Larrealignans A and B, novel lignan glycosides from the aerial parts of Larrea tridentata

Article abstract of DOI:10.1248/cpb.59.1467

Two new lignan glycosides, named larrealignans A (1) and B (2), and a known lignan (3) were isolated from the aerial parts of Larrea tridentata (Zygophyllaceae). The structures of 1 and 2 were determined on the basis of spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds (1-3) and aglycones (1a, 2a) of 1 and 2 were evaluated for their cytotoxic activities against HL-60 human leukemia cells.

Full text of DOI:10.1248/cpb.59.1467

December 2011
Regular Article
Chem. Pharm. Bull. 59(12) 1467—1470 (2011)
1467
Larrealignans A and B, Novel Lignan Glycosides from the Aerial Parts of
Larrea tridentata
Akihito YOKOSUKA,* Yukiko MATSUO, Maki JITSUNO, Kohei ADACHI, and Yoshihiro MIMAKI*
Laboratory of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences; 1432–1
Horinouchi, Hachioji, Tokyo 192–0392, Japan.
Received July 11, 2011; accepted September 13, 2011; published online September 14, 2011
Two new lignan glycosides, named larrealignans A (1) and B (2), and a known lignan (3) were isolated from
the aerial parts of Larrea tridentata (Zygophyllaceae). The structures of 1 and 2 were determined on the basis of
spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds (1—3) and aglycones (1a,
2a) of 1 and 2 were evaluated for their cytotoxic activities against HL-60 human leukemia cells.
Key words lignan; lignan glycoside; Larrea tridentata; Zygophyllaceae; cytotoxic activity
Larrea tridentata (SESSE. and MOC. Ex DC.) COVILLE. (Zy-
gophyllaceae) is an evergreen shrub that grows in the desert
area of the American continents. The aerial parts (leaves and
stems) of this plant are called Chaparral and are an alterna-
tive herbal medicine used for the treatment of various can-
cers, tuberculosis, menstrual pains, and diabetes in the
United States.1) Previously, we have reported 25 triterpene
glycosides from the aerial parts of L. tridentata.2) Further
phytochemical analysis of the MeOH extract of this plant has
resulted in the isolation of two new lignan glycosides, named
larrealignans A (1) and B (2), and a known lignan (3). In this
paper, we describe the structural elucidation of 1 and 2 on
the basis of spectroscopic analysis, including various two-di-
mensional (2D)-NMR spectroscopic studies, and the results
of hydrolytic cleavage. The cytotoxic activities of the isolated
compounds (1—3) and the aglycones (1a, 2a) of 1 and 2
against HL-60 human leukemia cells are also reported.
The aerial parts of L. tridentata (3.0 kg) were extracted
with MeOH under reflux conditions. After removal of the
solvent, the concentrated MeOH extract (940 g) was passed
through a porous-polymer polystyrene resin (Diaion HP-20)
column and successively eluted with MeOH in H2O (3 : 7),
MeOH–H2O (1 : 1), MeOH, EtOH, and EtOAc. The MeOH
eluate fraction (477 g) was subjected to column chromatogra-
phy on silica gel and octadecylsilanized (ODS) silica gel,
yielding compounds 1 (23.2 mg), 2 (14.5 mg), and 3
(11.2 mg). Compound 3 was identified by its physical and
spectroscopic data as (E)-4,4ꢀ-dihydroxy-7,7ꢀ-dioxolign-
8(8ꢀ)-ene.3)
Larrealignan A (1) was obtained as an amorphous solid
and showed an accurate [MꢁNa]ꢁ ion at m/z 973.3557 in the
high resolution (HR)-electrospray ionization (ESI)-time-of-
flight (TOF)-MS, corresponding to the empirical molecular
Chart 1
formula C42H62O24. The IR spectrum of 1 suggested the pres-
ence of hydroxy groups (3376 cmꢂ1) and aromatic rings
(1613, 1509 cmꢂ1). The UV spectrum showed an absorption and 5.59 (1H, d, Jꢃ7.3 Hz). Enzymatic hydrolysis of 1 with
maximum due to substituted aromatic rings (273 nm). The b-D-glucosidase in an HOAc/NaOAc buffer (pH 5.0) at room
1H-NMR spectrum of 1 contained signals for two 1,3,4- temperature yielded meso-nordihydroguaiaretic acid (meso-
trisubstituted aromatic rings at d 7.60 (1H, d, Jꢃ8.2 Hz), NDGA) ([a]D ꢄ0)4) and D-glucose. The above data suggest
7.50 (1H, br s), and 6.81 (1H, br d, Jꢃ8.2 Hz); 7.60 (1H, d, that 1 is a meso-NDGA tetraglucoside.
