pharmacometrics of 3-methoxypterostilbene: a component of traditional chinese medicinal plants

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 261468 11 pageshttpdxdoiorg1011552013261468

Research ArticlePharmacometrics of 3-MethoxypterostilbeneA Component of Traditional Chinese Medicinal Plants

Stephanie E Martinez Casey L Sayre and Neal M Davies

Faculty of Pharmacy Apotex Centre University of Manitoba Winnipeg MB Canada R3E 0T5

Correspondence should be addressed to Neal M Davies nealdaviesadumanitobaca

Received 9 February 2013 Accepted 26 March 2013

Academic Editor Srinivas Nammi

Copyright copy 2013 Stephanie E Martinez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

3-Methoxypterostilbene is a naturally occurring stilbene with potential in the treatment of diabetesThe preclinical pharmacokinet-ics and pharmacodynamics of 3-methoxypterostilbene were evaluated in the present studyThe right jugular veins of male Sprague-Dawley rats were cannulated The rats were dosed 10mgkg or 100mgkg of 3-methoxypterostilbene intravenously (IV) or orally(PO) respectively Serum and urine samples were analyzed using a previously validated reversed-phase HPLC method SerumAUC serum 119905

12 urine 119905

12 Cltotal and Vd for IV dosing were 481 plusmn 238 120583ghmL 189 plusmn 109 h 954 plusmn 151 h 478 plusmn 237 Lhkg

and 511 plusmn 038 Lkg respectively (mean plusmn SEM 119899 = 4) Serum AUC serum 11990512 urine 119905

12 Cltotal and Vd for PO dosing were

229 plusmn 446 120583ghmL 733 plusmn 891 h 206 plusmn 301 h 048 plusmn 0008 Lhkg and 520 plusmn 105 Lkg respectively (mean plusmn SEM 119899 = 4)Bioavailability of the stilbene was determined to be 506 plusmn 100 A 3-methoxypterostilbene glucuronidated metabolite wasdetected in both serum and urine 3-Methoxypterostilbene exhibited antidiabetic activity including 120572-glucosidase and 120572-amylaseinhibition as well as concentration-dependent antioxidant capacity similar to resveratrol 3-Methoxypterostilbene also exhibitedanti-inflammatory activity 3-Methoxypterostilbene appears to be a bioactive compound andmay be useful in reducing postprandialhyperglycemia

1 Introduction

3-Methoxypterostilbene (trans-331015840-5-trimethoxy-41015840-hydro-xystilbene) C

17H18O4 MW 286324 gmol (Figure 1)

is a naturally occurring stilbene [1 2] that can also beeasily synthesized using simple combinatorial synthesis[3ndash7] 3-Methoxypterostilbene is a structural analogue ofresveratrol which has demonstrated a myriad of potentialprohealth effects including anticancer cardioprotective anti-inflammatory neuroprotective and antiobesity properties[8]However it differs from resveratrol in its physicochemicalproperties The predicted octanol water partition coefficient(XLogP) for 3-methoxypterostilbene is 354 plusmn 049 [9] andthe experimentally determined XLogP for resveratrol is153 plusmn 001 [10]

3-Methoxypterostilbene has been isolated in two plantsused in traditional Chinese medicine The compound hasbeen found in Sphaerophysa salsula (also known as Swainsonasalsula) a shrub called ldquoku ma durdquo used for the treatment

of hypertension [1] 3-Methoxypterostilbene has also beenfound as an aglycone of a stilbene glycoside in the commonlyused Rheum palmatum (Chinese rhubarb) called ldquoda huangrdquoused to treat digestive disorders [2]

Due to 3-methoxypterostilbenersquos structural similarity toresveratrol and its presence in traditional Chinese medicinalplants 3-methoxypterostilbene may also possess potentialhealth benefits However information in the literature on thebioactivity of 3-methoxypterostilbene is scant In a reportseeking to identify biologically active piceatannol (anotherstructural analogue of resveratrol) analogs with greater sta-bility than piceatannol 3-methoxypterostilbene was reportedto demonstrate significantly greater activity in a 9 KB cyto-toxicity assay as well as a crown-gall plant antitumor (potatodisk) assay than piceatannol [3] 3-Methoxypterostilbeneproved to be as effective as resveratrol in the inhibitionof bacterial lipopolysaccharide-induced tissue factor expres-sion in human peripheral blood mononuclear cells in a

2 Evidence-Based Complementary and Alternative Medicine

H3CO

H3CO

OH

OCH3

Figure 1 Chemical structure of 3-methoxypterostilbene

study investigating resveratrol derivatives and coronary heartdisease [11] Several studies have also demonstrated that 3-methoxypterostilbene possesses greater apoptotic-inducingactivity than resveratrol in human leukemia-derived HL60cells [4 12 13]

With the ever-increasing natural products canon ofknowledge and growing concern for obesity-related diseasesthere has been much focus on the use of natural productsto aid in the management and treatment of type 2 diabetesResveratrol has been extensively studied in animals for itspotential to treat obesity and type 2 diabetes [8] Resveratrolappears to be able to increase insulin sensitivity in variousanimal models of insulin resistance [14ndash18] Additionallyanimal studies using models that consisted of geneticallyobese rats and mice with dietary induced obesity or chemi-cally induced diabetes found that resveratrol reduced bloodglucose levels which is important in the management oftype 2 diabetes and prediabetic patients [14 15 19ndash25]Thus structural analogues of resveratrol may also possessantidiabetic properties similar to those of resveratrol

To our knowledge there have been no studies evaluatingthe pharmacokinetics disposition or the in vivometabolismof 3-methoxypterostilbene other than that of a single ratpreviously reported by Martinez et al [9] The objectivesof the present study are to examine the pharmacokineticdisposition of 3-methoxypterostilbene as well as its in vivometabolism and antidiabetic properties To facilitate thisa reversed-phase high-performance liquid chromatographic(RP-HPLC) method was developed and validated for quan-tification of 3-methoxypterostilbene in biological matricesusing ultraviolet detection [9] Additionally the objectivesof this study were to investigate select biological activitiesof 3-methoxypterostilbene and perform a content analysison commercially available dried traditional Chinese herbsreported to contain 3-methoxypterostilbene using the pre-viously validated RP-HPLC method For the first time toour knowledge the preclinical pharmacokinetics antioxidantactivity cyclooxygenase-1 and -2 (COX-1 and -2) inhibitionand 120572-glucosidase and 120572-amylase inhibitory activity of 3-methoxypterostilbene are reported

2 Materials and Methods

21 Chemicals and Reagents 3-Methoxypterostilbene wasprovided by the Sabinsa Corporation (Piscataway NJ USA)and pinosylvin was purchased from Sequoia ResearchProducts Ltd (Oxford UK) HPLC-grade acetonitrileand water were purchased from J T Baker (PhillipsburgNJ USA) 120573-Glucuronidase type IX A (120573-glucuronidase)poly(ethylene glycol) (PEG) 400 dimethyl sulfoxide(DMSO) 120572-glucosidase from Saccharomyces cerevisiae4-nitrophenyl 120572-D-glucopyranoside 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) resveratrolibuprofen etodolac and 120572-amylase from porcine pancreastype VI-B were purchased from Sigma-Aldrich (St LouisMO USA) Amylase HR reagent was purchased fromMegazyme International Ireland (Wicklow Ireland)120573-Glucosidase from almonds was purchased from TokyoChemical Industry Co Ltd (Tokyo Japan) Dried Swainsonasalsula extract was provided by DaXingAnLing Snow LotusHerb Bio-technology Co Ltd (Heilongjiang China) anddried da huang (Chinese rhubarb) was purchased fromKwokShing Ent Ltd (Scarborough ON Canada) The antioxidantactivity kit and cyclooxygenase-1 and -2 inhibitor screeningkits were purchased from Cayman Chemical Company (AnnArbor MI USA)

