Phytochemistry Letters 8 (2014) 10–15

Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

New secondary metabolites from Dodonaea viscosa

Ahmad E. Mostafa a,b, Atef A. El-Hela b, Abd-Elsalam I. Mohammad b, Melissa Jacob a,
Stephen J. Cutler c, Samir A. Ross a,d,*
a
National Center for Natural Products Research, The University of Mississippi, University, MS 38677, USA
b
Department of Pharmacognosy, Faculty of Pharmacy, University of Al-Azhar, Cairo 11371, Egypt
c
Department of Medicinal Chemistry, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
d
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA

A R T I C L E I N F O A B S T R A C T

Article history: Bioassay guided fractionation and chemical investigation of the ethanolic extract of the aerial parts of
Received 8 October 2013 Dodonaea viscosa Linn. from Egypt, resulted in the isolation and identification of three new compounds,
Received in revised form 10 December 2013 including two new clerodane diterpenoids 1 and 2 and a new myo-inositol derivative 3, along with nine
Accepted 16 December 2013
known compounds 4–12. The structures of the isolated compounds were elucidated using 1D and 2D
Available online 19 January 2014
NMR experiments, HRESIMS, and comparison with literature data. All isolated compounds were
evaluated for their antibacterial, antifungal, antimalarial as well as antileishmanial activities. Compound
Keywords:
5 exhibited good antifungal activity against Cryptococcus neoformans with an IC50 value of 6.11 mg/ml.
Dodonaea viscosa
Sapindaceae
Compounds 10 and 12 showed moderate antileishmanial activity against Leishmania donovani with IC50
Diterpenoids values of 16.6 and 19.06 mg/ml, respectively.
Myo-inositol ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Antifungal
Antileishmanial

1. Introduction
Dodonaea viscosa Linn. is a shrub belonging to family
Sapindaceae, which is widely distributed in temperate regions
of Australia, Africa, Mexico, India, South America and Florida. The
plant is used in traditional medicine worldwide, administered
orally or as poultice to treat a great variety of ailments. It is
reported for the treatment of sore throat, cold, fever, rheumatism,
indigestion, ulcers, diarrhea, constipation, to expel roundworms, to
relieve itching, and to soothe toothaches and headaches (Rani et al.,
2009). Roots are ingested by women in East Africa to stimulate
milk production and to treat dysmenorrhea and irregular
menstruation (Rani et al., 2009). In India, seeds are used as a
fish poison (Wagner et al., 1987). The aqueous methanolic extract
of D. viscosa leaves has been reported to exert significant and
consistent antihyperlipidemic, hepatoprotective (Ahmad et al.,
2012) and hypoglycemic actions (Veerapur et al., 2010). Many
classes of secondary metabolites have been identified from D.
viscosa include terpenes, saponins, sterols, tannins and flavonoids
* Corresponding author at: National Center for Natural Products Research and
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi,
University, MS 38677, USA. Tel.: +1 662 915 1031; fax: +1 662 915 7989.
E-mail address: sross@olemiss.edu (S.A. Ross).
(Rani et al., 2009). The present work reports the isolation and
identification of three new compounds, including two clerodane
diterpenoids 1 and 2 and myo-inositol derivative identified as 1-L-1-
O-methyl-2-acetyl-3-P-Z-coumaryl-myo-inositol 3 (Fig. 1), together
with nine known compounds 4–12 identified as: 1-L-1-O-methyl-2-
acetyl-3-P-E-coumaryl-myo-inositol 4, pinocembrin 5, isokaemfer-
ide 6, 6-methoxy isokaemferide 7, 5,7-dihydroxy-3,6,40 -trimethoxy
flavone 8, penduletin 9, 5-hydroxy-3,6,7,40 -tetramethoxy flavone
10, aliarin 11 and 5,7-dihydroxy-30 -(4hydroxy-3methylbutyl)
3,6,40 -trimethoxyflavone 12 from D. viscosa aerial parts. Compound
5 exhibited good antifungal activity against Cryptococcus neoformans
with an IC50 value of 6.11 mg/ml. Compounds 10 and 12 showed
moderate antileishmanial activity against Leishmania donovani with
IC50 values of 16.6 and 19.06 mg/ml, respectively.
