Research Article Porcine Pancreatic Lipase Inhibitory ...Research Article Porcine Pancreatic Lipase Inhibitory Agent Isolated from Medicinal Herb and Inhibition Kinetics of Extracts

Research ArticlePorcine Pancreatic Lipase Inhibitory AgentIsolated from Medicinal Herb and Inhibition Kinetics ofExtracts from Eleusine indica (L) Gaertner

Siew Ling Ong Siau Hui Mah and How Yee Lai

School of Biosciences Taylorrsquos University No 1 Jalan Taylorrsquos 47500 Subang Jaya Malaysia

Correspondence should be addressed to How Yee Lai howyeelaitaylorsedumy

Received 4 July 2016 Revised 13 September 2016 Accepted 27 September 2016

Academic Editor Athar Ata

Copyright copy 2016 Siew Ling Ong et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Eleusine indica (Linnaeus) Gaertner is a traditional herb known to be depurative febrifuge and diuretic and has been reported withthe highest inhibitory activity against porcine pancreatic lipase (PPL) among thirty two plants screened in an earlier study Thisstudy aims to isolate and identify the active components that may possess high potential as an antiobesity agent Of the screenedsolvent fractions of E indica hexane fraction showed the highest inhibitory activity of 2701 plusmn 568 at 100120583gmL Bioactivity-guided isolation afforded three compounds from the hexane fraction of E indica namely 120573-sitosterol stigmasterol and luteinThestructures of these compounds were elucidated using spectral techniques Lutein showed an outstanding inhibitory activity againstPPL (5598plusmn104) with activity 60 higher than that of the reference drug OrlistatThe other compounds isolated and identifiedwere 120573-sitosterol (299plusmn080) and stigmasterol (268plusmn038)The enzyme kinetics of E indica crude methanolic extract on PPLshowed mixed inhibition mechanism

1 Introduction

Obesity is often defined as the excess accumulation ofbody fat resulting from a higher energy intake than energyexpenditure [1] In 2008 10 of men and 14 of women inthe world were obese compared with 5 of men and 8 ofwomen in 1980 [2] Rates of both overweight and obesity areprojected to increase in almost all countries with 15 billionpeople overweight in 2015 [2] Pancreatic lipase inhibition isone of the most widely studied mechanisms for antiobesitytreatment based on the principle that dietary fat will notbe directly absorbed by the intestine unless the fat has beensubjected to the action of pancreatic lipase [3 4]

Phytochemicals or bioactive compoundextract identi-fied from traditional medicinal plants had provided anexciting platform and opportunity for the development ofsafe and effective therapeutic drugs for the treatment of manymetabolic diseases [5] A review by Newman and Cragg(2007) [6] on the origin of drugs launched in the past 25 yearsshowed about half of the compounds that were successfulin clinical trials were derived from natural origin Despitemultiple research conducted in recent decades the potential

of antiobesity therapeutic drug of natural product origin isstill largely unexplored Previous screening study on thirtytwo plants reported strongest porcine pancreatic lipase (PPL)activity inE indica [7] and this has led to further investigationon this herb for potential antiobesity agent

E indica (Linnaeus) Gaertner (Poaceae) is an annualgrass native in the tropics and subtropical regions [8 9] It iscommonlywidespread asweed in rice field and is known to beresistant tomany herbicides (such as dinitroaniline) [10]Thisplant is commonly known as goosegrass wiregrass ldquorumputsambarirdquo or ldquorumput sambaurdquo in Malaysia [11] Its root istraditionally known to be depurative febrifuge diuretic andlaxative and thus is commonly used for treating hypertensioninfluenza oliguria and urine retention [8]The decoctions ofthe boiled whole plant are consumed for antihelminthic andfebrifuge treatment [12] The seed of E indica is sometimesused as famine food and in the treatment for liver complaints[13]

Several pharmacological properties on E indica havebeen reported including hepatoprotective effect [13] antiplas-modial and antidiabetic [14] antioxidant and antimicrobialactivity [8] anti-inflammatory [15] and cytotoxic effect

Hindawi Publishing CorporationJournal of PharmaceuticsVolume 2016 Article ID 8764274 9 pageshttpdxdoiorg10115520168764274

2 Journal of Pharmaceutics

towards several cancer cell lines [8 16] To date only onestudy reported the isolation of secondarymetabolites from Eindica where hexadecanoic acid and [[(2-aminoethoxy) hy-droxyphosphinyloxy]methyl]-12ndashethanediylester were iso-lated [17] Hence this paper is the first report on the kineticsof PPL enzyme inhibition by E indica and the bioactivity-guided isolation of a potent PPL inhibitory compound(lutein) from E indica

2 Materials and Methods

21 Plant Materials Extraction and Preparation of CrudeExtracts The whole plants of E indica (L) Gaertn werecollected from Persatuan Pengkaji Herba Tradisional NegeriSembilan (Pantai Negeri Sembilan coordinates 2∘4610158401310158401015840N101∘5910158404010158401015840E)This plant was authenticated byDr FadzureenaJamaludin from Forest Research Institute Malaysia (FRIM)the voucher specimen 00315 (collection date 11 February2015) is kept at the School of Biosciences Taylorrsquos University(Lakeside Campus)

The whole plant of E indica was cleaned from residualsoil freeze-dried and pulverised Analytical grade methanolwas added and the extracts were then filtered and pooled andthe solvent was evaporated off

22 Subextraction of the Main Extract The crude extract ofE indica was suspended in distilled water (1 10 wv) andsequentially extracted with solvents in increasing polarity(hexane chloroform ethyl acetate and butanol) three timeseach (1 1 vv) to obtain the respective solvent fractionsEach fraction was then assayed for porcine pancreatic lipaseinhibition activity

23 Porcine Pancreatic Lipase (PPL) Inhibition Assay Porcinepancreatic lipase (PPL) inhibitory assay was performed asdescribed by Bustanji et al (2011) [18] with minor modifica-tionThe enzyme solutions was prepared immediately beforeuse by suspending crude porcine pancreatic lipase powdertype II (Sigma EC 3113) in Tris-HCl buffer (50mM Tris150mM NaCl 1mM EDTA 10mM MOPS pH 76) to give aconcentration of 5mgmL (200 unitsmL) The solution wasthen centrifuged at 1500 rpm for 10 minutes and the clearsupernatantwas recoveredTheplant extract (100120583gmL)waspreincubated with 200 120583L of PPL solution for 5 minutes at37∘C before the addition of 5 120583L PNPB substrate solution(10mM in acetonitrile) The total reaction volume was madeto 1mL using the Tris-HCl buffer before measuring theabsorbance at 410 nm against blank using denatured enzymeThe denatured enzyme was prepared by boiling the enzymesolution for 5 minutes Orlistat was used as a reference drugThe extract was dissolved in DMSO at a final concentra-tion not exceeding 1 (vv) which will not affect enzymeactivity

The activity of the negative control was checked withand without the inhibitor The inhibitory activity (I) wascalculated according to the formula below [18]

119868 = (1 minus 119861 minus 119887119860 minus 119886) times 100 (1)

where A is the activity of the enzyme without inhibitor a isthe negative control without the inhibitor B is the activity ofthe enzyme with inhibitor and b is the negative control withinhibitor

24 Kinetic Study The inhibition mode of E indica meth-anolic crude extract on porcine pancreatic lipase (PPL)was assayed with increasing concentrations (20 40 60and 80 120583M) of synthetic substrate p-nitrophenyl butyrate(PNPB) in the presence and absence of two different con-centrations of the extracts (100 and 200 120583gmL) The modeof inhibition was determined by Lineweaver-Burk plot of thedata

25 Chromatography and Spectral Instrumentation

251 Thin Layer Chromatography (TLC) The TLC was per-formed on the TLC Silica Gel 60 coated with fluorescentindicator F254 Aluminium sheets (Merck) Samples werespotted and viewed under UV lamp at 254 nm and 365 nm

252 High Performance Liquid Chromatography (HPLC)The fingerprinting of the extracts was done on ShimadzuProminence Series coupled with photodiode array (PDA)detector SPD-M20A using either reversed-phase or normalphase settings