22 Chromatographic System and Conditions The HPLCsystemusedwas a ShimadzuHPLC (Kyoto Japan) consistingof two LC-10A pumps a SIL-10AF autoinjector a SPD-M10A photodiode array detector and a SCL-10A systemcontroller Data collection and integration were achievedusing Shimadzu EZ Start Class VP (version 74) software APhenomenex LunaC

18(2) (5 120583m 250times 460mm) columnwas

used The mobile phase consisted of acetonitrile and water(62 38 vv) that were filtered and degassed under reducedpressure prior to use Pinosylvin was used as the internalstandard Isocratic separation at ambient temperature using aflow rate of 105mLmin was employed Ultraviolet detectionwas carried out at 327 nm Validation indicated that theprecision of the assay was lt12 (RSD) and was within 12at the limit of quantification (LOQ) (005120583gmL)The bias ofthe assay was lt15 and was within 13 at the LOQ [9]

23 Animals and Surgical Procedures Male Sprague-Dawleyrats (asymp200 g) were obtained from Simonsen Laboratories(Gilroy CA USA) and allowed food (Purina Rat Chow5001) and water ad libitum upon arrival to the vivariumRats were housed in a temperature- and humidity-controlledfacility with a 12 h lightdark cycle Prior to the first day ofthe pharmacokinetic experiment the rats were anesthetizedusing isoflurane (IsoFlo Abbott Laboratories Abbot ParkIL USA) coupled with an oxygen regulator and monitoredby pedal reflex and respiration rate to maintain a surgicalplane of anesthesia The right jugular veins of the ratswere cannulated with sterile silastic cannulas (Dow Corn-ing Midland MI USA) After cannulation Intramedic PE-50 polyethylene tubing (Becton Dickinson and CompanyFranklin Lakes NJ USA) was exposed through the dorsal

Evidence-Based Complementary and Alternative Medicine 3

skin The cannulas were flushed with nonheparinized 09sterile saline solution The animals were placed in metaboliccages to recover and fasted overnight

Animal ethics approval was obtained from the Universityof Manitoba Office of Research Ethics and Compliance

24 Dosages No pharmacokinetics studies are reported inthe literature for 3-methoxypterostilbene Despite the paucityof 3-methoxypterostilbene pharmacokinetic studies in the lit-erature studies on other stilbenes have reported intravenous(IV) dosage ranges from 10 to 20mgkg [26ndash30] and from 50to 300mgkg for oral (PO) dosage [27 31 32] In keepingwith the literature doses of 10mgkg IV and 100mgkg POwere chosen

25 Pharmacokinetic Study Male Sprague-Dawley rats (119899 =8 average weight 200 g) were cannulated as described in theAnimals and Surgical Procedures section The animals wereplaced in metabolic cages following surgery where they wererecovered overnight and fasted for 12 h prior to dosing Onthe day of the experiment the animals were dosed with 3-methoxypterostilbene in PEG 400 either IV (10mgkg 119899 = 4)or PO (100mgkg 119899 = 4) After dosing a series of wholeblood samples (05mL) were collected at 0 1 and 15min and05 1 2 4 6 12 24 48 and 72 h and 0 025 05 1 2 4 612 24 48 and 72 h for IV and PO dosed rats respectivelyThe cannulas were flushed with 05mL 09 nonheparinizedsaline solution after each sample collectionThe samples werecollected into regular 20mL Eppendorf tubes centrifugedat 10000 rpm for 5min (Beckman Microfuge centrifugeBeckman Coulter Inc Fullerton CA USA) and the serumwas collected The serum was divided into two 01mLfractions and placed into regular 20mL Eppendorf tubeslabeled as free and total serum samples Samples were storedat minus20∘C until analyzed Urine samples were also collected at0 2 6 12 24 48 and 72 h following 3-methoxypterostilbeneadministration The volumes of urine produced by the ratswere recorded and two 01mL aliquots were collected intoseparate prelabeled regular polypropylene microcentrifugetubes and labeled as free and total urine samples and storedat minus20∘C until analyzed At 72 h after dose the animals wereeuthanized exsanguinated and serum was collected

26 Serum and Urine Sample Preparation Serum and urinesamples (01mL) were run in duplicate with or withoutthe addition of 20120583L of 500UmL 120573-glucuronidase andincubated in a shaking water bath at 37∘C for 2 h to liberateany glucuronide conjugates [33] The proteins present inthe serum samples were precipitated using 1mL of minus20∘Cacetonitrile Urine and serum samples were vortexed (VortexGenie-2 VWR Scientific West Chester PA USA) for 30 sand centrifuged at 10000 rpm for 5min The supernatantswere transferred to new labeled 20mL Eppendorfmicrocen-trifuge tubes The samples were evaporated to dryness by astream of nitrogen gas The residues were reconstituted with200120583L of mobile phase vortexed for 30 s and centrifuged at10000 rpm for 5min The supernatants were transferred to

HPLC vials and 100 120583L was injected into the HPLC systemfor each sample120573-Glucuronidase fromE coli type IX-A acts to specifically

cleave the sugar moiety attached to the parent compoundfrom the 3-methoxypterostilbene glucuronide back into theaglycone (3-methoxypterostilbene) The samples which didnot undergo enzymatic hydrolysis (free samples) were uti-lized to determine the concentration of the aglycone whereasthe samples that did undergo enzymatic hydrolysis (totalsamples) were used to determine the concentration of theaglycone originally present in the sample in addition to theconcentration of the glucuronidated metabolite cleaved backto 3-methoxypterostilbeneTherefore by subtracting the freesample concentration from the total sample concentrationthe concentration of the glucuronidated metabolite can becalculated without the use of an additional chromatographicrun and analysis

27 Pharmacokinetic Analysis Pharmacokinetic analysis wasperformed using data from individual rats for which themean and standard error of the mean (SEM) were calculatedfor each group Samples were analyzed using WinNonlinsoftware (version 10 Pharsight CorporationMountainViewCA USA) to calculate the pharmacokinetic parameters Theratsrsquo concentrations versus time points were subjected toa noncompartmental model The apparent elimination rateconstant (KE) was estimated from the slope of the log-linearphase of declining concentration versus time plot The half-life and rate of eliminated were determined by applying thepreviously described software with the specified parametersThe renal clearancewas calculated bymultiplying the fractionof compound excreted unchanged with total body clearanceTheplasma half-life (119905

12) was determined using the following

equation 11990512

= 0693KE To determine the fraction ofunchanged 3-methoxypterostilbene excreted (119891

119890) in urine

the total amount of urine was divided by the total doseadministered The renal clearance (CLrenal) was determinedby the equation CLrenal = 119891119890 times CLtotal

28 Content Analysis of Dried Traditional Chinese MedicinalPlants 3-Methoxypterostilbene has been reported to bepresent in two plants used in traditional Chinese medicinemdashSphaerophysa salsula (Swainsona salsula) and Rheum pal-matum (Chinese rhubarb) [1 2] Dried S salsula extractand dried Chinese rhubarb are both commercially availableproducts Chinese rhubarb was frozen under liquid nitrogenand then ground into a fine powder S salsula extract wasalready in powdered form Two 01 g of each product weremeasured and placed into 20mL Eppendorf tubes 15mLmethanol was added to each tube for extraction Tubes werevortexed for 30 seconds agitated for 3 h and centrifuged at10000 rpm for 5min One of the duplicates was treated toextract only aglycones (free) and the second of the duplicateswas treated to cleave any glycosides to aglycones (total) byusing 120573-glucosidase from almonds The supernatants fromthe free samples were transferred into new 20mL Eppendorftubes and 50 120583L of internal standard pinosylvin was addedSamples were vortexed for 30 s dried to completion under


a stream of nitrogen gas and stored at minus20∘C until analysisThe supernatants of the total samples were transferred tonew 20mL Eppendorf tubes dried to completion under astream of compressed nitrogen gas and reconstituted withPBS (200120583L at pH 74) 20120583L of 120573-glucosidase (750UmL inPBS at pH 74) was added and samples were incubated for48 h at 37∘C in a shaking water bath 120573-Glucuronidase actsby cleaving the glycosidic sugar moieties frequently presentin plant extracts as previously described [29] Acetonitrile(1mL) was added to stop the enzymatic reaction followedby the addition of internal standard (50120583L) Samples werecentrifuged at 10000 rpm for 5min and the supernatant wasdried to completion under a stream of compressed nitrogengas Both free and total samples were reconstituted in mobilephase (200120583L) and 100 120583Lwas injected into theHPLC underthe same conditions previously described

