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Journal of the Chinese Chemical Society, 2011, 58, 376-383

Flavonoid Composition in the Leaves of Twelve Litsea and Neolitsea Plants

Sheng-Fa Tsai and Shoei-Sheng Lee*
School of Pharmacy, College of Medicine, National Taiwan University, Taipei 10051, Taiwan, R.O.C.
Received January 14, 2011; Accepted January 18, 2011; Published Online February 11, 2011

This study was aimed to study the chemodiversity of flavonoids in the Formosan Litsea and Neolitsea plants. Applications of LC-SPE-NMR and LC/MS hyphenated techniques in analyzing polar constituents from the leaves of L. acuminata, L. hypophaea, N. acuminatissima, and N. konishii led to the identification of 13 known flavonoids and one new flavonol dioside, quercetin 3-O-(2-O-b-D-apiofuranosyl)-a-Lrhamnopyranoside. The quantity and variety of flavonoid composition in the leaves of 12 Litsea and Neolitsea plants were examined to enable more effective utilization of such bioactive ingredients. Of these, N. acuminatissima was found to contain the most quantity of flavonoids (ca. 0.24%, w/w, leaves). Keywords: Litsea; Neolitsea; Flavonoid glycosides; HPLC-SPE-NMR; LC-MS.

INTRODUCTION The Lauraceous genera Litsea and Neolitsea contain about 200 and 100 species, respectively. They distribute widely in tropical and subtropical Asia, Australia, New Zealand, North America and subtropical South America.1 So far, 17 Neolitsea species and 18 Litsea species have been recorded in Taiwan.2 Flavonoids have been reported to exhibit antioxida3,4 tive, cytotoxic,5 anti-inflammatory,6 and neuroprotective effects.7 Some in vitro and in vivo studies5-6,8 also disclosed a wide range of bioactivities for flavonoids. Flavonoids have been isolated from several Neolitsea and Litsea plants, including N. sericea var. aurata,9 L. glaucescens,10 L. japonica,11 and L. Chingpingensis.12 In this study, we applied high performance liquid chromatography-solid phase extraction-nuclear magnetic resonance (HPLC-SPENMR) hyphenated technique and LC/MS to identify flavonoids from leaves of L. acuminata, L. hypophaea, N. acuminatissima , and N. konishii first. In total, fourteen flavonoids were characterized. Next, the content of these flavonoids in the 12 species of the Neolitsea and Litsea genera (Table 1) were analyzed by HPLC. The fingerprints of the identified flavonoids from these 12 species provided chemical biodiversity for more effective utilization of flavonoid constituents. The following describes the out-

come of our effort on these aspects. RESULTS AND DISCUSSION The 12 Neolitsea and Litsea plants (Table 1) were collected at the same time and same place. The n-BuOH-soluble fractions of the EtOH extract of the leaves of these plants were further fractionated on a Sephadex LH-20 column to concentrate the UV-positive compounds, detected by TLC under UV lamp (254 and 366 nm), which were found to be flavonoid in this study (to be described later). Applications of HPLC-SPE-NMR and LC/MS in analyzing chemical constituents of these UV-positive subfractions from L. acuminata ( L -1) , L. hypophaea ( L -3) , N. acuminatissima (NL-1) and N. konishii (NL-5) (Figs. 1, 2 and 3) led to the identification of fourteen flavonoids in total (Fig. 4). The 1H NMR spectral data (Table 2) indicated an ABX system (H-2 , d 7.38~7.96, d, J = ~2.0 Hz; H-5 , d 6.93~6.96, d, J = ~8.5 Hz; H-6, d 7.33~7.66, dd, J = ~2.0, ~8.5 Hz), and an AX system (H-6, d ~6.25; H-8, d ~6.44; JAX = ~2.0 Hz) for a quercetin moiety (1-5 and 8-10), and an AA XX system (H-2 &6 , d 7.78~8.12; H-3 &5 , d 6.92~6.94; JAX = ~8.8 Hz), and an AX system (H-6, d ~6.25; H-8, d ~6.44; JAX = ~2.0 Hz) for a kaempferol moiety (6-7, 11-12). The presence of quercetin or kaempferol

