Post on 06-Jul-2020
RELATIVE BIOAVAILABILITY OF TWO DIFFERENT FORMULATIONS OF MILK THISTLE
By
WEN-YI LI
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY
UNIVERSITY OF FLORIDA
2018
© 2018 Wen-Yi Li
To my parents, sister, brother and family for their support and love
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ACKNOWLEDGMENTS
I would like to express my deepest gratitude and appreciation to my advisor
Dr. Reginald F. Frye for his training, guidance and support for my research work at the
University of Florida. I am very grateful to him for the great learning experiences and
professional development opportunities I was exposed to over the past three years.
Especially, I would like to thank him for giving me the opportunity to pursue my master’s
degree at the Department of Pharmacotherapy and Translational Research.
My sincerest indebtedness goes to my committee members, including Dr. John
S. Markowitz and Dr. Yan Gong for their help and guidance on my research project.
Specifically, I would like to thank Renee M. Hogan, ARNP, for her assistance on clinical
monitoring, data collection and conducting physical exams. I would like to express my
thankfulness to Rajesh Mohandas, MD, for providing clinical input and general medical
oversight to my study. Also, I am beholden to Guo Yu, PhD, a visiting scholar (Jiangsu,
China) in my lab who contributed to the PK data analysis and interpretation as well as
general encouragement to my research project. Lastly, I would like to thank Isagenix
International LLC (Gilbert, Arizona) for funding this project.
During my graduate studies, I worked as a graduate assistant for Dr. William
Cary Mobley and Dr. Heather R. Hardin. Here I want to thank them for their help and
advice on my work. In addition, I would like to thank all the people in the Department of
Pharmacotherapy and Translational Research for their great friendship and kindness
which created an enjoyable environment that I will bear in mind. Finally, I would like to
acknowledge the University of Florida, College of Pharmacy for supporting my graduate
work.
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Last but not the least, I would like to heartfeltly thank my parents, sister, brother
and other family members for their continuous inspiration, understanding and listening.
During my ups and downs, they are always the strongest source of care, support and
encouragement for me. I would like to dedicate this thesis to them for their endless love,
unlimited encouragement and unconditional support.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 10
ABSTRACT ................................................................................................................... 11
CHAPTER
1 INTRODUCTION .................................................................................................... 13
Dietary Supplements .............................................................................................. 13 Bioactivities of Milk Thistle in IsaGenesis and Product B ........................................ 15 Methods to Improve Silybin Bioavailability .............................................................. 17
Phytosomes ...................................................................................................... 17 Softgel Technology ........................................................................................... 18 Nanoparticles ................................................................................................... 19 Nanosuspensions ............................................................................................. 20 Nanoemulsions ................................................................................................. 21 Liposomes ........................................................................................................ 22
2 HYPOTHESIS ......................................................................................................... 27
Existing Problem in Herbal Supplements ................................................................ 27 Research Hypothesis .............................................................................................. 27 Objective ................................................................................................................. 28 Methods and Materials ............................................................................................ 28
Supplements and Chemicals ............................................................................ 28 Preparation of Stock Solutions, Calibrator Standards, and Quality Control
Samples ........................................................................................................ 29 Study Participants ............................................................................................ 29 Supplement Administration ............................................................................... 30 Sample Collection and Processing ................................................................... 30
Analytical Methods .................................................................................................. 31 Analysis of Silybin A and Silybin B from Oral Dosage Forms ........................... 31 Analysis of Silybin A and Silybin B from Human Plasma .................................. 31 Pharmacokinetic Analysis ................................................................................. 32 Statistical Analysis ............................................................................................ 33
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3 RESULTS ............................................................................................................... 36
Calibration and Linearity ......................................................................................... 36 Precision and Accuracy .......................................................................................... 36 Formulation Analysis............................................................................................... 36 Study Participants ................................................................................................... 36 Pharmacokinetic Analysis ....................................................................................... 37
4 DISCUSSION ......................................................................................................... 50
LIST OF REFERENCES ............................................................................................... 54
BIOGRAPHICAL SKETCH ............................................................................................ 60
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LIST OF TABLES
Table page 1-1 Definition of dietary ingredients .......................................................................... 24
1-2 Formulation comparison and content ................................................................. 25
3-1 Intra- and inter-run precision (CV, %) and accuracy (RE, %) for quality control (QC) samples in human plasma. ............................................................ 39
3-2 Comparison of silybin A and silybin B amounts in Product B and IsaGenesis .... 40
3-3 Pharmacokinetic parameters of silybin A and silybin B after single oral doses of Product B and IsaGenesis. ............................................................................. 41
3-4 P values of log-transformed Cmax and AUClast of silybin A and silybin B after single oral doses of Product B and IsaGenesis between male (n=6) versus female (n=6) subjects. ........................................................................................ 42
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LIST OF FIGURES
Figure page 1-1 Structures of silybin A and silybin B .................................................................... 26
2-1 Overview of a single-dose, randomized crossover pharmacokinetic assessment. CRC: Clinical Research Center. .................................................... 34
2-2 Representative ion chromatograms of two products from the study sponsor (Isagenix International, LLC) and one well-known milk thistle extract Legalon® (Madaus, GH) ..................................................................................... 35
3-1 Mean plasma concentrations of silybin A versus time profiles after oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects. ............................................................................................................. 43
3-2 Mean plasma concentrations of silybin B versus time profiles after oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects. ............................................................................................................. 44
3-3 Mean plasma concentrations of silybin (A and B) versus time profiles after oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects. ............................................................................................................. 45
3-4 Box plots of AUClast/Dose (h*ng/mL/mg) of silybin A. ......................................... 46
3-5 Box plots of Cmax/Dose (ng/mL/mg) of silybin A. ................................................. 47
3-6 Box plots of AUClast/Dose (h*ng/mL/mg) of silybin B. ......................................... 48
3-7 Box plots of Cmax/Dose (ng/mL/mg) of silybin B. ................................................. 49
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LIST OF ABBREVIATIONS
ALT Alanine aminotransferase
AST Aspartate aminotransferase
AUC Area under the concentration-time curve
AUCinf AUC from time 0 to infinity
AUClast AUC from time 0 to the last detectable concentration
BMI Body mass index
CAM Complementary and alternative medicine
Cmax The maximum plasma concentration
CRC Clinical Research Center
DSHEA Dietary Supplement Health and Education Act
EGCG Epigallocatechin-3-gallate
FDA Food and Drug Administration
GI Gastrointestinal
IS Internal standard
LC-MS/MS Liquid chromatography-tandem mass spectrometry
LLOQ Lower limit of quantitation
MTBE Methyl tert-butyl ether
PC Phosphatidylcholine
PE Pharmaceutical equivalents
PK Pharmacokinetic
SLNs Solid lipid nanoparticles
t1/2 Elimination half-life
Tmax Time to maximum plasma concentration
λz Terminal elimination rate constant
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science in Pharmacy
RELATIVE BIOAVAILABILITY OF TWO DIFFERENT FORMULATIONS OF MILK THISTLE
By
Wen-Yi Li
December 2018
Chair: Reginald F. Frye Co-chair: John S. Markowitz Major: Pharmaceutical Sciences
As the popularity of herbal dietary supplements has increased, more and more
multi-herb combination products have appeared on the dietary supplement market.
