Using in Vitro and in Vivo Models To Evaluate the Oral Bioavailability

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Using in vitro and in vivo models to evaluate the oral bioavailability of nutraceuticals Yuwen Ting, Qin Zhao, Chunxin Xia, and Qingrong Huang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2015 Downloaded from http://pubs.acs.org on January 28, 2015

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Journal of Agricultural and Food Chemistry

Title Header Models to Evaluate Nutraceutical Bioavailability

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Using in vitro and in vivo models to evaluate the oral bioavailability of nutraceuticals

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Yuwen Ting, Qin Zhao, Chunxin Xia and Qingrong Huang*

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Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey

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08901, USA

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* To whom correspondence should be addressed. Tel: (848)-932-5514. Fax: (732)-932-6776.

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Email: [email protected].

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ACS Paragon Plus Environment


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Abstract. Nutraceuticals are the bioactive compounds found in many dietary sources. Numerous

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publications reported their ability to prevent the development of degenerative diseases through

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modulation of the physiological and physiochemical processes in the living system. Having

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sufficient concentration at the target site of action is the most critical factor for nutraceuticals to

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deliver the health benefits. For consumers, it is commonly accepted to ingest these bioactive

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components through oral delivery route since it is convenient, cost-efficient, and allows flexible

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dosing schedule. Thus, it is important to understand the oral bioavailability of nutraceuticals in

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order to evaluate their qualifications as disease preventive agents and to calculate the required

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ingestion dosages. To predict the oral bioavailability of nutraceuticals, many in vitro and in vivo

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models have been developed to reduce the need of frequent human pharmacokinetic study, which

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is costly, time-consuming, and involves ethical complications. These models evaluate one or

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more of the influential factors that contribute to the oral bioavailability, and are efficient

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screening techniques with the potential of predicting the pharmacokinetic process in human. In

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order to accurately predict the human oral bioavailability, further research is not only required to

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develop a better correlation between the in vitro and in vivo models, but also an accurate scaling

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factor that takes into account of interspecies variations.

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Key

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bioaccessibility/ Caco-2/ metabolism/ bioavailability

words:

Nutraceutical/

pharmacokinetics/

TIM-1/

in

42 43 44 45

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vitro

digestion/

lipolysis/

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Introduction

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Bioactive compounds, widely found in variety of dietary sources, provide an excellent

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alternative to medicinal drugs for improving public health. The roles of these compounds to

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modulate the physiochemical reactions in the biological system were extensively studied. The

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term, nutraceutical, was then emerged to categorize the dietary compounds that could offer

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health functionalities in addition to simple satisfaction of the basic nutritional needs for survival

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of living organisms. Apart from drugs that support immediate relief of diagnosed pathological

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symptoms, nutraceuticals, on the other hand, are found more effective to prevent the

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development of degenerative illness, such as cardiovascular diseases and cancer (1, 2). Thus,

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chronic consumption of nutraceuticals becomes necessary since it is commonly used as disease

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preventive agent. In this case, oral administration, compare to other delivery routes, is the

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consumer-preferred method to utilize these bioactive ingredients because it is cheaper, more

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user-friendly, painless, and allowing flexible dosing schedule.

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Upon oral administration, the ingested nutraceuticals enter the gastrointestinal (GI) tact.

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Depending on the chemical structure of ingested compounds, complex physiological and

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physiochemical environment within the GI tract may variably affect the absorption of bioactives

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and could eventually prevent it from reaching the system circulation. As most of the

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nutraceutical merely subject to clinical trials, the biological activities of these bioactive

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ingredients are frequently characterized using in vitro evidences. According to the in vitro

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evaluations, the concentration required for nutraceuticals to produce meaningful bioactivities is

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usually in the micromolar (µM) range (3), which is at least an order of magnitude higher than the

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plasma concentration (< 1 µM) obtained from normal dietary intake (4). In order to obtain the

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desirable functionalities, appropriate concentration of the bioactive compound must reach the

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target site of action. To achieve sufficient system bioavailability, oral dosing concentration of

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nutraceutical must be selected to accommodate the fraction that will be eliminated by the

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physiological barriers.

