A new diarylheptanoid from the rhizomes of Zingiber mekongense

Article abstract of DOI:10.1016/j.fitote.2011.01.002

A new diarylheptanoid, (3S,5S)-3,5-diacetoxy-1,7-bis(3,4,5- trimethoxyphenyl)heptane (1), together with the known docosyl trans-ferulate (2), (1S,2S,4S)-p-menthan-1,2,4-triol (3), 5αH-eudesmane-4α,11-diol (4), 5αH-eudesmane-4β,11-diol (5), 4α,10β-dihydrox

Full text of DOI:10.1016/j.fitote.2011.01.002

Fitoterapia 82 (2011) 534–538
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
A new diarylheptanoid from the rhizomes of Zingiber mekongense
Arthittaya Chareonkla a, Manat Pohmakotr a, Vichai Reutrakul a, Chalobon Yoosook b, Jitra Kasisit b,
Chanita Napaswad b, Patoomratana Tuchinda a,
⁎
a
Department of Chemistry and Center for Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 October 2010
Accepted in revised form 24 December 2010
Available online 14 January 2011
A new diarylheptanoid, (3S,5S)-3,5-diacetoxy-1,7-bis(3,4,5-trimethoxyphenyl)heptane (1),
together with the known docosyl trans-ferulate (2), (1S,2S,4S)-p-menthan-1,2,4-triol (3), 5αH-
eudesmane-4α,11-diol (4), 5αH-eudesmane-4β,11-diol (5), 4α,10β-dihydroxy-1βH,5αH-
guaia-6-ene (guaianediol) (6), (+)-galanolactone (7), (E)-labda-8(17),12(13)-dien-15,16-
olide (8), labda-8(17),13(14)-dien-15,16-olide (9), 3,5-dihydroxy-7,4′-dimethoxyflavone (10)
and 3,5,3′-trihydroxy-7,4′-dimethoxyflavone (11) were isolated from the rhizomes of Zingiber
mekongense. Their structures were determined by spectroscopic methods. The stereochemistry
of 1 was proved through chemical conversion. Compounds 1, 4–7 and 9–11 exhibited anti-HIV-1
activities in the anti-syncytium assay using ΔTat/revMC99 virus and 1A2 cell line system, while
only compounds 7 and 11 were found active in the HIV-1 reverse transcriptase assay.
© 2011 Elsevier B.V. All rights reserved.
Keywords:
Zingiber mekongense
Zingiberaceae
Diarylheptanoid
Anti-HIV-1 activity
1. Introduction
ongoing search for anti-HIV-1 agents from plants [14,15], the
active hexane and ethyl acetate extracts from the rhizomes of
Zingiber mekongense Gagnep. belongs to the family
Zingiberaceae, which comprises 48 genera and 1200 species
worldwide [1]. Many species of Zingiber have been used in
traditional medicine. Ginger (Z. officinale) is frequently
prescribed for the treatment of vomiting, rheumatism,
duodenum and gastric ulcer in Chinese traditional medicine
[2], while the rhizomes of Z. cassumunar are used in Thailand
for treatment of bronchitis, asthma, and gastrointestinal
distress [3]. Z. zerumbet rhizomes are well known as a cure
of sores and swelling, worm infestation in children and loss of
appetite [4]. Numerous phytochemical studies of Zingibers
have led to the structure elucidation of many bioactive
compounds, such as gingerols and shogaol [5,6] as well as
diarylheptanoids [2,7,8] from Z. officinale, phenylbutanoid
[3,9–11] from Z. cassumunar, and a macrocyclic sesquiterpe-
noid zerumbone [4,12,13] from Z. zerumbet. As part of our
Z. mekongense were separated by chromatography, followed by
recrystallization to yield a new diarylheptanoid, (3S,5S)-3,5-
diacetoxy-1,7-bis(3,4,5-trimethoxyphenyl)heptane (1), along
with the known docosyl trans-ferulate (2) [16], (1S,2S,4S)-p-
menthan-1,2,4-triol (3) [17], 5αH-eudesmane-4α,11-diol (4)
[18], 5αH-eudesmane-4β,11-diol (5) [18], guaianediol (6)
[19], galanolactone (7) [20], (E)-labda-8(17),12(13)-dien-
15,16-olide (8) [21], labda-8(17),13(14)-dien-15,16-olide
(9) [22], 3,5-dihydroxy-7,4′-dimethoxyflavone (10) [23] and
3,5,3′-trihydroxy-7,4′-dimethoxyflavone (11) [24] (Fig. 1). We
herein described the isolation, structure elucidation, and anti-
HIV-1 activities of the compounds with sufficient amount.
