Printer Friendly

Investigation of cytochrome P450 inhibitory properties of maslinic acid, a bioactive compound from Olea europaea L., and its structure-activity relationship.

ABSTRACT

Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with [IC.sub.50] value at 46.1 and 62.3 [micro]M, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with [IC.sub.50] values more than 100 [micro]M. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with [K.sub.i] of 18.4, 98.7 and 66.3 [micro]M, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high [K.sub.i] values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs.

Keywords:

Cytochrome P450 (CYP)

Olea europaea

Maslinic acid

Oleanolic acid

Pentacyclic triterpenes.

Introduction

Olive oil is a regular dietary component of the different countries in the world, in particular bordering the Mediterranean Sea (Owen et al. 2004). It is recognized as a healthy food from a nutritional point of view because of its content in monounsaturated fatty acids as well as bioactive components, such as polyphenols and pentacyclic triterpenes (Ghanbari et al. 2012). Among the latter, maslinic acid (MA) is the most abundant pentacyclic triterpene acid in the fruit of Olea europaea L. (Lozano-Mena et al. 2012). Interestingly, MA has a similar chemical structure compared with oleanolic acid (OA) (Fig. 1), which is found in plants worldwide (Kim et al. 2004). Until now, MA has been reported to possess several biological activities, such as antitumor (Li et al. 2010; Reyes-Zurita et al. 2011; Reyes-Zurita et al. 2009), antiinflammation (Huang et al. 2011), antioxidation (Montilla et al. 2003), antiallodynic (Nieto et al. 2013) and antidiabetogenic activities (Guan et al. 2011). Moreover, MA also is a marked component of hawthorn fruit (Rigelsky and Sweet 2002), a well-known fruit in China, which is used to make many forms of food, including jams, jellies, fruit drinks and other soft drinks (Chen et al. 1995).

In recent years, much attention has been paid to MA because of its beneficial effects on human health. For example, an extract from the skin of olive fruits containing 73.3% MA and 25.8% oleanolic acid inhibits proliferation and induces apoptosis in HT-29 human colon cancer cells (Juan et al. 2006). In fact, the antitumor effect has also been reported in different human tumor cell lines, such as astrocytoma (Martin et al. 2007), pancreatic cancer cells (Li et al. 2010), prostate cancer cells (Park et al. 2013) and human breast cancer cells (Allouche et al. 2011). Previous studies in our lab have shown that MA together with TNF[alpha] could be new promising agents in the treatment of pancreatic cancer because MA can improve the antitumor activities of TNF[alpha] and inhibit pancreatic tumor growth and invasion (Li et al. 2010). In addition, we also reported that MA suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss by regulating RANKL-mediated NF-[kappa]B and MAPK signaling pathways (Li et al. 2011). Hence, MA is considered as a promising anutraceutical or candidate for drug development. However, the studies about the metabolic-related properties of MA are limited. In particular, the interactions between MA and the cytochrome P450 (CYP) enzymes remain uncertain. When people use MA and/or olive oil, or some food containing MA with Western drugs together, these may cause unwanted food-drug interactions via drug metabolizing CYP enzymes.

[FIGURE 1 OMITTED]

The aim of this study was to determine the effects of MA on several major human and rat CYP enzymes responsible for the metabolism and disposition of a large number of drugs currently used, including CYP1A2, CYP2C9(human)/2C11 (rat), CYP2D1 (rat)/2D6 (human), CYP2E1 and CYP3A2 (rat)/3A4 (human) isoforms. Enzyme kinetic studies using the model CYP probe substrates in the presence of various concentrations of MA were performed to investigate the mode of inhibition of the enzyme-substrate interactions. At the same time, it was also explored the structure-activity relationship of MA and OA with CYP activity in human liver microsomes.

Materials and methods

Chemicals and reagents

Pooled human liver microsomes and specific human CYP3A4 isoform were obtained from Corning Gentest Corporation (Woburn, MA, USA) and stored at -150[degrees]C until use. All the experimental procedures involving humans have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) guidelines. Phenacetin, paracetamol, metacetamol, oleanolic acid, tolbutamide, chlorpropamide, furafylline, dextromethorphan, 6[beta]-hydroxytestosterone, glucose 6phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PDH), [beta]-nicotinamide adenine dinucleotide phosphate (NADP), and tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 4-Hydroxytolbutamide, 6-hydroxychlorzoxazone and dextrorphan were obtained from Toronto Research Chemical (North York, Canada). Testosterone was purchased from Energy Chemical Co. (Shanghai, China). Corticosterone and ketoconazole were purchased from Tokyo Chemical Industry Co. (Shanghai, China). Chlorzoxazone was purchased from Alfa Aesar (Massachusetts, USA). Maslinic acid (purity >98%) was synthesized in our laboratory (Li et al. 2010). Acetonitrile and methanol (all HPLC grade) were purchased from Merck (Darmstadt, Germany). Acetic acid glacial (HPLC-grade) was purchased from TEDIA. Ethyl acetate (HPLC grade) was purchased from Fisher chemicals (Leicester, UK). Distilled water was purified in a Millipore system Milli Q.

Animals

Male Sprague-Dawley rats (200-250 g) were purchased from National Rodent Laboratory Animal Resources, Shanghai Branch of China. The animals were kept in animal holding room under standard conditions with 12 h light-dark cycles, with free access to rodent cubes and tap water. Animals were maintained according to the National Institutes of Health standards established in the 'Guidelines for the Care and Use of Experimental Animals', and all the experimental protocols were approved by the Animal Investigation Committee of the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University.

