NOTE
pubs.acs.org/jnp

Cytotoxic and Apoptosis-Inducing Activities of Limonoids from theSeeds of Azadirachta indica (Neem)
Takashi Kikuchi,† Koichi Ishii,† Taisuke Noto,† Akitomo Takahashi,† Keiichi Tabata,‡ Takashi Suzuki,‡ andToshihiro Akihisa*,††‡

College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan
School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
ABSTRACT: Thirty-ﬁve limonoids, including 15 of the azadiradione type (1-15), ﬁve of thegedunin type (16-20), four of the azadirachtin type (21-24), nine of the nimbin type (2533), and two degraded limonoids (34, 35), isolated from Azadirachta indica seed extracts, were
evaluated for their cytotoxic activities against ﬁve human cancer cell lines. Seven compounds (3,6, 7, 16, 18, 28, and 29) exhibited cytotoxic activity against one or more cell lines. Among thesecompounds, 7-deacetyl-7-benzoylepoxyazadiradione (7), 7-deacetyl-7-benzoylgeduin (18), and28-deoxonimbolide (28) exhibited potent cytotoxic activity against HL60 leukemia cells with
IC50 values in the range 2.7-3.1 μM. Compounds 7, 18, and 28 induced early apoptosis in HL60
cells, observed by ﬂow cytometry. Western blot analysis showed that compounds 7, 18, and 28
activated caspases-3, -8, and -9 in HL60 cells. This suggested that compounds 7, 18, and 28
induced apoptotic cell death in HL60 cells via both the mitochondrial- and the death receptor-mediated pathways. Futhermore,compound 7 was shown to possess high selective cytotoxicity for leukemia cells since it exhibited only weak cytotoxicity against anormal lymphocyte cell line (RPMI 1788).

cisplatin and 5-ﬂuorouracil, were evaluated against ﬁve humancancer cell lines by means of a thiazoyl blue tetrazolium bromide(MTT) assay, and the results are summarized in Table 1. Seven
compounds, 3, 6, 7, 16, 18, 28, and 29, exhibited IC50 values of
1.7-9.9 μM against one or more cancer cell lines. Thus, compound 3 was active against HL60 and CRL1579 cells, 7 against
HL60 and AZ521 cells, 16 against HL60 and SK-BR-3 cells, 28
against HL60, A549, AZ521, and SK-BR-3 cells, and 6, 18, and 29
against HL60 cells. In particular, the cytotoxic activities ofcompound 3 against HL60 and CRL1579 cells, 7 against HL60
and AZ521 cells, 16 against HL60 and SK-BR-3 cells, 18 against
HL60 cells, and 28 against HL60, A549, AZ521, and SK-BR-3
cells were observed to be superior to those of the reference compounds, cisplatin and/or 5-ﬂuorouracil, tested in the same assay.
On the basis of the results in Table 1, the following conclusions can be drawn about the structure-activity relationship ofthe compounds evaluated: (i) Azadiradione-type, gedunin-type,and nimbin-type limonoids possess in general potent or moderate
cytotoxic activity. (ii) Azadirachtin-type and degraded limonoids
possess relatively weak cytotoxic activity. (iii) Azadiradione-type
and gedunin-type limonoids with an acetoxy group at the C-7R
position are more active than those with a hydroxy group, as has
been observed for 1 vs 2, and 16 vs 17. Furthermore, thosecompounds with a benzoyloxy group at the C-7R position were
far more active than those with an acetoxy group, as observed for3 vs 1, 7 vs 6, and 18 vs 16. (iv) Gedunin-type limonoids with a
7R-acetoxy or a 7R-benzoyloxy group were more active than theT

he neem tree (Azadirachta indica A. Juss.; Meliaceae) is
indigenous to India, but is widely cultivated throughout thetropics.1 Since antiquity, various parts of the neem tree have beenused for food, medicine, and insecticidal purposes, and many
bioactive constituents including limonoids (tetranortriterpenoids)
have been isolated and identiﬁed.1-4 In the course of a search fornaturally occurring bioactive compounds,5,6 we have undertaken
a detailed investigation of the limonoid constituents of the nhexane7 and defatted MeOH extracts8 of A. indica seeds and haveshown that some limonoids exhibit potent inhibitory eﬀects
against melanogenesis in B16 melanoma cells, against 12-Otetradecanoylphorbol-13-acetate (TPA)-induced inﬂammationin mice, and against TPA-induced Epstein-Barr virus early antigen(EBV-EA) activation.7,8 We have also reported that azadirachtin
B (21) exerts a potent inhibitory eﬀect on in vivo two-stage mouseskin carcinogenesis.8 In a continuing study on the bioactivity of
the limonoid constituents of A. indica seeds, the cytotoxicity ofthese compounds has been evaluated. We report the cytotoxic
activities of 35 limonoids from A. indica seeds against ﬁve humancancer cell lines, namely, HL60 (leukemia), A549 (lung), AZ521(stomach), SK-BR-3 (breast), and CRL1579 (melanoma). In addition, the induction of apoptosis in HL60 by 7-deacetyl-7-benzoylepoxyazadiradione (7), 7-deacetyl-7-benzoylgedunin (18), and 28deoxonimbolide (28) and the mechanisms of the apoptotic cell
death were investigated.
Thirty-ﬁve limonoids of the azadiradione (1-15), gedunin(16-20), azadirachtin (21-24), and nimbin (25-33) types andtwo degraded limonoids (34, 35) were isolated from n-hexane7
and defatted MeOH extracts8 of A. indica seeds. The cytotoxic
activities of compounds 1-35 and two reference anticancer drugs,
Copyright r 2011 American Chemical Society andAmerican Society of Pharmacognosy

