Influences of processed rhubarbs on the activities of four CYP isozymes and the metabolism of saxagliptin in rats based on probe cocktail and pharmacokinetics approaches

Ethnopharmacological relevance

Cooked rhubarb and wine processed rhubarb are the processed rhubarbs of raw rhizomes from Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or Rheum officinale Baill. They are clinically used in traditional Chinese medicine to compose anti-diabetic formulas and remove pathogenic heat or toxin from the body.

Aim of the study

To elucidate potential influences processed rhubarbs might have on the activities of four cytochrome P450 (CYP) isozyme in rats (CYP1A2, CYP2C6, CYP2E1, and CYP3A1) and on the pharmacokinetics of saxagliptin.

Materials and methods

Relative activity estimation of four isozymes or influence on saxagliptin was carried out by comparing plasma pharmacokinetics of four respective substrates (theophylline for CYP1A2, tolbutamide for CYP2C6, chlorzoxazone for CYP2E1, and dapsone for CYP3A1) or saxagliptin between control and processed rhubarbs pretreated groups. Plasma concentrations of substrates and saxagliptin were quantified using UPLC-UV and UPLC-MS/MS methods, respectively.

Results

Wine processed rhubarb induced CYP1A2 activity; both the processed rhubarbs inhibited the CYP2C6 activity and induced CYP2E1; cooked rhubarb induced CYP3A1 activity. Both the processed rhubarbs reduced the absorbance and bioavailability, but increased the clearance of saxagliptin.

Conclusions

Processed rhubarbs can either induce or inhibit activities of CYP1A2, CYP2C6, CYP2E1, and CYP3A1, and modify the metabolism of saxagliptin. The results indicated that drug co-administrated with processed rhubarbs may need dose adjustment.     

The effects of lignan-riched extract of Shisandra chinensis on amyloid-β-induced cognitive impairment and neurotoxicity in the cortex and hippocampus of mouse

Ethnopharmacological relevance

The fruits of Schisandra chinensis (Trucz.) Baill. (Schisandraceae) which have been used as a tonic especially for kidney yin deficiency in Chinese traditional medicine are recently receiving attention for its preventive activity on age-related neurodegenerative diseases. A variety of studies demonstrated the cognitive-enhancing effects of Schisandra chinensis through animal tests and also in clinical trials.

Aim of study

In this study, we attempted to investigate the effects of the lignan-riched extract of Schisandra chinensis fruits (ESP-806) on neurotoxicity and memory impairment induced by Aβ_1–42 injection in mice.

Materials and methods

The fruits of Schisandra chinensis were extracted with the mixture of n-hexane:ethanol (9:1), which is riched with bioactive dibenzocyclooctadiene lignans, schizandrin, gomisin N, wuweigisu C. After oral treatment of ESP-806 (100 mg/kg body weight) followed by injection of Aβ_1–42 (2 μg/mouse, i.c.v.), novel object recognition and passive avoidance tests were evaluated. To verify the cognition enhancing effects of ESP-806, we examined the effects of ESP-806 on the activities of β-secretase and acetylcholinesterase, and the contents of Aβ and the reduced glutathione within the cortex and hippocampus of Aβ-injected mice.

Results

Oral treatment of ESP-806 (100 mg/kg body weight) significantly attenuated Aβ_1–42-induced memory impairment evaluated by behavioral tests. Furthermore, the treatment of ESP-806 attenuated the elevation of β-secretase activity accompanying the reduced level of Aβ_1–42 in the cortex and hippocampus of the brain. ESP-806 also significantly inhibited the acetylcholinesterase activity in the hippocampus and increased the content of the reduced glutathione in the cortex and hippocampus of mouse brain.

Conclusions

These data suggested that the extract of Schisandra chinensis fruits riched with dibenzocyclooctadiene lignans may be useful in the prevention and treatment of Alzheimer's disease.     

Gender-dependent pharmacokinetics of lignans in rats after single and multiple oral administration of Schisandra chinensis extract

Ethnopharmacological relevance

Schisandra chinensis (S. chinensis), a traditional Chinese medicine, has been widely used as sedatives and tonics in clinic. Schisandra lignans are believed to be the major bioactive components in S. chinensis. However, there is a lack of information about the effects of gender and repeated-dose on the pharmacokinetic properties of the schisandra lignans.

