The results of the XRD, FESEM, EDAX, FTIR, UV–Vis, TEM, XPS, and BET analyzes indicated that the catalyst BiOI/MOF was synthesized with excellent quality. Design of experiment (DOE), ANOVA statistical analysis, interaction of parameters and predicated optimum condition was done based on CCD. The effect of catalyst dose (0.25–0.5 mg/l), pH (4–8), reaction time (30–60 min) and O3 concentration (20–40 mN) at 10 mg/l of OTC on PCO/O3 process was optimized. Based on P-value and F-value coefficients  the model of OTC  and  coefficients, the model of COD removal was quadratic model. Under optimum condition pH 8.0, CD = 0.34 mg/l, RT = 56 min and O3 concentration = 28.7 mN, 96.2 and 77.2% of OTC and COD removed, respectively. The reduction of TOC was 64.2% in optimal conditions, which is less than the reduction of COD and OTC. The kinetics of reaction followed pseudo-first-order kinetic (R2 = 0.99). Synergistic effect coefficient was 1.31 that indicated ozonation, presence of catalyst and photolysis had a synergistic effect on OTC removal. The stability and reusability of the catalyst in six consecutive operating steps was acceptable and 7% efficiency decreased only. Cations , SO42− had no influence on performing the process, but other anions, organic scavengers, and nitrogen gas, had an inhibitory effect. Finally, the OTC degradation probably pathway includes direct and indirect oxidation that decarboxylation, hydroxylation, demethylation and were the main mechanism in OTC degradation.In this research, we synthesized BiOI/NH Environmental concerns have increased in recent years because of antimicrobial metabolites and their parents. Massive volumes of antibiotics are discharged to the environmental with the use of to human, animal, and aquaculture. It has been predicted that 100–200 thousand tons of antibiotics are consumed worldwide per year2. Human medicine, animal husbandry, and aquaculture use oxytetracycline (OTC) most frequently as a type of tetracycline antibiotic (TC)3. It is a complex, isoionic organic compound consisting of a sophisticated four-ring structure with multiple ionizable functional groups4. Because of confined absorption by humans and animals, about 27 to 75% of excess OTC as general structure is released into the environment5. Based on reports, OTC has been found in water, wastewater, soil, eggs, meat, and milk with variable concentrations ranged from ppb to ppm6. Until this point, Adsorption, Fenton regent, derivatives of Fenton oxidation, biological process, etc. have been used to remove and decompose OTC from aqueous9. As one of several degradation processes, photocatalytic ozonation (PCO/O3) is a subset of advanced oxidation processes (AOPs). It uses light, ozone molecules, and semiconductor catalysts in combination. Due to its attractive properties such as inexpensive, eco-friendliness, availability, suitable efficiency, and simple operation compared to other processes, it creates a fundamental function in the organic matter degradation10. It was shown that this integrated process enhances reactive oxygen species and subsequently enhances mineralization rates of resistant organic compounds, such as OTCs. The ozone molecule has just 2.07 V standard oxidation–reduction potential (ORP), while PCO/O3 process uses free oxidation radicals such as hydroxyl radical and reactive oxygen species (ROSs), that have higher than 2.0 eV ORP. In the photocatalytic process (PCO), the OTCs decomposition is restricted close the catalyst's surface, whilst in the integrated process, OTCs can be degraded to inorganic compounds such CO2 and H2O via ozone in solution11. The PCO/O3 process mechanism described by  generated that produces OH· and O3·− based on subsequent reactions. Newly, study about PCO/O3 process focalized in presentation of novel semiconductors. Some semiconductors like metal–organic framework (MOFs), g-C3N4, and slim band-gap oxide of metals families were used to expand the response spectrum of light until visible range12. The classic semiconductors like TiO2 and ZnO activated via UV-C (under 300 nm wavelength that is only 4% of sunlight spectrum)13. Recently, a growing attraction in MOFs has focused on photocatalysis for the photo-driven removal of pollutants14. There have been a variety of applications of them in recent studies, such as catalysis, chemical sensing, and adsorption15. Various MOF-based photocatalysis have recently been developed in environmental studies. The Ti-based amino-functioned MOF (NH2-MIL125 (Ti)) was chosen for some advantages, including non-toxicity, being cheap, photo/water stability, and visible-light absorption16. The BiOI/NH2-MIL125(Ti) catalyst was prepared to make a synergistic effect with outstanding photocatalytic activity17. Bismuth-based semiconductors as a novel form of nonpoisonous, consistent photocatalysis via high visible light absorbency are worthwhile, environment-friendly, and stable compounds18. Among these, BiOI has shown impressive photocatalytic activity due to its appropriate band gap (BG = 1.62–1.93 eV), exclusive layered structure, and Special electrical features. The high rate of recombination of BiOI restricts its practical application. It has been proposed that heterojunctions can overcome this disadvantage by increasing charge separation and reducing the photo-generated electrons and hole recombination19. One way to improve the efficiency of photocatalysis is to create a heterojunction structure. This method reduces the energy of the band-gap between the conduction band and the valence band, and as a result, the photocatalyst is activated in the range of visible and UV-A light. Also, the structure of catalyst BiOI/NH2-MIL125(Ti) is such that it overcomes the limitations of structures BiOI and NH2-MIL125(Ti) and finally becomes a catalyst with proper efficiency in the PCO/O3 process20. Alexsandra Valério et al., an investigation of the photocatalysis and ozonation process on the tetracycline degradation. In this study, the total organic carbon (TOC) removal reached above 90% after 3 h. Compared to the sum of the separated processes, the kinetics of reaction increase by 20%21. In this current study, for the first time, BiOI/NH2-MIL125(Ti) was used as a catalyst in the PCO/O3 process for the decomposition of OTC. This catalyst possesses a remarkable capacity to generate active free radicals because of its unique characteristics. Water and wastewater quality is degraded by synthetic organic compounds such as OTC due to their benzene rings and high carbon content. To assess the effectiveness of the PCO/O3 process in improving the quality of wastewater, besides examining the removal of OTC, chemical oxygen demand (COD) and TOC were also investigated. The principal aims of the current study were to synthesis of BiOI/NH2-MIL125(Ti) (in short BiOI/MOF) and optimization of parameters of PCO/O3 process for the degradation of OTC and COD. In a supplementary study, we investigated TOC decreasing, reaction kinetics, mechanisms that work together synergistically, catalyst's stability, organic co-existing and organic radical scavenger's effects, energy consumption and probably reaction path-way and by-products in PCO/O3 process.At the begging of the reaction chain Eq. , UV ligh OH· Eq. . Based o OH· Eq. , Hydroge2OH)2], potassium iodide [KI], bismuth nitrate pentahydrate [Bi (NO3)3·5H2O], N,N-dimethyl formamide [C3H7NO], tetra butyl titanate [C16H36O4Ti], 2-amino terephthalic acid [C8H7NO4], Oxytetracycline [C22H24N2O9], sodium hydroxide [NaOH], sodium bicarbonate [NaHCO3], sodium nitrate [NaNO3], oxalic acid [C2H2O4], acetonitrile [C2H3N], and methanol [CH3OH]. De-ionized water  was used for preparing solutions.All solvents and reagents bought from reputable companies . Ethylene glycol  was assessed to survey the crystal structure of the samples. Scherrer's equation  of BiOI is calculated using Eq. . Shimadzu JEM-1200 EX with 100 kV stimulating voltage was used to obtain transmission electron microscopy image (TEM). Nitrogen adsorption at 77 K was used to determine the volume, pore size, and surface area distribution of BiOI/MOF. In situ degassing of samples was conducted for 12 h at 200 °C under vacuum. Brunauer–Emmet–Teller (BET) applied to calculate surface areas according to the linear relationship between p/p0 and surface areas. An ESCALAB250XI electron spectrometer was used to analyze the XPS characterizations.The optic properties and structural features were examined using UV–Visible spectrum (UV–Vis DRS) was recorded by Agilent Cary 60 spectrophotometer. Tauc equations Eqs. , 15 were3 process runs were done in a 600-ml cylindrical unit with D = 10 cm and h = 20 cm dimensions. The quartz sheath with dimensions 4.5 × 16 cm was installed horizontally in the middle of the reactor. The UPVC box with 39.5 × 41.5 × 30 cm was used as a pilot unit  was installed in the middle of reactor, with 15 cm length . By passing oxygen-feed gas through an ozone generator , a steady flow of ozone gas was introduced into the reaction media by a micro-diffuser. O3 concentration adjusting via flow rate of feed oxygen. According to 2350 E method (iodometric) of the standard methods for the examination of water and wastewater, ozone generator capacity and ozone concentration in the reactor and output were determined26. The contents of the reactor during the PCO/O3 process was mixed by a magnetic stirrer. To measure OTC, 10 ml of each reaction mixture were filtered with 0.22 m syringe filters after being taken from the reactor and injected into high-performance liquid chromatography (HPLC). As the eventual results, mean values were calculated from three repetitions of each test. OTC, COD, and TOC degradation percentages in the PCO/O3 process were calculated using the following  at time t, respectively.PCO/O3 process for OTC, COD, and TOC removal was optimized at 10 mg/l OTC as an initial concentration via central composite design (CCD) as an approach in the response surface method. The range of variables is illustrated in Table The undertaking of PCO/OTable 27:The empirical second-order polynomial model of OTC removal via PCO/O 3 was described by Eq. 27:17\doc0 is the intercept, βi, βii, and βij are the linear, quadratic, and interaction effect coefficients of variables respectively, xi and xj are coded testing classes of the variables, k is the number of the independent variables, and e is the residual error. A coded value for each variable was calculated using Eq.  was performed to determine the interaction between response and factors. R2 and R2adj are correlation coefficients. In addition, R2 predicts were