obesity is often defined as the excess accumulation of body fat resulting from a higher energy intake than energy expenditure . in 2008 , 10% of men and 14% of women in the world were obese , compared with 5% of men and 8% of women in 1980 .
rates of both overweight and obesity are projected to increase in almost all countries , with 1.5 billion people overweight in 2015 .
pancreatic lipase inhibition is one of the most widely studied mechanisms for antiobesity treatment , based on the principle that dietary fat will not be directly absorbed by the intestine unless the fat has been subjected to the action of pancreatic lipase [ 3 , 4 ] .
phytochemicals or bioactive compound / extract identified from traditional medicinal plants had provided an exciting platform and opportunity for the development of safe and effective therapeutic drugs for the treatment of many metabolic diseases .
a review by newman and cragg ( 2007 )   on the origin of drugs launched in the past 25 years showed about half of the compounds that were successful in clinical trials were derived from natural origin . despite multiple research conducted in recent decades ,
previous screening study on thirty two plants reported strongest porcine pancreatic lipase ( ppl ) activity in e. indica   and this has led to further investigation on this herb for potential antiobesity agent . 

e. indica ( linnaeus ) gaertner ( poaceae ) is an annual grass native in the tropics and subtropical regions [ 8 , 9 ] .
it is commonly widespread as weed in rice field and is known to be resistant to many herbicides ( such as dinitroaniline ) .
this plant is commonly known as goosegrass , wiregrass ,  rumput sambari ,  or 
its root is traditionally known to be depurative , febrifuge , diuretic , and laxative and thus is commonly used for treating hypertension , influenza , oliguria , and urine retention .
the seed of e. indica is sometimes used as famine food and in the treatment for liver complaints .
several pharmacological properties on e. indica have been reported including hepatoprotective effect , antiplasmodial and antidiabetic , antioxidant and antimicrobial activity , anti - inflammatory , and cytotoxic effect towards several cancer cell lines [ 8 , 16 ] . to date , only one study reported the isolation of secondary metabolites from e. indica where hexadecanoic acid and [ [ ( 2-aminoethoxy ) hydroxyphosphinyloxy]methyl]-1,2ethanediylester were isolated .
hence , this paper is the first report on the kinetics of ppl enzyme inhibition by e. indica and the bioactivity - guided isolation of a potent ppl inhibitory compound ( lutein ) from e. indica .
the whole plants of e. indica ( l. ) gaertn . were collected from persatuan pengkaji herba tradisional negeri sembilan ( pantai , negeri sembilan , coordinates : 24613n , 1015940e ) .
fadzureena jamaludin from forest research institute malaysia ( frim ) ; the voucher specimen 003/15 ( collection date : 11 february 2015 ) is kept at the school of biosciences , taylor 's university ( lakeside campus ) .
the whole plant of e. indica was cleaned from residual soil , freeze - dried , and pulverised .
analytical grade methanol was added and the extracts were then filtered and pooled , and the solvent was evaporated off .
the crude extract of e. indica was suspended in distilled water ( 1  :  10 , w / v ) and sequentially extracted with solvents in increasing polarity ( hexane , chloroform , ethyl acetate , and butanol ) , three times each ( 1  :  1 , v / v ) , to obtain the respective solvent fractions .
porcine pancreatic lipase ( ppl ) inhibitory assay was performed as described by bustanji et al .
the enzyme solutions was prepared immediately before use , by suspending crude porcine pancreatic lipase powder type ii ( sigma , ec 3.1.1.3 ) in tris - hcl buffer ( 50  mm tris , 150  mm nacl , 1  mm edta , 10  mm mops , ph 7.6 ) to give a concentration of 5  mg / ml ( 200 units / ml ) .
the solution was then centrifuged at 1,500  rpm for 10 minutes and the clear supernatant was recovered .
the plant extract ( 100  g / ml ) was preincubated with 200  l of ppl solution for 5 minutes at 37c , before the addition of 5  l pnpb substrate solution ( 10  mm in acetonitrile ) .