2. Results and discussion
Compound 1 was obtained as yellow oil, whose molecular
formula was established to be C22H36O8 from the [MH] peak at
m/z 427.2283 (calcd. for C22H35O8, 427.2332), the [2MH] peak at
m/z 855.4680 (calcd. for C44H71O16, 855.4743), the [M+H]+ peak at
m/z 429.2402 (calcd. for C22H37O8, 429.2489), the [M+Na]+ peak at
m/z 451.2215 (calcd. for C22H36NaO8, 451.2307), and the [2M+Na]+
peak at m/z 879.4557 (calcd. for C44H72NaO16, 879.4719) in the HR-
ESIMS. The 1H NMR spectrum of 1 (Table 1) showed two methoxy

1874-3900/$ – see front matter ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2013.12.008
 A.E. Mostafa et al. / Phytochemistry Letters 8 (2014) 10–15 11

Fig. 1. Chemical structures of 1, 2 and 3.

signals resonating at d 3.50 (s, H-22) and 3.54 (s, H-21), two methyl showed correlations of H-14/C-16; H-15/C-16 and C-21; H-16/C-
signals at d 0.81 (s, H-20) and 1.59 (s, H-19), one doublet methyl 14, C-15 and C-22; H-21/C-15 and H-22/C-16. The 1H–1H COSY
signal at d 0.91 (J = 6.4 Hz, H-17), two doublet signals with a similar correlations (Fig. 2) of H-14/H-15 support the presence of a
coupling constant at d 4.58 (1H, J = 4.4 Hz, H-14) and 5.53 (1H, fragment of an oxidized furan ring that attached to other carbon in
J = 4.4 Hz, H-15), one signal at d 5.23 (1H, s, H-16) and one olefinic 13 position. The stereochemistry of methoxy and hydroxyl groups
proton at d 6.91 (1H, bs, H-3). The 13C NMR spectrum (Table 1) and in the furan ring are at the same orientation as it appeared clearly
DEPT 13C NMR spectrum for 1 showed two methoxy, three methyl, in NOE experiment (Fig. 3). Furthermore, the HMBC correlations
five methylene, seven methine and five quaternary carbons. The (Fig. 2) H-10/C-19; H-17/C-8 and C-9; H-19/C-4, C-5 and C-6; H-20/
chemical shift of CH3-19 was observed at d 17.8, CH3-20 appeared C-9, C-10 and C-11; as well as the 1H–1H COSY correlations (Fig. 2)
at d 18.7 while CH3-17 resonated at d 16.3. The 1H–1H and 1H–13C H-2/H-3; H-6/H-7; in addition to the close similarities of chemical
connectivities were supported by the correlation spectroscopy shifts from C1–C10 and C17–C20 for 1 with reported values for
(1H–1H-COSY) and HMQC spectra. The HMBC spectrum (Fig. 2) related compounds supported that 1 is possessing an ent-
clerodane diterpene skeleton (Ceñal et al., 1997; Cifuente et al.,
2002; Kuroyanagi et al., 1993). The chemical shift of C-6 was
Table 1
NMR spectroscopic data (400 MHz for dH, 125 MHz for dC) for 1 and 2(d in ppm, J in
observed at d 75.5 and the position of the hydroxyl group could be
Hz). assigned to such carbon, by observing its correlations; in HMBC
correlations, since there are correlations of H-19 (d 1.59) with C-4
Position 1a 2a 2b
(d 145.4), C-5 (d 45.9) and C-6 (d 75.5) were observed besides the
1
dH dC dC dH dC H–1H COSY correlation of H-6/H-7. The relative stereochemistry
1 1.73 m 18.0 18.1 1.81 m 18.2
2 1.87 m 27.9 27.9 2.26 28.2
3 6.91 bs 137.1 139.1 6.82 bs 141.3
4 145.4 143.6 142.9c
5 45.9 45.9 45.9
6 3.94 dd (4.4,10) 75.5 75.2 3.58 dd (5,10.8) 75.8
7 1.90 38.0 37.8 1.57 37.1
8 1.73 m 34.9 34.8 35.2
9 39.1 39.3 39.7
10 1.39 m 46.3 46.5 1.32 m 47.0
11 1.65, 2.04 32.6 36.9 1.57 37.4
12 1.91 m 27.6 19.6 19.6
13 81.8 134.6 134.6
14 4.58 d (4.4) 81.1 145.8 7.32 bs 147.5
15 5.53 d (4.4) 111.8 71.2 4.77 bs 72.1
16 5.23 s 109.8 175.0 176.8
17 0.91 d (6.4) 16.3 16.3 0.84 d (6.4) 16.0
18 176.2 175.1 176.8
19 1.59 s 17.8 17.6 1.18 s 17.0
20 0.81 s 18.7 18.3 0.73 18.1
21 3.54 s 56.5
22 3.50 s 55.4
a
HMBC Correlation
Spectra were recorded in deuterated pyridine.
