Bioactivity-guided Fractionation of Selected Botanticals

BIOACTIVITY-GUIDED FRACTIONATION
OF SELECTED BOTANICALS

11th International Conference
on the Science of Botanicals
Oxford, MS
April 16-19, 2012

A. Douglas Kinghorn1, Jie Li1,
Hee-Byung Chai1, and William J. Keller2
1College of Pharmacy, The Ohio State

University and 2Nature’s Sunshine
Products, Inc.

MR. JIE LI DR. HEE-BYUNG CHAI
THE OHIO STATE THE OHIO STATE
UNIVERSITY UNIVERSITY

DR. WILLIAM J.
KELLER
NATURE’S SUNSHINE
PRODUCTS, INC.

OUTLINE OF PRESENTATION

 Reminiscences of the Late Norm Farnsworth as
a Mentor and Colleague.

 Application of “Activity-guided Fractionation”
to Furnish Potential Cancer Chemopreventive
Principles of Several Botanicals: Black
Chokeberry (Aronia melanocarpa), Maqui
Berry (Aristotelia chilensis), Mangosteen
(Garcinia mangostana), and Noni (Morinda
citrifolia).

Late Dr. Norman R.
Farnsworth
University of Illinois at
Chicago
1930-2011

(Fong et al., J. Nat.
Prod. 69, 311, 2006)

SCHOOL OF PHARMACY, UNIVERSITY OF
LONDON (THE “SQUARE”; NOW UCL)

PROFESSOR
JAMES W.
FAIRBAIRN
(“PROF”)

PROFESSOR
FRED J. EVANS

PROFESSOR J. DAVID
PHILLIPSON

Bioactivity-guided Fractionation of Selected Botanticals

THE BEST DEPARTMENT OF PHARMACOGNOSY
AND PHARMACOLOGY IN THE WORLD, CA. 1977

Shown are the faculty, staff, postdoctorals, and graduate students

National Cooperative Drug Discovery Group (NCDDG) (Ohio State, UIC, Research
Triangle Institute, Bristol-Myers Squibb, National Cancer Institute) , circa 2005

CANCER CHEMOPREVENTION
A strategy of cancer control by administration of synthetic or natural
compounds to reverse or suppress the process of carcinogenesis
(Sporn et al., Fed. Proc., 35, 1332, 1976)

SOME PROMISING PLANT-DERIVED COMPOUNDS UNDER
DEVELOPMENT BY THE UNITED STATES NATIONAL CANCER INSTITUTE
AS POTENTIAL CANCER CHEMOPREVENTIVES

Agent(s) Phase Target Cancer(s)

Curcumin I/II/III Colon; Pancreatic
Genistein II Bladder; Breast; Kidney; Prostate; Skin
Indole-3-carbinol I/II/III Breast; Prostate
Perillyl alcohol I/II Breast; Leukemia; Pancreatic; Prostate
Soy isoflavones II/III Breast; Prostate
Tea (green) I/II Bladder; Esophageal; Cervical; Leukemia;
Liver; Lung; Prostate
Tea/epigallocatechin I Prostate
Resveratrol I/II Colon; Solid cancers
Vitamin C II Cervical; Ovarian; Uterine
Silibinin II Prostate

(http://www.clinicaltrials.gov; accessed April, 2008)

MAJOR STAGES IN NATURAL PRODUCT
DRUG DISCOVERY
Organism collection (after development of intellectual property
agreements).
Preparation of extracts (using standardized extraction scheme).
Initial bioassays (cell-based and target-based).
Biostatistics; data management; dereplication of leads; lead
prioritization).
Bioactivity-directed fractionation (= isolation of active compounds from
biomass using a decision tree based solely on bioactivity).
Structure elucidation of bioactive compounds.
Scale up and analog development of lead compounds.
Advanced bioassays; data management; biostatistics.
Lead optimization; pharmaceutical development.

