Separation and Purification Technology 52 (2007) 487–496

Extraction of hydrolysable tannins from Phyllanthus niruri Linn.:

Effects of solvents and extraction methods
Masturah Markom a,b,∗ , Masitah Hasan a , Wan Ramli Wan Daud b ,
Harcharan Singh c , Jamaliah Md Jahim b
a Department of Chemical Engineering, University Malaya, 50603 Kuala Lumpur, Malaysia
b Department of Chemical and Process Engineering, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
c Mewah Oils Sdn. Bhd, Lot 40, Section 4, Fasa 2A, Pulau Indah Industrial Park, 42920 Pulau Indah, Klang, Malaysia

Received 30 September 2005; received in revised form 2 June 2006; accepted 2 June 2006

Abstract
Effects of solvent types and extraction methods (solvent extraction (SE), supercritical fluid extraction (SFE) and pressurized water extraction
(PWE)) were investigated for effective recovery of bioactive hydrolysable tannins from Phyllanthus niruri Linn. Various organic and aqueous
solvents screened by Soxhlet method showed that the gallic acid and ellagic acid contents increased with water content whereas corilagin yield
reached a maximum value at 30% (v/v) ethanol in water. At a fixed temperature, solvent extraction by Soxhlet is the best method for gallic and ellagic
acid extractions, whereas pressurized methods are better for the corilagin extraction. Even though exhaustive extraction is achieved fastest by PWE,
SFE with the addition of ethanol–water cosolvent is superior in terms of low liquid solvent consumption and component fractionation produced.
Solvent polarity, solvent-to-solid ratio and contact time play significant roles in determining the most efficient method for tannin extraction.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Phyllanthus niruri; Solvent extraction; Supercritical fluid extraction; Sub-critical water; Ellagitannins

1. Introduction scriptase virus [5–7], enzymes processes peculiar to cancer cell’s

Phyllanthus niruri (Euphorbiacea) is an herbal plant indige-
nous to Malaysia and is locally known as ‘dukung anak’. It is
commonly found in tropical regions and in other countries, it is
known as ‘chanca piedra’ (Spanish), ‘paraparai mi’ (Paraguay),
‘quebra pedra’ (Brazil) or ‘punarnava’ (India). P. niruri is a pop-
ular folk medicine for treating kidney and gallbladder stones,
liver related diseases such as jaundice and liver cancer, viral
infections such as hepatitis and tuberculosis, malaria, diabetes
and fever [1].
In scientific studies, P. niruri was found to exhibit antispas-
modic, hypotensive, analgesic, antihepatotoxic, antihepatitis,
antimutagenic, antiviral and antibacterial properties. The aque-
ous and/or alcohol extracts were found to inhibit activity of
hepatitis B virus in vitro and in vivo [2–4], HIV-1 reverse tran-
∗ Corresponding author at: Department of Chemical and Process Engineering,
Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
Tel.: +60 3 89216114; fax: +60 3 89216148.
E-mail address: masturah@vlsi.eng.ukm.my (M. Markom).
replication and growth [8] and the formation of kidney stones
[9], lower the blood glucose levels [10], have the liver-protecting
(antihepatotoxic) properties in vivo and in vitro [11] and produce
analgesic effects in mice [12]. Other documented properties are
antimalarial [13] and lipid lowering activity [14].
The medicinal effects are attributed to the active components
present in P. niruri such as lignans, glycosides, flavonoids, alka-
loids, ellagitannins, terpenes, and phenylpropanoids [1]. Com-
mon lipids, sterols, and flavonols also occur in the plant. Two
tannin groups identified in P. niruri are hydrolysable tannins
(ellagitannins) and condensed tannins (flavonoids). The final
hydrolysis of ellagitannins yields ellagic acid and gallic acid
[15]. Chemical structures of active hydrolysable tannins, namely
geraniin and corilagin, gallic acid and ellagic acid, and con-
densed tannins such as flavon-3-ol and flavonol in P. niruri are
as shown in Figs. 1 and 2, respectively [5,16,17].
Most research on P. niruri was on the chemical screening,
identification and isolation, and the biological assay and phar-
macological studies [10,18,19]. However, not much study on the
effects of solvents have on the extraction of active components
from P. niruri has been reported. Notka et al. reported the effects

