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Abstract

The species Alpinia purpurata is scarcely cited as to ethnopharmacology and phytochemistry. This study aimed to analyze bioactive compounds through high-performance liquid chromatography (HPLC). Hydroalcoholic crude extract was obtained from A. purpurata dried leaves. Folin-Ciocalteau method was used to quantify total phenols, using gallic acid as standard. The obtained result was 15.6 mg GAE g-1. The crude extract was partitioned with the solvents ethyl acetate and butanol, followed by thin-layer chromatography (TLC) and HPLC. The flavonoids kaempferol-3-O-glucuronide and rutin were detected at a higher concentration in ethyl acetate and butanolic extracts. The butanolic extract contains the highest flavonoid percentage (94.3%). A. purpurata presents important flavonoids of therapeutic use, already verified for A. zerumbet. This is the first study verifying the presence of flavonoids in A. purpurata extracts.
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Recebido para publicação em 29/02/2008
Aceito para publicação em 06/10/2008
Detection of flavonoids in Alpinia purpurata (Vieill.) K. Schum. leaves using high-
performance liquid chromatography
VICTÓRIO, C.P.1*; KUSTER, R.M.2; LAGE, C.L.S.1
1 Laboratório de Fisiologia Vegetal, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro, Av. Carlos Chagas Filho, s/n, CCS, Bloco G, sala G2-050. Rio de Janeiro - RJ, 21941-902. Brazil.
*crispv@biof.ufrj.br 2 Laboratório de Fitoquímica, Núcleo de Pesquisas de Produtos Naturais (NPPN), Universidade
Federal do Rio de Janeiro, Av. Carlos Chagas Filho, s/n, CCS, Bloco H, Rio de Janeiro - RJ, 21941-902. Brazil.
ABSTRACT: The species Alpinia purpurata is scarcely cited as to ethnopharmacology and
phytochemistry. This study aimed to analyze bioactive compounds through high-performance
liquid chromatography (HPLC). Hydroalcoholic crude extract was obtained from A. purpurata
dried leaves. Folin-Ciocalteau method was used to quantify total phenols, using gallic acid as
standard. The obtained result was 15.6 mg GAE g-1. The crude extract was partitioned with the
solvents ethyl acetate and butanol, followed by thin-layer chromatography (TLC) and HPLC. The
flavonoids kaempferol-3-O-glucuronide and rutin were detected at a higher concentration in ethyl
acetate and butanolic extracts. The butanolic extract contains the highest flavonoid percentage
(94.3%). A. purpurata presents important flavonoids of therapeutic use, already verified for A.
zerumbet. This is the first study verifying the presence of flavonoids in A. purpurata extracts.
Key words: TLC, polyphenols, medicinal plants, rutin, Zingiberaceae
RESUMO: Detecção de flavonóides em folhas de Alpinia purpurata (Vieill.) K. Schum.
por cromatografia líquida de alta eficiência. A espécie Alpinia purpurata apresenta poucas
citações referentes a etnofarmacologia e fitoquímica. Este estudo propõe a análise de substâncias
bioativas através da técnica de cromatografia líquida de alta eficiência (CLAE). O extrato bruto
hidroalcóolico foi obtido a partir de folhas secas de A. purpurata. A quantificação de fenóis totais
foi realizada pelo método de Folin-Ciocalteau, usando ácido gálico como padrão. Como resultado,
foi verificado 15,6 mg EAG g-1. O extrato bruto foi particionado com os solventes acetato de etila
e butanol e depois analisado por cromatografia em camada delgada e CLAE. Nos extratos acetato
de etila e butanólico foi detectada a presença dos flavonóides kaempferol-3-O-glicuronídeo e
rutina, em maior concentração. O extrato butanólico contém a maior porcentagem de flavonóides
(94,3%). Esta espécie possui flavonóides importantes no uso terapêutico, já antes verificados
para a espécie A. zerumbet. Este é o primeiro trabalho que verifica a presença de flavonóides em
extratos de A. purpurata.
Palavras-chave: CCF, polifenóis, plantas medicinais, rutina, Zingiberaceae
INTRODUCTION
The genus Alpinia (Zingiberaceae family,
Alpinioideae subfamily, Alpinieae tribe) is native to
tropical and subtropical Asia (Kress et al., 2002).
Nowadays, it is cultivated in several places around
the world due to the attractive beauty of its
inflorescences and its therapeutic potential (Soares
de Moura et al., 2005; Victório, 2008). In addition,
these plants are important sources of raw material for
many useful products: foods, spices, medicines,
perfumes, dyes and fiber paper (Tomlinson, 1969).