Since the oral bioavailability of many phytochemicals is low, various dosage forms, such
as nanoemulsion formulations and phosphatidylcholine-complexed softgel formulations,
are being developed to improve the bioavailability of bioactive constituents. However,
there is scant clinical research on the effect of formulation on the bioavailability of
bioconstituents in multi-herb supplements in human subjects.
In the current study, we compared the bioavailability between two milk thistle-
containing dietary supplements, Product B, a standard powder-filled capsule and
IsaGenesis, a softgel formulation, by evaluating silybin A and silybin B as surrogate
markers for differences in absorption and bioavailability. For this randomized, crossover
PK study, 12 healthy study participants consumed a single-dose serving (2 capsules) of
each supplement separated by a washout period of at least one-week. Serial blood
samples were obtained at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h time points and analyzed by
Liquid chromatography-tandem mass spectrometry (LC-MS/MS).
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Both formulations exhibited rapid absorption and elimination of silybin A and
silybin B. However, the absorption, as indicated by mean Cmax values and mean plasma
AUC, were significantly higher for the IsaGenesis formulation. Relative to powder-filled
Product B, the IsaGenesis softgel formulation demonstrated 365% and 450% higher
mean Cmax, for silybin A and silybin B, respectively. On average, Tmax was reached at
least 1 h earlier with IsaGenesis relative to Product B for both silybin A and silybin B.
Our findings indicate that the IsaGenesis softgel formulation provides markedly higher
absorption and bioavailability of silybin A and silybin B in comparison with the powder-
filled capsule formulation, Product B.
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CHAPTER 1 INTRODUCTION
Dietary Supplements
In the “Dietary Supplement Health and Education Act (DSHEA)” of 1994, a
dietary supplement represents a product (other than tobacco) intended to supplement
the diet that contains one or more dietary ingredients [1]. DSHEA not only provides
regulations for dietary supplements, but also officially defines dietary ingredients as
shown in Table 1-1.
Over the past 15 years, dietary supplement use has grown steadily in the United
States and across the globe [2]. Herbal supplement sales accounted for nearly 20% of
the total dietary supplement sales in the U.S., and annual retail sales of these products
in the U.S. exceeded $7 billion in 2016 [3]. In developed countries, herbal supplement
or herbal medicine is generally referred to as a complementary and alternative medicine
(CAM). New CAM products are not required to undergo formal review like conventional
drugs regulated by the US Food and Drug Administration (FDA) [4, 5]. Accordingly,
many herbal supplements lack rigorous scientific evaluation relative to their safety and
effectiveness. Though CAM is regulated as food, consumers often regard herbal
supplements as medications for disease prevention and healthy aging [6]. For reasons
including desire for wellness, fitness, and disease prevention, as well as the perception
that natural alternatives are safer than conventional drugs, a substantial number of
individuals turn to herbal supplements.
As the popularity of herbal dietary supplements has increased, many more multi-
herb combination products have appeared on the dietary supplement market. But herbal
supplements usually differ in content of phytoconstituents from batch to batch and
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between different manufacturers. In addition, even with standardized active
constituents, there may be differences in other components, leading to variations in
bioactivity and bioavailability in humans [7]. A multi-herb supplement is a complex
mixture, and it cannot be assumed that individual components within a multi-herb
supplement will exert exactly the same effect(s) as they do in single-herb supplements.
In 2007, the Final Rule for Current Good Manufacturing Practices for Dietary
Supplements issued by the FDA provides standards to ensure the identity, purity,
quality, strength, and composition of dietary supplements, but it does not address
minimum requirements for quality and the inherent safety of the ingredients contained in
dietary products. Since dietary supplements or CAM products are regulated as food,
there is no guarantee that manufacturers register their commercial herbal products
before marketing [8, 9]. However, most of the evidence of positive effects of dietary
phytochemicals is inferred from in vitro models and most of the assessments of
bioactive constituents are conducted in single-herb supplements [10-14].
The bioavailability of many phytochemicals is low after oral administration,
therefore, different dosage forms, such as nanoemulsion formulations and
phosphatidylcholine-complexed softgel formulations, are being used to enhance the
bioavailability of bioactive constituents [15, 16]. To date, there is scant clinical research
on the effect of different formulations on the bioavailability of phytoconstituents in multi-
herb supplements in humans. Accordingly, the purpose of this study is to bridge the
knowledge gap regarding the influence of formulation on the bioavailability of individual
component in a multi-herb supplement.
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Bioactivities of Milk Thistle in IsaGenesis and Product B
In the current study, a softgel formulation, IsaGenesis, and a standard dry,
powder-filled formulation, Product B, are investigated to determine the impact of the
formulation on the bioavailability of bioactive constituents in these multi-herb
supplements. IsaGenesis and Product B both contain milk thistle extract as the main
ingredient, and both are mixtures of herbal extracts, vitamins and amino acids purported
to promote healthy aging and reduce oxidative damage. Besides milk thistle, other
herbal ingredients, such as green tea, horny goat weed, turmeric, and ashwagandha,
are incorporated into these two multi-herb dietary supplements (Table 1-2).
The milk thistle fruit has been utilized to treat liver and gallbladder disorders
since ancient times [17]. The crude extract obtained from crushed seeds of the plant
Silybum marianum, is termed silymarin, and contains a complex mixture of bioactive
constituents, including approximately 70% silymarin flavonolignans. These constituents
include silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B, and
silydianin, and silymarin flavonoids, including taxifolin and quercetin. Silymarin has
several pharmacological effects, including antioxidant, anticancer, and hepatoprotective
activity [18]. Among silymarin flavonolignans, silybin (silybin A and silybin B; Figure 1-1)
is the predominant and the most active constituent of silymarin. Silybin possesses
diverse bioactivities: hepatoprotection, antioxidant, chemopreventive and
neuroprotective effects, and it has been advocated for the treatment of alcohol-
associated liver diseases [19-21]. In animal experiments, silybin has been reported to
prevent the oxidative toxicities of alcohol, acetaminophen, and phalloidin [22]. These
findings imply that silybin, the predominant active constituent of silymarin, is a potent
liver protectant and an effective antioxidant in therapeutic applications. Given the
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chemical complexity of milk thistle extracts, Kroll, et al. addressed the importance of
proper nomenclature [23]. In this study, silybin represents two silybin diastereomers
(silybin A and silybin B), while silibinin is a roughly 1:1 ratio of silybin A and silybin B.