Aim of the study

The study was performed to investigate the influence of gender on the pharmacokinetics of schisandra lignans after administration of S. chinensis extract and to compare their pharmacokinetic behaviors between single and multiple administration.

Materials and methods

Two groups of rats (half male and half female) were received a single dose or multiple doses of S. chinensis extract, respectively. A liquid chromatography–tandem mass spectrometry method was developed and validated to determine the plasma concentrations of schisandra lignans.

Results

The pharmacokinetic parameters of schisandrin, schisandrol B, deoxyschisandrin, γ-schisandrin and schisantherin A were significantly different by gender difference. The t_1/2 of all the tested schisandra lignans in female rats were 2–9 times longer than the corresponding values in male rats. The C_max and AUC_0−t of these schisandra lignans except schisantherin A in female rats were 5–50 times higher than those in male rats. The pharmacokinetic profiles of schisandrin, schisandrol B, deoxyschisandrin and schisantherin A in both gender rats after multiple doses were similar to the corresponding profile after single dose.

Conclusion

All the tested schisandra lignans showed slower elimination and higher bioavailability in female rats after single or multiple administration of S. chinensis extract compared with male rats. Their pharmacokinetic profiles were not affected by repeated-dose except γ-schisandrin, which was eliminated more slowly in female rats after multiple administration.     

Inhibitory effects of cytochrome P450 enzymes CYP2C8, CYP2C9, CYP2C19 and CYP3A4 by Labisia pumila extracts

Ethnopharmacological relevance

Labisa pumila (LP), popularly known with its local name, Kacip Fatimah, is a well known herb grown in Indochina and Southeast Asia and is traditionally used to regain energy after giving birth in women. The propensity of LP to cause drug–herb interaction via cytochrome P450 (CYP) enzyme system has not been investigated.

Aim of the study

To evaluate the in vitro inhibitory effects of various LP extracts (aqueous, ethanol, dichloromethane (DCM) and hexane) on cytochrome P450 2C8 (CYP2C8), CYP2C9, CYP2C19 and CYP3A4 activities.

Materials and methods

Probe substrate-based high performance liquid chromatography (HPLC) methods were established for CYP2C9, CYP2C19 and CYP3A4 whereas a fluorescence-based enzyme assay was established for CYP2C8. The metabolite formations were examined after incubation of probe substrate with respective CYP isoform in the present or absent of LP extracts. The inhibitory effect of LP was characterized with kinetic parameters IC₅₀ and K_i values.

Results

LP extracts showed differential effect of CYP activities with the order of inhibitory potency as follows: dichloromethane>hexane>ethanol>aqueous. This differential effect was only observed in CYP2C isoforms but not CYP3A4. Both the hexane and DCM extracts exhibited moderate to potent inhibition towards CYP2C activities in different modes including non-competitive, competive and mixed-type. The DCM effect was notably strong for CYP2C8 and CYP2C9 showing K_i values of below 1 μg/ml. The selectivity of LP for CYP2C isoforms rather than CYP3A4 may be attributed to the presence of relatively small, lipophilic yet slightly polar compounds within the LP extracts.

Conclusions

The results of our study revealed that phytoconstituents contained in LP, particularly in hexane and dichloromethane extracts, were able to selectively inhibit CYP2C isoforms. The inactivation was characterized by low K_i values, in particular, in CYP2C8 and CYP2C9. These in vitro data indicate that LB preparations contain constituents that can potently inhibit CYP2C activities and suggest that this herb should be examined for potential pharmacokinetic drug interactions in vivo.     

Identification of a novel anti-inflammatory compound, α-cubebenoate from Schisandra chinensis

Aims of the study

Extracts of Schisandra chinensis have been used as an anti-fatigue and tonic agent. Because chronic fatigue syndrome is related to inflammatory and oxidative stress, we assessed whether Schisandra chinensis has anti-inflammatory constituents and studied the effect of a novel α-cubebenoate isolated from Schisandra chinensis.

Materials and methods

α-Cubebenoate was isolated from an extract of Schisandra chinensis fruits. The inductions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) by lipopolysaccharide (LPS) were quantified by RT-PCR and Western blotting in mouse peritoneal macrophages. Nitric oxide (NO) and prostaglandin E₂ (PGE₂) were also measured in the media by Griess reagent and EIA method. A mouse model of LPS-induced peritonitis was used to test the in vivo efficacy of α-cubebenoate.

