Vitamin K2 supplementation improves impaired glycemic homeostasis and insulin sensitivity for type 2 diabetes through gut microbiome and fecal metabolites.

BACKGROUND: There is insufficient evidence for the ability of vitamin K2 to improve type 2 diabetes mellitus symptoms by regulating gut microbial composition. Herein, we aimed to demonstrate the key role of the gut microbiota in the improvement of impaired glycemic homeostasis and insulin sensitivity by vitamin K2 intervention. METHODS: We first performed a 6-month RCT on 60 T2DM participants with or without MK-7 (a natural form of vitamin K2) intervention. In addition, we conducted a transplantation of the MK-7-regulated microbiota in diet-induced obesity mice for 4 weeks. 16S rRNA sequencing, fecal metabolomics, and transcriptomics in both study phases were used to clarify the potential mechanism. RESULTS: After MK-7 intervention, we observed notable 13.4%, 28.3%, and 7.4% reductions in fasting serum glucose (P = 0.048), insulin (P = 0.005), and HbA1c levels (P = 0.019) in type 2 diabetes participants and significant glucose tolerance improvement in diet-induced obesity mice (P = 0.005). Moreover, increased concentrations of secondary bile acids (lithocholic and taurodeoxycholic acid) and short-chain fatty acids (acetic acid, butyric acid, and valeric acid) were found in human and mouse feces accompanied by an increased abundance of the genera that are responsible for the biosynthesis of these metabolites. Finally, we found that 4 weeks of fecal microbiota transplantation significantly improved glucose tolerance in diet-induced obesity mice by activating colon bile acid receptors, improving host immune-inflammatory responses, and increasing circulating GLP-1 concentrations. CONCLUSIONS: Our gut-derived findings provide evidence for a regulatory role of vitamin K2 on glycemic homeostasis, which may further facilitate the clinical implementation of vitamin K2 intervention for diabetes management. TRIAL REGISTRATION: The study was registered at https://www.chictr.org.cn (ChiCTR1800019663).

Study on the action mechanism of Buyang Huanwu Decoction against ischemic stroke based on S1P/S1PR1/PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke is a common and frequent clinical disease. Recent studies have demonstrated that sphingolipid plays an important role in the pathological process of ischemic stroke. PI3K-Akt is a classic protective signaling pathway of cerebral ischemic injury. After acting on the S1P receptor, S1P can activate the downstream PI3K/Akt signaling pathway and play an anti-cerebral ischemia role. Buyang Huanwu Decoction (BHD) is a traditional Chinese medicine formula used to treat ischemic stroke. However, the mechanisms of BHD on ischemic stroke remain unclear based on S1P/S1PR1/PI3K/Akt signaling pathway. AIM OF THE STUDY: The present study is intended to investigate the action mechanism of BHD on ischemic stroke based on the S1P/S1PR1/PI3K/Akt signaling pathway from multiple perspectives. MATERIALS AND METHODS: The BHD lyophilized product was prepared by vacuum freeze-drying method, of which the chemical composition was determined by UPLC-Q-TOF/MS. The mouse permanent middle cerebral artery occlusion (pMCAO) model was established by the suture-occluded method. Male KM mice were randomly divided into seven groups: sham group, model group, FTY720 (positive control) group, BHD group, BHD + W146 (selective S1PR1 inhibitor) group, SEW2871 (selective S1PR1 agonist) group, and Calycosin group. Each group was administered continuously for 14 days and evaluated with modified neurological severity score (mNSS) and cerebral infarct volume on the 1st, 4th, 7th, and 14th days. The SphK1, SphK2, S1PR1, PI3K, Akt, and p-Akt protein in the prefrontal lobe, hippocampus, and striatum was quantified by Western blot and immunohistochemical (IHC) experiment respectively. The qRT-PCR method was employed to evaluate SphK1, SphK2, and S1PR1 mRNA expression in the above tissue. RESULTS: BHD and Calycosin both effectively improved mNSS scores with smaller infarct volumes. The SphK1 level in the prefrontal lobe, hippocampus, and striatum of mice in the BHD group was significantly lower, and SphK2, PI3K, and p-Akt in the hippocampus and striatum were significantly higher than those in the model group. BHD significantly decreased SphK1 mRNA expression in the prefrontal lobe, hippocampus, and striatum, and significantly up-regulated SphK2 mRNA and S1PR1 mRNA expression. Additionally, SphK1 protein expression levels of the prefrontal lobe, hippocampus, and striatum in the BHD group was significantly lower than model group, and SphK2, S1PR1, PI3K, Akt, and p-Akt protein expressions levels were increased obviously. Furthermore, SEW2871 can increase S1PR1 and Akt expression, and up-regulate SphK2 and S1PR1 mRNA expression. The effect of BHD on the expression of S1P/S1PR1/PI3K/Akt signaling pathway-related proteins and mRNA were weakened by BHD + W146. CONCLUSION: BHD and Calycosin significantly improved the symptoms of neurological deficits in pMCAO mice, reduced the cerebral infarction volume, up-regulated SphK2 and S1PR1 mRNA levels, enhanced SphK2, S1PR1, PI3K, Akt, p-Akt protein expression of the prefrontal lobe, hippocampus and striatum, and down-regulated SphK1 mRNA and protein expression, which may be helpful to clarify the mechanism of BHD through S1P/S1PR1/PI3K/Akt signaling pathway to protect against cerebral ischemic injury.

