Microchemical Journal 135 (2017) 10–15

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Microchemical Journal

journ al h om e pa ge : ww w . e l s e v i e r . c o m / l o c a t e / m i c r oc

Determination of bioactive phenolics in herbal medicines containing

Cynara scolymus, Maytenus ilicifolia Mart ex Reiss and Ptychopetalum
uncinatum by HPLC-DAD☆
Ramon Rodrigues Sá a, Rafael Amorim Matos a, Vagner Cardoso Silva a, Jamile da Cruz Caldas a,
Maria Celeste da Silva Sauthier a,b,d, Walter Nei Lopes dos Santos a,d,
Hemerson Iury Ferreira Magalhã es c, Aníbal de Freitas Santos Jú nior a,⁎
a
Universidade do Estado da Bahia, Rua Silveira Martins, 2555, Cabula, 41195-001 Salvador, BA, Brazil
b
Instituto Federal de Educação, Ciência e Tecnologia Baiano – Campus Governador Mangabeira, Rua Waldemar Mascarenhas, s/n, Portão (Estrada Velha da Chesf), 44350-000 Gov. Mangabeira,
BA, Brazil
c
Universidade Federal da Paraíba, Cidade Universitária, s/n, Castelo Branco, 58051-900, João Pessoa, Paraíba, Brazil
d
Universidade Federal da Bahia, UFBA, Instituto de Química, Departamento de Química Analítica, Campus de Ondina, Av. Adhemar de Barros, s/n, Ondina, 40170-290 Salvador, BA, Brazil

a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 March 2017 In this study, an HPLC-DAD method was developed and validated for the quantification of bioactive phenolics
Received in revised form 8 June 2017 in herbal medicines containing Cynara scolymus (Globe artichoke), Maytenus ilicifolia Mart ex Reiss
Accepted 21 July 2017 “Espinheira santa” and Ptychopetalum uncinatum “Marapuama”. The samples were lyophilized and 5.0 g of solid
Available online 21 July 2017 were extract- ed with 30 mL of methanol acidified with 100 μL of concentrated HCl, under magnetic stirring at 40
°C for 30 min. Separation was carried out on a C18 column with analytical solvents constituting a binary
Keywords: elution mixture, consisting of (A) ultrapure water (Millipore, USA), containing 1.0% acetic acid (v v−1) and
Bioactive phenolics (B) methanol (HPLC grade). Spectrophotometric detection was performed at a wavelength of 260 nm for
Herbal medicines vanillic acid; 280 nm for (+) – catechin and 330 nm for chlorogenic acid. The method to determine bioactive
Quality control
phenolics in herbal medicines showed adequate linearity, repeatability and accuracy. The limits of detection
HPLC-DAD
(LOD) and quantification (LOQ) were 0.025 μg g−1 and 0.031 μg g−1, respectively. The concentrations
(minimum–maximum in mg g−1) of chlorogenic acid (in samples containing C. scolymus) and vanillic acid (in
herbal medicines containing P. uncinatum “Marapuama”) ranged from 71.28 to 925.99 and 17.35 to 19.21,
respectively. The catechin content was
0.69 mg g−1 in Maytenus ilicifolia Mart ex Reiss “Espinheira santa”. Therefore, the results showed that the
devel- oped method is simple, less toxic, fast and reliable for the determination of bioactive phenolics in herbal
medicines.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
medicinal plants has been widely studied [4] due to their
pharmacolog- ical and antinutritional activities, besides the inhibition
The World Health Organization (WHO), in 1991, recognized the
of lipid oxida- tion and fungal proliferation, as well as their
im- portance of natural products and advocated the formulation of
participation in processes responsible for colour, astringency and
policies and regulations for the use of traditional medicines [1]. In this
smell in various foods [5]. Poly- phenols are secondary metabolites
context, the consumption of Medicinal Plants and Herbal Medicines
widely distributed in the plant king- dom and act as: antioxidants,
has been in- creasing, and the need for greater quality control by the
metal chelators, antimutagens, anticancer, antifungals and anti-
Pharmaceutical Industry becomes a relevant factor to ensure safety
allergic substances [6].
and efficacy [2,3].
Cynara scolymus L. (Asteraceae) “Globe artichoke”, Maytenus ilicifolia
The search for natural antioxidants has increased with the
Mart ex Reiss (Celastraceae) and Ptychopetalum uncinatum
discovery of the properties of components that are produced by
(Olacaceae) are popularly known in Brazil as “alcachofra”, “espinheira
plants through their secondary metabolism. The presence of
santa” and “marapuama”, respectively, and are grown in South
phenolic compounds in
America. “Globe ar- tichoke” is used in the treatment of: biliary and
hepatic diseases, eczema and rashes, constipation, diabetes mellitus
☆ Selected paper from the 4th Uruguayan Conference on Analytical Chemistry, 25-28 and dyslipidemia, as an antisclerotic agent [7,8]. Furthermore, it is
September, 2016, at the Convention Center-Torre de las Telecomunicaciones ANTEL, in popularly used as a coadjuvant in the treatment of anemia, scurvy and
Montevideo. rickets due to its iron, vitamin C and calcium content [9]. In the
* Corresponding author.
literature, studies have shown that leaves contain phenolic chemical
E-mail address: afjunior@uneb.br (A. de Freitas Santos Jú nior).
constituents, including
http://dx.doi.org/10.1016/j.microc.2017.07.009
0026-265X/© 2017 Elsevier B.V. All rights
reserved.
 R.R. Sá et al. / Microchemical Journal 135 (2017)