A. purpurata (Vieill.) K. Schum (red
inflorescence) is an herbaceous perennial plant,
internationally known in the ornamental plant market
as potted plant, landscape accent and cut flower
(Morón, 1987; Kress et al., 2002). An ethnobotanical
study developed in a community from Trujillo State,
Venezuela, investigated the use of its flowers as
decoction for cough (Bermúdez & Velásquez, 2002).
Rev. Bras. Pl. Med., Botucatu, v.11, n.2, p.147-153, 2009.
148
Zoghbi et al. (1999) analyzed A. purpurata essential
oil composition, which showed notable antibacterial
activity. However, studies about A. purpurata
phytotherapeutic potential are scarce; most scientific
works with this species are directed to the
improvement of its production as an ornamental plant,
including the evaluation of postharvest treatment as
an alternative to chemical insecticides, indications of
harvest and postharvest procedures, tolerance to
fumigation, biological control of pests, in vitro storage
of multiple shoots and micropropagation (Morón, 1987;
Dekkers et al., 1991; Illg & Faria, 1995; Hara et al.,
1997; Chen & Paull, 1998; Anderson & Gardner, 1999;
Gonzalez & Mogollon, 2001; Sangwanangkul et al.,
2008). The presence of A. purpurata in several parts
of Brazil allows easy access to it. Thus, this species
is an available resource to phytotherapeutic treatment.
Several species of the Zingiberaceae family present
antioxidant property mainly due to the considerable
presence of flavonoids such as rutin, quercetin,
alpinetin and different types of kaempferol in the genus
Alpinia (Table 1) (Williams & Harborne, 1977;
Mpalantinos et al., 1998; Vankar et al., 2006). Pugialli
et al. (1993) studied Zingiberaceae chemotaxonomy
and considered that flavonoids and their structural
variety are taxonomic markers. Zingiberaceae family
is at a higher level within the superorder Zingiberiflorae
due to the use of protection mechanisms of phenolic
hydroxyl groups: glycosylation and methylation.
Investigations of phytochemical compounds have been
important tools to study plant classification and
evolution (Kaplan & Gottlieb, 1982). This study
proposes a phytochemical approach to A. purpurata
based on the chemotaxonomy of the genus Alpinia,
which presents high therapeutic potential (Bleier &
Chirikdjian, 1972; Mendonça et al., 1998; Mpalantinos,
2001; Kim et al., 2006).
The aim of the present study was to evaluate
the phytochemistry of hydroalcoholic extracts from
A. purpurata leaves for the presence of the flavonoids
rutin, kaempferol-3-O-rutinoside and kaempferol-3-O-
glucuronide, reported in the scientific literature for their
therapeutic action.
MATERIAL AND METHOD
Plant material
A. purpurata leaf samples were collected from
plants growing in the city of Rio de Janeiro in the
Federal University of Rio de Janeiro (Rio de Janeiro
State, Brazil). The voucher specimen was identified
and deposited at the Herbarium of Rio de Janeiro
Botanical Garden under the accession number RB
433484.
Preparation of extracts and Fractions
A. purpurata leaves were collected from adult
plants in the morning; then, the plant material was
dried and ground in 70% ethanol for a week. After the
first extraction, leaves were kept in ethanol (100%)
until exhaustive extraction. Crude extracts were filtered
and dried through evaporation at 60ºC in a rotary
evaporator and through freeze-drying. From 1009 g
dried leaves, 112.5 g dried crude extract was obtained.
The yield was calculated as percentage, according
to the formula: (crude extract weight/plant material
weight) x 100. A 59.4g crude extract fraction was
resuspended in methanol:water (9:1, v/v) and
partitioned in different solvents of increasing polarity
range: hexane, dichloromethane, ethyl acetate and
n-butanol. Hexane partition was separated and
evaporated to dryness (4.1 g). The residue was dried
TABLE 1. Flavonoids reported for the genus Alpinia.
**The abstract did not cite.
Rev. Bras. Pl. Med., Botucatu, v.11, n.2, p.147-153, 2009.
149
using a rotary evaporator until methanol elimination;
then, the aqueous layer was further partitioned using
solvents and evaporated to dryness in order to obtain
dichloromethane (0.29 g), ethyl acetate (0.31 g) and
n-butanol (4.8 g) partitions. Each solvent extractor
(50 to 60 mL) was used five times.
Flavonoid standards
Flavonoids of the kaempferol class were
isolated from Alpinia zerumbet Roxb. and identified
using Nuclear Magnetic Resonance (NMR)
(Mpalantinos et al., 1998). Rutin was purchased from
Merck. Kaempferol-3-O-glucuronide had 82% purity,
kaempferol-3-O-rutinoside, 91%, and rutin, 98%.
