Shen, Nan Yu, Shubo Cai, Jie Zhou, Jiexin Sheng, Kang Liu, Heping Zhou, Youmin Guo, Gang Niu

2020, 10(2): 123-129.

Abstract(329) PDF(4)

Abstract:
To examine the feasibility of using a computer tool for stratifying the severity of Coronavirus Disease 2019 (COVID-19) based on computed tomography (CT) images. We retrospectively examined 44 confirmed COVID-19 cases. All cases were evaluated separately by radiologists (visually) and through an in-house computer software. The degree of lesions was visually scored by the radiologist, as follows, for each of the 5 lung lobes:0, no lesion present;1,<1/3 involvement;2,>1/3 and<2/3 involvement;and 3,>2/3 involvement. Lesion density was assessed based on the proportion of ground-glass opacity (GGO), consolidation and fibrosis of the lesions. The parameters obtained using the computer tool included lung volume (mL), lesion volume (mL), lesion percentage (%), and mean lesion density (HU) of the whole lung, right lung, left lung, and each lobe. The scores obtained by the radiologists and quantitative results generated by the computer software were tested for correlation. A Chi-square test was used to test the consistency of radiologist- and computer-derived lesion percentage in the right/left lung, upper/lower lobe, and each of the 5 lobes. The results showed a strong to moderate correlation between lesion percentage scores obtained by radiologists and the computer software (r ranged from 0.7679 to 0.8373, P < 0.05), and a moderate correlation between the proportion of GGO and mean lesion density (r=-0.5894, P<0.05), and proportion of consolidation and mean lesion density (r=0.6282, P<0.05). Computer-aided quantification showed a statistical significant higher lesion percentage for lower lobes than that assessed by the radiologists (x2 = 8.160, P = 0.004). Our experiments demonstrated that the computer tool could reliably and accurately assess the severity and distribution of pneumonia on CT scans.

Erucic acid from Isatis indigotica Fort. suppresses influenza A virus replication and inflammation in vitro and in vivo through modulation of NF-kB and p38 MAPK pathway

Xiaoli Liang, Yuan Huang, Xiping Pan, Yanbing Hao, Xiaowei Chen, Haiming Jiang, Jing Li, Beixian Zhou, Zifeng Yang

2020, 10(2): 130-146.

Abstract(107) PDF(4)

Abstract:
Isatis indigotica Fort. (Ban-Lan-Gen) is an herbal medicine prescribed for influenza treatment. However, its active components and mode of action remain mostly unknown. In the present study, erucic acid was isolated from Isatis indigotica Fort., and subsequently its underlying mechanism against influenza A virus (IAV) infection was investigated in vitro and in vivo. Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity. Erucic acid was found to exert inhibitory effects on IAV or viral (v) RNA-induced pro-inflam-matory mediators as well as interferons (IFNs). The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling. Furthermore, the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3 (ISGF-3), and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells. Additionally, IAV- or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid. In vivo, erucic acid administration consistently displayed decreased lung viral load and viral antigens expression. Meanwhile, erucic acid markedly reduced CD8+cytotoxic T lymphocyte (CTL) recruitment, pro-apoptotic signaling, hyperactivity of multiple signaling pathways, and exacerbated immune inflammation in the lung, which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1) strain infection. Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury, suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.

Metabolic profiling of four synthetic stimulants, including the novel indanyl-cathinone 5-PPDi, after human hepatocyte incubation

David Fabregat-Safont, Marie Mardal, Juan V. Sancho, Félix Hernández, Kristian Linnet, María Ibá(n)ez

2020, 10(2): 147-156.

Abstract(148) PDF(1)