(i) Reversed-phase Chromolith HighResolution RP-18endcapped 100ndash46mm (Merck) the mobile phaseused was solvent A acetonitrile and solvent B waterwith a standard flow rate of 10mLmin and injectionvolume of 20 120583L of 10mgmL extract the gradient ofthe mobile phase was as follows 0 to 50 A (0ndash45min)

(ii) Normal phase Phenomenex Luna Silica column (250times 46mm 100 A 5 120583m) the mobile phase used con-sists of solvent A hexane and solvent B 2-propanolwith a flow rate of 10mLmin and injection volumeof 20120583L of 10mgmL extract the gradient programwas as follows 100 A (0ndash5min) 100 to 0 A (5ndash25min)

253 Infrared Spectroscopy (FT-IR) The IR spectra weremeasured by Perkin Elmer Spectrum 100 using potassiumbromide pellet method

254 Gas ChromatographyndashMass SpectrometryUsing ElectronImpact Ionisation (GC-EI-MS) Mass spectra were recordedwith EIMS using a Direct Injection Probe on a ShidmadzuGC-MS QP 5050A Spectrometer GC-MS was performed toidentify the purity and molecular weight of compounds

255 UV-Visible Spectra The UV-Vis spectra were recordedon aThermo Scientific Genesys 10 UV Scanning Spectropho-tometer

256 Melting Point Melting points were recorded using amelting point probe Electrothermal IA 9000 Series

Journal of Pharmaceutics 3

18 fractionsH-9-13-1 to H-9-13-18

H-9-13-9

H-4 amp H-5

Hexaneacetone

H-9 25 fractionsH-9-1 to H-9-25

H-9-13

13 fractionsH-1 to H-13

120573-sitosterol (2300mg)

Stigmasterol (2506mg)

) Lutein (108mg)

Eleusine indica MeOHextract (2597 g)

Hexane (836 g)

Dichloromethane(22 g)

Ethyl acetate(30 g)

Butanol(209 g)

Water(1387 g)

Silica gel 60 (0063ndash0200 mm)



SiliaSphere PC 50120583m 60Aring

(1)

(3

(2)

HexaneCH2Cl2EAMeOH

HexaneCH2Cl2EAMeOH

HexaneCH2Cl2EAMeOH

Figure 1 Schematic flow of the bioactivity-guided isolation on E indica extract

257 Nuclear Magnetic Resonance (NMR) The spectra wereobtained from JEOL ECX500 FTNMR Spectrometer systemDeuterated chloroform (CDCl3) was used as the solventto dissolve the test samples Tetramethylsilane (TMS) wasused as internal standard for both 1H (500MHz) and13C (125MHz) The chemical shifts from the spectra wererecorded in ppm and coupling constants were given in Hertz(Hz)

26 Bioactivity-Guided Extraction and Isolation The activefraction was subjected to gravitational column chromatog-raphy packed with suitable packing materials the schematicflow is as shown in Figure 1Theweight of the selected packingmaterials introduced into the column was at least ten timesthe weight of the sample extract Sample was separated usingthe solvent system as stated in Figure 1

The isolated pure compounds were then characterisedand elucidated employing several spectral methods as statedin Section 25120573-sitosterol (1) White crystal mp 1345ndash1376∘C UV

(Hexane) 120582max nm (log 120576) 210 (817) 230 (54) IR 120592max cmminus1

3431 2937 1468 1382 1056 EIMS mz (rel int) 414 [M+]

329 (20) 145 (25) 107 (30) 105 (27) 91 (20) 93 (21) 95 (28)81 (28) 69 (27) 57 (51) 55 (36) 43 (100) 41 (32) 1H NMR(500MHz CDCl3) 120575 529 (m 1H H-3) 346 (m 1H H-6)13C NMR (125MHz CDCl3) 120575 1407 (C-5) 1218 (C-6) 791(C-3 amp C-13) 554 (C-14 amp C-17) 506 (C-9) 487 (C-24) 421(C-4) 395 (C-12) 390 (C-1) 384 (C-10) 372 (C-20) 334 (C-22) 327 (C-2) 312 (C-7 amp C-8) 298 (C-25) 275 (C-16) 268(C-23) 263 (C-15) 256 (C-28) 215 (C-11) 196 (C-26) 184(C-19 amp C-27) 178 (C-21) 155 (C-29) 149 (C-18)

Stigmasterol (2) White crystal mp 1680ndash1700∘C UV(Hexane) 120582max nm (log 120576) 210 (839) 230 (147) IR 120592max cm

minus13426 2936 1645 1465 1384 1052 and 959 EIMS mz (relint) 412 [M+] (61) 255 (52) 159 (56) 145 (61) 95 (58) 81(78) 69 (65) 55 (100) 1H-NMR (500MHz CDCl3) 120575 534(d 1H J = 46Hz H-6) 516 (m 1H H-22) 503 (m 1H H-23) 353 (m 1H H-3) 100 (s 3H H-19) 0916 (d 1H J =575Hz H-21) 0829 (m 9HH-26 H-27 H-29) 0685 (s 3HH-18) 13C NMR (125MHz CDCl3) 120575 1408 (C-5) 1384 (C-20) 1294 (C-21) 1218 (C-6) 719 (C-3) 569 (C-14) 562 (C-17) 513 (C-22) 502 (C-9) 424 (C-4) 423 (C-13) 399 (C-12) 398 (C-18) 374 (C-1) 366 (C-10) 320 (C-2) 317 (C-7)292 (C-8) 290 (C-16) 283 (C-25) 255 (C-23) 244 (C-15)


Table 1 Kinetic analyses of PPL inhibition by methanolic crude extract of E indica

Velocity of enzyme activity in different concentration of substrate [S] (120583M) 119881max (120583Mminminus1) 119870m (120583M)20 40 60 80

Control 0035 plusmn 0003 0026 plusmn 0005 0022 plusmn 0005 0019 plusmn 0003 6579 2636100 120583gmL ofmethanolic Eindica

0043 plusmn 0003 0029 plusmn 0001 0024 plusmn 0002 0023 plusmn 0004 6329 3370

200 120583gmL ofmethanolic Eindica


Values are expressed as mean plusmn SD

212 (C-11) 199 (C-27) 195 (C-26) 191 (C-19) 189 (C-28)123 (C-29) 121 (C-24)

Lutein (3) orange crystal mp 1738ndash1749∘C UV (Ace-tone) 120582max nm (log 120576) 266 426 (shoulder) 448 (628) 476(564) IR 120592max cm

minus1 3427 2926 1718 1465 1376 1261 EIMSmz (rel int) 568 [M+] 145 (49) 119 (96) 105 (100) 93 (54)91 (70) 1HNMR (500MHz CDCl3) 120575 657 (m 4H H-11 H-15 H-111015840 H-121015840) 634 (d 2H J = 149Hz H-12) 623 (m 2HH-14 H-141015840) 610 (m 5H H-8 H-10 H-71015840 H-81015840) 554 (s 1HH-4) 539 (dd 1H J = 104 92Hz H-7) 424 (s 1H H-3)398 (m 1H H-31015840) 236 (m 2H H-6 H-4eq1015840) 201 (dd 1H J= 103 92Hz H-4ax1015840) 196 (s 9H H-20 H-191015840 H-201015840) 190(s 3H H-19) 181 (dd 1H J = 57 69Hz H-2eq) 173 (s 3HH-181015840) 162 (s 12H H-18) 144 (t 1H J = 126 115Hz H-21015840)134 (dd 1H J = 57 69Hz H-2ax) 106 (s 6H H-161015840 H-171015840) 0838 amp 0987 (s 6H H-16 H-17) 13C NMR (125MHzCDCl3) 120575 1386 (C-81015840) 1381 (C-8) 1378 (C-5 C-121015840) 1376(C-12 C-61015840) 1366 (C-131015840) 1365 (C-13) 1358 (C-91015840) 1352 (C-9) 1327 (C-14 C-141015840) 1314 (C-101015840) 1309 (C-10) 1302 (C-151015840) 1301 (C-15) 1288 (C-7) 1262 (C-51015840) 1257 (C-71015840) 1250(C-4) 1249 (C-111015840) 1246 (C-11) 660 (C-3) 652 (C-31015840) 551(C-6) 485 (C-21015840) 447 (C-2) 426 (C-41015840) 372 (C-11015840) 341 (C-1) 303 (C-171015840) 296 (C-17) 288 (C-161015840) 244 (C-16) 228 (C-18) 217 (C-181015840) 132 (C-19) 129 (C-191015840 C-20) 128 (C-20)

