Nimbandiolactone-21 and nimbandioloxyfuran, two new 28-norlimonoids from the leaves of Azadirachta indica (Meliaceae)

Article abstract of DOI:10.1080/10286020.2018.1476498

From an EtOAc-soluble fraction of the leaves of Azadirachta indica, two new 28-norlimonoids named nimbandiolactone-21 (1) and nimbandioloxyfuran (2), together with nimbandiolactone-23 (3), were isolated. Their relative structures were elucidated based on

Full text of DOI:10.1080/10286020.2018.1476498

Journal of Asian Natural Products Research
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Nimbandiolactone-21 and nimbandioloxyfuran,
two new 28-norlimonoids from the leaves of
Azadirachta indica (Meliaceae)
Nhi Y Thi Nguyen, Phu Hoang Dang, Van Truong Thien Nguyen, Tuan Trong
Vo, Dao Anh Thi Nguyen, Minh Duc Huu Nguyen, Phuc Huu Dang & Quan Le
Tran
To cite this article: Nhi Y Thi Nguyen, Phu Hoang Dang, Van Truong Thien Nguyen, Tuan
Trong Vo, Dao Anh Thi Nguyen, Minh Duc Huu Nguyen, Phuc Huu Dang & Quan Le Tran
(2018): Nimbandiolactone-21 and nimbandioloxyfuran, two new 28-norlimonoids from the
leaves of Azadirachtaꢀindica (Meliaceae), Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2018.1476498
To link to this article: https://doi.org/10.1080/10286020.2018.1476498
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Journal of asian natural Products research, 2018
https://doi.org/10.1080/10286020.2018.1476498
Nimbandiolactone-21 and nimbandioloxyfuran, two new
28-norlimonoids from the leaves of Azadirachta indica
(Meliaceae)
Nhi Y Thi Nguyena, Phu Hoang Danga , Van Truong Thien Nguyena, Tuan Trong Vob,
Dao Anh Thi Nguyenb, Minh Duc Huu Nguyenb, Phuc Huu Dangc,d and Quan Le Trana
afaculty of chemistry, VnuhcM–university of science, ho chi Minh city, Vietnam; bfaculty of traditional
Medicine, university of Medicine and Pharmacy at ho chi Minh city, ho chi Minh city, Vietnam; ctheoretical
Physics research Group, advanced institute of Materials science, ton duc thang university, ho chi Minh city,
Vietnam; dfaculty of applied sciences, ton duc thang university, ho chi Minh city, Vietnam
ABSTRACT
ARTICLE HISTORY
received 27 March 2018
accepted 10 May 2018
From an EtOAc-soluble fraction of the leaves of Azadirachta indica,
two new 28-norlimonoids named nimbandiolactone-21 (1) and
nimbandioloxyfuran (2), together with nimbandiolactone-23 (3),
were isolated. Their relative structures were elucidated based on
NMR spectroscopic interpretation and biosynthetic consideration.
Nimbandioloxyfuran (2) and nimbandiolactone-23 (3) showed
potent α-glucosidase inhibitory activity, with the IC50 values of 46.2
and 38.7 μM, respectively.
KEYWORDS
Azadirachta indica;
Meliaceae; 28-norlimonoid;
α-glucosidase inhibitory
1. Introduction
Azadirachta indica A. Juss. (Meliaceae) is one of the most versatile medicinal plants, which
grows in India, Nepal, Pakistan, Bangladesh, Sri Lanka, Maldives, and Vietnam. In Vietnam,
the leaves of A. indica have been used as traditional medicines for the treatment of diabetes
[1]. e leaves extract of A. indica enhances the insulin signaling molecules and glucose
metabolism, which play a significant role in the management of diabetes mellitus type-2 [2].
Study on chemical constituents of A. indica has led to the isolation of various triterpenoids
CONTACT Phuc huu dang
danghuuphuc@tdt.edu.vn
the supplementary material for this paper is available online at https://doi.org/10.1080/10286020.2018.1476498.
© 2018 informa uK limited, trading as taylor & francis Group

