Granatumins A-G, limonoids from the seeds of a Krishna mangrove, Xylocarpus granatum

Article abstract of DOI:10.1021/np900625w

Seven new limonoids (1-7), named granatumins A-G, were isolated from seeds of an Indian mangrove (Xylocarpus granatum) collected from the wetlands of Krishna estuary, Andhra Pradesh. The known compounds khayasin T, tigloylseneganolide A, 6-deoxyswietenine

Full text of DOI:10.1021/np900625w

2110
J. Nat. Prod. 2009, 72, 2110–2114
Granatumins A-G, Limonoids from the Seeds of a Krishna Mangrove, Xylocarpus granatum
Min-Yi Li,†,3 Xiao-Bo Yang,‡ Jian-Yu Pan,§ Gang Feng,⊥ Qiang Xiao,| Jari Sinkkonen,° Tirumani Satyanandamurty,¶ and
Jun Wu*,†
Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164
West Xingang Road, Guangzhou 510301, People’s Republic of China, College of Chinese Medicinal Materials, Jilin Agricultural UniVersity,
Changchun 130118, People’s Republic of China, Institute of Medicinal Plant DeVelopment, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing 100193, People’s Republic of China, EnVironment and Plant Protection Institute, Academy of Tropical
Agriculture Sciences of China, Danzhou in Hainan ProVince, 571737, People’s Republic of China, Institute of Organic Chemistry, Jiangxi
Science & Technology Normal UniVersity, Nanchang 330013, People’s Republic of China, Graduate UniVersity of Chinese Academy of
Sciences, 19 Yuquan Road, Beijing 100049, People’s Republic of China, Laboratory of Organic Chemistry and Chemical Biology, Department
of Chemistry, UniVersity of Turku, Turku 20014, Finland, and GoVernment Degree College at Amadala Valasa, Srikakulam District,
Andhra Pradesh, India
ReceiVed October 5, 2009
Seven new limonoids (1-7), named granatumins A-G, were isolated from seeds of an Indian mangrove (Xylocarpus
granatum) collected from the wetlands of Krishna estuary, Andhra Pradesh. The known compounds khayasin T,
tigloylseneganolide A, 6-deoxyswietenine, swietemahonolide, febrifugin A, gedunin, xylogranatinin, phaseic acid, (2R,3R)-
3,4′,5,7-tetrahydroxyflavanone, and (E)-4-hydroxycinnamic acid were also isolated. The structures were established on
the basis of spectroscopic data. Granatumins A and B are mexicanolides with endo-conjugated ∆8,30 and ∆14,15 double
bonds, and granatumins F and G are polyhydroxylated phragmalins found previously in plants of the Meliaceae. Khayasin
T exhibited moderate insecticidal activity against fifth instar larvae of Brontispa longissima (Gestro) at a concentration
of 20 mg/L.
Limonoids, triterpene derivatives from a precursor with a 4,4,8-
trimethyl-17-furanylsteroid skeleton, have been found only in plants
of the order Rutales. They are classified by the type of four rings
in the intact triterpene nucleus, and these are usually oxidized and
designated as A, B, C, and D. The mangroves Xylocarpus granatum
and X. moluccensis are known for producing antifeedant limonoids,
especially mexicanolides and phragmalins. Previous investigations
on the seeds of these two species yielded an obacunol, two
phragmalins, three andirobins, and 14 mexicanolides, including
xyloccensins A-K.1-5 Previously we reported the isolation and
identification of eight 8,9,30-phragmalin ortho esters and 13
limonoids from the bark and seeds of a Chinese mangrove, X.
granatum.6-8 To date, 42 mexicanolides and 23 phragmalins have
been isolated from the wood, seeds, and fruits of X. granatum and
X. moluccensis.9 In the current paper we present the isolation and
characterization of seven new limonoids (1-7), five mexicanolides
and two phragmanlins named granatumins A-G, from seeds of
the Indian mangrove Xylocarpus granatum Ko¨nig (Meliaceae),
collected in the mangrove wetlands of Krishna estuary, Andhra
Pradesh, India, together with 10 known compounds, khayasin T,10
tigloylseneganolide A,11 6-deoxyswietenine,12 swietemahonolide,13
febrifugin A,14 xylogranatinin,15 gedunin,16 phaseic acid,17 (2R,
3R)-3,4′,5,7-tetrahydroxyflavanone,18 and (E)-4-hydroxycinnamic
acid.19 The structures of these compounds were established on the
basis of spectroscopic data or comparison with data in the literature.
