Dietary Supplements - American Chemical Society

Chapter 16

Bioavailability of Polymethoxyflavones 1

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on July 22, 2016 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch016

Shiming Li , Y u Wang , Slavik Dushenkov , and Chi-Tang H o

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Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520 WellGen Inc., 63 Dudley Road, New Brunswick, NJ 08901 2

One of the most influential factors of bioavailability of a nutrient or a drug is absorption. Both solubility and permeability affect absorption. Sufficient absorption of a drug or nutrient is necessary to elicit a therapeutic effect or for a successful nutritional usage. There are a variety of procedures to measure solubility and permeability. Prior to this report, bioavailability studies have been performed with flavonoids of all subclasses except the flavones and it was found that isoflavones had the best bioavailability relative to other flavonols studied. In the course of this work, we performed in vitro bioavailability assays of several polymethoxyflavones (PMFs) by measuring their solubility and permeability and concluded that P M F bioavailability is generally good owing to their lipophilic nature of the multiple methoxy groups on the P M F structure. This report represents the first systematic in vitro study of P M F bioavailability.

© 2008 American Chemical Society

Ho et al.; Dietary Supplements ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Background of Bioavailability Bioavailability of a drug or a nutrient is that amount of the drug or the nutrient which reaches the blood circulatory system and target tissues (1). The bioavailability is an overall effect of absorption, distribution, metabolism and excretion (ADME). Hence, its determination is the combined rate of these factors. Absorption describes a drug or a nutrient's ability to pass into the systemic circulation following oral administration. Metabolism is the rate that a drug or a nutrient is eliminatedfromthe systemic circulation, following its initial absorption. Distribution describes how well a drug or a nutrient reaches the target tissues. Excretion is the rate at which a drug or a nutrient is excreted from the systemic circulation (2). Figure 1 is a graphic description of the ADME process. Only the "unboundfraction"of a drug or a nutrient remains to reach the tissue and is able to interact with the molecular target (7,3).

Site of action bound * free

Tissue Reservoirs free ~ — ^ bound

Systemic Circulation

Absorption

Free Drug bound drug

|

Excretion

metabolites

Metabolism Figure 1. Bioavailability and ADME

One of the most influential factors of bioavailability of a nutrient or a drug is absorption, which can be defined as the product of solubility and permeability. Absorption is usually the first pharmacokinetic component studied (3). Solubility refers to aqueous solubility and is must include a specific pH range. A nutrient or a drug substance can only be absorbed from the gastrointestinal tract when it is in solution (2). Therefore, thefractionof drug absorbed into the portal vein is a function of the aqueous solubility of the substance. Permeability is a compound's ability to across through intestinal membranes into the blood circulation system (4). Both solubility and permeability affect absorption and they must be considered together, and not as independent parameters.



235 Compounds with high solubility and high permeability are usually well absorbed, whereas compounds with low solubility and low permeability are poorly absorbed. Compounds with mixed properties must be carefully characterized to ensure that they show sufficient absorption/permeability to elicit a therapeutic or nutritional use (7). It is reasonably straightforward to measure solubility but more difficult to measure permeability because permeability is less defined than solubility (4). The US Food and Drug Administration (FDA) accepts three absolute permeability criteria: (i) mass balance, absolute bioavailability, or intestinal perfusion studies in human subjects - costly methods usually reserved for compounds that are well along in the approval process; (ii) in vivo intestinal perfusion studies in animals; and (iii) in vitro permeation experiments across a monolayer of cultured human intestinal cells (Caco-2 cells) (2). Currently, the most productive methods for solubility measurements is LYSA (lyophilization solubility assay, reference 7). LYSA is a high throughput solubility measurement included in the Multi-dimensional Drug Optimization (MDO) system. The LYSA method has a relatively high throughput (hundreds of compounds per day) in comparison to low throughput solubility assays like THESA (thermodynamic solubility assay). Permeability represents the overall effects of influx and efflux in the body (Figure 2). Influx refers to a drug or nutrient being transported from the small intestine to the blood system by absorptive transporters such as multidrug resistence proteins (MRP1 and MRP3), , whereas efflux is defined as a process whereby the drug or nutrient is pumped outfromblood system to the intestine by a secretory transporter like breast cancer resistance protein (BCRP), Pglycoprotein and MRP2 (5). Cultured "intestine-like" cells such as monolayer cultured cells (Caco-2) have been used for many years to test drug and nutrient permeability. The Caco-2 cell monolayers are human carcinoma cell lines that have many enterocyte-like properties. They are used by the pharmaceutical industry to evaluate the oral absorption potential of drugs and/or to study their absorption mechanisms (7). Transport studies through Caco-2 cells provide information about: (i) intestinal permeability, (ii) transport mechanisms (paracellular or transcellular or active carrier), (iii) the role of intestinal metabolism, and (iv) the influence of P-glycoprotein efflux system (6). Caco-2 permeability studies can deliver valuable information in the early lead discovery of biologically active compounds and in the development phase for intestinal permeability and absorption prediction through membranes. Hence, Solubility and permeability are essential parameters in the prediction of absorption affecting the bioavailability of ingested substances (7,6)Another type of permeability test known as PAMPA (Parallel Artificial Membrane Permeation Assay) was developed by Roche scientists in 1998 (4,7). PAMPA, performed in microplates, measures permeation of a compound through a phospholipid-coated filter medium that mimics intestinal cell