b
Spectra were recorded in CD3OD. COSY Correlation
c 13
C NMR signals reported here were obtained indirectly from HMQC and HMBC
spectra. Fig. 2. HMBC and COSY correlations of 1.
12 A.E. Mostafa et al. / Phytochemistry Letters 8 (2014) 10–15
NOE Correlation
Fig. 3. NOE correlations of 1.
of 1 was determined by comparing with related compounds in the
literature, and on the basis of NOE experiments. No NOE was
observed between H-6 and the methyl groups CH3-19, indicating a
orientation of OH group at C-6. From all the above discussion,
compound 1 considered to be a new compound which is similar to
visclerodol acid (Huang et al., 2010), except that 1 contains
hydroxyl group at position 6, and the two ethoxy groups present in
visclerodol acid are replaced by two methoxy groups.
Compound 2 was obtained as colorless gummy solid whose
molecular formula was established to be C20H28O5 from the [MH]
peak at m/z 347.1920 (calcd. for C20H27O5, 347.1859), the [2MH]
peak at m/z 695.3876 (calcd. for C40H55O10, 695.3918), the [M]+ peak
at m/z 348.1959 (calcd. for C20H28O5, 348.1937), and the [2 M]+ peak
at m/z 696.3914 (calcd. for C40H56O10, 696.3997), in the HR-ESIMS.
The 1H NMR spectrum (Table 1), (recorded in deuterated methanol)
exhibited typical signals for tricyclic clerodane carbon skeleton
including a secondary methyl signal at d 0.84 (d, J = 6.4 Hz, H-17) and
two tertiary methyl signals at d 0.73 (s, H-20) and 1.18 (s, H-19). The
olefinic proton showed a signal at d 6.82 (1H, bs, H-3). The down-
fielded signal at d 7.32 (1H, bs, H-14) indicated the presence of an a-
substituted g-butenolide (Heymann et al., 1994). The 13C NMR
spectrum (Table 1) and DEPT 13C NMR spectrum for 2 showed the
presence of three methyl, six methylene, five methine and six
quaternary carbons. The chemical shift of CH3-19 was observed at d
17.0, CH3-20 appeared at d 18.1 while CH3-17 resonated at d 15.98.
The 1H–13C connectivity was supported by the correlation
spectroscopy HMQC and HMBC spectra. The chemical shift of C-6
was observed at d 75.8 and the position of the hydroxyl group could
be assigned to such carbon, by observing its HMBC correlations
(Fig. 4), since correlations of CH3-19 (d 1.18) with C-4 (d 142.9), C-5 (d
45.9) and C-6 (d 75.8) were observed. The oxy methylene protons at d
4.77 showed correlations with C-13 (d 134.6) and C-14 (d 147.5),
thereby confirming their presence at C-15 and subsequently the
carbonyl group at C-16 in a -substituted g-butenolide. In addition to
the close similarities of chemical shifts from C1–C10 and C17–C20
for 2 with reported values for related compounds supported that 2
possesses an ent-clerodane diterpene skeleton (the same as
compound 1) (Ceñal et al., 1997; Cifuente et al., 2002; Kuroyanagi
et al., 1993). The relative stereochemistry of 2 was determined by
comparing with those which reported in literature (Huang et al.,
2010; Niu et al., 2010; Oliveira et al., 2012; Omosa et al., 2010;
Simpson et al., 2011). Thus, 2 was recorded to be a new compound, the
only difference with the published before (Iqbal et al., 2004) is in the
stereochemistry of the methyl groups at the positions C-5, C-8 and C-9
and the hydroxyl group at the position C-6, which was confirmed as b
orientation, while in the present work it having a different
stereochemistry and was established as a-orientation and the
compound is: ()-6a-hydroxy-5a,8a,9a,10a-cleroda-3,13-dien-
16,15-olid-18-oic acid. In addition, the stereochemistry of the methyl
groups in both compounds 1 and 2 is similar to those of the clerodane
HMBC Correlation
Fig. 4. HMBC correlations of 2.