Results and Discussion
Compound 1 was obtained as a white, amorphous powder. Its
molecular formula of C31H36O8 was established by a quasi-molecular
ion peak in the HRTOFMS at m/z 559.2304 (calcd for [M + Na]+
559.2302), indicating that 1 had 14 degrees of unsaturation. The
1H and 13C NMR data (Tables 1 and 3) of 1 indicated that nine of
the 14 elements of unsaturation came from a ketone, three ester
functionalities, and five carbon-carbon double bonds. Therefore
the molecule was pentacyclic. The 13C NMR and DEPT experiments
revealed that 1 had six methyls (one OCH3 and five tertiary CH3
groups), four methylenes (one olefinic), 10 methines (five olefinic),
and 11 quaternary carbons (four carbonyls).
The 2D NMR studies (1H-1H COSY, HSQC, HMBC) (Figure
1) of 1 indicated the presence of a ketone (δC 214.2), a methoxy-
carbonyl group (δH 3.69 s, δC 52.0 CH3, 173.7), a methacryloyl
group [δH 2.03 s, 5.75 s, 6.24 s; δC 18.4 CH3, 127.1 CH2, 135.6
qC, 166.2 qC], and a ꢀ-furanyl ring [δH 6.48 br s, 7.43 br s, 7.51
br s; δC 110.2 CH, 143.2 CH, 141.4 CH, 120.2 qC]. An
R,ꢀ-unsaturated δ-lactone ring D, characterized by the following
NMR data [δH 5.15 s, 6.18 s; δC 79.7 CH, 112.4 CH, 37.5 qC,
160.6 qC, 164.9 qC], was confirmed by HMBC correlations between
H-15/C-13, H-15/C-14, H-15/C-16, H-17/C-13, and H-17/C-16. The
above NMR studies suggested that 1 was a mexicanolide. An
aliphatic methine group (δH 3.73 t, δC 48.9), having 1H-1H COSY
correlations to H-3 and H-30, was attributed to CH-2. Another
aliphatic methine group [δH 3.35 (d, J ) 9.5 Hz), δC 40.2], showing
a 1H-1H COSY correlation to H-6 and HMBC correlations to C-4,
C-6, and C-10, was assigned to CH-5. Two protons [δH 2.33 br s,
2.36 (d, J ) 9.5 Hz)], having HMBC correlations to C-5 and C-7,
were attributed to H2-6. An oxygenated methine group [δH 4.91
* To whom correspondence should be addressed. Tel: (86-20) 8445-
8442. Fax: (86-20) 8445-1672. E-mail: wwujun2003@yahoo.com.
† South China Sea Institute of Oceanology, Chinese Academy of
Sciences.
‡ Jilin Agricultural University.
(d, J ) 9.0 Hz), δC 78.5 CH], exhibiting a H-1H COSY cross-
1
§ Chinese Academy of Medical Sciences and Peking Union Medical
College.
peak to H-2 and HMBC correlations to C-2 and C-4, was assigned
to CH-3. A ∆8,30 double bond was suggested by HMBC correlations
between H-2/C-30, H-9/C-8, H-15/C-8, H-30/C-9, and H-30/C-14.
The results indicated that 1 was a mexicanolide with endo-
conjugated ∆8,30 and ∆14,15 double bonds. The strong HMBC
⊥ Academy of Tropical Agriculture Sciences of China.
| Jiangxi Science & Technology Normal University.
3 Graduate University of Chinese Academy of Sciences.
° University of Turku.
¶ Government Degree College at Amadala valasa.
