Daphnoretin (DAP) has various pharmacological activities, but its in vivo disposition after nanomicellar formulation remains unclear. This study compared the pharmacokinetics and tissue distribution of free DAP and two polymeric nanomicellar formulations, PP-DAP and GA-DAP, following intravenous administration. Plasma DAP concentrations in rats were determined by UPLC-MS/MS, and DAP concentrations in mouse tissues were determined by LC-MS/MS. Compared with free DAP, PP-DAP and GA-DAP showed higher systemic exposure, longer apparent elimination half-lives, and lower apparent clearance. The AUC0-∞ values of DAP, PP-DAP, and GA-DAP were 5474.14, 11,211.04, and 15,019.86 h · ng/mL, respectively, and the corresponding T1/2 values were 6.84, 9.24, and 9.90 h. DAP was detected in the heart, liver, spleen, lung, and kidney, with the nanomicellar formulations showing altered tissue distribution and GA-DAP exhibiting relatively sustained hepatic distribution. These findings suggest that nanomicellar formulation alters the in vivo disposition of DAP. However, free and micelle-associated DAP were not separately quantified, precluding direct characterization of in vivo drug release, and the validation range of the tissue quantification method was limited. Further studies are warranted to evaluate the in vivo behavior and liver-directed delivery potential of GA-DAP.

Background: Radix Wikstroemia indica (RWI) is one of the most commonly used drugs in Miao medicine. However, RWI is characterized by high toxicity. Objectives: In this study, we aimed to observe the effect of the “sweat soaking method” processed on reducing the hepatotoxicity of RWI and on cytochrome P450 (CYP) protein expression. Materials and Methods: The study focused on investigating the impact of both RWI raw products and RWI processed products on the proliferation of L-02 cells. To assess this, a cell counting kit-8 was employed. After the administration of RWI through oral gavage for a duration of 15 days in rats, the corresponding kits were utilized to determine the serum levels of the liver index factor. Furthermore, both hematoxylin and eosin (H&E) staining and western blot analysis were conducted to analyze the liver tissues. Results: Both the raw RWI and the processed RWI at high doses inhibited the proliferation of L-02 cells, but the cell viability of the concoction group was higher than that of the raw group. The liver tissues of rats in the control and processed product groups were normal, while those in the raw product groups showed different degrees of liver damage. Additionally, compared with the control group, the activity of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine transaminase (ALT) in the raw product groups was notably increased in a dose-dependent manner; the three enzyme activities of the processed group were lower than those of the raw product group. In addition, the expression levels of CYP3A4, 2C19, 2C9, 1A2, and 2E1 in the liver tissue of rats in the processed group were lower than those in the raw group, while the protein expression trend of CYP2D6 was irregular. Conclusion: The detoxication mechanism of RWI, after undergoing processing, appears to be associated with reduced protein expression of CYP1A2, CYP2E1, CYP2C9, CYP2C19, and CYP3A4, ultimately leading to decreased liver injury.