The Number of Intraoperative Intestinal Venous Circulating Tumor Cells Is a Prognostic Factor for Colorectal Cancer Patients.

Purpose: To assess the association between intestinal venous blood (IVB) circulating tumor cells (CTCs) and clinicopathological parameters in stage I-III colorectal cancer (CRC) patients. Methods: Participants were retrospectively retrieved, who were admitted to our hospital or took annual physical exams between December 1, 2015 and December 31, 2018. A negative enrichment-immunofluorescence in situ hybridization (NE-imFISH) technique was used to isolate and identify CTCs. Receiver operating characteristic (ROC) curves and Youden index values were used to determine the critical CTC cutoff value for the diagnosis of CRC. Kaplan-Meier and log-rank methods were used to conduct survival analyses, and multivariate Cox regression analyses were employed for multivariate corrections to comprehensively evaluate the value of CTCs in the diagnosis of CRC. Relationships between IVB CTCs, clinicopathological parameters, and prognosis were then analyzed based upon patient postoperative follow-up data. Results: In total, we retrieved 282 patients including 48 healthy controls, 72 patients with benign colorectal tumors, and 162 CRC patients. CRC patients exhibited significantly higher numbers of CTCs relative to control patients or those with benign disease. CTC numbers in CRC patient peripheral blood (PB) and IVB were closely associated with tumor node metastasis (TNM) staging (P < 0.01), carbohydrate antigen-125 (CA-125) levels (P < 0.001), and KRAS (Kirsten rat sarcoma virus oncogene) mutation status (P < 0.001). The disease-free survival (DFS) of patients in the CTC-negative group was significantly longer than that of patients in the CTC-positive group (24.60 ± 13.31 months vs. 18.70 ± 10.19 months, P < 0.05), with the same being true with respect to their overall survival (OS) (30.60 ± 12.44 months vs. 35.25 ± 11.57 months, P < 0.05). A multivariate analysis revealed that the detection ≥2 CTCs/3.2 ml was independently associated with poorer DFS and OS. CTC counts were independently predictive of CRC patients TNM staging, CA-125, and KRAS mutation status in both univariate and multivariate Cox proportional hazards regression analyses. Conclusion: CTCs are valuable biomarkers that can be monitored to predict CRC patient disease progression.

[Metabonomics research strategy based on microdialysis technique].

Metabonomics is the branch of systems biology. It has been widely used in the fields of diagnostic markers discovery, disease prognosis, drug action mechanism, drug efficacy and toxicity evaluation, traditional Chinese medicine syndromes differentiation. There are shortcomings in the conventional metabonomics research. Microdialysis technology is a new type of biosampling technology, and metabonomics research based on microdialysis technology is in the ascendant. In view of the particularity of microdialysis technology and its great differences from traditional sampling and pretreatment methods, the metabonomics process based on microdialysis technology has certain similarities with traditional metabonomics research, and its basic process has some particularity. Advantages and basic strategies of metabonomics research by microdialysis technology are systematically summarized for researchers' reference.

Effect of Low-Dose Vitamin K2 Supplementation on Bone Mineral Density in Middle-Aged and Elderly Chinese: A Randomized Controlled Study.

