Uric acid-lowering effect of harpagoside and its protective effect against hyperuricemia-induced renal injury in mice.

Hyperuricemia (HUA) is caused by increased synthesis and/or insufficient excretion of uric acid (UA). Long-lasting HUA may lead to a number of diseases including gout and kidney injury. Harpagoside (Harp) is a bioactive compound with potent anti-inflammatory activity from the roots of Scrophularia ningpoensis. Nevertheless, its potential effect on HUA was not reported. The anti-HUA and nephroprotective effects of Harp on HUA mice were assessed by biochemical and histological analysis. The proteins responsible for UA production and transportation were investigated to figure out its anti-HUA mechanism, while proteins related to NF-κB/NLRP3 pathway were evaluated to reveal its nephroprotective mechanism. The safety was evaluated by testing its effect on body weight and organ coefficients. The results showed that Harp significantly reduced the SUA level and protected the kidney against HUA-induced injury but had no negative effect on safety. Mechanistically, Harp significantly reduced UA production by acting as inhibitors of xanthine oxidase (XOD) and adenosine deaminase (ADA) and decreased UA excretion by acting as activators of ABCG2, OAT1 and inhibitors of GLUT9 and URAT1. Moreover, Harp markedly reduced infiltration of inflammatory cells and down-regulated expressions of TNF-α, NF-κB, NLRP3 and IL-1ß in the kidney. Harp was a promising anti-HUA agent.

Phomopsterone B Alleviates Liver Fibrosis through mTOR-Mediated Autophagy and Apoptosis Pathway.

Liver fibrosis is the initial pathological process of many chronic liver diseases. Targeting hepatic stellate cell (HSC) activation is an available strategy for the therapy of liver fibrosis. We aimed to explore the anti-liver fibrosis activity and potential mechanism of phomopsterone B (PB) in human HSCs. The results showed that PB effectively attenuated the proliferation of TGF-ß1-stimulated LX-2 cells in a concentration-dependent manner at doses of 1, 2, and 4 µM. Quantitative real-time PCR and Western blot assays displayed that PB significantly reduced the expression levels of α-SMA and collagen I/III. AO/EB and Hoechst33342 staining and flow cytometry assays exhibited that PB promoted the cells' apoptosis. Meanwhile, PB diminished the number of autophagic vesicles and vacuolated structures, and the LC3B fluorescent spots indicated that PB could effectively inhibit the accretion of autophagosomes in LX-2 cells. Moreover, rapamycin and MHY1485 were utilized to further investigate the effect of mTOR in autophagy and apoptosis. The results demonstrated that PB regulated autophagy and apoptosis via the mTOR-dependent pathway in LX-2 cells. In summary, this is the first evidence that PB effectively alleviates liver fibrosis in TGF-ß1-stimulated LX-2 cells, and PB may be a promising candidate for the prevention of liver fibrosis.

Design, synthesis and bioevaluation of 1,2,4-thiadiazolidine-3,5-dione derivatives as potential GSK-3ß inhibitors for the treatment of Alzheimer's disease.

Tideglusib is a non-competitive GSK-3ß inhibitor which contain 1,2,4-thiadiazolidine-3,5-dione moiety, and now mainly used for progressive supranuclear palsy due to the lack of some primary cognitive endpoints and secondary endpoints in a phase IIb trail for Alzheimer's disease. Additionally, insufficient evidence exists to support that there are obvious covalent bonds between Tideglusib and GSK-3ß. Targeted covalent inhibition strategy could improve the binding efficiency, selectivity and duration of kinase inhibitors. Based on the above premise, two series of targeted compounds with acryloyl warheads were designed and synthesized. The kinase inhibitory activity of the selected compound 10a with better neuroprotective effect improved 2.7 fold than that of Tideglusib. After the preliminary screening of GSK-3ß inhibition and neuroprotective activity, the mechanism action of the selected compound 10a was investigated in vitro and in vivo. The results confirmed that 10a with excellent selectivity among the whole tested kinases could significantly reduce the expressions of APP and p-Tau via increasing the level of p-GSK-3ß. The pharmacodynamic assay in vivo showed that 10a could markedly improve the learning and memory functions in AD mice induced by AlCl3 combined with d-galactose. At the same time, the damage of hippocampal neurons in AD mice was obviously reduced. Accordingly, the introduction of acryloyl warheads could increase the GSK-3ß inhibitory activity of 1,2,4-thiadiazolidine-3,5-dione derivatives, and the selected compound 10a deserves further research as an effective GSK-3ß inhibitor for the potential treatment of AD.