the total reaction volume was made to 1  ml using the tris - hcl buffer before measuring the absorbance at 410  nm against blank using denatured enzyme .
the extract was dissolved in dmso at a final concentration not exceeding 1% ( v / v ) which will not affect enzyme activity .
the inhibitory activity ( i% ) was calculated according to the formula below : ( 1)i%=1bbaa100,where a is the activity of the enzyme without inhibitor , a is the negative control without the inhibitor , b is the activity of the enzyme with inhibitor , and b is the negative control with inhibitor .
the inhibition mode of e. indica methanolic crude extract on porcine pancreatic lipase ( ppl ) was assayed with increasing concentrations ( 20 , 40 , 60 , and 80  m ) of synthetic substrate , p - nitrophenyl butyrate ( pnpb ) , in the presence and absence of two different concentrations of the extracts ( 100 and 200  g / ml ) .
the tlc was performed on the tlc silica gel 60 coated with fluorescent indicator f254 aluminium sheets ( merck ) .
samples were spotted and viewed under uv lamp at 254  nm and 365  nm .
the fingerprinting of the extracts was done on shimadzu prominence series coupled with photodiode array ( pda ) detector spd - m20a using either reversed - phase or normal phase settings : reversed - phase , chromolith highresolution rp-18 endcapped 1004.6  mm ( merck ) : the mobile phase used was solvent a , acetonitrile and solvent b , water with a standard flow rate of 1.0  ml / min , and injection volume of 20  l of 10  mg / ml extract ; the gradient of the mobile phase was as follows : 0% to 50% a ( 045  min).normal phase , phenomenex luna silica column ( 250  4.6  mm , 100   , 5  m ) : the mobile phase used consists of solvent a , hexane and solvent b , 2-propanol with a flow rate of 1.0  ml / min , and injection volume of 20  l of 10  mg / ml extract ; the gradient program was as follows : 100% a ( 05  min ) , 100% to 0% a ( 525  min ) .
reversed - phase , chromolith highresolution rp-18 endcapped 1004.6  mm ( merck ) : the mobile phase used was solvent a , acetonitrile and solvent b , water with a standard flow rate of 1.0  ml / min , and injection volume of 20  l of 10  mg / ml extract ; the gradient of the mobile phase was as follows : 0% to 50% a ( 045  min ) .
normal phase , phenomenex luna silica column ( 250  4.6  mm , 100   , 5  m ) : the mobile phase used consists of solvent a , hexane and solvent b , 2-propanol with a flow rate of 1.0  ml / min , and injection volume of 20  l of 10  mg / ml extract ; the gradient program was as follows : 100% a ( 05  min ) , 100% to 0% a ( 525  min ) .
the ir spectra were measured by perkin elmer spectrum 100 using potassium bromide pellet method .
mass spectra were recorded with eims using a direct injection probe on a shidmadzu gc - ms qp 5050a spectrometer .
the uv - vis spectra were recorded on a thermo scientific genesys 10 uv scanning spectrophotometer .
deuterated chloroform ( cdcl3 ) was used as the solvent to dissolve the test samples .
tetramethylsilane ( tms ) was used as internal standard for both h ( 500  mhz ) and c ( 125  mhz ) .
the chemical shifts from the spectra were recorded in ppm and coupling constants were given in hertz ( hz ) .
the active fraction was subjected to gravitational column chromatography packed with suitable packing materials ; the schematic flow is as shown in figure 1 .
the weight of the selected packing materials introduced into the column was at least ten times the weight of the sample extract .
the isolated pure compounds were then characterised and elucidated employing several spectral methods as stated in section 2.5 . 

