Effects of hydroxysafflor yellow A on the activity and mRNA expression of four CYP isozymes in rats

Ethnopharmacological relevance

In traditional therapy with Chinese medicine, hydroxysafflor yellow A (HSYA), a main active component isolated from the dried flower of Carthamus tinctorius L., is the principal efficiency ingredient of Safflor Yellow Injection. Now HSYA has been demonstrated to have good pharmacological activities of antioxidation, myocardial and cerebral protective and neuroprotective effects. The purpose of this study was to find out whether HSYA influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D4 and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied.

Materials and methods

A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), dextromethorphan (20 mg/kg) and midazolam (10 mg/kg), was given as oral administration to rats treated with short or long period of intravenous HSYA via the caudal vein. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC–MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time RT-PCR was performed to determine the effect of HSYA on the mRNA expression of CYP1A2, CYP2C11, CYP2D4 and CYP3A1 in rat liver.

Results

HSYA had significant inhibition effects on CYP1A2 and CYP2C11 in rats as oriented from the pharmacokinetic profiles of the probe drugs. Furthermore, HSYA had no effects on rat CYP2D4. However, CYP3A1 enzyme activity was induced by HSYA. The mRNA expression results were in accordance with the pharmacokinetic results.

Conclusions

HSYA can either inhibit or induce activities of CYP1A2, CYP2C11 and CYP3A1. Therefore, co-administration of some CYP substrates with HSYA may need dose adjustment to avoid an undesirable herb–drug interaction.     

Effect of Chinese herbs on CYP3A4 activity and expression in vitro

Ethnopharmacological relevance

Traditional Chinese Medicine (TCM) has become more popular among cancer patients in the Western world, who often use Chinese herbs as adjuvant therapy to reduce the adverse effects of conventional chemotherapy. However, pharmacokinetic (PK) interactions between Chinese herbs and anticancer drugs can occur and have dramatic consequences for these patients. Currently, only a few possible PK interactions between Chinese herbs and conventional Western drugs have been documented.

Aim of the study

Since the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) contributes to most of the PK interactions with (anticancer) drugs, the effect of four Chinese herbs (Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus) on the activity and expression of CYP3A4 was investigated in vitro.

Materials and methods

Ethanol and water–ethanol extracts of the four Chinese herbs were prepared from raw material. CYP3A4 inhibition was assessed by the use of Supersomes^™ in a fluorescence assay. Furthermore, CYP3A4 induction was evaluated in a human pregnane X receptor (hPXR)-mediated CYP3A4 reporter gene assay and a quantitative real time PCR assay, both in human colon adenocarcinoma-derived LS180 cells (LS180).

Results

Extracts of Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus inhibited CYP3A4 in human CYP3A4 Supersomes^™ (IC₅₀ values: 17–83 µg/mL). Oldenlandia diffusa and Rehmannia glutinosa significantly induced PXR-mediated CYP3A4 (p<0.001). Oldenlandia diffusa also significantly induced CYP3A4 mRNA levels (p<0.001 at 250 µg/mL).

Conclusions

Concomitant use of Oldenlandia diffusa and Rehmannia glutinosa could result in induction of CYP3A4, leading to a reduced efficacy of drugs that are CYP3A4 substrates and have a narrow therapeutic window. Because of the possible enhanced toxicity caused by CYP3A4 inhibition, clinical effects of CYP3A4 inhibition by Astragalus propinquus and Codonopsis tangshen must also be taken into account. In conclusion, herb–drug interactions between Chinese herbs and various CYP3A4 substrates can occur. Further research to investigate the clinical relevance of the interactions caused by Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus is required.     

Effects of Kang-Jia-Wan,a Chinese medicinal herb officinal,on apoptosis induction in goiter of rats

Ethnopharmacological relevance

Kang-Jia-Wan (KJW), a traditional Chinese herbal medicine, is widely used to treat goiter in the clinics in China.

Aim

The mechanisms by which KJW treats goiter are unclear. It is known that insufficient apoptosis of thyrocytes is involved in the formation of goiter. Here, we investigated whether KJW could induce apoptosis in goiter of rats given methimazole (MMI).