1383-5866/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2006.06.003
488 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496
Fig. 1. Chemical structures and molecular weights of hydrolysable tannins.
of three solvents (water, methanol, 50% ethanol) for the phar-
macological study on anti-HIV [5]. The researchers found that
the 50% ethanol extract was the most active in inhibiting the
replication of the reverse transcriptase virus. The 50% ethanol
extract was assayed to consist of 1.10% geraniin and 2.28%
(w/w) corilagin. De Souza et al. had reported on the quantity of
active gallic acid on the water extract using high performance
liquid chromatography (HPLC) technique, but no study on other
solvent or analysis of other components besides gallic acid were
carried out [20].
Therefore, the study of the solvent effects is very impor-
tant for the screening and solvent selection of the extraction,
fractionation and purification steps in the herbal processing. By
understanding the solvent properties, component (solute) prop-
erties and solvent–solute interaction, rapid fractionation and
isolation of desired components can be achieved. This paper
presents the results on the effects of various organic and aque-
ous solvents with different polarities on the extract yield and
the content of three hydrolysable tannins, namely gallic acid,
ellagic acid and corilagin. Qualitative and quantitative effects of
solvents using different extraction methods were investigated.
The extraction methods utilized are the solvent extraction and
Fig. 2. Basic chemical structures of: (a) flavon-3-ol and (b) flavonol.
the high-pressure extraction (supercritical fluid extraction and
pressurized water extraction).
2. Experimental method
2.1. Chemicals and standards
The reference standards (gallic acid and ellagic acid) were
both purchased from Sigma Chemicals (USA) at purity of 98%.
Isolated geraniin (unknown purity) was supplied by Prof. H.
Wagner (University of Munich, Germany). Analysis of corila-
gin was carried out by Nova Laboratories Sdn. Bhd. (Malaysia)
using their isolated and patented standard (purity of 98%). The
commercial P. niruri product, HEPAR-PTM (standardized to 4%
corilagin and 18% total flavonoid content) was obtained from
the same company.
All chemical reagents for the extraction and component anal-
ysis (n-hexane, petroleum ether, dichloromethane (DCM), chlo-
roform, acetone, methanol, ethanol, acetonitrile and phosphoric
acid) were of analytical grades. Ultra-pure water obtained using
ultra-filtration system (USF ELGA, UK) was utilized as the
extraction solvent, solvent mixtures and HPLC mobile phase.
Pure and industrial grade liquid CO2 (99.8%) was purchased
from Gas Pantai Timur (Malaysia).
2.2. Plant material
Dried and ground P. niruri samples were obtained from Nova
Laboratories Sdn. Bhd. (Malaysia). The sample contains stems
and aerial parts of the plant and has been used for the commercial
production of P. niruri product of HEPAR-PTM . The particle size
distribution (% w/w) determined by sieving was in the range of
45–212 ␮m (8%), 212–600 ␮m (35%), 600 ␮m–1.18 mm (43%)
and 1.18–3.35 mm (14%).
2.3. Solvent extraction
2.3.1. Soxhlet extraction (SE)
Five grams (±0.05) of plant sample was placed in a What-
man 25 mm × 100 mm cellulose thimble. The extraction using
standard Sohxlet method (BÜCHI Laboratechnik, Model B-811,
Switzerland) was carried out using 150 mL of solvent. The heat-
ing power was set to two (2) cycles per hour so that six (6)
cycles of extraction were achieved within 3 h of extraction time.
Various organic (7) and organic-aqueous solvents (6) with dif-
ferent polarities were investigated as listed in Table 1. For a
mixture of organic-aqueous solvent, the percentage indicates
the volume percentage of the organic solvent in the mixture (%
volume/volume or v/v).
The crude extract solutions obtained were concentrated and
dried using vacuum rotary evaporator (BÜCHI Laboratechnik,
Model R-144, Switzerland) at temperature 80 ◦ C or less to
remove the solvents. Higher temperatures were avoided to mini-
mize component degradation. All extracts were placed in a room
temperature condition before weighing gravimetrically to deter-
mine the yields.
 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496 489

Table 1
Solvent effects on the yield of Phyllanthus niruri extraction by Soxhlet method
Extraction solvent Snyder’s solvent Boiling point (◦ C) Extract yield (% g/g sample) Reference
polarity indexa
This work (±S.D.)b Literature

Organic
n-Hexane 0.1 69 1.8 (±0.1) 0.9c [21]
5.5d [22]
Petroleum ether 0.1 60 2.2
Dichloromethane 3.4 40 4.0
Chloroform 4.1 61 9.7
Acetone 5.4 56 3.9
Ethanol 5.2 78 11.6
Methanol 6.6 65 14.6 (±1.1) 17.1c [18]
Aqueous
70% acetone (70:30 v/v acetone–water) 6.5 84 18.5
70% ethanol (70:30 v/v ethanol–water) 8.2 90 20.8 [19]
50% ethanol (50:50 v/v ethanol–water) 7.9 94 22.5 [12]
30% ethanol (30:70 v/v ethanol–water) 7.1 97 26.4 (±1.9) 7–9c
20% ethanol (20:80 v/v ethanol–water) 6.3 98 27.1 21.6
Water 9.0 100 26.2 (±1.6)
Water (sample pre-extracted by n-hexane) 100 23.5
a Snyder’s solvent polarity index cited from [23]. The aqueous solvent mixture indexes were calculated from equation (I /100 × P ) + (I /100 × P ) where I
A A B B A
and IB are polarity index of solvents A and B, respectively, and PA and PB are percentage of solvents A and B, respectively, in the solvent mixture.
b Standard deviation of two or three replicates.
c Extraction was at room temperature.
d Dynamic maceration.

2.4. High-pressure extraction
2.4.1. Supercritical fluid extraction (SFE)
A lab-scale SFE unit was designed and assembled for the
extraction of P. niruri. The apparatus setup is as schematically
shown in Fig. 3. The supply system includes two motor-driven
piston pumps: pump 1 is for CO2 (Jasco, Model PU 1580, Japan)
and pump 2 is for cosolvent (Labtech, Jones Chromatography,
UK). The CO2 was chilled to 2 ◦ C using a chiller to maintain its
liquid state before it was pumped to the extractor. The extractor
consists of a 14 cm length × 1.5 cm internal diameter of high-
pressure stainless steel vessel filled with the plant sample. The
two ends were plugged with glass wool to hold the sample and
to eliminate the vessel’s dead volume. The extraction vessel is

Fig. 3. Schematic diagram of SFE system.