Since less than 50% of orally administrated silymarin (based on silybin) was
absorbed from the gastrointestinal (GI) tract due to its poor solubility in water and oil,
the oral bioavailability of silibinin (nearly 1:1 ratio of silybin A and silybin B) was
reportedly only 0.73% in rats [24-26]. Conspicuous PK variability of silymarin among
clinical trial outcomes has been reported, and low oral bioavailability of silymarin likely
leads to the inconsistency in clinical outcomes. Consequently, the oral bioavailability of
milk thistle-containing formulations is mostly considered limited and highly variable [27-
29]. Regarding low bioavailability of silybin, there are several possible causes: low
solubility, low permeability, extensive phase II metabolism, and rapid excretion [30].
Thus, methods to enhance the bioavailability of silybin have been pursued as the
popularity of herbal dietary supplements has increased. These methods to increase
silybin bioavailability include phytosomes, softgel technology, nanoemulsions, and
liposomes [22, 31, 32].
Since the ingredients contained in IsaGenesis and Product B have known
antioxidant properties, it is possible that these two plant-based supplements could
decrease oxidative stress and increase catalase concentrations in human subjects.
Catalase is the first identified antioxidant enzyme involved in cell defense against
oxidative damage by hydrogen peroxide (H2O2) and is the oldest known enzyme to
control cellular redox homeostasis [33].
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Sweazea et al. led a double-blind, placebo-controlled study to assess whether
the plant-based nutraceutical (Product B) can improve antioxidant status in healthy
subjects after 12 weeks [34]. Although markers of glucose and lipid regulation and most
biomarkers for antioxidant capacity were not significantly different, plasma catalase
levels were significantly augmented in the treatment group (+6.1 nmol/min/mL) as
compared to the control group (-10.3 nmol/min/mL).
Methods to Improve Silybin Bioavailability
Phytosomes
Since most bioactive constituents of plants are poorly absorbed and have limited
permeability, the clinical utility of these constituents is severely restricted [35]. A
phytosome is a biocompatible complex technology incorporating phytoconstituents or
standardized herbal extracts into phospholipids [36]. It has been observed that
complexation with phospholipid, mainly phosphotidylcholine, can significantly improve
the absorption and bioavailability of phytoconstituents [37]. Phytosomes serve as an
effective drug delivery system for enhancing therapeutic index and also can represent a
potent targeting agent to transport the encapsulated bioactive compounds to specific
sites for improving drug development. This phospholipid complex technique can also be
applied for conventional dosage forms and is generally known as a pharmacosome [37].
Because a phytosome possesses some advantages including enhanced
absorption, improved stability, and a lower therapeutic dose, its use has been a
noteworthy trend in newer drug delivery systems for bioconstituents and plant extracts
[22]. Silybin and phosphatidylcholine (PC) could form a 1:2 molecular complex-
Siliphos®, and the phytosme form was 4.6-times more bioavailable than non-phytosomal
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silymarin in healthy subjects [31]. Another study conducted in rats by Morazzoni et al.
found that the bioavailability of silybin phytosome was 10-times greater than that of
silybin alone, probably due to the effect of PC facilitating silybin movement across the
GI mucosa [38]. Moreover, after oral administration of silybin phytosome in
cholecystectomy patients, the mean area under the curve (AUC) for silybin phytosome
was over 4-fold greater than the mean AUC for non-phytosomal silybin [39].
Yan-yu et al. conducted a PK study in rats to compare the PK characteristics and
oral bioavailability of silybin phytosome complex and silybin-N-methylglucamine [40].
The PK parameter estimates of phytosomal silybin and silybin-N-methylglucamine in
rats were Cmax 126.72 and 104.29 ng/mL; Tmax 10 and 5 min; AUC0-inf 1020.33 and
235.81 ng/mL/h, respectively. After oral administration of silybin-phospholipid complex,
the oral bioavailability of phytosomal silybin in rats was much better than that of silybin-
N-methylglucamine because of the improvement of the lipophilicity of silybin phytosome.
These results indicate that silybin combined with phospholipids may augment the oral
bioavailability of silybin and improve its delivery into the liver.
Softgel Technology
Savio et al. performed a randomized, single-dose, cross-over study on silybin
bioavailability in a hard shell capsule and a softgel capsule in which silybin was mixed in
a 1:2 ratio with phosphatidylcholine [32]. After subjects consumed a softgel capsule, the
PK analysis showed that the mean values of Cmax and AUClast increased more than two-
fold in comparison to a hard shell capsule. The outcomes demonstrated that the soft
gelatin capsule could improve the bioavailability of silybin, probably because an oily
medium formulated softgel capsule worked as a physiological carrier to help silybin
cross the intestinal wall.
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Nanoparticles
Micronization and nanonization are two approaches to increase drug
bioavailability by increasing surface area. Particle size could be reduced to 2-5 µm in
micronization; whereas, nanonization is the process of minimizing the particle size to
10-1000 nm [16, 41]. Using nanonization, poorly soluble compounds could be made into
nanoparticles for increasing the dissolution rate and solubility. Due to the bioavailability-
improving ability of nanoparticles, they have been applied in the treatment of different
diseases.
Zhang et al. prepared silibinin-solid lipid nanoparticles (SLNs) by using stearic
acid (biodegradable lipid) and Brij-78 (surfactant) for slow release of silibinin. The in
vitro release results indicated that there was almost no drug release from their SLNs in
three days and that the SLNs with stearic acid and Brij-78 may possess extended-
release ability to avoid drug loss on delivering silibinin to the targeted liver cell [42].
Jun et al. studied the influence of SLNs on the oral bioavailability of silymarin
(based on silybin) in rats. Using three different particle sizes (150, 500, and 1000 nm) to
evaluate the impact of particle size on oral absorption, they found that the oral
bioavailability of the smallest SLNs (150 nm) was significantly higher than the other
SLNs. The AUC of 150 nm SLNs was 2.1- and 2.5-times larger than that of 500 and
1000 nm SLNs, respectively [43]. They also investigated the biodistribution of two kinds
of SLNs prepared by a hot and cold homogenization method. Based on the outcomes
from in vitro release experiments, they chose SLNs produced by cold homogenization
(cold-SLNs) for further biodistribution studies in mice. Compared with the milled
silymarin powder suspension, the relative bioavailability of cold-SLNs was 2.8-fold
higher. Apart from the kidneys, the exposure of cold-SLNs was higher in other organs
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than that of the suspension. These results showed that silymarin prepared as SLNs can
enhance the oral bioavailability of silymarin (based on silybin) and might be a promising
drug delivery system for silymarin [44].
Nanosuspensions
Nanotechnology, such as nanosuspensions, has been employed to increase the
solubility of poorly soluble phytochemicals. Nanosuspensions are defined as sub-micron
colloidal dispersion systems which consist of pure drug particles and surfactants or
stabilizers with a mean particle size between 10 and 1000 nm [45, 46]. Compared with
polymeric nanoparticles or liposomes, nanosuspensions possess 100% drug loading
[47]. Nanosuspensions demonstrate several benefits, including dissolution velocity
improvement, particle size reduction, and various administration routes [48]. Since poor
solubility and low oral bioavailability are the main obstacles with the application of
silybin, nanosuspensions have been used to address these problems in a few studies.