10.1021/np900625w CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/04/2009

Limonoids from the Seeds of a Krishna MangroVe
Journal of Natural Products, 2009, Vol. 72, No. 12 2111
Chart 1
Table 1. 1H NMR (500 MHz for 1, 3-5 and 400 MHz for 2) Data (δ) for Compounds 1-5 in CDCl3 (J in Hz)
position
1
2
3
4
5
2
3
5
6a
6b
8
9
11R
11ꢀ
12R
12ꢀ
14
3.73, t (7.5)
4.91, d (9.0)
3.35, d (9.5)
2.33, br s
3.65, m
3.67, dd (9.5, 2.0)
5.12, d (9.0)
3.44, d (8.5)
2.29, br s
3.14, m
3.56, m
4.90, d (9.0)
3.30, dd (9.0, 2.0)
2.33, br s
4.90 d (8.5)
3.77, d (9.0)
2.32, br s
2.35, d (9.0)
1.95, m
1.72, m
2.30, m
1.58, m
1.23, m
4.82, d (9.5)/4.85, d (9.5)
3.30, d (10.0)/3.25, d (9.5)
2.37, br s
2.36, d (9.5)
2.37, d (9.6)
2.34, d (9.5)
2.46, d (10.0)
2.29, m
1.75, m
1.48, m
1.29, m
1.94, m
2.30, m
1.74, m
1.49, m
1.30, m
1.90, m
1.99, m
1.74, m
1.49, m
1.26, m
2.02, m
2.23a
2.03, dd (12.5, 4.0)
1.77, m
1.40, m
1.69, m
1.89, m
1.67, m
2.23a
15R
15ꢀ
17
6.18, s
6.23, s
6.12, s
2.91, dd (19.0, 7.5)
2.36, d (19.0)
5.81, s
2.83, dd (18.5, 6.0)
2.75, br d (18.5)
5.43, s/5.55, s
1.11, s
5.15, s
1.04, s
5.14, s
1.05, s
5.22, s
1.15, s
18
0.97, s
19
1.20, s
1.19, s
1.08, s
1.09, s
1.15, s
21
22
23
28
7.51, br s
6.48, br s
7.43, br s
0.80, s
7.51, br s
6.48, br s
7.43, br s
0.82, s
7.51, br s
6.50, br s
7.45, br s
0.82, s
7.72, br s
6.47, br s
7.44, br s
0.84, s
6.07, br s/6.09, br s
6.20, br s/6.25, brs
0.85, s
29
0.84, s
0.78, s
0.82, s
0.79, s
0.81, s/0.82, s
30R
30ꢀ
7-OMe
3-Acyl-2′
3′
6.26, dd (6.0, 3.0)
6.30, dd (6.0, 3.0)
3.94, d (2.0)
1.80, m
2.82, dd (15.0, 7.0)
3.75, s
5.31, d (7.5)/5.34, br d (5.0)
3.69, s
3.69, s
2.53, m
1.57, m
1.78, m
1.01, t (7.5)
1.25, d (7.0)
3.71, s
3.75, s/3.69, s
6.98, m
5.75, s
6.24, s
2.03, s
7.07, m
6.88, m
4′
5′
a
1.93, d (7.0)
1.95, s
1.77, d (7.0)
1.82, s
1.80, d (6.5)
1.82, s/1.89, s
Overlapped signals assigned by H-1H COSY, HSQC, and HMBC spectra without designating multiplicity.
1
correlation from H-3 of the mexicanolide nucleus to the carbonyl
carbon (δC 166.2) of the methacryloyl group disclosed its location
at C-3.
ꢀ-orientation of H-5, and those between H-17/H-12ꢀ suggested the
ꢀ-orientation of H-17. Thus, the relative configuration of 1, named
granatumin A, was established as shown.