Ethnopharmacological relevance: To explore the differences in the anti-inflammatory efficacy and mechanisms of the Miao medicine, both raw and after processing, using the "sweat soaking method" of Radix Wikstroemia indica (RWI). Aim of the study: The purpose of this study was to explore the differences in the anti-inflammatory efficacy and mechanism of action before and after the processing of the Miao medicine (RWI) using the "sweat soaking method." Materials and methods: Network pharmacology technology was used to construct the "drug-component targetpathway-disease" network, and the main anti-inflammatory pathways of RWI were identified. Rat models of collagen-induced arthritis were established. The changes in body weight, swelling rate of the foot pad and ankle joint, arthritis index, thymus index, spleen index, pathological changes of the ankle joint, and the content of inflammatory cytokines (IL-1(i, IL-2, IL-6, IL-10, TNF-alpha, and NO) were used as indices to evaluate the effect of RWI on rats with collagen-induced arthritis before and after its processing. Plasma and urine samples were collected from the rats, and the potential biomarkers of, and metabolic pathways underlying the antiinflammatory effects of RWI before and after processing were identified using 1H-Nuclear magnetic resonance metabolomics combined with a multivariate statistical analysis. Results: Eleven key anti-inflammatory targets of IL6, IL-1(i, TNF, ALB, AKT1, IFNG, INS, STAT3, EGFR, TP53, and SRC were identified by network pharmacology. The PI3K-Akt signaling pathway, steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, tryptophan metabolism, and other pathways were mainly involved in these effects. Pharmacodynamic studies found that both raw and processed RWI products downregulated inflammatory factors in rats with collagen-induced arthritis and alleviated the pathological changes. A total of 41 potential pathways for the anti-inflammatory effects of raw RWI products and 36 potential pathways for the anti-inflammatory effects of processed RWI products were identified by plasma and urine metabolomics. The common pathways of network pharmacology and metabolomics were steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. Conclusions: The anti-inflammatory effect of RWI was mainly related to the regulation of steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. Finally, the "sweat soaking method" enhanced the anti-inflammatory effect of RWI.

Background Radix Wikstroemia indica (RWI) is one of the most commonly used drugs in Miao medicine. However, RWI is characterized by high toxicity. Objectives In this study, we aimed to observe the effect of the “sweat soaking method” processed on reducing the hepatotoxicity of RWI and on cytochrome P450 (CYP) protein expression. Materials and Methods The study focused on investigating the impact of both RWI raw products and RWI processed products on the proliferation of L-02 cells. To assess this, a cell counting kit-8 was employed. After the administration of RWI through oral gavage for a duration of 15 days in rats, the corresponding kits were utilized to determine the serum levels of the liver index factor. Furthermore, both hematoxylin and eosin (H&E) staining and western blot analysis were conducted to analyze the liver tissues. Results Both the raw RWI and the processed RWI at high doses inhibited the proliferation of L-02 cells, but the cell viability of the concoction group was higher than that of the raw group. The liver tissues of rats in the control and processed product groups were normal, while those in the raw product groups showed different degrees of liver damage. Additionally, compared with the control group, the activity of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine transaminase (ALT) in the raw product groups was notably increased in a dose-dependent manner; the three enzyme activities of the processed group were lower than those of the raw product group. In addition, the expression levels of CYP3A4, 2C19, 2C9, 1A2, and 2E1 in the liver tissue of rats in the processed group were lower than those in the raw group, while the protein expression trend of CYP2D6 was irregular. Conclusion The detoxication mechanism of RWI, after undergoing processing, appears to be associated with reduced protein expression of CYP1A2, CYP2E1, CYP2C9, CYP2C19, and CYP3A4, ultimately leading to decreased liver injury. Keywords Radix , Wikstroemia indica , sweat soaking method , cytochrome P450 , liver toxicity , processing mechanism

Background: In the research of the pharmacological activity of modern Chinese medicine, a large number of traditional Chinese medicines show anti-inflammatory activity. Asparagi radix is one of the anti-inflammatory drugs. With the deepening of people's research on Asparagi radix, its anti-inflammatory pharmacological effects have been gradually explored, but its specific anti-inflammatory active ingredients are still unclear. Therefore, it is necessary to screen and identify its specific anti-inflammatory active ingredients from Asparagi radix. At present, COX-2 is an important target for the treatment of chronic inflammation, and many drugs targeting this target have been marketed. However, some drugs have serious side effects, so it is very meaningful to study new COX-2 inhibitors. Methods: In this experiment, the fractions of different time periods are separated by high performance liquid chromatography-separation-trapping system (HPLC-CAD-FC). Then, ultra-high performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) and biological activity analysis techniques are used to identify the chemical composition and in vitro anti-inflammatory activity of each fraction. Finally, the data is integrated to screen out the substances with potential anti-inflammatory activity in Asparagi radix. Results: The results showed that the method established in this experiment can successfully isolate the active components of Asparagi radix, and four saponins with potential anti-inflammatory activity are identified. Conclusion: In summary, the analysis method established in this study can quickly and effectively screen out its anti-inflammatory active substances, and its potential anti-inflammatory active ingredients are mainly steroidal saponins.