490 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496
enclosed and temperature-controlled in a multi-purpose air cir-
culation oven (Shel Lab, USA). The pumped CO2 and cosolvent
were pre-mixed prior to entering the extraction vessel. The pre-
heater is placed before the extractor to ensure that the solvent
mixture has achieved the desired temperature before coming
into contact with the sample. A 7 ␮m in-line filter was installed
prior to the extraction vessel to prevent sample back-flow. A
back-pressure regulator (Jasco, Model BP-1580-81, Japan) was
employed to maintain the system pressure. It is consisted of an
automated relieve-valve, which was heated to 70 ◦ C to avoid
extract precipitation. Valve V5 reduced the pressure to atmo-
spheric pressure and released the extract to a collector, which
was immersed in a cold trap (2 ◦ C). Valves V1 to V5 controlled
the flow of the supercritical fluid extraction process.
In this study, 5 g (±0.05) P. niruri samples were used. Oper-
ating temperatures of 60 ◦ and 100 ◦ C, and pressure of 200 bar
were investigated with (10%, v/v) and without cosolvent. An
hour static extraction was allowed at the temperature and pres-
sure studied, followed by a dynamic extraction at a solvent flow
rate of 1.5 mL/min for 4 h. The extract fractions were collected
every 30 min and then placed in an air oven (Shel Lab, USA)
at 70 ◦ C for about 15–30 h to remove the remaining cosolvent.
When no cosolvent was present, the extracts were not heated to
avoid evaporation of volatile components. All extracts were then
placed at a room temperature condition before gravimetrically
weighing to determine the extract yields.
2.4.2. Pressurized water extraction (PWE)
Similar experimental setup to SFE system as shown in Fig. 3
was employed for the PWE. In this method, only pump 2 (cosol-
vent) was utilized as a pumping device. Five grams (±0.05) of
plant sample was extracted using water as the solvent. Operat-
ing temperatures of 60 and 100 ◦ C, and pressures of 100 and
150 bar were studied. Before starting the experiment, 30 min
static extraction was allowed at the temperature and pressure
studied, followed by a dynamic extraction until exhaustion at a
water flow rate of 1.5 and 3.0 mL/min. The extract fractions were
collected every 10 min, filtered using a Whatman filter paper (no.
1) and then dried in an air oven (Shel Lab, USA) at 70 ◦ C for
about 15–30 h. The extracts were then cooled down to a room
temperature before determining the yields.
2.5. HPLC analysis
2.5.1. Sample preparation
Reference standard solutions for gallic acid and ellagic acid
were prepared first. Gallic acid in water was prepared with a con-
centration ranging between 0.75 ␮g/mL and 1.2 mg/mL. Ellagic
acid was dissolved in 50% ethanol with concentrations from
2.5 ␮g/mL to 1 mg/mL. HEPAR-PTM and P. niruri extracts were
all dissolved in 50% ethanol at concentrations of 1–5 mg/mL.
Corilagin solution was prepared by the Nova Laboratories Sdn.
Bhd. (Malaysia) in appropriate dilution solvent at 0.5 mg/mL
concentration.
All of these solutions were ultra-sonicated at 60 ◦ C for 30 min
in order to remove air bubbles and to ensure all solids were com-
pletely dissolved before analysis by HPLC. Prior to injection, all
sample solutions were filtered through a 0.45 ␮m PVDF syringe
filter.
2.5.2. Detection and identification
Component analysis was carried out using high performance
liquid chromatography (HPLC) technique equipped with an
auto sampler and a UV/vis detector (Agilent Technologies, Ger-
many). The column used for the analysis was a reverse-phase
C18 Genesis with 250 mm × 4.6 mm i.d. and 4 ␮m particle
diameter (Jones Chromatography, UK). The chromatographic
separation was developed using a mobile phase of 0.1% phos-
phoric acid in water (solvent A) and acetonitrile (solvent B) with
a gradient of solvent B: 8–22% (35 min), 22–8% (10 min) at flow
rate of 1 mL/min. The injection volume was set at 20 ␮L and the
detection was in UV absorbance at 270 nm.
Chromatographic peaks were identified through comparison
with retention times of ellagic acid, gallic acid and corilagin
standards. Due to the unavailability of corilagin standard, few
samples were initially analyzed by Nova Laboratories Sdn. Bhd.
(Malaysia) using their purified corilagin standard (98%) and the
peak area was identified and calibrated to the one obtained by our
HPLC method. For the other reference standard solutions, they
were injected (in triplicate) at different concentrations and linear
regression analysis on the data of peak area versus concentration
was carried out. Linear calibrations with accuracy of more than
99.5% were obtained for all standards.
Single injection of solvent (blank) was made to determine
the solvent retention time. The contents of gallic acid, ellagic
acid and corilagin in the extracts were calculated based on the
corresponding peak areas and injected concentrations.
3. Results and discussion
3.1. Solvent effects on extract yield
The effects of organic and aqueous solvents on the extraction
yield and content of bioactive gallic acid, ellagic acid and cori-
lagin in P. niruri were studied using standard Soxhlet extraction
method. The amount of extract after removal of solvent is listed
in Table 1.
The results indicate that the P. niruri is most soluble in
polar solvents namely water (26.2%) and aqueous ethanol
(20.8–27.1%). This shows that most components in P. niruri
are hydrophilic or water-soluble. The color of the extracts were
dark brownish in water and brownish-green in aqueous ethanol
or aqueous acetone. Low extract yields were obtained in the