* Corresponding author. Tel: +886-2-23123456 ext. 88392; Fax: +886-2-23916127; E-mail: shoeilee@ntu.edu.tw

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Table 1. Weights of EtOH extract, BuOH fraction and UV-positive subfractions from 100 g leaves of 12 Litsea and Neolitsea plants Weight No. Plant species EtOH extract (g) 7.80 11.30 11.87 14.70 11.90 12.40 8.65 18.59 17.41 19.34 21.50 14.30 BuOH fractio (ng) 1.2 2.3 2.6 2.1 2.4 2.1 1.4 2.5 3.7 4.6 3.1 3.6 UV-(+)subfr. (mg) 460.8 1258.1 1154.4 1600.2 1622.4 1583.4 449.4 672.5 1668.7 1830.8 784.3 907.2

1 (L-1) 2 (L-2) 3 (L-3) 4 (L-4) 5 (L-5) 6 (NL-1) 7 (NL-2) 8 (NL-3) 9 (NL-4) 10 (NL-5) 11 (NL-6) 12 (NL-7)

Litsea acuminata (Blume) Kurata Litsea acutivena Hayata Litsea hypophaea Hayata Litsea lii Chang Litsea morrisonensis Hayata Neolitsea acuminatissima (Hayata) Kaneh & Sasaki Neolitsea buisanensis Yamam & Kamik Neolitsea daibuensis Kamik Neolitsea hiiranensis Liu & Liao Neolitsea konishii (Hayata) Kaneh & Sasaki Neolitsea parvigemms (Hayata) Kaneh & Sasaki Neolitsea sericea (Blume) Koidz

moiety in the corresponding compound was also confirmed by analyzing LC/ESI-MS (pos.), showing characteristic fragment ion at m/z 287 ([C15H10O6+H]+) for kaempferol or at m/z 303 ([C15H 10O 7+H]+ ) for quercetin (Fig. 1). The glycone moiety in each flavonoid glycoside (1-13) was determined by ESI-MS and the 1H NMR data which showed the characteristic signals for the anomeric protons and certain distinct protons such as Me-6 in the rhamnosyl residue. The isomeric hyperoside (3) and isoquercitrin (4), the former being 3- O -galactosylated and the latter being 3O-glucosylated, were distinguished by co-injection of the authentic sample, isoquercitrin, in HPLC analysis. Based

on such approaches, compounds 1-4, 6-8, 10-12, and 14 were identified as quercetin 3-O-[2-O-(b-D-xylopyrano-

Fig. 1. HPLC chromatogram of the UV-positive subfractions from N. konishii , monitored by UV (365 nm) and MS fragments at m/z 303 and 287. The key to peak numbering, see compound numbering.

Fig. 2. HPLC chromatograms of the UV-positive fractions from 12 lauraceous plants, monitored at UV 365 nm.

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Table 2. HPLC retention times (tR, min), ESI-MS, and 1H NMR data of flavonoids identified by HPLC-SPE-NMR (CD3CN, 400 MHz) Compd tR Quercetins
1 2 3 4 5 8 9 10 30.4 31.2 32.8 33.3 33.7 35.7 36.1 37.2

ESI-MS (pos.) m/z

597.0 [M+H]+, 465.0, 303.1 611.1 [M+H]+, 465.0, 303.0 465.0 [M+H]+, 303.0 465.0 [M+H]+, 303.0 567.0 [M+H]+, 435.0, 303.0 435.0 [M+H]+, 303.0 581.1 [M+H]+, 435.0, 303.0 449.0 [M+H]+, 303.0

H-NMR (H/ppm, mult, J/Hz) H-5 H-6 H-1 H-1 rha Me-6
1.06 d (6.2)

H-6

H-8

H-2

H-3

6.23 d (2.0) 6.45 d (2.0) 7.79 d (2.0) 6.26 d (2.0) 6.47 d (2.0) 7.75 d (2.0) 6.24 d (2.0) 6.48 d (2.0) 7.96 d (2.0) 6.26 d (2.0) 6.46 d (2.0) 7.96 d (2.0) 6.24 d (2.0) 6.44 d (2.0) 7.94 d (2.0) 6.26 d (2.0) 6.46 d (2.0) 7.94 d (2.0) 6.23 d (2.0) 6.42 d (2.0) 7.39 d (2.0) 6.23 d (2.0) 6.41 d (2.0) 7.38 d (1.9)

