ISOLATION AND IDENTIFICATION OF CAPSAICIN AND

ALLIED COMPOUND IN CHILLI

BY D. N. AWASTHI AND B. P. SINGH

(National Botanic Gardens, Lucknow)

Received Dec¢mber 18, 1972

(Communicated by Dr. L. D. Kapoor, F.A.Sc.)

ABSTRACT

The isolation and identification of capsaicin from fruits yielded two

substances. The white crystalline compound, which, after U.V. and
I.R. examination, exhibited the maximum absorption at 206 m/, and the
characteristics of long chain-n-alkyl, adjacent to a carboxyl group, was
identified as palmatic acid. The molecular formula was, later on, con-
firmed to C19HasO.,. The second, which was oily in nature, exhibited
the presence of capsaicin due to its maximum absorption in U.V. at 230
and 281-5 m/,. It also represented all the properties of capsaicin oleo-
resin, when compared chemically as well as physically with the commercial
oleoresin of capsaicin E. Merck Darmstadt, but could not be crystallized
even after chromatography over neutral and acidic alumina with various
solvents or after subjecting it to high vacuum distillation (10-4 mm).

I. INTRODUCTION

CHILLI (Capsicum annuum L.) is an important condiment grown for its fruits
in the plains and hills of India. It is rich in capsaicin (Claus, 1961 ; Balbaa,
1968). Capsaicin, responsible for pungency in chilli is a colourless, crystal-
line compound and ranges from 0-14 to 0.22°/o in chilli varieties (British
Pharmaceutical Codex, 1963). Methods for isolation, extraction and esti-
mation of capsaicin have been modified from time to time (Lapworth and
Royle, 1919 ; North, 1949 ; Karawya, et aL, •967). Sometimes allied
compounds are also found. Therefore in this experiment a modified method
for isolation and identification of capsaicin or its allied compound was
studied.

II. MATERIALS AND METHODS

The method of isolation and identilication, as described by kapworth

and Royle (1919) and Spath and Darling (1960), was very lengthy and time-
196
 I~olation & Identification of Capsaicin & Allied Compound in Chilli 197

consuming. The same has, therefore, been modified to extract capsaicin

easily and with more recovery.

After a number of preliminary experiments on solubility and related

chemical properties, the following method was applied to isolate and identify
the capsaicin or its allied compounds, if any.

1,250 gm of well-powdered chilli fruits were exhaustively soxhleted

with solvent ether. The fruits extract, thus obtained, was extracted three
to four times with 15 ml of 5 per cent KOH, which dissolved all the capsaicin
in the alakline solution.

The alkali solution containing capsaicin was divided in two parts. In

one part CO2 was passed till the solution had no colouration with phenolph-
thalein indicator, while in the other, sufficient quantity of NH4CI was added
and slightly warmed. This process converted all KOH into ammoniacal
form. Capsaicin was not soluble in both the solutions, either treated with
CO2 or NH4C1. Later on, these solutions were extracted separately with
ether and the ether solutions were concentrated. Residues of the extracts
were diluted with 50 mls methanol (norit) separately, filtered through What-
man 40 filter-paper and concentrated. The concentrates were kept in the
cold with dilute methanol and the resulting crystalline substances were
filtered under suction, and the crystallisates were dried under vacuum. The
mother liquors were again treated with charcoal and some more of the crystal-
line compound could be obtained in the same manner. To get the sub-
stances in a pure, white and crystalline form, fractions collected from both
the procedures, having similar melting points were mixed and recrystaUised
several times till there was no rise in the melting point. After final crystallisa-
tion the compound was purified by passing through neutral and acidic alumina
columns.

The mother liquors, thus collected and freed from solvent, were oily
in nature. This oily residue did not yield any crystalline compound even
after chromatography over neutral and acidic alumina with various solvents
or after subjecting it to high vacuum distillation (10-4) mm.

The crystalline compound was identified using U.V. spectrum, Infra-

red spectrum, melting point and microanalytical data. The mother liquor
was also examined spectroscopically.
Acad.--B 2
198 D. N. AWASTHI AND B. P. SINGH

III. RESULTS

The isolated pure, white and crystalline compound of 0.700 gm and

2-00 gm of oily mother liquor, which could not be crystallised, were exa-
mined for identification of capsaicin or its allied compound as given below:

The Crystalline Compound

U.V. spectrum.--In methanol, the compound shows the maximum
absorbanee at 206 m/~ and the lowest at 227 mt~ and 285 m/z (Fig. 1). This
confirms that the compound under examination is other than capsaicin,
which shows strong peaks at 230m/~ and 281.5tz.

o.o

o.i
0.2 tl~ . . . . . . . -- /
, ,,f
0.3 I /

0.5
0.~, I
~0.7
q

o
q
1,0
I.I
1.2
f,3
1.4
1.5
19(3 2OO 210 2::'0 230 240 2SO 260 270 2eO 2 ~ 300
WAVELENGTH (MILt.IMICRONS,)

FIG. 1. Ultra-violet spectrum for the ¢.-/~al line compound isolated from chilli fruits.