Zheng et al. evaluated the in vitro antitumor effect of a silybin nanosuspension on
human prostatic carcinoma PC-3 cell line compared with silybin solution formulation
[49]. Though silybin nanosuspension did not change the mechanism of silybin-induced
apoptosis, findings from this study indicated that silybin nanosuspension exerted higher
inhibition rates to PC-3 cells in comparison to silybin solution at the same concentration.
This phenomenon may be attributed to the silybin nanosuspension adhering to the
mucus layer to prolong biological activity and facilitating silybin transfer across the
epithelial cells.
Wang et al. performed in vitro and in vivo studies on the influence of silybin
nanosuspensions [50, 51]. They found that nanosuspensions had a significant impact
on drug transport across the Caco-2 cell monolayer and the enhanced permeability was
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due to the increased dissolution rate. After oral administration of silybin
nanosuspensions in beagles, the in vivo results showed that the silybin suspensions
significantly improved silybin’s bioavailability in comparison to the powdered
formulations.
Furthermore, they investigated the influence of particle size (637 ± 9.4 nm vs.
132 ± 4.8 nm) on the in vivo distribution and hepatoprotection. After intravenous
administration, the larger particle size was favorably targeted at liver and spleen. After
intravenous or oral administration, the silybin nanosuspensions demonstrated significant
hepatoprotective effects by lowering the levels of alanine transaminase (ALT), aspartate
transaminase (AST), and total bilirubin. When compared to the carbon tetrachloride
(CCl4) treated control group, hepatoprotective effects of the two silybin
nanosuspensions were confirmed by histopathological examination. These outcomes
suggested that the silybin nanosuspension could be used to improve the therapeutic
efficacy and drug target delivery of the silybin, and that silybin nanosuspensions with
smaller particle size exhibited a higher potential to increase oral bioavailability of silybin.
Nanoemulsions
An emulsion is a dispersion system constructed of oil phase, water phase, and
surfactants; the droplet size of the emulsion affects its target distribution [52]. Generally,
a nanoemulsion represents a droplet size from 10 to 100 nm [53]. Due to low oral
bioavailability of silymarin (based on silybin), incorporation of silymarin (based on
silybin) into a nanoemulsion formulation administered orally was employed to enhance
its solubility, oral bioavailability and therapeutic effects. A nanoemulsion could be
regarded as a sustained release formulation because the bioactive compound is
wrapped in the inner phase and avoids direct contact with the body [54]. Incorporation
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of herbal medicines into a nanoemulsion will not only sustain the effect of drugs and
increase the stability of herbal constituents, it may also enhance the penetrability of
drugs to the skin or mucous membranes for topical administration.
Parveen et al. evaluated the hepatoprotection of the nanoemulsion formulation of
silymarin against CCl4-triggered hepatotoxicity [55]. Through biochemical parameters
and histopathological properties, such as ALT, AST and liver histopathology,
hepatoprotective activity was assessed in Wistar rats. As compared to standard
silymarin, the nanoemulsion-treated rats demonstrated significant increase in total
albumin, globulin, and glutathione in tissues and decreased pyruvate transaminase,
glutamate oxaloacetate transaminase, and tissue lipid peroxides. The findings indicated
that a nanoemulsion formulation of silymarin-treated rats reversed liver toxicity induced
by CCl4 more effectively as compared to standard silymarin-treated rats. Though a
nanoemulsion formulation of silymarin appeared to be a promising way to improve the
effectiveness of silymarin in hepatoprotection, the PK properties of silybin within the
nanoemulsion formulation were not addressed in the study.
Liposomes
Liposomes are self-assembled, spherical particles featuring a lipid bilayer
characterized by amphiphilic properties [56]. Since polar lipids construct liposomal
bilayers, hydrophilic drugs are aggregated in the water section and lipophilic
compounds are entrapped in the lipid section. Therefore, hydrophilic or lipophilic
compounds can be encapsulated into liposomes without changing their chemical
structures. Given the unique characters, liposomes are able to boost the bioactivities of
drugs by augmenting compound solubility, enhancing bioavailability, and increasing
intracellular uptake [57, 58]. As a drug delivery system, liposomes are physiologically
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inert because of the natural phospholipid composition and able to enhance the
therapeutic index and safety of incorporated drugs by maintaining drug concentrations
for a long period and by carrying them to the target site.
Maheshwari et al. used the ethanol injection method to make liposomal vesicles
of silibinin [59]. The liposomal systems were investigated for hepatoprotective activity in
mice against CCl4-induced hepatotoxicity. The results of the in vivo studies showed
55.6% hepatoprotection by silibinin in liposomes in comparison to 33.1%
hepatoprotection by non-liposomal silibinin and 24.2% hepatoprotection by plain
liposomes. Owing to the phospholipid content, plain liposomes can demonstrate
hepatoprotective effects.
Yan-yu et al. compared the oral bioavailability and PK parameters of silymarin
proliposome with plain silymarin in beagles [60]. In this in vivo study, PK characteristics
of silybin within silymarin proliposome and plain silymarin were Cmax 472.62 and 89.78
ng/mL, Tmax both 30 min; and AUC0-inf 2606.21 and 697 ng/mL/h, respectively. Based on
the results, this research demonstrated that silymarin proliposome improved the oral
bioavailability of silybin and increased GI absorption of silybin in comparison to plain
silymarin.
