Mechanistic understanding of the inhibitory effect of cytochrome P450 3A4 and 3A5 by Wuzhi tablet (Schisandra sphenanthera extract) / 药学学报

Wuzhi tablet (WZ) is a prescribed herbal medicine extracted from Schisandra sphenanthera, which is widely used to protect the liver injury and drug-induced hepatotoxicity in clinical practices. Previous studies showed that WZ significantly increased the blood concentrations of tacrolimus, cyclosporine A, paclitaxel by inhibiting the cytochrome P450 3A (CYP3A)-mediated metabolism. CYP3A4 and CYP3A5 are the most important isoenzymes among the CYP3A subfamily. However, there are some differences in the catalytic and inhibitory activities between CYP3A4 and CYP3A5, which may lead to different risk of drug-drug and herb-drug interactions, and the risks may be further amplified in vivo. Currently, few reports have compared the herbal medicine inhibitory effects between CYP3A4 and CYP3A5 mediated metabolic reactions. Therefore, detailing the inhibitory effect of WZ on CYP3A4 and CYP3A5 will help understand and predict the potential herb-drug interaction. The results showed that WZ inhibited CYP3A4 and CYP3A5 in a NADPH-, time- and concentration- dependent manner. WZ showed more potent inhibition on CYP3A5 than CYP3A4. Cautions warranted when combining WZ with other therapeutic drugs to avoid the potential herb-drug interaction.

Wuzhi Tablet protects against APAP-induced liver injury at different pretreated intervals in mice / 药学学报

Acetaminophen (APAP, also known as paracetamol)-induced liver injury is the leading cause of drug-induced liver injury in the world. Wuzhi Tablet (WZ, an ethanol extract of Schisandra sphenanthera) is widely used in clinical practice to protect liver function. Our previous studies have shown that pretreatment with WZ for 3 days can significantly protect against APAP-induced liver injury; however, the effect of different intervals between APAP and WZ treatment on APAP-induced liver injury remains unclear. In this study, the change in liver injury indexes, APAP metabolites, and the activity of cytochrome P450 (CYP450) enzymes after treatment with WZ and APAP at different intervals were determined. The animal experiment was reviewed and approved by the Animal Ethics Committee of Sun Yat-sen University. The results show that 0 h, 0.5 h, and 2 h pretreatment with WZ significantly protected against APAP-induced liver injury in mice, as evidenced by a significant decrease in biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and malonaldehyde (MDA). WZ inhibited the metabolic activation of APAP mediated by CYP450 enzymes and reduced the formation of APAP metabolites. This study further demonstrates that pretreatment with WZ at different intervals (0 h, 0.5 h, and 2 h before APAP dosing) exerts a significant hepatoprotective effect against APAP-induced liver injury, and a single-dose of WZ inhibits the activity of CYP450 enzymes related to APAP metabolic activation, thereby protecting against APAP-induced hepatotoxicity.

Co-administration of Wuzhi tablet (Schisandra sphenanthera extract) alters tacrolimus pharmacokinetics in a dose- and time-dependent manner in rats.