Received: October 28, 2010
Published: March 07, 2011
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Journal of Natural Products

corresponding azadiradione-type limonoids against HL60 cells(6 vs 1 and 18 vs 3). (v) Defurylation at C-17 in azadiradionetype limonoids exerted almost no inﬂuence on the activity against
HL60 cells (1 vs 13). (vi) Substitution of a conjugated cyclopentenone-type D-ring of 13 by an R,β-unsaturated γ-lactonetype D-ring (34 and 35) reduced the activity against HL60 cells.
(vii) Hydroxylation at C-17(β) (5) or inversion of the stereochemistry at C-17 (8) of 1 exerted almost no inﬂuence on theactivity against HL60 cells. However, inversion of the stereochemistry at C-17 along with the hydroxylation at C-17(R) (9) of
1 reduced the activity. Hydroxylation at C-15 (4) of 1 also
reduced the activity against HL60 cells. (viii) A nimbin-type
limnoid with a hydroxy group at the C-6R position is more active
than that with an acetoxy group (26 vs 25). (ix) Substitution of a
cinnamoyl group at C-1 by a dihydrotigloyl or a tigloyl group for
nimbin-type limonoids reduced the activity against HL60 cells(29 vs 31 vs 30).
Compounds 7, 18, and 28 exhibited potent cytotoxic activities
against HL60 cells (IC50 3.1, 2.9, and 2.7 μM, respectively), andthese were evaluated for their apoptosis-inducing activity using
HL60 cells. HL60 cells were incubated with these test compounds for 8 and 24 h, and then the cells were analyzed by meansof ﬂow cytometry with annexin V-propidium iodide (PI) double
staining. Exposure of the membrane phospholipid phosphatidylserine to the external cellular environment is one of the earliest
markers of apoptotic cell death.9 Annexin V is a calcium-dependent phospholipid-binding protein with high aﬃnity for phosphatidylserine expressed on the cell surface. Propidium iodide(PI) does not enter whole cells with intact membranes and wasused to diﬀerentiate between early apoptotic (annexin V positive,

NOTE

PI negative), late apoptotic (annexin V, PI double positive), or
necrotic (annexin V negative, PI positive) cell death. The ratio of
early apoptotic cells (lower right) was increased after treatment
with 7 in HL60 cells for 8 h (38.7% vs 11.3% of negative control)and 24 h (51.8% vs 6.3% of negative control), and that of late
apoptotic cells (upper right) was increased after 24 h (15.5% vs
0.6% of negative control) (Figure 1A). In the case of compound
18, the ratio of early apoptotic cells was increased after treatment
for 24 h (41.4% vs 6.3% of negative control), and that of late
apoptotic cells (upper right) was increased after 24 h (45.7% vs
0.6% of negative control) (Figure 1B). After treatment with compound 28, the ratio of early apoptotic cells was increased after 8 h(10.9% vs 6.1% of negative control) and 24 h (18.7% vs 3.5% ofnegative control), and that of late apoptotic cells (upper right)
was increased after 24 h (28.1% vs 2.9% of negative control)(Figure 1C). These results revealed that most of the cytotoxic
activities of compounds 7, 18, and 28 against HL60 cells are due
to inducing apoptotic cell death.
Caspases are known to mediate the apoptotic pathway.10,11
Caspases-8 and -9, which are initiator caspases, appear to be theapical caspases activated in death receptor- and mitochondrial
stress-induced apoptotic cell death, respectively. Initiator caspases are responsible for either directly or indirectly activating
various eﬀector caspases, including caspases-3, -6, and -7, whichcontain short prodomains. Eﬀector caspases cleave a number ofstructural and regulatory proteins and are directly responsible formany of the apoptotic features, which are nuclear and cytoplasmic condensation, DNA fragmentation, cell membrane composition, and others.12 In order to clarify the mechanisms by which
compounds 7, 18, and 28 induce apoptotic cell death, activationof caspases-3, -8, and -9 was evaluated by Western blot analysis.
The levels of procaspases-8, -9, and -3 diminished, and then thelevels of cleaved caspases-8, -9, and -3 were increased after
treatment with compounds 7, 18, and 28 (Figure 2). These
results suggest that compounds 7, 18, and 28 induced apoptotic
cell death via both the mitochondrial- and the death receptormediated pathways.
Compounds 7, 18, and 28 and cisplatin were then tested fortheir cytotoxicity against a normal lymphocyte cell line, RPMI
1788, and their selectivity index (SI) value,13 which was determined by dividing the IC50 value for the normal cell line (RPMI
1788) by the IC50 value for the cancer cell line (HL60). As shownin Table 2, compounds 7, 18, and 28 exhibited SI values of 5.19,
0.69, and 1.00, respectively, greater than that of cisplatin (SI0.45). In particular, compound 7 exhibited the highest selective
cytotoxicity for leukemia cells since it exhibited only weakcytotoxicity for the RPMI 1788 cell lines.
From the results of the cytotoxicity evaluation of the limonoids from A. indica seed extracts, it appears that some of these
may be valuable anticancer lead compounds. Furthermore,compound 7, which induced apoptotic cell death in leukemia
and displayed high selective toxicity against leukemia, may beespecially promising in this regard. Although compounds 6,14,15
16,16-18 and 2819,20 have been reported previously to show
potent or moderate cytotoxicity against several cancer cell lines,
the mechanisms of cell death were not explored.

