J Nat Med DOI 10.1007/s11418-014-0834-z

NOTE

Phenolic constituents from stem bark of Erythrina poeppigiana and their inhibitory activity on human glyoxalase I Kiyomi Hikita • Hitoshi Tanaka • Tomiyasu Murata Kuniki Kato • Miyuki Hirata • Tatsuko Sakai • Norio Kaneda



Received: 6 December 2013 / Accepted: 24 March 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014

Abstract A novel isoflavone, erythgianin A (1), along with nine known compounds 2–10, was isolated from the stem bark of Erythrina poeppigiana (Leguminosae). The unusual isoflavone structure of 1, possessing a highly oxidized 300 ,400 -dihydroxy-200 -hydroxymethyl-200 -methyl200 ,300 -dihydropyrano substituent, was determined on the basis of spectroscopic analyses. All of the isolated compounds were evaluated for their in vitro inhibitory activity toward human glyoxalase I. Among the isolates, isolupalbigenin (10) with two prenyl groups showed the highest inhibitory activity. Keywords Erythrina poeppigiana  Leguminosae  Isoflavone  Erythgianin A  Glyoxalase I inhibitory activity

Introduction Glyoxalase I (EC 4.4.1.5: GLO I) catalyzes the reaction to transform hemimercaptal, formed from methylglyoxal (MG) and reduced glutathione, into S-D-lactoylglutathione, which is then converted to D-lactic acid by glyoxalase II K. Hikita  T. Murata  N. Kaneda (&) Laboratory of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Yagotoyama 150, Tempaku-ku, Nagoya 468-8503, Japan e-mail: [email protected] H. Tanaka (&)  K. Kato  M. Hirata Laboratory of Natural Product Chemistry, Faculty of Pharmacy, Meijo University, Yagotoyama 150, Tempaku-ku, Nagoya 468-8503, Japan e-mail: [email protected] T. Sakai Analytical Center, Meijo University, Yagotoyama 150, Tempaku-ku, Nagoya 468-8503, Japan

(GLO II) [1, 2]. The glyoxalase system, comprising GLO I and GLO II, is present in the cells of almost all organisms. MG, a side-product of glycolysis, is a highly reactive and cytotoxic carbonyl compound that can cause apoptosis. Elevated expression and higher enzyme activity of GLO I have been observed in many human malignant tissues including breast [3], colon [4], and lung [5] cancers. In addition, GLO I induces resistance of cancer cells to anticancer agents [6, 7], and elevated expression of GLO I in gastric cancer tissue is reported to be associated with tumor progression and advanced stages of the disease [8]. Thus, GLO I inhibitors are expected to be useful for inhibiting tumorigenesis through the accumulation of apoptosis-inducible MG in tumor cells. Natural flavonoids such as myricetin, quercetin, and luteolin, which possess a similar framework of C-4 carbonyl and C-5 hydroxyl groups that mimic MG transition-state structures, are highly effective inhibitors of recombinant human GLO I (hGLO I) [9]. Against this background, we aimed to assess Erythrina poeppigiana flavonoids for possible hGLO I inhibitory activity. E. poeppigiana belongs to the Leguminosae family and is distributed in South America, Africa, and Asia, and is planted as an ornamental plant in Okinawa prefecture, Japan. Previous phytochemical studies demonstrated that E. poeppigiana is rich in phenolic secondary metabolites, including arylbenzofuran, cinnamylphenols, isoflavanones, isoflavones, isoflavenes, and pterocarpans [10–13]. We and other group have previously reported that some of these phenolic compounds have antimicrobial activity against methicillin-resistant Staphylococcus aureus and Candida albicans [11, 12], and have binding activity to estrogen receptors [13]. In the present study, we describe the structural elucidation of a novel isoflavone, erythgianin A (1), along with nine known compounds 2–10 from the

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stem bark of E. poeppigiana cultivated in Okinawa, Japan and also evaluate the inhibitory activity of the isolates against purified recombinant hGLO I. Among these compounds, isolupalbigenin (10) exhibited the most potent inhibitory activity, which may have potential ability to induce elevated concentration of apoptosis-inducing MG in cultured tumor cells.