29 Antioxidant Capacity Determination The antioxidantcapacities of 3-methoxypterostilbene and resveratrol weremeasured through an assay that relied on the inhibition ofthe oxidation of ABTS (221015840-azino-di-[3-ethylbenzthiazolinesulphonate]) to ABTS∙+ by metmyoglobin The amountof ABTS∙+ can be monitored spectrophotometrically Thedegree of suppression of absorbance caused by the com-pound of interest is proportional to the concentration ofABTS∙+ which is expressed as Trolox equivalents (120583gmL)For this assay 3-methoxypterostilbene and resveratrol weredissolved in DMSO on the day of the experiment to yieldconcentrations of 1 5 10 50 and 100120583gmL No additionaldilution was employed To run the assay 10 120583L of sample wascombined with 10120583L metmyoglobin and 150120583L chromogenThen 40120583L of hydrogen peroxide working solution wasadded within 1min to all the samples The plate was coveredand incubated on a shaker for 5min at room temperature 923plusmn 1∘C) and the absorbance was measured at 750 nm usingthe SynergyHTmultiwell plate reader andGen5 data analysissoftware (Biotek Instruments Inc Winooski VT USA) Theassay was performed in quadruplet For more informationregarding the assay protocol please refer to the instructionsfor the kit (Antioxidant Assay Kit from Cayman ChemicalmdashCat no 709091)

210 Cyclooxygenase Inhibition Determination The inhibi-tion of COX-1 and -2 by 3-methoxypterostilbene was mea-sured through the use of a three-day commercial assay kitThe assay relied on the quantification of the prostanoidproduct based on enzyme immunoassay (EIA) which uses anonspecific antibody for prostaglandins (PGs) The constantconcentration of the PG-AChE-tracer and the varying con-centrations of the PGs available to bind to the antiserum isinversely proportional to the concentration of free PGs in thewell For this assay 3-methoxypterostilbene was dissolved inDMSOover the concentration range of 1ndash250120583gmL Ibupro-fen and etodolac COX-2 preferentialNSAIDs were dissolvedin DMSO and used as controls over the concentration rangeof 1ndash250120583gmL Reagents were prepared according to themanufacturerrsquos instructions that accompanied the kit TheELISA plate for this purpose was read at an absorbance of

415 nm within 10 minutes at room temperature (23 plusmn 1∘C)using the Synergy HT multiwell plate reader and Gen5 dataanalysis software (Biotek Instruments Inc Winooski VTUSA) The assay was performed in quadruplet For moreinformation regarding the assay protocol please refer to theinstructions for the kit (COX Inhibitor Screening Assay Kitfrom Cayman ChemicalmdashCat no 560131)

211 120572-Glucosidase Inhibition Determination Inhibition of120572-glucosidase was determined through a colorimetric assayadapted and modified from the method presented byTadera et al [34] The assay uses p-nitrophenyl-120572-D-glucopyranoside (PNPG) which is hydrolyzed specifically by120572-glucosidase into a yellow-colored product (p-nitrophenol)The absorbance at 410 nm of liberated p-nitrophenol wasmeasured For this assay 3-methoxypterostilbene and resver-atrol were dissolved in DMSO to create concentration rangesfrom 0 to 200120583gmL 160 120583L of 100mM phosphate buffer(pH 68) 25120583L of 20mM PNPG in phosphate buffer and10 120583L of stilbenes in DMSO were added to wells of a 96-well plate (10 120583L DMSO was added to the control wells)The plate was incubated at 30∘C for 5min and then 10 120583L ofthe buffer containing 002mgmL of enzyme was added toeach well The plate was further incubated for 5min 20120583Lof 325M sodium hydroxide was added to each well to stopthe reactionThe plate was immediately read at an absorbanceof 410 nm at room temperature (23 plusmn 1∘C) using the SynergyHT multiwell plate reader and Gen5 data analysis software(Biotek Instruments IncWinooski VT USA)The assay wasperformed in sextuplicate

Inhibition () was calculated as ((119860minus119861)119860)times100 where119860 was the average absorbance of the control wells and 119861 wasthe absorbance of the wells containing stilbenes

212 Alpha-Amylase Inhibition Determination Inhibition of120572-amylase was determined through a colorimetric assayalso adapted and modified from the method presented byTadera et al [34] A synthetic substrate nonreducing-end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) commer-cially prepared as amylase HR reagent which is hydrolyzedspecifically by 120572-amylase into p-nitrophenyl maltosaccha-ride is employed The 120572-glucosidase present in the assaythen converts the new substrate into p-nitrophenol andabsorbance at 410 nm is measured as previously stated inthe 120572-amylase assay 3-methoxypterostilbene and resveratrolwere dissolved in methanol to create concentration rangesfrom0 to 200120583gmL 100120583L of amylaseHR reagent (preparedfollowing directions accompanying the reagent MegazymeAmylase HR Reagent Cat no R-AMHR4) and 40 120583L ofstilbene in methanol were added to a 96-well plate (40 120583Lof methanol was added to the control wells) The plate wasincubated for 5 minutes at 37∘C and then 60 120583L of 01mgmL120572-amylase in 01M HEPES buffer (pH 69) was added tothe reaction mixture After further incubation at 37∘C for10min 20120583L of 325M sodium hydroxide was added toeach well to stop the reaction The liberated p-nitrophenolwas determined and the percent inhibition calculated as


01

1

10

100

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)

3-Methoxypterostilbene3-Methoxypterostilbene glucuronide

Con

cent

ratio

n (120583

gm

L)

(a)

1

10

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)


Con

cent

ratio

n (120583

gm

L)(b)

Figure 2 (a) 3-Methoxypterostilbene disposition in serum following intravenous administration (b) 3-Methoxypterostilbene disposition inserum following oral administration (119899 = 4 mean plusmn SEM)

described in the assay for 120572-glucosidase activity previouslydescribed The assay was performed in sextuplicate

213 Statistical Analysis Compiled data were presented asmean and standard error of the mean (mean plusmn SEM) Wherepossible the data were analyzed for statistical significanceusing Minitab 15 statistical software (Minitab Inc StateCollege PAUSA) Studentrsquos t-test was employed for unpairedsamples with a value of119875 lt 005 being considered statisticallysignificant

3 Results and Discussion

31 Pharmacokinetic Study Standard curves established lin-earity over the concentration range studied for the serumand urine samples Chromatograms showed no interfer-ence from endogenous components Total samples (incu-bated with 120573-glucuronidase from Escherichia coli type IX-A) verified the presence of a glucuronidated metabolitebased on the increase in 3-methoxypterostilbene (agly-cone parent compound) concentrations after enzymatichydrolysis in both serum and urine Glucuronidation of 3-methoxypterostilbene parallels previous rat and human stud-ies with resveratrol existing predominately in its conjugatedform in both plasma and urine [35]

The serum concentration versus time profile for IV-dosed 3-methoxypterostilbene demonstrates a rapid declinein concentration in the first hour representing a distribu-tion phase which was followed by a steady elimination upto 24 hours after which the serum concentrations werebelow detectable concentrations (005120583gmL) (Figure 2(a))3-Methoxypterostilbene dosed PO displayed rapid absorp-tion with an average 119879max of 30 minutes (Figure 2(b)) The

Table 1 Pharmacokinetic parameters of 3-methoxypterostilbene inthe rat

Pharmacokinetic parameter IntravenousMean plusmn SEM

OralMean plusmn SEM

AUCinf (120583gsdothmL) 481 plusmn 238 229 plusmn 446Vd120573(Lkg) 511 plusmn 0380 520 plusmn 105