’ EXPERIMENTAL SECTION
General Experimental Procedures. Nine limonoids, 2, 14, 20,
and 28-33, were isolated from the n-hexane extract of A. indica seeds,7with another 29 limonoids, 1, 3-13, 15-19, 21-27, 34, and 35, isolated
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NOTE

Table 1. Cytotoxic Activities on Five Human Cancer Cell Lines of Limonoids from Neem (Azadiracta indica A. Juss.) Seed
Extractsa
IC50 (μM)b,c
compound

HL60 (leukemia)

A549 (lung)

AZ521 (stomach)

SK-BR-3 (breast)

CRL1579 (melanoma)

azadiradione type1

azadiradione

14.7

>20

>20

15.9

>20

3
5

7-benzoylnimbocinol
17-hydroxyazadiradione

5.3
12.4

>20
>20

>20
>20

>20
>20

4.0
>20

6

epoxyazadiradione

9.3

>20

>20

12.3

>20

7

7-deacetyl-7-benzoylepoxyazadiradione

3.1

>20

8.4

>20

>20

8

17-epiazadiradione

12.6

>20

>20

>20

>20

9

17-epi-17-hydroxyazadiradione

>20

>20

>20

>20

19.1

11

azadiradionolide

>20

>20

>20

11.5

>20

13

desfuranoazadiradione

19.8

>20

>20

>20

>20

14
gedunin type1,3-diacetylvilasinin

15.3

>20

14.8

>20

>20

16

gedunin17

7-deacetylgedunin

18

7-deacetyl-7-benzoylgedunin

5.9

>20

16.9

8.3

>20

>20

>20

16.9

12.0

>20

2.9

>20

>20

>20

>20

nimbin type25

nimbin

>20

>20

>20

15.9

26

6-deacetylnimbin

11.6

>20

>20

>20

>20

28
29

28-deoxonimbolide
ohchinin acetate

2.7
9.9

9.3
>20

2.4
>20

1.7
>20

14.2
>20

31

20 ,30 -dihydrosalannin

17.1

>20

>20

>20

>20

cisplatin

4.2

>20

9.5

18.8

>20

5-ﬂuorouracil

9.1

>20

>20

>20

>20

reference compounds

Cells were treated with compounds (1  10-4 to 1  10-6 M) for 48 h, and cell viability was analyzed by the MTT assay. b The IC50 value is theconcentration of compound required to inhibit the growth of the cells by 50%. This was obtained based on triplicate assay results. c Compounds 2(nimbocinol), 4 (15-hydroxyazadiradione), 10 (7-acetyl-16,17-dehydro-16-hydroxyneotrichilenone), 12 (20,21,22,23-tetrahydro-23-oxoazadirone),
15 (3-acetyl-7-tigloylvalasinin lactone), 19 (nimolicinol), 20 (7-deacetyl-17-epinimolicinol), 21 (azadirachtin B), 22 (vepaol), 23 (23-epivepaol), 24 (3acetyl-11-methoxy-1-tigloylazadirachtinin), 27 (6-acetylnimbandiol), 30 (salannin), 32 (3-deacetylsalannin), 33 (17-defurano-17-oxosalannin), 34 (Rnimolactone), and 35 (β-nimolactone) exhibited IC50 values of >20 μM in all cell lines used.
aTable 2. Cytotoxicities of Compounds 7, 18, and 28 and Cisplatin against Leukemia (HL60) and Normal Lymphocyte (RPMI
1788) Cell Linesa
IC50 (μM)bcompound

