Chemical constituents of Osyris alba and their antiparasitic activities

Article abstract of DOI:10.1080/10286020.2010.502892

Phytochemical investigation of Osyris alba L. (Santalaceae) of Jordanian origin resulted in the isolation and identification of one new pyrrolizidine alkaloid, osyrisine (1), together with 16 other known compounds. The structures of all compounds were established on the basis of spectroscopic analysis. Osyrisine, catechin, and catechin-3-O-α- l-rhamnopyranoside exhibited a significant level of antiparasitic activity against two parasites, Entamoeba histolytica and Giardia intestinalis.

Full text of DOI:10.1080/10286020.2010.502892

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Chemical constituents of Osyris alba
and their antiparasitic activities
a
b
c
Hala I. Al-Jaber , Ibrahim M. Mosleh , Abdallah Mallouh , Omar
c
a
M. Abu Salim & Musa H. Abu Zarga
a
Chemistry Department , Faculty of Science, University of
Jordan , Amman, Jordan
b
Department of Biological Sciences , Faculty of Science,
University of Jordan , Amman, Jordan
c
Al-Kuds Medical Laboratories , Zarka, Jordan
Published online: 13 Sep 2010.
To cite this article: Hala I. Al-Jaber , Ibrahim M. Mosleh , Abdallah Mallouh , Omar M. Abu Salim
&
Musa H. Abu Zarga (2010) Chemical constituents of Osyris alba and their antiparasitic activities,
Journal of Asian Natural Products Research, 12:9, 814-820, DOI: 10.1080/10286020.2010.502892
To link to this article: http://dx.doi.org/10.1080/10286020.2010.502892
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Journal of Asian Natural Products Research
Vol. 12, No. 9, September 2010, 814–820
NOTE
Chemical constituents of Osyris alba and their antiparasitic activities
a
Hala I. Al-Jaber , Ibrahim M. Mosleh *, Abdallah Mallouh ,
b
c
c
Omar M. Abu Salim and Musa H. Abu Zarga
a
a
b
Chemistry Department, Faculty of Science, University of Jordan, Amman, Jordan; Department of
c
Biological Sciences, Faculty of Science, University of Jordan, Amman, Jordan; Al-Kuds Medical
Laboratories, Zarka, Jordan
(Received 14 March 2010; final version received 16 June 2010)
Phytochemical investigation of Osyris alba L. (Santalaceae) of Jordanian origin
resulted in the isolation and identification of one new pyrrolizidine alkaloid, osyrisine
(1), together with 16 other known compounds. The structures of all compounds were
established on the basis of spectroscopic analysis. Osyrisine, catechin, and catechin-3-
O-a-L-rhamnopyranoside exhibited a significant level of antiparasitic activity against
two parasites, Entamoeba histolytica and Giardia intestinalis.
Keywords: osyrisine; Osyris alba; pyrrolizidine alkaloids; catechin; catechin-3-O-a-
L-rhamnopyranoside; antiparasitic activity
1
.
Introduction
Analysis of natural sources is an alternative
strategy to develop such compounds.
The phytochemical investigation of plant
species used traditionally in the treatment
of the two diseases resulted in the isolation
and characterization of a variety of
bioactive chemical compounds [5].
Amoebiasis and giardiasis are among the
most important diarrheal infections in
several regions of the world. Metronida-
zole, the drug preferred by most clinicians
for the treatment of these infections, has a
mutagenic activity in eukaryotic cells [1]
and carcinogenicity in animals [2]. No
carcinogenic effect has yet been demon-
strated in humans [3], but its use during
pregnancy is avoided by many physicians.
Although drug resistance to Ent-
amoeba and Giardia does not appear to
be a serious problem, occasional reports of
failure with metronidazole and the reported
variations in drug sensitivities of isolates
Osyris alba (Santalaceae) is a widely
spread dioecious shrub growing wild in
southern Europe, North Africa, Southeast
Asia and in different localities in Turkey,
Jordan, and Palestine [6]. This plant is
known to be a root hemiparasite having a
wide range of hosts including forest trees,
perennial woody herbs, and fruit trees [7].
Previous phytochemical studies on this
plant revealed that it contains alkaloids,
carbohydrates, amino acids, fatty acids, and
phenolics [8]. An investigation concerning
the alkaloid constituents revealed the
concomitant occurrence of quinolizidine
[4] may be alarming. Because of the
limited number of drugs available against
anaerobic protozoal parasites, there is a
serious need for new active compounds.
*Corresponding author. Email: i.mosleh@ju.edu.jo
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286020.2010.502892
http://www.informaworld.com