2. Experimental
2.1. General
Melting points: digital melting point apparatus (Electro-
thermal). Optical rotations: JASCO DIP-370. UV: JASCO V-530.
FT-IR: Perkin Elmer 2000. 1H NMR (500 MHz), 13C NMR
(125 MHz), DEPT and 2D-NMR spectra: Bruker AV 500 in
⁎
Corresponding author. Tel.: +66 2 2015159; fax: +66 2 3547151.
E-mail address: scptc@mahidol.ac.th (P. Tuchinda).
0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2011.01.002

A. Chareonkla et al. / Fitoterapia 82 (2011) 534–538
535
Fig. 1. The structures of compounds 1–11 isolated from the hexane and ethyl acetate extracts of Z. mekongense.
CDCl3, otherwise stated. HR-TOF-MS: Micromass model VQ-
Tof2. EIMS 70 ev: Thermo Finnigan Polarlis Q. Column
chromatography (CC): silica gel 60 (Merck, 70–230 mesh)
or Sephadex LH 20 (Pharmacia). Preparative thin-layer
chromatography (Prep-TLC): Kieselgel 60 PF254 (Merck,
0.5 mm).
at room temperature. Removal of solvents yielded the crude
active hexane extract (cytotoxic and syncytium assays: IC50
56.5 μg/mL and EC50 21.4 μg/mL; HIV-1-RT assay: 32.0%
inhibition at 200 μg/mL) (94.5 g), active EtOAc extract
(cytotoxic and syncytium assays: IC50 137.1 μg/mL and EC50
30.3 μg/mL; HIV-1-RT assay: 73.1 % inhibition at 200 μg/mL)
(53.5 g) and inactive MeOH extract (inactive in both assays)
(173.4 g).
2.2. Plant material
The anti HIV-1 active hexane extract (93.0 g) was
subjected to Si-gel CC (CH2Cl2–hexane and MeOH–CH2Cl2
gradients) to afford frs. A1–A7. Separation of fr. A4 (20.4 g) by
Si-gel CC (CH2Cl2–hexane gradient) afforded frs. B1–B8. Fr. B2
(3.85 g) yielded 3 (4.7 mg) after addition of CH2Cl2–MeOH.
Fr. B4 (8.29 g) furnished frs. C1–C12 after Si-gel CC (Me2CO–
hexane gradient). Later Si-gel CC (1% MeOH–CH2Cl2) of fr. C3
(140.0 mg) gave frs. D1–D5. Fr. D1 (18.9 mg) after Si-gel
prep-TLC (5% Me2CO–hexane, 4 elutions) yielded 8 (2.8 mg,
Rf =0.40). Fr. D2 (95.4 mg) after Si-gel prep-TLC (5% Me2CO–
hexane, 4 elutions) afforded 9 (86.4 mg, Rf =0.39). Fr D4
(95.4 mg) after Si-gel prep-TLC (5% Me2CO–hexane, 4
The rhizomes of Z. mekongense were collected in Trat
Province, Thailand, in November 2007 and identified by
T. Santisuk. A voucher specimen (BK No. 63895) was
deposited at the Bangkok Herbarium, Plant Variety Protection
Division, Department of Agriculture, Ministry of Agriculture
and Co-operatives, Thailand.
