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Development of adsorptive (non-ionic) macroporous resins and their uses in

the purification of pharmacologically-active natural products from plant
sources
Jing Li* and Howard A. Chase
Received 7th July 2010
DOI: 10.1039/c0np00015a

Covering: up to the end of 2009

The development and characteristics of adsorptive (non-ionic) macroporous resins are described
together with an overview and evaluation of recently reported applications of these macroporous resins
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in the purification of various pharmacologically-active natural products from plant sources. The
versatility of adsorption using macroporous resins is demonstrated by the existence of numerous
successful separation processes for many different categories of compounds, including polyphenols,
glycosides and carotenoids. Adsorption by macroporous resins is considered to be superior to
conventional liquid–liquid or solid–liquid extraction due to their inherent characteristics such as
convenience, low operational cost, lower solvent consumption, and the absence of chemical residues in
the product. In order to improve the resolution of the separations, efforts have been made either to
combine the use of adsorptive macroporous resins with subsequent high performance column
chromatography, e.g. preparative high performance liquid chromatography (HPLC) and high speed
countercurrent chromatography (HSCCC), or to modify the surface chemistry and pore structure of
resins.

1 Introduction as a result of solvent wastage or high energy consumption.4,5

2 Adsorptive macroporous resins Therefore, in recent years, it has become important to develop
3 Applications of adsorptive macroporous resins a simple but efficient (as well as environmentally-friendly) tech-
3.1 Flavonoids/polyphenols nique to extract and purify pharmacologically-active natural
3.2 Glycosides products. Adsorption and desorption onto non-ionic macro-
3.3 Saponins porous resins has proved to be an efficient technique in this field
3.4 Taxol/taxoids due to its advantages, such as high adsorption capacity, low
3.5 Carotenoids operational expense and easy regeneration of the adsorbent.6–9
3.6 Serotonins Therefore, there have been increasing numbers of reports on the
3.7 Fatty alcohols application of macroporous resins to purify various pharmaco-
4 Approaches to improve the separation performance logically-active natural products, e.g. flavonoids, glycosides and
4.1 Description of strategies adopted carotenoids, from plant sources.
4.2 Combination with chromatography Following a brief description of their origin and historical
4.3 Modifications of resin chemistry development, this article addresses recently reported applications
5 Summary of adsorptive macroporous resins. In addition, examples of
6 References attempts to improve the resolution of separations, either
combined with high-resolution column chromatography or by
modification of the surface chemistry of the macroporous resins,
are presented.
1 Introduction
Pharmacologically-active natural products have gained unprec- 2 Adsorptive macroporous resins
edented popularity in recent decades.1–3 They have made great
contributions historically to drug development, and many of The term ‘‘adsorptive macroporous resins’’ in this review is used
them have had profound effects on our lives. However, recovery to describe the highly cross-linked, non-ionic (non-functional-
of natural products from plant sources by conventional solvent ized) resins that are characterized by their large number of
extraction has many pitfalls that may increase the operating cost permanent pores which are considered to be ‘‘spacious’’ (i.e. with
pore diameters of >50 A) 10 and are accessible to large molecules.
Their origin can be traced back to the earlier condensate polymers
Department of Chemical Engineering and Biotechnology, University of
Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK. E-mail: made by the copolymerization of formaldehyde with phenolic
jl426@cam.ac.uk; Fax: +44 (0)1223 331761; Tel: +44 (0)1223 334781 and aromatic amine derivatives in 1930s by B. A. Adams and E.

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L. Holmes.11 In subsequent years, investigations of the synthesis
and production of novel synthetic resins became a burgeoning
industry, and a new era of ion-exchange resins arrived.
Although these earlier condensate ion-exchange resins imme-
diately found application for the refining of sugar12 and partial
deionization of water, they were soon recognized to lack both
a sufficiently porous structure and a stable skeletal structure for
operation in rigorous environments. The follow-up work of G. F.
Mills and his colleagues gave rise to the macroporous phenolic
ion-exchange resins,13,14 which are considered to be the true
antecedents of present-day adsorptive macroporous resins. These
resins played an important role in the deionization of corn starch
hydrolysates for glucose and fructose manufacture, or were used
as adsorbents to upgrade both the colour and flavour of wines.15
At that time, another term – ‘‘macrorecticular resins’’16 – was
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introduced to describe the condensate ion-exchange resins made
under Adam’s patent or similar work. It was not until 1950s,
however, that these widely-used ‘‘macrorecticular resins’’ were
suspected to be macroporous, a suspicion was finally confirmed
following the development of appropriate analytical instruments
almost 20 years later.17 Although some modification work was
later performed on the structure of phenol–formaldehyde ion-
exchange resins, better polymer structures, known as gel-type
resins, have been obtained by using cross-linked polystyrenes.18,19
The copolymerization of styrene (S) with a small amount of
divinylbenzene (DVB) was first reported by Staudinger and
Huseman20 to yield gel resins that can swell but not dissolve in
solvents. Before long, various styrene–divinylbenzene (SDVB)
copolymers found applications as ion exchange resins due to the
fact that the degree of particle swelling can be adjusted according
to the content of DVB in the copolymerization.
In the 1960s, scientists began to realize that some copolymers
could be effectively used as adsorptive resins even in the absence
of functionalization,21 and resins of this type began to appear on
the market shortly after. At that time, these non-ionic adsorptive
resins were used to recover non-polar or less polar organic
Jing Li was born in 1982 and
studied bioengineering in Dalian
University of Technology, where
he received his Bachelor degree
in 2004. He became a Ph.D.
candidate in Cambridge
University under the supervision
of Professor Howard Chase in
2006, and was a recipient of an
EPSRC/Shell-sponsored Doro-
thy Hodgkin Postgraduate
Award for his Ph.D. duration.
His research interests focus on
Jing Li the various technologies for
separating biologically active
natural products from different
sources and their applications, especially the chemistry, biotech-
nology and applications of flavonoid-related natural products. In
2009, he was awarded the ‘‘Chinese Government Award for
Outstanding Students Abroad’’ by the China Scholarship Council.
1494 | Nat. Prod. Rep., 2010, 27, 1493–1510
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compounds from aqueous solutions. A general method for the
preparation of non-ionic macoporous resins was described in
patents by Davankov and Tsyurupa.22,23 Nowadays, these non-
ionic macroporous resins are usually manufactured from SDVB
or acrylic-based polymers in the presence of porogens, which are
usually miscible in the monomer mixture but are a poor solvent
for the copolymer – these include compounds such as toluene,
n-heptane, iso-octane and isobutanol. During the polymerization
process, the porogens penetrate into the interior space of
monomer mixture and are eventually removed by volatilisation
upon completion of the polymerization process, giving rise to
discrete macropores throughout the bead. The porogens can
perform as either a solvating agent or a non-solvating agent, or
even be incompatible with the resultant polymer network.24 The
resins are characteristically opaque in appearance, in contrast to
their clear gel-resin counterparts, due to fact that they may
contain up to 20% (w/w) DVB in their structure, and have
degrees of cross-linking of greater than 10%. The high content of
DVB results in a relatively rigid and stable structure that can
tolerate rigorous conditions such as high osmotic pressure,
oxidation and mechanical stress, although this structure also
results in decreased particle porosity.
It is well understood that the key technology during the
manufacture of such ‘‘macroporous’’ structures is the phase-
separation step. The phase-separation process, which can take
place either on the macroscale (macrosyneresis, deswelling), or
on a microscale (microsyneresis), is considered to be necessary
for the formation of a macroporous structure during the
copolymerization, in order to generate additional crosslinks to
stabilise the two-phase structure.24
Internal surface area, pore diameter and surface polarity are
the three key parameters that characterise an adsorptive
macroporous resin. The internal surface area for a dried resin
usually ranges from 100 to 1000 m2/g, with pore diameters
ranging from 100 to 300 A.  The polarity of the resins may vary
with the choice of monomer used in its synthesis or by additional
Howard Chase studied the
Natural Sciences Tripos at the
University of Cambridge,
specializing in Biochemistry,
and graduated with a B.A.
(Hons) degree in 1975. He
subsequently continued study at
Cambridge for the PhD degree,
researching into bacterial
microbiology. After a further
period of post-graduate
research, he transferred to the
department of Chemical Engi-
Howard Chase neering, where he initiated study
into the recovery of biological
products, and joined the
academic staff. He is currently a Chartered Engineer, Chartered
Chemist and Chartered Scientist, and is a Fellow of the Royal
Academy of Engineering. He is the Head of the School of Tech-
nology in the University of Cambridge.
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chemical treatment following polymerization. The adsorption
behaviour of a macroporous resin is dependent on its geometric
structure and can be controlled by varying the chemical
composition of the polymerization mixture during synthesis,
resulting in different surface chemistries on the adsorbent. The
history of their development and detail of almost every funda-
mental aspect of macroporous resins can be found in related
reviews.24–26
The rapid development of adsorptive macroporous resins in
the last two decades has led to an expansion of the uses for this
type of separation agent. Their major applications have been in
the refining of sugars, adsorption of gases, treatment of waste-
water, separation and enrichment of pharmacologically-active
natural products and the purification of other bioproducts.
Reviews of the use of these resins for the adsorption of gases,27
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and cancer.30 Inga edulis is a tree native to Central and South
America and is especially widely grown by indigenous Amazo-
nians. Its leaves are reported to possess beneficial effects such as
anti-inflammation activity.31 ()-Epicatechin (1, (2R,3R)-cate-
chin) and myricitrin (2, myricetin-3-O-a-L-rhamnopyranoside)
are two main polyphenolic components that may be linked to
this medicinal property.32 Silva et al. optimized the packed-bed
adsorption conditions for four macroporous adsorbents (XAD-
7, XAD-16, EXA-90, EXA-118) by varying variables.33 The
influences of three independent variables, proportion of water in
the hydroalcoholic extract solution, its pH value and the type of
resin employed were studied. The results indicated that two
conditions, the proportion of water in the hydroalcoholic extract
solution and the type of resin employed, strongly affected the
efficiency of adsorption; however, the pH value had no influence.

