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High-Performance Liquid Chromatography Analysis of Capsaicin Content in 16

Capsicum Fruits from Nepal

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DOI: 10.1089/jmf.2008.0187 · Source: PubMed

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JOURNAL OF MEDICINAL FOOD
J Med Food 12 (4) 2009, 908–913
Short Communication
# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089=jmf.2008.0187

High-Performance Liquid Chromatography Analysis of Capsaicin

Content in 16 Capsicum Fruits from Nepal
Bindu Thapa,1 Natasa Skalko-Basnet,1,2 Akihito Takano,3 Kazuo Masuda,3 and Purusotam Basnet1,4
1
The School of Pharmaceutical and Biomedical Sciences, Pokhara University, Pokhara, Nepal;
2
Department of Pharmacy and 4The National Research Center in Complementary
and Alternative Medicine, Faculty of Medicine, University of Tromsø,
Tromsø, Norway; and 3Showa Pharmaceutical University, Machida, Tokyo, Japan

ABSTRACT Capsicum fruit, a popular spice as chili pepper, is an important ingredient of the formulations used in
traditional medicines. Moreover, Capsicum fruit is listed as an official drug in several pharmacopoeias. Capsaicin, the most
abundant component in Capsicum fruit, exhibits its therapeutic and adverse effects in a dose-dependent manner. Therefore, the
known capsaicin content is the prerequisite for optimizing any formulation based on Capsicum fruit as a crude drug. We
studied 16 samples of Capsicum fruits grown at different altitudes in Nepal and determined their capsaicin content by high-
performance liquid chromatography. The capsaicin content was found to range from 2.19 to 19.73 mg=g of dry weight of
Capsicum fruits. Capsaicin content in pericarp was found to be higher than in seeds. No correlation was found between the
shape or size of the fruits and its capsaicin content. Our findings indicate that many of the formulations prepared from
Capsicum fruit, even as described in pharmacopoeias, may vary in their strength, therapeutic activity, and possible side effects
if the capsaicin content in Capsicum fruit is not standardized.

KEY WORDS:
capsaicin
Capsicum fruits
Capsicum tincture
high-performance liquid chromatography
pain

Solanaceae

INTRODUCTION Capsaicin, one of the major components found in the most

varieties of Capsicum fruits, is used as a therapeutic agent,
T he fruits of the genus Capsicum (Family Solana-
ceae) vary widely in size, shape, flavor, color, and
pungency. Capsicum fruit has been used worldwide as chili
and its main site of action is the transient receptor potential
vanilloid 1 receptor, which is expressed in primary sensory
neurons and vagal nerves.5 A hot and burning sensation is
peppers. It has also been used for centuries as a medicine in
elicited by capsaicin in a dose-dependent manner. However,
Ayurvedic preparations as an oil extract and is one of the
repeated and prolonged application of capsaicin causes the
major ingredients in Mayan therapeutic remedies. It is also
desensitization of sensory neurons, which contributes to
used as folk medicine in different tribes and ethnic com-
relief of pain.6,7
munities worldwide. In Western medicine, it is used as a
Capsaicin is widely applied clinically in painful diabetic
rubefacient in the form of Capsicum tincture. Pungency, the
neuropathy, chronic post-herpetic neuralgia, rheumatic dis-
organoleptic sensation of heat, is a major quality-determining
eases, post-mastectomy pain syndrome, cluster headache,
factor in Capsicum and is caused by the presence of one
hypersensitive vesical syndrome, hematuria syndrome,
or more of the 14 alkaloid compounds known as capsaici-
psoriasis, notalgia paresthetica, and painful panniculitis.7–10
noids.1,2 Capsaicinoids are acid amides of vanillylamine and
Capsaicin is used in the form of non-prescription (in the
C9–C11 branched-chain fatty acids. The two major capsai-
United States) or prescription (in the European Union)
cinoids present in the most varieties of Capsicum fruit are
topical analgesia.11
capsaicin [(E)-8-methyl-N-vanillyl-6-nonenamide] and di-
Although various methods have been reported to deter-
hydrocapsaicin (8-methyl-N-vanillyl-6-nonanamide), which
mine the capsaicin content3,4,12,13 and different varieties of
make up more than 91% of the total content of capsaici-
Capsicum powder are widely consumed all over the world,
noids.2–4
including Nepal, there are no data available on capsaicin
content of Nepalese Capsicum fruits. Some studies sug-
Manuscript received 17 July 2008. Revision accepted 6 December 2008. gested that capsaicin content varies in Capsicum fruits ac-
cording to the environment and genotypes.14,15 Our main
Address correspondence to: Purusotam Basnet, The National Research Center in Com-
plementary and Alternative Medicine, Faculty of Medicine, University of Tromsø, 9037
objective was to determine the capsaicin content in the
Tromsø, Norway, E-mail: purusotam.basnet@uit.no widely used different types of Capsicum fruits found in

