Identification and Quantification of Phenolic Acids in Macrotyloma uniflorum by Reversed Phase-HPLC
Extracts of Macrotyloma uniflorum plants were
examined as potential sources of phenolic compounds. Reversed phase high
performance liquid chromatography (RP-HPLC) with UV detection was employed
for the identification and quantification of the phenolic acids. Eight
phenolic acids, namely, 3, 4-dihydroxy benzoic, p-hydroxy benzoic,
vanillic, caffeic, p-coumeric, ferulic, syringic and sinapic acids
were isolated from an ethanolic extract of Macrotyloma uniflorum.
The most abundant phenolic acids were p-coumaric acid (8.95 mg
10-2 g of dry sample) and p-hydroxy benzoic acid (7.81
mg/100 g of dry sample).
Phenolic acids are a large family of secondary metabolites having hydroxyl
benzoic or hydroxyl cinnamic structures. They are widely distributed plant
constituents. They commonly occur as free acids and their esters, glycosides
and bound complexes and they are known to play important roles in plant
resistance to pathogens and herbivores, allelopathy, oxidative stress
and plant growth regulation. Their role concerns color and sensory characteristics
of plants as well as antioxidant properties of plant-based food (Moure
et al., 2001). This role in the organoleptic properties of foods
has been a big interest of analytical and food chemists (Canas et al.,
2003). Additionally, the content and profile of phenolic acids, their
effect on fruit maturation and prevention of enzymatic browning, and their
roles as food preservatives have been evaluated too (Robbins, 2003; Nurmi
et al., 2006). Recent interest comes from their potential protective
role against diseases that may be related to oxidative damage, such as
coronary heart diseases or cancers (Gomes et al., 2003; Nichenametla
et al., 2006).
Dietary polyphenols such as phenolic acids are considered to be powerful
antioxidants. Their antioxidant activity is much higher in vitro
than of well-known vitamin antioxidants (Tsao and Deng, 2004). Antioxidation
is, however, only one of the many mechanisms through which polyphenols
can exert their actions. Polyphenols have been reported demonstrate antimicrobial
(Taguri et al., 2006; Rauha et al., 2000), antiviral (Perez,
2003), antimutagenic (Lairon and Amiot, 1999), anticarcinogenic (Aaby
et al., 2004), anti-inflammatory (Dos Santos et al., 2006;
Parr and Bolwell, 2000), antiproliferative and vasodilatory actions (Lule
and Xia, 2005).
The recent methods dealing with the analysis of phenolics are summarized
in review articles (Naczk and Shahidi, 2004; Molnár-perl and Fuzfai,
2005; Rijke et al., 2006). It can be seen that the high performance
liquid chromatography (HPLC) occupies a leading position in the analysis
of phenolics. In general, HPLC separations are based on C18
reversed-phased columns and a binary solvent gradient. The mobile phase
usually consists of an aqueous solution of acid and an organic solvent
(acetonitrile or methanol).
Macrotyloma uniflorum Linn (Bengali name-Kurti kalai, English
name-horse gram, Family- Fabaceae) is a herbaceous plant with annual branches,
suberect or twining, leaflets 2.5-5 cm and widely distributed throughout
Bangladesh but abundant in Rajshahi and Dinajpur districts (Kirtikar and
Basu, 1998). It is famous for its medicinal uses because different parts
of the plants are used for the treatment of heart conditions, asthma,
bronchitis, leucoderma, urinary discharges and for treatment of kidney
stones (Ghani, 2003). Indeed, Macrotyloma uniflorum could play
a role in antioxidation (Reddy et al., 2005) as when this plants
were exposed to toxic levels of lead, several enzymes showed a pivotal
role against oxidative injury. Macrotyloma uniflorum has the greatest
potential for further utilization as nutraceuticals, forage and food for
malnourished and drought-prone areas of the world (Morris, 2008). Herbal
medicine is part and parcel of the much needed health care in most of
the developing countries including Bangladesh. A part of our investigations
on the medicinal plants, we investigated M. uniflorum and isolated
Kaempferol-3-O-β-D-glucoside, β-sitosterol and stigmasterol
(Kawsar et al., 2003) and we very recently reported the cytotoxicity
assessment (Kawsar et al., 2008a) and antimicrobial activities
(Kawsar et al., 2008b) of this plant. The aim of this study was
further conducted to identification and quantification of phenolic acids
detail in M. uniflorum plant by using RP-HPLC.