The relative configuration of 1 was established on the basis of
the NOESY interactions (Figure 2). The significant NOE interaction
from H-3 to H3-29, but not from H-3 to H-5, helped to establish
the 3R-H and the corresponding 3ꢀ-O-methacryloyl group. The axial
orientation of H-3 was supported by the value of its coupling
constant with H-2 (J ) 9 Hz). The NOE interactions between
H-12R/H3-18, H-9/H3-18, and H-9/H3-19 indicated their mutual cis
relationship and the R-orientation. Similarly, NOE interactions
between H-5/H-11ꢀ, H-5/H-30, and H-5/H3-28 indicated the
Compound 2 had the molecular formula C32H40O8, as established
by HRTOFMS (m/z 575.2609, calcd for [M + Na]+ 575.2615).
The NMR data of 2 were similar to those of 1, but revealed the
presence of a 2-methylbutyryl moiety [δH 1.01 (t, J ) 7.5 Hz),
1.25 (d, J ) 7.0 Hz), 1.57 m, 1.78 m, 2.53 m; δC 12.0 CH3, 17.1
CH3, 26.7 CH2, 41.4 CH, 175.7 qC] (Tables 1 and 3) in place of
the 3ꢀ-methacryloyl group of 1. The 2-methylbutyryl group was
1
corroborated by H-1H COSY correlations between H3-4′/H2-3′,
H2-3′/H-2′, and H-2′/H3-5′ and HMBC interactions between H3-

2112 Journal of Natural Products, 2009, Vol. 72, No. 12
Li et al.
Table 2. 1H NMR (400 MHz, CDCl3) Data (δ) for Compounds
6 and 7 (J in Hz)
CH3, 170.6 qC), a ∆14,15 double bond (δH 6.23 s; δC 120.6 CH,
162.3 qC) (Tables 2 and 3), and the absence of the 30-acetyl group
of xyloccensin Y (δH 2.19 s; δC 21.0 CH3, 171.4 qC in xyloccensin
Y). The HMBC correlation from H-12 (δ 4.89 dd, 13.2, 4.0) to the
acetyl carbon at δ 170.6 confirmed the location of the 12-acetyl
group. The existence of the ∆14,15 double bond was corroborated
by HMBC correlations between H-15/C-8, H-15/C-14, H-15/C-16,
H-17/C-14, and H3-18/C-14 (Figure 3). Moreover, the significant
NOE interactions observed in 6 from H-30 to H-2 and H-15, but
not from H-30 to H-5, H-11ꢀ, and H-17, established the 30R-H
and the corresponding 3ꢀ-OH group (Figure 4). Similarly, NOE
interactions from H-3 to Hpro-R-29, but not from H-3 to H-5, helped
to establish this 3R-H and the corresponding 3ꢀ-OAc group. Those
between H-5/H-12ꢀ, H-5/H-17, and H-12ꢀ/H-17 indicated their
mutual cis relationship and ꢀ-orientation (Figure 4). Therefore, the
structure of 6, named granatumin F, was established as shown.
position
6
7
2
3
5
6
11R
11ꢀ
3.07, d (12.4)
5.18, d (12.0)
2.61, br s
6.43, br s
2.23, dd (14.0, 4.0)
1.89, t (14.0)
4.89, dd (13.2, 4.0)
6.23, s
5.04, s
2.53, br s
6.43, br s
2.21a
1.89, t (14.0)
4.84, dd (13.5, 4.0)
6.31, s
12
15
17
5.92, s
5.95, s
18
1.64, s
1.65, s
19
1.37, s
1.41, s
21
22
23
28
7.44, br s
6.55, br s
7.38, br s
0.95, s
7.43, br s
6.55, br s
7.38, br s
0.95, s
Compound 7 had a molecular formula of C33H40O16, established
by HRTOFMS, which suggested the presence of an additional OH
group when compared to the structure of 6. Assignment of this
OH group to C-2 of 7 was corroborated by the downfield chemical
shift of this carbon in 7 (δC 76.0 qC) and HMBC correlations from
H-3 and H-30 to C-2. The relative configuration of 7 was the same
as that of 6, on the basis of NOE interactions between H-5/H-12ꢀ,
H-5/H-17, H-17/H-21, H-15/H3-18, H-21/H3-18, and H-22/H3-18.
Thus, granatumin G (7) was identified as 2-hydroxygranatumin F.