Abstract:
Synthetic cathinones are new psychoactive substances that represent a health risk worldwide. For most of the 130 reported compounds, information about toxicology and/or metabolism is not available, which hampers their detection (and subsequent medical treatment) in intoxication cases. The principles of forensic analytical chemistry and the use of powerful analytical techniques are indispensable for stab-lishing the most appropriate biomarkers for these substances. Human metabolic fate of synthetic cathinones can be assessed by the analysis of urine and blood obtained from authentic consumers;however, this type of samples is limited and difficult to access. In this work, the metabolic behaviour of three synthetic cathinones (4-CEC, 4-CPrC and 5-PPDi) and one amphetamine (3-FEA) has been evalu-ated by incubation with pooled human hepatocytes and metabolite identification has been performed by high-resolution mass spectrometry. This in vitro approach has previously shown its feasibility for obtaining excretory human metabolites. 4-CEC and 3-FEA were not metabolised, and for 4-CPrC only two minor metabolites were obtained. On the contrary, for the recently reported 5-PPDi, twelve phase I metabolites were elucidated. Up to our knowledge, this is the first metabolic study of an indanyl-cathinone. Data reported in this paper will allow the detection of these synthetic stimulants in intoxi-cation cases, and will facilitate future research on the metabolic behaviour of other indanyl-based cathinones.

Analysis of TRPA1 antagonist, A-967079, in plasma using high-performance liquid chromatography tandem mass-spectrometry

Obed A. Gyamfi, Nesta Bortey-Sam, Abigail B. Donkor, Carl W. White, Brian A. Logue

2020, 10(2): 157-163.

Abstract(123) PDF(3)

Abstract:
The noxious effects from exposure to toxic inhalation hazards (TIHs, such as isocyanates, chlorine, etc.) are known to be triggered by the activation of transient receptor potential ankyrin 1 (TRPA1) ion channel. Antagonists of TRPA1 have shown near complete attenuation of the noxious effects from TIH exposure. One of the TRPA1 antagonists, (1E,3E)-1-(4-fluorophenyl)-2-methyl-1-pentene-3-one oxime (A-967079), has shown impressive efficacy, high selectivity, high potency, and oral bioavailability. Although a vali-dated method to quantify A-967079 in biological matrices is vital for the further development of A-967079 as a therapeutic agent, no method for its analysis from any matrix is currently available. Hence, a rapid and simple HPLC-MS/MS method was developed and validated to quantify A-967079 in rabbit plasma. The method presented here features an excellent LOD of 25 nM and a wide linear range (0.05-200 μM), with good accuracy and precision (100 ± 10.5% and <14.2% relative standard deviation, respectively). The stability of A-967079 in plasma was excellent for most of the storage conditions evaluated. The method was successfully applied to determine A-967079 from treated animals and it may facilitate the development of this TRPA1 antagonist as a therapeutic agent against the noxious effects of TIH exposure.

Biophysical interactions between silver nanoparticle-albumin interface and curcumin

Vinod D. Jaiswal, P.M. Dongre

2020, 10(2): 164-177.

Abstract(122) PDF(2)

Abstract:
Active targeted drug delivery methods facilitate effective uptake of functionalized nanoparticles through receptor-mediated transcytosis. In recent years, albumin-nanoparticle interaction has been critically examined so that this functionalized nanoparticle can be efficiently loaded with drugs. The present investigation aims at understanding the adsorption of Bovine Serum Albumin (BSA) on Silver Nano-particle (SNP) surface, preparation of soft conjugates (SC) and hard conjugates (HC) of BSA-functionalized SNP (SNP-BSA), and their interaction with curcumin (CUR). HC contains tightly bound BSA whereas SC involves tightly and loosely bound BSA. Increase in the hydrodynamic radii of conjugates was observed upon SNP incubation with increased concentration of BSA. Three different SNP-BSA conjugate ratios were selected to study their interaction with CUR. Fluorescence spectroscopy showed a strong association between CUR and SNP:BSA conjugates. However, binding varied with a change in the conjugate ratio. Circular Dichroism (CD)/Fourier Transform Infrared (FTIR) spectroscopy revealed the alterations in the secondary structure of BSA upon CUR binding to the conjugates. Zeta potential data indicated stable conjugate formation. CUR in SNP:BSA conjugate was found to have a higher half-life as compared to the control. We believe that this is the first biophysical characterization report of conjugates that can be effectively extrapolated for targeted drug delivery.

Potential synergic mechanism of Wutou-Gancao herb-pair by inhibiting efflux transporter P-glycoprotein

Yufei He, Zihong Wei, Ying Xie, Xiulin Yi, Yong Zeng, Yazhuo Li, Changxiao Liu

2020, 10(2): 178-186.