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N. Y. T. NGUYEN ET AL.
Figure 1. structures of compounds 1–3.
and nortriterpenoids [3]. Limonoids (tetranortriterpenoids) are the characteristic compo-
nents of A. indica, which has shown the insecticidal, antibacterial, antifungal, antimalarial,
anticancer, and antiviral properties [4].
In a continued study on the screening of medicinal plants for α-glucosidase inhibitory
activity [5–11], we found that the EtOAc-soluble extract of the leaves of A. indica showed
α-glucosidase inhibitory activity with an IC50 value of 145.0 μg/ml. us, the bioactivity-
guided isolation was carried out to afford three 28-norlimonoids (1–3) including two new
ones named nimbandiolactone-21 (1) and nimbandioloxyfuran (2). Additionally, the α-glu-
cosidase inhibitory activities of the isolated compounds were evaluated.
2. Results and discussion
e powdered leaves of A. indica were refluxed with MeOH. e MeOH-soluble extract was
successively partitioned to yield petroleum ether, EtOAc, and n-BuOH-soluble fractions.
Further separation and purification of the EtOAc-soluble fraction led to the isolation of
three compounds nimbandiolactone-21 (1), nimbandioloxyfuran (2), and nimbandiolac-
tone-23 (3) [12] (Figure 1).
Compound 1, nimbandiolactone-21, showed the molecular formula C26H32O9, as
deduced from the positive HRESIMS at m/z 489.2160 [M + H]+. e 1H NMR spectrum
showed the presence of four methyl singlets (δH 1.17, 1.26, 1.56, and 1.72), a pair of AB
doublets of a 2-en-1-one system [δH 5.71 (d, J = 10.3 Hz, H-2) and 6.53 (d, J = 10.3 Hz, H-3)],
and one methoxy group (δH 3.67). e presence of a 23-hydroxy-21-butenolide moiety
was inferred by the 1H signals [δH 6.87 (H-22) and 6.03 (H-23)] [13]. e 13C and DEPT
NMR spectra showed resonances for three carbonyls (δC 171.7, 175.5, 202.4), three pairs
of olefinic carbons (δC 124.8, 152.6, 132.7, 149.3, 138.2, 143.8), one methoxy (δC 52.2), four
oxymethines (δC 67.1, 86.2, 87.5, 97.6), one oxygenated quaternary carbons (δC 71.3), three
methines (δC 38.9, 48.8, 50.2), two methylenes (δC 34.3, 39.7), two quaternary carbons (δC
47.9, 48.3), and four methyls (δC 13.1, 15.6, 17.4, 22.7) (Table 1). ese signals resembled
those of 28-norlimonoids possessing an opened C-ring and a 7,15-ether linkage [14]. e
presence of the C-6 hydroxy group was suggested based on the 1H–1H COSY correlations
between H-5/H-6/H-7. e HMBC correlations from H-17 to both olefinic carbons (δC
138.2, C-20 and 143.8, C-22) and one carbonyl (δC 171.7, C-21) indicated that the location
of a 23-hydroxy-21-butenolide group was at C-17. e presence of the opened C-ring