used. Then, In the optimal condition, organic, and inorganic radical scavengers effect, degradation kinetic based in difference OTC concentration, the synergistic effect on individual and combined processes  effect, degradation pathway, and intermediates production, changes in wavelength scanning in during the PCO/O3 process, and calculation of energy consumption were performed.Here, Y is OTC decrease (%),βsing Eq. , to enab2/g, carbon loading: 20%, pore volume: 1.25 ml/g, pH range 2–7.5, bonding type: monomeric) and a column. Temperatures of 4–25 °C and 353 nm, respectively, were kept constant for UV detection and column hold. Mobile phase consisted of 95:5 (v/v) mixture of acetonitrile and ultra-pure water (0.05% ammonia) at 1.0 ml/min flow rate. A 10 μl of OTC solution was injected at a retention time of 12 min. The TOC analysis was performed with Analytikjena's multi C/N 3100 TOC analyzer. As described in the standard method, COD was measured using the titrimetric approach 28. Residual catalyst and ROS were two interferers in COD test. So, the COD samples were initially passed through a 0.22 µ PTFE filter. Due to the rapid rate of decay, ROS are rapidly eliminated. The decay of the intermediates was studied by gas chromatography/mass spectrometry (GC–MS) . For the gas chromatography system, DB-5MS columns (30 m 0.5 lm film thickness) were used with high-purity (99.99%) helium as the carrier gas flowing at 1.0 ml/min. For temperature, the column was maintained at 35 °C for 1 min, increased to 300 °C at 7.0 °C/min, and held at 300 °C for 1 min. 10 cc of the sample was injected, with the injector and detector both set at 280 °C.Each sample was collected after the runs and processed through a 0.45 m micro-filter before injection into HPLC. The amount of OTC was measured using a HPLC  equipped with a UV–Vis detector , t is the reaction time (min) and kapp is the rate constant (1/min).Degradation of OTC in the optimum condition of PCO/Otion Eq. 29.19\doc30.The synergist effect of single mechanism such as photolysis, simple ozonation, catalytic ozonation, photocatalytic, and adsorption were investigated in an optimum condition of parameters. The synergist effect calculated via Eq. 30.20\doc3 process was tested in five runs. Also, the effect of inorganic and organic radical scavengers investigated in optimum condition via tert-butyl alcohol (t-BuOH or TBA), N2 gas, Oxalic Acid, NO3−, Cl−, SO42−, Mg2+, Ca2+ and HCO3−.The stability of catalyst in optimum condition of PCO/O3 process under optimum condition calculated by  of PCO/Od by Eq. 31.21\doc3 process.In this part of the study, we investigated mineralization, by products, intermediates, and degradation mechanism of OTC in optimum condition of PCO/O2-MIL125, and BiOI-MOF were analyzed through powder XRD patterns and the obtained finding are showed in  and tensile vibrations (–OH) of free water molecules on the surface of the BiOI, respectively. Similar results reported by Ai and coworkers34. The peaks between 500 and 1500 cm−1 for MOF shows characteristics of organic compounds 36.Five peaks at 571.7, 1309.5, 1382.2, 1627.4, and 3529.1 cm2O-BiOI) as a solvent37. Synthesis methods, chemicals, and solvents used affect the morphology of BiOI. The synthesized MOF is thin and disk-like. A MOF's structure changes when its organic-metal ratio changes. Research has shown that MOF structures change from rectangular to round as organic linkers increase. The nanoplate morphology of NH2-MIL125(Ti) was uniform in Zhang and coworkers' study38. BiOI-MOF is shown as a rod containing fine particles. The morphology of BiOI/MOF was similar in Du and coworkers' study17. It can be seen from the TEM image  composite has been distributed successfully on the surface of BiOI, forming a core–shell structure. In the . An overview of BiOI-MOF structure elements is presented in Table 18.The chemical state and surface composition of BiOI-MOF were determined using XPS Fig. . The XPS3 process  development and finding the optimal conditions for PCO/Oss Table . F-value2, R2adj, and R2 predict), this model proved exceptionally valid for predicting responses. There is a perfect correlation between R2, R2adj, and R2 predict, which suggests that the model has a high ability to predict responses40.Using the ANOVA, the results of OTC and COD Table  appeared41. The polynomial equation ascribed to the OTC and COD removal (%) was represented in terms of coded factors:The F-value of OTC, and COD is 2451/04, and 450/34 respectively. In the OTC and COD model, P-values of 0.0001 indicate a robust fit to the relationship between the experimental and predicted response values. A model discernment greater than 4.0 for the Adequate Precision ratio indicates a satisfactory modelA positive and a negative effect of the studied mediators on the removal of OTC and COD is illustrated in Eqs.  and 23)23). The 3 process highly depends on the pH of the solution, which has a substantial impact on the removal efficiency. Solution pH affects pollutant hydrolysis, ionization pollutants, catalyst surface characteristics, and activity rate of oxidants and reactive species, as well as the degradation route. Hence, the effects of pH (4–8) on OTC and COD removal efficiency were examined and illustrated in ; therefore, OTC molecules are in cationic and anionic forms at the pka below 3.18 and above 8.29, respectively43. Meanwhile, the pHzpc of the photocatalyst was 6.3, indicated that the photocatalyst surfaces act as protonates and non-protons in lower and higher, respectively. At lower pH levels, minimal adsorption was observed because of electrostatic repulsion between the positively charged catalyst and the protonated OTC molecules. Many studies have shown that contaminant decomposition occurs primarily through direct and indirect oxidation pathways in the ozonation process. OTC and ozone react directly at lower pH levels. As the pH of the solution was increased, ozone decomposition rate and free radical production increased, and free radicals with high oxidation potential were generated. These radicals demonstrate superior ability in OTC degradation. OTC molecules can react indirectly with free radicals in an alkaline environment, thus resulting in a higher removal efficiency. Asgari et al. investigated the ability of the photocatalytic ozonation process for ceftazidime removal. Based on this research, pH value at 11.0 was the optimum pH of solution44. The similar findings have been also reported by Espíndola et al.45. In this study, pH equal to 7.5 was reported as an optimum pH solution to oxytetracycline oxidation in the photocatalysis process.PCO/O of pH 4– on OTC a3 efficiency in OTC removal  increase the amount of ·OH radicals and the efficacy of OTC degradation46. When incoming air flow increases excessively, and the mass transfer rate from the gas phase to the liquid phase is limited, the efficacy of removing decreases. The concentration of ozone required for PCO/O3 depends on the type of process, the type of reaction reactor, the type of pollutant, and the specifications of the intermediate compounds48. In previous studies, a different concentration of ozone was reported as an optimum concentration. Yu et al.49 1.5 mg/l-min of ozone concentration, Heydari et al.50 11 mg/l of ozone concentration was reported as an optimum ozone concentration.Main important economic parameter in PCO/Oudy Fig. . The res3 effect in OTC and COD removal in 30 to 60 min range was investigated , UV/ catalyst (60%), and UV/O3 (66%) as binary processes exhibited more efficient OTC degradation than the individual processes. Ultimately, over 95% of the tetracycline concentration was removed through the fundamental process. This study confirms a synergistic effect between various processes, resulting in a more active degradation of OTC and an excess creation of super reactive radicals. However, when all variables were involved simultaneously, OTC removal performance significantly improved. Based on , photolysis , and sorption (7%) are ineffective in OTC degradation. Therefore, these mechanisms do not have satisfactory potential for degrading OTC individually. Od on Eq. , the SF 3 ability to remove pollutants. The influence of anions and cations on the PCO/O3 performance is illustrated in , Bicarbonate (HCO3− = 0.1 mM), and Chloride (Cl− = 0.1 mM) had inhibited PCO/O3 process efficiency. However, the PCO/O3 process was not affected by adding sulfate (SO42−). NO3−, HCO3− and Cl− ions use the ROS in the PCO/O3 process. Cl− ion readily adsorbs on the catalyst surface, preventing reactant molecules from adhering to it, reducing the reaction's efficiency. The process is not affected by the addition of Na+ or K+. A solution containing Mg2+ or Ca2+ promotes the removal of OTC. In Lu et al.46 and Asgari et al.44 similar finding reported. OTC removal was investigated using scavenging tests over PCO/O3 process to identify reactive oxidizing species. Free radical species  were evaluated by quenching tests for their effect on removing OTC52. OA acts as a scavenger of O2·- and e-—h+ pairs. TBA is the scavenger of O21 and OH·, and finally N2 gas is a scavenger of all ROSs. Based on results indicated that, performing PCO/O3 process in the presence of AO, it drops less, in the presence of TBA, the rate of decrease in process efficiency is higher, and N2 gas causes a significant decrease in efficiency. Therefore, O21 and OH· were the main active radicals. Similar finding reported in previous studies53.Many types of inorganic ions are found in practical wastewater. It is important to study how different inorganic ions can affect PCO/O in Fig. . Nitrate3 process. As shown in . Based on the review of previous studies, it was found that in most AOPs, the reaction kinetics follows pseudo-first-order kinetics55.On optimized conditions, the pseudo-first-order model Eq.  describe in Fig. , OTC deg3 process under optimum condition was 4.44 KWh. Based on previous studies, the EEC depends on various factors such as the power of the ozone generator, the power of the lamp, the reaction time, the volume of the reaction chamber and finally the efficiency of the process56. In Kang et al. study, 55 KWh/m3 reported as EEC10.In practical applications, energy consumption must be considered. The ozone generator and the UV lamp handled EEC in this process. The total energy consumption of was 220 W. According to Eq. , EEC of 3 formed by the cyclic cleavage of enolic acetylacetone. At the C4 target site, one methyl group of dimethylammonium group might be eliminated, resulting in intermediate P3 (m/z 448)57. By review of