6.94 d (8.5) 7.57 dd (2.0, 8.5) 5.34 d (6.0) 5.48 t (7.3) 6.93 d (8.5) 7.66 dd (2.0, 8.5) 5.00 d (7.3) 4.50 br. s 6.93 d (8.5) 7.53 dd (2.0, 8.5) 5.00 d (7.8) 6.94 d (8.4) 7.51 dd (2.0, 8.4) 4.99 d (7.8) 6.95 d (8.6) 7.52 dd (2.0, 8.6) 5.25 br. s 6.93 d (8.6) 7.52 dd (2.0, 8.6) 5.00 d (7.8) 6.96 d (8.3) 7.34 dd (2.0, 8.3) 5.49 br. s 5.04 d (1.7) 0.89 d (6.1) 6.94 d (8.3) 7.33 dd (1.9, 8.3) 5.42 br. s 0.85 d (5.5) 5.19 br. s

Kaempferols
6 7 11 12 34.5 35.5 38.8 41.3 617.2 [M+Na]+, 595.0, 449.0, 287.0 471.1 [M+Na]+, 449.0, 287.0 6.26 d (2.0) 6.47 d (2.0) 8.09 d (8.8) 6.93 d (8.8) 6.93 d (8.8) 419.0 [M+H]+, 287.0 433.0 [M+H]+, 287.0 6.26 d (2.0) 6.47 d (2.0) 8.09 d (8.8) 6.93 d (8.8) 6.93 d (8.8) 6.23 d (2.0) 6.41 d (2.0) 7.78 d (8.7) 6.94 d (8.7) 6.94 d (8.7) 8.09 d (8.8) 8.09 d (8.8) 7.78 d (8.7) 4.99 d (7.8) 5.02 d (6.9) 5.43 br. s 0.83 d (5.9) 6.26 d (2.0) 6.47 d (2.0) 8.12 d (8.9) 6.92 d (8.9) 6.92 d (8.9) 8.12 d (8.9) 5.00 d (7.3) 4.50 br. s 0.86 d (7.1)

other
13[a] 14
[b]

25.4 34.9

291.1 [M+H]+ 473.1 [M+Na]+, 303.0

5.88 d (2.2) 5.92 d (2.2) 5.93 d (2.0) 5.97 d (2.0)

6.93 br. s 6.97 br. s

4.10 br. s

6.80 br. s 6.86 br. s

6.80 br. s 6.86 br. s 3.89 br. s 1.09 d (6.2)

[a] 13: H2-4 at d 2.76 dd (4.4, 16.8) and 2.63 dd (2.3, 16.8). [b] 14: H-2 at d 5.11 d (10.8), H-3 at d 4.62 d (10.8).

syl)- b - D-glucopyranoside] ( 1 ), 13 rutin ( 2 ), 14 quercetin 3- O - b - D-galactopyranoside (hyperoside) ( 3 ), 15 isoquercitrin (4),14 kaempferol 3-O-rutinoside (nicotiflorin) (6),16 astragalin (7),17 quercetin 3-O-b-D-xylopyranoside (8),15 quercetin 3-O-a-L-rhamnopyranoside ( 10),9 kaempferol 3- O - b - D-xylopyranoside ( 11 ), 18 kaempferol 3- O - a- L rhamnopyranoside ( 12 ), 9 and (2 R, 3 R)-dihydroquercetin 3-O-a-L-rhamnopyranoside (14).9 It was noted that the signals of H-2 and H-6 in 10 and 12 were upfield shifted rela-

tive to the corresponding signals in the compounds with the same aglycone, such as 10 vs. 1-5 and 12 vs. 11, attributable to more steric effect caused by the 3-O-rhamnopyranosyl group than by other sugar units. Compound 13 was

Fig. 3. Representative 1 H NMR spectra of identified flavonids ( 1 , 3 , 4 , 5 , 9 and 11 ), adopted from HPLC-SPE-NMR.