Materials and methods

Fifty-six Wistar rats were randomly separated into four groups: normal, MMI, MMI + low-dose KJW and MMI + high-dose KJW. Except for the normal group (20 rats), all groups (each with 12 rats) were given 0.04% (w/v) MMI in their drinking water. One week later, the rats in MMI + low- and high-dose KJW groups were orally supplemented with KJW at 250 mg/kg d⁻¹ and 1000 mg/kg d⁻¹, respectively.

Results

After KJW treatment for 12 weeks, the relative thyroid weight (mg/100 g body weight) of the MMI + high-dose KJW group decreased significantly. Features of apoptosis were also apparent in thyroid tissues of rats given KJW treatment. Importantly, KJW markedly increased the caspase-3 and Fas protein expression, in a dose-dependent manner, in the thyroid specimens.

Conclusions

These results showed that KJW played a therapeutic role via apoptosis induction in the goitrous glands.     

Antihyperglycemic and hypolipidemic activity of aqueous extract of Cassia auriculata L. leaves in experimental diabetes

Ethnopharmacological relevance

Cassia auriculata L. (Caesalpiniaceae) is widely used from ancient period to treat diabetes mellitus. The leaves of Cassia auriculata are having potential in the development of drug for diabetes due to its antihyperglycemic and lipid-lowering activity.

Aim of the study

The present study was to evaluate antihyperglycemic and hypolipidemic activity of aqueous extract of Cassia auriculata leaves (CLEt) in streptozotocin (STZ)-induced mild diabetic (MD) and severe diabetic (SD) rats.

Materials and methods

Male Albino rats were rendered diabetic by STZ (45 mg/kg, intraperitoneally). CLEt was orally administered to MD and SD rats at 100, 200 and 400 mg/kg doses for 1 day to determine antihyperglycemic activity. The 400 mg/kg dose was administered daily for 3 weeks to assess glycemic control and hypolipidemic effect.

Results

CLEt showed dose dependant fall in fasting blood glucose (FBG). After 5 h of extract administration at 400 mg/kg dose, FBG was reduced by 13.9% and 17.4% in MD and SD rats respectively. After 3 weeks treatment, CLEt produced significant reduction in FBG and glycosylated haemoglobin (GHb) in both MD and SD rats. Serum lipid levels were reversed towards normal in extract fed MD and SD rats.

Conclusions

The results demonstrate that CLEt possesses potent antihyperglycemic and hypolipidemic activity in both MD and SD rats.     

Glycyrrhizin accelerates the metabolism of triptolide through induction of CYP3A in rats

Ethnopharmacological relevance

Triptolide (TP), a major active component of Tripterygium wilfordii, possesses various pharmacological activities with narrow therapeutic window and severe toxicities. Glycyrrhizin (GL), the principal bioactive ingredient of licorice root extract, has been reported to be concomitantly administered with TP in treatment of rheumatoid arthritis with the aim of potentiated efficacy and reduced toxicity. The aim of the study is to investigate the effect of GL on the pharmacokinetic profiles of TP and related mechanisms.

Materials and methods

Male and female Wistar rats were randomly divided into two groups: Control group and GL group (pretreated with GL at 100 mg/kg/day for seven consecutive days). After oral administration of TP at a single dose of 450 μg/kg, plasma concentrations of TP were determined using HPLC–MS/MS and pharmacokinetic parameters were calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 software. Since CYP3A is the primary isoform of cytochrome P450s responsible for the metabolism of TP, we further determined to what extent ketoconazole (KCZ), a potent CYP3A inhibitor, could influence the effect of GL on the pharmacokinetics of TP by comparing the pharmacokinetic profiles of TP in GL group (pretreated with GL) and GL+KCZ group (pretreated with both GL and KCZ), as well as verified whether pretreatment of GL could induce the activity of hepatic CYP3A by comparing the AUC parameters after intravenous administration of midazolam (MDZ), a typical probe drug for CYP3A, in rats pretreated with vehicle or GL.