removal of organic compounds and metal ions from water28 can Amongst all the resins tested, XAD-7 gave the highest adsorp-
be found in earlier publications. Research into the pharmaco- tion capacity at 239 mg per g of resin, and XAD-16 gave the
logical properties of natural products and strategies for their lowest at 16 mg per g of resin.
isolation and separation has intensified in recent years. Although
there are many different, well-documented technologies for
accomplishing such separations, adsorption processes using
macroporous resins have attracted increased attention because of
their unique adsorption properties and other advantages. The
latter include low operational cost, lower solvent consumption,
low amounts of unwanted chemical residues in the product,
together with the commercial availability of resins possessing
a variety of pore structures and internal surface areas. In spite of
many recent studies on the potential of macroporous resins in
this field, so far no comparative overview of the frequently-used
commercial resins, particularly in terms of their adsorption
performance, has been forthcoming. This review describes the Although it is well understood that the composition of the
adsorptive macroporous resins that feature in publications that extract solution and the type of resin will affect adsorption, it is
have appeared in the last decade on the purification of natural debatable as to what influence pH has on the adsorption of
products, and compares and evaluates their overall performance. polyphenols by macroporous resins. Conflicting conclusions on
this issue arose from early publications. Scordino et al. reported
that the adsorption of chrysanthemin (3, cyanidin 3-glucoside),
3 Applications of adsorptive macroporous resins a flavonoid that may have an antioxidative property that reduces
Adsorptive macroporous resins have been found to be useful for the risk of coronary heart disease,34 is not affected by pH vari-
the purification of numerous constituents of different classes of ations (from 1.0 to 4.5).35 However, most related references hold
pharmacologically-active natural products. Examples are pre- the opposite opinion. Huang et al. reported a maximum
sented below: adsorption capacity for phenols onto PVPTA (poly(p-vinyl-
phenyltrimethylamine)) and OPVPTA (oxidized poly(p-vinyl-
phenyltrimethylamine)) resins, two synthetic polymeric
3.1 Flavonoids/polyphenols adsorbents based on the structure of macroporous crosslinked
Polyphenols are a group of compounds that are usually found in chloromethylated poly(styrene-co-divinylbenzene) beads, in
plants, e.g. fruits, vegetables, berries, olives, walnuts, grapes and aqueous solution at pH 6.0,36 with a subsequent decrease of
tea leaves, and characterized by containing more than one phenol adsorption capacity as the pH was increased.
unit in each molecule. Flavonoids comprise the most abundant
group of the plant polyphenols; so far, several thousand flavo-
noids have been identified in plants. Flavonoids are said to have
various functions ‘‘in protecting against UV light (UV-B
screening pigments), in warding off pathogenic microorganisms
(phytoalexins) or pests (antifeedants), in the fertility and germi-
nation of pollen, in activating bacterial nodulation genes
(nitrogen fixation) and in regulating plant growth and enzyme
activity’’.29 Recent research suggests that many flavonoids have
potential antioxidant characteristics that may protect cells and
bodily chemicals against damage caused by free radicals and
reactive atoms, so as to reduce the risk of cardiovascular disease

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A similar phenomenon was reported by a Chinese research adsorption capacity went through a maximum as the pH was
group when they were separating flavonoids and polyphenols increased. Contrary to this result, Xu et al. observed that the
from the extracts of pigeon pea leaves and roots.37 Pigeon pea adsorption capacity for flavonoids increased gradually with
(Cajanus cajan) is one of the most popular and nutritionally- increasing pH (from 2 to 6.5) without passing through
valuable legumes grown worldwide, and contains high levels of a maximum value. It was deduced that dissociated H+ ions may
protein and the amino acids methionine, lysine and tryptophan. compete with nitrogen atoms in the flavonoid structure for the
In recent years, it was found that extracts of pigeon pea leaves active binding sites on the adsorbent, thus reducing the
exhibit notable antibiotic and anti-inflammatory effects.38 With formation of hydrogen bonds between the solute and adsor-
increasing understanding of the beneficial effects of flavonoids bent.44
on human health, the medicinal applications of pigeon pea leaf
extracts have been attributed to their significantly higher
luteolin content than in other plants. Luteolin (4, 2-(3,4-
dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) is one of the
most common flavones that are thought to play an important
role in the human body by acting as free-radical scavengers.39
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Eight macroporous resins (AB-8, NKA-9, NKA-2, D3520,

D101, H1020, H103 and AL-2) were employed by Fu et al. to
measure the static adsorption and desorption properties of
luteolin from the extracts of pigeon pea leaves,37 with AL-2
shown to produce the best performance. The eluent from the
column contained an almost 20-fold higher luteolin concen-
tration, compared to the feedstock, at 2.55% (w/w), and
a recovery yield of 78.5% was achieved. The highest adsorption
capacity for luteolin was reached at the pH 5 for each of the
three resins studied. In subsequent work, the same research
group employed eight resins (ADS-5, ADS-7, ADS-8, ADS-11,
ADS-17, ADS-21, ADS-31 and ADS-F8) to enrich vitexin and
isovitexin40 from pigeon pea leaves, and the static adsorption
results indicated that ADS-5 was the most appropriate resin for
this application. Vitexin (5, apigenin-8-C-glucoside) and
isovitexin (6, apigenin-6-C-glucoside) are a pair of isomeric
flavonoids present in pigeon pea leaves that have various
pharmacological properties, such as free-radical scavenging41
and antimicrobial activities.42 Following packed-bed adsorption
employing ADS-5 resin, the contents of vitexin and isovitexin
in the eluates were increased from 0.86 and 1.53% to 3.50 and
17.63%, respectively, which represent 4.1-fold and 11.5-fold
increases of their initial concentrations in the feedstock. The Canarium album is a fruit tree grown in tropical and
recovery yield for each component was 65.0% for vitexin and subtropical areas and is a member of the family of Burser-
74.0% for isovitexin. The adsorption capacities for vitexin and aceae. Recent studies have shown that the dried fruit of
isovitexin were found to reach their maximum values at pH 3.5 Canarium album is rich in phenolic compounds such as gallic
and 4, respectively. More recent work by Liu et al. reported the acid (9, 3,4,5-trihydroxybenzoic acid), ellagic acid (10, 2,3,7,8-
highest adsorption capacity for genistein (7, 40 ,5,7-trihydroxy- tetrahydroxychromeno[5,4,3-cde]chromene-5,10-dione) and
isoflavone) and apigenin (8, 40 ,5,7-trihydroxyflavone) at pH their derivatives (corilagin (11, 1-O-galloyl-3,6-O-hexahydroxy-
values of 4 and 4.5, respectively when they tried to separate diphenoyl-b-D-glucose), hyperin (12, quercetin-3-galactoside)
these two phenolic compounds from the extracts of pigeon pea and kaempferol 3-glucopyranoside) – compounds that have
roots.43 The pH of the solution was the parameter that most been shown to have medicinal functions such as anti-bacterial,
influenced the adsorption capacity due to its influence on the anti-inflammatory and detoxifying actions.45 He et al. inves-
adsorption mechanisms. The change of pH can affect the tigated the use of various macroporous resins (S-8, NKA-II,
nature of the interaction between flavonoids and the active AB-8, NKA and X-5) to separate and purify the phenolic
binding sites of the macroporous resins. To explain why the compounds present in crude extracts prepared by ethanol
adsorption capacities showed a maximum value, the researchers extraction.46 The researchers found AB-8 had a comparatively
concluded that the phenolic hydroxyl groups in the flavonoids high adsorption capacity and desorption capacity, and used it
dissociate to form the corresponding anions at a high pH, and for a dynamic packed-bed adsorption test. The operational
therefore the adsorption mechanism of polyphenols onto conditions were optimized and the resulting products were
macroporous resins is assumed to be associated with the obtained at a purity of 85% with a recovery yield of 75%. In
formation of hydrogen bonding.43 Though this assumption is this work, the authors considered the polarity of the resin to
reasonable when explaining the reduced adsorption capacity at be an important factor that influenced the adsorption
higher solution pH, it is difficult to understand why the capacity.