Results and discussion The CH2Cl2-soluble portion of the stem bark of E. poeppigiana was separated by silica gel column chromatography and preparative reversed phase HPLC to give the novel isoflavone 1 together with nine known compounds 2–10 (Fig. 1). Of the nine known compounds, compound 2 was identified as 5,40 -dihydroxy-200 -hydroxyisopropyldihydrofurano[4,5:7,8]-isoflavone, which was recently isolated from Ficus benjamina (Moraceae) [14]. Other compounds were identified as hydroxyerythrinin C (3) [15], erythrinin C (4) [16], erythrinin B (wighteone) (5) [17], M-Wi-2 (6) [18], erysubin B (7) [18], lupiwighteone (8) [19], laburnetin (9) [20], and isolupalbigenin (10) [21], based on comparison of their spectroscopic data with those of the authentic compounds or of reported values. The isolation of compound 2 was the first finding from

Leguminosae and we named the compound 2 erypoegin K as one of the series of erypoegin groups. Erythgianin A (1) was obtained as a colorless oil, [a]23 D -8.4 (c 0.10, MeOH) and assigned the molecular formula of C20H18O8 based on finding from HR-EIMS (m/z 386.1013 [M]?). The IR spectrum showed a conjugated carbonyl band (1670 cm-1) and a hydroxyl band (3393 cm-1). The UV spectrum and the NMR spectral data of C-2 (dH 8.22 and dC 154.5) (Tables 1, 2) indicated that 1 was an isoflavone derivative. The 1H-NMR spectrum revealed an aromatic proton (dH 6.45), a set of orthocoupled aromatic protons (dH 6.91 and 7.47) on a 4-hydroxybenzene moiety, and a hydrogen-bond hydroxyl group (dH 13.57), as well as a set of two aliphatic protons (dH 4.95 and 5.89), a methyl group (dH 1.42), and a hydroxymethyl moiety (dH 3.29 and 3.73) on a dihydropyran substituent. The presence of 4-hydroxybenzene moiety at the C-3 position was indicated by both the HMBC [correlation: H-20 (H-60 ) and C-3] and NOESY spectra [NOE interaction: H-2 and H-20 (H-60 )] as shown in Fig. 2. The 1H-NMR spectrum of 1 was closely similar to that of 7 except for the presence of signals of the two aliphatic protons (dH 4.95 and 5.89) in 1 instead of the olefinic protons (dH 5.75 and 6.77) in 7. The methyl group at the C-200 position was assigned using the HMBC technique (correlations: H-500 /C-200 , H-500 /C-300 , and H-500 /C-600 ).

5''

HO 4''

6''

2''

3''

6''

HOH2C 5''

O

8 7

9

O

2'' 3''

HO

6

OH

OH

HO

O

O

4'' 2''

O

O

2 1'

10

4''

O

HO 4'

OH

OH

1

O

3''

OH

O

OH

OH

3

2

HO O

O

HO

O

O

O

HO OH

O

OH

OH

O

O

OH

7

O

HO

OH

O

HO

OH

OH OH

8

O

9

O

HO

O

Fig. 1 Chemical structures of the isolated compounds (1–10) from Erythrina poeppigiana

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OH

6

O

OH

O

OH

5

4

HOH2C

OH

O

OH

OH

O

10

OH

J Nat Med Table 1 1H-NMR spectral data for compounds 1–3 and 7 in Me2COd6 (600 MHz)

Table 2 13C-NMR data for compounds 1–3 and 7 in Me2CO-d6 (150 MHz)

Position

1

2

3

7

Position

1

2

3

7

2

8.22 s

8.19 s

8.19 s

8.18 s

2

154.5

153.9

154.3

154.4

3

124.1

124.0

123.9

124.1

6.41 s

6.35 s

4

182.2

181.7

182.1

181.8

7.47 d (8.8)

