piperine , a nitrogenous pungent substance , is an alkaloid found in important and oldest spices , namely , piper nigrum ( black peppers ) and piper longum ( long peppers ) .
.   stated that piperine is naturally occurring organic compound that belongs to family piperaceae .
the fruits of piperine possess antidepressant effects , hepatoprotective effects , antioxidant activity , antitumour effects , antibacterial effects , and anticonvulsant effects [ 2 , 3 ] .
piperine also has the capability of reducing inflammation , relieving pain , improving digestion , and enhancing the bioavailability .
piperine is extensively used in medicinal field for years due to various medicinal properties including painkiller , antioxidant , and bioavailability enhancer . molecular imprinting technology ( mit )
is used to design molecular recognition materials because it is capable of mimicking natural recognition entities like antibodies and biological receptors [ 514 ] .
the original concept of molecular imprinting is developed by linus pauling in 1940s , but wulff and sarhan stimulated the interest in imprinting materials . according to vlatakis
et al . , in the early 1980s , the molecular imprinting polymers ( mips ) were successfully prepared by using noncovalent mit .
molecular imprinting is a universal method to produce polymers with high affinity binding sites for organic , inorganic , biological , and chemical molecules or ions .
mips allow the functional and crosslinking monomers to copolymerize in the presence of the target compound or known as template .
molecular imprinting polymers   can be prepared by various methods such as bulk polymerization , electropolymerization , suspension polymerization , emulsion polymerization , two - step polymerization , and precipitation polymerization .
zhou et al .   mentioned that the controlled / living radical polymerization ( crp ) is used to prepare mip microspheres as it permits more precise control over the molecular weight , composition , and end group functionality of the obtained polymers [ 2427 ] .
mips show excellent thermal and chemical stability and can be used in aggressive media . according to yan and row , mips have many advantages over their biological counterparts including inexpensive , simple preparation , storage stability , repeated operations without loss of activity , high mechanical strength , durability to heat and pressure , and applicability in harsh chemical media .
lai et al .   stated that mips have been used in important application such as chemical sensors [ 30 , 31 ] , capillary electrophoresis and electrochromatography , catalysis , hplc stationary phases [ 3439 ] , and solid - phase extraction ( spe ) . in this research noncovalent imprinting or self - assembly approach
the template and a functional monomer interact by noncovalent interactions in the prepolymerization mixture . according to spivak , noncovalent is simpler molecular imprinting method as compared to covalent and semicovalent because it involves synthetic steps toward the prepolymer complex . in this way interaction between the monomer and template
this method has been used to produce imprinted polymers of cinnamic acid . in this research as an application these imprinted polymers are used in extraction of piperine from spiked urine sample .
( united states ) , acrylic acid ( aa ) was bought from nippon shokubai co. ltd .
( japan ) , ethylene glycol dimethacrylate ( egdma ) was purchased from sigma - aldrich co. ltd .
( united states ) , acetonitrile ( acn ) was obtained from kunshan yalong trading co. ltd .
( china ) , 2 , 2-azobisisobutyronitrile ( aibn ) was obtained from sigma - aldrich co. ltd .
( united states ) , methanol ( meoh ) was obtained from nuasa kimia sejati co. ( indonesia ) , acetic acid ( ch3cooh ) was purchased from alpha chemika ( india ) , potassium bromide ( kbr ) was obtained from powder pack chem co. ( india ) , and hexane was obtained from seidler chemical co. ( united states ) .
scanning electron microscope ( jeol jsm-6390la ) was used to study the morphology of polymer particles .
shaker ( nb-101mt ) was used to allow the rebinding of polymer particles with template .
shimadzu lc-20a , a reversed - phase high performance liquid chromatography ( rp - hplc ) , was used to evaluate the batch binding of polymer particles .