Results

Our study revealed marked differences in pharmacokinetic profiling patterns of TP between male and female rats in the Control group; the plasma level of TP in males was far lower than that in females. After pretreatment with GL, the pharmacokinetic profiles of TP were significantly altered in both male and female rats; a remarkable decrease was found in the value of AUC_∞, MRT_∞ and t_1/2 in the GL group, compared with the Control group. But such a decrease was reversed by KCZ; compared with the GL group, the values of AUC_∞, MRT_∞ and t_1/2 were significantly increased in the GL+KCZ group. Pretreatment with GL notably increased the AUC_∞ of 1?-hydroxymidazolam (OH-MDZ) and the ratio of AUC_∞ of OH-MDZ to MDZ, demonstrating induction of the activity of CYP3A by GL.

Conclusion

Pretreatment with GL significantly accelerates the metabolic elimination of TP from the body mainly through induction of hepatic CYP3A activity. These results may help explain why toxicity of TP may be attenuated with concomitant use of GL.     

In vitro effects of active components of Polygonum Multiflorum Radix on enzymes involved in the lipid metabolism

Ethnopharmacology relevance

Raw and processed Polygoni Multiflori Radix (PMR and PMRP) are used in the prevention and treatment of non-alcoholic fatty liver disease (NAFLD), hyperlipidemia or related diseases. In our previous research, 2, 3, 5, 4′-tetrahydroxy-stilbene-2-O-β-d-glucoside (TSG) displayed the most important role in the total cholesterol (TC) lowering effect among all the chemical constituents of Polygonum multiflorum. Emodin and physcion displayed more favorable triglyceride (TG) reducing effects than TSG. However, there are few researches focus on the approach and mechanism of how do Polygonum multiflorum exhibit good lipid regulation activity. The targeted sites of active substances of Polygonum multiflorum are still not clearly elucidated. This research pays close attention to how major chemical components of Polygonum multiflorum affect the TC and TG contents in liver cells.

Materials and methods

In this research, a sensitive, accurate and rapid in vitro model, steatosis hepatic L02 cell, was used to explore target sites of active chemical substances of Polygonum multiflorum for 48 h. Steatosis hepatic L02 cell was exposed to emodin, physcion and TSG, respectively. The contents of four key enzymes in the pathway of synthesis and decomposition of TC and TG were investigated after exposure. Meanwhile, the contents of lipid transfer protein were also tested. The diacylgycerol acyltransferase 1 (DGAT1) controlled the biosynthesis of TG from free fatty acids while 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) limited the biosynthesis of TC. Hepatic triglyceride lipase (HTGL) and cholesterol 7α-hydroxylase (CYP7A) played the key role in the lipolysis procedure of TG and TC.

Results

The synthesis of TC and TG in steatosis L02 cells were apparently increased in the model group compared to the control group. Intracellular contents of HMG-CoA reductase and DGAT1 increased 32.33% and 56.52%, while contents of CYP7A and HTGL decreased 21.61% and 47.37%. Emodin, physcion and TSG all showed down-regulation effects on HMG-CoA reductase, while up-regulation effects on CYP7A. The most remarkable effect on HMG-CoA reductase was found on emodin. Emodin could reduce the DGAT1 content from 438.44±4.51 pg/mL in model group to 192.55±9.85 pg/mL (100 μm). The content of HTGL in 300 μm physcion group was 3.15±0.15 U/mL, which was more significantly effective than the control, lovastatin and fenofibrate group.

Conclusions

TSG could raise the content of CYP7A and then promote the lipolysis of cholesterol. Moreover, TSG also showed the best LDL-reducing effect. Emodin could inhibit HMG-CoA reductase and DGAT1, which were key enzymes in the synthesis of TC and TG. Physcion increased the content of HTGL, and then could boost the lipolysis of triglyceride. At the same time, physcion showed the best VLDL-reducing effect. In view of the above conclusions, we contributed the lipid regulation activity to an overall synergy of TSG, emodin and physcion.     

