Anti-babesial and anti-plasmodial compounds from Phyllanthus niruri

Article abstract of DOI:10.1021/np0497245

Bioassay-guided fractionation of boiled aqueous extracts from the whole plant of Phyllanthus niruri led to the isolation of 1-O-galloyl-6-O-luteoyl- α-D-glucose (1), with IC₅₀ values of 4.7 μg/mL against Babesia gibsoni and 1.4 μg/mL against Plasmodium falciparum in vitro. The known compounds β-glucogallin (2), quercetin 3-O-β-D-glucopyranosyl- (2→1)-O-β-D-xylopyranoside (3), β-sitosterol, and gallic acid were also isolated. Structures of these compounds were elucidated on the basis of their chemical and spectroscopic data.

Full text of DOI:10.1021/np0497245

J. Nat. Prod. 2005, 68, 537-539
537
Anti-babesial and Anti-plasmodial Compounds from Phyllanthus niruri
Subeki,† Hideyuki Matsuura,*,† Kosaku Takahashi,† Masahiro Yamasaki,‡ Osamu Yamato,‡ Yoshimitsu Maede,‡
Ken Katakura,‡ Sumiko Kobayashi, Trimurningsih,§ Chairul,§ and Teruhiko Yoshihara†
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan, Department of
Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan,
Transfusion Medicine, Hokkaido University, Sapporo 060-8648, Japan, and Research and Development for Biology,
Indonesian Institute of Sciences, Bogor 16122, Indonesia
Received August 23, 2004
Bioassay-guided fractionation of boiled aqueous extracts from the whole plant of Phyllanthus niruri led
to the isolation of 1-O-galloyl-6-O-luteoyl-R-D-glucose (1), with IC50 values of 4.7 µg/mL against Babesia
gibsoni and 1.4 µg/mL against Plasmodium falciparum in vitro. The known compounds â-glucogallin (2),
quercetin 3-O-â-D-glucopyranosyl-(2f1)-O-â-D-xylopyranoside (3), â-sitosterol, and gallic acid were also
isolated. Structures of these compounds were elucidated on the basis of their chemical and spectroscopic
data.
Canine babesiosis is a tick-borne hemolytic disease of
dogs caused by intraerythrocytic parasites, Babesia gibsoni
and Babesia canis. The disease occurs frequently in com-
panion animals and has become a serious problem clini-
cally. Babesia destroys red blood cells and induces severe
clinical symptoms, such as hemolytic anemia, fever, he-
moglobinuria, and marked splenomegaly. These symptoms
and the mechanisms of infection are very similar to those
of malaria. In addition, dogs that have recovered from the
disease commonly become chronic carriers and are thereby
a source of infection to other dogs.1-4 Anti-babesial drugs
used for the treatment of babesiosis are limited and usually
cause pronounced, severe, and drastic side effects.5-7 In
some cases, the drugs are not able to eliminate the
parasites completely. Therefore, an alternative chemo-
therapeutic agent with fewer side effects is urgently needed
for the treatment of B. gibsoni. One possible source of such
affordable treatment lies in the use of plant extracts.
Phyllanthus niruri L. (Euphorbiaceae) is a medicinal
plant widely distributed in Indonesia that is often used in
scribed in the Experimental Section. An extract (boiling
water) of the whole plant of Phyllanthus niruri (100 g) was
folk medicine to treat epilepsy, malaria, hypertension,
toothache, diarrhea, fever, tetanus, and urinary calculus.8
cooled and then partitioned using ethyl acetate. Purifica-
This plant has been intensively studied phytochemically.
tion of the ethyl acetate extract afforded â-sitosterol (7.6
mg), gallic acid (23.5 mg), and 1 (27.3 mg). The H2O-soluble
layer, using a series of chromatography techniques, gave
Constituents such as lignans, alkaloids, flavonoids, phe-
nols, and terpenes have been identified.9-12 In addition,
several pharmacologic experiments have also been re-
compounds 2 (34.5 mg) and 3 (34.2 mg).
ported.13,14 Despite the many phytochemical and pharma-
The isolated compounds, except for 1, were identified as
cologic investigations, there are no reports on the anti-
babesial activity of this plant. We therefore investigated
anti-babesial compounds of this plant and herein present
â-glucogallin (2),15 quercetin 3-O-â-D-glucopyranosyl-(2f1)-
O-â-D-xylopyranoside (3),16 â-sitosterol,17 and gallic acid,15
respectively, by comparison of their 1H and 13C NMR, mass
data on a novel polyphenolic glycoside, along with four
spectral data, and optical rotation values with those of
known related compounds that also possess some anti-
reported data.