Preparation of rat liver microsomes

In this study, male Sprague-Dawley rats (200-250 g) were fasted overnight and killed by cervical dislocation before removal of the liver. The liver was excised, rinsed with ice-cold saline (0.9% NaCl w/v), weighed and homogenized in a 0.05 mM Tris/KCl buffer (pH 7.4). The homogenate was centrifuged at 10,000 x g at 4[degrees]C for 30 min. The supernatant was then centrifuged at 105,000 x g at 4[degrees]C for 60 min. The pellet was reconstituted with 0.05 mM Tris/KCl buffer (pH 7.4) and stored at -150[degrees]C until used. The protein concentration of the liver microsomes was determined by a protein quantitative assay using bicinchoninic acid (Brown et al. 1989).

Assays of CYP1A2 activity in human liver microsomes

CYP1A2 activity was assessed by formation of paracetamol from phenacetin by the method reported previously (Wang et al. 2009). The oxidative metabolism of phenacetin was measured in a system consisting of an NADPH-generating system and microsomes according to the method specified below. The incubation mixture (final volume of 250 [micro]l in 0.1 M potassium phosphate buffer, pH 7.4) consisted of an NADPH-generating system (1.3 mM NADP, 3.3 mM G6P, 0.4 units/ml G6PDH, 3.3 mM magnesium chloride and 0.8 mg/ml pooled human liver microsomes). For inhibition study, 50 [micro]M phenacetin was used. The concentrations of MA and OA used were from 1.25 to 100 [micro]M. Furafylline, a selective CYP1A2 inhibitor, was used as positive control. The tubes were incubated in Eppendorf Themomixer at 800 rev/min, 37[degrees]C. The reaction was initiated by adding protein to incubation mixture. After 30 min, incubations were terminated by adding 250 [micro]l ice-cold acetonitrile. The tubes were then centrifuged in microcentrifuge at 13,000 x g for 12 min to precipitate protein. The supernatant was collected and metacetamol (15 [micro]l of 500 [micro]g/ml) was added as an internal standard. The whole mixture was then extracted with 500 [micro]l ethyl acetate at 1400 rev/min in Thermomixer for 30 min at 25[degrees]C. The tubes were then centrifuged at 8000 x g for 8 min. The organic layer was transferred to glass conical tube and evaporated at a heat block at 40[degrees]C under a gentle stream of nitrogen gas. The residue was dissolved in 120 [micro]l mobile phase, and then 50 [micro]l was used for HPLC analysis. HPLC analyses of paracetamol, metacetamol (internal standard) and phenacetin were performed on an Agilent 1260 series instrument with DAD detection at 249 nm. An Agilent reverse phase C18 column (Zorbax Eclipse XDB-C18, 4.6 x 150 [mm.sup.2], 5 [micro]m) with a C18 guard column in a gradient mobile phase containing acetonitrile and water (0.1% acetic acid) at room temperature and a flow rate of 1.0 ml/min. Condition for elution was as follows: 0-5 min, 5% acetonitrile; 5-10 min, 5% to 60% acetonitril; 10-13 min, 60% to 5% acetonitril; 13-15 min, 5% acetonitril. Under the experimental conditions, paracetamol, metacetamol (internal standard) and phenacetin were eluted at 7.4, 9.2, and 11.1 min, respectively.

Assays of CYP2C9 activity in human liver microsomes

CYP2C9 activity was assessed by formation of 4-hydroxytolbutamide from tolbutamide (Wang et al. 2010a). Pooled human liver microsomes (0.8 mg/ml) were incubated with NADPH-generating system (1.3 mM NADP, 3.3 mM G6P, 0.4 units/ml G6PDH, 3.3 mM magnesium chloride, in 0.1 M potassium phosphate buffer, pH 7.4). The incubation mixture was pre-incubated for 5 min at 37[degrees]C before adding NADPH and 60 min incubation was initiated by NADPH in an Eppendorf Thermomixer at 37[degrees]C thermomixer, at 800 rev/min. Tolbutamide (50 [micro]M) was used in inhibition studies. The final concentrations of MA and OA ranged from 1.25 to 100 [micro]M. Sulfaphenazole (5-50 [micro]M), a selective CYP2C9 inhibitor, was used as a positive control. The HPLC method for tolbutamide and 4-hydroxytolbutamide was previously described (Wang et al. 2010b). Chlorpropamide (500 [micro]g/ml, 10 [micro]l) was used as the internal standard for extraction and HPLC analysis.

Assays of CYP2D6 activity in human liver microsomes

CYP2D6 activity was assessed by the formation of dextrorphan from the CYP2D6 probe dextromethorphan (Sun et al. 2014). The incubation mixture (final volume 250 [micro]l, in 0.1 M potassium phosphate buffer containing 3.3 mM Mg[Cl.sub.2], pH 7.4) consisted of an NADPH-generating system (1.3 mM NADP, 3.3 mM G6P, 0.4 units/ml G6PDH), 25 [micro]M dextromethorphan and 0.8 mg/ml pooled human liver microsomes. The concentrations of MA and OA were present at 1.25-100 [micro]M. The reaction was initiated by adding NADP into incubation mixture and incubated for 60 min at 37[degrees]C. Incubations were terminated by adding 250 [micro]l ice-cold acetonitrile, centrifuged and the supernatant extracted with 500 [micro]l ethyl acetate. The organic layer was dried under a gentle stream of nitrogen, resuspended by mobile phase (87% 0.01 M Potassium phosphate buffer, 13% acetonitrile) for HPLC analysis. Dextrorphan, dextromethorphan and chlorpheniramine (internal standard) were separated through an Aligent ZORBAX Eclipse XDB-C18 column (4.6 x 150 [mm.sup.2], 5 [micro]m) maintained at ambient temperature and the flow rate was 1.0 ml/min. The mobile phase consisted of 0.01 M potassium phosphate buffer (pH 3.4, A) and acetonitrile (B) with a gradient elution as follows; 0-13 min, 13% B; 13-16 min, 13-60% B; 16-20 min, 60% B; 20-22 min, 60% to 13% B; 22-30 min, 13% B. Detection was conducted by DAD detector which was set at 217 nm.