Jꢃ8.2 Hz), 7.51 (1H, br s), and 6.81 (1H, br d, Jꢃ8.2 Hz),
The linkage positions of the glucosyl moieties to the agly-
two methyl groups at d 0.69 (3H, d, Jꢃ6.5 Hz) and 0.66 (3H, cone were solved by detailed analysis of the one-dimensional
d, Jꢃ6.5 Hz), and four anomeric protons at d 5.65 (1H, d, (1D) totally correlated spectroscopy (TOCSY) and 2D-NMR
1
Jꢃ7.9 Hz), 5.64 (1H, d, Jꢃ7.3 Hz), 5.60 (1H, d, Jꢃ7.6 Hz), spectra. The H-NMR subspectra of the individual glucosyl
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yokosuka@toyaku.ac.jp; mimakiy@toyaku.ac.jp

1468
Vol. 59, No. 12
units were obtained by using selective irradiation of non- Glcꢅꢅ) in 16,7) (Table 1). The b-orientations of the anomeric
overlapping proton signals in a series of 1D-TOCSY experi- centers of all of the glucosyl moieties were supported by the
1
ments.5) Subsequent analysis of the H–1H shift correlation relatively large J values of their anomeric protons (7.3—
spectroscopy (COSY) spectrum resulted in the sequential as- 7.9 Hz). In the 1H-detected heteronuclear multiple-bond con-
signments of all the proton resonances due to the four gluco- nectivity (HMBC) spectrum of 1, long-range correlations
syl units (Table 1). Using the 1H-detected heteronuclear mul- were observed between the anomeric proton (H-1) of Glcꢀ at
1
tiple-quantum coherence (HMQC) and H-detected hetero- dH 5.65 and C-3 of the aglycone at dC 148.50, H-1 of Glcꢅ at
nuclear single-quantum coherence (HSQC)-TOCSY spectra, dH 5.60 and C-4 of the aglycone at dC 146.93, H-1 of Glcꢆ at
the proton resonances were correlated with the correspond- dH 5.64 and C-3ꢀ of the aglycone at dC 148.63, and between
ing one-bond coupled carbon signals, which allowed the car- H-1 of Glcꢅꢅ at dH 5.59 and C-4ꢅ of the aglycone at dC
1
bon shifts to be completely assigned. The H- and 13C-NMR 147.02. Accordingly, the structure of larrealignan A (1) was
signals thus assigned were indicative of the presence of four elucidated as shown in Chart 1.