908
 HPLC ANALYSIS OF CAPSICUM FRUITS 909

Table 1. Capsaicin Content of Varieties of Capsicum Fruits Collected from Different Places in Nepal
Sample (voucher Capsaicin
number) Latin name Local name Place (date of collection) content (mg=g)a
A (312) C. annum Lwang Khursani Chitwan ( July 11, 2004) 10.27  1.06
B (313) C. annum Marchai Sukrabare Hatbazar, Biratnagar 3.60  0.71
(October 29, 2004)
C (314) C. annum Jire Khursani Tharpu, Tanahun (August 25, 2004) 8.91  0.39
(var. glabourculum)
D (315) C. annum Suryamukhi Malepatan, Pokhara ( June 24, 2004) 5.21  0.35
(var. Suryamukhi) Khursani
E (316) C. frutescens Dalle Khursani Parsevati, Syanja (October 10, 2004) 11.71  1.13
F (317) C. annum Khursani Gorkha ( July 25, 2004) 4.77  0.38
G (318) C. annum Chilli Silugudi (bought from Pokhara) 4.97  0.21
( June 20, 2004)
H (319) C. annum Khursani Methavurung, Syanja ( July 12, 2004) 6.01  0.83
I (320) C. annum Jire Khursani Bagmara, Kaski (October 15, 2004) 6.88  0.24
(var. glabourculum)
J (321) C. annum Khursani Khairenitar, Tanahun ( July 11, 2004) 4.54  0.86
K (322) C. frutescens Jyanmara Khursani Dakshinkali, Kathmandu ( July 11, 2004) 3.14  0.45
L (323) C. annum Nangi Marchai Rani, Biratnagar (October 25, 2004) 8.14  0.45
M (324) C. annum Khursani Jiling, Trishuli (October 07, 2004) 2.83  0.10
N (325) C. annum Khursani Bagmara, Kaski (October 20, 2004) 2.19  0.22
O (326) C. annum Nepali Khursani Lubu, Lalitpur (December 05, 2004) 2.24  0.11
P (327) C. annum Khursani Pidikhola, Syanja (August 12, 2004) 19.73  1.61
E (316)b C. frutescens Dalle Khurseni Parsevati, Syanja 4.34  0.01
K (322)b C. frutescens Jyanmara Khursani Dakshinkali, Kathmandu 0.98  0.13
M (324)b C. annum Khursani Jiling, Trishuli 0.53  0.24
O (326)b C. annum Nepali Khursani Lubu, Lalitpur 0.63  0.13
a
The quantity of capsaicin (mg) in Capsicum fruit (g of dry weight) was expressed as mean  standard deviation (n ¼ 3). The data were obtained by calculating the
HPLC peak area with the correlation equation (y ¼ 242,906x  11,177) and correlation coefficient (R2 ¼ 0.999).
b
Dried seeds of these samples were used for the determination of capsaicin content.

Nepal in order to utilize them as therapeutic agents with
minimal side effects. Determination of the capsaicin content
in each Capsicum fruit is needed to predict the potency and
efficacy of topical Capsicum tincture and oral Capsicum
powder, enabling the optimization of therapeutic outcome
and control over its side effects.
MATERIALS AND METHODS
Plant materials
The mature Capsicum fruits were harvested from differ-
ent places in Nepal (Table 1), and photographs of Capsicum
fruits are presented in Figure 1. All fruits were dried in the
shade. The dried fruits were freed from seeds, ground, and
stored in air-tight containers in the refrigerator until exam-
ination. Capsicum fruits were identified by comparing au-
thentic samples and also confirmed with the help of an
expert. The voucher sample of each material is recorded as
a specified voucher number in Table 1 and preserved in
the Museum of Natural Medicine, The School of Pharma-
ceutical and Biomedical Sciences, Pokhara University,
Pokhara, Nepal.
Reagents and materials
Standard capsaicin (purity 99%) was obtained from Wako
Pure Chemical Industries (Osaka, Japan). Acetonitrile and
methanol used were of high-performance liquid chroma-
tography (HPLC) grade. Double distilled water was used
throughout the experiment, and all other solvents and other
chemicals used were of analytical grade.
Apparatus
The HPLC system consisted of Shimadzu (Kyoto, Japan)
HPLC pump LC-9A, Shimadzu 6PD-6A ultraviolet spec-
trophotometer detector, and Shimadzu C-R5A Chromatopac
data processor. The column was RP18 Fluofix 3NW415
(4.6150 mm) joined with a precolumn.
Preparation of standard solutions
A stock solution of capsaicin was made by dissolving
2 mg of standard capsaicin in 2 mL of methanol. Working
solutions were prepared by diluting the stock solution to the
desired concentration with methanol.
Chromatographic conditions
The mobile phase consisted of 0.1% phosphoric acid=
acetonitrile (3:2 vol=vol). The temperature of the column
was maintained at 308C during the chromatographic sepa-
ration. The flow rate was 0.8 mL=minute, and the chroma-
tography was run for 25 minutes. The eluting compounds
were monitored at a wavelength of 281 nm.
910 THAPA ET AL.