2.3. Extraction and isolation
Air-dried and powdered rhizomes (2.3 kg) were sequen-
tially percolated with hexane, EtOAc and MeOH (6 L×4, each)

536
A. Chareonkla et al. / Fitoterapia 82 (2011) 534–538
Table 1
elutions) provided 2 (11.4 mg, Rf =0.32). Separation of fr. A6
(30.1 g) by Si-gel CC (EtOAc–hexane gradient) furnished frs.
E1–E12. Fr. E5 (1.82 g) gave 10 (51.9 mg) after addition of
CH2Cl2–MeOH. Fr. E6 (2.99 g) afforded 7 (45.4 mg) after Si-
gel CC (Me2CO–hexane gradient) and recrystallization from
EtOAc–hexane. Purification of fr. E7 (5.01 g) by Si-gel CC
(Me2CO–hexane gradient) yielded frs. F1–F5. Fr. F2
(633.1 mg) after Si-gel prep-TLC (1% MeOH–CH2Cl2, 2
elutions) gave 5 (107.0 mg, Rf =0.31). Fr. F3 (795.9 mg)
after Si-gel prep-TLC (40% EtOAc–hexane, 3 elutions) yielded
6 (31.7 mg, Rf =0.48). Fr. F4 (1.35 g) after Si-gel prep-TLC (1%
MeOH–CH2Cl2, 2 elutions) provided 1 (272.4 mg, Rf =0.43).
Fr. E8 (2.51 g) was separated by Si-gel CC (Me2CO–hexane
gradient) to yield frs. G1–G6. Fr. G3 (1.16 g) afforded an
additional amount of 1 (888.5 mg) after Si-gel prep-TLC (1%
MeOH–CH2Cl2, 2 elutions). Fr. E9 (2.04 g) was further
separated by Si-gel CC (Me2CO–hexane gradient) to obtain
frs. H1–H6. Fr. H2 (1.94 g) furnished 4 (769.8 mg) after
addition of CH2Cl2–hexane. Fr. H3 (243.0 mg) provided an
additional amount of 1 (102.0 mg) after Si-gel prep-TLC (1%
MeOH–CH2Cl2, 2 elutions). Purification of fr. A7 (12.6 g) by Si-
gel CC (EtOAc–hexane gradient) furnished frs. I1–I4. Fr. I2
(837.1 mg) yielded an additional amount of 4 (717. 3 mg)
after addition of CH2Cl2–hexane.
NMR data of compound 1 in CDCl3.
a
b
Position
δH mult.
δC
DEPT COSY
HMBC
J (Hz)
correlated H correlated H
1
2
2.56 m
1.88 dd
(14.1, 7.8)
4.98 quint
(6.3)
1.98 dt
(14.3, 7.2)
1.77 dt
(14.3, 5.8)
4.98 quint
(6.3)
32.0 CH2
35.7 CH2
2
1, 3
2, 3, 2′, 6′
1, 4a, 4b
3
70.5 CH
38.6 CH2
2, 4a, 4b
3, 4b, 5
3, 4a, 5
4a, 5b, 6
5, 7
1, 2, 4a, 4b, 5
2, 6
4a
4b
5
70.5 CH
35.7 CH2
32.0 CH2
3, 4a, 4b, 6, 7
4a, 4b, 7
6
1.88 dd
(14.1, 7.8)
2.56 m
–
7
6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5, 6, 2″, 6″
1, 2, 2′, 6′
1, 6′
2′, 3′–OCH3
2′, 6′, 4′–OCH3
6′, 5′–OCH3
1, 2′
6, 7, 2″, 6″
7, 6″
2″, 3″–OCH3
2″, 6″, 4″–OCH3
6″, 5″–OCH3
7, 2″
–
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
136.9
105.3 CH
C
6.38s
–
153.1
136.2
153.1
105.3 CH
136.9
105.3 CH
153.1
136.2
153.1
105.3 CH
21.1 CH3
170.6 C=O
21.1 CH3
170.6 C=O
56.0 CH3
60.8 CH3
56.0 CH3
56.0 CH3
60.8 CH3
56.0 CH3
C
C
C
–
–
6.38s
–
C
6.38s
–
C
C
C
–
The anti-HIV-1 active EtOAc extract (53.1 g) was fraction-
ated by Si-gel CC, eluting with Me2CO–hexane and MeOH–
Me2CO gradients to yield frs. J1–J9. Further separation by Si-
gel CC (MeOH–CH2Cl2 gradient) of fr. J5 (2.54 g) afforded frs.