Treatment with an extract of Terminalia superba Engler & Diels decreases blood pressure and improves endothelial function in spontaneously hypertensive rats

Ethnopharmacological relevance

The stem bark of Terminalia superba (TS) is widely used as a decoction by Cameroonian folk medicine for the treatment of hypertension. The aim of the present study was to evaluate the effect of a chronic treatment with a TS extract on spontaneously hypertensive rats (SHR) with respect to efficacy, biochemical mechanisms and safety.

Materials and methods

Eleven-week-old SHR and normotensive Wistar Kyoto rats (WKY) were daily treated by gavage with a methylene chloride extract of stem bark of Terminalia superba (TMSE, 150 mg/kg) or with the vehicle for 5 weeks. Systolic blood pressure (SBP) was measured weekly using the tail-cuff method. At the end of the treatment period, vascular function was assessed on isolated thoracic rings, urinary 8-iso-PGF_2α levels were measured and cytochrome P-450 3A (CYP 3A) activity was evaluated in liver microsomes.

Results

TMSE reduced SBP (P<0.001) in SHR but not in WKY rats. In SHR, the vasorelaxant response to acetylcholine was significantly improved by TMSE as a result of increased nitric oxide synthase (NO) activity and decreased superoxide anion production. In addition, TMSE reduced the vasoconstrictive effect of phenylephrine and improved the sensitivity of smooth muscle cells to NO. TMSE dramatically decreased 8-iso-PGF_2α levels in SHR. By contrast, TMSE did not affect all these parameters in WKY rats. Neither diuresis nor the hepatic CYP 3A activity was modified in both animal groups.

Conclusions

This study demonstrated that Terminalia superba has a potent antihypertensive activity in SHR which is partly due to endothelium-dependent and endothelium-independent effects as well as decreased oxidative stress. The data also provide evidence for the lack of herb-drug interaction through hepatic CYP 3A.     

Evaluation of hepatotoxicity and cholestasis in rats treated with EtOH extract of Fructus Psoraleae

Ethnopharmacological relevance

Fructus Psoraleae (FP) has been widely used to heal skin diseases as well as osteoporosis, osteomalacia, and bone fracture. There also exist many clinical reports about FP-induced hepatotoxicity associated with acute cholestatic hepatic injury. However, the FP-induced hepatotoxicity and the underlying mechanisms remain unclear.

Aims of the study

The present study aims to determine the hepatotoxicity of FP in Sprague-Dawley (SD) rats and to investigate the underlying mechanisms.

Materials and methods

Sprague-Dawley rats of both sexes were intragastrically administered with the EtOH extract of FP (EEFP) at doses of 1.875, 1.25 and 0.625 g/kg for 28 day. Body weight, relative liver weight, biochemical analysis, histopathology, the mRNA and protein expression of Cholesterol 7α-hydroxylase (CYP7A1), farnesoid X receptor (FXR), bile-salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance-associated protein 3 (MRP3) were evaluated to study the EEFP-induced hepatotoxicity and its underlying mechanisms.

Results

Many abnormalities were observed in the EEFP-treated groups including suppression of weight gain and food intake, change of some parameters in serum biochemistry, increased weight of liver, and decreased concentration of bile acid in bile. The mRNA and protein expression of CYP7A1, MRP3, MRP2, BSEP increased and the expression of FXR decreased in EEFP-treated female groups; the mRNA and protein of FXR and CYP7A1 decreased and that of the others remained the same in EEFP-treated male groups.

Conclusion

In conclusion, we provide evidence for the first time that EEFP can induce sex-related cholestatic hepatotoxicity, and that female rats are more sensitive to EEFP-induced hepatotoxicity, which involves the destruction of the biosynthesis and transportation of bile acid. Further investigation is still needed to uncover the mechanism of the sex-dimorphic EEFP-induced hepatotoxicity.     

Effects of aescin on cytochrome P450 enzymes in rats

Ethnopharmacological relevance

Aescin, the main active component found in extracts of horse chestnut (Aesculus hippocastanum) seed a traditional medicinal herb, is a mixture of triterpene saponins. It has been shown to be effective in inflammatory, chronic venous and edematous treatment conditions in vitro and in vivo, and is broadly used to treat chronic venous insufficiency. The purpose of this study was to find out whether aescin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied.