Purity was verified using three replicate injections of
standards into HPLC.
Evaluation of phenolic compound content
Total phenolic compounds were determined
using the Folin-Ciocalteau method. Hydroalcoholic
extracts were dissolved in ethanol (70%) at 1mg mL-1.
A 0.5 mL aliquot of diluted extract and 2 mL Folin-
Ciocalteau reagent (10%) were added after 3 min,
together with 2 mL of 7.5% sodium carbonate, and
mixed. The mixture was homogenized and incubated
at 50oC for 30 min. Absorbance was measured at 740
nm in a spectrophotometer using gallic acid as
standard. Two controls were used: (1) Folin-Ciocalteau
+ sodium carbonate and (2) crude extract solution.
Phenolic compounds in crude extracts were quantified
through the regression equation of calibration curves:
y = 0.0229x + 0.0968 (R2 = 0.9993), and expressed
as mg gallic acid equivalents (GAE) per 1 g dried
leaves. All measurements were done in triplicate. The
analyses included A. purpurata and A. zerumbet
samples collected in June (2006) and February (2007).
Thin-layer chromatography (TLC)
Aliquots of standards, and crude, ethyl
acetate and butanolic extracts were spotted on TLC
plate (silica gel 60 F254 nm, Merck) and developed in
ethyl acetate, formic acid and distilled water (65/20/
15, v/v/v) mobile phase. TLC was observed under UV
spectrum at 254 and 360 nm before and after spraying
with NP/PEG reagent. The flavonoid standards of rutin
(Rf = 0.69), kaempferol-3-O-rutinoside (Rf = 0.76) and
kaempferol-3-O-glucuronide (Rf = 0.83) were verified
in the extracts after concomitant running with
standards (Victório et al., 2007).
High-performance liquid chromatography
(HPLC) Crude, ethyl acetate and butanolic extracts
were dissolved in methanol (70%) at 20 mg mL-1 and
filtered under vacuum; HPLC-UV analyses were
performed in a Shimadzu apparatus equipped with
SPD-M10A diode array detector, LC-10AD pump and
CBM-10 interface. Data were obtained and processed
in a reversed phase column (Lichrosorb RP-18, 25
cm x 5 mm), at room temperature. Separation was
done in the following mobile phase: MilliQ water +
0.1% phosphoric acid (A) and methanol (B): 1-10 min
(30% B); 20 min (40% B); 60 min (100% B). The
prepared mobile phase was degassed using ultrasonic
agitation. After 61 min, the gradient was recycled to
the initial conditions and held for 10 min before a new
injection. The flow rate was kept constant at 1 mL
min-1 and peaks were detected at 254 nm and 360
nm. All chemicals used in the analysis, such as
methanol and phosphoric acid, were of HPLC grades
and were purchased from Merck. MilliQ water was
used in HPLC mobile phase and sample preparation.
Standards were dissolved in 70% methanol at 1 mg.mL-
1 and analyzed in the same elution. Injections were
done in triplicate. Linearity was observed in the
concentration range 0.0078 - 0.0625 mg mL-1 rutin and
0.01325 - 0.25 mg mL-1 kaempferol-3-O-glucuronide.
Flavonoids in the extracts were quantified against
calibration curves of standards, where y is the peak
area and x the concentration in mg mL-1 (Figure 1).
FIGURE 1. Calibration curves of rutin and kaempferol-3-O-glucuronide standards.
Rev. Bras. Pl. Med., Botucatu, v.11, n.2, p.147-153, 2009.
150
Flavonoid detection
Flavonoids were detected through retention
times (RT), ultraviolet spectrum compared with
flavonoid standard spectrum (Figure 2) and coinjection
with authentic samples analyzed under the same
conditions. For coinjection, a mixture (1:1, v/v) of
extracts at 20 mg mL-1 and standard at 1 mg mL-1
was prepared. The purity of each flavonoid peak in
Figures 4 and 5 was assessed by comparing the UV
spectra at upslope and downslope inflexion points for
both wavelengths (254 and 360 nm).
RESULT AND DISCUSSION
Phenolic compounds present antioxidant
activity; thus, they are considered important therapeutic
agents. As already known, antioxidants reduce the
effects of excessive free radical production in critically
ill patients affected by diseases like cancer,
cardiovascular disturbances, and brain dysfunction
(Atoui et al., 2005). Folin-Ciocalteau method can quantify
the presence of flavonoids and other phenolic
compounds in plant material. There were significant
dif ferences in total phenolic content (p<0.03)
between A. purpurata and A. zerumbet in both
evaluated months (Figure 3). A. zerumbet was used
for comparison since it is greatly employed in folk
medicine and has been extensively reported
concerning phytochemistry and phytotherapy.