Assays of CYP2El activity in human liver microsomes

CYP2E1 activity was assessed by the formation of 6-hydroxychlorzoxazone from the CYP2E1 probe chlorzoxazone (Wang et al. 2010a). Pooled human liver microsomes (0.8 mg/ml) were incubated with incubation buffer (0.05 M potassium phosphate buffer containing 3.3 mM magnesium chloride, pH 7.4), NADPH-generation system (1.3 mM NADP, 3.3 mM G6P, 0.4 units/ml G6PDH) and 50 [micro]M probe substrate chlorzoxazone in a total volume of 250 [micro]l. The concentrations of MA and OA used were from 1.25 to 100 [micro]M. The incubation mixture was pre-incubated for 5 min at 37[degrees]C before adding NADP. Incubation for 60 min was initiated by NADP at 37[degrees]C thermomixer at 800 rev/min. The reaction was terminated by adding 250 [micro]l ice-cold acetonitrile. The mixture was centrifuged and the supernatant was extracted with 500 [micro]l ethyl acetate. The organic layer was dried under a gentle stream of nitrogen. The residue was reconstituted with 200 [micro]l mobile phase (87% potassium phosphate buffer, 13% acetonitrile) and 20 [micro]l was injected for HPLC analysis. Phenacetin (500 [micro]g/ml, 25 [micro]l) was used as the internal standard for extraction and HPLC analysis. The incubation mixture was separated through an Aligent ZORBAX Eclipse XDB-C18 column (4.6 x 150 [mm.sup.2], 5 [micro]m) at flow rate of 1 ml/min with a gradient elution (A: potassium phosphate buffer (0.01 M, pH 3.4); B: acetonitrile) as follows: 0-5 min, 18% B; 5-9 min, 18-35% B; 9-14 min, 35-18% B; 14-15 min, 18% B. Aligent Technologies 1260 series HPLC system with a DAD detector set at 287 nm was used for analysis.

Assays of CYP3A4 activity in human liver microsomes

CYP3A4 activity was assessed by formation of 6[beta]-hydroxytestosterone from testosterone by the method reported previously (Wang et al. 2010a; Wang and Yeung 2012). The oxidative metabolism of testosterone was measured in a system consisting of an NADPH-generating system and microsomes according to the method specified below. The incubation mixture (final volume of 250 [micro]l) consisted of an NADPH-generating system (1.3 mM NADP, 3.3 mM G6P, 0.4 units/ml G6PDH, 3.3 mM magnesium chloride and 0.5 mg/ml human liver microsomes, in 0.1 M potassium phosphate, pH 7.4). Testosterone (50 [micro]M) was used in inhibition study and in enzyme kinetic studies (50-150 [micro]M). The concentrations of MA and OA used were from 1.25 to 100 [micro]M. Ketoconazole (0.5-10 [micro]M), a CYP3A4-selective inhibitor, was used as positive control. The analysis for testosterone and its metabolites was performed on a Waters reverse C18 column (Symmetry 4.6 x 150 [mm.sup.2], 5 [micro]m) with Phenomenex Security Guard column in a gradient mobile phase containing acetonitrile and water (0.1% acetic acid) at room temperature and a flow rate of 1.0 ml/min. Condition for elution was as follows: 0-3 min, 25% acetonitrile; 3-8 min, 25-50%: 8-13 min, 50% acetonitril; 13-15 min, 50-25% acetonitril. 6P-hydroxytestosterone and testosterone were detected at 245 nm. The retention times of 6f)-hydroxytestosterone, corticosterone (internal standard) and testosterone were 6.4,9.4 and 11.7 min, respectively.

Assays of CYP1A2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 activities in rat liver microsomes

In this study, the effects of MA on the above-mentioned CYPs activities were also investigated in rat liver microsomes, employing phenacetin (CYP1A2), tolbutamide (CYP2C11), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A2) as the probe substrates. The assays of CYP1A2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 activities in rats were assessed by the methods reported previously in our laboratory (Sun et al. 2014; Wang and Yeung 2011).

Statistical analysis

Enzyme kinetic and inhibition parameters estimation as well as statistical analyses were performed using GraphPad Prism 5.0 (GraphPad software Inc., CA, USA). Michaelis constant ([K.sub.m]) and maximal velocity ([V.sub.max]) values were obtained from Michaelis-Menten non-linear regression equation. The data were fitted to Michaelis-Menten model and further analyzed using Lineweaver-Burk plot and Dixon plot. Primary Lineweaver-Burk plot (obtained by reciprocal of reaction velocities versus reciprocal of substrate concentrations) and Dixon plot (obtained by reciprocal of reaction velocities versus inhibitor concentrations) were used to determine the quality of fit to a specific inhibition model. The inhibition constant ([K.sub.i]) value was obtained by the secondary plot of Lineweaver-Burk plot (obtained by the slopes of the regression lines in the Lineweaver-Burk plot versus inhibitor concentrations). [IC.sub.50] values (concentration of inhibitor to cause 50% inhibition of original enzyme activity) were determined by GraFit where appropriate using the following equation:

V = [V.sub.0]/1 + [(I/[IC.sub.50]).sup.S]

where [V.sub.0] is uninhibited velocity, V is observed velocity, S is slope factor and I is inhibitor concentration. [IC.sub.20] values (concentration of inhibitor to cause 20% inhibition of original enzyme activity) were calculated similarly, with [IC.sub.20] replacing [IC.sub.50] in the equation. All data were expressed as the mean [+ or -] SEM. One-way analysis of variance was used to estimate the significance of differences. There was statistical significance between control and test groups if p < 0.05.