non-polar solvent such as n-hexane (1.8%) and petroleum ether
(2.2%), and the color was observed to be yellowish with a tinge of
green. The extraction yields of other solvents were in between
(4.0–14.6%) and the extracts were observed to be green (dif-
ferent intensity for different solvent extracts). The green color
might have been caused by the presence of chlorophylls.
It is shown that solvent polarity plays an important role. The
net molecular polarities of solvents are measured by their dipole
moments. The polarities and boiling points of solvents used are
listed in Table 1 and the dipole moments for some of the sol-
vents are listed in Table 2. Other properties of solvents (except
 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496
Table 2
Properties of solvents [24]
Solvent name Dipole moment Dielectric constant
(Debye)
Acetone 2.88 20.49
Chloroform 1.04 4.71
DCM 1.60 8.93
Ethanol 1.69 24.85
n-Hexane 0.00 1.88
Methanol 1.70 32.61
Water 1.87 78.36
a Is equal to square of Hildebrandt–Scott solubility parameter.
for petroleum ether) used in this study, such as the dielectric
constant, cohesive energy, viscosity and surface tension are also
given. With the exception of acetone, the extract yield increases
with the solvent polarity. The addition of water in acetone and
ethanol tremendously increases the extract yield. It is also found
that the highest yield can be achieved at 20% ethanol. It is signif-
icantly higher than by using pure ethanol and is slightly higher
than by using water since both the polar and less polar com-
pounds were co-extracted together. Extraction of P. niruri by
using two immiscible solvents, non-polar n-hexane (1.8% yield)
followed by very polar water (23.6% yield), indicates that each
solvent extracted different groups of compounds. The total yield
of the two-step process is very close to the yield using only water
(26.2%) in a single-step process. Therefore, it can be concluded
that water extracts both non-polar and polar components at its
boiling point. Table 1 also shows that the yields obtained for
other solvents are not far different from the yields obtained by
other researchers, provided that the extraction conditions are
relatively similar.
The separation of component by a solvent depends on the
polarity of both solvent and component [21]. According to Bar-
wick, a single solvent might or might not be selective for the
separation of two components as shown in Fig. 4. For exam-
ple, if the desired compound x is non-polar, it can be selectively
extracted using solvent with polarity P 1. On the other hand,
if the more polar compound y is desired, P x solvent can be
Fig. 4. Separation of compound x and y as a function of solvent polarity.
P x = polarity of compound x, P y = polarity of compound y [21].
491
Cohesive energy Viscosity (mPa) Surface tension
densitya (J mol/mL) (cal/mol A2 )
362.07 0.31 33.77
332.00 0.54 38.39
400.22 0.41 39.15
618.87 1.07 31.62
200.76 0.30 25.75
808.26 0.54 31.77
2095.93 0.89 104.70
used to remove compound x first, followed by solvent P 2 to
extract compound y. However, for plant materials that consist
of multi-components with complex interactions, 100% recov-
ery of individual component is probably not achieved since a
single solvent may not be selective for a single compound.
It should be noted that Soxhlet extraction only works at the
boiling points of the solvents. At boiling point of the solvent,
its surface tension and viscosity are greatly reduced compared
to at a lower temperature, therefore the solvent can reach the
active sites inside the matrix far more easily. The tempera-
ture effects of solvent had been studied by Xu and Godber
for the extraction of ␥-oryzanol from rice bran using hexane,
ethyl acetate, isopropanol and different solvent combinations
[22]. They discovered that temperature was significant for the
solvent that modified the matrix but not for the solvent govern-
ing the extraction of solute. Therefore, provided that the basis
of solvent-to-solid ratio and contact time (residence time) are
the same, solvent comparison and effects on the extraction of
hydrolysable tannins using Soxhlet can be attributed more sig-
nificantly to the solvent polarity than to the temperature.
3.2. Solvent effects on component content
The presence of four major component peaks was consis-
tently detected in the HPLC chromatograms of HEPAR-PTM
and some of the P. niruri extracts. The chromatogram of stan-
dardized HEPAR-PTM extract is shown in Fig. 5. Gallic acid (1),
corilagin (3) and ellagic acid (4) were identified in the sample
by comparison with the external standards. Gallic acid, corilagin
and ellagic acid have a retention time of 5.4, 18.9 and 31.2 min,
respectively. By comparison to the standard chemical fingerprint
of HEPAR-PTM [25], the chromatogram obtained in this study
shows similar chemical profile, with the highest peak identified
as corilagin. Component A, however, was not identified in this
study.
Even though geraniin has been identified as the most active
hydrolysable tannin in P. niruri, the presence of geraniin could
not be verified in this study based on the HPLC method devel-
oped. It is possible that the HPLC method used is not suitable
for the geraniin detection or that the geraniin obtained might
have been hydrolyzed and thus, might result in lower purity and
produce several artifacts. Hydrolysis and alcoholysis to simpler
components such as corilagin, brevifolin carboxylic acid, gal-
lic acid and ellagic acid might have occurred in the presence of
492 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496