CLhepatic (Lhkg) 471 plusmn 233 0480 plusmn 00800CLrenal (Lhkg) 0760 plusmn 0460 00100 plusmn 0000CLtotal (Lhkg) 478 plusmn 237 0480 plusmn 00800119891119890() 164 plusmn 0950 124 plusmn 016011990512

(h) serum 189 plusmn 109 733 plusmn 89111990512

(h) urine 954 plusmn 151 206 plusmn 301MRT (h) 260 plusmn 150 105 plusmn 131Bioavailability (F) 100 506 plusmn 100

glucuronidated metabolite in both routes of administrationappeared to display multiple peaking which is suggestiveof enterohepatic recycling as indicated by an increase inserum concentration around 4 h after dose Enterohep-atic recycling has previously been reported for resveratrol[27 36]

Table 1 summarizes the pharmacokinetic parametersexhibited by 3-methoxypterostilbene at an IV dose of10mgkg and a PO dose of 100mgkg Noncompartmentalanalysis in WinNonlin software (ver 10) was used to modelboth serum and urine data The total serum clearanceof 3-methoxypterostilbene was determined to be 478 plusmn237 Lhkg for IV dosing and 0480 plusmn 00800 for PO dosingThe mean fraction excreted in urine unchanged (119891

119890) was


0

20

40

60

80

100

120

140

160

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(a)

0

50

100

150

200

250

300

350

400

450

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(b)

10

100

1000

0 6 12 18 24 30 36 42 48 54 60Mid time (h)

Rate

of u

rinar

y ex

cret

ion

(120583g

hr)


(c)

00

01

10

100

1000

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)


(d)

Figure 3 (a) Cumulative 3-methoxypterostilbene and glucuronidated metabolite (120583g) excreted in urine over 72 h after intravenousadministration (b) Cumulative 3-methoxypterostilbene and glucuronidated metabolite (120583g) excreted in urine over 72 h after oraladministration (c) Rate of excretion (120583gh) of 3-methoxypterostilbene and glucuronidated metabolite in urine over 72 h after intravenousadministration (d) Rate of excretion (120583gh) of 3-methoxypterostilbene and glucuronidated metabolite in urine over 72 h after oraladministration (119899 = 4mean plusmn SEM)

164 plusmn 0950 for IV and 124 plusmn 0160 for PO indi-cating that 3-methoxypterostilbene is mainly excreted vianonrenal routes Renal clearance (CLrenal) was measured at0760 plusmn 046 Lhkg for IV and 00100 plusmn 000100 Lhkgfor PO and hepatic clearance (CLhepatic = CLtotal minusCLrenal) was determined to be 471 plusmn 233 Lhkg for IVand 0480 plusmn 90800 Lhkg for PO assuming that nonrenalclearance is hepatic clearance The volume of distribution of3-methoxypterostilbene is 511 plusmn 0380 Lkg IV and 520 plusmn100 Lkg PO which is greater than total body water sug-gesting that 3-methoxypterostilbene is highly distributed intotissues The mean area under the curve (AUC) representingthe total amount of exposure in the serum over time was481 plusmn 238 120583gsdothmL for IV and 229 plusmn 446 120583gsdothmL for POThe serumconcentration of 3-methoxypterostilbene declinedvery slowly with a mean elimination half-life of 189 plusmn 109 hfor IV and 733 plusmn 891 h for PO The oral bioavailability for

3-methoxypterostilbene was determined to be 5060020 plusmn100

Reported bioavailability of resveratrol in rats rangesfrom 20 to 388 [27 32] and lt1 in humans [37] witha wide variability in pharmacokinetic parameters amongindividuals [38] Species-dependent rapid conjugation withhigher glucuronidation rates and affinity in humans maylimit stilbene bioavailability and show expressed differencesin pharmacokinetics between rodent and human studiesPoor bioavailability in humans is a potential limitation in theuse of resveratrol as a therapeutic agent hence the interestin structural analogs 3-Methoxypterostilbene bioavailabil-ity in rats has been determined to be 506 plusmn 100The melting point range of 3-methoxypterostilbene wasexperimentally determined to be 885ndash912∘C The reportedmelting point range of resveratrol is 253ndash255∘C [39] There-fore 3-methoxypterostilbene has a lower crystallinity than


0

20

40

60

80

100

120

140

160

180

200

Baseline

3-MethoxypterostilbeneResveratrol

1 5 10 100

Trol

ox eq

uiva

lent

(120583g

mL)

lowast

lowast

lowast

lowastlowast

lowast

Dagger

Dagger

50

(120583gmL)

Figure 4 Antioxidant capacity (119899 = 4 mean plusmn SEM) of 3-methoxypterostilbene and resveratrol at 1 10 50 and 100120583gmLdissolved in DMSO lowastSignificantly greater than baseline (DMSO)sample (119875 lt 005) DaggerSignificantly different from resveratrol at thesame concentration (119875 lt 005)

resveratrol The low crystallinity of 3-methoxypterostilbeneis responsible for its increased dissolution over resvera-trol Furthermore the aqueous solubility of resveratrol issim30mgmL [40] and the predicted aqueous solubility usingAlogPs [41] of 3-methoxypterostilbene is 8ndash88mgL Theincreased bioavailability of 3-methoxypterostilbene in ratscompared to resveratrol is likely due to its lower crystallinityresulting in enhanced dissolution over resveratrol and maybe in part due to differences in solubility and absorbancemechanisms which will be examined in further studiesThe increased bioavailability of 3-methoxypterostilbene overresveratrol in rats may extend to the greater bioavailabil-ity of 3-methoxypterostilbene than that of resveratrol inhumans

Analysis of urine samples for both routes of admin-istration displayed the presence of the parent compound3-methoxypterostilbene and the glucuronidated metabolitepreviously identified in the serum (Figures 3(a) and 3(b))Thetotal cumulative urinary excretion plot (Figure 3(a)) indicatesthat 3-methoxypterostilbene is excreted predominantly in theaglycone form for PO administration and almost equallyin the aglycone and glucuronidated metabolite form for IVdosing The glucuronidated metabolite appears to be mostlyexcreted by 12 h after dose for both routes of administra-tion while 3-methoxypterostilbene (aglycone) appeared tobe predominately excreted by 12 h after dose for IV butsteadily increased in excretion even at 72 h after dose forPO administration The half-life of 3-methoxypterostilbenein urine was determined to be 954 plusmn 141 h for IV and206 plusmn 301 h for PO The rate of urinary excretion plot(Figure 3(b)) indicates that 3-methoxypterostilbene and its

glucuronidated metabolite have similar rates of excretionas indicated by their parallel slope (minusKE2303) for IVadministration but the glucuronidated metabolite appears tohave a greater rate of excretion over the aglycone after POadministration

The total dose of 3-methoxypterostilbene administeredwas 10mgkg for IV and 100mgkg PO The average weightof the rats in this experiment was sim200 g Each rat receivedsim2mg of 3-methoxypterostilbene IV and 20mg POThe plotsof cumulative amount excreted in urine for both the aglyconeand glucuronidated metabolite forms excreted (sim98120583g and80 120583g resp for IV and 341 120583g and 182 120583g resp for PO) arevery small compared to the overall dose administered (sim2mgand 20mg for IV and PO resp) This further suggests that 3-methoxypterostilbene is eliminated predominately by nonre-nal routes As previously mentioned119891

119890was 164plusmn0950 for

IV and 124plusmn0160 for PO and thereforeCLrenal was 0760 plusmn0460 Lhkg for IV and 00100 plusmn 000100 Lhkg for POExcretion via nonrenal routes for 3-methoxypterostilbeneagrees with literature reports of nonrenal excretion of otherstilbenes [27 30]

