Synechocystis salina is a cyanobacterium that has biotechnological potential thanks to its ability to synthesize several bioactive compounds of interest. Therefore, this study aimed to find optimal conditions, in terms of temperature (15–25 °C), pH (6.5–9.5), and NaCl concentration (10–40 g·L−1), using as objective functions the productivities of biomass, total carotenoids, total PBPs, phycocyanin (PC), allophycocyanin (APC), phycoerythrin (PE), and antioxidants (AOXs) capacity of Synechocystis salina  strain LEGE 06155, based in factorial design resorting to Box-Behnken. The model predicted higher biomass productivities under a temperature of 25 °C, a pH of 7.5, and low NaCl concentrations (10 g·L−1). Maximum productivities in terms of bioactive compounds were attained at lower NaCl concentrations (10 g·L−1) (except for PE), with the best temperature and pH in terms of carotenoids and total and individual PBPs ranging from 23–25 °C to 7.5–9.5, respectively. PE was the only pigment for which the best productivity was reached at a lower temperature (15 °C) and pH (6.5) and a higher concentration of NaCl (≈25 g·L−1). AOX productivities, determined in both ethanolic and aqueous extracts, were positively influenced by lower temperatures (15–19 °C) and higher salinities (≈15–25 g·L−1). However, ethanolic AOXs were better recovered at a higher pH (pH ≈ 9.5), while aqueous AOXs were favored by a pH of 8. The model showed that biomass production can be enhanced by 175% (compared to non-optimized conditions), total carotenoids by 91%, PC by 13%, APC by 50%, PE by 130%, and total PBPs by 39%; for AOX productivities, only water extracts exhibited a  improvement of 1.4%. This study provided insightful information for the eventual upgrading of Synechocystis salina biomass in the biotechnological market. The second-order polynomial was established as follows:1, β2, and β3 are linear coefficients; γ1, γ2, and γ3 are two-factor interaction coefficients; ⍵1, ⍵2, and ⍵3 are quadratic coefficients; and A, B, and C are independent variables, i.e., temperature, pH, and [NaCl], respectively.In which Y is the predicted response; α0 is a constant (intercept); βThe polynomial equation was also expressed as surface and contour plots; the fit adequacy of the quadratic model and the associated regression coefficient, r, was determined for every objective function. In addition, the best conditions estimated for each response were scored regarding their desirability. This function varies from 0 to 1, with the highest score representing a completely desirable or ideal response value.S. salina was cultivated as per the conditions associated with the central point of the design, i.e., 25 g·L−1 of [NaCl], a pH of 8, and 20 °C. Each run was established with an initial optical density (OD) of 0.1 (OD = λ680 nm − λ750 nm), and the experiments were carried out in two L-flat bottom round flasks (1.8 L of working volume) maintained in batch over 22 d. The light was provided by fluorescent lamps  with an intensity of 150 µmolphotons·m−2·s−1 and an L/D cycle of 16 h/8 h. Agitation was provided via continuous air bubbling, with an airflow of 0.75 Lair·L−1·min−1. To maintain pH in the medium, different buffer solutions were added to a final concentration of 30 mM MES buffer  was used to keep the pH at 6.5, HEPES buffer  was used to keep the pH at 8, and, finally, CHES buffer  was used to keep the pH at 9.5. The pH was duly adjusted at the beginning of each experiment, with a tolerance range of ±0.5, and was periodically monitored  with a pH meter .In all runs, pre-inocula were established over 22 days with BG11 culture medium. To standardize the adaptation of the cyanobacterium to each condition tested, The biomass was evaluated by the determination of the OD and dry weight (DW); hence, culture samples were collected regularly . The OD was obtained in a microplate spectrophotometer reader  at a wavelength of λ = 680–750 nm for each biological replicate and assayed in triplicate.−1, was diluted up to 10 g·L−1 NaCl, as described elsewhere  of 25 and 40 g·Llsewhere . All DW x, mg·L−1·d−1) was determined using DW experimental data and estimated based on Px (t) = (Xmax − X0)/(t − t0), where X0 is the biomass at the beginning of the experiment (time t0) and Xmax denotes the maximum biomass concentration attained at time t.The maximum biomass productivity (Pv/v) and water, respectively, on days 0, 1, 4, 6, 8, 11, 15, 18, 20, and 22. These extracts were also used to ascertain the antioxidant capacity, as described in For the total carotenoids and PBPs, a sequential extraction was performed with ethanol ; the supernatant was discarded, and 3 mL of ethanol was added to the pellet with glass beads. Each sample was extracted in a Precellys homogenizer , using a 6 min cycle at 8000 rpm . Ethanolic extracts were centrifuged at 2744× g for 10 min, kept under dimmed light, and stored at −4 °C until pigment content and antioxidant capacity determination.To analyze the total carotenoid content, 3 mL aliquots of different culture conditions were harvested and centrifuged  according to Lichtenthaler and Buschmann (2001) . The absg for 8 min and then kept under dark conditions before the analysis for pigment content and antioxidant capacity.For the extraction of total PBPs, the remaining pellet of ethanolic extraction was added with 3 mL of water, and then samples were subjected to vortexing for 20 s. The extract was centrifuged at 2744× The determination of the PBP content, namely phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE), was via the spectrophotometric measurement of absorbance at λ = 562, 615, and 652 nm, followed by the application of Bennett and Bogorad′s equations , see Equ−1·d−1.The PBP productivities were expressed in mg·La were duly determined.For the run that achieved the highest total carotenoid productivities, the profile and content in carotenoids and chl g for 10 min. A volume of 5 mL of acetone (100%) and 100 µL of the internal standard solution of trans-β-Apo-8′-carotenal ] was added to the pellet to control the process of extraction and carotenoid quantification. Samples were accordingly subjected to Precellys homogenization at 8000 rpm for 6 min and then centrifuged (2744× g for 10 min); the supernatant was kept at −20 °C, until further analysis.Regarding carotenoid HPLC analysis, 10 mL of biomass was first harvested and centrifuged at 2744× ® 250-4 C18-reversed-phase column  , while the mobile phase was ethyl acetate (solvent A) and 9:1 (v/v) acetonitrile:water (solvent B). The overall flow rate was set at 1 mL·min−1, with a column pressure of 3000 bar, a constant temperature of 25 ± 2 °C, and a gradient regarding solvents A and B over time as follows: 0–31 min (0–60% A); 31–36 min (60% A); 36–38 min (60–100% A); 38–43 min (100% A); 43–50 min (100–0% A); and 50–55 min (0% A). Spectral data from all peaks were collected within the range of 250–750 nm.The identification and quantification of carotenoids by HPLC were performed as described elsewhere . Acetonia were identified via UV-visible photodiode spectra and comparison of the retention times (RT) with those of standard zeaxanthin  with an RT of 13.2 min, chl a ) with an RT of 24.6 min, echinenone with an RT of 25.2 min, and β-carotene ) with an RT of 32.8 min, performed with the aid of calibration curves prepared with such standards. Three biological replicates were analyzed, and the results were expressed in µg·gDW−1. Total carotenoid productivities were expressed in mg·L−1·d−1.The carotenoids and chl •+ assay was performed using a microplate spectrophotometer reader  according to Guedes et al. (2013)  for productivity regarding biomass, pigments , and antioxidant capacity. For all parameters analyzed, the chosen model was statistically significant, with a p-value < 0.001; the underlying equations for the predicted responses are shown in 2 ranged from 0.72–0.98  favors biomass; however, it can also be observed that biomass productivity is likely to increase at lower temperatures, when combined with a low pH   A. On thee., 6.5) B, and the., 6.5) C. The mo−1·d−1. The surface response graphs showed that intermediate pH levels and a higher temperature positively influence the productivity of carotenoids  and the combination of a high pH and high temperature (≈23 °C) contribute to increasing PBP productivity  was observed to have a significant impact on PC productivity , the combination of a high pH (9.5) and a temperature (25 °C) impacts positively upon APC productivity , temperature and pH exhibit interaction effects in opposite directions , the combination of a lower temperature and higher pH leads to higher productivities , the interaction between temperature and pH is observed at intermediate levels and results in a higher productivity concerning AOX-water compounds (−1) are better in terms AOX-water compound productivity, even though no true local maximum is found   .−1, with a maximum predicted productivity of 637.43 ± 20.56 mg·L−1·d−1 (desirability = 0.974). When compared to the non-optimized conditions, the maximum predicted value represented an increase of ≈175% , as estimated by the model. Compared to the conditions prevailing at the central point, the total carotenoid productivity increased by ≈90.7% , which entails a 39% increase in total PBP productivity . A comparison with the central value resulted in an increase of ca. 19% in this case . A comparison between the experimental results taken at the central value and the predicted value resulted an increase in APC maximum productivity by 50% . Compared to the non-optimized conditions, the predicted response increased by 130%.The best conditions determined to achieve higher PE productivities require lower temperatures and a lower pH combined with a slightly higher [NaCl] than in the case of PC and APC; in this case, T = 15 °C, pH = 6.5, and [NaCl] ≈ 25 g·LTE·L−1·d−1 (desirability = 1). Compared to the intermediate experimental point, the maximum productivity of EtOH-soluble AOX compounds corresponds to a 6%-increase.Concerning the best conditions to increase the productivity of AOX compounds in ethanolic extracts, the model provided the following: T = 15 °C, pH ≈ 9.5, and [NaCl] ≈ 15%, with a predicted maximum of 2.24 ± 0.18 mg−1, with a predicted value of 1.47 ± 0.09 mgTE·L−1·d−1 (desirability = 0.988)  see . The imp988) see .S. salina to attain higher productivity in terms of carotenoids essentially coincide with those prevailing during one of the runs . Hence, the carotenoid profile and content  were β-carotene, zeaxanthin, and the unidentified carotenoid , as were other unidentified carotenoids (6.88%)  see .This study provided an insight into the best conditions in terms of temperature, pH, and [NaCl] associated with the highest productivities of several compounds of interest within the biotechnological field , namely, total carotenoids, total PBPs, and antioxidant compounds in ethanolic and aqueous extracts.The conditions found for each objective function are different from each other. It is well recognized that the best conditions to attain high productivities in terms of biomass do not necessarily coincide with the best conditions to achieve the highest productivities in terms of pigments or antioxidant compounds .S. salina was seen to efficiently adapt to physicochemical variations imposed upon the culture, as its growth was not inhibited within the studied ranges. The combination of a high temperature (25 °C), neutral levels of pH (7.5), and lower [NaCl] (10 g·L−1) appeared to favor biomass maximum productivity.Temperature, pH, and [NaCl] influence several metabolic activities, including the function of intracellular enzymes, nutrient uptake, photosynthesis , or membrane stability, and ultimately growth and maximum biomass accumulation . HoweverS. salina, 25 °C maximized biomass productivity. In fact, for other strains of Synechocystis , 30 °C and a pH of 7–8 were recommended for their cultivation  of 40 g·L−1 and a pH of 8); however, its growth was compromised and the culture collapsed (data not shown). It is worth mentioning that Box–Behnken is a model that has to be applied at equidistant levels for the studied factors. This means that, despite observing interesting results at 15º C , we decided to go for 20 °C because it is a mild