Background: RWI has an analgesic effect and is related to metabolites such as 4-pyridoxic acid, l-glutamic acid, and agmatine. It is involved in arginine and proline metabolism, arginine biosynthesis, and alanine, aspartate, and glutamate metabolism. In network pharmacology, there were 404 common targets between RWI and pain diseases, and eight core targets were screened, including SRC, STAT3, and HSP90AA1. GO functional enrichment analysis found that RWI had effects on molecular processes such as protein phosphorylation and response to xenobiotic stimulus, cell composition such as receptor complex and membrane raft, and molecular functions such as enzyme binding. KEGG pathway enrichment analysis obtained 193 pathways. Arginine proline metabolism and nitrogen metabolism are involved in the same pathway as metabolomic analysis.Purpose: To explore the analgesic effect and therapeutic mechanism of RWI processed by "Sweat soaking method".Materials and methods: The torsion experiment was carried out with acetic acid. The metabolomic analysis of serum samples was carried out based on 1H-NMR technology, and the intersection targets of RWI and pain diseases were screened by network pharmacology for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.Results: RWI has an analgesic effect and is related to metabolites such as 4-pyridoxic acid, l-glutamic acid, and agmatine. It is involved in arginine and proline metabolism, arginine biosynthesis, and alanine, aspartate, and glutamate metabolism. In network pharmacology, there were 404 common targets between RWI and pain diseases, and eight core targets were screened, including SRC, STAT3, and HSP90AA1. GO functional enrichment analysis found that RWI had effects on molecular processes such as protein phosphorylation and response to xenobiotic stimulus, cell composition such as receptor complex and membrane raft, and molecular functions such as enzyme binding. KEGG pathway enrichment analysis obtained 193 pathways. Arginine proline metabolism and nitrogen metabolism are involved in the same pathway as metabolomic analysis.Conclusion: RWI has an analgesic effect, and its therapeutic mechanism mainly involves arginine and proline metabolism.

Background Asparagi Radix (AR) is one of the widely used Traditional Chinese Medicines (TCMs) for clinical applications, owning the effects of clearing the lungs and promoting body fluid, nourishing yin, and moistening the lung. To reduce the loss of active ingredients and improve production efficiency, the integrated processing technology of primary processing was used to obtain the decoction pieces of AR. However, there are no specific processing methods and index ingredients of AR in Chinese Pharmacopoeia (2020 edition). Materials and Methods This study aimed to establish a method of content determination of protodioscin and asparagine by high-performance liquid chromatography with charged aerosol detector (HPLC-CAD) as the index of process optimization. Furthermore, Box-Behnken design was used to optimize the integrated processing technology of primary processing. Results The result showed the contents of protodioscin and asparagine could reach 0.2678% and 0.4114%, respectively, both above the traditional process. After verification, the actual value (99.56) and predicted value (101.15) were similar, indicating that the integrated technology was feasible. In particular, the optimized process parameters of boiling time, drying time, and temperature were 25 min, 12 h, and 60°C, respectively. Conclusion In summary, these research findings may provide a reference for the primary processing and quality control of AR.

