AUGUST0 Section

de Alimentos,

Department0

TREJO-GONZALEZ

de lngenierh

Bioquimica,

lnstituto

Politknico

A NEW METHOD FOR THE DETERMINATION CAPSAICIN IN CAPSICUM FRUITS

YH3 0I

INTRODUCTION THE MEXICAN PEOPLE are large consumers of chili, fruit of the genus Capsicum. Chili peppers are used mostly as unripe fruits as a spice, especially in sauces. Sometimes the whole fruit is cooked to prepare some dishes or dried and ground to provide color and flavor, the pungent flavor being determined by the capsaicin content. Chemically, capsaitin is N-(3-methoxy-4-hydroxybenzyl)-8methylnon-trans-6-enamide and the structure is shown in Figure 1. However, Benett and Kirby (1968), Kosuge and Furuta (1970) and Masada et al. (1971) have reported that capsaicin is actually a mixture of at least five closely related vanillyl amides. A precise and accurate method for the quantitative determination of capsaicin has not been reported even though numerous scientific investigators have worked on the determination of this compound. Most calorimetric methods for capsaicin determination are based on the color reactions of capsaicin with vanadium oxytrichloride, a method first developed by von Fodor (193 1) and modified by other investigators, Tice (1932), Prokhorova and Prozorovskaya (1939) and Hayden and Jordan (1941). Ting and Barrons (1942) and later van Blaricom and Martin (1947) developed calorimetric methods using synthetic color standards. North (1944) used a method based on the reaction of capsaicin with phosphotungstic-phosphomolybdic acid. Nogrady (1943) developed a procedure based on the titration of capsaicin with picric acid. Other investigators have combined chromatographic and colorimetric techniques to quantitate capsaitin, including Heusser (1964), Biichi and Hippenmeier (1948), Suzuki et al. ( 1957) and Spanyir and Blazovich (1969). Rios and Duden (197 1) have reported a method utilizing chromatography and fluorescence spectrophotometry to determine capsaicin content. Todd and Perun (1961), Morrison (1967), Kosuge and Furuta (1970), Hartman (1970) and Masada et al. (1971) have used gas-liquid chromatography (GLC) for the detection of capsaicin. Sensory methods have also been used, including methods developed and modified by Nelson (19 19), Wirth and Gathercoal (1924), Munch (1930) 342-JOURNAL

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HO-

,CH3 -CHz-NH-C-ICH2)~-CH=CH-CH 8 Fig.

\

National,

WILD-AL

TAMIRANO

de Ciencias Medico

Biolbgicas

17, D. F. Mekico

OF

ing the aqueous layer, the ether layer was evaporated to dryness in the flash-evaporator. The purified oily residue remaining was dissolved in isopropanol, collected in a volumetric flask, and total volume brought up to 25 ml with isopro-

e”3

1 -Capsaicin.

and Newman (1953), but these methods have been shown to be even less accurate and reproducible than the chemical methods. The purpose of this study was to develop a simple, accurate and reproducible method for the quantitative measurement of capsaicin.

EXPERIMENTAL Materials and equipment

The fresh green chili peppers, Cap&urn annuum L. variety Veracruz S-69 (Serrano), were obtained from the Horticulture Dept., INIA, Mexico. All chemicals used were of analytical reagent grade. Methanol, isopropanol and chloroform were of spectrophotometric grade (Uvasol), purchased from Merck-Mexico, S.A. The capsaicin used as a standard was obtained from K & K Lab.. New York. The absorbent charcoal No. 55159261 was obtained from Harleco Dare, Mexico. The samples were ground in a Sorvall Omnimixer Homogenizer. Evaporation was done in a Flash-evaporator from Buchler Instruments. Ultraviolet absorption spectra were determined on a Coleman-Hitachi Recording Spectrophotometer Model EPSjT. The UV absorbance at 281 nm was determined by a Zeiss Spectrophotometer Model PMQ II. Procedure Capsaicin isolation and calibration of standard curve. 20g of finely ground chili peppers