(Clark, In Foye’s Principles of Medicinal Chemisry, 5th Edn., Williams, D.A.;
Lemke, T.L., Eds., Lippincott Williams & Wilkins: Baltimore, 2002, p. 24)

TYPES OF ASSAYS USED FOR POTENTIAL NATURAL PRODUCT
CANCER CHEMOPREVENTIVE AGENTS

 SHORT-TERM IN VITRO BIOASSAYS BASED ON FACTORS
ASSOCIATED WITH TUMOR INITIATION, PROMOTION AND
PROGRESSION Antimutagenicity
Tumor Initiation: Antioxidant acitivity
Phase II enzyme induction (quinone
reductase induction)

Antioxidant activity
Tumor Promotion: Cyclooxygenase inhibition
Aromatase inhibition
Ornithine decarboxylase inhibition
Aromatase inhibition
Tumor Progression: Antiestrogenic activity
Receptor assays with luciferase
Differentiation-inducing agents
(Modified from Pezzuto et al., In Cancer Chemoprevention, Vol. 2:
Strategies for Cancer Chemoprevention, G. Kelloff et al., eds., Humana
Press, Inc., Totowa, NJ, 2005; p. 3)

BLACK CHOKEBERRY
(ARONIA MELANOCARPA) (ROSACEAE)
 A deciduous shrub originating from the eastern parts of
North America and East Canada, which has been introduced
to Europe.
 Has a long tradition of use in European and North American
folk medicine. Now used for the production of jam, juice,
tea, wine, and as a natural colorant.
 Consumed in part because of its high antioxidant capacity
when compared to other berries and fruits.

(Kulling et al., Planta
Med., 74, 1625, 2008;
Valcheva-Kuzmanova et
al., Folia Med., 48, 11, 2006) www.vitarroz.com www.seedman.com/fruit.htm

BIOLOGICAL ACTIVITIES OF BLACK
CHOKEBERRY CONSTITUENTS OR EXTRACTS
 Antioxidant activities (phenolics)
 Chemopreventive potential via antimutagenic activity,
immunomodulatory activity, and inhibition of carcinogenesis and
cancer cell proliferation (flavonoid-rich fruit extract, isolated
anthocyanins)
 Anti-inflammatory activities in vitro and in rats (fruit extract or
juice)
 Miscellaneous: hepatoprotective, gastroprotective, and
cardioprotective effects; antibacterial and antiviral effects; and
antidiabetic effects
Key references:
(Oszmianski et al., Eur. Food Res. Technol., 221, 809, 2005; Sueiro et al., J. Food Sci.,
71, C480, 2006; Ding et al., J. Biol. Chem., 281, 17359, 2006; Zapolska-Downar et
al., Eur. J. Nutr., DOI: 10.1007/s00394-011-0240-1; Borissova et al., Acta Physiol.
Pharmacol. Bulg., 20, 25, 1994; Valcheva-Kuzmanova et al., Exp. Toxicol. Pathol.,
56, 195, 2004; Simeonov et al., Folia Med., 44, 20, 2002)

HYDROXYL RADICAL-SCAVENGING AND QUINONE REDUCTASE
INDUCTION ACTIVITIES OF ISOLATES OF BLACK CHOKEBERRY FRUITS
 From the results obtained, the following four isolates (hyperoside, 1;
neochlorogenic acid methyl ester, 4; quercetin, 9; protocatechuic acid, 14)
showed potent activities in the .OH-scavenging and QR induction assays:

MAQUI BERRY
(ARISTOTELIA CHILENSIS) (ELAEOCARPACEAE)
 A native South America evergreen
shrub.
 This species produces small edible
purple or black berries that are eaten
fresh or used for juice, jams, and wine-
making.
www.healthytastychow.com
 The leaves and fruits are astringent and
have been used in Chilean folk
medicine as anti-diarrheal, anti-
inflammatory, antihamorrhagic, and as
a febrifuge.
(Schreckinger et al., J. Med. Food, 13, 233, 2010;
Escribano-Bailon et al., www.vitarroz.com
Phytochem. www.seedman.com/fruit.htm
http://superberries.co.uk
Anal., 17, 8, 2006)

