Isolation of Piperine from Black Pepper

Mitchell Cardow

Chem 213, Spring 2018

Introduction

The extraction of natural products is important to the medicine and drug industries because many

of the current drugs on the market are synthesized from natural products.2 Extraction of compounds form

a natural product is a less expensive and more efficient method of obtaining a chemical to be used in drugs

rather than the synthesis of a compound.2 The natural products extracted from living organisms are often

used as a starting point to discover new compounds that can be used in drug development.4 The extraction

done in this experiment was the extraction of Piperine (Scheme 1) from the natural compound, black

pepper.

Scheme 1. Chemical structure of Piperine.1

Piperine is an alkaloid in black pepper, which comes from peppercorns. Black pepper is spicy due

to the presence of piperine in the natural compound.3 Piperine is an important natural product to be

extracted because it can be used many different ways in the medicine and drug industries. Piperine can be

useful to treat several various types of cancers, since it is one of the bioenhancers to date. A bioenhancer

does not have its own pharmacological effect when used in a dosage alone but it increases the

bioavailability and bio efficiency of a drug when combined in a dosage.3 So piperine ca be used to

increase the affectability of drugs used to reduce the growth of tumors in the body. 3 Piperine can also be

used to help stroke victims learn how to swallow food again due to its chemical structure it is a very
pungent smelling, and tasting chemical which increases the biological response of swallowing and

sniffing.1 Piperine has many uses in the medicine and drug industry but can also be used as an insecticide

that can be used against mosquito larvae to reduce mosquito population.1

The method of isolation of Piperine used was to reflux finely grounded black pepper in ethanol,

then let the piperine precipitate out with ten percent potassium hydroxide by weight in ethanol in the fridge

for a couple of days.1 The piperine product that precipitated out overnight was then purified using

recrystallization to produce pure piperine in good yield.3 There are many other ways to extract piperine

from black pepper using either glacial acetic acid or dichloromethane which is still refluxed and then still

precipitated out of solution, but all methods of extraction produces a similar yield of product so this

experiment used ethanol to reflux the black pepper to isolate piperine.3

Purpose

The purpose of this experiment was to isolate piperine from black pepper. Recrystallization was

used to purify the solid product. Recrystallization is used by organic chemists to purify solids with trace

amounts of impurities trapped within the crystals. The isolated piperine product was then characterized

by melting point, IR, mass spectrometry, 60 MHz and 400 MHz 1HNMR analysis.

Results and Discussion

Black pepper contains piperine and other alkaloids like piperanine and piperettine, piperine had to

be isolated from these other alkaloids.1 Piperine was isolated by reflux of black pepper with ethanol, which

was then refrigerated overnight in ten percent potassium hydroxide in ethanol and water to precipitate out.

The products was purified by recrystallization and was characterized by melting point, IR, MS and

1HNMR analyses. Piperine was refluxed with ethanol because it is soluble in it and when heated the

piperine went into solution while the other alkaloids with a higher molecular weight and less polar

structure they stayed as a solid in the black pepper. The solid black pepper was filtered out to leave the

remaining solution of piperine and ethanol which was concentrated leaving a brown oil. To get piperine
to precipitate out potassium hydroxide in ethanol with water was used and since piperine is only slightly

soluble in water it had to be cooled to increase the amount of piperine that were to fall out of solution.

The solid that precipitated out of the solution was purified using recrystallization since it was a

solid product and only had a trace amount of impurities, this made recrystallization a suitable means of

purification. The recrystallization was done with warm acetone to completely dissolve the solid piperine

and then water was added slowly so piperine would slowly recrystallize to form pure piperine solid.

The purification afforded shiny yellow flakey crystals with a yield of 2.3% which is a good yield

since there is only fractional amounts of piperine present in black pepper.1 The percent recovery from the

recrystallization of pure piperine product was 85% recovery, this suggests that trace amounts of impurities

were present in the crude crystal product. The melting point of the pure piperine product had a range of

129.8-132.9 oC which was accurate compared to the expected melting point of 131.5 oC.1 This suggests a

pure product because if, the other major alkaloid present in black pepper, piperanine was present in the

product the melting point would be depressed since the melting point of piperanine was 80 oC.6

The 400 MHz 1HNMR graph (Figure 2) showed the TMS peak is at 0 ppm and an acetone peak

is present at 2.18 ppm and a water peak at 1.25 ppm, which both of these could have been present in the

NMR tube. The most upfield doublet peak with a range of 1.66-1.58 ppm and an integration value of 5.99

represents the six protons furthest from the nitrogen on the piperdine ring, these protons are the most

upfield because their electron density is the most shielded on a sp3 hybridize carbon. The doublet peak

with a range of 3.63-3.51 ppm with an integration value of 3.61 represents the four protons closest to the

nitrogen on the piperdine ring and are still far upfield because they are on a sp3 hybridized carbon. The

singlet peak at 5.97 ppm with an integration value of 1.75 represents the two protons in between the two

oxygen atoms these are also somewhat upfield since they are on a sp3 hybridized carbon and their electron

density is shielded by having more s character. The doublet peak with a range of 6.46-6.41 ppm with an

integration value of .81 represents the single hydrogen on the aromatic ring adjacent to the alkene chain