were placed in the extraction shell of a Soxhlet extractor and isopropanol was percolated through the sample until no more green color appeared in the percolated isopropanol. The extract was cooled and adjusted to a volume of 200 ml with isopropanol. Sg of charcoal was added to the colored extract. The slurry was heated to boiling for the duration of 3 min, filtered and the charcoal residue washed several times with isopropanol. The clear extract was then evaporated to dryness in the flash-evaporator. The oil residue was dissolved in petroleum ether and transferred to a separatory funnel and washed with distilled water. After discard38 (1973)

and CARLOS Escuela National

panol. The absorbance was read at 281 nm against an isopropanol blank. Capsaicin standard solutions containing 10, 20, 30, 40 and 50 pg/ml capsaicin were prepared in isopropanol and their absorbance values recorded at 281 nm. A standard curve plotting absorbance against pg capsaicin/ml was prepared and the quantity of capsaicin in the samples was determined from this standard curve. Paper chromatography. The ascending technique of paper chromatography was used exclu-

sively. 50 ~1 of each extract was spotted on Whatman #l chromatographic paper 1.5 cm from the end of strips 2.5 cm wide x 17.5 cm

long. Each strip was hung in a 30

x

200 mm

corked test tube by means of hooked metallic

pins inserted in the cork stopper. The developing solvent system was chloroform-methanolacetic acid (95:1:5 v/v). Approximately 5 ml of the developing solvent was used and the paper was equilibrated with the solvent 15 min and when the solvent front reached a line 2.5 cm

from the top of the paper, the strip was removed and air-dried. The chromatograms were examined under a 254 nm wavelength UV

lamp. Capsaicin fluoresced and was located at the solvent front. The capsaicin was then eluted

from the chromatograms with 2 ml spectrophotometric grade isopropanol. A spectrophotometric blank was prepared from an unspotted region of a blank chromatogram.

RESULTS & DISCUSSION CHARCOAL was used to clean up the chili extracts. Adding less than 5g of charcoal per 20g of ground chili pepper in a volume of 200 ml isopropanol resulted in a yellow-green extract. Absorbance at 281 nm was too high when using less than 5g, indicating that impurities were absorbing at that wavelength as well as capsaicin. By using 5g of charcoal the extract appeared colorless and the differences between replications in absorbance at 281 nm were minimal. After drying the extract, the oily residue was slightly colored. It was found that water extracted these colored substances from capsaicin in petroleum ether. From the solvent systems described in the literature, the following were tested for the capacity to separate capsaicin from other substances: butanol-isopropanolacetic acid (10:75:25 v/v); chloroformmethanol-acetic acid (95: I :5 v/v>; light

1

DETERMINATION Table 2-Determination of capsaicirP

0.5

O F CAPSAICIN-343 of percent

Capsaicin added Capsaicin found

recovery

% Recovery

20 a/ml

19.8 pg/ml 99.0 18.0 90.0 17.9 89.5 17.3 86.5 17.9 89.5 18.2 f 0.95 aRelative standard deviation = 5.2%; Relative error = 9.0%; Total error = 19.4%

0.1

0.3

)g

Fig. P-Czpsaicin

Table samples

Sample no. 1 2 3 4

5 6 I 8 9

10

1-Capsaicin

CAPSAICIN

standard

contents

from

Fresh wt k)

Dry wt (!a

29.90 27.68 23.36 23.58 24.17 23.74 26.50 26.50 23.30 22.98

3.06 2.78 2.39 2.58 2.35 2.34 2.59 2.63 2.12 2.06

IIll

/

curve at 281 nm.

the extracts

of

10 chili

pepper

Capsaicin contenta amt in mg % on dry wt basis 9.1 12.0 11.7 11.3 10.8 11.2 11.5 13.5 9.6 9.1

0.30 0.43 0.49 0.43 0.48 0.47 0.44 0.5 1 0.45 0.47 0.45 i-O.06

aRelative standard deviation = 13.3%

petroleum (boiling range 60-l O O ’C)-benzene-methanol-ethanol (10:2: 1: 1 v/v); and isopropanol. The best separation was achieved with the chloroform-methanolacetic acid solvent system. Using this system, capsaicin moved with the solvent front leaving the interfering substances in the vicinity of the origin. The separation of capsaicin under these experimental conditions was relatively rapid, taking approximately 20 min. The position of capsaicin on the chromatograms was initially established using the following sprays: (1) equal volumes, mixed immediately before use, of iron (III) chloride (1% in water) and potassium ferricyanide (1% in water). This

resulted in a reaction yielding a blue color in the capsaicin region; (2) 0.5% solution of Fast Blue Salt B followed by O.lN NaOH, yielding a red-brown color as the indicator of capsaicin. Neither method is specific for capsaicin, as other compounds containing phenolic groups may also give color reactions. Since these reactions are destructive, the detection of capsaicin for quantitative measurement was done using the 254 nm UV lamp. The spots of fluorescence at the solvent front and also at the origin were cut out and eluted in isopropanol. The spectra of the isopropanol eluents were measured in the region of 200-300 nm. In comparing the spectra of pure

capsaicin with capsaicin from the chili extracts, the same absorption maxima were found, one peak at 281 nm and another at 231 nm. Although the compounds at the origin of the chromatogram fluoresced under UV light, they did not exhibit absorption maxima at these two wavelengths. It was then considered that these other compounds would not interfere with capsaicin measurement if they were not separated from capsaicin by chromatography. Therefore, the chromatography of the extract was eliminated as part of the clean-up procedure. The wavelength of 281 nm was chosen for the capsaicin quantitation because a very distinct peak is obtained at that wavelength. The absorbance of different concentrations of pure capsaicin in isopropanol followed the Lambert-Beer law, as shown by the standard curve presented in Figure 2. At 281 nm a molar absorptivity of 3070 was obtained. The capsaicin contents determined in the extracts of 10 chili pepper samples from the same lot are presented in Table 1. The average capsaicin content was 0.45% on a dry weight basis, with a standard deviation of 0.06. To determine the percent recovery, 500 pg of pure capsaicin was added to ground chili samples. W h e n the samples were carried through the extraction procedure and the charcoal clean-up, the added capsaicin was recovered to the extent of 86.5-99.0% with a standard deviation among 5 replicate analyses of 0.95 pg/ml or 5.2% (Table 2).

CONCLUSIONS THE PROCEDURE for the capsaicin quantitation from green chili pepper extracts reported here has shown precision to a satisfactory degree, accuracy and simplicity. It was not necessary to utilize chromatographic techniques as has been reported by other investigators. The method is therefore well suited for the determination of capsaicin-containing extracts from fruits of various varieties of Capsicum. The capsaicin content determined in the Veracruz S-69 variety (Serrano) was, on the average, 0.45% on a dry weight basis. This value falls within the

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range reported by Suzuki et al. (1957) with Mombassa chili peppers containing 0.80% capsaicin and the mild variety, Abyssinia, yielding 0.08% capsaicin, and within the range of values reported by Govindarajan and Ananthakrishna (1970) for Mysore variety yielding 0.12% capsaitin, and Guntur variety, yielding 0.07%.

REFERENCES Benett, D.J. and Kirby, G.W. 1968. Constitution and biosynthesis of capsaicin. J. Chem. Sot. (c): 442. Brauer, 0. and Schoen, W. 1962. Determination of the flavor-forming components, specifically of capsaicin, in paprika fruits. Angewandte Botanik 36: 25. Biichi. J. and Hippenmeier, F. 1948. Zur wertbestimmung van rein-cap&cm und capsaitin-haltiaen drown und orenar&en. Pharm. Acta Heiv. 23: 327. - Govindarajan, VS. and Ananthakrishna. S.M. 1970. Observations on the seDaration of CCIDsaicin from Capsicum and its oleoresin. J. Food Sci. Tech. 7: 212. Hartman, K.T. 1970. A rapid gas-liquid chromatographic determination for capsaicin in Cap&urn spices. J. Food Sci. 35: 543. Hayden, A. and Jordan, C.B. 1941. Report of the vanadium oxytrichloride calorimetric method for the determination of capsaicin in Capsicum. J. Am. Pharm. Assoc. 30: 107. Heusser. b. 1964. Die bewertung einigen arznei-