Fig. 5. HPLC chromatogram of HEPAR-PTM . The column used was a reverse-phase C18 Genesis 250 mm × 4.6 mm i.d., 4 ␮m particle diameter. A mobile phase of
0.1% phosphoric acid in water (solvent A) and acetonitrile (solvent B) with a gradient of solvent B: 8–22% (35 min), 22–8% (10 min) at flow rate of 1 mL/min. The
injection volume was set at 20 ␮L and the detection was in UV absorbance at 270 nm. Components detected are gallic acid (1), component A (2), corilagin (3) and
ellagic acid (4).

water and alcohols at high temperatures [15]. The presence of
geraniin was previously confirmed by the NMR method [18,19]
after multi-steps fractionation and isolation processes.
The concentrations or contents (% w/w of extract) of ellagic
acid, gallic acid and corilagin in the extracts using Soxhlet
extraction method are shown in Fig. 6. No tannins were detected
in n-hexane and petroleum ether extracts, and trace amounts
were detected in chloroform and DCM extracts. In the herbal
extraction, n-hexane and petroleum ether were used mainly
for removing non-polar lipids and unwanted glycosides [26,27]
while chloroform and DCM were mostly used for extraction or
isolation of low polarity lignans and glycosides [28].
Similar to the extract yield, the highest overall tannin contents
were found in water and aqueous ethanol extracts. Increasing the
water content in ethanol resulted in higher contents of ellagic and
gallic acid as seen in Fig. 6. The highest yields for both compo-
nents were obtained using pure water in the Soxhlet extraction.
On the other hand, corilagin content was found highest in 30%
(v/v) ethanol in water. Higher content of water did not further
increase the total extract yield but a further increase in the gallic
and ellagic acid contents and reduction in the corilagin content
were observed. The Soxhlet results also showed that the com-
position of HEPAR-PTM components was close to that of 70%
ethanol extract.
From Table 2, it is also interesting to note that the tannin
components could only be extracted by a solvent with a dielec-
tric constant value of more than 20 (acetone, ethanol, methanol
and water). Solvent with a lower dielectric constant could not
extract the tannins. For example, even though dichloromethane
and chloroform have almost the same cohesive energies and
resulted in similar or higher extract yields to that of acetone, the
tannin contents obtained are far less or undetectable (Fig. 6).
On the other hand, water has much higher viscosity and sur-
face tension, which are usually not desired in extraction since

Fig. 6. Component contents in Phyllanthus niruri extracted by different solvents in comparison with the standardized HEPAR-PTM extract.
 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496
they hinder solvent absorption into the active sites inside the
matrix. However, the dielectric constant and cohesive energy are
significantly higher and therefore, water molecules are strongly
bonded to the polar tannin components. Increasing the solvent
temperature to boiling point could also reduce the viscosity and
surface tension and further increases the extraction yield.
In order for a component to dissolve in solvent, the solubil-
ity parameters of both substances must be similar. Solubility
parameter of a solute can be determined from the original sol-
ubility equation developed by Hildebrandt and Scott [29]. The
solubility of solute in a solvent is not only dependent upon the
polarity (dipole moment) but also on the hydrogen bonding,
electrostatic bonding and conformation (dihedral angle). The
preferential of solvents to donate/accept electron (nucleophilic)
and to donate/accept H+ (electrophilic) with the components is
also crucial. All of these factors are important for explaining the
solvent–solute and solvent–solvent interactions. For example,
Saidman and co-workers proposed that flavone interacts with
the solvent ethanol through the interaction of carbonyl group
of the drug with two solvent molecules [30]. It was determined
that the carbonyl oxygen atom (ketone group) in the flavone
contains higher electronic density compared to the intracyclic
oxygen atom (ether group). The interaction between the solute
and solvent also resulted in the overall increase of dipole moment
for the solution.
Properties of the tannins however, are not extensively stud-
ied and so far, no published thermodynamic and physical data
are available for geraniin or corilagin. Provided that the boiling
point, the molecular weight and the specific gravity are avail-
able, the critical properties (i.e. critical temperature, pressure
and volume) and the acentric factor can be estimated by corre-
lations proposed by Watanasiri et al. [31]. These properties are
required to calculate the dipole moments of components.
Table 3
Number of functional groups in hydrolysable tannins in decreasing polarity
Functional group Chemical structurea
Carboxylic acid
Hydroxyl
Ketone
Ester
Ether
a Adopted from Ref. [33].
493
The solubility of solute in the solvent can be roughly esti-
mated by the functional groups present in the chemical structure
of the tannin component. Table 3 gives the type and number
of functional groups in decreasing order of polarity. Gallic acid
contains carboxylic acid group and is most soluble in polar sol-
vent such as water. Other tannin components do not have this
functional group and therefore, the polarity is based on the next
polar group (hydroxyl). If other less polar functional groups
are not taken into account, geraniin is the next polar compound
followed by corilagin and ellagic acid. However, the number
and proportion of these groups compared to the more polar
functional groups might play significant roles in increasing or
decreasing the solubility in a solvent. Therefore, gallic acid is
probably the most polar followed by the ellagic acid, corilagin
and finally geraniin.
In this study, we can deduce that components containing
carboxylic acid and hydroxyl groups are preferably extracted
by water through the hydrogen bonding. The carboxylic acid,
ketone and ester groups have the nucleophilic ability (electron
donor) in the carbonyl group and can react with both water and
alcohol. Ether is the least polar and requires less polar solvent.
However, since the tannin compounds have more than one func-
tional group, each compound is selectively soluble in different
water to ethanol ratio. For example, water is preferable for the
extraction of phenolic acids but the addition of ethanol (30%,
v/v) is preferred for the extraction of corilagin.
The results of this study also confirm the work by others
that in the herbal extraction, water and aqueous solvents were
preferably used for the extraction of tannins and polyphenols
[27,32]. The extraction yield, however, is not only influenced by
the solvent type (chemical characteristics) but also by the sol-
vating strength, solvent-to-solid ratio and contact time (physical
characteristics) at different extraction conditions.
Number of functional group
Gallic acid Ellagic acid Corilagin Geraniin
1 0 0 0
3 4 11 13
0 0 0 1
0 2 3 5
0 0 1 2
494 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496

