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Vol. 15(1), pp. 45-55, January, 2021
DOI: 10.5897/JMPR2020.7044
Article Number: F90BDCA65864
ISSN 1996-0875
Copyright © 2021
Author(s) retain the copyright of this article Journal of Medicinal Plants Research
http://www.academicjournals.org/JMPR
Full Length Research Paper
Development and validation of a new method to
quantify vitexin-2''-O-rhamnoside on
Passiflora L. extracts
Lorenna C. da Rosa1*, Monica R. P. Siqueira1, Francisco J. R. Paumgartten1, Georgia
Pacheco2, Elisabeth A. Mansur de Oliveira2 and Davyson de L. Moreira1,3
1
Laboratório de Toxicologia Ambiental, Departamento de Ciências Biológicas, Escola Nacional de Saúde Pública,
Fundação Oswaldo Cruz, Rio de Janeiro RJ, Brazil.
2
Núcleo de Biotecnologia Vegetal, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de
Janeiro, Rua São Francisco Xavier, 524, PHLC, sala 505 – Maracanã, Rio de Janeiro, Brazil.
3
Departamento de Produtos Naturais, Instituto de Tecnologia em Fármacos, Fundação Oswaldo Cruz,
Rio de Janeiro RJ, Brazil.
Received 22 September, 2020; Accepted 18 December, 2020
The genus Passiflora L. is the most representative of the Passifloraceae family and includes about 500
species. The aim of this work was to analyze three different passion fruit species in relation to vitexin-
2´´-O-rhamnoside content by high-performance liquid chromatography coupled to a diode-array
ultaviolet detector. Samples were prepared by water infusion (10% w/v), dried by lyophilization, and
stored in amber vials at -20°C. The method for quantification of vitexin-2´´-O-rhamnoside was developed
and validated using a Shimadzu Class-VP liquid phase chromatograph. Quantification of vitexin-2´´-O-
rhamnoside was done with an Ascentis-phenyl supelco column (250 mm x 4.6 mm i.d. x 5 µm), mobile
phase composed of ultrapure water (pH = 3.0) (Solvent A)/ acetonitrile (Solvent B), in gradient elution
mode, flow rate of 1.4 mL/min, and UV detection at 340 nm. The adopted method showed great linearity,
precision, accuracy, detection (LOD), and quantification (LOQ) limits, recovery and robustness. Total
analysis time was 16 min. This method has clear advantages when compared to those found in the
literature, since the use of a silica-based phenyl column allowed the best chromatographic resolution,
resulting in better LOD and LOQ. Vitexin-2''-O-rhamnoside content was higher in P. foetida (7.21%),
followed by P. setacea (3.66%), and P. alata (2.89%).
Key words: Vitexin-2''-O-rhamnoside quantity, high-performance liquid chromatography (HPLC) new method,
passion fruit.
INTRODUCTION
Passion fruit is the common name of several species representative of the Passifloraceae family. This family
from the genus Passiflora L., which is the most has 16 genera and about 700 species, 576 of which
*Corresponding author. E-mail: rosalorenna1@gmail.com. Tel: +55 21 3882-9018.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License46 J. Med. Plants Res.
belong to the genus Passiflora, native to tropical and alkaloids, considered its main chemical constituents.
subtropical America (Bernardes et al., 2020). A great Simple phenols, saponins, flavonoids, and cyanogenic
number of Passiflora species are native to Brazil, glycosides are also found in extracts of P. foetida
occurring mainly in the Cerrado biome that is threatened (Dhawan et al., 2004). The chemical standardization of
by human action (Gadioli et al., 2017). Passiflora species herbal medicines are required to guarantee their
have been studied for their sedative, anxiolytic, anti- effectiveness and may be carried out by the use of
inflammatory, antimicrobial, analgesic, healing, developed and validated analytical methods to detect and
antioxidant, gastroprotective, and antitumor effects quantify chemical markers, as well as by pharmacological
(Dhawan et al., 2004; Siebra et al., 2018). In this work, a assays (Carvalho et al., 2008). In the genus Passiflora,
newly developed and validated method to quantify C-glycosylated flavonoids are among the most frequently
vitexin-2´´-O-rhamnoside, the chemical marker of this cited chemical constituents, along with saponins. These
medicinal species (1, Figure 1) is proposed in Passiflora secondary metabolites have a wide distribution in the
alata Curtis (Brazil, 2011). In addition, in order to test the genus, therefore qualitative and quantitative differences
new method, we also quantified (1) in extracts of P. have been reported among some Passiflora species
setacea DC. and P foetida L. Passiflora alata Curtis is (Dhawan et al., 2004; Pereira et al., 2004). For example,
commonly known as “sweet passion fruit”. It is a native apigenin, vitexin, and homorientin, were found in
and endemic species from Brazil and occurs in the Passiflora species, while saponins are present especially
Atlantic Forest, Cerrado, and Amazon biomes. It is widely in P. alata and P. edulis (Yoshikawa et al., 2000;
distributed throughout the Brazilian territory (Bernacci et Reginatto et al., 2001; Dhawan et al., 2004). Hence, due
al., 2015) and its fruits are commercially exploited, being to their high prevalence, structural diversity, chemical