Materials and methods

A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg), was given as oral administration to rats treated with a single dose or multiple doses of intravenous aescin via the caudal vein. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC–MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time RT-PCR was performed to determine the effects of aescin on the mRNA expression of CYP1A2, CYP2C9, CYP2E1 and CYP3A4 in rat liver.

Results

Treatment with a single dose or multiple doses of aescin had inductive effects on rat CYP1A2, while CYP2C9 and CYP3A4 enzyme activities were inhibited. Moreover, aescin has no inductive or inhibitory effect on the activity of CYP2E1. The mRNA expression results were in accordance with the pharmacokinetic results.

Conclusions

Aescin can either inhibit or induce activities of CYP1A2, CYP2C9 and CYP3A4. Therefore, caution is needed when aescin is co-administration with some CYP1A2, CYP2C9 or CYP3A4 substrates in clinic, which may result in treatment failure and herb-drug interactions.     

A new TCM formula FTZ lowers serum cholesterol by regulating HMG-CoA reductase and CYP7A1 in hyperlipidemic rats

Ethnopharmacological relevance

Based on a theory of Chinese Medicine, Regulating Gan (liver) to lower lipids that is in brief to regulate the lipid metabolic related factors in the liver will improve serum lipid profile, we have developed Fufang Zhenzhu Tiao Zhi (FTZ) which includes eight herbs that are quality assured. FTZ has been developed with the potential to correct abnormal lipid metabolism. This Chinese herbal medicine has been prescribed for 20 years, which has been issued patent and clinically proven for use in the treatment of dyslipidemia.

Aim of the study

To investigate the cholesterol-lowering effect and the mode of action of FTZ extract on high lipid diet induced hyperlipidemic rats.

Materials and methods

The FTZ was prepared by alcohol and water extraction of eight herbs that have been quality-controlled according to the protocol. The cholesterol-lowering effect of FTZ was evaluated on SD rats fed with high-lipid diet. RT-PCR and western blot were used to analyze the gene expression of cholesterol metabolism-related enzymes including HMG-CoA reductase and cholesterol 7α-hydroxylase (CYP7A1) in the livers of the rats. The activity of HMG-CoA reductase and CYP7A1 were assessed by colorimetrical method and by quantification of the cholesterol metabolite of CYP7A1 using HPLC analysis respectively.

Results and conclusions

FTZ significantly decreased the levels of serum total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), whilst elevated the serum high-density lipoprotein cholesterol (HDL-C) and decreased serum atherogenic index (A.I.) values in high lipid diet induced hyperlipidemic rats. Furthermore, FTZ showed significant antihyperlipidemic effect by at least three pathways in the high lipid diet induced hyperlipidemic rats: (1) upregulating the gene expression and activity of CYP7A1 which promotes the conversion of cholesterol into bile acid; (2) downregulating the gene expression and activity of HMG-CoA reductase to reduce de novo synthesis of cholesterol; (3) increasing the cholesterol excretion from feces. In these three pathways, HMG-CoA reductase and CYP7A1 are two pivotal enzymes in lipid cholesterol metabolism and are expressed mainly in hepatic cells, which support our new TCM treatment strategy: Modulating Liver to Treat Hyperlipemia.     

Pharmacokinetic changes of unbound theophylline are due to plasma protein binding displacement and CYP1A2 activity inhibition by baicalin in rats

Ethnopharmacological relevance

Baicalin is one of the major bioactive constituents of Scutellariae Radix, the root of Scutellariae baicalensis Georgi and possesses a wide variety of pharmacological properties.

Aim of the study

To elucidate the effect of baicalin on the pharmacokinetics of theophylline in rats, focusing on plasma protein binding displacement and inhibition effect on CYP1A2 in vivo and in vitro.

