Ruellia angustiflora (Nees) Lindau ex Rambo: extraction and characterization of phenolic compounds and evaluation of antiradical, photoprotective and antimicrobial activities

Abstract Ruellia angustiflora is a shrub popularly known as flower of fire, used in traditional medicine as a healing agent. This study aims to verify the presence of characteristics of pharmaceutical interest in extracts from the root, stem and leaves of this species. The ideal time for ultrasound-assisted extraction is 60 min with a solid-liquid ratio of 1% (w/v). The kinetics of the reaction of the extracts with DPPH is biphasic, and IC50 values are related to the total phenolic compounds. Five phenolic compounds were identified in the extracts, where ferulic acid, rutin and quercetin showed to be the main responsible for the antiradical activity. The extracts displayed considerable photoprotective activity, mainly Ra-LE (SPF = 23). Ra-RE showed an inhibition halo similar to erythromycin against Streptococcus pyogenes. Taken together, these data reveal that Ruellia angustiflora is a source of phenolic compounds and has relevant antiradical, photoprotective and antibacterial activities. Graphical Abstract


Introduction
The use of plants for their medicinal properties is as old as mankind itself.Besides helping to relieve pain and promote the cure of illnesses, plant extracts are also able to prevent the onset of several diseases (Rahman et al. 2019).Brazil is an extremely diverse country, accounting for 10 to 20% of the known species in the world.The ethnic, cultural and socioeconomic plurality contributes to the country's strong tradition in the use of medicinal plants (Pilon et al. 2017).Despite the increase in the number of research in the area, there are few examples of products containing Brazilian plant species, which represents an enormous economic potential to be explored (Valli et al. 2018).
Belonging to the Acanthaceae family, Ruellia angustiflora (Nees) Lindau ex Rambo is a shrub present in the Pampa biome that occurs mainly in southern Brazil and in certain regions of Paraguay, Argentina and uruguay (Hammes et al. 2021).This species is popularly known as flower of fire, and it is often cultivated due to its beautiful red flowers (Silva et al. 2019).In traditional medicine, Ruellia angustiflora is used as a wound healing agent (Alice et al. 1995), and this activity has already been demonstrated in an animal model (Pires et al. 2021a).In addition, the extracts show antiparasitic activity (Pires et al. 2020) and an absence of cytotoxicity and genotoxicity (Pires et al. 2021b).However, this species is still very little investigated regarding its therapeutic potential.
Thus, the aim of this study is to determine the optimal conditions to obtain extracts from the root, stem and leaves of Ruellia angustiflora, and identify the main phenolic compounds present in them.Furthermore, considering its healing activity, it aims to evaluate the antiradical, photoprotective and antibacterial activities of the extracts, in order to verify their potential as a natural additive in the development of cosmetic formulations for skin care and antiaging.

Ultrasound assisted extraction (UAE) of phenolic compounds
During the uAE process, the content of phenolic compounds obtained increases over time according to a two-step mechanism.In the first step, the soluble components on the surface of the matrix undergo rapid dissolution.After that, the extraction yield slowly increases, as the mass transfer from the plant matrix to the solvent occurs by diffusion and osmotic processes (Şahin and Samli 2013).These phenomena can be noted in Figure S1, which shows that the extraction rates were higher at the beginning of the process, and after 60 min they remained practically stable.Thus, this was the time defined for obtaining the extracts, in order to optimize the process and avoid possible degradation.
Another important variable that strongly influences the extraction yield of phenolic compounds in the uAE is the solid/liquid ratio.When changes occur in the plant material and the phenomenon of mass transfer increases, it is possible to obtain more soluble compounds.Consequently, solvent saturation can be reached if used in low amounts.An increase in the proportion of solvent prevents its saturation and increases the extraction yield (Paz et al. 2015).However, it also generates an increase in solvent consumption and cost.Therefore, evaluating the extraction rates, extracts yield and cost-effectiveness, a solid/liquid ratio of 1% (w/v) was chosen to carry out additional tests (Figure S1, Table S1).

Content of total phenolic compounds and total flavonoids
Phenolic compounds are considered the most important and diverse secondary metabolites in the plant kingdom (Haminiuk et al. 2012).Table S2 presents the content of phenolic compounds and total flavonoids of Ruellia angustiflora extracts, and it can be seen that Ra-RE has a significantly higher amount of phenolic compounds (244.2 ± 2.1 mg GAE/g).The root is one of the main organs responsible for the accumulation of compounds with medicinal value in plants (Li et al. 2020), and some authors also reported to have found a higher content of phenolic compounds in root extracts when compared to extracts from other parts of plants (Srećković et al. 2020;Bakhouche et al. 2021).Regarding the flavonoids, Ra-LE had the highest content (157.4 ± 1.8 mg RE/g).Similar results were found by Cheong et al. (2013) when determining the flavonoid content of the methanolic extract of the leaf (140.29 mg/g) and stem (113.71mg/g) of Ruellia tuberosa L.