Fig. 4. Structures of the identified flavonoids.

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identified as epicatechin,19 based on ESI/MS and the characteristic signals for H2-4 as shown in Table 2. Compounds 5 and 9 had the molecular formula C25H26O15 and C26H28O15, respectively, as deduced from HR-ESI-MS. Their ESI-MS (pos.) showed two fragments at m/z 449.0 ([M+H-132]+ in 5 ; [M+H-132]+ in 9 ) and 303.0 ([M+H-132-132]+ in 5; [M+H-132-146]+ in 9), indicating 5 and 9 to be dioside and the former (5) comprising of two pentosyl residues and the latter (9) comprising of one pentosyl and one deoxyhexosyl residues. The sugar residues in 5 were determined to be 2-O-(b-apiofuranosyl)a-arabinopyranosyl by analyzing 1H and 13C NMR data as shown in Table 3 and by comparison of the reported 13 C-NMR data in the literatures.20,21 The assignments of these data were confirmed by 2D NMR spectral analyses. The 13C NMR data for the proton bearing carbons were obtained from DEPT and HSQC due to the limited amounts of material. The sugar linkage was supported by the NOESY spectrum which showed the correlation of ara- p H-1 ( d 5.31) api H-2 (d 3.94) api H-1 (d 5.27) ara-p H-2 (d 4.05). Thus, 5 was established as quercetin 3-O-(2-O-bapiofuranosyl)-a-arabinopyranoside.21 The complete 1H

NMR assignment of 5 (Table 3) was made for the first time by analyzing 2D NMR spectra (COSY, NOESY and HSQC). The assignment for the sugar protons in 9 was verified by COSY spectral analysis. The presence of a b-apiofuranosyl moiety was identified by the highly deshielded anomeric carbon at d 111.9.22 The rhamnosyl unit was also readily identified by the characteristic 1H NMR signals, verified by COSY spectral analysis, especially for H-1 (d 5.39, d, J = 1.6 Hz) and Me-6 (d 0.97, d, J = 6.2 Hz), and the MS fragment at m/z 303 [M+H-132-146]+. The 13C NMR assignment for the proton bearing carbons in 9 was made based on HSQC analysis and that for the quaternary api C-3 (d 80.3) was made based on its correlation to the api H2-5. It was found that the rha C-2 (d 79.1) was downfield shifted relative to that in a mono-oside such as quercetin 3-O-a23 L-rhamnopyranoside (d 71.9), indicating the glycosylation effect and pointing out the position of the sugar linkage. Such suggestion was supported by comparison of 13C NMR data with the model compound betonyoside F, 24 a phenylethanoid trioside containing a terminal dioside identical to 9. Such sugar linkage was also confirmed by the

Table 3. 1H and 13C NMR Data of 5 and 9 (d/ppm, m, J/Hz) (CD3OD, AV-600) 5 Position 6 8 2 5 6 a-L-ara-p 1 2 3 4 5 b-D-api 1 2 4 5 dC[a] 99.4 (d) 94.2 (d) 116.6 (d) 116.0 (d) 122.7 (d) 101.0 (d) 76.3 (d) 71.9 (d) 67.4 (d) 64.2 (t) dH 6.19 d (2.2) 6.38 d (2.2) 7.57 d (2.0) 6.89 d (8.3) 7.54 dd (2.0, 8.3) 5.32 d (4.6) 4.05 dd (4.6, 6.5) 3.83 dd (3.5, 6.5) 3.80 ddd (3.2, 3.5, 6.6) 3.34 dd (3.2, 11.8) 3.82 dd (6.6, 11.8) 5.27 d (1.9) 3.94 d (1.9) 3.70 d (9.7) 3.91 d (9.7) 3.56 d (11.5) 3.61 d (11.5) Position 6 8 2 5 6 a-L-rha 1 2 3 4 5 6 b-D-api 1 2 3[b] 4 5 dC[a] 99.5 (d) 94.4 (d) 116.5 (d) 116.1 (d) 122.4 (d) 102.4 (d) 79.1 (d) 71.4 (d) 73.4 (d) 71.7 (d) 17.5 (q) 111.9 (d) 77.5 (d) 80.3 (s) 74.7 (t) 65.2 (t) 9 dH 6.20 d (2.0) 6.36 d (2.0) 7.32 d (2.1) 6.91 d (8.3) 7.29 dd (2.1, 8.3) 5.39 d (1.6) 4.20 d (1.6, 3.4) 3.84 d (3.4, 9.8) 3.28 m 3.57 dt (3.4, 6.2) 0.97 d (6.2) 5.10 d (2.5) 3.90 d (2.5) 3.67 d (9.7) 3.81 d (9.7) 3.51 d (11.8) 3.53 d (11.8)

