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Journal of Medicinal Plants Research Vol. 5(13), pp. 2729-2735, 4 July, 2011
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Isolation and characterization of antimicrobial,
anti-inflammatory and chemopreventive flavones from
Premna odorata Blanco
Lunesa C. Pinzon1*, Mylene M. Uy1, Kung Hong Sze2, Mingfu Wang3 and Ivan Keung Chu2
1Department of Chemistry, College of Science and Mathematics, MSU-Iligan Institute of Technology, Iligan City,
Philippines.
2Department of Chemistry, Faculty of Science, University of Hong Kong, Hong Kong SAR.
3School of Biological Sciences, Faculty of Science, University of Hong Kong, Hong Kong SAR.
Accepted 5 April, 2011
Premna odorata Blanco (Verbenaceae) is a native tree of the Philippines where its leaves are used
traditionally for vaginal irrigation and tuberculosis. It is one of the seven components of a
commercialized Philippine herbal preparation called “Pito-Pito”. Its medicinal uses, however, have not
been scientifically validated. This tree is not commonly cultivated and thrive in the less accessible
limestone forests of the Philippines. Solvent partitioning and fractionation of the ethanolic crude extract
of the leaves isolated two yellow amorphous powders. The identities of these compounds were
determined by LC/MS/MS and NMR spectroscopic analyses, and their spectra were compared with
literature data. The isolates were flavone aglycones which were the widespread acacetin and the non-
widespread diosmetin. These flavones were isolated from the P. odorata for the first time ever. They
had been reported by earlier studies to exhibit medicinal properties as antimicrobial, anti-inflammatory
and chemopreventive. Thus, the current study has provided a scientific evidence of the medicinal
properties of the leaves of P. odorata that could become the popular basis for the plant’s sustainable
use, conservation and cultivation.
Key words: Premna odorata Blanco (Verbenaceae), “pito-pito”, “alagau”, antimicrobial, anti-inflammatory,
chemopreventive, flavones, diosmetin, acacetin.
INTRODUCTION
Premna odorata Blanco (Verbenaceae) is a native of
temperate and tropical Asia which includes the
Philippines. It is also known by a few other scientific
names such as, P. curranii H. Lam., P. oblongata Miq.
var. puberula H. Lam., P. pubescens Blume. var. odorata
H. Lam., P. serratifolia Blanco and P. vestita Schauer. It
has many Philippine names, but is more popularly known
as “alagau” and “agbau”. In the Philippines, the decoction
of the leaves is used for vaginal irrigation and
*Corresponding author. E-mail: lunesa.pinzon@g.msuiit.edu.ph
Tel:/Fax: (63)-(63)-221-4068.
tuberculosis (Quisumbing, 1978). It is one of the seven
components of a commercialized Philippine herbal
preparation called “Pito-Pito”. The ethnomedicinal uses of
P. odorata, however, have not been scientifically
validated. Apart from its uncommonly known
ethnomedicinal uses, this tree does not have much
economic value and is generally cut and replaced with
plants that are perceived to be more profitable. For this
reason, even though it is a native plant, it is not generally
cultivated and not commonly found in populated areas,
but thrive in the less accessible secondary limestone
forests of the Philippines. The isolation of bioactive and
medicinal compounds from the leaves of P. odorata
would provide a scientific evidence of the medicinal
2730 J. Med. Plant. Res.
properties of the plant. The validated medicinal
properties, in turn, could become the popular motivation
for the plant’s sustainable use, conservation and
cultivation. The current study was able to isolate two
flavone aglycones, diosmetin and acacetin, from the
crude EtOH extract of the leaves.