29pro-S
29pro-R
30
7-OMe
3-OAc-2′
6-OAc-2′′
12-OAc-2′′′
2.30, d (10.8)
1.44, d (10.8)
4.42, br s
3.74, s
2.02, s
2.20, s
2.18, d (10.5)
1.68, d (10.5)
4.41, br s
3.74, s
2.06, s
2.21, s
1.55, s
1.55, s
a Overlapped signals assigned by 1H-1H COSY, HSQC, and HMBC
spectra without designating multiplicity.
The obtained mexicanolides were tested for insecticidal activity
using a conventional leaf disk method against the fifth instar larvae
of Brontispa longissima (Gestro). Khayasin T, previously reported
to have significant insecticidal activity against the leafcutting ants
Atta sexdens rubropilosa,25 exhibited moderate insecticidal activity
at a concentration of 20 mg/L. Its lethal rates against the fifth instar
larvae of B. longissima at exposure times of 48, 72, and 96 h were
17.4%, 27.8%, and 41.5%, respectively. The other compounds were
less active. However, lethal rates of toosendanin at the same
concentrations were 0%, 7.4%, and 7.4%, respectively.
4′/C-3′, H3-4′/C-2′, H3-5′/C-2′, H3-5′/C-1′, and H-2′/C-1′. The
HMBC cross-peak from H-3 [4.90 (d, J ) 9.0 Hz)] to a carbonyl
carbon placed the 2-methylbutyryl group at C-3. The significant
NOE interaction observed in 2, from H-3 to H3-29, but not from
H-3 to H-5, established the 3ꢀ-orientation of the 2-methylbutyryl
group. The absolute configuration at C-2′ in the 2-methylbutyryl
group was determined as S on the basis of the positive specific
rotation of the corresponding acid [[R]25 +16 (c 0.05, Me2CO)],
D
obtained from alkaline hydrolysis of 2.20-22 Thus, granatumin B
(2) was identified as 3-O-2S-methylbutyryl 3-demethacryloylgrana-
tumin A.
Experimental Section
Compound 3 had the molecular formula C32H38O9, as established
by HRTOFMS. The NMR data of 3 were similar to those of
swietemahonolide,13 except for the presence of a ∆14,15 double bond
(δH 6.12 s; δC 118.8 CH, 160.9 qC). The existence of this double
bond was supported by HMBC correlations between H-15/C-8,
H-15/C-13, H-15/C-14, H-15/C-16, and H-30/C-14. Moreover, the
R-orientation of the 8,30-epoxy ring in 3 was confirmed by the
NOE interaction between H-30/H-15. Therefore, granatumin C (3)
was concluded to be 14,15-dedihydroswietemahonolide.
The NMR data of 4 (C32H42O8 by HRTOFMS) were similar to
those of swietemahonolide,13 except for the lack of the 8,30-epoxy
ring [δH 3.22 (d, J ) 2.5 Hz); δC 60.7, 63.4 in swietemahonolide],
which was confirmed by carbon multiplicities observed in the DEPT
experiment. Significant NOE interactions observed in 4 between
H-9/H3-19, H-14/H3-18, and H-8/H-9 established their R-orientation.
Thus, granatumin D (4) was identified as 8,30-deepoxyswietemaho-
nolide.
General Experimental Procedures. Optical rotations were recorded
on a Polaptronic HNQW5 automatic high-resolution polarimeter
(Schmidt & Haensch Co. Ltd.). UV spectra were obtained on a Beckman
DU-640 UV spectrophotometer, and MALDITOFMS spectra were
measured on a Bruker APEX II spectrometer in positive ion mode.
NMR spectra were recorded in CDCl3 using Bruker AV-400 or AV-
500 spectrometers with TMS as the internal standard. Preparative HPLC
was carried out on ODS columns (250 × 10 mm i.d. and 250 × 4.6
mm i.d.,YMC) with a Waters 2998 photodiode array detector. For CC,
silica gel (200-300 mesh) (Qingdao Mar. Chem. Ind. Co. Ltd.) and
RP C18 gel (Cosmosil C18-PREP 140 µm, Nacalai Tesque, Kyoto,
Japan) were used.