目的:研究SD大鼠灌胃给予紫云英苷后,各主要组织中紫云英苷的浓度和分布情况,探讨各组织的分布特征.方法:大鼠灌胃给予紫云英苷后分别于0.5 h、1 h、6 h时间点取心、肝、脾、肺、肾、胃、血浆,以槲皮素-3-0-葡萄糖酸苷为内标,采用UPLC-MS/MS方法测定各组织及血浆中紫云英苷含量.结果:建立的UPLC-MS/MS方法中,紫云英苷在0.1～100 ng/mL浓度范围内线性关系良好,定量下限为0.1 ng/mL,其专属性、基质效应、回收率、精密度与准确度以及稳定性等方法学考察均满足要求.大鼠灌胃给药紫云英苷后,在血浆、胃、心组织中可以广泛分布,给药0.5 h后,血液里面的紫云英苷浓度最高,灌胃1 h及6 h之后,紫云英苷浓度最高的是胃,血浆及心脏次之.结论:大鼠各组织样品中内源性物质对紫云英苷含量测定无干扰,方法学考察符合要求.实验结果表明各脏器都能吸收紫云英苷,而主要吸收紫云英苷的器官是胃.

Background: Radix Wikstroemia indica is a traditional Chinese medicine (TCM) used as anti-inflammatory and anti-tumor drug. However, it has serious hepatotoxicity, "Sweat soaking method" processed could effectively decrease its hepatotoxicity. Objective: The objective of this study is to study the effects of Radix Wikstroemia indica on six kinds of cytochrome P450(CYP450) isozymes of rat liver microsomes before and after processing, and to study the mechanism of Radix Wikstroemia indica processed by the "Sweat soaking method" to reduce liver toxicity in rats. Methods: In this study, the effects of Radix Wikstroemia indica and processed Radix Wikstroemia indica on the six main CYP450 isoforms (2E1, 1A2, 2C6, 2D1, 2C11, and 3A1) were investigated in vitro. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we carried out in vitro enzymatic studies in liver microsomal incubation systems via UPLC-MS/MS. Results: The results showed that the established UPLC-MS/MS method was precise and reliable. Compared with the blank group, the activities of six enzymes in the RWI and PRWI groups were higher than those in the blank group. At the same dose, the enzyme activities of CYP2E1, CYP1A2, CYP2C6, CYP2C11, and CYP3A1 increased with the increase in dose, and the enzyme activities of the RWI group were higher than those of the PRWI group. The enzyme activities of CYP2E1 and CYP1A2 in the Radix Wikstroemia indica group were significantly increased compared with the blank group, CYP3A1 in the RWI high-dose group was higher than that in the blank group and PRWI group with statistical differences (p<0.05 or p<0.01). Conclusion: The processed Radix Wikstroemia indica could reduce liver injury, and its detoxication mechanism might be related to the decrease in enzyme activity of CYP1A2, CYP2E1 and CYP3A1.

Background Asparagi Radix (AR) is one of the widely used Traditional Chinese Medicines (TCMs) for clinical applications, owning the effects of clearing the lungs and promoting body fluid, nourishing yin, and moistening the lung. To reduce the loss of active ingredients and improve production efficiency, the integrated processing technology of primary processing was used to obtain the decoction pieces of AR. However, there are no specific processing methods and index ingredients of AR in Chinese Pharmacopoeia (2020 edition). Materials and Methods This study aimed to establish a method of content determination of protodioscin and asparagine by high-performance liquid chromatography with charged aerosol detector (HPLC-CAD) as the index of process optimization. Furthermore, Box-Behnken design was used to optimize the integrated processing technology of primary processing. Results The result showed the contents of protodioscin and asparagine could reach 0.2678% and 0.4114%, respectively, both above the traditional process. After verification, the actual value (99.56) and predicted value (101.15) were similar, indicating that the integrated technology was feasible. In particular, the optimized process parameters of boiling time, drying time, and temperature were 25 min, 12 h, and 60°C, respectively. Conclusion In summary, these research findings may provide a reference for the primary processing and quality control of AR. Keywords , , Box-Behnken Design , HPLC-CAD , integrated processing technology of primary processing , response surface methodology