consumed in nature due to their sweet taste. This plant is stability, and the availability of qualitative and quantitative
also used worldwide ornamentally and in folk medicine. analysis methods, flavonoids can be used as chemical
Phytochemical prospection of P. alata revealed the markers and can provide the authentication, to detect
following constituents: C-glycosyl flavonoids (Birk et al., alterations and to provide differentiation between
2005; Pacheco et al., 2016), β-carbolinic alkaloids taxonomically specific Passiflora species. The current
(Machado et al., 2010), steroid and triterpene saponins knowledge on chemistry and pharmacology of the genus
(Reginatto et al., 2001; Birk et al., 2005), as well as Passiflora L. indicates its potential for the development of
steroids and triterpenes (Reginatto et al., 2001). anxiolytic and hypnotic/sedative phytomedicines
Flavonoids and saponins are its major constituents and (Gosmann et al., 2011). Thus, as Passiflora species are
have been isolated from aerial parts (Reginatto et al., important in the study of the development of new
2001; De-Paris et al., 2002). Passiflora setacea DC. is anxiolytics and antidepressants, in addition to their
also native to Brazil (Rinaldi et al., 2017), occurring in the current use in the treatment of these clinical disorders
Cerrado and Caatinga biomes and in environments with (such as P. alata, P. edulis and P. incarnata)
high solar incidence (Ataíde et al., 2012). It is an (Phytotherapeutic form the Brazilian Pharmacopoeia,
herbaceous climbing species (Braga et al., 2004). It has 2011), there are many other species that have not been
great potential for fresh consumption due to the pleasant studied, including P. foetida and P. setacea, two species
aroma and sweet taste of its fruits (Ataíde et al., 2012). of passion fruit from Brazil.
Its chemical constituents are mainly alkaloids and Previous published methods to quantify vitexin-2''-O-
flavonoids. Other compounds, such as saponins, rhamnoside (Table 1) include silica-based C18 columns
cyanogenic glycosides, steroids, lignin, fatty acids, and as a stationary phase and mixtures of three or more
tannins, are frequently cited in the literature (Dhawan et solvents to compose the mobile phases. Based on these
al., 2004; Gosmann et al., 2011). P. setacea has been information, the present study aimed to develop and
domesticated and a new cultivar was developed by the validate a new quantification method for vitexin-2''-O-
Brazilian Agricultural Research Corporation (do Cerrado, rhamnoside (1, Figure 1), the chemical marker of P.
2015), called P. setacea cv. „BRS Pérola do Cerrado‟ (do alata, to standardize the extracts in relation to the content
Cerrado, 2015; De Carvalho et al., 2018), which of this flavonoid.
produces sweated fruits and it was used in this work.
Passiflora foetida L., commonly known as stinking
passion fruit, is native to South America and West India MATERIALS AND METHODS
and has widespread in tropical regions around the world Plant collection
(Shuayprom et al., 2016). It can be found in riverbeds,
forests, and coastal vegetation (Melo Filho et al., 2018). Leaves and fruits from Passiflora alata Curtis (GPS S22o85'34"
This plant is used to treat asthma and to stimulate blood W42o23'66") and leaves from Passiflora foetida L. (GPS S22o51'47"
flow in the pelvic region and uterus (Bernardes et al., W42o59'52") were collected from cultivation in private areas in the
2020). Pharmacological studies showed anti-inflammatory, cities of Iguaba Grande and São Gonçalo, Rio de Janeiro State,
respectively, in Mach 2019. Leaves from Passiflora setacea cv.
analgesic, antihistaminic, antidepressant, antioxidant, BRS Pérola do Cerrado (GPS N2°45'30.6" W60°43'50.1") were
antitumor, antimicrobial, and immune modulatory effects, collected in March 2019 from the cultivation fields of the Brazilian
which have been associated with the presence of Agricultural Research Corporation (EMBRAPA), Boa Vista, Roraimada Rosa et al. 47
Table 1. HPLC-UV methods for analysis of vitexin 2''-O-rhamnoside in plant matrices described in the literature.
Matrices Detector Mobile phase / condition / LOQ, LOD Column Ref
Shim-pack VPODS C18 column
THF / ACN / phosphoric acid 0.05% (20: 3: 77, v / v / v)
Crataegus (250 mm × 4.6 mm i.d. × 5 μm)
/ isocratic; flow rate at 1 mL/ min / LOQ = 2 ng/ mL/
leaves and DAD-UV and a Shimpack GVP-ODS C18
LOD = 0.6 ng/mL/ Temperature 25°C/ VT2R tR = 10.97 [1]
fruits guard column (10 mm × 4.6 mm
min/ total run time = 25 min
i.d. × 5μm)
(A) ACN/ THF (95:5, v/v) and (B) phosphoric acid 1%
(v/v)/ gradient: 13-18% (A) at 0-11 min, 18-19% (A) at
11-25 min, 19-20% (A) at 25-30 min, 20-22% (A) at 30-
35 min, 22-25% (A) at 35-40 min, 25-28% (A) at 40-45
Crataegus [2]
min, 28-30% (A) at 45-50 min, 30-32% (A) at 50-55 Diamonsil C18 column (150 mm ×
pinnatifida DAD-UV
min, 100% (A) at 55-65 min, and then returned to initial 4.6 mm i.d. × 5 μm).
leaves
condition for a 5 min re-equilibration/ flow rate at 1
mL/min / LOQ = 1,992 ng/mL/ LOD = 49.8 ng/ mL.