terminal b-D-glucopyranosyl moieties (Glcꢀ, Glcꢅ, Glcꢆ, and
Larrealignan B (2) was found to have a molecular formula
Table 1. 1H- and 13C-NMR Chemical Shift Assignments of 1 and 2 in C5D5N
1
2
Position
1H
J (Hz)
13C
Position
1H
J (Hz)
13C
1
2
3
4
5
6
7a
b
8
137.36
120.42
148.50
146.93
119.33a)
123.97
38.98
1
2
3
4
5
6
7a
b
8
137.72
121.11
148.99
147.46
119.81
124.46
39.10
7.50 br s
7.58 d
1.5
7.60 d
8.2
8.2
7.62 d
6.89 dd
2.26 m
2.72 m
1.78 m
0.77 d
8.5
8.5, 1.5
6.81 br d
2.15 m
2.66 m
1.68 m
0.69 d
39.79b)
15.80
39.70
16.17
9
6.5
9
7.0
1.5
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
7ꢀa
b
137.42
120.53
148.63
147.02
119.37a)
124.00
38.72
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
7ꢀa
b
136.31
114.04
149.65
146.13
116.40
121.77
39.02
7.51 br s
6.91 d
7.60 d
8.2
8.2
7.56 d
8.0
8.0, 1.5
6.81 br d
2.12 m
2.67 m
1.68 m
0.66 d
6.77 dd
2.22 m
2.75 m
1.75 m
0.77 d
8ꢀ
9ꢀ
39.47b)
15.87
8ꢀ
9ꢀ
3ꢀ-OMe
39.70
16.27
56.11
104.51
75.22
78.19
71.31
78.80
62.40
6.5
7.9
6.5
7.6
3.82 s
5.66 d
Glcꢀ
Glcꢅ
1
2
3
4
5
6a
b
1
2
3
4
5
6a
b
5.65 d
103.90
74.96
78.02
71.04
78.59
62.05
Glcꢀ 1
4.32 m
4.34 m
4.35 m
4.02 m
4.50 br d
4.36 m
5.60 d
4.31 m
4.33 m
4.34 m
4.04 m
4.52 br d
4.37 br d
5.64 d
4.32 m
4.34 m
4.35 m
4.02 m
4.50 br d
4.36 m
5.59 d
4.30 m
4.32 m
4.33 m
4.03 m
4.52 br d
4.38 br d
2
3
4
5
6a
b
4.35 m
4.34 m
4.34 m
4.02 m
4.50 br d
4.37 dd
5.58 d
4.34 m
4.33 m
4.33 m
4.02 m
4.53 br d
4.40 dd
5.68 d
4.38 dd
4.37 dd
4.36 dd
4.14 m
4.55 br d
4.40 dd
10.9
7.6
11.1
11.1, 4.1
7.6
104.09
74.96
77.96
71.04
78.59
62.14
Glcꢅ 1
104.58
75.22
78.29
71.31
78.84
62.40
2
3
4
5
6a
b
11.4
11.4
7.3
10.8
10.8, 3.2
7.5
7.5, 9.5
9.5, 9.5
9.5, 9.5
Glcꢆ 1
104.02
74.96
78.02
71.04
78.59
62.05
Glcꢆ 1
102.48
74.97
78.29
71.27
78.54
62.40
2
3
4
5
6a
b
2
3
4
5
6a
b
11.2
7.3
11.0
11.0, 3.8
Glcꢅꢅ 1
104.12
74.96
77.96
71.04
78.59
62.14
2
3
4
5
6a
b
11.3
11.3
a, b) Assignments may be interchangeable.

December 2011
1469
of C37H54O19 as determined by HR-ESI-TOF-MS analysis (Sigma, St. Louis, MO, U.S.A.); penicillin G sodium salt, and streptomycin
1
(m/z 825.3144 [MꢁNa]ꢁ). The H-NMR spectrum of 2 con-
sulfate (Gibco, Grand Island, NY, U.S.A.). All other chemicals used were of
biochemical reagent grade.
Plant Material The aerial parts of L. tridentata were obtained from
Richters, Ontario, Canada. A small amount of the sample is preserved in our
tained signals for three anomeric protons at d 5.68 (d,
Jꢃ7.5 Hz), 5.66 (d, Jꢃ7.6 Hz), and 5.58 (d, Jꢃ7.6 Hz), as
well as signals for two 1,3,4-trisubstituted aromatic rings at d
laboratory (07-003-LR).
Extraction and Isolation The aerial parts of L. tridentata (3.0 kg of dry
weight) were extracted with hot MeOH (77 l). After removing the solvent,
the MeOH extract (940 g), which showed cytotoxic activity against HL-60
cells (IC50 6.8 mg/ml), was passed through a Diaion HP-20 column and suc-
cessively eluted with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc.
The MeOH, EtOH, and EtOAc eluate fractions exhibited cytotoxic activity
against HL-60 cells (IC50 2.7, 2.4, 11.0 mg/ml respectively). Column chro-
matography (CC) of the MeOH eluate portion (477 g) on silica gel and elu-
tion with a stepwise gradient mixture of CHCl3–MeOH–H2O (19 : 1 : 0;
9 : 1 : 0; 40 : 10 : 1), and finally with MeOH alone, gave six fractions (I—VI).
