Drug Research Articles

Ethno-Taxonomical Studies of some Ayurvedic Drugs

Ethno-taxonomical explanation of some plants forming constituents of Ayurvedic drugs has been provided in the present paper.

Species identification of biological ingredients in herbal product, Gurigumu-7, based on DNA barcoding and shotgun metagenomics.

Accurate identification the species composition in mixtures poses a significant challenge, especially in processed mixtures comprising multiple species, such as those found in food and pharmaceuticals. Therefore, we have attempted to utilize shotgun metabarcoding technology to tackle this issue. In this study, the method was initially established using two mock samples of the Mongolian compound preparation Gurigumu-7 (G-7), which was then applied to three pharmaceutical products and 12 hospital-made preparations. A total of 119.72 Gb of raw data sets were obtained through shotgun metagenomic sequencing. By combining ITS2, matK, and rbcL, all the labeled bio-ingredients specified in the G-7 prescription can be detected, although some species may not be detectable in all samples. The prevalent substitution of Akebia quinata can be found in all the pharmaceutical and hospital samples, except for YN02 and YN12. The toxic alternative to Akebia quinata, Aristolochia manshuriensis, was exclusively identified in the YN02 sample. To further confirm this result, we validated it in YN02 using HPLC and real-time PCR with TaqMan probes. The results showed that aristolochic acid A (AAA) was detected in YN02 using HPLC, and the ITS2 sequence of Aristolochia manshuriensis has been validated in YN02 through qPCR and the use of a TaqMan probe. This study confirms that shotgun metabarcoding can effectively identify the biological components in Mongolian medicine compound preparation G-7. It also demonstrates the method's potential to be utilized as a general identification technique for mixtures containing a variety of plants.

Oregano Essential Oil in Livestock and Veterinary Medicine.

With a growing global concern over food safety and animal welfare issues, the livestock and veterinary industries are undergoing unprecedented changes. These changes have not only brought challenges within each industry, but also brought unprecedented opportunities for development. In this context, the search for natural and safe products that can effectively replace traditional veterinary drugs has become an important research direction in the fields of animal husbandry and veterinary medicine. Oregano essential oil (OEO), as a natural extract, is gradually emerging in the fields of animal husbandry and veterinary medicine with its unique antibacterial, antioxidant, and multiple other biological activities. OEO not only has a wide antibacterial spectrum, effectively fighting against a variety of pathogenic microorganisms, but also, because of its natural properties, helps us to avoid traditional veterinary drugs that may bring drug residues or cause drug resistance problems. This indicates OEO has great application potential in animal disease treatment, animal growth promotion, and animal welfare improvement. At present, the application of OEO in the fields of animal husbandry and veterinary medicine has achieved preliminary results. Studies have shown that adding OEO to animal feed can significantly improve the growth performance and health status of animals and reduce the occurrence of disease. At the same time, pharmacokinetic studies in animals show that the absorption, distribution, metabolism, and excretion processes of OEO in animals shows good bioavailability. In summary, oregano essential oil (OEO), as a substitute for natural veterinary drugs with broad application prospects, is gradually becoming a research hotspot in the field of animal husbandry and veterinary medicine. In the future, we look forward to further tapping the potential of OEO through more research and practice and making greater contributions to the sustainable development of the livestock and veterinary industries.

Determination of Sedative and Anesthetic Drug Residues in Aquatic Food Products Using Solid Phase Extraction (SPE) and Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)

The residues of sedative and anesthetic drugs in aquatic products are an important factor affecting food safety. In order to screen the residues of sedative and anesthesia drugs in aquatic products, a rapid and sensitive method was established using solid phase extraction (SPE) and high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). The samples were extracted by 80% aqueous acetonitrile by an alumina column and separated by a C18 column. The mass spectra were determined in positive ion mode. The standard curves were matrix matched and quantified by the external standard method. The results showed that the linear ranges of the drugs in aquatic products were from 0.5 to 100 μg kg−1. The correlation coefficient (R 2) was greater than 0.990 and the detection limits were between 0.001 and 0.322 μg kg−1. The average recovery rate and relative standard deviation were between 78.64% and 112.15% and from 0.41%-8.26%. Overall, the developed method showed promising results for the simultaneous qualitative and quantitative determination of the drug residues in aquatic products.