Results

α-Cubebenoate (5–10 μg/ml) inhibited the inductions of iNOS and COX-2 in mouse peritoneal macrophages at the mRNA and protein levels. LPS-induced productions of NO and PGE₂ were inhibited by α-cubebenoate (5–10 μg/ml). In addition, α-cubebenoate inhibited the LPS-induced activation of JNK, but not those of ERK and p38 MAPK in mouse peritoneal macrophages. Furthermore, in the LPS-induced in vivo peritonitis model, α-cubebenoate (1 mg/kg) strongly inhibited the accumulation of polymorph nuclear lymphocytes in the peritoneal cavity.

Conclusion

α-Cubebenoate inhibited LPS-induced expression of iNOS and COX-2 in a concentration-dependent manner, thereby suppressing productions of NO and PGE₂ in vitro in peritoneal macrophages. α-Cubebenoate also inhibited LPS-induced accumulation of polymorph nuclear lymphocytes in LPS-induced peritonitis model in vivo. α-Cubebenoate may act as an anti-fatigue constituent of Schisandra chinensis through anti-inflammation and could be of therapeutic use as a treatment for inflammatory diseases.     

Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes

Ethnopharmacological relevance

The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes.

Material and methods

A cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression.

Results

The cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Cl_int) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment.

Conclusion

We demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.     

The mechanism of vasorelaxation induced by Schisandra chinensis extract in rat thoracic aorta

Aim of the study

Schisandra chinensis (SC) is a known medical herb for the treatment of cardiovascular symptoms associated with menopausal symptoms in Korea. However, the pharmacological action mechanisms involved have not been well studied. This study was aimed to investigate the vascular effects of SC in rat thoracic aorta.

Materials and methods

We isolated the hexane, chloroform, and methanol extracts from SC and evaluated their vasodilatory effects in the rat thoracic aorta.

Results

Hexane extracts of SC (SCHE, 5 × 10⁻⁵ to 10⁻³ g/L) caused a concentration-dependent relaxation in both endothelium-intact and -denuded aortas. The relaxant effect of SCHE on the endothelium-intact aorta was more prominent than on the endothelium-denuded aorta. The former was significantly attenuated by L-NAME, a nitric oxide synthase inhibitor, and ODQ, a soluble guanyl cyclase inhibitor, but not by tetraethylammonium, a nonselective blocker of K⁺ channels, and indomethacin, a cyclooxygenase inhibitor. Furthermore, SCHE caused nitrite production as well as eNOS activation in aortic segments, suggesting implication of NO signal pathway in SCHE-induced relaxation. In endothelium-denuded aorta, SCHE-induced vasorelaxation was also attenuated by calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, but not by ML-9, a MLC kinase inhibitor, suggestive of implication of MLC phosphatase activation. Phenylephrine-enhanced MLC phosphorylation ratio was significantly attenuated by SCHE, which was recovered to the control level by pretreatment with calyculin A.

Conclusions

Taken collectively, these findings suggest that the vascular relaxation evoked by SCHE was mediated by not only endothelium dependent NO pathway but also direct effect on vascular smooth muscle cell via dephosphorylation of MLC.     

Identification and characterization of potent CYP2D6 inhibitors in lotus leaves

Ethnopharmacological relevance

The herb of lotus (Nelumbo nucifera) leaves is a commonly used traditional Chinese herbal medicine that is utilized for the treatment of sunstroke, to assuage thirst, and to cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the herb exhibits various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-HIV, anti-microbial, and anti-hypoglycemic activities. Currently, the herb is becoming more popular in China as a “tea drink” or as a main ingredient of some herbal formulations, which implies that the herb and/or its products are now more likely to be concurrently administered with conventional medicines for losing body weight and reducing blood lipids. However, its potential inhibitory effect on human cytochrome P450 (CYP) has not been systemically investigated to date. The present study was performed to assess the potential inhibitory effects of lotus leaf alcoholic extract (LAE), its major fractions, and its main compounds on five CYP isoenzymes (CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in vitro.