Fraction VI was chromatographed on silica gel eluted with CHCl3–MeOH–
H2O (40 : 10 : 1; 20 : 10 : 1; 7 : 4 : 1) and ODS silica gel eluted with
MeOH–H2O (11 : 9; 2 : 1; 8 : 2) to afford 1 (23.2 mg) and 2 (14.5 mg). The
EtOH eluate portion (40.7 g) on silica gel and elution with a stepwise gradi-
ent mixture of CHCl3–MeOH (1 : 0; 19 : 1; 9 : 1), and finally with MeOH
alone, gave 12 fractions (i—xii). Fraction x was chromatographed on ODS
silica gel CC eluted with MeOH–H2O (3 : 2; 7 : 3) to afford 3 (11.2 mg).
Larrealignan A (1): Amorphous solid. [a]D25 ꢂ21.1 (cꢃ0.10, MeOH).
HR-ESI-TOF-MS m/z: 973.3557 [MꢁNa]ꢁ (Calcd for C42H62O24Na:
7.62 (1H, d, Jꢃ8.5 Hz), 7.58 (1H, d, Jꢃ1.5 Hz), and 6.89
(1H, dd, Jꢃ8.5, 1.5 Hz); 7.56 (1H, d, Jꢃ8.0 Hz), 6.91 (1H, d,
Jꢃ1.5 Hz), and 6.77 (1H, dd, Jꢃ8.0, 1.5 Hz), two methyl
groups at d 0.77 (3H, d, Jꢃ7.0 Hz) and 0.77 (3H, d,
Jꢃ6.5 Hz), and one methoxy group at d 3.82 (3H, s). Enzy-
matic hydrolysis of 2 with b-D-glucosidase yielded D-glucose
and an NDGA derivative (2a),8) which gave meso-tetra-O-
methyl-NDGA (2b, [a]D ꢄ0)9) by treatment of 2a with
trimethylsilyldiazomethane (TMS-CH2N2). The above data
suggest that 2 is a triglucoside of 2a. Using the same proce-
dures as described for 1, all the 13C-NMR signals for the glu-
cosyl moieties of 2 were assigned to the three terminal b-D-
glucopyranosyl units (Glcꢀ, Glcꢅ, and Glcꢆ). In the HMBC
spectrum of 2, long-range correlations were observed be-
tween H-1 of Glcꢀ at dH 5.66 and C-3 of the aglycone at dC
148.99, H-1 of Glcꢅ at dH 5.58 and C-4 of the aglycone at dC
147.46, and between H-1 of Glcꢆ at dH 5.68 and C-4ꢀ of the
aglycone at dC 146.13. Thus, the structure of larrealignan B
(2) was characterized as shown in Chart 1 or its antipode in
regard to the C-8 and C-8ꢀ configurations.
973.3529). UV lmax (MeOH) nm (log e): 273 (3.48). IR nmax (film) cmꢂ1
:
3376 (OH), 2925 (CH), 1613 and 1509 (aromatic rings), 1454, 1267, 1070.
1H-NMR (600 MHz, C5D5N) and 13C-NMR (125 MHz, C5D5N) spectro-
scopic data, see Table 1. The CD spectrum showed no Cotton effect in the
600—200 nm region.
Enzymatic Hydrolysis of 1 Compound 1 (4.1 mg) was treated with b-
D-glucosidase (EC 3.2.1.21, Sigma, 31.4 mg) in HOAc/NaOAc buffer (pH
5.0, 5 ml) at room temperature for 44 h. The reaction mixture was diluted
with H2O (5 ml) and extracted with EtOAc (10 mlꢇ3). After concentration
of the EtOAc-soluble phase, it was chromatographed on silica gel eluted
with CHCl3–MeOH (19 : 1) to yield meso-NDGA (1a; 0.9 mg). The H2O-
soluble phase was chromatographed on silica gel eluted with
CHCl3–MeOH–H2O (7 : 4 : 1) to yield a sugar fraction (2.0 mg). The sugar
fraction was analyzed by HPLC under the following conditions: column,
Capcell Pak NH2 SG80 Å (4.6 mm i.d.ꢇ250 mm, 5 mm, Shiseido, Tokyo,
Japan); solvent, MeCN–H2O (17 : 3); flow rate, 1.0 ml/min; detection, OR.