caffeine-like acids (chlorogenic acid, 1,5-dicapheoilquinic acid and drugstores in Salvador, Bahia, Brazil. The formulations did not contain
cinnarine), flavonoids (scolymoside and cynaroside), sesquiterpenes excipients, according to
(Cynaropricrin) in a smaller amount, and various aliphatic acids,
espe- cially hydroxyacids (lactic, glycolic, malic and
hydroxymethylacrylic acids) [10,11,12].
Studies with Maytenus ilicifolia Martius ex Reiss began in the
1960′s,
stimulated by its effectiveness in the treatment of ulcers and cancer,
in addition to its antioxidant effect [13,14,15,16]. In the literature,
phyto- chemical studies using High performance liquid
chromatography (HPLC) showed that leaves containing phenolic
compounds, quercetin and kaempferol; condensed tannins (e.g.
catechin, epicatechin, and epi- gallocatechin) as monomers and
triterpenes, such as friedelin, reduce gastric hypersecretion [17],
motility and gastric emptying [18], in addi- tion to presenting a
hypotensive effect [19].
Ptychopetalum uncinatum is native to the Amazon rainforest,
region- ally known as “marapuama, muiratã , muirapuama, pau-
homem and liriosma"” There are hypotheses about its secondary plant
metabolism, and data on its phytochemical composition and
pharmacological func- tions are not yet fully elucidated. However, few
studies report its use for sexual dysfunction and stimulating action on
the central nervous system, attributed to the presence of alkaloids,
tannins and essential oils [20,21,22].
Currently, monitoring the quality of pharmaceutical solid oral dos-
age forms (capsules and tablets) has gained greater importance to
phar- maceutical companies and regulatory authorities. Several
herbal products are in oral solid form and, despite the use of this test
in the quality control of synthetic drugs, their use in the evaluation of
natural products has not yet been widely adopted [23,24,25].
Although the use of Globe artichoke and “Espinheira santa” is
wide-
spread in medicine and public health in Brazil and other countries of
South America, as well as the high consumption of Ptychopetalum
uncinatum in regions of Brazil, in vitro monitoring of bioactive phenolics
by HPLC-DAD was proposed. In this study, quantification methods by
HPLC were developed and validated in order to evaluate gelatin
capsules of Maytenus ilicifolia Mart ex Reiss and Ptychopetalum
uncinatum, besides plant drugs and tablets containing Cynara scolymus
L. “Globe artichoke” purchased from the market, using the release of
two phenolic acids (Chlorogenic and vanillic acids) and catechin,
quantified by HPLC-DAD.

2. Experimental

2.1. Reagents and plant materials

Analytical grade reagents were used in the development of this

study and all solutions were prepared using high purity water
obtained from a Milli-Q water purification system (Millipore, Bedford,
USA). All laboratory material was decontaminated in a 10% (v v −1)
HNO3 solu- tion bath for 24 h and rinsed with high-purity water.
Subsequently, all materials were dried under clean-air conditions at
room temperature. All solvents and reagents were of the highest
commercially available purity grade.
Catechin (Sigma-Aldrich® C1251 lot: BCBH43V assigned purities
of 98.9), Chlorogenic and vanillic acids (Sigma-Aldrich®) were used.
HPLC grade methanol and acetic acid were obtained from Vetec. All
chemicals used were of analytical grade with no further purification.
Hydrochloric acid (HCl) was purchased from Quimex® (Merck,
Brazil). The solutions were prepared from reference stock solutions
(1000.0 μg mL−1), from rates and appropriate dilutions necessary to
prepare ref- erence solutions with a final concentration ranging from
1.0 to 50.0 μg mL−1. The solutions were stored in previously
decontaminated poly- ethylene bottles.
The samples of herbal medicines, commercially available in the
form of gelatin capsules (Maytenus ilicifolia Mart ex Reiss and
Ptychopetalum uncinatum) and tablets (Cynara scolymus L.) “Globe
artichoke” contain- ing the dry extract, were obtained from local
 R.R. Sá et al. / Microchemical Journal 135 (2017)
the label. Plant drugs (Cynara scolymus L. “Globe artichoke” and
Ptychopetalum uncinatum) were purchased at fairs in Salvador,
Bahia, Brazil. All samples were originally stored in closed plastic
containers until analysis.