diterpenoids isolated from different species of the genus Dodonaea
and were established in literature (Huang et al., 2010; Niu et al., 2010;
Oliveira et al., 2012; Omosa et al., 2010; Simpson et al., 2011) (Fig. 5).
Compound 3 was obtained as white crystalline solid, its
molecular formula was determined as C18H22O9, from the [MH]
 peak at m/z 381.1143 (calcd. for C18H21O9, 381.1185), the [M]+
peak at m/z 382.1159 (calcd. for C18H22O9, 382.1263), the [M+Na]+
peak at m/z 405.1138 (calcd. for C18H22NaO9, 405.1160) and the
[2M+Na]+ peak at m/z 787.2348 (calcd. for C36H44NaO9, 787.2423) in
the HR-ESIMS. The 1H NMR and 13C NMR spectral data of 3 and 4
(Table 2) are similar to each other except in the position of a and b
protons of the coumaryl moiety. There were appeared in 3 as doublet
protons (J = 12 Hz) at d 6.92 and 5.76, While in 4 they were appeared
as doublet protons (J = 16 Hz) at d 7.59 and 6.26 for H-70 and H-80 .
The data of 4 compared favorably with the published data of trans-
isomer of 1-L-O-methyl-2-acetyl-3-p-coumaryl-myo-inositol (Mata
et al., 1991), the position of the same protons appeared as doublet
protons (J = 15 Hz) at d 7.61 and 6.22. The 13C NMR and DEPT 13C
NMR of 3 and 4 showed the presence of two methyl, twelve methine
and four quaternary carbons. Thus by comparing all of those data
with the previously published data (Mata et al., 1991), we suggest
that 3 should be a cis-isomer of 1-L-O-methyl-2-acetyl-3-p-coumaryl-
myo-inositol, while 4 is the trans-isomer of the same compound.
Compounds 5–12 were identified by using 1H NMR, 13C NMR,
DEPT-135, HMQC and HMBC as well as HRESIMS experiments, as:
pinocembrin 5 (Agrawal, 1989), isokaemferide 6 (Agrawal, 1989),
6-methoxy isokaemferide 7 (Agrawal, 1989), 5,7-dihydroxy-3,6,40 -
trimethoxy flavone 8 (Agrawal, 1989), penduletin 9 (Azizuddin
et al., 2010), 5-hydroxy-3,6,7,40 -tetramethoxy flavone 10 (Sachdev
and Kulshreshtha, 1983), aliarin 11 (Muhammad et al., 2012) and
5,7-dihydroxy-30 -(4hydroxy-3methylbutyl) 3,6,40 -trimethoxyfla-
vone 12 (Muhammad et al., 2012). Compound 5 exhibited good
antifungal activity against Cryptococcus neoformans with an IC50
value of 6.11 mg/ml. Compounds 10 and 12 showed moderate
antileishmanial activity against Leishmania donovani with IC50
values of 16.6 and 19.06 mg/ml, respectively.
3. Experimental
3.1. General experimental procedures
NMR spectra were recorded on a Bruker Avance DRX-500
instrument at 500 (1H) and 125 MHz (13C), and a Varian Mercury
400 MHz spectrometer at 400 (1H) and 100 MHz (13C). The
HRESIMS spectra were measured using a Bruker Bioapex-FTMS
with electrospray ionization (ESI). The UV spectra were recorded
 A.E. Mostafa et al. / Phytochemistry Letters 8 (2014) 10–15 13

Fig. 5. Chemical structures of compounds 4–12.