134.5137.6c ; uv ( hexane ) max  nm ( log   ) : 210 ( 817 ) , 230 ( 54 ) ; ir max  cm : 3431 , 2937 , 1468 , 1382 , 1056 ; eims m / z ( 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 ) ; h nmr ( 500  mhz , cdcl3 ) :   5.29 ( m , 1h , h-3 ) , 3.46 ( m , 1h , h-6 ) ; c nmr ( 125  mhz , cdcl3 ) :   140.7 ( c-5 ) , 121.8 ( c-6 ) , 79.1 ( c-3 & c-13 ) , 55.4 ( c-14 & c-17 ) , 50.6 ( c-9 ) , 48.7 ( c-24 ) , 42.1 ( c-4 ) , 39.5 ( c-12 ) , 39.0 ( c-1 ) , 38.4 ( c-10 ) , 37.2 ( c-20 ) , 33.4 ( c-22 ) , 32.7 ( c-2 ) , 31.2 ( c-7 & c-8 ) , 29.8 ( c-25 ) , 27.5 ( c-16 ) , 26.8 ( c-23 ) , 26.3 ( c-15 ) , 25.6 ( c-28 ) , 21.5 ( c-11 ) , 19.6 ( c-26 ) , 18.4 ( c-19 & c-27 ) , 17.8 ( c-21 ) , 15.5 ( c-29 ) , 14.9 ( c-18 ) . 

168.0170.0c ; uv ( hexane ) max  nm ( log   ) : 210 ( 839 ) , 230 ( 147 ) ; ir max  cm : 3426 , 2936 , 1645 , 1465 , 1384 , 1052 and 959 ; eims m / z ( rel . int . ) : 412 [ m ] ( 61 ) , 255 ( 52 ) , 159 ( 56 ) , 145 ( 61 ) , 95 ( 58 ) , 81 ( 78 ) , 69 ( 65 ) , 55 ( 100 ) ; h - nmr ( 500  mhz , cdcl3 ) :   5.34 ( d , 1h , j = 4.6  hz , h-6 ) , 5.16 ( m , 1h , h-22 ) , 5.03 ( m , 1h , h-23 ) , 3.53 ( m , 1h , h-3 ) , 1.00 ( s , 3h , h-19 ) , 0.916 ( d , 1h , j = 5.75  hz , h-21 ) , 0.829 ( m , 9h , h-26 , h-27 , h-29 ) , 0.685 ( s , 3h , h-18 ) ; c nmr ( 125  mhz , cdcl3 ) :   140.8 ( c-5 ) , 138.4 ( c-20 ) , 129.4 ( c-21 ) , 121.8 ( c-6 ) , 71.9 ( c-3 ) , 56.9 ( c-14 ) , 56.2 ( c-17 ) , 51.3 ( c-22 ) , 50.2 ( c-9 ) , 42.4 ( c-4 ) , 42.3 ( c-13 ) , 39.9 ( c-12 ) , 39.8 ( c-18 ) , 37.4 ( c-1 ) , 36.6 ( c-10 ) , 32.0 ( c-2 ) , 31.7 ( c-7 ) , 29.2 ( c-8 ) , 29.0 ( c-16 ) , 28.3 ( c-25 ) , 25.5 ( c-23 ) , 24.4 ( c-15 ) , 21.2 ( c-11 ) , 19.9 ( c-27 ) , 19.5 ( c-26 ) , 19.1 ( c-19 ) , 18.9 ( c-28 ) , 12.3 ( c-29 ) , 12.1 ( c-24 ) . 

173.8174.9c ; uv ( acetone ) max  nm ( log   ) : 266 , 426 ( shoulder ) , 448 ( 628 ) , 476 ( 564 ) ; ir max  cm : 3427 , 2926 , 1718 , 1465 , 1376 , 1261 ; eims m / z ( rel . int . ) : 568 [ m ] , 145 ( 49 ) , 119 ( 96 ) , 105 ( 100 ) , 93 ( 54 ) , 91 ( 70 ) ; h nmr ( 500  mhz , cdcl3 ) :   6.57 ( m , 4h , h-11 , h-15 , h-11 , h-12 ) , 6.34 ( d , 2h , j = 14.9  hz , h-12 ) , 6.23 ( m , 2h , h-14 , h-14 ) , 6.10 ( m , 5h , h-8 , h-10 , h-7 , h-8 ) , 5.54 ( s , 1h , h-4 ) , 5.39 ( dd , 1h , j = 10.4 , 9.2  hz , h-7 ) , 4.24 ( s , 1h , h-3 ) , 3.98 ( m , 1h , h-3 ) , 2.36 ( m , 2h , h-6 , h-4eq ) , 2.01 ( dd , 1h , j = 10.3 , 9.2  hz , h-4ax ) , 1.96 ( s , 9h , h-20 , h-19 , h-20 ) , 1.90 ( s , 3h , h-19 ) , 1.81 ( dd , 1h , j = 5.7 , 6.9  hz , h-2eq ) , 1.73 ( s , 3h , h-18 ) , 1.62 ( s , 12h , h-18 ) , 1.44 ( t , 1h , j = 12.6 , 11.5  hz , h-2 ) , 1.34 ( dd , 1h , j = 5.7 , 6.9  hz , h-2ax ) , 1.06 ( s , 6h , h-16 , h-17 ) , 0.838 & 0.987 ( s , 6h , h-16 , h-17 ) ; c nmr ( 125  mhz , cdcl3 ) :   138.6 ( c-8 ) , 138.1 ( c-8 ) , 137.8 ( c-5 , c-12 ) , 137.6 ( c-12 , c-6 ) , 136.6 ( c-13 ) , 136.5 ( c-13 ) , 135.8 ( c-9 ) , 135.2 ( c-9 ) , 132.7 ( c-14 , c-14 ) , 131.4 ( c-10 ) , 130.9 ( c-10 ) , 130.2 ( c-15 ) , 130.1 ( c-15 ) , 128.8 ( c-7 ) , 126.2 ( c-5 ) , 125.7 ( c-7 ) , 125.0 ( c-4 ) , 124.9 ( c-11 ) , 124.6 ( c-11 ) , 66.0 ( c-3 ) , 65.2 ( c-3 ) , 55.1 ( c-6 ) , 48.5 ( c-2 ) , 44.7 ( c-2 ) , 42.6 ( c-4 ) , 37.2 ( c-1 ) , 34.1 ( c-1 ) , 30.3 ( c-17 ) , 29.6 ( c-17 ) , 28.8 ( c-16 ) , 24.4 ( c-16 ) , 22.8 ( c-18 ) , 21.7 ( c-18 ) , 13.2 ( c-19 ) , 12.9 ( c-19 , c-20 ) , 12.8 ( c-20 ) .
significance of difference from the control was determined by tukey 's post - hoc test ( one - way anova ) p value < 0.05 using spss software ( version 16.0 ) .
the inhibition mode of ppl by e. indica methanolic extract at 100  g / ml and 200  g / ml was analysed by double - reciprocal lineweaver - burk plot as shown in figure 2 .
kinetic parameters calculated from the double reciprocal trend lines showed that both the maximal velocity of the ppl enzyme - substrate extract reaction ( vmax ) and the affinity ( km ) were affected by the extract concentration , hence indicating a mixed mode inhibition .
the michaelis - menten parameters are tabulated in table 1 , where the michaelis - menten constant ( km ) of ppl with synthetic substrate pnpb was 26.36  m and maximal velocity ( vmax ) was 65.79  m  min .
the mixed mode inhibition exhibited by ppl indicates that the formation of enzyme - substrate complex was possible with the inhibitor binding at a distinct site from the active site resulting in reduction in the complex affinity , thus explaining the increase in km .
similar inhibition mode was observed in levisticum officinale methanolic extract and regular cocoa extract against porcine pancreatic lipase [ 19 , 20 ] .
the methanolic crude extract of e. indica was then partitioned via liquid - liquid fractionation , to yield five ( 5 ) solvent extracts , that is , hexane , dichloromethane , ethyl acetate , butanol , and water .
table 2 shows that the hexane extract from e. indica possessed the highest ppl inhibitory activity , that is , 27.01  5.68% .
although e. indica dichloromethane extract recorded a comparable value of 25.57  3.26% ppl inhibitory activity , due to its low yield from the partition ( 1.18% ) , this extract was not further tested .
the hplc chromatograms of all e. indica methanolic extract and solvent fractions are shown in figure 3 .
six major peaks were detected in elution profile of the crude methanolic extract of e. indica ( figure 3(a ) ) .
no peak was detected in hexane fraction ( figure 3(b ) ) due to the incompatibility of reverse phase column with the nature of the fraction , which was highly nonpolar .
the hexane fraction was later optimised and run with luna 5  m silica ( phenomenex ) , a normal phase column coupled with normal phase mobile phase , where six major peaks were detected as shown in figure 4 .
a cluster of eight peaks were eluted in the second half end of the chromatogram in dichloromethane fraction ( figure 3(c ) ) due to its more nonpolar nature .
most of the peaks from this cluster ( peak 8 to peak 15 ) were visible in the chromatogram of crude methanolic extract in figure 3(a ) , but the concentration of these compound present in the crude extract was too low to be detected as the major peaks .
figures 3(d ) and 3(e ) which showed the elution profile of ethyl acetate and butanol extracts , respectively , recorded peak 4 to peak 7 which corresponded to the major peaks as detected in elution profile of crude methanolic extract where they represented elution of semipolar compounds at the midrange of the chromatograms .
the most polar compounds were eluted at the beginning of the spectrum ( retention time less than 2.5 minutes ) in the aqueous fraction ( figure 3(f ) ) , which were also detected as one of the major peaks in the crude extract .
high ppl inhibitory activity was detected in hexane ( 27.01  5.68% ) and dichloromethane ( 25.57  3.26% ) fractions from e. indica ( table 2 ) .
chromatogram of the hexane fraction ( figure 4 ) showed presence of six ( 6 ) major components ( numbers 1 to 6 ) while chromatogram of the dichloromethane fraction ( figure 3(c ) ) showed eight major components ( numbers 8 to 15 ) .
attempts were made to identify the components by running with ten standards , namely , rutin , quercetin , neringenin , caffeic acid , chlorogenic acid , coumaric acid , gallic acid , esculin , kaempferol , and myricetin . however , none of the peaks ' retention time matched those of the standards . as such
, the components eluted from the hexane and dichloromethane fractions could not be identified from the chromatograms .
nevertheless , these chromatographic fingerprints obtained from the methanolic crude and all solvent fractions may provide a simple guide for future identification and comparison of e. indica l. even if the geographical location and/or season of collection are different .
the hexane fraction of e. indica was further separated into eleven ( 11 ) fractions .  
figure 5 shows h-9 ( 4.21  g ) recorded the highest ppl inhibitory activity , that is , 39.35  0.24% , which was obtained from the elution with 75% dichloromethane/25% ethyl acetate .
the remaining fractions obtained from this column recorded less than 20% of ppl inhibitory activity . alongside with the separation of h-1 to h-11
, two common sterols were isolated from the fractions h-4 and h-5 , on elution with 90% dichloromethane/10% ethyl acetate .
-sitosterol ( 1 ) was isolated as a white flower - liked crystalline powder while stigmasterol ( 2 ) was in white needle - like crystals .
the detailed physical properties of  -sitosterol and stigmasterol will be discussed in the later part of this paper .
both compounds were found to possess very low ppl inhibition activity , that is , 2.99  0.80% ( -sitosterol ) of inhibition at 100  g / ml ( 242  m ) and 2.68  0.38% ( stigmasterol ) of inhibition at 100  g / ml ( 243  m ) , respectively .
fraction h-9 ( 4.21  g ) was subjected to further separation due to its high ppl inhibitory activity and sufficient yield .  
figure 6 shows the fraction yield and ppl inhibitory activity of twenty five ( 25 ) subfractions of h-9 .
highest ppl inhibitory activity was captured in h-9 - 13 ( 32.15  5.11% ) , followed by h-9 - 14 ( 29.26  1.08% ) , where the yield was 0.32  g and 0.47  g , respectively .
subfractions of h-9 - 13 , h-9 - 17 , and h-9 - 21 recorded ppl inhibitory activity of more than 20% while other subfractions were either promoted or showed less than 20% ppl inhibition . both h-9 - 13 and h-9 - 14
were selected for further isolation due to their higher yield ( 323.6  mg and 465.3  mg , resp . ) .
as both h-9 - 13 and h-9 - 14 showed comparable ppl inhibition as well as similar tlc spotting profile ( data not shown ) , both subfractions were combined for further isolation and the results of the ppl inhibitory activity of the resultant isolated subfractions are shown in figure 7 .
highest ppl inhibitory activity was shown by h-9 - 13 - 9 ( 108.8  mg ) , that is , 52.08  2.93% , which was about 18% higher than that recorded by orlistat ( 34.49  5.39% ) .
fraction h-9 - 13 - 10 recorded the second highest ppl inhibition , 34.96  4.87% , which was similar to that of orlistat .
the tlc profile from h-9 - 13 - 9 showed one prominent yellow spot where further isolation was then done , resulting in the isolation of lutein ( 3 ) ( 10.8  mg ) from this fraction .
this isolated pure compound ( lutein ) was subjected to few analysis including melting point analysis , uv , gc - ms ( for the determination of molecular weight ) , ft - ir ( determination of functional group ) , and nmr ( structural elucidation ) .
the characterised lutein was found to possess very strong ppl inhibitory activity , that is , 55.98  1.04% at 100  g / ml ( 176  m ) , which was 21.5% higher than that recorded in orlistat ( 34.49  5.39% ) .
to date , this is the first study to reveal the potential of lutein as a strong ppl inhibitory agent .
the white crystals of  -sitosterol ( 1 ) had melting point of 134.5137.6c [ lit .
134 - 135c ] and also showed ir absorptions at 3431 ( 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 with the molecular ion peak at m / z 414 . similarly , stigmasterol ( 2 ) was isolated as white crystals with melting point of 168.0170.0c ( lit 170c ) , and its mass spectral data suggested the molecular formula as c29h48o .
the ir absorption of stigmasterol was similar to that of  -sitosterol with an additional absorption band at 1645  cmindicative of an olefin group .
sterols normally absorb in the range of 190210  nm , due to the transitions of  to  .
the h nmr spectrum of  -sitosterol ( 1 ) had detected multiplet of signals at   3.21 of h-3 which belonged to the hydroxymethyl group at c-3 , and another pair of multiplet resonated at   4.61 which is assigned to the olefinic proton at h-6 .
the c nmr spectrum showed a total of 29 carbon signals were captured , which supported the molecular formula obtained in eims .
these signals consist of a quaternary methine at c-5 (  145.3 ) , an olefinic methine at c-6 (  121.8 ) , as well as several methyl group at c-18 , c-19 , c-21 , c-26 , c-27 , and c-29 (  14.9 , 18.4 , 17.8 , 19.6 , 18.4 , and 15.5 , resp . ) . and methylene group was located at   39.0 ( c-1 ) ,   32.7 ( c-2 ) ,   42.1 ( c-4 ) ,   31.2 ( c-7 ) ,   21.5 ( c-11 ) ,   39.5 ( c-12 ) ,   26.3 ( c-15 ) ,   27.5 ( c-16 ) ,   33.4 ( c-22 ) ,   26.8 ( c-23 ) , and   25.6 ( c-28 ) .