Results

Effect of MA on CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 activities in pooled human liver microsomes

To investigate whether the MA affects the catalytic activity of human CYP enzymes, the probe reaction assays were conducted with various concentrations of MA. Specific inhibitors of CYP1A2, 2C9, 2D6, 2E1 and 3A4 were used as positive controls. Results showed that MA did not significantly affect CYP2E1-mediated 6-hydroxychlorzoxazone formation, only weakly inhibited CYP1A2-mediated paracetamol formation ([IC.sub.20] = 63.1 [micro]M), CYP2C9-mediated 4-hydroxytolbutamide formation ([IC.sub.20] = 61.6 [micro]M) and CYP2D6-mediated dextrorphan formation ([IC.sub.20] more than 100 [micro]M) (Fig. 2A, Table 1). MA inhibited CYP3A4-mediated 6[beta]-hydroxytestosterone formation ([IC.sub.20] = 11.6 [micro]M; [IC.sub.50] = 46.1 [micro]M) concentration-dependently in pooled human liver microsomes (Fig. 3A, Table 1). Therefore, further metabolic studies using specific human CYP3A4 isoform were conducted. Fig. 3B shows that MA inhibited 6[beta]-hydroxytestosterone formation ([IC.sub.20] = 4.03 [micro]M; [IC.sub.50] = 62.3 [micro]M) concentration-dependently in human CYP3A4 isoform.

Effect of OA on CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 activities in pooled human liver microsomes

The structural difference between MA and OA is only at one hydroxyl (-OH) group in C-2 position of triterpenic acid (Fig. 1). To explore the structure-activity relationship of MA and OA with CYP activity, this study also investigated the effects of OA on the catalytic activity of human CYP enzymes in pooled human liver microsomes. Data showed that OA did not significantly affect CYP2D6 and CYP2E1 activities, only weakly inhibited CYP1A2 activity by 12.8% at the highest concentration (100 [micro]M) (Fig. 2B). In addition, OA inhibited CYP2C9 and CYP3A4 activities in a concentration-dependent manner with [IC.sub.20] values at 81.0 and 13.3 [micro]M, respectively (Fig. 2B, Table 1).

Effect of MA on CYP1A2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 activities in rat liver microsomes

MA weakly inhibited CYP1A2-mediated paracetamol formation and CYP2D1-mediated dextrorphan formation only at the highest concentration 100 [micro]M (Fig. 4). MA also inhibited CYP2C11-mediated 4-hydroxytolbutamide formation ([IC.sub.20] = 11.9 [micro]M), CYP2E1-mediated 6-hydroxychlorzoxazone formation ([IC.sub.20] = 9.57 [micro]M) and CYP3A2-mediated 6[beta]-hydroxytestosterone formation ([IC.sub.20] = 8.77 [micro]M) in rat liver microsomes (Fig. 4). Moreover, MA at the highest concentration used (100 [micro]M) inhibited rat liver CYP2C11, CYP2E1 and CYP3A2 activities at 47.9%, 34.9% and 49.6%, respectively.

[FIGURE 2 OMITTED]

Enzyme inhibition kinetic analysis

To further characterize the inhibition of human CYP3A4, rat CYP2C11 and CYP3A2 activities by MA, enzyme inhibition kinetic experiments were carried out with various substrate concentrations in the presence and absence of MA. From the primary Lineweaver-Burk plot linear transformation of reciprocal of enzyme reaction velocities versus reciprocal of substrate concentrations, Figs. 5A, 6A and 7A show the straight lines intersected on the common point in the first quadrant. At the same time, Dixon plot linear transformation of reciprocal of enzyme reaction velocities versus inhibitor concentration showed the straight lines intersected on the second quadrant (Figs. 5C, 6C and 7C). These indicated competitive inhibition of MA on CYP3A4, 3A2 and 2C11. The [K.sub.i] values of MA on human CYP3A4, rat CYP3A2 and 2C11 were obtained from the secondary Lineweaver-Burk plot for K, with values of 18.4, 66.3 and 98.7 [micro]M, respectively (Figs. 5B, 6B and 7B). Moreover, OA also competitively inhibited human CYP3A4 activity with the [K.sub.i] value at 111.9 [micro]M by analyzing the data via the Lineweaver-Burk plot and Dixon plot (Fig. 8).

[FIGURE 3 OMITTED]

The inhibitory effect of MA and OA on human CYP3A4 was comparable to ketoconazole, a specific human CYP3A4 inhibitor ([K.sub.i] = 0.02 [micro]M). In addition, the inhibitory effects of MA on rat CYP3A2 and 2C11 were also comparable to ketoconazole and sulfaphenazole, the selective rat CYP3A2 and 2C11 inhibitor, with [K.sub.i] values of 0.65 and 11.8 [micro]M, respectively.

Discussion

New knowledge relating to interactions between drugs and diet is steadily accumulating. In this context, pharmacokinetic interactions encompass not only drug-drug interactions, but also interactions involving several foods and even beverages. A well-known example is that of grapefruit juice (Bailey et al. 1998; Uno and Yasui-Furukori 2006). This beverage, commonly consumed by the general population, is an inhibitor of the intestinal CYP3A4 isoform responsible for the first-pass biotransformation of many drugs (Chan et al. 1998; Dahan and Amidon 2009; Schmiedlin-Ren et al. 1997). The possible interactions that might occur would lead to increased serum levels and/or decreased clearance, increasing the risk of overdosing. In fact, foods that contain some substance can affect drug absorption, metabolism and excretion. For example, foods that contain the substance tyramine slow down enzymes that metabolize monoamine oxidase (MAO) inhibitors (a type of antidepressant medication) and can cause dangerously high blood pressure (Blob et al. 2007). Cytochrome P450 monooxygenases belong to a superfamily of haemproteins that play an important role in catalyzing the oxidation of many endogenous and exogenous substances. Most drugs undergo deactivation by CYP, either directly or by facilitated excretion from the body. In addition, CYP plays a primary role in food-drug and drug-drug interactions that can result in drug toxicity, reduced pharmacological effect and adverse drug reactions (Bibi 2008). At the same time, many foods and drugs also have the ability to affect CYP expression and activity that may have the potential to interfere with other drug metabolism (Bibi 2008). The relative abundance and significance of individual CYP enzyme in human drug metabolism include CYP1A, CYP2C, CYP2D and CYP3A isoform families. Induction or inhibition of the CYP enzymes, after exposure to different drugs and chemicals in the food, is directly linked to a number of drug-induced toxicity and drug interactions leading to treatment failure (Sun et al. 2014). Furthermore, CYP-associated metabolic studies in vitro have been considered cost-effective for predicting the potential drug-drug interactions, which is one of the major attritions in drug development and recommended by the US Food and Drug Administration (FDA). To our knowledge, this study is the first to investigate the effects of MA on the activity of human liver CYP isoforms including CYP1A2, 2C9, 2D6, 2E1 and 3A4.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