temperature. Then, accordingly, 25 °C was the chosen temperature for the model. Further temperatures were not tested close to 30 °C  because similar results in terms of collapse could occur. Additionally, from an industrial point of view, temperatures above 25 °C can have higher costs at production level, which could be economically unfeasible.The optimum temperature for growth and biomass production are variable; in the case of tivation . To defiAnabaena cylindrica can grew between 9 and 33 °C, but its optimal temperature was established at 31 °C under an irradiance of 100 μmolphotons·m−2·s−1.Usually, the optimum growth rate is achieved by increasing the temperature to its optimum level; this is a strain-dependent factor because the range of growth temperatures and the optimal growth temperature depend on the microorganism at stake . For insArthrospira platensis and Arthrospira fusiformis underwent, in turn, a drop in their accumulated biomass when subjected to low (15 °C) and high (40 °C) temperatures, but with maximum biomass productivity at 32 °C (up to 2.4 g·L−1) and 37 °C (up to 2.3 g·L−1), respectively. Rapid shifts in temperature may redirect metabolic processes and thus change the biochemical composition of cells. This will create high stress; if the capacity to acclimate to new conditions is limited, growth and biomass production may cease, causing, in extreme cases, the culture tent to collapse through the inhibition of its photosynthetic apparatus  significantly reduced the maximum biomass productivities (p < 0.05) of S. salina, even when compared to the mid-point conditions . However, the results suggest that this strain can adapt to high [NaCl] because the cultures did not collapse. High concentrations of Na+ and Cl- in the medium have been claimed to destabilize ion cell homeostasis and consequently lead to osmotic stress, with severe repercussions regarding growth and biomass accumulation [Thermosynechococcus sp. CL-1 used in a CO2 fixation process and subjected to [NaCl] of 8, 18, and 29 g·L−1, decreased its biomass productivities from 90.31 to 49.58 and 6.46 mg·L−1·h−1, respectively ) or pH  (10 g·L]) or pH ,34. Thisntophyll . Howeverntophyll . In cyanntophyll ,38,39. Dntophyll  showed t. (2016) , who stu−1) together with temperature (20–30 °C) and pH (6–9). The optimal concentration was set at 10 g·L−1, combined with a pH of 9, a temperature of 20 °C, and a carotenoid productivity of 2.04 mg·L−1·d−1. Regarding the three studied factors, the NaCl concentration had the least impact in terms of carotenoid production. In another study, Synechocystis sp. CCNM 250 was tested for different concentrations of NaCl , and reached a higher carotenoid content (7.05 mg·gDW−1) under a low [NaCl] (0.2 M ≈ 10 g·L−1) [A higher [NaCl] did not trigger a response in terms of carotenoid productivity, as would be expected from studies elsewhere ,31. The S. salina was established under the best conditions predicted by the model. Its profile was unveiled and was similar to that reported by Assunção et al. (2021)  of 25 g·L−1, using Z8 for culture medium.The carotenoid profile of . (2021)  in terms. (2021) ,42. In t. (2021) , their sa and photosynthetic reaction centers  .For high productivities of APC, a lower [NaCl] , with a decrease at 20 °C (23.6%) and 40 °C (38%)  exhibited an important role in PBP productivity. According to Lee et al. (2021)  increasi. (2020)  subjectentration . On the  of PBPs ,53. An e of PBPs .S. salina cells can be subjected to various conditions in terms of temperature, pH, and [NaCl], it was decided that we would explore the radical scavenging capacity of lipophilic and hydrophilic antioxidants, expressed in Trolox maximum productivity equivalents, in both ethanolic and water extracts, under every experimental condition.Cyanobacteria can easily adapt to the most extreme environmental stresses in Nature because their complex metabolism is susceptible to adjustment as a protective strategy against abiotic stresses . One of these strategies is the production of secondary metabolites, such as antioxidants, phenolic compounds, or PUFAs, which constitute the first line of defense against ROS-induced cell damage, including both enzymatic and non-enzymatic mechanisms ,55. Sinc−1. An increase of 6% in AOX-EtOH productivity was estimated when compared to the mid-point condition. Several studies showed that a shift in abiotic factors such as light, temperature, salinity, or pH favor the accumulation of lipophilic antioxidants, in particular carotenoids, certain polyphenols, and PUFAs  ≈ 16 g·Lnd PUFAs ,57,58,59hocystis . In the  content , as thos content ,62.−1). It is possible that these extracts, richer in PBPs, possess other types of antioxidant-related proteins and phenolic compounds  that are prone to occur under mild conditions .On the other hand, the best conditions to achieve maximum productivity in terms of AOX compounds present in water extracts were not so different from that obtained with the control , as a tool can help pinpoint the interaction effects between several abiotic parameters as well as identify their individual influence. This is considered a reliable statistical tool which has already been employed in many cyanobacterial and microalgal biomass and metabolite optimization-related studies ,64,65,66S. salina in different ways. In terms of biomass, [NaCl] appears to have a higher preponderance than other factors. Regarding pigments and antioxidants, the influence of different factors was disparate. It would appear that lower salinity (10 g·L−1) and alkaline pH are relevant when maximizing pigment productivities (except PE), but both temperature and pH, ranging from 23–25 °C and 8–9.5, respectively, differ for carotenoids, total PBPs, and each PBP individually. The pigment that stood out was PE, for which the best productivity conditions are influenced by a low temperature (15 °C) and pH (6.5) under a higher degree of salinity (up to ≈26 g·L−1). On the other hand, lower temperatures (15–19 °C) and higher salinities (16–25 g·L−1) influence the AOX compound productivities in both ethanolic and aqueous extracts. However, lipophilic compounds are influenced by a higher pH (≈9.5), while aqueous antioxidants are favored by an intermediate pH (=8).A Box–Behnken factorial design was therefore employed to ascertain the interaction effects between temperature, pH, and [NaCl] on the productivities of biomass, pigments, and antioxidant compounds, as found in ethanolic and aqueous extracts. It confirmed that the three factors influence the metabolism of It is worth mentioning that the optimal conditions for metabolite productivity may not coincide with their maximum content in the cyanobacterium cell; however, the accumulation of the final product within a short period of time is relevant in the context of bioprocess decision-making over a high content of pigment because this can imply longer cultivation with larger process-associated costs.S. salina. The model could predict a 2.7-fold increase in biomass productivity under optimized conditions, whilst for pigments, PE stood out, with a 2.3-fold increase, as did carotenoids and total PBPs, with 1.90- and 1.39-fold increases, respectively. For lipophilic antioxidants, a 1.1-fold increase was found when compared to the non-optimized conditions; only for AOX-water compounds did the prediction entail a marginal improvement of 1.38%.Overall, the Box–Behnken model predicted the best conditions to enhance biomass, pigment production, and antioxidant capacity productivities of strain Nevertheless, further fundamental studies are still needed to complement this information since other abiotic factors, such as light, nutrient availability, or even biotic factors , are relevant to the overall effective cost of upstream processing. An ideal, large-scale scenario of a biorefinery process, in which biomass use is maximized and associated with the co-production of several value added-compounds in a single batch, could be an option to alleviate some of the costs associated with the overall cyanobacterial bioprocess. This would, accordingly, aid competitiveness in the nutraceutical and food markets.


Scenedesmus sp. was used to treat municipal landfill leachate via batch and continuous cultivation modes to protect the environment and explore sufficient biomass production for bioethanol production using Saccharomyces cerevisiae. Physicochemical characteristics of leachate were determined for the phases before, during, and after the process. Batch and continuous cultivation were used to treat raw leachate to determine optimum conditions for treatment. Then, the biomass of Scenedesmus sp. with and without sonication was used as a substrate for ethanol production. Sonication was carried out for biomass cell disruption for 20 min at a frequency of 40 kHz. Through batch cultivation mode, it was found that pH 7 was the optimum condition for leachate treatment. Continuous cultivation mode had the highest removal values for COD, phosphorus, and carbohydrate, namely 82.81%, 79.70%, and 84.35%, respectively, among other modes. As for ethanol production, biomass without sonication with 9.026 mg·L−1 ethanol, a biomass concentration of 3.300 µg·L−1, and pH 5 were higher than biomass with sonication with 5.562 mg·L−1 ethanol, a biomass concentration of 0.110 µg·L−1, and pH 5. Therefore, it is evident that the leachate has the potential to be treated by Scenedesmus sp. and converted to bioethanol in line with the concept of sustainable materials.Sustainable material sources are an important agenda to protect the environment and to meet human needs. In this study,  Landfilling has become the most common and vital means of municipal solid waste disposal globally . Landfill functions as a huge anaerobic bioreactor, covering a wide range of chemical, physical, and biological systems . It may In recent years, more leachate treatment technologies have been developed in several countries, which can be grouped into chemical, physical, and biological treatments, and are highly dependent on the leachate physicochemical conditions to determine the most appropriate treatment to adopt . In MalaOwing to its numerous advantages, leachate treatment using algae has generated great interest among researchers in terms of its high energy production capacity, particularly biogas, in addition to its high growth rate and ease of cultivation. Compared to terrestrial raw materials, algae can grow 5 to 10 times in favorable conditions and also has higher production rates . In addiScenedesmus sp. Among some microalgae, Scenedesmus sp. can achieve the maximum biomass concentration even though it is cultivated under heterotrophic conditions, as reported by [Scenedesmus sp. also possesses beneficial features such as rapid growth, CO2 fixation, and the ability to grow in wastewaters and accumulate lipids, which act as the basis for its selection in microalgae studies. Moreover, yeast is another microorganism beneficial for assisting ethanol production from algae-based leachate treatment. Yeast is known to have the capability of removing organic matter from municipal wastewater, eliminating additional nutrient removal steps, and reducing treatment costs [One of the most promising algal species used as feedstock for ethanol production is orted by . A heterorted by . Scenedent costs . It alsoScenedesmus sp. for landfill leachate treatment and, subsequently, the treated leachate was then used as a substrate for fermentation by yeast, namely Saccharomycescerevisiae, for ethanol production. This study also examined the effect of two cultivation modes, namely batch and continuous, for raw leachate treatment. This study aimed to find a sustainable treatment for landfill leachate that can contribute to energy research. The combination of the use of algae and yeast with leachate as a substrate could indicate the potential of producing ethanol in a sustainable manner from renewable energy sources.Several studies have reported on the use of combined microalgae and yeast cultivation in treating wastewater and producing valuable products. For example, this approach has been used for a pilot-scale urban wastewater treatment and biodiesel production , yeast i3 per day. The characteristics of the Ampang Jajar landfill site are as follows: 2.9 