Saposhnikovia divaricata (Turcz.) Schischk., is one of the most common botanicals in Asian countries, now basically comes from cultivation, resulting in the market price being only 1/10 to 1/3 of its wild counterpart, so improving the quality of cultivated Radix Saposhnikoviae is of urgency. Nitric oxide (NO) plays a crucial role in generating reactive oxygen species and modifying the secondary metabolism of plants. This study aims to enhance the quality of cultivated Radix Saposhnikoviae by supplementing exogenous NO. To achieve this, sodium nitroprusside (SNP) was utilized as an NO provider and applied to fresh roots of S. divaricata at concentrations of 0.03, 0.1, 0.5, and 1.0 mmol/L. This study measured parameters including the activities of antioxidant enzymes, secondary metabolite synthesis enzymes such as phenylalanine ammonia-lyase (PAL), 1-aminocyclopropane-1-carboxylic acid (ACC), and chalcone synthase (CHS), as well as the contents of NO, superoxide radicals (O2·−), hydrogen peroxide (H2O2), malondialdehyde (MDA), and four secondary metabolites. The quality of Radix Saposhnikoviae was evaluated with antipyretic, analgesic, anti-inflammatory effects, and inflammatory factors. As a result, the NO contents in the fresh roots were significantly increased under SNP, which led to a significant increase of O2·−, H2O2, and MDA. The activities of important antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were found to increase as well, with their peak levels observed on the 2nd and 3rd days. PAL, ACC, and CHS activities were also significantly enhanced, resulting in the increased secondary metabolite contents of Radix saposhnikoviae in all groups, especially the 0.5 mmol/L SNP. The four active ingredients, prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol, increased by 88.3%,325.0%, 55.4%, and 283.8%, respectively, on the 3rd day. The pharmaceutical effects of Radix Saposhnikoviae under 0.5 mmol/L SNP were significantly enhanced. Exogenous SNP can induce the physiological response of S. divaricata under adverse conditions and significantly improve the quality of Radix Saposhnikoviae.The ecological significance of secondary metabolites is to improve the adaptive ability of plants. Secondary metabolites, usually medicinal ingredients, are triggered by unsuitable environment, thus the quality of medicinal materials under adversity being better. The quality of the cultivated was heavily declined due to its good conditions. Radix Saposhnikoviae, the dried root of  This medicinal plant is rich in various bioactive components, including chromone, coumarin, and polysaccharides. The pharmacological activities of Radix Sposhnikoviae are diverse and valuable, such as antipyretic, analgesic, anti-inflammatory, rheumatic, anti-tumor, and antioxidant2. With the increase in population and farmland, as well as years of over-harvesting, the wild resources of S. divaricata have been depleted and cannot meet the market requirements of more than 5000t per year hence the cultivated have become a primary source, which accounts for more than 70% of the market share3. The transformation from wild to cultivated has also led to a serious decline in quality, with the active ingredient content reduced by half4 and the price being only 1/3 to 1/10 of that of wild, making the improvement of the cultivated Radix Saposhnikoviae quality to an urgent issue5. The quality of herbs directly influences their healing effects. Today, researchers emphasize evaluating various plant constituents to understand their pharmacological effects and explore their potential healing properties6.Radix Sposhnikoviae is a widely used herbal medicine in Asian countries, derived from the root and rhizome of the plant S7, while moderate ecological stress is an indispensable condition for producing high-quality herbal medicine. Photosynthesis in plants is the process of biosynthesis, CO2 and H2O into energy-rich organic matter through the absorption of light energy, electron transfer, photosynthetic phosphorylation, and carbon assimilation while releasing O2. Reactive oxygen species (ROS), including O2·−, H2O2, ·OH, etc., are hyperactive oxygen-containing compounds produced during this aerobic metabolism in organisms: the released O2 is easily reduced to the chemically hyperactive O2·−, then transformed into ·OH, H2O2, etc. It has been confirmed that under severe ecological stress, plants generally experience a significant increase in ROS levels8, resulting in various physiological and metabolic alterations9, further leading to damage in essential macromolecules, such as proteins, DNA and biofilm10. During the protracted revolution, organisms evolved antioxidant enzyme systems such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) et al. to eliminate excess ROS and lessen their damage. Nevertheless, unlike mobile organisms, plants cannot avoid adversity by moving, and large amounts of ROS will inevitably be produced, making it difficult for plants to adapt to adversity solely through antioxidant enzymes. To enhance their adaptability to the environment, plants have evolved secondary metabolism as a supplement. Chemical bonds such as –S–S-bonds are indispensable for enzymes to maintain the corresponding structure and carry out catalysis. Because excess ROS can affect the formation of these chemical bonds, it can affect metabolism by the altered the activity of enzymes, and become the medium of the environment influencing metabolism. It has been reported that ROS can promote the change of plant antioxidant enzymes and secondary metabolites at a certain concentration9. Secondary metabolites are significant in plant adaptation to ecological stress, and these metabolites often serve as the active ingredients of herbal medicine11. Numerous studies have demonstrated that the high amount of ROS under ecological stress can elevate the contents of plant secondary metabolites12, for example, the active ingredient contents of Scutellaria baicalensis increased by more than 50% when exposed to Na2S2O4 (a carrier substance for O2·−). Similarly, the quality of Fructus schisandrae chinensis was significantly improved when treated with H2O2, etc.14.It is worth noting that unfavorable environmental conditions can contribute to variations in the quality of herbal medicine2·−15, making it capable of exhibiting the effects of ROS. Additionally, NO has four main physiological effects: (1) Reducing oxidative damage: NO can rapidly convert the higher activity O2·− to low activity ONOO· for plants18, with a reaction rate of 5 × 109 mol L−1 s−1. Meanwhile, it can significantly reduce the conversion of O2·− to ·OH, the destruction of the latter being about thousands of times more than O2·−. (2) Metabolic regulation: NO can regulate enzyme activities through protein modification by binding to cysteine residues, hemoglobin, or iron-sulfur centers, as well as through ONOO˙ formation and tyrosine residue nitration17. (3) Regulation of cellular redox environment: NO maintains metabolic homeostasis by decreasing GSH/GSSG20. (4) Eliminate ROS. NO is highly active and quickly reacts with O2, H2O2, etc., making it less destructive. As a result, NO has a broader advantage. NO can improve the antioxidant capacity of chickpea plants, the antioxidant and wound-healing ability of sweet potato roots, and more22. Additionally, NO and ROS can synergistically improve plant resistance and alleviate the damage caused by ecological stress23. Sodium nitroprusside (SNP), as an exogenous NO donor, is capable of generating both NO and ROS in organisms24 and leads to oxidative stress. Still, it also contributes to the imbalance of ROS and potential damage to cells and tissues25. On the positive side, SNP can also improve the tolerance of plants to ecological stress, with total phenols and flavonoids being significantly elevated in tomatoes26. Additionally, for marjoram herb, SNP treatment substantially enhances secondary metabolite content and essential oil yield27. Notably, plants' fresh medicinal parts possess a complete secondary metabolism, and SNP can also directly influence isolated roots. In this study, the application of SNP elucidated the formation of Radix Saposhnikoviae quality by inducing a physiological response under ecological stress. This approach opens up new possibilities for producing high-quality Radix Saposhnikoviae and represents a promising avenue for further research and development in herbal medicine.NO is a small molecule free radical with a high solubility in water and lipids, allowing it to diffuse across biological membranes into adjacent tissue cells freely. Through processes such as oxidation–reduction of electron carrier, ATP synthase, changes in mitochondrial