Ambient oven temperature/ VT2R tR = 20.3 min. total
run time = 70min
(A) ACN, (B) MeOH and (C) TFA 0.05% (v/v) /
gradient: 0-20 min (75% C:15% B:10% A); 20-25 min
HiChrom C18 column (250 mm ×
Echinodorus (65% C:20% B:15% A); 20-25 min(55% C:25% B:20%
4.6 mm i.d. × 5 μm) and a pre- [3]
scaber and E. DAD-UV A); 25-30 min (75% C:15% B:10% A); flow rate at 1
column Kromasil C18 (3.0 mm ×
grandiflorus mL/min/ LOQ = 2,050 ng/ mL; LOD = 680 ng/ mL.
4.6 mm i.d.).
temperature N/A; VT2R tR = 11.2 min; total run time =
30 min
THF/ACN/ MeOH /phosphoric acid 0.05% (pH 5.0) Diamonsil C18 column (250 mm ×
(18:1:1:80 v/v/v/v) / isocratic; flow rate at 1.0 mL/min/ 4.6 mm i.d. 5 μm) and a Shim-
Crataegus [4]
DAD-UV LOQ = 100 ng/ mL; LOD = 300 ng /mL. Temperature at pack GVP-ODS C18 guard column
pinnatifida 20 °C; tR = 27 min; total run time = N/A (10 mm × 4.6 mm i.d × 5 μm).
(A) phosphoric acid 0.01% and (B) THF/ACN/ 2-
propanol (8:4:1, % v/v/v) / gradient: 0-12 min, 15-18%
Crataegus B; 12-22 min, 18-20% B, 22-23 min, 20-75% B, 23-25 [5]
Phenomene× C18 column, (150
leaves and DAD-UV min, 75% B; 25-25.5: 75-15% B, 25.5-30.5 min 15% B
mm × 4.6 mm i.d. × 2.6 μm).
flower with a post-run time of 5 min; flow rate at 0.4 mL/ min/
LOQ = 1, 200 ng/mL; LOD = 450 ng/mL/ temperature
at 25°C/ VT2R tR = 12-13 min; total run time = 25 min
LOQ = Limit of quantification; LOD = Limit of detection; Condition = Isocratic or Gradient and flow rate; ACN = Acetonitrile; THF = Tetrahydrofuran;
MeOH = Methanol; TFA = Trifluoro acetic acid. VT2R = vitexin-2''-O-rhamnoside. [1] Wang et al., 2011; [2] Ying et al., 2009; [3] Strada et al. 2017; [3]
Cheng et al. 2007; [5] Mudge et al. 2016.
State, and gently provided by Dr. Fábio Gelape Faleiro. Passiflora Quantification of vitexin -2''-O-rhamnoside
alata and Passiflora setacea cv. BRS Pérola do Cerrado were in
the fruiting period. All studied species were registered at Genetic For the development and validation of the analytical method for
Heritage Management Council under the code AD898E6. quantification of vitexin-2''-O-rhamnoside in the extracts, serial
dilutions from a stock solution of the standard (Fluka - Analytical
Standard - Lot 101455326) in methanol (200 µg/mL) were prepared
Extract preparation on the day of use.
All fresh plant material were frozen in liquid nitrogen, fragmented
and subjected to extraction by ultrapure water infusion (Milliq- Development of the analytical method
Millipore®) for 30 min, at 10% (w/v), according to the
Phytotherapeutic Form of the Brazilian Pharmacopoeia, (2011). The Initially, for the development of the analytical method, it was
aqueous extracts were then dried by lyophilization (Christ - Model: necessary to establish the mobile and stationary phases, according
Gamma 2-16 LSCplus) and stored in amber vials, protected from to HPLC analytical development procedures and the availability of
light, at -20°C, until use. The samples were solubilized in ultrapure laboratory materials. Determination of analytical performance
water immediately before use. parameters included retention time (tR), signal symmetry and48 J. Med. Plants Res.
Figure 1. Chemical structure of vitexin-2”-O-rhamnoside (1).
Table 2. Elution gradient to quantify vitexin-2''-O-rhamnoside in Passiflora extracts.
Time (min) Solvent A Solvent B
0 95 5
1 90 10
2 85 15
7 85 15
10 60 40
13 60 40
13.01 95 5
16 95 5
Solvent A: Ultrapure acidified water (pH = 3.0; glacial acetic acid); Solvent B: Acetonitrile
(HPLC grade, Tedia, Brazil).
retention factor or capacity (α). All methodology for the development Linearity (obtained at three different days, in the concentration
and analytical validation were performed at the Laboratory of range of 0.5; 2.0; 1.0, 7.5, 15, 30, 40, 50 and 100 µg/mL); Precision
Environmental Toxicology, ENSP/ FIOCRUZ, in a Shimadzu® (performed intra-day and inter-day to a concentration near of the
Class-VP coupled to DAD-UV detector, equipped with SCL-10AVP limit of quantitation - 0.5 µg/mL - and an intermediate value of
controller, DGU-14A degasser, LC-10AD VP binary pump, CTO- analytical curve - 40 µg/mL); Accuracy (obtained from experimental
10ASVP oven, DAD SPDM10AVP detection system. The data relative to nominal data); detection (LOD) and quantification
chromatograms were manipulated using Shimadzu Class VP® (LOQ) limits (obtained from successive dilutions and recording of
software, version 6.1. Analytical test conditions were done with signals in the chromatogram, by the ratio N/S 3 and 10,
stationary phases Supelco Ascentis-phenyl column (250 mm × 4.6 respectively); recovery (assessed at the concentration of 30 µg/mL)
mm i.d. x 5 µm, particle size) or Supelcosil C-18 column (250 mm × and robustness (evaluated at 30 µg/mL from small variations in
4.6 mm i.d. x 5 µm, particle size). Mobile phases were composed of analytical parameters).
ultrapure water (MilliQ deionized)/ acetonitrile (HPLC grade, Tedia,
Brazil) or ultrapure water (MilliQ deionized)/ methanol (HPLC grade,
Tedia, Brazil), in isocratic or gradient elution mode. Results RESULTS
represent mean ± standard deviation.
Development of the analytical method
Validation of the methodology
From the standard methanol solution (200 µg/mL) and
Analytical method for the quantification of vitexin-2''-O-rhamnoside variable mobile phase conditions assays, analytical
was validated as specified in the Guide for Validation of parameters were defined for the quantification of vitexin-
Bioanalytical and Analytical Methods of the National Health 2''-O-rhamnoside by HPLC-DAD-UV. All conditions tested
Surveillance Agency (ANVISA, 2017) and in the national standards for the analysis of this flavonoid, which showed the best
for analytical validation of The National Institute of Metrology, capacity or retention factor (α = 1.9), signal symmetry (~
Standardization and Industrial Quality/Brazil (INMETRO, 2016). The
evaluated parameters to determine the analytical performance were:
1.0), and selectivity were: Supelco Ascentis-phenyl
Selectivity (obtained from solvent injection and observation of column (250 mm × 4.6 mm i.d. × 5 µm, particle size);
signals in the vitexin-2''-O-rhamnoside chromatographic window; mobile phase in gradient (Table 2) composed byda Rosa et al. 49
ultrapure acidified water (pH = 3.0; glacial acetic acid) / the method (Table 3). Thus, the developed method was
acetonitrile HPLC grade (HPLC grade, Tedia, Brazil); flow considered accurate since limits ranged from 85 to 115%
rate at 1.4 mL/ min; oven temperature at 50°C; initial (INMETRO, 2016; ANVISA, 2017).
average pressure of 135 bar; standard injection volume
of sample 20 µL; wavelength monitoring at λ 340 nm; and
injector wash solvent methanol HPLC grade (Tedia, Limits of detection and quantification
Brazil). Under these conditions, vitexin-2''-O-rhamnoside
(1) showed a retention time (tR) of 12.70 to 13.28 min Limits of detection (LOD) and quantitation (LOQ) for
and total analysis time of 16 min. vitexin-2''-O-rhamnoside were 100 and 200 ng/mL,
Vitexin-2''-O-rhamnoside (1) had higher wavelength (λ) respectively. These values were obtained by the
absorption at 260 nm but as many other compounds successive dilution technique and represent an N/S of 3
absorbing in this UV region, we chose to quantify (1) at λ (LOD) and 10 (LOQ).
340 nm for more selectivity gain.
Recovery
Validation of the analytical method for quantification
of vitexin-2''-O-rhamnoside Data regarding recovery were evaluated at the
concentration of 40 µg/mL. Recovery was greater than
Selectivity 95%, therefore, within legal valid specifications
(INMETRO, 2016).
The selectivity of the method was demonstrated from
blank sample analysis (pure methanol) obtained by
HPLC-DAD-UV at λ 340 nm. The chromatograms Robustness
obtained by injection of pure methanol or extracts
showed no interferences in the chromatographic window Robustness results done at 30 µg/mL are shown in Table
of vitexin-2''-O-rhamnoside standard (tR = 12.70 - 13.88 4. There was no difference between the means of the
min). The chromatogram obtained for (1), analytical areas in the tested concentration, which indicates that the
standard, is shown in Figure 2a. developed method was robust (INMETRO, 2016; ANVISA,
2017).
Linearity
Quantification of vitexin-2''-O-rhamnoside by HPLC-
Linearity was demonstrated from three analytical curves DAD in Passiflora samples
of the vitexin-2''-O-rhamnoside standard, obtained on
three different days. The linear correlation was positive, The results of HPLC-DAD-UV quantification of vitexin-2''-
with an average of r = 0.9925 ± 0.0007, in the O-rhamnoside in P. alata, P. foetida, and P. setacea cv.
concentration range of 0.5 to 100 µg/mL. Residual BRS Pérola do Cerrado samples are presented in Table
analysis showed a homoscedastic distribution. The 5.
formula to calculate the vitexin-2''-O-rhamnoside content The content of vitexin-2''-O-rhamnoside in the aqueous
concentration was (µg/ mL) = (ABS - 42219)/21362. extract of P. alata leaves, at a concentration of 1 mg/mL,
was 28.92 ± 0.72 µg/mL, which is equivalent to 2.89% of
Precision the lyophilized extract. Figure 2b shows the
chromatographic profile obtained by HPLC-DAD-UV of
Precision was determined intra-day (morning and this sample (λ 340 nm). The retention time (tR) of vitexin-
afternoon on the same day) and inter-day (three different 2''-O-rhamnoside determined in this chromatogram was
days) at 0.5 and 40 µg/mL (low and medium 13.20 min.
concentrations). The results showed that the RSD was Lyophilized samples of the P. alata endocarp and pulp
below the limit (15%) and showed no variation between were solubilized in water at a concentration ten times
the intra-day averages when compared with the inter-day greater than that tested with leaves. Still, the
average (Table 3). Therefore, the developed method was chromatogram of these extracts showed no signal for
precise (INMETRO, 2016; ANVISA, 2017). vitexin-2''-O-rhamnoside for both samples (Figures 2c
and d). Therefore, it was not possible to quantify vitexin-
2''-O-rhamnoside in P. alata pulp and endocarp samples.
Accuracy A sample of the aqueous extract of P. foetida at a
concentration of 1 mg/mL was analyzed by HPLC-DAD-
An analysis of six different concentrations ranging from UV and the chromatographic profile (λ 340 nm) is shown
7.5 to 100 µg/mL, comprising low, medium, and high in Figure 2e. The glycosylated flavonoid vitexin-2''-O-
concentrations, were used to determine the accuracy of rhamnoside (tR = 13.16 min) content in the sample was50 J. Med. Plants Res.
da Rosa et al. 51 Figure 2. Chromatograms of vitexin-2”-O-rhamnoside standard and aqueous extractsof Passiflora species. Vitexin-2”-O-rhamnoside in methanol solution at 25 µg/mL; (a) aqueous extracts of Passiflora alata; (b) leaves (1 mg/mL), (c) pulp (10 mg/mL) and (d) endocarp (10 mg/mL); aqueous extracts of (e) Passiflora foetida and (f) Passiflora fsetacea cv. BRS Perola do Cerrado leaves (1 mg/mL). Arrows indicate signals for vitexin- 2”-O-rhamnoside.
52 J. Med. Plants Res.
Table 3. Analytical method precision and accuracy.
Precision Accuracy
Concentration Intra-day Inter-day
(µg/mL) AVR AVR Experimental
SD RSD% SD RSD% Calculated** µg/mL
(mAU) (mAU) ABS (mAU)
100 2135280 97.98 -2.02 -2.02 97.98
50 1224548 55.35 5.35 10.69 110.69
40 865059 9199 1.06 865059 3592 0.42 857566 38.17 -1.83 -4.58 95.42
30 704923 31.02 1.02 3.41 103.41
15 351536 14.48 -0.52 -3.47 96.53
7.5 185300 6.70 -0.80 -10.69 89.31
**Calculated concentration from the analytical curve: Concentration µg/ mL = [ABS (mAU) - 42219 ± 1239] / 21362 ± 20; [ ] = difference in concentration; % = difference in percentage.
AVR = Average; SD = standard deviation; RSD = relative standard deviation.
72.08 ± 1.85 µg/mL, equivalent to 7.21% of the phase was cost-effective because most of it to ensuring the reliability of repeated
lyophilized extract. consists of MilliQ deionized water that is obtained measurements taken over the same day and over
The content of vitexin-2''-O-rhamnoside (tR = directly from the laboratory. different days, as well as to assess how
13.49) in the aqueous extract of P. setacea cv. A new approach with the stationary phase was experimental data relates to expected (theoretical)
BRS Pérola do Cerrado was calculated as 36.64 ± used, employing a Supelco Ascentis-phenyl data. The developed method reported here is
2.04 µg/mL, which corresponds to 3.66% of the column (250 mm × 4.6 mm i.d. × 5 μm, particle precise because the precision test results showed
lyophilized extract (Figure 2f). size). To the best of our knowledge and as shown an RSD less than 15% for measurements made
Considering these results, the vitexin-2''-O- in Table 1, this is the first time that a Silica-based on the same day and on different days and there
rhamnoside content was higher in extracts from column modified with phenyl groups was used to was no variation between the intra-day and inter-
leaves of P. foetida (7.21%), followed by P. quantify vitexin-2''-O-rhamnoside. This approach day chromatographic run averages.