New limits proposed for the management of non-mutagenic impurities

Multiple international guidelines exist that describe both quality and safety considerations for the control of the broad spectrum of impurities inherent to drug substance and product manufacturing processes. However, regarding non-mutagenic impurities (NMI) the most relevant ICH Q3A/B guidelines are not applicable during early phases of drug development leading to confusion about acceptable limits at this stage. Thus, there is need for more flexible approaches that ensure that patient safety remains paramount, while taking into consideration the limited duration of exposure. An EFPIA survey, which collected quantitative data from different types of studies applied to qualify impurities in accordance with ICH Q3A, shows that no toxicities could be attributed to any of the 467 impurities at any tested level in vivo. This data combined with earlier published toxicological datasets encompassing drug substances and intermediates, food related substances and chemicals provide convincing evidence that for NMIs, the application of a generic 5 mg/day limit for an exposure duration <6 months, and a 1 mg/day generic limit for life-long exposure, provides sufficient margins to ensure patient safety. Hence, application of these absolute limits to trigger qualification studies (instead of the relative limits described in Q3A/B), is considered warranted. This approach will prevent conduct of unnecessary dedicated impurity qualification studies and the resulting use of animals.

Cyclic olefin copolymer (COC) as a promising biomaterial for affecting bacterial colonization: investigation on Vibrio campbellii

Cyclic olefin copolymer (COC) has emerged as an interesting biocompatible material for Organ-on-a-Chip (OoC) devices monitoring growth, viability, and metabolism of cells. Despite ISO 10993 approval, systematic investigation of bacteria grown onto COC is a still not documented issue.This study discusses biofilm formations of the canonical wild type BB120 Vibrio campbellii strain on a native COC substrate and addresses the impact of the physico-chemical properties of COC compared to conventional hydroxyapatite (HA) and poly(dimethylsiloxane) (PDMS) surfaces.An interdisciplinary approach combining bacterial colony counting, light microscopy imaging and advanced digital image processing remarks interesting results. First, COC can reduce biomass adhesion with respect to common biopolymers, that is suitable for tuning biofilm formations in the biological and medical areas. Second, remarkably different biofilm morphology (dendritic complex patterns only in the case of COC) was observed among the examined substrates. Third, the observed biofilm morphogenesis was related to the interaction of COC with the conditioning layer of the planktonic biological medium. Fourth, Level Co-occurrence Matrix (CGLM)-based analysis enabled quantitative assessment of the biomass textural fractal development under different coverage conditions. All of this is of key practical relevance in searching innovative biocompatible materials for pharmaceutical, implantable and medical products.

The politics and governance of drug production in public-private partnerships: Brazil’s response to hepatitis C

ABSTRACT The local manufacture of advanced pharmaceutical products has been a long-standing objective of health and industry policy in many developing countries, including in Latin America. This strategy has been applied to fight epidemics such as HIV/AIDS, malaria, and the COVID-19 pandemic. However, we still know little about the politics and governance that enable such arrangements, especially when there is no consent from the originator company. This study focuses on the case of Brazil, a country that is well-known for its health-industry policy, which includes the local production of direct-acting antivirals (DAAs), a new treatment for hepatitis C. We seek to explain the factors that have contributed to Brazil’s successful production of generic versions of DAAs, and, later, to the decision by the Ministry of Health (MoH) to procure drugs from multinational pharmaceutical companies rather than from local laboratories. A lack of support for domestic production by important stakeholders, the patent holder’s attempt to block domestic production and the MoH’s adoption of more modern treatment guidelines under a different procurement logic all created an unfavourable environment for local production and procurement of DAAs. Our study draws implications for middle-income countries that wish to produce drugs domestically without voluntary license agreements.