hectares, clay as lining material, 800 kg/m3 density of compacted waste, 2.3–2.5 m/year of average rainfall, and 1.3–1.4 m/year of evaporation [In this study, leachate was collected from Ampang Jajar Municipal Solid Waste (MSW) Collection Station, Permatang Pauh, Penang . The landfill has been in operation since 2005; it receives around 500 tons of municipal solid wastes daily, including 68% of organic materials, and the volume of leachate generated in this landfill is estimated to be approximately 270 mporation . ApproxiScenedesmus sp. was collected from a river in Johor, Malaysia. The algae samples were collected using a 25 μm planktonic net and then stored in sterilized glass bottles until being transferred to the laboratory via a portable refrigeration box, before they were subjected to isolation. In the current study, Scenedesmus sp. was isolated using BBM medium containing 1.5% agar. Spread plates were inoculated with 0.1–0.2 mL of water sample and incubated for 5–7 days at 25 °C, 60 µmol photons m−2 s−1, and a 14:10 h light:dark photoperiod [In this study, toperiod . To prevAfter sampling, the leachate samples were analyzed for pH, chemical oxygen demand (COD), carbohydrate content, nitrogen, and phosphorus content. Analysis of pH was conducted immediately using a pH meter at the laboratory. The COD analysis, phosphorus content, and total Kjeldahl nitrogen (TKN) were conducted following the standard method of APHA (2005). For COD analysis, the reading was taken with a wavelength of 620 nm. For total Kjeldahl nitrogen (TKN) analysis, the samples were digested with sulfuric acid at 380 °C. The digested sample was then added with 10 mL of distilled water and the solution was distilled into 25 mL of 4% boric acid solution containing 5 drops of methyl red indicator with 35% sodium hydroxide using a steam distillation unit . The boric acid receiving solution was titrated with 0.02 M of hydrochloric acid solution. The volume of hydrochloric acid required for the solution to turn pink or purple was recorded. 2SO4, previously prepared by mixing 500 mL of concentrated acid with 200 mL of water, and cooled in the refrigerator before mixing. A standard glucose solution was prepared by adding 100 mg of glucose to 100 mL of distilled water. Then, 10 mL of this stock was diluted with 100 mL of distilled water to produce a working standard. This standard was used to prepare standard tubes with the following concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mL, with ‘0’ acting as the blank. All volumes were made up to 1 mL with distilled water if necessary. Two mL of leachate was then hydrolyzed in a boiling tube by it placing in a boiling water bath for 3 h after adding 5 mL of 2.5N HCL. Thereafter, the tubes were allowed to cool and then neutralized with solid sodium carbonate until the effervescence ceased. The samples were then made up to 100 mL and centrifuged. Approximately 1 mL aliquots of supernatant were taken for further analysis. Four mL of Anthrone reagent was added to all tubes (standards and samples) and heated for 8 min in a boiling water bath. The tubes were cooled rapidly, and the absorbance was read at a wavelength of 630 nm using a DR2800 spectrophotometer. Approximately 200 mg of Anthrone reagent was dissolved in 100 mL of H−1. The aeration flow rate was 0.5 mL/min and the temperature inside the bioreactors was 28 ± 2 °C. Since Scenedesmus sp. can grow mixotrophically and heterotrophically and grows well under light conditions, it tends to divide preferably under dark conditions by binary or multiple fission to produce daughter cells, and this seems to have a significant implication for the overall productivities of microalgal cultures. The duration and intensity of light directly affect the growth and photosynthesis of microalgae.In this experiment, batch and continuous modes were used. The geometry and size of the bioreactors were the same. The glass vessel was connected with some tubes, one of them for aeration and others for sampling in a batch bioreactor. In a continuous bioreactor, with a 800 mL level, there is media output to remove the excess media from this level; it is also connected with a peristaltic pump for the input of raw leachate with different dilution rates, as shown in Scenedesmus sp. In the batch bioreactor, sterile leachate was filtered and transferred into four bioreactors, each with a volume of 800 mL. Each bioreactor was set at different pH, namely 6.5, 7, 7.5, and 8. The pH of the leachate was adjusted using H2SO4 (1 mol) and NaOH (1 mol). The inoculum was prepared before being transferred to the leachate; the strain (Scenedesmus sp.) was inoculated in BBM media at an initial pH of 7.4 ± 0.1 and grown photo-autotrophically at 28 ± 2 °C, with a 12/12 h photoperiod (light/dark cycle) and 60 µmol photons m−2 s−1 irradiance given by two 40-watt daylight fluorescent lights. Then, 10% of inoculum was prepared to be transferred to 800 mL of leachate. Algae cultivation was monitored for 15 days during all phases A is the lag phase, B is the exponential phase, C is the stationary phase, and D is the death phase, as shown in In this study, leachate was used as the culture medium for the cultivation of For the continuous bioreactor, the bioreactor was run for 33 days. pH 7 was set for the continuous bioreactor. After 15 days, fresh leachate was added to the bioreactor at intervals, and reactions were observed at varying flow rates to compare various treatment results. The first flow rate was 2 mL/min and ran from day 15 to day 18. The second flow rate was 2.5 mL/min, which was from day 19 to day 22. Meanwhile, the third flow rate was 3 mL/minute from day 29 to day 33. The growth of biomass was divided into four phases: A is the lag phase, B is the exponential phase, C is the stationary phase, and D is the death phase, as shown in The treated leachate was centrifuged at 4500 rpm for 15 min and then separated into two parts, namely supernatant and biomass. The biomass was then further divided into two parts: one part was subjected to sonication while the other was without sonication. Sonication was conducted for 20 min at a frequency of 40 kHz. Biomass samples were freeze-dried at −47 °C for 48 h. After that, the biomass was weighed (0.165 g) and divided into five equal parts to obtain different concentrations. These parts were then added to different volumes of water  to form different biomass concentrations for use in ethanol production.−1) to determine the most appropriate concentration for S. cerevisiae growth and subsequently ethanol production. Each sample dilution was then prepared in three different pH values, namely 5.0 (acidic), 6.5 , and 9.0 , to determine the optimum pH conditions for S. cerevisiae growth and ethanol production. Fermentation was conducted in anaerobic conditions for the conversion of biomass to ethanol. Biomass algae without and with sonication were placed in sterile storage vessels in a laminar flow cabinet in sterile conditions to prevent contamination. Each biomass group  was prepared in five different concentrations  S. cerevisiae culture. The fermentation process was conducted in the incubator at 30 °C for 24 h. Sampling was conducted for 24 h. The fermentation vials were taken out from the incubator and the OD readings were measured immediately. The concentration of ethanol produced in each sample was measured using gas chromatography (Agilent 5820-A) with a split/splitless inlet with a temperature of 290°, a flame ionization detector (FID), and a capillary column .Before inoculation, the medium was injected with nitrogen gas using a gauge needle. The fermentation vials used were autoclaved at 121 °C for 15 min. The standard inoculum (YEPG) was prepared from −1 COD, 4.350 mg·L−1 phosphorus, 245.00 mg·L−1 nitrogen, and 0.812 mg·L−1 carbohydrates. The raw leachate in this study can be considered as young leachate as it indicated high COD (>5000 mg·L−1), low nitrogen content (<400 mg·L−1), and low pH . A high COD value was recorded, which could be due to the high pollutant levels and the humic acid substrates present, which are unable to be stabilized by the microorganism. From the physicochemical analysis, it was found that the raw leachate was in an acidic condition at pH 5.25 containing 6583.00 mg·LIn general, leachate composition is dependent on the landfill nature, waste composition, and age .Scenedesmus sp. Optimum pH will intensify the growth of biomass production and the removal of carbohydrates [Scenedesmus sp. growth and induced the best removal percentage for COD, carbohydrate, and other parameters.The pH values of four leachate samples were regulated in batch mode for 15 days. The removal of COD, phosphorus, carbohydrate, and nitrogen was monitored. hydrates . Thus, a−1. In contrast, pH 8 had the highest biomass of 0.669 g·L−1. The composition of algal biomass is a vital indicator in assessing the potential of algae as a biofuel source [2. This inhibits cell growth and leads to low biomass/growth rates [Nevertheless, although pH 7 exhibited the best values for physicochemical parameters, it recorded the lowest biomass of 0.560 g·Ll source . pH affeth rates .−1, despite demonstrating the lowest COD, carbohydrate, phosphorus, and nitrogen removal. In contrast, the dilution rate of 3 mL/min had the lowest biomass of 0.498 g·L−1 but had the highest COD, carbohydrate, phosphorus, and nitrogen removal. The optimum dilution rate will provide optimum biomass and percentage consumption or removal of carbohydrates [For continuous cultivation, three different flow rates were examined at pH 7. The flow rate, which is the speed of the support media in comparison to the culture broth, affects the algal growth . Continuhydrates .Continuous mode for algae cultivation demonstrated higher biomass production compared to batch mode. Moreover, continuous mode treatment also indicated higher COD, carbohydrate, phosphorus, and nitrogen removal compared to batch mode. Batch reactor treatment is more difficult to apply for large-scale industrial purposes. The continuous mode is considered to be a less expensive option, capable of producing better results compared to the batch cultivation mode . SimilarS. cerevisiae was selected due to its potential to produce a high ethanol yield . Different pH conditions and biomass concentrations were used to show their influence on the growth of S. cerevisiae and ethanol production. pH is one of the factors that affects the nutrient uptake of an organism, cell aggregation, and cell membrane destabilization . For this reason, optimum pH should be maintained in algal growth media to achieve maximum biomass and carbohydrate production [oduction .S. cerevisiae. The study found that S. cerevisiae was able to convert algal biomass to ethanol efficiently, with results that encourage the use of algal biomass as feedstock for ethanol production. In addition, the authors in [−1. They concluded that the variation in bioethanol yield and concentration was due to the pre-treatment steps used. Similarly, they also obtained a better bioethanol yield and concentration as compared to previous studies. Microorganisms have many nutrient-rich elements in their bodies that can be utilized for other valuable products or chemicals.The results of this study are similar to those of Gohain et al. (2021), who investigated the quantitative conversion of algal biomass to ethanol via fermentation by thors in  comparedNitrogen, phosphorous, and carbohydrate are the primary products of photosynthesis and thus store metabolic energy temporarily. The highest carbohydrate removal of 46% was obtained in this study. On another note, the authors in  also achS. cerevisiae under sonication and without sonication. From Based on the results obtained, sonication was found to increase the concentration of dissolved nutrients such as carbohydrates, phosphorus, and nitrogen. Sonication could be the main reason for decreasing nutrient removal through fermentation . For thi−1, while carbohydrate removal was the highest at 0.165 µg·L−1. At pH 6.5, carbohydrate removal was the highest at 0.110 µg·L−1. For biomass with sonication, only concentrations of 3.30 µg·L−1 and 2.20 µg·L−1 indicated slight nitrogen removal of 4.84% and 3.56%, respectively, at pH 5 and 9. Meanwhile, for biomass without sonication, higher