using a Varian 50 Bio spectrophotometer. The optical rotation was
recorded using an AUTOPOL IV (Automatic polarimeter). Column
chromatographic separation was performed on silica gel 60 (0.04–
0.063 mm) and Sephadex LH-20 (0.25–0.1 mm, Aldrich). TLC was
performed on precoated TLC plates with silica gel 60 F254 (0.2 mm,
Merck). Semipreparative HPLC (Waters delta prep 4000) was
performed using Waters, Atlantis T31 RP-18 (5 m, 250, 10 mm),
Atlantis1 RP-18 (5 m, 250, 20 mm).
3.2. Plant material
The aerial parts of D. viscosa were collected in May 2011 from
Orman garden, Giza, Egypt. The plant was authenticated by Dr.
Mohammad El-Gebaly, Professor of Plant Taxonomy, Cairo
University, Egypt. A voucher specimen (DV 5) has been deposited
in the Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar
University, Cairo, Egypt.
3.3. Extraction and isolation
The air-dried powdered aerial parts of D. viscosa (1.65 kg) were
exhaustively extracted by maceration with 70% EtOH (10 L  3), at
room temperature. The combined ethanolic extracts were concen-
trated under reduced pressure to afford 200 g residue. The residue
was subjected to vacuum liquid chromatography (VLC) on silica gel
(1500 g, 45 cm, 15 cm) using n-hexane, n-hexane–EtOAc (50:50),
14 A.E. Mostafa et al. / Phytochemistry Letters 8 (2014) 10–15

Table 2 NMR spectroscopic data see (Table 1); HR-ESIMS m/z 347.1920
NMR spectroscopic data (500 MHz for dH, 125 MHz for dC) for 3 and 4 (d in ppm, J in
[MH] (calcd. for C20H27O5, 347.1859), m/z 695.3876 [2MH]
Hz).
(calcd. for C40H55O10, 695.3918), m/z 348.1959 [M]+ (calcd. for
Position 3a 4a C20H28O5, 348.1937) and m/z 696.3914 [2M]+ (calcd. for C40H56O10,
dH dC dH dC 696.3997). [a]D20 48 (c = 0.25, MeOH).
1 3.29 80.9 3.29 80.9
1-L-O-Methyl-2-acetyl-3-p-cis-coumaryl-myo-inositol, 3:
2 5.75 68.8 5.70 68.8 white crystalline solid; for 1H NMR and 13C NMR spectroscopic
3 3.63 73.6 3.63 73.5 data see (Table 2); HR-ESIMS m/z 381.1143 [MH] (calcd. for
4 4.83 73.1 4.78 73.4 C18H21O9, 381.1185), m/z 382.1159 [M]+ (calcd. for C18H22O9,
5 3.34 76.1 3.32 76.0
382.1263), m/z 405.1138 [M+Na]+ (calcd. for C18H22NaO9,
6 3.79 72.1 3.75 72.2
7 3.43 s 58.3 3.36 58.3 405.1160) and m/z 787.2348 [2M+Na]+ (calcd. for C36H44NaO9,
8 171.9 171.9 787.2423). [a]D20 +0.88 (c = 0.1, MeOH).
9 2.04 s 20.6 2.05 s 20.7
10 127.5 126.9 3.5. Antimicrobial assay
20 7.67 d (8) 133.8 7.61 d (9) 131.3
30 6.78 d (8) 115.8 6.71 d (8.5) 116.9
40 160.1 161.7 All isolated compounds were tested for antimicrobial activity
50 6.78 d (8) 115.8 6.71 d (8.5) 116.9 against Staphylococcus aureus ATCC 29213, methicillin-resistant S.