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The determination of oral bioavailability is then as important as to identify the potential

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therapeutic uses of neutraceuticals. Even though it is more precise to evaluate oral bioavailability

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directly using human subjects and clinical testing, it is frequently challenged by the costly

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consequences involving safety and ethical considerations. Thus, many investigators choose to

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alternatively study the pharmacokinetic profile of bioactives using animal models. The in vivo

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animal models provide valuable information to determine the oral bioavailability in a conclusive

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manner. Unlike the man-made machinery that can be examined step-by-step, in vivo models

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provide only unilateral end result determining the fraction of ingested dosages that are available

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to the systemic circulation without providing enough explanation to the events that lead to it.

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Compared to in vivo pharmacokinetic model, in vitro methods developed to mimic the

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events occurred after oral administration offer opportunities to study the effect of different

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physical and chemical parameters that could play critical roles in the systemic availability of

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bioactive components. In addition, the easy-to-perform in vitro tools are commonly used for

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rapid prediction on the oral bioavailability of bioactive compounds before performing an in vivo

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study that are more costly, time-consuming, and may subject to ethical complications. In this

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perspective, in vitro and in vivo methods for predicting the oral bioavailability of bioactive

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components will be summarized and compared in relation to their relevance to the living system.

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Later, the discussion on the potential of using in vitro and in vivo methods for the rapid

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bioavailability screening of future bioactive ingredient and their related oral formulations will

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also be included.

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Oral bioavailability

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A sufficient dosage at the target site of action is critical for nutraceutical to generate the

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desirable biological activities. That is, until compound concentration becomes greater than the

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threshold dose, there could be no desirable health promoting effect found. To obtain the

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appropriate concentration at target site, it is important to determine the bioavailability of the

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bioactive compounds. Bioavailability is a principal pharmacokinetic term describing the fraction

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of the administered dose reaches the system circulation without being altered. As system

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concentration obtained from intravenous injection was defined as 100% bioavailability,

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nutraceuticals administered through other delivery routes generally present lower bioavailability

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with incomplete absorption due to the physiological or physiochemical barriers.

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In specific for the system concentration obtain after oral administration, oral

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bioavailability is greatly depend on the ability of nutraceuticals to survive the structural

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alteration from pre-absorption degradation and post-absorption metabolism. The pre-absorption

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events such as pH instability, ionic interaction, enzymatic degradation, and rapid GI elimination

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rate could significantly decrease the amount of ingested compound available for the gut wall

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absorption. Once taken up by the intestinal enterocytes, the bioactive compound is then subjected

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to extensive structural transformation first by intestinal and then by hepatic metabolism. Taking

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into the account of all events occurred after oral administration (Figure 1), oral bioavailability

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(Foral) can be summarized and defined by following equation(5, 6).

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= × ×

Eq. 1

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In this mathematical expression, FB is defined as the fraction of ingested dose that

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become accessible for intestinal absorption. The second component of the equation, FT, describes

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the fraction of unchanged compound being transported into the portal system. The third

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component to determine the oral bioavailability, FM, outlined the fraction of ingested dose that

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survive the metabolic activities as it travels to various systemic organs, such as liver, heart, and

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lung. Hence, unlike intravenous injection that all applied dose reaches the system circulation, the

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system bioavailability of orally ingested nutraceutical may be reduced to various degree

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depending on their specific chemical structure that influences the abovementioned

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physiochemical parameters.

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In vitro models for predicting oral bioavailability

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Oral bioavailability is positively correlated with the bioaccessbility for GI absorption (FB),

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the gut wall transport coefficient (FT), and the resistibility to the systemic metabolism (FM). Base

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on the aforementioned theory, in vitro and in vivo models developed for predicting the oral

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bioavailability of nutraceuticals generally focus on evaluating one or more of the factors.