membrane potential, and complex reverse electron transport, NO can produce OS. divaricata, identified by Prof. Xiang-Cai Meng of Heilongjiang University of Chinese Medicine, were collected in Daqing City, Heilongjiang Province, China, collected in October 2022 and immediately wrapped in plastic to keep fresh. The cultivated Radix Saposhnikoviae (FF20221001) has been deposited in a publicly available herbarium of Heilongjiang University of Chinese Medicine. And we complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.All the plant experiments complied with relevant institutional, national, and international guidelines and legislation. Cultivated Radix Saposhnikoviae (FF20221001) collection was done with permission. Fresh 3-year-old roots of cultivated LC-2010A High-performance liquid chromatography (Japan), 752 UV–visible spectrophotometers , CP225D 0.1 mg electronic balance , SD40 ice machine , TGL-16LM desktop high-speed refrigerated centrifuge , DKZ-3B water bath thermostat oscillator , Julabo TW20 digital display thermostatic water bath , QL-902 vortex oscillator , Thermo enzyme standard instrument , DHG-9015A blast dryer  Shimadzu.2·− Free Radical Determination Kit ; Protein Quantification (TP) Determination Kit (20220220), H2O2 Determination Kit (20220903), Superoxide Dismutase (SOD) Determination Kit (20220624), Catalase (CAT) Determination Kit (20220903), Peroxidase (POD) Determination Kit (20220616), Malondialdehyde (MDA) Determination Kit (20220422), Phenylalanine Ammonia-Lyase (PAL) Determination Kit (20220806) (Nanjing Jiancheng Bioengineering Research Institute); 1,3-Phosphoglyceric Acid (202211), NO Determination Kit (202211), ACC Determination Kit (202303), CHS Determination Kit (202303) ; Prim-O-glucosylcimifugin (AF21092803), Cimifugin (AF21092804), 4′-O-β-d-glucosyl-5-O-methylvisamminol (AFBG 1328), Sec-O -glucosylhamaudol (AF20081401) were purchased from Chengdu Aofei Biotechnology Co., Ltd., with a purity greater than 98%; HPLC-grade methanol (R142190); Sodium nitroprusside ; Rats and mice was purchased from Liaoning Changsheng Biotechnology Co., Ltd. by the Drug Safety Evaluation Center of Heilongjiang University of Chinese Medicine in Liaoning Changsheng Biotechnology; Dry yeast , Glacial acetic acid , xylene , and physiological saline  were also used; Mouse Tumor Necrosis Factor-α (TNF-α) ELISA Kit (202303), Mouse Interleukin-6 (IL-6) ELISA Kit (202303) , etc.OS. divaricata were mixed evenly and then divided into 5 groups. SNP aqueous solution with 0.00, 0.03, 0.10, 0.50, and 1.0 mmol/L was sprayed, respectively, until the droplet was about to drop, that is, saturation, to ensure that the dosage could be controlled and uniform. The pre-test results showed that several active ingredients peaked on the 3rd day, suggesting that the desired effect of substance SNP was anticipated on the 2nd day. To match this expectation, we applied SNP only on the first two days and switched to distilled water afterward. The 0.30 g × 30 samples of fresh root phloem were taken from each group per day, and three repeat samples for each index were used to determine activities of SOD, POD, CAT, PAL, CHS, ACC, and the contents of malondialdehyde (MDA), 1,3-diphosphoglyceric acid , and NO. Fresh root phloem of each group was taken 0.10 g × 10 samples to determine O2·− and H2O2. The above samples were sealed in aluminum foil and stored in a refrigerator at – 80 °C. At the same time, at least six fresh roots were taken from each group, homogeneous per sample, more than 30 g × 3, dried in an oven at 55 °C, then crushed and sieved through 60-mesh sieve for the determination of the contents of prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol.The collected fresh roots of The NO contents in fresh roots were determined with a plant NO ELISA assay kit.2·− and H2O2 using the O2·− assay kit and the plant H2O2 assay kit, respectively.The homogenized protein contents. The fresh roots were determined with the protein quantification (TP) assay kit Bradford method and the OThe MDA contents were determined with TBA using a malondialdehyde kit.The activities of antioxidant enzymes were determined with SOD, CAT, and POD assay kits, respectively.The contents of primary metabolite 1,3-DPG were determined with a plant 1,3-DPG ELISA detection kit, the activities of PAL ACC and CHS using a PAL, Acetyl coenzyme A carboxylase (ACC), and chalcone synthase (CHS) ELISA kits, respectively.O-glucosylcimifugin and 2.25 mg of 4′-O-β-d-glucosyl-5-O-methylvisamminol into a 5 mL volumetric flask, and methanol was added to the scale to prepare 0.38 mg/mL and 0.45 mg/mL of the control solution. In addition, accurately weigh 1.03 mg of cimifugin and 1.05 mg of sec-O-glucosylhamaudol into a 25 mL volumetric flask, and methanol was added to the scale to make up 0.042 mg/mL, and 0.021 mg/mL of the control solution. Preparation of sample solution: Accurately weigh 1.5 g of the three groups of Radix Saposhnikoviae into a 50 mL conical flask, 50 mL of methanol was added, and the mass was weighed and extracted by heating reflux at 70 °C for 2 h in a water bath. After cooling, weigh it again, balance the weight loss, shake well, and filter. The filtrate was then passed through a microporous membrane (0.45 μm), and the contents of prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol were determined with HPLC.Preparation of standard solution: Accurately weigh 1.88 mg of prim-18 chromatographic column ; column temperature: 40 °C; mobile phase: methanol (A)-aqueous solution (B); elution gradient: 0–8 min, 30–50% A; 8–18 min, 50–70% A; volume flow rate: 1.0 mL/min; detection wavelength: 254 nm, injection volume: 10 μL.Diamonsil CO-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol were 1.35%, 1.63%, 1.25%, and 1.67%, respectively, indicating the precision was high.The intra-day precision was calculated by taking the test solution under 2.7.1 and measuring it 6 times according to the method under 2.7.2; the inter-day precision was calculated by analyzing it for three consecutive days. The RSDs of the inter-day precision of prim-O-glucosyl cimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methylvisamminol, and sec-O-glucosyl hamaudol, respectively, indicating good repeatability of the analytical method.The test solution under 2.7.1 was taken and determined with the method under item 2.7.2 to obtain the RSDs of the test solutions were 0.28%, 2.75%, 1.07%, and 2.79% for prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methyl visamminol, and sec-O-glucosylhamaudol were 1.76%, 1.78%, 0.79%, and 1.64%, respectively, which indicated that the stability of the test solution was good within 12 h. The test solution was analyzed under 2.7.2 for 0 h, 3 h, 6 h, 9 h, and 12 h after preparation.The RSDs of peak areas of prim-O-glucosylcimifugin, cimifugin, 4′-O-β-d-glucosyl-5-O-methyl visamminol, and sec-O-glucosylhamaudol controls.The average recoveries were 98.60%, 101.08%, 100.23%, and 99.90% with RSDs of 2.69%, 2.44%, 2.02%, and 1.80%, respectively, when the known content of Radix Saposhnikoviae from Daqing was added with a prim-The medicinal efficacy of Radix Saposhnikoviae is closely related to the content of secondary metabolites, so the optimal group of Radix Saposhnikoviae with the combined increase of the four chromones, was selected as the high-quality Radix Saposhnikoviae herb group, i.e., 0.5 mmol/L SNP treatment.The ordinary Radix Saposhnikoviae group (0-day group) and the high-quality Radix Saposhnikoviae group (0.5 mmol/L SNP group) were dried to constant weight, taken 9.0 g, added 10 times the amount of water, soaked for 1 h, decocted for 1 h, filtered, and the extraction was repeated three times and the filtrates were combined. Concentrated to 0.045 g botanicals/mL, and 2.0 mL solution was given by gavage to each rat and concentrated 0.065 g botanicals/mL for mice at 0.2 mL each.Animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH guidelines) and ARRIVE guidelines and approved by the Ethical Committee of Heilongjiang University of Chinese Medicine .In male SD rats, 180 ± 20 g, the basal body temperature was first measured 3 times a day for 3 days. For rats with an average anal temperature of 36.85 ± 0.37 °C, 2.0 mL/100 g of 15% yeast suspension were injected subcutaneously, and those whose body temperature increased by > 0.8 °C were selected as the test rats. The rats were randomly divided into four groups: saline group, model group, ordinary Radix Saposhnikoviae group, and high-quality Radix Saposhnikoviae group, with 10 rats in each group. The saline and model groups were administrated at 2.0 mL of saline daily, and the Radix Saposhnikoviae group was administrated at 0.045 g botanicals/mL for 2.0 mL daily for seven days. At the 3rd hour after the last administration, the rats were subcutaneously injected with 2.0 mL/100 g 15% yeast suspension, and their rectal temperature was measured at 0.5, 1, 2, 3, 4, and 5 h after injection.Male Kunming mice, weighing 18 ± 2 g, were randomly divided into four groups: saline group, model group, ordinary Radix Saposhnikoviae group, and high-quality Radix Saposhnikoviae group, 10 mice in each group. 