A cutting-edge electrochemical sensing platform for the simultaneous determination of the residues of antimicrobial drugs, rifampicin and norfloxacin, in water samples

BackgroundThe widespread use and abuse of antibiotics has resulted in the pollution of water sources with antibiotic residues, posing a threat to human health, the environment, and the economy. Therefore, a highly sensitive and selective method is required for their detection in water samples. Herein, advanced ultrasensitive electrochemical sensor platform was developed by integrating gold-silver alloy nanocoral clusters (Au–Ag-ANCCs) with functionalized multi-walled carbon nanotube-carbon paste electrode (f-MWCNT-CPE) and choline chloride (ChCl) nanocomposites for simultaneously determining the residues of antimicrobial drugs, rifampicin (RAMP) and norfloxacin (NFX), in water samples. ResultsThe developed sensor (Au–Ag-ANCCs/f-MWCNTs-CPE/ChCl) was extensively characterized using several analytical (UV–Vis, FT-IR, XRD, SEM, and EDX) and electrochemical (EIS, CV, and SWV) techniques. It exhibited outstanding performance in a wide linear range, from 14 pM to 115 μM for RAMP, and from 0.9 nM to 200 μM for NFX, with a limit of detection (LOD, 3σ/m, S/N = 3, n = 5) and a limit of quantification (LOQ, 10σ/m, S/N = 3, n = 5) values of 2.7 pM and 8.85 pM for RAMP, and 0.14 nM and 0.47 nM for NFX, respectively. The sensor also exhibited exceptional reproducibility, stability, and resistance to interference. SignificanceThe developed sensor was effectively utilized to determine RAMP and NFX residues in hospital wastewater, river, and tap water samples, yielding recoveries within the range of 96.8–103 % and relative standard deviations below 5 %. Generally, the proposed sensor demonstrated remarkable performance in detecting the target analytes, making it an ideal tool and the first of its kind for addressing global antibiotic residue pollutants in water sources.

Ferromanganese oxide-functionalized TiO2 for rapid catalytic ozonation of PPCPs through a coordinated oxidation process with adjusted composition and strengthened generation of reactive oxygen species

Ferromanganese oxide (MFOx) was first utilized to functionalize TiO2 and an MFOx@TiO2 catalyst was developed for catalytic ozonation for rapid attack of pharmaceutical and personal care products (PPCPs) with adjusted reactive oxygen species (ROSs) composition and strengthened ROSs generation. Unlike Al2O3, which strongly relied on adsorption and was significantly influenced by MFOx loading, synergistic catalytical effects of MFOx and TiO2 were observed, and optimal MFOx doping of 2 wt% and MFOx@TiO2 dosage of 500 ppm were obtained for catalyzing ozonation. In ibuprofen (IBP) degradation, MFOx@TiO2-catalyzed ozonation (MFOx@TiO2/O3) obtained 2.0-, 4.7- and 6.9-folds the kobs of TiO2/O3, MFOx/O3 and bare ozonation (B/O3). Stronger O3 decomposition was observed by MFOx@TiO2 over bare TiO2 with the participation of redox pairs Fe(II)/Fe(III) and Mn(II)/Mn(III)/Mn(IV) and increased surface oxygen vacancies (SOVs) from 9.8 % to 33.7 % was detected. The results revealed that Fe(II), Mn(II) and Mn(III) with low valance accelerated Ti(III) generation from Ti(VI), obtaining an unprecedented high Ti(III) composition occupying 35.3 % of the total Ti atoms. Ti(III) catalyzed the direct reduction of SOVs-O2 to •O2–, and it accelerated the formation of Ti(VI)-OH and Ti(VI)-O which catalyzed O3 decomposition into •O2–. •O2– was found to primarily initiate IBP degradation with nucleophilic addition and dominated over 66 % IBP removal. The enhanced •O2– generation further strengthened •OH and 1O2 production. MFOx@TiO2/O3 obtained 17 %, 21 % and 30 % higher TOC removal over TiO2/O3, MFOx/O3 and B/O3, respectively. Acute toxicity tests confirmed the effective toxicity control of organics by MFOx@TiO2/O3 process (inhibition rate: 10.9 %). Degradation test of atenolol and sulfamethoxazole confirmed the catalytic effects of MFOx@TiO2. MFOx@TiO2 performed strong resistance to water matrix in application test and showed good stability and reusability. The study proposed an effective catalyst for strengthening the ozonation process on PPCPs degradation and provided an in-depth understanding of the mechanisms and characteristics of the MFOx@TiO2 catalyst and MFOx@TiO2/O3 process.