Materials and methods

The study was a randomized, three-period crossover design. Nine rats were given saline (control) or 450 mg/kg baicalin (dosage regimen A or B). Dosage regimen A was administered once at 0 h. Dosage regimen B was divided into three dosages (225,112.5, 112.5 mg/kg) and was given at 0, 2 and 4 h, respectively. Then theophylline (5 mg/kg, i.v.) was administered immediately. The effect of baicalin on CYP1A2 activity was determined by metabolism of phenacetin in vitro and plasma protein binding of theophylline was determined by ultrafiltration.

Results

C_max decreased from (12.4±1.6) to (8.7±0.9) and (8.6±2.0) mg/L, T_1/2 increased by 116 and 96%, V_d increased by 51 and 49% for total theophylline in rats treated with dosage regimen A and B of baicalin, respectively. C_max was significantly increased, V_d decreased by 43 and 29% for unbound theophylline in rats treated with dosage regimen A and B of baicalin, respectively (P<0.01). T_1/2 of unbound theophylline increased by 104% only in rats treated with dosage regimen B. No significant effects on the CL and AUC of both total and unbound theophylline were observed in the rats treated with dosage regimen A, but the CL decreased and AUC increased for total theophylline and CL decreased for unbound theophylline in the group treated with dosage regimen B (P<0.05). Correlation analysis showed that the mean unbound theophylline (%) and mean baicalin concentration was in good correlation (P<0.01). Baicalin decreased metabolism of phenacetin and exhibited a mixed-type inhibition in rat liver microsomes, with a K_i value of 88.1 μM in vitro. Moreover baicalin was a competitive displacer of theophylline from plasma protein in vitro.

Conclusions

The changes in C_max, T_1/2, CL and AUC of theophylline due to baicalin may be attributed to two mechanisms, plasma protein binding displacement and CYP1A2 activity inhibition.     

Triptolide promotes generation of FoxP3+ T regulatory cells in rats

Ethnopharmacological relevance

Triptolide (TPT), a component of the Chinese herb Triptergium wilfordii, has potent immunosuppressive and anti-inflammatory activity and is used clinically in recipients of kidney transplantation.

Aim of the study

This work aimed to investigate the effect of TPT on the differentiation of regulatory T lymphocytes (Tregs) from CD4+ cells in rats.

Materials and methods

MACS-purified rat CD4+ cells were costimulated with anti-CD3 and anti-CD28 in the presence of TGF-β to induce the expression of FoxP3, which was detected by flow cytometry. TPT and cyclosporine A (CsA) were separately added into the cultures to observe the effect on the expression of FoxP3. Kidney transplantation was performed in rats that either received no treatment or were treated with TPT after transplantation.

Results

TPT treatment enhanced the expression of FoxP3 in CD4+ cells, whereas CsA inhibited the FoxP3 expression. In the rat kidney transplantation model, the recipient rats treated with TPT survived longer than the control rats (18–19.83 vs 6.83 days, P < 0.05). Meanwhile, the FoxP3+ T cells in the spleens of treated rats were higher than those from the untreated rats (12.4% vs 4.7%, P < 0.05).

Conclusions

These data suggest that TPT may promote the differentiation of CD4+ cells to FoxP3+ Tregs. This would be at least one of the pathways responsible for the immunosuppressive activity of TPT.     

Andrographolide inhibits the expression and metabolic activity of cytochrome P450 3A4 in the modified Caco-2 cells

Aim of the study

The aim of this study is to examine the effects of andrographolide on intestinal enzyme cytochrome P450 3A4 (CYP3A4) and predict whether oral administration of andrographolide-containing remedy leads to herb–drug interaction.

Materials and methods

Caco-2 cells are treated with 1α, 25-dihydroxyvitamin D3 for 3 wks to induce the expression of CYP3A4, and then andrographolide (1, 10, 100 μM) is added and treated for 72 h. Upon the further 4-h testosterone (250 μM) or nifedipine (200 μM) treatment, the basolateral medium samples and the Caco-2 monolayers are collected for analyses.

Results

Andrographolide (1, 10, 100 μM) significantly down-regulates the mRNA level and protein level of CYP3A4, and inhibits nifedipine oxidation and testosterone 6β-hydroxylation.

