Anti-tumor activity and relative mechanism of ethanolic extract of Marsdenia tenacissima (Asclepiadaceae) against human hematologic neoplasm in vitro and in vivo

Ethnopharmacological relevance

Marsdenia tenacissima, a traditional Chinese medicinal herb, endemic to Yunnan Province is widely used to treat cough, asthma, expectorant, esophageal cancer, gastric cancer, lung cancer, and hepatocellular carcinoma. The aim of this study was to evaluate in vitro and in vivo anti-hematologic neoplasm potential of the ethanolic extract of this herb (crude ethanolic extract of Marsdenia tenacissima, CME) and by using different assays to elucidate its possible mechanism of action.

Materials and methods

The cytotoxicity of CME on tumor cells and peripheral blood mononuclear cells (PBMCs) was evaluated using MTT and apoptosis assays. We also tested the effect of CME on colony formation inhibition and cell cycle distribution of tumor cells. The protein expressions of Cyclin D1, Bax, Bcl-2, caspase-3 and caspase-9 were detected through Western blotting. In vivo anti-tumor effect was evaluated by measuring tumor volume changes, measuring tumor weight, evaluation of tumor microvessel density (MVD) and TUNEL staining by using immunohistochemistry staining in tumor models of nude mice.

Results

Marsdenia tenacissima ethanolic extract exhibited effects of proliferation inhibition and induction of apoptosis on human hematologic neoplasm tumor cells in vitro, as well as hematologic neoplasm growth in vivo.

Conclusion

This study clearly indicated that the ethanolic extract of Marsdenia tenacissima displayed strong anti-tumor effects against hematologic neoplasm cells and could induce tumor cells apoptosis in vitro and in vivo, and also had a significant anti-angiogenic effect in vivo against tumor cell apoptosis. Its multi-mechanism of action might be associated with the cell cycle (G0/G1) arrest, induction of apoptosis through up-regulation protein expressions of Bax, caspase-9 and caspase-3 genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes, a decrease in tumor microvessel density and an increase of TUNEL-positive cells in vivo. These findings provided the molecular theoretical basis of clinical application.     

In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica

Ethnopharmacological relevance

Centella asiatica (CA) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa, and Oceanic countries and traditionally used for wound healing and maintaining normal blood pressure.

Aim of the study

The present study was carried out to examine the potential modulatory effects of three commercially available active components (asiaticoside, asiatic acid and madecassic acid) and four extracts (aqueous, ethanol, dichloromethane and hexane) of CA on three major cDNA-expressed human cytochrome P450 (CYP) isoforms.

Materials and methods

High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6β-hydroxylase assays were developed to probe activities of CYP2C9, CYP2D6 and CYP3A4, respectively. Probe substrates were incubated with or without each active component and extract for each isoform, followed by examination of the kinetics parameters, IC₅₀ and K_i, to characterize modulatory effects.

Results

CYP2C9 was more susceptible to inhibitory effects by CA extracts compared to CYP2D6 and CYP3A4. Moderate degree of inhibition was observed in ethanol (K_i = 39.1 μg/ml) and dichloromethane (K_i = 26.6 μg/ml) extracts implying potential risk of interaction when CYP2C9 substrates are consumed with CA products. The two extracts however showed negligible inhibition towards CYP2D6 and CYP3A4 (IC₅₀'s of 123.3 μg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC₅₀'s of 117.9 μg/ml and above). Among the active constituents investigated, asiatic acid and madecassic acid appeared to selectively inhibit CYP2C9 and CYP2D6 more than CYP3A4. Of particular interest is the potent inhibitory effect of asiatic acid on CYP2C9 (K_i = 9.1 μg/ml). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Inhibitions of asiatic acid with the other isoforms and that of madecassic acid with all isoforms were only moderate (K_i's ranged from 17.2 to 84.4 μg/ml). On the other hand, the IC₅₀ values for asiaticoside were high (1070.2 μg/ml or above) for all three isoforms, indicating negligible or low potential of this compound to modulate CYP enzymatic activity.

Conclusion

Centella asiatica extracts and active constituents inhibited CYP2C9, CYP2D6 and CYP3A4 activities with varying potency with CYP2C9 being the most susceptible isoform to inhibition. Significant inhibition was observed for asiatic acid and CA ethanol and dichloromethane extracts, implying involvement of semipolar constituents from CA in the effect. This study suggested that CA could cause drug–herb interactions through CYP2C9 inhibition.     

in vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction

Ethnopharmacological relevance

Codiaeum variegatum, grown in many varieties, has been widely used as a houseplant based on its brightly decorative foliage. In addition, a variety of this plant has been used for a long time against bloody diarrhea by the local population in Cameroon.