K1–K6. Fr. K2 (73.9 mg) provided 10 (41.5 mg) after
recrystallization from MeOH–CH2Cl2. Fr. J6 (3.69 g) furnished
frs. L1–L6 after separation by Si-gel CC (Me2CO–hexane
gradient). Fr. L3 (79.2 mg) yielded an additional amount of
10 (58.8 mg) after recrystallization from MeOH–CH2Cl2. Fr. L5
(2.78 g) was separated by Si-gel CC (Me2CO–hexane gradi-
ent) to give frs. M1–M5. Further purifications of frs. M2
(353.4 mg) and M3 (2.23 g) by Sephadex LH-20 CC (50%
MeOH–CH2Cl2) provided 4 (116.8 mg) and 1 (36.4 mg),
respectively. Separation of fr. J7 (3.50 g) by Si-gel CC
(Me2CO–hexane gradient) afforded frs. N1–N4. Compound
11 (330.4 mg) was obtained from fr. N3 (1.87 g) by
recrystallization from CH2Cl2–MeOH. Separation of fr. J8
(9.99 g) by Si-gel CC (CH2Cl2–hexane gradient) yielded frs.
O1–O6. Fr. O2 (2.41 g) provided an additional amount of
11 (322.2 mg) after recrystallization from CH2Cl2–MeOH.
(3S,5S)-3,5-diacetoxy-1,7-bis(3,4,5-trimethoxyphenyl)
heptane (1), pale yellow oil; [α]25869+4.9° (c 0.50, CHCl3); UV
(EtOH) λmax (log ε) 230 (4.93 sh), 269 (4.06)nm; IR (CHCl3)
1731, 1592, 1509, 1464, 1422, 1374, 1330, 1240, 1185, 1131,
–
6.38s
3–OCOCH3 2.04s
3, 3–OCOCH3
–
5–OCOCH3 2.04s
5, 5–OCOCH3
3′–OCH3
4′– OCH3
5′– OCH3
3″–OCH3
4″–OCH3
5″–OCH3
3.86s
3.82s
3.86s
3.86s
3.82s
3.86s
–
–
–
–
–
–
a
1H NMR (500 MHz).
13C NMR (125 MHz).
b
(3 mL) and extracted with EtOAc (5 mL×3). The EtOAc layer
was washed with water, brine and dried over anhydrous
Na2SO4. After filtration and solvent removal, the product 1a
(30.1 mg, 83%) was obtained.
2.4.2. Esterification of 1a to afford bis-p-bromobenzoate 1b
To a solution of 1a (19.0 mg, 0.0409 mmol) in dry CH2Cl2
(1 mL) containing dimethylaminopyridine (DMAP, 25.0 mg,
0.2046 mmol) as a catalyst was added dropwise a solution of
p-bromobenzoyl chloride (44.9 mg, 0.2046 mmol) in dry
CH2Cl2 (1 mL), and the mixture was stirred at room
temperature for 3 h. The reaction mixture was diluted with
H2O (5 mL), then extracted with CH2Cl2 (5 mL×3), washed
with water, brine and dried over anhydrous Na2SO4. After
filtration and removal of solvent, it was purified by Si-gel
prep-TLC (30% Me2CO–hexane) to give bis-p-bromobenzoate
1b (21.7 mg, 64%).