Three New Selaginellin Derivatives from Selaginella pulvinata and Their α-Glucosidase Inhibitory Activity.

Three new selaginellin derivatives, selaginpulvilins V-X (1-3), together with seven known analogs (4-10) were isolated from whole plants of Selaginella pulvinata. Their structures were determined by extensive spectroscopic methods including 1D and 2D NMR, HR-ESI-MS and chemical derivatization method. Compound 1 represents a rare example of naturally occurring selaginellin with an alkynylphenol-trimmed skeleton. Biological evaluation showed that compounds 2, 6 and 8 displayed moderate inhibition against α-glucosidase with IC50 values of 3.71, 2.04 and 4.00 µM, respectively.

The Discovery of Indole-2-carboxylic Acid Derivatives as Novel HIV-1 Integrase Strand Transfer Inhibitors.

As an important antiviral target, HIV-1 integrase plays a key role in the viral life cycle, and five integrase strand transfer inhibitors (INSTIs) have been approved for the treatment of HIV-1 infections so far. However, similar to other clinically used antiviral drugs, resistance-causing mutations have appeared, which have impaired the efficacy of INSTIs. In the current study, to identify novel integrase inhibitors, a set of molecular docking-based virtual screenings were performed, and indole-2-carboxylic acid was developed as a potent INSTI scaffold. Indole-2-carboxylic acid derivative 3 was proved to effectively inhibit the strand transfer of HIV-1 integrase, and binding conformation analysis showed that the indole core and C2 carboxyl group obviously chelated the two Mg2+ ions within the active site of integrase. Further structural optimizations on compound 3 provided the derivative 20a, which markedly increased the integrase inhibitory effect, with an IC50 value of 0.13 µM. Binding mode analysis revealed that the introduction of a long branch on C3 of the indole core improved the interaction with the hydrophobic cavity near the active site of integrase, indicating that indole-2-carboxylic acid is a promising scaffold for the development of integrase inhibitors.

Discovery of 2-(isoxazol-5-yl)phenyl 3,4-dihydroxybenzoate as a potential inhibitor for the Wnt/ß-catenin pathway.

Carnosic acid could disrupt the ß-catenin/BCL9 protein-protein interaction and inhibit ß-catenin dependent transcription, thereby reduce the incidence of colorectal cancer induced by abnormal activation of Wnt/ß-catenin signalling pathway. However, its activity was weak (IC50 for SW480: 28.2 ± 2.05 µM) and total synthesis was difficult. During the structural simplification of natural products, S0 was revealed to be the basic pharmacophore of carnosic acid. Subsequent structural optimization of S0 led to the discovery of S11 as a possible anticancer agent with prominent proliferation inhibition effect (IC50 for SW480: 9.56 ± 0.91 µM) and best selectivity index (SI = 3.0) against Wnt hyperactive cancer cells. Futher mechanism investigation through TOP/FOP dual luciferase reporter assay, immunofluorescence, co-immunoprecipitation, microscale thermophoresis, downstream oncoprotein expression and cell apoptosis showed that compound S11 could significantly inhibit the proliferation of SW480 cells via obvioudsly decreasing the nucleus translocation of ß-catenin and effectively disrupting ß-catenin/BCL9 protein-protein interaction. Additionally, cell migration, molecule docking, in vitro stability and solubility assays were also conducted. Overall, S11 was worthy of in-depth study as a potential inhibitor for the Wnt/ß-catenin pathway and its discovery also proved that the structural simplification of natural products was still one of the effective methods to find new lead compounds or candidate drugs.

Deciphering the chemical constituents of Shengjiang Xiexin decoction by ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry and the impact of 20 characteristic components on multidrug resistance-associated protein 2 in the vesicular transport assay.