2.2. Sample preparation

The extracts were prepared in triplicate by adding 30 mL of

metha- nol acidified with 100 μL of concentrated hydrochloric acid
to 0.5 g of herbal medicine mass and stirring for 30 min on a shaker
table (QUIMIS Q-225-M) at 100 rpm. The extracts were filtered and
concentrated on a rotary evaporator (IKA RV 10) at 40 °C and 40
mbar. The resulting ma- terial was solubilized in 1.5 mL of
methanol, stored at −20 °C and fil- tered through a PTFE (0.45 μm)
syringe filter prior to HPLC and spectroscopic determinations.

2.3. Chromatographic instruments and conditions

LC-DAD analysis was performed on a Shimadzu liquid

chromatogra- phy system (Shimadzu Scientific Instruments, Japan),
consisting of a high-pressure quaternary pump (LC-20 CE,
Shimadzu); Degasser vacu- um; Manual high-pressure injection
valve (20 μL injection loop) and a photodiode array (PDA) detection
system (SPD-20A, Shimadzu). The liquid chromatograph was
equipped with a “Licrhospher®” RP 18 col- umn (Agilent), 5 μm, 4.6
× 250 mm, controlled by the LC-System software.
LC separation was carried out at 40 °C with analytical solvents,
con- stituting a binary elution mixture consisting of methanol (HPLC
grade), with (A) 1.0% acetic acid (v v −1) and (B) (methanol).
Chromatography was carried out for 40 min at a constant flow rate
of 1.0 mL min −1. The gradient program was as follows: 0–10 min,
100% A; 10–20 min, 70% A; 20–30 min, 10% A; 30–37 min, 70% A
and 37–40 min, 100% A.
Plants produce various chemicals and some can be used as
chemical markers. From the data obtained in the scientific
literature, an active metabolite was studied per herbal medicine:
chlorogenic acid (CGA) in Cynara scolymus; Vanillic acid (VA) in
Ptychopetalum uncinatum and catechin in Maytenus ilicifolia Mart ex
Reiss. In a preliminary qualitative analysis, they were tested at
different wavelengths (nm), in accordance with the literature. UV
detection was performed at 260 nm for vanillic acid; 280 nm for
(+)–catechin and 330 nm for chlorogenic acid. Identi- fication and
qualitative analyses were performed by comparison with standard
spectra at each retention time (Tr) [26].

2.3.1. Validation of chromatographic conditions

All samples and standards were analyzed in triplicate and
precision was evaluated by the performance of intra-day and inter-
day assays by six replicated injections of the standard solutions.
Limits of detection (LOD) and quantification (LOQ), detected as the
injection concentration provided peak heights 3- and 10-fold the
signal-to-noise ratio (s/n), were acquired. The analytical method for
the quantification of catechin, chlorogenic and vanillic acids was
validated by HPLC, according to Anvisa [27,28] and ICH Guidelines
[29]. In order to demonstrate wheth- er the method was adequate, it
was validated through the analysis of stability, linearity, precision
and accuracy parameters.

2.3.1.1. Stability and specificity. The stability of solutions and samples

of catechin, chlorogenic and vanillic acids was evaluated for 24 h at
room temperature, kept at 37 °C for 2 h in 0.01 and 0.1 mol L −1 HCl,
by checking changes in the analytical signal after analyzed by HPLC-
DAD. Specificity was determined by analyzing the chromatographic
profile (retention time) of standard compounds at 0.1 mol L−1 HCl
(blank), to verify the presence of interference. The formulations did
not contain excipients.

2.3.1.2. Linearity. Aliquots of 1000.0 μg mL−1 reference solutions of

cat- echin, chlorogenic and vanillic acids, prepared with 0.1 mol
L−1 HCl,
 R.R. Sá et al. / Microchemical Journal 135 (2017)

were transferred to volumetric flasks to obtain the final phenolic in Ptychopetalum uncinatum were slighty higher in the
concentrations of 1.0; 5.0; 15.0; 20.0; 25.0; 30.0; 40.0 and 50.0 μg capsules. The
mL−1. Each solution was prepared in triplicate. Linearity was evaluated
by linear regression, which was calculated by the least square
regression method. A good lin- earity was obtained for all observed
lines (r = 0.9995–0.9999).

2.3.1.3. Precision and accuracy. Repeatability and intermediate

precision were used to assess the precision of the method.
Repeatability was eval- uated through relative standard deviation
(RSD) from the recovery data at 100% level [30] on two different
days, and intermediate precision through RSD inter-day, using HPLC-
DAD.
A recovery study was conducted by adding known amounts (5 μg)
of
each reference solution of catechin, chlorogenic and vanillic acids to a
sample. Data recovery was performed in triplicate.

