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 CHAITHE 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. FarnsworthUniversity 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
THE BEST DEPARTMENT OF PHARMACOGNOSYAND 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 CHEMOPREVENTIONA 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 CHEMOPREVENTIVESAgent(s) Phase Target Cancer(s)Curcumin I/II/III Colon; PancreaticGenistein II Bladder; Breast; Kidney; Prostate; SkinIndole-3-carbinol I/II/III Breast; ProstatePerillyl alcohol I/II Breast; Leukemia; Pancreatic; ProstateSoy isoflavones II/III Breast; ProstateTea (green) I/II Bladder; Esophageal; Cervical; Leukemia; Liver; Lung; ProstateTea/epigallocatechin I ProstateResveratrol I/II Colon; Solid cancersVitamin C II Cervical; Ovarian; UterineSilibinin 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 effectsKey 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 etal., 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 beenreported to occur in various plant parts ofmangosteen, 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 7.4aONOO is the scavenging activity of authentic peroxynitrite and generatedperoxynitrite from SIN-1 (IC50: M). Values of ONOO scavenging/inhibitory activitiesexpressed 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 140.4Pharmacokinetic parameters were AUMC0-6 (ng*hr*hr/ml) 791.3 63.84determined after a single intravenousdose of 2 mg/kg -mangostin in rats (n = AUMC0-∞ (ng*hr*hr/ml) 2335 434.28), with data calculated using non-compartmental analysis. Disposition MRT (hr) 1.85 0.39biphasic (fast distribution; slow Vz (L/kg) 6.82 1.10elimination).. 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 (Chitchumroonchokchai et al., J. Nutr., 142, 675, 2012)
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. (Chitchumroonchokchai et al., J. Nutr., 142, 675, 2012)
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 CITRIFOLIAThe following compound classes have been reportedto occur in various plant parts of noni, with effectfrom 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) 28.7aCD = Concentration required to double quinone reductase induction. bIC50 = Concentrationfor 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: SUMMARYBotanical dietary supplements have become widely available inthe United States since the passage of the Dietary SupplementHealth and Education Act (DSHEA) in 1994. Their widespreaduse has had a major impact on biomedical research andpharmacy practice.Fractionation studies on selected “botanicals” have yielded quitepotent biologically active compounds, although generallyoccurring at low concentrations. Some of these are useful leads aspotential cancer chemopreventive agents.The fruits of mangosteen (G. mangostana) and noni (M. citrifolia)have already yielded interesting bioactive compounds. Theconstituents of these botanical dietary supplements seem to offerparticular promise for further investigation.
ACKNOWLEDGMENTSPhytochemical Studies Biological TestingThe Ohio State University The Ohio State UniversityDr. Young-Won Chin Dr. Marcy P. BalunasDr. Ye Deng Dr. Young-Won ChinDr. Hyun-Ah Jung Ms. Chureeporn Chitchumroonchokchai (Human Nutrition)Dr. Alison D. Pawlus Dr. Alison D. Pawlus (at University of Illinois atDr. Baoning Su Chicago) Dr. Bin Su (Dr. Robert W. Brueggermeier)Collaborators – The Ohio Collaborator – UniversityState University of FloridaDr. Steven Clinton Dr. Veronika ButterweckDr. Mark Falia Supply of BotanicalsDr. 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.