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Bioaccessibility. Once ingested from mouth, dietary components enter the GI tract, which is

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essentially a compartment separating from other interior organs and circulating system with a

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semipermeable barrier. To get to the inner system, nutraceutical must be first absorbed by the

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enterocytes. Thus, it is most critical that these bioactives are available at the absorption contact

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area. The bioaccessibility of a nutraceuitcal is then determined by quantifying the amount of

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ingested dose reaches the absorption site with its intact structure. GI tract, specialized for the

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digestion, consists of several sections that each has a distinct physical and physiochemical

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environment responsible for the breakdown of diverse dietary components.

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Nutraceutical, just as all other dietary ingredients, go through the entire GI transition

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process, during which it may be chemically degraded and/or enzymatically metabolized to the

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product structure with totally different chemical property. In other words, nutraceutical must

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survive all the degradation activities within the GI tract while being solubilized, or at least

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dispersed, in the aqueous lumen in order to become bioaccessible for absorption. To determine

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the bioaccessibility of a bioactive component, numbers of in vitro models, from simple to

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complex, are designed based on the separable nature of GI tract. Namely, effect of pH, ionic

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interaction, enzyme activities, temperature, and luminal solubility on the compound

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bioaccessibility can be studied individually or systematically using suitable tests.

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Chemical tests evaluating the stability of nutraceutical at physiological relevant ranges of

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temperature (37°C), pH (2 - 7.5), and ionic composition are commonly performed methodologies

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to initially assess the oral dosage efficiency(7-14). However, the events occurs in the actual

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human GI tract is a rather complex biochemical process than just few simple chemical reactions.

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Enzyme activities not only directly cause the structural change, such as bond cleavage and sugar

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moiety substitution, but also interfere with the absorption kinetic when other dietary components,

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such as lipid, protein, and starch are present (10, 15-18).

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As an example, the absorption of hydrophobic compound is significantly enhanced when

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lipid is present due to the improved aqueous solubility after being incorporated into the mixed

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micelle, which is the vehicle-like structure formed after lipid digestion (5, 19-21). In vitro pH-

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stat model simulating the digestion environment in the small intestine, the major site for lipid

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digestion, is an especially suitable analytical tool to evaluate the change in bioaccessibility for

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hydrophobic compounds when lipid is ingested either as part of the food matrix or oral

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formulations(22, 23). The single-factored in vitro chemical models is preferred by many

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investigators for initial screening of dosing efficiency since it is relatively simple to perform,

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cost-effective, rapid, and allow large sample through-put.

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In addition to methodologies that contain only single factor, multi-factored models that

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combine two or more bioaccessibility-influential factors are also available (Figure 2). For

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example, the in vitro digestion model may simulate the physiochemical condition first in the

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stomach and then the small intestine(24). However, the in vitro models discussed above,

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regardless of number of factor addressed, are considered as static model due to their inability to

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reproduce the dynamic in vivo physical conditions including the peristalsis motion of GI tract,

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integration of all the influential physiological factors, transitional change in physiological

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environment, and elimination from the absorption site. Consequently, the bioaccessibility

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predicted by static models usually overestimate the oral efficiency (25).

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To take the dynamic factor into account, a sophisticated instrument, TIM-1

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gastrointestinal simulation system, featuring a continuous in vitro digestion process was

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developed by TNO quality of life, The Netherlands. Physiological and physiochemical factors,

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such as body temperature, peristalsis movement, gastric and intestinal residence time, pH,

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luminal composition, and elimination, are closely approximated by an automated computer

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system(26). The bioaccessible fraction is determined by continuous sampling from jejunum and

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ileum sections over a pre-determined time period(25, 27). Another advantage to use an open-

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compartment system, in which the unabsorbed portion is eliminated and collected as efflux,

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allows estimation of loss due to degradation since it can be quantified by subtracting the total

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recovered portion from the original input dosage. TIM system, by far, is the most sophisticated

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and precise instrument for bioaccessiblity estimation of dietary components.

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Transport coefficient. To travel across the intestinal lining, nutraceuticals may undertake either

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the transcellular or paracellur route. When transcellular route is assumed, nutraceutical from the

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gastric lumen is first absorbed into the enterocytes through either passive diffusion that is driven

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by concentration gradient or active transportation through the transcellular protein channels.