Abstract(175) PDF(3)

Abstract:
Wutou-Gancao herb-pair is extensively used to attenuate the toxicity and enhance the efficacy of aconite. In this study, potential synergic mechanism of the herb pair was investigated by utilizing multiple ap-proaches. In silico and in vitro Caco-2 cell models were applied to study the potential binding mode of bioactive ingredients existing in liquorice with P-glycoprotein (P-gp), as well as the inhibition effects on P-gp. Additionally, anti-inflammatory activity of aconitine (AC) combined with active ingredients of liquorice, as well as pharmacokinetic patterns of AC after co-administration was investigated. Anti-inflammatory effect of AC (1 mg/kg) in rats was enhanced in combination with bioactive ingredients of liquorice (10 mg/kg). In the meanwhile, the exposure of AC in vivo was altered, in terms of Cmax and AUC. For instance, the Cmax and AUC were increased to 1.9 and 1.3 folds, respectively, when used in combination with liquiritigenin. The in silico study revealed the potential binding mode with outward facing conformation of P-gp. The resulting data obtained from transport of rhodamine-123 (Rh-123) across Caco-2 cell monolayer further indicated that the function of P-gp was inhibited by chemicals in liquorice. The synergic effect was therefore proposed to be attributed to inhibition of P-gp by liquorice since AC has been demonstrated to be the substrate of P-gp. The resuls revealed that potential synergic mechanism of Wutou-Gancao herb-pair by inhibiting function of key efflux transporter P-gp to enhance the exposure of AC in systematic circulation, and further the anti-inflammatory effect, which helps clarify the compatibility rationale of these two herbs.

Inhibition of cell proliferation and migration by Oroxylum indicum extracts on breast cancer cells via Rac1 modulation

Benjaporn Buranrat, Sirintra Noiwetch, Tippaporn Suksar, Aphimook Ta-ut

2020, 10(2): 187-193.

Abstract(137) PDF(4)

Abstract:
In this study, we investigated how Oroxylum indicum leaf and fruit extracts affect the viability and migration of MCF-7 breast cancer cells and the mechanisms of action responsible for these effects. MCF-7 cells treated with the extracts were examined using the sulforhodamine B, colony formation and caspase 3 activity assays, and by Western blotting. O. indicum extracts were found to inhibit MCF-7 cell growth in a concentration-and time-dependent manner, with 48 h IC50 values of 57.02 ± 2.85μg/mL and 131.3 ± 19.2μg/mL for leaf and fruit extracts, respectively. Further, the O. indicum leaf extract caused a reduction in MCF-7 cell viability, induction of MCF-7 cell apoptosis and ROS formation, and an increase in caspase 3 activity. Also, the two extracts inhibited MCF-7 cell migration and reduced both MMP 9 and ICAMP1 gene expression and MMP9 protein expression. Additionally, O. indicum extracts greatly reduced expression of the cell cycle regulatory protein Rac1 in the mevalonate pathway. In summary, O. indicum leaf and fruit extracts reduce breast cancer cell growth, cell viability and cell migration. O. indicum constituents could, therefore, be useful for augmenting the activity of chemotherapeutic drugs employed to treat breast cancer.

Prediction of fibril formation by early-stage amyloid peptide aggregation

Jiaojiao Hu, Huiyong Sun, Haiping Hao, Qiuling Zheng

2020, 10(2): 194-199.

Abstract(72) PDF(2)

Abstract:
Amyloid fibrils are found in systemic amyloidosis diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes. Currently, these diseases are diagnosed by observation of fibrils or plaques, which is an ineffective method for early diagnosis and treatment of disease. The goal of this study was to develop a simple and quick method to predict the possibility and speed of fibril formation before its occurrence. Oligomers generated from seven representative peptide segments were first isolated and detected by ion-mobility mass spectrometry (IM-MS). Then, their assemblies were disrupted using formic acid (FA). Interestingly, oligomers that showed small ion intensity changes upon FA addition had rapid fibril formation. By contrast, oligomers that had large ion intensity changes generated fibrils slowly. Two control peptides (aggregation/no fibrils and no aggregation/no fibrils) did not show changes in their ion intensities, which confirmed the ability of this method to predict amyloid formation. In summary, the developed method correlated MS intensity ratio changes of peptide oligomers on FA addition with their amyloid propensities. This method will be useful for monitoring peptide/protein aggregation behavior and essential for their mechanism studies.

2020 Vol. 10, No. 2