. 0.5  mmol of template ( piperine ) , 2  mmol of monomer ( aa ) , 8  mmol of cross - linker ( egdma ) , 75  ml of porogen ( acn ) , and 0.011  g of initiator ( aibn ) were added into 150  ml conical flask , respectively .
the mixture was sonicated for 10 minutes in order to remove bubbles and allow complete dissolution . then
, the conical flask containing mixture was placed in a bucket of ice cubes and the reaction mixture was purged with nitrogen gas for 15 minutes .
ice cubes were used in this experiment to allow a suitable environment for noncovalent interactions between piperine and acrylic acid .
the polymerization was conducted for 6 hours , initially temperature was maintained at 60c for the first three hours , and later temperature was raised up to 80c and maintained for another three hours in order to complete the polymerization .
the produced polymer particles were extracted out by using the centrifugation at 5000  rpm for 10  min .
the template was removed by washing the mips successively in the mixture of methanol and acetic acid ( 9  :  1 , v / v ) until the template was not detected by rp - hplc at 270  nm . the hplc was conducted by using the c18 column ( 250  4  mm , 5  m ) with the mobile phase consisting of acetonitrile , distilled water , and acetic acid in the ratio of 60  :  39.5  :  0.5 , v / v / v , respectively .
the flow rate was set at 0.6  ml / min with uv detection at 270  nm and injection volume was set at 20  l .
the nonimprinted polymeric particles ( nips ) were prepared in the same way without the addition of the template molecule .
the similar procedure was used for the synthesis of different molecular imprinted polymers of piperine with varying composition of aa and egdma ( table 1 ) by precipitation polymerization , namely , mip 2 , mip 3 , and mip 4 for mips as well as nip .
a series of 150  ml five conical flasks containing 0.5  g of the mip ( mip 1 , mip 2 , mip 3 , and mip 4 ) and nip beads were added with a 75  ml of acetonitrile containing 0.5  mmol of piperine .
the conical flasks were shaken on the shaker at 100  rpm and the samples were collected at different time intervals ( 0 , 30 , 60 , 90 , 120 , 150 , 240 , and 360 minutes ) .
the collected samples were centrifuged at 5000  rpm for 10 minutes in order to remove any suspended particles and supernatant was used for further analysis .
the binding capacity of mips and nip of piperine was calculated   by using the following equation:(1)binding capacity%=cicfci100,where ci is the initial piperine concentration in the solution and cf is the final piperine concentration in the solution .
a 150  ml conical flask containing 0.5  g of the mip 3 beads was added and a solution of 75  ml of acetonitrile containing equal concentration ( 0.5  mmol ) of piperine and caffeine .
both of the conical flasks were shaken on the shaker at 100  rpm and the samples were collected at different time intervals ( 0 , 30 , 60 , 90 , 120 , 150 , 240 , and 360 minutes ) .
after shaking at the appropriate time intervals , all the samples were centrifuged at 5000  rpm for 10 minutes .
the distribution ratios ( ml  g ) of piperine between the mips or nip in the porogen ( acetonitrile ) were determined   by the following equation:(2)distribution ratio :  kd = cicfvcim , where ci is the initial piperine concentration in the solution , cf is the final piperine concentration in the solution , v is the volume of porogen ( acn ) used , and m is the mass of mip / nip used .
the selectivity coefficients for piperine relative to binding competitor caffeine for mip 3 and nip can be calculated   by(3)selectivity coefficient :  kpiperine / caffeinesel = kdpiperinekdcaffeine , where kd is the batch binding assay of mip / nip for piperine and kd is the batch binding assay of mip / nip for caffeine .
the relative selectivity coefficient ( k ) was determined   by the following equation:(4)k=kmip  3knip .
urine sample was first centrifuged and filtered and then spiked with a piperine to get a concentration of 50  ppm .
after this 50  ml of spiked urine sample was added in flask containing 0.5  g of mip 3 .
synthesis of microsphere imprinted polymers is a very crucial step in order to produce uniform shape and size of particles .
previous studies revealed   that various preparation methods have been carried out for the preparation of polymer microspheres   such as the synthesis of polymer microspheres by dispersion and emulsion polymerization , where the surfactants in aqueous solution   and stabilizers in organic solution   are crucial to stabilize the polymer phase and prevent the aggregation of particles .
precipitation polymerization can form polymer microspheres with constant size and shape that can lead to narrow dispersion , without the need for any added surfactant or stabilizer [ 4649 ] . in this research
we have successfully produced imprinted polymer microspheres by precipitation method in the acetonitrile ( porogen ) .
ir analysis is an important chemical characterization method to detect the functional groups present in a compound .
the ft - ir spectra of different mips and nip are shown in figure 1 .
based on figure 1 , small peak in the range of 3519.29  cm to 3613.06  cm attributed to the vibration mode of o 
the bands in the range of 2926.75  cm to 2996.02  cm and 2854.76  cm to 2957.24  cm showed the vibration mode of c 
h stretching of aliphatic compound as well as asymmetric and symmetric ch2 stretching in mips and nip .