Journal of Asian Natural Products Research
815
and pyrrolizidine alkaloids in O. alba [6].
The use of the plant O. alba in tradi-
tional medicine seems to be rare and its
use against dysentery has never been
mentioned in the literature. However,
some Jordanian traditional healers claim
the successful use of this plant in the
treatment of amoebic dysentery. The
presence of different types of alkaloids in
this plant seemed to support its unusual use
in traditional medicine in Jordan. Driven
by these findings, bioactivity-guided frac-
tionation of extracts of the whole plant
material was performed and the activity of
each preparation on axenic strains of
Entamoeba histolytica and Giardia intes-
tinalis (¼lamblia) was tested.
and 2-octenoic acid (14); b-sitosteryl gluco-
side (15); triterpenoids ursolic acid (16) and
oleanolic acid (17); and p-hydroxybenzoic
acid (4). All these compounds, except for
mannitol (3), chysin A (5), and p-hydro-
xybenzoic acid (4), are reported for the first
time from O. alba.
The new compound osyrisine (1)
(Figure 1) was isolated as a yellow oil
from the bioactive fractions AV and BIV.
The high-resolution mass spectra (HR-
MS) of compound 1 showed a molecular
þ
ion peak at m/z 226.1802 [M þ H] ,
corresponding to the molecular formula
C H NO . The IR spectrum of com-
pound 1 revealed the presence of ester
1
3
23
2
2
1
1
carbonyl at 1732 cm . The H NMR
(
(
CDCl ) spectrum of this compound
3
Table 1) showed resonances for two
2
.
Results and discussion
methyl groups, one of which appeared as
a triplet at d 0.84 (J ¼ 7.4 Hz) while the
other as a doublet at d 1.07 (J ¼ 7.0 Hz).
The appearance of several aliphatic signals
in the range d 1.2–2.8 was indicative of a
bicyclic pyrrolizidine nucleus. An inter-
2
.1 Bioguided fractionation
The crude methanol extract of the dried,
ground, and defatted plant material was
partitioned according to the procedure
described in Section 3 into hexane (C), an
aqueous methanol (A), butanol (B), and
water (W) extracts. The aqueous methanol
1
esting feature of the H NMR spectrum of
this compound was the two signals located
(
A) and butanol (B) extracts were found to
at d 4.04 (dd, J ¼ 8.1, 11.1 Hz, H-9 ) and
a
be bioactive. Both extracts were fractio-
nated as described in Section 3 and
antiparasitic activity tests were performed
for each fraction. The results showed that
fractions AV, BIV–BVII were active.
Chromatography of these fractions
afforded six compounds identified as
catechin (2a) [8], engeletin (6) [9], chysin
A (5) [6], osyrisine (1), catechin-3-O-a-L-
rhamnopyranoside (2b) [10], and mannitol
4.14 (dd, J ¼ 7.0, 11.1 Hz, H-9 ), which
b
were assigned to the diastereotopic protons
1
3
of the oxymethylene group. The C NMR
spectrum of osyrisine revealed the pre-
sence of 13 carbon signals including one
ester carbonyl carbon at d 176.6, one
oxymethylene carbon at d 63.9, one
nitrogen-bearing methine carbon at d
66.4, and two nitrogen-bearing methylene
carbons at d 53.7 and 55.8. The remaining
(
3), respectively. The structures of all
these compounds were determined by their
spectral data including EI-MS, FAB-MS,
UV, IR, 1D and 2D NMR.
O
The bio-inactive fractions AII–AIV and
BIII afforded 12 known compounds ident-
ified as: the flavonoids, salvigenin (7),
pachypodol (8) [11], kumatakillin (9) [12],
penduletin (10) [12], jecidiene (11) [13],
and kampferol-7-methyl ether (12) [13];
two fatty acids: dotriacontanoic acid (13)
O
H
N
1
Figure 1. Structure of osyrisine (1).