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During the in-and-out passage through the enterocytes, cellular metabolism significantly reduces

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the fraction of intact compound being release into the portal circulation (28). Apart from the

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transcellular route, small molecules (Mw < 200) permeate through the membrane paracellularly

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through tight junction and do not go through the enterocytes. Nutraceuticals utilized paracellular

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route escaped from the intestinal metabolism and retain the aglycone structure when streaming

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into the portal blood (28, 29).

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Hence, the rate of compound transported from GI tract into the portal system is depended

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on its permeability to the cellular membrane and resistibility to intestinal metabolism. Unlike

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bioaccessiblity is determined majorly by the physiochemical events in the GI lumen and can be

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easily simulated using in vitro chemical studies, transport coefficient cannot be characterized

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using only the chemical experiment. Gastrointestinal model that include a dialysis step after the

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in vitro digestion is the most simplified in vitro experiment for measuring the membrane

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transport rate of dissolved compound(30). Since no biological system was involved, dialysis

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method that allows permeation of small molecules over concentration gradient can only be used

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for estimating the fraction of compound that is transported through the paracellualr route.

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The intestinal walls composed of single layer of epithelial cells forms the rate-limiting

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barrier to the transport of bioactive compounds. Cell culture model (Figure 3) of properly

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differentiated Caco-2 human colon carcinoma cell into phenotype with the characteristic

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physiochemical functions of the enterocytes is the commonly used in vitro method for

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elucidating the transport mechanism of nutraceutical (31-35). Caco-2 monolayer allows effective

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in vitro prediction of compound permeability and absorption while taking into account all the

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profound physical, chemical, and biological events during the intestinal transportation(36).

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Moreover, Caco-2 monolayer model is also a well-developed model to effectively evaluate the

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lymphatic transport (alterative transport route into the system circulation) of lipid-soluble

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compounds when included in lipid-based oral formulations(37-39). However, the transport

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studies by Caco-2 monolayer still have numbers of disadvantages since the consistency of this

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study is highly depended on the quality of the monolayer. Factors such as cell condition, passage

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number, length of cultivation, circumstance under which the study is performed, and the

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transport unit varies among laboratories(40). Thus, the absolute transport coefficient value

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generated by different laboratories deviate accordingly and could not be compared directly.

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Moreover, the fact that Caco-2 is human colon carcinoma cell with different physiological

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characteristics than the normal intestinal cell, such as lacking mucin, biofilm, and other epithelial

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cell types, can also cause unrealistic prediction of transport coefficient value in human(24).

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In addition to the cell culture model, single-pass intestinal perfusion (SPIP) is an in situ animal

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model that provides enhanced approximation of intestinal physiology and allows the study of

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difference in the absorption behavior at various intestinal regions (41). The experimental

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parameters, such as the compound concentration and the flow rate through the perfused section,

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are closely controlled to ensure the intra- and inter- experimental comparability. In this model,

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the compound permeation efficiency is quantified as the concentration difference between the

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inlet and outlet of perfusate normalized by volume decrease due to water absorption(42).

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However, permeation rate quantified by the lost of compound in the perfusate retain a

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shortcoming of being unable to differentiate between the metabolized compound and the

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unchanged fraction, which is the main parameter for calculating the transport coefficient.

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Resistibility to systemic metabolism. Once being transported into the portal system, absorbed

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component is then conveyed to liver where they will be facing the rigorous first-pass metabolic

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activities. Due to the high density of the metabolic related cytochromes and enzymes, liver is the

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principal facility for biotransformation of xenobiotic and is thought to be the one of major rate-

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limiting factors that reduce the system bioavailability of orally ingested nutraceuticals (43).

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Cytochrome P-450 (CYPs) represent a family of membrane associated proteins that is variably

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expressed in different organ tissues (44, 45) and is responsible for the metabolism of more than

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75% of endogenous compounds (46). The major biotransformation of nutraceuticals by CYP450

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monooxygenase includes oxidation, hydroxylation and demethylation (47, 48). The isozymes

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mainly involve in the metabolism of bioactive compounds are CYP1A1, CYP2B, and CYP3A

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(43, 49, 50).