27 Statistical Analysis All results were expressed as mean plusmnstandard deviation Significance of difference from thecontrol was determined by Tukeyrsquos post-hoc test (one-wayANOVA) p value lt 005 using SPSS software (version 160)

3 Results and Discussion

31 Kinetic Analysis The inhibitionmode of PPL by E indicamethanolic extract at 100120583gmL and 200120583gmLwas analysedby double-reciprocal Lineweaver-Burk plot as shown inFigure 2 Kinetic parameters calculated from the doublereciprocal trend lines showed that both the maximal velocityof the PPL enzyme-substrate extract reaction (119881max) andthe affinity (119870m) were affected by the extract concentrationhence indicating a mixed mode inhibition The Michaelis-Menten parameters are tabulated in Table 1 where theMichaelis-Menten constant (119870m) of PPL with syntheticsubstrate PNPB was 2636 120583M and maximal velocity (119881max)was 6579 120583Mminminus1 The mixed mode inhibition exhibitedby PPL indicates that the formation of enzyme-substrate

0000

0010

0020

0030

0040

0050

0060

0000 0020 0040 0060

1[V

]

1[S]

Control

minus0060 minus0040 minus0020minus0010

100120583gmL200120583gmL

Figure 2 Lineweaver-Burk plot of E indica methanolic extractagainst PPL at two different concentrations (100 and 200120583gmL)Bar indicates the standard deviation

complex was possible with the inhibitor binding at a distinctsite from the active site resulting in reduction in the complexaffinity thus explaining the increase in 119870m Similar inhibi-tion mode was observed in Levisticum officinale methanolicextract and regular cocoa extract against porcine pancreaticlipase [19 20]

The methanolic crude extract of E indica was then parti-tioned via liquid-liquid fractionation to yield five (5) solventextracts that is hexane dichloromethane ethyl acetatebutanol and water All solvent extracts were then assayedfor their inhibitory activity against PPL Table 2 shows thatthe hexane extract from E indica possessed the highest PPLinhibitory activity that is 2701 plusmn 568 Although E indicadichloromethane extract recorded a comparable value of2557plusmn 326PPL inhibitory activity due to its low yield fromthe partition (118) this extract was not further tested

32 Fingerprinting of Solvent Extracts from E indica TheHPLC chromatograms of all E indica methanolic extractand solvent fractions are shown in Figure 3 Six major peakswere detected in elution profile of the crude methanolicextract of E indica (Figure 3(a)) No peak was detectedin hexane fraction (Figure 3(b)) due to the incompatibilityof reverse phase column with the nature of the fractionwhich was highly nonpolar The hexane fraction was later


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Figure 3 HPLC profiles of different fractions of E indica (a) crudemethanolic extract (b) hexane fraction (c) dichloromethane fraction (d)ethyl acetate fraction (e) butanol fraction and (f) aqueous fraction using Chromolith HighResolution RP-18 (Merck) HPLC column (reversephase) at 254 nm using the gradient mobile phase 0 to 50 acetonitrile 100 to 50 deionised water (0 to 45 minutes)

Table 2 Yield and PPL inhibitory activity of E indica crudemethanolic extract and solvent fractions

Yield (g100 gcrude extract)

PPL inhibition()

Crude methanolic extract mdash 2505 plusmn 358cHexane fraction 3416 plusmn 753 2701 plusmn 568cDichloromethane fraction 098 plusmn 047 2557 plusmn 326cEthyl acetate fraction 140 plusmn 047 1038 plusmn 273bButanol fraction 972 plusmn 236 523 plusmn 229aAqueous fraction 5278 plusmn 437 418 plusmn 191aOrlistat mdash 3449 plusmn 539dValues are expressed as mean plusmn SD 119899 = 3 values The concentration of thetested extract against PPLwas 100 120583gmL Statistically significant effects werecompared using one-way ANOVA (Tukeyrsquos post-hoc test) where differentletters in superscripts indicated significance at 119901 lt 005

optimised and run with Luna 5 120583m Silica (Phenomenex) anormal phase column coupled with normal phase mobilephase where six major peaks were detected as shown inFigure 4 A cluster of eight peaks were eluted in the secondhalf end of the chromatogram in dichloromethane fraction(Figure 3(c)) due to its more nonpolar nature Most of thepeaks from this cluster (peak 8 to peak 15) were visible in thechromatogramof crudemethanolic extract in Figure 3(a) butthe concentration of these compound present in the crudeextract was too low to be detected as the major peaks Figures3(d) and 3(e) which showed the elution profile of ethyl acetateand butanol extracts respectively recorded peak 4 to peak 7which corresponded to themajor peaks as detected in elutionprofile of crude methanolic extract where they represented

1 2 34 5

61 2 3

4 5

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0

(min)

(mAU

) 254nm 4nm (100)

300

275

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225

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10075

50

25

00

Figure 4 HPLC profiles of E indica hexane fraction using Luna5 120583mSilica (Phenomenex) HPLC column (normal phase) at 254 nmusing the gradientmobile phase 100hexane for 5minutes followedby 100 to 0 of hexane 0 to 100 2-propanol (5 to 25 minutes)

elution of semipolar compounds at the midrange of thechromatograms The most polar compounds were eluted atthe beginning of the spectrum (retention time less than 25minutes) in the aqueous fraction (Figure 3(f)) which werealso detected as one of the major peaks in the crude extract

High PPL inhibitory activity was detected in hexane(2701 plusmn 568) and dichloromethane (2557 plusmn 326) frac-tions from E indica (Table 2) Chromatogram of the hex-ane fraction (Figure 4) showed presence of six (6) majorcomponents (numbers 1 to 6) while chromatogram of thedichloromethane fraction (Figure 3(c)) showed eight majorcomponents (numbers 8 to 15) Attempts were made to iden-tify the components by running with ten standards namelyrutin quercetin neringenin caffeic acid chlorogenic acidcoumaric acid gallic acid esculin kaempferol andmyricetinHowever none of the peaksrsquo retention time matched thoseof the standards As such the components eluted from thehexane and dichloromethane fractions could not be identi-fied from the chromatograms Nevertheless these chromato-graphic fingerprints obtained from the methanolic crude


b ce f

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1

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus20

minus40

minus60

minus80

PPL inhibition percentage ()Weight (g)

Figure 5 The fraction yield and PPL inhibitory activity of H-1 toH-11 Bar indicates the standard deviation Statistically significanteffects were compared using one-way ANOVA (Tukeyrsquos post-hoctest) where different letters indicated significance at 119901 lt 005

and all solvent fractions may provide a simple guide forfuture identification and comparison of E indica L evenif the geographical location andor season of collection aredifferent

33 Bioactivity-Guided Extraction The hexane fraction of Eindica was further separated into eleven (11) fractions Fig-ure 5 shows H-9 (421 g) recorded the highest PPL inhibitoryactivity that is 3935 plusmn 024 which was obtained from theelution with 75 dichloromethane25 ethyl acetate Theremaining fractions obtained from this column recorded lessthan 20 of PPL inhibitory activity

Alongside with the separation of H-1 to H-11 two com-mon sterols were isolated from the fractions H-4 and H-5on elution with 90 dichloromethane10 ethyl acetate 120573-sitosterol (1) was isolated as a white flower-liked crystallinepowder while stigmasterol (2) was in white needle-likecrystals The detailed physical properties of 120573-sitosterol andstigmasterol will be discussed in the later part of this paperBoth compounds were found to possess very low PPL inhibi-tion activity that is 299 plusmn 080 (120573-sitosterol) of inhibitionat 100 120583gmL (242120583M) and 268 plusmn 038 (stigmasterol) ofinhibition at 100120583gmL (243120583M) respectively

Fraction H-9 (421 g) was subjected to further separa-tion due to its high PPL inhibitory activity and sufficientyield Figure 6 shows the fraction yield and PPL inhibitoryactivity of twenty five (25) subfractions of H-9 Highest PPLinhibitory activity was captured in H-9-13 (3215 plusmn 511)followed byH-9-14 (2926plusmn 108) where the yieldwas 032 gand 047 g respectively Subfractions of H-9-13 H-9-17 andH-9-21 recorded PPL inhibitory activity of more than 20while other subfractions were either promoted or showed lessthan 20 PPL inhibition