8
16
H.I. Al-Jaber et al.
1
13
Table 1. H and C NMR spectral data for
compound 1 in CDCl
3
.
4'
3
'
No.
dH (J in Hz)
2.62 (1H, m)
1.63 (2H, m)
2.73 (1H, br t, J ¼ 9.4)
3.21 (1H, dt, J ¼ 6.3, 10.0)
2.52 (1H, dt, J ¼ 6.4, 10.0)
3.42 (1H, br t, J ¼ 10.0)
1.87 (3H, m)
dC
O
1'
2'
5'
1
2
3a
40.2
27.0
53.7
9
O
H
3
5
b
7
55.8
1
3
a
8
6
2
5b
N
6
7
8
, 7
a
26.8
26.4
66.4
63.9
5
b
1.69 (1H, m)
3.78 (1H, m)
Figure 2. Main HMBC correlations in 1.
9a
4.04 (1H, dd, J ¼ 8.1, 11.1)
9
b
4.14 (1H, dd, J ¼ 7.0, 11.1)
0
1
2
3
–
2.33 (q, J ¼ 7.0)
1.43 (1H, m)
176.6
41.1
26.2
0
0
of the pyrrolizidine moiety in osyrisine (1)
were comparable to those of known
pyrrolizidine alkaloids [6,14].
a
0
3
b
1.61 (1H, m)
0
4
0.84 (3H, t, J ¼ 7.4)
1.07 (3H, t, J ¼ 7.0)
11.7
16.6
The alkaline hydrolysis of compound 1
gave 1-hydroxymethyl pyrrolizidine moi-
0
5
2
D
5
ety with an optical rotation of ½aꢀ þ 7.5
(
agreement with the optical rotation of the
c ¼ 0.2, MeOH), which is in good
carbon signals were assigned to two
methyl, four methylene, and one methine
carbons. Further analysis of COSY,
HMBC, and HMQC spectra of compound
known pyrrolizidine alkaloid (þ)-labur-
2
D
5
nine (½aꢀ þ 9.1). These results confirm
the stereochemistry of the pyrrolizidine
nucleus in osyrisine as (1S, 8R) [15].
1
suggested that the compound consisted
of a pyrrolizidine moiety (C-1–C-9)
esterified to 2-methylbutanoic acid (C-
Further confirmation of the structure of
osyrisine (1) was gained from its mass
spectrum, which showed the correct
molecular ion peak at m/z 225 and a base
peak at m/z 124 resulting from the loss of
the ester moiety from the molecular ion.
0
0
1
–C-5 ) through the hydroxymethylene
group (C-9). The interesting long-range
correlations observed in the HMBC
spectrum (Figure 2) between the oxy-
methylene protons at C-9 with the
0
carbonyl carbon (C-1 ) and the methine
2
.2 Antiparasitic activity
carbons C-1 and C-8 confirmed the
location of the ester linkage at C-9. The
chemical shifts for all protons and carbons
The IC50 values for the three com-
pounds, osyrisine (1), catechin (2a), and
Table 2. The biological activity of compounds 1, 2a, 2b and metronidazole against the parasites,
E. histolytica and G. intestinalis, and cancer (Hep-2) and nonmalignant (Vero) cells.
IC ^ SD (growth as in negative
0
IC50 ^ SD mg/ml
control) mg/ml
Compounds
E. histolytica
G. intestinalis
Hep-2 cells
Vero cells
1
7.10 ^ 0.36
17.67 ^ 2.5
29.67 ^ 2.5
0.80 ^ 0.04
7.57 ^ 0.35
19.00 ^ 1.73
29.67 ^ 2.01
0.86 ^ 0.02
597.00 ^ 11.53
444.33 ^ 5.51
596.00 ^ 6.93
186.33 ^ 8.62
596.66 ^ 7.64
299.33 ^ 8.62
596.00 ^ 3.46
194.67 ^ 5.03
2
2
a
b
Metronidazole