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Lonicera japonica is a species of honeysuckle native to eastern
Asia and is a significant source of food for deer, rabbits and some
other wildlife. Chlorogenic acid (13, 3-(3,4-dihydroxy-
cinnamoyl)quinic acid), a dominant polyphenolic compound
found in honeysuckle, has been confirmed to have the ability to
suppress significantly the N-nitrosating reaction, and, as a result,
plays an important role by acting as an antioxidant, antitumor
and anti-carcinogenic agent.47 Zhang et al. developed an efficient
preparative separation method for the enrichment of chlorogenic
acid from crude honeysuckle extracts using adsorption onto
macroporous resins.48 Nine macroporous resins (HPD-300,
HPD-450, HPD-500, HPD-700, HPD-750, HPD-850, X-5, AB-8
and NKA-II) were compared and evaluated. HPD-850 was
selected for further packed-bed trials, as it demonstrated the best
adsorption capacity and desorption ratio among the resins
tested. After packed-bed adsorption performed under optimized
operational conditions, the chlorogenic acid content in the eluent
obtained was increased from 11.2 to 50.0%, with a recovery yield
of 87.9%. However, the researchers observed that the solution
pH had no obvious impact on the adsorption capacity when the
pH was lower than 3. Subsequently, the adsorption capacity
decreased greatly with an increase of pH value. This is due to the
enhanced hydrogen bonding between chlorogenic acid and the
adsorbent. Furthermore, chlorogenic acid is found to be more
stable in water when the pH is around 3.48
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Licorice (Glycyrrhiza uralensis) is a flowering plant and is one
of the most important medicinal herbs in traditional Chinese
medicine. Two important groups of compounds, glycyrrhizic
acid (14) and licorice flavonoids, were shown to possess various
therapeutic effects such as antiviral, anti-inflammatory and
possibly anticancer activities.49 An earlier publication on the
separation of active ingredients from licorice by macroporous
resins by Fu et al.50 employed four common macroporous resins
(XDA-1, LSA-10, D101 and LSA-20) and investigated the
adsorption performances for the two groups of compounds. The
work suggested that XDA-1 showed the best adsorption char-
acteristics for both glycyrrhizic acid and licorice flavonoids. In
addition, XDA-1 demonstrated a higher affinity for glycyrrhizic
acid than licorice flavonoids. A one-step packed-bed adsorption
using XDA-1 resin was then used to separate the two compo-
nents from crude licorice extracts. The two ingredients could be
eluted from the bed separately, with the enriched licorice flavo-
noids (21.9% purity) obtained at a 74.8% recovery yield and
enriched glycyrrhizic acid (65.6% purity) at 52.0% recovery yield.
The authors attributed the very high adsorption capacity of
XDA-1 to its ‘‘higher surface area, optimum average pore
diameter and appropriate surface functional polarity’’. However,
this conclusion is not fully proven since no formal relationship
between these factors and the observed adsorption capacity of
the resins was revealed, although it would be natural to correlate
these particular properties with the overall adsorption perfor-
mance. In addition, the adsorption capacity was observed to
have a maximum value at pH 5, and subsequently decreased with
increasing pH (from pH 5 to 8).
3.2 Glycosides
Glycosides are molecules in which a sugar is bound to a non-
carbohydrate moiety. Glycosides consist of many sub-classes,
e.g. flavonoid glycosides, cyanogenic glycosides and saponin
glycosides. Glycosides usually exist in various plants in the form
of precursors, which can be hydrolyzed to the active glycosides
by enzymes and then play numerous important roles in living
organisms.
Golden root (Rhodiola rosea) is a plant that is grown in the
cold regions of the world and has been found to be effective for
improving mood and alleviating depression.51 Salidroside (15, 4-
hydroxyphenethyl)-b-D-glucopyranoside) is a phenylethanoid
glycoside compound found in the extracts of golden root, and
has been identified as a promising candidate responsible for the
anti-depressant, anti-diabetic and antioxidative effects.52 Ma
et al. made efforts to find a simple and efficient method to
Nat. Prod. Rep., 2010, 27, 1493–1510 | 1497