Aim of the study

In our previous study, the aqueous extract of this plant and an isolated sub-fraction exhibited significant anti-amoebic activity on axenic culture of Entamoeba histolytica. Due to the medicinal value of these extracts, we promptly initiated to investigate their genotoxic and mutagenic potential in order to assure their safe and rationale usage in traditional healthcare system.

Material and methods

Both extracts were incubated with L5178Y mouse lymphoma cells, primary hepatic cells and HepG2 human hepatocellular carcinoma cells and their genotoxicity and mutagenicity were evaluated by quantifying DNA damage and chromosomal aberrations through comet assay, micronucleus assay and mouse lymphoma mutation assay.

Results

The aqueous extract of Codiaeum variegatum is not cytotoxic up to 2000 µg/mL while the amoebicidal fraction is significantly cytotoxic (≤ 40–55%) on L5178Y mouse lymphoma and HepG2 cells at concentrations higher than 500 µg/mL. Besides, no significant DNA damage and induction of micronucleus formation were identified at concentrations up to 2000 µg/mL. Moreover, the mutagenic potential of these extracts after short (4 h) and long term (24 h) treatment, revealed no significant gene mutation induction.

Conclusion

The aqueous extract of Codiaeum variegatum and the amoebicidal fraction SF9B are neither genotoxic on non-competent or metabolic competent cell lines, nor mutagenic in mouse lymphoma mutation assay and therefore they could be safely used at lower doses for medicinal purpose.     

Comparative metabolism of Radix scutellariae extract by intestinal bacteria from normal and type 2 diabetic mice in vitro

Ethnopharmacological relevance

Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of disease, especially the complicated and chronic ones. In TCMs, many ingredients which are known to have biological effects just pass through the gut, they do not get into the bloodstream. Study on interactions of these active ingredients with the intestinal bacteria is very helpful to unravel how TCM works. Radix scutellariae was widely used alone or in combination with other medicinal herbs to the treatment of type 2 diabetes mellitus for a long time in China even in Asia. Additionally, the incidence of type 2 diabetes is closely related to the changes of intestinal flora. In this paper, the metabolism of baicalin in Radix scutellariae extract by normal and type 2 diabetic mice intestinal bacteria were firstly investigated.

Materials and methods

Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/QTOF-MS) technique combined with Metabolynx^TM software was used for analysis of the metabolic profile of baicalin in Radix scutellariae extracts by the intestinal bacteria from normal and type 2 diabetic mice.

Results

The amount of baicalin?s aglycone (baicalein) in type 2 diabetic mice samples were remarkably more than that in normal mice samples and oroxylin A only existed in type 2 diabetic mice samples. Intestinal bacteria produced not only a small amount of baicalein, but also some conjugates such as hydrogenated baicalin and methylated baicalin.

Conclusions

We proposed that β-d-glucuronidases contributed to the deglycosylation prior to absorption. Intestinal bacteria from pathological state mice produced more baicalein, which was well absorbed contributing to the treatment of type 2 diabetes. Additionally, the pharmacological effects of oroxylin A were associated with type 2 diabetes. Hence, the production of metabolites of baicalin might influence the effects of traditional medicines. Thus the study on the metabolism of baicalin by intestinal bacteria from normal and type 2 diabetic mice was of great importance to understanding the effects of traditional medicines. Furthermore, this work demonstrated the potential of the ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry approach with MetaboLynx for quite rapid, simple, reliable and automated identification of metabolites of natural products.     

北柴胡糖基转移酶基因BcUGT3,BcUGT6的表达分析及其原核表达

该研究测定了北柴胡中2个糖基转移酶基因BcUGT3和BcUGT6在不同组织中的转录水平及MeJA处理后不定根中的转录水平。BcUGT3在根、叶、花和果中的转录水平无明显差异,但均高于茎中的转录水平。BcUGT6在叶中转录水平最高,在花中最低。以未处理的为对照,BcUGT6在MeJA处理后2 h,8 h,24 h,2 d,4 d的北柴胡不定根中,转录水平均提高近2倍,表明BcUGT6的转录受MeJA影响较小。BcUGT3转录水平随处理时间的延长不断增加,4 d时,达7倍左右。利用载体pET-28a（+）,进行了2个基因的原核表达。IPTG诱导后,宿主菌表达出了目的蛋白,并获得了纯化蛋白。为后续开展这2个基因的功能研究奠定了基础。     

Exploring the possible metabolism mediated interaction of Glycyrrhiza glabra extract with CYP3A4 and CYP2D6

The rhizome of Glycyrrhiza glabra L. (licorice) is used very widely in Indian and Chinese traditional medicine, and it is a popular flavor ingredient of drinks, sweets and candies. Its medicinal uses include treating bronchitis, dry cough, respiratory infections, liver disorders and diabetes. Glycyrrhizin is normally considered to be its biologically active marker, so a rapid RP‐HPLC method was developed for the quantitative estimation of glycyrrhizin in the extract. The effect of the standardized extract and its marker on drug metabolizing enzymes was evaluated through CYP3A4 and CYP2D6 inhibition assays to evaluate the safety through its drug interaction potential. The inhibition of CYP3A4 and CYP2D6 isozymes was analysed by the fluorescent product formation method. In the CYP450‐CO assay, the interaction potential of the standardized extract and pooled microsomes (percentage inhibition 23.23 ± 1.84%), was found to be less than the standard inhibitor. In the fluorimetric assay, G. glabra extracts showed higher IC₅₀ values than their positive inhibitors, ketoconazole and quinidine for CYP3A4 and CYP2D6, respectively. Furthermore, the interaction potential of the plant extract was greater than the pure compound. The results demonstrate that G. glabra and its principle bioactive compound, glycyrrhizin, when co‐administered with conventional medicines showed only a weak interaction potential with drug metabolizing enzymes. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Effect of an extract of Andrographis paniculata leaves on inflammatory and allergic mediators in vitro

Aim of study

Andrographis paniculata has been known to possess widespread traditional application in the treatment of allergy and inflammatory diseases. In the current study, we sought to examine the effects of an extract of Andrographis paniculata leaves on inhibition of lipopolysaccharide (LPS) induced [nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1beta (IL-1 beta), and interleukin-6 (IL-6)] and calcimycin (A23187) induced [leukotriene B₄ (LTB₄), thromboxane B₂ (TXB₂) and histamine] mediators in diverse cell based models.

Materials and methods

Effect of an extract of Andrographis paniculata leaves (AP) was studied on inhibition of LPS induced NO, PGE₂, IL-1 beta and IL-6 in J774A.1 murine macrophages; A23187 induced LTB₄ and TXB₂ in HL-60 promyelocytic leukemic cells and histamine in RBL-2H3 rat basophilic leukemia cells.

Results and conclusion

AP illustrated significant alleviation of pro-inflammatory, inflammatory, and allergic mediators. However, no inhibition was observed against histamine release. This outcome has been summed up to deduce that AP is fairly potent in attenuating the inflammation by inhibiting pro-inflammatory (NO, IL-1 beta and IL-6), inflammatory (PGE₂ and TXB₂) and allergic (LTB₄) mediators.     

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.     

天王补心丸中生地黄、玄参、天冬和麦冬水提液对大鼠肝CYP450酶含量及其亚型CYP3A, CYP2E1和CYP1A2活性的影响

目的:研究天王补心丸中滋阴类药味生地黄、玄参、天冬和麦冬水提物对大鼠肝肝药酶(CYP450)含量及对亚型CYP3A,CYP2E1,CYP1A2活性的影响,探讨CYP450在天王补心丸代谢中的作用.方法:大鼠灌胃给予生地黄、玄参、天冬和麦冬水提物7d后取肝脏称重并制备肝微粒体,紫外分光光度法测定肝微粒体细胞色素b5(Cytb5),P450的含量及CYP3A的活性,高效液相法测定CYP2E1和CYP1A2的活性.结果:与空白对照组比较,各给药组大鼠肝指数无显著变化;生地黄组CYP450含量极显著减少(P＜0.01),CYP3A活性极显著增加(P＜0.01),CYP1A2活性显著增加(P＜0.05);玄参组CYP450含量减少(P＜0.05),CYP3A和CYP1A2活性均极显著增加(P＜0.01);天冬组Cytb5含量增加(P＜0.05),CYP2E1活性增加(P＜0.05),CYP1A2活性极显著增加(P＜0.01);麦冬组cytb5,CYP450含量无统计学差异,CYP3A活性增加(P＜0.05),CYP2E1活性增加(P＜0.05),CYP1A2活性极显著增加(P＜0.01).结论:天王补心丸中生地黄、玄参降低大鼠肝脏CYP450酶含量并均诱导CYP3A和CYP1A2活性,天冬诱导CYP2E1和CYP1A2活性,麦冬诱导CYP3A,CYP2E1和CYP1A2活性.由于抑制CYP450活性,减少对其他药代谢,滋阴药组在天王补心丸全方配伍中可增强其他各功能组比如补血养血、宁心安神类药味的作用,为探讨天王补心丸的配伍机制提供了理论基础.     

Aqueous extract of Taxus Chinensis (Pilger) Rehd inhibits lung carcinoma A549 cells through the epidermal growth factor receptor/mitogen-activated protein kinase pathway in vitro and in vivo

Objective

To explore the anticancer mechanism of aqueous extract of Taxus Chinensis (Pilger) Rehd (AETC).

Methods

The serum pharmacological method was used to avoid interference from administration of the crude medicinal herbs. Eight purebred New Zealand rabbits were used for preparation of serum containing various concentrations of AETC. Forty-eight Balb/c-nu mice were used for in vivo experiments. The effects of serum containing AETC on the proliferation of A549 cells and expression levels of the epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway-related proteins in vitro were investigated. Additionally, the effects on the growth of A549 xenografts in nude mice, and expression levels of the EGFR/MAPK pathway-related proteins in the xenografts, were investigated.

Results

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the serum containing AETC significantly decreased the viability of A549 cells in a dose-dependent manner. Western blot showed that the serum containing various concentrations of AETC strongly reduced the levels of phospho-Jun N-terminal kinase (p-JNK) and phospho-extracellular signal-regulated kinasel/2 (ERK1/2) while it increased the level of p-p38. However, no significant effects on the expression levels of JNK, ERK1/2, and p38 MAPK were found. In addition, an anticancer effect from AETC was observed in vivo in the Balb/c-nu mice bearing A549 xenografts.

Conclusion

AETC has significant effects on the growth of A549 xenografts and on the activity of the EGFR/MAPK pathway. Therefore, AETC may be beneficial in lung carcinoma treatment.     

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.     

HPV E6 down-regulation and apoptosis induction of human cervical cancer cells by a novel lipid-soluble extract (PE) from Pinellia pedatisecta Schott in vitro

Aim of the study

To evaluate the cytotoxicity and apoptosis induction effects of a novel lipid-soluble extract (PE) from Pinellia pedatisecta Schott on CaSki, HeLa and HBL-100 cells. Particularly, the effect of PE on HPV E6 gene expression was tested, and the mechanism of its apoptosis induction effect was also studied.

Materials and methods

Cell viability was measured by the MTT assay. DAPI staining and flow cytometric analysis (FCM) were used to identify apoptotic cells in PE-treated CaSki, HeLa, and HBL-100 cells. Expression of the HPV E6 gene in CaSki and HeLa cells was detected by real-time RT-PCR and western blot analysis. Apoptosis-associated genes were examined by RT-PCR and western blot analysis in CaSki cells.

Results

PE inhibited the growth of CaSki and HeLa cells in a time- and dose-dependent manner, but it had no obvious inhibiting effect on HBL-100 cells except at a relatively high dose (500 μg/mL). PE could induce apoptosis in CaSki and HeLa cells in a time-dependent manner but not in HBL-100 cells. HPV E6 mRNA and protein were decreased significantly by PE. Caspase-8, caspase-3, Bax, P53 and P21 mRNAs as well as proteins were increased while Bcl-2 mRNA and protein were decreased significantly by 24 h of PE treatment.

Conclusions

PE can function as a tumor suppressor by inducing apoptosis in human cervical cancer cells but it has little side effect on normal cells. It probably acts via mitochondria-dependent and death receptor-dependent apoptotic pathways. HPV E6 may be the key target of its action.     

黄芩、柴胡及其配伍提取物体外抗柯萨奇 B3m病毒及对细胞活性的影响

目的 :观察黄芩、柴胡及其配伍提取物体外抗柯萨奇B3m病毒(CVB_3m)及对细胞活性的影响。 方法 :采用微量细胞培养法,以细胞病变法和MTT法判定细胞活性,观察黄芩、柴胡及其配伍提取物以及黄芩两种不同提取制剂体外抗CVB_3m病毒和对细胞活性的影响,确定各组药物的抑制病毒指数和药物对细胞的预防和治疗保护率。 结果 :黄芩提取物浓度为1.563 mg ·mL^-1时,抑制病毒指数大于2,而柴胡组和配伍组的抑病毒指数均小于2;3组药物都具有不同程度的预防保护作用,而柴胡组和配伍组的预防保护作用尤为明显,其预防保护率明显高于黄芩组,有显著性差异( P＜0.05);在病毒感染后,3组药物对细胞均具有不同程度的治疗保护作用,黄芩组和配伍组表现明显高于柴胡组,差异具有显著性(P＜0.05)。 结论 :黄芩提取物具有明显的直接灭活病毒作用;3组药物都具有不同程度的预防保护作用和治疗保护作用,柴胡组和配伍组预防给药保护细胞作用明显;黄芩提取物和配伍提取物组的治疗保护作用明显;配伍组具有预防和治疗保护双重作用,但没有表现出配伍后作用增强的优势。     

Antihyperglycemic effect of a natural chicoric acid extract of chicory (Cichorium intybus L.): A comparative in vitro study with the effects of caffeic and ferulic acids

Ethnopharmacological relevance

In Eurasia folk medicine, roots of chicory (Cichorium intybus L.) have been reported to exert antidiabetic benefits. In vitro, a natural chicoric acid extract (NCRAE) from Cichorium intybus root has been shown to increase insulin secretion by pancreatic β-cells and glucose uptake by muscle cells.

Materials and methods

In vitro experiments were designed to compare the effects of two hydroxycinnamic acids, caffeic and ferulic acids, to those obtained with NCRAE (50 and 100 µg.mL⁻¹) on the three major tissues implicated in glycemic regulation (pancreas, muscle and liver). In vivo experiments were performed in Wistar rats submitted to a daily intraperitoneal injection of NCRAE (3, 15 or 30 mg kg⁻¹) for 4 days. On the fourth day, an intraperitoneal glucose tolerance test (IPGTT; 1 g kg⁻¹) was carried out.

Results

Our results show that the three compounds we used are able each to induce an original response. Caffeic acid mainly promotes a decrease in hepatic glycogenolysis. Ferulic acid elicits a clear increase of insulin release and a reduction of hepatic glycogenolysis. However, this compound induces an inhibition of muscle glucose uptake. NCRAE provokes an increase of insulin release and glucose uptake without any effect on hepatic glycogenolysis. We could also show that none of these compounds implicates hepatic glucose 6-phosphatase in contrast to chlorogenic acid, known as an inhibitor of glucose 6-phosphatase and which is able to decrease glucose output from hepatocytes. Our results point out that NCRAE is able to decrease blood glucose without any effect hepatic effect. Our in vivo experiments bring evidence that 4 daily IP administrations of NCRAE improve IP glucose tolerance in a dose-dependent manner and mainly via an insulin sensitizing effect.

Conclusions

We conclude that NCRAE presents an antihyperglycemic effect essentially due to a peripheral effect on muscle glucose uptake.     

In vitro Inhibition of Human CYP1A2, CYP2D6, and CYP3A4 by Six Herbs Commonly Used in Pregnancy

Black elderberry, cranberry, fennel, ginger, horsetail, and raspberry leaf, herbs frequently used in pregnancy, were investigated for their in vitro CYP1A2, 2D6, and 3A4 inhibitory potential. Aqueous or ethanolic extracts were made from commercially available herbal products, and incubations were performed with recombinant cDNA‐expressed human CYP enzymes in the presence of positive inhibitory controls. Metabolite formation was determined by validated LCMS/MS or HPLC methodologies. IC₅₀ inhibition constants were estimated from CYP activity inhibition plots using non‐linear regression. The most potent inhibition was shown for fennel towards CYP2D6 and 3A4 with respective IC₅₀ constants of 23 ± 2 and 40 ± 4 µg/ml, horsetail towards CYP1A2 with an IC₅₀ constant of 27 ± 1 µg/ml, and raspberry leaf towards CYP1A2, 2D6, and 3A4 with IC₅₀ constants of 44 ± 2, 47 ± 8, and 81 ± 11 µg/ml, respectively. Based on the recommended dosing of the different commercial herbal products, clinically relevant systemic CYP inhibitions could be possible for fennel, horsetail, and raspberry leaf. In addition, fennel and raspberry leaf might cause a clinically relevant inhibition of intestinal CYP3A4. The in vivo inhibitory potential of these herbs towards specific CYP enzymes should be further investigated. Copyright © 2013 John Wiley & Sons, Ltd.