Conclusion

Oral administration of andrographolide likely leads to reduction of the metabolic activity of intestinal CYP3A4, therefore herb preparations containing andrographolide may result to herb–drug interactions in combination therapy.     

Effects of unprocessed versus vinegar-processed Schisandra chinensis on the activity and mRNA expression of CYP1A2, CYP2E1 and CYP3A4 enzymes in rats

Ethnopharmacological relevance

Schisandra chinensis (SC) is a well-known traditional Chinese herbal medicine that has been used in clinical practices for thousands of years. However, the differences between the effects of unprocessed and vinegar-processed Schisandra chinensis (VSC) on cytochrome P450 (CYP450) activities are poorly understood.

Aim of the study

To evaluate the differences between processed and unprocessed SC on the metabolism of CYP1A2, CYP2E1 and CYP3A4 substrates in rats using a cocktail method based on a developed and validated HPLC method. We also investigate the influence of processing on the levels of CYP mRNA.

Materials and methods

Three probe substrates (theophylline, dapsone and chlorzoxazone) were delivered simultaneously into rats treated with single or multiple doses of processed or unprocessed SC extract. The plasma concentrations of the three probes were profiled by HPLC, and their corresponding pharmacokinetic parameters were calculated. Real-time RT-PCR was performed to determine the effects of processed and unprocessed SC on the mRNA expression of CYP1A2, CYP2E1 and CYP3A4 in the liver.

Results

Treatment with single or multiple doses of either extract of SC induced CYP3A4 enzyme activity and inhibited CYP1A2 enzyme activity in rats. Furthermore, the inhibitory effect of SC was more potent after vinegar processing than without vinegar processing. CYP2E1 enzyme activity was induced after treatment with a single dose but was inhibited after multiple doses. The mRNA expression results were in accordance with the pharmacokinetic results.

Conclusions

These results provide useful scientific data for the safe clinical application of either extract of SC in combination with other drugs, which should lack the side effects induced by other herb–drug interactions.     

Effects of Bu-Zhong-Yi-Qi-Tang on hepatic drug-metabolizing enzymes and plasma tolbutamide concentration in rats

Ethnopharmacological relevance

Bu-Zhong-Yi-Qi-Tang (BT) is the dry powder derived from the aqueous extract of a mixture of 10 medicinal herbs. It is a traditional Chinese medicine being used for the treatment of various immune-related diseases.

Aim of the study

To investigate the effect of BT on hepatic drug-metabolizing enzymes and its effect on plasma concentrations of tolbutamide, a substrate of CYP2C, in rats.

Materials and methods: Exp 1

Thirty-two male Wistar rats were divided into four groups. Rats were fed a control diet and a control diet containing 1, 2.5 and 5% (w/w) of BT, respectively, for eight weeks. The activities of the major CYP and Phase II conjugating enzymes in rat liver microsomes as well as the antioxidant system in rat liver were assessed. Exp 2: Male Wistar rats were fed a control diet or a control diet containing 2.5% of BT, respectively, for eight weeks. A single 20-mg/kg oral dose of tolbutamide was then administered to each rat. Plasma samples were collected from each rat at 0.5, 1, 2, 4 and 8 h after dosing. The concentrations of tolbutamide and glucose level in plasma were determined by high-performance liquid chromatography-mass spectrometer (HPLC/MS) and enzymatic method, respectively.

Results

Significant decrease in microsomal CYP2C-catalyzed diclofenac 4-hydroxylation in the liver of rats fed the BT diet was observed. Increased UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) activities were also observed in the liver of rats fed the diet containing 2.5 and 5% of BT. Immunoblot analyses also showed decreases of CYP2C11 proteins in the liver of BT fed rats. In addition, rats fed the 2.5% BT diet for eight weeks had no effects on the disposition of tolbutamide and reduction of glucose level in plasma after orally administered of tolbutamide.

Conclusions

Rats fed the BT diet for eight weeks may decrease CYP2C enzyme activity and protein expression and increase Phase II conjugating enzyme activities in liver. However, BT may not affect the disposition and efficacy of tolbutamide.     