Diosmetin or 5,7,3’-trihydroxy-4’-methoxyflavone was
classified as a non-widespread flavone (Valant-Vetschera
and Wollenweber, 2006). Many earlier studies have
reported its bioactivities. It inhibited 3H-dopamine uptake
in control and differentiated neuroblastoma cells and in
small-cell lung carcinoma cells. It also inhibited 3H-
serotonin uptake in both cell types. These inhibitory
effects could be responsible for the increased vascular
tone observed in vivo after treatment with diosmetin and
its glycoside, diosmin, as vasotonic agents (Sher et al.,
1992). As an antimicrobial agent, diosmetin exhibited the
MIC at 25 µg/mL against B. subtilis and the MIC at 50
µg/mL against the fungus Trichophyton rubrum, the most
common cause of athlete’s foot (Meng et al., 2000). It
down-regulated the enzyme cyclooxygenase-2 and so,
could be an anti-inflammatory agent (Lopez-Posadas et
al., 2008). It increased osteoblast differentiation and so,
could be a potential agent for treating osteoporosis (Hsu
and Kuo, 2008). At 60 µM, it exhibited 55% inhibition of
the enzyme α-phosphatidylinositol-3-kinase (Agullo et
al., 1997). At 6 µM, it exhibited 70% inhibition of the
enzyme 17-β hydroxysteroid dehydrogenase type 1. As
a chemopreventive compound, diosmetin directly
inhibited the activity of the enzyme cytochrome P450 1A1
(Ciolino et al., 1998). It blocked apoptosis that was
induced by dimethylbenz(a)anthracene (Ciolino et al.,
2002). It exerted cytostatic effects on cell cycle
progression and proliferation of breast cancer cells
(Androutsopoulos et al., 2009).
Acacetin or 5,7-dihydroxy-4’-methoxy-flavone was
classified as a widespread flavone (Valant-Vetschera and
Wollenweber, 2006). Several bioactivites of acacetin
have been reported by earlier studies. As an antifungal
agent, acacetin had a comparable potency with the
antifungal drug Amphotericin B against Candida glabrata
KCTC 7219, Candida tropicalis KCTC 7725 and C.
tropicalis KCTC 7212 (Rahman and Monn, 2007). It was
reported to be a promising agent for the treatment of
atrial fibrillation (Li et al., 2008).
As a chemopreventive agent, acacetin had
antiproliferative effect on human liver cancer cell line
HepG2 (Hsu et al., 2004a) and in human nonsmall cell
lung cancer A549 cells (Hsu et al., 2004b). It was capable
of preventing inflammation-associated tumorigenesis
(Pan et al., 2006). It induced apoptosis of human breast
cancer MCF-7 (Shim et al., 2007). It inhibited the invasion
and migration of human prostate cancer DU145 (Shen et
al., 2009). It inhibited TPA-induced MMP-2 and u-PA
expressions of human lung cancer cells (Fong et al.,
2010).
MATERIALS AND METHODS
Plant material
Green, healthy and mature leaves of P. odorata were collected
during the pre-flowering phase of a single tree in Luinab, Iligan City,
Philippines; with geographical c oordinates and elevation as: 8°
14’
29.73” N; 124° 16’ 03.87” E; 36 m elevation. The identification of
the plant was done by Prof. Carmelita Garcia-Hansel, a botanist
from the Mindanao State University, Marawi City, Philippines. A
voucher specimen (Voucher No. 1051) is deposited at the Natural
Science Museum of MSU-Iligan Institute of Technology, Iligan City,
Philippines. Figure 1 shows the leaves of P. odorata at its pre-
flowering phase.
Chemicals
Except for the technical grade 95% EtOH, the various brands of
solvents used were of analytical or HPLC grade.
Extraction and isolation
Air-dried leaves, weighing 5 k, were soaked in absolute ethanol for
48 h. The crude ethanolic extract was filtered, c oncentrated with a
rotary evaporator at temperatures below 55°C, suspended in water,
and solvent-partitioned sequentially using Hex, DCM and EtOAc.
Isolation of the two flavones was done by two rounds of gravity
open-column chromatography using Amberlite XAD16 and silica gel
(200 to 300 mesh). W ith Amberlite XAD16, chromatography was by
gradient elution using mixtures of 95% EtOH and water; starting
with EtOH:H2O (10:90), increasing EtOH by 10%, and ending with
95% EtOH. W hen silica gel was used, gradient elution was done
with Hex, DCM, EtOAc and their mixtures; starting with Hex-DCM
(50:1). Thin layer chromatography (TLC) was done to monitor the
fractionation. The developed chromatograms were visualized with
5% sulfuric acid in ethanol, with which the isolated flavonoids were
yellow.