60 7.67 d (8) 133.8 7.61 d (9) 131.3 aureus ATCC 33591 (MRS), Escherichia coli ATCC 35218, Pseudomo-
70 6.92 d (12) 146.0 7.59 d (16) 147.8
nas aeruginosa ATCC 27853, Mycobacterium intracellulare ATCC
80 5.76 d (12.5) 116.0 6.26 d (16) 114.5
90 167.3 168.5 23068, Candida albicans ATCC 90028, Candida glabrata ATCC 90030,
a
Candida krusei ATCC 6258, Cryptococcus neoformans ATCC 90113,
Spectra were recorded in CD3OD.
and Aspergillus fumigatus ATCC 204305 (Bharate et al., 2007;
Radwan et al., 2007). Ciprofloxacin and Amphotericin B were used
EtOAc, EtOAc–MeOH (95:5), EtOAc–MeOH (50:50) and MeOH, as positive controls for bacteria and fungi, respectively.
each 2.0 L to give six fractions (A–F). Fr. C (1.5 g) was subjected to
CC (silica gel, 40 g, 50 cm, 5 cm, eluted with n-hexane–EtOAc 3.6. Antileishmanial assay
mixtures in a manner of increasing polarities) to obtain six
fractions (C1–C6). Fr. C3 was subjected to CC (sephadex LH-20, All isolated compounds were tested for antileishmanial activity
45 cm, 1 cm) eluted with MeOH, to obtain compounds: 5 (2 mg), 8 in vitro against a culture of Leishmania donovani promastigotes;
(3 mg) and 10 (4 mg). Fr. C6 was subjected to CC (sephadex LH-20, Pentamidine and Amphotericin B were used as a positive controls
45 cm, 1 cm) eluting with MeOH, to afford compounds: 6 (2 mg), 7 (Ma et al., 2004).
(13 mg) and 9 (5 mg). Fr. E (22 g) was subjected to VLC on silica gel
(500 g, 45 cm, 15 cm) using DCM, DCM–MeOH (80:20), DCM– 3.7. Antimalarial assay
MeOH (50:50) and MeOH, each 1 L to give four fractions (E1–E4).
Fr. E2 (7.5 g) was subjected to VLC on silica gel (200 g, 45 cm, All isolated compounds were tested for antimalarial activity in
15 cm) using n-hexane–EtOAc (50:50), EtOAc, EtOAc–MeOH vitro against chloroquine sensitive (D6, Sierra Leone) and resistant
(75:25), EtOAc–MeOH (50:50) and MeOH, each 1 L to give five (W2, Indo China) strains of Plasmodium falciparum by measuring
fractions (e1-e5). Fr. e2 (4 g) was subjected to CC (silica gel, 180 g, plasmodial LDH activity as described earlier (Makler and Hinrichs,
50 cm, 5 cm, eluted with DCM–MeOH mixtures in a manner of 1993). Chloroquine was used as positive control.
increasing polarities) to give 14 subfractions (1–14). Subfr. 4
(300 mg) was subjected to repeated CC on silica gel and sephadex 4. Conclusions
to afford 2 (5 mg). Subfr. 6 (870 mg) was subjected to repeated CC
on silica gel and sephadex to afford 1 (2 mg). Subfr. 9 (133 mg) was Three new compounds: 13,14 dihydroxy-15,16 dimethoxy-()-
subjected to subsequent purification on preparative RP-C18 HPLC 6a-hydroxy-5a,8a,9a,10a-cleroda-3-en-18-oic acid 1, ()-6a-
gradually eluted with mixture of MeOH and H2O, using Atlantis1 hydroxy-5a, 8a,9a,10a-cleroda-3,13-dien-16,15-olid-18-oic acid
RP-18 (5 m, 250, 20 mm) at a flow rate 17.0 ml/min and detection 2, 1-L-O-methyl-2-acetyl-3-p-cis-coumaryl-myo-inositol 3, to-
at lmax 270 nm to yield compounds: 11 (14 mg) and 12 (5 mg). gether with nine known compounds were isolated from the aerial
Subfr. 11 (64 mg) was subjected to subsequent purification on parts of D. viscosa L. growing in Egypt and evaluated for their
semipreparative RP-C18 HPLC gradually eluted with mixture of antibacterial, antifungal, antimalarial as well as antileishmanial
MeOH and 0.1% formic acid in H2O, using Atlantis1 RP-18 (5 m, activities. Compound 5 exhibited good antifungal activity against
250, 10 mm) at a flow rate 5.0 ml/min and detection at lmax Cryptococcus neoformans with an IC50 value of 6.11 mg/ml.
310 nm to give compounds: 3 (2 mg) and 4 (2 mg). Compounds 10 and 12 showed moderate antileishmanial activity
against Leishmania donovani with IC50 values of 16.6 and 19.06 mg/
3.4. Spectral data ml, respectively.

13,14 dihydroxy-15,16 dimethoxy-()-6a-hydroxy-5a, Acknowledgments

8a,9a,10a-cleroda-3-en-18-oic acid 1: yellow oil; for 1H NMR
and 13C NMR spectroscopic data see (Table 1); for HR-ESIMS m/z
429.2402 [M+H]+ (calcd. for C22H37O8, 429.2489), m/z 427.2283
[MH] (calcd. for C22H35O8, 427.2332), m/z 855.4680 [2MH]
(calcd. for C44H71O16, 855.4743), m/z 429.2402 [M+H]+ (calcd. for
C22H37O8, 429.2489), m/z 451.2215 [M+Na]+ (calcd. for
C22H36NaO8, 451.2307) and m/z 879.4557 [2M+Na]+ (calcd. for
C44H72NaO16, 879.4719). [a]D20 +28 (c = 0.1, MeOH).
()-6a-Hydroxy-5a, 8a,9a,10a-cleroda-3,13-dien-16,15-
olid-18-oic acid, 2: colorless gummy solid; for 1H NMR and 13C
We are grateful to the Egyptian Government and National
Center for Natural Products Research, University of Mississippi for
financial support. We also thankful for Dr. Baharthi Avula for
HRMS, Drs. Babu Tekwani and Surendra Jain for antileishmanial,
Shabana Khan and John Trott for antimalarial assays, and Marsha
Wright for antimicrobial testing. This investigation was conducted
in part in a facility constructed with support from the Research
facilities improvement Program C06 RR-14503-01, the NIH, NIAID,
Division of AIDS, Grant No. AI 27094, and the United States
 A.E. Mostafa et al. / Phytochemistry Letters 8 (2014) 10–15
Department of Agriculture ARS specific cooperative Agreement No.
58-6408-1-603.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2013.12.
008.
References
Agrawal, P.K., 1989. Carbon-13 NMR of Flavonoids. Elsevier Science Ltd.
Ahmad, M., Mahmood, Q., Gulzar, K., Akhtar, M.S., Saleem, M., Qadir, M.I., 2012.
Antihyperlipidemic and hepatoprotective activity of Dodonaea viscosa leaves
extracts in alloxan-induced diabetic rabbits (Oryctolagus cuniculus). Pak. Vet. J.
32, 50–54.
Azizuddin, Makhmoor, T., Choudhary, M.I., 2010. Radical scavenging potential of
compounds isolated from Vitex agnus-castus. Turk. J. Chem. 34, 119–126.
Bharate, S.B., Khan, S.I., Yunus, N.A., Chauthe, S.K., Jacob, M.R., Tekwani, B.L., Khan,
I.A., Singh, I.P., 2007. Antiprotozoal and antimicrobial activities of O-alkylated
and formylated acylphloroglucinols. Bioorg. Med. Chem. 15, 87–96.
Ceñal, J.P., Giordano, O.S., Rossomando, P.C., Tonn, C.E., 1997. Neoclerodane diter-
penes from Baccharis crispa. J. Nat. Prod. 60, 490–492.
Cifuente, D.A., Borkowski, E.J., Sosa, M.E., Gianello, J.C., Giordano, O.S., Tonn, C.E.,
2002. Clerodane diterpenes from Baccharis sagittalis: insect antifeedant activity.
Phytochemistry 61, 899–905.
Heymann, H., Tezuka, Y., Kikuchi, T., Supriyatna, S., 1994. Constituents of Sindora
sumatrana MIQ. III. New trans-clerodane diterpenoids from the dried pods.
Chem. Pharm. Bull. 42, 1202–1207.
Huang, Z., Jiang, M.-Y., Zhou, Z.-Y., Xu, D., 2010. Two new Clerodane diterpenes from
Dodonaea viscosa. J. Chem. Sci. 65 (1) 83–86.