separate and purify salidroside from extracts of golden root at
preparative scale.53 They investigated the static adsorption and
desorption properties and kinetics of salidroside on five different
macroporous adsorbents (D101, HPD-200, AB-8, HPD-600 and
ADS-17). The performance of HPD-200 surpassed the other
tested resins and was therefore used for further optimization of
packed-bed separation processes. After two adsorption and
desorption packed-bed runs of the crude extracts, a product
containing salidroside at a purity of 92.2% was obtained with
a recovery yield of 48.8%. Finally, a lamellar crystalline form of
salidroside at 99.0% purity was obtained by subsequent enrich-
ment of the eluate obtained using 10% ethanol in a second cycle
of packed-bed purification. The packed-bed adsorption process
employed in this work was stated to be less expensive and more
efficient than conventional methods. In more recent work, Ma
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et al. purified rosavin (16, 3-phenyl-2-propenyl-6-O-a-L-arabi-
nopyranosyl-b-D-glucopyranoside) from the same plant, which is
a dominant phenylpropanoid glycoside that is responsible for
some immunostimulating and hepatoprotective properties.54
Five macroporous resins (D101, HPD-200, AB-8, XDA-6 and
LSA-10) were used in this study, and adsorption isotherms were
determined and desorption performance was assessed. HPD-200
was selected as showing the best performance for the isolation of
rosavin. Following the use of an optimized packed-bed process,
the content of rosavin in the product was increased from 0.69 to
11.6% with a recovery yield at 82.5%.
Polygonum multiflorum is a herb that is widely used in China
due to its folk-documented medicinal efficacy of rejuvenating
the human body. Recent studies have indicated that SG (17,
2,3,5,40 -tetrahydroxystilbene-2-O-b-glucopyranoside), a major
stilbene glycoside isolated from Polygonum multiflorum, has
many medicinal uses and potential health benefits for chole-
sterol reduction and the reduction of age-related deterioration
by acting as an antioxidant and free-radical scavenger.55 With
the aim of developing a novel and rapid method based on
macroporous resins to separate SG from the crude extracts of
Polygonum multiflorum, Lv et al. investigated the adsorption
performance of 15 macroporous resins (AB-8, NKA, NKA-II,
NKA-9, HPD-100, HPD-500, HPD-700, HPD-800, D-101,
DM-11, DM-130, DM-301, HZ-801, Z-841 and DA-201), and
found that HPD-100 gave the best performance.56 The optimi-
zation of parameters affecting adsorption, such as sample
concentration, pH value and flow rate, was carried out on
a packed bed filled with HPD-100 resin. A final product with
a purity of 81.9% (w/w) was recovered from a scaled-up column
at a recovery yield of 74.7%.
1498 | Nat. Prod. Rep., 2010, 27, 1493–1510
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Erigeron breviscapus is a plant used to treat cardiovascular
disease in Chinese medicine. In recent studies, its therapeutic
effects on dilating blood vessels, increasing cerebral flood flow
and inhibiting platelet aggregation have been attributed to the
major bioactive ingredient of scutellarin (18, scutellarein-7-O-b-
57
D-glucuronide). Gao et al. reported an efficient method that
separated this flavone glycoside from crude extracts of Erigeron
breviscapus using macroprous resins.58 Several resins in the HPD
series (HPD-100, HPD-300, HPD-400, HPD-450, HPD-500,
HPD-600, HPD-700 and HPD-800) were tested in their work,
and HPD-800 offered the best performance in both adsorption
and desorption processes. Using a packed bed filled with HPD-
800 resin, the content of scutellarin of the eluate increased nearly
16-fold, from 2.61 to 41.0% (w/w) together with a recovery yield
of 95.0%.
3.3 Saponins
Saponins are a category of grouped amphipathic glycosides that
are usually found as secondary metabolites in natural sources.59
Saponins are characterized by foaming properties that are caused
by the combination of a hydrophobic (fat-soluble) sapogenin and
a hydrophilic (water-soluble) sugar part. Most saponins are
derived from plants, e.g. vegetables, herbs and beans, but some
bioactive saponins have also been reported to have been sepa-
rated from marine organisms.60 Different saponins may have
different health benefits, such as cholesterol reduction,61
cardiovascular disease prevention,62 and antitumor and antioxi-
dant activity.63 Saponins are usually classified into steroidal
saponins (only found in monocotyledonous angiosperms) and
triterpenoid saponins (mainly found in dicotyledonous angio-
sperms).64 ‘Tianqi’ (Panax pseudoginseng) is a herb grown in
Japan and China that has a very favourable reputation in folk
medicine for the treatment of blood disorders and blood
deficiency. Two groups of steroidal saponins, PTS (19, 20(S)-
protopanaxatriol-type saponins) and PDS (20, 20(S)-proto-
panaxadiol-type saponins), contained in the root of Panax
pseudoginseng have been demonstrated to have anticarcinogenic
and protective properties on the cardiovascular and cerebro-
vascular system.65 Wan et al. tried to establish a simple method
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for the preparative separation of PTS and PDS from crude

extracts of Panax pseudoginseng root.66 They studied the
adsorption properties of four macroporous resins (D-101, DA-
201, DM-301 and DS-401) towards PTS and PDS, and suggested
that DS-401 showed the best adsorption behaviour. DS-401 was
then used for optimization of the dynamic adsorption process in
a packed bed. PTS and PDS were successfully eluted with 30 and
80% (v/v) aqueous ethanol solution, respectively. Following
isolation using a packed bed, PTS and PDS were obtained at
purities of 88.2% and 92.6% (w/w) and with recovered yields of
80.2 and 82.3%, respectively.
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3.4 Taxol/taxoids

Taxol/taxoids are a group of diterpene compounds with

a common tricyclic pentadecane skeleton that possess strong
antitumor properties and a unique mechanism of action on
microtubules.71 A family of these compounds has been approved
by the US FDA to be used in the treatment of various cancers.72
Paclitaxel (23) and docetaxel (24) are two important taxoids
currently being evaluated at the clinical-trial stage for their
possible treatment of some types of tumour such as ovarian,
liver, lung and breast cancers.73 These pharmaceutical substances
have to be extracted from the stem bark of various yew trees of
the genus Taxus, which not only grow slowly but also yield very
little bark.71 Although semi-synthesis is regarded nowadays as
the most effective way to overcome the shortage of the active
compounds, there continue to be publications reporting the
Centella asiatica is a herbaceous plant grown in some parts of isolation of taxoids from plants by macroporous resins. This is
Asia, and is used as a folk medicinal herb in Asiatic countries. because the taxoids in other chemical forms, e.g. 10-DAB III (25,
Triterpenoid saponins contained in Centella asiatica are a group 10-deacetylbaccatin III) and 7-xyl-10-DAT (26, 7-xylosyl-10-
of compounds of the pentacrylic family. Among them, made- deacetyl paclitaxel), are much more abundant in natural sources
cassoside and asiaticoside67 are two major compounds that are
reported to have beneficial effects such as antioxidative, anti-
inflammatory and anticancer actions.68 Jia et al. employed five
macroporous resins (HPD-100, HPD-300, X-5, AB-8 and D101)
to enrich and purify madecassoside (21) and asiaticoside (22)
from crude extracts of Centella asiatica, and obtained prepara-
tions containing triterpenoid saponins at an overall purity of
80%.69 Of the resins used in the preliminary tests, HPD-100
showed the highest static adsorption and desorption capacities
and the fastest rates of adsorption. Columns packed with HPD-
100 were then selected to optimize the separation process, and the
dynamic adsorption and desorption properties were assessed.
After a one-step packed-bed purification process, the final
contents of madecassoside and asiaticoside in the eluted fractions
were increased from 3.9 and 2.0% to 39.7 and 21.5% respectively,
i.e. about ten times higher than in the pre-column extracts. The
recovery yields for product were 70.4 and 72%. However, in very
recent work to enrich the oleanane-type triterpenoid saponins
(OTSs) from Gypsophila pacifica by various chromatographic
materials, MCI-gel (Mitsubishi Chemical Co.) showed higher
capacities for both adsorption and desorption processes than
macroporous resins and octadecylsilyl (ODS) materials.70