FIG. 1. Various Capsicum fruits collected from different places in Nepal.

Extraction and capsaicin content determination
Each powder sample was put in a Petri dish and kept in
the oven at 608C for 1 hour to make it dry. One gram of each
powder sample was transferred into the volumetric flask
(25 mL) and filled with methanol. Volumetric flasks were
kept in the water bath at 508C for 4 hours, and the entire
contents were left overnight at room temperature. The final
volume was adjusted to 25 mL by addition of methanol. The
samples were centrifuged and passed through a Millipore
(Billerica, MA) nylon filter (pore size, 0.45 mm). The sam-
ples were packed in the vials and kept in the refrigerator
until examination. Extraction of seeds of some Capsicum
fruits was also performed in the same way as described
above. The sample solution or standard capsaicin solution
(20 mL) was directly injected into the HPLC apparatus.
 HPLC ANALYSIS OF CAPSICUM FRUITS 911

RESULTS
Calibration
The determination of capsaicin in Capsicum fruit extract
was performed using the external standard method. The
calibration graphs were expressed as chromatographic peak
areas of standard capsaicin versus corresponding concen-
trations of the standard in the concentration range of 50–
1,000 mg=mL.

Limit of detection (LOD)

The LOD and the lower limit of quantification (LOQ)
were 0.709 mg and 2.365 mg per sample, respectively. The
LOD is defined as three times the standard error of the least
square calibration curve divided by the slope, and the LOQ
is defined as 10 times the standard error of the calibration
curve divided by the slope.16 The calibration curve was
found to be linear (y ¼ 242,906x  11,177) with a correla-
tion coefficient of 0.999.