babesial activity. Because of similar symptoms and infec-
Compound 1 was isolated as a yellow powder. Fast atom
tion mechanisms, anti-plasmodial activity is also discussed.
bombardment mass spectrometry (FABMS) of 1 showed an
ion peak [M - H]- at m/z 633, corresponding to the
Results and Discussion
molecular formula C27H21O18. The IR spectrum displayed
characteristic absorptions for hydroxyl groups (3387 cm-1),
Bioassay-guided fractionations were carried out during
carbonyl functions (1719 cm-1), and aromatic rings (1618
the isolation procedure for anti-babesial compounds. Anti-
cm-1). The 1H and 13C NMR spectra of 1 were partially
similar to those of 2, particularly the resonances derived
from galloyl and pyranose moieties (Table 1). Gas chroma-
tography/mass spectrometry (GC/MS) analysis of the N,O-
bis(trimethylsilyl)acetamide derivative of the methanolysis
product of 1 revealed that the pyranose was glucose. The
linkage of glucose and galloyl moieties was determined to
babesial activity was measured using the methods de-
* Author for correspondence. Tel: +81-11-706-2495. Fax: +81-11-706-
2505. E-mail: matsuura@chem.agr.hokudai.ac.jp.
† Division of Applied Bioscience, Hokkaido University.
‡ Department of Veterinary Clinical Sciences, Hokkaido University.
Transfusion Medicine, Hokkaido University.
§ Indonesian Institute of Sciences.
10.1021/np0497245 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/29/2005

538 Journal of Natural Products, 2005, Vol. 68, No. 4
Subeki et al.
Table 1. 1H NMR (δ) Assignments for 1 and 2 (CD3OD, 500
MHz)
Table 2. 13C NMR (δ) Assignments for 1 and 2 (CD3OD, 125
MHz)
H
1
2
C
1
2
1
2
3
4
5
6
6.37 d (J ) 3.7 Hz)
3.99 m
5.59 d (J ) 7.8 Hz)
3.26-3.44 m
1
94.9
69.3
96.0
74.1
2
4.81 m
3.26-3.44 m
3
71.5
78.2
4.47 m
3.26-3.44 m
4
62.4
71.1
4.53 ddd (J ) 2.1, 5.0, 11.4 Hz) 3.26-3.44 m
5
76.1
78.8
4.15 dd (J ) 5.0, 11.1 Hz)
4.96 dd (J ) 2.1, 11.1 Hz)
3.66 dd (J ) 5.1, 11.9 Hz)
6
64.9
62.3
3.83 dd (J ) 2.0, 11.9 Hz)
1′
120.5
110.9
146.3
140.4
146.3
110.9
166.7
116.7a
145.3b
137.6c
145.9c
108.3
125.4a
170.1
117.2d
145.4b
138.2e
145.5e
110.9
125.3d
168.5
120.7
110.6
146.5
140.3
146.5
110.6
167.1
2′, 6′ 7.06 s
5′′
5′′′
7.08 s
2′
6.66 s
6.69 s
3′
4′
5′
6′
7′
1′′
2′′
3′′
4′′
5′′
6′′
7′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
a-e Assignments may be interchanged within each column.
Table 3. Anti-babesial and Anti-plasmodial Activities of
Compounds Isolated from Phyllanthus niruri against Babesia
gibsoni and Plasmodium falciparum in Vitroa
Figure 1. Important heteronuclear multiple bond correlations (HMBC)
of compound 1.
IC50 (µg/mL)
be between the C-1 of the glucose moiety and the C-7′ of
the galloyl moiety because of the cross-peak observed in
HMBC spectra, and the linkage was determined to be
R-glucose due to the JC-H coupling constant value (J )
175.9 Hz ).18 This was also supported by the coupling
constant between H-1 and H-2 (J ) 3.7 Hz). Because the
cross-peaks of the HMBC spectrum appeared, as shown in
Figure 1, it was determined that compound 1 had a luteoyl
moiety, and the linkage between the glucose and luteoyl
moieties was deduced to be between C-6 of the glucose
moiety and C-7′′ of the luteoyl moiety due to the cross-peak
of the HMBC spectrum. To confirm the luteoyl moiety,
compound 1 was treated with NaOMe in MeOH followed
by treatment with acetic anhydride in pyridine. The usual
workup was performed to give a colorless oil, which was
purified by PTLC to afford 4 and 5. Compound 4 was
determined to be ellagic acid tetraacetate19 by comparing
its spectroscopic data with those of the acetylated deriva-
tive derived from authentic ellagic acid. Compound 5 was
determined to be methyl gallate triacetate by comparing
its spectroscopic data with those reported.20 Therefore,
compound 1 was determined to be 1-O-galloyl-6-O-luteoyl-
R-glucose.