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1h (500 Mhz) and 13c (125 Mhz) nMr spectral data for compounds 1 and 2 in cdcl3.
Nimbandiolactone-21 (1)
Nimbandioloxyfuran (2)
Position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
202.4,c
124.8,ch
152.6,ch
71.3,c
50.2,ch
67.1,ch
87.5,ch
47.9,c
–
5.71 d (10.3)
6.53 d (10.3)
–
2.72 d (11.5)
4.28 dd (11.5, 3.0)
4.05 d (3.0)
–
202.4,c
124.9,ch
151.8,ch
71.0,c
50.7,ch
67.8,ch
86.7,ch
47.7,c
–
5.74 d (10.0)
6.51 d (10.0)
–
2.62 d (12.0)
4.24 dd (12.0, 3.5)
3.97 d (3.5)
–
2
3
4
5
6
7
8
9
38.9,ch
48.3,c
34.3,ch2
2.49 dd (5.2, 3.3)
–
2.87 dd (16.8, 5.2)
39.0,ch
47.9,c
34.1,ch2
2.60 dd (5.5, 3.8)
–
2.92 dd (16.8, 5.5)
10
11
2.15 dd (16.8, 3.3)
2.21 dd (16.8, 3.8)
12
13
14
15
16
175.5,c
132.7,c
149.3,c
86.2, ch
39.7,ch2
–
174.2,c
132.0,c
150.4,c
86.7, ch
36.6,ch2
–
–
–
–
–
5.23 brs
2.25–2.27 m
5.42–5.45 m
2.35 dd (12.0, 6.5)
2.02–2.06 m
3.50 d (8.5)
1.72 brs
1.17 s
1.79 ddd (12.0, 8.5, 8.5)
2.89 d (8.5)
1.87 s
17
48.8,ch
13.1,ch3
15.6,ch3
138.2,c
60.4,ch
14.6,ch3
15.9,ch3
79.2,c
105.0,ch
76.8,ch
109.3,ch
23.8,ch3
17.7,ch3
51.8,ch3
56.8,ch3
55.9,ch3
18
19
1.10 s
20
–
–
21
171.7,c
–
4.79 s
22
143.8,ch
97.6,ch
22.7,ch3
17.4,ch3
52.2,ch3
6.87 brs
6.03 brs
1.56 s
3.67 d (4.0)
4.84 d (4.0)
1.58 s
23
29
30
1.26 s
3.67 s
1.29 s
12-oMe
21-oMe
23-oMe
4-oh
3.72 s
3.48 s
3.54 s
4.57 s
containing a C-11 methoxycarbonyl group was indicated based on the HMBC correlations
from H-9, H-11, and MeO-12 to the C-12 carbonyl (δC 175.5) (Figure 2). us, the structure
of nimbandiolactone-21 (1) was assigned as 17-desfuran-17-(23-hydroxybut-20(22)-ene-
21,23-γ-lactone)nimbandiol. Due to the small isolated amount of 1, it was not possible to
record NOE enhancements. Nimbandiol, a 28-norlimonoid with furan moiety which was
isolated from Azadirachta indica, could be transformed to the secondary metabolite with
lactone-21 (1) or lactone-23 ones (3) under the photooxidation conditions [15]. us, the
relative configuration of 1 was suggested similar to that of nimbandiol derivatives [16]. e
relative configurations at C-23 of 1 and C-21 of 3 remained undetermined.
Compound 2, nimbandioloxyfuran, showed the molecular formula C28H40O11 based on
the positive HRESIMS at m/z 553.2699 [M + H]+. Comparison of the 1H and 13C NMR data
of 2 with those of 1 suggested that these compounds only differed from each other due to
the presence of a fully O-substituted furanyl moiety [δH 4.79 (s, H-21), 3.67 (d, J = 4.0 Hz,
H-22), 4.84 (d, J = 4.0 Hz, H-23); δC 79.2 (C-20), 105.0 (C-21), 76.8 (C-22), 109.3 (C-23)]
(Table 1). e presences of the 7,15-ether linkage and the opened C-ring were confirmed
based on the 13C NMR chemical shifs of C-7 and C-15 at δC 86.7. e locations of two
methoxy groups were determined at C-21 and C-23 based on their HMBC correlations to
two corresponding acetal carbons at δC 105.0 and 109.3 (Figure 2). e NOESY correlations
between Me-18/Me-19/Me-29/Me-30, H-6/Me-19, H-7/Me-30, and H-17/Me-18 suggested

4
N. Y. T. NGUYEN ET AL.
Figure 2. connectivities (bold lines) as deduced by 1h–1h cosY, and significant hMBc (solid arrows) and
noesY (dashed arrows) correlations observed for new compounds.
their β-orientation. e C-9 methoxycarbonylmethyl group was confirmed as β-orientation
by the NOESY correlations between H-11/Me-19/Me-30. e NOESY correlations between
H-15/H-9/H-5 were supportive of their α-orientation. e relative configuration of the
O-substituted furanyl moiety was established based on the NOESY correlations between
H-17/H-21, H-21/MeO-23, H-23/MeO-21, and H-22/H-23 (Figure 2). e relative configu-
ration at C-20 remained unresolved based on the NOESY spectrum. Compound 2 was opti-
cally inactive; thus, it comprised a racemic mixture. e structure of nimbandioloxyfuran
(2) was assigned as 17-desfuran-17-(2,3-dihydroxy-1,4-dimethoxy-1,4-epoxybutan-2-yl)
nimbandiol.
All isolated compounds were tested for their α-glucosidase inhibitory activity.
Compound 1 was inactive with an IC50 value > 100 μM. Compounds 2 and 3 possessed
α-glucosidase inhibitory activity, with the IC50 values of 46.2 and 38.7 μM, respectively.
Nimbandiolactone-23 (3) showed the most potent effect, which was nine times more active
than acarbose (IC50, 360 μM).
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on an A. Krüss Optronic polarimeter P3000 (Krüss
Optronic GmbH, Hamburg, Germany). e UV–vis absorbances were measured with a
Shimadzu UV-1800 spectrophotometer (Shimadzu Pte., Ltd., Singapore). e IR spectra were
measured with a Shimadzu IR-408 infrared spectrometer (Shimadzu Pte., Ltd., Singapore,
Singapore). e NMR spectra were recorded on a Bruker Avance III 500 spectrometer
(Bruker BioSpin AG, Bangkok, ailand) with TMS as an internal standard, and the chem-
ical shifs are expressed as δ values. HRESIMS data were acquired on Bruker micrOTOF
QII (Bruker Singapore Pte., Ltd., Singapore, Singapore) mass spectrometer. Column chro-
matography was carried out using silica gel 60, 0.06–0.2 mm (Scharlau, Barcelona, Spain)