Shengjiang Xiexin decoction, a traditional Chinese medical formula, has been utilized to alleviate the delayed-onset diarrhea induced by irino tecan. However, the chemical constituents of this formula and the activities of its constituents remain unclear. In this study, ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry was employed to comprehensively analyze the chemical constituents of Shengjiang Xiexin decoction. A total of 270 components, including flavonoids, coumarins, triterpenoids, alkaloids, diarylheptanoids and others, were identified or characterized. Multidrug resistance-associated protein 2 is an efflux transporter responsible for regulating drug absorption. A total of 20 characteristic components from the formula were selected to evaluate their effects on the function of multidrug resistance-associated protein 2 using the vesicular transport assay. Glycyrrhizic acid and glycyrrhetinic acid were identified as potential multidrug resistance-associated protein 2 inhibitors, while 9 flavonoid aglycones increased the uptake of the substrate [3 H]-estradiol 17-ß-glucuronide in the vesicles. This was the first systematic investigation of the chemical constituents from Shengjiang Xiexin decoction and the effect of its characteristic components on the transporter. The results offered a basis for further exploring the detoxification mechanisms of this formula and its interactions with other drugs.

Insights into the Inhibitory Mechanism of Viniferifuran on Xanthine Oxidase by Multiple Spectroscopic Techniques and Molecular Docking.

Viniferifuran was investigated for its potential to inhibit the activity of xanthine oxidase (XO), a key enzyme catalyzing xanthine to uric acid. An enzyme kinetics analysis showed that viniferifuran possessed a strong inhibition on XO in a typical anti-competitive manner with an IC50 value of 12.32 µM (IC50 for the first-line clinical drug allopurinol: 29.72 µM). FT-IR and CD data analyses showed that viniferifuran could induce a conformational change of XO with a decrease in the α-helix and increases in the ß-sheet, ß-turn, and random coil structures. A molecular docking analysis revealed that viniferifuran bound to the amino acid residues located within the activity cavity of XO by a strong hydrophobic interaction (for Ser1214, Val1011, Phe914, Phe1009, Leu1014, and Phe649) and hydrogen bonding (for Asn768, Ser876, and Tyr735). These findings suggested that viniferifuran might be a promising XO inhibitor with a favorable mechanism of action.

Ent-Abietane Diterpenoids from Euphorbia fischeriana and Their Cytotoxic Activities.

The roots of Euphorbia fischeriana have been used as a traditional Chinese medicine for the treatment of tuberculosis and ringworm. In the current study, diterpenoids from the ethyl acetate extract of the roots E. fischeriana and their cytotoxic effects against five cancer lines were investigated. Two new ent-abietane diterpenoids, euphonoids H and I (1-2), as well as their two analogues (3-4) were first isolated from this source. The structures of the two new compounds were elucidated on the basis of spectroscopic data and quantum chemical calculation. Their absolute configurations were assigned via ECD spectrum calculation. The isolated compounds were evaluated for their antiproliferative activities against five cancer cell lines. Compounds 1 and 2 exhibited significant inhibitory effects against human prostate cancers C4-2B and C4-2B/ENZR cell lines with IC50 values ranging from 4.16 ± 0.42 to 5.74 ± 0.45 µM.

Systematically Exploring the Chemical Ingredients and Absorbed Constituents of Polygonum capitatum in Hyperuricemia Rat Plasma Using UHPLC-Q-Orbitrap HRMS.

Polygonum capitatum as an ethnic medicine has been used to treat urinary tract infections, pyelonephritis and urinary calculi. In our previous study, P. capitatum was found to have anti-hyperuricemia effects. Nevertheless, the active constituents of P. capitatum for treating hyperuricemia were still unclear. In this study, an ultra-high-performance liquid chromatography coupled to quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to comprehensively detect the chemical ingredients of P. capitatum and its absorbed constituents in the plasma of hyperuricemia rats for the first time. Xcalibur 3.0 and Compound Discoverer 2.0 software coupled to mzCloud and ChemSpider databases were utilized for qualitative analysis. A total of 114 chemical components including phenolics, flavonoids, tannins, phenylpropanoids, amino acids, amides and others were identified or tentatively characterized based on the exact mass, retention time and structural information. Compared to the previous P. capitatum study, an additional 66 different components were detected. Moreover, 68 related xenobiotics including 16 prototype components and 52 metabolites were found in the plasma of hyperuricemia rats. The metabolic pathways included ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. This work may provide important information for further investigation on the active constituents of P. capitatum and their action mechanisms for anti-hyperuricemia effects.

