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Isolation of Piperine from Black Pepper

Introduction

Extraction of natural products is important to medicine and the pharmaceutical

industry, because large proportion of natural products in drug discovery was based on the

diverse structures and carbon skeletons of natural products. For the future, extractions of

natural products are significant sources for new drugs and are good lead compounds

suitable for further modification during drug development.3For this experiment, we will

isolate piperine from black pepper.

Black pepper is well known for its applications ranging from flavoring, and its

multiple medicinal uses, particularly in gastrointestinal disorders, such as constipation,

diarrhea, and indigestion.4

In addition, pepper has been recognized to have antibacterial,

antihistaminic, digestive enzyme stimulatory, immune enhancing, and chemopreventive

effects as well.4The quality of pepper is contributed by two components: Piperine that

contributes the spiciness and volatile oil that is responsible for the aroma and flavor.

Piperine was first isolated by Oersted in 1819 as a yellow crystalline substance and

discovered that Piperine the major alkaloids for the spicy component present in pepper.

There are five minor alkaloids possessing pungency in pepper extracts, which are

piperettine, piperanine, piperylin A, piperolein B and pipercine.2They all have similar

chemical functional groups but different molecular weights, and different structures.

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Figure 1 Chemical structures of five analogues of

piperinePiperine (C17H19O3N; m.p 128-130) is a

weak base, and when it is hydrolyzed with

HNO3 or any other aqueous alkali, it yields a

volatile base piperidine (C5H11N). Piperine is

highly sensitive to light, thus the reaction

should be carried out accordingly.2Piperine has

also been discovered as possessing anti-

asthmatic, anti-inflammatory and anti-

hypertensive, antitumor, antibacterial, and even

analgesic activities.4In addition, piperine

exhibits a chemo-protective effect, where it shows a protective effect against radiation in

radiotherapy. Moreover, piperine is also used as a bioavailability enhancer in many drug

formulations, thus the high demand for pure piperine leads to more efficient way to

extract pure piperine from black pepper.3

For this experiment piperine is isolated from the black pepper by refluxing with

ethanol and KOH. However there is more than one way to extract piperine from black

pepper. For example, by using the chemical properties of piperine, piperine can be

isolated from black pepper. One of the properties is that piperine is soluble in

dichloromethane and in cold ethanol. Thus, instead of refluxing with ehthanol, black

pepper can be refluxed with CH2Cl2 and with saxhelt (C2H5OH 95%). With extraction

involving refluxing, 2 principal alkaloid amide products, piperine and piperanine can be

obtained. Recrystallization from ethanol produces pure piperine solube in cold ethanol (pf

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128C) and pure piperanine solube in hot ethanol (pf 135C).5

In addition, it is also

known that combining butyl benzene sulfonate as hydrotrope with a surfactant in aqueous

solutions isolates piperine from black pepper. This method is proven to increase the

percentage extraction of piperine when mixed with sodim dodecyl sulfate (SDS) as

compared to the hydrotrope alone.3

For this lab, piperine will be isolated by refluxing in ethanol and KOH. And the

obtained piperine will be purified via recrystallization. Typical recoveries are between

2.5% to 10%. The purity of the extracted piperine will be analyzed by taking a melting

point of the purified product, and obtaining data from IR, 60MHz 1H NME, 400MHz

NMR, and MS.

Experiment

Piperine. Grinded black pepper (12.5g, ) and placed in 100 mL round bottom

flask to reflux it with Ethanol (50mL, ) for 90 minutes. The resulting solution was filtered

out via Buchner porcelain funnel. The liquid was concentrated with stream of nitrogen

and a warm water bath. The resulting residue was dissolved in 10% by weight KOH in

ethanol (12.5mL, ). The solution was cooled in an ice bath, and distilled water was added

drop-wise (20mL, ). The produced precipitate was collected via vacuum filtration. For the

recrystallization, collected solute was dissolved in 3:2 Acetone: Hexane solution (5mL).

The crystalized product was collected via vacuum filtration (0.069g, 0.552%). mp 130-

131C;1H NMR (60MHz, CDCl3) (ppm) 1.620 (s, 6H), 3.8 (s, 4H), 5.972 (s, 2H),

6.542-6.986 (m, 6H), 7.267 (m, 1H);1H NMR (400MHz, CDCl3) (ppm) 1.2527-1.6868,

2.1723, 3.5259-3.6377, 5.9762, 6.4240-6.4605, 6.6934-6.7882, 6.8782-6.8983, 6.9023-

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6.9844, 7.2698-7.3999; IR (ATR) max(cm-1

) 3008.67, 2938.56, 2855.48, 1631.21,

1610.63, 1580.62, 1489.05, 1436.73, 1247.75, 1131.01, 1111.08; MS 285 (MW), 201,

173, 143, 115, 84, 77, 55, 41, 28.