236 Intestinal Lumen

Epithelial Cells

Blood Supply

Mucosal (Apical) Surface


Influx

Secretory Transport

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Figure 2. Influx and efflux pathways

structures. The idea behind the PAMPA assay is to predict human intestinal permeability. Among the three possible pathways through a membrane (paracellular, transcellular and active transport), 80%-90% of all small molecular drugs permeability is transcellular. Transcellular permeation is based on passive diffusion, driven by a concentration gradient between donor and acceptor. The small intestine (duodenum, jejunum and ileum) is the major absorption site with the largest absorption area. The PAMPA assay mimics these absorptive conditions using an artificial phospholipidmembrane (4,7). Many samples per day can be analyzed by the PAMPA system

Absorption and Bioavailability of Flavonoids To elucidate the role of dietary flavonoids in human health, it is essential to know the concentrations and forms present in the plasma and tissues after ingestion of these flavonoids (6). Absorptionfromthe small intestine is more efficient than that of the colon. Good absorption from small intestines leads to higher plasma values. Upon absorptionfromthe small intestine, flavonoids are then conjugated with glucuronic acid or sulfate, or even Omethylated (6). Most flavonoids, except catechins, are usually present in the diet as p-glycosides. Glycosides were thought to be too hydrophilic to be well absorbed by passive diffusion in the small intestine (6). Research results have shown that the sugar moiety of quercetin plays an important role in increasing the bioavailability of



237 quercetin (8). The plasma concentrations and kinetics after single dose administration of someflavonoidsin humans are summarized in tabular form by Hollman (P). The table showed that catechins and EGCG are quickly absorbed. The bioavailability of catechins was similar. Overall, isoflavones showed the highest bioavailability among the studied flavonols. Although bioavailability studies have been performed with flavonoids of all subclasses, with the exception of the flavones (P), there seems to be a lack of understanding or consideration of the importance of bioavailability in the in vitro studies, which are subsequently used for the design of in vivo experiments. Bioavailability should be carefully considered in the design and interpretation of in vitro and in vivo experiments (10,11). It is important to understand the correlation between in vitro and in vivo bioavailability, ensuring that the latter can be predicted, based on the datafromin vitro experiments.

Absorption and Bioavailability of PMFs With the increased interests in exploring the health benefits of PMFs, the bioavailability study of PMFs has lagged behind. Ohigashi and his colleagues investigated the in vitro absorption of nobiletin and luteolin and found that nobiletin preferably accumulated in a differentiated Caco-2 cell monolayer while luteolin did not (12). The conclusion can be drawnfromthis in vitro experiment that nobiletin has a much higher permeability and a tendency to accumulate in the intracellular compartment relative to luteolin. The same research group conducted an in vivo study of nobiletin in SD male rats and concluded that nobiletin has the distinct property to accumulate in a wide range of organs including the stomach, small and large intestines, liver, and kidney during the 1 to 4 hour periods following single dose administration. In contrast, luteolin and its conjugates were predominantly detected in the gastrointestinal tract (73).

Recent in vitro Bioavailability Studies of PMFs We performed a bioavailability study of some PMFs by measuring their solubility and permeability with the goal of further understanding the cause of their absorption and bioavailability properties. The high throughput screening procedure, LYSA, was used to measure the solubility of PMFs. Some permeability data were also obtained from both PAMPA and Caco-2 experiments. From the data it was found that the solubility of PMFs is fairly low, but their permeability is quite high (Table I). As a net result, the absorption profile of PMFs is high, possibly contributing to the high relative bioavailability of PMFs. Although multiple methoxy groups are hydrophobic, contributing to


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the poor solubility of PMFs the lipophilic nature of methoxy groups are closely related to the good permeability of PMFs (14).