CYP1A2 is one of the major P450 enzymes in the human liver which accounts for approximately 13% of the total content of this enzyme group (Faber et al. 2005). CYP1A2 plays an important role in the metabolism of several clinically used drugs, including theophylline, clozapine and tacrine, and foodborne procarcinogens such as polycyclic aromatic hydrocarbons or imidazoquinoline derivatives (Faber et al. 2005). Murine and human CYP1A2 showed about 72% amino acid sequence homology with common catalytic activity (Ikeya et al. 1989). Our previous study also found that the Sprague-Dawley rat liver microsomes were useful for assessing drug-drug interaction in the hepatic first-pass metabolism responsible for CYP1A2 at the early drug-discovery stage (Wang and Yeung 2012). This study shows that MA only weakly inhibited CYPlA2-mediated paracetamol formation ([IC.sub.50] > 100 [micro]M) in human and rat liver microsomes. Therefore, MA was a weak CYP1A2 inhibitor and the potential drug-drug interactions with CYP1A2 would be low.

Human CYP2C9 is a major CYP enzyme involved in the metabolism of a wide range of therapeutic agents, including non-steroidal anti-inflammatory drugs, oral anticoagulants and oral hypoglycaemic agents (Rettie and Jones 2005). More than 100 currently used drugs are known substrates of CYP2C9 which corresponds to 10-20% of commonly prescribed drugs, such as meloxicam, suprofen, tolbutamide and S-warfarin. In addition, CYP2C9 also contributes to the metabolism of fatty acids, prostanoids and steroid hormones (Kirchheiner and Brockmoller 2005). This study shows that MA was a weak human CYP2C9 inhibitor with [IC.sub.20] at 61.6 [micro]M and [IC.sub.50] more than 100 [micro]M. The rat forms of the human CYP2C9 equivalent CYP2C isoforms include CYP2C6 and CYP2C11, with CYP2C11 being a homolog of the human CYP2C9 with 77% homology (Wang et al. 2010b). In the male SD rats, the CYP2C11 isoform is more important since it is the more abundantly expressed CYP, in a way equivalent to CYP3A4 in humans. Recently, many nature compounds isolated from herbs have been identified as inhibitors of CYP2C11 enzyme. For example, tanshinones from Danshen and celastrol from Trypterygium wilfordii Hook F. competitively inhibited rat CYP2C11 activity, which is also mainly responsible for S-warfarin hydroxylase (Sun et al. 2014; Wang and Yeung 2011). In this study, it is clear from the in vitro inhibition kinetic studies in rat liver microsomes that MA can act as competitive inhibitor to rat CYP2C11 with a of 98.7 [micro]M. Thus, MA was a weak CYP2C11 inhibitor, compared with sulfaphenazole, a selective rat CYP2C11 inhibitor ([K.sub.i] = 30.8 [micro]M) in this study.

The CYP3A subfamily is one of the dominant CYP enzymes in both the liver and extra-hepatic tissues such as intestine, and plays an important role in the oxidation of xenobiotics and contributes to the biotransformations of about 60% of currently used therapeutic drugs (Zhou et al. 2005). Human CYP3A4 is one of the most abundant drug-metabolizing P450 isoforms in human liver microsomes, which accounts for approximately 40% of the total P450 (Zhou et al. 2005). In fact, characterization of the CYP3A4 isoform responsible for the metabolism of drugs is important for the identification of potential drug-drug or food-drug interactions in humans. This study shows that MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with [IC.sub.50] value at 46.1 and 62.3 [micro]M, respectively. Further enzyme kinetic study showed MA was a weak competitive human CYP3A4 inhibitor with the [K.sub.i] value of 18.4 [micro]M, compared with ketoconazole, a specific human CYP3A4 inhibitor ([K.sub.i] = 0.02 [micro]M). In addition, MA also competitively inhibited rat CYP3A2 activity, which is the major contributor to testosterone 6[beta]-hydroxylation in male rats, with the [K.sub.i] value of 66.3 [micro]M. The different [K.sub.i] values in humans and rats may be due to species difference in CYP3A isoenzymes. Moreover, human CYP3A4 is also abundantly expressed in intestine, accounting for approximately 80% of total intestine P450 (Paine et al. 2006). Previous studies reported MA inhibited human intestinal CYP3A4 activity with [IC.sub.50] value at 7.4 [micro]M (Kim et al. 2011). However, the mechanisms of interaction of MA with intestinal CYP3A4 in humans remain uncertain. Further system study in vivo is needed to identify the interactions of MA with CYP3A4 in humans.

