Development and validation of a new method to quantify vitexin-2''-O-rhamnoside on Passiflora L. extracts

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.


INTRODUCTION
Passion fruit is the common name of several species from the genus Passiflora L., which is the most representative of the Passifloraceae family. This family 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 License belong to the genus Passiflora, native to tropical and subtropical America (Bernardes et al., 2020). A great number of Passiflora species are native to Brazil, occurring mainly in the Cerrado biome that is threatened by human action (Gadioli et al., 2017). Passiflora species have been studied for their sedative, anxiolytic, antiinflammatory, antimicrobial, analgesic, healing, antioxidant, gastroprotective, and antitumor effects (Dhawan et al., 2004;Siebra et al., 2018). In this work, a newly developed and validated method to quantify vitexin-2´´-O-rhamnoside, the chemical marker of this medicinal species (1, Figure 1) is proposed in Passiflora alata Curtis (Brazil, 2011). In addition, in order to test the new method, we also quantified (1) in extracts of P. setacea DC. and P foetida L. Passiflora alata Curtis is commonly known as "sweet passion fruit". It is a native and endemic species from Brazil and occurs in the Atlantic Forest, Cerrado, and Amazon biomes. It is widely distributed throughout the Brazilian territory (Bernacci et al., 2015) and its fruits are commercially exploited, being consumed in nature due to their sweet taste. This plant is also used worldwide ornamentally and in folk medicine. Phytochemical prospection of P. alata revealed the following constituents: C-glycosyl flavonoids (Birk et al., 2005;Pacheco et al., 2016), β-carbolinic alkaloids (Machado et al., 2010), steroid and triterpene saponins (Reginatto et al., 2001;Birk et al., 2005), as well as steroids and triterpenes (Reginatto et al., 2001). Flavonoids and saponins are its major constituents and have been isolated from aerial parts (Reginatto et al., 2001;De-Paris et al., 2002). Passiflora setacea DC. is also native to Brazil (Rinaldi et al., 2017), occurring in the Cerrado and Caatinga biomes and in environments with high solar incidence (Ataíde et al., 2012). It is an herbaceous climbing species (Braga et al., 2004). It has great potential for fresh consumption due to the pleasant aroma and sweet taste of its fruits (Ataíde et al., 2012). Its chemical constituents are mainly alkaloids and flavonoids. Other compounds, such as saponins, cyanogenic glycosides, steroids, lignin, fatty acids, and tannins, are frequently cited in the literature (Dhawan et al., 2004;Gosmann et al., 2011). P. setacea has been domesticated and a new cultivar was developed by the Brazilian Agricultural Research Corporation (do Cerrado, 2015), called P. setacea cv. "BRS Pérola do Cerrado" (do Cerrado, 2015;De Carvalho et al., 2018), which 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 and has widespread in tropical regions around the world (Shuayprom et al., 2016). It can be found in riverbeds, forests, and coastal vegetation (Melo Filho et al., 2018). This plant is used to treat asthma and to stimulate blood flow in the pelvic region and uterus (Bernardes et al., 2020). Pharmacological studies showed anti-inflammatory, analgesic, antihistaminic, antidepressant, antioxidant, antitumor, antimicrobial, and immune modulatory effects, which have been associated with the presence of alkaloids, considered its main chemical constituents. Simple phenols, saponins, flavonoids, and cyanogenic glycosides are also found in extracts of P. foetida (Dhawan et al., 2004). The chemical standardization of herbal medicines are required to guarantee their effectiveness and may be carried out by the use of developed and validated analytical methods to detect and quantify chemical markers, as well as by pharmacological assays (Carvalho et al., 2008). In the genus Passiflora, C-glycosylated flavonoids are among the most frequently cited chemical constituents, along with saponins. These secondary metabolites have a wide distribution in the genus, therefore qualitative and quantitative differences have been reported among some Passiflora species (Dhawan et al., 2004;Pereira et al., 2004). For example, apigenin, vitexin, and homorientin, were found in Passiflora species, while saponins are present especially in P. alata and P. edulis (Yoshikawa et al., 2000;Reginatto et al., 2001;Dhawan et al., 2004). Hence, due to their high prevalence, structural diversity, chemical stability, and the availability of qualitative and quantitative analysis methods, flavonoids can be used as chemical markers and can provide the authentication, to detect alterations and to provide differentiation between taxonomically specific Passiflora species. The current knowledge on chemistry and pharmacology of the genus Passiflora L. indicates its potential for the development of anxiolytic and hypnotic/sedative phytomedicines (Gosmann et al., 2011). Thus, as Passiflora species are important in the study of the development of new anxiolytics and antidepressants, in addition to their current use in the treatment of these clinical disorders (such as P. alata, P. edulis and P. incarnata) (Phytotherapeutic form the Brazilian Pharmacopoeia, 2011), there are many other species that have not been studied, including P. foetida and P. setacea, two species of passion fruit from Brazil.
Previous published methods to quantify vitexin-2''-Orhamnoside (Table 1) include silica-based C18 columns as a stationary phase and mixtures of three or more solvents to compose the mobile phases. Based on these information, the present study aimed to develop and validate a new quantification method for vitexin-2''-Orhamnoside (1, Figure 1), the chemical marker of P. alata, to standardize the extracts in relation to the content of this flavonoid.  Table 1. HPLC-UV methods for analysis of vitexin 2''-O-rhamnoside in plant matrices described in the literature.

Extract preparation
All fresh plant material were frozen in liquid nitrogen, fragmented and subjected to extraction by ultrapure water infusion (Milliq-Millipore®) for 30 min, at 10% (w/v), according to the Phytotherapeutic Form of the Brazilian Pharmacopoeia, (2011). The aqueous extracts were then dried by lyophilization (Christ -Model: Gamma 2-16 LSCplus) and stored in amber vials, protected from light, at -20°C, until use. The samples were solubilized in ultrapure water immediately before use.

Quantification of vitexin -2''-O-rhamnoside
For the development and validation of the analytical method for 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 on the day of use.

Development of the analytical method
Initially, for the development of the analytical method, it was necessary to establish the mobile and stationary phases, according to HPLC analytical development procedures and the availability of laboratory materials. Determination of analytical performance parameters included retention time (tR), signal symmetry and   Linearity (obtained at three different days, in the concentration range of 0.5; 2.0; 1.0, 7.5, 15, 30, 40, 50 and 100 µg/mL); Precision (performed intra-day and inter-day to a concentration near of the limit of quantitation -0.5 µg/mL -and an intermediate value of analytical curve -40 µg/mL); Accuracy (obtained from experimental data relative to nominal data); detection (LOD) and quantification (LOQ) limits (obtained from successive dilutions and recording of signals in the chromatogram, by the ratio N/S 3 and 10, respectively); recovery (assessed at the concentration of 30 µg/mL) and robustness (evaluated at 30 µg/mL from small variations in analytical parameters).

Development of the analytical method
From the standard methanol solution (200 µg/mL) and variable mobile phase conditions assays, analytical parameters were defined for the quantification of vitexin-2''-O-rhamnoside by HPLC-DAD-UV. All conditions tested for the analysis of this flavonoid, which showed the best capacity or retention factor (α = 1.9), signal symmetry (~ 1.0), and selectivity were: Supelco Ascentis-phenyl column (250 mm × 4.6 mm i.d. × 5 µm, particle size); mobile phase in gradient ( (1) showed a retention time (tR) of 12.70 to 13.28 min and total analysis time of 16 min. Vitexin-2''-O-rhamnoside (1) had higher wavelength (λ) absorption at 260 nm but as many other compounds absorbing in this UV region, we chose to quantify (1) at λ 340 nm for more selectivity gain.

Selectivity
The selectivity of the method was demonstrated from blank sample analysis (pure methanol) obtained by HPLC-DAD-UV at λ 340 nm. The chromatograms obtained by injection of pure methanol or extracts showed no interferences in the chromatographic window of vitexin-2''-O-rhamnoside standard (tR = 12.70 -13.88 min). The chromatogram obtained for (1), analytical standard, is shown in Figure 2a.

Linearity
Linearity was demonstrated from three analytical curves of the vitexin-2''-O-rhamnoside standard, obtained on three different days. The linear correlation was positive, with an average of r = 0.9925 ± 0.0007, in the concentration range of 0.5 to 100 µg/mL. Residual analysis showed a homoscedastic distribution. The formula to calculate the vitexin-2''-O-rhamnoside content concentration was (µg/ mL) = (ABS -42219)/21362.

Precision
Precision was determined intra-day (morning and afternoon on the same day) and inter-day (three different days) at 0.5 and 40 µg/mL (low and medium concentrations). The results showed that the RSD was below the limit (15%) and showed no variation between the intra-day averages when compared with the inter-day average (Table 3). Therefore, the developed method was precise (INMETRO, 2016;ANVISA, 2017).

Accuracy
An analysis of six different concentrations ranging from 7.5 to 100 µg/mL, comprising low, medium, and high concentrations, were used to determine the accuracy of da Rosa et al. 49 the method (Table 3). Thus, the developed method was considered accurate since limits ranged from 85 to 115% (INMETRO, 2016;ANVISA, 2017).

Limits of detection and quantification
Limits of detection (LOD) and quantitation (LOQ) for vitexin-2''-O-rhamnoside were 100 and 200 ng/mL, respectively. These values were obtained by the successive dilution technique and represent an N/S of 3 (LOD) and 10 (LOQ).

Recovery
Data regarding recovery were evaluated at the concentration of 40 µg/mL. Recovery was greater than 95%, therefore, within legal valid specifications (INMETRO, 2016).

Robustness
Robustness results done at 30 µg/mL are shown in Table  4. There was no difference between the means of the areas in the tested concentration, which indicates that the developed method was robust (INMETRO, 2016;ANVISA, 2017).
The content of vitexin-2''-O-rhamnoside in the aqueous 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 the lyophilized extract. Figure 2b shows the chromatographic profile obtained by HPLC-DAD-UV of this sample (λ 340 nm). The retention time (tR) of vitexin-2''-O-rhamnoside determined in this chromatogram was 13.20 min.
Lyophilized samples of the P. alata endocarp and pulp were solubilized in water at a concentration ten times greater than that tested with leaves. Still, the chromatogram of these extracts showed no signal for 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.
A sample of the aqueous extract of P. foetida at a concentration of 1 mg/mL was analyzed by HPLC-DAD-UV and the chromatographic profile (λ 340 nm) is shown in Figure 2e. The glycosylated flavonoid vitexin-2''-Orhamnoside (tR = 13.16 min) content in the sample was  72.08 ± 1.85 µg/mL, equivalent to 7.21% of the lyophilized extract. The content of vitexin-2''-O-rhamnoside (tR = 13.49) in the aqueous extract of P. setacea cv. BRS Pérola do Cerrado was calculated as 36.64 ± 2.04 µg/mL, which corresponds to 3.66% of the lyophilized extract (Figure 2f).

DISCUSSION
This newly developed and validated method for the quantification of vitexin-2''-O-rhamnoside (1) employed acidified ultrapure water (MilliQ deionized) and acetonitrile (HPLC grade) in gradient mode as a mobile phase. The best capacity of retention factor (α = 1.9), signal symmetry (~ 1.0), and selectivity of this new method are great parameters to quantify this Cglucosyl-flavonoid. Preparation of the mobile phase was cost-effective because most of it consists of MilliQ deionized water that is obtained directly from the laboratory.
A new approach with the stationary phase was used, employing a Supelco Ascentis-phenyl column (250 mm × 4.6 mm i.d. × 5 μm, particle size). To the best of our knowledge and as shown in Table 1, this is the first time that a Silica-based column modified with phenyl groups was used to quantify vitexin-2''-O-rhamnoside. This approach introduced clear advantages for the new method, including a great separation factor (1.9) and signal symmetry (1.0) that is reflected in the obtained LOD and LOQ.
The linearity of the method was excellent, as the correlation coefficient was 0.9925 ± 0.0007, considering a concentration range of 0.5 to 100 µg/mL. This is quite wide and ranges from 2.5 to 200 times the LOQ. Dispersion of the points of the analytical curves was homoscedastic, without outliers.
The precision and accuracy of the method were within the parameters established in the standards (INMETRO, 2016;ANVISA, 2017). The accuracy and precision of an analytical method are critical to ensuring the reliability of repeated measurements taken over the same day and over different days, as well as to assess how experimental data relates to expected (theoretical) data. The developed method reported here is precise because the precision test results showed an RSD less than 15% for measurements made on the same day and on different days and there was no variation between the intra-day and interday chromatographic run averages.
Regarding accuracy, when comparing the expected (theoretical) with the observed (experimental) values, the results showed that the greatest variation was from 89.31% to 7.5 μg/mL and 110.69% to 50 μg/mL, within the recommended limits (85-115%). Thus, the developed method can be considered accurate and precise (INMETRO, 2016;ANVISA, 2017).
The recovery of the method was excellent, greater than 90%, according to the validation standards (INMETRO, 2016;ANVISA, 2017). Another determining factor in method validation was robustness. The method was robust because the slight variations in the vitexin-2''-O-rhamnoside signal area (ABS in mAU), such as decreased flow   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 by 50 to 47°C, and increased pH of the aqueous phase from 3.0 to 3.5, did not influence the average of ABS areas. Identification (LOD, 100 ng/mL) and quantification (LOQ, 200 ng/mL) limits were low on the ng/mL scale. In the case of an UV detector, detection is expected to be on the nanogram scale (10 -9 ). Compounds with a great molar extinction coefficient, for example those with chromophores that absorb strongly in UV light, will have good detection sensitivity. Thus, the detector employed proves to be efficient for the detection and quantification of vitexin-2''-O-rhamnoside.
Previously published methods for the analysis of vitexin-2''-O-rhamnoside available in the literature (Table  1), present, in part, a greater total analysis time. Additionally, the mobile phases used are more toxic and harmful to the chromatographic system. Some of these methods also use more than one organic solvent, while our method uses only one (acetonitrile). The lack of 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., 2011). Some LOQ are extremely high, in the scale of µg/mL (Ying et al., 2009;Mudge et al., 2016;Strada et al., 2017). These LOQ can compromise the vitexin-2''-Orhamnoside quantification.

Conclusion
We presented a newly developed and validated method to quantify vitexin-2''-O-rhamnoside in passion fruit extracts. The method described here has clear advantages when compared to other previously reported methods, mainly for the use of a silica-based phenyl column that allowed the best chromatographic parameters. Additionally, the new validated method employs a mixture of acidified ultrapure water and acetonitrile, which is more cost-effective than those previously published. The validated method was tested to quantify and standardize the extracts of vitexin-2''-O-rhamnoside in three different Passiflora species. Results showed higher content of this bioactive flavonoid in P. foetida (7.21%) and P. setacea cv. BRS Pérola do Cerrado (3.66%) than in the pharmacopeical species P. alata (2.89%).

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest to disclose.