Infra-red spectrum.--As shown in Fig. 2, the compound is optically

inactive. In the infra-red regions, peaks are at 3000, 2500, 1700, 1435, 1296
and 940 cm-1. These obviously indicate a carboxylic acid dimer, and the
evenly spaced absorptions in the range 1350-1180cm-1 are characteristic
of long-n-alkyl chains. Further, the band 1418 cm-1 indicates a CH2 group-
ing adjacent to a carboxyl group. The bands 733 and 725 cm-~ indicate
methylene chain rocking. The bands between 1350 and l l 8 0 c m -~ show
long-chain-n-alkyl compounds and the evenly spaced bands are characteristic
of chain length.
 Isolation & Identification of Capsaiein & Allied Compound in Chilli 199

According to the relation between the number of bands and the chain
length, there were six bands with intervals of 20 cm-1. The compound thus
appears to be palmatie acid which was confirmed through a mixed melting
point determination as well as superimposable infra red spectra.

4000 3000 2000 150< CM "1 JO00 vo0 800 700

IOO

~o
z
~ 6e
"x
/h
ffl

V
z 4C

20

O
3 4 5 6 7 ~ 9 I0 II 12, 13 14 15
WAVELENGTH (MICRONS)

FIG. 2. Infra-red spectrum for the crystalline compound isolated fiom chilli fruits.

Melting point.--The melting point of the compound remained constant

between 56-57 ° C.

Micro-analysis.--The analytical data confirmed to the molecular for-

mula C,H3302.

The Mother Liquor

The mother liquor yielded 2.00 grn of oily substance. This was exa-
mined in U.V. spectra at 230 m/~ and 281.5 m/~ for the presence of capsaicin.
The results obtained clearly showed the presence of capsaicin (Fig. 3) in
strong concentration. This substance represents all the properties of cap-
saicin oleoresin and should, therefore, be considered as the oleoresin of
eapsaicin. The oleoresin was then tried for crystallisation from methanol
and n-hexane, but the results were not conclusive and definite. The sub-
stance, after passing through acidic and neutral alumina columns and sub-
sequent crystallisation, also gave unsuccessful results. It was further distilled
in high vacuum (10 -4 mm) and then tried for crystallisation, but the attempts
were again not successful.
200 n . N . AWASTH! AND B. P. SINGH

IV. DlSCUSSlON
The isolation and identification of capsaicin from fruits yielded two
substances. The white crystalline compound, which, after U.V. and I.R.
examination, exhibited the maximum absorption at 206 m~ and the charac-
teristics of long chain-n-alkyl, adjacent to a carboxyl group, was identified
as palmatic acid. The molecular formula was, later on, conformed to
C19HasO2. The second, which was oily in nature, exhibited the presence of
capsaicin due to its maximum absorption in U.V. at 230 and 281.5 mtz. It
also represented all the properties of capsaicin oleoresin, when compared
chemically as well as physically with the commercial oleoresin of capsaicin
E. Merck Darmstadt, but could not be crystallised even after chromato-
graphy over neutral and acidic alumina with various solvents or after subject-
ing it to high vacuum distillation (10 4 mm).

0.0 .. / . ~ ::;'-':.~- . . . . . . . .
o,,

0,2 ....
" - " ~ .. ' ................... ' ,f//'"

0.3 i

o.5
O,e
i /
/ : /"
'

//
°" /

< I.° /
i

1.2 /

i.c_ 200 250 300 350 390

WAVELENGTH (MILLIMICRONS)

FtG. 3. Ultra-violet spectrum for the Oleoresin of capsaicin isolated from chilli fruits.

V. ACKNOWLEDGEMENTS

Authors are grateful to Dr. L. B. Singh and Dr. V. N. Sharma of National

Botanic Gardens, Lucknow. Authors are also thankful to Dr. L. D. Kapoor
for reading the, m:~nuscriot.
 Isolation & Identification of Capsaicin & Allied Compound in Chilli 201
Balbaa, S. I., Karawya,
M. S. and Girgis, A. N.
British Pharmaceutical Codex
Claus, E. D.
Karawya, M. S., Balbaa, S. I.
Girgis A. N. and
Youssef, N. Z.
Lapworth and Royle, F. A . . .
Ncrth, H.
Spath, E. and Darlirg, S. F.
VI. REFERENCES
" The capsaicin content of Capsicum fruits at different stages of
maturity," Lloydia, 1968, 31, 272-74.
The Council of Pharmaceutical Society of Great Britain,
London, 1963.
Pharmacognosy, 4th ed., Henry, Kimpton, London, pp. 277,
1961.
" A micro-method for the determination of capsaicin in Capsicum
fruits," Analyst (Lond.), 1967, 92, 581-83.
" The constitution of capsaicin," J. Chem. Soc., 1919, 115,
1109-16.
"Colorimetric determination of capsaicin in oleoresin of Cap-
sicum." Analyst. Chem., 1949, 21, 934-36.
"Synthesis of capsaicin," Acta. physiol, hung, 1930, 11, 1937-
49.