Material and methods

Five probe substrates were incubated with human liver microsomes in the presence or absence of the LAE, the alkaloid fraction (AF), the flavonoid fraction (FF), or the individual aporphine alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and 2-hydroxy-1-methoxyaporphine (HMA). After the incubation, the relative metabolites of the substrates were analyzed using LC–MS/MS.

Results

The results showed that the LAE strongly inhibited CYP2D6 with an IC₅₀ value of 12.05 µg/mL and weakly inhibited other isoenzymes. In addition, FF was found to weakly inhibit CYP2D6, whereas AF exerted a markedly higher inhibitory effect on CYP2D6 activity with an IC₅₀ value of 0.96 µg/mL. The three aporphine alkaloids isolated from the AF (NF, N-NF, and HMA) significantly inhibited CYP2D6 with IC₅₀ values of 3.78, 3.76, and 3.15 µM, respectively. Their Lineweaver–Burk plots and Dixon plots showed that NF, N-NF, and HMA competitively inhibited CYP2D6 activity with K_i values of 1.88, 2.34, and 1.56 µM, respectively.

Conclusion

The study revealed that the alkaloid compounds in lotus leaves exert a potent inhibitory effect on CYP2D6 isoenzyme. The possible drug interactions of the leaves and their preparations with conventional medicines should thus be taken into account.     

Modulatory effects of Kaempferia parviflora extract on mouse hepatic cytochrome P450 enzymes

Ethnopharmacological relevance

Kaempferia parviflora is a herbal plant, the extracts of which are commonly used as alternative medicines. It widely uses as aphrodisiac, anti-inflammation, anti-microbacterial, and anti-peptic ulcer.

Aim of the study

In order to obtain an effective utilization and safety of the herb, the influence of Kaempferia parviflora on hepatic CYP450 metabolizing enzymes including CYP1A1, CYP1A2, CYP2B, CYP2E1, and CYP3A was investigated.

Materials and methods

The impact of Kaempferia parviflora on CYP450 both in vitro and in vivo was examined by using ethoxyresorufin O-dealkylation, methoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, p-nitrophenol hydroxylation, and erythromycin N-demethylation assays, respectively.

Results

In vitro studies using non-induced mouse hepatic microsomes in the presence or absence of Kaempferia parviflora extract showed that Kaempferia parviflora extract altered CYP1A1, CYP1A2, CYP2B, and CYP2E1 activities by non-competitive, mixed-competitive, competitive, and uncompetitive mechanisms, respectively. Among these enzymes, CYP1A2 was affected by Kaempferia parviflora based on the highest value of V_max (15.276 ± 0.206 nmol/min) and lowest of K_i value (0.008 ± 0.002 μg/ml). In addition, the plant extract also modulated CYP2B activity based on the low K_m value (1.599 ± 0.147 pmol). For in vivo studies, mice were orally treated with 250 mg/kg of Kaempferia parviflora extract for 7, 14, and 21 days. The results demonstrated that Kaempferia parviflora extract significantly induced CYP1A1, CYP1A2 enzyme activities following short-term treatment. CYP2B enzyme activities were markedly increased all Kaempferia parviflora extract treatment timepoints, whereas Kaempferia parviflora extract significantly enhanced CYP2E1 activity only after long-term treatment. However, Kaempferia parviflora extract did not affect the CYP3A enzyme activity.

Conclusions

Kaempferia parviflora extract modulated several CYP450 enzyme activities, thus, its utilization with drugs or other herbs should raise concern for potential drug–herb interactions.     

Biochemical mechanism of Wu-Zi-Yan-Zong-Wan,a traditional Chinese herbal formula,against alcohol-induced oxidative damage in CYP2E1 cDNA-transfected HepG2 (E47) cells

Aim of the study

Wu-Zi-Yan-Zong-Wan (WZ) is a traditional Chinese herbal formula which is commonly used for treating patients with “Yang deficiency”. In the present study, the effect of WZ on ethanol-induced toxicity in CYP2E1 cDNA-transfected HepG2 (E47) cells was investigated.

Materials and Methods

WZ extract was obtained by extracting the herbal powder with 50% ethanol (v/v, in water) and the effect of the extract on ethanol-induced toxicity was investigated in cultured cells.