previous studies, it was found that based on the process used, different intermediate compounds and different reaction paths may occur in the degradation of OTC.To identify the intermediates produced during OTC degradation, GC–MS experiments were conducted to reveal the degradation pathway. According to the results analysis and previous relative studies, two main pathways were identified for OTC degradation: direct oxidation and indirect oxidation. During direct oxidation, mineralization plays a minimal role. Indirect oxidation destroys OTC through hydroxylation, demethylation, and decarbonylation Fig. . OTC's aTo conclude, Table 3 concentration (20–40 mN) at 10 mg/l of OTC on PCO/O3 process optimized. Based on F-value and P-value coefficients, the model of OTC  and COD  removal was quadratic. Under optimum condition pH 8.0, CD = 0.34 mg/l, RT = 56 min and O3 concentration = 28.7 mN, 96.2 and 77.2% of OTC and COD removed, respectively. TOC decreasing under optimum condition was 64.2% that lower than OTC and COD. The kinetic of reaction follows pseudo-first-order kinetics (R2 = 0.99). Synergistic effect coefficient was 1.31 that indicated ozonation, presence of catalyst and photolysis had a synergistic effect on OTC removal. The stability and reusability of catalyst in six consecutive operating was acceptable and 7% efficiency decreased only. Cations , SO42− had no effect on the efficiency of the process, but other anions, organic scavengers and N2 gas, had an inhibitory effect on the efficiency of the process. EEC under optimum condition 4.44 KWh/m3 calculated. Finally, the OTC degradation probably pathway includes direct and indirect oxidation that Hydroxylation, Demethylation, and Decarboxylation were the fundamental mechanism in OTC degradation.In this study, BiOI-MOF was synthesized by solvo-thermal method. Based on results of XRD, FESEM, EDAX, FTIR, UV–Vis spectra, TEM, BET, and XPS analysis indicated that synthesis of BiOI/MOF was done with best character. Design of experiment, ANOVA statistical analysis, interaction of parameters and predicated optimum condition was done based on CCD. The effect of pH of solution (4–8), catalyst dose (0.25–0.5 mg/l), reaction time (30–60 min) and O


A sensitive and rapid ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was hereby developed for the determination of seven components, namely, glycyrrhizic acid, glycyrrhetinic acid, dehydrotumulosic acid, isoliquiritin, liquiritin, atractylenolide III, and cinnamic acid, in the plasma of rats after the oral administration of Ling-Gui-Zhu-Gan decoction (LGZGD). Besides, this very method was methodologically validated for specificity, linearity, inter-day and intra-day precision, accuracy, matrix effect, extraction recovery, and stability. It was also successfully used for the first time to compare the pharmacokinetic characteristics of the seven components after oral administration of LGZGD to normal rats and non-alcoholic fatty liver disease (NAFLD) rats. The results indicated significant differences between the pharmacokinetic characteristics of normal and NAFLD rats. To further reveal the different pharmacokinetic behaviors, the expressions of enzymes and transporters in the liver of normal and NAFLD rats were detected using UPLC-MS/MS. In the NAFLD rats, UDP-glucuronosyltransferase 1-1 (UGT1A1) and nine transporters were significantly inhibited and a positive correlation was observed between them and the AUC of the major components. The present results indicate that the pharmacokinetic differences between the normal and NAFLD rats might be attributed to the significant lower expression levels of both the metabolic enzyme UGT1A1 and nine transporter proteins in the NAFLD rats than in the normal rats. Meanwhile, UGT1A1 and the nine transporter proteins might be used as potential biomarkers to assess the ameliorative effect of LGZGD on NAFLD, which could provide useful information to guide the clinical application of LGZGD. Treatise on Febrile Diseases and Synopsis of the Golden Chamber drafted by Zhongjing Zhang in the Han dynasty (Poria cocos (Schw.) Wolf (Fuling), Cinnamomum cassia Presl (Guizhi), Atractylodes macrocephala Koidz. (Baizhu), and Glycyrrhizae Radix et Rhizoma (Gancao), in the ratio 4:3:3:2 (w/w/w/w)  is a common chronic liver disease featuring an increasing incidence year after year. The lack of safe and effective drugs for thorough treatment has made related cardiovascular diseases a major problem of chronic diseases . To this dynasty , which iw/w/w/w) . LGZGD fw/w/w/w) . In recew/w/w/w) . LGZGD hw/w/w/w) . Besidesw/w/w/w) , which pin vivo processes of drugs. Previous studies on LGZGD have focused on phytochemistry and pharmacology, but pharmacokinetic studies of LGZGD components in rats have been rarely reported. Pharmacological studies have shown that the main active compounds in Glycyrrhizae Radix et Rhizoma include glycyrrhizic acid (Poria cocos (Schw.) Wolf extracts protect the liver from acute ethanol insult (Poria cocos (Schw.) Wolf is dehydrotumulosic acid  were all procured from Hongyong Biotechnology Co., Ltd. . Sulfatase was procured from Helix pomatia . The peptides LTIIPQDPILFSGSLR, GVALPETIEEAENLGR, AAATEDATPAALEK, TFQFPGDIESSK, LLLSGFQEELR, STALQLIQR, NTTGALTTR, EENLGITK, SVQPELK, and TYPVPFQR, as well as stable isotope-labeled internal standards (≥98% purity) were synthesized by Bankpeptide Biological Technology, Co., Ltd. . The ProteoExtract Native Membrane Protein Extraction Kit was purchased from Merck KGaA . The BCA Protein Assay Kit and In-Solution Tryptic Digestion Kit were obtained from Pierce Biotechnology . Formic acid dedicated to mass spectrometry (99% purity); ammonium bicarbonate (98% purity); and sodium deoxycholate (98% purity) were purchased from Sinopharm Chemical Reagent Co., Ltd. . Acetonitrile and methanol, both MS grades, were obtained from Merck KGaA .LGZGD, consisting of Fuling: 120 g, Guizhi: 90 g, Baizhu: 90 g, and Gancao: 60 g, was soaked in 2,880 mL of deionized water  for 0.5 h and reflux extracted for 1.5 h. The right amount of aromatic water was collected for the first time. Then, 2,880 mL of deionized water was added for reflux extraction for another 1.5 h. The extract solutions were mixed and concentrated to an appropriate amount, and LGZGD extract powder was obtained by spray drying. The inlet air temperature of the spray dryer was controlled at 180°C ± 5°C; the outlet air temperature was 90°C–100°C; the dry paste moisture was below 5%; the atomization frequency was 200 Hz; and the extract yield was 17.56%. The aromatic water was taken and β-cyclodextrin inclusion was added to it, and the inclusion compound was mixed with the extract powder. The content of the analyte in the LGZGD extract was determined by the UPLC-MS/MS method. The amount of glycyrrhizic acid, glycyrrhetinic acid, dehydrotumulosic acid, isoliquiritin, liquiritin, atractylenolide III, and cinnamic acid in the content was 5360.3, 16.4, 6.0, 290.5, 6991.5, 141.8, and 739.2 μg/g, respectively.Male Sprague–Dawley rats, weighing 200–220 g, SPF grade, were purchased from Shanghai Sippr-BK laboratory animal Co. Ltd.  and raised in the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine. The rats were kept in an environmentally controlled condition: on a 12:12 h light–dark cycle (lights on at 7:00 a.m.) with regulated temperature and humidity, at a temperature of 22°C–24°C and a relative humidity of 60–65%. In the experiment, the rats in the normal group were fed a certified standard rat diet and tap water, while those in the model group were fed a high-fat diet for 8 weeks to construct the NAFLD model . The animal experiment program was approved by the Animal Committee of Shanghai University of Traditional Chinese Medicine (License No.: PZSHUTCM201016001).18 column . The analytes were separated using the gradient elution method, and the mobile phase was composed of water (0.05% V/V formic acid) (A) and acetonitrile (B) at a flow rate of 0.4 mL/min . The multiple reaction-monitoring parameters are shown in An Agilent 1290 UPLC system was combined with an Agilent 6460 series MS/MS system  to quantify the analytes in the ESI-positive ionization mode. The mass spectrum conditions were as follows: a capillary voltage of 4,000 V; a gas flow rate of 10 L/min; a nebulizer of 30 psi; a gas temperature of 350°C; and a Delta EMV(+) of 200 V. Five microliters of the samples was injected into an Agilent SB-CA plasma sample of 50 µL was placed in a 1.5 mL centrifuge tube with 10 µL of schisandrin (IS) solution (50 ng/mL) and mixed for 5 min. The samples were successively extracted with 500 µL of ethyl acetate and treated with 250 µL of methanol, which were then mixed for 5 min and centrifuged at 4°C for 10 min . All the supernatant was absorbed into another 1.5 mL centrifuge tube, dried under a gentle stream of nitrogen at 37°C, and then added with 50 µL of 50% methanol for reconstruction. After being centrifuged at 4°C for 10 min , 5 µL of the supernatant was extracted and injected into a chromatographic column for analysis, and the chromatograms were recorded.The bioanalytical method was validated based on the current US FDA Bioanalytical Method Validation Guidance for Industry , with vaThe specificity of endogenous interference was examined by chromatograms of six batches of blank rat plasma, blank plasma with the seven components and internal standard, and plasma samples after the oral administration of LGZGD for 4 h.2) least squares linear regression. The LLOQ was defined as the lowest quantifiable calibration concentration with an acceptable accuracy within ±20%, which was hereby determined at the lowest concentration with a signal-to-noise ratio (S/N) of 10.The calibration curve was obtained by measuring the blank plasma samples with analytes at eight concentration levels and by analyzing the peak area ratio of the components to IS using weighted  quality control samples on the same day and three batches of samples for three consecutive days, respectively. The accuracy was expressed as a relative error percentage , while the intra-day and inter-day precisions were evaluated as a relative standard deviation percentage (RSD).At three quality control (QC) levels, the extraction recovery was determined by comparing the ratio of the peak area to the internal standard in the blank plasma sample with the analyte added before extraction and after extraction. The matrix effect was measured at three QC levels by comparing the peak areas of the extracted blank plasma samples with the analyte (six batches of rat plasma) and the corresponding pure standard solution.Stability refers to the ability of the components to retain their chemical properties in the matrix. To analyze the stability of all components in the plasma, six replicates of QC samples at three concentrations were hereby prepared under each condition. The stability of the QC samples at three concentrations was analyzed under different storage conditions: −80°C for 30 days, after three freeze–thaw cycles, and 24 h in an auto sampler at 4°C.The rats were randomly divided into the normal group and the model group. According to the preliminary experiment, the dose was adjusted to about eight times the normal dose . The norEight weeks after modeling, the blood samples were collected in the coagulation vessels of the model group and the normal group, which were then centrifuged at 4°C for 10 min  to obtain the serum samples. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were determined to evaluate the modeling efficiency. Besides, the liver samples were isolated and stored in 4% paraformaldehyde for histological examination. Serum TG, TC, HDL-C, and LDL-C were measured by using a chemical analyzer . The liver tissue blocks fixed with 4% of formaldehyde solution were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and observed under an optical microscope.Herein, a comprehensive study was conducted to detect the expressions of UGT1A1 and nine other transporters in the liver of the NAFLD model group and normal rats, which were successfully determined. Previous methods of protein quantification were immunological, which however require antibodies that are frequently unavailable, and even when available, are often of uncertain specificity. Additionally, the UPLC-MS/MS method was hereby used to detect the expression level of metabolic enzymes, which is provided with advantages in absolute accuracy, stability, operability, and efficiency when compared with the traditional quantitative methods .18 column, 2.1 mm × 50 mm, 1.8 µm particles) and eluted at 0.4 mL/min with a gradient elution of water (with 0.05% v/v formic acid) (A) and acetonitrile (B) . According to previous experiments by the research group of The Agilent 1290 series UPLC system and Agilent 6460 series MS/MS system  were hereby combined to quantify the signature peptides in the ESI-positive ionization mode. The conditions of mass spectrometry were as follows: a capillary voltage of 2,000 V; a gas flow of 8 L/min; a nebulizer of 30 psi; a gas temperature at 300°C; and a Delta EMV(+) of 400 V. Five milliliters of the sample was injected into the column  and 5 mM ammonium bicarbonate buffer). Finally, the protein samples were digested with 10 µL of trypsin. The ratio of protein to trypsin was 25:1 (w/w). After incubation at 37°C for 24 h, the digestion reaction was terminated by 60 µL of IS cocktail. The samples were centrifuged at 18,000 g for 10 min at 4°C, and 5 µL of the sample was injected in the UPLC-MS/MS system.Total membrane proteins were isolated from the liver tissue using the Native Membrane Protein Extraction Kit protocols (three replicates), and the protein concentration was detected by the BCA Protein Assay Kit. The preparation process was based on the previous experiments by the present researchers . In simpCmax), time to maximum concentration (Tmax), half-life (t1/2), area under the curve (AUC0-t), and total area under the curve (AUC0-∞), were hereby calculated using the DAS 3.2.8 software. Meanwhile, the pharmacokinetic data of the normal and model rats were compared using the SPSS software (version 21.0). Besides, Spearman rank bivariate correlation coefficient was used to determine the correlation between AUC values and UGT1A1 and nine other transporters. All the calculated data were expressed as mean ± SD. When p < 0.05, the difference was considered significant and statistically significant; and when p < 0.01 or <0.001, the difference was considered very significant.Important pharmacokinetic parameters, which included the maximum plasma concentration (p < 0.05) and the LDL-C (p < 0.01) were significantly increased, but no significant difference was observed between TG and HDL-C. Besides, liver pathological sections were observed under the microscope  of each analyte and IS and the corresponding nominal concentration (x-axis) of the analyte was plotted by weighted (1/x2) least square linear regression. All standard curves showed a good linearity with the correlation coefficient (r > 0.9952). The calibration curves, linear ranges, LLOQ, and correlation coefficients of the seven components are shown in The relationship curve between the peak area ratio , respectively.The quantitative results of UGT1A1 and the nine transporters are shown in The AUC is an important indicator of drug absorption. In this study, a significant decrease in UGT1A1 and the nine other transporters was observed in the NAFLD rats. In order to further understand the reasons for the pharmacokinetic differences, a correlation analysis between the expression levels of the active ingredients and UGT1A1 and transporters in the model group and the normal group was conducted, and the results are shown in p < 0.05) were significantly correlated with the exposure amount of the seven components in vivo, respectively, while those of NTCP  (p < 0.05) and OATP1A1  (p < 0.05) were significantly correlated with the exposure amount of glycyrrhetinic acid, dehydrotumulosic acid, isoliquiritin, liquiritin, atractylenolide III, and cinnamic acid in vivo, respectively, and those of MDR1 were significantly correlated with the AUC values of glycyrrhizic acid (ρ = 0.681), glycyrrhetinic acid (ρ = 0.772), dehydrotumulosic acid (ρ = 0.657), isoliquiritin (ρ = 0.845), atractylenolide III (ρ = 0.802) (p < 0.01), and cinnamic acid (ρ = 0.748) (p < 0.05). In addition, the expression levels of NTCP and OATP1A1 were significantly correlated with the AUC values of glycyrrhetinic acid (ρ = 0.721 and ρ = 0.867), dehydrotumulosic acid (ρ = 0.685 and ρ = 0.733), isoliquiritin (ρ = 0.661 and ρ = 0.758), liquiritin (ρ = 0.685 and ρ = 0.685), atractylenolide III (ρ = 0.770 and ρ = 0.867) (p < 0.01), and cinnamic acid (ρ = 0.818 and ρ = 0.939) (p < 0.01). The expression levels of OCT1, MATE1, and MRP2 were found related to the AUC values of glycyrrhetinic acid , dehydrotumulosic acid , isoliquiritin , atractylenolide III , and cinnamic acid  (p < 0.05).The expression levels of BSEP  (p < 0.01), dehydrotumulosic acid (ρ = 0.794) (p < 0.01), atractylenolide III (ρ = 0.685) (p < 0.05), and cinnamic acid (ρ = 0.673) (p < 0.05), while those of OATP1A2 were significantly correlated with the AUC values of glycyrrhetinic acid (ρ = 0.733) (p < 0.05), dehydrotumulosic acid (ρ = 0.697) (p < 0.05), and cinnamic acid (ρ = 0.733) (p < 0.05).Besides, the expression levels of OATP1A4 were significantly correlated with the AUC values of glycyrrhetinic acid (ρ = 0.855) (As mentioned previously, NAFLD is a common chronic liver disease  and the 0-∞ and Cmax of glycyrrhizic acid, glycyrrhetinic acid, dehydrotumulosic acid, isoliquiritin, liquiritin, atractylenolide III, and cinnamic acid in the plasma of NAFLD rats were significantly lower than those of normal rats. This is basically the same trend in the normal group and the NAFLD rats. The mean residence time of atractylenolide III, glycyrrhizic acid, glycyrrhetinic acid, and liquiritin was increased in the NAFLD group when compared with the normal group. The mean retention time of isoliquiritin and dehydrotumulosic acid was decreased. In the rat model of NAFLD, the scavenging rates of seven analytes were increased when compared with the normal group, while the in vivo absorption of atractylenolide III, dehydrotumulosic acid, and glycyrrhetinic acid was enhanced. The absorption of glycyrrhizic acid, liquiritin, and isoliquiritin was inhibited when compared with that in the normal group. The pharmacokinetics of the main bioactive components of Ilex hainanensis extract after oral administration to normal and NAFLD rats were also studied (Cmax of chlorogenic acid, kaempferol 7-O-β-D-glucoside, and ilexgenin A was decreased significantly under the plasma concentration–time curve. Kaempferol 7-O-β-D-glucoside and ilexgenin A increased the MRT in NAFLD rats. This pharmacokinetic feature is similar to the hereby observed phenomenon.Liquid–liquid extraction can effectively reduce the endogenous effect. In the early stage of the experiment, organic reagents were used to precipitate protein, but the recoveries of dehydrotumulosic acid and glycyrrhizic acid were not higher than 50%. Methanol, ethyl acetate, and methanol combined with ethyl acetate were subsequently investigated, and the recovery of glycyrrhizic acid and dehydrotumulosic acid was found to be the highest when the samples were extracted with 500 µL ethyl acetate and treated with 250 µL methanol successively. The pharmacokinetics of seven LGZGD components in the normal and NAFLD rats were successfully compared by using the UPLC-MS/MS method. The pharmacokinetic behavior of the seven components was significantly different between the normal group and the fatty liver model rats. The AUC studied . The AUCChanges of enzymes and transporters in the rat liver directly affect drug absorption and metabolism . StudiesIt has also been shown that hepatic cytochrome P450 expression is significantly higher in NAFLD rats than in normal rats, which led to faster metabolism of the drug in the liver of NAFLD rats, , consistAn efficient and stable UPLC-MS/MS method was used to simultaneously determine seven components in rat plasma, which were successfully applied to study the pharmacokinetics of LGZGD in normal rats and NAFLD rats. It is also the first time to compare the pharmacokinetic characteristics of the seven components between normal and NAFLD rats after LGZGD administration. After LGZGD administration, the pharmacokinetic profiles of the seven compounds were significantly altered in NAFLD rats when compared to normal rats, which might be attributed to the decreased expression of UGT1A1 metabolizing enzymes and the nine transporters in NAFLD rats. The results indicate the applicability of UGT1A1 and the nine other transporters as potential biological markers for evaluating the ameliorative effects of LGZGD in NAFLD. Additionally, it is suggested that in hepatic pathological states, attention should be paid to the effects of abnormal drug metabolism on hepatic drug enzymes to avoid hepatotoxicity. Overall, the hereby obtained information can provide a meaningful basis for the clinical development of a dosing regimen for the treatment of NAFLD.


