Flavonoid Composition in the Leaves of Twelve

7/29/2019 Flavonoid Composition in the Leaves of Twelve

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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 ofL. acuminata, L. hypophaea,N. acuminatissima,andN. konishii led to the identification

of 13 known flavonoids and one new flavonol dioside, quercetin 3- O-(2-O-b-D-apiofuranosyl)-a-L-

rhamnopyranoside. 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 antioxida-

tive,3,4

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-SPE-

NMR) 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 12Neolitsea andLitseaplants (Table 1) were col-

lected at the same time and same place. The n-BuOH-solu-

ble fractions of the EtOH extract of the leaves of these

plants were further fractionated on a Sephadex LH-20 col-

umn 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).

The1H 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 AAXX 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 moi-

ety (6-7, 11-12). The presence of quercetin or kaempferol

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

* Corresponding author. Tel: +886-2-23123456 ext. 88392; Fax: +886-2-23916127; E-mail: [email protected]


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moiety in the corresponding compound was also confirmed

by analyzi ng LC/ESI-MS (pos.), showing characteristic

fragment ion at m/z287 ([C15H10O6+H]+

) for kaempferol or

at m/z 303 ([C15H10O7+H]+

) for quercetin (Fig. 1). The

glycone moiety in each flavonoid glycoside (1-13) was de-

termined by ESI-MS and the 1H NMR data which showed

the characteristic signals for the anomeric protons and cer-

tain distinct protons such as Me-6 in the rhamnosyl resi-

due. The isomeric hyperoside (3) and isoquercitrin (4), the

former being 3-O-galactosylated and the latter being 3-

O-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-

Flavonoids in the Leaves of Twelve Litsea and Neolitsea Plants J. Chin. Chem. Soc., Vol. 58, No. 3, 2011 377

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)

BuOH

fractio

(ng)

UV-(+)-

subfr.

(mg)

1 (L-1) Litsea acuminata (Blume) Kurata 7.80 1.2 460.8

2 (L-2) Litsea acutivena Hayata 11.30 2.3 1258.1

3 (L-3) Litsea hypophaea Hayata 11.87 2.6 1154.4

4 (L-4) Litsea lii Chang 14.70 2.1 1600.2

5 (L-5) Litsea morrisonensis Hayata 11.90 2.4 1622.4

6 (NL-1) Neolitsea acuminatissima (Hayata) Kaneh & Sasaki 12.40 2.1 1583.4

7 (NL-2) Neolitsea buisanensis Yamam & Kamik 8.65 1.4 449.4

8 (NL-3) Neolitsea daibuensis Kamik 18.59 2.5 672.5

9 (NL-4) Neolitsea hiiranensis Liu & Liao 17.41 3.7 1668.710 (NL-5) Neolitsea konishii (Hayata) Kaneh & Sasaki 19.34 4.6 1830.8

11 (NL-6) Neolitsea parvigemms (Hayata) Kaneh & Sasaki 21.50 3.1 784.3

12 (NL-7) Neolitsea sericea (Blume) Koidz 14.30 3.6 907.2

Fig. 1. HPLC chromatogram of the UV-positive sub-

fractions from N. konishii, monitored by UV

(365 nm) and MS fragments at m/z303 and 287.

The key to peak numbering, see compound

numbering.

Fig. 2. HPLC chromatograms of the UV-positive frac-

tions from 12 lauraceous plants, monitored at

UV 365 nm.


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syl)-b-D-glucopyranoside] (1),13

rutin (2),14

quercetin

3-O-b-D-galactopyranoside (hyperoside) (3),15

isoquer-

citrin (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 (2R,3R)-dihydroquercetin

3-O-a-L-rhamnopyranoside (14).9

It was noted that the sig-

nals 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, attribut-

able to more steric effect caused by the 3- O-rhamnopyr-anosyl group than by other sugar units. Compound 13 was

378 J. Chin. Chem. Soc., Vol. 58, No. 3, 2011 Tsai and Lee

Fig. 3. Representative 1H NMR spectra of identified

flavonids (1, 3, 4, 5, 9 and 11), adopted from

HPLC-SPE-NMR. Fig. 4. Structures of the identified flavonoids.

Table 2. HPLC retention times (tR, min), ESI-MS, and1H NMR data of flavonoids identified by HPLC-SPE-NMR (CD3CN, 400 MHz)

Compd tR ESI-MS (pos.)1H-NMR (H/ppm, mult, J/Hz)

Quercetins m/z H-6 H-8 H-2 H-3 H-5 H-6 H-1 H-1 rha Me-6

1 30.4 597.0 [M+H]+, 465.0, 303.1 6.23 d (2.0) 6.45 d (2.0) 7.79 d (2.0) 6.94 d (8.5) 7.57 dd (2.0, 8.5) 5.34 d (6.0) 5.48 t (7.3)

2 31.2 611.1 [M+H]+, 465.0, 303.0 6.26 d (2.0) 6.47 d (2.0) 7.75 d (2.0) 6.93 d (8.5) 7.66 dd (2.0, 8.5) 5.00 d (7.3) 4.50 br. s 1.06 d (6.2)

3 32 .8 46 5.0 [M+H]+, 303.0 6.24 d (2.0) 6.48 d (2.0) 7.96 d (2.0) 6.93 d (8.5) 7.53 dd (2.0, 8.5) 5.00 d (7.8)

4 33 .3 46 5.0 [M+H]+, 303.0 6.26 d (2.0) 6.46 d (2.0) 7.96 d (2.0) 6.94 d (8.4) 7.51 dd (2.0, 8.4) 4.99 d (7.8)

5 33.7 567.0 [M+H]+, 435.0, 303.0 6.24 d (2.0) 6.44 d (2.0) 7.94 d (2.0) 6.95 d (8.6) 7.52 dd (2.0, 8.6) 5.25 br. s 5.19 br. s

8 35 .7 43 5.0 [M+H]+, 303.0 6.26 d (2.0) 6.46 d (2.0) 7.94 d (2.0) 6.93 d (8.6) 7.52 dd (2.0, 8.6) 5.00 d (7.8)

9 36.1 581.1 [M+H]+, 435.0, 303.0 6.23 d (2.0) 6.42 d (2.0) 7.39 d (2.0) 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)

10 37 .2 44 9.0 [M+H]+, 303. 0 6. 23 d (2.0) 6.41 d (2. 0) 7. 38 d (1. 9) 6. 94 d (8. 3) 7 .33 dd (1.9, 8. 3) 5 .42 br. s 0. 85 d (5. 5)

Kaempferols

6 34.5617.2 [M+Na]+, 595.0, 449.0,

287.06.26 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)

7 35.5 471.1 [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) 8.09 d (8.8) 4.99 d (7.8)

11 38 .8 41 9.0 [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) 8.09 d (8.8) 5.02 d (6.9)

12 41 .3 43 3.0 [M+H]+, 28 7.0 6.23 d (2 .0 ) 6 .4 1 d (2.0) 7.78 d ( 8.7) 6 .9 4 d (8.7) 6.94 d ( 8.7) 7 .7 8 d (8.7) 5.43 br . s 0.83 d (5.9)

other

13[a] 25.4 291.1 [M+H]+ 5.88 d (2.2) 5.92 d (2.2) 6.93 br. s 4.10 br. s 6.80 br. s 6.80 br. s

14[b] 34 .9 47 3.1 [M+Na]+, 303.0 5.93 d (2.0) 5.97 d (2.0) 6.97 br. s 6.86 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).


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identified as epicatechin,19

based on ESI/MS and the char-

acteristic 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), indi-

cating 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 analyzing1H and

13C NMR data as

shown in Table 3 and by comparison of the reported13C-NMR data in the literatures.20,21 The assignments of

these data were confirmed by 2D NMR spectral analyses.

The13

C NMR data for the proton bearing carbons were ob-

tained 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-b-

apiofuranosyl)-a-arabinopyranoside.21

The complete1H

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 veri-

fied by COSY spectral analysis. The presence of a b-apio-

furanosyl moiety was identified by the highly deshielded

anomeric carbon at d 111.9.22

The rhamnosyl unit was also

readily identified by the characteristic1H 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),andthe

MS fragment at m/z 303 [M+H-132-146]+

. The13

C 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-a-

L-rhamnopyranoside (d 71.9),23

indicating the glycosyla-

tion effect and pointing out the position of the sugar link-

age. Such suggestion was supported by comparison of13

C

NMR data with the model compound betonyoside F,24

a

phenylethanoid trioside containing a terminal dioside iden-

tical to 9. Such sugar linkage was also confirmed by the


Table 3. 1H and 13C NMR Data of5 and 9 (d/ppm, m, J/Hz) (CD3OD, AV-600)

5 9

Position dC[a] dH Position dC

[a] dH

6 99.4 (d) 6.19 d (2.2) 6 99.5 (d) 6.20 d (2.0)

8 94.2 (d) 6.38 d (2.2) 8 94.4 (d) 6.36 d (2.0)

2 116.6 (d) 7.57 d (2.0) 2 116.5 (d) 7.32 d (2.1)

5 116.0 (d) 6.89 d (8.3) 5 116.1 (d) 6.91 d (8.3)

6 122.7 (d) 7.54 dd (2.0, 8.3) 6 122.4 (d) 7.29 dd (2.1, 8.3)

a-L-ara-p a-L-rha

1 101.0 (d) 5.32 d (4.6) 1 102.4 (d) 5.39 d (1.6)

2 76.3 (d) 4.05 dd (4.6, 6.5) 2 79.1 (d) 4.20 d (1.6, 3.4)

3 71.9 (d) 3.83 dd (3.5, 6.5) 3 71.4 (d) 3.84 d (3.4, 9.8)

4 67.4 (d) 3.80 ddd (3.2, 3.5, 6.6) 4 73.4 (d) 3.28 m5 64.2 (t) 3.34 dd (3.2, 11.8) 5 71.7 (d) 3.57 dt (3.4, 6.2)

3.82 dd (6.6, 11.8) 6 17.5 (q) 0.97 d (6.2)

b-D-api b-D-api

1 110.2 (d) 5.27 d (1.9) 1 111.9 (d) 5.10 d (2.5)

2 77.7 (d) 3.94 d (1.9) 2 77.5 (d) 3.90 d (2.5)

4 74.9 (t) 3.70 d (9.7) 3[b] 80.3 (s)

3.91 d (9.7) 4 74.7 (t) 3.67 d (9.7)

5 65.5 (t) 3.56 d (11.5) 3.81 d (9.7)

3.61 d (11.5) 5 65.2 (t)3.51 d (11.8)

3.53 d (11.8)

[a] Data from DEPT-135 (150 MHz) and HSQC (600 MHz). [b] The chemical shift of api C-

3 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 of9 showing a positive and a negative Cotton

effect (CE) at 255 and 339 nm, respectively, being consis-

tent with that of quercetin 3-O-a-L-rhamnoside [(+)-CE at

249 nm and (-)-CE at 339 nm]25

from our collection,9

sup-

ported the rhamnosyl residue in 9 to be a-L-form. Accord-

ingly, the apiosyl moiety should be b-D-form to make the1H and

13C NMR spectral data of the diosyl moiety consis-

tent 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. Complete13

C NMR assignment,

however, was not achieved due to the limited amounts of

material.

Quantitation of rutin, isoquercitrin, quercetin 3-O-

a-L-rhamnopyranoside and kaempferol 3-O-a-L-

rhamnopyranoside

The standard curves and regression equations of au-

thentic rutin (2), isoquercitrin (4), quercetin 3-O-a-L-

rhamnopyranoside (10), and kaempferol 3-O-a-L-rhamno-

pyranoside (12) were established by HPLC analysis of a se-

ries 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 con-

tents of 12 flavonol glycosides were calculated using the

regression equations of the authentic sample for com-

pounds with the same chromophore, such as 12, containing


Table 5. The contents of 12 flavonoids in UV-positive subfractions (A)[a] and 100 g of leaves (B)[b] of 12 lauraceous plants

L-1 L-2 L-3 L-4 L-5 NL-1 NL-2 NL-3 NL-4 NL-5 NL-6 NL-7Compd

A B A B A B A B A B A B A B A B A B A B A B A B

1[c] 2.3 0.3 3.8 0.6 42.5 7.8

2 239.4 11.0 99.0 12.5 16.0 2.6 12.5 2.0 493.9 78.2 66.2 3.0 254.8 17.1 7.8 1.3 8.1 1.5 300.3 23.6 387.6 35.2

3[d] 635.5 29.3 178.1 22.4 7.9 0.9 114.6 18.3 430.4 69.8 365.5 57.9 22.5 1.0 178.0 32.6 44.1 3.5

4 91.8 4.2 137.5 17.3 17.3 2.0 43.6 7.0 20.1 3.3 48.5 7.7 43.0 1.9 30.2 2.0 122.7 20.5 165.4 30.3 161.9 12.7 36.5 3.35[c] 146.2 6.7 12.5 1.6 42.0 4.8 2.0 0.3 58.8 9.5 1.9 0.1 71.4 13.1 14.1 1.1

6[e] 28.8 1.3 25.6 3.2 32.1 5.1 8.3 0.4 31.0 2.1 0.9 0.2 28.9 2.3 38.5 3.5

7[e] 58.2 2.7 11.3 1.4 2.2 0.4 45.1 7.3 33.1 5.2 7.4 0.3 94.4 17.3

8[d] 39.3 1.8 21.5 2.7 0.6 0.1 28.7 4.7 56.7 9.0 38.3 7.0 16.4 1.3

9[c] 7.0 1.1 16.8 2.7 1.9 0.1 146.3 26.8

10 178.7 8.2 205.2 25.8 98.8 11.4 24.3 3.9 71.3 11.6 387.5 61.4 414.6 18.6 1 294.6 87.1 169.0 28.2 63.4 11.6 610.1 47.9 770.3 6 9.9

11[e] 6.6 0.3 1.1 0.2 4.9 0.8 27.2 5.0 2.4 0.2

12 5.9 0.3 25.5 3.2 3.2 0.4 1.5 0.2 64.1 10.1 9 5.2 4.3 161.9 10.9 7.4 1.2 10.6 1.9 61.5 4.8 49.5 4.5

Total 1430. 4 65. 8 718.5 90. 4 169.8 19. 6 213.5 34. 2 686.3 111.3 1486. 3 235.4 661 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 of4 in Table 4 Mw; [e] data obtained using regression equation of 12 in Table 4 Mw.

Table 4. Characteristic parameters for regression equations of four authentic flavonol glycosides, obtained by HPLC analysis

Intra-day precision[c] Inter-day precision[c]

Compd A[a] B[a] LOD[b] LOQ[b] r2

tR[d] peak area[d] tR

[d] peak area[d]

2 1661.75 6.05 19.9545 1.5125 0.0147 0.0211 0.9999 0.10 0.85 0.10 0.78

4 1622.95 6.05 9.9236 1.4384 0.0088 0.0150 0.9999 0.10 0.44 0.14 1.22

10 1214.15 3.55 0.4277 1.4947 0.0040 0.0040 0.9999 0.11 0.50 0.16 1.03

12 1102.80 4.20 14.7632 1.4662 0.0174 0.0267 0.9999 0.12 1.14 0.15 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., %.


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a kaempferol moiety, for6, 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) con-

tained the most variety of flavonoids, each 12 in total. Ex-

cept forL. hypophaea, hyperoside (3) was found to be the

main component in the other fourLitsea 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 ofNeolitsea

plants except for N. konishii. In addition, epicatechin (13)

was detected only in six Neolitsea plants but not in Litsea

plants andN. 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 demon-

strated to be a powerful method for rapid screening of both

known and new natural products. Moreover, the estab-

lished HPLC method in analyzing flavonoid composition

could be applied to the exploration of such bioactive ingre-

dients 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 chromatogra-

phy (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); HPLC-

SPE-NMR: an Agilent 1100 HPLC, equipped with a photo-

diode array detector, a Knauer K120 HPLC pump (makeup

pump) (Berlin, Germany), a Prospekt 2 solid-phase extrac-

tion unit (Bruker & Spark, Emmen, Holland) containing 96

HySphere Resin GP cartridges (10 2 mm, Spark, Emmen,

Holland), a nitrogen separator (Bruker) for flushing car-

tridge, 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 ofN. 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 H2O

(130 mL) and then partitioned against CH2Cl2, EtOAc and

BuOH (water saturated), each 130 mL 3. The BuOH-sol-

uble 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 sub-

fractions, 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 col-

umn and conditions including eluent, flow rate, volume per

tube as described above for that of N. konishii. The UV-

positive 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% TFAaq, 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 wa-

ter, 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. The1H NMR spectra were recorded using a

multiple solvent suppression pulse program for residual

protons and water signals in the D-so lvent. All spectra

were measured at 300 K, and the1H chemical shift was ref-

erenced 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 pro-

gram 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 wereacquired in the positive mode over a scan range of 50 to

1000 Da.

Separation of compounds 5 and 9 via Semi-prepara-

tive HPLC

The n-BuOH soluble fraction (2.0 g out of 4.6 g) of

the EtOH extract of the leaves ofN. konishii was subjected

to Sephadex LH-20 CC (high 48 cm, O.D. 4.0 cm; MeOH-

H2O 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-H2O

(19.5:80.5) with a flow rate of 2.6 mL/min, monitored at

UV280nm, togive 5 (0.2 mg, tR23.6 min) and 9 (0.2 mg, tR

29.5 min).

Quercetin 3-O-(2-O-b-D-apiofuranosyl)-a-L-arabino-

pyranoside (5)

UV (MeOH) 256, 356 nm;1H- and

13C-NMR (CD3OD)

(Table 3); ESI/MS (pos.) m/z567.0 (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-rhamno-

pyranoside (9)

UV (MeOH) (log e) 263 (4.35), 353 (4.16); CD [39

mM (est. from UV), MeOH] [q]339 9790, [q]255 +11280;1H- 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 kaemp-

ferol-3-O-a-L-rhamnopyranoside (12) were prepared by

dilution of the stock standard solutions to reach the concen-

trations 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. Quantifica-

tion was carried out by calculation of every peak area inte-

grated at 365 nm. The peak area values were plotted against

the corresponding amount of sample (in nmol). A linear re-

lationship within this test range for each compound was

obtained (Table 4).

ACKNOWLEDGEMENTS

We thank Mr. Won-Bing Chen and Mr. Jer-Tone Lin,

Fu-shan Research Center, Taiwan Forestry Research Insti-

tute, 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.

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Flavonoid Composition in the Leaves of Twelve

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