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Table 1-1. Definition of dietary ingredients Clause Description A a vitamin B a mineral C an herb or other botanical D an amino acid E a dietary substance for use by man to supplement the diet by increasing the
total dietary intake F a concentrate, metabolite, constituent, extract, or combination of any
ingredient described in clause A, B, C, D, or E
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Table 1-2. Formulation comparison and content IsaGenesis (2 softgel capsules) Product B (2 capsules)
Ingredient Amount Ingredient Amount Milk Thistle (Silybum marianum PE 55% silymarin) 320 mg
Milk Thistle (Silybum marianum PE 80% silymarin) 250 mg
Green Tea (35% EGCG) 64 mg Green Tea (98% polyphenols) 35 mg Horny Goat Weed (5:1 PE) 60 mg Horny Goat Weed 30 mg Turmeric (95% curcuminoids) 64 mg Turmeric (95% curcuminoids) 20 mg Pomegranate Juice (4:1 PE) 30 mg Pomegranate (punica granatum PE) 29 mg Ashwagandha (Withania somnifera 4:1 PE) 27.5 mg
Ashwagandha (Withania somnifera 4:1 PE) 50 mg
Grape Seed (80% oligomeric proanthocyanidins) 20 mg
Grape Seed (80% oligomeric proanthocyanidins) 20 mg
Bacopa (Bacopa monnieri whole plant 25:1 PE) 10 mg Bacopa (Bacopa monnieri PE 50%) 9 mg Knotweed root 62 mg - R-α-Lipoic Acid 12 mg - Black Tea (5:1 PE) 4 mg Black Tea (PE 50% polyphenols) 4 mg White Tea (30% EGCG) 4 mg White Tea (PE 90% polyphenols) 4 mg Blueberry 18 mg Blueberry 7 mg Raspberry 10 mg Red Raspberry 7 mg N-Acetyl Cysteine 18 mg - L-Glutathione 10 mg - Quercetin 8 mg - Bilberry 14 mg Bilberry 11 mg
Panax Ginseng (25:1 PE) 10 mg Panax Ginseng (Root PE 80% ginsenosides) 12 mg
Mucuna Pruriens (4:1 PE) 6 mg - Harada (Terminalia chebula fruit) 8 mg Harada (Terminalia chebula fruit) 8 mg Boswellia (20:1 PE) 18 mg Boswellia (PE 65% boswellic acid) 7 mg Maca 6 mg Maca 7 mg Shilajit 6 mg Shilajit (20% fulvic acid) 5 mg - Resveratrol 28 mg Ascorbic Acid 13 mg Ascorbic Acid 7.5 mg Acacia Bark (12:1 PE) 4 mg - Goldthread (5:1 PE) 6 mg - d-α-tocopherol 5 IU - Vitamin B12 12 mg - Canola Lecithin 10 mg - Vitamin A 2 mg - Flaxseed Oil 250 mg - Lutein 1 mg - Zeaxanthin 0.33 mg - Phosphatidylcholine 200 mg - PE=Pharmaceutical Equivalents EGCG=epigallocatechin-3-gallate
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Figure 1-1. Structures of silybin A and silybin B
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CHAPTER 2 HYPOTHESIS
Existing Problem in Herbal Supplements
Though herbal supplement use has been an emerging trend over the last several
decades, there are few studies conducted in multi-herb supplements at this time [61,
62]. Soare et al. performed a randomized, single-blind controlled trial to evaluate the
metabolic and cardiovascular effects of consumption of 10 dietary supplements each
day over 6 months. The clinical study showed that endothelial function, blood pressure,
insulin and inflammatory cytokines in healthy subjects are not influenced after a 6-
month use of these supplements. The low oral bioavailability of phytochemicals
contained in these dietary supplements might be a likely explanation for the lack of
therapeutic effects. Since many bioactive phytochemicals demonstrate relatively low
oral bioavailability, numerous biopharmaceutical approaches, including complexation
with phospholipids and nanotechnology-based drug delivery systems, have been
employed to enhance the bioavailability of bioactive phytochemicals contained in dietary
supplements.
Research Hypothesis
Owing to low oral bioavailability of many bioactive phytochemicals, softgel
formulations are considered promising dosage forms to enhance bioavailability of poorly
soluble phytochemicals. A softgel formulation, IsaGenesis, and a standard dry-
powdered formulation, Product B, have been chosen for the present study. We
hypothesized that the softgel formulation, IsaGenesis, can augment the bioavailability of
the selected bioactive constituents, silybin A and silybin B.
28
Objective
Since milk thistle extract is the most predominant ingredient in these two
formulations chosen for study, and since silybin A and silybin B are the most abundant
flavonolignans of milk thistle extract, the two silybin diastereomers have been chosen as
surrogate PK biomarkers for elucidating differences in bioavailability with the respective
formulations. The purpose of this study is to test our hypothesis by evaluating the
relative bioavailability of silybin A and silybin B with the respective formulations in
healthy study participants.
Methods and Materials
Supplements and Chemicals
Two milk thistle-containing oral dosage forms, Product B (Lot #041403615) and
IsaGenesis (Lot #025400816), were provided by the study sponsor (Isagenix
International, LLC). Product B containing 125 mg milk thistle extract/capsule (80%
silymarin) was manufactured by Arizona Nutritional Supplements (Chandler, AZ),
whereas IsaGenesis containing 160 mg milk thistle extract/softgel capsule (55%
silymarin) was manufactured at Best Formulations (City of Industry, CA). Compared
with the powder-filled Product B, a notable pharmaceutical difference in IsaGenesis is
the incorporation of flaxseed oil and phosphatidylcholine to enhance the absorption and
bioavailability of phytochemicals.
Authentic analytical reference standards of silybin A and silybin B were
purchased from Phytolab GmbH & Co. (Vestenbergsgreuth, Germany). Naringenin, the
analytical internal standard (IS), was obtained from SAFC Supply Solutions (St. Louis,
MO). Liquid chromatography-mass spectrometry (LC-MS) grade ammonium acetate,
29
methanol, formic acid, and methyl tert-butyl ether (MTBE) were all purchased from
Sigma-Aldrich (St. Louis, MO).
Preparation of Stock Solutions, Calibrator Standards, and Quality Control Samples
Stock solutions of silybin A, silybin B and the IS naringenin were prepared in
methanol at concentrations of 1 mg/mL and were used to prepare calibration standards
and quality control (QC) samples. All stock and working solutions were stored at -20°C
until use. Calibrator solutions were prepared by adding the desired amount of working
solutions to 180 µL of blank human plasma. The calibrator concentrations were 1, 5, 10,
25, 50, 80, and 100 ng/mL plasma for silybin A and silybin B. The lower limit of
quantitation (LLOQ) was considered to be the lowest calibration standard concentration.
QC samples were similarly prepared at concentrations of 1 (LLOQ), 3, 15, and 75
ng/mL. All calibration standards and QCs were stored at -20°C until use.
Calibration standards over the concentration range of 1-100 ng/mL were
analyzed in duplicate for three runs. The standards were considered acceptable if both
the precision, expressed as the coefficient of variation (CV), and the accuracy,
presented as the percent relative error (RE), were within ± 15%. The LLOQ was
acceptable if the CV and RE were within ± 20%.
Study Participants
Twelve healthy participants (6 male and 6 female) were recruited in the study.
Each participant granted written IRB-approved informed consent and underwent a
health assessment. The inclusion criteria were healthy nonsmokers within the age
interval of 18 and 50 years, as well as body mass index (BMI) between 18.5 and 28
kg/m2. All participants were required to have no clinically significant diseases or history
30
of a medical condition. To avoid pregnancy during the entire protocol, female subjects
were required to have negative urine pregnancy test before enrollment and during study
participation. No prescription or over-the-counter medications, and no botanical,
nutritional supplements (vitamins and energy drinks) were allowed during study
participation.
Supplement Administration
Two study visits were scheduled and conducted at the UF Clinical Research
Center (CRC). The washout period was at least 7 days between the two visits (Figure 2-
1). Based on the randomization sequence, participants were administrated two capsules
of Product B or two capsules of IsaGenesis with 240 mL water at room temperature.
Depending on product labeling, two capsules represent a recommended serving of the
two respective formulations. After administration of each formulation, participants
remained fasting for an additional 4 hours to minimize any food effect on absorption. On
both study visits, subjects were provided the same meal to minimize any potential food
influence on these multi-herb supplements.
Sample Collection and Processing
Blood samples were collected via indwelling venous catheters and a total of nine
blood samples were obtained over an 8-hour period. Blood collection occurred
immediately prior to formulation administration (0 h) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8 hrs.