Previous studies indicated a positive effect of vitamin K2 (VK2) supplementation on bone turnover biomarkers and bone mineral density (BMD), but the doses varied, and few studies have focused on the difference between VK2 supplementation alone and in combination with calcium and vitamin D3. The aim of this study was to explore a low and effective dose of VK2 for improving BMD, and to examine whether the co-supplementation of VK2, calcium and vitamin D3 would bring greater effects. In this trial, a total of 311 community-dwelling men and postmenopausal women aged 50 and 75 years were randomly assigned to four groups, receiving placebo, 50 µg/day, 90 µg/day or co-supplementation with calcium (500 mg/day) and vitamin D3 (10 µg/day) for 1 year. At the endpoint, the bone loss of femoral neck was significantly lower in postmenopausal women in the two 90 µg groups (treatment × time, p = 0.006) compared with placebo, but no effects in men. Serum biomarkers cOC/ucOC ratio increased in the intervention groups (treatment × time, p < 0.001). VK2 supplementation in dose of 90 µg/day performed a significant effect on reducing bone loss in postmenopausal women, but in combination with calcium and vitamin D3 brought no additional effects.Trial registration This trial was registered at http://www.chictr.org.cn as chiCTR1800019240.

[Brain lipidomics of intervention effect of Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma on ischemic stroke based on UPLC-Q-TOF-MS technique].

The aim of this paper was to investigate the intervention effect of Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma on brain lipid metabolism in rats with ischemic stroke. The ischemic stroke rat model was established by middle cerebral artery occlusion( MCAO) method. The brain tissues were collected after the last administration with Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma decoction lyophilizate. UPLC-Q-TOF-MS was used to carry out the brain lipidomics study. The lipidomics data were processed with the OPLS-DA model to find out the lipid regulation effect of Salviae Miltiorrhizae Radix et Rhizoma combined with Chuanxiong Rhizoma on ischemic stroke. The results showed that Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma decoction lyophilized powder can significantly alleviate brain lipidomics profiles in middle cerebral artery occlusion model rats. Eleven differential lipid metabolites in ischemic stroke model were identified. In this experiment,the protective effects of Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma decoction lyophilized powder on cerebral ischemia injury was verified,which might be related to the regulation of abnormal lipid metabolism.

Research progress on berberine with a special focus on its oral bioavailability.

The natural product berberine (BBR) has become a potential drug in the treatment of diabetes, hyperlipidemia, and cancer. However, the oral delivery of BBR is challenged by its poor bioavailability. It is necessary to improve the oral bioavailability of BBR before it can be used in many clinical applications. Understanding the pharmacokinetic characteristics of BBR will enable the development of suitable formulas that have improved oral bioavailability. The key considerations for BBR are how to enhance the drug absorption and to avoid the intestinal first-pass effect. This review summarizes the pharmacological activities of BBR and analyzes the factors that lead to its poor oral bioavailability. In particular, the therapeutic potential of BBR in new indications from the aspect of oral bioavailability is discussed. In conclusion, BBR is a promising drug candidate for metabolic disorders and cancer but faces considerable challenges due to its poor oral bioavailability.

Effect of Xiongbing compound on the pharmacokinetics and brain targeting of tetramethylpyrazine.

OBJECTIVES: To investigate the effect of the Xiongbing compound (XBC) on the pharmacokinetics and brain targeting of tetramethylpyrazine (TMP). METHODS: Three microemulsions containing the same TMP concentration were prepared. XBC microemulsions were made from Rhizoma ligustric Chuanxiong extracts, borneol and TMP. TMP microemulsions were made with TMP only. Borneol microemulsions contained borneol and TMP. Microdialysis with high performance liquid chromatography (HPLC) was used to measure the concentration of TMP in the blood and striatum after intravenous (i.v.) or intragastric (i.g.) administration of the three different microemulsions. KEY FINDINGS: The pharmacokinetics of free TMP concentration in the blood and the striatum fit a first-order rate, open two-compartment model after intravenous and intragastric microemulsion administration. The maximal concentration (C(max) ) and area under curve (AUC) values in the XBC microemulsion i.v. group were significantly higher than that in the TMP microemulsion and borneol microemulsion i.v. groups. After XBC microemulsion i.g. administration, the t(½), mean residence time (MRT) and AUC of TMP in both plasma and brain tissues were greater than those with TMP microemulsion and borneol microemulsion administration. The relative brain targeting efficiency of TMP for the XBC microemulsion i.v and i.g. groups relative to the TMP microemulsion and borneol microemulsion groups were greater than 1. CONCLUSION: XBC microemulsion can enhance TMP oral bioavailability, brain targeting and tissue distribution, mainly through a synergistic action of Rhizoma ligustric Chuanxiong extracts and borneol.