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As CYPs is most abundantly expressed in liver, isolated liver microsomes are usually

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employed in the in vitro metabolic model that characterizes the biotransformation of

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nutraceutical. During the metabolic test, compounds are incubated with liver microsome and the

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resulting product composition is usually identified by selected analytical instrument. In vitro

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metabolism model is effective method to evaluate the metabolic stability of nutraceuticals as it

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allow rapid, low-cost, and large through-put sampling. However, the use of animal liver

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microsome to predict human metabolite composition is somewhat unreliable since the expression

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of CYPs is variable among animal species and is also highly dependent on the incubation and

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sampling interval.

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In vivo pharmacokinetic model and correlation to human oral bioavailability

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The bioavailability of orally ingested compound is the product of a complex process that

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integrates series of physiological or physiochemical reactions. In vitro methods designed to

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simulate an aspect of physiochemical process are useful tools for characterization and prediction

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of the in vivo pharmacokinetics in early screening of bioactive components. Since the

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bioavailability is affected by multiple factors, in vitro tests conducted in isolation usually present

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inadequate in vitro-in vivo correlations (IVIVC). Using the combinations of two or more in vitro

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models, the precision of estimating the oral bioavailability will increase since more influential

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factors are addressed. However, even the TIM-1 system being the most complex and closest

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simulation of pre-absorption events in the human GI tract still cannot perfectly predict the oral

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bioavailability due the lacking of biological factors, such as enterocytes absorption, systemic

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metabolism, and volumes of body distribution. Thus, even though in vivo pharmacokinetic study

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is time consuming, costly, and sometimes inhumane, it is still considered as the best described

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technique to predict human oral bioavailability. Since completely reproducing the

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pharmacokinetic process using in vitro model is overly complicated and unrealistic, in vivo

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animal model offers a living system imitating the full dynamic physiological and physiochemical

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events during the absorption, distribution, metabolism, and elimination of orally ingested

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nutraceuticals. Even though the mechanism that underlines the pharmacokinetic process cannot

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be fully explained, analyzing the timed serum profile from the in vivo studies provide useful

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information on the oral bioavailability of ingested compounds as well as the metabolite profile.

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Although animal data, especially pharmacokinetic profile of rodents, have been widely

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used to predict oral bioavailability in human, the correlation in the absorption of bioactive

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compounds between animals and human has not been extensively studied. The variations in the

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body weight, organ size, hepatic and renal blood flow, metabolism, distribution, and elimination

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rate result in significantly different pharmacokinetic profiles among different animal species. As

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an example, the pharmacokinetic parameters, such as peak concentration time (Tmax), Peak serum

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concentration (Cmax), total area under curve (AUC), and urinary excretion, of quercetin deviate

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accordingly when different in vivo models were used (table 1). Developed according to the body

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weight and physiological parameters, allometric scaling was applied to in vivo animal data to

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better reflect the actual pharmacokinetic process in human(51, 52). However, due to the

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complexity of physiological events in the living system, methods or scaling to 100% correlate

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the in vivo data to human oral bioavailability is still not available (53).

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Besides physiological factors of modeling systems, the pharmacokinetic profile is also

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significantly affected when different experimental designs were applied. For oral ingestion, the

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most influential parameter to the bioavailability is the physical and chemical characteristic of

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dosing formulation. By using different dosing media, the absorption of nutraceuticals can

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deviate appreciably due to change in the bioaccessibility, a positively correlated parameter to the

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system bioavailability. In particular for hydrophobic compound, oral absorption is improved

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when its solubility, the important factor for compound to be available for gut wall absorption,

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become higher in the dosing medium (54-56). In support of this idea, Piskula et al. found the

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oral bioavailability of quercetin was highest when solubilized in 100% propylene glycol,

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followed by 25% propylene glycol solution and then water (55). Inspired by the finding that

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better bioaccessibility leads to higher absorption rate, various oral delivery systems was designed

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to improve the pharmacokinetic profile of nutraceuticals with diverse chemical properties(5, 6).

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As an example, the oral bioavailability of hydrophobic compound, such as curcumin (table 2), is

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improved by the oral delivery system that enhanced its aqueous solubility. On the other hand,

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delivery systems that could enhance the luminal stability and membrane permeability will lead to

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better oral bioavailability of EGCG, a hydrophilic compound which could be rapidly degraded in

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the GI tract (Table 3).

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Due to the lack of consensus in the selection of animal models, dosing formulation, and

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delivery system, the in vivo pharmacokinetic data published by different investigators can rarely

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be comparable. Thus, the prediction of human oral bioavailability is difficult without systematic

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collation of available pharmacokinetic data. Despite of being the best pre-clinical model to

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evaluate the oral bioavailability of bioactive compounds, pharmacokinetic study itself does not

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provide enough mechanistic information to allow efficient data organization. In this sense, in

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vitro test should be collectively conducted to study the pharmacokinetic behavior of

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nutraceuticals.

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Prediction of human oral bioavailability

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To prevent the development of degenerative diseases, nutraceuticals are desirable to have

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good oral dosage efficiency since it is usually consumed either as part of (functional food) or

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supplementary (dietary supplement) to the diet. Oral bioavailability of bioactive compound, as

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discussed in earlier sections, is majorly influenced by its bioaccessibility in the GI lumen,

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transport rate across the gut wall, and resistibility to the systemic metabolism. In order to better

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understand their contribution to the oral bioavailability individually and conjointly, these

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parameters could be evaluated by using proper in vitro and in vivo modeling systems since

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evaluating the pharmacokinetic profile using human subject is expensive and may involve ethical

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issues. Thus, it is further desirable that these modeling systems could eventually allow accurate

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prediction of the pharmacokinetic events in human body. Even though in vitro studies

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conveniently examine the factors that contribute to oral bioavailability, it cannot accurately

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predict human pharmacokinetic profile without proper correlation to the in vivo system and the

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application of allometric scaling, which is calculated with regard to the interspecies difference.

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To take full advantages of the pre-clinical modeling system to realistically predicting human oral

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bioavailability of nutraceutical, the mathematical expression of oral bioavailability could be re-

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write as follow:

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= × × × ×

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Eq. 2

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The mathematical equation reveals a future perspective, in which the evaluation of

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human oral bioavailability can be projected using only the in vitro modeling systems. In this

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equation, the bioaccessibility (FB), transport rate (FT), and resistibility to systemic metabolism

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(FM) can all be determined with appropriate in vitro studies. The other two components represent

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the correlation factors of in vitro and in vivo modeling system (CA) and interspecies scaling (CS).

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To determine CA and CS, a consistent experimental procedure is required when carrying out

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pharmacokinetic study using both animal and human subjects. The in vivo animal models serve

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as an important bridging factor that links the in vitro data to the actual event occurring in human

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body.

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In order to accurately calculating CA and CS, there are definitely much more work need to

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done and problems to be solve. However, the benefit from successfully developing such

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mathematical relationship between in vitro measurement and human pharmacokinetic process is

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numerous. First of all, the cost and disadvantages of using models involve living creatures could

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be minimized. Secondly, factors that change the pharmacokinetic process can be easily taken

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into account by conducting in vitro testing of affected parameters. Third, rapid estimation of

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required dosage for disease prevention is much more reliable and realistic to the actual value in

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human subject. Fourth, the effectiveness of oral formulation to enhance bioavailability can be

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efficiently determined without going through complicated evaluations. Overall, integration of

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modeling systems covering all aspect of physiological and physiochemical reactions that

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contribute to the oral bioavailability of nutraceticals offers the possibility to further elucidate

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human pharmacokinetic process with much more simplified and efficient procedure.

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51. Boxenbaum, H., Interspecies Scaling, Allometry, Physiological Time, and the Ground Plan of Pharmacokinetics. J. Pharmacokinet Biop. 1982, 10, (2), 201-225. 52. Dedrick, R.; Bischoff, K. B.; Zaharko, D. S., Interspecies correlation of plasma concentration history of methotrexate (NSC-740). Cancer Chemother. Rep. 1970, 54, (2), 95-101. 53. Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C.; MacIntyre, F.; Rance, D. J.; Wastall, P., The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. J. Pharmacol. Exp. Ther. 1997, 283, (1), 46-58. 54. Vallejo, F.; Larrosa, M.; Escudero, E.; Zafrilla, M. P.; Cerda, B.; Boza, J.; Garcia-Conesa, M. T.; Espin, J. C.; Tomas-Barberan, F. A., Concentration and Solubility of Flavanones in Orange Beverages Affect Their Bioavailability in Humans. J. Agr. Food Chem. 2010, 58, (10), 6516-6524. 55. Piskula, M. K.; Terao, J., Quercetin's solubility affects its accumulation in rat plasma after oral administration. J. Agr. Food Chem. 1998, 46, (10), 4313-4317. 56. Das, S.; Lin, H. S.; Ho, P. C.; Ng, K. Y., The impact of aqueous solubility and dose on the pharmacokinetic profiles of resveratrol. Pharm. Res. 2008, 25, (11), 2593-2600. 57. Chen, X.; Yin, O. Q.; Zuo, Z.; Chow, M. S., Pharmacokinetics and modeling of quercetin and metabolites. Pharm. Res. 2005, 22, (6), 892-901. 58. Hou, Y.; Chao, P.; Ho, H.; Wen, C.; Hsiu, S., Profound difference in pharmacokinetics between morin and its isomer quercetin in rats. J. Pharm. Pharmacol. 2003, 55, (2), 199203. 59. Reinboth, M.; Wolffram, S.; Abraham, G.; Ungemach, F. R.; Cermak, R., Oral bioavailability of quercetin from different quercetin glycosides in dogs. Brit. J. Nutr. 2010, 104, (2), 198-203. 60. Cermak, R.; Landgraf, S.; Wolffram, S., The bioavailability of quercetin in pigs depends on the glycoside moiety and on dietary factors. J. Nutr. 2003, 133, (9), 2802-2807. 61. Gugler, R.; Leschik, M.; Dengler, H., Disposition of quercetin in man after single oral and intravenous doses. Eur. J. Clin. Pharmacol. 1975, 9, (2-3), 229-234. 62. Goldberg, D. M.; Yan, J.; Soleas, G. J., Absorption of three wine-related polyphenols in three different matrices by healthy subjects. Clin. Biochem. 2003, 36, (1), 79-87. 63. Moon, Y. J.; Wang, L.; DiCenzo, R.; Morris, M. E., Quercetin pharmacokinetics in humans. Biopharm. Drug Dispos. 2008, 29, (4), 205-217. 64. Li, J.; Jiang, Y. Y.; Wen, J.; Fan, G. R.; Wu, Y. T.; Zhang, C., A rapid and simple HPLC method for the determination of curcumin in rat plasma: assay development, validation and application to a pharmacokinetic study of curcumin liposome. Biomed. Chromatogr. 2009, 23, (11), 1201-1207. 65. Liu, A. C.; Lou, H. X.; Zhao, L. X.; Fan, P. H., Validated LC/MS/MS assay for curcumin and tetrahydrocurcumin in rat plasma and application to pharmacokinetic study of phospholipid complex of curcumin. J. Pharmaceut. Biomed. 2006, 40, (3), 720-727. 66. Hu, L. D.; Jia, Y. H.; Niu, F.; Jia, Z.; Yang, X.; Jiao, K. L., Preparation and Enhancement of Oral Bioavailability of Curcumin Using Microemulsions Vehicle. J. Agr. Food Chem. 2012, 60, (29), 7137-7141. 67. Liu, Z. F.; Chiu, M.; Wang, J.; Chen, W.; Yen, W.; Fan-Havard, P.; Yee, L. D.; Chan, K. K., Enhancement of curcumin oral absorption and pharmacokinetics of curcuminoids and curcumin metabolites in mice. Cancer Chemoth. Pharm. 2012, 69, (3), 679-689.