Journal of Asian Natural Products Research
817
catechin-3-O-a-L-rhamnopyranoside (2b),
and the standard drug, metronidazole,
against the tested parasites are shown in
Table 2. Among the purified compounds,
the new pyrrolizidine alkaloid, osyrisine,
was the most potent (around 7 mg/ml).
Although its activity was around eight
times less than the standard drug, its cyto-
toxicity against Hep-2 cell lines and non-
malignant (Vero) cells was around three
times less (Table 2). The other two purified
biologically active compounds were also
non-cytotoxic to the two cell lines at con-
centrations much greater than the anti-
parasitic concentrations (Table 2). This
cytotoxicity pattern of the three biologi-
cally active compounds indicates poten-
tially safe alternative therapeutic agents
against the susceptible pathogens [16].
Interestingly, the three compounds
exhibited a similar activity against
G. intestinalis and E. histolytica (Table 2),
indicating that each compound affects both
the parasites by a similar mechanism of
action. Regardless of this mechanism, the
importance of such biologically active,
non-cytotoxic compounds, especially
osyrisine (1), lies in their potential
contribution to overcome any emerging
problem of resistance of pathogens to the
standard drug [5]. It is noteworthy that the
low cytotoxicity of osyrisine 1 is consistent
with the previous reports about the non-
cytotoxicity of pyrrolizidine alkaloids with
a saturated necine nucleus [17].
HR-MS were measured in positive ion
mode using electrospray ionization (ESI)
technique on a Bruker APEX-2 instrument.
The optical rotation was recorded on a
Perkin-Elmer polarimeter 141. Column
chromatography was performed on silica
gel 60 (0.063–0.200 mm; Fluka, Stein-
heim, Germany) or silica gel S (Ridel de-
ha e¨ n, Seelze-Hannover, Germany). Purifi-
cation of the compounds was achieved by
routine TLC on silica gel G-UV254 glass
plates (0.25 mm; Macherey-Nagel, Easton,
PA, USA). Compounds were visualized
under UV light and spraying with sulfuric
acid–anisaldehyde spray reagent followed
by heating at 1208C. Alkaloids were
detected by spraying with iodoplatinic
acid spray reagent.
3.2 Plant material
O. alba was collected during the flowering
period in July 2001 from the Salt area
(25 km northwest of Amman, Jordan).
The plant was identified by Prof. Sawsan
Al-Oran, Department of Biological
Sciences, University of Jordan, Amman,
Jordan. A voucher specimen of the plant has
been kept in the herbarium of the Depart-
ment of Biological Sciences, University of
Jordan.
3.3 Extraction and isolation
The dried plant (7.0 kg) was defatted by
soaking in petroleum ether and then was
repeatedly soaked in methanol at room
temperature (50 liters, four times, 7 days
each). The residue obtained upon removal
of MeOH under reduced pressure (1.0 kg)
was partitioned between chloroform and
water. The dried chloroform layer was
further partitioned between hexane and
10% aqueous methanol, furnishing the
aqueous methanol extract (A, 125 g) and
hexane extract (C, 100 g). The water layer
was extracted with n-butanol to afford the
butanol extract (B, 375.4 g) and the water
extract (W, 355.6 g).
3
.
Experimental
3
.1 General experimental procedures
The IR spectra were recorded on a
Thermo-Nicolet Nexus 870 FT-IR spec-
1
trophotometer. H NMR spectra were
recorded on a Bruker DPX-300 MHz
spectrometer with TMS as an internal
1
3
standard. C NMR spectra were recorded
at 75.5 MHz. EI-MS were recorded using a
Finnigan MAT TSQ-70, JMS 600 or
MAT-112 S spectrometer at 70 eV and
ion source temperature equal to 2008C.