1004, and 830 cm−1 1H NMR data (500 MHz, CDCl3), 13C
;
NMR data (125 MHz, CDCl3) and 2D NMR data, see Table 1;
EIMS (70 ev) m/z 548 [M]+ (65%), 488 (14%), 233 (36%), 195
(25%), 182 (100%), and 181 (46%); HR-TOF-MS m/z 571.2556
[M+Na]+ (calcd for C29H40O10Na 571.2519).
2.4. Chemical conversion of 1 to 1a and 1b
2.4.1. Hydrolysis of 1 to afford 1a
(3S,5S)-3,5-dihydroxy-1,7-bis(3,4,5-trimethoxyphenyl)
heptane (1a), pale yellow oil; [α]25839+2.8° (c 0.50, CHCl3); UV
(EtOH) λmax (log ε) 230 (5.00), 270 (4.03) nm; IR (CHCl3):
3495, 1591, 1509, 1464, 1421, 1332, 1240, 1184, 1130, 1004,
and 829 cm−1; 1H NMR (CDCl3, 500 MHz) δ: 1.66 (2H, m, H-4),
To a solution of 1 (42.7 mg, 0.0779 mmol) in MeOH (5 mL)
was added an aqueous KOH solution (20.4 mg, 0.365 mmol)
in H2O (2 mL). After stirring for 1 h at room temperature, the
solution was neutralized with a saturated NH4Cl solution

A. Chareonkla et al. / Fitoterapia 82 (2011) 534–538
537
1.70 (1H, br s, OH), 1.78 (4H, m, H-2 and H-6), 2.70 (4H, m, H-1
and H-7), 3.02 (1H, br s, OH), 3.84 (6H, s, 4′-OMe and 4″-OMe),
3.88 (12H, s, 3′-OMe, 3″-OMe, 5′-OMe and 5″-OMe), 3.94 (2H,
parallel, in duplicate; the result was expressed as 50%
inhibitory concentration (IC50).
m, H-3 and H-5), and 6.44 (4H, s, H-2′, H-2″, H-6′ and H-6″); 13
C
NMR (CDCl3, 125 MHz) δ: 32.1 (C-1), 39.9 (C-2), 72.4 (C-3),
43.2 (C-4), 72.4 (C-5), 39.9 (C-6), 32.1 (C-7), 21.1 (3-OCOCH3
and 5-OCOCH3), 170.6 (3-OCOCH3 and 5-OCOCH3), 137.6 (C-1′
and C-1″), 105.4 (C-2′ and C-2″), 153.2 (C-3′ and C-3″), 136.4
(C-4′ and C-4″), 153.2 (C-5′ and C-5″), and 105.4 (C-6′ and C-
6″); HR-TOF-MS m/z 487.2222 [M + Na]+ (calcd for
3. Results and discussion
Compound 1 was shown to possess a molecular formula
C
29H40O10 as determined by HR-TOF-MS, exhibiting [M+Na]+
ion peak at m/z 571.2556 (calcd for C29H40O10Na, 571.2519).
The FT-IR spectrum suggested the presence of C=O of ester at
C25H36O8Na 487.2308).
1731 cm−1 and aromatic C=C at 1592, 1509 and 1464 cm−1
.