3.3. Comparison of extraction methods

Ellagic acid
A study of different extraction methods is especially impor-

Component content (% g/g extract)

17.48
12.97
10.82
5.74
6.48
6.90
5.52
5.94
6.75
7.31
5.10
6.22
8.91
4.17
tant for the determination of efficient extraction of solute from
the complex matrices such as plants. The comparison was

Corilagin
made between the solvent extraction by Soxhlet (SE), and

2.64 (4)
high-pressure extraction methods by pressurized water extrac-

2.96
3.71
3.52
3.19
2.42
2.23
3.45
2.82
3.67
4.09
2.45
2.35
4.11
tion (PWE) and supercritical fluid extraction (SFE). Water was
employed as a cosolvent in SFE but it acted as the main solvent

Gallic acid
in other extraction methods. Similarly, 30%-, 50%- and 70%-
ethanol were also utilized as cosolvents in SFE and as main

1.15
0.89
0.56
0.18
0.48
0.41
0.38
0.45
0.34
0.84
0.27
0.33
0.65
0.21
solvents in SE.
The extractions were carried out for 3 h in solvent extraction

Total yield (%
and until exhaustion for the high-pressure extractions. Many

g/g sample)
variables involved in each method, therefore, direct compar-
ison among the methods cannot be made. In this study, the

26.2
26.4
22.5
20.8
17.8
22.5
20.6
8.5
17.5
26.6
19.7
23.2
27.0
–
comparison is made based on the solvent-to-solid ratio, con-
tact time (residence time) and extraction time for comparable

Total residence
extraction conditions, yields and component contents as shown
in Table 4. For SFE and PWE, the residence times for both static

–: information is not available. Number in bracket is the standardized corilagin content reported by Nova Laboratories Sdn. Bhd. (undated).
time (h)
and dynamic extractions were accounted for in the 25 mL ves-

3.0
3.0
3.0
3.0
3.8
3.8
3.8
3.8
3.8
3.8
0.6
0.6
0.8
sel. Semi-batch extractions are assumed for all methods. The

–
residence time was determined to be 0.5 h per cycle or a total of
3 h for Sohxlet, 3.8 h for SFE and 0.6–0.8 h for PWE. In SFE,
Liquid solvent-to-solid

the non-tannins were initially fractionated in the first 0.5–3 h

ratio (×103 m3 /kg)

followed by a tannin extraction in another 1–3.5 h, depending

upon the extraction conditions employed. Solvent-to-solid ratio
(m3 /kg) is calculated based on the amount of solvent/cosolvent
that is in contact with the plant sample.
7.2
7.2
7.2
7.2
7.2
7.2
30
30
30
30

36
36
18
–
At a fixed temperature of 100 ◦ C, PWE at pressure of 100 bar
is better compared to SE using water in terms of less contact
Total solvent/cosolvent

time and total extraction time for a similar or less solvent-to-solid

a Liquid solvent was used as cosolvent mixed with supercritical carbon dioxide at 10% (v/v).
volume (×106 m3 )

ratio. In PWE, the reduction in flow rate (from 3.0 to 1.5 mL/min)
caused the contact time to increase and therefore, increased the
extraction efficiency. Due to a lower water flow rate, equilib-
rium and mass transfer is better achieved at a longer residence
150
150
150
150
36
36
36
36
36
36
180
180
90
–
time. Therefore, even though the extract yield is approximately
equal, the corilagin content (4.11%) in PWE is 39% higher than
Extraction

in the SE (2.96%). It is also possible that the polarity of water

time (h)

is reduced at the sub-critical condition, therefore similar sol-

3.0
3.0
3.0
3.0
4.0
4.0
4.0
4.0
4.0
4.0
1.0
1.0
1.0

ubility parameter to that of corilagin could be achieved. The

–

sub-critical water had also been found to increase the solubility

Comparison of different solvents and extraction methods
Solvent/cosolvent

of relatively polar solutes in the extraction of natural products

such as catechin and epicatechin [34]. In PWE, different solvat-
30% ethanol
50% ethanol
70% ethanol

30% ethanol
50% ethanol
70% ethanol
50% ethanol
50% ethanol

ing strengths can be achieved by changing water properties from

Water

Water

Water
Water
Water

normal to sub-critical to supercritical water [35]. In SE, higher

type

corilagin content could only be achieved by adding ethanol into

–

water.
P (bar)