BIOLOGICAL ACTIVITIES OF MAQUI
BERRY CONSTITUENTS OR EXTRACTS
 Antioxidant activities (fruit extract or juice, phenolic
extract, and 3-hydroxyindole derivatives)
 Chemopreventive effects and induction of apoptosis in
cancer cell lines (cyanidin 3-O-glucoside, delphinidin 3-O-
glucoside and other anthocyanins, and gallic acid)
 Anti-inflammatory activities (leaf extract)
 Miscellaneous: analgesic, cardioprotective, α-
glucosidase/α-amylase inhibitory, nematicidal, and
antiviral activities.
Key references:
(Cespedes et al., Food Chem., 107, 820, 2008; Cespedes et al., Z. Naturforsch.,
64c, 759, 2009; Munoz et al., J. Pharm. Pharmacol., 63, 849, 2011; Rubilar et al., J.
Agric. Food Chem., 59, 1630, 2011; Schreckinger et al., J. Med. Food, 13, 233,
2010; Ohno et al., Anti-cancer Drugs, 10, 845, 1999; Hou et al., Curr. Mol. Med., 3,
149, 2003)

HYDROXYL RADICAL-SCAVENGING AND QUINONE REDUCTASE
INDUCTION ACTIVITIES OF ISOLATES OF MAQUI BERRY
 From the results obtained, twelve compounds (1-12) showed potent antioxidant
activity in the .OH-scavenging assay. Five compounds (3, 7, 9, 11, and 14) exhibited
relatively high QR-inducing activity. Compounds 3 (gallic acid methyl ester), 9
(protocatechuic acid), 7 (hyperoside), and 11 (guaijaverin) are shown below:

GARCINIA MANGOSTANA L. (MANGOSTEEN)
(CLUSIACEAE)
 Garcinia mangostana L.
(Clusiaceae), commonly
known as “Mangosteen”, is
one of the most widely
recognized tropical fruits.
 Known as the “queen of
fruits” and used in
Southeast Asian traditional
medicine.
 In Thai folk medicine, the
pericarp of Mangosteen
has been used for many
years to healing skin
infections and wounds and
for relief of diarrhea.

MANGOSTEEN AS A BOTANICAL
DIETARY SUPPLEMENT

 Now one of top-selling botanical dietary
supplements in the U.S., sales of over $200
million in 2008 (Sloan, Nutraceutical
World, 13, 16, 2010).
 Mangosteen is used as a constituent of products
sold in the United States for its antioxidant
effects.
 Botanical products are standardized against α-
mangostin (ca. 1% w/w yield from pericarps)
and -mangostin (ca. 0.25% w/w yield from
pericarps).
 Quite large amounts of the xanthone α-
mangostin may be ingested in mangosteen juice.

CHEMICAL CONSTITUENT PROFILE OF GARCINIA
MANGOSTANA
 The following compound classes have been
reported to occur in various plant parts of
mangosteen, as published by the end of 2007:
Xanthones 68

Flavonoids 5

Benzophenones 3

Triterpenoids and Sterols 7

Miscellaneous 3

(Chin and Kinghorn, Mini-Rev. Org. Chem., 5, 355, 2008)