Purification of the Isolates
Chromatographic fractions that showed yellow spots on their TLC
plates were pooled together, evaporated dry and resuspended in
MeOH to yield an orange colloidal mixture. The resulting colloidal
mixture was suction filtered, using MeOH to wash down the non-
targeted plant c omponents, thus leaving behind the purified pale
yellow isolate. The purification process was repeated until the
resulting colloidal mixture was no longer orange, but dark brown.
The collected filtrate was evaporated f or subsequent
chromatography. The purified isolate was also collected, dried in a
vaccuum dessicator, weighed and spectroscopically analyzed.
HPLC
HPLC was used to assess the purity of isolates. The analytical
HPLC system used consisted of Shimadzu LC-20AT series
pumping system, SIL-20A automatic injector, SPD-M20A UV visible
detector set at 285 nm and Class-Vp chromatography data station
software with the analytical column, Ultimate XB-C18 column (250
× 4.6 mm, 5 µm) which was purchased from Welch Materials, Inc.
of Shanghai, People’s Republic of China. The analyses were
carried out on an Ultimate XB-C18 column ( 250 × 4.6 mm, 5 µm).
The mobile phase was 0.2% formic acid (solvent A) and acetonitrile
Pinzon et al. 2731
Figure 1. Leaves of P. odorata.
(solvent B). The gradient elution was as follows: 20% B for 0 to 10
min, 20 to 40% B for 10 to 20 min, 40 to 60% B for 20 to 30 min, 60
to 80% B for 30 to 40 min, 80 to 95% B. The post-running time was
10 min. Flow-rate was set at 1.0 mL/min. The UV-Vis detector was
set at 254 nm. The column was maintained at room temperature.
LC/MS/MS analysis
1100 LC system with one well-plate auto sampler. Samples were
separated on a 150 x 4.60 mm, 5 micron, Jupiter C18 column
(Phenomenex). The mobile phase consisted of 0.5% formic acid in
water (A) and 0.5% formic acid in acetonitrile (B). Separations were
effected by a gradient using a flow rate of 0.2 mL/min as follows:
Starting with 5% B, 0 to 5 min; 5 to 50% B, 5 to 15 min; 50 to 80%
B, 15 to 25 min; 80% B, 25 to 35 min.
Reversed-phase liquid chromatography was performed on agilent
column eluent was directed into a QTrap hybrid triple-quadrupole
mass spectrometer equipped with a turbo ionspray source (QTrap
2000, Applied Biosyst ems/MDS Sciex). Samples were run in
positive ion mode using optimized parameters as follows: ion spray
voltage is 5 kV, source temperature was 450°C, nebulizer gas was
25 (arbitrary units), curtain gas was 10 (arbitrary units), collision gas
was 35 (arbitrary units). CID spectr a were acquired using
information-dependent acquisition (IDA); a full scan over a mass
range of m/z 100 to 1000 as a survey scan, followed by two MS/MS
scans of the most abundant peaks over a mass range of m/z 50 to
1000. Data were acquired using the Analyst 1.4.1® software.
NMR Spectroscopy
The purified isolates were dissolved in DMSO-d6. The 1H ( 500
MHz), 13C (125 MHz), DEPT-135, and 2 D-NMR spectra (1H-1H
COSY, 1H-1H TOCSY, 1H-1H NOESY and HETCOR) were
acquired on a Bruker DRX500 NMR spectrometer equipped with a
BBIz 5mm probe. The 2D-HMBC spectra were acquired on a
Bruker AVANCE 600 instrument with cyroprobe. TOCSY spectra
were recorded with spin-locking times of 70 ms. The NOESY
spectra were recorded using mixing times of 300 m. A total of 2048
complex data points with 512 complex increments were collected
for each 2D-NMR experiment. The spectra were processed within
the Xwin-NMR software, where HETCOR and HMBC raw data were
zero filled to 4 K x 2 K prior to Fourier transformation. Spectra were
plotted with the Xwin-plot software.