All samples were drawn in heparinized tubes (BD Vacutainer) and stored on ice until
centrifugation at 4°C. Because the stability of phytoconstituents varies within biological
matrices, the samples were not kept on ice for longer than 15 minutes before
centrifugation. Plasma samples were obtained by centrifugation of blood samples. The
resulting plasma was acidified with 10 µL of 1M acetic acid/mL plasma as a
31
preservative, transferred to labeled storage tubes, and frozen immediately at -70°C until
LC-MS/MS analysis.
Analytical Methods
Analysis of Silybin A and Silybin B from Oral Dosage Forms
A comparison of silybin A and silybin B content of the two respective formulations
was performed before the PK study. The content of two capsules (1 serving) of each
formulation was weighed, placed into a 10 mL volumetric flask, and dissolved in pure
analytical grade methanol. A 1 mL aliquot of the resulting solution was removed and
further diluted to an appropriate concentration suitable for LC-MS/MS analysis.
Analysis of Silybin A and Silybin B from Human Plasma
A high-performance liquid chromatographic tandem mass spectrometric assay
was utilized in our laboratory for simultaneously analyzing the free (nonconjugated)
flavonolignans: silybin A and silybin B. This method was mainly based on our previously
published method for simultaneously detecting flavonolignans in human plasma [29,
63].
In short, 200 µL of plasma, 100 ng/mL naringenin and 2 mL of MTBE were
added. The samples were shaken at 200 cycles/min for 10 min and then centrifuged at
3000 g for 10 min at room temperature. The organic phase was then transferred to
clean glass tubes. The supernatant was evaporated to dryness under a gentle stream of
nitrogen at 40°C, and the remaining residue was reconstituted with 100 µL of mobile
phase, 20 µL of which were injected for analysis.
The LC-MS/MS system consisted of a Shimadzu HPLC system (Shimadzu,
Tokyo, Japan) and an Applied Biosystems-Sciex API 3000 quadrupole mass
spectrometer (Foster City, CA, USA). The analytical column was Phenomenex Luna 5µ
32
C18 column (100A 250 mm x 2 mm, 5 μm, Torrance, CA) and C18 guard column (4 mm x
20 mm, SecurityGuard, Torrance, CA). The mobile phase was 50% methanol and 50%
water containing 0.1% formic acid and 10 mM ammonium acetate and delivered at a
flow rate of 0.25 mL/min. The MS was operated in negative ion mode using turbo
electrospray ionization. In the multiple reaction monitoring mode, the following
transitions were monitored: M/Z: 481.2→125.0 for silybin A and silybin B, and M/Z:
271.1→151.0 for naringenin (internal standard) in this method. Measurement of
individual silymarin constituents (i.e., silybin A and silybin B) was achieved by using the
following parameters: curtain gas, 8 psi; nebulizer gas (gas 1), 12 psi; CAD gas, 6 psi;
TurboIonSpray (IS) voltage, -4500 V; entrance potential, -10 V; collision cell exit
potential, -6 V; declustering potential, -80 V; collision energy, 40 eV for m/z: 481.2>125
and 26 eV for m/z: 271.1>151.0; source temperature, 400 °C; and dwell time, 200 ms.
Data was analyzed by AB Sciex Analyst software, version 1.4.2 (AB Sciex, Toronto,
Canada). Representative ion chromatograms of IsaGenesis and Product B from the
study sponsor (Isagenix International, LLC) and one well-known milk thistle extract
Legalon® (Madaus, GH) are shown in Figure 2-2.
Pharmacokinetic Analysis
Plasma concentrations of silybin A and silybin B were determined in 12 healthy
participants completing the entire protocol of a single dose, randomized crossover
clinical trial. The PK parameters for silybin A and silybin B were estimated by a non-
compartmental analysis using WinNonlin 6.1 (Pharsight, Mountain View, CA). The
maximum plasma concentration (Cmax) and time to maximum plasma concentration
(Tmax) were obtained directly from the plasma concentration-time data. According to the
linear trapezoidal rule, the area under the plasma concentration-time curve from time 0
33
to infinity (AUCinf) and AUC from time 0 to the last detectable concentration (AUClast)
were estimated. The terminal elimination rate constant (λz) was calculated by linear
least-squares regression of the terminal portion of the plasma concentration time curve,
and the elimination half-life (t1/2) was then estimated using the formula t1/2 = 0.693/ λz.
Regarding relative bioavailability, the following calculation: relative bioavailability (F) =
(AUClast /Dose) IsaGenesis / (AUClast /Dose) Product B *100%, was used to estimate relative
bioavailability between these two formulations.
Statistical Analysis
For this crossover trial, G*Power software 3.1 was used for a sample size
calculation [64]. Based a two-tailed paired t-test with alpha level of 0.05, in order to
detect an effect size of 1 with 80% power, a total sample size of 10 is needed.
Therefore, our sample size (twelve particpants) would provide enough power to identify
effect sizes that are smaller than one in PK parameter differences between Product B
and IsaGenesis. Results are presented as median and interquartile range for Tmax and
mean ± SD for other PK parameters. To evaluate potential differences in peak and
systemic exposure of silybin diastereomers, paired sample t-test is applied to assess
the log-transformed Cmax and AUClast of silybin A and silybin B after oral administration
of either Product B or IsaGenesis. Statistical analysis was performed by SAS 9.4 (Cary,
NC) and a P-value ≤ 0.05 is considered statistically significant.
34
Figure 2-1. Overview of a single-dose, randomized crossover pharmacokinetic
assessment. CRC: Clinical Research Center.
Single Dose Randomized Crossover Pharmacokinetic Assessment
Screening/ Informed Consent
28 Days CRC
(1 Day) Sample collection
CRC (1 Day)
Sample collection Wash-out (7 Days)
IsaGenesis (capsule x 2)
Product B (capsule x 2)
Product B (capsule x 2)
IsaGenesis (capsule x 2)
35
Figure 2-2. Representative ion chromatograms of two products from the study sponsor
(Isagenix International, LLC) and one well-known milk thistle extract Legalon® (Madaus, GH)
36
CHAPTER 3 RESULTS
Calibration and Linearity
Calibration curves determined by plotting the analyte-to-IS peak area ratio vs.
concentration were linear over the range of 1-100 ng/mL for silybin A and silybin B. The
range of concentrations was selected based on anticipated concentrations of analytes in
human plasma within the two study visits. The correlation coefficients (r2) from three
independent experiments were ≥ 0.99 for silybin A and silybin B.
Precision and Accuracy
The intra- and inter-run precision (CV, %) and accuracy (RE, %) assessed for
silybin A and silybin B are shown in Table 3-1. The estimated precision and accuracy
met the acceptance criteria for silybin A and silybin B.
Formulation Analysis
The results of Product B and IsaGenesis capsule analysis are shown in Table 3-
2. These values were used to correct overall silybin exposure to the research
participants. In Table 3-2, both formulations contain roughly equivalent amounts of
silybin A and silybin B.