Iqbal, K., Malik, A., Mukhtar, N., Anis, I., Khan, S.N., Choudhary, M.I., 2004. a-
Glucosidase inhibitory constituents from Duranta repens. Chem. Pharm. Bull.
52, 785–789.
15
Kuroyanagi, M., Uchida, K., Ueno, A., Satake, M., Shimomura, K., 1993. Neo-clerodane
type diterpenes from Baccharis trinervis. Phytochemistry 34, 1377–1384.
Ma, G., Khan, S.I., Jacob, M.R., Tekwani, B.L., Li, Z., Pasco, D.S., Walker, L.A., Khan, I.A.,
2004. Antimicrobial and antileishmanial activities of hypocrellins A and B.
Antimicrob. Agents Chemother. 48, 4450–4452.
Makler, M.T., Hinrichs, D.J., 1993. Measurement of the lactate dehydrogenase
activity of Plasmodium falciparum as an assessment of parasitemia. Am. J. Trop.
Med. Hyg. 48, 205–210.
Mata, R., Contreras, J.L., Crisanto, D., Pereda-Miranda, R., Castañeda, P., Del Rio, F.,
1991. Chemical studies on Mexican plants used in traditional medicine, XVIII.
New secondary metabolites from Dodonaea viscosa. J. Nat. Prod. 54, 913–917.
Muhammad, A., Anis, I., Khan, A., Marasini, B.P., Choudhary, M.I., Shah, M.R., 2012.
Biologically active C-alkylated flavonoids from Dodonaea viscosa. Arch. Pharm.
Res. 35, 431–436.
Niu, H.-M., Zeng, D.-Q., Long, C.-L., Peng, Y.-H., Wang, Y.-H., Luo, J.-F., Wang, H.-S., Shi, Y.-
N., Tang, G.-H., Zhao, F.-W., 2010. Clerodane diterpenoids and prenylated flavo-
noids from Dodonaea viscosa: original article. J. Asian Nat. Prod. Res. 12, 7–14.
Oliveira, S.Q.d., Almeida, M.T.R.d., Maraslis, F., Silva, I.T., Sincero, T.C.M., Palermo, J.A.,
Cabrera, G.M., Caro, M.S.B., Simões, C.M.O., Schenkel, E.P., 2012. Isolation of three
new ent-labdane diterpenes from Dodonaea viscosa Jacquin (Sapindaceae): pre-
liminary evaluation of antiherpes activity. Phytochem. Lett. 5, 500–505.
Omosa, L.K., Derese, S., Midiwo, J.O., Yenesew, A., Peter, M.G., Heydenreich, M., 2010.
neo-Clerodane diterpenoids from the leaf exudate of Dodonaea angustifolia.
Phytochem. Lett. 3 (4) 217–220.
Radwan, M.M., Manly, S.P., Ross, S.A., 2007. Two new sulfated sterols from the
marine sponge Lendenfeldia dendyi. Nat. Prod. Commun. 2, 901–904.
Rani, SandhyaM., Rao, PippallaS., Mohan, K., 2009. Dodonaea viscosa Linn.—an
overview. Asian J. Pharm. Res. Health Care 97–112.
Sachdev, K., Kulshreshtha, D.K., 1983. Flavonoids from Dodonaea viscosa. Phyto-
chemistry 22, 1253–1256.
Simpson, B.S., Claudie, D.J., Gerber, J.P., Pyke, S.M., Wang, J., McKinnon, R.A., Semple,
S.J., 2011. In vivo activity of benzoyl ester clerodane diterpenoid derivatives
from Dodonaea polyandra. J. Nat. Prod. 74, 650–657.
Veerapur, V., Prabhakar, K., Kandadi, M., Srinivasan, K., Unnikrishnan, M., 2010.
Antidiabetic effect of Dodonaea viscosa aerial parts in high fat diet and low dose
streptozotocin-induced type 2 diabetic rats: a mechanistic approach. Pharm.
Biol. 48, 1137–1148.
Wagner, H., Ludwig, C., Grotjahn, L., Khan, M.S.Y., 1987. Biologically active saponins
from Dodonaea viscosa. Phytochemistry 26, 697–701.