Both H-9-13 and H-9-14 were selected for further iso-lation due to their higher yield (3236mg and 4653mgresp) As both H-9-13 and H-9-14 showed comparable PPLinhibition as well as similar TLC spotting profile (datanot shown) both subfractions were combined for further

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()

minus20

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minus05

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minus25

minus30


Figure 6 The fraction yield and PPL inhibitory activity of H-9-1 toH-9-25 Bar indicates the standard deviation Statistically significanteffects were compared using one-way ANOVA (Tukeyrsquos post-hoctest) where different letters indicated significance at 119901 lt 005

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minus20

02010minus01minus02minus03minus04minus05minus06minus07minus08minus09minus1

Figure 7 The fraction yield and PPL inhibitory activity of H-9-13-1 to H-9-13-18 Bar indicates the standard deviation Statisticallysignificant effects were compared using one-way ANOVA (Tukeyrsquospost-hoc test) where different letters indicated significance at 119901 lt005

isolation and the results of the PPL inhibitory activity of theresultant isolated subfractions are shown in Figure 7

Highest PPL inhibitory activity was shown by H-9-13-9(1088mg) that is 5208plusmn 293whichwas about 18higherthan that recorded by Orlistat (3449 plusmn 539) Fraction H-9-13-10 recorded the second highest PPL inhibition 3496 plusmn487 which was similar to that of Orlistat

The TLC profile from H-9-13-9 showed one prominentyellow spot where further isolation was then done resultingin the isolation of lutein (3) (108mg) from this fractionThis isolated pure compound (lutein) was subjected to fewanalysis includingmelting point analysis UVGC-MS (for the


1

1

2

2

3

3

45

5

4

6

6

7

7

8

8

10

10

12

3

45

67

810

9

9

919

19

1911

11

1112

12

1218

18

18

13

13

13

14

14

14

17

17

1720

20

20

16

16

16

15

15

15

27 27

23 2324 2425 2526 26

28 2829 29

2221 21

22

HO HO

OH

HO

1 2

320998400

1199840013998400

14998400 8998400

17998400

1099840012998400

1998400 299840039984005998400

4998400

6998400

79984009998400

15998400

16998400

18998400

19998400

Figure 8 Chemical structures of 120573-sitosterol (1) stigmasterol (2) and lutein (3)

determination of molecular weight) FT-IR (determinationof functional group) and NMR (structural elucidation)The characterised lutein was found to possess very strongPPL inhibitory activity that is 5598 plusmn 104 at 100 120583gmL(176 120583M) which was 215 higher than that recorded inOrlistat (3449 plusmn 539) To date this is the first study toreveal the potential of lutein as a strong PPL inhibitory agent

34 Isolation of Compounds from E indica The structuresof the isolated compounds were shown in Figure 8 Thewhite crystals of 120573-sitosterol (1) had melting point of 1345ndash1376∘C [lit 134-135∘C [21]] and also showed IR absorptions at3431 (O-H stretching) 2937 (aliphatic C-H stretching) 1468(C-H2 bending) 1382 (C-H3 bend) and 1056 (=CH bend)EIMS has confirmed the molecular formula C29H50O withthe molecular ion peak atmz 414 Similarly stigmasterol (2)was isolated as white crystals with melting point of 1680ndash1700∘C (lit 170∘C [22]) and its mass spectral data suggestedthemolecular formula as C29H48OThe IR absorption of stig-masterol was similar to that of 120573-sitosterol with an additionalabsorption band at 1645 cmminus1indicative of an olefin groupBoth 120573-sitosterol and stigmasterol recorded UV absorptionmaxima at 210 nm Sterols normally absorb in the range of190ndash210 nm due to the transitions of 120587 to 120588lowast [23]

The 1H NMR spectrum of 120573-sitosterol (1) had detectedmultiplet of signals at 120575 321 of H-3 which belonged to thehydroxymethyl group at C-3 and another pair of multipletresonated at 120575 461 which is assigned to the olefinic protonat H-6 The 13C NMR spectrum showed a total of 29 carbonsignals were captured which supported the molecular for-mula obtained in EIMSThese signals consist of a quaternarymethine at C-5 (120575 1453) an olefinic methine at C-6 (120575 1218)as well as several methyl group at C-18 C-19 C-21 C-26 C-27 and C-29 (120575 149 184 178 196 184 and 155 resp) Andmethylene group was located at 120575 390 (C-1) 120575 327 (C-2) 120575421 (C-4) 120575 312 (C-7) 120575 215 (C-11) 120575 395 (C-12) 120575 263(C-15) 120575 275 (C-16) 120575 334 (C-22) 120575 268 (C-23) and 120575

256 (C-28) Both proton and carbon signals obtained werematched and in agreement with literature [24]

In the 1H NMR spectrum of stigmasterol (2) therewas presence of methyl singlets at 120575 069 (H-18) and 120575100 (H-19) multiplets at 120575 516 (H-22) and 120575 503 (H-23) signified olefinic protons which were not found in 120573-sitosterol Similarly 13C NMR had revealed 29 carbon peakswhere olefinic carbons were identified at 120575 1408 (C-5) 1205751218 (C-6) 120575 513 (C-22) and 120575 255 (C-23) methyl carbonswere detected at 120575 398 (C-18) 120575 191 (C-19) 120575 1294 (C-21)120575 195 (C-26) 120575 199 (C-27) and 120575 123 (C-29) methylenecarbons were detected at 120575 374 (C-1) 120575 320 (C-2) 120575 424(C-4) 120575 317 (C-7) 120575 212 (C-11) 120575 399 (C-12) 120575 244 (C-15) 120575 290 (C-16) and 120575 189 (C-28) Both spectra data werematched and in agreement with literature [25]

Lutein (3) was isolated as orange crystals with a meltingpoint of 1738ndash1749∘C and maxima wavelengths recorded at426 448 and 476 nm which were in agreement with the dataof the literature [26 27] Mass spectrum of lutein given themolecular ion at mz 568 followed by fragments in mz 550is attributed to the loss of a hydroxy group 1H NMR spectradata revealed hydroxyl protons resonated at 120575 424 (H-3) and120575 398 (H-31015840) olefinic protons at 120575 424 (H-3) 120575 554 (H-4)120575 539 (H-7) 120575 610 (H-8) 120575 610 (H-10) 120575 657 (H-11) 120575 634(H-12) 120575 623 (H-14) 120575 657 (H-15) 120575 610 (H-71015840 and H-81015840)120575 657 (H-111015840 andH-121015840) and 120575 623 (H-141015840) allylic protons at120575 162 (H-18) 120575 190 (H-19) 120575 196 (H-20) 120575 173 (H-181015840) and120575 196 (H-191015840 and H-201015840) methyl singlets at 120575 0838 120575 0987(H-16 andH-17) and 120575 106 (H-161015840 andH-171015840)The 13CNMRspectra data revealed olefinic carbons resonated at 120575 1250 (C-4) 120575 1378 (C-5) 120575 1288 (C-7) 120575 1381 (C-8) 120575 1352 (C-8)120575 1352 (C-9) 120575 1309 (C-10) 120575 1246 (C-11) 120575 1376 (C-12)120575 1365 (C-13) 120575 1327 (C-14) 120575 1301 (C-15) 120575 1262 (C-51015840)120575 1376 (C-61015840) 120575 1257 (C-71015840) 120575 1386 (C-81015840) 120575 1358 (C-91015840)120575 1314 (C-101015840) 120575 1249 (C-111015840) 120575 1378 (C-121015840) 120575 1366 (C-131015840) 120575 1327 (C-141015840) and 120575 1302 (C-151015840) hydroxyl attachedcarbon resonated at 120575 660 (C-3) and 120575 652 (C-31015840) methyl


carbons were detected at 120575 244 (C-16) 120575 296 (C-17) 120575 228(C-18) 120575 132 (C-19) 120575 128 (C-20) 120575 288 (C-161015840) 120575 303 (C-171015840) 120575 217 (C-181015840) and 120575 129 (C-191015840ampC-201015840) andmethylenecarbons resonated at 120575 447 (C-2) 120575 485 (C-21015840) and 120575 426(C-41015840) Both 1H and 13CNMR values were in agreement withpublished values [26]