ETHNOPHARMACOLOGY RELEVANCE: Tacrolimus is a well-known potent but expensive immunosuppressant. We previously clarified the herb-drug interaction between tacrolimus and Wuzhi tablet (WZ), a prescribed drug of ethanol extract of Schisandra sphenanthera, and showed the ideal effect of WZ on maintaining therapeutic level of tacrolimus and reducing the total drug expense. However, WZ possesses a biphasic effect on regulating CYP3A (the major metabolizing enzyme of tacrolimus), which could induce the mRNA and protein expression after long-term treatment while transiently inhibit the activity of CYP3A. In clinic, clinicians are confused about the relationship between the blood concentration of tacrolimus and the dose and the duration of pretreatment of WZ. Therefore, the effects of the pretreatment time and the dose of WZ on the pharmacokinetics of tacrolimus is urgently needed to be clarified to better combine the use of WZ and tacrolimus in clinic. AIM OF THE STUDY AND METHOD: This study aimed to investigate the effects of the pretreatment time and the dose of WZ on the pharmacokinetics of tacrolimus in rats. RESULTS AND CONCLUSIONS: After pretreated rats with WZ for 0, 0.5, 2, 6, 12 or 24 h, the area under the curve (AUC) of tacrolimus was 2.27 ± 0.59, 1.87 ± 1.14, 2.86 ± 0.64, 1.62 ± 0.70, 1.54 ± 1.06 and 1.12 ± 0.69-fold of that of the tacrolimus alone group, respectively. The ratio of AUC of tacrolimus to that of the co-administration group with 0, 62.5, 125, 250, 500 or 750 mg/kg of WZ was 1.00: 1.07: 1.44: 2.60: 2.32: 2.42, respectively. These findings suggested that WZ increased tacrolimus AUC in a pretreatment time- and dose-dependent manner. In line with the in vivo findings, WZ extract inhibited CYP3A activity in a pre-treatment time- and concentration-dependent manner in human liver microsomes. In conclusion, the pharmacokinetics of tacrolimus was significantly affected by the pretreatment time and the dose of WZ. Oral pretreatment with WZ for 0-2 h or co-dosing of 250 mg/kg of WZ most significantly increased the blood concentration of tacrolimus. These findings would be helpful for guiding the reasonable use of WZ and tacrolimus in clinic.

Targeted bile acids and gut microbiome profiles reveal the hepato-protective effect of WZ tablet (Schisandra sphenanthera extract) against LCA-induced cholestasis.

Cholestasis is caused by the obstacle of bile formation or secretion and can develop into severe liver diseases. We previously reported the ethanol extract of Schisandra sphenanthera (Wuzhi tablet, WZ) can significantly protect against lithocholic acid (LCA)-induced intrahepatic cholestasis in mice, partially due to the activation of PXR pathway and promotion of liver regeneration. However, the effect of WZ on the bile acids profile and gut microbiome in cholestastic mice remain unknown. In this study, the effect of WZ against LCA-induced liver injury was evaluated and its effect on the bile acids metabolome and gut microbiome profiles in cholestastic mice was further investigated. Targeted metabolomics analysis was performed to examine the change of bile acids in the serum, liver, intestine and feces. The change of intestinal flora were detected by the genomics method. Targeted metabolomics analysis revealed that WZ enhanced the excretion of bile acids from serum and liver to intestine and feces. Genomics analysis of gut microbiome showed that WZ can reverse LCA-induced gut microbiome disorder to the normal level. In conclusion, WZ protects against LCA-induced cholestastic liver injury by reversing abnormal bile acids profiles and alteration of gut microbiome.

Effect of six bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) on P-glycoprotein and its herb-drug interaction with digoxin / 药学学报

This study was aimed to investigate the effects of six Schisandra lignans of Wuzhi tablet (WZ, a preparation of ethanol extract of Schisandra sphenanthera) on the pharmacokinetic process of digoxin (DG, a classical P-gp substrate) after intravenous and oral administration in rats. The effect of Schisandra lignans on the transportion of DG in Caco-2 cells was further elucidated. Our data showed that the plasma concentrations of DG were increased to different extent following co-administration of schisandrin A, schisandrin B, schisandrol B and schisantherin A, respectively. Schisandrol B showed the most potent effect among the six lignans. However, schisandrin C and schisandrol A showed little effect on pharmacokinetic of DG. Schisandrol B led to 99.0% (P < 0.05) and 109.2% (P < 0.05) increase in the AUC after orally or intravenously administered of DG, suggesting that co-administration of schisandrol B induced a more potent effect on increasing hepatic bioavailability of DG than that of intestinal. Furthermore, in vitro transport experiment showed that schisandrin A, schisandrin B, schisandrol B and schisantherin A inhibited P-gp-mediated efflux of DG, suggested that these lignans inhibited the P-gp-mediated efflux of DG. In conclusion, the exposure of DG in rats was increased when co-administered with Schisandra lignans, and schisandrol B showed the strongest effect. The dramatic increase in oral bioavailability of digoxin in the presence of schisandrol B may be due to the inhibition of hepatic/renal P-gp activity.