Role of IT on the Effectiveness of Logistics and Supply Chain Management Systems in the Pharmaceutical Sector in Saudi Arabia Vision 2030

The main objective of this study is to evaluate how IT might improve the efficacy and efficiency of supply chain management and logistics systems in the pharmaceutical sector because the pharmaceutical industry is experiencing a transformation by the lofty objectives outlined by Saudi Arabia Vision 2030. The study was conducted using 150 executives working in the Saudi Arabian pharmaceutical industry's logistics and supply chain division. An open-ended survey questions were used to determine how IT technology affects pharmaceutical logistics and supply chain using non-probability sampling, logistics managers' LinkedIn accounts were selected for the sample, irrespective of the organization's size, location, or gender. SPSS statistical tools were used to analyse the data. It was found that the integration of IT tools and systems has emerged as a crucial enabler in this process. By harnessing the power of data analytics, IoT (Internet of Things), and automation, pharmaceutical companies can gain real-time insights into their supply chains. This, in turn, allows them to develop agile responses to changing market dynamics and customer expectations. Such strategic decisions, driven by IT, enable businesses to adapt quickly, reorganize their operations, and offer value-added services tailored to the specific needs of the market. Moreover, the creative application of IT solutions facilitates efficient inventory management, demand forecasting, and tracking of pharmaceutical products from manufacturing facilities to end consumers results also show that companies may simply increase the pace of the supply chain with the aid of innovative chances in major choices and judgments.

Supercoiled DNA percentage: A key in-process control of linear DNA template for mRNA drug substance manufacturing

The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high quality in-vitro-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of polyA tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity, and in vitro transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through Oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template CMC (Chemistry, Manufacturing, and Controls) and process development for mRNA drug substance.

A Cross-Sectional Descriptive Study: Proportion of Authenticity and Complete Product Information of FarmaTag Hologram on Registered Pharmaceutical Products among Selected Mainstream Medicines’ Sellers in the State of Sabah (ProvoS)

FarmaTag Hologram is a two-dimensional QR code sticker that is required on all registered pharmaceutical products (RPP) in Malaysia. By scanning the QR code, the authenticity and product information of the hologram are displayed. However, FarmaTag holograms with incomplete product information may be susceptible to being misused by manufacturers/distributors/suppliers (MDS) for different pharmaceutical products, unregistered pharmaceutical products, or counterfeit products. In addition to misusing, these callous acts may lead to consumer distrust, doubt, and a lack of confidence in the authenticity of RPP. A cross-sectional descriptive study was conducted among Selected Mainstream Medicines’ Sellers in the State of Sabah between January 2022 and September 2022 to evaluate the proportion of authenticity and complete product information of FarmaTag Hologram on registered pharmaceutical products (RPP) available among selected Mainstream Medicines’ Sellers (MMS) in the state of Sabah. The total sample size was 5048 registered pharmaceutical products. There was no formal sample size calculation performed because this was the first study to explore this topic. All samples being selected from MMS were authentic (100%), which included 31.7% (n = 1600) from private medical clinics, 18.8% (n = 948), from retail pharmacies, and 49.5% (n = 2500) from Non-Pharmacy Drug Stores (NPDS). However, only 268 (5.3%) samples were found to have complete product information. The findings of this study provide preliminary information to relevant authorities on the authenticity and complete product information of FarmaTag Hologram on registered pharmaceutical products among selected MMS in the state of Sabah.