Cannabis is a multifaceted plant with numerous therapeutic properties on one hand, and controversial psychotropic activities on the other hand, which are modulated by CB1 endocannabinoid receptors. Δ9-Tetrahydrocannabinol (Δ9-THC) has been identified as the main component responsible for the psychotropic effects, while its constitutional isomer cannabidiol (CBD) has shown completely different pharmacological properties. Due to its reported beneficial effects, Cannabis has gained global popularity and is openly sold in shops and online. To circumvent legal restrictions, semi-synthetic derivatives of CBD are now frequently added to cannabis products, producing "high" effects similar to those induced by Δ9-THC. The first semi-synthetic cannabinoid to appear in the EU was obtained through cyclization and hydrogenation of CBD, and is known as hexahydrocannabinol (HHC). Currently, there is limited knowledge regarding HHC, its pharmacological properties, and its prevalence, as it is not commonly investigated in routine toxicological assays. In this study, synthetic strategies were explored to obtain an excess of the active epimer of HHC. Furthermore, the two epimers were purified and individually tested for their cannabinomimetic activity. Lastly, a simple and rapid chromatographic method employing a UV detector and a high-resolution mass spectrometer was applied to identify and quantify up to ten major phytocannabinoids, as well as the HHC epimers, in commercial cannabis samples. Δ9-Tetrahydrocannabinol (Δ9-THC), which was identified by Raphael Mechoulam in the early 1960s, is the primary phytocannabinoid responsible for the psychotropic effects of cannabis. The constitutional isomer of Δ9-THC, cannabidiol (CBD), is the second most abundant phytocannabinoid found in cannabis and does not exhibit any narcotic activity. CBD is predominantly present in cannabis cultivated for industrial purposes, commonly referred to as hemp varieties. Other so-called “minor” phytocannabinoids have been isolated and characterized, and some of them have demonstrated euphoriant activity similar to Δ9-THC. Among the recently discovered THC-type phytocannabinoids, such as Δ9-tetrahydrocannabiphorol (Δ9-THCP)2, Δ9-tetrahydrocannabihexol (Δ9-THCH)3, and Δ9-tetrahydrocannabibutol (Δ9-THCB)4, the heptyl homologue has exhibited an unequivocal cannabinomimetic activity even higher than Δ9-THC in animal experiments2.Cannabis is the most widely used illicit drug, with an estimated 4% of the population (15–64 years old) in 2019 reported to have used it, according to the World Drug Report 20219-THC as the main psychotropic component of cannabis, chemical and pharmaceutical research focused on modifying the lead compound to obtain more potent analogues5. Additionally, the discovery of the endocannabinoid receptors CB1 and CB2 has led to the identification of new scaffolds, enabling the development of increasingly potent synthetic cannabinoids (SCs). However, such compounds, which have limited therapeutic utility, are generally synthesized in illegal laboratories and sold for recreational use. Recently, over 250 synthetic compounds targeting the endocannabinoid system have been developed in clandestine laboratories6. These compounds are typically added to products often referred to as “Spice” or “K2” and distributed to end-users7. In contrast to THC, which has a relatively large safety margin when used recreationally and requires higher doses to cause serious adverse effects8, the use of SCs has been documented as completely unsafe, with a range of dangerous side effects9.After the identification of Δ10. These varieties can have high levels of CBD, which lacks cannabinomimetic properties and has been recognized as a potent antiepileptic drug11. However, when CBD is treated with acids, it cyclizes to form Δ9-THC and Δ8-THC12. Both THC isomers are subject to international restrictions and are scheduled under Schedule II of the United Nations Convention on Psychotropic Substances13. To address the marketing of controlled substances, the development of semi-synthetic derivatives of CBD has become a widely employed strategy in the USA. The first semi-synthetic cannabinoid (SSC) reported in the EU and monitored as a novel psychoactive substance (NPS) by the EU Early Warning System since October 21, 2022, is hexahydrocannabinol, better known as HHC14. HHC (hexahydrocannabinol) was first described in 1940 by Adams15, but it has only recently gained attention from toxicologists and analysts16. Despite its long history, few experimental studies have investigated its effects, particularly in humans. HHC is often sold as a “legal” alternative to illegal THC, and it is commonly sprayed onto or mixed with cannabis products marketed as “legal highs”14.Low-THC cannabis varieties (containing up to 0.3% THC) have recently been legalized in the USAR and 9S, while the configuration at C10a and C6a remains unchanged. Early studies on HHC’s biological activity in animals, although compromised by the low purity of the material used, indicated that HHC exhibited lower marijuana-like activity compared to THC18. Subsequent studies using better-characterized material revealed that the R epimer is the one responsible for cannabimimetic activity, while the S epimer is either devoid of or has less psychotropic effect19.HHC has three stereogenic centres, which theoretically give rise to eight stereoisomers. However, in practice, HHC is consistently found as two epimers: 920. The study found that the epimer with the equatorial methyl substituent ((R)-HHC) induced severe stupor, ataxia, immobility, and other effects indicative of high potency, even at low doses. The epimer with the axial methyl group ((S)-HHC) induced drowsiness and decreased motor activity at higher doses20. However, the authors noted that the compound used was not pure, and impurities of the other epimer could have influenced the results20.The psychotropic activity of HHC epimers was evaluated in a study by Mechoulam et al. in 1980, which involved administering the individual epimers of HHC to rhesus monkeysR epimer in certain industrial hemp products16. Therefore, it is important to investigate the formation of one epimer over the other starting from CBD and analyze the epimeric ratio in HHC-containing cannabis products. The present study aims to evaluate the epimeric excess resulting from different synthetic strategies and determine the concentrations of both epimers in commercial HHC-containing cannabis products. To achieve these goals, high-performance liquid chromatography coupled with a diode array detector and high-resolution mass spectrometry (HPLC–DAD-HRMS) was employed. In addition to HHC epimers, ten other phytocannabinoids commonly found in cannabis extracts were analyzed, including CBD, Δ9-THC, Δ8-THC, CBG, CBC, their corresponding carboxylated native precursors (cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabigerolic acid (CBGA), and cannabichromenic acid (CBCA)), and the THC oxidation product cannabinol (CBN) , while Δ8-THC was obtained when CBD was treated with p-toluensulfonic acid (pTSA) for a longer duration (18 h)21. Subsequent reduction of the double bond on the terpene group resulted in a mixture of the two HHC epimers. Hydrogenation of Δ9-THC yielded an excess of the S epimer compared to the R epimer (approximately 2:1 ratio), whereas hydrogenation of Δ8-THC gave a 3:1 epimeric ratio in favour of the R epimer21.The synthesis of HHC epimers was carried out by subjecting CBD to acidic treatment. Depending on the reaction conditions, cyclization and hydrogenation of CBD can lead to the formation of either ΔpTSA for specific durations, followed by hydrogenation without purification of the intermediates. HHC was obtained as the product in both reactions, but with different epimeric ratios depending on the reaction conditions. Specifically, the reaction with HCl for 2 h resulted in a 57:43 S/R ratio, while the reaction with pTSA for 18 h yielded a 61:39 R/S ratio -HHC and (9R)-HHC, were isolated using semi-preparative HPLC, achieving purity grades exceeding 95% and 99%, respectively, which were sufficient for conducting the biological tests. UV, MS, and NMR analyses were performed to characterize both epimers. The UV spectra were found to be superimposable  of a fragment at m/z 193.1223 in HESI + mode. This fragment likely corresponds to the resorcinol group attached to one carbon atom of the reduced terpene moiety, with the oxygen atom no longer included in the pyran ring -HHC, while the second eluted epimer corresponds to (9R)-HHC -HHC/(R)-HHC mixture with a 3:1 ratio, demonstrating that the signals indicated as “major” corresponded to the S isomer, while those indicated as “minor” corresponded to the R isomer22. The findings of Gaoni et al. and Archer's work support the characterization of the HHC epimers based on the NMR spectra. Gaoni et al. suggested that the isomer labeled as VIa, which exhibited a proton signal at C10a (C3 in the old numbering system) appearing “as a broad doublet centered at 2.85 ppm”, corresponded to the isomer with the methyl group at C9 (C1 in the old numbering system) in the axial position ((S)-HHC). Conversely, the “broad doublet centered at 3.05 ppm” in structure VIb corresponded to the isomer with the methyl group at C9 in the equatorial position ((R)-HHC)21. In the current study, a peculiar shift in the NMR spectrum was observed for the hydrogen attached to C10α, with a change from 2.85 ppm in (S)-HHC to 3.03 ppm in (R)-HHC. Additionally, the hydrogen atom of C10a exhibited a shift from 2.67 ppm in the S epimer to 2.49–2.37 ppm in the R epimer. These observed shifts are consistent with the data reported in the previously mentioned works22, further confirming the assignment of the stereochemistry of the HHC epimers in this study.The individual HHC epimers, , while the R epimer was found in the range of 17.13% to 26.46%.The method was applied to analyze all phytocannabinoids and HHC epimers in ethanolic extracts of two hemp biomass samples (HHC-1 and HHC-2), one HHC hashish sample (HHC-3), and a pure HHC sample (HHC-4) . Extraction of the analytes was performed using ethanol, as previous studies have demonstrated its superior ability to extract this class of compounds9-THC and Δ8-THC, with the former being predominant. On the other hand, THCA and CBCA were present in trace amounts. The cannabinoid concentrations in the analyzed samples are reported in Table All extracts exhibited a prominent presence of CBDA and CBD, along with lower amounts of CBGA, indicating that the inflorescence originated from industrial hemp varieties. Specifically, CBDA ranged from 2.8 to 7.18%, and CBD ranged from 1.20 to 2.60%. CBGA was also detected in low concentrations, ranging from 0.06 to 0.26%. Additionally, CBN was consistently present in all samples, with concentrations ranging from 0.05 to 0.21%. All samples showed the presence of both Δm/z 319.2629 and [M-H]- at m/z 317.2488, displaying higher intensity in positive ionization mode . This compound, with a predicted molecular formula of C21H34O2, presented a characteristic fragment at m/z 139.1480, which differed by two hydrogen atoms from that of HHC (137.1324) and by four hydrogen atoms from CBD- and THC-like cannabinoids (135.1174). This fragment could likely be attributed to the terpene moiety. Based on the chromatographic data, which showed a longer retention time compared to CBD, and the HRMS fragmentation pattern, a CBD-like structure with all saturated C–C bonds on the terpene moiety could be proposed. As its retention time fell between THC and HHC species, the presence of an additional free hydroxyl group could explain its