SUMMARY OF BIOLOGICAL ACTIVITIES OF MANGOSTEEN
CONSTITUENTS/EXTRACTS
 Antioxidant (especially potent are -mangostin and -mangostin)
 Infectious disease related
Antibacterial
Antifungal
Antimalarial
HIV-protease and reverse transcriptase inhibition
 Cancer related
Aromatase inhibition (-mangostin is active in both a
microsomal and a cell-based assay)
Cytotoxicity for cancer cells
Inhibition of sphingomyelinase
Inhibition of topoisomerases
In vivo cancer chemoprevention
 Inflammation related
Inhibition of COX, iNOS, IKK; in vivo carageenan-induced
paw
 Miscellaneous
Ca2+ATP-ase inhibitor
H1-receptor antagonist
(Balunas et al., J. Nat. Prod., 71, 1161, 2008; Chin and Kinghorn, Mini-Rev. Org.
Chem., 5, 355, 2008)

XANTHONES ISOLATED FROM THE PERICARP OF
MANGOSTEEN

PEROXYNITRITE ANTIOXIDANT ACTIVITY OF
ISOLATED XANTHONES
IC50 (M)a
Compound
Authentic ONOO SIN-1 derived ONOO
1* (new) 4.6 10.0
3 (smeathxanthone) 2.2 9.7
4 (-mangostin) 12.2 <0.5
5 (-mangostin) 8.0 3.1
6 (8-deoxygartanin) N.A.c 11.9
7 (gartanin) 9.1 9.3
8 (mangostinone) N.A. N.A.
9 (tovophyllin A) N.A. N.A.
10 (cudraxanthone G) N.A. 3.2
DL-penicillamineb 3.1 7.4
aONOO is the scavenging activity of authentic peroxynitrite and generated
peroxynitrite from SIN-1 (IC50: M). Values of ONOO scavenging/inhibitory activities
expressed as the mean of three experiments. bDL-Penicillamine used as positive control.
cN.A.: no activity within the tested concentration (5-100 M).

(Jung et al., J. Agric. Food Chem., 54, 2077, 2006)

PHARMACOKINETICS OF PURE -MANGOSTIN
AFTER INTRAVENOUS ADMINISTRATION

Parameters Mean SE
C0 (ng/ml) 17880 4432
Ke (1/hr) 0.26 0.03
t1/2 (hr) 2.97 0.38
AUC0-6 (ng*hr/ml) 1233 157.1
AUC0-∞ (ng*hr/ml) 1372 140.4

Pharmacokinetic parameters were AUMC0-6 (ng*hr*hr/ml) 791.3 63.84
determined after a single intravenous
dose of 2 mg/kg -mangostin in rats (n = AUMC0-∞ (ng*hr*hr/ml) 2335 434.2
8), with data calculated using non-
compartmental analysis. Disposition
MRT (hr) 1.85 0.39
biphasic (fast distribution; slow Vz (L/kg) 6.82 1.10
elimination)..
CL (L/hr/kg) 1.54 0.12
(Li et al., Mol. Nutr. Food Res. 55, S67,
2011)

PHARMACOKINETICS OF PURE -MANGOSTIN
AFTER ORAL ADMINISTRATION

 On oral administration of a single dose of 20 mg/kg -mangostin to
rats, the bioavailability was so low (ca. 0.4%) that it was not
possible to obtain a full concentration-time profile.
 As a result of this study, it was concluded that it is questionable if
the in vitro effects of -mangostin occur in an in vivo setting.
(Li et al., Mol. Nutr. Food Res. 55, S67, 2011)

DETERMINATION OF BIOAVAILABILITY IN HUMANS
OF XANTHONES FROM MANGOSTEEN JUICE
 The bioavailability of mangosteen xanthones using human
subjects has received comparatively little attention.
 In a preliminary study, Kondo et al. administered ca. 60 mL
of a supplement (mangosteen; aloe vera; green tea;
multivitamins) to 20 fasted healthy human volunteers, and
it was concluded that -mangostin is bioavailable
(observed Cmax at tmax of ca. 1 hour) (Kondo et al. J. Agric.
Food Chem., 57, 8788, 2009).
 In a study carried out at The Ohio State University, the
bioavailability of xanthones was investigated in ten healthy
adults (five females; five males), who consumed a single
dose of 100% mangosteen juice along with a typical fast-
food (high-fat) breakfast, which was supplemented with
canola oil and soybean oil (Chitchumroonchokchai et al. J.
Nutr., 142, 675, 2012).

PROFILE OF XANTHONE CONTENT IN THE
MANGOSTEEN JUICE USED IN THE STUDY
 When analyzed by Content Percentage of
Xanthones in juice total xanthones
HPLC, the 100% (µM) identified (%)
mangosteen juice garcinone C 291 + 11.2 5.5

used in the study garcinone D 520 + 10.9 10.2
garcinone E 239 + 18.5 5.1
provided 5.3 + 0.1
α-mangostin 3190 + 123 59.9
mM total β-mangostin 121 + 9.3 2.3
xanthones, with α- -mangostin 356 + 4.3 6.5
mangostin being 8-deoxygartanin 176 + 4.5 3.1
the most abundant gartanin 157 + 6.9 2.8
tovophillin B 50 + 2.9 1.1
(59.9%), as
9-hydroxycalabaxanthone 193 + 7.4 3.6
indicated in the Totals 5290 + 166 100
table. Values are means + SD; n = 5 independent replicates

(Chitchumroonchokchai et al., J. Nutr., 142, 675, 2012)

PHARMACOKINETIC PARAMETERS OF FREE AND
CONJUGATED α-MANGOSTIN IN SERUM
 There was marked variation Participant AUC (nM/L x h) Cmax (nM/L) Tmax (h)

in values of AUC (area under Females

the curve, 762-4029 nM x h), 1 1605 78 3

Cmax (maximum 2 962 57 8

concentration, 42-450 nM), 3 4029 450 3
4 1484 132 2
and Tmax (time to the
5 1249 79 3
maximum concentration, 2-8
Mean + SD 1870 + 1230 159 + 165 3.8 + 2.4
h) for α-mangostin in the
Males
serum, during the 24-h
1 1004 42 2
collection period.
2 771 44 8
 The serum AUC, Cmax, and 3 1798 154 4
Tmax values of α-mangostin 4 816 42 2
were similar in female and 5 762 49 2
male participants. Mean + SD 1030 + 440 66 + 49 3.6 + 2.6


CONCLUSIONS FROM THE OHIO STATE BIOAVAILABILITY
STUDY ON XANTHONES FROM MANGOSTEEN JUICE
 Both free and conjugated (glucuronidated/sulfated) xanthones
were detected in serum and urine of healthy adult participants.
 There was marked variation in the AUC, Cmax, and Tmax for α-
mangostin in serum samples during the 24-h collection period.
 The total xanthones in the 24-h urine samples ranged from 0.9-
11.2 μM and accounted for ca. 2.0 ± 0.3% (range 0.3-3.4%) of the
ingested dose of mangosteen juice.
 There were no significant differences between female and male
participants in mean pharmacokinetic values of α-mangostin in
serum and urinary total xanthones.
 Xanthones in mangosteen juice were absorbed when ingested
along with a high fat meal, although the release of xanthones
from the juice during digestion may be somewhat limited.

NONI (MORINDA CITRIFOLIA L.)
(RUBIACEAE)
 Occurs in tropical and
subtropical regions

http://www.nps.gov/kaho/KAHOckLs/KAHOplnt/noni.htm
 Small evergreen tree or
shrub
 Flowers perfect with five-
lobed, small white
corollas
 5 to 10 cm long fleshy,
syncarpous fruit
 Unpleasant taste and
butyric acid odor when
ripe
 All parts (fruit, leaf, bark,
flower, and seed) have
been used medicinally
 Roots and bark are used
as a dye
(Pawlus and Kinghorn, J. Pharm. Pharmacol. 59, 1587, 2007)

CHEMICAL CONSTITUENT PROFILE OF MORINDA
CITRIFOLIA
The following compound classes have been reported
to occur in various plant parts of noni, with effect
from mid-2007:
Anthraquinones 45