Supply Chain Management in the Indian Pharmaceutical Industry: Challenges, Innovations, and Future Directions

Supply chain management (SCM) plays a crucial role in the pharmaceutical industry by ensuring the efficient flow of pharmaceutical products from manufacturers to end-users. The Indian pharmaceutical industry, renowned for its generic drug manufacturing capabilities, faces unique challenges and opportunities in SCM due to factors such as complex regulatory requirements, diverse market dynamics, and evolving global standards. This paper examines the intricacies of SCM in the Indian pharmaceutical sector, exploring key challenges, innovative practices, and future directions to enhance efficiency, transparency, and resilience across the supply chain.

Contrasting the validation parameters and greenness of normal-phase and reverse-phase stability-indicating HPTLC methods for lemborexant analysis

There are very limited analytical procedures to determine lemborexant (LMB) in dosage forms and biological materials. However, the literature does not provide any “high-performance thin-layer chromatographic (HPTLC)” assays to determine LMB. In an effort to analyze LMB in commercial pharmaceutical tablets more precisely, accurately, and sustainably than with the normal-phase-HPTLC (NP-HPTLC) approach, a sensitive and greener reverse-phase-HPTLC (RP-HPTLC) method was developed and validated. For NP-HPTLC, an acetone-petroleum ether (40:60 v/v) developing system was used. However, ethanol–water (85:15 v/v) was the developing system for RP-HPTLC. Four different techniques, including the National Environmental Method Index (NEMI), Analytical Eco-Scale (AES), ChlorTox, and Analytical GREENness (AGREE), were used to evaluate the greenness of both procedures. LMB measurement was linear in the 50–500 and 20–1000 ng/band ranges, respectively for NP and RP procedures. RP procedure was more robust (uncertainties = 0.90–0.95 %), accurate (recoveries = 98.24–101.57 %), precise (uncertainties = 0.87–1.00 %), linear (20–1000 ng/band), sensitive (LOD = 0.92 ng/band and LOQ = 2.76 ng/band), and greener over NP procedure. The results of greenness assessment using NEMI (all four circles green), AES (93), ChlorTox (0.88 g), and AGREE (0.89) demonstrated that the RP strategy was greener than NP strategy and all other reported HPLC methods. The fact that both techniques can assess LMB when its degradation products are present implies that they both have characteristics that point to stability-indicating features. 89.24 % and 98.79 %, respectively, were the assay results for LMB in pharmaceutical tablets when utilizing the NP and RP procedures. Based on all validation and greenness metrics, it was found that RP procedure was better than the NP procedure. As a result, it is possible to assess LMB in pharmaceutical products using RP procedure.

Toward a unified understanding of drug-drug interactions: mapping Japanese drug codes to RxNorm concepts.

Linking information on Japanese pharmaceutical products to global knowledge bases (KBs) would enhance international collaborative research and yield valuable insights. However, public access to mappings of Japanese pharmaceutical products that use international controlled vocabularies remains limited. This study mapped YJ codes to RxNorm ingredient classes, providing new insights by comparing Japanese and international drug-drug interaction (DDI) information using a case study methodology. Tables linking YJ codes to RxNorm concepts were created using the application programming interfaces of the Kyoto Encyclopedia of Genes and Genomes and the National Library of Medicine. A comparative analysis of Japanese and international DDI information was thus performed by linking to an international DDI KB. There was limited agreement between the Japanese and international DDI severity classifications. Cross-tabulation of Japanese and international DDIs by severity showed that 213 combinations classified as serious DDIs by an international KB were missing from the Japanese DDI information. It is desirable that efforts be undertaken to standardize international criteria for DDIs to ensure consistency in the classification of their severity. The classification of DDI severity remains highly variable. It is imperative to augment the repository of critical DDI information, which would revalidate the utility of fostering collaborations with global KBs.