2.0 mL of saline was administrated daily for the saline group and the model group, and 0.2 mL 0.065 g botanicals/mL was administrated for the Radix Saposhnikoviae group. This operating process was done once a day for 7 consecutive days. At 3rd hour after the last administration, 0.2 mL 0.6% of acetic acid was injected intraperitoneally, and the number of twists in mice within 15 min after the injection was observed, and the twist inhibition rate was calculated.Inhibition rate = [(numbers of twists in the control group − numbers of twists in assay group)/numbers of twists in control group] × 100%.Animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH guidelines) and approved by the Ethical Committee of Heilongjiang University of Chinese Medicine . Male Kunming mice weighing 18 ± 2 g were randomly divided into four groups: saline group, model group, ordinary Radix Saposhnikoviae group, and high-quality Radix Saposhnikoviae group, 10 mice for each group. 2.0 mL of saline was administrated daily for the saline group and the model group, and 0.2 mL was administrated for each Radix Saposhnikoviae group according to 0.65 g of botanicals/kg, once a day for 7 days. At the 3rd hour after the final administration, 0.03 mL of xylene was evenly applied to the anterior and posterior sides of the right ear of each mouse for 1 h to induce inflammation.Both ears were cut along the baseline of the auricle, and the tissue of the same part of the left and right ear was weighed by punching with an 8 mm punch, and the swelling rate and swelling inhibition rate of the auricle of mice were calculated.Swelling inhibition rate (%) = (mean swelling rate in the control group − mean swelling rate in the medicated group)/mean swelling rate in the control group × 100%The contents of TNF-α and IL-6 were determined using the double antibody sandwich method with TNF-α and IL-6 kits.P < 0.05 indicated statistically significant differences, while P < 0.01 indicated extremely statistically significant differences.Microsoft Office Excel 2007 and SPSS 26.0 software were used to process the data, and GraphPad Prism software was used to make graphs. The measurement data were expressed as , ARRIVE guidelines and approved by the Ethical Committee of Heilongjiang University of Chinese Medicine .S. divaricata on the first day and an increase on the 2nd and the 3rd day, reaching a peak on the 3rd day, with a rise of 55.0% compared with the 0-day (P < 0.01). As shown in Fig. Compared with the 0-day, there was no significant trend in the distilled water group and no significant trend in the 0.03, 0.1, and 1.0 mmol/L treatment groups except for a slight decrease on the 1st day. The 0.5 mmol/L SNP group caused a slight decrease in NO content in the fresh roots of 2·− contents in the fresh roots of S. divaricata, showing a trend of increasing and then decreasing. The distilled water group showed no significant trend. The 0.03 and 1.0 mmol/L SNP groups peaked on the 2nd day. The 0.1 and 0.5 mmol/L treatment groups peaked on the 3rd day. The 0.5 group showed the most significant increase, with an increase of 135.3% compared with the 0-day (P < 0.01). As shown in Fig. Compared with the 0-day control, different concentrations of SNP all increased the O2O2 contents in the fresh roots of S. divaricata increased significantly, increasing and then decreasing from the 0 to the 4th day. The 1.0 mmol/L treatment group peaked on the 2nd day, and all other treatment groups peaked on the 3rd day. The 0.5 mmol/L treatment group showed a 985.1% increase (P < 0.01). As shown in Fig. Compared with the 0-day control, except for the distilled water group, the other groups showed no significant trend, the other HS. divaricata rose with concentrations of SNP, except for the distilled water group. The MDA contents increased as the concentration of SNP increased, with the 1.0 mmol/L treatment group reaching a peak on the 1st day and all other treatment groups reaching a peak on the 2nd day, with the most significant increase of 133.6% in the 0.5 mmol/L treatment group compared with the 0-day (P < 0.05). As shown in Fig. Compared with the 0-day control, the MDA contents in the fresh roots of P < 0.05). As shown in Fig. Compared with the 0-day control, the antioxidant enzyme activities of all treatment groups showed a trend of increasing and then decreasing, except for the distilled water group and the 1.0 mmol/L treatment group, with a minor variation in antioxidant enzyme activities. SOD peaked on the 2nd day, with a 27.5% increase in the 0.5 mmol/L treatment group. CAT and POD activity peaked on the 3rd day, with 281.6% and 297.1% increases in the 0.5 mmol/L group, respectively  and peroxidase (POD), work together to convert H2O2 into H2O and O230. SOD peaked on the 2nd day, converting O2·− into H2O2, and H2O2 contents were significantly increased in the 0.1 and 0.5 mmol/L SNP-treated groups on the 3rd day. Meanwhile, CAT and POD activities also elevated, especially in the 0.5 mmol/L group (P ˂ 0.05). With the interaction of CAT and POD, H2O2 contents decreased on the 4th day  is a key and limiting enzyme that catalyzes the carboxylation of acetyl-CoA to form propionyl-CoA. The other is the shikimic acid pathway, in which phenylalanine ammonia-lyase (PAL), a key enzyme for flavonoids, catalyzes phenylalanine to form cinnamic acid. Coumaric acid under cinnamate 4-hydroxylase (C4H), followed by coumaric acyl-CoA under 4-coumaroyl-CoA ligase (4CL). Based on this, the propionyl-CoA and the coumaric acyl-CoA undergo a series of reactions under chalcone synthase and form chromone compounds36. As shown in Fig. The main secondary metabolites of 37. By applying SNP to the fresh roots, the activities of ACC, PAL, and CHS, the key enzymes of secondary metabolite biosynthesis, were increased in each treatment group, among them, the activities of CHS and ACC in the 0.5 mmol/L SNP group increased by 117.8% and 44.1% on the 2nd day and 3rd day, respectively  was reduced by 0.11–0.13 °C. The inhibition rate of twisting body and ear swelling in mice was increased by 14.45% and 38.78%, the inhibition rate being 1.5 and 3.1 times of the ordinary botanicals, respectively. The contents of IF-6 and TNF-α were reduced by 17.52% and 18.00%, and the inflammatory factor content fell into the original level of the blank group, which indicated that the efficacy of the high-quality Radix Saposhnikoviae group was significantly improved.Botanicals contain a wide variety of active ingredients, and there are significant variations in the contents, activities, and bio-availabilities of various active ingredients45. For example, Cerium oxide nanoparticles increase NO production in rice leaves under salt stress and enhance nitrate reductase activity and nitrate reductase activity46. Similarly, multilayer nanotubes have shown the ability to modulate NO production and improve salt tolerance in oilseed rape47. SNP is an injectable agent for the treatment of hypertension and acute heart failure, the application to herb quality improvement is also in very low concentrations and amounts. Besides, the SNP only increases the level of intrinsic ingredients of S. divaricata under drought stress without producing other toxic components. Therefore, it is safe to use SNP. SNP as an inducer of secondary metabolism has promising applications.The nano-materials as delivery carriers have been used for biological research for decades, and some significant breakthroughs have been madeO-β-d-glucosyl-5-O-methyl visamminol, and sec-O-glucosylhamaudol increased by 88.3%, 325.0%, 55.4%, and 283.8%, respectively. Based on this, the efficacy of Radix saposhnikoviae was enhanced. This study is based on a common strategy for plants to adapt to ecological stresses, applying SNP to improve the quality of Radix Saposhnikoviae can provide a new pathway for producing other high-quality medicinal herbs.The exogenous NO donor SNP can increase the levels of ROS, resulting in the enhancement of antioxidant enzyme activities and secondary metabolite contents in fresh roots. The antioxidant enzymes and secondary metabolites work together and reduce the damage to the plant body caused by ROS. In the 0.5 mmol/L SNP-treated group, prim-O-glucosylcimifugin, cimifugin, 4′-Supplementary Information 1.Supplementary Information 2.