Fluorescence
The purified isolates were dissolved in DMSO and analyzed for
2732 J. Med. Plant. Res.
Table 1. Various spectroscopic data on the isolates.
Spectroscopic analyses Isolate ID1 Isolate ID2
ESI MS
(Positive ion mode); (M+H), Most abundant peak at m/z 301.242. Most abundant peak at m/z 285.0697.
Fluorescence (in DMSO): 544.0 nm (excitation) 404.0 nm (excitation)
574.0 nm (relaxation) 468.0 nm (relaxation)
1H-nmr
(in DMSO-d6, 500 MHz)
δ 12.935 (1 H, s), 9.563 (1 H, broad s), 7.559
(1 H, d, J = 8.0 Hz), 7.441 (1 H, s), 7.110 (1
H, d, J = 8.5 Hz),), 6.762 (1 H, s), 6.498 (1 H,
s), 6.224 (1 H, s), 3.884 (3 H, s)
δ12.91 (1 H, s), 8.03 (2 H, d, J = 2.1 Hz), 7.00
(2 H, d, J = 2.1 Hz), 6.85 (1 H, s), 6.49 (1 H, d,
J = 1 Hz), 6.19 (1 H, d, J = 1 Hz), 3.71 (3 H, s)
13C-nmr
(in DMSO-d6, 125 MHz)
δ 181.777, 164.301, 163.639, 161.520,
157.417, 123.067, 118.812, 112.99, 112.266,
103.821, 103.580, 98.981, 94.028, 55.852.
δ 181.717, 164.470, 163.242, 162.282,
161.430, 157.352, 128.290, 122.845, 114.567,
103.647, 103.516, 98.942, 94.048, 55.538.
DEPT-135
(in CDCl3, 500 MHz)
(+ ; for CH- and C-types)
δ118.564, 112.729, 112.005, 103.324,
98.729, 93.783, 55.599.
(+ ; for CH- and C-types)
δ128.033, 114.308, 103.260, 98.654, 93.774,
55.280.
fluorescence with J asco FP-777 Spectrofluorometer.
RESULTS
Solvent partitioning of EtOH crude extract of the leaves (5
k) resulted in four fractions weighing 73 g of the Hex
semicrude extract, 92 g of the DCM semicrude extract,
24 g of the EtOAc semicrude extract, and 125 g of the
aqueous semicrude extract. On the first round of
adsorption chromatography of the DCM semicrude
extract with Amberlite XAD16, more retained fractions
showed a yellow spot on their TLC plates. These
fractions were pooled together and through the
subsequent purification process, a total of 500 mg of a
yellow powder was collected. This powder was
designated as isolate ID1 and was later spectroscopically
identified as the flavone aglycone diosmetin. Table 1
shows various spectroscopic data on isolate ID1. The Rf
of isolate ID1 with EtOAc was 0.75. The collected filtrate
resulting from the purification of isolate ID1 was dried and
chromatographed for the second time by normal-phase
partition chromatography. The more retained fractions
showed a yellow spot on their TLC plates. These
fractions were pooled together and through the
subsequent purification process, a total of 50 mg of a
yellow powder was collected. The collected yellow
powder weighed 50 mg and was designated as isolate
ID2. It was later spectroscopically identified as the
flavone aglycone acacetin.
(Table 1) shows various spectroscopic data on the
isolates. The Rf value of isolate ID2 with EtOAc was 0.86.