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than paclitaxel, and can be transformed to paclitaxel or its
derivatives by chemosynthesis or biosynthesis methods.74,75
Chinese yew (Taxus chinensis) is a large evergreen tree wide-
spread in China containing taxoids well known for their
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remarkable anticancer activity. Fu et al. studied the adsorption
and desorption behaviour of pure 10-DAB III and 7-xylosyl-10-
deacetyl paclitaxel on seven selected macroporous resins (AB-8,
ADS-17, ADS-21, ADS-31, ADS-8, H1020 and NKA-II), and
found that AB-8 demonstrated an outstanding adsorption
capacity for both compounds.76 The column proved to be
effective for the separation and enrichment of 10-DAB III (25)
and 7-xyl-10-DAT (26), with the product contents being
increased by 62.4-fold and 8.5-fold, and recovery yields of 85.9
and 52.8%, respectively, being obtained. The enrichment of
samples by macroporous resins in this study was superior to
conventional liquid–liquid extraction, and optimized conditions
for adsorption and desorption processes were given.76 Recently,
Sun et al. enriched four taxoids, 10-DAB III (25), 7-xyl-10-DAT
(26), cephalomannine (27) and paclitaxel (23), from extracts of
the needles of Taxus chinensis by macroporous resin column
chromatography.77 The above seven macroporous resins were
again compared, with AB-8 again showing the best performance
for all the four taxoids, whose contents in eluates were increased
by 7.63-, 3.68-, 6.18- and 6.55-fold compared to those in the
extracts. The recovery yields of the four components were 95.0,
77.3, 88.1, and 95.3%. These two publications also contain
information on the separation and enrichment of taxoids at
a preparative scale.76,77
3.5 Carotenoids
Carotenoids are a large family of organic pigments that are
mainly found in the chloroplasts and chromoplasts of various
plants and photosynthetic algae, fungi and bacteria. Carotenoids
usually serve two roles in the plants and micro-organisms:
adsorbing light energy for photosynthesis and protecting
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chlorophyll from photodamage.78 Most carotenoids present in
food consumed by humans are antioxidants that are able to
scavenge free-radicals in humans and are beneficial for the
immune system.79 Nowadays, the majority of carotenoids are
extracted from orange peel, sweet potato and carrot using
various combinations of cellulase and pectinase enzymes.80 Other
extraction methods include the application of microwaves81 and
the use of superficial CO2 extraction coupled with membrane
filtration.82
Gardenia (Gardenia jasminoides) is an evergreen fragrant
flowering plant widely cultivated in gardens in South and East
Asia. Crocins, which are a family of mono- and di-glucosyl esters
of crocetin, are natural carotenoid pigments extracted from
Gardenia jasminoides with the potential of replacing synthetic
colourants in food products.83 Crocins (28) are also confirmed to
have a variety of pharmacological effects including neuro-
protection,84 antitumor activity,85 and protection from cardio-
vascular disease.86 Yang et al. investigated the adsorption
performance of various macroporous resins and have made
efforts to understand the mechanism of the adsorption process,87
and similar isolation work has been carried out on a preparative
ODS column.88 Pulverised fruits of Gardenia jasminoides were
first extracted with water and then partially purified by micro-
filtration using ceramic membranes. The characteristics of the
adsorption of crocin onto 10 poly-SDVB resins (XAD-1180,
XAD-1600, EXA-32, EXA-45, EXA-50, EXA-117, EXA-118,
HP-20, AB-8 and HPD-100) from the extracts were investigated.
XAD-1180, HP-20, HPD-100A and AB-8 were found to be
superior to others in terms of adsorption capacity and selectivity.
The rates of adsorption were found to fit well to either pseudo-
second-order or first-order kinetics. The rate-limiting steps in the
adsorption process were attributed to intraparticle diffusion and
boundary-layer effects. After further analysis of its thermody-
namics, the authors suggested that adsorption was an exothermal
process that was controlled by physical rather than chemical
adsorption mechanisms.87
3.6 Serotonins
Serotonins are a family of chemicals produced by neurons that
are responsible for regulating heart rate, body temperature and
mood. Though serotonins are primarily found in humans and
other animals, many of their derivatives can also be isolated from
insects, fungi and plants. Serotonin derivatives are a class of
alkaloids featuring a serotonin moiety bound to a phenyl-
propanoid moiety via an amide bond. CS (29, N-(p-coumaroyl)
serotonin) and FS (30, N-feruloyl serotonin) are two well-known
bioactive components that have been reported to possess
cathartic effects, anti-tyrosinase activity and antitumour
activity.89 These two serotonins have been enriched from extracts
of Chinese safflower seed. Safflower (Carthamus tinctorius) has
been grown widely in China for the purpose of using its seeds as
colouring and flavourings, and more recently as a source of
serotonin derivatives. Jin et al. studied and evaluated the
adsorption and desorption characteristics of six common mac-
roporous resins (D-312, 860021, DM-131, HZ-801, AB-8 and
XDA-1) in their possible use for the separation of CS and FS
from the extracts of safflower seeds.90 XDA-1 was shown to
possess the best static adsorption and desorption behaviour. The
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researchers then established the optimum operational conditions
for this separation process on a packed bed filled with XDA-1
resin. After one cycle of adsorption and desorption, the content
of CS and FS increased from 2.0 and 1.7% in the crude extracts to
24.1 and 22.4% in the final products, with recovery yields of 77.2
and 68.5%, respectively.
3.7 Fatty alcohols
Solanesol (31) is a 45-carbon trisesquiterpenoid fatty alcohol
predominantly found in tobacco leaves that has been shown to
have anticancer, antiviral, antifungal and anti-HIV properties.91
Du et al. developed a preparative-scale method to purify sol-
anesol from the crude extracts of tobacco leaves.92 Five macro-
porous resins (including SP207, SP700, SP825, SP850 and HP20)
were employed to test the adsorption and desorption character-
istics at different temperatures, and results showed that SP850
gave the best performance. Further optimization of operational
parameters, such as feed concentration, flow rate, bed height,
elution solvent composition, and elution mode (isocratic or
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stepwise) were carried out on a column packed with SP850. The
regeneration of SP850 was tested, and the throughput when
operated using the optimal conditions was estimated. It was
concluded that SP850 possesses good ability for the separation of
solanesol from the crude extracts of tobacco leaves.
The adsorption capacities summarized in Table 1 have been
evaluated under their various reported operational conditions
and have been fitted to either Langmuir or Freundlich isotherms.
As shown in the table, it is very difficult to compare the
adsorption performance of various different macroporous resins
due to a lack of consistency in the adsorption conditions used in
the experimental work described in the literature.
4 Approaches to improve the separation performance
The selected examples described above clearly demonstrate the
versatility of adsorption onto macroporous resins for the sepa-
ration and enrichment of natural products from plant sources. In
most of the examples using single-step packed-bed adsorption
columns filled with macroporous resins, the compound of
Nat. Prod. Rep., 2010, 27, 1493–1510 | 1501

interest can be recovered at an overall yield of 70–80%. However,
the purity of the isolated product may vary from 10 to 90%,
depending on the complexity of the composition of the crude
extract. The interactions between macroporous resins and target
compounds are attributed to the selectivity of macroporous
resins, and many factors may be included in the separation
process, e.g., electrostatic forces, hydrogen bonding, complex
formation and size-sieving actions. The differences in molecular
weight, polarity or shape of different compounds may lead to
differences in their resultant affinity for the resins. The likelihood
of non-specific adsorption of other compounds to macroporous
resins therefore makes it difficult to obtain high-resolution
separations and high degrees of purification of the target
compound from complex samples.
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4.1 Description of strategies adopted
More recently, efforts have been made to improve the resolution
of separations that can be achieved using macroporous resins.
One strategy is simply to employ purification using macroporous
resins as an effective pre-separation step and then to follow it by
a subsequent high-resolution column purification process, e.g.
HSCCC (high speed countercurrent chromatography) or
preparative grade HPLC. Following preparation of a crude
extract, a packed bed of macroporous resin can be used to isolate
groups of compounds with similar overall properties. Several
compounds belonging to the same chemical family are likely to
be present in the various elution fractions from the packed bed,
and analytical HPLC can be employed to locate the target
compounds of interest. Subsequently, the individual members
within a particular chemical family can finally separated from
each other by preparative HPLC or HSCCC.
In practice, the specific parts of the plant that contain the
highest amounts of the compounds of interest are dried and
pulverized to form powders which are extracted with aqueous
ethanol solution. The crude extract is then evaporated to dryness
to form a concentrate that is usually redissolved in water. This
method of extraction is thought to be relatively friendly to the
environment since only ethanol, as opposed to less biodegradable
solvents, has been used in the process. Column chromatography
is been performed using a packed bed of macroporous resin.
After loading the aqueous concentrate onto the packed bed, de-
ionized water is usually used to remove the water-soluble
components such as pigments and polysaccharides that are
weakly retained on macroporous resins. Secondly, step-gradients
of aqueous ethanol are used to elute the target compounds into
several ‘‘group compound fractions’’ prior to further purification
using preparative HPLC and HSCCC.
So far, many researchers have preferred to use the resins D101
and AB-8 for the column chromatography. D101 and AB-8 are
two cheap but efficient macroporous resins that can be used for
most of the applications in this field. D101 is a non-polar resin
found to be suitable for the adsorption for most flavonoid
molecules. By contrast, AB-8 is weakly polar and has mostly
proved to be appropriate for various glycosides. The extensive
uses of these two resins are the result of an appropriate balance
between their overall surface area and pore size. There is no
doubt that the surface area of an adsorbent will strongly affect its
adsorption capacity; however, the pore size can play an
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important role in the ease in which adsorbates can enter or leave
the adsorbent particle. The surface area and pore size of an
adsorbent are determined by the properties of materials and
process of synthesis. Generally, the smaller the pore diameter, the
higher the surface area, and vice versa. For the adsorption
processes involving macroporous resins, the optimal ratio of the
pore diameter to the adsorbate diameter was found to be in the
range 2–6.93 It is difficult for the adsorbates to diffuse from
the bulk aqueous solution into the pores if the pore diameter is
too small. However, adsorbates already adsorbed within the
pores will tend to leave again if the pore diameter is too large. In
addition, the interior of a particle does not contain a larger
surface area for adsorption when there is too large a pore
diameter.
The molecular structures of the flavonoid and glycoside
backbones are shown in Fig. 1. Based on data for bond lengths
and bond angles,94 the diameter of flavonoids and glycosides are
estimated to be at least 1.0 nm, depending on what kind and how
many functional groups are attached to their backbones.
Considering that in practice the molecules of interest are usually
considerably larger than the backbone itself and that, for
glycosides, a number of sugar groups are often attached onto the
molecule, the molecular diameter can be estimated as being
around 2.0 nm. Both D101 and AB-8 resins possess average pore
diameters near to 13 nm, which are about 6 times the molecular
diameters of flavonoids and glycosides. The pore sizes of these
two resins are thus appropriate for their reported use in the
purification of flavonoids and glycosides. In addition, the
glycoside molecule will be much more polar if more than one
sugar group is attached to the backbone, and this will strengthen
the adsorption process to the weakly polar AB-8 resin. Mean-
while, both D101 and AB-8 resins are SDVB resins in which p–p
interactions are proposed to take place between the hydrophobic
phenyl ring of the matrix and the aromatic ring of the flavonoid
and glycoside molecules (Fig. 2).
Fig. 1 Molecular structures of flavonoid (left) and glycoside (right)
backbones.
Fig. 2 Schemes for proposed p–p interaction between an aromatic ring
and SDVB resins.
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Table 1 Comparison of various macroporous resins showing their physical properties and their adsorption behaviour of natural products