New Meroterpenoid and Isocoumarins from the Fungus Talaromyces amestolkiae MST1-15 Collected from Coal Area.

Three new compounds including a meroterpenoid (1) and two isocoumarins (8 and 9), together with thirteen known compounds (2-7, 10-16) were isolated from the metabolites of Talaromyces amestolkiae MST1-15. Their structures were identified by a combination of spectroscopic analysis. The absolute configuration of compound 1 was elucidated on the basis of experimental and electronic circular dichroism calculation, and compounds 8 and 9 were determined by Mo2(OAc)4-induced circular dichroism experiments. Compounds 7-16 showed weak antibacterial activities against Stenotrophomonas maltophilia with MIC values ranging from 128 to 512 µg/mL (MICs of ceftriaxone sodium and levofloxacin were 128 and 0.25 µg/mL, respectively).

Design, Synthesis, and Biological Evaluation of N14-Amino Acid-Substituted Tetrandrine Derivatives as Potential Antitumor Agents against Human Colorectal Cancer.

As a typical dibenzylisoquinoline alkaloid, tetrandrine (TET) is clinically used for the treatment of silicosis, inflammatory pulmonary, and cardiovascular diseases in China. Recent investigations have demonstrated the outstanding anticancer activity of this structure, but its poor aqueous solubility severely restricts its further development. Herein, a series of its 14-N-amino acid-substituted derivatives with improved anticancer effects and aqueous solubility were designed and synthesized. Among them, compound 16 displayed the best antiproliferative activity against human colorectal cancer (HCT-15) cells, with an IC50 value of 0.57 µM. Compared with TET, 16 was markedly improved in terms of aqueous solubility (by 5-fold). Compound 16 significantly suppressed the colony formation, migration, and invasion of HCT-15 cells in a concentration-dependent manner, with it being more potent in this respect than TET. Additionally, compound 16 markedly impaired the morphology and motility of HCT-15 cells and induced the death of colorectal cancer cells in double-staining and flow cytometry assays. Western blot results revealed that 16 could induce the autophagy of HCT-15 cells by significantly decreasing the content of p62/SQSTM1 and enhancing the Beclin-1 level and the ratio of LC3-II to LC3-I. Further study showed that 16 effectively inhibited the proliferation, migration, and tube formation of umbilical vein endothelial cells, manifesting in a potent anti-angiogenesis effect. Overall, these results revealed the potential of 16 as a promising candidate for further preclinical studies.

Sinensiols H-J, three new lignan derivatives from Selaginella sinensis (Desv.) Spring.

One new lignan sinensiol H (1) and two new bisnorlignans, sinensiols I and J (2 and 3), along with three known compounds were isolated from the whole plants of Selaginella sinensis. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopy as well as high-resolution mass spectrometry. The absolute configuration of 1 was established by ECD calculation. Compounds 2 and 3 represent rare examples of naturally occurring 9,9'-bisnorlignans. All the isolated compounds were assayed for their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages.

Pseudosterins A-C, Three 1-Ethyl-3-formyl-ß-carbolines from Pseudostellaria heterophylla and Their Cardioprotective Effects.

Pseudostellaria heterophylla is used in China not only as a functional food but also as an herb to tonify the spleen, enhance immunity, and treat palpitation. Our previous investigation showed that a fraction enriched in glycosides obtained from the roots of P. heterophylla possessed pronounced protective effects on H9c2 cells against CoCl2-induced hypoxic injury. However, the active compounds responsible for the observed effects were still unknown. In the current investigation, pseudosterins A-C (1-3), three new alkaloids with a 1-ethyl-3-formyl-ß-carboline skeleton, together with polydatin, have been isolated from the active fraction. Their structures were elucidated on the basis of spectroscopic analysis and quantum chemical calculations. The four compounds showed cardioprotective effects against sodium hydrosulfite-induced hypoxia-reoxygenation injury in H9c2 cells, with the three alkaloids being more potent. This is also the first report of alkaloids with a ß-carboline skeleton isolated from P. heterophylla as cardioprotective agents.