110.2 (d) 77.7 (d) 74.9 (t) 65.5 (t)

[a] Data from DEPT-135 (150 MHz) and HSQC (600 MHz). [b] The chemical shift of api C3 was assigned based on the correlation to api Hs-5 in the HMBC spectrum (600 MHz) (see Supplementary data).

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NOSEY spectrum, showing the key NOE correlation of api H-1 (d 5.10) rha H-2 (d 4.16) rha H-1 (d 5.39). The CD spectrum of 9 showing a positive and a negative Cotton effect (CE) at 255 and 339 nm, respectively, being consistent with that of quercetin 3-O-a-L-rhamnoside [(+)-CE at 249 nm and (-)-CE at 339 nm]25 from our collection,9 supported the rhamnosyl residue in 9 to be a-L-form. Accordingly, the apiosyl moiety should be b-D-form to make the 1 H and 13C NMR spectral data of the diosyl moiety consistent with those reported for the terminal diosyl residue in betonyoside F.24 Therefore, 9 was elucidated as quercetin 3-O -(2-O -b-D-apiofuranosyl)-a-L-rhamnopyranoside, a new flavonol glycoside. Complete 13C NMR assignment, however, was not achieved due to the limited amounts of material.

Quantitation of rutin, isoquercitrin, quercetin 3-Oa-L-rhamnopyranoside and kaempferol 3-O-a-Lrhamnopyranoside The standard curves and regression equations of authentic rutin ( 2 ), isoquercitrin ( 4 ), quercetin 3- O- a- L rhamnopyranoside (10), and kaempferol 3-O-a-L-rhamnopyranoside (12) were established by HPLC analysis of a series of different concentrations between 0.25 and 250 mg/mL (injection amounts 0.0045.781 nmol) (Table 4). Each concentration was tested and quantified in triplicate. Based on HPLC chromatograms (Fig. 2) obtained from the leaves of 12 Neolitsea and Litsea plants studied, the contents of 12 flavonol glycosides were calculated using the regression equations of the authentic sample for compounds with the same chromophore, such as 12, containing

Table 4. Characteristic parameters for regression equations of four authentic flavonol glycosides, obtained by HPLC analysis Compd 2 4 10 12 A[a] 1661.75 6.05 1622.95 6.05 1214.15 3.55 1102.80 4.20 B[a] 19.9545 1.5125 9.9236 1.4384 0.4277 1.4947 14.7632 1.4662 LOD[b] 0.0147 0.0088 0.0040 0.0174 LOQ[b] 0.0211 0.0150 0.0040 0.0267 r2 0.9999 0.9999 0.9999 0.9999 Intra-day precision[c] tR[d] 0.10 0.10 0.11 0.12 peak area[d] 0.85 0.44 0.50 1.14 Inter-day precision[c] tR[d] 0.10 0.14 0.16 0.15 peak area[d] 0.78 1.22 1.03 0.10

[a] Linear regression lines (y = ax + b), where y is the peak area detected at 365 nm, x is the concentration (0.004 5.781 nmole, n = 3) of the analytes, and a and b are the respective slope and intercept of the calibration curve. The correlation coefficient is r2. [b] LOD (the limit of detection) = (b + 3sb)/a and LOQ (the limit of quantification) = (b + 10sb)/a, where a is the slope of the calibration curve; b is the intercept; and sb is the standard deviation associated with the intercept. [c] n = 3. [d] R.S.D., %.
Table 5. The contents of 12 flavonoids in UV-positive subfractions (A)[a] and 100 g of leaves (B)[b] of 12 lauraceous plants Compd 1 2 3[d] 4 5[c] 6[e] 7[e] 8[d] 9[c] 10 11[e] 12 Total
[c]