DISCUSSION
The ESI mass spectra (positive ion mode) of isolate ID1
showed that the most abundant peak was at m/z 301.35
(M+H), so the molecular ion must be 300 and was
calculated for the molecular formula of C16H12O6. Two
likely flavonoidal structures were initially suggested; a
flavone and a flavonol. The 1H-nmr spectra of ID1 was
not much of a help to distinguish between a flavone or a
flavonol. Its 13C-nmr spectra however, clearly
distinguished between the two types of flavonoids. The
13C-nmr spectra of ID1 showed no peaks around δ136 to
139.0 ppm that might correspond to C-3 in flavonols, nor
peaks at δ172 to 177 for C-4. On the other hand,
characteristic peaks of flavones for the C-3 at δ103.0 to
111.8 and C-4 at δ177.3 to 184.0 (Bohm, 1998) were
shown on the spectra of isolate ID1. Literature search
showed that the NMR spectra of isolate ID1 were
basically the same as that of diosmetin. Only one peak
(Table 2) was observed for the two hydroxyl protons at C-
5 and C-7. This was because, in general, when a
compound contained several hydroxyl protons, only one
signal at the average position was observed due to rapid
exchange. However, a separate peak for the hydroxyl
proton at C-3’ was detected. This could be because the
C-3’ hydroxyl proton was far enough from those at C-5
and C-7 and, as in most cases with DMSO, the exchange
could be slow (Pretsch et al., 2000). Table 3 shows the
carbon-proton correlations in the isolate ID1 based on its
HMBC spectra and Figure 2 shows the long-range
couplings of C-4 and C-4’ with various protons. The ESI
mass spectra (positive ion mode) of isolate ID2 showed
Pinzon et al. 2733
Table 2. Carbon and proton chemical shifts of isolate ID1 (Diosmetin) in DMSO-d6.
Carbon shifts; 125 MHz
(δ
δ δ
δ ppm)
Carbon
position
Proton shifts; 500 MHz
( δ
δδ
δ
ppm)
Number of
proton (s)
Proton
position
181.777 C-4 *12.935; s 1 H 5-OH
164.301 C-2 1 H 7-OH
163.639 C-7 9.563; br s 1 H 3’-OH
161.520 C-5 7.559; d, (J = 8 Hz) 1 H H-6’
157.417 C-9 7.441; br s 1 H H-2’
151.257 C-4’ 7.110; d, (J = 8.54 Hz) 1 H H-5’
146.869 C-3’ 6.762; s 1 H H-3
123.067 C-1’ 6.498; s 1 H H-8
118.812 C-6’ 6.224; s 1 H H-6
112.992 C-2’ 3.884; s 3 H 4’-OCH3
112.266 C-5’
103.821 C-10
*only one peak at the average position was observed for the two hydroxyl protons at C-5 and C-7 due to rapid exchange.
Table 3. The c arbons and protons of the isolate ID1 (DIOSMETIN) with long-
range couplings or HMBC correlations.
Carbon position HMBC correlations (600 MHz)
C-4 OH-5 ; H-3 ; H-8 ; H-6
C-5 OH-5 ; H-6
C-9 H-8
C-4’ H-6’ ; H-2’ ; H-5’
C-3’ H-6’ ; H-2’ ; H-5’
C-1’ H-2’ ; H-5’ ; H-3
C-6’ H-2’
C-2’ H-6’
C-5’ H-6’
C-10 OH-5 ; H-8 ; H-6
C-3 OH-5 ; H-3
C-6 OH-5 ; H-8 ; H-6
C-8 H-8 ; H-6
that the most abundant peak was at m/z 285.07 (M+H),
so the molecular ion must be 284 and was calculated for
the molecular formula of C16H12O5. The 13C-nmr spectra
also showed the characteristic carbon chemical shifts for
C-3 and C-4 of a flavone (Bohm, 1998). Library search
showed that isolate ID2 had basically the same spectra
with acacetin. Only one peak (Table 4) was observed for
the two hydroxyl protons at C-5 and C-7 due to rapid
exchange. The structure of diosmetin (Figure 3) contains
one more hydroxyl group than that of acacetin. This extra
hydroxyl group at C-3’ position makes diosmetin more
polar than acacetin. The greater polarity of diosmetin
over acacetin is manifested by its lower Rf value with
EtOAc of 0.75, while that of acacetin is 0.86.