19



527 528 529 530 531 532 533 534 535 536 537 538 539 540

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68. Kakkar, V.; Singh, S.; Singla, D.; Kaur, I. P., Exploring solid lipid nanoparticles to enhance the oral bioavailability of curcumin. Mol. Nutr. Food Res. 2011, 55, (3), 495-503. 69. Wan, S. X.; Sun, Y. Q.; Qi, X. X.; Tan, F. P., Improved Bioavailability of Poorly WaterSoluble Drug Curcumin in Cellulose Acetate Solid Dispersion. AAPS Pharmscitech 2012, 13, (1), 159-166. 70. Pietta, P.; Simonetti, P.; Gardana, C.; Brusamolino, A.; Morazzoni, P.; Bombardelli, E., Relationship between rate and extent of catechin absorption and plasma antioxidant status. Biochem. Mol. Biol. Int. 1998, 46, (5), 895-903. 71. Dube, A.; Nicolazzo, J. A.; Larson, I., Chitosan nanoparticles enhance the plasma exposure of (-)-epigallocatechin gallate in mice through an enhancement in intestinal stability. Eur. J. Pharm. Sci. 2011, 44, (3), 422-426.

20


Page 21 of 27

541 542 543


Table 1: Pharmacokinetic profile different among animal species.

Phenolics

Model

Dose

Quercetin

SD Rats SD Rats

10 mg/kg 50 mg/kg 100 mg/kg 70.6mg/kg 50 mg/kg 50 – 65 mg/kg 10 mg/70 kg 500 mg

Dogs Pigs Human Human Human

Tmax (hr) 0.078 1.2 2 0.5 3

Cmax (µmol/L) 0.69 4.9 9.5 0.23 1.19 < 0.001 0.16 – 0.45 0.05

AUC (µmol/Lhr) 0.2 48.44 80.3 2.59 6.55 0.21

544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575

21


Urinary excretion (%) 2.9 – 7.0 1.18

Ref. (57) (58) (59) (60) (61) (62) (63)


576 577 578

Page 22 of 27

Table 2: Effect of oral formulation on the oral bioavailability of curcumin. Results

Ref.

Liposome

Functional Mechanisms solubility

bioavailability

(64)

Phospholipid complex

solubility

bioavailability

(65)

Microemulsion

solubility

bioavailability

(66)

Nanoemulsion

solubility

bioavailability

(67)

Solid lipid nanoparticle

solubility stability

bioavailability

(68)

Solid dispersion (with cellulose acetate)

solubility

bioavailability longer elimination time

(69)

Formulations

579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603

22


Page 23 of 27

604 605 606


Table 3: Effect of oral formulation on the oral bioavailability of EGCG. Formulations Phospholipid complex Chitosan-nanoparticle

Functional Mechanisms permeability stability permeability stability paracellular transport

Results

Ref.

bioavailability

(70)

bioavailability

(71)

607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627

23



Page 24 of 27

628 629 630 631

Figure 1: Process of orally ingested compound becomes bioavailable and factors that influence

632

the oral bioavailability.

633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654

24


Page 25 of 27


655 656 657

Figure 2: Common in vitro methods to evaluate the bioaccessibility of orally ingested

658

nutraceuticals. Single factor model simulate only the digestion activity with small intestine.

659

Multi-factor model simulated the dynamic GI digestion events in both stomach and small

660

intestine.

661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 25



Page 26 of 27

685 686 687 688 689

690 691 692 693

Figure 3: Transport coefficient evaluated using Caco-2 monolayer model. Caco-2 monolayer

694

simulated the transport activity of intestinal epithelium utilizing both paracellular and

695

transcellular transport route.

696 697

26


Page 27 of 27

698 699 700


TOC

701 702

27


Using in Vitro and in Vivo Models To Evaluate the Oral Bioavailability

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