Plant Material. The seeds of X. granatum were collected in
September 2007 at the mangrove wetlands in Krishna estuary, Andhra
Pradesh, India. The identification of the plant was performed by one
of the authors (T. S.). A voucher sample (No. IndianXM-02) is
maintained in the Herbarium of the South China Sea Institute of
Oceanology.
Compound 5, a white, amorphous powder, had the molecular
formula C32H40O10. The NMR data of 5 were similar to those of
febrifugin A,14 except for the different γ-hydroxybutenolide group
substituted at C-17. The NMR data of the γ-hydroxybutenolide
group in 5, characterized by two broad proton singlets at δH 6.07/
6.09 (H-21) and 6.20/6.25 (H-22) and by resonances at δC 163.2
(C-20), 97.5/99.3 (C-21), 120.5/122.0 (C-22), and 169.4 (C-23),
were the same as those of kihadanin A.23 The appearance of pairs
of most proton and carbon resonances in the NMR spectra of 5
suggested the presence of C-21 epimers. Thus, the structure of
granatumin E (5) was elucidated as shown.
Extraction and Isolation. Dried seeds (10.0 kg) of X. granatum
were extracted three times with 95% EtOH at room temperature. The
extract was concentrated under reduced pressure, followed by suspen-
sion in H2O and extraction with EtOAc. The resulting EtOAc extract
(328 g) was chromatographed on silica gel and eluted using a
CHCl3-MeOH system (100:0-5:1) to yield 229 fractions. Fractions
63 to 70 (25.0 g) were combined and further purified by RP C18 CC
(MeCN-H2O, 50:50-100:0) to afford 82 subfractions. Subfractions
30 and 31 were combined and subjected to preparative HPLC (YMC-
Pack ODS-5-A, 250 × 20 mm i.d. and 250 × 4.6 mm i.d.,
MeOH-H2O, 50:50 to 55: 45) to yield compounds 1 (3 mg), 2, (5
mg), 3 (3 mg), 4, (3 mg), 5 (2 mg), 6 (3 mg), and 7 (2 mg), khayasin
T10 (4 mg), tigloylseneganolide A11 (4 mg), 6-deoxyswietenine12 (6
mg), swietemahonolide13 (5 mg), febrifugin A14 (2 mg), xylogranati-
nin15 (30 mg), gedunin16 (5 mg), phaseic acid17 (2 mg), (2R,3R)-
Compound 6 had the molecular formula C33H40O15. The NMR
data of 6 were similar to those of xyloccensin Y (C33H42O15),24
except for the presence of a 12-acetyl group (δH 1.55 s; δC 19.9

Limonoids from the Seeds of a Krishna MangroVe
Journal of Natural Products, 2009, Vol. 72, No. 12 2113
Table 3. 13C NMR (125 MHz for 1, 3-5 and 100 MHz for 2, 6-7) Data (δ) for Compounds 1-7 in CDCl3
position
1 (δ, mult.)
2 (δ, mult.)
3 (δ, mult.)
4 (δ, mult.)
5 (δ, mult.)
216.4, qC
48.5/48.6, CH
77.0, CH
38.4/38.6, qC
42.0/42.3, CH
33.2/33.3, CH
176.4, qC
137.5/137.9, qC
56.0/56.4, CH
49.4, qC
6 (δ, mult.)
7 (δ, mult.)