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
and LiChroprep® RP-18, 40−63 μm (Merck KGaA, Darmstadt, Germany). Analytical and
preparative TLC were carried out on precoated Kieselgel 60F254 or RP18 plates (Merck KGaA,
Darmstadt, Germany). Organic solvents were of the highest grade available (RCI Labscan
Limited, Bangkok, ailand). p-Nitrophenyl-α-D-glucopyranoside and α-glucosidase were
obtained from Sigma-Aldrich Pte Ltd (Singapore). NaH2PO4, Na2HPO4, and Na2CO3 were
purchased from Merck KGaA (Darmstadt, Germany). Acarbose 95% was purchased from
Acros Organics (ermo Fisher Scientific, Geel, Belgium).
3.2. Plant material
e leaves of Azadirachta indica were collected at Phuoc Dinh commune, Ninh Phuoc
District, Ninh uan Province, Vietnam, in March 2010 and were identified by MSc. Viet
Hoang, Department of Biology and Biotechnology, VNUHCM – University of Science. A
voucher specimen (DOC2010-N-AI) has been deposited at the Department of Organic
Chemistry, Faculty of Chemistry, VNUHCM – University of Science.
3.3. Extraction and isolation
e powdered leaves of A. indica (7.5 kg) were macerated with MeOH, and the MeOH-
soluble extract (1.3 kg) was suspended in H2O and successively partitioned with petro-
leum ether, EtOAc, and n-butanol to give the petroleum ether (470 g), EtOAc (125 g), and
n-butanol (138 g)-soluble fractions. e EtOAc-soluble fraction was subjected to silica gel
column chromatography (12 × 150 cm) and eluted with MeOH–CHCl3 (v/v, 0:100 → 30:70)
mixture, to obtain 16 fractions (Fr.1−16). Fr.7 (5.5 g) was chromatographed over a silica
gel column (4 × 60 cm) with MeOH–CHCl3 mixture and purified by RP-18 silica gel with
H2O–MeOH (1:1), to afford 3 (9.0 mg). Fr.9 (4.8 g) was separated over a silica gel column
(4 × 60 cm) with MeOH–CHCl3 mixture and purified by RP-18 silica gel with H2O–MeOH
(1:1.5), to obtain 1 (2.5 mg). Fr.10 (2.4 g) was passed over a silica gel column (4 × 60 cm)
with MeOH–CHCl3 mixture, to yield 2 (6.0 mg).
3.3.1. Nimbandiolactone-21 (1)
White amorphous solid;[ꢀ]2D5 + 100.7 (c 0.1, MeOH); IR (KBr) νmax 2926, 1724, 1650, 1245,
1038 cm−1; 1H (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1;
HRESIMS: m/z 489.2160 [M + H]+ (calcd for C26H33O9, 489.2125).
3.3.2. Nimbandioloxyfuran (2)
White amorphous solid; [ꢀ]2D5 + 0 (c 0.1, MeOH); IR (KBr) νmax 2922, 1732, 1645, 1462,
1026 cm−1; 1H (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1;
HRESIMS: m/z 553.2699 [M + H]+ (calcd for C28H41O11, 553.2649).
3.4. α-Glucosidase inhibitory assay
e inhibitory activity of α-glucosidase was determined according to the previous method
[9]. 3 mM p-nitrophenyl-α-D-glucopyranoside (25 μl) and 0.2 U/ml α-glucosidase (25 μl)
in 0.01 M phosphate buffer (pH = 7.0) were added to the sample solution (625 μl) to start
the reaction. Each reaction was carried out at 37 °C for 30 min and stopped by adding 0.1 M

6
N. Y. T. NGUYEN ET AL.
Na2CO3 (375 μl). Enzymatic activity was quantified by measuring absorbance at 401 nm.
One unit of α-glucosidase activity was defined as an amount of enzyme liberating p-nitro-
phenol (1.0 μM) per min. e IC50 value was defined as the concentration of α-glucosidase
inhibitor that inhibited 50% of α-glucosidase activity. Acarbose, a known α-glucosidase
inhibitor, was used as positive control.
Supplementary material
1D, 2D NMR, and MS spectra for compounds 1–3.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Phu Hoang Dang
http://orcid.org/0000-0002-4989-9315
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