7.46 d (8.8)

5

160.6

164.3

160.0

157.6

6

109.7

94.4

113.3

106.0

6

6.22 s

8

6.45 s

20

7.47 d (8.8)

7.45 d (8.8)

30

6.91 d (8.8)

6.91 d (8.8)

6.91 d (8.8)

6.91 d (8.8)

7

167.5

167.7

167.9

160.6

50

6.91 d (8.8)

6.91 d (8.8)

6.91 d (8.8)

6.91 d (8.8)

8 9

89.4 160.3

104.8 153.6

89.4 160.0

95.3 158.2

60

7.47 d (8.8)

7.45 d (8.8)

7.47 d (8.8)

7.46 d (8.8)

200 300

4.95 d (5.9)

4.87 dd (9.5, 8.1)

4.45 d (2.9)

3.27 dd (15.4, 9.5)

5.56 br s

5.75 d (10.3)

3.32 dd (15.4, 8.1) 400

5.89 d (5.9)

500

1.42 s

00

6

3.29 d (9.5)

6.77 d (10.3) 1.30 s 1.26 s

1.30 s 1.25 s

1.43 s 3.62 dd (11.7, 6.6)

600

3.73 d (9.5)

5-OH

13.57 s

13.24 s

13.47 s

13.42 s

OH

8.56 br s

8.57 br s

8.55 br s

8.61 br s

OH

4.41 br s

3.84 s

4.73 br s

4.20a dd (6.6, 5.9)

OH

2.85 br s

a

10

107.3

106.2

106.8

106.6

10

122.8

123.0

122.9

122.9

20

131.2

131.2

131.2

131.1

0

3

116.0

115.9

116.0

116.0

40

158.6

158.5

158.5

158.5

50

116.0

115.9

116.0

116.0

60

131.2

131.2

131.2

131.1

200

79.5

92.7

100.6

82.0

300

95.4

27.1

70.1

126.4

00

4

79.9

71.4

71.1

117.4

500

19.0

25.5

26.0

23.6

600

75.8

26.0

25.5

68.7

3.67 dd (11.7, 5.9)

3.85 br s

00

6 -OH, disappeared after addition of deuterium oxide

The dihydropyran ring fused to the C-6 and C-7 positions was assigned from the HMBC spectrum, indicating correlations between H-8/C-6, H-8/C-7, H-400 /C-7, and OH-5/C6. The relative stereochemistry at the C-200 , C-300 , and C-400 positions was decided from NOE difference experiments, in which irradiation of d 4.95 (H-300 ) showed 6 and 2 % enhancements for d 5.89 (H-400 ) and d 1.42 (H-500 ), respectively. When each signal of H-400 and H-500 was irradiated, 3 % enhancement was observed for the signal at H-300 (Fig. 3). Additional proof for the stereochemistry was obtained using the NOESY spectrum, in which the NOE interaction between H-300 and H-500 was observed. These results indicated that the methyl group at the C-200 position and two carbinolic protons at the C-300 and C-400 positions have the same orientation. Consequently, the structure of erythgianin A was established to be (-)rel-(2R,3R,4R)-5,40 -dihydroxy-(300 ,400 -dihydroxy-300 ,400 -dihydro)-2 00 -hydroxymethyl-200 -methylpyrano-[500 ,600 :6,7]-isoflavone (1). However, the absolute configuration was not