Gallic acid and ellagic acid contents were also found higher
1
1
1
1
200
200
200
200
100
200
150
100
100
–

in SE compared to PWE. Higher degree of hydrolysis process

might have occurred, possibly due to the direct contact of the
T (◦ C)

extract collector (solvent reservoir) with the hot plate during the
100

100
100
b.p.
b.p.
b.p.
b.p.
60
60
60
60
60

60

extraction. On the other hand, the extract was collected at room

temperature in PWE process. Thus, even though the hydrolysis
HEPAR-PTM

might have occurred in the extractor, most of the energy was

Method
Table 4

used to free the solutes from the plant matrix. Re-extraction

PWE
SFEa
SE

of the used plant sample in PWE by Soxhlet did not extract any
 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496
more ellagic or gallic acid, thus confirming that the high contents
of phenolic acids in the SE were resulted from the hydrolysis
process.
Even though high temperature is preferred to break the
solute–matrix interaction bonds and to increase the solute
volatility, it may also cause thermal degradation for compo-
nents that are sensitive to heat and should be avoided if pos-
sible. Therefore, high-pressure extractions at lower temperature
(60 ◦ C) were also investigated (Table 4). SFE at 200 bar and
PWE at 150 bar are compared. Higher pressure than 150 bar
in PWE results in no further increase in yield and component
contents. Almost similar results are obtained for both methods
indicating that water cosolvent at 10% (v/v) in SFE, which has
a lower liquid solvent-to solid ratio of 0.0072 m3 /kg compared
to 0.036 m3 /kg in PWE, could give the same yield as the PWE.
However, PWE is four times faster than SFE due to larger enter-
ing water volume and shorter residence time. Similarly, very
short extraction times (5–20 min) were required for the pressur-
ized liquid extraction of other medicinal herbs [36].
As mentioned earlier, the addition of ethanol in water could
increase the corilagin content in SE method. Therefore, 30%-,
50%- and 70%-ethanol as cosolvents (10%, v/v) in SFE were
investigated as well at both 60 ◦ and 100 ◦ C. The extract yield
was found to increase with a reduced concentration of ethanol in
the cosolvent and was highest at 30% ethanol. In contrast to 30%
ethanol in SE, in SFE the corilagin content was found highest
using 50% ethanol cosolvent.
At 60 ◦ C, in order to get the same amount of extract obtained
using water cosolvent, the addition of 50% (v/v) ethanol in water
could reduce the operating pressure required from 200 bar to
100 bar. Furthermore, the extract yield (20.6%) and corilagin
content (3.45%) of SFE with 50% ethanol at pressure of 200 bar
and at a lower temperature (60 ◦ C) is comparable to SE using
50% ethanol (22.5%, 3.52%) at the boiling point of the solvent.
Therefore, the operating temperature could be reduced in the
SFE.
At 200 bar and 10% (v/v) of 50% ethanol cosolvent, increas-
ing to higher temperature (100 ◦ C) led to an increase in the
extract yield (26.6%), which is similar to the yield of SE using
water (26.2%). At this temperature, all component contents are
higher than the SFE at 60 ◦ C. Even though similar temperature
to SE was achieved, similar or higher phenolic acid contents than
the ones in SE could not be achieved due to the same reasons
as mentioned earlier for PWE. Higher corilagin content (4.09%)
than SE, however could be obtained, which is comparable to that
of PWE. Table 4 shows that the residence time in SFE (3.8 h)
is slightly higher compared to SE (3 h) but the solvent-to-solid
ratio required is far less in SFE.
Another advantage of SFE with water and ethanol–water
cosolvents investigated is that the extraction of less polar com-
ponents (volatiles, lipids, flavonoids and chlorophylls) could
be enhanced and fractionated prior to the hydrolysable tannins
compared to SFE without cosolvent or SFE with only organic
cosolvents such as methanol or ethanol (Fig. 7). The increased
yield might have been caused by the modification of the plant
matrix and the enhanced desorption of the less polar compounds
with the presence of water. Due to a low solubility of water in
495
Fig. 7. Cosolvent type effect in SFE at 200 bar and 60 ◦ C. The dotted lines
indicate the fractionation of hydrolysable tannins.
CO2 , two phases might co-exist together and result in a fractiona-
tion, with the vapor phase governed mainly by CO2 that extracted
the less polar compounds and the liquid phase governed mainly
by water, which extracted the hydrolysable tannins.
It can be concluded that SFE with ethanol–water cosolvent
has the most efficient liquid solvent utilization for the extraction
of hydrolysable tannins. However, if time is the controlling factor