HL60

RPMI1788(leukemia)(normal lymphocyte)

SIc

3.1
2.9

16.1
2.0

5.19
0.69

7
18

7-deacetyl-7-benoylepoxyazadiradione
7-deacetyl-7-benzoylgedunin

28

28-deoxonimbolide

2.7

2.7

1.00

cisplatind

4.2

1.9

0.45

Cells were treated with compounds (1  10-4 to 1  10-6 M) for 48 h, and cell viability was analyzed by the MTT assay. b IC50 based on triplicate assay
results. c SI refers to the selectivity index, which was obtained by dividing the IC50 value for the normal cells by the IC50 value for the cancer cells.
dReference compound.
afrom the defatted MeOH extract of A. indica seeds.8 Chemicals andreagents were purchased as follows: fetal bovine serum (FBS), Roswell
Park Memorial Institute (RPMI) 1640 medium, antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin), and nonessential aminoacid (NEAA) from Invitrogen Co. (Auckland, New Zealand), Dulbecco’s modified Eagle’s medium (D-MEM), Eagle's minimum essential
medium (MEM), and MTT from Sigma-Aldrich Japan Co. (Tokyo,
Japan), and rh Annexin V/FITC kit (Bender MedSystems) from Cosmo

Bio Co. Ltd. (Tokyo, Japan). All other chemicals and reagents were ofanalytical grade.
Cell Cultures. Cell lines HL60 (leukemia), A549 (lung), AZ521(stomach), SK-BR-3 (breast), and CRL1579 (melanoma) were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). A normal cellline, RPMI 1788 (lymphocytic), was obtained from Health Science
Research Resources Bank (Osaka, Japan). Three cell lines, HL60, SKBR-3, and CRL1579, were grown in RPMI 1640 medium, while A549
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NOTE

Figure 2. Western blot analysis of HL60 cells treated with 30 μM eachof compounds 7, 18, and 28. The results are from one representative
experiment among three runs, which showed similar patterns to one
another.

Cytotoxicity Assays. Cytotoxicity assays were performed according to methodology previously reported.21,22 Briefly, the cell lines cited
above [each 3  103 cells/well] were treated with test compounds for
48 h, and then MTT solution was added to each well. After incubationfor 3 h, the blue formazan generated was solubilized with 0.04 M HCl
in 2-propanol. The absorbances at 570 nm (top) and 630 nm (bottom)
were measured with a microplate reader (Tecan Japan Co., Ltd., Kawasaki,
Japan).
Annexin V-Propidium Iodide (PI) Double Staining. Annexin
V-PI double staining was performed according to the method previously reported.21 Apoptosis was detected using an rh annexin V/FITC
kit. HL60 (1  105 cells) were exposed to each test compound (final
concentration: 30 μM). To prepare the cell sample for flow cytometry,cells were washed with annexin-binding buffer and stained with annexin
V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for
10 min. Cell samples were analyzed using a flow cytometer (Cell Lab
Quanta SC; Beckman Coulter K.K., Tokyo, Japan) with the FL1 andFL2 ranges for annexin V-FITC and -PI, respectively.
Western Blotting. Western blot analysis was performed according
to the method previously reported.21,22 Briefly, HL60 (4  105 cells)
were exposed to test compound (final concentration: 30 μM) for 8 and24 h. Cells were collected and lysed. Lysates of total protein were separated
by 15% sodium dodecyl sulfate-polyacrylamide gels and transferred topolyvinylidene difluoride membranes. After blocking, the membranes wereincubated with anticaspase-3 (Sigma-Aldrich Japan Co., Tokyo, Japan) andanticaspase-8, anticleaved caspase-3, anticaspase-9, and anti-β-actin(Cell Signaling Technology, Beverly, MA) primary antibodies at 4 °C
overnight. The blots were then detected with enhanced chemiluminescence plus Western blotting detection system (GE Healthcare, Chalfont
St. Giles, U.K.) .

’ AUTHOR INFORMATION
Corresponding Author*Tel: þ81-3-3259-0806. Fax: þ81-3-3293-7572. E-mail: akihisa@chem.cst.nihon-u.ac.jp.

Figure 1. Compounds 7, 18, and 28 induced apoptosis against
HL60 cells. (A) HL60 cells were cultured with compound 7 (30μM) for 8 and 24 h. (B) HL60 cells were cultured with compound 18(30 μM) for 8 and 24 h. (C) HL60 cells were cultured withcompound 28 (30 μM) for 8 and 24 h. Each value is the mean ofthree experiments.

’ ACKNOWLEDGMENT
This work was supported, in part, by a Grant-in-Aid from theJapan Society for the Promotion of Science (No. 20590015),
2008-2010.
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