setacea cv. BRS Pérola do Cerrado (3.66%), and introduced clear advantages for the new method, Regarding accuracy, when comparing the
P. alata (2.89%). including a great separation factor (1.9) and signal expected (theoretical) with the observed
symmetry (1.0) that is reflected in the obtained (experimental) values, the results showed that the
LOD and LOQ. greatest variation was from 89.31% to 7.5 μg/mL
DISCUSSION The linearity of the method was excellent, as the and 110.69% to 50 μg/mL, within the
correlation coefficient was 0.9925 ± 0.0007, recommended limits (85-115%). Thus, the
This newly developed and validated method for considering a concentration range of 0.5 to 100 developed method can be considered accurate
the quantification of vitexin-2''-O-rhamnoside (1) µg/mL. This is quite wide and ranges from 2.5 to and precise (INMETRO, 2016; ANVISA, 2017).
employed acidified ultrapure water (MilliQ 200 times the LOQ. Dispersion of the points of the The recovery of the method was excellent,
deionized) and acetonitrile (HPLC grade) in analytical curves was homoscedastic, without greater than 90%, according to the validation
gradient mode as a mobile phase. The best outliers. standards (INMETRO, 2016; ANVISA, 2017).
capacity of retention factor (α = 1.9), signal The precision and accuracy of the method were Another determining factor in method validation
symmetry (~ 1.0), and selectivity of this new within the parameters established in the standards was robustness. The method was robust because
method are great parameters to quantify this C- (INMETRO, 2016; ANVISA, 2017). The accuracy the slight variations in the vitexin-2''-O-rhamnoside
glucosyl-flavonoid. Preparation of the mobile and precision of an analytical method are critical signal area (ABS in mAU), such as decreased flowda Rosa et al. 53
Table 4. Robustness tested parameters for vitexin-2´´-O-rhamnoside in water solution (30 µg/mL).
Condition
Parameter
0 1 2 3
Flow rate mL/min A a A A
Aqueous solutions pH B B b B
Oven temperature °C C C C c
30 751236** 796376 792564 771434
30 754687 808372 791943 770708
30 724819 816627 795809 754687
30 751236 796376 792564 771434
Average 743581 807125 793439 765610
SD 16339 10183 2076 9466
RSD% 2.20 1.26 0.26 1.24
Condition: 0 = best chromatographic condition: A = 1.4, B = 3.0, C = 50; 1: a = 1.3, B = 3.0, C = 50; 2: A= 1.4, b = 3.5, C = 50;
3: A= 1.4, B = 3.0, c = 47. **ABS measured in mAU. SD = standard deviation; RSD = relative standard deviation.
Table 5. Quantification of vitexin-2''-O-rhamnoside by HPLC-DAD in Passiflora samples.
Species Plant material Content (µg/ mL) Percentage % (w/ w)
Leaves 28.92 ± 0.72 2.89
P. alata Pulp * - -
Endocarp * - -
P. foetida Leaves 72.08 ± 1.85 7.21
P. setacea cv. BRS Pérola do Cerrado Leaves 36.64 ± 2.04 3.66
All samples were tested at 1 mg/ mL exception for * (10 mg/mL).
rate from 1.4 to 1.3 mL/min, decreased oven temperature al., 2017). These LOQ can compromise the vitexin-2''-O-
by 50 to 47°C, and increased pH of the aqueous phase rhamnoside quantification.
from 3.0 to 3.5, did not influence the average of ABS
areas.
Identification (LOD, 100 ng/mL) and quantification Conclusion
(LOQ, 200 ng/mL) limits were low on the ng/mL scale. In
We presented a newly developed and validated method
the case of an UV detector, detection is expected to be
to quantify vitexin-2''-O-rhamnoside in passion fruit
on the nanogram scale (10-9). Compounds with a great
extracts. The method described here has clear
molar extinction coefficient, for example those with
advantages when compared to other previously reported
chromophores that absorb strongly in UV light, will have
methods, mainly for the use of a silica-based phenyl
good detection sensitivity. Thus, the detector employed
column that allowed the best chromatographic parameters.
proves to be efficient for the detection and quantification
Additionally, the new validated method employs a mixture
of vitexin-2''-O-rhamnoside.
of acidified ultrapure water and acetonitrile, which is more
Previously published methods for the analysis of
cost-effective than those previously published. The
vitexin-2''-O-rhamnoside available in the literature (Table
validated method was tested to quantify and standardize
1), present, in part, a greater total analysis time.
the extracts of vitexin-2''-O-rhamnoside in three different
Additionally, the mobile phases used are more toxic and
Passiflora species. Results showed higher content of this
harmful to the chromatographic system. Some of these
bioactive flavonoid in P. foetida (7.21%) and P. setacea
methods also use more than one organic solvent, while
cv. BRS Pérola do Cerrado (3.66%) than in the
our method uses only one (acetonitrile). The lack of
pharmacopeical species P. alata (2.89%).
sensibility needs attention, since those published LOD
and LOQ are higher than those obtained in this new
method (except for Crataegus dosage, Wang et al., CONFLICT OF INTEREST
2011). Some LOQ are extremely high, in the scale of
µg/mL (Ying et al., 2009; Mudge et al., 2016; Strada et The authors declare that there is no conflict of interest to54 J. Med. Plants Res.
disclose. quádrupla aptidão: consumo in natura, processamento industrial,
ornamental e funcional. Available at:< Available at: http://www. cpac.
embrapa.
br/publico/usuarios/uploads/lancamentoperola/foldertecnico. pdf>.
ACKNOWLEDGEMENTS Accessed on: Jan, 5.
Gadioli IL, Cunha MS, De Carvalho, MV, Costa AM, Pineli LL (2017).
Asystematic review on phenolic compounds in Passiflora plants:
The authors are grateful to FAPERJ (Fundação de
Exploring biodiversityfor food, nutrition, and popular medicine. Critical
Amparo à Pesquisa do Estado do Rio de Janeiro), Reviews in Food Science and Nutrition,
process number E-26/202.719/2017, for providing the 58(5). https://doi.org/10.1080/10408398.2016.1224805
scholarship to the first author. Gosmann G, Provensi G, Comunello LN, Rates SMK (2011).
Composição química e aspectos farmacológicos de espécies de
Passiflora L. (Passifloraceae). Brazilian Journal of Biosciences
9(s1):88-99.
REFERENCES http://www.ufrgs.br/seerbio/ojs/index.php/rbb/article/view/1607
INMETRO (2016). DOQ-CGCRE-008 - Orientação sobre Validação de
Anvisa R (2017). Guia para validação de métodos analíticos e Métodos Analíticos. Revisão 02. - Instituto Nacional de Metrologia,
bioanalíticos. Diário Oficial da União, Brasília, Brazil, July 24, 2017. Qualidade e Tecnologia.
0
N. 166, https://www.in.gov.br/materia/- http://www.inmetro.gov.br/Sidoq/Arquivos/CGCRE/DOQ/DOQ-
/asset_publisher/Kujrw0TZC2Mb/content/id/19194581/do1-2017-07- CGCRE8_02.pdf Access in January 2020.
25-resolucao-rdc-n-166-de-24-de-julho-de-2017-19194412 Access in Machado MW, Stern Neto C, Salgado J, Zaffari J, Barison A, Campos
November 2020. FR, Corilo YE, Eberlin MN, Biavatti MW (2010). Search for alkaloids
Ataíde EM, Oliveira JC, Ruggiero C (2012). Florescimento e frutificação on callus culture of Passiflora alata. Brazilian Archives of Biology and
do maracujazeiro silvestre Passiflora setacea D. C. cultivado em Technology 53(4):901-910. https://doi.org/10.1590/S1516-
Jaboticabal, SP. Revista Brasileira de Fruticultura 34:377-381. 89132010000400020.
https://doi.org/10.1590/S0100-29452012000200009 Melo Filho AA, Kinuko ÂK, Ribeiro PRE, Melo ACGR, Fernández IM,
Bernacci LCC, Cervi CA, Milward-De-Azevedo MA, Nunes TS, Imig DC, Santos RC, Chagas EA, Chagas PC (2018). Chemical Composition,
Mezzonato AC (2015). Passifloraceae in Lista de Espécies da Flora Antioxidant and Biological Activity of Leaves Passiflora foetida.
do Brasil. Chemical Engineering Transactions 64:241.
http://www.floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB12508201. https://doi.org/10.3303/CET1864041
Access in January 2020. Mudge EM, Liu Y, Lund JA, Brown PN (2016). Single-
Bernardes PM, Nicoli CF, Alexandre RS, Guilhen JHS, Praça-Fontes laboratoryvalidation for the determination of flavonoids in
MM, Adésio F, Ferreira MFS (2020). Vegetative and reproductive hawthornleaves and finished products by LC–UV. Planta Medica
performance of species of the genus Passiflora. Scientia 82(17):1487-1492. http://dx.doi.org/ 10.1055/s-0042-118463
Horticulturae 265:109193. Pacheco G, Simão MJ, Vianna MG, Garcia RO, Vieira MLC, Mansur E
https://doi.org/10.1016/j.scienta.2020.109193 (2016). In vitroconser-vation of Passiflora- a review. Scientia
Birk CD, Provensi G, Gosmann G, Reginatto FR, Schenkel EP (2005). Horticulturae 211:305-311.
TLC Fingerprint of Flavonoids and Saponins from Passiflora Species. https://doi.org/10.1016/j.scienta.2016.09.004
Journal of Liquid Chromatography and Related Technologies Pereira CAM, Yariwake J H, Lanças FM, Wauters JN, Tits M, Angenot
28(14):2285-2291. https://doi.org/10.1081/JLC-200064212 LA (2004). HPTLC densitometric determination of flavonoids from
Braga MF, Junqueira NTV, Faleiro FG, Almeida DA, Cabral GA, Sousa Passiflora alata, P. edulis, P. incarnata and P. caerulea and
AATC, Resende AM (2004). Desempenho agronômico de um clone comparison with HPLC method. Phytochemical Analysis 15(4):241-
de maracujazeiro azedo propagado por estaquia e enxertia em 248. https://doi.org/10.1002/pca.778
estacas enraizadas de um híbrido F1 de Passiflora edulis f. flavicarpa Phytotherapeutic Form of the Brazilian Pharmacopoeia, 1 ed., Ministério
comercial x P. setacea. In: Congresso Brasileiro de Fruticultura, 18. da Saúde. Agência Nacional de Vigilância Sanitária. RDC n° 60, de
Florianópolis, SC. Anais Jaboticabal: Sociedade Brasileira de 10 de novembro de 2011. Diário Oficial, Brasília, November 11,
Fruticultura. 2011.
Access in 17 January 2020. ario-de-Fitoterapicos-da-Farmacopeia-Brasileira-sem-marca.pdf
Carvalho ACB, Balbino EE, Maciel A, Perfeito JOS (2008). Situação do Access in November 2020.
registro de medicamentos fitoterápicos no Brasil. Brazilian Journal of Reginatto FH, Kauffmann C, Schripsema J, Guillaume D, Gosmann G,
Pharmacognosy 18(2):314-319. https://doi.org/10.1590/S0102- Schenkel EP (2001). Steroidal and triterpenoidal glucosides from
695X2008000200028 Passiflora alata. Journal of the Brazilian Chemical Society 12(1):32-
De Carvalho MVO, Oliveira L, Costa AM (2018). Effect of training 36. https://doi.org/10.1590/S0103-50532001000100003
system and climate conditions on phytochemicals of Passiflora Rinaldi MM, Costa AM, Faleiro FG, Junqueira NTV (2017).
setacea, a wild Passiflora from Brazilian savannah. Food Chemistry Conservação pós-colheita de frutos de Passiflora setacea DC.
266:350-358. https://doi.org/10.1016/j.foodchem.2018.05.097 submetidos a diferentes sanitizantes e temperaturas de
Cheng FS, Qiu F, Huang J, He J (2007). Simultaneous determinationof armazenamento. Brasilian jornal of food technology, 2: e2016046.
vitexin-2‟‟-O-glucoside, vitexin-2‟‟-O-rhamnoside, rutin, andhyperoside https://doi.org/10.1590/1981-6723.4616
in the extract of hawthorn (Crataegus pinnatifida Bge.) leaves by RP- Shuayprom A, Sanguansermsri D, Sanguansermsri P, Fraser IH,
HPLC with ultraviolet photodiode arraydetection. Journal of Wongkattiya N (2016). Quantitative determination of vitexin in
Separation Science 30:717- Passiflora foetida Linn. leaves using HPTLC. Asian Pacific Journal of
721. https://doi.org/10.1002/jssc.200600353 Tropical Biomedicine 6(3):216-220.
De-Paris F, Petry RD, Reginatto FH, Gosmann G, Quevedo J, https://doi.org/10.1016/j.apjtb.2015.11.006
Salgueiro JB, Kapczinski F, Ortega GG, Schenkel EP (2002). Siebra ALA, Oliveira LR, Martins AOBPB, Siebra DC, Albuquerque RS,
Pharmacochemical study of aqueous extracts of Passiflora alata Lemos ICS, Delmondes GA, Tintino SR, Figueredo FG, Costa JGM,
Dryander and Passiflora edulis Sims. Acta Farmaceutica Bonaerense Coutinho HDM, Menezes IRA, Felipe CFB, Kerntopf MR (2018).
21(1):5-8. Potentiation of antibiotic activity by Passiflora cincinnata Mast. front
Dhawan K, Dhawan S, Sharma A (2004). Passiflora: a review update of strains Staphylococcus aureus and Escherichia coli. Saudi journal
Journal of Ethnopharmacology 94(1):1-23. of biological sciences 25(1):37-43.
https://doi.org/10.1016/j.jep.2004.02.023 https://doi.org/10.1016/j.sjbs.2016.01.019
do Cerrado BP (2015). Cultivar de maracujazeiro silvestre com Strada CL, Lima KC, Silva VC, Ribeiro RV, Dores EFGC, Dall'Oglio EL,da Rosa et al. 55 Schmeda-Hirschmann G, Carollo CA, Martins DTO, Sousa Júnior PT Yoshikawa K, Katsuta S, Mizumori J, Arihara S (2000). Four (2017). Isovitexin as marker and bioactive compound in the cycloartane triterpenoids and six related saponins from Passiflora antinociceptive activity of the Brazilian crude drug extracts of edulis. Journal of Natural Products 63(9):1229-1234. Echinodorus scaber and E. grandiflorus. Revista Brasileira de https://doi.org/10.1021/np000126+ Farmacognosia 27(5):619-626. https://doi.org/10.1016/j.bjp.2017.05.011 Wang C, Wang Y, Liu H (2011) Validation and application by HPLC for simultaneous determination of vitexin-2″-O-glucoside, vitexin-2″-O- rhamnoside, rutin, vitexin, and hyperoside. Journal of Pharmaceutical Analysis 1(4):291-296. https://doi.org/10.1016/j.jpha.2011.09.003 Ying X, Wang R, Xu J, Zhang W, Li W, Zhang C, Li F (2009). HPLC Determination of Eight Polyphenols in the Leaves of Crataegus pinnatifida Bge. var. major. Journal of Chromatographic Science 47:201-205. https://doi.org/10.1093/chromsci/47.3.201
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