The chromatographic profile of A. purpurata
shows rutin and kaempferol-3-O-glucuronide peaks,
similarly to those for A. zerumbet (Figure 4) in opposite
proportions. These flavonoids were verified in ethyl
FIGURE 2. UV spectra of (A) rutin (RT= 32.5 min) and (B) kaempferol-3-O-glucuronide (RT= 35.2 min).
FIGURE 3. Comparison of total phenolic content extracted from Alpinia zerumbet and A. purpurata collected in two
different periods of the year. A (total phenols in mg GAE g-1 dried leaves), B (June, 2006) and C (February, 2007).
Rev. Bras. Pl. Med., Botucatu, v.11, n.2, p.147-153, 2009.
151
acetate and butanolic extracts between 20 and 40
min (Figure 5). The main difference was detected
between 40 and 60 min, when A. zerumbet crude
extract showed more compounds in the
chromatographic profile than A. purpurata (Figure 4).
The flavonoid kaempferol-3-O-rutinoside was not
detected through TLC or HPLC in the extracts. HPLC
and TLC methods were reproducible. UV spectrum of
peaks 1 and 3 of the chromatogram (Figure 5) are
characteristic of flavonoids; however, for an accurate
identification, they should be isolated in SEPHADEX
column using polar solvents from ethyl acetate and
butanolic fractions, followed by structural elucidation
using the spectroscopy technique. Rutin and
kaempferol-3-O-glucuronide were isolated from A.
zerumbet and also detected in A. purpurata at the
following concentrations, respectively: 17.8 and 8.7
mg g-1 dried leaves (ethyl acetate) and 356 and 85.5
FIGURE 4. Comparison between chromatographic
profiles of crude extracts from Alpinia purpurata (A)
and A. zerumbet (B): rutin (1) and kaempferol-3-O-
glucuronide (2). Values obtained at 254 nm.
FIGURE 5. Chromatographic profile of Alpinia
purpurata: crude extract (A), butanolic extract (B) and
ethyl acetate extract (C). Rutin (2) and kaempferol-3-
O-glucuronide (4). Values obtained at 254 nm.
TABLE 2. Quantitative analysis of flavonoids rutin and kaempferol-3-O-glucuronide in extracts of dried leaves of
Alpinia purpurata. Values obtained at 254 nm.
Data represent mean of triplicates.
Rev. Bras. Pl. Med., Botucatu, v.11, n.2, p.147-153, 2009.
152
phytoterapeutic resources.
ACKNOWLEDGMENT
The authors are grateful to Coordination for
the Improvement of Higher Education Personnel
(CAPES) for the fellowship to the first author and
PROAP/PROEX for financial support.
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... Typical TPC contribute more to plant AA are mainly phenolic acids and flavonoids. The matrix difference among plant species from different geographical origin and variance in genetics and cultivation conditions make the correlation analysis between AA with TPC and TFC a challenging work [21,23,28,29,31,35,36]. We adopted the Pearson correlation coefficient (PCC), also referred to as Pearson's r, to express the strength and direction of the linear relationship of correlation. ...
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... Micropropag., Lavras, v.4, n.2, p. 92-98, 2008 used for cough in Venezuela and presents antibacterial activity (BERMÚDEZ & VELÁSQUEZ, 2002;ZOGHBI et al., 1999). Recent studies have showed the phytochemical potential of this species (VICTÓRIO et al., 2007(VICTÓRIO et al., , 2008. ...
... Leaves of A. purpurata from field and in vitro systems were dried and macerated in 70% ethanol for 45 min using ultrasonic bath according to Victório (2008). Crude extracts were filtered and dried by evaporation at 60ºC in rotary evaporator and by lyophilization. ...
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... K. Schum and Alpinia galanga (L) Willd. In pharmacological tests, galangal extract exhibits promising potentials, which has a reduced blood pressure effect [2], antioxidant activity, anticancer [3], antibacterial, antiviral, antifungal, antiparasitic [4], antiinflammatory [5], antitumor, analgesics, and antiflatulence [6]. Phytochemical test results from red galangal ethanol extract contain alkaloid compounds, phenols, flavonoids, and tannins [5]. ...
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... According to Kochuthressia et al. [27] , [20,21,28] , which present antimicrobial properties [29] . albicans, but a marked inhibition of other fungi, such as C. neoformans, F. pedrosoi, T. rubrum, M. canis and M. gypseum. ...
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... The samples were subsequently stored in separate sample bottles for further study. The modified method of Victório et al. (2009) was applied for the extraction of plant material. The samples (2 gm each) were extracted with 20 ml methanol at room temperature for 24-h with occasional shaking. ...
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