higher hydrophilicity compared to HHC, while the complete saturation of the terpene moiety could account for its higher lipophilicity compared to THC. This compound is likely to derive from the hydrogenation of residual CBD starting material in the reaction mixture, known as H4CBD. The spectroscopic data aligns with the findings reported by Collins et al., who synthesized and characterized both HHC and H4CBD.Isomers of HHC could be tentatively identified in all extracts by HRMS, along with other oxidation derivatives of HHC. Since no standard was available for these compounds, only the molecular formula could be hypothesized . Interestingly, the sample of pure HHC exhibited a unique peak at 12.96 min with a precursor ion [M + H] + at R)-HHC and (9S)-HHC was evaluated using a tetrad of behavioral tests on mice. These tests assess spontaneous activity, catalepsy (immobility index), analgesia, and changes in rectal temperature -HHC at a dose of 10 mg/kg significantly reduced the spontaneous activity of mice in the open field test compared to the vehicle-treated group. The distance covered by mice in the open field was significantly decreased by (9R)-HHC , whereas (9S)-HHC did not affect locomotion  -HHC administration increased the latency of moving from the catalepsy bar, indicating a decrease in cataleptic behaviour. Although the difference was not statistically significant compared to the vehicle-treated group, there was a trend towards reduced catalepsy  for (9R)-HHC -HHC demonstrated a significant antinociceptive effect, indicating analgesic properties. The latency to respond to the hot plate stimulus was significantly increased by (9R)-HHC  compared to the vehicle-treated group (13.768 ± 2.367 s) -HHC did not induce catalepsy  or analgesia .In the hot plate test, (9 s) Fig. d. In conR)-HHC and (9S)-HHC showed a trend towards decreasing body temperature, indicating hypothermic effects. However, the difference was not statistically significant compared to the vehicle-treated mice. (9R)-HHC exhibited a greater trend towards decreasing body temperature  compared to (9S)-HHC , but further analysis is needed to establish statistical significance -HHC possesses cannabimimetic activity, as evidenced by decreased locomotor activity, decreased catalepsy, increased analgesia, and a trend towards hypothermia. In contrast, (9S)-HHC did not significantly affect these behavioural parameters.These results suggest that  or “spice” have emerged as illegal drugs. These SCs are structurally distinct from Δ9-THC and have been marketed as alternatives to cannabis.Since the discovery of Δ11. The World Health Organization (WHO) has suggested that CBD should not be classified as a scheduled substance, as it lacks the intoxicating properties associated with THC or other SCs27.In recent years, there has been a resurgence in the cultivation of cannabis for industrial purposes, particularly due to the high concentrations of CBD, which is non-psychoactive and possesses various therapeutic properties. CBD is used in medicinal products for the treatment of certain types of childhood epilepsyIn some European countries, the recreational use of industrial hemp inflorescence as a substitute for high-THC cannabis has become a widespread phenomenon known as “light cannabis”. However, the legal status of such products remains unclear in European legislation. In parallel, the USA have allowed the legal marketing of all cannabis products with THC levels below 0.3% with the Farm Bill Act in 2018.9-THC and Δ8-THC. By removing the double bond at either the C9 or C8 position, a new stereogenic center is generated, resulting in either the R or S epimer. Mechoulam had suggested that it is possible to obtain an excess of the R epimer from Δ8-THC and an excess of the S epimer from Δ9-THC21.Alongside the emergence of SCs, there has been the appearance of semi-synthetic cannabinoids (SSCs) in the United States. These compounds are derived from chemical modifications of cannabinoids extracted from cannabis. One such compound, HHC, is the hydrogenation product of ΔR epimer has a higher affinity for the CB1 receptor, while the S epimer has poor affinity19. This suggests that different epimeric mixtures with varying intensities of biological effects can be obtained based on the synthetic procedure.In vitro studies have indicated that the 9-THC or Δ8-THC under acidic conditions, depending on factors such as reaction time, catalyst type, and reaction conditions21. As a result, the production of Δ8-THC and Δ9-THC as semi-synthetic products from CBD extracted from hemp has increased14. However, both Δ8-THC and Δ9-THC are controlled substances and not legally marketed. Therefore, the subsequent reduction of the double bond to form semi-synthetic HHC has allowed for a legal alternative that offers similar psychoactive effects to THC. HHC is currently being sprayed onto hemp products and openly sold to provide the desired “high” effects of cannabis.CBD has been shown to isomerize to either ΔR epimer were consistently higher than those of the S epimer in all samples.The scientific studies on HHC are limited, highlighting the need for further investigation. The researchers’ work demonstrates that HHC can be obtained from CBD, and the reaction conditions can selectively favour the production of one epimer over the other. Using a validated analytical method, the presence of HHC was identified in two commercial samples of hemp biomass (HHC-1 and HHC-2) and one hemp hashish sample (HHC-3). The concentrations of the 9-THC and Δ8-THC in the biomass extracts and pure HHC mixture were below 0.5%, while the HHC hashish sample contained around 2% of these phytocannabinoids. The presence of Δ9-THC and Δ8-THC could be attributed to residues from the HHC synthesis process, although a natural origin cannot be ruled out. The relatively high levels of HHC compared to CBD and THC in the samples suggest its exogenous origin rather than being naturally occurring28.The hemp biomass samples also contained significant concentrations of other phytocannabinoids, particularly CBDA and CBD, indicating that they were derived from industrial hemp. The levels of ΔR epimer was consistently found in excess of the S epimer, it is likely that HHC was synthesized starting from CBD with Δ8-THC as the reaction intermediate, rather than using Δ9-THC. Analysis of the pure HHC sample (HHC-4) revealed the absence of CBDA and CBD, but the presence of CBN, Δ8-THC, and Δ9-THC as impurities alongside the main component HHC. The R epimer was slightly in excess, indicating a similar synthetic route.Furthermore, since the R)-HHC significantly affected two out of the four behaviours in the tetrad test: hypolocomotion (reduced spontaneous activity) and analgesia (pain relief). These findings suggest a partial CB1-mediated psychotropic effect. However, the potency of HHC in inducing the tetrad effects was lower than that of Δ9-THC and Δ9-THCP, a recently discovered THC analog.To assess the cannabimimetic activity of HHC, the tetrad test in mice was conducted. Due to the limited availability of the two epimers, only one dose (10 mg/kg) was tested, similar to the dose used for THC in the tetrad test. The results showed that ; dichloromethane (CH2Cl2); cyclohexane (CE); chloroform (CDCl3); ethanol (EtOH). Stock solutions (1 mg/mL) of certified reference cannabinoid standards of CBDA, Δ9-THCA, CBGA, CBCA, CBD, CBG, CBC, CBN, as well as of Δ9-THC and Δ8-THC (500 µg/mL) were bought from Cayman Chemical . Stock solutions (1 mg/mL) of CBDH, Δ9-THCH, (9R)-HHC and (9S)-HHC were obtained by properly diluting the pure compounds in-house synthesized.Ethanol 96%, acetonitrile (ACN), and formic acid (FA) were all LC–MS grade and were bought from Sigma Aldrich (USA). Ultrapure water was obtained with a water purification system . Chemicals and solvents employed in the synthetic process were reagent grade and used without further purification. The following abbreviations for common organic solvents were adopted: diethyl ether  of the European Union Treaty (GUCE 26/10/2012).Samples of hemp biomass (HHC-1 and HHC-2), HHC hashish (HHC-3) and pure HHC were purchased from Baked Bologna . The use of 2CO3 and subject to hydrogenation with an H-Cube ThalesNano flow reactor  according to the following experimental conditions: 3 mm 10% Pd/C cartridge, 30 °C, 20 bar, 1 mL/min. The crude product mixture showed a 57:43 S/R HHC mixture along with other by-products. The solvent was evaporated and the crude of the reaction was purified with semipreparative HPLC–UV . An isocratic elution was employed with mobile phase 80% ACN (with 0.1% FA) and 20% MilliQ water (with 0.1% FA) at a flow rate of 7.5 mL/min. The UV trace was followed at 228 nm and the compounds of interest were obtained with a purity higher than 95% (12 mg for (9R)-HHC and 8 mg for (9S)-HHC).CBD (0.7 mmol) was dissolved in 20 mL of absolute EtOH containing 0.05% HCl and refluxed for 2 h. The resulting crude was neutralized with a saturated solution of Na2Cl2, pTSA  was added at room temperature, under a nitrogen atmosphere. The reaction was stirred in the same conditions for 48 h. After that, the mixture was diluted with Et2O and washed with a saturated solution of NaHCO3. The organic layer was collected, washed with brine, dried over anhydrous Na2SO4 and concentrated. The obtained crude was subject to hydrogenation as described for the synthetic procedure b to give a crude product with a 61:39 R/S HHC mixture The two epimers were purified with semi-preparative HPLC as reported above (11 mg for (9S)-HHC and 8 mg for (9R)-HHC).To a solution of CBD (0.7 mmol) in 15 mL of anhydrous CH1H and 100.62 MHz for 13C) or a Bruker 600 spectrometer (working at 600.130 MHz for 1H and 150.902 MHz for 13C). Monodimensional spectra were acquired with a spectral width of 8278 Hz (for 1H NMR) and 23.9 kHz (for 13C NMR), a relaxation delay of 1 s and a number of transients of 32 and 1024 for 1H NMR and 13C NMR, respectively. NMR spectra were acquired in CDCl3 and chemical shifts (δ) were registered in ppm with respect to that of the residual solvent (δ = 7.26 ppm for 1H and δ = 77.20 ppm for 13C); coupling constants are reported in Hz, splitting patterns are expressed as singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), quintet (qnt), multiplet (m), broad signal (b).NMR spectra were recorded either on a Bruker 400  δ 6.25 , 6.07 , 4.61 , 2.92–2.79 , 2.67 , 2.46–2.38 , 2.11 , 1.68–1.61 , 1.58–152 , 1.46–1.44  1.36 , 1.33–1.28 , 1.13 , 1.09 , 0.88 . 13C NMR  δ 155.38, 154.77, 142.65, 110.63, 110.19, 107.79, 77.03, 50.09, 36.33, 35.59, 32.40, 31.77, 30.76, 29.49, 28.06, 27.76, 27.10, 23.26, 22.73, 19.30, 18.98, 14.20.1H NMR  δ 6.25 , 6.08 , 4.64 , 3.03 , 2.49–2.37 , 1.87–1.80 , 1.67–1.60 , 1.59–1.52 , 1.46–1.44 , 1.36 , 1.34–1.24 , 1.16–1.07 , 1.06  0.94 , 0.88 , 0.78 . 