2.3.1.4. Detection and quantification limits. The figures of merit for

each procedure were evaluated based on the limits of detection (LOD)
and quantification (LOQ) under robust HPLC-UV–Vis/PDA conditions.
The method detection limits (LOD) were calculated using 3σ criterion,
that is, the concentration equivalent to three times the standard
deviation (3σ) of the signal (n = 10) of the reagent blank solution.

3. Results and discussion

To develop the method and identify peaks (catechin, chlorogenic

and vanillic acids), 20 μL of standard stock solution (1000 μg mL−1)
were injected into the HPLC system. Methanol was then used as an or-
ganic solvent and the peak was symmetric, with a retention time free
from interferences. In the chosen gradient program, bioactive
phenolics elute at 9.86; 10.60 and 12.46 min for catechin, chlorogenic
and vanillic acids, respectively. The UV-spectrum of this peak shows
bands at differ- ent wavelengths: 260 nm for vanillic acid; 280 nm for
gallic acid and (+)-catechin and 330 nm for chlorogenic acid. These
data on retention time and UV-spectrum were used to identify and
confirm these bioac- tive phenolic peaks in samples.

3.1. Analytical performance

At the chosen concentration range (from 1 to 50 μg mL−1 bioactive

phenolics), linearity was kept with r = 0.9996. The limit of quantifica-
tion in the final extract was estimated through linear coefficient stan-
dard deviation. The limits of detection (LOD) and quantification
(LOQ) obtained for chlorogenic and vanillic acids and (+)-catechin
were 0.020; 0.025 and 0.031 μg g−1, respectively. In precision assays,
all stan- dard deviations were below 0.5% average in three aliquots.
Intra (RSD b 6%) and inter-day (RSD b 10%) precision were obtained.
The devel- oped calibration curves were considered stable, since
relative standard derivation (RSD) values of the slope were all lower
than 2.0%.
In this extraction method, an addition standard was used for
bioac- tive phenolics in herbal medicines. Firstly, 5 μg of each bioactive
pheno- lic compound (catechin, chlorogenic and vanillic acids) were
added to a sample, which was previously analyzed and had no
quantifiable bioac- tive phenolics, a standard solution and then
extraction was carried out; analysis was performed using HPLC. This
analysis shows that the method was efficient to extract bioactive
phenolics from this kind of matrix (Fig. 1). To ensure that there is not
any content of bioactive phe- nolics in the samples after extraction,
the solid residue from the first treatment was retreated with 100 μL
of concentrated hydrochloric acid and solubilized in 1.5 mL of
methanol, injected into the HPLC sys- tem and no peak at 4.5 min was
detected. The obtained mean recovery was in the range of 95.2 to
100.9% for bioactive phenolics, which indicat- ed the good accuracy of
the method.
All samples had some bioactive phenolics, as shown in Table 1.
The highest content of chlorogenic acid was obtained for (Cynara
scolymus L.) “Globe artichoke” (plant drug). The levels of this bioactive
 R.R. Sá et al. / Microchemical Journal 135 (2017)
contents of vanillic acid were higher for (Cynara scolymus L.) properties related to the polyphenolic fraction as antioxidants and
“Globe ar- tichoke” (tablet) and similar for Ptychopetalum substrates for
uncinatum (gelatin cap- sule and plant drug). The gelatin capsules,
containing Maytenus ilicifolia Mart ex Reiss, showed contents of
catechin below those found by Cipriani et al. [31].
Each of the analyzed extracts displayed a similar
chromatographic profile, revealing several major components and
a number of minor peaks. Fig. 2 shows the chromatographic
profiles (UV / DAD spectrum) for catechin, chlorogenic and
vanillic acids, in the analysis of herbal medicines.
Bioactive phenolics are an object of interest in plants. In the
litera- ture, it was possible to find papers where bioactives in
Ptychopetalum olacoides were analyzed. Colombo et al. [32]
developed a method for the determination of vanillic acid in
commercial extracts of Ptychopetalum olacoides (commercial
water-soluble dry powder, com- mercial fluid and commercial dry
extract). They used a Phenomenex Luna phenyl-hexyl column and
a mobile phase consisting of 1% aqueous acetic acid-acetonitrile,
with linear gradient elution from 5 to 100% ace- tonitrile in 40
min, at a flow rate of 1.0 mL·min−1. The method is based on the use
of acetonitrile instead of methanol which, unfortunately, is not the
best idea. In this study, the novelty of the method is based on the
use of Methanol, since it is cheaper and less toxic than acetonitrile.
The range obtained for vanillic acid was of 0.02 to 4.90 (mg
vanillic acid/g extract) at a wavelength of 261 nm. In this study, a
range be- tween 17.35 and 19.21 (mg vanillic acid/g extract) was
obtained. The analysis of active substances is essential to evaluate
the safety and effi- cacy of the use of herbal medicines; however,
the chemical composition may vary due to climatic and agronomic
conditions of cultivation, such as plant organ, storage conditions,
geographical location, impact of air pollution and food production
processes [33].
Vistuba et al. [34] evaluated the qualitative profile of different
flavo- noids in an extract of Maytenus ilicifolia Martius ex Reissek by
HPLC-MS / MS and observed that most of the tannins were
derived from catechins and epicatechins. Therefore, the
determination of the catechin content in dried extracts of
Maytenus ilicifolia Martius ex Reissek is an alterna- tive to evaluate
the qualitative profile of the tannic compounds present in the
sample. Cipriani et al. [31] identified catechin and epicatechin in
leaves of Maytenus ilicifolia Martius ex Reissek by GC–MS,
quantified by HPLC, and found 1.6 and 1.7% dry weight,
respectively. In this study, catechin levels were found in the
range of 0.3937 to 0.7489 mg g−1, corresponding to about 0.1%.
This fact represents the need to extend quality control tests for
catechin determination in the analyzed samples, since laboratories
did not declare the active ingredi- ent content on the label.
The results for the determination of catechin in the
formulations containing Maytenus ilicifolia Martius ex Reissek,
showed that catechin contents in the gelatin capsules from three
different laboratories had great variation. Phytotherapeutic A
showed a higher concentration of catechin, compared to the other
herbal products. Baggio et al. [17] re- ported that extracts
enriched with flavonoids and characterized by the presence of
(2%) catechin, (3.1%) epicatechin and (27%) galactitol pre- sented
better reduction in gastric secretion. These data show the impor-
tance of determining these markers, since herbal medicines based
on Maytenus ilicifolia Martius ex Reissek are indicated for the
treatment of stomach disorders such as ulcers and gastritis [35].
The consumption of Medicinal Plants and Herbal Medicines has
been
increasing, and the need for greater control of their quality by the
Phar- maceutical Industry, becomes a relevant factor to ensure
their safety and efficacy. In Brazil, in 2006, the Ministry of Health
approved the Na- tional Policy on Integrative and Complementary
Practices (PNPIC), in the Unified Health System (SUS) and
included Maytenus ilicifolia Martius ex Reissek “Espinheira santa”
for the treatment of stomach dis- orders (gastritis and ulcer) and
other pharmacological activities (anti- inflammatory,
antispasmodic, antacid and healing) [36].
Many studies have focused on its health and antioxidant
 R.R. Sá et al. / Microchemical Journal 135 (2017)