nitrogen removal was recorded at 3.30 µg·L−1, 2.20 µg·L−1, and 0.165 µg·L−1. The values of nitrogen removal were 11.01%, 7.68%, 6.04%, 6.90%, and 8.21% at different pH values.For phosphorus and carbohydrate, samples without sonication had higher removal than samples with sonication. At pH 9, phosphorus removal was the highest at 3.30 µg·L−1 was found in a biomass concentration of 3.300 µg·L−1 at pH 5 when fermentation occurred in samples without sonication. Microalgae, such as Scenedesmus sp. used in this study, are known to be extremely useful in the production of bioethanol. For fermentation, S. cerevisiae is considered one of the most effective yeasts to serve this purpose. It induces high bioethanol yields and can resist inhibitory components that may interfere with the fermentation process, resulting in high bioethanol concentrations. The optimum pH range for S. cerevisiae is 3.5 to 5 [Leachate is rich in a variety of nutrients that could be utilized to produce other valuable chemicals. Treated leachate is also a promising medium for bioethanol production. The continuous mode at pH 7 indicated optimum biomass and the removal of other undesired nutrients. In this study, fermentation was performed at three different pH values , and with and without sonication. The results for ethanol production are depicted in 3.5 to 5  or 4 to 3.5 to 5 . This is−1 without sonication at pH 5 showed the highest ethanol concentration of 9.020 mg·L−1. The biomass concentration of 0.110 µg·L−1 at pH 6.5 obtained the lowest ethanol concentration of 1.19 mg·L−1. In contrast, the highest ethanol concentration of 5.56 mg·L−1 was obtained by the 0.11 µg·L−1 biomass concentration with sonication at pH 5, while, at a similar pH, the lowest ethanol concentration of 0.17 mg·L−1 was obtained at the 3.300 µg·L−1 biomass concentration. These results suggested that higher biomass concentrations without sonication favored higher ethanol concentrations, while lower biomass concentrations with sonication achieved higher ethanol concentrations. This was because S. cerevisiae will produce high ethanol concentrations with less or no oxygen (anaerobic) [S. cerevisiae.aerobic) . The son−1 COD, 4.35 mg·L−1 phosphorus, 245.00 mg·L−1 nitrogen, and 0.812 mg·L−1 carbohydrates. For batch cultivation, pH 7 was the optimum condition for leachate treatment. Continuous cultivation indicated the highest removal of COD (82.81%), phosphorus (79.70%), and carbohydrate (84.35%). For ethanol production, the highest ethanol concentration of 9.020 mg·L−1 was obtained from a biomass concentration of 3.300 µg·L−1 at pH 5 when fermentation occurred in samples without sonication. Under sonication, the biomass concentration of 3.30 µg·L−1 at pH 5 exhibited the highest ethanol concentration of 9.020 mg·L−1. Based on the results obtained, the combined use of Scenedesmus sp. and S. cerevisiae for leachate treatment and ethanol production is promising to be established as a sustainable approach in maximizing waste management.Algae demonstrates great potential for the production of various bioproducts such as bioethanol, biodiesel, and chemicals. According to the physicochemical characteristics of raw leachate, it recorded pH 5.25, 6583.30 mg·L


Tanreqing capsules (TRQCs) and Tanreqing injections (TRQIs) are widely used in the treatment of respiratory diseases. In this study, a simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous quantification of the main components of Tanreqing, which include chlorogenic acid, ursodeoxycholic acid, chenodeoxycholic acid, and baicalin, in beagle dog plasma to compare their pharmacokinetic parameters.3  column using a gradient elution with (A) 0.1% (v/v) formic acid aqueous solution and (B) acetonitrile. Six healthy beagles were divided into two groups, and a crossover, comparative pharmacokinetic study of TRQC (0.09 g/kg) and TRQI (0.5 mL/kg) after a single-dose administration or daily doses over 7 days was carried out. One group was administrated a single dose of TRQC and followed continuously for 7 days, whereas the other group was treated with TRQI in the same way.Plasma samples were pretreated with protein precipitation. Chromatographic separation was performed on Waters Acquity UPLC HSS TThe calibration curves were linear over the ranges of 2.00–1000.00 ng/mL for baicalin, 10.00–5000.00 ng/mL for ursodeoxycholic acid, 1.00–500.00 ng/mLfor chenodeoxycholic acid and chlorogenic acid, respectively. The relative standard deviation of both intra-day and inter-day accuracy is less than 11.23%. The average extraction recovery of all compounds was greater than 82.21%. The major pharmacokinetic parameters of the four compounds were not significantly different between the two formulations (P > 0.05).The measured levels of the four major components of TRQCs and TRQIs were comparable in these dogs, providing a reference for the clinical application of TRQCs instead of TRQIs.The online version contains supplementary material available at 10.1186/s13020-022-00690-9. Guided by traditional Chinese medicine (TCM) theory, traditional Chinese medicine injections (TCMIs) are preparations of active substances extracted from single or combined Chinese or natural medicines using modern scientific techniques and methods . AccordiTanreqing is a traditional Chinese medicine compound preparation that comprises five herbs, Scutellaria baicalensis Georgi, Selenaretos thibetanus Cuvier, Saiga tatarica Linnaeus., Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. It has significant anti-inflammatory, antiviral, antibacterial, and immunomodulatory activity, among other effects, and is widely used in the treatment of respiratory diseases , 7. In oSince it first became commercially available in 2003, TRQI has been recommended by many clinical guidelines and treatment protocols. Since 2019, TRQI has been listed in the COVID-19 Diagnosis and Treatment Guidelines  of China for the treatment of COVID-19 patients with critical symptoms , 18. TheIn this study, a two-cycle, crossover design was used to compare the key pharmacokinetic parameters between TRQC (0.09 g/kg) and TRQI (0.5 mL/kg) administered as a single dose, followed by seven consecutive days of administration in six healthy beagles. A LC–MS/MS method was developed to measure the levels of the main components of TRQC and TRQI in these dogs after treatment to provide a reference for the theoretical basis of replacement of TRQIs with TRQCs.All reference standards including baicalin (Lot: D0321AS), ursodeoxycholic acid (Lot: O0424AS), chenodeoxycholic acid (Lot: S0221AS), chlorogenic acid (Lot: N0805AS), and the internal standard (IS) puerarin (Lot: J1003AS) were provided by Dalian Meilun Biological Co., Ltd. . Their purity is greater than or equal to 98%. HPLC-grade methanol and acetonitrile were provided by Merck Company . Analytical-grade formic acid (≥ 95%) was purchased from McLean Biochemical Technology Co., Ltd. . The water was double-distilled and was purchased from Watsons . All other reagents used in the experiment were of analytical grade. TRQI (Lot: 1905101) and TRQC (Lot: 1907321) were provided by Shanghai Kaibao Pharmaceutical Co., Ltd. .An Agilent 1290-6460A ultra-high performance liquid chromatography–tandem mass spectrometry system, which includes the G4220A binary pump, G1969-80230 vacuum degasser, G4226A autosampler, and G1316C column oven, was used in our experiments, and the raw data acquirement and processing were carried out using MassHunter software .3 column  at a flow rate of 0.3 mL/min. The column temperature was kept at 35 °C, and the temperature in the autosampler was 4 °C. A gradient elution procedure was used with 0.1% formic acid aqueous solution as solvent A and acetonitrile as solvent B. The gradient elution consisted of 0–1 min, 10–55% B; 1–2.5 min, 55–70% B; 2.5–3.5 min, 70–95% B; 3.5–4.5 min, 95% B. The injection volume was 5 μL with a needle wash for 3 s using 5% methanol aqueous solution. The equilibration time between injections was 2 min.Chromatographic separation of all four compounds was performed on Waters Acquity UPLC HSS TElectrospray ionization (ESI) source and negative ionization mode were used for analyte ionization, and the data were collected in multiple reaction monitoring (MRM) mode. The ionization source parameters for MS were as follows: gas temperature, 350 °C; gas flow, 10 L/min; nebulizer, 40 psi; capillary, 4000 V; MS1 heater, 100 °C; MS2 heater, 100 °C. The optimized MRM conditions for all analytes are shown in Table Reference standards for chlorogenic acid, ursodeoxycholic acid, chenodeoxycholic acid, and baicalin were precisely weighed at 2.00 mg, 2.02 mg, 2.01 mg, and 2.02 mg, respectively, and were then dissolved using methanol to obtain stock solutions at 1.00 mg/mL, which were stored at – 80 °C. The stock solution of the IS (puerarin) was prepared and stored using the same method at a concentration of 1.00 mg/mL.The stock solutions of all analytes were further mixed and diluted with methanol/water 10:90, v/v) solution to prepare the working solutions. An aliquot of 50 μL combined working solution was added to 450 μL blank plasma to obtain the following calibration standards: 1.00, 2.00, 5.00, 10.00, 50.00, 100.00, and 500.00 ng/mL for chlorogenic acid and chenodeoxycholic acid; 10.00, 20.00, 50.00, 100.00, 500.00, 1000.00, and 5000.00 ng/mL for ursodeoxycholic acid; and 2.00, 4.00, 10.00, 20.00, 100.00, 200.00, and 1000.00 ng/mL for baicalin. The quality control (QC) samples were prepared separately in the same way at 2.00, 10.00, 100.00 ng/mL of chlorogenic acid and chenodeoxycholic acid; 20.00, 100.00, 1000.00 ng/mL of ursodeoxycholic acid; and 4.00, 20.00, 200.00 ng/mL of baicalin. The QC samples were stored in a refrigerator at – 80 °C until retrieval  was added, and then the sample was vortex-mixed for 3 min. The mixture was centrifuged at 20,000 ×This study was carried out in six beagles , that were purchased from Junda Pet Hospital . This experimental protocol was approved by the Experimental Animal Ethics Committee of the Naval Military Medical University .g for 5 min, and each supernatant was collected and aliquoted. After a 7-day wash out period, the dogs in each group were administrated the other Tanreqing formulation in a crossover design, and blood samples were collected and processed in the same way.The six healthy beagles were divided into two groups: beagles 1–3 were all male and were assigned to the first group, and beagles 4–6 were all females and assigned to the second group. The two-cycle, crossover study was designed with a 7-day wash out period. For the single-administration process, the dogs in the first group were injected intravenously with 0.5 mL/kg TRQI , and blood samples were collected at 0, 5, 10, 15, 20, 30, and 45 min and then at 1, 2, 4, 6, 8, 12, and 24 h  after injection. For each blood sample, 1 mL of blood was collected from the forelimb vein in heparin-treated tubes. The dogs in the second group were orally administrated 0.09 g/kg TRQC . All blood samples were centrifuged at 4000 × . After the seventh administration, blood samples were collected at 0, 10, 20, and 40 min and then at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h  and were processed as described in “After the end of the single-dose experiment, the dogs in group one were given 0.5 mL/kg injection of TRQI once per day for 7 days following the wash out period, and the dogs in group two were given 0.09 g/kg TRQC once per day for 7 dayst test.All data obtained were analyzed using Drug and Statistics (DAS) 2.0 software (Chinese Society of Pharmacology), and a non-compartment analysis was used to calculate plasma pharmacokinetic parameters. The principal components of TRQI and TRQC were compared with an analysis of variance (ANOVA) and/or a two-tailed student-18 column , the Waters Xbridge SB-C18 column , and the Waters Acquity UPLC HSS T3 column , among others, for their ability. The HSS T3 column had better separation effects and a shorter analysis time. The analysis time was optimized from 15 to 10 min. Second, we tested the influence of different column temperatures on chromatographic separation and found that lower column temperatures resulted in a longer analysis time. The analysis time was optimized from 10 to 5 min, which shortens the analysis cycle [, respectively. Water plus 0.1% (v/v) formic acid (phase A) and acetonitrile (phase B) resulted in the best peak shape and retention time.To obtain better separation effects and higher sensitivity, we first optimized the chromatographic column, mobile phase, and mass spectrometry conditions and investigated the Agilent Zorbax SB-CThe presence of endogenous isomers ursodeoxycholic acid and chenodeoxycholic acid in this experiment made the chromatographic separation of these two compounds difficult. In this study, based on their different retention times, we used their corresponding standards to identify their chromatographic peaks.We first investigated different pretreatment methods of the biological samples, such as solid-phase extraction, protein precipitation, ultrafiltration, and so on. Solid-phase extraction and ultrafiltration were both affected by plastic adsorption, resulting in a low recovery (< 50%), and the matrix effects are strong and vary greatly (RSD% > 15%). For protein precipitation, we analyzed solvent mixtures containing methanol, acetonitrile, and different proportions of formic acid and ammonium hydroxide as additives. However, it is found that different ratios of additives have great impact on the experimental results. Ultimately, we found that precipitation with a mixture that included methanol and acetonitrile  plus 0.1% formic acid resulted in high extraction recovery and steady matrix effects, while other ratio of additives gave unsteady recovery and tailing peaks.The methodology validation of this study was carried out in accordance with the guidance of the 2020 Chinese Pharmacopoeia. The validation criteria were based on previous reports , includiSpecificity was assessed by comparing blank plasma samples from each of the six dogs, blank plasma spiked with IS, lower limit of quantification (LLOQ) sample, and real sample. We then examined the retention time of the chlorogenic acid, ursodeoxycholic acid, chenodeoxycholic acid, and baicalin, in addition to that of the IS, puerarin. There was no substantial interference in the retention time of the four analytes or IS Fig. .Fig. 1Tyχ2 weighting factor. The linear correlation coefficients were > 0.98 for all analytes. The deviations of the back-calculations of the seven calibration standards were all ± 15% . The parameters of the four calibration curves are shown in Table The combined working solutions of the four compounds  were diluted in blank plasma  to generate a series of calibration standards. The concentrations were 1.00, 2.00, 5.00, 10.00, 50.00, 100.00, and 500.00 ng/mL for chlorogenic acid and chenodeoxycholic acid; 10.00, 20.00, 50.00, 100.00, 500.00, 1000.00, and 5000.00 ng/mL for ursodeoxycholic acid; and 2.00, 4.00, 10.00, 20.00, 100.00, 200.00, and 1000.00 ng/mL for baicalin. The calibration standards were prepared in three replicates for each concentration, and LC–MS/MS analysis was carried out over 3 days. The best linearity and least-squares residuals for the calibration curves were achieved with a 1/The QC samples and LLOQ samples were used to evaluate the inter- and intra-day precision and accuracy. These tests were carried out along with the linearity assessment with five replicates. The results showed good precision and accuracy, with intra-day and inter-day precision < 11.23% and accuracy within ± 14.00%. All data are summarized in Table The stability, which includes short-term stability (6 h at room temperature), long-term stability (− 80 °C for 30 days in the refrigerator), and freeze–thaw stability (three cycles from − 80 °C to room temperature), were assessed using QC samples at three concentration levels in three replicates. As shown in Table To examine the extraction recovery and the matrix effects, we used the high and low concentrations of QC samples to prepare six replicates at each of the two concentrations. The peak areas from the spiked samples were compared with the peak areas obtained from the spiked post-extraction samples at the same concentration, and the extraction recovery was determined. The matrix effects were calculated based on the ratio of the peak area of each spiked post-extraction sample to that of solvent-substituted sample at the same concentration. The results are shown in Table We used this LC–MS/MS method to carry out a pharmacokinetic study of TRQC and TRQI and measure the concentrations of the four main components in plasma samples from the beagles. There was a huge difference in the administration route of TRQI and TRQC. As it is difficult for an individual to take nine capsules once a day, we first confirmed that a dose of three capsules of TRQC was comparable to one-third of a 20-mL injection of TRQI with respect to the main pharmacokinetic parameters. Based on the conversion ratio and pre-experimental validation (data not shown), the dose in this study was calculated as 0.5 mL/kg for TRQI and 0.09 g/kg for TRQC.1/2 values for ursodeoxycholic acid and chenodeoxycholic acid in another study were, respectively, 14 and 33 min [1/2 value of baicalin was 216 min, four times longer than that previously reported (48 min). There are large differences in the results of these studies. Feng et al. [1/2 values among three doses of TRQI  were not significantly different (p > 0.05). In addition, the area under the curve (AUC) values for baicalin were relatively high in that study, which demonstrated that TRQI and TRQC administration results in higher exposure levels of baicalin than the other components. Finally, the distribution and metabolism of baicalin in rat occurred relatively rapidly owing to a higher clearance rate (1.46 L/h/kg) and volume of distribution (1.07 L/kg) compared with the values for the three other components. In our study, the t1/2 values for TRQCs and TRQI were comparable for a single dose, and the Tmax and Cmax of TRQI were shorter and higher, respectively, than those of TRQC. We note that after dosing over a 7-day period, the t1/2 for all compounds in TRQC-treated dogs was approximately twice that of TRQI-treated dogs. Thus, TRQCs have a relatively longer half-life in vivo and may have an advantage with respect to an extended dosing interval. In summary, the exposure concentrations of TRQC and TRQI in vivo were roughly similar. In our study, there was no significant difference in the AUC0–24 h and AUC0–∞ between the two formulations (P > 0.05). To the best of our knowledge, this is the first experiment in which the pharmacokinetic parameters of an injectable and capsule formulation from natural herbs have been compared. As both TRQIs and TRQCs had similar pharmacokinetic parameters, but TRQCs do not induce a severe adverse reaction, for the most part, as compared with TRQIs, TRQCs may be an effective alternative to TRQIs.The pharmacokinetic parameters of six healthy beagles after a single-dose intravenous injection of TRQI or a single-dose oral administration of TRQCs were estimated using a non-compartmental model Tables  and 7. Rd 33 min , which ad 33 min  study (5g et al.  found th. However, the revelation of the metabolic behavior of chlorogenic acid in this study can provide certain reference value for the subsequent research.The results of the study showed that the metabolism of baicalin in vivo was faster after intravenous injection of TRQI. In addition, ursodeoxycholic acid and chenodeoxycholic acid had obvious enterohepatic circulation. In mice, the metabolism of baicalin is slow, the metabolism of ursodeoxycholic acid and chenodeoxycholic acid is fast, and only chenodeoxycholic acid is associated with enterohepatic circulation. Thus these different results may reflect the different animals used in these studies. For TRQC in dogs, the oral availability of baicalin was small, which is consistent with a previous study . HoweverIn this study, a rapid, sensitive, and convenient LC–MS/MS method was established and validated. This method has the advantages of a shorter running time and simpler pretreatment method and has been successfully applied to the quantification of four active components of Tanreqing in plasma samples from beagles. The exposure levels of the main components of TRQC and TRQI were largely consistent between both administration methods in these dogs. As compared with TRQI, TRQC resulted in a similar exposure among the beagles when the amount of prescription medicinal materials was increased four folds. TRQC may be an alternative for TRQI based on their comparable pharmacokinetics and lesser side effects of TRQC, although further study is needed to elucidate the clinical efficacy of TRQC as an alternative to TRQI.Additional file 1:Table S1. Optimization of sample pretreatment. Table S2. The optimization of mobile phase constitution. Table S3. Optimization of the LC–MS/MS conditions.


The rapid and accurate detection of thyroid-stimulating hormone (TSH) receptor antibodies has always been an urgent need for the clinical diagnosis and management of Graves’ disease (GD). We aimed to evaluate the use of an automated thyroid-stimulating immunoglobulin (TSI) bridge immunoassay in the diagnosis of GD and to analyze the relationship between TSI and the degree of hyperthyroidism.A total of 227 new-onset GD patients, 29 Hashimoto thyroiditis, 43 non-autoimmune thyroid diseases and 37 euthyroid controls were consecutively recruited. All participants accepted the measurement of their serum thyroid function and thyroid-associated antibodies, including TSI being measured by an Immulite 2000 bridge immunoassay and TSH receptor autoantibodies (TRAb) being measured by a third-generation Roche electrochemiluminescence immunoassay. The quantitative consistency between the TSI and TRAb detection methods was analyzed by using Passing-Bablok regression and Bland–Altman plots. The diagnostic performance for GD was assessed by receiver operating characteristic (ROC) curve analysis.r = 0.8099). According to the cut-off values proposed by the manufacturers , the positive rates of TSI and TRAb in new-onset GD patients were 96.92% and 95.15%, respectively. Both TSI and TRAb levels positively correlated with FT4 levels  and FT3 levels  in new-onset GD patients. The ROC analysis showed that the optimal TSI cut-off value was 0.577 IU/L for GD diagnosis in this Chinese population, with a sensitivity of 96.92% and a specificity of 97.25%, respectively. The optimal TRAb cut-off value of was 1.38 IU/L, with a sensitivity of 96.92% and a specificity of 99.08%. There were no significant differences between the cut-off values obtained through the ROC analysis and those provided by the manufacturer for both TSI and TRAb when calculating their sensitivity and specificity in diagnosing GD. Among the 8 newly diagnosed GD cases with discordant qualitative antibody results, TSI was more likely than TRAb to match the clinical diagnosis of GD (6 TSI-positive vs. 2 TRAb-positive patients).Among 227 GD patients , the quantitative TSI was positively correlated with TRAb (The automated TSI bridge immunoassay was positively correlated with thyroxine levels in new-onset GD patients and was more likely to be consistent with the clinical diagnosis of GD than with that of TRAb. The positive Immulite 2000 TSI cut-off value of 0.577 IU/L for GD diagnosis in the Chinese population were close to the value recommended by the manufacturer.The online version contains supplementary material available at 10.1186/s12902-022-01114-3. Graves’ disease (GD) is the most common cause of hyperthyroidism. It usually occurs in women of childbearing age and is one of the main factors that contribute to adverse pregnancy outcomes , 2. The As a pathogenic antibody, the TSH receptor autoantibody (TRAb) is very important in the diagnosis and treatment of GD , 8. HoweIn recent years, a new TSI assay, using a pair of recombinant human TSH receptors in a bridging format to capture and detect thyroid-stimulating autoantibodies, has been confirmed by multiple studies to have good diagnostic performance and cost-effectiveness , 16. Witn = 149 .According to a recent study  showing n = 227), Hashimoto’s disease (n = 29), non-autoimmune thyroid diseases (n = 43) , and euthyroid control (n = 37) groups. The diagnosis of GD was based on Chinese thyroid disease diagnosis and treatment guidelines [We screened 336 consecutive thyroid disease patients and 37 