Identification of D-glucose present in the sugar fraction was carried out by
comparison of the retention time and optical rotation with that of an authen-
tic sample. tR (min): 16.45 (D-glucose, positive optical rotation).
A number of lignan glycosides have been isolated from the
plant kingdom. Although lignan bisdesmosides have been re-
ported from Eucomia ulmoides10) (Eucommiaceae), Sedum
sarmentosum11) (Crassulaceae), and Daphne pseudomeze-
reum12) and D. feddei13) (Thymelaeaceae), larrealignans A (1)
and B (2) are believed to be the first representatives of lignan
tetra- and trisdesmosides, respectively.
The isolated compounds (1—3) and the aglycones (1a, 2a)
of 1 and 2 were evaluated for their cytotoxic activities against
HL-60 cells. Compounds 1—3 did not exhibit cytotoxicity
even at a sample concentration of 20 mM. However, 1a and 2a
showed moderate cytotoxicity with respective IC50 values of
7.0 and 4.9 mM, whereas etoposide used as a positive control
gave an IC50 value of 0.33 mM.
meso-NDGA (1a): Amorphous solid. [a]D26 ꢄ0 (cꢃ0.10, MeOH). 1H-
NMR (400 MHz, CD3OD): d 6.70 (2H, d, Jꢃ8.0 Hz, H-2, 2ꢀ), 6.63 (2H, d,
Jꢃ2.0 Hz, H-5, 5ꢀ), 6.50 (2H, dd, Jꢃ8.0, 2.0 Hz, H-6, 6ꢀ), 2.68 (2H, dd,
Jꢃ13.3, 5.0 Hz, H-7b, 7bꢀ), 2.21 (2H, dd, Jꢃ13.3, 9.2 Hz, H-7a, 7aꢀ), 1.74
(2H, m, H-8, 8ꢀ), 0.85 (6H, d, Jꢃ6.7 Hz, Me-9, 9ꢀ). The CD spectrum
showed no Cotton effect in the 600—200 nm region.
Experimental
Optical rotations were measured using a JASCO P-1030 (Tokyo, Japan)
automatic digital polarimeter. IR spectra were recorded on a JASCO Fourier
transform (FT)-IR 620 spectrophotometer, UV spectra on a JASCO V-630
spectrophotometer, and circular dichroism (CD) spectra on a JASCO J-720
instrument, respectively. NMR spectra were recorded on a Bruker DPX-500
spectrometer (500 MHz for 1H-NMR, Karlsruhe, Germany) or a Bruker
DRX-600 spectrometer (600 MHz for 1H-NMR) using standard Bruker
pulse programs. Chemical shifts are given as d values with reference to
tetramethylsilane (TMS) as an internal standard. HR-ESI-TOF-MS data was
recorded on a Waters-Micromass LCT mass spectrometer (Manchester,
U.K.). Diaion HP-20 (Mitsubishi-Chemical, Tokyo, Japan), silica gel (Fuji-
Silysia Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque, Kyoto,
Japan) were used for column chromatography. TLC was carried out on Silica
gel 60 F254 (0.25 mm thick or 0.5 mm thick, Merck, Darmstadt, Germany)
and RP-18 F254S (0.25 mm thick, Merck) plates, and spots were visualized by
spraying with 10% H2SO4 followed by heating. HPLC was performed with a
system composed of a CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010
controller (Tosoh), a RI-8010 (Tosoh) and a Shodex OR-2 (Showa-Denko,
Tokyo, Japan) detector, and a Rheodyne injection port. The following mate-
rials and reagents were used for cell culture assay; microplate reader, Spec-
Larrealignan B (2): Amorphous solid. [a]D26 ꢂ26.8 (cꢃ0.10, MeOH).