Schisandra sphenanthera extract (Wuzhi Tablet) protects against chronic-binge and acute alcohol-induced liver injury by regulating the NRF2-ARE pathway in mice.

Alcohol abuse leads to alcoholic liver disease and no effective therapy is currently available. Wuzhi Tablet (WZ), a preparation of extract from Schisandra sphenanthera that is a traditional hepato-protective herb, exerted a significant protective effect against acetaminophen-induced liver injury in our recent studies, but whether WZ can alleviate alcohol-induced toxicity remains unclear. This study aimed to investigate the contribution of WZ to alcohol-induced liver injury by using chronic-binge and acute models of alcohol feeding. The activities of ALT and AST in serum were assessed as well as the level of GSH and the activity of SOD in the liver. The expression of CYP2E1 and proteins in the NRF2-ARE signaling pathway including NRF2, GCLC, GCLM, HO-1 were measured, and the effect of WZ on NRF2 transcriptional activity was determined. We found that both models resulted in liver steatosis accompanied by increased transaminase activities, but that liver injury was significantly attenuated by WZ. WZ administration also inhibited CYP2E1 expression induced by alcohol, and elevated the level of GSH and the activity of SOD in the liver. Moreover, the NRF2-ARE signaling pathway was activated by WZ and the target genes were all upregulated. Furthermore, WZ significantly activated NRF2 transcriptional activity. Collectively, our study demonstrates that WZ protected against alcohol-induced liver injury by reducing oxidative stress and improving antioxidant defense, possibly by activating the NRF2-ARE pathway.

Metabolic mapping of Schisandra sphenanthera extract and its active lignans using a metabolomic approach based on ultra high performance liquid chromatography with high-resolution mass spectrometry.

Schisandra sphenanthera, the dried ripe fruit of Schisandra sphenanthera Rehd. et Wils, is widely used as a restorative, tonic and nutrition in many countries. Wuzhi tablet, an ethanol extract preparation of Schisandra sphenanthera, is a well-known herbal medicine widely used in China. Our previous studies show that Wuzhi tablet and its active lignans significantly protect liver injury. However, its metabolic profile remains unknown in vivo and in vitro. In this study, ultra high performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry based metabolomics was employed to decipher the metabolic map of Wuzhi tablet and its active lignans. Serum (2 h) and urine (24 h) samples after a 700 mg/kg single oral dose of Wuzhi tablet, and mice liver microsome samples after incubation with its active lignans were collected and analyzed. The data were further analyzed using metabolomics and metabolite identification software. In total, 33 metabolites in vivo and 34 metabolites in vitro were identified, and six among them were new metabolites. The major metabolic reactions encompassed demethylation, hydroxylation, dehydrogenation, and epoxidation. Taken together, in vitro and in vivo studies revealed the metabolic profile of Wuzhi tablet and its active lignans and demethylation and hydroxylation were their major metabolic pathways.

Simultaneous Determination of 10 Lignanoids in Wuzhi Tablets by RP-HPLC / 中国药房

OBJECTIVE:To establish a method for content determination of schizantherin E,gomisin J,angeloylgomisin H,schisantherin A,schisantherin B,schisanhenol,anwuligan,schizandrin A,schizandrin B and schizandrin C in Wuzhi tablets.METHODS:RP-HPLC method was adopted.The determination was performed on Symmetry C18 column with acetonitrile-0.1％ phosphoric acid solution (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelength was set 225 rm,and the column temperature was 30 ℃.The sample size was 10 μL.RESULTS:The linear ranges of schizantherin E,gomisin J,angeloylgomisin H,schisantherin A,schisantherin B,schisanhenol,anwuligan,schizandrin A,schizandrin B and schizandrin C were 2.25-67.5ng(r=0.999 6),2.1-63 ng(r=0.999 8),28-840 ng(r=0.999 9),124.6-3 738 ng(r=0.999 9),22.7-681 ng(r=0.999 9),32.7-981 ng(r=0.999 9),47-1 410 ng(r=0.999 9),208-6 240 ng(r=0.999 9),5.36-160.8 rig(r=0.999 9),4.48-134.4 ng(r=0.999 8).The limits of quantitation were 14.17,13.32,9.33,11.37,14.62,19.88,14.66,12.50,16.40,13.55 rg.The limits of detection were 4.62,4.60,3.08,3.76,4.81,6.74,4.93,4.16,5.86,5.03 ng.RSDs of precision,stability and reproducibility tests were less than 3.0％;the recoveries were 96.36％-100.00％(RSD=1.83％,n=6),95.00％-100.00％(RSD=2.07％,n=6),95.00％-98.00％(RSD=1.22％,n=6),95.37％-98.91％ (RSD=1.29％,n=6),95.62 ％-103.71％ (RSD=2.85％,n=6),97.33％-102.67％ (RSD=2.00％,n=6),95.00％-99.33％ (RSD=1.75％,n=6),97.24％-104.93％ (RSD=2.63％,n=6),95.00％-97.50％ (RSD=1.42％,n=6),96.00％-102.00％ (RSD=2.45％,n=6),respectively.CONCLUSIONS:The developed method is accurate,sensitive and reproducible,and it can be used for content determination of 10 lignanoids in Wuzhi tablets.

Effect of long-term treatment of Wuzhi tablet on the expression and activity of cytochrome P450 3A in rats / 药学学报

The study aims to evaluate the effect of long-term pretreatment of the rat with Wuzhi tablet (WZ) on hepatic and intestinal CYP3A mRNA and protein expression and activity. Male Sprague-Dawley rats were orally administered of midazolam (2 mg·kg-1) with or without 14 days of pretreatment of WZ (0.25 g·kg-1) to determine CYP3A activity. Meanwhile, RNA and protein of rats liver and intestine samples were prepared 24 h after the last dose of 14 days of WZ treatment to determine CYP3A mRNA and protein expression. Long-term treatment of WZ increased the mRNA expression of hepatic Cyp3a1, Cyp3a9 and intestinal Cyp3a9 by 54.6%, 188.3% (PP<0.05), respectively;and increased the protein expression of hepatic CYP3A by 43.2%. However, after long-term treatment of WZ, the AUC of orally administered of midazolam in the WZ group was increased 29.9% (WZ pretreatment group) and 154.2% (WZ coadministered group) compared to that of control group. In conclusion, long-term treatment of WZ increased the mRNA and protein expression of CYP3A, while could inhibit the activity of CYP3A.

Determination of hepatic and small intestinal distribution of lignans of Wuzhi tablet in rats by liquid chromatography tandem mass spectrometry method / 药学学报

This study was aimed to determine the hepatic and small intestinal distribution of active lignans in rats after treated with Wuzhi tablet (WZ, Schisandra sphenanthera extract) by LC-MS/MS method. Male Sprague-Dawley rats were sacrificed at 0.25, 1.5, 4, 6, 10, 24 h after an oral administration of WZ, and then hepatic and small intestinal samples were collected for analysis. The results showed that concentrations of lignans in liver and small intestine of rats were decreased with WZ pretreated time. The concentrations of all lignans in rat liver and small intestine at 0.25 h were the highest after a single oral administration. All lignans was undetectable in all tissues 24 h after oral dosing, suggesting lignans of WZ were eliminated rapidly in rats. The concentrations of schisandrin A, schisandrol B and schisantherin A in small intestine were much higher than those in the liver, suggesting the effect of WZ on the intestinal metabolism enzyme might be more potent than that on the liver. In short, the current results suggest that lignans of WZ were not accumulated in rat liver and small intestine. The concentrations of lignans of WZ in small intestine were much higher than those in liver.

Protective effect of Wuzhi tablet (Schisandra sphenanthera extract) against cisplatin-induced nephrotoxicity via Nrf2-mediated defense response.

Cisplatin is a potent anti-cancer agent for various types of tumors. However, the clinical use of cisplatin is often limited by its nephrotoxicity. This study reports that WZ tablet (WZ, a preparation of an ethanol extract of Schisandra sphenanthera) mitigates cisplatin-induced toxicity in renal epithelial HK-2 cells and in mice. Pretreatment of HK-2 cells with WZ ameliorated cisplatin-induced cytotoxicity caused by oxidative stress, as was demonstrated by reductions in the levels of reactive oxygen species (ROS) and increased levels of glutathione (GSH). WZ facilitated the nuclear accumulation of the transcription factor NF-E2-related factor 2 (Nrf2) and the subsequent expression of its target genes such as NAD(P)H: quinine oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1) and glutamate cysteine ligase (GCL). Protective effects of WZ on cisplatin-induced nephrotoxicity were also observed in mice. WZ attenuated cisplatin-induced renal dysfunction, structural damage and oxidative stress. The nuclear accumulation of Nrf2 and its target genes were increased by WZ treatment. Taken together, these findings demonstrated WZ have a protective effect against cisplatin-induced nephrotoxicity by activation of the Nrf2 mediated defense response, which is of significant importance for therapeutic intervention in cisplatin induced renal injury.

Effect of Tacrolimus on the pharmacokinetics of bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) and the potential roles of CYP3A and P-gp.

We recently reported that Wuzhi tablet (WZ), a preparation of the ethanol extract of Wuweizi (Schisandra sphenanthera), had significant effects on blood concentrations of Tacrolimus (FK506) in renal transplant recipients and rats. The active lignans in WZ are schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, schisantherin A, and schisantherin B. Until now, whether the pharmacokinetics of these lignans in WZ would be affected by FK506 remained unknown. Therefore, this study aimed to investigate whether and how FK506 affected pharmacokinetics of lignans in WZ in rats and the potential roles of CYP3A and P-gp. After a single oral co-administration of FK506 and WZ, the blood concentration of lignans in WZ was decreased by FK506; furthermore, the AUC of schisantherin A, schisandrin A, schisandrol A and schisandrol B was only 64.5%, 47.2%, 55.1% and 57.4% of that of WZ alone group, respectively. Transport study in Caco-2 cells showed that these lignans were not substrates of P-gp, suggesting decreased blood concentration of lignans by FK506 was not via P-gp pathway. Metabolism study in the human recombinant CYP 3A showed that these lignans had higher affinity to CYP3A than that of FK506, and thus had a stronger CYP3A-mediated metabolism. It was concluded that the blood concentrations of these lignans were decreased and their CYP3A-mediated metabolisms were increased in the presence of FK506 since these lignans had higher affinity to CYP3A.

Effect of Wuzhi tablet (Schisandra sphenanthera extract) on the pharmacokinetics of cyclosporin A in rats.

In our previous reports, Wuzhi tablet (an herbal preparation of ethanol extract of Wuweizi (Schisandra sphenanthera)) can significantly increase the blood concentration of tacrolimus and paclitaxel in rats by inhibiting the CYP3A-mediated metabolism and the P-gp-mediated efflux. Cyclosporin A (CsA), a well-known immunosuppressant agent, is also a substrate of CYP3A and P-gp. Therefore, this study aimed to investigate whether and how WZ affects pharmacokinetics of CsA in rats. The AUC0-48 h and Cmax of CsA were increased by 40.1% and 13.1%, respectively, with a single oral co-administration of WZ and high dose of CsA (37.8 mg/kg). Interestingly, after a single oral co-administration of WZ and low dose of CsA (1.89 mg/kg), the AUC0-36 h and Cmax of CsA were dramatically increased by 293.1% (from 1103.2 ± 293.0 to 4336.5 ± 1728.3 ng.h/mL; p < 0.05) and 84.1% (from 208.5 ± 67.9 to 383.1 ± 92.5 ng/mL; p < 0.05), respectively. The CL/F was decreased from 1.7 L/h/kg to 0.5 L/h/kg. Thus, the effect of WZ on high dose of CsA was not significant, but pharmacokinetic parameters of CsA at low dose were significantly influenced by co-administration of WZ. The herb-drug interaction should be taken into consideration at this situation.