32 Content Analysis of Dried Traditional Chinese MedicinalPlants Evaluation of the S salsula extract and dried Rpalmatum indicated that only the S salsula extract containeddetectable levels of 3-methoxypterostilbene The aglyconeconcentration for the S salsula extract was determinedto be 0842120583gg and the total concentration of 3-methox-ypterostilbene (aglycone and glycoside) was determined tobe 0853 120583gg indicating that 3-methoxypterostilbene existsprimarily in its aglycone form in S salsula extract Despitethe report that 3-methoxypterostilbene exists as an aglyconeof a stilbene glycoside in R palmatum [2] the compoundwas not detected as an aglycone or glycoside in the com-mercially available dried Chinese rhubarb It is suspectedthat 3-methoxypterostilbene may be detectable in othercommercially available Chinese rhubarb samples and thatplant variation likely accounts for the lack of detection in thissample

33 Antioxidant Capacity of 3-Methoxypterostilbene Figure 4reports the antioxidant capacity of 3-methoxypterostilbenein units of Trolox equivalents (120583gmL) The baseline (DMSOonly) samples have a low antioxidant capacity (812 plusmn747 120583gmL or 0325 plusmn 00299mM) 3-Methoxypterostilbenedemonstrates amodest concentration-dependent antioxidantactivity with an antioxidant capacity at 1 120583gmL of 986 plusmn137 120583gmL (0394 plusmn 00546mM) Trolox equivalents anda capacity at 100120583gmL of 174 plusmn 270 120583gmL (0695 plusmn000940mM) Trolox equivalents This indicated that 3-methoxypterostilbene prevents oxidation at comparable lev-els to Trolox if not better (as seen at lower concentrations)3-Methoxypterostilbene demonstrated significantly greateractivity to the baseline samples from 10 to 100 120583gmL (119875 lt005) and only showed significant difference from resveratrolactivity at the two highest concentrations tested 50 and100 120583gmL (119875 lt 005)


0

20

40

60

80

100

120In

hibi

tion

()

Ibuprofen3-MethoxypterostilbeneEtodolac

1 10 250

lowastdagger

(120583gmL)

(a)

0

10

20

30

40

50

60

Inhi

bitio

n (

)

lowastlowast

dagger


1 10 250 (120583gmL)

(b)

Figure 5 (a) Cycloxoygenase I inhibition activity of 3-methoxypterostilbene at 1 10 and 250 120583gmL (b) Cycloxoygenase II inhibition activityof 3-methoxypterostilbene at 1 10 and 250 120583gmL lowastSignificantly greater activity than 1 120583gmL ibuprofen (119875 lt 005) lowastlowastSignificantly greateractivity than 250 120583gmL ibuprofen (119875 lt 005) daggerSignificantly greater activity than 1 120583gmL etodolac (119875 lt 005) DaggerSignificantly greater activitythan 250 120583gmL etodolac (119875 lt 005) (119899 = 4 mean plusmn SEM)

The ability of 3-methxoypterostilbene to work as anantioxidant is of paramount importance as the wide rangeof health benefits of polyphenols are thought to result atleast in part from their antioxidant capacity For examplethe ability of resveratrol to limit the start and progression ofatherosclerosis is associated with the compoundrsquos ability toinhibit lipid oxidation of polyunsaturated fatty acids [42] Itis likely that the health benefits of 3-methoxypterostilbenewill also be associated with its antioxidant capacity In theliterature it is well known that the number and position ofthe hydroxyl groups on stilbenes are critical for bioactivityand t antioxidant activity [43ndash46] A computer model ofantioxidant activity of hydroxystilbenes suggests that 3-methoxypterostilbene is one of the most potent 34 hydrox-ystilbenes modeled and has greater potency than resveratrol[47]

34 Cyclooxygenase Inhibitory Activity Figure 5 reportsthe cyclooxygenase inhibitory activity of 3-methoxypter-ostilbene and two NSAIDs etodolac and ibuprofen 3-Me-thoxypterostilbene appears to have greater activity againstCOX-1 than COX-2 Figure 5(a) demonstrates the positiveconcentration-dependent inhibitory activity of 3-methox-ypterostilbene against COX-1While 3-methoxypterostilbenedid not demonstrate significantly greater (119875 lt 005)COX-1 inhibition activity than the two NSAIDs at lowerconcentrations the high concentration (250120583gmL) of3-methoxypterostilbene demonstrated significantly greateractivity (119875 lt 005) than the NSAIDs at low doses Figure 5(a)

details the COX-2 inhibitory activity of 3-methox-ypterostilbene At the concentration of 10120583gmL 3-methox-ypterostilbene demonstrated statistically greater inhibition(119875 lt 005) than etodolac at 1 120583gmL and ibuprofen at250120583gmL Several stilbenes including resveratrol areknown to be preferential COX-2 inhibitors [28]

35 Antidiabetic Activity

351 Alpha-Glucosidase Activity Figure 6 reports thealpha-glucosidase inhibition of 3-methoxypterostilbene andresveratrol Resveratrol shows a clear positive concentration-dependent inhibition relationship whereas 3-methox-ypterostilbene does not appear to exhibit greater inhibitionat higher concentrations 120572-Glucosidase inhibition activityis only statistically different (119875 lt 005) between the twostilbenes at the two highest concentrations tested (100 and200120583gmL)120572-Glucosidase is an enzyme found in the small intes-

tine which hydrolyzes 14-120572-bonds of disaccharides intoglucose Inhibition of 120572-glucosidase suppresses postprandialhyperglycemia by lowering the rate of glucose absorption viadelayed carbohydrate digestion and extended digestion time120572-Glucosidase inhibitors are useful in maintaining glycemiccontrol of prediabetic and type two diabetic patients Thelower concentrations of 3-methoxypterostilbene tested maybe biologically achievable resulting in moderate inhibitionof 120572-glucosidase comparable to that of resveratrol andreduction of postprandial hyperglycemia could be seen


0

10

20

30

40

50

60

70

80In

hibi

tion

()

3-Methoxypterostilbene

Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)

Figure 6 Alpha-glucosidase inhibition activity of 3-methox-ypterostilbene and resveratrol (119899 = 6 mean plusmn SEM) lowastSignificantlydifferent from resveratrol at the same concentration (119875 lt 005)

Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10

Figure 7 Alpha-amylase inhibition activity of 3-methoxypteros-tilbene and resveratrol (119899 = 6 mean plusmn SEM) lowastSignificantly differentfrom resveratrol at the same concentration (119875 lt 005)

352 Alpha-Amylase Activity 120572-Amylase inhibition activ-ity by 3-methoxypterostilbene and resveratrol is shownin Figure 7 Both resveratrol and 3-methoxypterostilbenedisplay relatively weak inhibition of 120572-amylase and bothstilbenes show a slightly negative concentration inhibi-tion relationship At the highest concentrations tested 3-methoxypterostilbene appears to increase activity of 120572-amylase At lower biologically relevant concentrations (1-50120583gmL) there is no significant difference between inhibi-tion activity of 3-methoxypterostilbene and resveratrol

In humans 120572-amylase is an enzyme predominately foundin the pancreas and saliva Like 120572-glucosidase 120572-amylasehydrolyzes 120572-14-glycosidic bonds but acts on polysaccha-rides Inhibition of 120572-amylase also reduces postprandialhyperglycemia and 120572-glucosidase inhibitors may be used totreat type 2 diabetes The modest inhibitory activity of 3-methoxypterostilbene at biologically relevant levels suggeststhat it may be as effective as resveratrol at helping reducepostprandial hyperglycemia

4 Conclusions

In summary the pharmacokinetics in rats and the invitro metabolism of 3-methoxypterostilbene was evalu-ated for the first time 3-methoxypterostilbene demon-strates improved bioavailability compared to resveratrol3-Methoxypterostilbene demonstrated antioxidant activitycomparable to resveratrol at biologically relevant concen-trations This stilbene also showed strong COX-1 inhibitioncomparable to twoNSAIDs andmoderate inhibition of COX-2 3-Methoxypterostilbene demonstrated moderate antidia-betic activity via inhibition of 120572-glucosidase and 120572-amylasecomparable to resveratrol Further exploration of the phar-macodynamics of 3-methoxypterostilbene is under way todemonstrate utility in reducing postprandial hyperglycemiaadiposeness cardiac hypertrophy and inflammation