HR-ESI-TOF-MS m/z: 825.3144 [MꢁNa]ꢁ (Calcd for C37H54O19Na:
825.3157). UV lmax (MeOH) nm (log e): 275 (3.55). CD lmax (MeOH) nm
(De): 272 (ꢂ1.0). IR nmax (film) cmꢂ1: 3389 (OH), 2926 (CH), 1593 and
1
1512 (aronatic rings), 1454, 1265, 1072. H-NMR (600 MHz, C5D5N) and
13C-NMR (125 MHz, C5D5N) spectroscopic data, see Table 1.
Enzymatic Hydrolysis of 2 Compound 2 (10.0 mg) was subjected to
enzymatic hydrolysis as described for 1 to give 2a (2.5 mg) and a sugar frac-
tion (4.3 mg). HPLC analysis of the sugar fractions under the same condi-
tions as in the case of 1 showed the presence of D-glucose. tR (min): 16.45
(D-glucose, positive optical rotation).
Compound 2a: Amorphous solid. [a]D25 ꢁ13.0 (cꢃ0.07, MeOH). 1H-
NMR (400 MHz, CD3OD): d 6.72 (1H, d, Jꢃ8.0 Hz, H-5ꢀ), 6.71 (1H, d,
Jꢃ8.0 Hz, H-5), 6.67 (1H, d, Jꢃ1.9 Hz, H-2ꢀ), 6.66 (1H, d, Jꢃ2.0 Hz, H-2),
6.60 (1H, dd, Jꢃ8.0, 1.9 Hz, H-6ꢀ), 6.53 (1H, dd, Jꢃ8.0, 2.0 Hz, H-6), 3.84
(1H, s, OMe), 2.76 (1H, dd, Jꢃ13.3, 5.0 Hz, H-7bꢀ), 2.68 (1H, dd, Jꢃ13.5,
6.0 Hz, H-7bꢀ), 2.31 (1H, dd, Jꢃ13.5, 8.7 Hz, H-7a), 2.23 (1H, dd, Jꢃ13.3,
9.5 Hz, H-7aꢀ), 1.78 (1H, m, H-8), 1.75 (1H, m, H-8ꢀ), 0.88 (3H, d,
Jꢃ6.8 Hz, Me-9), 0.88 (3H, d, Jꢃ6.8 Hz, Me-9ꢀ). The CD spectrum showed
tra Classic (Tecan, Salzburg, Austria); 96-well flat-bottom plate (Iwaki no Cotton effect in the 600—200 nm region.
Glass, Chiba, Japan); JCRB 0085 HL-60 cells (Human Science Research
Resources Bank, Osaka, Japan); fetal bovine serum (FBS) (Bio-Whittaker,
Walkersville, MD, U.S.A.); RPMI 1640 medium, etoposide, cisplatin, and 3-
Methylation of 2a TMS-CH2N2 in hexane (2.0 M, 0.5 ml) was added to
a solution of 2a (2.0 mg) in MeOH (4.0 ml), and then the mixture was stirred
at room temperature for 15 h. The reaction mixture was subjected to prepara-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), tive TLC using hexane–acetone (1 : 2) as the solvent to yield meso-tetra-O-

1470
Vol. 59, No. 12
methyl-NDGA (2b, 2.0 mg).9)
5) Yokosuka A., Kawakami S., Haraguchi M., Mimaki Y., Tetrahedron,
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Cell Culture and Assay for Cytotoxic Activity against HL-60 Cells
The cell growth was measured with an MTT reduction assay as described in
a previous paper.14) Briefly, HL-60 cells were maintained in RPMI 1640
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glutamine, 100 unit/ml penicillin G sodium salt, and 100 mg/ml streptomycin
sulfate. The cells (4ꢇ104 cells/ml) were continuously treated with each com-
pound for 72 h, and cell growth was measured with an MTT reduction assay
procedure. A dose–response curve was plotted for 1a and 2a, and the con-
centration giving 50% growth inhibition (IC50) was calculated.
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