The Pennsylvania State University

Isolation of Piperine from Black Pepper

Raad Bassam A Mulla
Chem 213-02
FFR#1(revised)






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Introduction:
Natural products are compounds found within natural source
1
, such as plants and animal.
However, according to Mcmurry
2
, natural products are secondary metabolites, which are small
molecules living in the organism without affecting it in a major way, like nucleus acids, which
are essential for the survival of the organisms
19
. The importance of natural products convey on
its variety of uses, since natural products are used in pharmaceutical industry, food production
and pesticides
3
. The techniques commonly used to purify natural products are: Column
chromatography, extraction and distillation. In this paper, the efficiency of the extraction method
in synthesizing natural products will be evaluated.
Extraction is a very common laboratory procedure used when isolating or purifying a
product.
5
its one of the ancient techniques that humans developed to extract liquid from solid
natural product
6
. The most famous example is the making of coffee, in which water is used as the
liquid
6
. The caffeine, because of its molecular weight and polarity, will dissolve in the hot water
and be removed. Solid-liquid extraction will be discussed today regarding extracting piperine
from black pepper. Black pepper is a common hot spice. In addition, its quite used in the field of
traditional medicine. For an example, it is used for digestive disorders, such as indigestion,
vomiting, diarrhea, and flatulence
7
. Furthermore, in modern medicine, its used in the treatment
of cigarette withdrawal symptoms
10
. Pepper is made up of 5-9% of alkaloids that include
piperine, which is responsible for the hot taste of the pepper
4
. Piperine shows low solubility in
water, but ethanol and other organic solvent are suitable for solving this substance. In recent
decades, piperine came into the focus of pharmaceutical research because its antibacterial,
antioxidant, anti-inflammatory
11
.
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Figure 1. Chemical structure of Piperine
18