Conflict of Interests

The authors declare no financial conflict of interest

Acknowledgments

The authors would like to acknowledge the University ofManitoba Graduate Fellowships from the University of Man-itoba awarded to Stephanie E Martinez and Casey L Sayrea Manitoba Graduate Scholarship awarded to Stephanie EMartinez a Pfizer Canada Centennial Pharmacy ResearchAward awarded to Stephanie E Martinez and a Duff RoblinFellowship to Casey L Sayre The authors would also like tothank the Sabina Corporation for their support

References

[1] Z J Ma X Li N Li and J H Wang ldquoStilbenes from Sphae-rophysa salsulardquo Fitoterapia vol 73 no 4 pp 313ndash315 2002

[2] Y Kashiwada G I Nonaka I Nishioka et al ldquoStudies onrhubarb (Rhei rhizome) XIV Isolation and characterizationof stilbene glucosides from Chinese rhubarbrdquo Chemical andPharmacetucial Bulletin vol 36 no 4 pp 1545ndash1549 1988

[3] M T Gill R Bajaj C J Chang D E Nichols and J LMcLaughlin ldquo33rsquo5rsquo-Tri-O-methylpiceatannol and 43rsquo5rsquo-tri-O-methylpiceatannol improvements over piceatannol in bioactiv-ityrdquo Journal of Natural Products vol 50 no 1 pp 36ndash40 1987

[4] M Roberti D Pizzirani D Simoni et al ldquoSynthesis andbiological evaluation of resveratrol and analogues as apoptosis-inducing agentsrdquo Journal of Medicinal Chemistry vol 46 no 16pp 3546ndash3554 2003


[5] R Amorati M Lucarini V Mugnaini G F Pedulli MRoberti and D Pizzirani ldquoAntioxidant activity of hydroxystil-bene derivatives in homogeneous solutionrdquo Journal of OrganicChemistry vol 69 no 21 pp 7101ndash7107 2004

[6] V Jerkovic F Nguyen S Nizet and S Collin ldquoCombinatorialsynthesis reversed-phase and normal-phase high-performanceliquid chromatography elution data and liquid chromatogra-phypositive atmospheric pressure chemical ionization tandemmass spectra of methoxylated and glycosylated resveratrolanaloguesrdquo Rapid Communications in Mass Spectrometry vol21 no 15 pp 2456ndash2466 2007

[7] H S Lee B W Lee M R Kim and J G Jun ldquoSyntheses ofresveratrol and its hydroxylated derivatives as radical scavengerand tyrosinase inhibitorrdquo Bulletin of the Korean ChemicalSociety vol 31 no 4 pp 971ndash975 2010

[8] O Vang N Ahmad C A Baile et al ldquoWhat is new for an oldmolecule systematic review and recommendations on the useof resveratrolrdquo PLoS ONE vol 6 no 6 article e19881 2011

[9] S E Martinez C L Sayre and N M Davies ldquoAnalysis of 3-methoxypterostilbene in biological fluids by high-performanceliquid chromatography application to pre-clinical pharmacoki-neticsrdquo Biomedical Chromatography vol 27 no 1 pp 67ndash722013

[10] C Privat J P Telo V Bernardes-Genisson A Vieira J PSouchard and F Nepveu ldquoAntioxidant properties of trans-120576-Viniferin as compared to stilbene derivatives in aqueous andnonaqueousmediardquo Journal of Agricultural and FoodChemistryvol 50 no 5 pp 1213ndash1217 2002

[11] G Kaur M Roberti F Raul and U R Pendurthi ldquoSuppres-sion of human monocyte tissue factor induction by red winephenolics and synthetic derivatives of resveratrolrdquo ThrombosisResearch vol 119 no 2 pp 247ndash256 2007

[12] A J Gescher ldquoResveratrol from red grapesmdashpedestrianpolyphenol or useful anticancer agentrdquo Planta Medica vol 74no 13 pp 1651ndash1655 2008

[13] I G Tsygankova and S M Zhenodarova ldquoThe structuremdashactivity correlation in a series of stilbene derivatives and relatedcompounds the inducers of apoptosisrdquo Russian Journal ofGeneral Chemistry vol 81 no 5 pp 913ndash919 2011

[14] J A Baur J K Pearson N L Price et al ldquoResveratrol improveshealth and survival of mice on a high-calorie dietrdquo Nature vol444 no 7117 pp 337ndash342 2006

[15] L Rivera R Moron A Zarzuelo and M Galisteo ldquoLong-term resveratrol administration reducesmetabolic disturbancesand lowers blood pressure in obese Zucker ratsrdquo BiochemicalPharmacology vol 77 no 6 pp 1053ndash1063 2009

[16] M Lagouge C Argmann Z Gerhart-Hines et al ldquoResver-atrol improves mitochondrial function and protects againstmetabolic disease by activating SIRT1 and PGC-1120572rdquo Cell vol127 no 6 pp 1109ndash1122 2006

[17] J Shang L L Chen F X Xiao H Sun H C Ding and HXiao ldquoResveratrol improves non-alcoholic fatty liver disease byactivating AMP-activated protein kinaserdquo Acta PharmacologicaSinica vol 29 no 6 pp 698ndash706 2008

[18] A Gonzalez-Rodriquez J A Mas Gutierrez S Sanz-Gonzalezet al ldquoInhibition of PTP1B restores IRS1-mediated hepaticinsulin signaling in IRS2-deficient micerdquo Diabetes vol 59 no3 pp 588ndash599 2010

[19] J P Huang S S Huang J Y Deng C C Chang Y J Day and LMHung ldquoInsulin and resveratrol act synergistically preventingcardiac dysfunction in diabetes but the advantage of resveratrol

in diabetics with acute heart attack is antagonized by insulinrdquoFree Radical Biology and Medicine vol 49 no 11 pp 1710ndash17212010

[20] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

[21] P Palsamy and S Subramanian ldquoResveratrol a naturalphytoalexin normalizes hyperglycemia in streptozotocin-nicotinamide induced experimental diabetic ratsrdquo Biomedicineand Pharmacotherapy vol 62 no 9 pp 598ndash605 2008

[22] G Ramadori L Gautron T Fujikawa C R Vianna J KElmquist and R Coppari ldquoCentral administration of resver-atrol improves diet-induced diabetesrdquo Endocrinology vol 150no 12 pp 5326ndash5333 2009

[23] J C Milne P D Lambert S Schenk et al ldquoSmall moleculeactivators of SIRT1 as therapeutics for the treatment of type 2diabetesrdquo Nature vol 450 no 7170 pp 712ndash716 2007

[24] K K R Rocha G A Souza G X Ebaid F R F Seiva A CCataneo and E L B Novelli ldquoResveratrol toxicity effects onrisk factors for atherosclerosis and hepatic oxidative stress instandard and high-fat dietsrdquo Food and Chemical Toxicology vol47 no 6 pp 1362ndash1367 2009

[25] Y H Kim Y S Kim S S Kang G J Cho and W S ChoildquoResveratrol inhibits neuronal apoptosis and elevated Ca2+calmodulin-dependent protein kinase II activity in diabeticmouse retinardquo Diabetes vol 59 no 7 pp 1825ndash1835 2010

[26] M Asensi I Medina A Ortega et al ldquoInhibition of cancergrowth by resveratrol is related to its low bioavailabilityrdquo FreeRadical Biology and Medicine vol 33 no 3 pp 387ndash398 2002

[27] J F Marier P Vachon A Gritsas J Zhang J P Moreau andM P Ducharme ldquoMetabolism and disposition of resveratrol inrats extent of absorption glucuronidation and enterohepaticrecirculation evidenced by a linked-rat modelrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 302 no 1 pp369ndash373 2002

[28] K A Roupe C M Remsberg J A Yanez and N M DaviesldquoPharmacometrics of stilbenes seguing towards the clinicrdquoCurrent Clinical Pharmacology vol 1 no 1 pp 81ndash101 2006

[29] K A Roupe J A Yanez X W Teng and N M Davies ldquoPhar-macokinetics of selected stilbenes rhapontigenin piceatannoland pinosylvin in ratsrdquo Journal of Pharmacy and Pharmacologyvol 58 no 11 pp 1443ndash1450 2006

[30] C M Remsberg J A Yanez Y Ohgami K R Vega-VillaA M Rimando and N M Davies ldquoPharmacometrics ofpterostilbene preclinical pharmacokinetics and metabolismanticancer antiinflammatory antioxidant and analgesic activ-ityrdquo Phytotherapy Research vol 22 no 2 pp 169ndash179 2008

[31] S Zhou R Yang Z Teng et al ldquoDose-dependent absorptionand metabolism of frans-polydatin in ratsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 11 pp 4572ndash4579 2009

[32] I M Kapetanovic M Muzzio Z Huan et al ldquoPharmacoki-netics oral bioavailability and metabolic profile of resveratroland its dimethylether analog pterostilbene in ratsrdquo CancerChemotherapy and Pharmacology vol 68 no 3 pp 593ndash6012011

[33] C Y Yang S Y Tsai P D L Chao H F Yen T M Chienand S L Hsiu ldquoDetermination of hesperetin and its conjugatemetabolites in serum and urinerdquo Journal of Food and DrugAnalysis vol 10 no 3 pp 143ndash148 2002


[34] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[35] E Wenzel and V Somoza ldquoMetabolism and bioavailability oftrans-resveratrolrdquo Molecular Nutrition and Food Research vol49 no 5 pp 472ndash481 2005

[36] N M Davies J K Takemoto D R Brocks and J A YanezldquoMultiple peaking phenomena in pharmacokinetic dispositionrdquoClinical Pharmacokinetics vol 49 no 6 pp 351ndash377 2010

[37] T Walle ldquoBioavailability of resveratrolrdquo Annals of the New YorkAcademy of Sciences vol 1215 no 1 pp 9ndash15 2011

[38] M Vaz-da-Silva A I Loureiro A Falcao et al ldquoEffect of foodon the pharmacokinetic profile of trans-resveratrolrdquo Interna-tional Journal of Clinical Pharmacology and Therapeutics vol46 no 11 pp 564ndash570 2008

[39] A Amri J C Chaumeil S Sfar andC Charrueau ldquoAdministra-tion of resveratrol what formulation solutions to bioavailabilitylimitationsrdquo Journal of Controlled Release vol 158 no 2 pp182ndash193 2012

[40] ldquoResveratrolrdquo Product information No R5010 Sigma SaintLoius Mo USA 2013 httpwwwsigmaaldrichcometcmedialibdocsSigmaProduct Information Sheet1r5050pisPar 0001Filetmpr5010pispdf

[41] I V Tetko J Gasteiger R Todeschini et al ldquoVirtual computa-tional chemistry laboratorymdashdesign and descriptionrdquo Journalof Computer-AidedMolecular Design vol 19 no 6 pp 453ndash4632005

[42] L Fremont ldquoBiological effects of resveratrolrdquo Life Sciences vol66 no 8 pp 663ndash673 2000

[43] S Stojanovic H Sprinz and O Brede ldquoEfficiency and mech-anism of the antioxidant action of trans-resveratrol and itsanalogues in the radical liposome oxidationrdquo Archives of Bio-chemistry and Biophysics vol 391 no 1 pp 79ndash89 2001

[44] J G Fang M Lu Z H Chen et al ldquoAntioxidant effects ofresveratrol and its analogues against the free-radical-inducedperoxidation of linoleic acid in micellesrdquo Chemistry vol 8 no18 pp 4191ndash4198 2002

[45] Y J Cai J G Fang L P Ma L Yang and Z L Liu ldquoInhibitionof free radical-induced peroxidation of rat liver microsomes byresveratrol and its analoguesrdquo Biochimica et Biophysica Actavol 1637 no 1 pp 31ndash38 2003

[46] K B Harikumar and B B Aggarwal ldquoResveratrol a multitar-geted agent for age-associated chronic diseasesrdquo Cell Cycle vol7 no 8 pp 1020ndash1037 2008

[47] S Rayne C D Goss K Forest and K J Friesen ldquoQuantitativestructure-activity relationships for estimating the aryl hydro-carbon receptor binding affinities of resveratrol derivativesand the antioxidant activities of hydroxystilbenesrdquo MedicinalChemistry Research vol 19 no 8 pp 864ndash901 2010


H3CO

H3CO

OH

OCH3

Figure 1 Chemical structure of 3-methoxypterostilbene

study investigating resveratrol derivatives and coronary heartdisease [11] Several studies have also demonstrated that 3-methoxypterostilbene possesses greater apoptotic-inducingactivity than resveratrol in human leukemia-derived HL60cells [4 12 13]

With the ever-increasing natural products canon ofknowledge and growing concern for obesity-related diseasesthere has been much focus on the use of natural productsto aid in the management and treatment of type 2 diabetesResveratrol has been extensively studied in animals for itspotential to treat obesity and type 2 diabetes [8] Resveratrolappears to be able to increase insulin sensitivity in variousanimal models of insulin resistance [14ndash18] Additionallyanimal studies using models that consisted of geneticallyobese rats and mice with dietary induced obesity or chemi-cally induced diabetes found that resveratrol reduced bloodglucose levels which is important in the management oftype 2 diabetes and prediabetic patients [14 15 19ndash25]Thus structural analogues of resveratrol may also possessantidiabetic properties similar to those of resveratrol

To our knowledge there have been no studies evaluatingthe pharmacokinetics disposition or the in vivometabolismof 3-methoxypterostilbene other than that of a single ratpreviously reported by Martinez et al [9] The objectivesof the present study are to examine the pharmacokineticdisposition of 3-methoxypterostilbene as well as its in vivometabolism and antidiabetic properties To facilitate thisa reversed-phase high-performance liquid chromatographic(RP-HPLC) method was developed and validated for quan-tification of 3-methoxypterostilbene in biological matricesusing ultraviolet detection [9] Additionally the objectivesof this study were to investigate select biological activitiesof 3-methoxypterostilbene and perform a content analysison commercially available dried traditional Chinese herbsreported to contain 3-methoxypterostilbene using the pre-viously validated RP-HPLC method For the first time toour knowledge the preclinical pharmacokinetics antioxidantactivity cyclooxygenase-1 and -2 (COX-1 and -2) inhibitionand 120572-glucosidase and 120572-amylase inhibitory activity of 3-methoxypterostilbene are reported

2 Materials and Methods

21 Chemicals and Reagents 3-Methoxypterostilbene wasprovided by the Sabinsa Corporation (Piscataway NJ USA)and pinosylvin was purchased from Sequoia ResearchProducts Ltd (Oxford UK) HPLC-grade acetonitrileand water were purchased from J T Baker (PhillipsburgNJ USA) 120573-Glucuronidase type IX A (120573-glucuronidase)poly(ethylene glycol) (PEG) 400 dimethyl sulfoxide(DMSO) 120572-glucosidase from Saccharomyces cerevisiae4-nitrophenyl 120572-D-glucopyranoside 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) resveratrolibuprofen etodolac and 120572-amylase from porcine pancreastype VI-B were purchased from Sigma-Aldrich (St LouisMO USA) Amylase HR reagent was purchased fromMegazyme International Ireland (Wicklow Ireland)120573-Glucosidase from almonds was purchased from TokyoChemical Industry Co Ltd (Tokyo Japan) Dried Swainsonasalsula extract was provided by DaXingAnLing Snow LotusHerb Bio-technology Co Ltd (Heilongjiang China) anddried da huang (Chinese rhubarb) was purchased fromKwokShing Ent Ltd (Scarborough ON Canada) The antioxidantactivity kit and cyclooxygenase-1 and -2 inhibitor screeningkits were purchased from Cayman Chemical Company (AnnArbor MI USA)