38 (1973)

pflan+r bzw. deren zubereitungen mit hilfe der dunnschicht-chromatographie. f,n “ChromatOgraDhie unter besonderer berucksichtigung der papierchromatographie.” Verlag Merck, Darmstadt. Kosuge, S. and Furuta, M. 1970. Studies on the pungent principle of Capsicum. 14. Chemical constitution of the pungent principle. Agr. Biol. Chem. (Japan) 34: 248. Masada, Y., Hashimoto, K.. Inoue, T. and Suzuki, M. 1971. Analysis of the pungent principles of Capsicum annuum by combined gas chromatography-mass spectrometry. J. Food Sci. 36: 858. J.I. 1967. Gas chromatographic Morrison. method for measuring pungency in Capsicum suites. Chem. Ind. (London) 42: 1785. Munch, J.C. 1930. The bioassay of Eapsaicin. U.S.P.X. J. Assoc. Offic. Agric. Cbem. 13: 383. Nelson, E.K. 1919. The constitution of capsaitin, the pungent principle of Capsicum. J. Am. Chem. Sot. 41: 1115. Newman, A.A. 1953. Natural and synthetic pepper flavoring substances. Chem. Products 16: 413. Nogrady. G. 1943. Determination of the spiciness of paprika by thf: fluorescence titration $xpsaicin. Kiserletugyi Kozlemenyek 46: North, H. 1944. Calorimetric determination of capsaicin in oleoresin of Capsicum. Anal. Chem. 21: 934. Prokhorova. N.T. and Prozorovskaya, L.L. 1939. Determining capsaicin in pepper. Doklady Vseoyuz Akad. Sel’skokhoz Navk im V I Lenina 16: 41. Rios, V.M. and Duden, R. 1971. Dunnschichtchromatographische isolierung und remis-

sionsphotometrische bestimmung van capsaicin. Lebensm.-Wiss. v Technol. 4: 97. Spanyk, P. and Blazovich, M. 1969. A thinlayer chromatographic method for the determination of caps&in in ground paprika. Analyst 94: 1084. Suzuki, J., Tausig. F. and Morse, R. 1957. Some observations on red pepper. 1. A new method for the determination of pungency in red pepper. Food Technol. 11: COO. Tice. L.F. 1932. A simDlified and more efficient method for the extraction of capsaicin together with the calorimetric methods for its determination in Capsicum fruit and oleoresin. Am. J. Pharm. 105: 320. Ting. S.V. and Barrens, K.C. 1942. Chemical test for the pungency in peppers. Proc. Am. Sot. Hort. Sci. 40: 504. Todd, P.H. and Perun, C. 1961. Gas-liquid chromatrographic analysis of Capsicum amides. Food Technol. 15: 270. van Blaricom. L. and Martin, J.A. 1947. Permanent standards for the chemical test for pungency in peppers. Proc. Am. Sot. Hart. Sci. 50: 297. van Fodor. K. 1931. New reaction for capsaitin. S. Unter. Lebens 61: 94. Wirth. E.H. and Gathercoal. E.N. 1924. ReDort of’the Scoville organoleptic method for- the valuation of capsaicin. J. Am. Pharm. Assoc. 13: 217. Ms received 5119172: revised 10/2/72; accepted 10/4/72. The authors express their appreciation to Dr. J.A. Laborde for the samples of green chili peppers; Miss M.E. Merino for tech&al assistance: Mrs. R.M. Panaborn and Dr. V.L. Sinaleton for critical reviews of the paper; and to Mrs. M. Cantwell for preparation of the paper.