Equilibrium
Surface Pore Particle isotherm Isotherm
Resin Matrix  size (mm)
area (m2/g) diameter (A) Solute model used parameters Ref.

AB-8, weakly polar SDVB 480–520 130–140 300–1250 Crocin 28 Freundlich kF ¼ 0.2309, 1/n ¼ 0.9989 87
Salidroside 15 Langmuir qm ¼ 14.17 (mg/g) 53
Freundlich kF ¼ 11.11, 1/n ¼ 0.3290
Rosavin 16 Langmuir qm ¼ 5.802 (mg/g) 54
Freundlich kF ¼ 16.16, 1/n ¼ 0.4690
7-Xyl-10-DAT 26 Langmuir qm ¼ 18.38 (mg/g) 76
Freundlich kF ¼ 50.11, 1/n ¼ 0.2864
10-DAB III 25 Langmuir qm ¼ 12.14 (mg/g)
Freundlich kF ¼ 19.28, 1/n ¼ 0.4197
ADS-5 SDVB 520–600 250–300 300–1250 Vitexin 5 Freundlich kF ¼ 4.837, 1/n ¼ 0.2125 40
Isovitexin 6 Freundlich kF ¼ 5.610, 1/n ¼ 0.1842
Genistein 7 Langmuir qm ¼ 73.86 (mg/g) 43
Freundlich kF ¼ 364.55, 1/n ¼ 0.4891
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Apigenin 8 Langmuir qm ¼ 9.19 (mg/g)

Freundlich kF ¼ 160.61, 1/n ¼ 0.4710
ADS-17, Acrylic 250–300 90–150 300–1250 Salidroside 15 Langmuir qm ¼ 4.96 (mg/g) 53
moderately polar Fruendlich kF ¼ 3.190, 1/n ¼ 0.3810
AL-2, polar SDVB 550–600 150–250 300–1250 Luteolin 4 Langmuir qm ¼ 434.8 (mg/g) 37
D101, non-polar SDVB 480–550 90–150 300–1250 Salidroside 15 Langmuir qm ¼ 14.48 (mg/g) 53
Freundlich kF ¼ 11.62, 1/n ¼ 0.3080
Rosavin 16 Langmuir qm ¼ 5.944 (mg/g) 54
Freundlich kF ¼ 16.05, 1/n ¼ 0.4590
PTS 19 Freundlich kF ¼ 107.6, 1/n ¼ 0.0878 66
PDS 20 Freundlich kF ¼ 136.5, 1/n ¼ 0.1244
Licorice flavonoids Freundlich kF ¼ 56.1, 1/n ¼ 0.2656 50
Glycyrrhizic acid 14 kF ¼ 190.1, 1/n ¼ 0.5372
DM11, non-polar SDVB $ 500 70–80 315–1250 SG 17 Langmuir qm ¼ 454.55 (mg/g) 56
Freundlich kF ¼ 1.135, 1/n ¼ 0.01274
DM-301, SDVB $ 330 140–170 300–1250 SG 17 Langmuir qm ¼ 333.33 (mg/g) 56
weakly polar Freundlich kF ¼ 1.190, 1/n ¼ 0.01405
DS-401, SDVB $ 480 120–140 300–1250 PTS 19 Freundlich kF ¼ 118.2, 1/n ¼ 0.1174 66
weakly polar PDS 20 Freundlich kF ¼ 117.6, 1/n ¼ 0.1717
EXA-32 SDVB 600 520 N/A crocin 28 Freundlich kF ¼ 0.4240, 1/n ¼ 0.9780 87
EXA-45 SDVB 1000 76 N/A Crocin 28 Freundlich kF ¼ 5.16  107, 1/n ¼ 2.0944
EXA-50 SDVB 1000 114 N/A Crocin 28 Freundlich kF ¼ 2.04  107, 1/n ¼ 2.2644
EXA-117 SDVB 570 160 N/A Crocin 28 Freundlich kF ¼ 0.4417, 1/n ¼ 0.8606
EXA-118 SDVB 1200 180 N/A Crocin 28 Freundlich kF ¼ 0.2576, 1/n ¼ 0.9891
HP-20 SDVB 600 520 300–1250 Crocin 28 Freundlich kF ¼ 0.3912, 1/n ¼ 0.9509
HPD-100, SDVB 650–700 85–90 300–1250 Crocin 28 Freundlich kF ¼ 0.2141, 1/n ¼ 0.9648
non-polar Madecassoside 21 Langmuir qm ¼ 268.7 (mg/g) 69
Freundlich kF ¼ 438.1, 1/n ¼ 0.2486
Asiaticoside 22 Langmuir qm ¼ 164.8 (mg/g)
Freundlich kF ¼ 279.8, 1/n ¼ 0.697
SG 17 Langmuir qm ¼ 588.2 (mg/g) 56
Freundlich kF ¼ 1.114, 1/n ¼ 0.01240
Puerarin 77 Freundlich kF ¼ 22.76, 1/n ¼ 0.4753 109
HPD-200, SDVB 700–750 85–90 300–1250 Salidroside 15 Langmuir qm ¼ 14.49 (mg/g) 53
non-polar Freundlich kF ¼ 11.63, 1/n ¼ 0.3190
Rosavin 16 Langmuir qm ¼ 5.920 (mg/g) 54
Freundlich kF ¼ 16.09, 1/n ¼ 0.4390
HPD-500, polar SDVB 500–550 100–120 300–1200 SG 17 Langmuir qm ¼ 370.37 (mg/g) 56
Freundlich kF ¼ 1.162, 1/n ¼ 0.01316
HPD-600, polar SDVB 500–550 100–120 300–1250 Salidroside 15 Langmuir qm ¼ 4.920 (mg/g) 53
Freundlich kF ¼ 3.790, 1/n ¼ 0.2710
HPD-800, SDVB 700–750 90–110 300–1200 Scutellarin 18 Langmuir qm ¼ 42.52 (mg/g) 58
moderately polar Freundlich kF ¼ 8.273, 1/n ¼ 0.810
HPD-850, polar SDVB 1100–1300 85–95 300–1200 Chlorogenic acid 13 Langmuir qm ¼ 114.16 (mg/g) 48
Freundlich kF ¼ 110.78, 1/n ¼ 0.3760
LSA-10, Methacrylic 500–540 85–90 300–1250 Rosavin 16 Langmuir qm ¼ 3.511 (mg/g) 54
moderately polar Freundlich kF ¼ 6.245, 1/n ¼ 0.4200
Licorice flavonoids Freundlich kF ¼ 44.9, 1/n ¼ 0.2140 50
Glycyrrhizic acid 14 kF ¼ 141.8, 1/n ¼ 0.4312
LSA-20, non-polar Methacrylic 420–500 85–90 300–1250 Licorice flavonoids Freundlich kF ¼ 78.0, 1/n ¼ 0.2887
Glycyrrhizic acid 14 kF ¼ 217.2, 1/n ¼ 0.2774
NKA II, polar SDVB 160–200 145–155 300–1250 SG 17 Langmuir qm ¼ 588.20 (mg/g) 56
Freundlich kF ¼ 1.120, 1/n ¼ 0.01283
XAD-7 Acrylic 450 90 250–840 Total phenolics Langmuir qm ¼ 239 (mg/g) 33

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Table 1 (Contd. )

Equilibrium
Surface Pore Particle isotherm Isotherm
Resin Matrix  size (mm)
area (m2/g) diameter (A) Solute model used parameters Ref.