Experimental Conditions and Reagents Preparation of PMF samples: 5,6,7,3',4'-pentamethoxyflavone, tangeretin, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone, and 3-hydroxynobiletin were isolatedfromsweet orange peel. 3'-Hydroxy-5,6,7,4'-tetramethoxyflavone and Gardenin A were purchased from Indofine, Inc. (Hillsborough, NJ). 3,5,6,7,8,3',4',5-octamethoxyflavone was synthesized from Gardenin A according to a published procedure (14). 3'hydroxy-5,6,7,8,4'-pentamethoxyflavone, 4'-hydroxy-5,6,7,8,3'-pentamethoxyflavone, and 3\4'-hydroxy-5,6,7,8-tetramethoxyflavone were synthesized according to a reported procedure (14). A total of 12 PMFs were weighed and dissolved in 0.1 mL of DMSO, which is the stock solution of PMFs (10 mM). Reagents and solvents: Phosphate buffer (pH 6.5), EDTA solution, sterile PBS solution, cell culture media, trypsin, and non-essential amino acids were purchasedfromFisher Scientific (Fairlawn, NJ).

LYSA Procedure DMSO stock solutions of PMFs were diluted to 0.05, 0.1, 0.2 and 0.5 mM for preparation of a four point calibration curve. The OD (Optical Density) maxima ( was determined by direct U V measurement for each PMF. In a similar manner, sample solutions of PMFs were also prepared in duplicate from the DMSO stock solutions (10 mM). The DMSO was then removed with a centrifuge vacuum evaporator. After evaporation of DMSO, the solid PMFs were dissolved in a 0.05 M phosphate buffer (pH 6.5), stirred for one hour and then shaken for two hours. After standing overnight,, the solutions were filtered using a microtiter filter plate. The spectra of the filtrate and its 1/10 dilution were measured with the U V microplate reader and solubility was calculated for each PMF studied.

PAMPA Procedure PAMPA is an automated assay which is based on a 96-well microplate. The permeation of drugs is measured using a "sandwich" construction (this needs to be fixed?) (4,7). Afilterplateis coated with a phospholipid membrane and


239 placed into a donor plate containing a drug/buffer solution. The filterplate is next filled with buffer solution (acceptor). The donor concentration is measured at the start of the experiment (reference, t ) and compared with the donor and acceptor concentrations after a certain time te . The drug concentration analysis is based on UV spectroscopy. UV absorption of the samples, compared between the initial reading of the assay (tstart: reference) and at the end (te : donor, acceptor), allow the determination of a sample distribution between donor, membrane and acceptor. Once the permeation time (tend - W ) is known, a permeation rate constant can be calculated. The unit of this constant is 10" cm/s, is indicative of a kinetic value. The detailed experimental procedure of PAMPA measurement is illustrated in references 4 and 7. start

nd

nd


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Caco-2 Experimental Procedure Cell culture: Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD). Caco-2 cells at passage number 28 to 40 were used in these studies. All cells were grown in Dulbecco's modified Eagle's medium containing 90% Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% nonessential amino acids, 100 units/ml penicillin, and 100 pg/ml streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% C 0 . Culture medium was changed every other day, and cells were passed every 3 to 5 days by trypsinization with 0.05% trypin and 0.53 mM EDTA at 37 °C for 10 min (75). Directional transport assays. Directional transport assays were performed essentially as described by Irvine, et al. (7(5). Briefly, Caco-2 cells were seeded in Transwell plates at a density of 6.25 X 10 cells/cm . The cells were then fed withfreshmedium every other day and cultured for 21 to 25 days. Prior to the assay, cells were rinsed with transport medium (Hank's balanced solution, pH 7.4, containing 10 mM Hepes). Cells were equilibrated in the transport medium at 37 °C for 30 min. The transepithelial electrical resistance (TEER) of cell monolayers was measured using "chopstick" electrodes (World Precision Instrument, Sarasonta, FL). The TEER values were determined and corrected by subtracting the resistance of blank filters. 2

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Result and Discussion In this study we demonstrated the overall bioavailability of PMFs are good. The PMFs have excellent permeability but poor aqueous solubility. In the LYSA assay, any number that is greater than 200 pg/mL is considered soluble. The number between 100 pg/mL and 200 pg/mL is considered medium


240 Table I. Solubility and Permeability Data of PMFs LASA

Name

Structure

Sinensetin

(Mg/ ml)

Caco-2 (P p)

PAMPA

(cm/s xKT ) 6

ap

B to A (cm/s xlQ- )

Ratio (A to B)/ (B to A)

nd

nd

nd

nd

nd

nd

802

535

0.7

A toB Permea (cm/s bility xKT ) 6

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M

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