1
2
3
4
5
6
7
8
9
214.2, qC
48.9, CH
78.5, CH
39.0, qC
40.2, CH
32.9, CH2
173.7, qC
136.2, qC
54.1, CH
51.9, qC
214.3, qC
49.1, CH
78.0, CH
38.9, qC
40.5, CH
33.0, CH2
173.7, qC
136.0, qC
53.5, CH
51.8, qC
214.2, qC
48.6, CH
77.1, CH
39.7, qC
41.8, CH
32.7, CH2
173.5, qC
60.3, qC
219.6, qC
48.9, CH
76.9, CH
39.8, qC
41.8, CH
32.8, CH2
174.1, qC
36.6, CH
51.1, CH
50.3, qC
82.1, qC
48.3, CH
77.6, CH
46.0, qC
43.5, CH
71.6, CH
171.8, qC
70.6, qC
83.8, qC
76.0, qC
86.4, CH
43.8, qC
43.6, CH
71.5, CH
171.9, qC
72.1, qC
55.9, CH
49.0, qC
78.9, qC
49.9, qC
78.0, qC
48.8, qC
10
11
12
13
14
15
16
17
18
19
20
21
22
23
28
29
30
21.8, CH2
32.9, CH2
37.5, qC
21.4, CH2
32.4, CH2
37.5, qC
21.0, CH2
33.1, CH2
39.1, qC
17.8, CH2
34.5, CH2
34.4, qC
21.1, CH2
32.1, CH2
68.0, CH
43.3, qC
32.1, CH2
67.9, CH
43.3, qC
34.5/34.8, CH2
36.9/37.7, qC
45.6/46.3, CH
29.5, CH2
167.3, qC
75.5/75.7, CH
21.0, CH3
16.4, CH3
163.2, qC
97.5/99.3, CH
120.5/122.0, CH
169.4, qC
20.0, CH3
23.2, CH2
124.1/124.5, CH
53.1, CH3
166.8/167.1, qC
127.3, qC
140.2/140.4, CH
14.8, CH3
11.9, CH3
160.6, qC
112.4, CH
164.9, qC
79.7, CH
22.4, CH3
15.6, CH3
120.2, qC
141.4, CH
110.2, CH
143.2, CH
22.2, CH3
21.0, CH3
128.9, CH
52.0, CH3
166.2, qC
135.6, qC
127.1, CH2
18.4, CH3
160.6, qC
112.2, CH
164.8, qC
79.8, CH
22.3, CH3
15.7, CH3
120.2, qC
141.5, CH
110.2, CH
143.2, CH
21.8, CH3
21.3, CH3
129.0, CH
52.0, CH3
175.7, qC
41.4, CH
26.7, CH2
12.0, CH3
17.1, CH3
160.9, qC
118.8, CH
163.9, qC
79.3, CH
21.3, CH3
15.8, CH3
119.8, qC
141.7, CH
110.4, CH
143.3, CH
22.7, CH3
22.7, CH3
61.9, CH
52.1, CH3
166.6, qC
127.9, qC
140.0, CH
14.8, CH3
12.3, CH3
41.2, CH
31.9, CH2
170.2, qC
78.3, CH
22.6, CH3
18.2, CH3
121.2, qC
141.3, CH
109.8, CH
143.1, CH
22.3, CH3
22.9, CH3
25.8, CH2
52.2, CH3
167.0, qC
128.1, qC
138.8, CH
14.6, CH3
12.0, CH3
162.3, qC
120.6, CH
164.8, qC
78.6, CH
15.9, CH3
14.3, CH3
121.3, qC
142.1, CH
110.4, CH
142.8, CH
15.7, CH3
44.8, CH2
66.2, CH
53.2, CH3
170.9, qC
21.6, CH3
161.7, qC
120.9, CH
164.8, qC
78.6, CH
16.0, CH3
14.5, CH3
121.2, qC
142.1, CH
110.4, CH
142.9, CH
15.6, CH3
41.1, CH2
68.0, CH
53.3, CH3
170.3, qC
21.6, CH3
7-OMe
3-acyl-1′
2′
3′
4′
5′
6-OAc-1′′
169.1, qC
21.1, CH3
170.6, qC
19.9, CH3
169.2, qC
21.1, CH3
170.7, qC
19.9, CH3
2”
12-OAc-1′′′
2′′′
3,4′,5,7-tetrahydroxyflavanone18 (3 mg), and (E)-4-hydroxycinnamic
Granatumin C (3): white, amorphous powder; [R]25D +20 (c 0.22,
acid19 (3 mg).