Freundlich kF ¼ 14.446, 1/n ¼ 0.3366

Total flavonoids Langmuir qm ¼ 64 (mg/g)
Freundlich kF ¼ 5.6828, 1/n ¼ 0.3461
XAD-16 SDVB 630 210 250–840 Total phenolics Langmuir qm ¼ 16 (mg/g)
Freundlich kF ¼ 3.4488, 1/n ¼ 0.2749
Total flavonoids Langmuir qm ¼ 4 (mg/g)
Freundlich kF ¼ 0.9417, 1/n ¼ 0.3302
XAD-1180 SDVB 700 400 250–840 Crocin 28 Freundlich kF ¼ 0.2714, 1/n ¼ 0.9938 87
XAD-1600 SDVB 800 150 250–840 Crocin 28 Freundlich kF ¼ 0.3493, 1/n ¼ 0.9927
XDA-1, Acrylic 800–1000 85–95 300–1250 CS 29 Langmuir qm ¼ 18.1 (mg/g) 90
non-polar Freundlich kF ¼ 28.71, 1/n ¼ 0.2475
FS 30 Langmuir qm ¼ 13.8 (mg/g)
Freundlich kF ¼ 22.00, 1/n ¼ 0.2415
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Licorice flavonoids Freundlich kF ¼ 145.8, 1/n ¼ 0.4259 50

Glycyrrhizic acid 14 kF ¼ 191.8, 1/n ¼ 0.1347
XDA-6, polar Acrylic 450–500 120–160 300–1250 Rosavin 16 Langmuir qm ¼ 3.103 (mg/g) 54
Freundlich kF ¼ 5.434, 1/n ¼ 0.4040

4.2 Combination with chromatography Patrinia villosa, with the purities of each compound being higher
than 96%.98
There now follows a description of some typical examples of the
Peng et al. developed an efficient method to isolate and purify
purification of bioproducts from plant sources adopting
two glycosides, curculigoside (44) and curculigoside B (45), from
a strategy in which adsorption is followed by a chromatographic
Curculigo orchioides by preparative HSCCC, after two rounds of
separation technique. Four flavonoid glycosides, namely orientin
extraction with 60% aqueous ethanol and pre-treatment by
(32), vitexin, quercetin-3-O-neohesperidoside (33) and one novel
adsorption onto D101.99 The contents of these two components
compound, were purified by HSCCC and preparative HPLC
in the semi-purified samples obtained after initial clean-up by
from Trollius ledebouri following extraction by ethanol and
D101 were reported to be 5.09 and 26.1%, respectively. This
pretreatment by D101 resin.95 Yang et al. established an efficient
research group also employed this simple but efficient strategy
method to separate and purify phenolic acids from the herb
again to enrich schizandrin (46) and gomisin A (47) from
Smilax china by HSCCC after pretreatment using a D101 resin
column.96 These researchers reported a purity of over 98% for
each of the five phenolic acids, including gallic acid (9), proto-
catechuic acid (34), caffeic acid (35), gentisic acid (36) and trans-
o-coumaric acid (37). It was claimed that this was the first
successful separation of the last three compounds.
Three flavonoids, epimedokoreanoside I (38), icariin (39) and
icariside II (40), each at a purity of over 98%, were obtained in
a one-step HSCCC separation from Epimedium koreamum by
Liu et al.,97 and D101 was used for the initial sample preparation.
D101 combined with preparative HSCCC was also employed in
the isolation of three flavonoids, bolusanthol B (41), tetrapterol I
(42) and a novel compound named 5,7,20 ,60 -tetrahydroxy-6,8-
di(g,g-dimethylallyl)flavanone (43), from the crude extract of

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Schisandra chinensis.100 Two isomeric diterpenoid alkaloids,
namely 13-dehydro-1b,2a,6b-trihydroxyhetisine (48) and 13-
dehydro-2a,3b,6b-trihydroxyhetisine (49), were successfully
separated from the roots of Aconitum coreanum by preparative
HSCCC, after partial purification using D101.101 Overall purities
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of both compounds were reported to be higher than 95%.
Zhang et al. purified four active ingredients of the flavone C-
glycoside family from an ethanol aqueous extraction of bamboo
leaves. These ingredients were orientin (32), homoorientin (50),
vitexin (5) and isovitexin (6), and these are thought to have
therapeutic value as a result of their strong antioxidant activity.
A column containing AB-8 macroporous resin was used for
initial purification followed by further purification using
a preparative HPLC column.102 Peng et al. used AB-8 in a similar
manner to isolate four novel (51, 52, 53 and 54) and two known
flavonoids (42 and 55) from the leaves of Patrinia villosa.103
The uses of other extraction strategies and resins have also
been reported to be effective. D101 resin was used by Chen et al.
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to purify salvianolic acids (56, 57) from sonicated crude water
extracts of ‘danshen’ (Salvia miltiorrhiza) in conjunction with
semi-preparative HSCCC.104 Similarly, sonication during
extraction, column chromatography with D101, and subsequent
use of HSCCC was also reported to be effective for the purifi-
cation of eight active compounds (58, a-spinasterol; 59, b-sitos-
terol; 60, lupenone; 61, 24-methylene cycloartanoll; 62,
3-O-palmitoyl-b-sitosterol; 63, 3-O-b-D-glucosyl-palmitoyl-b-
sitosterol; 64, nonacosan-10-ol; 65, eicosanoic acid) from the root
of the Chinese herb Adenophora tetraphylla.105 AB-8 has been used
in similar processes to purify three flavonoid glycosides, iso-
quercitrin (66), hyperoside (67) and astragalin (68), from the leaves
of Nelumbo nucifera,106 while NKA-12 resin has been used to isolate
five flavonoid glycosides including ononin (69), calycosin-7-O-b-D-
glucoside (70), (6aR, 11aR)-9,10-dimethoxypterocarpan-3-O-b-D-
glucoside (71), calycosin-7-O-b-D-glucoside 600 -O-acetate (72) and
(3R)-20 -hydroxy-30 ,40 -dimethoxyisoflavan-7-O-b-D-glucoside (73)
from an extract of Radix Astragali.107 In the latter example, ethyl
acetate was used for extraction. The preparative isolation and
purification of three steviol glycosides, stevioside (74), rebau-
dioside A (75) and rebaudioside C (76), has been achieved by
HSCCC, following pre-purification of a crude aqueous solution
of Stevia rebaudiana using chromatography on two sequential
columns containing LSA-21 and D941 macroporous resins,
respectively.108
4.3 Modifications of resin chemistry
In addition, efforts have been made to modify the surface
chemistry and pore structure of certain macroporous resins in
order to enhance their selectivity. Yang et al. covalently coupled
oligo-b-cyclodextrin (CDP) onto 12 polystyrene-based macro-
porous resins (AB-8, S-8, NKA-9, NKA-II, HPD-100, HPD-
450, HPD-600, YWD01B, YWD03F, YWD04, YWD06 and
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D-201) and investigated their batch adsorption performance with
puerarin (77, daidzein 8-C-glucoside).109 The latter is an iso-
flavonoid extracted from the root of Pueraria lobata that
is believed to be effective in relieving fever and treating cardio-
vascular diseases.110 Enhanced selectivity of adsorption was
observed for a number of these adsorbents following derivati-
zation of the base matrices with CDP, and in particular, CDP-
derivatized HPD-100 showed the highest adsorption and
desorption capacities. A one-step packed-bed adsorption
procedure using this derivatized resin was used to purify puerarin
from a crude extract, and the final product was obtained at
a purity of 95.3% with a recovery yield of 86.7% under optimal
mobile-phase conditions.
Ongoing efforts have been made to synthesize novel macro-
porous resins derivatised with various functional groups to
control the structure of resins and to observe their influence on the
adsorption performance. Xu et al. synthesized a series of macro-
porous methacrylate–divinylbenzene (MA-DVB) resins with
different DVB contents and investigated the relationship between
1506 | Nat. Prod. Rep., 2010, 27, 1493–1510
View Article Online
surface chemistry and adsorption performance.7 When purifying
Ginkgo biloba extracts, they found the surface chemistry affects
the adsorption selectivity not only for the flavonol glycosides, but
also for the terpene lactones (including ginkgolides and biloba-
lides). Selectivity for both classes of components increased with
increasing content of the methacrylate moiety. In order to deter-
mine the effect of different porogenic agents on selectivity, several
solvents have been used as porogenic agents during the synthesis
of macroporous adsorbents. However, the use of these porogenic
agents only produced adsorbents that showed good adsorption
selectivity for ginkgolides but had almost no selectivity for
flavonol glycosides and bilobalides. Later, this group developed
a novel resin that consisted of gelatin in the presence of poly(vinyl
alcohol) used as a supporting matrix so as to improve the resin’s
mechanical strength.44 This novel resin demonstrated high
adsorption affinity towards flavonol glycosides but not towards
terpene lactones. The difference in affinity for these two classes of
compounds is consistent with the hypothesis that the adsorption
mechanism involves hydrogen bonding.
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This laboratory has also reported the synthesis of a family of
weak hydrophobic resins derivatised with several different
functional groups.111 Amide groups were introduced in the resin
because of their ability to form hydrogen bonds with adsorbates.
It was found that the hydrophobic affinity of the resin decreased
when the content of DVB was decreased. In particular, when the
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View Article Online
DVB content was reduced to 8% (w/w), the resin could no longer
adsorb terpene lactones. However, this resin could still adsorb
the flavonol glycosides efficiently as a result of hydrogen
bonding. It was suggested that in addition to hydrogen bonding,
hydrophobic interactions may also make an important contri-
bution to the adsorption process. Shi et al. verified the presence
Nat. Prod. Rep., 2010, 27, 1493–1510 | 1507

of hydrophobic interactions by introducing quaternary ammo-
nium groups (e.g. –N+(CH3)3) into a conventional AB-8 resin
for use in the purification of Stevia glycosides from Stevia
rebaudiana.112 Before the introduction of quaternary ammonium
groups, the relative amounts of the five Stevia glycosides that
were adsorbed onto the underivatised resin were almost the same
as those present in the feedstock. However the modified AB-8
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resin adsorbed different relative amounts of these five glycosides.
Lower proportions of rebaudioside A and rebaudisside C, which
were the largest of the five glycosides, were adsorbed onto the
resin. The introduction of quaternary ammonium groups into
the resin made the more accessible sites more hydrophilic, and
thus it was more difficult for the larger glycosides to adsorb onto
the modified resin.
Although adsorption by macroporous resins in packed-bed
mode can give good purification efficiency, it still has the
following disadvantages. Firstly, clarification of feedstock prior
to application to the packed-bed column is usually an expensive
process that consists of multiple operational steps. Each unit
operation in this scheme usually leads to the loss of active
ingredients, as well as increasing both the operational time and
the process costs. Secondly, in packed-bed mode, the mass
transfer of active ingredients normally only occurs within a small
region of the packed column. Although the mass transfer zone
moves progressively along the bed as the adsorbent in this region
becomes saturated, the adsorbent located upstream or down-
stream of the region does not participate in the adsorption
process. If the column is too long, the time needed for the
movement of mass transfer zone through the bed will be much
longer and the pressure drop across the column much higher.
Thirdly, as macroporous resins are particles with poor heat
transfer characteristics, it is difficult to remove the heat generated
during the exothermic adsorption process. An increase in the
column temperature will in return decrease the adsorption effi-
ciency for further adsorbate.
Recent work by Li and Chase has shown that the successful
application of macroporous resins in expanded-bed mode for the
purification of flavonoids from Ginkgo biloba leaves may over-
come several of the disadvantages mentioned above.113,114
Expanded-bed adsorption (EBA) is a separation technique that
can greatly reduce the overall process time by reducing the
number of different operational steps needed by directly puri-
fying target substances from particulate-containing feed-
stocks.115 In addition, as the feedstock is pumped upward
through the expanded bed of adsorbent, the zone for contact and
mass transfer between the adsorbent and adsorbates in the
expanded bed is much wider than that in a packed bed. In
addition, enhanced liquid flow between the adsorbent particles
will also help to remove the heat generated during the adsorption
process. Initially, Li et al. confirmed the feasibility of employing
EBA procedures based on the use of Amberlite XAD 7HP
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View Article Online
macroporous resin.113 In a second step, they utilized three
different isolation strategies (liquid–liquid extraction, packed-
bed adsorption and EBA) to recover flavonoids from Ginkgo
biloba leaves and compared their performance.114 The results
demonstrated that, among the three techniques investigated, the
best overall technical and economic performance was achieved
using EBA. However, expanded beds containing a modified
XAD 7HP resin exhibiting lower axial dispersion coefficients,
and hence higher numbers of theoretical plates, are expected to
improve further the performance of macroporous resins in
expanded-bed mode.
5 Summary
Purification and enrichment of natural products from plant
sources has received increased attention in recent years due to the
increasing demand for healthcare products from non-conven-
tional sources. There are a large variety of technologies available
for accomplishing this purpose, but adsorption onto macro-
porous resins is particularly attractive due to factors such as
convenience, low operational cost, lowered solvent consumption,
and low amounts of residual chemicals in the final product. The
selectivity of these resins can be adjusted by modifications of
their surface chemistry and pore size distributions. This paper
has reviewed recent progress on the use of various commercially-
available macroporous resins for the purification of valuable
components from plants and has evaluated their performance in
such separations. Adsorption of target compounds onto mac-
roporous resins from the crude extracts following solvent
extraction has proved to be an effective and efficient strategy for
the purification of these natural products, with most of the
recovery yields being reported at more than 70%. However, the
purity of the products may vary from 10 to 90% depending on
the complexity of the composition of the crude extracts. There-
fore, a widely used strategy is to combine adsorption onto
a macroporous resins with preparative grade HPLC or HSCCC
column chromatography, in order to improve the separation
efficiency. Another strategy adopted by some researchers is to
modify the surface chemistry and pore structure by coupling
appropriate groups onto the macroporous resins to obtain
a matrix with higher efficiency. However, there is not much work
describing the effects of chemical modification or the correlation
between the adsorbent structure and adsorption performance.
Although preparative HPLC or HSCCC chromatography
systems offer certain advantages, namely high purification, high
selectivity, and high reproducibility, they may be difficult or
uneconomical to scale-up in practice. It is reasonable to expect
that more efforts will be made on the modification and optimi-
sation of packed-bed process equipment or on the surface
chemistry and pore structure of the resins, and recent work by
Yang et al.109 is a good example of this present trend. In addition,
EBA tends to be a promising novel technique for use in this field.
Extensive investigations should also be made to study the
detailed mechanisms of such adsorption processes. The selection
of appropriate modifying agents is an important step in the
development of purpose-designed purification processes
employing macroporous resins. Ideal macroporous resins are
expected to have appropriate pore diameter, polarity and
internal surface area for the target molecules to be separated
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from the complex mixture of compounds present in crude
extracts. More research is needed on both the structural modi-
fication of commercially available resins, and the synthesis of
novel resins, in order to produce resins that show specific inter-
actions with the target molecules during adsorption and subse-
quent desorption.
The use of adsorptive macroporous resins either as a stand-
alone system, or as part of a pre-treatment process, contributes
significantly to the purification and enrichment of valuable
natural products from plant sources, and their adoption
undoubtedly presents many benefits for preparative and indus-
trial-scale purposes. The information presented in the above
examples can be extrapolated to predict design and operational
parameters for scaled-up packed-bed adsorption systems utilis-
ing macroporous resins. Further investigations of the scaling-up
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of adsorption processes using macroporous resins and its asso-
ciated economics is an important target for future research. It is
hoped that the body of information presented here can be
exploited widely, in order that the extensive range of therapeutic
outcomes that are manifested by compounds present in plants
can actually benefit mankind, as a result of the ability to effec-
tively isolate the bioactive ingredients.
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