(3S,5S)-3,5-di(4-bromobenzoyloxy)-1,7-bis(3,4,5-tri-
The 1H NMR spectrum of 1 in CDCl3 (Table 1) displayed an
aromatic singlet at δ 6.38 (4H), two methoxy singlets at δ 3.86
(12H) and 3.82 (6H), an acetoxymethyl singlet at δ 2.04 (6H),
together with four sets of signals belonging to the five
methylene groups at δ 2.56 (4H, m), 1.98 (1H, dt, J=14.3
and 7.2 Hz), 1.88 (4H, dd, J=14.1, 7.8 Hz) and 1.77 (1H, dt,
J=14.3 and 5.8 Hz), and two methine protons at δ 4.98 (quint,
J=6.3 Hz). The 13C NMR spectrum of 1 (Table 1) showed
twelve signals for twenty-nine carbons of which the carbon
types were determined by DEPT spectra. The above informa-
tion is consistent to those of oxygenated diarylheptanoids with
two acetoxy groups at positions 3 and 5 [28]. The 1H and 13C
assignments were performed through analysis of 2D spectra
(see Table 1). The locations of six methoxy groups were
assigned at C-3′, C-4′, C-5′, C-3″, C-4″ and C-5″, as the HMBC
correlations between these carbon signals to the methoxy
protons were observed. The connections of C-1 and C-7 to the
aromatic rings were indicated by the correlations of C1/H-2′,
H-6′; C-7/H2″, H-6″; C-1′/H-1, H-2, and C-1″/H-6, H-7. The
assignments within the C7-chain was confirmd by the
correlations of C-1/H-2, H-3; C-2/H-1, H-4a, H-4b; C-3/H-1,
H-2, H-4a, H4b, H-5; C-4/H-2, H-6; C-5/H-3, H-4a, H-4b, H-6,
H-7; C-6/H-4a, H-4b, H-7; and C-7/H-5, H-6. The locations of
two acetoxy groups at C-3 and C-5 were assigned due to
correlations between acetoxycarbonyl carbons to H-3 and H-5.
The absolute configurations at C-3 and C-5 in 1 were
determined by applying the CD exciton chirality method to
its bis-p-bromobenzoate derivative 1b [29,30]. Compound 1b
was prepared by deacetylation of 1 with an aqueous solution of
KOH in MeOH at room temperature for 1 h to give the
dihydroxy derivative 1a (83% yield), followed by the reaction
with p-bromobenzoyl chloride in the presence of N,N-
dimethylaminopyridine (DMAP) in dry CH2Cl2 at room
temperature to afford 1b (64% yield). The CD spectrum of 1b
exhibited exciton split Cotton effects at λext 247.5 nm,
Δε+12.8 and λext 238.5 nm, Δε−14.3 at 27 °C in EtOH,
which are consistent with a positive chirality [29,30]. There-
fore, the absolute configurations were determined as 3S and
5S, and the structure of 1 was assigned as (3S,5S)-3,5-
diacetoxy-1,7-bis(3,4,5-trimethoxyphenyl)heptane.
methoxyphenyl)heptane (1b), amorphous solid; [α]25879+1.9 (c
0.22 CHCl3); CD (3.5×10−4 M, EtOH) nm (Δε) 247.5 (+12.8),
238.5 (−14.3); UV (EtOH) λmax (log ε) 241 (4.51), 271 (3.72)
nm; IR (CHCl3): 1716, 1591, 1509, 1464, 1422, 1399, 1272,
1240, 1174, 1130, 1070, and 848 cm−1 1H NMR (CDCl3,
;
500 MHz) δ: 2.02 (1H, m, H-4b), 2.06 (4H, m, H-2 and H-6),
2.31 (1H, m, H-4a), 2.63 (4H, m, H-1 and H-7), 3.78 (6H, s, 4′-
OMe, 4″-OMe), 3.80 (12H, s, 3′-OMe, 5′-OMe, 3″-OMe, 5″-
OMe), 5.32 (2H, m, H-3 and H-5), 6.34 (4H, s, H-2′, H-6′, H-2″
and H-6″), 7.54(4H, d, J=8.5 Hz, H-3″′, H-5″′, H-3″″ and H-5″″),
and 7.82 (4H, d, J=8.5 Hz, H-2″′, H-6″′, H-2″″ and H-6″″); 13
C
NMR (CDCl3, 125 MHz) δ: 32.0 (C-1), 36.0 (C-2), 72.0 (C-3),
38.7 (C-4), 72.0 (C-5), 36.0 (C-6), 32.0 (C-7), 136.6 (C-1′), 105.3