1
Me2CO); UV (MeCN) λmax 221.6 nm; H and 13C NMR data (Tables
Granatumin A (1): white, amorphous powder; [R]25 +68 (c 0.1,
1 and 2); HRTOFMS m/z 589.2412 (calcd for C32H38O9Na [M + Na]+,
589.2408); HRTOFMS m/z 567.2599 (calcd for C36H47O11 [M + H]+,
567.2589).
D
1
Me2CO); UV (MeCN) λmax 214.5, 283.2 nm; H and 13C NMR data
(Tables 1 and 2); HRTOFMS m/z 559.2304 [calcd for C31H36O8Na [M
+ Na]+, 559.2302]; HRTOFMS m/z 537.2509 (calcd for C35H45O11
[M + H]+, 537.2483).
Granatumin D (4): white, amorphous powder; [R]25D -131 (c 0.28,
1
Me2CO); UV (MeCN) λmax 215.7 nm; H and 13C NMR data (Tables
Granatumin B (2): white, amorphous powder; [R]25D +46 (c 0.56,
1 and 2); HRTOFMS m/z 577.2786 (calcd for C32H42O8Na [M + Na]+,
577.2772).
1
Me2CO); UV (MeCN) λmax 210.1, 284.1 nm; H and 13C NMR data
(see Tables 1 and 2); HRTOFMS m/z 575.2609 (calcd for C32H40O8Na
Granatumin E (5): white, amorphous powder; [R]25D -101 (c 0.1,
[M + Na]+, 575.2615).
1
Me2CO); UV (MeCN) λmax 214.5 nm; H and 13C NMR data (Tables
Absolute Configuration of the 2-Methylbutyryl Group of Grana-
tumin B (2). The hydrolysis of 2 with 6% KOH in water yielded
2-methylbutyric acid. The absolute configuration of its C-2 was
suggested to be S by comparison of its specific rotation [[R]25D +16 (c
0.05, Me2CO)] with those reported in the literature.20-22
1 and 2); HRTOFMS m/z 607.2520 (calcd for C32H40O10Na [M + Na]+,
607.2514).
Granatumin F (6): white, amorphous powder; [R]25D +35 (c 0.33,
1
Me2CO); UV (MeCN) λmax 206.2 nm; H and 13C NMR data (Tables
1 and 2); HRTOFMS m/z 699.2263 (calcd for C33H40O15Na [M + Na]+,
699.2259).
Figure 1. Selected 1H-1H COSY and HMBC correlations for
granatumin A (1).
Figure 2. Diagnostic NOE correlations for granatumin A (1).

2114 Journal of Natural Products, 2009, Vol. 72, No. 12
Li et al.
Acknowledgment. Financial support for this work from the Impor-
tant Project of the Chinese Academy of Sciences (KSCX2-YW-R-093,
KZCX2-YW-216), the National High Technology Research and
Development Program of China (863 Program) (2007AA09Z407), the
National Natural Science Foundation of China (20772135), and the
Research Foundation for Young Talents from the South China Sea
Institute of Oceanology, Chinese Academy of Sciences (M.-Y.L.,
SQ200802) is gratefully acknowledged. M.-Y.L. wishes to thank the
Centre for International Mobility (CIMO) and Chinese Academy of
Sciences for a scholarship. We are grateful to Mr. T. Srikanth (Acharya
Nagarjuna University, Guntur, Andhra Pradesh, India) for help and
assistance in the collection and identification of the seeds of X.
granatum. We are also thankful to Mr. Mokana Srinivas for his
assistance in the field work in collection of the seeds of X. granatum.
1
Supporting Information Available: HRTOFMS and H and 13C
Figure 3. Selected 1H-1H COSY and HMBC correlations for
granatumin F (6).
NMR spectra of compounds 1-7. This material is available free of
charge via the Internet at http://pubs.acs.org.
References and Notes
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Figure 4. Diagnostic NOE correlations for granatumin F (6).
Granatumin G (7): white, amorphous powder; [R]25D +39 (c 0.12,
1
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Insecticidal Bioassay. Brontispa longissima (Gestro), popularly
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of each compound for three seconds and then air-dried for five minutes.
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NP900625W