Flavonoids 10

Iridoids 18

Lignans 11

Triterpenoids and Sterols 12

Miscellaneous >38

(Pawlus and Kinghorn, J. Pharm. Pharmacol. 59, 1587, 2007)

QUINONE REDUCTASE-INDUCING ACTIVITY OF ISOLATED
CONSTITUENTS OF NONI FRUITS

Compound CDa, M (g/mL) IC50b, M (g/mL) CIc
1d 0.009 (0.0027) >69.9 (>20) >7770
2 1.67 (0.52) >66.6 (>20) >39.9
L-sulforaphane* 0.34 (0.061) 9.77 (1.73) 28.7

aCD = Concentration required to double quinone reductase induction. bIC50 = Concentration
for 50% inhibition of cell viability. cCI = Chemopreventive Index. d Only 2 mg of 1,3,6-
trihydroxy-2-methoxyanthraquinone(1) isolated from ca. 9 kg dried noni fruits. * = Control.

1 (new) 2
(Pawlus et al., J. Nat. Prod. 68, 1720, 2005)

INHIBITORY EFFECT OF NONI FRUITS ON RAT
ESOPHAGEAL TUMORIGENESIS
 Male F344 rats were fed a diet of 5% w/w dried
powdered fruits of M. citrifolia (noni), and six other dried
fruits, including black raspberry (Rubus occidentalis), in a
comparison study.
 The standard carcinogen, NMBA (N-
nitrosomethylbenzylamine was used to treat rats for five
weeks, and the experiment was terminated after 35
weeks.
 All seven fruits had similar effects on reducing
esophageal tumor incidence, size, and multiplicity, and
also reduced the levels of two serum cytokines.
 Noni fruits have lower levels of anthocyanins and
ellagitannins than black raspberries.
(Stoner et al., Pharm. Res. 27, 1138, 2010)

SEARCH FOR BIOLOGICALLY ACTIVE PRINCIPLES OF
BOTANICAL DIETARY SUPPLEMENTS: SUMMARY

Botanical dietary supplements have become widely available in
the United States since the passage of the Dietary Supplement
Health and Education Act (DSHEA) in 1994. Their widespread
use has had a major impact on biomedical research and
pharmacy practice.
Fractionation studies on selected “botanicals” have yielded quite
potent biologically active compounds, although generally
occurring at low concentrations. Some of these are useful leads as
potential cancer chemopreventive agents.
The fruits of mangosteen (G. mangostana) and noni (M. citrifolia)
have already yielded interesting bioactive compounds. The
constituents of these botanical dietary supplements seem to offer
particular promise for further investigation.

ACKNOWLEDGMENTS

Phytochemical Studies Biological Testing
The Ohio State University The Ohio State University
Dr. Young-Won Chin Dr. Marcy P. Balunas
Dr. Ye Deng Dr. Young-Won Chin
Dr. Hyun-Ah Jung Ms. Chureeporn Chitchumroonchokchai
(Human Nutrition)
Dr. Alison D. Pawlus Dr. Alison D. Pawlus (at University of Illinois at
Dr. Baoning Su Chicago)
Dr. Bin Su (Dr. Robert W. Brueggermeier)
Collaborators – The Ohio Collaborator – University
State University of Florida
Dr. Steven Clinton Dr. Veronika Butterweck
Dr. Mark Falia Supply of Botanicals
Dr. Gary D. Stoner Nature’s Sunshine Products, Inc.,
Spanish Fork, Utah
Faculty start-up funding from the Molecular Carcinogenesis and
Chemoprevention Program of The Ohio State University Comprehensive
Cancer Center (to A.D. Kinghorn), and an unrestricted gift from Nature’s
Sunshine Products, Inc. are gratefully acknowledged.

College of Pharmacy and
OSU Comprehensive Cancer Center

Bioactivity-guided Fractionation of Selected Botanticals

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