MATERIALS AND METHODS
Macrotyloma uniflorum (Fabaceae) was collected from the village,
Susunda of Muradnagar, Comilla, Bangladesh in March 2002. The botanical
identification and voucher specimen was deposited at the Bangladesh National
Herbarium (BNH) (DACB Accession No. 28264). The whole plants were cleaned,
air-dried and followed by drying in an oven at 40 °C. The dried plants
were powdered by grinding in a cyclotec-grinding machine (200 mesh). The
powdered plant (100 g) was extracted as shown in Fig. 1.
The plant powdered (100 g) was treated with ethanol (99%, 3x500 mL,
8 h, Fig. 1) at room temperature (25 °C). The ethanol
extract (A) was collected by suction through a Buchner funnel. The residue
was air dried for 8 h followed by drying at 40 °C for 18 h in an oven
and ground to a powder. This was further extracted with aqueous 80% ethanol
(2x500 mL, 30 min) at reflux temperature (B). The combined ethanol extract
(A+B) was evaporated to remove ethanol. The final volume was adjusted
to 500 mL by adding water and was distributed between chloroform to remove
lipids and water (3x500 mL).
Isolation of Phenolic Acids
The water fraction was adjusted to pH 2.5 by adding 1 M HCl. It was
then extracted with ethyl acetate (3x200 mL) and the combined extract
was evaporated after drying with anhydrous sodium sulphate (EA-1). The
residual aqueous solution was divided into two portions. To one portion
sodium hydroxide solution (2 M) was added to a final concentration of
1M. after standing for 20 h at room temperature (22 °C), the solution
was adjusted to pH 2.5 and extracted with ethyl acetate (EA-2). The other
portion was treated with pectinase (Sigma, P5146 from Aspergillus niger)
for 10 h, adjusted to pH 2.5 and extracted with ethyl acetate (3x100 mL)
(EA-3). EA-2 and EA-3 were dried and evaporated as described for EA-1.
Part of the ethanol-insoluble residue was treated with 1 M sodium hydroxide
for 6 h under nitrogen at room temperature. The mixture was neutralized
with 2 M sulphuric acid and filtered and the filtrate was adjusted to
pH 2.5 and extracted with ethyl acetate (EA-4) as described above. The
yields of the ethyl acetate extracts which contained free (EA-1), ester-linked
(EA-2) and glycosidically linked (EA-3) phenolic acids extractable with
aqueous ethanol and those bound to polysaccharides and/or lignin (EA-4)
are given in Table 1-4 along with
the amounts of the eight identified phenolic acids.
HPLC Analysis of Phenolic Acid Fractions
The extracts (EA-1 to EA-4) were dissolved in 10 mL ethanol in a measuring
flask. Portions (2 mL) of each were transferred to a small conical flask
and evaporated to dryness by flushing with nitrogen. The dried extracts
were dissolved in the mobile phase (2 mL, 30% MeOH in 0.01M phosphate
buffer, pH 2.8) containing p-methoxyphenyl acetic acid (0.45 mg,
internal standard) and were filtered. The filtrates were analyzed by HPLC
on Supelco C18 column using 0.01 M phosphate buffer (pH 2.8):
MeOH, 70:30 v/v for 15 min to 60:40 for 25 min with a flow rate of 1 mL
min-1. Compounds were identified by comparison of retention
times and UV spectra with those of appropriate standards analyzed under
the same conditions. Quantitative determination was performed at 320 nm
for cinanamic acid derivatives (ferulic, p-oumaric, caffeic and
sinapic acids) and 254 nm for benzoic acid derivatives (p-hydroxy
benzoic, 3, 4-dihydroxybenzoic, vanillic and syringic acids).
RESULTS AND DISCUSSION
It is very important to determine phenols in plants, both qualitatively
and quantitatively. A number of analytical methods have been proposed
for the separation and determination of phenolic compounds. Most of these
protocols are based on a high performance liquid chromatography (HPLC)
technique with UV spectrophotometry because derivatization is not required
prior to analysis (Mattila and Kumpulanen, 2002; Justesen and Knuthsen,
2001; Merken and Beecher, 2000). Before HPLC analysis, hydrolysis of glycosides
or esters was necessary, so as to determine phenolic content, since a
considerable fraction is in bound form (Lee and Widmer, 1996). Extraction
was performed with a mixture of 80-90% aqueous methanol or ethanol. They
have a protective role and can prevent phenolic compounds from being oxidized
by enzymes, such as phenoloxidases (Harborne, 1998). Columns employed
to separate phenolics are almost exclusively reversed-phase. This system
is a high resolution chromatographic technique widely used for simultaneous
separation and quantification of phenolic substances.