Effects of Campomanesia xanthocarpa on biochemical,hematological and oxidative stress parameters in hypercholesterolemic patients

Ethnopharmacological relevance

In Southern Brazil, the plant Campomanesia xanthocarpa Berg. (Myrtaceae), popularly known as “guavirova”, has been empirically used for its potential effect in reducing blood cholesterol levels.

Aim of the study

Since there are no scientific data confirming its popular use, the aim of the present study was to investigate the effect of Campomanesia xanthocarpa on biochemical, hematological, anthropometrical and oxidative stress parameters in hypercholesterolemic patients.

Materials and methods

Thirty three patients were selected according to total cholesterol (TC) levels: 200–240 mg/dL, undesirable level (UL), and >240 mg/dL, hypercholesterolemic level (HL). UL or HL patients were randomly divided into control group (CG), which received placebo capsules, and experimental group 250 (EG 250) or 500 (EG 500), which received either 250 or 500 mg of encapsulated Campomanesia xanthocarpa. All groups received a cholesterol restriction diet and capsules once a day. The biochemical (TC, triglycerides, HDL, LDL and VLDL), hematological (hematocrit and hemoglobin), anthropometrical (weight and abdominal circumference) and oxidative stress (protein carbonyl) parameters were measured before, 45 and 90 days after the treatment started.

Results

There was no alteration on biochemical, hematological, anthropometric or oxidative stress parameters in UL patients of all groups. However, a significant decrease in TC and LDL levels was observed in HL patients from EG 500 group (reduction of 28 ± 3% and 45 ± 4% to levels before treatment) in relation to CG group patients (reduction of 12 ± 2% and 29 ± 4%). Moreover, a significant reduction in oxidative stress was observed in HL patients of EG 250 (51 ± 12%) and EG 500 groups (34 ± 18%) when compared to levels before treatment. A positive correlation between plasma oxidative stress PC and TC levels was observed. Finally, we found that Campomanesia xanthocarpa extract possesses anti-oxidant properties and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitory activity in vitro.

Conclusion

Confirming its popular use, the treatment with Campomanesia xanthocarpa encapsulated reduced blood TC and LDL levels in hypercholesterolemic patients.     

Protective effect of Danhong injection on cerebral ischemia–reperfusion injury in rats

Ethnopharmacological relevance

Danhong injection (DH), a Chinese medical product, is used extensively for the treatment of cerebrovascular diseases such as acutely cerebral infarction in clinic.

Aim of the study

To explore the protective effect and the relevant mechanisms of DH on cerebral ischemia–reperfusion (I/R) injury.

Materials and methods

Cerebral I/R injury was induced through four-vessel occlusion (4-VO) or middle cerebral artery occlusion (MCAO). Adult male SD rats were randomly divided into six kinds of groups: normal control group, sham-operated group, I/R injury group, DH-treated groups at doses of 0.5 ml/kg, 1.0 ml/kg and 2.0 ml/kg. The effects of DH on murine neurological deficits and cerebral infarct volume, 6-keto-prostagladin F_1α (6-keto-PGF_1α) level, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in brain tissue, as well as the activities of plasma tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) after I/R were evaluated. Moreover, the expressions of Bcl-2 and Bax protein were detected by immunohistochemistry.

Results

There was no significant difference between the control group and the sham-operated group based on the measurement indicators. Compared with the vehicle-treated group, rats treated with DH showed dose dependent reductions in brain infarction size, and improvement of neurological outcome. The level of 6-keto-PGF_1α and the activities of SOD and plasma t-PA were enhanced significantly, whereas the level of MDA and the activity of plasma PAI were declined significantly. The immunohistochemical staining results also revealed that the expression of Bcl-2 protein was up-regulated and that of Bax protein was down-regulated when exposed to DH.

Conclusion

DH demonstrates a strong ameliorative effect on cerebral I/R damage in rats by its anticoagulant, antithrombotic, antifibrinolytic and antioxidant activities. Furthermore, suppressing apoptosis through regulating Bcl-2 and Bax protein expressions should be another potential mechanism by which DH exerts its neuroprotective function.