euthyroid controls aged 18–70 years-old from April 2020 to April 2021. The participants were divided into the new-onset GD group (idelines . Partici4) 11.45–23.17 pmol/L, and free triiodothyronine (FT3) 3.50–6.50 pmol/L, TPOAb < 60 IU/mL and TGAb < 15 IU/mL. TRAb was measured using electrochemiluminescence immunoassay by a Cobas e601 . The measuring range for TRAb was 0.30–40 IU/L and the cut-off value provided by the manufacturer was 1.75 IU/L. According to the manufacturer’s quality control, the coefficient of variation (CV) was calculated according to the formula CV = SD/Mean × 100%. The intra-assay CV was 2.29% and the inter-assay CV was 2.65%.In our laboratory, the serum concentrations of thyroid hormones and thyroid-related antibodies including thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibody (TGAb) were detected by a supersensitive electrochemiluminescence immunoassay , with reference ranges for: TSH 0.55–4.78 μIU/mL, free thyroxine  or the gonadotropin receptor aa 261–329. The captured receptor was fixed on a microtiter plate and bound to one antibody arm of TSI. The signal receptor, which consists of TSHR (aa 21–261) and secretory alkaline phosphatase (SEAP), binds to the other antibody arm of TSI. The amount of TSI bound was determined by the intensity of enhanced chemiluminescence development by the reaction of SEAP with luminescent substrate. This assay detected TSI with an intra-assay CV of 3.46% and an inter-assay CV of 2.69%, and a CV of 4.34% at a higher TSI concentration (18.9 IU/L) and 4.17% at a lower concentration (0.87 IU/L), respectively. The measuring range for TSI was 0.10–40 IU/L and the cut-off value suggested by the manufacturer was 0.55 IU/L.P-value < 0.05 was defined as statistically significant.Statistical analyses and data processing were performed using the SPSS 24.0 Medcalc19.6 and GraphPad Prism 8 software. Continuous variables were described as the mean ± standard deviation or median (quartile range). Categorical data were described by case number and percentage. The Mann–Whitney U test was used to compare quantitative data between groups. The Chi-square test or Fisher's exact test was used to compare qualitative data between groups. The qualitative consistency of the two detection methods was evaluated by Kappa test and was explained as follows: a kappa value > 0.80 was considered to indicate great agreement, 0.60 to 0.80 substantial, 0.40 to 0.60 moderate, < 0.40 fair. The quantitative consistency between TSI and TRAb detection methods was analyzed using Passing-Bablok regression and Bland–Altman scatter plot after excluding individuals whose test results exceeded the detection limit with TRAb or TSI. The receiver operating characteristic (ROC) curve was drawn using MedCalc and the area under the curve (AUC) and its 95% confidence interval (CI) were calculated. The diagnostic cut-off point was taken as the value of TSI and TRAb at the maximum Youden index (sensitivity + specificity -1). A Totally, data from 336 participants were analyzed in this study, including 227 patients with new-onset GD, 29 with Hashimoto’s disease, 43 with non-autoimmune thyroid disease and 37 controls without a history of thyroid disease. The main characteristics and levels of TSI and TRAb in each group were shown in Table In 227 patients with new-onset GD, the median levels of serum TSI and TRAb were 7.10 IU/L and 8.79 IU/L, respectively. The TSI was positive (> 0.55 IU/L) for 96.92% patients in GD group, except for 7 patients. Similarly, 95.15% of GD patients presented positive TRAb (> 1.75 IU/L), except for 11 patients. In line with expectations, the percentages of non-GD patients that was positive for TSI and TRAb were both very low. The distribution of TSI and TRAb level in each group was shown in Figure S4 and FT3 and the titers of TSI and TRAb . The antibody titers arranged by different levels of thyroid function listed as categorical variables in Table 4 or FT3 and TSI or TRAb. Compared with subjects in the lower quartile levels of thyroid function (FT4 < 29.20 pmol/L and/or FT3 < 9.60 pmol/L), the percentage of GD patients with upper quartile TSI or TRAb levels was significantly higher in patients with severe hyperthyroidism (FT4 > 66.48 pmol/L and/or FT3 > 28.51 pmol/L) .In 210 newly diagnosed GD patients with a simultaneous determination of thyroid function and TSI/TRAb, a positive correlation was shown between the serum levels of FT4, FT3 and TSH, they were divided into lower quartile groups (< P25), middle quartile groups (P25-P75), and upper quartile groups (> P75), respectively    or TSI (< 0.10 IU/L or TSI > 40 IU/L), we used Passing-Bablok regression analysis to analyze the consistency of TSI and TRAb detections. The bridge immunoassay TSI positively correlated with TRAb, with a correlation coefficient of 01) Fig. . GeneralP = 0.289). Similarly, there was no difference in the specificity of TSI > 0.55 IU/L and TRAb > 1.75 IU/L in diagnosing GD (96.33% vs. 99.08%) (P = 0.250) . We selected the optimal clinical decision-making point (TSI 0.577 IU/L) by the maximum Youden index to distinguish patients with untreated GD from those with other thyroid diseases and control group, with a sensitivity 96.92% (95% CI: 93.70%-98.80%) and a specificity 97.25% (95% CI: 92.20%-99.40%), respectively. A TRAb of 1.38 IU/L was the best cut-off value for GD diagnosis, showing 96.92% sensitivity (95% CI: 93.70%-98.80%) and 99.08% specificity (95% CI: 95.00%-100.00%).The AUC for the TSI assay was 0.991 (95% CI: 0.974–0.998) and was not significantly different from that for the TRAb assay  (P = 1.0000). Although there was no significant difference, the sensitivity (96.9%) of the cut-off value for TRAb 1.38 IU/L calculated in this analysis was slightly lower than 1.75 IU/L provided by the manufacturers (95.2%). Among 227 patients with a new clinical diagnosis of GD, 5 were identified as antibody-negative for TRAb 1.75 IU/L. If the TRAb cut-off point of 1.38 IU/L from our analysis was used for re-assessment, 4 of the 5 cases were antibody-positive.In the current study, there was no significant difference between the new cut-off value for TSI of 0.577 IU/L and the manufacturer-suggested point of 0.55 IU/L in terms of the sensitivity, specificity, and positive rate of TSI in GD patients, Hashimoto's disease patients, non-autoimmune thyroid diseases patients, and healthy controls  and excellent concordance . According to the cut-off value proposed by the manufacturer, the positive rates of TSI and TRAb in new-onset GD patients were 96.92% and 95.15%, respectively, which suggests that the presence of TSH receptor antibody can be used as a key indicator of GD. In line with the results of other studies, the sensitivity and specificity of TSI and TRAb for GD diagnosis were both high and had no significant differences. However, in comparison with TRAb, the qualitative analysis of discordant cases showed that the TSI assay is more consistent with a clinical diagnosis of GD.4 and TSI in a study using bioassays to detect TSI [4 showed a more significant correlation with TSI than with TRAb. In the present study, we found that both TSI and TRAb levels positively correlated with FT4 and FT3 levels in new-onset GD patients, which is consistent with the results reported by Allelein et al. [The correlation between TRAb or TSI and thyroid functions was controversial in previous studies. Kabadi et al. found that there was no significant correlation between the levels of FTtect TSI . In the tect TSI , FT4 shon et al. . The corUsing an ROC curve analysis, a new TSI cut-off value (0.577) IU/L for GD diagnosis with a sensitivity of 96.9% and a specificity of 97.2% was obtained in this study. This new cut-off value was very close to the cut-off value (0.55 IU/L) provided by the manufacturer, as well as the cut-off values reported in several studies  –17. CorrAfter a previous study on the use of the Immulite 2000 TSI assay for GD diagnosis , we repoIn conclusion, both TSI and TRAb assays had relatively high diagnostic accuracy for GD. The new-marketed automated TSI detection assay had a higher concordance rate with clinical GD diagnosis and was expected to be promoted in clinical practice.Additional file 1:Figure S1. TSI and TRAb levels in the different groups enrolled in the study. GD, Graves’disease; HS Hashimoto’s disease; NATD, non-autoimmune thyroid diseases.Additional file 2:Table S1. Qualitative method comparison of TSI and TRAb assays. Table S2. Diagnostic consistency analysis of TSI and TRAb for GDAdditional file 3:Table S3. Description of GD patients with negative TRAb/TSI.Additional file 4:Table S4. The association between thyroid volume with TSI/TRAb levels in GD patients.


Objective: To observe the antioxidative effects of N- xanthine oxidase inhibitors (NAY) in vitro and in vivo models of hyperuricemia and explore the mechanism.Methods: A classical experimental method of acute toxicity and a chronic toxicity test were used to compare the toxic effects of different doses of NAY in mice. The hyperuricemia mouse model was established by gavage of potassium oxonate in vivo. After treatment with different doses of NAY  and allopurinol , observe the levels of uric acid (UA), creatinine (CRE), and urea nitrogen (BUN) in urine and serum, respectively, and detect the activities of xanthine oxidase in the liver. The hyperuricemia cell model was induced by adenosine and xanthine oxidase in vitro. The cells were given different doses of NAY  and allopurinol (100 μmol/L). Then the culture supernatant UA level of the medium was measured. The next step was to detect the xanthine oxidase activity in the liver and AML12 cells, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammatory factors in the kidney and serum of mice. Western blot was used to detect xanthine oxidase protein expression in mouse liver tissue and AML12 cells, ASC, Caspase-1, NLRP3, GLUT9, OAT1, and OAT3 protein expression in mouse kidney tissue and HK-2 cells. Hematoxylin–eosin staining was used to stain the liver and kidney tissues of mice and observe the tissue lesions.Results: NAY had little effect on blood routine and biochemical indexes of mice, but significantly reduced the serum UA level. NAY significantly reduced the level of UA in hyperuricemia mice and cells by inhibiting xanthine oxidase activity and reduced the levels of TNF-α, IL-6, and other inflammatory factors in serum and kidney of mice. NAY can inhibit inflammation by inhibiting the NLRP3 pathway. In addition, NAY can downregulate GLUT9 protein expression and upregulate OAT1 and OAT3 protein expression to reduce the UA level by promoting UA excretion and inhibiting UA reabsorption.Conclusion: These findings suggested that NAY produced dual hypouricemic actions. On the one hand, it can inhibit the formation of UA by inhibiting xanthine oxidase inhibitors activity, and on the other hand, it can promote the excretion of UA by regulating the UA transporter. It provides new ideas for the development of hyperuricemia drugs in the future. UA is tetc. , which letc.  for the In humans, the kidneys play a key role in maintaining circulating UA concentrations, as more than 70% of UA excretion in the body is accomplished through the kidneys . HoweverStructure–activity relationship (SAR) analysis and molecular modeling studies demonstrated that the 9,10-anthraquinone moiety located at the outer region of XO active pocket and performed a role of the lipophilic fragment as in the case of the isobutoxy group of febuxostat . We synt2Cl2 (40 ml) and stirred at 25°C for 15 min, then EDCI  were added immediately. The reaction mixture was then stirred at 25°C overnight. After the reaction is complete, the mixture was concentrated under reduced pressure, then alcohol (10 ml) was added and poured into cold water (200 ml), filtered, and dried, and the solid was used directly without further purification (The synthetic route to target compound 3 (NAY) is shown in fication .The solification .ad libitum. A total of 12 SPF grade SD rats, male, were provided by the Experimental Animal Management Department of Shanghai Institute of Family Planning Science. (Permit ID: SCXK(HU)2018-0006, certificate No.: 20180006026509). Before administration, the rats would be adaptively fed for 1 week in the Experimental Animal Center Laboratory of China