decided due to the limited availability. Compound 1 was a rare example of highly oxidized dihydropyranoisoflavone, possessing a 300 ,400 -dihydroxy-200 -hydroxymethyl-200 -methyl200 ,300 -dihydropyrano substituent, which might arise from epoxidation of a double bond on a pyran group of erysubin B (7), followed by hydrolysis of the transitory epoxide. Erypoegin K (2) was obtained as a colorless oil, [a]23 D ± 0 (c 0.10, Me2CO), and assigned the molecular formula of C20H18O6 (m/z 354.1100 [M]?) from HREIMS, of which the HMBC spectrum showed correlation between H-300 and C-8, indicating the dihydrofuran ring fused to the C-7 and C-8 positions as shown in Fig. 2. Based on the [a]D value of isoflavones with 1-hydroxy-1methylethyldihydrofuran moiety [22], erypoegin K was shown to be a racemic compound. Compound (3) was isolated in a racemic form, [a]23 D ± 0 (c 0.10, MeOH), and assigned the molecular formula of C20H18O7 (m/z 370.1069 [M]?) from HR-EIMS. The presence of the 3-hydroxy-2-(1-hydroxy-1-methylethyl)-2,3dihydrofuran substituent was exhibited from both the 1 H-1H COSY (correlation: H-200 /H-300 ) and HMBC experiments (correlations: H-500 /C-200 , H-500 /C-400 , and H-600 /C400 ) (Fig. 2). The attachment of the dihydrofuran group fused to the C-6 and C-7 positions was determined on the basis of the HMBC technique, which showed correlations between H-200 /C-7 and H-300 /C-7. The stereochemistry of compound 3 was assigned to be a trans configuration (C-200

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HOH2C

O

O

Table 3 IC50 values of compounds 1–10 against hGLO I activity

2

7

5"

6 3"

2'

3 1'

4"

HO

OH

OH

4'

O

OH

IC50 values are defined as the concentration of compounds 1– 10 required to reduce hGLO I activity by 50 % compared to vehicle alone. Values were obtained from Fig. 4. BBG was used as a positive control

1 5"

HO 4" 2"

6"

3"

8

O

ND not determined

O

Compound

IC50 (lM)

1

95.2

2

109.4

3

ND

4

92.5

5

5.0

6

39.7

7

37.5

8

9.3

9

20.2

10

4.1

BBG

65.0

7 6

OH

O OH

2 5"

HO

4"

O

6"

O

2"

7 6

H

3"

HO OH

O OH

3 H H H

H 1H-1H COSY C HMBC H NOESY

Fig. 2 Key 1H-1H COSY, HMBC, and NOESY correlations for compounds 1–3 OH

The unambiguous assignment of the 1H- and 13C-NMR spectroscopic signals of 1–3 was accomplished by analyses of the 1H-1H COSY, HMQC, HMBC, and NOESY spectra. We evaluated the dose-dependency of the isolates 1–10 against hGLO I inhibitory activity and determined their IC50 values. A known inhibitor, S-p-bromobenzylglutathione (BBG) [23], was used as a positive control. Results are summarized in Table 3 and Fig. 4. Compound 10 showed the highest inhibitory activity on hGLO I at 4.1 lM, followed by 5 (5.0 lM), 8 (9.3 lM), 9 (20.2 lM), 7 (37.5 lM), 6 (39.7 lM), 4 (92.5 lM), 1 (95.2 lM), and 2 (109.4 lM) in that order. Compound 3 exhibited almost no inhibitory activity at 100 lM. Thus, the IC50 value of compound 3 was not determined. Compounds 5 and 8 with a prenyl group on an A-ring strongly inhibited hGLO I activity, and 10 with an additional prenyl group on the B-ring displayed even stronger hGLO I inhibitory activity. Thus, the presence of free prenyl groups on A- and B-rings can be suggested to become the factor which increases the inhibitory activity of isoflavone derivatives. Compound 10 with two prenyl groups on A- and B-rings might be expected to become a potent leading compound for the development of anticancer agents.

Hb 6''

Ha Irr.

O

5"

Irr.

O

Experimental

2''

H

3''

General procedures

4''

HO H

OH

O

OH

1

OH

Irr.

Fig. 3 Difference NOEs in compound 1

and C-300 ) from the NOESY spectrum, which showed correlations between H-300 /H-500 and H-300 /H-600 . Thus, compound 3 was identified as hydroxyerythrinin C [15].