and not the solvent volume, PWE is recommended due to shorter
extraction and residence times.
4. Conclusions
The screening by various extraction solvents showed that
most components in P. niruri are hydrophilic or water-
soluble. The active hydrolysable tannins (gallic acid, ellagic
acid and corilagin) were preferably extracted using water or
ethanol–water mixtures. Due to the nature of water at sub-critical
conditions, it was found that pressurized water extraction (PWE)
had the highest overall extract and corilagin yield in the shortest
extraction time compared to Soxhlet or SFE method. However,
SFE with water and ethanol–water cosolvents gave an interesting
result since fractionation between the less polar compounds and
the hydrolysable tannins was possible. SFE was also superior in
terms of minimum liquid solvent consumption. Therefore, opti-
mization of operating parameters in both PWE and SFE with
the selected cosolvent mixture should be further studied for the
most efficient extraction of P. niruri with reduced processing
steps.
Acknowledgements
The research was funded by Malaysian Ministry of Sci-
ence, Technology and Innovation (MOSTI) under Intensified
Research in Priority Area (IRPA) grant no. 09-02-02-0091-
EA234 and by University Malaya under Vote F (0168/2003A).
The main author also thank Mr. N.L. Phang (Nova Laborato-
ries Sdn. Bhd., Malaysia) and Prof. H. Wagner (University of
496 M. Markom et al. / Separation and Purification Technology 52 (2007) 487–496
Munich, Germany) for providing free HEPAR-PTM capsules and
geraniin standard, respectively.
References
[1] Database for Chanca Piedra (Phyllanthus niruri). Available online from
http://www.rain-tree.com/chanca.htm (accessed on August 2003).
[2] S.P. Thyagarajan, S. Subramanian, T. Thirunalasundari, P.S.
Venkateswaran, B.S. Blumberg, Lancet 2 (1988) 764.
[3] P.S. Venkateswaran, I. Millman, B.S. Blumberg, Proc. Natl. Acad. Sci.
U.S.A. 84 (1987) 274.
[4] J. Liu, H. Lin, H. McIntosh, Viral Hepat. 8 (2001) 358.
[5] F. Notka, G.R. Meier, R. Wagner, Antiviral Res. 1795 (2002) 1.
[6] T. Ogata, AIDS Res. Hum. Retroviruses 8 (1993) 1937.
[7] J. Qian-Cutrone, J. Nat. Prod. 59 (1996) 196.
[8] N.V. Rajeshkumar, J. Ethnopharmacol. 81 (2002) 17.
[9] A.H. Campos, N. Schor, Nephron 81 (1999) 393.
[10] I.B. Jaganath, N.L. Teik, Herbs: The Green Pharmacy of Malaysia, MARDI,
Serdang, Malaysia, 2000, pp. 78–80.
[11] K.V. Syamsundar, B. Singh, R.S. Thakur, A. Hussain, Y. Kiso, H. Hikino,
J. Ethnopharmocol. 14 (1985) 41.
[12] A.R.S. Santos, V.C. Filho, R.A. Yunes, J.B. Calixto, Gen. Pharmacol. 26
(1995) 1499.
[13] L. Tona, N.P. Ngimbi, M. Tsakal, K. Mesia, K. Cimanga, S. Apers, T. De
Bruyne, L. Pieters, J. Totté, A.J. Vlietinck, J. Ethnopharmocol. 68 (1999)
193.
[14] A.K. Khanna, F. Rizvi, R. Chander, J. Ethnopharmacol. 82 (2002) 19.
[15] P. Luger, M. Weber, S. Kashino, Y. Amakura, T. Yoshida, T. Okuda, G.
Beurskens, Z. Dauter, Acta Cryst. B 54 (1998) 687.
[16] M. Naczk, F. Shahidi, J. Chromatogr. A 1054 (2004) 95.
[17] X. He, J. Chromatogr. A 880 (2000) 203.
[18] K. Ishimaru, K. Yoshimatsu, T. Yamakawa, H. Kamada, K. Shimomura,
Phytochemistry 31 (1992) 2015.
[19] H. Ueno, S. Horie, Y. Nishi, H. Shogawa, M. Kawasaki, S. Suzuki, T.
Hayashi, M. Arisawa, M. Shimizu, M. Yoshizaki, N. Morita, J. Nat. Prod.
51 (1988) 357.
[20] T.P. De Souza, M.H. Holzschuh, M.I. Lionco, G.G. Ortega, P.R. Perovick,
J. Pharm. Biomed. Anal. 30 (2002) 351.
[21] J. Barwick, Trends Analyt. Chem. 16 (1997) 293.
[22] Z. Xu, J.S. Godber, J. Am. Oil Soc. 77 (2000) 547.
[23] Properties of Solvents in Various Sorbents. Available online from
http://home.planet.nl/∼skok/techniques/hplc (accessed on February 17
2004).
[24] C. Gu, H. Li, R.B. Gandi, K. Raghavan, Int. J. Pharm. 283 (2004)
117.
[25] Nova Laboratories Sdn. Bhd., Sepang, Malaysia, undated.
[26] H.H. Ang, Y. Hototsuyanagi, H. Fukaya, K. Takeya, Phytochemistry 59
(2002) 833.
[27] Y.H. Choi, J. Kim, K. Yoo, Chromatographia 56 (2002) 753.
[28] C. Chang, Y. Lien, C.S. Karin, C. Liu, S. Lee, Phytochemistry 63 (2003)
825.
[29] J.H. Hildebrandt, R.L. Scott, The Solubility of Nanelectrolytes, Reinhold,
New York, 1950.
[30] E. Saidman, A. Yurquina, R. Rudyk, M.A.A. Molina, F.H. Ferretti, J. Mol.
Struct. (Theochem.) 585 (2002) 1.
[31] S. Watanasiri, V.H. Owens, K.E. Starling, Ind. Eng. Chem. Proc. Des. Dev.
24 (1985) 294.
[32] C. Yang, Y. Xu, W. Yao, J. Agric. Food Chem. 50 (2002) 846.
[33] J. McMurray, Organic Chemistry, Brooks/Cole, United States, 2004.
[34] Z. Piñeiro, M. Palma, C.G. Barroso, J. Chromatogr. A 1026 (2004) 19.
[35] S.B. Hawthorne, C.B. Grabanski, E. Martin, D.J. Miller, J. Chromatogr. A
892 (2000) 421.
[36] B. Benthin, H. Danz, M. Hamburger, J. Chromatogr. A 837 (1999) 211.