22 Chromatographic System and Conditions The HPLCsystemusedwas a ShimadzuHPLC (Kyoto Japan) consistingof two LC-10A pumps a SIL-10AF autoinjector a SPD-M10A photodiode array detector and a SCL-10A systemcontroller Data collection and integration were achievedusing Shimadzu EZ Start Class VP (version 74) software APhenomenex LunaC

18(2) (5 120583m 250times 460mm) columnwas

used The mobile phase consisted of acetonitrile and water(62 38 vv) that were filtered and degassed under reducedpressure prior to use Pinosylvin was used as the internalstandard Isocratic separation at ambient temperature using aflow rate of 105mLmin was employed Ultraviolet detectionwas carried out at 327 nm Validation indicated that theprecision of the assay was lt12 (RSD) and was within 12at the limit of quantification (LOQ) (005120583gmL)The bias ofthe assay was lt15 and was within 13 at the LOQ [9]

23 Animals and Surgical Procedures Male Sprague-Dawleyrats (asymp200 g) were obtained from Simonsen Laboratories(Gilroy CA USA) and allowed food (Purina Rat Chow5001) and water ad libitum upon arrival to the vivariumRats were housed in a temperature- and humidity-controlledfacility with a 12 h lightdark cycle Prior to the first day ofthe pharmacokinetic experiment the rats were anesthetizedusing isoflurane (IsoFlo Abbott Laboratories Abbot ParkIL USA) coupled with an oxygen regulator and monitoredby pedal reflex and respiration rate to maintain a surgicalplane of anesthesia The right jugular veins of the ratswere cannulated with sterile silastic cannulas (Dow Corn-ing Midland MI USA) After cannulation Intramedic PE-50 polyethylene tubing (Becton Dickinson and CompanyFranklin Lakes NJ USA) was exposed through the dorsal











equation 11990512


119890) in urine












01

1

10

100

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)


Con

cent

ratio

n (120583

gm

L)

(a)

1

10

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)


Con

cent

ratio

n (120583

gm

L)(b)









OralMean plusmn SEM







119890) was


0

20

40

60

80

100

120

140

160

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(a)

0

50

100

150

200

250

300

350

400

450

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(b)

10

100

1000

0 6 12 18 24 30 36 42 48 54 60Mid time (h)

Rate

of u

rinar

y ex

cret

ion

(120583g

hr)


(c)

00

01

10

100

1000

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)


(d)






0

20

40

60

80

100

120

140

160

180

200

Baseline


1 5 10 100

Trol

ox eq

uiva

lent

(120583g

mL)

lowast

lowast

lowast

lowastlowast

lowast

Dagger

Dagger

50

(120583gmL)











0

20

40

60

80

100

120In

hibi

tion

()


1 10 250

lowastdagger

(120583gmL)

(a)

0

10

20

30

40

50

60

Inhi

bitio

n (

)

lowastlowast

dagger


1 10 250 (120583gmL)

(b)









0

10

20

30

40

50

60

70

80In

hibi

tion

()


Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)


Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10




4 Conclusions




Acknowledgments


References





























































equation 11990512


119890) in urine












01

1

10

100

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)


Con

cent

ratio

n (120583

gm

L)

(a)

1

10

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)


Con

cent

ratio

n (120583

gm

L)(b)









OralMean plusmn SEM







119890) was


0

20

40

60

80

100

120

140

160

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(a)

0

50

100

150

200

250

300

350

400

450

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(b)

10

100

1000

0 6 12 18 24 30 36 42 48 54 60Mid time (h)

Rate

of u

rinar

y ex

cret

ion

(120583g

hr)


(c)

00

01

10

100

1000

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)


(d)






0

20

40

60

80

100

120

140

160

180

200

Baseline


1 5 10 100

Trol

ox eq

uiva

lent

(120583g

mL)

lowast

lowast

lowast

lowastlowast

lowast

Dagger

Dagger

50

(120583gmL)











0

20

40

60

80

100

120In

hibi

tion

()


1 10 250

lowastdagger

(120583gmL)

(a)

0

10

20

30

40

50

60

Inhi

bitio

n (

)

lowastlowast

dagger


1 10 250 (120583gmL)

(b)









0

10

20

30

40

50

60

70

80In

hibi

tion

()


Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)


Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10




4 Conclusions




Acknowledgments


References




























































01

1

10

100

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)


Con

cent

ratio

n (120583

gm

L)

(a)

1

10

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)


Con

cent

ratio

n (120583

gm

L)(b)









OralMean plusmn SEM







119890) was


0

20

40

60

80

100

120

140

160

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(a)

0

50

100

150

200

250

300

350

400

450

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(b)

10

100

1000

0 6 12 18 24 30 36 42 48 54 60Mid time (h)

Rate

of u

rinar

y ex

cret

ion

(120583g

hr)


(c)

00

01

10

100

1000

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)


(d)






0

20

40

60

80

100

120

140

160

180

200

Baseline


1 5 10 100

Trol

ox eq

uiva

lent

(120583g

mL)

lowast

lowast

lowast

lowastlowast

lowast

Dagger

Dagger

50

(120583gmL)











0

20

40

60

80

100

120In

hibi

tion

()


1 10 250

lowastdagger

(120583gmL)

(a)

0

10

20

30

40

50

60

Inhi

bitio

n (

)

lowastlowast

dagger


1 10 250 (120583gmL)

(b)









0

10

20

30

40

50

60

70

80In

hibi

tion

()


Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)


Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10




4 Conclusions




Acknowledgments


References




















































0

20

40

60

80

100

120

140

160

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(a)

0

50

100

150

200

250

300

350

400

450

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)

(b)

10

100

1000

0 6 12 18 24 30 36 42 48 54 60Mid time (h)

Rate

of u

rinar

y ex

cret

ion

(120583g

hr)


(c)

00

01

10

100

1000

0 6 12 18 24 30 36 42 48 54 60 66 72Time (h)

Tota

l am

ount

excr

eted

(120583g)


(d)






0

20

40

60

80

100

120

140

160

180

200

Baseline


1 5 10 100

Trol

ox eq

uiva

lent

(120583g

mL)

lowast

lowast

lowast

lowastlowast

lowast

Dagger

Dagger

50

(120583gmL)











0

20

40

60

80

100

120In

hibi

tion

()


1 10 250

lowastdagger

(120583gmL)

(a)

0

10

20

30

40

50

60

Inhi

bitio

n (

)

lowastlowast

dagger


1 10 250 (120583gmL)

(b)









0

10

20

30

40

50

60

70

80In

hibi

tion

()


Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)


Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10




4 Conclusions




Acknowledgments


References




















































0

20

40

60

80

100

120

140

160

180

200

Baseline


1 5 10 100

Trol

ox eq

uiva

lent

(120583g

mL)

lowast

lowast

lowast

lowastlowast

lowast

Dagger

Dagger

50

(120583gmL)











0

20

40

60

80

100

120In

hibi

tion

()


1 10 250

lowastdagger

(120583gmL)

(a)

0

10

20

30

40

50

60

Inhi

bitio

n (

)

lowastlowast

dagger


1 10 250 (120583gmL)

(b)









0

10

20

30

40

50

60

70

80In

hibi

tion

()


Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)


Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10




4 Conclusions




Acknowledgments


References




















































0

20

40

60

80

100

120In

hibi

tion

()


1 10 250

lowastdagger

(120583gmL)

(a)

0

10

20

30

40

50

60

Inhi

bitio

n (

)

lowastlowast

dagger


1 10 250 (120583gmL)

(b)









0

10

20

30

40

50

60

70

80In

hibi

tion

()


Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)


Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10




4 Conclusions




Acknowledgments


References




















































0

10

20

30

40

50

60

70

80In

hibi

tion

()


Resveratrol

1 10 100

lowast

lowast

50 200 (120583gmL)


Inhi

bitio

n (

)

lowast

lowast


Resveratrol

1 10 100 50 200 (120583gmL)

minus10

minus30

minus50

minus70

50

30

10




4 Conclusions




Acknowledgments


References



















































pharmacometrics of 3-methoxypterostilbene: a component of traditional chinese medicinal plants

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