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Optical rotations were measured on a JASCO P-2000 digital polarimeter. UV and IR spectra were obtained on a JASCO V-560 and a JASCO FT/IR-4200 spectrophotometer, respectively. NMR spectra were recorded on a JEOL JNM ALPHA-600 instrument (1H: 600 MHz, 13C: 150 MHz) using TMS as an internal standard. EIMS and HR-EIMS were recorded on a JEOL JMS-700 mass spectrometer. Column chromatography was carried out on Merck silica gel (230–400 mesh; Darmstadt, Germany).

J Nat Med

Product Chemistry, University.

Relative activity (% of control)

a 100 80 compound compound compound compound BBG

60 40

1 2 3 4

20 0 0

20

40

60

80

100

Concentration of compound (µ M)

Relative activity (% of control)

b 100 80 compound 5 compound 6 compound 7 BBG

60 40 20 0 0

20

40

60

80

100

Concentration of compound (µ M) Relative activity (% of control)

c 100 80 compound 8 compound 9 compound 10 BBG

60 40 20 0 0

20

40

60

80

100

Concentration of compound (µ M)

Fig. 4 Inhibitory effect of compounds 1–10 and BBG on hGLO I a compounds 1–4, b compounds 5–7 and c compounds 8–10. Dose dependency of the compounds on hGLO I inhibition at 0–100 lM was measured as described in ‘assay of hGLO I activity’. Data represent the mean (±SE) of at least three independent experiments. Control is vehicle (DMSO) alone, and BBG was used as a positive control

Reversed phase HPLC was performed on a Shimadzu LC8A apparatus equipped with a Shimadzu SPD-20AV UV detector (Kyoto, Japan), using a Develosil ODS-10 column (20 mm 9 250 mm, 10 lm; Nomura Chemical Co, Aichi, Japan). Spots on TLC plates (Merck silica gel 60 F254) were detected with a UV lamp or by staining with I2 vapor. Plant material Stem bark of E. poeppigiana was collected in Okinawa Prefecture, Japan, in July 2002. A voucher specimen (No. 020722) was identified by one of the authors (Prof. H. Tanaka) and was deposited at the Department of Natural

Faculty

of

Pharmacy,

Meijo

Extraction and isolation Fine stem bark (8.88 kg) was macerated with Me2CO (2 9 18 L) and solvent was removed to give a dark brown residue, which was divided into three parts for n-hexane-, CH2Cl2-, and EtOAc-soluble extractions. The CH2Cl2soluble fraction (84.5 g) was applied to silica gel column chromatography and eluted with CHCl3-Me2CO (40:1, 10:1, 1:1), Me2CO, and then CHCl3-MeOH (10:1, 1:1) (each volume, 500 mL) to afford 36 fractions (A1–A36). Fr. A26 (1.38 g) was purified by repeated silica gel column chromatography using n-hexane-Me2CO (2:1) to yield 10 (181 mg). Fr. A28 (27.7 g) was subjected to silica gel column chromatography using CHCl3-Me2CO (10:1.5, 3:1) (each volume, 200 mL) to give 42 fractions (B1–B42). Frs. B14–B21 (2.73 g) were chromatographed on silica gel column using benzene-EtOAc (5:1) to provide 26 fractions (C1–C26). Frs. C5 and C6 (78 mg) were separated by silica gel column chromatography using CHCl3-Me2CO (20:1) to yield 5 (9.2 mg) and 8 (8.8 mg). Frs. C7–C9 (110 mg) were purified by silica gel column chromatography using n-hexane-Me2CO (3:1) to produce 9 (6.5 mg). Frs. C23 and C24 (250 mg) were combined to give a crude solid, which was recrystallized from EtOAc to yield 4 (14.3 mg). The resulting filtrate (21.8 mg) was separated by preparative HPLC (30 % CH3CN in H2O, 6 mL/min) to yield 2 (6.6 mg, tR 153 min) and 4 (15.9 mg, tR 163 min). Frs. B22–B32 (3.15 g) were chromatographed on silica gel column using benzene-EtOAc (5:1, 1:1), followed by silica gel column using n-hexane-Me2CO (2:1) to yield 6 (16 mg) and 7 (30 mg). Fr. A29 (8.0 g) was subjected to silica gel column chromatography using CHCl3-Me2CO (10:1.5, 3:1) to give a crude oil (955 mg), which was chromatographed on silica gel column using n-hexaneMe2CO (1.5:1, 1:1), followed by silica gel column using benzene-EtOAc (2:1) to afford an oil (21.1 mg), which was purified by preparative HPLC (30 % CH3CN in H2O, 6 mL/min) to yield 1 (5.3 mg, tR 75 min). Fr. A33 (1.67 g) was subjected to silica gel column chromatography using n-hexane-Me2CO (1.5:1, 1:1) to provide a crude oil (189 mg), which was rechromatographed on silica gel column using benzene-EtOAc (2:1) to give a brown oil (25.2 mg). The brown oil was purified by preparative HPLC (30 % CH3CN in H2O, 6 mL/min) to yield 3 (6.1 mg, tR 39 min). Erythgianin A (1) Colorless oil; [a]23 D -8.4 (c 0.10, MeOH); UV kmax (MeOH) nm (log e): 261 (4.90), 206 (4.94); IR mmax (KBr)