L-1 A B A

L-2 B
0.3 12.5 22.4 17.3 1.6 3.2 1.4 2.7

L-3 A B A

L-4 B
0.6 2.6

L-5 A
12.5

NL-1 B
2.0

NL-2 A
66.2 22.5 43.0 1.9

NL-3 A B

NL-4 A
7.8

NL-5 A
42.5 8.1 178.0

NL-6 A B

NL-7 A B

B
3.0 1.0 1.9 0.1 0.4 0.3

B
1.3

B
7.8 1.5 32.6 30.3 13.1 0.2 17.3 7.0 26.8 11.6 5.0 1.9

2.3 239.4 11.0 99.0

3.8 16.0 7.9 17.3 42.0 0.9 114.6 2.0 4.8 43.6 2.0

493.9 78.2 365.5 57.9 48.5 7.7

254.8 17.1

300.3 23.6 44.1 3.5

387.6 35.2

635.5 29.3 178.1 91.8 146.2 28.8 58.2 39.3 4.2 137.5 6.7 1.3 2.7 1.8 12.5 25.6 11.3 21.5

18.3 430.4 7.0 0.3 20.1 58.8

69.8 3.3 9.5

30.2

2.0 122.7

20.5 165.4 71.4

161.9 12.7 14.1 28.9 1.1 2.3

36.5

3.3

32.1 2.2 0.6 0.1 7.0 1.1 3.9 0.4 45.1 28.7 16.8 71.3 1.1 7.3 4.7 2.7 11.6 0.2 0.2 33.1 56.7

5.1 5.2 9.0

8.3 7.4

31.0

2.1

0.9 94.4 38.3

38.5

3.5

16.4

1.3

1.9 387.5 61.4 414.6 4.9 0.8 95.2 661

0.1 18.6 1294.6 87.1 169.0 28.2

146.3 63.4 27.2

178.7 6.6 5.9

8.2 205.2 0.3 0.3 25.5

25.8

98.8

11.4

24.3

610.1 47.9

770.3 69.9 2.4 0.2 4.5

3.2

3.2

0.4 19.6 213.5

1.5

64.1 10.1

4.3

161.9 10.9

7.4

1.2

10.6

61.5

4.8

49.5

1430.4 65.8 718.5

90.4 169.8

34.2 686.3 111.3 1486.3 235.4

29.7 1772.5 119.2 306.9

51.2 846.5 155.1 1237.3 97.2 1284.8 116.6

[a] ng/10 mg of the UV-positive subfractions obtained from Sephadex LH-20 fractionation of the BuOH soluble fraction (see Experimental section); [b] mg of 100 g leaves; [c] data obtained using regression equation of 2 in Table 4 Mw; [d] data obtained using regression equation of 4 in Table 4 Mw; [e] data obtained using regression equation of 12 in Table 4 Mw.

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a kaempferol moiety, for 6, 7 and 11. The results are shown in Table 5. Among 5 Listea plants, L. hypophaea (L-3) was found to contain the least quantity and variety of flavonoids (Table 5). L. acuminata (L-1) and L. acutivena (L-2) contained the most variety of flavonoids, each 12 in total. Except for L. hypophaea, hyperoside (3) was found to be the main component in the other four Litsea plants. Among the seven Neolistea plants, N. konishii (NL-5) contained the most variety of flavonoids, altogether 13 identified (Table 5 and Fig. 2). Quercetin 3-O-a-L-rhamnopyranoside (10) was found to be the main component in most of Neolitsea plants except for N. konishii. In addition, epicatechin (13) was detected only in six Neolitsea plants but not in Litsea plants and N. acuminatissima (NL-1). Of these 12 plants, N. acuminatissima was found to be the most abundant in flavonoids (ca. 0.24%, w/w, dry leaves) (Table 5). This study reveals that the flavonoid contents in Neolitsea and Litsea plants are highly diversified. The combination of HPLC-SPE-NMR and LC/MS was demonstrated to be a powerful method for rapid screening of both known and new natural products. Moreover, the established HPLC method in analyzing flavonoid composition could be applied to the exploration of such bioactive ingredients in natural resources. EXPERIMENTAL Instrumental The physical data were obtained using the following instruments. UV (MeOH): lmax nm, Hitachi (Tokyo, Japan) 150-20 Double Beam spectrophotometer; 1H, 13C, and 2D NMR spectra: Bruker AV-400 and AV-600 spectrometers (Burker, Rheinstetten, Germany); MS: Esquire 2000 ion trap and MicrOTOF orthogonal ESI-TOF (HR-ESI-MS) mass spectrometers (Bruker, Daltonik, Bremen, Germany), both with electrospray ion source; Column chromatography (CC): Sephadex LH-20 (Pharmacia); HPLC: Agilent 1100 system, Phenomenex Prodigy ODS-3 (anal.: 250 4.6 mm, 5 mm; semi-prep.: 250 10 mm, 5 mm); HPLCSPE-NMR: an Agilent 1100 HPLC, equipped with a photodiode array detector, a Knauer K120 HPLC pump (makeup pump) (Berlin, Germany), a Prospekt 2 solid-phase extraction unit (Bruker & Spark, Emmen, Holland) containing 96 HySphere Resin GP cartridges (10 2 mm, Spark, Emmen, Holland), a nitrogen separator (Bruker) for flushing cartridge, and an AV-400 NMR spectrometer equipped with a 30 mL inverse probe (Bruker); Thin layer chromatography:

silica gel plates (KG60-F254, Merck), visualized under UV 254 and 366 nm, and by anisaldehyde spray reagent. Chemicals and reagents Acetonitrile, methanol (CAS and chromatographic grade), CHCl3, CH2Cl2, EtOAc, BuOH, trifluoroacetic acid (TFA) were purchased from Mallinckrodt (KY, USA). Acetonitrile-d3 (99.8%) was purchased from Cambridge Isotope Lab., Inc. (Andover, MA, USA) and methanol-d4 (99.8%) from Merck (Germany). Deionized water was obtained from a Barnstead water purification system (Dubuque, IA, USA). Authentic rutin (2), isoquercitrin (4), quercetin 3-O-a-L-rhamnopyranoside (10), and kaempferol 3-O-a-L-rhamnopyranoside (12), were obtained from our lab collection. Plant Material The leaves of five Litsea and seven Neolitsea plants (Table 1) were collected in September 2005 at the Fu-shan Research Center, Taiwan Forestry Research Institute (TFRI), Yilan County, Taiwan, and authenticated by Mr. Jer-Tone Lin, Associate Researcher, TFRI. The voucher specimens (NTUSP9409Ls/NLs) were deposited in the herbarium of that institute. Extraction and Isolation All dry leaves of the 12 Litsea and Neolitsea plants (Table 1) were extracted, partitioned, chromatographed, and monitored in the same manner. A typical procedure was described as follows. The dried leaves of N. konishii (NL-5) (100 g) were powdered and macerated in 95% EtOH (1 L 3). The EtOH extract (6.2 g), obtained after evaporation under reduced pressure at 45 C, was suspended in H 2O (130 mL) and then partitioned against CH2Cl2, EtOAc and BuOH (water saturated), each 130 mL 3. The BuOH-soluble fraction (100 mg out of 4.6 g) was chromatographed over a Sephadex LH-20 column (high 30 cm, O.D. 1.5 cm; MeOH, flow rate 1 mL/min, 3 min/tube) to give five subfractions, combined on the basis of TLC examination (CHCl3-MeOH-H2O 9:4:1, lower layer; UV 254 nm), of which subfr.-5 (tubes 10-16, 39.8 mg) was found to be UV-positive and rich in flavonoids. The BuOH extracts from other species were fractionated using the same column and conditions including eluent, flow rate, volume per tube as described above for that of N. konishii. The UVpositive subfractions, detected by TLC under UV-lamp were combined for further analysis. LC-SPE-NMR analysis An aliquot of the UV-positive subfractions (10 mL,