both proton and carbon signals obtained were matched and in agreement with literature . in the h nmr spectrum of stigmasterol ( 2 ) , there was presence of methyl singlets at   0.69 ( h-18 ) and   1.00 ( h-19 ) ; multiplets at   5.16 ( h-22 ) and   5.03 ( h-23 ) signified olefinic protons which were not found in  -sitosterol . similarly ,
c nmr had revealed 29 carbon peaks where olefinic carbons were identified at   140.8 ( c-5 ) ,   121.8 ( c-6 ) ,   51.3 ( c-22 ) , and   25.5 ( c-23 ) ; methyl carbons were detected at   39.8 ( c-18 ) ,   19.1 ( c-19 ) ,   129.4 ( c-21 ) ,   19.5 ( c-26 ) ,   19.9 ( c-27 ) , and   12.3 ( c-29 ) ; methylene carbons were detected at   37.4 ( c-1 ) ,   32.0 ( c-2 ) ,   42.4 ( c-4 ) ,   31.7 ( c-7 ) ,   21.2 ( c-11 ) ,   39.9 ( c-12 ) ,   24.4 ( c-15 ) ,   29.0 ( c-16 ) , and   18.9 ( c-28 ) .
lutein ( 3 ) was isolated as orange crystals with a melting point of 173.8174.9c and maxima wavelengths recorded at 426 , 448 , and 476  nm , which were in agreement with the data of the literature [ 26 , 27 ] .
mass spectrum of lutein given the molecular ion at m / z 568 , followed by fragments in m / z 550 , is attributed to the loss of a hydroxy group .
h nmr spectra data revealed hydroxyl protons resonated at   4.24 ( h-3 ) and   3.98 ( h-3 ) ; olefinic protons at   4.24 ( h-3 ) ,   5.54 ( h-4 ) ,   5.39 ( h-7 ) ,   6.10 ( h-8 ) ,   6.10 ( h-10 ) ,   6.57 ( h-11 ) ,   6.34 ( h-12 ) ,   6.23 ( h-14 ) ,   6.57 ( h-15 ) ,   6.10 ( h-7 and h-8 ) ,   6.57 ( h-11 and h-12 ) , and   6.23 ( h-14 ) ; allylic protons at   1.62 ( h-18 ) ,   1.90 ( h-19 ) ,   1.96 ( h-20 ) ,   1.73 ( h-18 ) , and   1.96 ( h-19 and h-20 ) ; methyl singlets at   0.838 ,   0.987 ( h-16 and h-17 ) , and   1.06 ( h-16 and h-17 ) .
the c nmr spectra data revealed olefinic carbons resonated at   125.0 ( c-4 ) ,   137.8 ( c-5 ) ,   128.8 ( c-7 ) ,   138.1 ( c-8 ) ,   135.2 ( c-8 ) ,   135.2 ( c-9 ) ,   130.9 ( c-10 ) ,   124.6 ( c-11 ) ,   137.6 ( c-12 ) ,   136.5 ( c-13 ) ,   132.7 ( c-14 ) ,   130.1 ( c-15 ) ,   126.2 ( c-5 ) ,   137.6 ( c-6 ) ,   125.7 ( c-7 ) ,   138.6 ( c-8 ) ,   135.8 ( c-9 ) ,   131.4 ( c-10 ) ,   124.9 ( c-11 ) ,   137.8 ( c-12 ) ,   136.6 ( c-13 ) ,   132.7 ( c-14 ) , and   130.2 ( c-15 ) ; hydroxyl attached carbon resonated at   66.0 ( c-3 ) and   65.2 ( c-3 ) ; methyl carbons were detected at   24.4 ( c-16 ) ,   29.6 ( c-17 ) ,   22.8 ( c-18 ) ,   13.2 ( c-19 ) ,   12.8 ( c-20 ) ,   28.8 ( c-16 ) ,   30.3 ( c-17 ) ,   21.7 ( c-18 ) , and   12.9 ( c-19 & c-20 ) ; and methylene carbons resonated at   44.7 ( c-2 ) ,   48.5 ( c-2 ) , and   42.6 ( c-4 ) .