Neurotoxicity of Some Environmental Pollutants to Zebrafish

The aquatic environment encompasses a wide variety of pollutants, from plastics to drug residues, pesticides, food compounds, and other food by-products, and improper disposal of waste is the main cause of the accumulation of toxic substances in water. Monitoring, assessing, and attempting to control the effects of contaminants in the aquatic environment are necessary and essential to protect the environment and thus human and animal health, and the study of aquatic ecotoxicology has become topical. In this respect, zebrafish are used as model organisms to study the bioaccumulation, toxicity, and influence of environmental pollutants due to their structural, functional, and material advantages. There are many similarities between the metabolism and physiological structures of zebrafish and humans, and the nervous system structure, blood–brain barrier function, and social behavior of zebrafish are characteristics that make them an ideal animal model for studying neurotoxicity. The aim of the study was to highlight the neurotoxicity of nanoplastics, microplastics, fipronil, deltamethrin, and rotenone and to highlight the main behavioral, histological, and oxidative status changes produced in zebrafish exposed to them.

Analysis of non-mutagenic substances in the context of drug impurity assessment – Few are potent toxicants

ICH Q3A/B guidelines provide qualification thresholds for impurities or degradation products in new drug substances and products. However, the guidelines note that certain impurities/degradation products may warrant further safety evaluation for being unusually potent or toxic. The purpose of this study was to confirm that especially toxic non-mutagenic compounds are rare and to identify classes of compounds that could warrant lower qualification thresholds. A total of 2815 compounds were evaluated, of which 2213 were assessed as non-mutagenic. For the purpose of this analysis, compounds were considered potent when the point of departure was ≤0.2 mg/kg/day based on the qualification threshold (1 mg/day or 0.02 mg/kg/day for a 50 kg human) in a new drug substance, with an additional 10-fold margin. Only 54 of the entire set (2.4%) would be considered potent based on this conservative potency analysis, confirming that the existing ICH Q3A/B qualification thresholds are appropriate for the majority of impurities. If the Q3A/B threshold, without the additional 10-fold margin is used, 14 compounds (0.6%) are considered “highly potent”. Very few non-mutagenic structural classes were identified, including organothiophosphates and derivatives, polychlorinated benzenes and polychlorinated polycyclic aliphatics, that correlate with potential high potency, consistent with prior publications.

Synthesis of myrcene using a hybrid cell-free reaction system

Myrcene is a typical monoterpene with applications as pharmaceutical product, biofuel, and so forth. However, only traces of myrcene accumulate in plants, and in vivo production of monoterpene in microbial cells also contains multiple restrictive factors. To alleviate this limitation, a hybrid cell-free metabolic engineering (CFME) system was developed in this study and a series of CFME strategies were employed for the engineering of myrcene synthase from glucose. First, a convenient CFME system was established, in which Pichia pastoris and Escherichia coli lysates enriched with MVA pathway and myrcene synthase respectively were used as “plug and play” modules and combined. After optimization, the myrcene titer was significantly improved by 119.26%. Second, rational design of myrcene synthase was performed by conserved residues modification and substrate tunnel engineering. Myrcene synthase variant N238S outperformed the wild-type enzyme in CFME system, and the myrcene titer value reached 56.80 mg/L. Molecular dynamics simulations provided significant mechanistic insights into the enhanced catalytic capability and myrcene production. This work highlights the feasibility of hybrid CFME system to connect tandem module and sustain complex metabolism for myrcene biomanufacturer. It also illustrates the value of the cell-free approach as a starting point for the rational engineering of monoterpene synthase.