Phenolic acids are considered as allelopathic compounds and these play
important role in biological process (Chung et al., 2001). Like
other plants, Cajanus cajan (Nahar et al., 1990), Fissistigma
rubiginosum (Nahar et al., 2001), Ficus racemosa (Islam
et al., 2001), Macrotyloma uniflorum was also found to contain
several phenolic acids in varying amounts (Table 1-4).
Extractable phenolic acids were isolated in three batches as free (EA-1),
ester linked (EA-2) and glycosidically bound (EA-3) (Fig.
1) from aerial part of M. uniflorum plant. The latter two batches
were released, respectively by alkali and pectinase treatment. Non-extractable
bound phenolic acids (EA-4) were isolated by extraction of the ethanol-insoluble
plant residue with alkali. The extractable glycosidically linked fraction
EA-3 contain highest amount of phenolic acids among the other three fractions.
p-Coumaric acid was the major component followed by p-hydroxy
benzoic acid of that fraction. The non-extractable bound phenolic acid
fraction (EA-4) contained five different phenolic acids namely 3, 4-dihydroxy
benzoic, p-hydroxy benzoic, p-coumaric, ferulic and sinapic
acids. p-Hydroxy benzoic acid (0.69 mg/100 g of plant dry material)
was the major component of EA-4 (Table 4). Small amount
of 3,4-dihydroxy benzoic acid was found only in bound phenolic acid fraction
EA-4. 3,4-Dihydroxy benzoic, p-hydroxy benzoic, p-coumaric
and syringic acids were found to present in the extractable glycosidically
linked phenolic acid fraction, EA-3 whereas, p-coumaric acid (8.95
mg/100 g of plant dry material) was the major component and syringic acid
(0.11 mg/100 g of plant dry material) was the minor component of EA-3.
Extractable ester linked fraction, EA-2 contain only two phenolic acids,
p-coumaric- and ferulic acids whereas amount of p-coumaric
acid was higher than ferulic acid. Free phenolic acid fraction, EA-1 contained
six phenolic acids namely, p-hydroxy benzoic, p-coumaric,
caffeic, ferulic, vanillic and syringic acids. But p-coumaric
acid (2.17 mg/100 g of plant dry material) was the major and syringic
acid (0.05 mg/100 g of plant dry material) was the minor component of
EA-1 fraction. Vanillic and caffeic acids were absent in all the fractions
|| Fractionation scheme of extraction and isolation of
The identified and quantified phenolic acids displayed in Fig.
2-3 shows the structures and chromatogram of phenolic
acids, respectively. A good resolution, with sharp peaks was achieved
for all the phenolic compounds within 20 min when the solvent system and
the chromatographic conditions reported in the materials and methods section
were employed. Typical phenolics that possess antioxidant activity are
known to be mainly phenolic acids and flavonoids. Phenolic acids are a
major class of phenolic compounds, widely occurring in the plant kingdom
especially in fruits and vegetables. Selected phenolics in several species,
separated and identified by the RP-HPLC method are shown in Table
1-4. Considerable variation was found in phenolic
compounds of different species. Because of the diversity and complexity
of the natural mixtures of phenolic compounds in hundreds of herb extracts,
it is rather difficult to characterize every compound and elucidate its
structure, but it is not difficult to identify major groups and important
aglycones of phenolic compounds. Many medical herbs and species have been
studied and to some extent their phenolic chemistry is known (Cai et
Antioxidant activity of M. uniflorum is a result of phenolic
acid, especially caffeic and p-coumaric, acid content. The 3,4-position
of dihydroxylation on the phenolic ring in caffeic acid showed increased
antioxidant activity as compared to p-coumaric acid (Kim and Lee,
2004). Caffeic acid is expected to have higher antioxidant activity because
of additional conjugation in the propenoic side chain, which might facilitate
the electron delocalization, by resonance, between the aromatic ring and
|| Structures of the identified and quantified phenolic
The specific aims were to isolation of phenolic acids in M. uniflorum
plant and eight phenolic acids were identified and quantified by using
quantitative reversed phase HPLC method. This study shows that this plant,
rich in phenolic acids could be a good source of natural antioxidants.
The authors are indebted to the Chairman of the Department of Chemistry,
University of Dhaka to give the opportunity to carry out this present
study. We are also thankful to Prof. M. Salar Khan of Dhaka University
for identification of the plant.
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