Common plant sterols like 120573-sitosterol and stigmasterolare important in cellular and developmental mechanismsin plants [28] In therapeutic treatment via dietary optionsfood products supplemented with plant sterols helped in thereduction of plasma cholesterol and the risk of atherosclerosis[29] However the cholesterol lowering effect may not beattributed to the inhibition via pancreatic lipase since thePPL inhibition activity of both phytosterols obtained wasless than 2 Weak PPL inhibition activity of 120573-sitosteroland stigmasterol isolated from Alpinia zerumbet had alsobeen reported by Chompoo et al (2012) [30] with IC50value of 9999 plusmn 186120583gmL and 12505 plusmn 476 120583gmLrespectively in comparison with the inhibition shown bycurcumin (IC50 = 492 plusmn 021120583gmL) and quercetin (IC50= 1860 plusmn 086120583gmL) which were used as positive controlsin their study In our study 120573-sitosterol and stigmasterolwere recorded with weak PPL inhibitory activity of only30 plusmn 08 and 27 plusmn 04 at 100 120583gmL respectively (ie242120583M and 243 120583M) in contrast with that obtained fromOrlistat (345 plusmn 54 at 100 120583gmL) which were compara-tively lower than that recorded in literature (ie 50 PPLinhibition at 100120583gmL) [30] This may be due to differentexperimental settings where concentrations of both subtrateand enzyme concentrations used were different in bothstudies

Lutein a member under the xanthophyll family is theprincipal carotenoid in greens leaves and yellow flowersThe bioavailability of xanthophyll is highly dependent on thematrix due to its hydrophobicity of the long carbon skeletonsand thus dietary lipids are required to facilitate the dispersionof lutein where similar matrix was adapted by pancreaticlipase [31] Another study demonstrated xanthophyll esterhydrolysed by cholesteryl esterase but not triacylglycerollipase (pancreatic lipase) and thus ruled out the possibilityof xanthophyll as a competitive substrate [32 33] To datethe mechanism and the inhibitory effect of xanthophyll onpancreatic lipase are still largely unknown based on theliterature findings thatmay lead to postulation that luteinmaybe a noncompetitive inhibitor by binding on the allostericsite of pancreatic lipase Further study is thus required tofully understand the kinetic interactions between lutein andpancreatic lipase

4 Conclusions

Bioactivity-guided isolation on hexane extract of E indicahas led to isolation and elucidation of potent PPL inhibitoryagent lutein (3) with strong inhibitory activity of 5598 plusmn104 alongside with the isolation of two other commonsterols (1) 120573-sitosterol and (2) stigmasterol To date this isthe first report on the pancreatic lipase inhibitory activity oflutein

Abbreviations

EI Eleusine indicaPPL Porcine pancreatic lipaseHPLC High performance liquid chromatographyIR InfraredNMR Nuclear magnetic resonance

Competing Interests

Theauthors wish to confirm that there is no known conflict ofinterests associated with this publication and there has beenno significant financial support for this work that could haveinfluenced its outcome

Acknowledgments

The authors wish to thank the Taylorrsquos Research GrantScheme TRGS22011SOBS012 and Postgraduate ResearchScholarship Programme for financial supports

References

[1] N N Finer ldquoPharmacotherapy of obesityrdquo Best Practice ampResearch Clinical Endocrinology amp Metabolism vol 16 no 4pp 717ndash742 2002

[2] World Health Organization (WHO) Obesity and Overweight2015 httpwwwwhointmediacentrefactsheetsfs311en

[3] R B Birari andK K Bhutani ldquoPancreatic lipase inhibitors fromnatural sources unexplored potentialrdquo Drug Discovery Todayvol 12 no 19-20 pp 879ndash889 2007

[4] J W Yun ldquoPossible anti-obesity therapeutics from naturemdashareviewrdquo Phytochemistry vol 71 no 14-15 pp 1625ndash1641 2010

[5] R Birari V Javia and K K Bhutani ldquoAntiobesity and lipid low-ering effects ofMurraya koenigii (L) spreng leaves extracts andmahanimbine on high fat diet induced obese ratsrdquo Fitoterapiavol 81 no 8 pp 1129ndash1133 2010

[6] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007

[7] S-L Ong S Paneerchelvan H-Y Lai and N K Rao ldquoIn vitrolipase inhibitory effect of thirty two selected plants inMalaysiardquoAsian Journal of Pharmaceutical and Clinical Research vol 7 no2 pp 19ndash24 2014

[8] A S Al-Zubairi A B Abdul S I Abdelwahab C Y Peng SMohan and M M Elhassan ldquoEleucine indica possesses antiox-idant antibacterial and cytotoxic propertiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID965370 6 pages 2011

[9] M H Ricardus and T S Myrna ldquoTwo new records fromLebanon Chamaesyce nutans (Lag) Small (Eiphorbiaceae) andEleusine indica (L) Gaertner (Poaceae)rdquo Turkish Journal ofBotany vol 31 pp 341ndash343 2007

[10] A I Yemets L A Klimkina L V Tarassenko and Y B BlumeldquoEfficient callus formation and plant regeneration of goosegrass[Eleusine indica (L) Gaertn]rdquo Plant Cell Reports vol 21 no 6pp 503ndash510 2003

[11] T K Lim ldquoEleusine indicardquo in Edible Medicinal and Non-Medicinal Plants pp 228ndash236 Springer Canberra Australia2016


[12] K Julius ldquoA preliminary survey of traditional medicinal plantsin the West Coast and Interior of Sabahrdquo Journal of TropicalForest Science vol 10 no 2 pp 271ndash274 1997

[13] M Iqbal and C Gnanaraj ldquoEleusine indica L possessesantioxidant activity and precludes carbon tetrachloride (CCl4)-mediated oxidative hepatic damage in ratsrdquo EnvironmentalHealth and Preventive Medicine vol 17 no 4 pp 307ndash315 2012

[14] J E Okokon C S Odomena I Effiong J Obot and JA Udobang ldquoAntiplasmodial and antidiabetic activities ofEleusine indicardquo International Journal of Drug Development andResearch vol 2 no 3 pp 493ndash500 2010

[15] G O De Melo M F Muzitano A Legora-Machado et al ldquoC-glycosylflavones from the aerial parts of Eleusine indica inhibitLPS-inducedmouse lung inflammationrdquo PlantaMedica vol 71no 4 pp 362ndash363 2005

[16] P Hansakul C Ngamkitidechakul K Ingkaninan S Sireer-atawong and W Panunto ldquoApoptotic induction activity ofDactyloctenium aegyptium (L) PB and Eleusine indica (L)Gaerth Extracts on human lung and cervical cancer cell linesrdquoSongklanakarin Journal of Science and Technology vol 31 no 3pp 273ndash279 2009

[17] I O Alaekwe V I E Ajiwe A C Ajiwe and G N AningoldquoPhytochemical and anti-microbial screening of the aerial partsof Eleusine indicardquo International Journal of Pure amp AppliedBioscience vol 3 no 1 pp 257ndash264 2015

[18] Y Bustanji M Mohammad M Hudaib K Tawaha and IM Al-Masri ldquoScreening of some medicinal plants for theirpancreatic lipase inhibitory potentialrdquo Jordan Journal of Phar-maceutical Sciences vol 4 no 2 pp 81ndash88 2011

[19] A Gholamhoseinian B Shahouzebi and F Sharifi-FarldquoInhibitory effect of some plant extracts on pancreatic lipaserdquoInternational Journal of Pharmacology vol 6 no 1 pp 18ndash242010

[20] Y Gu W J Hurst D A Stuart and J D Lambert ldquoInhibitionof key digestive enzymes by cocoa extracts and procyanidinsrdquoJournal of Agricultural and Food Chemistry vol 59 no 10 pp5305ndash5311 2011

[21] V S Chaturvedula and I Prakash ldquoIsolation of Stigmasteroland 120573-Sitosterol from the dichloromethane extract of Rubussuavissimusrdquo International Current Pharmaceutical Journal vol1 no 9 pp 239ndash242 2012

[22] D R LideCRCHandbook of Chemistry and Physics CRCPressTaylor amp Francis Boca Raton Fla USA 88th edition 2007