The 1:2 ratio of griseofulvin and HP-γ-cyclodextrin in the complex was determined based on its NMR study. After complexation with HP-γ-cyclodextrin, griseofulvin’s water solubility was increased 477 times compared with that of griseofulvin alone, which is the best result thus far. The complex showed 90% of griseofulvin release in vitro in 10 min, in an in vivo dog pharmacokinetic study; the Cmax was increased from 0.52 µg/mL to 0.72 µg/mL, AUC0–12 was increased from 1.55 μg·h/mL to 2.75 μg·h/mL, the clearance was changed from 51.78 L/kg/h to 24.16 L/kg/h, and the half-life time was changed from 0.81 h to 1.56 h, indicating the obtained griseofulvin complex can be a more effective drug than griseofulvin alone.Since griseofulvin was marketed as a non-polyene antifungal antibiotic drug in 1958, its poor water solubility has been an issue for its wide applications, and over the last sixty years, many attempts have been made to increase its water solubility; however, a significant result has yet to be achieved. Through supercritical carbon dioxide-assisted cyclodextrin complexation with the addition of a trace amount of water-soluble polymer surfactant, the griseofulvin inclusion complex with HP- Griseofulvin, structured as 1′S,6′R)- 7-chloro- 2′,4,6-trimethoxy- 6′-methyl- 3H,4′H-spiro ene]-3,4′-dione, as shown in ′S,6′R- 7Griseofulvin processed into 3~5 μm particles by using co-precipitation, freeze-drying, or solid dispersion shows water solubility improvement. When its particle size decreases, the solubility will increase ; therefoα-cyclodextrin, β-cyclodextrin, dimethyl-β-cyclodextrin, and γ-cyclodextrin were formed in complexes with griseofulvin by using kneading, spray, physical mixing, or precipitation methods, γ-cyclodextrin was found to give the best result for an increase in water solubility  is the intrinsic solubility of griseofulvin and the slopes of initial straight lines were obtained from phase solubility diagrams.The association constants of the complexation were calculated as γ-cyclodextrin, and it was found that the water solution method through supercritical carbon dioxide assistance is a more efficient method than others for comparing the water solubility of griseofulvin.Spray drying, grinding, the ultrasonic method, the microwave reaction, using an autoclave reactor, and the water solution method were attempted to prepare the inclusion complex of griseofulvin with HP-γ-cyclodextrin of 1:1, 1:2, or 1:3 at 40 °C, 50 °C, or 60 °C for 10 h, 15 h, or 20 h with stirring speed at 800 r/min, 1000 r/min, or 1200 r/min, respectively, were tried to make the inclusion complex in the mixture of water and acetonitrile with the assistance of supercritical carbon dioxide through statistical orthogonal design strategy. After filtration and lyophilization, the inclusion complex from each condition was obtained for HPLC analysis, and the best complex was obtained from the 1:1 molar ratio of griseofulvin and HP-γ-cyclodextrin at 50 °C for 20 h under 1200 r/min stirring speed as a solid with 81% of inclusion yield and 2% of inclusion rate. The water solubility of griseofulvin in the complex was 2.95 mg/mL which is 343-fold higher than that of griseofulvin (0.0086 mg/mL).The inclusion conditions with different molar ratios of griseofulvin and HP-γ-cyclodextrin as two types of complexes exist in aqueous solutions; after adding a small amount (0.25%) of water-soluble polymers or organic salts such as hydroxypropyl methylcellulose, sodium salicylate, cellulose, sodium carboxymethyl, polyethylene glycol 4000, polyvinylpyrrolidone, hexadimethrine bromide, sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane, sodium acetate, and benzalkonium chloride, the cyclodextrin complexation reaction mixture was worked up and analyzed by HPLC. It was found that the water solubility of griseofulvin in these complexes with a trace amount of water-soluble polymers and organic salts was in the range of 2.50 mg/mL to 4.105 mg/mL, and hydroxypropyl methylcellulose could increase the water solubility of griseofulvin the most to 4.105 mg/mL, which is 477-fold higher than that of griseofulvin, and from a water solubility standpoint, this result is thus far the best one  × 100%.The inclusion yield can be obtained from the following formula: inclusion yield (%) = [griseofulvin inclusion complex (mg)/griseofulvin (mg) + HP-The inclusion rate can be obtained from the formula: inclusion rate (%) = [griseofulvin in inclusion complex (mg)/griseofulvin (mg)] × 100%.γ-cyclodextrin (1000 mg), or the complex of griseofulvin with HP-γ-cyclodextrin (containing 100 mg of griseofulvin) were used for testing, respectively. From the medium (degassed water: 900 mL) at 37 °C with 100 r/min rotating speed, samples (3 mL for each) at 2, 5, 10, 15, 20, 30, 45, and 60 min were collected for HPLC analysis and followed by the addition of 3 mL of water after each collection, respectively.The griseofulvin (100 mg), the physical mixture of griseofulvin (100 mg) and HP-Healthy dogs (12) around 5 kg were randomly divided into two groups as the griseofulvin group and the complex group, each group containing three male and three female dogs. After fasting for 12 h, water was forbidden for 1 h before dosing. A single oral dose of griseofulvin or its complex was administered to each dog at 15 mg/kg in 10 mL of water. After drug administration, the dog was fasted for food for 4 h and for water for 1 h. The blood samples (2 mL) were collected with disposable EDTA (ethylenediaminetetraacetic acid) anticoagulation syringes at 0, 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 72 h, respectively. After being centrifuged at 4000 r/min for 10 min, the plasma samples were stored at −80 °C in a refrigerator.The plasma sample (0.5 mL) in 1 mL of acetonitrile was vortexed in a 2 mL centrifuge tube for 3 min, centrifuged at 13,000 r/min for 10 min, and the supernatant was collected in a glass tube. The precipitate was extracted with 0.5 mL of acetonitrile two times, and the combined supernatants were centrifuged and dried under nitrogen at 37 °C, 0.5 mL of the mobile phase was added, and the mixture was vortexed for 4 min, filtered, and analyzed by HPLC by using 60:40 of acetonitrile/water with 0.1% acetic acid as the mobile phase with a 1.0 mL/min of flow rate on an Inertsil ODS-3 C18 (250 mm × 4.6 mm) column by UV detector at 290 nm at 30 °C. The recovery rate of griseofulvin in plasma was 92.32 ± 0.43%~108.75 ± 6.12%, the intra-assay coefficient of variation was 1.11~3.73%, and the inter-assay coefficient of variation was 0.07~5.63%.The solutions of griseofulvin in the mobile phase (50 µL) with different concentrations from 0.5, 1, 2.5, 5, 10, 25, 50 to 100 µg/mL were added to 450 µL of the canine blank plasma in a centrifuge tube. After vortexing in a vortexer for 4 min, each sample was analyzed by HPLC, and based on the peak areas and concentrations, the standard curve of griseofulvin in dog plasma was obtained.The solutions of griseofulvin in the mobile phase (50 µL) with the concentrations of 2, 50 and 100 µg/mL were added into 450 µL of canine blank plasma, each mixture was vortexed for 3 min, obtaining the griseofulvin solutions in plasma with low, medium, and high concentrations as 0.2, 1, and 5 µg/mL, respectively, three samples were prepared for each concentration, and each sample was analyzed by HPLC three times to obtain the intra-day coefficient of variation. The experiments were repeated for three days to obtain the inter-day coefficient of variance.Each experiment was carried out in triplicate to obtain the average value, and results are expressed as the mean ± standard deviation. Each point in the pharmacokinetic curve represents the average of six experiments. GraphPad Prism 5.0 software was selected for statistical analysis, including data processing and graphical presentation. Two-tailed t-tests were used to assess the significance of differences in pharmacokinetic parameter values between dogs given pure griseofulvin and dogs given the inclusion complex. An AP value of 0.05 was considered significant for statistical analysis.γ-cyclodextrin was prepared through supercritical carbon dioxide assisted complexation with addition of a tract amount of hydroxypropyl methylcellulose and confirmed by FTIR, NMR, X-ray, and ESM. The ratio of griseofulvin and HP-γ-cyclodextrin in the complex was determined as 1:2 based on the proton NMR data. Through the complexation, the water solubility of griseofulvin was increased 477 times compared with that of griseofulvin alone, which is thus far the best result. The complex showed more than 90% of griseofulvin was released in 10 min, its in vivo pharmacokinetic studies showed significant improvement in pharmacokinetic properties comparing with griseofulvin, the Cmax was increased from 0.52 µg/mL to 0.72 µg/mL, AUC0–12 was increased from 1.55 μgh/mL to 2.75 μgh/mL, the clearance was changed from 51.78 L/kg/h to 24.16 L/kg/h, and the half-life time was changed from 0.81 h to 1.56 h, which indicated this griseofulvin complex can be used as a potential more effective antifungal, antibiotic, or anti-cancer drug than griseofulvin alone.In conclusion, the griseofulvin inclusion complex with HP-


Brucella abortus 2308 (Ba) at 14- and 28-days post-infection. Serum levels of six cytokines  were assessed by FACS, while the number of bacteria in the spleen was measured via CFU. Serotonin levels in the hippocampus were analyzed via HPLC, and behavioral tests were conducted to assess strength, equilibrium, and mood. Our results showed that mice infected with Brucella abortus 2308 and treated with ImiP for six days (Im6Ba14) had significantly different outcomes compared to infected mice (Ba14) at day 14 post-infection. The mood was enhanced in the forced swimming test (FST) (p < 0.01), tail suspension test (TST) (p < 0.0001), and open-field test (p < 0.0001). Additionally, there was an increase in serotonin levels in the hippocampus (p < 0.001). Furthermore, there was an improvement in equilibrium (p < 0.0001) and muscle strength (p < 0.01). Lastly, there was a decrease in IL-6 levels (p < 0.05) and CFU count in the spleen (p < 0.0001). At 28 days, infected mice that received ImiP for 20 days (Im20Ba28) showed preservation of positive effects compared to infected mice (Ba28). These effects include the following: (1) improved FST (p < 0.0001) and TST (p < 0.0001); (2) better equilibrium (p < 0.0001) and muscle strength (p < 0.0001); (3) decreased IL-6 levels (p < 0.05); and (4) reduced CFU count in the spleen (p < 0.0001). These findings suggest the potential for ImiP to be used as an adjuvant treatment for the symptoms of brucellosis, which requires future studies.Brucellosis infection causes non-specific symptoms such as fever, chills, sweating, headaches, myalgia, arthralgia, anorexia, fatigue, and mood disorders. In mouse models, it has been associated with increased levels of IL-6, TNF-α, and IFN-γ, a decrease in serotonin and dopamine levels within the hippocampus, induced loss of muscle strength and equilibrium, and increased anxiety and hopelessness. Imipramine (ImiP), a tricyclic antidepressant, is used to alleviate neuropathic pain. This study evaluated the effects of ImiP on Balb/c mice infected with  Brucella-infected mice using imipramine (ImiP). This is a tricyclic antidepressant with serotonin transporter blocking activity, which allows for the restoration of serotonin levels in the hippocampus and behavioral symptoms with an immunomodulatory effect [Leishmania donovani infection in animal models [B. abortus 2308 infection for the treatment of brucellosis-associated symptoms.For at least a decade, research has suggested that treatment-resistant mood disorders occur secondarily to microbial infections, either through direct neural infection or chronic neuroinflammation as a mediator ,5,6,7,8.y effect ,26. Prevl models ,28,29 anl models ,31,32. Sl models ,31,32 anl models ,35,36,37l models ,35,36,37Brucella abortus 2308 (referred to as Ba14 and Ba28); infection with B. abortus 2308 and treated with imipramine (referred to as Im6Ba14 and Im20Ba28); and a group treated with imipramine only (referred to as ImiP6 and ImiP20) , forelimb grip strength test (FST) , p < 0.0001), tail suspension test (TST) , p < 0.0001), and serotonin levels , p < 0.0001). In FST and TST, Ba14 and Ba28 mice exhibited increased immobility time compared to the corresponding Ctr group throughout the follow-up period (TST 133.70 ± 21.74 vs. 66.90 ± 8.81 p < 0.0001 on day 14 and 155.20 ± 20.71 vs. 65.60 ± 6.80 on day 28 p < 0.0001 and FST 130.90 ± 13.23 vs. 74.40 ± 3.20 on day 14 and 155.00 ± 21.56 vs. 65.33 ± 7.08 p < 0.0001). No differences in immobility time were found when comparing Ctr14 vs. Imip6 and Ctr 28 vs. ImiP20. Taken together, these results suggest that Ba14 and Ba28 mice exhibit a lack of motivation  [p < 0.0001 on day 14 and 9.83 ± 0.90 vs. 12.93 ± 1.58 p < 0.01 on day 28). At the same time, no differences were found when comparing ImiP6 vs. Ctr14 and ImiP20 vs. Ctr28. In contrast to the Ba groups, Im6Ba14 and Im20Ba28 mice significantly improved their FST scores compared to the Ba groups on day 14 (130.90 ± 13.23 vs. 99.40 ± 17.09 p < 0.01) and on day 28 (155.00 ± 21.56 vs. 75.60 ± 20.60 p < 0.0001). A similar phenomenon is observed in the TST, where the ImBa groups improved their scores compared to the Ba groups (67.60 ± 8.43 vs. 133.70 ± 21.74 p < 0.0001 at day 14 and 77.60 ± 20.30 vs. 155.20 ± 20.71 p < 0.0001 at day 28). In the hippocampus, serotonin concentrations were improved at day 14 in Im6Ba14 compared to Ba14 (6.62 ± 0.71 vs. 15.46 ± 3.35 p < 0.001), and although no significant differences in serotonin levels were detected between Im20Ba28 and Ba28, the TST and FST parameters are equivalent to the values presented by the Ctr