and another proton on the aromatic ring since because it is the most shielded sp2 hybridized carbon on
molecule. The multiplet peak with a range of 6.78-6.72 ppm with an integration value of 2.70 represents

the three hydrogens on the alkene chain closest to the aromatic ring. The other proton on the alkene chain

next to the carbonyl group is represented by the most downfield multiplet peak with a range of 7.41-7.37

ppm and an integration value of .76 because its electron density is the most removed by the carbonyl

making it desheilded causing it to be the most downfield The doublet peak with a range of 6.89-6.88 ppm

with an integration value of .788 represent the proton on the aromatic ring in-between the ester group and

the other carbon on the aromatic ring with a proton since it is the only doublet in the aromatic region that

is the least shielded of its electron density. The last singlet peak at a 6.98 ppm with an integration value

of .765 represents the lone proton on the aromatic ring in between the ester group and the alkene chain

since it is the only singlet proton in the aromatic region. If there was any other alkaloid present like

piperanine there would be more peaks upfield representing the more shielded protons on the more sp3

hybridized carbons present on the molecule. Therefore, the 1HNMR analysis of piperine shows a highly

pure product other than the acetone that could have been present in NMR tube and water that was not fully

evaporated from the product.

The IR analysis (Figure 3) also provided evidence that a pure product was obtained since there is

a peak at 3478 cm-1 which represents the carbon hydrogen bond on the aromatic ring. The signal at 2922

cm-1 which represents the carbon hydrogen bond on the alkene section of the molecule. The next peak

identifies at 1627 cm-1 represents the carbon carbon double bonds on both the aromatic ring and the alkene

chain on the piperine molecule. The peak at 1608 cm-1 represents the secondary amide functional group

present on the priperine molecule. The signal at 1491 cm-1 represents the carbon hydrogen scissor bond

that is present on all of the sp3 hybridized carbons on the molecule. The peak at 1156 cm-1 represents the

carbon bonded to oxygen as for the two ether functional groups present in the piperine molecule.

Therefore, IR analysis proves that a pure piperine was obtained.

The last anlaysis of the piperine crystal product was mass spectrometry (Figure 4) which also

provided significant evidence that a pure piperine product was obtained since the molecular weight shown
by the mass spectrometry was 285 grams per mole which matches with the literature data given.1 If there

were any of the piperanine alkaloid still present in the crystal product the molecular weight would be

larger at 287 grams per mole. Therefore, the MS also shows that a pure product was produced.

In conclusion, the isolation of piperine from black pepper produced a highly pure crystal product

with a yield of 2.3% and the IR, MS, and 400 MHz 1HNMR show that the product had a high purity which

was suggested by the narrow range of the melting point of 129.8-132.9 oC. The MS displayed a molecular

weight of 285 grams per mole which is accurate compared to the literature data.1 The IR only had peaks

for the ether and secondary amide functional groups with the addition to the aromatic ring, alkene chain

and sp3 hybridized carbons. The NMR data showed the correct number of protons present in piperine, and

all of this data combine provide sufficient evidence that a pure piperine product was obtained. Error in

this experiment could have occurred from the escape of vapors during reflux which could lower the yield

of product. So, in future experiments a different solvent during reflux could be used like dichloromethane,

to extract more piperine from the black pepper to produce a higher yield. Also, an increased time for reflux

to extract as much piperine as possible from black pepper to alos produce a higher yield.

Experimental

Piperine. Grinded black pepper (12.5g) was refluxed in ethanol (10 mL) for 90 minutes. Upon completion,

the mixture was vacuum filtered. The residue was dissolved in 10% by weight potassium hydroxide in

ethanol (12.5 mL). Water (100mL) was slowly added and was sealed, and then refrigerated till the next

lab period. The precipitate was vacuum filtered until completely dry. Then recrystallized (acetone, water)

which afforded piperine (.289g, 2.31%) as a yellow shiny powder. MP 129.8-132.9 oC; 1HNMR (400

MHz, CDCL3)  (ppm) 7.41-7.28 (m, 1H), 6.98 (s, 1H), 6.89-6.88 (d, 1H), 6.78-6.72 (m, 3H), 6.46-6.41

(d, 1H), 5.97 (d, 2H), 3.63-3.51 (d, 4H), 1.66-1.58 (d, 6H); IR max (cm-1) 3468, 2922, 1627, 1608, 1491,

1156; MS(m/z) 285.

(1) Epstein, W.W.; Netz, D.F.; Seidel, J.L. Isolation of piperine from black pepper. J. Chem. Ed. 1993,

70, 598‐599.

(2) Chintoju, N.; Kondru, P.; Kathula, R. L.; Remella, R. Importance of Natural products in Modern

History. Res. Rev. J. Hosp. Clin. Pharm. 2015, 1, 5-10.

(3) Shingate, P. N.; Dongre, P.P.; Kanur, D. M. New Method development for extraction and isolation of

Piperine. Int. J. Pharm. Res. 2013, 8, 3165-3170.

(4) Ji, H. F.; Li, X. J.; Chang, H. J. Natural Products and Drug Discovery. EMB. Rep. 2009, 10.3, 194-

200.

(5) Piperine; SDS No.P49007; Sigma-Aldrich. www.sigmaaldrich.com (accessed 4/16/18)

(6) Piperanine; SDS No. SMB00097; Sigma-Aldrich. www.sigmaaldrich.com (accessed 4/16/18)