Discovery of tetrandrine derivatives as tumor migration, invasion and angiogenesis inhibitors.

Metastatic progression of cancer is a complex and clinically daunting process, with migration, invasion and angiogenesis being the key features. Tetrandrine (TET) is a typical dibenzylisoquinoline alkaloid with promising anti-tumor activity. In our previous work, a number of TET derivatives were designed and synthesized with obvious anti-proliferation activities against cancer cells, however, the anti-metastatic effects of these compounds were not evaluated. In the current investigation, five TET derivatives (8, 18, 32, 71, and 72) with pronounced anti-proliferative activities (IC50 values of 1.00, 1.91, 3.43, 3.78, and 1.93 µM, respectively) against human umbilical vein endothelial cells (HUVECs) were screened out. Scratch assays showed that these compounds significantly suppressed the migration of HUVECs and induced their apoptosis. Among them, derivatives 8 and 72 obviously inhibited the proliferation, colony formation and invasion of HCT-15 cells. Tube formation assays revealed that 4 µM of 8 or 72 remarkably inhibited the tube forming capacity of HUVECs. Moreover, 8 and 72 surpressed the formation of filopodia in HUVECs and severely impaired their motility. Both compounds effectively inhibited the angiogenesis in the zebrafish model with low toxicities in vivo. These results indicated that TET derivatives 8 and 72 are promising anti-metastatic inhibitors.

Design, synthesis, and biological evaluation of 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent HIV-1 Vif inhibitors.

Viral infectivity factor (Vif) is one of the accessory protein of human immunodeficiency virus type I (HIV-1) that inhibits host defense factor, APOBEC3G (A3G), mediated viral cDNA hypermutations. Previous work developed a novel Vif inhibitor 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)thio)benzamide (1) with strong antiviral activity. Through optimizations on the two side branches, a series of compound 1 derivatives (2-18) were designed, synthesized and tested in vitro for their antiviral activities. The biological results showed that compound 5 and 16 inhibited the virus replication efficiently with EC50 values of 9.81 and 4.62 µM. Meanwhile, low cytotoxicities on H9 cells were observed for the generated compounds by the MTT assay. The structure-activity relationship of compound 1 was preliminarily clarified, which gave rise to the development of more potent Vif inhibitors.

Auriculatone Sulfate Effectively Protects Mice Against Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP) overdose is very common worldwide and has been widely recognized as the leading cause of drug-induced liver injury in the Western world. In our previous investigation, auriculatone, a natural product firstly obtained from Aster auriculatus, has demonstrated a potent protective effect against APAP-induced hepatotoxicity in HL-7702 cells. However, the poor water solubility and low bioavailability restrict its application. Auriculatone sulfate (AS) is a sulfated derivative of auriculatone with highly improved water-solubility. Hepatoprotective effects against APAP-induced liver injury (AILI) showed that intragastric pretreatment with AS at 50 mg/kg almost completely prevented mice against APAP-induced increases of serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and ATPase. Histological results showed that AS could protect the liver tissue damage. In addition, AS pretreatment not only significantly retained hepatic malondialdehyde and the activities of glutathione, superoxide dismutase, and glutathione peroxidase at normal levels, but also markedly suppressed the increase of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 levels in mouse liver caused by overdose APAP. Immunohistochemical analysis showed that AS obviously attenuated the expression of CD45 and HNE in liver tissue. Further mechanisms of action investigation showed that inhibition of cytochrome P450 3A11 (CYP 3A11) and CYP2E1 enzymatic activities (but not that of CYP1A2) was responsible for APAP bioactivation. In conclusion, AS showed a hepatoprotective effect against AILI through alleviating oxidative stress and inflammation and inhibiting CYP-mediated APAP bioactivation. It may be an effective hepatoprotective agent for AILI and other forms of human liver disease.

Screening and analysis of potentially active components in Shenxiong glucose injection using UHPLC coupled with photodiode array detection and MS/MS.