Human CYP2D6 enzyme is involved in the biotransformation of 30% of drugs on the market, although it is expressed at a low level in liver accounting for about 4% of total P450 (Madani et al. 1999). In addition, only one isoform, CYP2D6, is expressed in various tissues including the liver, kidney, placenta, brain, breast, lung and intestine in humans (Huang et al. 1997). The rat and human CYP2D isoforms share a high sequence identity (>70%), and CYP2D1 is the rat ortholog of human CYP2D6 (Venhorst et al. 2003). This study shows MA only slightly inhibited human CYP2D6 and rat CYP2D1 at the highest concentration 100 [micro]M. Thus, MA would appear to have a low potential to cause drug-drug interaction with CYP2D6. The effects of MA on the metabolism of model probe substrates of CYP2E1 were also carried out in this study. CYP2E1 is the only gene of this subfamily. In humans, CYP2E1 accounts for approximately 6% of total P450 in the liver and is involved in the metabolism of 2% of the drugs on the market (Zuber et al. 2002). This study shows that MA did not affect CYP2E1-mediated 6-hydroxychlorzoxazone formation in vitro in human liver.

Previous studies revealed that oleanolic acid (OA), a well-known triterpenic acid, competitively inhibited CYP1A2 and CYP3A4 activities in human liver microsomes, with [IC.sub.50] ([K.sub.i]) values of 143.5 (74.2) and 78.9 (41.0) [micro]M, respectively (Kim et al. 2004). Table 1 shows the inhibition of CYP isoforms by MA and OA in human liver microsomes in this study. Although MA and OA showed no or weak inhibition on CYP1A2, 2C9, 2D6 and 2E1 activities, MA presented more potent inhibitory effect on CYP3A4 than OA with [IC.sub.50] ([K.sub.i]) at 46.1 (18.4) [micro]M. Furthermore, this study provided a clue for structure-activity relationship of CYP3A4 activity of MA. The structural difference between MA and OA is only at one hydroxyl (-OH) group in C-2 position of triterpenic acid (Fig. 1), but this resulted in the different potent inhibition of CYP3A4. MA as a competitive inhibitor prevents the binding of the substrate by binding reversibly the active site of CYP3A4 enzyme. The presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with OA may magnify its competitive inhibition on CYP3A4 via iron chelating and/or hydrogen bonding interaction. However, this mechanistic alternative to the inhibition of CYP3A4 remains unclear. Further systematic study is needed to identify the potential relationship of structure-activity.

The concentration of MA in commercial table olives ranges from 287.1 [+ or -] 66.6 to 1318.4 [+ or -] 401.0 mg/kg depending upon the variety and the method of processing (Lozano-Mena et al. 2012). The amount of MA in the oil is much lower than in the fruit, and its concentration depends upon the oil extraction process. In extra virgin olive oil with acidity inferior to 1%, MA can be found at 64.2 [+ or -] 8.1 mg/kg, whereas it increases to 193.9 [+ or -] 14.0 mg/kg in virgin olive oil (Perez-Camino and Cert 1999). Previous studies reported that not only the single oral administration of MA at 1000 mg/kg to mice did not produce any signs of morbidity or mortality, but also the repeated daily oral administration of 50 mg/kg of MA for 28 days did not induce any sign of toxicity during the experimental period (Sanchez-Gonzalez et al. 2013). In particular, the dose 50 mg/kg evaluated in the repeated oral administration for 28 days of MA corresponds to approximately 125 times the amount that may be consumed by a person eating 40 g or 10-medium sized olives and 33 g of olive oil a day (Sanchez-Gonzalez et al. 2013). Moreover, the plasma [C.sub.max] values of MA in rats treated with an oral dose (50 mg/kg)) were less than 4 [micro]M (Lozano-Mena et al. 2012). Therefore, the rat plasma concentrations were much lower than the [IC.sub.50] and [K.sub.i] values determined in this study. Thus, the potential for MA to cause the possible toxicity by inhibition of these CYP isoforms would be low. Furthermore, due to species difference in ADME/toxicity and CYP expression, further system study in vivo is needed to identify the interactions of MA with CYP isoforms in humans.

Conclusions

The data in this study demonstrate that the MA was not only a weak competitive inhibitor of CYP3A4 in humans, but also a weak competitive inhibitor of CYP2C11 and 3A2 in rats, with [K.sub.i] of 18.4, 98.7 and 66.3 [micro]M, respectively. In addition, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with OA could magnify its competitive inhibition on human CYP3A4 activity. The relatively high [K.sub.i] values of MA for CYP2C and 3A would suggest a low potential for MA to cause the possible toxicity by inhibition of these CYP isoforms. Moreover, MA and some food containing MA, such as olive oil, also would have a low potential to cause drug-drug and/or food-drug interactions involving CYP enzymes. This study may be also helpful for the development and application of MA as a promising agent. However, the drug-drug interaction potential should be confirmed by further in vivo studies. Further systematic studies in humans in vivo are also needed to identify the interactions of MA with cytochrome P450 enzymes.

Conflict of interest statement

The authors have declared no conflict of interest.

ARTICLE INFO

Article history: Received 4 July 2014

Revised 20 August 2014

Accepted 15 October 2014

Acknowledgements

This work was supported in part by grants from the National Natural Science Foundation of China (No. 81301908), and the Science and Technology Commission of Shanghai Municipality (Nos. 12XD1406100, 13ZR1412600 and 14DZ2270100).

References

Allouche, Y., Warleta, F., Campos, M., Sanchez-Quesada, C., Uceda, M., Beltran, G., et al., 2011. Antioxidant, antiproliferative, and pro-apoptotic capacities of pentacyclic triterpenes found in the skin of olives on MCF-7 human breast cancer cells and their effects on DNA damage. J. Agric. Food Chem. 59, 121-130.

Bailey, D.G., Malcolm, J., Arnold, O., Spence, J.D., 1998. Grapefruit juice-drug interactions. Br. J. Clin. Pharmacol. 46, 101-110.

Bibi, Z., 2008. Role of cytochrome P450 in drug interactions. Nutr. Metab. 5, 27.

Blob, L.F., Sharoky, M., Campbell, B.J., Kemper, E.M., Gilmor, M.G., VanDenberg, C.M., et al., 2007. Effects of a tyramine-enriched meal on blood pressure response in healthy male volunteers treated with selegiline transdermal system 6 mg/24 hour. CNS Spectr. 12, 25-34.