groups. Thus, our results show that administration of ImiP to Ba-infected mice (ImBa groups) improves hippocampal serotonin levels and depression-like behavior. The OF anxiety test showed that the ImiP groups exhibited increased exploratory behavior at day 14 (60.70 ± 7.30 vs. 43.70 ± 5.39 p < 0.001), although the intensity of the phenomenon decreased by day 28 (56.20 ± 10.88 vs. 40.00 ± 4.87 p < 0.05) compared to Ctr14 and Ctr28, respectively. As previously published, infected Ba mice exhibited decreased exploratory behavior  compared to their Ctr counterparts. Im6Ba14 mice showed exploratory behavior equivalent to that of their Ctr14 at day 14, better than that of Im20Ba28 mice at the end of the follow-up (22.80 ± 7.00 vs. 40.00 ± 4.87 p < 0.0001). Nevertheless, the Im20Ba28 group showed an exploratory behavior like that of Ba28, lower than that of controls (22.80 ± 7.00 vs. 40.00 ± 4.87 p < 0.001) and ImiP 20 (22.80 ± 7.00 vs. 56.20 ± 10.88 p < 0.0001). Thus, the effect of ImiP is likely to improve anxious behavior in infected mice in the short term. The mean ± SD values obtained from the analyses of the mice can be found in the The mean ± standard deviation (SD) of the behavioral test results and hippocampal serotonin levels for each group are shown in ehavior) . Reducedp < 0.0001) and the forelimb grip strength test (FGST) . As previously reported [p < 0.0001) and Ba 28 with Ctr28 (25.90 ± 1.37 vs. 17.30 ± 0.94 p < 0.0001), while the presence of muscle weakness in the FGST was also higher during clinical follow-up when comparing infected mice with control mice  (p < 0.0001) and Im20Ba28 (21.00 ± 1.49 vs. 25.90 ± 1.37 p < 0.0001) mice compared to Ba14 and Ba28 groups, respectively. Similarly, imipramine treatment improved muscle strength in mice in the Im6Ba14 (0.43 ± 0.07 vs. 0.30 ± 0.03 p < 0.01) and Im20Ba28 (0.49 ± 0.03 vs. 0.33 ± 0.04 p < 0.0001) groups compared with Ba14 and Ba28, respectively, so that their performance was equivalent to that of controls and much better than that observed in Ba. Thus, our results suggest that the administration of ImiP to B. abortus 2308-infected mice can help restore balance and muscle strength. The mean ± SD values obtained from the analyses of the mice can be consulted in the Statistical differences were found in the motor balance and control test (MBCT)  . The Imip < 0.0001) was found, and no changes in circulating levels were detected via imipramine administration (Crt vs. ImiP), while Im6Ba20 mice showed significantly lower circulating levels (p < 0.01)—similar to the Im20Ba28 group (p < 0.0001)—compared to Ba mice groups. Im20Ba28 mice showed a significantly greater decrease in serum TNF-α levels than Im6Ba14 mice (p < 0.0001). In both cases, TNF-α levels did not return to baseline levels when compared to the Ctr groups (Im6Ba14 vs. Ctr14 p < 0.0001 and Im20Ba28 vs. Ctr28 p < 0.0001). In the case of IL-6  p < 0.0001), IL-12  p < 0.01), and IFN-γ  p < 0.01), the Ba and ImBa groups were compared to determine whether imipramine treatment modifies cytokine concentrations. No changes were found in the serum concentration of IL-12 and IFN-γ when Ba and ImBa groups were compared; however, a decrease in the concentration of IL-6 was observed when comparing the Im6Ba14 and Ba14 groups (p < 0.0001), while at day 28, IL-6 was ND in the Im20Ba28 group  and were not published. In the same way, we obtained values below the detection limit in the Ctr, ImiP6 and ImiP20 groups for IL-6, IL-12, and IFN-γ, which were also declared as ND; in all cases, these values were not considered in the statistical analysis . A signiroup see .p < 0.0001) and dendritic cells  p < 0.0001) in the spleen of the studied groups. As expected during active infection, an increase in splenic macrophage numbers was found in the Ba14 and Ba28 groups of macrophages compared to Ctr14 and Ctr28  and dendritic cells  (p < 0.0001) and ImiP20 (60.40 ± 20.80 vs. 134.30 ± 24.36 p < 0.0001) groups when compared to the respective control group. The mean ± SD values obtained from the analyses of the mice can be found in the Statistical differences were found in the number of macrophages   day 28) . When co day 28) . It is ip < 0.0001). The Im6Ba14 group had a lower number of B. abortus 2308 CFU in the spleen when compared to the Ba14 group  and Im20Ba28 vs. the Ba28 group , as shown in B. abortus 2308, as in the work of Mukherjee [Brucella [Our results showed statistical differences in the number of CFU in the spleen  Brucella . We are Brucellosis is a debilitating and disabling disease that causes severe chronic pain, anxiety, and depression, affecting the quality of social and work life lives . AntibioBrucella abortus 2308 infection in mice induced a significant increase in circulating levels of IL-6, IL-12, TNF-α, and IFN-γ; an increase in the number of macrophages and dendritic cells in the spleen; a decrease in hippocampal serotonin levels by day 14; and loss of physical strength, balance, and hopelessness, as previously reported by our group [Our results show that ur group . Changesur group . The intur group .Depression may be a chronic outcome of infectious diseases ,5,6,7,8.The mechanisms by which ImiP improves motivation are fairly well understood. It increases extracellular concentrations of neurotransmitters after inhibiting serotonin, norepinephrine, and dopamine reuptake ,44,45,46B. abortus 2308 to consolidate infection in mice. B. abortus 2308 traverses the host cell membrane using the VirB complex and lipid rafts [B. abortus 2308 from entering target cells [Escherichia coli and Yersinia enterocolitica [Leishmania donovani [Our results suggest that ImiP administration in ImBa mice modulates some aspects of the immune response, as described previously . Accordiid rafts . Howeveret cells , making et cells . Macropiet cells , so it ccolitica , antimalcolitica , and in donovani . Howeverdonovani . Our resB. abortus 2308 CFU in the spleen. This fact partially explains the difference in the intensity of the phenomenon. There was a reduction in CFU because IFN-γ levels had decreased in both the ImBa and Ba groups by day 28 of follow-up; however, a greater number of splenic macrophages was observed.Our results show that the mice in the ImBa groups had equivalent IFN-γ levels to those in the infected group during follow-up. This apparent dissonance could be explained by the elevated levels of extracellular serotonin detected at 14 days. These were associated with ImiP administration and could have triggered the release of IFN-γ , whose lOur data showed a significant decrease in IL-6 levels in ImBa mice; a similar fact has been described previously, as ImiP administration reduces the secretion of IL-1β, TNF-α and IL-6 by macrophages . IL-6 haB. abortus 2308 .n = 80; 18–21 g body weight) supplied by Ferandhel, Mexico, were used in the study and housed in groups of five per cage. Upon arrival, the mice were allowed to