Since the mode of malarial infection and replication is
similar to that of B. gibsoni, we also investigated anti-
plasmodial activities of compounds 1-3, â-sitosterol, and
gallic acid. The results of anti-babesial and anti-plasmodial
testing of 1-3, â-sitosterol, and gallic acid are given in
Table 3. Compound 1 had the strongest activity, with IC50
values of 4.7 µg/mL against B. gibsoni and 1.4 µg/mL
against P. falciparum. Diminazene aceturate (product
name: Ganazeg)5-7 and chloroquine21,22 are effective drugs
for babesia and malaria symptoms, respectively, but there
are side effects. In the case of chloroquine, the most serious
problem is emergence of chloroquine-resistant P. falci-
parum. The isolated compounds were less active relative
compound
Babesia gibsoni Plasmodium falciparum
1
2
3
4.7 ( 0.8
7.5 ( 0.6
35.2 ( 1.9
61.6 ( 2.8
13.4 ( 2.0
0.6 ( 0.04
1.4 ( 0.1
4.6 ( 0.4
11.0 ( 1.3
32.0 ( 3.7
14.8 ( 1.9
â-sitosterol
gallic acid
diminazene aceturate
chloroquine
0.04 ( 0.001
a Data are expressed as means ( standard deviation in triplicate
analyses.
to the standard drugs, diminazene aceturate (IC50: 0.6 µg/
mL) and chloroquine (IC50: 0. 04 µg/mL). The host red blood
cells were not affected by the compounds at the test
concentrations. Parasites of B. gibsoni and P. falciparum
treated with the test compounds demonstrated stagnation
in the ring forms, including size reduction of the nucleus
and disappearance of the parasite cell cytoplasm. On the
other hand, nontreated parasites in the erythrocytes
demonstrated typical petaloid and schizont forms after 3
days of incubation with clear cytoplasm and nuclei in the
parasite cells. These findings may provide the basis for
further understanding of B. gibsoni and P. falciparum
infections and contribute toward the development of new
and effective treatments against these parasites.
Experimental Section
General Experimental Procedures. Melting points were
measured on a Yazawa micro melting point apparatus. IR
spectra were recorded on a Perkin-Elmer 2000 Series FT-IR
spectrometer. FABMS and HRFABMS were obtained on a
JEOL JMS-AX500 mass spectrometer. 1H and 13C NMR
spectra were recorded on a Bruker AM-500 FT-NMR (500
MHz) and a JEOL JNM-EX 270 FT-NMR (67.5 MHz) spec-
trometer, respectively. Optical rotations were determined on
a JASCO DIP-370 digital polarimeter. GC/MS analysis was

Anti-babesial and Anti-plasmodial Compounds
Journal of Natural Products, 2005, Vol. 68, No. 4 539
performed using Shimadzu QP-5000. Column chromatography
was performed on silica gel 60 (Spherical, 70-140 mesh ASTM,
Kanto Chemical), Sephadex LH-20 (Pharmacia), and Diaion
HP-20 (Mitsubishi). Silica gel 60 F254 precoated plates (Merck)
were used for analytical TLC and pTLC. Authentic ellagaic
acid was purchased from Wako Pure Chem. Co. Ltd. (Tokyo,
Japan).
Plant Material. The whole plant of Phyllanthus niruri L.
was collected from Palangkaraya District in Central Kaliman-
tan, Indonesia, in July 2000. The plant was identified by Dr.
Irawati at the Herbarium Bogoriense, Indonesia, and a
voucher specimen (CK.E. 13712) was deposited at the Her-
barium.