Analysis of capsaicin content in Capsicum fruits

The placenta of all fruits and seeds of some of them were
extracted separately. The color of the extracts corresponded
to the surface color of the Capsicum fruits. The chromato-
gram of a typical Capsicum extract is shown in Figure 2. The
peak just after capsaicin is likely to be dihydrocapsaicin.3
Capsaicin peak identification in the extract was done by
matching the retention time with that of standard capsaicin,
which was further confirmed by co-injecting standard cap-
saicin together with the extract. Quantitative analysis of
capsaicin in Capsicum fruits was the result of triplicate
experiments. The results of analysis of Capsicum fruits
collected from different parts in Nepal and date of collection
are presented in Table 1. The analysis of Capsicum fruits by
HPLC demonstrated variability in capsaicin content. The
total capsaicin content ranged from 2.19 to 19.73 mg=g.
Capsaicin content in seeds of some of detected samples was
less than that of the placenta of the same sample (Table 1).
DISCUSSION
To date, capsaicinoids have been described only in the
fruits of the genus Capsicum. The less spicy varieties of
Capsicum have capsaicinoid concentrations that range from
0.003% to 0.01% in the dry weight of fruit. The concen-
tration of capsaicinoids in the mildly hot spicy varieties
ranges from 0.01% to 0.3%; very hot spicy varieties are
characterized by a content of capsaicinoids in excess of
0.3% of the total weight, and this can reach up to 1%.12
Other data available on capsaicin content of Capsicum
grown in different parts of the world suggest that the cap-
saicin content in Capsicum varies from 0.0% in sweet
pepper to as high as 1.86% in Indian chili, which is the
highest record reported up to now.17 In our experiment, we
found capsaicin content in Capsicum fruit as to be as high as
1.97% (sample P, Table 1). Capsaicin content among all
samples analyzed ranged from 2.19 mg=g (0.22% in sample
FIG. 2. A typical HPLC chromatogram of (A) standard capsaicin
and (B) a representative chromatogram of Capsicum fruit extract.
HPLC conditions were as follows: column, Fluofix (4.6150 mm);
column temperature, 308C; mobile phase, 0.1% phosphoric acid=
acetonitrile (3:2 vol=vol); detector, ultraviolet at 281 nm; flow rate,
0.8 mL=minute; chart speed, 5 mm=minute.
N, Table 1) to 19.73 mg=g (1.97% in sample P, Table 1) of
dry fruit. The lowest concentration was observed in the
elongated type with twisted round tip Capsicum fruit,
whereas the highest concentration was detected in Capsicum
fruit of which the shape is round but tapers to a sharp, abrupt
point (Fig. 1).
The capsaicin content did not appear to be correlated with
the altitude of environment where Capsicum fruits were
grown. Samples A, B, G, and L (Table 1) were collected
from the Terai region, a low-altitude land (100–300 m above
sea level), whereas other samples were collected from hilly
regions up to 1,500 m above sea level. The capsaicin content
was found to be higher in placenta than in seeds (Table 1),
which supports the proposal that capsaicinoids are synthe-
sized in the placenta and that their presence in other parts of
fruits appears to be result of leakage and diffusion from the
placenta to the periderm and seeds.4
With respect to any correlation between fruit size and
pungency, the capsaicin content in Capsicum fruit had once
912 THAPA ET AL.
been considered to be inversely proportional to the size of
the fruit.18 However, our experiment showed capsaicin
content in Capsicum fruit not to be related to the size of the
fruit. The fruit with the highest capsaicin content was among
the largest fruits (19.73 mg=g), whereas the smallest Cap-
sicum fruit had the third highest capsaicin content (10.27
mg=g) (Fig. 1).
In Nepal, chili is generally harvested from the beginning
of autumn to late autumn. Harvesting time widely varies
because of the type of chili cultivated by the farmers and
climate variation due to the sharp altitude variation. In this
study, we are unable to correlate the level of pungency with
regard to the time of harvesting.
Capsicum frutescens is generally considered as a hotter
chili in Nepal. In this study, samples E and K were identified
as C. frutescens because of their morphologically similari-
ties and plant identification; however, their capsaicin con-
tents differed by almost 400%. Similarly, the capsaicin
content among Capsicum annum was found to vary from
2.19 mg=g in sample N to 19.73 mg=g in sample P. There-
fore, in order to confirm whether C. frutescens found in
Nepal is hotter than C. annum, further study would be
needed.
A variety of capsaicin concentration has been used topi-
cally in humans ranging from 0.025% to 0.075% in oint-
ments and creams.8–10 These rather low doses may not
be fully sufficient to relieve pain; on the other hand, at
increased concentrations, a capsaicin-associated burning
sensation might negate the beneficial and therapeutic
effects.19
Although the amount of capsaicin to cause lethal toxicity
is higher than the highest possible levels of human dietary
exposure based on animal experiment, orally administered
capsaicin has several physiological effects on the gastro-
intestinal tract, which are dependent on the dose of capsai-
cin. Ingestion of capsaicin in large doses has been reported
to cause histopathological and biochemical changes, includ-
ing acute erosion of gastric mucosa and hepatic necrosis,9
whereas several experiments have shown that intragastic
application of capsaicin at low concentration (range, 0.13–
160 mM) protects against gastric ulceration induced by eth-
anol or acidified aspirin.20 Knowledge of the quantitative
capsaicin content in Capsicum fruits enables us to use the
appropriate amount of locally available Capsicum powder
as a daily dietary additive sufficient to prevent and cure
gastrointestinal disorders with minimum side effects.
As predicted, we found variability in the capsaicin con-
tent in different Capsicum fruits from Nepal. Therefore, the
variability in capsaicin content in different Capsicum fruits
may contribute to the unpredictability in efficacy of topi-
cally applied Capsicum tincture or conventional semisolid
drug dosage forms as mentioned in the Japanese Pharma-
copoeia.21 It might be failure to induce desired therapeutic
effects and sometimes potential to elicit adverse physio-
logical responses, as the biological effects of capsaicin are
known to be dose-dependent and the effective dose for an-
algesia is proposed to be in between 0.025% to 0.075%. The
great variation in the capsaicin profile in Capsicum fruits,
the limited knowledge on its minor constituents, and the
differences in potency between natural capsaicinoids require
standardization of Capsicum fruits prior to manufacturing of
drug formulations. In addition, determination of capsaicin
content in Capsicum fruits enables us to recommend a daily
dose as a food additive, which would be a very cheap and
available preventive nutrient in gastrointestinal disorders.
ACKNOWLEDGMENTS
The authors are indebted to Prof. Dr. Radheshyam
Kayastha, Tribhuvan University, Nepal for his help in
identification of some samples. This work was partially
supported by a Pokhara University research grant, for which
the authors express their gratitude.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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