The purpose of this experiment was to isolate Piperine form black pepper via solid-liquid
extraction using ethanol and KOH. Recrystallization was used to purify the product.
1
HNMR
(400 MHZ, 60 MHZ), IR, MP and MS were utilized to identify the product and determine its
purity.
Experimental:
Piperine. Grinded black pepper (12.61g) was refluxed with ethanol (50 ml) for 90 minutes.
Upon completion, the solution was vacuum filtered using Buchner porcelain funnel. The solvent
was removed, yield a white residue. The residue was dissolved in a 10% by weight KOH in
ethanol (12.5 ml). Upon complete dissolution of the residue, H
2
O (100 ml) was added to form a
yellowish precipitation. The precipitated produect was collected using vacuum filtration and
washed with ether (8 ml). Recrystallization (3:2 hexane/acetone) gave piperine (1.081g, 8.07%)
as yellowish crystals. Mp 125-131
o
C;
1
HNMR (400 MHz, CDCl3) (ppm) 7.4(q, 1H), 6.9(s, 1H),
6.89(d, 1H), 6.7(m, 3H), (d,1H), 6.40(d, 1H), 5.9(s, 2H), 3.5(d, 4H), 1.64(m,4H), 1.59(m,4H) ;IR
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(ATR)
v
max(cm
-1
) 3469.39, 2862.02, 1625.78, 1608.49, 1441.9, 1156.5 ;MS(M/Z), MW=285,
N-C
5
H
10
=201.
Results and Discussion, Conclusions:
Isolating piperine from black pepper was done by extracting it with ethanol and potassium
hydroxide. After that, the product was purified using recrystallization by dissolving it in a 3:2 hot
hexane/acetone.
1
H NMR, IR, 400 H NMR, MP and MS were utilized to identify the desired
product and verify that no major contaminates were present in the product. Piperine is insoluble
in water, slightly soluble in alcohol, and soluble in chloroform, benzene, and acetic acid.
According to (Epstein, W., Netz, D., & Seidel, J.), there were five major alkaloids in black
pepper. After refluxing, two layers were observed, solid and liquid. The liquid layer was the
polar one. Pepperine is the least polar. This leaves four alkaloids left. Another major factor that
determined which alkaloid was going to be present in the liquid phase was MW, in which the
lower the MW, the more likely for the alkaloid to dissolve in the liquid layer. Looking at the four
alkaloids MW: piperine (MW=285), piperanine (MW=311.37), piperettine (MW=287),
piperacid (MW=355), we find that piperine had the lowest MW. Lastly, piperine was
recrystallized to eliminate any contamination.
From the IR spectra, there were strong signs of the presence of several functional groups that
made up piperine. The most notable was the carbonyl group at 1606.49 cm
-1
. Furthermore, Ar-H
stretch at 3469 cm-1, R
2
NC=O stretch at 1625cm
-1
, ether stretch at 1156 cm
-1
and C-H at
2862.02cm
-1
. These characteristics show strong evidence that the desired product has been
obtained. In addition, it didnt show great contamination.
From the
1
H NMR spectra of piperine, peaks were difficult to be identified without the 400 H
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NMR spectra. However, it was helpful showing that the piperine was clean, except for water,
which didnt dry completely after recrystallization. To be noted, the annotation on the
s
H NMR
was done after observing 400 H NMR.
From the 400 H NMR spectra of the product, 9 key peaks were observed representing 9
chemically distinct hydrogens, namely H
a
, H
b
, H
c
, H
d
, H
e
, H
f
, H
g
, H
h
, H
i
and H
2
O. The total
number of hydrogen atoms was 19 .Hydrogens A and B, multiplets, located at (1.5902-1.6701)
ppm were methylene bridge hydrogens, which typically peaks at (1.2-1.4), but due to the N atom,
the peaks moved to down field. Hydrogen C, multiplet at 3.55 ppm and having an integration
value of 4 was consistent with the methylene bridge in the N-ring. Hydrogen D, a singlet, was
also a methylene bridge but the two oxygen atoms right next to it, increased its ppm value by
almost 5 ppm. Hydrogen E, a doublet, represent the methine group next to the N-ring. Hydrogens
F, G, H and I all represented methine groups. Although the peaks of H
f
werent that clear, they
were very close to be identified as doublet of doublets, which gave the notion that piperine was
para-substituted, which was partially true because it was meta-substituted too, and this junk of
peaks at H
f
proved the conclusion. The last peak was the H
2
O peak, a singlet, which was at 4.8
ppm, due to the fact that the time between the end of purification and NMR testing was shorter
than required.
The Melting point was a strong indication of peperine presence, it was recorded at 125-
131
o
C, which is a range that contains the literature value (
130oC
).
Finally, the MS was also very helpful to proving that piperine was obtained since it
recorded the MW as 285, which is the exact value obtained in a different literature
12
.
Overall, the experiment was very successful. Although the recovery percentage is small
(8.07%), but the fact was that s alkaloids represented only 9% of the total mass of the pepper,
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can explain the low percentage. In addition, the NMR was helpful in identifying different
hydrogens but with uncertainty. Thats why a 400 NMR was needed. To improve the results in
the future, a nitrogen stream should be used to dry the product to eliminate any H
2
O contaminant
before running any test. Also, an increase in the time of the refluxing process would produce
better percentages of recovery.
References:
1
Samuelson G (1999). Drugs of Natural Origin: A Textbook of Pharmacognosy. Taylor &
Francis Ltd,.ISBN 9789186274818.
2
McMurry, John. Organic Chemistry. Belmont, CA: Thomson, 2008. pp. 164.
3
Leung, A. Y., Ed. Encyclopedia of Common Natural Ingredients Used in Food, Drugs and
Cosmetics; John Wiley
4
Epstein, W., Netz, D., & Seidel, J. (1993). Isolation of piperine from black pepper
American Chemical Society. pp 598-599. Revised by Sheryl Rummel (online)
http://pubs.acs.org/doi/pdf/10.1021/ed070p598
5
J. Chem. Ed. 1993, 70,598
6
Bioorganic & Medicinal Chemistry Letters, Volume 23, Issue 20, 15 October 2013, Pages
55525557
7
Tetrahedron , Volume 64, Issue 49, 1 December 2008, Pages 1112911135
8
Extraction Theory and General Procedure (Adapted from Mohrig, pp. 57-64, 72-77.) (online)
http://academics.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Tec
hniques/Extraction/extraction_n.html
9
Peperine, pierettine, piperanine piperacid. Sigma Aldrich.
10
Natural Standard: The Authority on Integrative Medicine. www.naturalstandard.com
1
1 F. Tausig, JI Suzuki and RE Morse; 1956; Food Technology, Volume 10; pp. 151
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154.
12
K. G. Raghuveer and Ananthakrishna, S. M.; 1980; Journal of Food Science and
Technology, Volume 17; pp. 268-272
13
P.V. Karsha and O.B. Lakshmi; 2010; Indian Journal of Natural Products and
Resources, Volume 1; pp. 213-215
14
R.S. Vijayakumar et al.; 2004; Redox Report, Volume 9; pp. 105-110
15
J. S. Bang et al.; 2009; Arthritis Research & Therapy, Volume 11; pp. 1-9
16
K. Srinivasan; 2007; Critical Reviews in Food Science and Nutrition, Volume 47; pp.
735-748
17
A. B. Wood et al.; 1988; Flavour and Fragrance Journal, Volume 3; pp. 55-64

18
J Med Microbiol February 2011 vol. 60 no. 2 223-229 (online)
http://jmm.sgmjournals.org/content/60/2/223/F1.expansion
19
Richard J. P. Cannell, How to Approach the Isolation of a Natural Product, Vol. 4 Natural
Products lsolatron, Edited by R J P Cannell 0 Humana Press Inc , Totowa, NJ
(online)
http://catbull.com/alamut/Bibliothek/How_to_Approach_the_Isolation_of_a_Product.pdf










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