Study Participants
Twelve healthy subjects (six males and six females) completed the entire
protocol, and the mean age and weight of these participants is 24.1 ± 3.7 years and
67.9 ± 11.6 kg (mean ± SD), respectively. All participants tolerated the two formulations
well; none of them experienced any unanticipated adverse events during the entire
clinical trial.
37
Pharmacokinetic Analysis
After a single oral dose of either Product B or IsaGenesis, the PK results showed
that the two surrogate markers, silybin A and silybin B, were rapidly absorbed and
eliminated. The corresponding PK parameter estimates of silybin A and silybin B
evaluated in this study are presented in Table 3-3. Figures 3-1 and 3-2 depict mean
plasma concentrations versus time profiles after oral administration of silybin A and
silybin B, respectively; mean plasma concentrations versus time profiles of silybin
(silybin A and silybin B) are presented in Figure 3-3.
Because the total amounts of silybin A and silybin B are unequal in Product B
and IsaGenesis, a dose correction was made by multiplying by a factor of 0.88 (176/200
based on different silymarin exposure per serving) to compare directly data with
IsaGenesis to the powdered formulation-Product B (Table 1-2). This adjustment was
made assuming the absorption and disposition of silybin A and silybin B are linear. To
provide further comparison between the two respective formulations, the mean plasma
concentrations versus time profiles after dose adjustment of IsaGenesis are presented
in Figures 3-1 to 3-3.
Over the 8-hour blood-sampling period, the silybin A and silybin B PK profiles
differed markedly between IsaGenesis and Product B, as depicted in Figures 3-1 and 3-
2. The IsaGenesis softgel formulation displayed a more rapid rise in plasma
concentration and achieved higher peak concentrations (Cmax) of silybin A and silybin B
over the first 6-hour period in comparison with Product B. Furthermore, IsaGenesis
manifested an earlier median time to maximum plasma concentration (Tmax). For silybin
A and silybin B, the Tmax values presented in Table 3-3 were 2 h for the IsaGenesis
softgel formulation versus 3 h for the Product B formulation. Although the content of
38
silybin is similar, the softgel formulation resulted in a 365% and 450% larger Cmax, for
silybin A and silybin B, respectively, when compared with the Product B formulation.
Additionally, the dose-normalized Cmax for silybin A was 1.17 ± 0.67 ng/mL/mg versus
0.32 ± 0.23 ng/mL/mg and for silybin B was 0.72 ± 0.57 ng/mL/mg versus 0.16 ± 0.14
ng/mL/mg, respectively for IsaGenesis versus Product B. The values of relative
bioavailability (F) shown in Table 3-3 demonstrate that absorption was higher with
IsaGenesis compared with Product B by 284% for silybin A and 334% for silybin B.
Regarding inter-individual differences and variability in concentration-time curves,
Figures 3-4 through 3-7 are box plots exemplifying the discrepancy in these two
formulations in connection with dose-normalized AUClast and Cmax of both silybin
isomers. The minimum, first quartile, median, third quartile, and maximum values of
AUClast per dose and Cmax per dose are shown in these box plots. Regarding intra-
individual differences, our study design did not permit an evaluation because each
volunteer received each formulation only once. Notably, participants who demonstrated
lower overall absorption and lower concentrations of silybin A and silybin B did so with
the two respective formulations indicating some physiologic differences in
gastrointestinal tract rather than formulation issues [65, 66].
In view of reported gender differences in silybin A and silybin B
pharmacokinetics, we found no statistically significant difference in Cmax and AUClast of
silybin A and silybin B between male and female participants in this study (P > 0.05;
Table 3-4). However, gender differences in the pharmacokinetics of silybin A and silybin
B cannot be determined or excluded given the limited sample size and large inter-
individual variability in the present study.
39
Table 3-1. Intra- and inter-run precision (CV, %) and accuracy (RE, %) for quality control (QC) samples in human plasma.
Analyte Nominal concentration (ng/mL)
Intra-run Inter-run Precision
(%) Accuracy
(%) Precision
(%) Accuracy
(%)
Silybin A 1 (LLOQ) 4.99 7.10 9.77 6.08
3 (Low QC) 2.48 1.28 6.46 -2.61
15 (Medium QC) 3.89 4.00 8.21 -0.22
75 (High QC) 3.47 5.56 10.75 0.09
Silybin B 1 (LLOQ) 2.92 11.67 12.82 7.93
3 (Low QC) 1.94 -0.17 3.41 -2.72
15 (Medium QC) 2.76 -0.22 5.43 -3.33
75 (High QC) 7.17 1.00 7.37 3.36
40
Table 3-2. Comparison of silybin A and silybin B amounts in Product B and IsaGenesis
Formulation Silybin A content
(mg) Silybin B content
(mg) Silybin A and B content
(mg) IsaGenesis 55.1 87.5 142.6 Product B 48.7 80.3 129.0
Each sample is taken as 1/10 from the mixture of two capsules (1 serving) and diluted (10,000-fold) to a concentration in LC-MS/MS detection range.
41
Table 3-3. Pharmacokinetic parameters of silybin A and silybin B after single oral doses of Product B and IsaGenesis.
Pharmacokinetic Parameter
Silybin A for Product B
Silybin A for IsaGenesis
Silybin B for Product B
Silybin B for IsaGenesis
Dose (mg) 48.7 55.1 80.3 87.5 Tmax (h) 3.0 (1.5-6) 2.0 (1.0-4.0) 3.0 (1.5-4.0) 2.0 (0.5-4.0) t1/2 (h) 1.98 ± 1.64 1.30 ± 0.32 1.82 ± 1.20 1.28 ± 0.34 Cmax (ng/mL) 15.7 ± 11.4 64.4 ± 37.1 12.9 ± 11.4 62.8 ± 49.8 Cmax/Dose (ng/mL/mg) 0.32 ± 0.23 1.17 ± 0.67 0.16 ± 0.14 0.72 ± 0.57
AUClast (ng×h/mL) 37.2 ± 30.3 118.8 ± 55.9 30.2 ± 31.6 111.0 ± 74.3
AUClast/Dose (ng×h/mL/mg) 0.76 ± 0.62 2.16 ± 1.01 0.38 ± 0.39 1.27 ± 0.85
AUCinf/Dose (ng×h/mL/mg) 0.96 ± 0.62 2.24 ± 1.04 0.52 ± 0.40 1.31 ± 0.84
F (IsaGenesis/Product B)* 284.21% 334.21%
The Tmax values are expressed as median (range) and others are mean ± S.D. (n=12). *Relative bioavailability (F) values in Table 3-2 of 284% (silybin A) and 334% (silybin B) represent the relative bioavailability of the respective silybin isomers in IsaGenesis compared with that in Product B.
42
Table 3-4. P values of log-transformed Cmax and AUClast of silybin A and silybin B after single oral doses of Product B and IsaGenesis between male (n=6) versus female (n=6) subjects.