[23] P Acuna-Johnson and A C Oehlschelager ldquoIdentificationof sterols and biologically significant steroids by ultravioletand infrared spectroscopyrdquo in Analysis of Sterols and OtherBiologically Significant Steroids pp 267ndash284 Academic PressSan Diego Calif USA 1989

[24] F Jamaluddin S Mohamed and Md Nordin Lajis ldquoHypogly-caemic effect of Parkia speciosa seeds due to the synergisticaction of 120573-sitosterol and stigmasterolrdquo Food Chemistry vol 49no 4 pp 339ndash345 1994

[25] D Xie L Wang H Ye and G Li ldquoIsolation and production ofartemisinin and stigmasterol in hairy root cultures ofArtemisiaannuardquo Plant Cell Tissue and Organ Culture vol 63 no 2 pp161ndash166 2000

[26] M A El-Raey G E Ibrahim and O A Eldahshan ldquoLycopeneand Lutein a review for their chemistry and medicinal usesrdquoJournal of Pharmacognosy and Phytochemistry vol 2 no 1 pp245ndash254 2013

[27] A Z Mercadante A Steck and H Pfander ldquoCarotenoids fromguava (Psidium guajava L) isolation and structure elucidationrdquo

Journal of Agricultural and Food Chemistry vol 47 no 1 pp145ndash151 1999

[28] Y Sheng and X-B Chen ldquoIsolation and identification of anisomer of 120573-sitosterol by HPLC and GC-MSrdquo Health vol 1 no3 pp 203ndash206 2009

[29] L Calpe-Berdiel J C Escola-Gil and F Blanco-Vaca ldquoNewinsights into the molecular actions of plant sterols and stanolsin cholesterol metabolismrdquo Atherosclerosis vol 203 no 1 pp18ndash31 2009

[30] J Chompoo A Upadhyay S Gima M Fukuta and S TawataldquoAntiatherogenic properties of acetone extract ofAlpinia zerum-bet seedsrdquoMolecules vol 17 no 6 pp 6237ndash6248 2012

[31] E Kotake-Nara and A Nagao ldquoAbsorption and metabolism ofxanthophyllsrdquoMarine Drugs vol 9 no 6 pp 1024ndash1037 2011

[32] D E Breithaupt A Alpmann and F Carriere ldquoXanthophyllesters are hydrolysed in the presence of recombinant humanpancreatic lipaserdquo Food Chemistry vol 103 no 2 pp 651ndash6562007

[33] C Chitchumroonchokchai and M L Failla ldquoHydrolysis ofzeaxanthin esters by carboxyl ester lipase during digestionfacilitates micellarization and uptake of the xanthophyll byCaco-2 human intestinal cellsrdquoThe Journal of Nutrition vol 136no 3 pp 588ndash594 2006

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


towards several cancer cell lines [8 16] To date only onestudy reported the isolation of secondarymetabolites from Eindica where hexadecanoic acid and [[(2-aminoethoxy) hy-droxyphosphinyloxy]methyl]-12ndashethanediylester were iso-lated [17] Hence this paper is the first report on the kineticsof PPL enzyme inhibition by E indica and the bioactivity-guided isolation of a potent PPL inhibitory compound(lutein) from E indica

2 Materials and Methods

21 Plant Materials Extraction and Preparation of CrudeExtracts The whole plants of E indica (L) Gaertn werecollected from Persatuan Pengkaji Herba Tradisional NegeriSembilan (Pantai Negeri Sembilan coordinates 2∘4610158401310158401015840N101∘5910158404010158401015840E)This plant was authenticated byDr FadzureenaJamaludin from Forest Research Institute Malaysia (FRIM)the voucher specimen 00315 (collection date 11 February2015) is kept at the School of Biosciences Taylorrsquos University(Lakeside Campus)

The whole plant of E indica was cleaned from residualsoil freeze-dried and pulverised Analytical grade methanolwas added and the extracts were then filtered and pooled andthe solvent was evaporated off

22 Subextraction of the Main Extract The crude extract ofE indica was suspended in distilled water (1 10 wv) andsequentially extracted with solvents in increasing polarity(hexane chloroform ethyl acetate and butanol) three timeseach (1 1 vv) to obtain the respective solvent fractionsEach fraction was then assayed for porcine pancreatic lipaseinhibition activity

23 Porcine Pancreatic Lipase (PPL) Inhibition Assay Porcinepancreatic lipase (PPL) inhibitory assay was performed asdescribed by Bustanji et al (2011) [18] with minor modifica-tionThe enzyme solutions was prepared immediately beforeuse by suspending crude porcine pancreatic lipase powdertype II (Sigma EC 3113) in Tris-HCl buffer (50mM Tris150mM NaCl 1mM EDTA 10mM MOPS pH 76) to give aconcentration of 5mgmL (200 unitsmL) The solution wasthen centrifuged at 1500 rpm for 10 minutes and the clearsupernatantwas recoveredTheplant extract (100120583gmL)waspreincubated with 200 120583L of PPL solution for 5 minutes at37∘C before the addition of 5 120583L PNPB substrate solution(10mM in acetonitrile) The total reaction volume was madeto 1mL using the Tris-HCl buffer before measuring theabsorbance at 410 nm against blank using denatured enzymeThe denatured enzyme was prepared by boiling the enzymesolution for 5 minutes Orlistat was used as a reference drugThe extract was dissolved in DMSO at a final concentra-tion not exceeding 1 (vv) which will not affect enzymeactivity

The activity of the negative control was checked withand without the inhibitor The inhibitory activity (I) wascalculated according to the formula below [18]

119868 = (1 minus 119861 minus 119887119860 minus 119886) times 100 (1)

where A is the activity of the enzyme without inhibitor a isthe negative control without the inhibitor B is the activity ofthe enzyme with inhibitor and b is the negative control withinhibitor

24 Kinetic Study The inhibition mode of E indica meth-anolic crude extract on porcine pancreatic lipase (PPL)was assayed with increasing concentrations (20 40 60and 80 120583M) of synthetic substrate p-nitrophenyl butyrate(PNPB) in the presence and absence of two different con-centrations of the extracts (100 and 200 120583gmL) The modeof inhibition was determined by Lineweaver-Burk plot of thedata

25 Chromatography and Spectral Instrumentation

251 Thin Layer Chromatography (TLC) The TLC was per-formed on the TLC Silica Gel 60 coated with fluorescentindicator F254 Aluminium sheets (Merck) Samples werespotted and viewed under UV lamp at 254 nm and 365 nm

252 High Performance Liquid Chromatography (HPLC)The fingerprinting of the extracts was done on ShimadzuProminence Series coupled with photodiode array (PDA)detector SPD-M20A using either reversed-phase or normalphase settings

(i) Reversed-phase Chromolith HighResolution RP-18endcapped 100ndash46mm (Merck) the mobile phaseused was solvent A acetonitrile and solvent B waterwith a standard flow rate of 10mLmin and injectionvolume of 20 120583L of 10mgmL extract the gradient ofthe mobile phase was as follows 0 to 50 A (0ndash45min)

(ii) Normal phase Phenomenex Luna Silica column (250times 46mm 100 A 5 120583m) the mobile phase used con-sists of solvent A hexane and solvent B 2-propanolwith a flow rate of 10mLmin and injection volumeof 20120583L of 10mgmL extract the gradient programwas as follows 100 A (0ndash5min) 100 to 0 A (5ndash25min)

253 Infrared Spectroscopy (FT-IR) The IR spectra weremeasured by Perkin Elmer Spectrum 100 using potassiumbromide pellet method

254 Gas ChromatographyndashMass SpectrometryUsing ElectronImpact Ionisation (GC-EI-MS) Mass spectra were recordedwith EIMS using a Direct Injection Probe on a ShidmadzuGC-MS QP 5050A Spectrometer GC-MS was performed toidentify the purity and molecular weight of compounds

255 UV-Visible Spectra The UV-Vis spectra were recordedon aThermo Scientific Genesys 10 UV Scanning Spectropho-tometer

256 Melting Point Melting points were recorded using amelting point probe Electrothermal IA 9000 Series


18 fractionsH-9-13-1 to H-9-13-18

H-9-13-9

H-4 amp H-5

Hexaneacetone


H-9-13




) Lutein (108mg)


Hexane (836 g)


Ethyl acetate(30 g)

Butanol(209 g)

Water(1387 g)





(1)

(3

(2)

HexaneCH2Cl2EAMeOH

HexaneCH2Cl2EAMeOH

HexaneCH2Cl2EAMeOH






3431 2937 1468 1382 1056 EIMS mz (rel int) 414 [M+]












212 (C-11) 199 (C-27) 195 (C-26) 191 (C-19) 189 (C-28)123 (C-29) 121 (C-24)






0000

0010

0020

0030

0040

0050

0060

0000 0020 0040 0060

1[V

]

1[S]

Control


100120583gmL200120583gmL






35

6 7

42

1

100500(m

AU)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

254nm 4nm (100)

(a)

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

200100

0

254nm 4nm (100)

(b)

8 9 1011

12

13 14

15

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

500250

0

254nm 4nm (100)

(c)

6

7

5

4

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

20001000

0

254nm 4nm (100)

(d)

3

45

6 7

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

(mAU

) 500250

0

254nm 4nm (100)

(e)

11

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

0255075

(min)

(mAU

) 254nm 4nm (100)

(f)




PPL inhibition()



1 2 34 5

61 2 3

4 5

6

300200100

0

(min)

(mAU

) 254nm 4nm (100)

300

275

250

225

200

175

150

125

10075

50

25

00





b ce f

cdf

a

cd

hg

d

0

20

40

0

20

40

60

80

100

H-1

H-2

H-3

H-4

H-5

H-6

H-7

H-8

H-9

H-1

0

H-1

1

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus20

minus40

minus60

minus80








cdef

cdef

defg

ij

abcd

hijfghi

aabcd

a

ab

ghi

l

klkl jk kl

defghdefghi

defg

jk

abc

efghi efghi

bcde

00

05

10

0

10

20

30

40

50

60

H-9

-1H

-9-2

H-9

-3H

-9-4

H-9

-5H

-9-6

H-9

-7H

-9-8

H-9

-9H

-9-1

0H

-9-1

1H

-9-1

2H

-9-1

3H

-9-1

4H

-9-1

5H

-9-1

6H

-9-1

7H

-9-1

8H

-9-1

9H

-9-2

0H

-9-2

1H

-9-2

2H

-9-2

3H

-9-2

4H

-9-2

5 Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus10

minus30

minus05

minus10

minus15

minus20

minus25

minus30



b b bcd

def fg gh h

j

i

defg

debc b

a

bd

0

10

20

30

40

50

60

70

80

H-9

-13-

1H

-9-1

3-2

H-9

-13-

3H

-9-1

3-4

H-9

-13-

5H

-9-1

3-6

H-9

-13-

7H

-9-1

3-8

H-9

-13-

9H

-9-1

3-10

H-9

-13-

11H

-9-1

3-12

H-9

-13-

13H

-9-1

3-14

H-9

-13-

15H

-9-1

3-16

H-9

-13-

17H

-9-1

3-18

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()


minus10

minus20







1

1

2

2

3

3

45

5

4

6

6

7

7

8

8

10

10

12

3

45

67

810

9

9

919

19

1911

11

1112

12

1218

18

18

13

13

13

14

14

14

17

17

1720

20

20

16

16

16

15

15

15

27 27

23 2324 2425 2526 26

28 2829 29

2221 21

22

HO HO

OH

HO

1 2

320998400

1199840013998400

14998400 8998400

17998400

1099840012998400

1998400 299840039984005998400

4998400

6998400

79984009998400

15998400

16998400

18998400

19998400












4 Conclusions


Abbreviations


Competing Interests


Acknowledgments


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


18 fractionsH-9-13-1 to H-9-13-18

H-9-13-9

H-4 amp H-5

Hexaneacetone


H-9-13




) Lutein (108mg)


Hexane (836 g)


Ethyl acetate(30 g)

Butanol(209 g)

Water(1387 g)





(1)

(3

(2)

HexaneCH2Cl2EAMeOH

HexaneCH2Cl2EAMeOH

HexaneCH2Cl2EAMeOH






3431 2937 1468 1382 1056 EIMS mz (rel int) 414 [M+]












212 (C-11) 199 (C-27) 195 (C-26) 191 (C-19) 189 (C-28)123 (C-29) 121 (C-24)






0000

0010

0020

0030

0040

0050

0060

0000 0020 0040 0060

1[V

]

1[S]

Control


100120583gmL200120583gmL






35

6 7

42

1

100500(m

AU)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

254nm 4nm (100)

(a)

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

200100

0

254nm 4nm (100)

(b)

8 9 1011

12

13 14

15

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

500250

0

254nm 4nm (100)

(c)

6

7

5

4

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

20001000

0

254nm 4nm (100)

(d)

3

45

6 7

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

(mAU

) 500250

0

254nm 4nm (100)

(e)

11

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

0255075

(min)

(mAU

) 254nm 4nm (100)

(f)




PPL inhibition()



1 2 34 5

61 2 3

4 5

6

300200100

0

(min)

(mAU

) 254nm 4nm (100)

300

275

250

225

200

175

150

125

10075

50

25

00





b ce f

cdf

a

cd

hg

d

0

20

40

0

20

40

60

80

100

H-1

H-2

H-3

H-4

H-5

H-6

H-7

H-8

H-9

H-1

0

H-1

1

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus20

minus40

minus60

minus80








cdef

cdef

defg

ij

abcd

hijfghi

aabcd

a

ab

ghi

l

klkl jk kl

defghdefghi

defg

jk

abc

efghi efghi

bcde

00

05

10

0

10

20

30

40

50

60

H-9

-1H

-9-2

H-9

-3H

-9-4

H-9

-5H

-9-6

H-9

-7H

-9-8

H-9

-9H

-9-1

0H

-9-1

1H

-9-1

2H

-9-1

3H

-9-1

4H

-9-1

5H

-9-1

6H

-9-1

7H

-9-1

8H

-9-1

9H

-9-2

0H

-9-2

1H

-9-2

2H

-9-2

3H

-9-2

4H

-9-2

5 Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus10

minus30

minus05

minus10

minus15

minus20

minus25

minus30



b b bcd

def fg gh h

j

i

defg

debc b

a

bd

0

10

20

30

40

50

60

70

80

H-9

-13-

1H

-9-1

3-2

H-9

-13-

3H

-9-1

3-4

H-9

-13-

5H

-9-1

3-6

H-9

-13-

7H

-9-1

3-8

H-9

-13-

9H

-9-1

3-10

H-9

-13-

11H

-9-1

3-12

H-9

-13-

13H

-9-1

3-14

H-9

-13-

15H

-9-1

3-16

H-9

-13-

17H

-9-1

3-18

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()


minus10

minus20







1

1

2

2

3

3

45

5

4

6

6

7

7

8

8

10

10

12

3

45

67

810

9

9

919

19

1911

11

1112

12

1218

18

18

13

13

13

14

14

14

17

17

1720

20

20

16

16

16

15

15

15

27 27

23 2324 2425 2526 26

28 2829 29

2221 21

22

HO HO

OH

HO

1 2

320998400

1199840013998400

14998400 8998400

17998400

1099840012998400

1998400 299840039984005998400

4998400

6998400

79984009998400

15998400

16998400

18998400

19998400












4 Conclusions


Abbreviations


Competing Interests


Acknowledgments


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of









212 (C-11) 199 (C-27) 195 (C-26) 191 (C-19) 189 (C-28)123 (C-29) 121 (C-24)






0000

0010

0020

0030

0040

0050

0060

0000 0020 0040 0060

1[V

]

1[S]

Control


100120583gmL200120583gmL






35

6 7

42

1

100500(m

AU)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

254nm 4nm (100)

(a)

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

200100

0

254nm 4nm (100)

(b)

8 9 1011

12

13 14

15

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

500250

0

254nm 4nm (100)

(c)

6

7

5

4

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

20001000

0

254nm 4nm (100)

(d)

3

45

6 7

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

(mAU

) 500250

0

254nm 4nm (100)

(e)

11

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

0255075

(min)

(mAU

) 254nm 4nm (100)

(f)




PPL inhibition()



1 2 34 5

61 2 3

4 5

6

300200100

0

(min)

(mAU

) 254nm 4nm (100)

300

275

250

225

200

175

150

125

10075

50

25

00





b ce f

cdf

a

cd

hg

d

0

20

40

0

20

40

60

80

100

H-1

H-2

H-3

H-4

H-5

H-6

H-7

H-8

H-9

H-1

0

H-1

1

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus20

minus40

minus60

minus80








cdef

cdef

defg

ij

abcd

hijfghi

aabcd

a

ab

ghi

l

klkl jk kl

defghdefghi

defg

jk

abc

efghi efghi

bcde

00

05

10

0

10

20

30

40

50

60

H-9

-1H

-9-2

H-9

-3H

-9-4

H-9

-5H

-9-6

H-9

-7H

-9-8

H-9

-9H

-9-1

0H

-9-1

1H

-9-1

2H

-9-1

3H

-9-1

4H

-9-1

5H

-9-1

6H

-9-1

7H

-9-1

8H

-9-1

9H

-9-2

0H

-9-2

1H

-9-2

2H

-9-2

3H

-9-2

4H

-9-2

5 Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus10

minus30

minus05

minus10

minus15

minus20

minus25

minus30



b b bcd

def fg gh h

j

i

defg

debc b

a

bd

0

10

20

30

40

50

60

70

80

H-9

-13-

1H

-9-1

3-2

H-9

-13-

3H

-9-1

3-4

H-9

-13-

5H

-9-1

3-6

H-9

-13-

7H

-9-1

3-8

H-9

-13-

9H

-9-1

3-10

H-9

-13-

11H

-9-1

3-12

H-9

-13-

13H

-9-1

3-14

H-9

-13-

15H

-9-1

3-16

H-9

-13-

17H

-9-1

3-18

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()


minus10

minus20







1

1

2

2

3

3

45

5

4

6

6

7

7

8

8

10

10

12

3

45

67

810

9

9

919

19

1911

11

1112

12

1218

18

18

13

13

13

14

14

14

17

17

1720

20

20

16

16

16

15

15

15

27 27

23 2324 2425 2526 26

28 2829 29

2221 21

22

HO HO

OH

HO

1 2

320998400

1199840013998400

14998400 8998400

17998400

1099840012998400

1998400 299840039984005998400

4998400

6998400

79984009998400

15998400

16998400

18998400

19998400












4 Conclusions


Abbreviations


Competing Interests


Acknowledgments


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


35

6 7

42

1

100500(m

AU)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

254nm 4nm (100)

(a)

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

200100

0

254nm 4nm (100)

(b)

8 9 1011

12

13 14

15

1

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

500250

0

254nm 4nm (100)

(c)

6

7

5

4

(mAU

)

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

20001000

0

254nm 4nm (100)

(d)

3

45

6 7

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

(min)

(mAU

) 500250

0

254nm 4nm (100)

(e)

11

425

400

375

350

325

300

275

250

225

200

175

150

125

10075

50

25

00

0255075

(min)

(mAU

) 254nm 4nm (100)

(f)




PPL inhibition()



1 2 34 5

61 2 3

4 5

6

300200100

0

(min)

(mAU

) 254nm 4nm (100)

300

275

250

225

200

175

150

125

10075

50

25

00





b ce f

cdf

a

cd

hg

d

0

20

40

0

20

40

60

80

100

H-1

H-2

H-3

H-4

H-5

H-6

H-7

H-8

H-9

H-1

0

H-1

1

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus20

minus40

minus60

minus80








cdef

cdef

defg

ij

abcd

hijfghi

aabcd

a

ab

ghi

l

klkl jk kl

defghdefghi

defg

jk

abc

efghi efghi

bcde

00

05

10

0

10

20

30

40

50

60

H-9

-1H

-9-2

H-9

-3H

-9-4

H-9

-5H

-9-6

H-9

-7H

-9-8

H-9

-9H

-9-1

0H

-9-1

1H

-9-1

2H

-9-1

3H

-9-1

4H

-9-1

5H

-9-1

6H

-9-1

7H

-9-1

8H

-9-1

9H

-9-2

0H

-9-2

1H

-9-2

2H

-9-2

3H

-9-2

4H

-9-2

5 Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus10

minus30

minus05

minus10

minus15

minus20

minus25

minus30



b b bcd

def fg gh h

j

i

defg

debc b

a

bd

0

10

20

30

40

50

60

70

80

H-9

-13-

1H

-9-1

3-2

H-9

-13-

3H

-9-1

3-4

H-9

-13-

5H

-9-1

3-6

H-9

-13-

7H

-9-1

3-8

H-9

-13-

9H

-9-1

3-10

H-9

-13-

11H

-9-1

3-12

H-9

-13-

13H

-9-1

3-14

H-9

-13-

15H

-9-1

3-16

H-9

-13-

17H

-9-1

3-18

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()


minus10

minus20







1

1

2

2

3

3

45

5

4

6

6

7

7

8

8

10

10

12

3

45

67

810

9

9

919

19

1911

11

1112

12

1218

18

18

13

13

13

14

14

14

17

17

1720

20

20

16

16

16

15

15

15

27 27

23 2324 2425 2526 26

28 2829 29

2221 21

22

HO HO

OH

HO

1 2

320998400

1199840013998400

14998400 8998400

17998400

1099840012998400

1998400 299840039984005998400

4998400

6998400

79984009998400

15998400

16998400

18998400

19998400












4 Conclusions


Abbreviations


Competing Interests


Acknowledgments


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


b ce f

cdf

a

cd

hg

d

0

20

40

0

20

40

60

80

100

H-1

H-2

H-3

H-4

H-5

H-6

H-7

H-8

H-9

H-1

0

H-1

1

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus20

minus40

minus60

minus80








cdef

cdef

defg

ij

abcd

hijfghi

aabcd

a

ab

ghi

l

klkl jk kl

defghdefghi

defg

jk

abc

efghi efghi

bcde

00

05

10

0

10

20

30

40

50

60

H-9

-1H

-9-2

H-9

-3H

-9-4

H-9

-5H

-9-6

H-9

-7H

-9-8

H-9

-9H

-9-1

0H

-9-1

1H

-9-1

2H

-9-1

3H

-9-1

4H

-9-1

5H

-9-1

6H

-9-1

7H

-9-1

8H

-9-1

9H

-9-2

0H

-9-2

1H

-9-2

2H

-9-2

3H

-9-2

4H

-9-2

5 Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()

minus20

minus10

minus30

minus05

minus10

minus15

minus20

minus25

minus30



b b bcd

def fg gh h

j

i

defg

debc b

a

bd

0

10

20

30

40

50

60

70

80

H-9

-13-

1H

-9-1

3-2

H-9

-13-

3H

-9-1

3-4

H-9

-13-

5H

-9-1

3-6

H-9

-13-

7H

-9-1

3-8

H-9

-13-

9H

-9-1

3-10

H-9

-13-

11H

-9-1

3-12

H-9

-13-

13H

-9-1

3-14

H-9

-13-

15H

-9-1

3-16

H-9

-13-

17H

-9-1

3-18

Frac

tion

wei

ght (

g)

Inhi

bitio

n of

PPL

()


minus10

minus20







1

1

2

2

3

3

45

5

4

6

6

7

7

8

8

10

10

12

3

45

67

810

9

9

919

19

1911

11

1112

12

1218

18

18

13

13

13

14

14

14

17

17

1720

20

20

16

16

16

15

15

15

27 27

23 2324 2425 2526 26

28 2829 29

2221 21

22

HO HO

OH

HO

1 2

320998400

1199840013998400

14998400 8998400

17998400

1099840012998400

1998400 299840039984005998400

4998400

6998400

79984009998400

15998400

16998400

18998400

19998400












4 Conclusions


Abbreviations


Competing Interests


Acknowledgments


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


1

1

2

2

3

3

45

5

4

6

6

7

7

8

8

10

10

12

3

45

67

810

9

9

919

19

1911

11

1112

12

1218

18

18

13

13

13

14

14

14

17

17

1720

20

20

16

16

16

15

15

15

27 27

23 2324 2425 2526 26

28 2829 29

2221 21

22

HO HO

OH

HO

1 2

320998400

1199840013998400

14998400 8998400

17998400

1099840012998400

1998400 299840039984005998400

4998400

6998400

79984009998400

15998400

16998400

18998400

19998400












4 Conclusions


Abbreviations


Competing Interests


Acknowledgments


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





4 Conclusions


Abbreviations


Competing Interests


Acknowledgments


References








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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