Improving the Current European Pharmacopoeia Enantio-Selective HPLC Method for the Determination of Enantiomeric Purity in Atorvastatin Calcium Salt Drug Substance

Atorvastatin (ATV) is a well-established lipid-lowering agent. ATV has two stereogenic centers, and of the four possible stereoisomers, only the (3R,5R) form is used therapeutically. The European Pharmacopoeia (EP) monograph 2022 for ATV calcium salt describes a normal-phase high-performance liquid chromatography (HPLC) method for the determination of enantiomeric purity in both drug substance and working standard samples, based on a 150 mm × 4.6 mm Chiralpak AD-H column. The main problems with this method are the very long analysis time and the high solvent consumption. Here, an alternative chromatographic protocol was developed using the Chiralpak AD-3 column (250 mm × 4.6 mm) packed with 3 μm silica particles instead of the 5 μm silica particles of the Chiralpak AD-H chiral stationary phase and characterized by the same polysaccharide selector, amylose-tris(3,5-dimethylphenylcarbamate). Using a mobile phase consisting of a mixture of n-hexane-ethanol-formic acid 90:10:0.1 (v/v/v) as the mobile phase and setting the flow rate and column temperature to 1.0 mL min−1 and 35 °C, respectively, a simultaneous stereo-selective separation was achieved within 35 min without observing any overlap between the enantiomeric impurity peak and peaks related to other ATV impurities. Compared to HPLC EP conditions, the analysis time to elute all the potentially related substances was faster and significantly less mobile phase volume was required. The linearity of the method has been demonstrated in the range of 4.4 μg mL−1 to 1000 μg mL−1 (R2 &gt; 0.999). At a concentration of 4.4 μg mL−1, which is 0.075% of the test solution (5.8 mg mL−1, as ATV free acid), the signal-to-noise ratio was found to be 20.

Protective efficacy of Eimeria maxima EmLPL and EmTregIM-1 against homologous challenge in chickens

Chicken coccidiosis has inflicted significant economic losses upon the poultry industry. The primary strategies for preventing and controlling chicken coccidiosis include anticoccidial drugs and vaccination. However, these approaches face limitations, such as drug residues and resistance associated with anticoccidial drugs, and safety concerns related to live vaccines. Consequently, the urgent development of innovative vaccines, such as subunit vaccines, is imperative. In previous study, we screened 2 candidate antigens: Eimeria maxima lysophospholipase (EmLPL) and E. maxima regulatory T cell inducing molecule 1 (EmTregIM-1). To investigate the immune protective effect of the 2 candidate antigens against Eimeria maxima (E. maxima) infection, we constructed recombinant plasmids, namely pET-28a-EmLPL and pET-28a-EmTregIM-1, proceeded to induce the expression of recombinant proteins of EmLPL (rEmLPL) and EmTregIM-1 (rEmTregIM-1). The immunogenic properties of these proteins were confirmed through western blot analysis. Targeting EmLPL and EmTregIM-1, we developed subunit vaccines and encapsulated them in PLGA nanoparticles, resulting in nano-vaccines: PLGA-rEmLPL and PLGA-rEmTregIM-1. The efficacy of these vaccines was assessed through animal protection experiments. The results demonstrated that rEmLPL and rEmTregIM-1 were successfully recognized by anti-E. maxima chicken sera and His-conjugated mouse monoclonal antibodies. Immunization with both subunit and nano-vaccines containing EmLPL and EmTregIM-1 markedly mitigated weight loss and reduced oocyst shedding in chickens infected with E. maxima. Furthermore, the anticoccidial indexes (ACI) for both rEmLPL and PLGA-rEmLPL exceeded 160, whereas those for rEmTregIM-1 and PLGA-rEmTregIM-1 were above 120 but did not reach 160, indicating superior protective efficacy of the rEmLPL and PLGA-rEmLPL formulations. By contrast, the protection afforded by rEmTregIM-1 and PLGA-rEmTregIM-1 was comparatively lower. Thus, EmLPL is identified as a promising candidate antigen for vaccine development against E. maxima infection.