Discussion

Piperine was isolated from the black pepper by refluxing the black pepper with

ethanol (95%), and dissolving the residue in 10% by weight KOH in ethanol. Isolated

crude product was purified by recrystallization. The crude product was dissolved in

approximately 5 mL of hot 3:2 ratio of acetone and hexane solution. To speed up the

recrystallization process, drops of cold distilled water were added as well. As a result the

recrystallized product yielded 0.552% of the starting material; the typical recoveries are

between 2.5% to 10% of the starting material. Most of the products could have been lost

during the recrystallization and while collecting the product from the solution to the

filter; not giving enough time for the solutes to crystallize will lead to loss of product as

well. One other loss of product may have occurred while forming a precipitate by adding

cold distilled water into the mixture of concentrated reflux solution and 10% KOH. If not

enough cold water was added, then the amount of product will be less than the theoretical

yield, or the expected amount. Moreover, loss of product may also have occurred while

transferring the precipitate into Hirsch funnel for vacuum filtration. To determine the

structure and the purity of the synthesized product, melting point, IR, NMR, and MS data

were analyzed.

To determine the purity of the isolated product, melting point of the product can

be compared with the melting point of the pure substance. The melting point of the pure

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piperine is 131-135C.6The collected melting point was 130-131C, which is close to

the pure substance, just a little less than expected. This result highly suggests that the

isolated piperine has an identical melting point property with the pure compound. To

further investigate the purity of the compound, IR data was collected.

IR has been used to analyze the functional groups of the isolated product and also

to check the purity of the isolated product. From the data, the peak at 3008.67cm-1

indicates the presence of hydrogen from benzene ring, the peak at 2938.56 cm-1

suggests

the presence of alkene (R2-C=C-R-H), and peak at 2855.48 cm-1

may suggest the

presence of C-H group. Furthermore, the peak at 1631.21 cm-1

indicates the presence of

carbonyl group attached to the tertiary amine group (O=C-N-R2). The peak at

1580.62cm-1

indicates the presence of benzene group, peak at 1489.05 cm-1

suggests the

presence of H2-C-R2 scissor bend group, and the peak at 1436.73 cm-1

indicates the

presence ofCH2- group. In addition, the strongest peak at 1247.75 strongly suggests the

presence of C-O vinyl group, and the peaks at 1131.01 cm-1

and 1111.08 cm-1

indicates

the presence of C-O bonding group. Surprisingly, the presence of alkene group with trans

isomerism (RHC=CHR) at approximately 1675 cm-1

is missing in the IR data. It is

unclear why it is missing a peak at that region when it should be there. Nonetheless, it is

difficult to determine whether or not the isolated product is piperine, since the rest of the

alkaloids that are in black pepper (piperanine, and piperettine) also share the common

functional groups of the piperine. Thus, to further analyze the structure of the isolated

product, the product was analyzed under two types of NMR instrument, 60MHz and

400MHz.

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To further analyze the structure of the isolated product of piperine, two different

NMR data have been collected: 1H 60MHz NMR, and 1H 400MHz NMR. Since three

different alkaloids that are found in black pepper consist different numbers of carbons

and hydrogens, NMR will help determine the isolated productsstructure among the three

different alkaloids (piperine, piperanie, and piperettine). According to the 1H 60MHz

NMR data, the isolated product has a peak at 1.620 ppm with approximately six

hydrognes, which could represent the hydrogens attached on the five-carbon ring

structure with nitrogen on it. A peak at 3.8 ppm with four hydrognes could suggest the

remaining four hydrognes on the five-carbon ring structure with nitrogen on it. The

chemical shift is higher than the previous six hydrognes due to the nitrogen atom nearby.

A singlet peak at 5.972 ppm with approximately two hydrognes indicates the hydrogens

from the carbon nearby two C-O groups. The chemical shift value is higher than expected,

since two oxygens are affecting the electron density of the carbon. The remaining seven

hydrogens are clustered around peaks at 6.542-7.267 ppm, these hydrogens could be from

the benzene ring and from the two trans isomerism alkene groups. Generally with higher

resolution, like using 400MHz NMR, the clustered peaks can be magnified and

chemically distinct hydrogens can be distinguished. By looking at the structure of the

piperine, there are total eleven chemically distinct hydrogens, and total 19 hydrogens.

For a better resolution of the NMR data, 1H 400MHz NMR data was collected.