common plant sterols like  -sitosterol and stigmasterol are important in cellular and developmental mechanisms in plants . in therapeutic treatment via dietary options , food products supplemented with plant sterols helped in the reduction of plasma cholesterol and the risk of atherosclerosis .
however , the cholesterol lowering effect may not be attributed to the inhibition via pancreatic lipase since the ppl inhibition activity of both phytosterols obtained was less than 2% .
weak ppl inhibition activity of  -sitosterol and stigmasterol isolated from alpinia zerumbet had also been reported by chompoo et al .
( 2012 )   with ic50 value of 99.99  1.86  g / ml and 125.05  4.76  g / ml , respectively , in comparison with the inhibition shown by curcumin ( ic50 = 4.92  0.21  g / ml ) and quercetin ( ic50 = 18.60  0.86  g / ml ) which were used as positive controls in their study . in our study ,  -sitosterol and stigmasterol were recorded with weak ppl inhibitory activity of only 3.0  0.8% and 2.7  0.4% at 100  g / ml , respectively , ( i.e. , 242  m and 243  m ) in contrast with that obtained from orlistat ( 34.5  5.4% at 100  g / ml ) , which were comparatively lower than that recorded in literature ( i.e. , 50% ppl inhibition at 100  g / ml ) .
this may be due to different experimental settings where concentrations of both subtrate and enzyme concentrations used were different in both studies .
lutein , a member under the xanthophyll family , is the principal carotenoid in greens , leaves , and yellow flowers .
the bioavailability of xanthophyll is highly dependent on the matrix due to its hydrophobicity of the long carbon skeletons , and thus dietary lipids are required to facilitate the dispersion of lutein , where similar matrix was adapted by pancreatic lipase .
another study demonstrated xanthophyll ester hydrolysed by cholesteryl esterase , but not triacylglycerol lipase ( pancreatic lipase ) , and thus ruled out the possibility of xanthophyll as a competitive substrate [ 32 , 33 ] . to date , the mechanism and the inhibitory effect of xanthophyll on pancreatic lipase are still largely unknown , based on the literature findings that may lead to postulation that lutein may be a noncompetitive inhibitor by binding on the allosteric site of pancreatic lipase .
further study is thus required to fully understand the kinetic interactions between lutein and pancreatic lipase .
bioactivity - guided isolation on hexane extract of e. indica has led to isolation and elucidation of potent ppl inhibitory agent lutein ( 3 ) with strong inhibitory activity of 55.98  1.04% , alongside with the isolation of two other common sterols : ( 1 )  -sitosterol and ( 2 ) stigmasterol . to date
 
 eleusine indica ( linnaeus ) gaertner is a traditional herb known to be depurative , febrifuge , and diuretic and has been reported with the highest inhibitory activity against porcine pancreatic lipase ( ppl ) among thirty two plants screened in an earlier study . 
 this study aims to isolate and identify the active components that may possess high potential as an antiobesity agent . of the screened solvent fractions of e. indica 
 , hexane fraction showed the highest inhibitory activity of 27.01  5.68% at 100  
 g / ml . bioactivity - guided isolation afforded three compounds from the hexane fraction of e. indica , namely ,  -sitosterol , stigmasterol , and lutein . 
 the structures of these compounds were elucidated using spectral techniques . 
 lutein showed an outstanding inhibitory activity against ppl ( 55.98  1.04% ) , with activity 60% higher than that of the reference drug orlistat . 
 the other compounds isolated and identified were  -sitosterol ( 2.99  0.80% ) and stigmasterol ( 2.68  0.38% ) . 
 the enzyme kinetics of e. indica crude methanolic extract on ppl showed mixed inhibition mechanism . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions