J Nat Med DOI 10.1007/s11418-015-0912-x

ORIGINAL PAPER

Acyl flavonoids, biflavones, and flavonoids from Cephalotaxus harringtonia var. nana Noriko Komoto1 • Takahisa Nakane2 • Sachiko Matsumoto3 • Shusuke Hashimoto3 Osamu Shirota4 • Setsuko Sekita2,4 • Masanori Kuroyanagi4,5



Received: 22 January 2015 / Accepted: 4 April 2015  The Japanese Society of Pharmacognosy and Springer Japan 2015

Abstract A methanol extract of the leaves of Cephalotaxus harringtonia var. nana and its ethyl acetate (EtOAc)soluble fraction demonstrated strong antitumor activity against A549 and HT-29 cell lines. The EtOAc-soluble fraction was purified by column chromatography and highperformance liquid chromatography (HPLC) using a reverse-phase column to yield three novel acyl flavonoids and a biflavonoid, along with 15 other known compounds that included flavonoids, biflavonoids, and other phenolics. The structures of the new compounds were elucidated using spectral data from HR-MS and NMR, including twodimensional NMR studies, as (2R,3R)-3-O-eicosanoyltaxifolin (1), (2R,3R)-3-O-docosanoyltaxifolin (2), (2R,3R)-3-O-tetracosanoyltaxifolin (3), and 6-methyl40 ,7,700 -tri-O-methylamentoflavone (4). The isolated compounds, including the known compounds, were tested for possible antitumor activity; some of the biflavones were & Osamu Shirota [email protected] & Masanori Kuroyanagi [email protected] 1

Healthcare Research Institute, Wakunaga Pharmaceutical Co. Ltd., 1624 Shimokotachi, Kodach, Akitakata, Hiroshima 739-1195, Japan

2

Present Address: Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan

3

Yakult Central Institute for Microbiological Research, 1976 Yaho, Kunitachi, Tokyo 186-8650, Japan

4

Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan

5

Present Address: School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan

found to be active. The potent antitumor activity of the extract was attributed to Cephalotaxus alkaloids, such as homoharringtonine (20). Keywords Cephalotaxus harringtonia var. nana  Cephalotaxaceae  Antitumor activity  Acyl flavone  Biflavone  Flavone

Introduction Cancer is one of the main causes of mortality globally. Many studies have screened for active antitumor compounds from plant sources. Many types of natural products have been isolated as antitumor compounds, and their chemical structures have been elucidated. Two antitumor compounds, paclitaxel and camptothecin, which are derived from woody plants, have been used clinically. In the course of our research isolating biologically active compounds from woody plants in Japan, we have reported several antioxidative compounds from Alnus japonica [1], antitumor-promoting active compounds from Chaenomeles sinensis [2], and active neurite outgrowth promoting compounds from Chamaecyparis obtuse [3]. One hundred methanol (MeOH) extracts of woody plants collected in the Hiroshima area were tested for antitumor activity, and some samples were selected for a study on the isolation of antitumor constituents. Of these, the MeOH extract of Cephalotaxus harringtonia var. nana (Haiinugaya in Japanese) had the strongest antitumor activity. Many Cephalotaxus alkaloids with a characteristic skeleton were isolated from Cephalotaxus plants [4–7], some with very potent antitumor activities against various types of tumor cells. C. harrigtonia var. nana was also studied, and the investigation resulted in the identification of several novel

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alkaloids [8–10]. The activity of nonalkaloid fractions was of particular interest. A MeOH extract of C. harrigtonia var. nana was fractionated into ethyl acetate (EtOAc)-, nbutanol (n-BuOH)-, and water-soluble fractions. All fractions exhibited activity. The EtOAc-soluble fraction contained flavonoids, but only traces of alkaloids, while the nBuOH- and water-soluble fractions contained large amounts of alkaloids. Thus, the isolation and structural elucidation of the compounds from the EtOAc-soluble fraction were performed, yielding three novel acyl flavones and a biflavone, along with several known biflavonoids and flavonoids. The present study outlines the isolation and structural elucidation of the novel acyl flavones and a biflavone and describes their antitumor activities (Fig. 1).

Results and discussion Over the course of screening woody plants for antitumor compounds, a MeOH extract of the needle-like leaves of C. harrigtonia var. nana showed strong antitumor activity against human lung (A549) and human colorectal (HT-29) cancer cell lines; the growth inhibition activities were IC50 0.165 and 0.229 lg/ml, respectively. The MeOH extract was partitioned into EtOAc and n-BuOH, successively, to yield EtOAc-, n-BuOH-, and water-soluble fractions, all of which showed potent activity. The n-BuOH- and watersoluble fractions showed the presence of alkaloids after TLC analysis using Dragendorff’s reagent. The EtOAcsoluble fraction was successively purified employing various chromatographic procedures, as mentioned in the ‘‘Experimental’’ section, to yield three novel acyl flavones (1–3) and a biflavone (4), along with 15 known compounds, 5–19. Part of the n-BuOH-soluble fraction was purified by HPLC using an ODS column to yield an alkaloid (20). The other known compounds were identified

Fig. 1 Structure of compounds 1–4

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based on MS and NMR spectral data, as follows: taiwanhomoflavone-A (5) [11], sequoiaflavone (6) [12], ginkgetin (7) [13], 7,700 -di-O-methylamentoflavone (8) [14], 40 ,7,700 tri-O-methylamentoflavone (9) [15], 40 ,4¢¢¢,7,700 -tetra-Omethylamentoflavone (10) [13], naringenin (11), aromadendrin (12), sinensin-7-O-b-D-glucopyranoside (13), apigenin (14), quercitrin (15), quercetin-30 -O-b-D-glucopyranoside (16) [16], quercetin-3-O-a-L-rhamnopyranoside (17), 2-(3,4-methylenedioxyphenyl)propan-1,3-diol (18) [17], methyl protocatechuate (19), and homoharringtonine (20) [18]. Compound 1 was obtained as pale yellow viscous oil. The HR-FAB-MS of 1 showed a pseudo-molecular ion at m/z 599.3569 ([M?H]?, C35H51O8), consistent with the molecular formula C35H50O8. The IR spectrum showed absorptions at 3401 (OH), 2920, 2851, 1732 (aliphatic ester carbonyl), and 1643 (flavone carbonyl) cm-1. The UV spectrum showed maxima at 232 nm (e 11,500), 284 nm (e 11,000), 297 nm (e 10,900), and 332 nm sh (e 5600). The 1 H-NMR spectrum showed the presence of a long-chain alkyl group [dH 0.87 (3H, t, J = 6.5 Hz), 1.56 (2H, br q, J = 7.5 Hz), 2.33 (1H, dt, J = 15.5, 7.0 Hz), 2.40 (1H, dt, J = 15.5, 7.5 Hz), and 1.26 (2nH, overlap)], methine protons at C-2 and C-3 of a flavanonol moiety [dH 5.63 (1H, d, J = 12.0 Hz), 6.43 (1H, d, J = 12.0 Hz)], a 3,4dihydroxyphenyl group [d 7.65 (1H, d, J = 2.5 Hz), 7.29 (1H, d, J = 8.0 Hz), 7.23 (1H, dd, J = 8.0, 2.5 Hz)], and two m-coupled H groups [dH 6.46 (1H, d, J = 2.0 Hz), 6.34 (1H, d, J = 2.0 Hz)]. The 13C-NMR spectrum showed five oxygen-bearing aromatic carbons (dC 169.3, 166.0, 163.6, 148.6, 147.5), other aromatic carbons (dC 127.5, 120.3, 116.5, 116.2, 101.7, 97.7, 96.5), two oxygen-bearing methine carbons (dC 82.1, 72.7), a carbonyl group (dC 192.7), ester carbonyl group (dC 172.5), and a long-chain alkyl group (dC 34.1, 32.1, 29–30, 25.3, 22.9, 14.3). These results indicated that 1 is an ester linkage between a

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long-chain fatty acid and a 30 ,40 ,5,7-tetrahydroxyflavanonol. The linkage position of the acyl group and positions of functional groups were determined from the HMBC correlation as follows: H-3 (dH 6.43, 1H, d, J = 12.0 Hz) showed a correlation with the ester carbonyl carbon, C-10, 10 , and 100 (dC 101.8, 127.5, 172.4); H-2 (dH 5.63, 1H, d, J = 12.0 Hz) with C-4, 20 , and 60 (dC 192.7. 116.2, 120.3) as shown in Fig. 2. This indicated that 1 was a 3-O-acyl-flavanonol derivative. The relative configuration at C(2) and C(3) of the flavanonol moiety was confirmed to be trans from the large vicinal coupling constant (J2,3 = 12.0 Hz). The absolute configuration was determined from CD experiments. The CD spectrum of 1 showed a positive Cotton effect ([h] ?4477) at 326 nm and a negative Cotton effect ([h] -25074) at 292 nm, demonstrating that 1 has a 2R configuration [19] and therefore possesses a (2R,3R) configuration. The acyl part was determined to be eicosanoic acid from the molecular formula. Thus, the structure of 1 was determined to be (2R,3R)-3-Oeicosanoyltaxifolin. Compounds 2 and 3 were obtained as pale yellow viscous oils. HR-FAB-MS of 2 and 3 showed a pseudomolecular ion at m/z 627.3887 ([M?H]?, C37H55O8) and m/z 655.4229 ([M?H]?, C39H59O8), respectively. These data are consistent with the molecular formulas C37H54O8 and C39H58O8, respectively. The 1H-NMR and 13C-NMR spectra of 2 and 3 showed almost similar signal patterns to that observed for compound 1, but their 1H-NMR spectra showed overlapping methylene protons with higher integrals than that observed for 1. Compounds 2 and 3 possess more two and four methylene units than 1, as confirmed by their larger molecular weights (m/z ?28 and m/z ?56, respectively) compared to the M? of 1. These results indicated that 2 and 3 are homologs with longer acyl chains compared to 1. The HMBC correlations between H–C (3) and the ester carbonyl carbons were also observed in 2 and

3, similar to that observed in 1 (Fig. 2). The coupling constants between H-2 and H-3 also showed large values (J = 12.0 Hz), and the Cotton effects in the CD of 2 and 3 showed results {[h]327 ?5156, [h]292 -28125, and [h]326 ?2321, [h]293 -18545, respectively} similar to that of 1. Thus, the absolute configurations of 2 and 3 are 2R,3R [19], and their structures were determined to be (2R,3R)-3O-docosanoyltaxifolin and (2R,3R)-3-O-tetracosanoyltaxifolin, respectively. Compounds 1, 2, and 3 were hydrolyzed under an acidic condition (1 N HCl in MeOH) to yield polar and nonpolar parts. The polar parts were identical to each other and were purified into a compound that was identified as taxifolin by means of HR-MS and NMR spectra, including 2D-NMR. The nonpolar parts were analyzed via HR-ESI-MS and were identified as methyl eicosanoate, methyl docosanoate, and methyl tetracosanoate. Thus, the structures of 1, 2, and 3 were unambiguously confirmed. Compound 4 was obtained as a pale yellow powder. The HR-FAB-MS of 4 showed a pseudo-molecular ion at m/z 595.1626 ([M?H]?, C34H27O10), consistent with the molecular formula C34H26O10. The IR spectrum showed absorptions at 3320, 2960, 1655, 1605, and 1500 cm-1. The UV spectrum showed absorptions at 215 nm (e 51,900), 272 nm (e 39,000), and 323 nm (e 35,700). The 1 H-NMR spectrum showed three methoxy groups [dH 3.79 (3H, s), 3.75 (3H, s), 3.74 (3H, s)], a C-methyl group on the aromatic ring [dH 2.27 (3H, s)], a p-oxyphenyl group [dH 7.78 (2H, d, J = 8.5 Hz), 7.14 (2H, d, J = 8.5 Hz)], 1,2,4substituted benzene ring [dH 8.43 (1H, d, J = 2.5 Hz), 8.13 (1H, dd, J = 9.0, 2.5 Hz), 7.34 (1H, d, J = 9.0 Hz)], and four singlet aromatic protons [dH 7.16 (1H, s), 6.75 (1H, s), 6.98 (1H, s), 6.84 (1H, s)]. The 13C-NMR spectrum showed the presence of three methoxy groups (dC 56.1 9 2, 56.4), a methyl group (dC 7.7), ten oxygen-bearing sp2 carbons (dC 164.9, 164.2, 163.8, 163.3, 163.1, 162.9, 161.3, 159.0,

Fig. 2 Key HMBC correlations (H ? C) of compounds 1–4

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156.5, 154.6), etc. These facts indicated that compound 4 is an amentoflavone-type biflavone with three O-methyl groups and a C-methyl group. The positions of functional groups were determined by HMBC correlations as follows: the hydrogen-bonding hydroxyl-H (dH 13.7) at C-5 showed correlation with C-6 (dC 108.8); H-600 (dH 6.84, 1H, s) with C-700 , 800 , and 1000 (dC 163.3, 105.4, 105.5); the methyl group at C-6 (dH 2.27, 3H, s) with C-5, 6, and 7 (dC 159.0, 108.8, 163.8); the methoxy group (dH 3.75, 3H, s) with C-700 (dC 163.3); the methoxy group (dH 3.74, 3H, s) with C-7 (dC 163.8); the methoxy group (dH 3.79, 3H, s) with C-40 (dC 161.3); H-20 (dH 8.43, 1H, d, J = 2.5 Hz) with C-40 , 60 , and 800 (dC 161.3, 128.6, 105.4). (Fig. 2). These Table 1 13C and 1H-NMR spectral data of 4 and 9 measured in pyridine-d5

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results indicated the bonding positions of functional groups and dimeric position of both flavone units. Furthermore, the 1H- and 13C-NMR data of 4 were almost identical to those of 9, except for the A-ring of the upper flavone part as shown in Table 1. Thus, the structure of 4 was determined to be 6-methyl-40 ,7,700 -tri-O-methylamentoflavone. Compounds isolated from C. harringtonia var. nana were tested for their antitumor activity against A549 and HT-29 cell lines. Biflavones 4, 5, 7, and 8 showed moderate antitumor activity, and an alkaloid, homoharringtonine (20), showed strong activity. However, the acyl flavones 1–3 exhibited no activity (Table 2). These results indicated that the antitumor activity of C. harringtonia var.

Position

dC

Compound 4 dH (J in Hz)

2

164.2



164.3



3

105.1

7.16 (1H, s)

105.0

7.12 (1H, s)

4

183.3



183.3



5

159.0



158.2

6

108.8



98.8

7

163.8



165.9

8

90.4

9

156.5

6.75 (1H, s) –

dC

92.9 162.6

Compound 9 dH (J in Hz)

– 6.70 (1H, d, J = 2.2) – 6.59 (1H, d, J = 2.2) –

10

105.4



105.9



10

122.2



123.5



20

131.7

8.43 (1H, d, J = 2.5)

131.7

8.41 (1H, d, J = 2.4)

0

3 40

122.7 161.3

– –

122.7 161.3

– –

50

112.0

7.34 (1H, d, J = 9.0)

111.9

7.31 (1H, d, J = 8.8)

60

128.6

8.13 (1H, dd, J = 9.0, 2.5)

128.8

8.10 (1H, dd, J = 8.8, 2.4)

200

164.9



164.8



00

3

103.6

6.98 (1H, s)

103.5

6.96 (1H, s)

400

182.9



182.8



500

163.1



163.1



600

96.1

700

163.3



163.1



800

105.7



105.3



900

154.6



154.6



1000

105.5



105.5



1¢¢¢

122.7



122.1



2¢¢¢,6¢¢¢

128.8

7.78 (2H, d, J = 8.5)

128.8

7.76 (2H,d, J = 8.8)

3¢¢¢,5¢¢¢

117.0

7.14 (2H, d, J = 8.5)

116.9

7.13 (2H, d, J = 8.8)

4¢¢¢ CH3 at C-6

162.9 7.7

– 2.27 (3H, s)

162.9 –

– –

OCH3 at C-7

56.1

3.74 (3H, s)

55.9

3.68 (1H, s)

OCH3 at C-40

56.4

3.79 (3H, s)

56.4

3.76 (1H, s)

OCH3 at C-700

56.1

3.75 (3H, s)

56.1

3.72 (3H, s)

6.84 (1H, s)

96.0

6.82 (1H, s)

OH at C-5



13.7 (1H, br s)



13.6 (1H, s)

OH at C-500



14.0 (1H, s)



14.0 (1H, s)

J Nat Med Table 2 Antitumor activity of the compounds isolated from C. harringtonia var. nana Compounds

A549 IC50 (lg/ml)

HT29 IC50 (lg/ml)

1

[10

[10

2

[10

[10

3 4

[10 [10

[10 0.93

5

[10

4.8

7

3.9

0.75 3.0

8

[10

20

\0.0046

0.0074

SN-38a

0.0082

0.00078

DTXa

0.00024

0.00017

a

Positive controls. SN-38: The active metabolite of irinotecan. DTX: Docetaxel

nana depends on the presence of alkaloids, while the activity of the EtOAc-soluble fraction partially depends on the biflavones.

Experimental General experimental procedures HPLC was carried out using a reverse-phase column (Mightysil C-18, Kanto Chemical Co., Ltd.) and CH3CN/ H2O containing 0.1 % trifluoroacetic acid. Silica gel 60 N (Kanto Chemical Co., Ltd.) was used for column chromatography. Analytical and preparative TLC was carried out on precoated Kieselgel 60F254 (Merck). UV and IR spectral data were obtained by employing U-2001 (Hitachi) and FT-IR spectroscopy (Perkin Elmer). Optical rotations were measured with a JASCO P-1010 polarimeter at 25 C. CD spectra were measured on a JASCO J-820 spectropolarimeter at 25 C. HR-FAB-MS data were obtained from a JEOL HX110 mass spectrometer. NMR spectra were recorded on a JEOL a-500 (1H-NMR: 500 MHz; 13C-NMR: 125 MHz) spectrometer using CDCl3 and pyridine-d5 as a solvent. Chemical shifts are shown as d values (ppm) with tetramethylsilane (TMS) as an internal standard. Plant material The needle-like leaves of C. harringtonia var. nana were collected from the northern part of Hiroshima Prefecture in September 2005. The plant species were identified by Mr. Toshihiro Sano, Hiroshima Prefectural Forestry Research Center. The plant voucher (voucher no. MK20050920-1) was deposited at the Natural Product Laboratory, Faculty

of Life and Environmental Sciences, Prefectural University of Hiroshima. Extraction and isolation The dried and powdered leaves of C. harringtonia var. nana (2.3 kg) were extracted with MeOH under reflux. The MeOH solution was evaporated under reduced pressure to yield a MeOH extract (305 g). The MeOH extract (300 g) was partitioned between water and EtOAc to give an EtOAc-soluble fraction (66 g) and water layer. The water layer was extracted with n-BuOH to give n-BuOH-soluble (44 g) and water-soluble (141 g) fractions. The EtOAc-, nBuOH-, and water-soluble fractions showed potent antitumor activity. The EtOAc-soluble fraction contained many compounds, while the n-BuOH- and water-soluble fractions showed the presence of alkaloids. There have been many reports on the alkaloid components of this plant; thus, in the present study, we aimed to isolate the constituents of the nonalkaloid fraction (EtOAc-soluble fraction). The EtOAc extract (60 g) was subjected to silica gel column chromatography (CC) using a gradient CHCl3– MeOH solvent system to yield ten fractions: Fr. 1 (5.3 g), Fr. 2 (12.3 g), Fr. 3 (6.4 g), Fr. 4 (2.1 g), Fr. 5 (2.0 g), Fr. 6 (6.5 g), Fr. 7 (6.2 g), Fr. 8 (5.1 g), Fr. 9 (18.7 g), and Fr. 10 (17.2 g). Fraction 3 (3.5 g) was purified by HPLC using a C-8 column and 70 % CH3CN to yield compounds 4 (13 mg), 9 (26 mg), and 10 (50 mg). Fraction 4 (2 g) was purified by filtration to yield a crystalline compound, which was purified by HPLC to yield compound 4 (41 mg). Fraction 6 (3 g) underwent HPLC purification using an ODS column and 55 % CH3CN to yield compounds 5 (27 mg), 7 (680 mg), 11 (8 mg), 18 (65 mg), and 19 (35 mg). Then the column was washed with 100 % CH3CN to yield a viscous liquid. This liquid was purified by HPLC using an ODS column and 100 % CH3CN to give compounds 1 (22 mg), 2 (32 mg), and 3 (21 mg). Fraction 7 (5.4 g) was chromatographed on a silica gel column using a gradient CHCl3–MeOH solvent system to generate five fractions: Fr-7-1–Fr-7-5. Fraction 7-3 (2 g) was purified by HPLC using an ODS column and 45 % CH3CN to yield compounds 6 (49 mg), 7 (37 mg), 8 (23 mg), and 12 (53 mg). Fraction 8 (500 mg) was purified by HPLC using an ODS column and 35 % CH3CN to yield compounds 12 (8 mg), 13 (10 mg), and 14 (15 mg). Fraction 9 underwent silica gel CC and HPLC employing an ODS column, successively yielding compounds 15 (10 mg), 16 (20 mg), and 17 (30 mg). One gram of the n-BuOH-soluble fraction was treated with 3 % tartaric acid aqueous solution and EtOAc, and the resultant H2O layer was made alkaline with Na2CO3 and extracted with CHCl3 to give an alkaloid fraction (53 mg) that was purified by HPLC using an ODS column

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J Nat Med Fig. 3 Structure of compounds 5–20

(Capcell PAK C18, Shiseido) and 30 % CH3CN/0.03 M (NH4)2CO3 to yield compound 20 (18 mg) (Fig. 3). (2R,3R)-3-O-Eicosanoyltaxifolin (=(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxochroman-3-yl eicosanoate; 1). Amorphous solid. UV (MeOH): 232 (4.06), 284 (4.04), 297 (4.04), 332sh (3.75). CD (MeOH) ([h]: 326 (?4477), 292 (-25074). IR (KBr): 3401 (OH), 1732 (ester C=O), 1643 (C=O). 1H-NMR (CDCl3): dH 0.87 (3H, t, J = 6.5 Hz, H-2000 ), 1.26 (2nH, br s, H-400 –H-1900 ), 1.56

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(2H, br q, J = 7.5 Hz, H-300 ), 2.33 (1H, dt, J = 15.5, 7.0 Hz, H-200 ), 2.40 (1H, dt, J = 15.5, 7.5 Hz, H-200 ), 5.63 (1H, d, J = 12.0 Hz, H-2), 6.34 (1H, d, J = 2.0 Hz, H-8), 6.43 (1H, d, J = 12.0 Hz, H-3), 6.46 (1H, d, J = 2.0 Hz, H-6), 7.23 (1H, dd, J = 8.0, 2.5 Hz, H-60 ), 7.29 (1H, d, J = 8.0 Hz, H-50 ), 7.65 (1H, d, J = 2.5 Hz, H-20 ). 13CNMR (CDCl3): dC 14.3 (C-2000 ), 22.9 (C-1900 ), 25.3 (C-300 ), 29 – 30 (C-400 – 1700 ), 32.1 (C-1800 ), 34.1 (C-200 ), 72.7 (C-3), 82.1 (C-2), 96.5 (C-8), 97.7 (C-6), 101.7 (C-10), 116.2 (C-

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20 ), 116.5 (C-50 ), 120.3 (C-60 ), 127.5 (C-10 ), 147.5 (C-30 ), 148.6 (C-40 ), 163.6 (C-9), 166.0 (C-5), 169.3 (C-7), 172.4 (C-100 ), 192.7 (C-4). HR-FAB-MS: 599.3569 ([M?H]?, C35H51O8?; calcd. 599.3584). (2R,3R)-3-O-Docosanoyltaxifolin (=(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxochroman-3-yl docosanoate; 2). Amorphous solid. UV (MeOH): 232 (4.06), 284 (4.04), 297 (4.03), 332sh (3.75). CD (MeOH) ([h]): 327 (?5156), 292 (-28125). IR (KBr): 3435 (OH), 1737 (ester C=O), 1643 (C=O). 1H-NMR (CDCl3): dH 0.87 (3H, t, J = 6.5 Hz, H-2200 ), 1.25 (34H, br s, H-400 –H-2100 ), 1.57 (2H, br q, J = 7.0 Hz, H-300 ), 2.33 (1H, dt, J = 15.5, 7.0 Hz, H-200 ), 2.40 (1H, dt, J = 15.5, 7.5 Hz, H-200 ), 5.64 (1H, d, J = 12.0 Hz, H-2), 6.34 (1H, d, J = 2.0 Hz, H-8), 6.43 (1H, d, J = 12.0 Hz, H-3), 6.47 (1H, d, J = 2.0 Hz, H-6), 7.23 (1H, dd, J = 7.5, 2.0 Hz, H-60 ), 7.29 (1H, d, J = 7.5 Hz, H-50 ), 7.65 (1H, d, J = 2.0 Hz, H-20 ); 13CNMR (CDCl3): dC 14.3 (C-2200 ), 22.9 (C-2100 ), 25.3 (C-300 ), 29–30 (C-400 –1900 ), 32.1 (C-2000 ), 34.1 (C-200 ), 72.7 (C-3), 82.1 (C-2), 96.6 (C-8), 97.8 (C-6), 101.8 (C-10), 116.2 (C20 ), 116.5 (C-50 ), 120.3 (C-60 ), 127.5 (C-10 ), 147.5 (C-30 ), 148.6 (C-40 ), 163.6 (C-9), 165.0 (C-5), 169.3 (C-7), 172.4 (C-100 ), 192.7 (C-4). HR-FAB-MS: 627.3887 ([M?H]?, C37H55O8?; calcd. 627.3897). (2R,3R)-3-O-tetracosanoyltaxifolin (= (2R,3R)-2-(3,4dihydroxyphenyl)-5,7-dihydroxy-4-oxochroman-3-yl tetracosanoate; 3). Amorphous solid. UV (MeOH): 232 (4.06), 284 (4.04), 297 (4.03), 332sh (3.75). CD (MeOH) ([h]): 326 (?2321), 293 (-18545). IR (KBr): 3401 (OH), 1736 (ester C = O), 1643 (C = O). 1H-NMR (CDCl3): dH 0.87 (3H, t, J = 7.0 Hz, H-2400 ), 1.25 (38H, br s, H-400 –H-2300 ), 1.57 (2H, br q, J = 7.0 Hz, H-300 ), 2.33 (1H, dt, J = 15.5, 7.0 Hz, H-200 ), 2.40 (1H, dt, J = 15.5, 7.5 Hz, H-200 ), 5.64 (1H, d, J = 12.0 Hz, H-2), 6.34 (1H, d, J = 2.0 Hz, H-8), 6.43 (1H, d, J = 12.0 Hz, H-3), 6.47 (1H, d, J = 2.0 Hz, H-6), 7.23 (1H, dd, J = 8.0, 2.5 Hz, H-60 ), 7.29 (1H, d, J = 8.0 Hz, H-50 ), 7.65 (1H, d, J = 2.5 Hz, H-20 ); 13CNMR (CDCl3): dC 14.3 (C-2400 ), 22.9 (C-2300 ), 25.3 (C-300 ), 29–30 (C-400 –2100 ), 32.1 (C-2200 ), 34.2 (C-200 ), 72.7 (C-3), 82.1 (C-2), 96.6 (C-8), 97.8 (C-6), 101.8 (C-10), 116.2 (C20 ), 116.5 (C-50 ), 120.3 (C-60 ), 127.5 (C-10 ), 147.5 (C-30 ), 148.6 (C-40 ), 163.6 (C-9), 165.0 (C-5), 169.3 (C-7), 172.4 (C-100 ), 192.7 (C-4). HR-FAB-MS: 655.4229 ([M?H]?, C39H59O8?; calcd. 655.4210). 6-Methyl-7,40 ,700 -tri-O-methylamentoflavone (=5-hydroxy-2-{3-[5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4oxo-4H-chromen-8-yl]-4-methoxyphenyl}-7-methoxy-6methyl-4H-chromen-4-one; 4). Pale yellow powder. [a]23 D = –5.2 (c = 0.04, CH3CN). IR (KBr): 3320 (OH), 1655 (C=O), 1605 (arom.). UV (CH3CN): 215 (51900), 272 (39000), 323 (35700). CD (CH3CN) ([h]): 325 (-1657), 305 (?308). NMR spectra: see Table 1. HRFAB-MS: 595.1626 ([M?H]?, C34H27O10?; calcd.

595.1604). HR-ESI-MS: 593.1349 [M-H]-, C34H25O10-; calcd. 593.1448). Hydrolysis of compounds 1–3 and identification of the hydrolysis products Compounds 1, 2, and 3 (each ca 5 mg) were hydrolyzed by 1 N HCl/MeOH under reflux for 1 h. The reaction solutions were poured into water and extracted with EtOAc. The EtOAc solutions were washed with sat. NaHCO3 aq. and water, successively, and were evaporated in vacuo. MeOH (0.2 ml) was added to the dried products and filtered to give MeOH-soluble polar parts. The residual parts were resolved with EtOAc to give nonpolar parts. The three polar parts showed the same spots on TLC (silica gel, CHCl3:MeOH = 6:1) and the same peaks on HPLC (Mightysil RP-18 GP). The polar parts were purified by HPLC to yield a pure compound (2.5 mg) that was measured via ESI-MS and NMR and identified as taxifolin. The three nonpolar parts from comp. 1, 2, and 3 were analyzed via ESI-MS to yield pseudmolecular ion, m/z 349.3069 [M ? Na]?, m/ z 377.3382 [M ? Na]?, and m/z 405.3700 [M ? Na]?, respectively, which were C21H42O2 for methyl eicosanoate, C23H46O2 for methyl docosanoate, and C25H52O2 for methyl tetracosanoate. Cell lines and culture conditions Human lung and colorectal cancer cell lines, A549 and HT29, were obtained from ATCC (Lockville, MD, USA). A549 and HT-29 cells were maintained in Dulbecco’s modified Eagle’s medium (D-MEM) (D6046, Sigma) and D-MEM/F-12 medium (D8062, Sigma) with 10 % heatinactivated fetal bovine serum (FBS) and 5 mg/ml of gentamicin, respectively, at 37 C in a humidified atmosphere containing 5 % CO2. Growth-inhibition assay A 190-ll volume of an exponentially growing cell suspension (1 9 104 cells/1.9 ml) was seeded in a 96-well microtiter plate, and 10 ll of each drug at various concentrations was then added 24 h after the seeding of tumor cells. Drugs were initially dissolved in DMSO and diluted to each concentration with culture medium. After incubation for 96 h at 37 C, 10 ll of TetraColor ONE (Seikagaku Biobusiness Co., Tokyo, Japan) was added to each well, and the plates were incubated for a further hour at 37 C. After incubation, the optical density was measured at 450 nm with a microplate reader (SPECTRA max PLUS, Molecular Devices, CA, USA), and the concentration causing 50 % inhibition of cell proliferation (IC50) was

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calculated from a linear regression analysis of the linear portion of the growth curves.

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Acyl flavonoids, biflavones, and flavonoids from Cephalotaxus harringtonia var. nana.

A methanol extract of the leaves of Cephalotaxus harringtonia var. nana and its ethyl acetate (EtOAc)-soluble fraction demonstrated strong antitumor a...
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