Background: Daphnoretin, as a known bicoumarin compound that contained various pharmacological activities, was isolated from Wikstroemia indica C.A. Mey (RWI). Objective: The study aims to investigate the pharmacokinetic characteristics of daphnoretin from RWI ethanol extracts in rat plasma and to determine daphnetin in rat plasma and various tissues by a rapid, reliable and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method. Methods: The UPLC-MS/MS method was established. Daphnoretin and IS (buspirone) were chromatographed on an agilent Zorbax XDB-C18 column (2.1 mm × 50 mm, 3.5 μm), and Gradient elution of acetonitrile-0.15% formic acid in aqueous solution. Quantification was performed using electrospray ionization in positive ion multiple reaction monitoring mode of the transitions m/z 353.1→179.1 for daphnoretin and m/z 386.3→122.3 for IS. Results: Good linearity between 5-10000 ng/mL for cyperidin in plasma and tissue samples (r ≥ 0.99) was resulted. The accuracies of plasma and tissue homogenates ranged from-3.31% to 9.00%, and the precision was less than 5.78%. After that, the validated method was successfully applied to the pharmacokinetics and tissue distribution study of daphnoretin after oral administration of ethanol extract from the roots of RWI to rats. Conclusion: Daphnoretin was well absorbed in the systemic circulation after oral administration and was widely distributed in tissues, with the highest concentration in lung tissue. This study is beneficial to the development and utilization of RWI and provides a reasonable reference for its clinical administration.

Based on metabolomics, to study the mechanism of Radix Wikstroemia indica (RWI) "Sweat soaking method" processing detoxification. The raw drug group and processed products was given raw RWI and processed RWI respectively by gavage. The control group was given the same amount of 1% sodium carboxy methyl cellulose distilled water by gavage. After 7 days of continuous gavage, blood samples were collected. The blood samples of rats in each group were analyzed by 1H-NMR technology to explore the changes of endogenous metabolism and the possible metabolic pathways to rats before and after processing. Compared with the control group, the raw RWI could significantly reduce 16 metabolites and increase 10 metabolites. The processed RWI can increase the levels of most metabolites that decrease to the raw RWI, such as 13 metabolites such as alanine, L-glutamine, L-valine, L-serine, betaine and glutamic acid; At the same time, the metabolites that increased in the level of crude products were down-regulated, such as asparagine, lactic acid, 2hydroxyisobutyric acid, sucrose, glucose and D-glucose. Compared with raw products, RWI treated with "Sweat soaking method" can reversely regulate or reduce amino acid, choline metabolism, energy and carbohydrate metabolism, thereby reducing hepatotoxicity and nephrotoxicity.

目的 研究苗药了哥王提取物对斑马鱼的毒性作用.方法 以斑马鱼胚胎模型为对象,经10、20、40μg/mL了哥王提取物暴露处理后,检测并记录1 min内自主抽动次数、10 s内心率及其畸形、死亡发生情况;以斑马鱼模型为对象,经10～100μg/mL了哥王提取物暴露处理24、48、72 h后,计算各时间点了哥王提取物对斑马鱼的半数致死浓度(LC50);经低、中、高质量浓度(27、37、51μg/mL)了哥王提取物暴露处理后,观察斑马鱼肝脏表型和细胞凋亡、脂质沉积情况,检测其肝组织中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)活性.结果 与空白组比较,了哥王提取物10、20、40μg/mL组斑马鱼胚胎的自主抽动次数、畸形率(10μg/mL组除外)、死亡率均显著升高,胚胎心率(10、20μg/mL组除外)均显著降低(P<0.05);了哥王提取物干预24、48、72 h对斑马鱼的LC50分别为39.850、28.300、21.490μg/mL;了哥王提取物低、中、高质量浓度组斑马鱼的肝脏区域透明度降低、形态膨大,细胞凋亡和脂质沉积均有所增加,肝组织中ALT、AST、LDH活性均较空白组显著升高(P<0.05).结论 了哥王提取物对斑马鱼胚胎具有发育毒性,对斑马鱼具有肝损伤毒性.