Parameter Silybin A for Product B
Silybin A for IsaGenesis
Silybin B for Product B
Silybin B for IsaGnesis
log (Cmax) 0.30 0.55 0.40 0.43 log (AUClast) 0.53 0.54 0.60 0.48
43
Figure 3-1. Mean plasma concentrations of silybin A versus time profiles after oral
administration of single doses of Product B and IsaGenesis in 12 healthy subjects.
44
Figure 3-2. Mean plasma concentrations of silybin B versus time profiles after oral
administration of single doses of Product B and IsaGenesis in 12 healthy subjects.
45
Figure 3-3. Mean plasma concentrations of silybin (A and B) versus time profiles after
oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects.
46
Figure 3-4. Box plots of AUClast/Dose (h*ng/mL/mg) of silybin A.
47
Figure 3-5. Box plots of Cmax/Dose (ng/mL/mg) of silybin A.
48
Figure 3-6. Box plots of AUClast/Dose (h*ng/mL/mg) of silybin B.
49
Figure 3-7. Box plots of Cmax/Dose (ng/mL/mg) of silybin B.
50
CHAPTER 4 DISCUSSION
To evaluate biological activities and to interpret prospective health-promoting
benefits of dietary supplements in humans, it is crucial to know the bioavailability of
phytochemicals administrated as dietary supplements. This information is important to
discover whether promising in vitro findings are likely to be achieved in vivo based on
relative exposure. In other words, the putative bioconstituents must be sufficiently
penetrated or delivered to their target site of biological activity.
To date, a comprehensive evaluation of biodisposition is lacking for most dietary
supplements and relatively little clinical PK research has been conducted. In view of the
fact that significant PK differences among many milk thistle-containing supplements
have been reported, it is essential to perform a comparative PK study before marketing.
Additionally, the bioavailability of most oral formulations is usually considered low and
highly inconsistent [29]. Major reasons for the poor bioavailability of silymarin
constituents include low aqueous solubility, extensive pre-systemic metabolism, and
poor permeability across intestinal cells.
In order to compare the pharmacokinetics of two multi-herb dietary supplement
formulations, a single-dose crossover PK study was completed. Based on the
randomization sequence, participants were administered either two capsules of Product
B or two softgel capsules of IsaGenesis on alternate clinic visits. In the present study,
two flavonolignans of silymarin, silybin A and silybin B, served as surrogate PK
biomarkers for characterizing the absorption and relative bioavailability of these two
formulations. As with previous clinical PK studies on silybin, the peak concentration
(Cmax) and systemic exposure (AUCinf) were significantly higher for silybin A compared
51
with silybin B [27, 29]. The observed variability results from the known stereoselective
metabolism of silybin isomers with faster metabolism of silybin B [67].
The PK evaluation suggested that on average, silybin A and silybin B were
rapidly absorbed and eliminated with the two respective formulations. The absorption of
silybin A and silybin B was greater with IsaGenesis compared with Product B. Indeed,
after dose normalization of silybin A and silybin B, the IsaGenesis formulation exhibited
365% and 450% higher mean Cmax values, respectively, compared with the Product B
formulation. Moreover, the Cmax values of IsaGenesis for silybin A fitted the range of
values from our previous investigation on a well characterized silymarin formulation [29].
Regarding Tmax, both silybin A and silybin B achieved the Cmax at least 1 hour earlier
with IsaGenesis compared with Product B (Table 3-3). The estimated t1/2 of silybin A
and silybin B appeared to differ between the two formulations; both silybin A and silybin
B have a shorter t1/2 with the IsaGenesis formulation compared with the Product B
formulation (Table 3-3), the reasons for which are unclear.
Although the systemic concentrations of silybin A and silybin B achieved after
oral administration are relatively low compared to the in vitro concentrations typically
used to assess biological activities, there remains the possibility that these silybins may
exhibit their bioactivities at relatively low systemic concentrations.
Additionally, inter-subject PK differences were observed with both formulations.
In view of box plots in Figures 3-4 to 3-7, there seems to be a greater amount of inter-
individual variability in the silybins with the IsaGenesis formulation.
There are a few limitations to the current study. First, the respective milk thistle-
containing supplements were not single herbal extract formulations because they
52
included multiple herbal constituents from different plant sources. Secondly, there is no
potential effect of concomitant drugs on the results because of exclusion criteria for
participants. Thirdly, the two dosage forms did not contain the same amounts of
silymarin content (silybin A and silybin B) as well as other botanical extracts. But, the
uneven amounts of silybin A and silybin B administered in IsaGenesis and Product B
were normalized by dose for PK comparisons.
Regarding the pharmacokinetics of the silybins administered as part of a multi-
herb supplement in humans, there is scant research at this time and the possible
influence of other included bioconstituents on the disposition of silybin A and silybin B is
still an open question. The potential of synergistic or competitive effects on absorption
and metabolism among various bioconstituents administered as part of multi-herb
formulation versus the administration of a single extract or extract component from a
natural product has been documented [61, 68, 69]. Concerning silybin A and silybin B
systemic exposure, the IsaGenesis formulation obviously improved the absorption of the
two silybin isomers, however, it must be emphasized that these findings cannot simply
be projected to other constituents incorporated into these multi-herb supplements
without directly assaying plasma for representative bioactive constituents.
In the present study, flaxseed oil and phosphatidylcholine were excipients of
IsaGenesis but not Product B. As an n-3 fatty acid source, flaxseed oil was a potential
diluent for the fat-soluble bioconstituents but was not included with the particular
intention to improve bioavailability. With respect to the phosphatidylcholine content, the
IsaGenesis softgel formulation was not the same as a silybin-phosphatidylcholine
complex for some primary silymarin formulations used in previous studies [70].
53
However, recognizing the plenitude of phosphatidylcholine in cellular membranes and
the established role of phospholipids as excipients for different pharmaceutical
applications including as emulsifiers and solubilizers, its inclusion through a micro-
coating manufacturing process may enhance the absorption of lipophilic bioconstituents
[30]. Although IsaGenesis appeared to provide greater overall exposure to silybin A and
silybin B, whether this resulted from phosphatidylcholine inclusion remains speculative
at this time and requires further study.
Comprehensively, the IsaGenesis softgel formulation showed greater overall
systemic exposure to silybin A and silybin B compared with the Product B formulation.
Further study is needed to characterize the pharmacokinetics of other phytoconstituents
(e.g., withaferin A, withanolide A, bacopaside I, and icariin) in IsaGenesis and to
investigate the potential herb-drug interactions of taking a multi-herb supplement in
healthy subjects.
54
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BIOGRAPHICAL SKETCH
Wen-Yi Li was born in Taiwan. He received his master’s degree from the
University of Florida in the fall of 2018. Before he joined the clinical pharmaceutical
graduate program in the Department of Pharmacotherapy and Translational Research
at the College of Pharmacy in 2015, he received his master’s degree in pharmaceutical
sciences at National Defense Medical Center and then worked as a pharmacist and an
adjunct lecturer for several years.