The scientifically validated therapeutic properties of
both Diosmetin and Acacetin, as reported by other earlier
studies, are not directly related to the traditional use of
the leaves of P. odorata as a vaginal wash and for the
treatment of tuberculosis. The medicinal compounds that
might be directly related to the traditional use of P.
odorata could be present in the other semicrude extracts
and in the other fractions from the DCM semicrude
extract. Finding Diosmetin and Acacetin in the leaves
makes it reasonable to report that P. odorata is indeed a
medicinal plant. Diosmetin, in particular, has been
commercially available for years as the glycoside
diosmin. It is indicated as a vasotonic agent for the
treatment of varicose veins, hemmorrhoids and other
venous diseases. It is marketed under several
brandnames. Veno-active drugs (VAD) based on
diosmetin were a subject of an international consensus
statement among medical specialists on hemorheology
and microcirculation. The final resolution was that the
VAD are safe and effective and may be applied in chronic
2734 J. Med. Plant. Res.
Figure 2. Long-range couplings of C-4 and C-4’ with various protons based
on the HMBC spectra of the isolate ID1 (Diosmetin).
Table 4. Carbon and proton chemical shifts of isolate ID2 (Acacetin) in DMSO-d6.
Carbon shifts; 125
MHz (δ
δδ
δ
ppm)
Number of
carbon(s)
Carbon
position
Proton shifts;
500 MHz (δ
δ δ
δ ppm)
Number of
proton(s)
Proton
position
181.717 1C C-4 *12.91; s 1 H 5-OH
164.470 1C C-7 1 H 7-OH
163.242 1C C-2 8.03; d,(J = 2.1 Hz) 2 H H-2’,6’
162.282 1C C-4’ 7.11; d,(J = 2.1 Hz) 2 H H-3’,5’
161.430 1C C-5 6.85; s 1 H H-3
157.352 1C C-9 6.49; d,(J = 1 Hz) 1 H H-8
128.290 2C C-2’, C-6’ 6.19; d, (J = 1 Hz) 1 H H-6
122.845 1C C-1’ 3.71; s 3 H 4’-OCH3
114.567 2C C-3’, C-5’
103.647 1C C-10
103.516 1C C-3
98.942 1C C-6
94.048 1C C-8
55.538 1C 4’-OCH3
*only one peak at the average position was observed for the two hydroxyl protons at C-5 and C-7 due to rapid exchange.
venous disease, or chronic venous insufficiency, when
symptomatic (Ramelet et al., 2005). Diosmetin, being not
widespread in nature and commercially available as a
nutraceutical, renders the plant too valuable to ignore
medicinally and agriculturally. The plant itself should be
extensively promoted as a medicinal plant, so that it
would be sustainably used, conserved and cultivated.
CONCLUSION AND RECOMMENDATIONS
The presence of Diosmetin and Acacetin, which are
antimicrobial, anti-inflammatory and chemopreventive, in
the leaves supports the use of P. odorata as a medicinal
plant. Diosmetin, Acacetin and the unidentified
compounds in the other fractions could be investigated
for significant bioactivities, particularly in relation to the
plant’s ethnomedicinal use. Moreover, an efficient and
healthy method of extracting and purifying Diosmetin or
Diosmin from the leaves of P. odorata could be
developed.
ACKNOWLEDGEMENTS
We thank Carmelita Garcia-Hansel for the identification of
Pinzon et al. 2735
Figure 3. Structures of diosmetin and acacetin.
the sample; Zhongping Zheng for overseeing the isolation
and running the HPLC; Yun Sang Tang for running the
NMR; Pak-Wing Kong and Chun-Ming Ng for running the
LC/MS/MS; Steven Siu, Wei-Ting Chu; Gene Gagabe
and Evelyn Creencia for facilitating access to library
materials; Jeannette Kunz and Alexes Daquinag for
helping in the enzyme-inhibitory tests; the Philippine
Department of Agriculture-Bureau of Agricultural
Research for the research grant. L.C. Pinzon gratefully
acknowledges the study-leave grant from MSU-IIT and
the scholarship grant from the Commission on Higher
Education.
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