The residue from fraction B was subjected to PTLC (CHCl3-
MeOH-H2O, 50:40:10) to give compound 2 (34.5 mg). The
MeOH-H2O (7:3) eluate was evaporated to give a residue (215
mg), which was subjected to column chromatography on a
Sephadex LH-20 column, eluted with CHCl3-MeOH-H2O (50:
40:10) to give three fractions (D-G). Fraction D was purified
by PTLC (CHCl3-MeOH-H2O, 50:40:10) to yield compound
3 (34.2 mg).
GC/MS Experiment. GC/MS analysis of the glycoside part
in compounds was performed according to the procedure of
Hiradate et al.26
Preparation of Ellagic Acid Tetraacetate (4) and
Methyl Gallate Triacetate (5). NaOMe (5.4 mg, 0.1 mmol)
was added to a stirred solution of 1 (10 mg, 0.015 mmol) in
MeOH (2 mL) at room temperature, and the reaction mixture
was stirred for 30 min. Excess Amberlite 120B resins were
added to the reaction mixture. The resins were filtrated off,
and the volatile components of the resulting solution were
removed to give an oil. A solution of acetic anhydride (0.5 mL)
was added to a stirred solution of the oil in pyridine (1 mL),
and the reaction mixture was stirred further for 12 h. The
usual workup was performed to give a residue, which was
purified by PTLC using MeOH-CHCl3 (3:97) as a solvent to
afford 4 (1.2 mg) and 5 (2.0 mg).
Anti-babesial Assay. The in vitro assay against Babesia
gibsoni is given in detail in a previous paper.23 Standard drug
used was diminazene aceturate (Ganazeg).
Anti-plasmodial Assay. Chloroquine-susceptible P. falci-
parum strain FCR-3 was maintained in a culture medium
according to a modified method of Trager and Jensen.24
Parasitized red blood cells (pRBCs) were synchronized with
5% D-sorbitol25 and then were washed three times with RPMI
1640 medium. The pRBCs were resuspended in RPMI 1640
medium supplemented with 10% human serum, 25 mM Hepes,
25 mM gentamicin, and Na2CO3 to achieve 0.25-0.50% para-
sitemia. The assay was performed in a 24-well culture plate
with each well containing 500 µL of synchronous pRBCs
suspension and 20 µL of test compound solution. Two wells
per plate without test compound served as controls to monitor
parasite growth. After 24 h of incubation under a 5% CO2
atmosphere at 37 °C, the control wells were checked. When a
significant percentage (20%) of schizonts appeared, the culture
plate was removed from the incubator. Thin smear specimens
stained with Giemsa solution were made from each well. The
effects of test compounds on parasite growth were expressed
as IC50, which were calculated from a simple graph of the
inhibition of schizonts against the concentration of test
compounds. Chloroquine was used as the standard.
Acknowledgment. The authors are grateful to Mr. Kenji
Watanabe and Dr. Eri Fukushi for measuring the NMR and
mass spectra.
References and Notes
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Isolation of 1, â-Sitosterol, and Gallic Acid. The EtOAc
layer (2.8 g) was chromatographed on a silica gel column,
eluted with CHCl3 (500 mL), MeOH-CHCl3 (3:97, 500 mL),
MeOH-CHCl3 (1:4, 500 mL), and MeOH (500 mL), succes-
sively. The CHCl3 eluate was evaporated to yield a residue
(138 mg), which was subjected to column chromatography on
silica gel, eluted with MeOH-CHCl3 (1:99) to give five frac-
tions (H-L). Fraction J was purified by PTLC (CHCl3-MeOH,
98:2) to yield â-sitosterol (7.6 mg). The MeOH-CHCl3 (1:4)
eluate was evaporated to yield a residue (329 mg), which was
subjected to column chromatography on silica gel, eluted with
MeOH-CHCl3 (1:4) to give five fractions (M-Q). Fraction O
was purified by PTLC using MeOH-CHCl3 (1:49) to yield gallic
acid (23.5 mg). Fraction P was purified by PTLC eluted with
MeOH-CHCl3 (3:7) to yield compound 1 (27.3 mg).
1-O-Galloyl-6-O-luteoyl-r-glucose (1): yellow powder; mp
204-205 °C; [R]D -152.3° (c 0.1, MeOH); IR (KBr) cm-1 3387,
2935, 1719, 1618, 1522, 1448, 1350, 1208, 1032; 1H and 13C
NMR spectral data, see Tables 1 and 2; FABMS m/z 633 [M -
H]+; HRFABMS m/z 633.0707 [M - H]+ (calcd for C27H21O18
requires 633.0726).
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NP0497245