strong peaks at 1726.11  cm to 1736.42  cm indicated the presence of c = o of acrylic acid .
the vibration mode of c = c stretching of aromatic compound can be found within 1635.37  cm to 1636.84  cm .
the ch2 bending at 1450.82  cm to 1452.70  cm indicated the presence of alkane group in mips and nip .
n stretching of mips and nips after leaching was detected at 1388.44  cm to 1390.06  cm .
peaks of mips and nip at 1253.88  cm to 1259.61  cm showed the vibration mode of o  ch2o symmetric stretching . small band at 1035.88  cm to 1050.59  cm in mips and nip explained the presence of symmetric stretching of = c  o  c . the vibration mode of c 
h stretching of aliphatic compound can be observed at 2954.09  cm to 2985.19  cm .
sem analysis is a very important morphological study for polymer particles that provides the idea about the shape and size .  
, uniform size of imprinted polymers can be formed by using a noncovalent imprinting approach by precipitation polymerization .
arabzadeh and abdouss   stated that interaction between monomer and template could be another factor that contributed to uniform size distribution with clean surfaces .
research conducted by park et al .   mentioned that there are various factors that affect the production of uniform polymer microspheres including volume of solvent , reaction of solvent , and presence of template ion .
excess solvent or porogen used in the synthesis of polymer particles will produce highly uniform polymer microspheres with imprinted binding sites .
rp - hplc was used to evaluate the binding efficiency of mips and nip of piperine .  
figure 3 depicts the binding capacity of different mips and nip at different time intervals .
mip 3 showed the highest binding capacity ( 84.94% ) , followed by mip 2 ( 75.86% ) , mip 1 ( 69.40% ) , and mip 4 ( 60.80% ) .
mip 3 contains a higher amount of monomer ratio as compared to mip 1 and mip 2 but mip 4 contains a higher amount of cross - linker . in this study
increasing amount of monomer would produce specific interaction sites with the piperine and hence rebinding efficiency was also increased .
but the increase in amount of cross - linker has produced a reverse effect as can be seen in mip 4 .
if we compare the mips with nip it is clear from figure 3 the binding capacity is low .
this can be conferred that nip does not contain any binding site complimentary with the piperine . in order to evaluate the properties of mip of piperine as a sensing material ,
two compounds ( piperine and caffeine ) were tested using both mip 3 and nip .
the distribution ratio of piperine in both mip 3 and nip was higher than the distribution ratio of caffeine in both mip 3 and nip , resulting in higher selectivity coefficient of piperine than that of caffeine in both mip 3 and nip ( table 2 ) .
the results indicate that the imprinted polymer has got complimentary binding sites or cavities with the piperine as compared to caffeine .
the extensive use of piperine in medicine and spices has generated this idea to first use the selected mip 3 in the extraction of piperine from urine .
this will provide us a way forward to expand the application of these imprinted polymer particles . from this study
it was found that about 81.18% of piperine was successfully extracted from the spiked urine sample .
the binding efficiencies of mips and nip of piperine were evaluated by batch binding assay .
mip 3 exhibited the highest binding capacity ( 84.94% ) as compared to nip ( 40% ) .
 molecularly imprinted polymer ( mip ) microspheres for piperine were synthesized by precipitation polymerization with a noncovalent approach . in this research piperine was used as a template , acrylic acid as a functional monomer , ethylene glycol dimethacrylate as a cross - linker , and 2,2-azobisisobutyronitrile ( aibn ) as an initiator and acetonitrile as a solvent . 
 the imprinted and nonimprinted polymer particles were characterized by using fourier transform infrared spectroscopy ( ft - ir ) and scanning electron microscopy ( sem ) . 
 the synthesized polymer particles were further evaluated for their rebinding efficiency by batch binding assay . 
 the highly selected imprinted polymer for piperine was mip 3 with a composition ( molar ratio ) of 0.5  :  3  :  8 , template  :  monomer  :  cross - linker , respectively . 
 the mip 3 exhibits highest binding capacity ( 84.94% ) as compared to other imprinted and nonimprinted polymers . the extraction efficiency of highly selected imprinted polymer of piperine from spiked urine was above 80% . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion