16 Fr» Vol. 50, No. 1

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Examination of Original Plant of Jamaica Quassia Extract, a Natural Bittering Agent, Based on Composition of the Constituents

Atsuko T6961, ῍,Naoki SJ<>BDID1,Kyoko S6ID1,Takumi A@>N6B61,Masaharu AH6CDB62, Young Sook YJC 3,Takeshi Y6B6O6@>1 and Kenichi T6C6BDID1

1 National Institute of Health Sciences: 1ῌ18ῌ1 Kamiyoga, Setagaya-ku, Tokyo 158ῌ8501, Japan; 2 Nagoya City Public Health Research Institute: 1ῌ11 Hagiyama-cho, Mizuho-ku, Nagoya 467ῌ8615, Japan; 3 School of Life Science, Tokyo University of Pharmacy and Life Sciences, 1432ῌ1 Horinouchi, Hachioji, Tokyo 192ῌ0392, Japan; ῍ Corresponding author

Jamaica quassia extract is a natural bittering agent used as a food additive in Japan. The main constituents of the extract have already been reported to be quassin and neoquassin. In this study, the di#erences in composition of the constituents among four Jamaican quassia extract products were analyzed by LC/MS. The results showed that the four products have similar compositions of their minor constituents, as well as their main constituents. We isolated four of the minor constituents that were commonly included in the four products, and identified them as 11 -dihydro-12-norneoquassin, canthin-6-one, 4-methoxy-1-vinyl-b-carboline and 4,9-dimethoxy-1- vinyl-b-carboline. The List of Existing Food Additives in Japan mentions that Jamaica quassia (Picrasma excelsa)isthe original plant from which Jamaica quassia extract is produced. However, we presume that Jamaica quassia extract may actually be made from appropriate plants other than Picrasma excelsa,since P. excelsa is listed as an endangered species by the International Union for Conservation of Nature and Natural Resources. We prepared hot water extracts from two other species of plants, Quassia amara (American quassia, Surinam quassia) and P. quassioides (‘Nigaki’ in Japanese), and investigated their constituents by LC/MS. The results showed that the compositions of the constituents in the Jamaica quassia extract products resembled those in the extract derived from Q. amara. These findings suggest that Jamaica quassia extract products are probably made from Q. amara. (Received July 29, 2008)

Key words: ῲ῰ῨΎῧ Jamaica quassia extract; FGῷ food additive; bittering agent; ῲ῰ῨΎῧ Picrasma excelsa; ῧ῱ῳ῭KL Quassia amara; ῭KL Picrasma quassioides

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1.5 mg/mL ῧῑ῟ῌ 10 mL ῳῡ῍ῧῘ῟῍ ῔῎ῒῑ

LC ῎ ῴῌ XTerra RP18 (2.1 mm i. d. ῕ 150 1. LC/MS ῏ῒΐῙ῜ῐῗ῞Ί῕ mm, 5 mm, Waters); ῌ 0.2 mL/min; ῴῌ ´38&ῴῴ῿ῼ῏ῠῨ;ῤῌ qῡŽqῧ‚ 30; Ὸ´ Ῠῌ ῌ  90 : 10(0 Ῐῦ|ῗῳῙ‘`Ὺῌ LC/MS ῧῬ`ῡῳ“ῡ῍ min)!85 : 15(7ῑ22 min)!50 : 50(80ῑ95 min); $ Fig. 2aῑd ῧῩῌ PDA ῧῬ΅ UV 200ῑ600 nm ῤ+ %&'ῐ *&+,ῌ 200ῑ600 nm; +Ίῒῌ 257 ῟}~ῳ”῟῍ 4 ; (AῑD) ‘•ΰῧῖ|ΰΰ—˜ nm.  37 ῡ (compound 1) ῖῬV 40 ῡῥ 41 ῡ (compound MS ῎ -ῐ.ῌ 120; /Ῠ῝ῌ 350; / 2) Ῠ ῐῷῘ™š῞῰ῌ {῰›῰ῷῼ ῒFig. 1 œΐ Ῠ῝῵. ῌ 400 L/hr; Ὼῐ῵.ῌ 50 L/hr; ῶ3 ῖῬV 'ῷῼ ῒ16 €‚ƒ rῠΐῒFig. 1 ῐ῕5ῌ 3.0 kV; Ὼῐ῕5ῌ 30 V (ESI pos.); . žΐ Ῠΰ—˜ῌ UV .Ÿῷ (Fig. 3A-a, b) ῖῬV ῶ3῞6ῌ m/z 100ῑ1,000; SIM (Selected Ion Moni- MS .Ÿῷ (Fig. 3B-a, b) ῥ ῎῟῝ῥῗ῭ῌ com- toring) [M῔H]῔ (ESI pos.). pound 1 ῳῷῼῌ compound 2 ῳ 'ῷῼ ῒ2 5. ῝ῗ῞ compounds 3῍6 ῐῚ῟ῌῘῙ ‚ƒ rῠΐ ῥ"ΐ῟῍ $¡rῠῘ´38&ῴῴ῿ῼ ´38&ῴῴ῿ῼ῏ῠ; ῒ; Bΐ 25 g ῳ>? ῏ῠ;Ῠ¢qῡῤ†ῡ῍ $¡rῠῨ ῐῷῤ£῟ ῾ 300 mL ῧῨA῟ῌ BῨCῡῳD5EF῟ῌ ῖ῭ Ῡ 4 ;ῤ¤¥'῟Ὶῌ ‚Ῡ™š῞῰ῦῗῡ (Fig. ῰I῞ῳK; 300 mL ῧῨALῌ M 300 mL ῨOῶ 2aῑd)῍ §ῌ qῡῧῢ|ΰΎqῡ῟©ῳ“ῡ῍ Fig. Ώῌ ῷRRS ῌ >?῾ ῖῬVWX ῐ ῤ[\ 2eῑh Ῡ Fig. 2aῑd ῳ{῰›῰ª«῟ΰ”῟ΎῨῤ† ]ῌ]ῡ῜῏῟῍ ῷRRS ῏]ῳD5Ὶ^῟ῌ `῍ ¬­Ῠ( Ῠqῡ ῐῷῘ+῞῰Ῐῌ {ῨῷR ῼῴΌ ῴ ῒῗ`῎ 45 mm; ῒ῞῎ 300 mm; a 8 ῸῩ 4 ;ῤ¤¥'῟Ὶ‚Ῡ™š῞῰ῦῗῡ b῎ ῷRRS ῌcX ῐ 1:0!1:1, cX ῐ Ὶ ῍ ῐῷῤ£Ῐ῟©῔«Ῑ|¢*ῦqῡ ῐῷῳ῞῭ ῳ. ῿ef&gῧh῜ 8 ῌjῤῨῌ k 2,200 ῧῡC‘`ῧ,+-ῳ“ῡῥ῝῱ῌ ΰ—˜ 21 ῡῖῬ mLΐ ῧ῟῍ ]ῳῦ 10 mL lῢῡC῟ῌ LC/MS V 35 ῡῨ ῐῷῧῩ.­Ῠ¡rῠῘ°ῦῡΰῨ῟ΰῖ ῤn Xῐ῟ῦῘ῭ῌ qῡ compounds 3ῑ6 ῘῨ῟ ΅ῌ ῡ῟ΰ±`ῥ² ¡rῠ†΅Ῠ ῐῷῤ£Ῡ³῞ MCῡῳrῲtῌ D5EF῟ῌ MqῡῨuῡCῡῳῖ ῗῡ῍ {῝ῤῌ ῐῷῤ£Ῐ῟©῔«ῙῚῌ ῗῢ ῐῷ ῍ ῖ῭῰uῡCῡῳvwῨῡx LC/MS ῤῡx ῧ´§῰`qῡῘ¢ῧ² Ῠ¡rῠῤ†`ῥµ¶῞῰ ῳy΅ῢ῟ῌ u compounds 3ῑ6 ῳῖ῍ ῞῭ῧῌ {῰ ῐῷ 3 ῑ 6 (compounds 3 ῑ 6) ῧ ῐῥ῟῍ Com- ῭ῳῡx ODS ῴῳῩ| HPLC ῧῬ΅K;῟ῌ com- pounds 3ῑ6 Ῡῌ {῰›῰ΰ—˜ῠΊῤῦῚῌ UV ῖ pound 3 (2.2 mg), compound 4 (1.7 mg), compound 5 ῬV MS .Ÿῷ Ύῌ 4 ;ῤ ῎‘`῝ῥῘῙ῞ (1.3 mg) ῖῬV compound 6 (1.5 mg) ῳῖ῍ ῦῖῌ NMR AῨ}~ῌ compound 3 Ῡῌ 16 €‚ƒ 2 „ ῒ3A ῖῬV 3Bΐ Ῠ rῠῤ†`῝ῥῘῡῗῡ ῒL wΐ῍ ῞ LC/MS ῖ

ῡx LC ῎ ῴῌ XTerra Prep RP18 (19 mm i.d. ῕100 mm, 5 mm, Waters); ῌ 10 mL/min; ῴ ῌ 30; abῌ ῌcX ῐ 7:3 (compound 3) ῖῬV 5:5(compound 5)ῌ ῌ  9:1(0 min)!8:2(7ῑ25 min)!5:5(60ῑ75 min) ῒcompound 4 ῖῬV compound 6ΐ῏ c&ῷ῿eῨ῝ῌ ῌ  8:2(v/v) (1.0 mL/min)ῌ $ %&'ῐ *&+,ῌ 210ῑ500 nm; +Ίῒῌ 257 nm. Fig. 2. HPLC chromatograms of four products of Jamai- MS ῎ -ῐ.ῌ 120; /Ῠ῝ῌ 350; / ca quassia extract Ῠ῝῵. ῌ Ὼῐ῵. ῶ3  350 L/hr; 50 L/hr; a) product A; b) product B; c) product C; d) ῐ῕5ῌ 4.0 kV; Ὼῐ῕5ῌ 30 V (ESI pos.); .ῶ3 product D; e)ῌh) magnified views of a)ῌd), respec- ῞6ῌ m/z 100ῑ600; SIM (Selected Ion Monitoring) tively 1:quassin; 2: neoquassin isomers; Detection: UV ῔ ῔ +ῖῬVῡx ῵ῐ [M H] (ESI pos.); (ESI pos.), 200ῌ600 nm by photodiode array detector m/z 379 (compound 3), m/z 221 (compound 4), m/z The LC conditions: column, XTerra RP18 (2.1 ῕ ῌ 225 (compound 5), m/z 255 (compound 6)῍ mm i.d. 150 mm, 5 mm, Waters); solvent, H2O CH3CN 90 : 10(0 min)!85 : 15(7ῌ22 min)!50 : 50(80ῌ95 min); flow rate, 0.2 mL/min. The details are described in Experimental section. February 2009 ‹ŒΎ῭ῪῩ121< ῝’ “ ” 19

ῧῚ῍ UV (Fig. 3A-cῐf), MS (Fig. 3B-cῐf) KΰM 1H- KΰM ῷ  ῤῌ ῗῚΎ῭ῪῩ 13C-NMR ῬO5΅ῴῨCRῗῌ STU2)ῐ7) ,ῺVKΰ  4  !Ὼ῟῔ῦ&'()*῕,-.ῖῧῚ῍ MWX<ΏῘ[\] NMR <ΰῦ COSY, HMQC, HMBC 2. Ὶῖ῜ (compounds 3ῌ6) ῍ῗῙ KΰM NOESY ^. ῢ`aῗE´BbcῨῷ 012ῲ῏5 3ῐ6 4 ῟ 8ῗ῞ ῖῧῌ ῜ ῼΰe

Fig. 4. Structures of isolated minor constituents in Jamaica quassia extract 20 — » Vol. 50, No. 1

Fig. 5. HPLC chromatograms of Jamaica quassia extract Fig. 6. Relative peak areas of compounds 3, 4, 5 and and hot water extracts from standard plants 6 in Jamaica quassia extract and hot water a) quassin reagent isolated from Q. amara;b) extracts from standard plants on LC/MS chroma- hot water extract from Q. amara;c)Jamaica tograms quassia extract (product B); d) hot water extract a) quassin reagent isolated from Q. amara;b) from P. quassioides;e)hot water extract from P. hot water extract from Q. amara;c)Jamaica quassioides (Japanese Pharmacopoeial Product) quassia extract (product B); d) hot water extract The LC conditions were the same as described from P. quassioides;e)hot water extract from P. in the legend of Fig. 2, except for detection quassioides (Japannese Pharmacopoeial Product) condition (UV 257 nm). The detection of compounds 3, 4, 5 and 6 were 1:quassin; 2: neoquassin isomers carried out by LC/MS in SIM (ESI pos.) mode, m/z 379, 221, 225 and 255, respectively.
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