13C NMR  δ 155.15, 154.83, 142.74, 110.44, 110.23, 107.77, 77.14, 49.30, 39.16, 35.71, 35.58, 33.03, 31.75, 30.76, 28.24, 27.94, 22.78, 22.73, 19.22, 14.21.A Vanquish Core system  with binary pump, vacuum degasser, thermostated autosampler and column compartment, and diode array detector (DAD) was interfaced to an Exploris 120 Orbitrap mass analyzer with a heated electrospray ionization source (HESI). The chromatographic separation was achieved on a Poroshell 120 EC C18   with an isocratic elution at 70% ACN for 20 min and a washing step at 98% ACN for 3 min. The column was re-equilibrated at 70% ACN for further 3 min for a total run time of 26 min at a constant flow rate of 0.5 mL/min.29. The mass analyzer was operated in both full scan (FS) and data-dependent acquisition (DDA) mode. In FS mode the resolution was set at 60,000 FWHM , the RF lens level at 70%, the maximum injection time 54 ms, the m/z range at 150–750, and the isolation window at m/z 1.2. In DDA mode the resolution was set at 30,000 FWHM, the maximum injection time at 22 ms, the m/z range at 50–750, the isolation window at m/z 1.2, and the stepped normalized collision energy (NCE) at 20–40-10029. The injection volume was 5 µL. The analyses were acquired with Xcalibur 3.0 and processed using Chromeleon 7 for the UV traces and TraceFinder 54.0 for the MS traces .The HESI parameters were optimized in previous works for cannabinoids: spray voltage 4200 kV and 3800 kV for HESI + and HESI- mode respectively, sheath gas 70 au, auxiliary gas 5 au, sweep gas 0.5 au, ion transfer tube temperature 390 °C and vaporizer temperature 150°C24. The extracts HHC-1 and HHC-2 from hemp inflorescence were analysed after 100 × dilution with mobile phase, while the extract HHC-3 from HHC hashish was 1000 × diluted. The sample of pure HHC (HHC-4) was injected at the concentration of 10 µg/mL obtained by dissolving 10 mg of the sample in 1 mL of ACN and preparing serial 10 × dilutions with mobile phase up to the desired concentration.Calibration solutions of all phytocannabinoid standards were prepared by diluting the stock solutions with ACN to get the final concentrations indicated in Table m/z of the precursor ion in both HESI + and HESI- mode was extracted with a 5-ppm error from the HRMS TIC and used for calibration.Quantification of cannabinoids was accomplished with both UV and MS data. The UV chromatograms were extracted at 228 nm. The exact n = 4). They were divided into two groups: one group received (9R)-HHC at a dose of 10 mg/kg dissolved in a vehicle  via intraperitoneal (i.p.) administration, and the other group received only the vehicle as a control. The same animals were used for all four behavioural tests.In the experiment, male C57BL6/J mice at 7 weeks old were used (R)-HHC on hypomotility (measured using the open field test), hypothermia (measured by monitoring body temperature), antinociception , and catalepsy (assessed through the bar test) were assessed. These tests were performed following the procedures described by Metna-Laurent et al.30.The effects of  followed by Tukey’s multiple comparisons test.Body temperature. The measurement of body temperature was performed after immobilizing the mouse. A probe was gently inserted 1 cm into the rectum, and the temperature was recorded once it stabilized. The probe was cleaned with 70% ethanol and dried with a paper towel between each mouse to prevent cross-contamination.Open field. The open field test was conducted 30 min after administering the drug (or vehicle). The apparatus used for the test was cleaned with a 70% ethanol solution before each behavioural session. Naïve mice were randomly assigned to different treatment groups. The mice were placed in an open field arena (dimensions: length × width × height: 44 × 44 × 30 cm), and their ambulatory activity  was recorded and analyzed for a duration of 15 min. An automated behavioural tracking system called Any-maze, specifically the Video-tracking software by Ugo Basile, was used to record and analyze the behaviours.Bar test. The bar test was conducted to assess catalepsy. A glass rod measuring 40 cm in length and 0.4 cm in diameter was horizontally elevated 5 cm above the surface. The mouse’s forelimbs were positioned on the bar while the hind legs remained on the floor of the cage, ensuring that the mouse was not lying down on the floor. The chronometer was started when the mouse held onto the bar with both forelimbs, and it was stopped when the mouse descended from the bar  or after 5 min (cut-off time). Catalepsy was measured as the duration in seconds that the mouse held the elevated bar.Hot plate. Each mouse was placed on a hot plate (Ugo Basile), which was kept at the constant temperature of 52 °C. Licking of the hind paws or jumping were considered as a nociceptive response (NR) and the latency was measured in s 85 min after drug or vehicle administration, taking a cut-off time of 30 s to prevent tissue damage. The hot plate test was performed to evaluate antinociceptive effects. Each mouse was placed on a hot plate set at a constant temperature of 52 °C. The licking of hind paws or jumping were considered nociceptive responses (NR), and the latency to respond was measured in seconds. The measurement was taken 85 min after drug or vehicle administration, with a cut-off time of 30 s to prevent tissue damage.In the described experiments, male C57BL/6 mice from Charles River  were used. The mice weighed between 18 and 20 g. The mice were acclimated to the laboratory conditions for at least 1 week before the start of the experiments. The laboratory maintained a 12-h light/dark cycle with the lights turning on at 6:00 A.M. The temperature in the facility was maintained at 20–22 °C, and the humidity was kept at 55–60%. The mice were housed in cages with three mice per cage. They had access to standard chow and tap water ad libitum, meaning they could eat and drink freely.31. Animal care and welfare were the responsibility of trained personnel who adhered to the relevant Italian and European regulations. Every effort was made to minimize the number of animals used in the experiments and to prevent any unnecessary suffering or harm to the animals during the course of the study.  and the European Commission  regarding the protection of animals used for research purposes. The experimental methods described in the study also followed the ARRIVE guidelines, which provide recommendations for reporting animal researchdy. Fig. .Supplementary Information.


Streptococcus agalactiae is a highly contagious pathogen that causes bovine mastitis, leading to significant economic losses. This study aimed to (1) identify and characterize S. agalactiae strains responsible for bovine mastitis by examining their phenotypic and genotypic characteristics in Thai dairy-intensive farming areas and (2) determine their susceptibility profiles to antimicrobial agents.S. agalactiae isolates obtained from clinical and subclinical mastitis cases from 13 dairy herds located in the central region of Thailand were examined. To confirm the identity of the bacterial pathogens, conventional microbiological procedures recommended by the National Mastitis Council (NMC) and the VITEK® 2 system were employed.In total, 100 S. agalactiae using the NMC procedure, whereas 94 isolates were identified as S. agalactiae using the VITEK® 2 system. Finally, the S. agalactiae-specific gene dlt S was identified in all the examined isolates using polymerase chain reaction. Capsular polysaccharide (CPS) typing revealed that all strains belonged to CPS type Ia. Multilocus sequence typing identified 33 selected isolates as sequence type 103. Random amplified polymorphic DNA (RAPD) typing yielded 43 RAPD types, with 6 RAPD clusters identified. These results demonstrated a high level of genetic diversity among S. agalactiae within the studied herds. RAPD analysis suggested that specific S. agalactiae strains could persist in dairy farms for 2–12 months. Furthermore, antimicrobial susceptibility testing was performed using the broth microdilution method. Most strains demonstrated susceptibility to ampicillin, penicillin, penicillin/novobiocin, cephalothin, oxacillin, ceftiofur, and erythromycin.All 100 isolates were successfully identified as S. agalactiae isolates responsible for bovine mastitis in the central region of Thailand. The rapid identification of S. agalactiae and application of molecular typing methods can provide valuable epidemiological information regarding S. agalactiae causing mastitis in dairy farms. The antimicrobial susceptibility of S. agalactiae indicates that antimicrobial treatment for control and eradication could be a successful protocol. Our findings revealed that a single clonal strain of S. agalactiae affected the 13 studied farms. Further research is needed to explore the feasibility of vaccine development and application.This study revealed the phenotypic and genotypic characteristics of  The use of beta-lactam antimicrobials is the recommended treatment strategy for S. agalactiae mastitis  and acceptable reproducibility when analyzing isolates simultaneously  103 strain  No. IBC 1931051.2.2.S. agalactiae isolates were obtained from 58 lactating cows diagnosed with clinical (n = 21) and subclinical (n = 37) mastitis between 2016 and 2019. Clinical mastitis cases were assessed by a veterinarian through visual examination, which involved evaluating milk abnormalities and udder abnormalities including warmth, redness, and swelling. Subclinical mastitis was determined using the California mastitis test, resulting in positive findings. Quarter culture analysis led to the identification of S. agalactiae in one quarter (n = 33), two quarters (n = 11), three quarters (n = 11), and all quarters (n = 3) of infected cows  Laboratory Handbook on Bovine Mastitis . The sample plates were incubated with carbon dioxide at 37°C overnight. After incubation, a pure colony was observed on the plate. Subsequently, it was subcultured for the second round, and a single pure colony was selected for further analysis.2.3.S. aureus ATCC 25923  and S. agalactiae ATCC 12400 (CPS type Ia), ATCC 13813 (CPS type II), and ATCC 31475 (CPS type III).The following bacterial reference strains were obtained from the American Type Culture Collection , which were used for laboratory quality control: 2.4.® Blood & Tissue Kit  according to the instruction protocol. The concentration and purity of the extracted DNA were measured using a spectrophotometer . The extracted DNA was used as the template for all polymerase chain reaction (PCR) assays. All DNA templates were collected and stored at −20°C for further analysis.DNA extraction was performed in a BSL-2 cabinet. DNA was extracted from the pure culture using DNeasy2.5.® 2 system  following the manufacturer’s protocol; and (3) conventional genotypic molecular PCR using specific primers for the dlt s gene (dlts-F (5′-AGGAATACCAGGCGATGAACCGAT-3′) and dlts-R (5′-TGCTCTAATTCTCCCCTTATGGC-3′) were used. PCR was performed following the protocol used by Poyart et al. , 1 μL of the forward primer, 1 μL of the reverse primer, 2 μL of the DNA template, and 8.5 μL of distilled water (DW). The PCR mixtures underwent amplification in a DNA thermal cycler , involving an initial denaturation step for 5 min at 95°C, followed by 35 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension cycle at 72°C for 5 min. The PCR products and a 1-kb DNA size marker were analyzed in a 1.5% agarose gel, stained with nucleic acid gel stains  at 100 V for 1 h. Finally, the agarose gel was visualized and photographed using a UV transilluminator. To confirm the identification of S. agalactiae, a PCR amplicon size of 952 bp was expected.The 100 isolates were identified using three methods: (1) conventional microbiological techniques following the NMC Laboratory Handbook on Bovine Mastitis . Brieflyt s gene , 35. Thet et al. . Each PC2.6.Molecular characterization assays, including CPS, MLST, and RAPD fingerprint typing, were employed to assess the genotypic diversity and relationships among the isolates.2.6.1.cps genes. This technique was previously introduced by Poyart et al. (® Flexi Buffer (PROMEGA), 1 μL of the forward primer, 1 μL of the reverse primer, 2 μL of the DNA template, and 8.5 μL of DW. In the case of a negative control, 10.5 μL of DW was used. Positive controls included the reference S. agalactiae strains ATCC 12400 (type Ia), ATCC 13813 (type II), and ATCC 31475 (type III). The PCR mixtures were amplified in a DNA thermal cycler  with an initial denaturation step for 5 min at 95°C, followed by 35 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension cycle at 72°C for 5 min. The PCR products were analyzed on a 1.5% agarose gel, stained with nucleic acid gel stain , and subjected to gel electrophoresis at 100 V for 1 h. Finally, the agarose gel was visualized and photographed using a UV transilluminator.The CPS type of the studied isolates was determined through PCR assays targeting nine t et al. . The seq2.6.2.Thirty-three isolates were selected for MLST to represent both clinical and subclinical mastitis cases from all the studied herds. Therefore, at least one isolate per clinical and subclinical mastitis case within a herd was selected. Additionally, isolates from repeated mastitis cases were included .S. agalactiae was performed as described by Jones et al.  was used to determine the ST. A comparative analysis of geographic regions based on ST, using the eBURST program, led to the designation of clusters or clonal complexes.MLST for s et al. . Nested 2.6.3.® Flexi Buffer (PROMEGA), 0.4 μM of primer, and 50 ng of DNA template. Primers for OPS-11 (5′-AGTCGGGTGG-3′) were used in this study. The PCR mixtures were subjected to a DNA thermal cycler , beginning with an initial denaturation step for 5 min at 94°C, followed by 35 cycles at 94°C for 30 s, 55°C for 1 min, 72°C for 5 min, and a final extension cycle at 72°C for 5 min. The reference S. agalactiae ATCC 13813 strain was used as a control, and DW was used for the negative control. All amplified products and a 1-kb DNA size marker were analyzed in a single batch on a 2% agarose gel, stained with nucleic acid gel stain , at 70 V for 180 min. Finally, the agarose gel was visualized and photographed using a UV transilluminator.RAPD fingerprints were determined according to the protocol described by Martinez et al. . The reaS. agalactiae isolates. Isolates with ≥80% similarity were considered to belong to the same genotypic pattern or cluster. The discriminatory power (D) was calculated using the Hunter–Gaston formula  was used. The CMV1AMAF panel comprised the following 10 antimicrobials in a serial two-fold dilution: ampicillin, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin/novobiocin combination, pirlimycin, sulfadimethoxine, and tetracycline. The range of concentrations of antimicrobials used in the CMV1AMAF plate is shown in S. aureus ATCC 29213 was employed as the laboratory control strain. MIC was defined as the lowest concentration of an antimicrobial that inhibited the visible growth of an isolate. The MIC breakpoints (μg/mL) were derived from the Clinical and Laboratory Standards Institute (CLSI) guidelines for S. agalactiae (CLSI 2019 and 2020) supplement VET08 ED4 or M100 ED30. The MIC breakpoints were interpreted as susceptible, resistant, and intermediate according to CLSI VET08. MIC50 and MIC90 represent the concentrations at which 50 and 90% of the studied isolates were inhibited, respectively.Antimicrobial susceptibility was assessed using the broth microdilution method. Minimum inhibitory concentrations (MICs) were determined through broth microdilution, employing the semi-automatic Sensititre™ system from Thermo Fisher Scientific3.3.1.n = 88). Furthermore, all isolates produced positive results in the CAMP test but negative results in the esculin hydrolysis and catalase tests.All 100 isolates were assessed for their phenotypic characteristics, including the presence of beta hemolysis on blood agar for bacterial colony growth . Using the VITEK® 2 system, the identification time for S. agalactiae ranged from 4.60 to 7.80 h.The VITEKS. agalactiae isolates exhibited positive reactions in the fermentation and acid production tests, including d-galactose, lactose, d-maltose, d-mannose, N-acetyl-d-glucosamine, lactose, phosphatase, and alanine arylamidase. Miscellaneous tests such as polymyxin, bacitracin, and novobiocin also yielded positive results. However, the S. agalactiae isolates tested negative in the fermentation and acid production tests for d-amygdalin, d-xylose, d-sorbitol, d-mannitol, d-raffinose, phosphatidylinositol phospholipase C, l-aspartate arylamidase, beta-galactopyranosidase, alpha-mannosidase, l-pyroglutamyl arylamidase, and urease.All 94 identified S. agalactiae isolates were classified into 37 bionumber types, with a discriminatory power of 0.915. Among the isolates identified using this system, the majority belonged to bionumber type 1 (n = 25) (data not shown).Furthermore, the 94 dlt S gene confirmed that all the studied isolates belong to S. agalactiae. This confirmation was achieved through three bacterial identification procedures, all of which confirmed that our studied isolates were indeed S. agalactiae.Genotypic identification using the specific 3.2.3.2.1.n = 100). In the MLST study involving the 33 selected isolates, only one multilocus ST (ST 103) was identified in all 13 herds, indicating a consistent correlation between the CPS and MLST genotypes and confirming the existence of a single S. agalactiae clone in our study.All isolates were classified as CPS type Ia . The resulting RAPD fingerprints exhibited the varied number of bands, ranging from 3 to 7, with sizes spanning 300–2,200 bp. A dendrogram was constructed based on these RAPD profiles, and a similarity analysis revealed 43 distinct RAPD types among the 100 isolates .n = 34) and 32% (n = 30) of the studied isolates , D (n = 12), E (n = 30), and F (n = 9) were consistently identified throughout this period. Clusters A and B (n = 5) were observed in 2016 and 2019 only . Subclusters grouped pathogens with 100% identical RAPD types, indicating a close genetic relationship.According to RAPD typing, In two subcluster groups, isolates were obtained from the same cow but different quarters during specific periods. In herd I, subcluster E35.1 was isolated from the left front quarter of a cow named “dangnoi,” while subcluster E35.2 was isolated from the left hind quarter of the same cow. Similarly, in herd F, subcluster F37.1 was isolated from the right front quarter of a cow named “aumpun,” and subcluster F37.2 was isolated from the left hind quarter of the same cow.Furthermore, one subcluster was able to infect at least two cows simultaneously in several herds .Several subclusters, including C13.1–C13.3, C16.1–C16.3, and C18.1–C18.3, were able to cause mastitis occurrences for up to 2–7 months. In herd F, subcluster E  was observed, with two cows exhibiting an association with these subclusters over 6 months. Similarly, in herd D, subcluster C (comprising C11.1 and C11.3) was identified, with two cows showing an association with these subclusters over 12 months.S. agalactiae. RAPD genotype clusters may be related to the VITEK® 2 bionumber phenotype. Only the following six cases demonstrated a relationship between the RAPD cluster and VITEK® 2 bionumber type: cow “5543” (RAPD clusters C13.1 and C18.2 related to VITEK® 2 bionumber type 1), cow “58007” (clusters E26.1 and E27.2 related to bionumber type 1), cow “58041” , cow “gene” (clusters E31.1 and E31.2 related to bionumber type 1), cow “jumba” (clusters E31.1 and E31.2 related to bionumber type 1), and cow “som” (clusters C10.3 and C14 related to bionumber type 20)  .3.3.S. agalactiae isolates obtained from 2016 to 2019 were tested against 10 antimicrobial drugs. 50 and MIC90 values. Both MIC50 and MIC90 for ampicillin, penicillin, penicillin/novobiocin, cephalothin, ceftiofur, erythromycin, oxacillin, and pirlimycin were 0.125, 0.125, 1/2, 2, 0.5, 0.25, 2, and 0.5 μg/mL, respectively. The MIC50 and MIC90 for tetracycline were 1 and 8 μg/mL, respectively, whereas those for sulfadimethoxine were 128 and 256 μg/mL, respectively.The 100 S. agalactiae isolates were resistant to tetracycline (33%) and sulfadimethoxine (50%).All isolates exhibited susceptibility to ampicillin, penicillin/novobiocin, cephalothin, oxacillin, and pirlimycin. Moreover, 99% of the isolates were susceptible to penicillin, followed by erythromycin (99%) and ceftiofur (98%). Notably, 4.S. agalactiae, which causes bovine mastitis among animals in dairy-intensive farming areas in the central region of Thailand. Knowledge on mastitis caused by this pathogen is useful for the application of effective control and prevention strategies.This study identified and characterized S. agalactiae, commonly known as Group B streptococcus, is a causative pathogen of bovine mastitis and plays a crucial role in the control and eradication of IMM infections caused by S. agalactiae. The standard method for diagnosing S. agalactiae involves bacterial culture of milk samples on blood agar, following the procedure established by the NMC , Finland , Brazil , Germany , and Iran (types II and III)  , 52–54.S. agalactiae in this area. However, our study was conducted in the central region of Thailand, which is known for its high-density farming areas, and revealed a significant genetic similarity among S. agalactiae isolates from herds. This finding suggests the potential transmission of a dominant clone, likely due to inadequate adoption of within-herd biosecurity measures and cow-to-cow transmission during milking   . Howeverame herd .S. agalactiae demonstrated genetic relatedness among infected quarters within the same cow, suggesting a pattern of pathogen transmission. This finding also implies contagious behavior, indicating the ability of the pathogen to transmit from cow to cow within the same herd and to circulate within the herd for extended periods when control and prevention strategies may be less effective. The occurrence of direct transmission between mammary glands during the milking procedure serves as a strong indicator of the effectiveness of the biosecurity program implemented in infected herds, as supported by a previous study   . The redal agent . InapproS. agalactiae. In a previous study, IMM treatment of S. agalactiae with cefquinome achieved a high cure rate in 14–21 days. However, in cases without antimicrobial treatment, new infections were reported in the initially negative culture or previously uncured quarters group. Furthermore, the untreated quarters exhibited high levels of S. agalactiae shedding  have shown significant antibacterial activity against both gram-positive and gram-negative bacteria associated with bovine mastitis in Thailand, including hia coli . FurtherS. agalactiae as the causative agent of mastitis in dairy-intensive farming areas in Thailand. We elucidated both the phenotypic and genotypic characteristics of S. agalactiae isolates responsible for bovine mastitis. Rapid identification of S. agalactiae and the application of molecular typing methods can provide valuable epidemiological insights into mastitis caused by S. agalactiae in dairy farms. Our findings revealed a single clone with CPS type Ia and ST 103, indicating the transmission and circulation of this pathogen in herds with contagious behavior. Beta-lactam antimicrobials have proven to be effective and should be judiciously selected for a robust treatment protocol. Treating all quarters of infected cows is essential for the effective control and eradication of mastitis in the evaluated herds.To the best of our knowledge, this study investigated The original contributions presented in the study are included in the article/All procedures have been reviewed and approved by the Institutional Biosafety Use protocol  No. IBC 1931051.KA and SW designed the study, conducted statistical analyses, and drafted the manuscript. SW, TB, and SS performed laboratory analyses. SW assembled the data and interpreted the results. All authors contributed to the article and approved the submitted version.