acclimate for 21 days before the start of the experiments. They were kept in temperature (21 °C)- and light -controlled rooms and housed in cages with microfilters at the animal facility of the Department of Immunology of the Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional. The mice had free access to sterilized water and food  always. All procedures, such as water, food, and bedding changes, were performed in a biosafety cabinet (Nuaire Class II Type A/B3) to avoid contamination with external agents. The study design included eight groups of 10 mice each, as shown in B. abortus 2308 intraperitoneally (IP). In the groups receiving imipramine, imipramine administration started on day 8 after IP administration of B. abortus 2308 or PBS. In all cases, mice were sampled at day 14 or 28 post-IP administration; four groups were followed for 14 days, and the remaining ones were followed for 28 days. Groups were identified as follows: 14- and 28-day controls (Ctr14 and Ctr28); 14- and 28-day B. abortus 2308-infected mice (Ba14 and Ba28); 14- or 28-day B. abortus 2308-infected and 6- or 20-ImiP-treated mice (Im6Ba14 and Im20Ba28); and 6- and 20-day ImiP-treated mice (ImiP6 and ImiP20). On day 0, Ctr14, Ctr28, ImiP6, and ImiP20 received a single intraperitoneal administration of either phosphate buffer saline  or 1 × 106 colony-forming units (CFU) of B. abortus 2308 suspended in 100 μL PBS inside a biosafety cabinet (Nuaire Class II Type A/B3). Behavioral tests (see below) were performed, and blood samples were collected inside a biosafety cabinet on days 14 or 28 after IP infection/inoculation. All mice were then sacrificed by cervical dislocation, and brain samples were dissected (see below). Animal handling and experimental protocols were reviewed and approved by the local ethics committee (Registry No. ENCB/CEI/077/2020).Six- to eight-week-old female BALB/c mice  were ground and dissolved in sterile water to the required concentration of 15 mg/kg/day . ImiP  at room temperature for 10 min. Serum was then collected, aliquoted (100 μL), and stored at −80 °C until use. Mice were then subjected to cervical dislocation before decapitation. The brain was removed from the skull, and the hippocampus was dissected as previously described [On the day of completion of clinical follow-up, blood samples were collected from the facial vein of the mice and centrifuged . The resulting cell suspension was washed once in PBS (3 mL), centrifuged, and resuspended at room temperature. A total of 20 µL of the cell suspension was then taken and sequentially diluted (1:10) in 1M PBS to a final dilution of 10−4. Then, 20 µL samples of each dilution were plated in duplicate on tryptic soy agar (TSA) for 48 h at 37 °C. The number of Brucella CFU was estimated by multiplying the number of colonies by the inverse dilution factor.On the day of completion of clinical follow-up, the spleen was mechanically removed, minced in a Petri dish filled with 5 mL PBS (1M), and passed through a cell strainer , negative lineage  , PE-CF594 anti-mouse CD11c, and V500 anti-mouse CD11b . Cells were washed with PBS and analyzed on a FACS-Aria III cytometer  on the same day of collection for a total of one million events. Autofluorescence and compensation controls were used for each fluorochrome. The analysis strategy was as follows: detritus and double events were eliminated by comparing area to size forward scatter; then, granulocytic cells were selected in a plot of side scatter versus forward scatter. From this region, CD11c+, I-A/I-E+ double-positive cells were selected. Macrophages were CD11b-positive and dendritic cells were Lin-, MHC-II+, and CD 11c+ (see Ammonium chloride potassium (ACK) buffer was used to eliminate erythrocytes from the spleen cell suspension. The cell suspension was then adjusted to 1 × 1011c+ see .Serum samples were used to estimate IL-6, IL-12, TNF-α, IFN-γ, MCP-1, and IL-10 concentrations using a mouse inflammation kit , according to the manufacturer’s instructions. Samples were analyzed on a FACSARIA III flow cytometer . The manufacturer’s limits of detection for cytokines were as follows: 5 pg/mL for IL-6; 17.5 pg/mL for IL-10; 52 pg/mL for MCP-1; 2.5 pg/mL for IFN-γ; 10.7 pg/mL for IL-12; and 7.3 for TNF-α.g at 4 °C for 10 min, and the supernatants were collected, aliquoted (200 μL), and stored at −80 °C until used.The hippocampus was homogenized via sonication (≈5 mg tissue) in 200 μL serotonin (5HT) extraction buffer containing 5% citric acid, 200 mM sodium phosphate, 2.5 mM L-cysteine, and 2.5 mM EDTA. Then, 50 μL of 0.4 M perchloric acid was added to precipitate proteins after incubating the homogenate at 20 °C for 20 min. Finally, homogenates were centrifuged at 12,000× 18, 1 mL, Cat. 702001; J.T. Baker). Briefly, we used 0.1% trifluoracetic acid (TFA) in water and 0.1% TFA in acetonitrile as mobile phase A (MP-A) and B (MP-B), respectively. The column was then loaded with 250 μL supernatant, followed by a wash with 250 μL MP-A. Finally, 250 μL MP-B was used to elute serotonin.Serotonin concentration was enriched using a solid phase extraction (SPE) column  was equilibrated with 100% MP-A; then, 90 μL of each sample was injected in duplicate. All runs were performed with at a column temperature of 30 °C. Chromatographic runs were performed in a linear gradient as follows: 0–3 min 100% MP-A; 3–33 min 0–10% MP-Bl; 33–38 min 10–20% MP-B; and 38–39 min 20–30% MP-B. Then, 30% MP-B was maintained until 43 min to clean the column, and at 43–45 min, the column was switched back to 100% MP-A. Finally, 100% MP-A was maintained until 60 min to equilibrate the column for the next injection. The serotonin peak was detected at min 26 with the following detector parameters: gain, 100; attenuation, 32; response, 20 s; excitation, 280 nm; and emission, 315 nm.Serotonin chromatograms were obtained via reversed-phase HPLC (RP-HPLC) on a JASCO HPLC system . The system was controlled by ChromNav . The column . Datasets were subjected to the D’Agostino and Pearson omnibus normality test. In all cases, if the distribution was normal, Welch’s ANOVA test was used, and the value of W was described in the text. When the distribution was non-Gaussian, the Brown–Forsythe ANOVA test was used, and the F* value was described in the text. In both cases, the Dunnett T3 post hoc test was used. In Orogastric administration of imipramine reduced serum IL-6 levels and bacterial counts in the spleen, restored serotonin levels in the hippocampus to levels equivalent to controls, increased motivation scores, and improved physical fitness in the subjects. The results suggest that treatment with imipramine for 6 days is more effective. Thus, imipramine may be a pharmacological agent worth considering as an addition to the pharmacological options for the treatment of brucellosis as it enhances the immune response and psychoimmune resilience at a safe dose.