Shenxiong glucose injection, a pharmaceutical preparation containing a water extract of the roots of Salvia miltiorrhizae and ligustrazine hydrochloride, is widely used in clinical to treat cardiovascular diseases in China. The chemical components of the water extract have been reported and the cardioprotective effects of the injection have been evaluated. However, the chemical constituents of the injection and their correlations with its pharmacological effects have not been established. In this study, 13 chemical constituents of the injection have been identified or characterized by ultra-high performance liquid chromatography with diode array detection and electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Besides, the potentially active compounds of this preparation that directly act on cardiac cells have been screened by cell extraction and ultra high performance liquid chromatography targeted multiple reaction monitoring. As a result, eight potentially active compounds, danshensu (1), ligustrazine hydrochloride (4), salvianolic acid I/H (7), lithospermic acid (8), salvianolic acid D (9), rosmarinic acid (10), salvianolic acid B (12), and salvianolic acid C (13), were obtained and structurally characterized from the 11 target compounds used for screening. The liquid chromatography with quadrupole time-of-flight mass spectrometry and liquid chromatography with multiple reaction monitoring tandem mass spectrometry combination method has demonstrated its potency for the screening, detection, and structural identification of bioactive compounds in a complex matrix.

Discovery and structure-activity relationship of auriculatone: A potent hepatoprotective agent against acetaminophen-induced liver injury.

Acetaminophen (APAP, paracetamol) overdose has been the most frequent cause of drug-induced liver failure. APAP-induced liver toxicity can be fatal in many cases even with treatment of the clinically used N-acetylcysteine (NAC), and the need for novel therapeutic agents is apparent. Through evaluating the hepatoprotective effects of the co-occurring substances present in oleanolic acid tablets which have been used in China for decades as an adjuvant therapy for acute and chronic hepatitis, auriculatone was found to protect HL-7702 cells from APAP-induced liver injury comparable to NAC at the concentration of 10µM. Structure activity relationship on auriculatone and its analogs showed that absence of the C17 carboxyl group of auriculatone was essential to achieve good hepatoprotective activity, and that the C3-OH, C16 carbonyl and C12-C13 olefinic group were critical for retaining the exceptional activity of auriculatone. Any modifications in the current investigation were all detrimental to the hepatoprotective activity. Docking and drug-metabolizing activity studies demonstrated that CYP3A4 was likely the main target of auriculatone, and that auriculatone elicited the hepatoprotective effect possibly through inhibiting CYP3A4's metabolism of APAP to the toxic metabolite NAPQI. The work may pave the way for the use of auriculatone in the treatment of APAP-induced liver injury.

A UPLC-MS/MS Method for Simultaneous Determination of Free and Total Forms of a Phenolic Acid and Two Flavonoids in Rat Plasma and Its Application to Comparative Pharmacokinetic Studies of Polygonum capitatum Extract in Rats.

The principal active constituents of Polygonum capitatum are phenolic acids and flavonoids, such as gallic acid, quercitrin, and quercetin. The aim of this study was to develop and validate a method to determine the three constituents and the corresponding conjugated metabolites of Polygonum capitatum in vivo and to conduct pharmacokinetic studies on the herb, a well-known Miao medicinal plant in China. Gallic acid, quercitrin, and quercetin were analysed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). Protein precipitation in plasma samples was performed using methanol. For the determination of total forms of analytes, an additional process of hydrolysis was conducted using ß-glucuronidase and sulphatase. The analytes were separated on a BEH C18 column (50 mm × 2.1 mm; i.d., 1.7 µm) and quantified by multiple reaction monitoring (MRM) mode. The linear regression showed high linearity over a 729-fold dynamic range for the three analytes. The relative standard deviations of intra- and inter-day measurements were less than 9.5%, and the method was accurate to within -11.1% to 12.5%. The extraction recoveries for gallic acid, quercitrin, and quercetin were 94.3%-98.8%, 88.9%-98.8%, and 95.7%-98.5%, respectively. All samples were stable under short- and long-term storage conditions. The validated method was successfully applied to a comparative pharmacokinetic study of gallic acid, quercitrin, and quercetin in their free and total forms in rat plasma. The study revealed significantly higher exposure of the constituents in total forms for gallic acid and quercetin, while quercitrin was detected mainly in its corresponding free form in vivo. The established method was rapid and sensitive for the simultaneous quantification of free and total forms of multiple constituents of Polygonum capitatum extract in plasma.