Pharmaceutical University. During the experiment, the rats had normal diet and water and being maintained with the circadian rhythm cycle. The experimental plan was approved by the Animal Protection and Ethics Committee of China Pharmaceutical University.SPF male and female KM mice  were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Permit ID: SCXK(SU)2020-0009, certificate No.: 202103492). Animals were under a normal 12-h/12-h light/dark schedule with the lights on at 07:00 a.m. They were housed at room temperature (22 ± 2°C) with relative humidity (55 ± 5%) and given a standard chow and water Alpha mouse liver 12 (AML12) and human renal proximal tubule (HK-2) cells were provided by Cell Bank of Chinese Academy of Sciences . The cells were cultured according to the reference culture conditions provided by Shanghai Cell Bank.Fetal bovine serum (FBS), phosphate balanced saline (PBS), BCA protein assay kit, 4% paraformaldehyde Solution, mouse IL-1β, IL-6, TNF-α, and NLRP3 ELISA Kits were from Nanjing Senbega Biotechnology Co., Ltd. . Penicillin–streptomycin were obtained from MCE. Adenosine and xanthine oxidase were purchased from Sigma-Aldrich Chemical Company . Potassium oxonate and allopurinol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . UA, BUN, CRE, and XOD determination kits were purchased from Nanjing Jiancheng Biotechnology Research Institute . RPMI-1640 and DMEM/F12 medium and PageRuler prestained protein ladder were from Thermo Fisher Technology Co., Ltd. . Anti-GLUT9, anti-OAT1, anti-OAT3, anti-caspase 1, anti-β-actin, anti-GAPDH, and HRP-conjugated affinipure goat antirabbit IgG were purchased from Proteintech Group, Inc. . Anti-XOD was purchased from Abcam , anti-NLRP3 and anti-ASC were purchased from Affinity . Protease inhibitor cocktail and RIPA buffer were purchased from New Cell and Molecular Biotech Co., Ltd. . PVDF membrane was purchased from Bio-Rad Laboratories, Inc. . Both SDS-PAGE running buffer (powder) and SDS-PAGE transfer buffer (powder) were bought from Servicebio Technology Co., Ltd. . PAGE Gel Fast Preparation Kits were purchased from Shanghai Epizyme Biomedical Technology Co., Ltd. . Primary antibody dilution buffer, secondary antibody dilution buffer, and blotting grade were purchased from Beyotime Biotechnology Co., Ltd. .The method of A total of 30 male and 30 female mice were selected randomly and then divided into 12 groups of five mice each. Female and male rats were divided into six dose groups. The dosage of the six dose groups were 0, 50, 100, 500, 1000, and 2500 mg/kg, respectively. All drug administration was performed consecutively for 7 days after which the animals were sacrificed.The blood was taken from the eyes of mice and sacrificed by neck amputation, then the heart, liver, spleen, lung, kidney, and brain tissues of mice were separated quickly on the ice platform, and the tissues were weighed. Tissue weight was used to calculate the organ coefficient. The dissected organs were fixed in paraformaldehyde for later pathological sections. One drop of mouse blood was taken into an anticoagulant tube for the detection of blood routine, and the remaining blood was centrifuged at 3000 rpm for 10 min to obtain serum for the detection of biochemical indexes such as uric acid.Serum UA, CRE, BUN, and other biochemical levels and blood routine were determined by the hospital. Blood cells were counted using a hematology analyzer, and mice serum biochemical indexes were measured using an automated blood biochemistry analyzer.The liver and kidney tissues fixed in paraformaldehyde were dehydrated to be transparent. Then put the transparent tissue block into the melted paraffin, and embed it after the paraffin is completely immersed in the tissue block. Fix the embedded wax block on the microtome and cut it into 5 μm thin slices. The thin slice is ironed in hot water, then pasted on the glass slide, and dried. Before staining, paraffin wax in the sections was removed with xylene, then soaked with high to low concentration alcohol, and finally soaked in distilled water for 2 min. The dewaxed and rehydrated slices were stained in hematoxylin solution for several minutes and then washed with running water. Then put the slices into 1% hydrochloric alcohol (5–10 s) and rinse with tap water, after rinsing, place the slices in distilled water. After 2 min, the slices were removed and dehydrated with high to low concentration alcohol. After dehydration, the slices were stained with alcohol eosin solution for 2–3 min. After dehydration by alcohol, xylene was used to make the slices transparent. Finally, the transparent slices were dripped with gum and sealed with a cover glass.Ninety SPF male KM mice (20 ± 2 g) were maintained in a normally controlled breeding room with standard laboratory food and water for 1 week prior to the experiments. The uricase inhibitor potassium oxonate was used to induce hyperuricemia in mice, according to previous reports .Potassium oxonate (model drug), NAY (drug to be tested), and allopurinol (positive drug) were dissolved or suspended in double steaming water. The drug dose used is expressed in “mg/kg.” The dose given for each dose is converted based on the body surface area conversion method. Male Kunming mice were randomly divided into each group with eight mice, namely, blank control group, model control group (250 mg/kg potassium oxonate), extract model administration groups , and positive control allopurinol model administration group (10 mg/kg). Except the normal control mice, others were orally administered by 250 mg/kg potassium oxonate once daily for seven consecutive days to induce hyperuricemia. From day 1 to day 7, it was administered daily at 9:00 a.m., mice were first given potassium oxonate, after 1 h, positive drug group was given allopurinol by gavage, experimental groups were given NAY by gavage, and normal control group was treated with distilled water. After 7 days’ treatment, diets were removed from the cages 12 h before the animals were sacrificed. The blood was allowed to clot for approximately 1 h at room temperature and centrifuged at 3,000 rpm for 10 min to obtain serum. Serum was divided into three portions, one to detect biochemical markers such as UA, one to detect inflammatory factors, and the remaining stored at−20°C as a backup. The livers and kidneys of mice were taken, some were immersed in paraformaldehyde, and the rest were stored at –80°C for subsequent detection.The levels of serum UA, CRE, and BUN were measured by using the method recommended in the purchase kit. The levels of serum TNF-α, IL-6, and NLRP3 were measured by using the method recommended in the ELISA kits.0.1 g mouse liver tissue was weighed accurately, and 0.9 g 0.9% normal saline was added. The mixture was mechanically homogenized under the condition of ice water bath to prepare 10% homogenate and centrifuged at 3000 rpm for 10 min to obtain supernatant for determination. The levels of XOD activities in liver were determined by the colorimetric method, using commercially available kits.Accurately weigh 0.1 g of mouse kidney tissue, and then add 0.9 g PBS (PH7.4) to grind. Homogenize the specimen thoroughly with a homogenizer and centrifuge at 3000 rpm for 20 min for collecting the supernatant. The collected supernatant was divided into two parts, one to detect inflammatory factors and the other remained frozen at—20°C for further use. The levels of kidney TNF-α, IL-6, IL-1β, and NLRP3 were detected by using ELISA kits. The BCA kit was used to measure the concentration of renal protein and correct the expression level of renal inflammatory factors.The kidney tissue (100 mg) was mixed with radio immunoprecipitation assay , containing phenylmethanesulfonyl fluoride (PMSF) and cocktail for homogenization. The homogenate was transferred into a 1.5 ml tube and placed on ice for 30 min. Then, centrifugation was carried out at 14,000 r/min for 10 min. The supernatant was collected into a 1.5 ml tube and stored at -80°C. A fraction of supernatant was mixed with loading buffer, followed by boiling for 10 min. The mixture was allowed to cool to room temperature and stored at -20°C after centrifugation. The protein concentration was determined with the BCA method. The protein sample was mixed with 1×loading buffer at a ratio of 1:1. Electrophoresis was performed at a constant voltage of 80 V for 120 min until the protein bands reaching the bottom of the gel. Then, the proteins were transferred onto the polyvinylidene fluoride membrane at a constant voltage for 1 h. The membrane was blocked in non-fat milk for 2 h at room temperature. Then wash the membrane in Tris-HCl Tween (TBST) thrice (5 min for each). The next step was treating the membrane with first antibody  at 4°C overnight. After washing in TBST thrice (5 min for each), the membrane was treated with HRP-conjugated secondary antibody (1:10000) at 37°C for 2 h. After washing in TBST thrice (5 min for each), chemiluminescent substrate was added onto the membrane followed by incubation in dark for 5 min, and the protein bands were detected by an ECL kit and analyzed by ImageJ software. The protein expression of ASC, Caspase-1, NLRP3, GLUT9, OAT1, and OAT3 in each group was normalized to that of β-actin.Liver tissues were electrophoresed, transmembrane and other experiments were performed according to the same methods as appeal, the only difference was the use of XOD primary antibody to treat the membrane overnight at 4°C.2/95% humidified air at 37°C.AML12 cells were cultured according to the cell supplier’s instruction with slight modifications in DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 lg/ml) under atmosphere of 5% CO2. The culture medium was changed every other day.HK-2 cells were cultured in RPMI 1640 medium with 10% FBS, 100 μg/ml streptomycin, and 100 units/mL penicillin. The cells were incubated at 37°C in a humidified atmosphere supplemented with 5% CO5 cells/ml. 100 µL of cell suspension was added to each well of the 96-well plate and then 100 µL of medium was added to each well. Then, the mixture was gently shaken and then cultured in a 5% CO2 cell incubator at 37°C for 24 h.AML12 cells and HK-2 cells in the logarithmic growth phase were taken and placed in suspension. After being evenly blown, the cells were counted with a cell counter, and the concentration of cell fluid was diluted and adjusted to 102 at 37°C cell culture box for 24 h. After 24 h, remove the media containing drugs, rinse with PBS for three times, and add CCK-8 detection solution. After adding CCK-8 detection solution, the 96-well plate was placed in a 5% CO2 cell culture box at 37 °C for 3 h. After 3 h of incubation, the enzyme plate was detected at 450 nm to calculate the cell survival rate.After 24 h, the cell culture medium was sucked out, washed with PBS for three times, and the incomplete medium with NAY was added. The final concentration of NAY was 1, 500, 250, 125, 67.5, and 0 μmol/L, which was put into 5% COIn vitro hyperuricemia model of AML12 cells and HK-2 cells was induced by adenosine and xanthine.5 cells/mL, and 1 ml of each well was inoculated in a medium dish. The cells were placed in a 5% (V/V) CO2 cell incubator at 37 °C for 24 h. 24 h later, the cell supernatant was discarded, washed with PBS for three times, and the adenosine solution prepared with the current basic medium without fetal bovine serum as the solvent was added. The dosage of adenosine was 1.5 mmol/L, and the cells were placed in the cell culture box for further incubation for 24 h. The culture dish was taken out, 0.005 IU xanthine oxidase (XO) was added to each well, and incubated in the cell incubator for 24 h, and then the hyperuricemia liver cell model was obtained.AML12 cells in logarithmic growth were digested with trypsin, and cell suspension was made by pipette blowing. After counting with a blood counting plate, the cells were diluted to 102, 37°C cell incubator. The cells in the logarithmic growth phase were inoculated on a 24-well plate at a concentration of 105 cells/ml. The cells were cultured in a 37 °C incubator for 24 h. 24 h later, discard the culture medium, and wash cells by PBS for three times. Add 2.5 mmol/L adenosine solution prepared from the incomplete medium to each well and incubate in the incubator for 24 h. Then 