[Impact on absorption of berberine and palmatine in different compatibilities of Wuji pill].

OBJECTIVE: To observe the impact on absorption of berberine and palmatine in different compatibilities of Wuji pill by the perfused rat intestine-liver preparation. METHOD: Use L9 (3(4)) orthogonal design table, establish the perfused rat intestine-liver preparation, the twelve Wuji pill compatibilities duodenal administrated, collect the perfusate at different times points for LC-MS detection, calculate the absorbed score, Ka. RESULT: Evodiae Fructus and the absorption score, Ka of berberine and palmatine are inverse correlated. The most superior portion which promote the absorption is Coptidis Rhizoma-Evodiae Fructus-Paeoniae Radix Alba 3:1:3. CONCLUSION: Evodiae Fructus suppressed the absorption of berberine and palmatine. With the different portion the absorption also have big different.

Quality evaluation of a herbal prescription through quantification of 40 components by HPLC-ESI-MS/MS.

INTRODUCTION: The Kang-nao-shuai (KNS) capsule is a combined herbal prescription used in the treatment of insomnia, amnesia, neurasthenia, age-related dementia and brain injuries. Multiple constituents are considered to be responsible for the therapeutic effects of this herbal prescription. However, the quality control of the multicomponents is limited. OBJECTIVE: To establish a liquid chromatography-electrospray ionisation-mass spectrometry method for the analysis of 40 constituents in KNS capsules. METHODOLOGY: The optimal chromatographic conditions were achieved on an Agilent C18-column with a gradient elution that consisted of methanol and 0.1% formic acid in water. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer in positive and negative mode respectively using multiple-reaction monitoring. RESULTS: A total of 40 constituents including organic acid, flavonoid, quinone, terpene, alkaloid and saponin were quantified, most of the 40 components were determined for the first time in the KNS capsule. A quantitative HPLC-ESI-MS/MS method allowing the quantification of 40 marker compounds was optimised and validated for linearity, precision, accuracy, stability, specificity and limits of detection and quantification. The method was successfully applied to analyse 10 batches of KNS capsule. CONCLUSION: The established method is simple and can be used as a tool for quality evaluation and control of this natural product.

[Preparation of xiongbing microemulsion and its quality evaluation].

OBJECTIVE: To study the preparation of xiong-bing microemulsion (XB ME) and its quality evaluation. METHOD: XB ME was prepared by using the methods of titration to prepare the Pseudo-ternary phase diagram. The stability and size distribution of XB ME were tested. The contents of TMP and FA in ME were determined by HPLC method. RESULT: The character of XB ME was stable. The average diameter was 17.6 nm, and a uniform distribution of particle size were achieved. The solubility of TMP in ME was 2.6 g x L(-1), the methodological average recovery was 97.0% with RSD of 1.5% (n=6); The solubility of FA in ME was 0.3 g x L(-1), the methodological average recovery was 95.9% with RSD of 1.9% (n=6). CONCLUSION: The results indicated that the quality of XB ME was stable. The method to determine the contents of TMP and FA was accurate and reliable.

[Studies on pharmacokinetics of evodiamine and rutaecarpine in rats plasma after oral administration extracts of euodiae fructus].

OBJECTIVE: Develop an LC-MS method to determine evodiamine and rutaecarpine in rats plasma simultaneously. The method was employed to investigate pharmacokinetics of evodiamine and rutaecarpine. METHOD: Blood samples were collected in different time after oral administrated with the extracts of Euodiae Fructus, the plasma concentration of evodiamine and rutaecarpine was determined by LC-MS, pharmacokinetic parameters were calculated by WinNonlin 5.1 software. RESULT: The linear ranges of evodiamine and rutaecarpine were 0.5-100 microg x L(-1) (r = 0.995 9), 1-200 microg x L(-1) (r = 0.999 3) respectively. The average recovery were exceeded 76% (n = 5), the precision of inner-day and inter-day were less than 15%. The pharmacokinetics parameters AUC, t1/2, CL _F of evodiamine were: (2 215.24 +/- 414.49), (4 230.62 +/- 753.77), (13 219.21 +/- 3 740.95) min x ng(-1) x mL(-1); (146.57 +/- 38.38), (114.38 +/- 14.65), (163.37 +/- 8.83) min; (184 607.29 +/- 32 502.21), (192 878.22 +/- 31 897.37), (19 3224.63 +/- 62 278.74) mL x min(-1). The pharmacokinetics parameters AUC, t1/2, CL_F of rutaecarpine were (2 283.53 +/- 298.51), (4 424.84 +/- 276.95), (14 239.93 +/- 3648.27) min x ng(-1) x mL(-1); (167.10 +/- 15.82), (131.58 +/- 20.07), (144.41 +/- 13.65) min; (1 177 340.54 +/- 2 4942.21), (181 262.92 +/- 11 162.22), (177 508.10 +/- 52 611.80) mL x min(-1). CONCLUSION: The method described in this report has high sensitivity and selectivity, and was suitable for pharmacokinetic studies of evodiamine and rutaecarpine. The kinetic process of evodiamine and rutaecarpine in rats in vivo were all yielded to be one-compartment model.

[Effect of different compatibility of Wuji Wan on bile excretion of representative ingredient].

OBJECTIVE: To research the representative ingredient excretion in bile using the different compatibilities of Wuji Wan, in order to indicate the regularity of compatibility. METHOD: L9 (3(4)) orthogonal design table was used, in addition 9 simples, altogether 18 compatibilities. After duodenal administration bile at different time spot was collected for LC-MS detection. RESULT: The excretion of Evodiae Fructus was negative correlated with that of berberine and palmatine in bile. The excretion of Paeoniae Radix Alba was positive correlated with that of berberine and palmatine in bile. The excretion of Coptidis Rhizoma, Paeoniae Radix Alba was negative correlated with that of evodiamine, rutaecarpine; meanwhile, the most superior proportion was also caculated which promote the representative ingredient excrete from bile. CONCLUSION: Evodiae fructus suppresses representative ingredients of Coptidis Rhizoma excrete through bile. Paeoniae Radix Alba promote suppresses representative ingredients of Coptidis Rhizoma excrete through bile. Coptidis Rhizoma, paeoniae Radix Alba suppresses representative ingredients of Evodiae Fructus excrete through bile. Coptidis Rhizoma, Evodiae Fructus suppresses representative ingredients of Paeoniae Radix Alba excrete through bile.

[Rat in vitro plasma-binding percentage determination of sinomenine hydrochloride by using microdialysis method and ultrafiltration].

OBJECTIVE: To assess feasibility of plasma protein-binding percentage by using microdialysis method. METHOD: Sinomenine hydrochloride was selected as model drug. The in vitro rat plasma protein-binding percentage of three kinds of concentration was determinated by using microdialysis sampling tool and HPLC-UV. At the same time the classical study method, such as ultrafiltration method, was also used to determinate the protein-binding percentage to compare the difference between the two methods. RESULT: The in vitro rat plasma protein-binding percentage of sinomenine hydrochloride using microdialysis method was about 26% and kept relative stable at the concentration range. There was no significant difference between the two methods. CONCLUSION: The results shows that the binding percentage is relative low. Microdialysis method is a new method for the study of protein-binding degree.

[The pharmacokinetics study on Sinomenine transdermal patch on anaesthetized Beagle dogs].

OBJECTIVE: To explore the pharmacokinetic of Sinomenine transdermal patch. METHODS: The plasma drug concentration of Beagle dogs was determined after administration with HPLC-UVD as analysis tools. The pharmacokinetics parameters were fitted with kinetica software package. RESULTS: The exclusive analysis method was established with the following pharmacokinetics parameters: T (peak) = 8 h, Cmax = 366 ng/mL, MRT = 13 h. The pharmacokinetic characteristics was accordance with one-order rate and two-compartment model. CONCLUSION: The method is preferable to be applied to the pharmacokinetics research and further applied pharmacological study which will play a reference role in clinical application.

[Pharmacokinetics study on paeoniflorin in radix paeoniae alba extract by LC-MS].

OBJECTIVE: To develop a LC-MS method to determine paeoniflorin concentration in rats plasma. The method was applied to investigate pharmacokinetics of paeoniflorin in rats in vivo. METHOD: Blood samples were collected at different time after oral administration of Radix Paeoniae Alba extract at doses of 0.2, 0.4, 0.8 g x kg(-1). The paeoniflorin concentration in plasma was determined by LC-MS method. Pharmacokinetic parameters were fitted by WinNonlin 5.1 software package. RESULT: The linear range and the average recovery of paeoniflorin were 2.5-500 microg x L(-1) (r = 999 4) and more than 80% (n = 5) , respectively. The inner- and inter-days precision were both less than 15%. The T1/2 was similar. The relationship between dose and AUC showed good linearity. CONCLUSION: The method described in this report has high sensitivity and selectivity, and was suitable for pharmacokinetic study of paeoniflorin. The kinetic process of paeoniflorin in palsma showed two-compartment model after oral administration of Radix Paeoniae Alba extract at doses of 0.2, 0.4, 0.8 g x kg(-1) to rats.

[Absorption of extractive Radix Paeoniae Alba in rat everted gut sacs and its interaction with P-glycoprotein].

OBJECTIVE: To research the intestinal absorption characteristics of paeoniflorin in extractive Radix Paeoniae Alba in the different intestinal segment, and the interaction with P-glycoprotein. METHOD: Paeoniflorin, a representative component in extractive Radix Paeoniae Alba, on the intestinal absorption was studied in vitro using everted gut sacs model and detected by HPLC method. The absorption characteristics was evaluated by the absorption parameter. RESULT: The absorption of paeoniflorin was linearity at different intestine segment and dose, and the square of regrees correlation coefficient exceed 0.9 (R2 > 0.9), which consistent with zero order rate process. The Kalpha of paeoniflorin showed a dose-dependent increase along with the raised dose of extractive Radix Paeoniae Alba, indicated it was a mechanism of passive absorption. The absorption rate was jejunum > ileum > colon. Verapamil (100 micromol x L(-1)), a inhibitor of the P-glycoprotein, can remarkable increase the absorption of the paeoniflorin in ileum (P < 0.05). After administer the extractive Radix Paeoniae Alba for 5 days, the extraction of Rho123 is significantly increase in ileum (P < 0.01). CONCLUSION: The intestinal absorption of paeoniflorin is zero order rate process and passive absorption. Paeoniflorin is a substrate of P-glycoprotein, and extractive Radix Paeoniae Alba could induce the expression of the P-glycoprotein.

[Absorption of extractive Rhizoma Coptidis in rat everted gut scas].

OBJECTIVE: Using in vitro everted gut seas to research the intestinal absorption of the extractive Rhizoma Coptidis at the different intestinal section and the different density. METHOD: Berberine (BER) and palmatine (PAL) which are representative compositions of the extractive Rhizoma Coptidis in everted gut seas are detected by HPLC, and calculated the absorption parameter to describe the character of absorption. RESULT: The absorption of BER and PAL is linearity in different intestine and different dose, and the square of coefficient correlation exceed 0.9, which consistent with zero order rate process. The K(a) of BER and PAL increases along with the raised dosage of the extractive Rhizoma Coptidis (P < 0.05), indicated it is the passive absorption. The absorption of BER and PAL in the jejunum is the most quick, the ileum and colon are slower. CONCLUSION: In the different dosage of extractive Rhizoma Coptidis, the absorption of BER and PAL Conforms to the zero order rate process at the different intestine, and is the passive absorption.

[Determination in vitro of rat plasma protein binding rate of sinomenine by using microdialysis method].

AIM: To determine in vitro the rat plasma protein binding rate by using microdialysis method. METHODS: The binding rate was determined by using microdialysis probe as sampling tools and zero-net flux method as calibrating method. The regression equation was made by the difference of concentrations between the dialysis sample and the perfusate. The x-intercept of regression equation was the free drug concentration (Cf). The plasma protein binding rate was calculated by using the following equation: f = ( C0 - Cf)/C0. RESULT: The binding rate was kept relatively stable in the studied concentration range. CONCLUSION: It is feasible that the plasma protein binding rate can be determined by using microdialysis method.