Results

The treatment with WZ extract (12.5–200 μg/mL) for 24 h dose-dependently protected against ethanol-induced toxicity in E47 cells, as evidenced by the enhanced cell viability and decreased extent of lactate dehydrogeanse leakage. The cytoprotection against ethanol-induced toxicity was associated with decreases in the extents of reactive oxygen species production and lipid peroxidation, as well as increases in mitochondrial reduced glutathione and membrane potential. In addition, WZ extract treatment also suppressed the formation of DNA fragments in ethanol-intoxicated E47 cells.

Conclusions

WZ extract was found to protect against the ethanol-induced toxicity in E47 cells, possibly by virtues of its antioxidant activity.     

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.     

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.     

Effects of Schisandra chinensis Baillon (Schizandraceae) on lipopolysaccharide induced lung inflammation in mice

Ethnopharmacological relevance

Schisandra chinensis Baillon (Sc), an anti-inflammatory herb that has been used in traditional Chinese medicine for thousands of years, is frequently used to treat upper respiratory tract infections.

Aim of the study

This study was conducted to evaluate the ability of a water extract of Sc to prevent airway inflammation both in vitro and in vivo.

Materials and methods

Human lung alveolar epithelial-derived A549 cells were stimulated with to interleukin-1β, tumor necrosis factor-α, and interferon-γ (IL-1β, TNF-α, and INF-γ; cytokine mixture; CM) and treated with Sc extracts. They were then evaluated using nitric oxide (NO), IL-8 and monocyte chemotactic protein-1 (MCP-1) secretions. In the in vivo study, BALB/c mice were challenged with lipopolysaccharide (LPS) to induce acute airway inflammation. After this challenge, the mice were treated with Sc extracts (10, 50 and 100 mg/kg) by oral administration, and inflammatory cells in the bronchoalveolar lavage (BAL) fluid were counted. IL-6 and TNF-α secretions were measured using an enzyme-linked immunosorbent assay. Lung tissues of the LPS treated mice were prepared and stained with hematoxylin and eosin (HE) for histological examination.

Results

In the A549 cells, Sc extracts dose-dependently and significantly inhibited CM-induced NO production and reduced IL-8 and MCP-1 secretions. Sc extracts efficiently suppressed neutrophil and macrophage infiltrations of lung tissues and increased IL-6 and TNF-α levels in BAL fluid in LPS-instilled BALB/c mice. In addition, Sc extracts treatment inhibited pathologic progress in the lung tissues, as confirmed by H&E staining. These findings indicate that Sc extracts could be potentially useful for the treatment of acute lung inflammation and acute lung injury.     

Anti-diabetic activity of Swertia corymbosa (Griseb.) Wight ex C.B. Clarke aerial parts extract in streptozotocin induced diabetic rats

Ethnopharmacologicals relevance

Swertia corymbosa locally called as Shirattakuchi have a long history of use in Ayurveda herbal preparations in Indian traditional system of medicine. It has been used in folklore medicine for the treatment of diabetes.

Aim of the study

The present study aimed to investigate the effect of the methanolic extract of Swertia corymbosa (SC) in diabetic and to analyze its chemical composition by HPLC–ESI/MS that may correlate with their pharmacological activities.

Materials and methods

The in vitro anti-diabetic activity of the extracts was measured by using α-glucosidase and α-amylase enzyme inhibitory activity. The methanolic extract of Swertia corymbosa were administered orally (125, 250 and 500 mg/kg, for 28 days) to streptozotocin-induced diabetic rats. Hypoglycemic effects, oral glucose tolerance test, change in body weight and lipid profile, biochemical analysis and histopathological examination were assessed. High-performance liquid chromatography–electrospray ionization/mass spectrometry (HPLC–ESI/MS) method was also developed to analyze the chemical composition.

Results

In vitro anti-dabetic study, the methanol extract of SC is found to be a potent inhibitor of α-glucosidase and α-amylase activity. Oral administration of SC and standard drug for 28 days caused a significant decrease in the concentrations of blood glucose level, total cholesterol (TC), serum triglycerides (TGs), low-density lipoprotein-cholesterol (LDL-C), malondialdehyde (MDA) and significant increase in the concentrations of high density lipoprotein-cholesterol (HDL-C), serum insulin and body weight. Furthermore, activities of antioxidative enzymes, including SOD, GPx, GSH and CAT were enhanced dosed dependently with SC. Histopathological studies of the pancreas showed the regeneration of the β-cells by extract which were earlier necrosed by streptozotocin. Ten major compounds such as loganic acid (1), swertiamarin (2), sweroside (3), gentiopicroside (4), isovitexin (5), amoroswertin (6), amarogentin (7), gentiacaulein (8), decussatin (9) and swertianin (10) were analyzed by HPLC–ESI/MS system.