8
18
H.I. Al-Jaber et al.
Fractions A and B were successively
CHCl (3 £ 20 ml). The organic layer was
3
subjected to CC and the isolated com-
pounds were purified by TLC. The
aqueous methanol extract (A, 125 g) was
chromatographed on a silica gel S (63–
collected and evaporated under vacuum
affording impure alkaloid. Final purifi-
cation was achieved on TLC using the
solvent mixture hexane–diethyl amine
(90:10, v/v) to give the pure alkaloid as a
yellow oil (50 mg). Similarly, the same
compound was obtained from fraction BIV
following the same procedure (30 mg).
2
30 mesh; Fluka) column eluted with a
gradient of MeOH/CHCl3 of increasing
polarity to give five fractions (AI–AV).
Each fraction was purified by a combi-
nation of CC, TLC, and recrystallization
using suitable solvent systems for elution.
Fraction AII (2.3 g) afforded compound 13
3.3.1 Osyrisine (1)
(361 mg). Fraction AIII (32.33 g) afforded
a mixture of compounds 16 and 17
Yellow oil (80 mg). [a ] þ 45.2
D
(c ¼ 0.001, MeOH). IR (film): 1732
1
(CvO). H NMR spectral data: see
(164 mg), 7 (36 mg), 14 (12 mg), 8
(11 mg), 9 (6 mg), 10 (43 mg), and 11
(15 mg). Fraction AIV (6.4 g) afforded
1
Table 1, C NMR spectral data, see
3
þ
Table 1. EI-MS m/z (%): 225 [M] (17),
124 (100), 122 (11), 95 (10), 83 (37), 55
compound 12 (145 mg). Fraction AV
7.0 g) afforded compounds 15 (1000 mg)
and 1 (50 mg).
The butanol extract (B, 375.4 g)
(
(16), 41 (43); HR-ESI-MS: 226.1806
þ
[M þ H]
(calcd for C H NO ,
13 24 2
226.1802).
precipitated a white pinkish solid upon
treatment with distilled methanol. The
solid was collected and the mother liquor
was chromatographed following the same
procedure described for fraction A to
give eight fractions (BI–BVIII). Each
fraction was further purified by a
combination of column chromatography
and TLC using suitable solvent systems.
Fraction BIII (2.0 g) afforded compound
3
.3.2 Alkaline hydrolysis of 1
Compound 1 (7 mg) was hydrolyzed by
refluxing with 5.0 ml of 0.10 N NaOH in
5.0 ml methanol for 3 h. The mixture was
cooled, diluted with water, and extracted
with CHCl3 (3 £ 4 ml). The chloroform
layer was evaporated to give 2 mg of 1-
hydroxymethyl pyrrolizidine alkaloid. The
optical activity of the product was
4 (45 mg). Fraction BIV (7.6 g) afforded
compounds 2a (400 mg), 6 (145 mg), 1
2
5
½aꢀ þ 7.5.
D
(
(
30 mg), and 5 (75 mg). Fractions BV
29 g) and BVI (15.0 g) afforded com-
3
.4 Biological materials
pound 2b (900 and 100 mg, respectively).
Fraction BVII (15.0 g) afforded com-
pound 3 (2870 mg).
Namely, fraction AV (7.0 g) was
introduced into a silica gel column
E. histolytica HK-9 strain (ATCC number
30015) was cultured in LYI-S-2 medium
supplemented with antibiotics. G. intesti-
nalis WB strain (ATCC number 30957)
was grown in a modified YI-S medium
with antibiotics. Both parasites were
cultivated in 15 ml screw-capped borosili-
cate glass tubes containing 13 ml of
medium. The tubes were incubated on a
158 horizontal slant at 36–378C. Culture
maintenance and subculturing were per-
formed as described previously [18].
Entamoeba and Giardia were harvested
(
34.5 £ 4.8 cm) packed in ethyl acetate
and eluted with a gradient of ethyl
acetate/methanol of increasing polarity.
Then, the mobile phase was changed into
CHCl –MeOH mixture (85:15, v/v) satu-
3
rated with ammonia to elute the alkaloid.
The crude fraction containing the alkaloid
was evaporated, treated with 10% aqueous
ammonia solution and then extracted with

Journal of Asian Natural Products Research
819
from confluent cultures by the chilling of
the tubes in ice for 5–10 min to detach
cells, followed by centrifugation at 800 g
for 5 min.
medium in each well was then replaced
with 150 ml of fresh medium. Solutions of
the plant preparations and metronidazole
were dissolved in DMSO, prepared in
medium, and filter-sterilized. Then,
150 ml of twofold serial dilutions of each
3
.5 Antiparasitic activity
of the plant preparations and the reference
drug starting at a concentration of
2000 mg/ml in the culture medium were
prepared in the plates. After 48 h incu-
bation, the number of cells in each well
was determined using a hemacytometer.
Each tested plant preparation or metronid-
azole was assayed in duplicate in each of
three independent experiments. In each
assay, the negative controls (without any
compound or reference drug) were
included in duplicates. IC0 was the
concentration at which the number of
cells in the tube was more than or equal to
The antiamoebic and antigiardial activities
of the plant fractions and metronidazole
(
the reference drug) were tested as
previously described [19]. Briefly, the
plant preparations and metronidazole were
dissolved in dimethyl sulfoxide (DMSO),
then in medium and filter-sterilized. Two-
fold dilutions ranging from 50 to
0
.2 mg/ml were prepared in a final volume
of 15 ml to exclude air from the tube. Each
tube was inoculated with 20,000 cells of
the parasite under testing (Entamoeba or
Giardia). Each one of the plant prep-
arations was assayed in duplicate in each
of three independent experiments. In each
assay, the appropriate controls were
performed, including one without any
plant preparation and another with
metronidazole as the positive control.
The biological activity of the plant
preparations was evaluated by counting
the parasites in each tube using the
standard hemacytometer. In each count,
trypan blue was employed to distinguish
live from dead parasites [20]. The 50%
inhibitory concentration (IC ) was
that of the negative control (IC ¼ no
0
inhibition of cell growth).
Acknowledgements
This research was supported by a joint grant
from the Deanship of Scientific Research
and Hamdi Mango Center for Scientific
Research (HMCSR), University of Jordan.
We wish to thank Ms Lina Al-Natsheh
for technical assistance and maintenance of
cultures.
5
0
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tested plant preparations or metronidazole
which cuts the number of parasites to half
that in the negative control.
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