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cm-1: 3393, 1670, 1620, 1572, 1516, 1260, 1212; 1H and 13 C-NMR (Me2CO-d6): Tables 1 and 2, respectively; EIMS m/z: 386 [M]? (11), 285 (14), 279 (32), 256 (41), 243 (16), 228 (17), 213 (22), 199 (12), 185 (34), 167 (100), 161 (53); HR-EIMS m/z: 386.1013 [M]? (calculated for C20H18O8: 386.1002). Differential NOE: Irradiation at d 4.95 (H-300 )—6 and 2 % enhancement at d 5.89 (H-400 ) and 1.42 (H-500 ), respectively; irradiation of H-400 —3 % enhancement of H-300 ; irradiation at H-500 —5 and 3 % enhancement at d 3.29 (H-600 a) and H-300 , respectively; irradiation of H-600 a—30 and 3 % enhancement of H-600 b and H-500 , respectively; irradiation at d 3.73 (H-600 b)— 27 % enhancement of H-600 a. Erypoegin K (2) Colorless oil; [a]23 D ± 0 (c 0.10, Me2CO); UV kmax (EtOH) nm (log e): 266 (4.37), 203 (4.41); IR mmax (KBr) cm-1: 3435, 1654, 1625, 1558, 1541, 1508, 1398, 1255; 1H and 13 C-NMR (Me2CO-d6): Tables 1 and 2, respectively; EIMS m/z: 354 [M]? (69), 321 (51), 295 (100), 268 (16), 59 (20), 44 (26); HR-EIMS m/z: 354.1100 [M]? (calculated for C20H18O6: 354.1103). Hydroxyerythrinin C (3) Colorless oil; [a]23 D ± 0 (c 0.10, MeOH); UV kmax (MeOH) nm (log e): 262 (4.90), 214 sh (4.95), 203 (4.98); IR mmax (KBr) cm-1: 3405, 1663, 1626, 1579, 1456, 1370, 1270, 1214, 1071; 1H and 13C-NMR (Me2CO-d6): Tables 1 and 2, respectively; EIMS m/z: 370 [M]? (3), 352 (18), 337 (42), 334 (18), 294 (100), 176 (29), 148 (11), 44 (6), 43 (12); HR-EIMS m/z: 370.1069 [M]? (calculated for C20H18O7: 370.1052).