(C-2′), 153.1 (C-3′), 136.3 (C-4′), 153.1 (C-5′), 105.3 (C-6′),
56.0 (3′-OCH3 and 5′ -OCH3), 60.7 (4′ -OCH3), 136.6 (C-1″),
105.3 (C-2″), 153.1 (C-3″), 136.3 (C-4″), 153.1 (C-5″), 105.3 (C-
6″), 56.0 (3″ -OCH3 and 5″ -OCH3), 60.7 (4″ -OCH3), 165.3
(2×OCOAr), 128.2 (C-1″′ and C-1″″), 131.0 (C-2″′ and C-2″″),
131.7 (C-3″′ and C-3″″), 128.9 (C-4″′ and C-4″″), 131.7 (C-5″′
and C-5″″), and 131.0 (C-6″′ and C-6″″); HR-TOF-MS m/z
851.1166[M+Na]+ (calcdfor C39H42O10Br729Na, 851.1042)and
853.1150 (calcd for C39H42O10Br821Na, 853.1022).
2.5. Bioassays for anti-HIV-1 activity
2.5.1. Anti-HIV-1 reverse transcriptase assay
The test was carried out in a 96-well micrometer plate in
duplicate using the tannin-free supernatant of the extract or
compound in DMSO at a final concentration of 200 μg/mL as
described by Tan et al. [25]. An appropriate amount of HIV-1
reverse transcriptase (Amersham Pharmacia Biotech Asia
Pacific Ltd., Hong Kong) employed in the reaction mixture
was standardized with fagaronine chloride. Fagaronine
chloride and nevirapine were used as positive controls and
DMSO without the extract as a negative control. The results
from duplicate wells were averaged and the percent of
inhibition was calculated. A 50% inhibitory concentraction
(IC50) was estimated from dose–response data.
2.5.2. Cell-based assay
The known compounds 2–11 were identified by compar-
ison of their physical properties and spectroscopic data with
those reported in the literature [16–24].
Compounds 1, 1a, 1b, 4–7 and 9–11 were tested, employing
HIV-1 RT assay as described previously [25] and the syncytium
assay using ΔTat/RevMC99 and 1A2 cell line system [26,27]. The
results (Table 2) indicated that all natural compounds showed
significant anti-HIV-1 activities in the syncytium assay, except
the modified compounds 1a and 1b. Compound 7 was most
active with IC50 315.6 μM and EC50 26.1 μM. The results also
The syncytium assay using ΔTat/revMC99 virus and 1A2 cell
line system [26,27] was used. The experiment was carried out
in triplicate, starting at final concentrations of 3.9–125 μg/mL
or 7.8–250 μg/mL. Virus control and cell control wells
contained neither extract nor the virus; cytotoxicity control
wells containing cells with the extract/compound and a
positive control, i.e., azidothymidine, AZT, were included. The
result was expressed as 50% effective concentration (EC50).
Colorimetric cytotoxic assay using XTT was also carried out in

538
A. Chareonkla et al. / Fitoterapia 82 (2011) 534–538
Table 2
References
Anti-HIV-1 activities of the isolated and modified compounds.
Compound Cytotoxic and syncytium assays HIV-1 RT assay
SI Activity % inhibition at IC50
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IC50
(μM)
EC50
(μM)
200 μg/mL
(μM)
1
119.6
N269.1
N301
630.7 277.1
N520
N1048.8 266.8
315.6
368.3
N795.4
N757
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N3.9
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be against RT, with IC50 211.3 μM and 96.6 μM, respectively.
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This research project is supported by Mahidol University, the
Commission on Higher Education (CHE-RES-RG) and the Center
for Innovation in Chemistry (PERCH-CIC).