0.005 IU/mg XO solution was added to each well in the 24-well plate, and the supernatant was collected after incubation for 12 h. The absorbance was measured on the microplate reader using the UA kit.According to the establishment method of HK-2 cells, hyperuricemia model was reported in the literature , and HK-After cell safety range test, the final dose concentration range was determined to be 50–200 μmol/L.2 cell incubator. Logarithmic growth cells were made into cell suspension with trypsin. The cells were inoculated into 24-well plates at the concentration of 105 cells/ml and cultured in a cell incubator at 37°C and 5% CO2 for 24 h. After the cells adhered to the wall, the medium in 24-well plates was discarded and washed with PBS for three times. NAY solution  and allopurinol solution (100 μmol/L) prepared with serum-free medium were added to the administration group, and serum-free medium was added to the blank control and model groups. After 24 h, the model and administration groups were washed with PBS for three times. The model and administration groups were added with 2.5 mmol/L adenosine solution prepared by serum-free medium and incubated in incubator for 24 h. Then 0.005 IU/mg XO solution was added to each well in 24-well plates. After incubation for 12 h, collect the supernatant to detect the level of UA and XOD activity of AML12 cells.AML12 cells were cultured in complete medium at 37 °C and a 5% CO2 cell incubator at 37 °C in complete medium. The cells in the logarithmic growth phase were inoculated on a 24-well plate at a concentration of 105 cells/ml and cultured in a 5% CO2 cell incubator at 37°C for 24 h. After 24 h, the culture medium was discarded and washed with PBS for three times. The blank control and model groups were added with incomplete medium, and the drug administration groups were added with NAY  and allopurinol solution (100 μmol/L) in an incubator for culture. After 24 h, the model and drug administration groups were washed with PBS for three times, and the model and drug administration groups were added with 2.5 mmol/L adenosine solution prepared with incomplete whole medium and incubated in an incubator for 24 h. Then add 0.005 IU/mg XO solution to each well in the 24-well plate. After 12 h of incubation, the supernatant was collected to detect the UA level.Hk-2 cells were cultured in a 5% COThe culture, induction, and administration of hyperuricemia HK-2 cells were the same as described earlier. Finally, the supernatant of the culture medium was collected to detect the levels of inflammatory factors. ELISA kits were used to detect the supernatant TNF-α, IL-6, IL-1β, and NLRP3 levels of the culture medium.The total protein of HK-2 cells and AML12 cells were extracted with total protein extraction kit, and the concentration was determined by the BCA kit. Electrophoresis, membrane transfer, antibody incubation, and other operations were performed in accordance with the Western blot experiment procedures. The protein bands were quantified by ImageJ software and normalized to individual β-actin expression levels.A total of 12 SD rats were randomly divided into four groups with three in each group; the four groups were intravenous NAY group (5 mg/kg), intravenous NAY group (50 mg/kg), intravenous allopurinol group (5 mg/kg), and allopurinol group (50 mg/kg). The volume of intravenous administration was 1 ml/250 g, and the volume of oral administration was 1 ml/100 g. Whole blood was collected at 2, 5, 10, 20, 30, 60, 120, 240, 360, 480, 720, and 1440 min after intravenous administration. The rats’ blood was transferred to the heparinized test tube. The whole blood was centrifuged at 8,000 rpm for 5 min, and the plasma was separated and stored in a refrigerator at -80°C for testing.The HPLC-MS/MS method was established to determine the concentrations of NAY and allopurinol in rat plasma, respectively, and WinNonlin software was used to calculate the pharmacokinetic parameters.https://www.rcsb.org/) database (https://www.uniprot.org/), and then modeled by AlphaFold Protein Structure Database   was downloaded from the PDB (database . Amino a.ac.uk/) . The act.ac.uk/) . The sof.ac.uk/) , and the.ac.uk/) .50) of the extract was estimated to be more than 5,000 mg/kg. The body weight gain showed no significant difference between the control and treated groups. The macroscopic observation showed no remarkable pathological changes for the tested organs.During the acute toxicity test, there were no deaths or any signs of toxicity observed after administration of NAY at 2,500 mg/kg, which was the no-observed-adverse-effect level (NOAEL). The dose of 5,000 mg/kg led to the death of one female and one male mouse within 24 h and on day 7, respectively. Therefore, the median lethal dose . Compared with the normal group, serum UA decreased significantly in all dose groups . The CRE levels of male mice also decreased in all dose groups, but the CRE levels of female mice did not change significantly compared with the normal group. It is speculated that male mice are highly sensitive to NAY. CRE levels are lower in female mice than in male mice, so the reduction may not be significant.p < 0.001). Except for the mice in the 50 mg/kg dose group, the heart coefficients of the female mice in the other dose groups were significantly lower than those in the normal group. The spleen coefficient of male mice in each dose group also decreased in varying degrees compared with the normal group.The relevant data of coefficients of organs in mice after 1 week treatment with NAY are shown in As shown in As shown in The hyperuricemia model was successfully established by intragastric administration of potassium oxonate. As shown in p < 0.001). In p < 0.001).Finally, we can see from p < 0.05).The hyperuricemia model was successfully established by intragastric administration of potassium oxonate. As shown in p < 0.01). The contents of UA, CRE, and BUN in the urine of mice were increased at each dose of the NAY administration group. The effect of the drug is dose-dependent, and high-dose NAY has the most significant effect on promoting excretion .As can be seen from Hyperuricemia can cause inflammatory reaction in mice. As shown in p < 0.01). In p < 0.05). In p < 0.001), and in p < 0.001).As shown in p < 0.001). In p < 0.001).As shown in p < 0.05) compared with normal control mice.The effects of NAY and allopurinol (positive control) on liver protein levels of XOD and renal protein levels of ASC, Caspase-1, NLRP3, GLUT9, OAT1, and OAT3 in hyperuricemia are shown in p < 0.05, p < 0.01, and p < 0.001, respectively) compared with the model control group as shown in p < 0.01, Caspase-1, p < 0.05; 20 mg/kg: ASC and Caspase-1, p < 0.001, NLRP3, p < 0.05; 40 mg/kg: SC and Caspase-1, p < 0.001, NLRP3, p < 0.05). As shown in p < 0.01). As shown in p < 0.05, p < 0.01, and p < 0.01, respectively). But as shown in p < 0.05).NAY could downregulated the expression levels of liver XOD protein in hyperuricemia mice in a dose-dependent manner  and the level of supernatant UA was significantly increased (p < 0.001), which proved that the hyperuricemia in vitro model was successfully constructed. Compared with the model group, both the effect of the allopurinol group on reducing XOD viability and the level of supernatant UA were obvious (p < 0.001). In p < 0.01). In p < 0.001; 100 μmol/L: p < 0.01; 200 μmol/L: p < 0.01).As shown in p < 0.001), and the hyperuricemia cell model was successfully constructed. The level of UA in the positive drug group and NAY group was significantly decreased . Moreover, NAY decreased UA levels in a dose-dependent manner, and the highest reduction was observed in the high-dose group.The results in p < 0.01 or p < 0.001).The content of inflammatory factors in supernatant of HK-2 cells in each group was detected by ELISA as shown in p < 0.01). In p < 0.01). The moderate and high-dose NAY groups also had a IL-6 level reduction . In p < 0.01). In p < 0.05; 100 μmol/L and 200 μmol/L: p < 0.01).In The effects of NAY and allopurinol on the expression of XOD protein in AML12 cells and ASC, Caspase-1, NLRP3, GLUT9, OAT1, and OAT3 protein in HK-2 cells are shown in p < 0.05), and compared with the model group, the expression level of XOD protein in the allopurinol administration and NAY administration groups was significantly reduced .As shown in p < 0.05), and NAY can alleviate the overexpression of ASC, Caspase-1, and NLRP3 proteins .In p < 0.05), and each dose of NAY could inhibit the increase of this protein expression level, and the inhibition degree was dose-dependent .In p < 0.01; 100 μmol/L, p < 0.05; OAT3: p < 0.05).In 2 = 0.9923, with a quantitative range of 5–5,000 ng/ml. Main pharmacokinetic parameters of NAY in rats are shown in 2min = 30576.33 ± 6801.86 ng/ml), the drug was more slowly metabolized in blood, and the drug body exposure was greater . After intragastric administration, the concentration showed a second peak at 8 h. However, the second peak of plasma concentration did not occur in the intravenous injection group, so there may be no hepatoenteric circulation , indicating that multiple sites of NAY may be absorbed in rats after intragastric administration.The blood concentration time profiles of NAY after intravenous and intragastric administration in rats are shown in 2 = 0.9930, and the quantitative range was 5–10,000 ng/ml. Main pharmacokinetic parameters of NAY in rats are shown in 2min = 5280.84 ± 686.48 ng/ml), the drug metabolism in blood was fast, and the drug exposure in vivo was small (AUC0-∞ = 1590.90 ± 138.60 h μg/L). The drug had a short half-life and a large elimination rate constant .The blood concentration time profiles of allopurinol after intravenous and intragastric administration in rats are shown in We docked febuxostat, the original ligand of XOD, to ensure the accuracy of the docking method. We docked febuxostat, the original ligand of XOD, to ensure the accuracy of the docking method. As shown in in vivo by hypoxanthine-guanine phosphoribose transferase, xanthine invertase, and phosphoribose pyrophosphoamide conversion enzyme. Exogenous is the metabolism of purines in food into UA in the body. UA is excreted mainly by the kidneys. If the body produces too much UA or excretes too little UA from the kidney (urine), it will lead to the ultra-saturated state of urate in the extracellular fluid, which is a very insidiously harmful metabolic disease of human health. The serum UA level is not only directly related to gout, recent studies have found that hyperuricemia is often associated with traditional metabolic cardiovascular risk factors, such as hypertension, hyperlipidemia, diabetes, obesity, and insulin resistance and is an independent risk factor for cardiovascular disease , also known as 9,10-anthraquinone, refers to AQ compounds with the carbonyl group at C50 > 5,000 mg/kg BW in rats and mice.  in the body and is mainly synthesized in the liver . It is min vitro and in vivo, and the experimental results show that NAY had different regulatory effects on GLUT9, OAT1, and OAT3. As a member of the glucose transporter family, GLUT9 can accelerate the reabsorption of UA by transporting glucose (UA transporters were identified as potential therapeutic targets for hyperuricemia. Previous studies have shown that 70% of urate is mainly dependent on the renal urate transport system . The met glucose . As expeThe NLRP3 inflammasome, a protein complex discovered in 2002 by in vivo and in vitro experiments. The present results show that this NAY significantly inhibits XOD activity while reducing serum UA, CRE, and urea BUN levels, as well as regulating the expression of urate transporters GLUT9, OAT1, and OTA3. The NLRP3 inflammatory signaling pathway was intervened by blocking the activation of Caspase-1, inhibiting inflammatory responses. Moreover, the compounds exhibited significant protective effects against liver and kidney injury in PO-induced hyperuricemia mice, and the effect showed a little dose-dependent, the optimal dose and specific action targets of drugs need to be further studied. While having good efficacy and low toxicity ratio of the drugs, this compound has good development prospects as a future antihyperuricemia drug.The therapeutic mechanism of NAY on hyperuricemia mice is shown in