Chuck of multeplet peaks at 1.2527-1.6868 ppm has total 6 hydrogens. This seems to be

representing the hydrogens attached on the para and meta side of the nitrogen carbon

ring. I believe these chunks of multiplet should be separated into two distinct integrations

of hydrogens, 4H and 2H. The peaks at 3.529-3.6377ppm with 4 hydrogens are

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representing the hydrogens attached on the ortho side of the nitrogen carbon ring. The

strongest peak at 5.9762ppm indicates the presence of two hydrogens on the carbon,

which is bonded to two oxygens. Because of these two oxygens, the -CH2 chemical shift

value was higher than the expected value. Next the huge chunk of multiplets at 6.5ppm-

7.4ppm in 60MHz NMR data is now broken down to five distinct multiplet peaks in

400MHz NMR. This huge chunk of multiplets ranging from 6.4240ppm to 7.3999ppm

can be divided into two categories: one with the benzene ring, and the other with the

alkene group. Since the normal NMR range for alkene group is from 4.5ppm to 6.5ppm,

any peaks within that range will be considered as the part of the alkene group. Anything

above 6.5ppm will be considered for the benzene ring group. I believe there are four

chemically distinct hydrogen groups in the alkene chain with four hydrognes. However,

the NMR data shows only two distinct hydrogen groups. Thus, I am guessing the

multiplet peaks at 6.6934ppm -6.7882ppm should be separated into three peaks with

integrated 1 hydrogen for each peaks. The rest of the peaks located above 6.5ppm,

corresponds to the hydrogens located on the benzene ring. Since the benzene ring of the

piperine consists three chemically distinct hydrogen groups, there should be three

different peaks, each with integration value of 1 hydrogen. In conclusion, there is a high

possibility that the isolated product is piperine, because the NMR structure seems to be

matching with the pure piperine structure with total 19 hydrogens.

Since black pepper consists three different types of alkaloids (piperine, piperanine,

and piperettine) and all of the alkaloids have different molecular mass, mass spectrum

will be the best way to analyze the isolated product. According to the data from the mass

spectrum, the isolated products molecular weight was 285 moles per gram. The highest

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peak (also known as the base peak) was 201m/z, which represents the fragment of the

piperine structure, which does not contain the O-C-O group. The next highest peak is

115m/z, which is 86m/z less than the base peak. With this result, I assume the fragmented

structure is the five-carbon ring containing nitrogen in it and the C=O group branching

out from the nitrogen. By analyzing each peaks and matching the fragmented structures

to it, as a result the data has constructed a piperine structure. This data result highly

suggests that the isolated product indeed is piperine.

From the IR, NMR, and the MS data, it is safe to conclude that the

isolated product is piperine. From the IR data no trace of impurity was

found, but it is difficult to determine whether or not the isolated product is piperine.

Since the rest of the alkaloids that are in black pepper (piperanine, and piperettine) also

share the common functional groups of the piperine, to further analyze the structure of

the isolated product, the product was analyzed under two types of NMR instrument,

60MHz and 400MHz. From the NMR, it was clear that there the isolated product is

piperine, because the NMR structure seems to be matching with the pure piperine

structure with total 19 hydrogens. Lastly, since black pepper consists three different types

of alkaloids (piperine, piperanine, and piperettine) and all of the alkaloids have different

molecular mass, mass spectrum will be the best way to analyze the isolated product.

According to the data from the mass spectrum, the isolated products molecular weight

was 285 moles per gram. By analyzing each peaks and matching the fragmented

structures to it, as a result the data has constructed a piperine structure. This data result

highly suggests that the isolated product indeed is piperine. Thus, it is safe to say that the

isolated product is piperine.

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Reference

1) McNamara, F., Randall, A., Gunthorpe, M.. Effects of piperine, the pungentcomponent of black pepper, at the human vaniloid receptor (TRPV1).Br J

Pharmacol. 2005March; 144()6: 781-790. doi:10.1038/sj.bip.0706040.

2) Ravindran, P.N., Kallupurackal, J.A.. Black Pepper Indian Institute of SpicesResearch, Kerala.

3) Padalkar, K.V., Gaikar, V.G.. Extraction of Piperine from Piper Nigrum (BlackPepper) by Aqueous Solutions of Surfactant and Surfactant+Hydrotrope

Mixtures Separation Science and Technoogy.43:11-12, 2008.

4) Mehmood, M.H., Gilani, A.H.. Pharmacological Basis for the Medicinal Use ofBlack Pepper and Piperine in Gastrointestinal DisordersJournal of Medicinal

Food. 13 (5) 2010, 1086-1096.

5) Dahmani, M., Et-Tauham, A., Al-Deyah, S.S., Hammouti, B., Bouyazor, A..Extraction of PiperineInternational Journal of Electro-Chemistry(5), 2010. pp.

1060-1069.

6) PiperineSigma-Aldrich.http://www.sigmaaldrich.com/catalog/product/aldrich/P49007?lang=en&region=

US