Brown, R.E., Jarvis, K.L., Hyland, K.J., 1989. Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem. 180, 136-139.

Chan, W.K., Nguyen, L.T., Miller, V.P., Harris, R.Z., 1998. Mechanism-based inactivation of human cytochrome P450 3A4 by grapefruit juice and red wine. Life Sci. 62, 135-142.

Chen, J.D., Wu, Y.Z., Tao, Z.L., Chen, Z.M., Liu, X.P., 1995. Hawthorn (shan zha) drink and its lowering effect on blood lipid levels in humans and rats. World Rev. Nutr. Diet 77, 147-154.

Dalian, A., Amidon, G.L., 2009. Grapefruit juice and its constituents augment colchicine intestinal absorption: potential hazardous interaction and the role of p-glycoprotein. Pharm. Res. 26, 883-892.

Faber, M.S., Jetter, A, Fuhr, LL, 2005. Assessment of CYP1A2 activity in clinical practice: why, how, and when?. Basic Clin. Pharmacol. Toxicol. 97, 125-134.

Ghanbari, R., Anwar, F.. Alkharfy, K.M., Gilani, A.H., Saari, N., 2012. Valuable nutrients and functional bioactives in different parts of olive (Olea europaea L)-A review. Int. J. Mol. Sci. 13, 3291-3340.

Guan, T., Qian, Y., Tang, X., Huang, M., Huang, L., Li, Y., et al., 2011. Maslinic acid, a natural inhibitor of glycogen phosphorylase, reduces cerebral ischemic injury in hyperglycemic rats by GLT-1 up-regulation. J. Neurosci. Res. 89, 1829-1839.

Huang, L., Guan, T., Qian, Y., Huang, M., Tang, X., Li, Y., et al., 2011. Anti-inflammatory effects of maslinic acid, a natural triterpene, in cultured cortical astrocytes via suppression of nuclear factor-kappa B. Eur. J. Pharmacol. 672, 169-174.

Huang, Z., Fasco, M.J., Kaminsky, L.S., 1997. Alternative splicing of CYP2D mRNA in human breast tissue. Arch. Biochem. Biophys. 343, 101-108.

Ikeya, K., Jaiswal, A.K., Owens, R.A., Jones, J.E., Nebert, D.W., Kimura, S., 1989. Human CYP1A2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1A2 mRNA expression. Mol. Endocrinol. 3, 1399-1408.

Juan, M.E., Wenzel, U., Ruiz-Gutierrez, V., Daniel, H., Planas, J.M., 2006. Olive fruit extracts inhibit proliferation and induce apoptosis in HT-29 human colon cancer cells. J. Nutr. 136, 2553-2557.

Kim, E., Sy-Cordero, A., Graf, T.N., Brantley, S.J., Paine, M.F., Oberlies, N.H., 2011. Isolation and identification of intestinal CYP3A inhibitors from cranberry (Vaccinium macrocarpon) using human intestinal microsomes. Planta Med. 77, 265-270.

Kim, K.A., Lee, J.S., Park. H.J., Kim, J.W., Kim, C.J., Shim, I.S., Kim, N.J., Han. S.M., Lim, S., 2004. Inhibition of cytochrome P450 activities by oieanolic acid and ursolic acid in human liver microsomes. Life Sci. 74, 2769-2779.

Kirchheiner, J., Brockmoller, J., 2005. Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin. Pharmacol. Ther. 77, 1-16.

Li, C., Yang, Z., Li, Z., Ma, Y., Zhang, L., Zheng, C., et al., 2011. Maslinic acid suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss by regulating RANKL-mediated NF-kappaB and MAPK signaling pathways. J. Bone Miner. Res. 26, 644-656.

Li, C., Yang, Z., Zhai, C, Qiu, W., Li, D., Yi, Z., et al., 2010. Maslinic acid potentiates the anti-tumor activity of tumor necrosis factor alpha by inhibiting NF-kappaB signaling pathway. Mol. Cancer 9, 73.

Lozano-Mena, G., Juan, M.E., Garcia-Granados, A., Planas, J.M., 2012. Determination of maslinic acid, a pentacyclic triterpene from olives, in rat plasma by high-performance liquid chromatography. J. Agric. Food Chem. 60, 10220-10225.

Madani, S., Paine, M.F., Lewis, L., Thummel, K.E., Shen, D.D., 1999. Comparison of CYP2D6 content and metoprolol oxidation between microsomes isolated from human livers and small intestines. Pharm. Res. 16, 1199-1205.

Martin, R., Carvalho-Tavares, J., Ibeas, E., Hernandez. M., Ruiz-Gutierrez, V., Nieto, M.L., 2007. Acidic triterpenes compromise growth and survival of astrocytoma cell lines by regulating reactive oxygen species accumulation. Cancer Res. 67, 3741-3751.

Montilla, M.P., Agil, A, Navarro, M.C., Jimenez, M.I., Garcia-Granados, A, Parra, A., et al., 2003. Antioxidant activity of maslinic acid, a triterpene derivative obtained from Olea europaea. Planta Med. 69, 472-474.

Nieto, F.R., Cobos, E.J., Entrena, J.M., Parra, A., Garcia-Granados, A, Baeyens, J.M., 2013. Antiallodynic and analgesic effects of maslinic acid, a pentacyclic triterpenoid from Olea europaea. J. Nat. Prod. 76, 737-740.

Owen, R.W., Haubner, R., Wurtele, G., Hull, E., Spiegelhalder, B., Bartsch, H., 2004. Olives and olive oil in cancer prevention. Eur. J. Cancer Prev. 13, 319-326.

Paine, M.F., Hart, H.L., Ludington, S.S., Haining, R.L., Rettie, A.E., Zeldin, D.C., 2006. The human intestinal cytochrome P450 pie. Drug Metab. Dispos. 34, 880-886.

Park, S.Y., Nho, C.W., Kwon, D.Y., Kang, Y.H., Lee, K.W., Park, J.H., 2013. Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1 alpha signalling. Br.J. Nutr. 109, 210-222.

Perez-Camino, M.C., Cert, A, 1999. Quantitative determination of hydroxypentacyclic triterpene acids in vegetable oils. J. Agric. Food Chem. 47, 1558-1562.

Rettie, A.E., Jones, J.P., 2005. Clinical and toxicological relevance of CYP2C9: drug-drug interactions and pharmacogenetics. Annu. Rev. Pharmacol. Toxicol. 45, 477-494.

Reyes-Zurita, F.J., Pachon-Pena, G., Lizarraga, D., Rufino-Palomares, E.E., Cascante, M., Lupianez. J.A., 2011. The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism. BMC Cancer 11, 154.

Reyes-Zurita, F.J., Rufino-Palomares, E.E., Lupianez, J.A., Cascante, M., 2009. Maslinic acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29 human colon-cancer cells via the mitochondrial apoptotic pathway. Cancer Lett. 273, 44-54.

Rigelsky, J.M., Sweet, B.V., 2002. Hawthorn: pharmacology and therapeutic uses. Am. J. Health Syst. Pharm. 59, 417-422.

Sanchez-Gonzalez, M., Lozano-Mena, G., Juan, M.E., Garcia-Granados, A, Planas, J.M., 2013. Assessment of the safety of maslinic acid, a bioactive compound from Olea europaea L. Mol. Nutr. Food Res. 57, 339-346.

Schmiedlin-Ren, P., Edwards, D.J., Fitzsimmons, M.E., He, K., Lown, K.S., Woster, P.M., et al., 1997. Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents, decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins. Drug Metab. Dispos. 25, 1228-1233.

Sun, M., Tang, Y., Ding, T., Liu, M., Wang, X., 2014. Inhibitory effects of celastrol on rat liver cytochrome P450 1A2, 2C11, 2D6, 2E1 and 3A2 activity. Fitoterapia 92, 1-8.

Uno, T., Yasui-Furukori, N., 2006. Effect of grapefruit juice in relation to human pharmacokinetic study. Curr. Clin. Pharmacol. 1, 157-161.

Venhorst, J., ter Laak, A.M., Commandeur, J.N., Funae, Y., Hiroi, T., Vermeulen, N.P., 2003. Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities. J. Med. Chem. 46, 74-86.

Wang, X., Cheung, C.M., Lee, W.Y., Or, P.M., Yeung, J.H., 2010. Major tanshinones of Danshen (Salvia miltiorrhiza) exhibit different modes of inhibition on human CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities in vitro. Phytomedicine 17, 868-875.

Wang, X., Lee, W.Y., Or, P.M., Yeung, J.H., 2009. Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates. Phytomedicine 16, 712-725.

Wang, X., Lee, W.Y., Or, P.M., Yeung, J.H., 2010. Pharmacokinetic interaction studies of tanshinones with tolbutamide, a model CYP2C11 probe substrate, using liver microsomes, primary hepatocytes and in vivo in the rat. Phytomedicine 17, 203-211.

Wang, X., Yeung, J.H., 2011. Inhibitory effect of tanshinones on rat CYP3A2 and CYP2C11 activity and its structure-activity relationship. Fitoterapia 82, 539-545.

Wang, X., Yeung, J.H., 2012. Investigation of cytochrome P450 1A2 and 3A inhibitory properties of Danshen tincture. Phytomedicine 19, 348-354.

Zhou, S., Yung Chan, S., Cher Goh, B., Chan, E., Duan, W., Huang, M., et al., 2005. Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs. Clin. Pharmacokinet. 44, 279-304.

Zuber, R., Anzenbacherova, E., Anzenbacher, P., 2002. Cytochromes P450 and experimental models of drug metabolism. J. Cell Mol. Med. 6, 189-198.

Min Sun (a), (1), Yu Tang (a), (1), Tonggui Ding (a), Mingyao Liu (a,b), Xin Wang (a), *

(a) Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China

(b) Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TK 77030, USA

* Corresponding author at: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China. Tel.: +86 21 2420 6564; fax: +86 21 5434 4922.

E-mail address: usxinwang@gmail.com, xwang@bio.ecnu.edu.cn (X. Wang).

(1) These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.phymed.2014.10.003
Table 1
Inhibition of CYP-catalyzed reactions by MA and OA in human liver
microsomes.

         Maslinic acid

         [IC.sub.50]                [K.sub.i]    Type of
         ([IC.sub.20]) ([micro]M)   ([micro]M)   inhibition

CYP1A2   >100 (63.1)                --           --
CYP2C9   >100 (61.6)                --           --
CYP2D6   --                         --           --
CYP2E1   --                         --           --
CYP3A4   46.1 (11.6)                18.4         Competitive

         Oleanolic acid

         [IC.sub.50]                        [K.sub.i]    Type of
         ([IC.sub.20]) ([micro]M)           ([micro]M)   inhibition

CYP1A2   [much greater than] 100 (> 100)    --           --
CYP2C9   >100 (81.0)                        --           --
CYP2D6   --                                 --           --
CYP2E1   --                                 --           --
CYP3A4   >100 (13.3)                        111.9        Competitive
COPYRIGHT 2015 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Sun, Min; Tang, Yu; Ding, Tonggui; Liu, Mingyao; Wang, Xin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jan 15, 2015
Words:7333
Previous Article:Original mechanisms of antipsychotic action by the indole alkaloid alstonine (Picralima nitida).
Next Article:In vitro anti-hyperglycemic activity of 4-hydroxyisoleucine derivatives.
Topics:

Terms of use | Copyright © 2017 Farlex, Inc. | Feedback | For webmasters