Intracerebral gene delivery (GD) provided a significant improvement among AADCD patients .Aromatic We describe the clinical, biological, and imaging evolution of two AADCD patients ages >10 years after GD.Eladocagene exuparvovec, a recombinant adeno‐associated virus containing the human complimentary DNA encoding the AADC enzyme, was administered into bilateral putamen by stereotactic surgery.l‐6‐[18F] fluoro‐3, 4‐dihydroxyphenylalanine uptake was increased at 1 month, persisting at 1 year compared to baseline.Eighteen months after GD, patients showed improvement in motor, cognitive and behavioral function, and in quality of life. Cerebral Two patients with a severe form of AADCD had an objective motor and non‐motor benefit from eladocagene exuparvovec injection even when treated after the age of 10 years, as in the seminal study. Eladocagene exuparvovec was available under a temporary authorization for compassionate use, according to the article L.5121‐12 of the Public Health Code, in Montpellier University Hospital. Baseline and post‐GD assessment schedule of the patients and neurosurgical procedure according to the fabricant recommendations have been validated by the French Drug Agency as daily care including expenses support.11 viral genomes delivered as four intraputaminal injections of 80 μL conducted at a rate of 3 μL/min. Protocols for surgery, pharmacy, storage, and shipment of eladocagene exuparvovec have been previously described.Each patient received a total of 1.8 × 10l‐6‐[18F] fluoro‐3, 4‐dihydroxyphenylalanine (18F‐DOPA) positron emission tomography (PET) were performed before GD and 1 month and 1 year after GD. Cerebrospinal fluid analysis  was performed before GD and 18 months after GD. Anti–adeno‐associated virus type 2 titers were monitored at baseline and at 3, 6, 12, and 18 months after GD.Clinical data were collected in the medical file and at each follow‐up visit, specifically to motor function , cognitive function , sleep and mood disturbances, irritability, comfort, wellbeing, and ease of caregiving according to Caregiver Priorities and Child Health Index of Life with Disabilities (CPCHILD) questionnaire, gastrointestinal and feeding difficulties, weight, respiratory infections, pharmacological changes, dyskinesia, and oculogyric crisis (Pearson's score).11 viral genomes delivered as four intraputaminal injections) at ages 10.7 years and 11.4 years, respectively. Past medical history is summarized in Table Patient 1 and 2 benefited from GD  resulted in increased in body weight of both patients; respiratory infections in patient 2 significantly improved Table .No serious adverse events were reported during the surgical procedure or follow‐up. Patient 2, with a known, well‐controlled epilepsy, exhibited a cluster of brief focal seizure during a non‐infectious febrile peak 36 h after GD. Moderate and transient dyskinesia occurred in both patients from 4 weeks following GD; they were controlled with low doses of tetrabenazine in patient 1 and by tapering down dopaminergic drugs in patient 2 , but not of the serotonin metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) level in patient 2 at last follow‐up compared to baseline. Dopamine levels remained stable in patient 1  and disappearance of OCG and dystonia following intraputaminal administration of rAAV‐hAADC gene therapy.We report the first European compassionate experience of intraputaminal delivery of eladocagene exuparvovec in two children >10 years old with a severe form of AADCD. This well‐tolerated treatment resulted in consistent improvement of motor and non‐motor function. Follow‐up was characterized by early improvement in sleep and mood disturbances, alertness, and communication skills, especially expression abilities. Both patients managed to hold up their head before 8 months. Spontaneous movements were dramatically enriched, allowing the patients to reach/grab objects; increased Gross Motor Function Measure‐88 Item score highlight motor outcome. This progress, as well as resolution of OGC, and ability to feed orally resulted in remarkable improvement of quality of life. Previous studies showed faster and greater progress in young patients, especially regarding gross motor progress and fine motor skills (ability to reach/grab or eat with hands),Side effects were limited, dyskinesia occurred as reported in other studies;18F‐DOPA is rapidly washed out from putaminal neurons if not metabolized into dopamine, evidence of recovery of a certain level of cerebral AADC activity is provided by recovery of putaminal uptake of 18F‐DOPA for both patients 1 year after GD, albeit a partial decrease in uptake compared with uptake 1 month after GD for patient 2. These results are similar to those of Hwu et al,18F‐DOPA PET in providing indication of the biological effectiveness of GD.Because DDC gene delivery have not been assessed in animal models, therefore, scientific experimental data concerning CSF dopamine metabolites as reliable biomarkers of AADC activity restauration after gene delivery are lacking. The value of CSF biomarkers as a relevant clue for AADC activity restauration and/or prognosis indicator of good motor outcome will need to be further studied with standardized timepoints of assessement.Restoration of AADC activity is also supported by increased CSF levels of HVA in patient 2. Patient 1's HVA CSF dosage was not increased from baseline to last follow‐up. Although a strong correlation has been reported between the post‐treatment HVA level and motor outcome,As in previous series of AADCD patients who received gene delivery, clinical assessment of the two patients was unblinded, and part of evaluation was based on caregivers rating ;Robust evidence corroborates the absence of the degenerative process in AADC.(1) Research project/study: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the First Draft, B. Review and Critique.S.S.: 1C, 3B.P.C.: 1C, 3B.S.S.: 1C, 3B.I.R.T.: 1C, 3B.C.M.: 1C, 3B.P.M.: 1C, 3B.A.L.: 1C, 3B.D.G.: 1C, 3B.G.C.: 1C, 3B.D.M.G.: 1B, 1C, 3B.D.P.: 1C, 3B.J.B.: 1C, 3B.M.A.N.M.: 1C, 3B.A.Q.: 1C, 3B.G.B.: 1C, 3B.C.S.: 1C, 3B.B.D.: 1C, 3B.E.D.: 1C, 3B.M.G.: 1C, 3B.S.T.: 1C, 3B.A.Z.: 1C, 3B.N.L.: 1C, 3B.V.K.: 1C, 3B.E.R.: 1B, 1C, 3B.M.C.F.H.: 1C, 3A.G.P.; 1C, 3A.T.R.: 1A, AB, 1C, 3B.A.R.: 1A, AB, 1C, 3B.Ethical Compliance Statement: The authors confirm that the approval of an institutional review board was not required for this work. All legal representative of the patients provided written and oral informed consent according to the Declaration of Helsinki to perform the procedure, collect and share the data including videos. All authors confirm that they have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.Funding Sources and Conflicts of Interest: The authors have no funding source to declare and no conflicts of interest to declare.Financial Disclosures for the Previous 12 Months: E.R. received honorarium for speech from Orkyn, Aguettant, Elivie and for participating in an advisory board from Merz‐Pharma. He received research support from Merz‐Pharma, Orkyn, Aguettant, Elivie, Ipsen, Everpharma, Fondation Desmarest, AMADYS, Fonds de dotation Patrick Brou de Laurière, Agence Nationale de la Recherche, Société Française de Médecine Esthétique, Dystonia Medical Reasearch Foundation. T.R. received honorarium for advisory board from PTC Therapeutics. A.R. received honorarium for advisory board from PTC Therapeutics. The authors declare that there are no additional disclosures to report.Table S1. Characteristics and past medical history of patients.Table S2. Quantification of the specific putaminal uptake at baseline, 1 month, and 1 year after GD.Click here for additional data file.