DPPH radical scavenging activity
The DPPH radical method has long been used to measure the free radical scavenging activity of fruits, vegetables and plant extracts.However, the lack of parameters standardization such as reaction time makes it difficult to compare the antiradical activity of different samples (Menezes et al. 2021).Besides that, the kinetics of the radical reaction with the antioxidant must be observed, since using a fixed incubation time in the assay does not consider the radical scavenging activities for molecules that present a slow reaction (Mishra et al. 2012).
For most compounds that have antiradical properties, the reaction with DPPH has two phases.First, there is a fast decrease of absorbance in the initial minutes by fast subtraction of the hydrogen atom, and then a slower phase begins due to the involvement of degradation products (Pyrzynska and Pękal 2013).This behavior was found for Ra-RE, Ra-SE and Ra-LE (Figure S2), and may occur due to the presence of compounds with different reaction kinetics in the extracts.Moreover, Figure S3 presents the percentages of DPPH radical scavenged with different concentrations of extracts according to their reaction time.Considering that the consumption of the DPPH radical by the extracts does not reach equilibrium even after 120 min, the IC 50 values for different reaction times, between 3 and 120 min are described in Table S3.It is possible to observe that the IC 50 values vary considerably, decreasing over time, in a linear mode.Ra-RE and Ra-LE present very similar IC 50 values.Ra-SE showed higher values, and its lower content of total phenolic compounds may help to explain this result, since this relationship has already been established (Jiménez et al. 2015).

Identification of phenolic compounds
Five phenolic compounds were identified in Ra-SE, Ra-LE and, to a greater extent, in Ra-RE (Figures S4 and S5).The major signs were seen for ferulic acid (2) and rutin (3).Ferulic acid has numerous pharmacological activities, such as antioxidant, anti-inflammatory, photoprotective, antimicrobial and anticancer (Sauce et al. 2021).It has been widely used in the cosmetic industry as an antioxidant and to prevent skin aging, as it acts in the protection of important cell structures such as keratinocytes and fibroblasts (Zduńska et al. 2018).Some experiments performed on diabetic rats have shown that ferulic acid accelerates wound healing (Ghaisas et al. 2014;Bairagi et al. 2018).In addition, it provides a whitening, moisturizing and erythema reduction effect, being considered a promising active in formulations for topical application (Zduńska-Pęciak et al. 2022).The compounds identified here were previously detected in the species (Pires et al. 2021b) and found in the characterization of other species belonging to the Acanthaceae family (Cassola et al. 2019;Basit et al. 2022).It is worth mentioning that several environmental factors can influence the composition of secondary metabolites produced by a plant, such as temperature, carbon dioxide, lighting, soil fertility and soil salinity (Pant et al. 2021).

DPPH-HPLC experiment
The DPPH-HPLC experiment has been used to perform rapid screening of antioxidants, especially for plant extracts.After the reaction with the DPPH radical, the chromatographic peak areas of compounds that exhibit antiradical activity were reduced or disappeared in the chromatogram.In contrast, for compounds that do not show antiradical activity, the peak areas remained practically unchanged (Wang et al. 2017).The chromatograms of the extracts before and after the reaction with DPPH are shown in Figure S4.It can be seen that peaks 2 and 3 had a significant reduction in their areas, and peak 4 totally disappeared after the reaction with DPPH, demonstrating that these compounds are the main contributors to the antiradical activity.on the other hand, peaks with retention time close to 29 min had an increase after reacting with the radical, these being probably residual DPPH or reaction products.Peaks 1 and 5 had no reduction in their areas after the addition of the DPPH solution, which suggests that they did not contribute to the antiradical activity demonstrated by the extracts.

Photoprotective activity and determination of sun protection factor (SPF)
Exposure to ultraviolet radiation is associated with a variety of damaging effects, such as early photoaging and skin cancer induction.The synthetic uV filters available have some limitations as they can generate negative effects on the skin (Saewan and Jimtaisong 2015).Thus, plant extracts can be considered as alternatives, since flavonoids and other phenolic compounds have light absorption properties in the uVA (320-400 nm) and uVB (290-320 nm) ranges, and may act as a natural sunscreen (Alencar Filho et al. 2016).Figure S6 presents the absorption profile of the Ruellia angustiflora extracts and the standards rutin and ferulic acid.The uV spectra of the extracts exhibited an important absorption in the uVA and uVB regions, especially Ra-LE.The wavelengths of maximum absorption for the three plant extracts showed to be similar, with peaks around 290 nm and 340 nm.According to international recommendations (Food and Drug Administration 2021), adequate sunscreen should have an SPF of 15 or higher to provide maximum benefit.Thus, Ra-RE (19.91 ± 0.05), Ra-SE (15.77 ± 0.02) and mainly Ra-LE (23.09 ± 0.03) met this requirement and could be considered as active ingredients in photoprotective formulations (Figure S7).
Ferulic acid demonstrated a high SPF value even at a low concentration (18.26 ± 0.6 at 25 mg/L).In this sense, Peres et al. (2018) found a synergistic effect in the association between ferulic acid and uV filters in a formulation, with an increase in SPF in vivo of 37% after the addition of the phenolic compound.In this study, rutin showed an SPF of 15.49 ± 0.2 at 100 mg/L.Choquenet et al. (2008) found that rutin (10% w/w) as an oil-in-water emulsion have a SPF similar to homosalate, a known uV filter.Taken together, these data reaffirm the potential of phenolic compounds as photoprotective agents.

Antibacterial activity
Some types of skin infections are commonly treated with broad-spectrum antibiotics.However, the use of these drugs can lead to deleterious effects on the skin's natural flora and result in bacterial resistance (Pinho et al. 2014).Thus, the investigation of new antimicrobial agents is essential and should be treated as a priority in the pharmaceutical field.As can be seen in Table S4, Ra-RE, Ra-SE and Ra-LE exhibited significant inhibition halos against Streptococcus pyogenes, and Ra-RE demonstrated antimicrobial activity comparable to erythromycin.Regarding the activity against Staphylococcus aureus, only Ra-RE presented the formation of inhibition halos.S. aureus and S. pyogenes are the main agents responsible for superficial skin infections and are often associated with cases of impetigo in humans (Abrha et al. 2020).Infections by both bacteria can range from self-limiting skin infections to potentially serious complications such as cellulitis, osteomyelitis and sepsis (Taiaroa et al. 2021).
In general, the main mechanism suggested for the antimicrobial activity of phenolic compounds involves their interaction with the cytoplasmic membrane of bacteria, producing changes in its integrity and fluidity (Álvarez-Martínez et al. 2021).Therefore, the relevant results observed mainly by Ra-RE may be a consequence of its higher total phenolic content.However, the extracts did not reveal satisfactory results against Gram-negative strains of Escherichia coli, Salmonella enterica and Pseudomonas putida.In fact, Gram-negative bacteria are less susceptible to antimicrobial agents because their outer cell membranes contain lipoproteins and lipopolysaccharides capable of regulating their access to underlying structures (Siddiqui et al. 2016).
Different species of Ruellia have already reported antimicrobial activity, such as Ruellia prostrata Poir., whose extract indicated dose-dependent antibacterial activity against Gram-positive and Gram-negative bacteria in the concentration range of 62.5 to 500 mg/mL (Akhter et al. 2022).The essential oils of Ruellia asperula (Mart.ex Nees) Lindau and Ruellia paniculata L. demonstrated antimicrobial potential against streptococci causing oral infections (Vasconcelos et al. 2021).other studies also report the antimicrobial activity in extracts of Ruellia tuberosa L. (Vasantharaj et al. 2019) and Ruellia patula Jacq.(Ramadevi et al. 2016).The results described here for Ruellia angustiflora reinforce the presence of antibacterial activity in this plant genus.

Experimental
See supplementary material.

Conclusions
The extraction of phenolic compounds from the root, stem and leaves of Ruellia angustiflora proved to be efficient and cost-effective at 60 min of extraction, with a solid-liquid ratio at 1% (w/v).Five phenolic compounds were identified in the extracts, with ferulic acid being the main component.Through the HPLC-DPPH experiment, it was verified that ferulic acid, rutin and quercetin were the main responsible for the antiradical activity.The kinetics of the reaction of the extracts with the DPPH radical was biphasic, and the IC 50 values were associated with the content of phenolic compounds.The extracts also demonstrated relevant photoprotective activity, especially Ra-LE, which showed the highest SPF.Antimicrobial activity was reported against bacteria that cause skin infections, mainly for Ra-RE, that showed an inhibition halo similar to erythromycin against S. pyogenes.Thus, these data reveal the potential of Ruellia angustiflora as an active ingredient for the development of functional cosmetics.