Fig. 1. Overlay of spiked sample (rose) and standard stock solution (green/blue/black) chromatograms. (Tr = retention time) for chlorogenic acid; vanillic acid and catechin.

Table 1
Concentration of bioactive phenolics (mg g−1) and in Cynara scolymus L. “Globe artichoke”, Ptychopetalum uncinatum “Marapuama” and Maytenus ilicifolia Mart ex Reiss “Espinheira-
Santa”.

Sample AC AV CT

Cynara scolymus L. “Globe artichoke” (tablet) 71.28 ± 0.32 49.93 ± 0.86 –

(92.1% ± 0.15)* (93.0% ± 2.21)*
Cynara scolymus L. “Globe artichoke” (vegetal drug) 925.99 ± 55.76 10.67 ± 0.40 –
(90.3% ± 3.03)* (94.8% ± 1.76)*
Ptychopetalum uncinatum (gelatin capsules) 60.09 ± 0.64 17.35 ± 0.53 –
(97.4% ± 0.33)* (95.9% ± 0.54)*
Ptychopetalum uncinatum (vegetal drug) 41.50 ± 0.42 19.21 ± 0.26 –
(95.1% ± 0.82)* (98.3% ± 0.37)*
Maytenus ilicifolia Mart ex Reiss (gelatin capsules) – – 0.69 ± 0.54**
(96.9% ± 0.45)*

CT = catechin, AC = chlorogenic acid and AV = vanillic acids/* = recovery, % (means ± SD)/** = average of three herbal medicines (gelatin capsules) analyzed (means ± SD).
 R.R. Sá et al. / Microchemical Journal 135 (2017)

Fig. 2. Representative HPLC-DAD chromatographic profile of commercial extracts showing peaks corresponding to the isolated standards: (A) chlorogenic acid in samples containing C.
scolymus “Globe artichoke” – Black: plant drug and Pink: herbal medicine (tablets); (B) catechin in samples containing Maytenus ilicifolia Mart ex Reiss “Espinheira-Santa”– Blue:
Herbal medicine 1; Black: Herbal medicine 2 and Pink: Herbal medicine 3; (C) vanillic acid in samples containing P. uncinatum “Marapuama”– Black: plant drug and Pink: herbal
medicine (gelatin capsules).

oxidative browning reactions [37]. The leaves of Cynara scolymus L.

trifluoroacetic acid; solvent A) and 95% acetonitrile (containing 0.1%
“Globe artichoke” have been recognized since ancient times for their
trifluoroacetic acid; solvent B). Lutz et al. [10] compared the chemical
beneficial and therapeutic effects (promotion of blood circulation,
composition and antioxidant properties of mature and baby
mobilization of energy reserves, induction of choleresis, inhibition of
artichokes, raw and after cooking. The content of chlorogenic acid
cholesterol biosynthesis and LDL oxidation, besides significant
was determined by HPLC. The mobile phase was a mixture of (A): H2O
antibac- terial, antifungal and antioxidant activities, as well as strong
adjusted to pH 2.4 with 99% acetic acid, and (B): acetonitrile. The
hepatopro- tective effects). These actions could be strictly related to
range of 112.73 to
the polyphenolic fraction [38].
456.30 mg/100 g was found for chlorogenic acid.
Fratianni et al. [38] evaluated the total phenol content
The method of this study proposes the use of methanol and water
(chlorogenic acid, coumaric acid, ferulic acid, apigenin, luteolin and
as solvents, reducing toxicity, according to the principles of
cynarin) in Cynara scolymus L. “Globe artichoke” in Italy by HPLC. The
sustainable green Chemistry. The range was of 71.28 to 925.99 mg g−1
authors found a significant amount of chlorogenic acid (5.50 to 8.14
chlorogenic acid for “Globe artichoke” (tablet) and Globe artichoke
μM g−1). The mobile phase included HPLC-grade water (containing
(plant drug), re- spectively, and 41.50 to 60.09 mg g−1 for
0.1%
Ptychopetalum olacoides, marketed in Bahia-Brazil. This plant is grown
worldwide and numerous
 R.R. Sá et al. / Microchemical Journal 135 (2017)
commercial and local varieties adapted to different environments, can
differ in chemical composition, especially of the polyphenolic fraction,
and hence exhibit different nutraceutical and pharmacological
properties.
4. Conclusions
In recent decades, the consumption of Medicinal Plants and Herbal
Medicines has been increasing, and the need for greater control of
their quality by the Pharmaceutical Industry, becomes relevant to en-
sure safety and efficacy.
The proposed method meets the requirements of green and
sustain- able chemistry, using less toxic solvents at a lower cost. The
analytical performance showed that the method was satisfactory,
considering the figures of merit and the parameters that evaluate
separation efficiency. Phenolic bioactives were separated in b 13 min
and the results indicate that Cynara scolymus L. “Globe artichoke”
Artichoke, Ptychopetalum olacoides “Marapuama” and Maytenus
ilicifolia Martius ex Reissek “Espinheira Santa”, marketed in Salvador-
Bahia-Brazil (plant and phytotherapeutic drugs), are sources of
antioxidant species. The results showed that the analyzed samples
presented variations in chlorogenic acid, catechin and vanillic acid
levels, which may represent a risk to patient therapy.
Acknowledgements
The authors are grateful for the financial support received from
Fundaçã o de Amparo a Pesquisa do Estado da Bahia (FAPESB) and
Conselho Nacional de Desenvolvimento Científico e Tecnoló gico
(CNPq). Research Group: “Biopharmaceutics and Drugs”, State
Universi- ty of Bahia (UNEB).
References
[1] World Health Organization (WHO), Regional Office for the Western Pacific, Re-
search Guidelines for Evaluating the Safety and Efficacy of Herbal Medicines,
WHO, Manila, 1993.
[2] T.M.S. Moreira, H.R.N. Salgado, R.C.L.R. Pietro, Brazil in the context of quality
control of medicinal plants, Rev. Bras. Farm. 20 (2010) 435–440.
[3] A.C.B. Carvalho, J.P.S. Perfeito, L.V. Costa e Silva, L.S. Ramalho, R.F.O. Marques, D.
Silveira, Regulation of herbal medicines in Brazil: advances and perspectives, Braz.
J. Pharm. Sci. 47 (2011) 467–473.
[4] N.O. Rêgo Jú nior, L.G. Fernandez, R.D. Castro, L.C. Silva, S.A. Gualberto, M.L.A.
Pereira,
M.V. Silva, Bioactive compounds and antioxidant activity of crude extracts of
caatinga plant species, Braz. J. Food Technol. 14 (2011) 50–57.
[5] H. Peleg, K.K. Bodine, A.C. Noble, The influence of acid on astringency of alum
and phenolic compounds, Chem. Senses 23 (1998) 371–378.
[6] L.G. Malta, E.P. Tessaro, M. Eberlin, G.M. Pastore, R.H. Liu, Assessment of antioxidant
and antiproliferative activities and the identification of phenolic compounds of ex-
otic Brazilian fruits, Food Res. Int. 53 (2013) 417–425.
[7] M. Asadi-Samani, N. Kafash-Farkhad, N. Azimi, A. Fasihi, E. Alinia-Ahandani, M.
Rafieian-Kopaei, Medicinal plants with hepatoprotective activity in Iranian folk
medicine, Asian Pac. J. Trop. Biomed. 5 (2015) 146–157.
[8] R. Mocelin, M. Marcon, G.D. Santo, L. Zanatta, A. Sachett, A.P. Schö nell, F. Bevilaqua,
M. Giachini, R. Chitolina, S.M. Wildner, M.M.M.F. Duarte, G.M.M. Conterato, A.L.
Piato, D.B. Gomes, W.A. Roman Junior, Hypolipidemic and antiatherogenic
effects of Cynara scolymus in cholesterol-fed rats, Rev. Bras. Farm. 26 (2016)
233–239.
[9] E. Vamanu, A. Vamanu, S. Nita, S. Colceriu, Antioxidant and antimicrobial
activities of ethanol extracts of Cynara scolymus (Cynarae folium, Asteraceae
family), Trop. J. Pharm. Res. 10 (2011) 777–783.
[10] M. Lutz, C. Henríquez, M. Escobar, Chemical composition and antioxidant properties
of mature and baby artichokes (Cynara scolymus L.), raw and cooked, J. Food
Compos. Anal. 24 (2011) 49–54.
[11] R.S. Costa, E.F. Ozela, W.L.R. Barbosa, N.L. Pereira, J.A.C. Silva, Physical, chemical and
physicochemical characterization of the spray-drying of Cynara scolymus L.
(Asteraceae), Rev. Bras. Farm. 90 (2009) 169–174.
[12] ESCOP Monographs, The scientific foundation for herbal medicinal products, Euro-
pean Scientific Cooperative on Phytotherapy, Hederae helicis Folium, Ivy Leaf, 2nd
editionThieme Verlag, Stuttgart, 2003.
[13] T.R. Cipriani, C.G. Mellinger, L.M. Souza, C.H. Baggio, C.S. Freitas, M.C.A. Marques,
P.A.J. Gorin, G.L. Sassaki, M. Iacomini, Polygalacturonic acid: another anti-ulcer
polysaccharide from the medicinal plant Maytenus ilicifolia, Carbohydr. Polym. 78
(2009) 361–363.
[14] P.M. Costa, P.M.P. Ferreira, V.S. Bolzani, M. Furlan, V.A.F.M. Santos, J. Corsino, M.O.
Moraes, L.V. Costa-Lotufo, R.C. Montenegro, C. Pessoa, Antiproliferative activity of
pristimerin isolated from Maytenus ilicifolia (Celastraceae) in human HL-60 cells,
Toxicol. In Vitro 22 (2008) 854–863.
[15] J.C.R. Vellosa, N.M. Khalil, V.A.F. Formenton, V.F. Ximenes, L.M. Fonseca, M. Furlan,
I.L. Brunetti, O.M.M.F. Oliveira, Antioxidant activity of Maytenus ilicifolia root bark,
Fitoterapia 77 (2006) 243–244.
[16] R.S. Oliveira, S.C. Cunha, W. Colaço, Review of Maytenus ilicifolia Mart. Ex
Reissek, Celastraceae Contribution to the study of pharmacological properties,
Rev. Bras. Farm. 19 (2009) 650–659.
[17] C.H. Baggio, C.S. Freitas, G.M. Otofuji, T.R. Cipriani, L.M. Souza, G.L. Sassaki, M.
Iacomini, M.C.A.M. Marques, S. Mesia-Vela, Flavonoid-rich fraction of Maytenus
ilicifolia Mart. Ex. Reiss protects the gastric mucosa of rodents through
inhibition of both H+, K+ - ATPase activity and formation of nitric oxide, J.
Ethnopharmacol. 113 (2007) 433–440.
[18] C.H. Baggio, C.S. Freitas, B. Mayer, A.C. Santos, A. Twardowschy, F.B. Potrich, T.R.
Cipriani, L.M. Souza, G.L. Sassaki, M. Iacomini, M.C. Marques, S. Mesia-Vela,
Musca- rinic-dependent inhibition of gastric emptying and intestinal motility by
fractions of Maytenus ilicifolia Mart ex. Reissek, J. Ethnopharmacol. 123 (2009)
385–391.
[19] S. Crestani, Y.D. Rattmann, T.R. Cipriani, L.M. Souza, M. Iacomini, C.A. Kassuya,
M.C. Marques, J.E. Silva-Santos, A potent and nitric oxide-dependent hypotensive
effect induced in rats by semi-purified fractions from Maytenus ilicifolia, Vasc.
Pharmacol. 51 (2009) 57–63.
[20] S.E. Drewes, J. George, F. Khan, Recent findings on natural products with erectile-
dysfunction activity, Phytochemistry 62 (2003) 1019–1025.
[21] I.R. Siqueira, C. Fochesatto, I.L.S. Torres, A.L. da Silva, D.S. Nunes, E. Elisabetsky, C.A.
Netto, Antioxidant activities of Ptychopetalum olacoides (“muirapuama”) in rat
brain, Phytomedicine 14 (2007) 763–769.
[22] P.M.A. Salomã o, S.K.F. Barroso, M.C.L. Marcellino, Effects of Marapuama
(Ptychopetalum olacoides Bentham) on reserpine-induced motor changes in mice,
Salusvita 31 (2011) 105–116.
[23] A.F.V.B. Leal, M.O. Berreta, J.R. de Paula, D.G.A. Diniz, M.T.F. Bara, Dissolution
profiles of herbal medicines containing Maytenus ilicifolia Mart ex Reiss, Lat. Am. J.
Pharm. 31 (2012) 1108–1116.
[24] R. Capasso, A.A. Izzo, L. Pinto, T. Bifulco, C. Vitobello, N. Mascolo, Phytotherapy and
quality of herbal medicines, Fitoterapia 71 (2000) S58–S65.
[25] A.F. Santos Jú nior, R.A. Matos, E.M.J. Andrade, W.N.L. dos Santos, H.I.F. Magalhã es,
F.N. Costa, M.G.A. Korn, Multielement determination of macro and micro
contents in medicinal plants and phytomedicines from Brazil by ICP OES, J. Braz.
Chem. Soc. 28 (2017) 376–384.
[26] O. Sagdic, I. Ozturk, O. Ozkan, H. Yetin, L. Ekici, M.T. Yilmaz, RP-HPLC–DAD
analysis of phenolic compounds in pomace extracts from five grape cultivars:
evaluation of their antioxidant, antiradical and antifungal activities in orange
and apple juices, Food Chem. 126 (2011) 1749–1758.
[27] ANVISA, Resolution RDC-899, 29/05/2003. Guide for Validation of Analytical and
Bioanalytical Methods. National Health Surveillance Agency. Anvisa, Brasília, Brazil,
2003.
[28] ANVISA, Resolution RDC-31, 11/08/2010. Pharmaceutical Equivalence Studies and
Comparative Dissolution Profile. National Health Surveillance Agency. Anvisa,
Brasília, Brazil, 2010.
[29] International Conference on Harmonisation (ICH), Validation of analytical
proce- dures: text and methodology Q2 (R1)Available at:
http://www.ich.org/fileadmin/ Public_Web_Site/ICH_Products/Guidelines/
2005.
[30] The United States Pharmacopoeia, 33rd Rev., U.S.P. Convention, Rockville, 2011.
[31] T.R. Cipriani, C.G. Mellinger, L.M. de Souza, C.H. Baggio, C.S. Freitas, M.C.A. Marques,
P.A.J. Gorin, G.L. Sassaki, M. Iacomini, Acidic heteroxlans from medicinal plants
and their anti-ulcer activity, Carbohydr. Polym. 74 (2008) 274–278.
[32] R. Colombo, A.N.L. Batista, G.C.C. Bomfim, R.C.R. Burgos, A.J. Cavalheiro, V.S. Bolzani,
D.H.S. Silva, M.C.H. Reimberg, Validated high-performance liquid
chromatographic method for the standardisation of Ptychopetalum olacoides
Benth., Olacaceae, com- mercial extracts, Braz. J. Pharmacogn. 20 (2010) 781–
788.
[33] F.M. Al-Barwani, E.A. Eltayeb, Antifungal compound from induced Conium
maculatum L. plants, Biochem. Syst. Ecol. 32 (2004) 1097–1108.
[34] J.P. Vistuba, M. Piovezan, M.G. Pizzolatti, A.M. Rebelo, M.S. Azevedo, L. Vitali, A.C.
Costa, G.A. Micke, Increasing the instrumental throughput of gas
chromatography method using multiple injections in a single experimental run:
application in deter- mination of friedelan-3-ol and friedelin in Maytenus
ilicifolia, J. Chromatogr. A 25 (2013) 159–164.
[35] H. Lorenzi, F.J.A. Matos, Medicinal Plants in Brazil: Native and Exotic, 2nd ed
Nova Odessa, São Paulo, 2008.
[36] BRAZIL, Ministry of Health, National Policy on Integrative and Complementary
Prac- tices in SUS - PNPIC-SUS: Attitude to Increase Access, Brasília, Ministry of
Health, 2006.
[37] V. Lattanzio, A. Cardinali, D. Di Venere, V. Linsalata, S. Palmieri, Browning
phenom- ena in stored artichoke (Cynara scolymus L.) heads: enzymic or
chemical reaction? Food Chem. 50 (1994) 1–7.
[38] F. Fratianni, M. Tucci, M. Palma, R. Pepe, F. Nazzaro, Polyphenolic composition in
dif- ferent parts of some cultivars of globe artichoke (Cynara cardunculus L. var.
scolymus (L.) Fiori), Food Chem. 104 (2007) 1282–1286.