Conclusions

These results demonstrate that SC aerial parts of methanolic extract is an effective anti-diabetic and antioxidant activities which provides the scientific proof for the folklore medicine.     

In vitro modulatory effects of Andrographis paniculata, Centella asiatica and Orthosiphon stamineus on cytochrome P450 2C19 (CYP2C19)

Ethno pharmacological relevance

Andrographis paniculata (AP), Centella asiatica (CA) and Orthosiphon stamineus (OS) are three popular herbs traditionally used worldwide. AP is known for the treatment of infections and diabetes and CA is good for wound healing and healthy skin while OS is usually consumed as tea to treat kidney and urinary disorders. Interaction of these herbs with human cytochrome P450 2C19 (CYP2C19), a major hepatic CYP isoform involved in metabolism of many clinical drugs has not been investigated to date.

Aim of the study

In this study, the modulatory effects of various extracts and major active constituents of AP, CA and OS on CYP2C19 activities were evaluated.

Materials and methods

S-Mephenytoin, the CYP2C19 substrate probe, was incubated in the presence or absence of AP, CA and OS components. The changes in the rate of metabolite (hydroxymephenytoin) formation were subsequently determined by a high-performance liquid chromatography (HPLC)-based enzyme assay to characterize the modulatory effects.

Results

Among the herbal extracts studied, AP ethanol extract and CA dichloromethane extract exhibited mixed type inhibition towards CYP2C19 with K_i values of 67.1 and 16.4 μg/ml respectively; CA ethanol extract and OS petroleum ether extract competitively inhibited CYP2C19 activity (K_i = 39.6 and 41.5 μg/ml respectively). Eupatorin (a major active constituent of OS) was found to significantly inhibit CYP2C19 by mixed type inhibition (K_i = 7.1 μg/ml or 20.6 μM).

Conclusions

It was observed that AP, CA and OS inhibited CYP2C19 activity with varying potency. While weak inhibitory effect was observed with AP, moderate to strong inhibition was observed with CA dichloromethane extract and eupatorin, the major OS constituent. Therefore care should be taken when these CA and OS components are co-administered with CYP2C19 substrates (such as omeprazole, proguanil, barbiturates, citalopram, and diazepam).     

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.     

Up-regulatation of CYP3A expression through pregnent X receptor by praeruptorin D isolated from Peucedanum praeruptorum Dunn

Ethnopharmacological relevance

Qianhu, the dried roots of Peucedanum praeruptorum DUNN (Umbelliferae), is a well-known traditional Chinese medicinal herb which was officially listed in the Chinese Pharmacopoeia. Praeruptorin D (PD) is one of the major active constituents of Peucedanum praeruptorum Dunn (Qianhu). The Pregnane X receptor (PXR) is an orphan nuclear receptor and plays a pivotal role in the activation of human cytochrome P450 3A4 (CYP3A4) gene.

Aim of the study

The purpose of this study was to investigate the effect of PD on the PXR-mediated transactivation of CYP3A4, and thus to predict potential herb–drug interactions between PD, Qianhu, and the other co-administered drugs that metabolized by CYP3A4.

Materials and methods

The effect of PD on the Cyp3a11, mPXR mRNA expression in mice primary hepatocytes was measured using real-time PCR. The gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells after transfected with PXR expression plasmids were determined by real-time PCR, Western blot analysis, and LC–MS/MS based CYP3A4 substrate assay.

Results

The results revealed that the level of Cyp3a11 gene expression in mice primary hepatocytes was significantly increased by PD, but PD cannot induce the mPXR gene expression. On the other hand, CYP3A4 mRNA, protein expression and functional activity in PXR-over-expression LS174T cells were significantly increased by PD through PXR-mediated pathway; conversely, no significant change was found in the untransfected cells.

Conclusions

These findings suggest that PD can significantly up-regulate CYP3A4 expression and activity via the PXR-mediated pathway and this should be taken into consideration to predict any potential herb–drug interactions when PD and Peucedanum praeruptorum Dunn are co-administered with other drugs.