(69)Nippon Shokuhin Kogyo Gakkaishi Vol.37, No.7, 565～568 (1990) 〔Research Note〕565 component of brown or black mustard3) and Separation and Quantitation of Allyl possesses antimicrobial activity4). Isothiocyanatein Brown Mustard and Cinnamon or cassia is the dried bark of Cinnamaldehyde in Cinnamon by Cinnamonum zeylanicum Blume, C. loureirii Reverse-phase High Performance Nees, C. burmanni Blume or C. cassia Blume5). Liquid Chromatography Their main aromatic component is cinnamal- dehyde (CA)5) which possesses antimicrobial Kaori KANEMARU*, Tomohisa TAKAYA* activity6). While gas chromatography has been and TeijiroMIYAMOTO* used to analyze pungent or volatile components *Faculty ofScience of Living in spices7)～10), only a few references11)～13) are , Osaka available which noted their separation and City University,3-3-138, Sugimoto, Sumiyoshi-ku, Osaka 558 quantitation by high-performance liquid chro- matography (HPLC). The object of this paper is to extract AIT in brown mustard and CA in cinnamon and determine them by reverse-phase A reverse-phase high performance liquid HPLC in order to elucidate the role of the chromatography (HPLC) was developed to principal pungent compounds in these spices on separate and quantitate allyl isothiocyanate antimicrobial activities. (AIT), a pungent principle of brown mus- 1. Materials and Methods tard, and cinnamaldehyde (CA), an aromat- Reagents: Standard AIT was purchased from is principle of cinnamon. These compounds Tokyo Kasei Kogyo Co., Ltd. (Tokyo). Stan- were extracted from ground spices with 70% dard CA was purchased from Wako Pure (v/v) ethanol, separated on a Hitachi-gel # 3011-O by reverse-phese HPLC using me- Chemical Ind. Co., Ltd. (Tokyo). Standard AIT thanol as a mobile phase with a flow rate of solutions and standard CA solutions were 1.00ml/min and detected by ultraviolet prepared in methanol. AIT was used immedi- absorption at (190～400)nm. (1) AIT had ately after preparation as it is labile. a UV absorption maximum at 245nm and Sample preparation: Mustard (Asaoka Spice RT was 2.39min. AIT content of mustard Co., Inc,,�Tokyo)was defatted with n-hexane extract (20% w/v) was 1.808±0.019mg/ml after grinding the seeds in a mortar with a and in terms of defatted mustard, it was pestle. Sample (3g) was slurried with 2ml equivalent to 9.040±0.095mg/g. (2) CA had distilled water and incubated for an hour at a UV absorption maximum at 286nm and 37℃ for the satisfactory liberation of AIT. RT 2.80min. CA content of cinnamon extract (20% w/v) was 6.810±0.014mg/ml, Then 10ml 85% (v/v) ethanol was mixed with and in cinnamon, it was 34.050±0.070mg/g. the slurry, and AIT was extracted for an hour at 37℃ from the mixture. Spices have been used primarily as flavoring Cinnamon (McCormick Spice Co., Inc,, Bal- and seasoning agents in food and beverages, timore, MD) was ground to fine powder in a and they have also been used for food protec- microblender. CA was extracted from 3g tion and prevention,as they possesssignificant sample with 12ml 70% (v/v) ethanol for an antioxidant1)and antimicrobialactivities2). hour at 37℃. Brown mustard, from the seeds of Brassica These ethanolic suspensions were centrifuged juncea L., contains glucosinolate, sinigrin at 3000rpm for 30min to prepare 20% (w/v) which is found in black mustard, B. nigra extracts. L. The pungency of mustard is primarily due HPLC procedure: 1. Pretreatment: These to isothiocyanatesreleased from glucosinolates extracts were pretreated with SEP-PAK C18 by hydrolytic action of naturally occurring cartridge (Waters Assoc., Milford, Mass.) myrosinase3). Allyl isothiocyanate (AIT) lib- regenerated by washing with methanol and erated from sinigrin is a major pungent then distilled water. Procedures of the pre- 566日 本 食 品 工 業 学 会 誌 第37巻 第7号 1990年7月(70) treatment were as follows. Extract of mus- spice extract. This was accomplished on a tard was diluted with distilled water in the Hitachi-gel # 3011-O (4_??_×150mm) column ratio of 1:4, and 4ml volume of the diluted kept at 45℃. The chromatographic system solution was loaded onto the cartridge. It was used was a Hitachi 655-11 liquid chromato- washed with 4ml 30% (v/v) methanol, then graph equipped with Rheodyne injector and AIT was eluted with methanol and 10ml Otsuka Denshi MCPD-3500 detector. Mobile volume of eluate was collected. The eluate phase was methanol with a flow rate of 1.00 was filtered through a 0.45μm Millipore filter ml/min. Detection was based on UV absorp- and 5μl of the filtrate was injected in to the tion scanning from 190 to 400nm, visualization HPLC column. Extract of cinnamon was was accomplished with NEC personal computer . diluted with distilled water in the ratio of 2. Results and Discussion 1:9, and 1ml of the diluted solution was Separation and Quantitation of AIT: Stan- treated as similarly as that of mustard except dard AIT had a UV absorption maximum at that the cartridge was not washed with 30% 245nm and a retention time at the peak was (v/v) methanol and 25ml volume of eluate was 2.39min. Fig.1 shows three dimensional chro- collected. matograms of standard AIT and a mustard 2. HPLC conditions: Reverse-phase HPLC extract. On the chromatogram, AIT in the was applied to separate AIT or CA in each mustard extract was satisfactorily separated under these conditions. As a UV absorption maximum and a retention time of the mustard extract agreed with those of standard AIT, the peak found on the chromatogram of sample extract was assumed to be that of AIT. The Fig.1 Three dimensional chromatograms Fig.2 Reverse-phase HPLC chromato- of reverse-phase HPLC of standard grams of standard allyl isothiocy- allyl isothiocyanate (a) and brown anate; injection level 0.75/μg (a) mustard extract (b). and brown mustard extract (b). Conditions: column; Hitachi-gel Conditions: column; Hitachi-gel # 3011-O, mobile phase; methanol, # 3011-O, mobile phase; methanol, flow rate; 1.00ml/min, detector flow rate; 1.00ml/min, detector wavelength; 200～400nm. wavelength; 245nm. (71)KANEMARU et al.: HPLC of Components of Mustard and Cinnamon567 wasabi Wasabia japonica. Its AIT content was chromatograms at 245nm of standard AIT and the mustard extract are shown in Fig.2. The 2.911±0.005mg/g of dry matter (n=5). recovery test of AIT by treatment of the Separation and Quantitation of CA: CA in cartridge at the dose of 5mg was performed. cinnamon extract was separated favorably Mean recovery value was 98.7%. The coeffi- using methanol as mobile phase. Fig.3 shows cient of variation was 0.2% (n=6). The three dimensional chromatograms of standard recovery test of AIT in this overall process was CA and a cinnamon extract. The peak of the also performed. Before extraction withetha- extract had a UV absorption maximum at nol 25mg AIT was added in paste containing 286nm and its retention time was 2.80min. 3g mustard, then the paste was treated in the They agreed closely with those of standard CA. same manner as above. Mean recovery value Both chromatograms at 286nm are shown in of AIT was 96.5%. The coefficient of variation Fig.4. Mean recovery value of CA by treat- was 0.3% (n=5). From peak area of absorp- ment of the cartridge at the dose level of tion at 245nm by MCPD-3500 Integrator, AIT 10mg was 99.3% and its coefficient of varia- content of the mustard extract (20% w/v) was tion was 0.3% (n=6). The recovery test of determined as 1.808±0.019mg/ml (n=5) which CA in this overall process was as follows. was equivalent to 9.040±0.095mg/g of defat- Before extraction, 100mg CA was mixed with ted mustard. Furthermore determination of 3g cinnamon. Mean recovery value of CA was AIT was satisfactorily applied to stem of 98.5%, its coefficient of variation was 0.2% (n =5) . CA content of cinnamon extract (20% w/v) was 6.810±0.014mg/ml (n=5), it is equivalent to 34.050±0.070mg/g of cinnamon. Gas chromatography (GC) is suitable for Fig.4 Reverse-phase HPLC chromato- Fig.3 Three dimensional chromatograms grams of standard cinnamaldehyde; of reverse-phase HPLC of standard injection level 0.14μg (a) and cin- cinnamaldehyde (a) and cinnamon namon extract (b). Conditions: extract (b). Conditions: column; column; Hitachi-gel # 3011-O, mo- Hitachi-gel # 3011-O, mobile phase; bile phase; methanol, flow rate; methanol, flow rate; 1.00ml/min, 1.00ml/min, detector wavelength; detector wavelength; 200～400nm. 286nm. 568日 本 食 品工 業 学 会 誌 第37巻 第7号 1990年7月(72) determing components in head space gas as and RAJAPAKSE, L.S.: J. Sci. Food Agric., KOJIMA14) reported. However HPLC in this 25, 1211 (1974). 8) SENANAYAKE, U.M., LEE, T.H. and WILLs, paper may be more suitable than GC for B.H.: J. Agric. Food Chem., 26, 822 determinig antimicrobial components in spices, (1978). as it is to determine UV spectrum in addition 9) KAMEOKA, H. and HASHIMOTO, S.: Nippon to retention time of components in the sample Nogeikagaku Kaishi, 54, 535 (1980). and easier than GC-MS. KOJIMA et al.10) have 10) KoJIMA, M., HAMADA, H. and TOSHIMITSU, reported that AIT content was about 1% (w/w) N.: Nippon Shokuhin Kogyo Gakkaishi, in black mustard and it contained slightly 33, 199 (1986). 3-butenyl, 3-methyl-thiopropyl, β-phenethyl 11) ARCHER, A.W.: J. Chromatogr., 351, 595 and sec-butyl isothiocyanates, etc. Brown (1986). mustard in this paper contained 0.9% (w/w) 12) BARANOWSKI, J.D.: J.