BL21 (DE3). Expression of recombinant protein was carried out by incubating at 37 °C with 1 mM isopropyl-b-Dthiogalactopyranoside. After incubation and subsequent centrifugation, the bacterial pellet was homogenized on ice using an ultrasonic homogenizer in 30 mM Tris HCl buffer, pH 7.5, containing 1 mM dithiothreitol. The homogenate was centrifuged at 543,200 g for 15 min at 4 °C, and the supernatant was applied on a Ni-chelating affinity column (HisTrapTM HP; GE Healthcare, Uppsala, Sweden) preequilibrated with a binding buffer consisting of 20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole, pH 7.4. After washing the column with the binding buffer, the enzyme was eluted with an elution buffer consisting of 20 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, pH 7.4. The purified enzyme was in a solution of 50 mM imidazole, pH 7.2, 20 % glycerol, 20 mM glutathione, 5 mM manganese sulfate, and was stored at -30 °C. Assay of hGLO I activity hGLO I activity was measured according to the method described by [24], which was based on the observed increase of optical density at 240 nm due to the formation of S-D-lactoylglutathione (1 OD = 3.37 mM-1 cm-1). Prior to initiating the enzyme reaction, 7.9 mM MG and 1 mM reduced glutathione were mixed, and the solution was allowed to stand at 25 °C for 90 min to form hemimercaptal at equilibrium. The enzyme was then added to the equilibrated substrate solution along with the test compounds or DMSO as a vehicle, and the increase of optical density at 240 nm was followed for 2 min at 25 °C. Acknowledgments We thank Dr. Takeo Sakai for help with the difference NOE experiments.

Erysubin B (7) Colorless oil; [a]23 D ± 0 (c 0.1, MeOH); UV (MeOH) kmax nm (log e): 283 (4.57), 226 (4.33), 203 (4.46); IR (KBr) mmax cm-1: 3380, 1650, 1620; 1H and 13C-NMR (Me2CO-d6): Tables 1 and 2, respectively; EIMS m/z: 352 [M]? (5), 321 (100), 203 (5); HR-EIMS m/z: 352.0940 [M]? (calculated for C20H16O6: 352.0947). Expression and purification of hGLO I hGLO I cDNA, which was prepared by PCR from a human lung cDNA library (BioChain, Hayward, CA, USA), was subcloned into the bacterial expression vector pET-14b (Novagen, Darmstadt, Germany) at Nde I and Sal I sites to construct a plasmid pET-hGLO I, which can produce the recombinant 6 9 His-tagged fusion protein for hGLO I. The pET-hGLO I was introduced into Escherichia coli

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References 1. Racker E (1951) The mechanism of action of glyoxalase. J Biol Chem 190:685–696 2. Thornalley PJ (1990) The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life. Biochem J 269:1–11 3. Rulli A, Carli L, Romani R, Baroni T, Giovannini E, Rosi G, Talesa V (2001) Expression of glyoxalase I and II in normal and breast cancer tissues. Breast Cancer Res Treat 66:67–72 4. Ranganathan S, Tew KD (1993) Analysis of glyoxalase-I from normal and tumor tissue from human colon. Biochim Biophys Acta 1182:311–316 5. Sakamoto H, Mashima T, Sato S, Hashimoto Y, Yamori T, Tsuruo T (2001) Selective activation of apoptosis program by Sp-bromobenzylglutathione cyclopentyl diester in glyoxalase I-overexpressing human lung cancer cells. Clin Cancer Res 7:2513–2518 6. Sakamoto H, Mashima T, Kizaki A, Dan S, Hashimoto Y, Naito M, Tsuruo T (2000) Glyoxalase I is involved in resistance of

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7. 8.

9.

10.

11.

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Phenolic constituents from stem bark of Erythrina poeppigiana and their inhibitory activity on human glyoxalase I.

A novel isoflavone, erythgianin A (1), along with nine known compounds 2-10, was isolated from the stem bark of Erythrina poeppigiana (Leguminosae). T...
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