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100 mg/mL) from 12 lauraceous species obtained from the procedure described above were analyzed on an analytical RP-18 column, delivered with ACN-0.1% TFA aq, 7% to 33% in 44 min, to 95% in 3 min, both linear gradient, and 95% for 6 min; flow rate 0.5 mL/min; UV detection 280 and 365 nm. The postcolumn eluates were diluted with water, supplied by a makeup pump (flow rate 1.2 mL/min), and each compound peak was passed through a HySphere resin GP cartridge. The loaded cartridges were flushed by dry nitrogen for 20 min, then the compound in each dried cartridge was eluted by acetonitrile-d3 into a 30-mL inverse NMR probe. The 1H NMR spectra were recorded using a multiple solvent suppression pulse program for residual protons and water signals in the D-solvent. All spectra were measured at 300 K, and the 1H chemical shift was referenced to a residual signal of CD3CN at dH 1.93 ppm. LC-DAD-MS analysis The UV-positive subfractions (10 mL, 1 mg/mL) as indicated above were analyzed using the same HPLC program for HPLC-SPE-NMR. A small ratio (5%) of LC flow was directed into DAD and MS via a splitter (1:20). The temperature of the ESI interface heated capillary was 300 C. The nebulizer gas (N2) pressure was set to 15 psi and the dry gas (N2) flow of 5 L/min was used. MS data were acquired in the positive mode over a scan range of 50 to 1000 Da. Separation of compounds 5 and 9 via Semi-preparative HPLC The n-BuOH soluble fraction (2.0 g out of 4.6 g) of the EtOH extract of the leaves of N. konishii was subjected to Sephadex LH-20 CC (high 48 cm, O.D. 4.0 cm; MeOHH2O 7:3) to give a fraction containing 5 and 9 (6.9 mg), which was subjected to semi-preparative RP-18 HPLC, sampling 2.3 mg/50 mL each run, eluted by MeOH-H 2O (19.5:80.5) with a flow rate of 2.6 mL/min, monitored at UV 280 nm, to give 5 (0.2 mg, tR 23.6 min) and 9 (0.2 mg, tR 29.5 min). Quercetin 3-O-(2-O-b-D-apiofuranosyl)-a-L-arabinopyranoside (5) UV (MeOH) 256, 356 nm; 1H- and 13C-NMR (CD3OD) (Table 3); ESI/MS (pos.) m/z 567.0 (87%) ([M+H]+), 435.0 (30%), 303.0 (100%); HR-ESI/MS (neg.) [M-H]- m/z 565.1193 (C25H26O15H, calcd. 565.1199). Quercetin 3-O-(2-O-b-D-apiofuranosyl)-a-L-rhamnopyranoside (9) UV (MeOH) (log e) 263 (4.35), 353 (4.16); CD [39

mM (est. from UV), MeOH] [q]339 9790, [ q]255 +11280; H- and 13C-NMR (CD3OD) (Table 3); ESI-MS (pos.) m/z 581.1 (46%) ([M+H]+ ), 435.0 (39%), 303.0 (100%); HR-ESI-MS (neg.) [M-H]- m/z 579.1356 (C26H28O15H, calcd. 579.1355). Calibration curves The standard solutions of rutin (2), isoquercitrin (4), quercetin-3- O - a-L-rhamnopyranoside ( 10 ) and kaempferol-3-O-a-L-rhamnopyranoside (12) were prepared by dilution of the stock standard solutions to reach the concentrations in the range of 0.25250 mg/mL. Triplicate 10 mL injections for every concentration, each corresponding to 0.0045.781 nmol of samples, were analyzed under the chromatographic conditions described above. Quantification was carried out by calculation of every peak area integrated at 365 nm. The peak area values were plotted against the corresponding amount of sample (in nmol). A linear relationship within this test range for each compound was obtained (Table 4).
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ACKNOWLEDGEMENTS We thank Mr. Won-Bing Chen and Mr. Jer-Tone Lin, Fu-shan Research Center, Taiwan Forestry Research Institute, for assisting in plant collection and authentication, and the National Science Council, Taiwan (Grants No. NSC 94-2320-B-002-089 and NSC 95-2320-B-002-033) for financial support. REFERENCES
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