Identification, Quantification, and Characterization of the Phenolic Fraction of Brunfelsia grandiflora: In Vitro Antioxidant Capacity

Brunfelsia grandiflora is an ancient plant widely used for its promising medicinal properties, although little explored scientifically. Despite being a rich source of phenolic compounds responsible in part for the proven anti-inflammatory activity, its characterization has not been carried out to date. The present work deals with the exhaustive identification and quantification of its phenolic fraction, along with its antioxidant activity. Decoction resulting from the bark as fine powder was filtered and lyophilized, and polyphenols were extracted from the resulting product by aqueous-organic solvents. Seventy-nine polyphenols were identified using LC-MSn. Hydroxycinnamates was the most abundant group of compounds (up to 66.8%), followed by hydroxycoumarins (15.5%), lignans (6.1%), flavonols (5.7%), phenolic simples (3.1), gallates (2.3%), flavanols (0.3%), and flavanones (0.2%). About 64% of the characterized phenols were in their glycosylated forms. The quantification of these phytochemicals by LC-QToF showed that this medicinal plant contained 2014.71 mg of phenolic compounds in 100 g dry matter, which evidences a great antioxidant potency determined by ABTS and DPPH assays. Therefore, Brunfelsia grandiflora represents an important source of polyphenols which supports its therapeutic properties scientifically proven.


Introduction
Healing with medicinal plants is as old as humanity itself. Awareness of medicinal plant usage is a result of the many years of struggles against illnesses due to which man learned to pursue drugs in barks, seeds, fruit bodies, and other parts of the plants. The need to integrate the knowledge of traditional medicine with scientific medicine, based on experience and observation, makes it necessary to validate therapeutic action and establish the correct uses of plant resources. This is the case of Brunfelsia glandiflora, a traditional native remedy employed against rheumatism, arthritis, fevers, and snake bites in the upper Amazon region [1]. Brunfelsia glandiflora is a plant belonging to the Solanaceae family and family and the Brunfelsia genus, traditionally known as Chiric sanango, chiricaspi chacrudo; hu-ha-hai, sanango, mucapari, and chirihuayusa [2].
Brunfelsia glandiflora is a glabrous shrub up to five meters high, with tough bark, alternate leaves, apically leafy or scattered on flowering branches, 15-20 cm long, 5-8 cm wide. It has cymose inflorescence, pedicellate flowers 3.5-4 cm long, which are purple and white with tubular, campanulate corolla with five large lobes, and short calyx 1.5-2 cm long. Anthers are free from the stigma, small, obtuse, appendicular at the base, and superior bicarpelar ovary (Figure 1). Fruit in berry is ovate-rounded. It grows in the Andean mountainous area between Venezuela and Bolivia. It is distributed at the height of 200 m in Peru, above sea level, in the low and high Amazon areas (Regions of Loreto, Ucayali, Madre de Dios, and Cuzco) [2,3]. Brunfelsia grandiflora species known as "Chiric Sanango" is mainly sold in the medicinal plant markets, especially in the Amazon regions and in the capital Lima, from wild populations or home gardens, similar to other medicinal species. Our ancestors commonly used woody vascular plants, mainly their bark, and today this part of the plant is renowned as a source of antioxidants with potential health-promoting properties. There are few scientific publications on the pharmacological action of B. grandiflora, but the one described in the traditional medicine of the Peruvian Amazon refers to the aqueous maceration of the root of Brunfelsia grandiflora, which is used as a drink against arthritis, syphilis, bone pain, ovarian pain, fatigue and as an antipyretic. The infusion of the leaves against arthritis and rheumatism is another form of common use. Some reports mention that the bark decoction is applied to burns, to areas of the body affected by leishmaniasis, and as a healing agent, although its narcotic effects have also been reported [1,2,4,5].
The few pharmacological effects of B. grandiflora described above could be due to the presence of secondary metabolites such as polyphenolic acid compounds. One of these compounds could be scopoletin [6], with known anti-inflammatory activity, which would justify the effect of B. grandiflora against rheumatism, arthritis, body pain, headache, and joint and muscle pain. On the other hand, the hallucinogenic and narcotic properties associated with B. grandiflora would be mediated by brunfelsamidine, cuscohygrin, scopolamine, scopoletin, and esculetin, this one last used in oncology as an antiproliferative. Furthermore, the effects of brunfelsamidine and cuscohygrine in the fields of anesthesiology have been demonstrated [7][8][9].
The main objective of this work was to identify for the first time the phenolic composition of this medicinal plant to know the chemical structures of these phytochemicals that There are few scientific publications on the pharmacological action of B. grandiflora, but the one described in the traditional medicine of the Peruvian Amazon refers to the aqueous maceration of the root of Brunfelsia grandiflora, which is used as a drink against arthritis, syphilis, bone pain, ovarian pain, fatigue and as an antipyretic. The infusion of the leaves against arthritis and rheumatism is another form of common use. Some reports mention that the bark decoction is applied to burns, to areas of the body affected by leishmaniasis, and as a healing agent, although its narcotic effects have also been reported [1,2,4,5].
The few pharmacological effects of B. grandiflora described above could be due to the presence of secondary metabolites such as polyphenolic acid compounds. One of these compounds could be scopoletin [6], with known anti-inflammatory activity, which would justify the effect of B. grandiflora against rheumatism, arthritis, body pain, headache, and joint and muscle pain. On the other hand, the hallucinogenic and narcotic properties associated with B. grandiflora would be mediated by brunfelsamidine, cuscohygrin, scopolamine, scopoletin, and esculetin, this one last used in oncology as an antiproliferative. Furthermore, the effects of brunfelsamidine and cuscohygrine in the fields of anesthesiology have been demonstrated [7][8][9].
The main objective of this work was to identify for the first time the phenolic composition of this medicinal plant to know the chemical structures of these phytochemicals that are behind the renowned biological properties of Brunfelsia grandiflora. Additionally, polyphenol content and antioxidant capacity will be determined to evaluate the magnitude of this phytochemical fraction in Brunfelsia grandiflora.

Results and Discussion
Ever since ancient times, people have looked for drugs in nature to face different diseases. Brunfelsia grandiflora is an excellent example of folk medicine used for ages with successful results against rheumatism, arthritis, cold, tiredness, pain of ovaries, sexual potency, pain in bones, laziness, and cancer of uterus [2], although limited scientific studies confirm these effects [10,11]. Even in our time, when there is increasing awareness of the importance of diet quality to prevent chronic disease, and although the main sources of phenolic compounds are fruits and vegetables, more and more studies refer to woody vascular plants, especially bark [12], directing the interest to the traditional herbal as a source of antioxidants with potential health-promoting properties. This situation points out the importance of considering these medicinal plants as an adjuvant to deal with prevalent diseases and hence the adequacy of properly characterizing their phytochemical composition. Phenolic compounds are ubiquitously distributed phytochemicals found in most plant sources with recognized health benefits [13], and as far as we know, the phenolic fraction in Brunfelsia grandiflora has never been characterized. In the present work, the identification and quantification of the phenolic fraction were assessed in a lyophilized extract obtained from the Brunfelsia grandiflora bark. Additionally, total phenolic content by Folin-Ciocalteu and the antioxidant capacity was carried out.

Total Phenolic Content and Antioxidant Capacity
The total phenolic content determined by the Folin-Ciocalteu assay and its antioxidant potency developed by FRAP and DPPH assays are summarized in Table 1. IC 50 values determined by both FRAP and DPPH assays are included. The evaluated extract had about 3% of the phenolic content of the dry matter. Additionally, the antioxidant ability of the Brunfelsia grandiflora bark was tested by two methods (DPPH and ABTS) that measure the ability of antioxidants contained in this medicinal plant to scavenge the DPPH and ABTS, respectively, and based on an electron transfer and the reduction of a colored oxidant. The IC 50 (half-maximal inhibitory concentration) was calculated as the concentration of sample necessary to decrease by 50% the initial absorbance of DPPH and ABTS. Both methods showed a very high radical scavenging, 2.55 and 4.55 µg/mL for DPPH and ABTS, respectively. These values agree with the high polyphenol amount determined with the spectrophotometric Folin-Ciocalteu method. Recently, the antioxidant capacity of an herbal remedy (HR) was compared with that of a crude hydroalcoholic extract (CHE) obtained from Brunfelsia uniflora (Pohl) D. Don roots [14]. IC 50 values determined by the ABTS assay showed significantly higher values (1678.00 ± 11.26 µg/mL and 3441.00 ± 36.05 µg/mL for HR and CHE, respectively) than that determined for Brunfelsia grandiflora bark (4.55 µg/mL). Likewise occurred with the IC 50 determined by DPPH for HR and CHE, where the values of 37,698.00 ± 3437.00 µg/mL and 68,452.00 ± 5155.00 µg/mL of HR and CHE, respectively, were much higher than that obtained for Brunfelsia grandiflora (2.55 µg/mL), which suggested a substantially higher antioxidant activity of our medicinal plant than that evaluated in this article. Borneo et al. [15] characterized the antioxidant capacity by DPPH of 15 Asteraceae plant species from Cordoba (Argentina) in relation to their phenol content determined by the Folin-Ciocalteu assay. Phenolic content ranged from 11.3 to 54.4 mg/g, and their IC 50 values from 198 to 2009 µg/mL, which were higher than that determined for ascorbic acid, BHT, and quercetin (11.5, 15.3, and 14.8 µg/mL, respectively). Brunfelsia grandiflora showed higher antioxidant potency than the Argentinian plants and, more importantly, well-known antioxidants such as ascorbic acid, BHT, and quercetin. A recent study developed by Rebolledo et al. [16] with the Peruvian peppertree Schinus areira L. from Chile observed that the methanolic extracts were highly rich in both polyphenols (>195 mg/g dw~19.5%) and antioxidant activity (IC 50 > 476 mg/mL; >273 mg ascorbic acid/g dw (DPPH); >301 mg ascorbic acid/g dw (FRAP)) and were in line with that described in the present manuscript. Therefore, the high antioxidant potency of Brunfelsia grandiflora bark highlights the potential of this plant for pharmacological use.

LC-QToF Identification of the Phenolic Fraction of Brunfelsia grandiflora
Seventy-six phenolic compounds were identified in Brunfelsia grandiflora based on their relative retention time, mass spectra and commercial standards. Table 2 shows the retention time (RT), molecular formula, accurate mass of the molecular ion [M − H] − after negative ionization, and MS 2 fragments of the main compounds identified in Brunfelsia grandiflora by LC-QToF.  The presence of scopoletin has been mentioned in the few studies carried out with Brunfelsia grandiflora [6], which belong to hydroxycoumarin group. This compound was identified thanks to its MS spectra ([M − H] − at m/z 191.0350 and fragment ions at m/z 148, 120, and 104). Also belonging to coumarins, it was identified esculetin and its glycosylated derivative (esculin) at 5.7 and 3.7 min, respectively. The first one showed a quasimolecular ion at m/z 177.0193 and fragment ions at m/z 149, 133, and 105 compatible with its chemical structure, and the glycosylated derivative ([M − H] − at m/z 339.0722) showed as the main fragment ion that corresponds to its free precursor, esculetin (m/z 177) ( Table 2).
Some of the identified compounds showed a chemical structure belonging to gallates, such as methyl-and ethyl-gallate, as well as galloyl glucose. Three isomers of methylgallates at 3.6, 6.6, and 9.  (Table 2).
An important group of phenolic compounds identified in Brunfelsia grandiflora belonged to hydroxycinnamic acids. Three isomers of caffeoylquinic acids were identified at 4.7, 5.  (Table 2).
A minor group of the identified compounds corresponded to flavanols, such as gallocatechin and methyl-gallocatechin. Both commercial standards and MS spectra allowed their unequivocal identification at 6.1 and 14.2 min, respectively ( Table 2).
Seven flavanones were also identified in the lyophilized extract from Brunfelsia grandiflora bark. Eriodictyol and two glycosylated derivatives were characterized at 10.2, 12.3, and 16.2 min, respectively. Commercial eriodictyol facilized its identification along with its MS spectra, while eriodyctiol-O-glucoside showed well-suited MS spectra ([M − H] − at m/z 449.1089 and fragment ions at m/z 287 and 255 corresponding to eriodictyol). Likewise, naringenin and two glycosylated derivatives at 15.9, 12.1, and 18.0 min, respectively, were characterized based on their compatible MS spectra. Hesperetin was also present in Brunfelsia grandiflora bark, although no glycosylated derivative was identified.
Belonging to the flavonols group, two compounds with the same molecular formula (C 27 H 30 O 15 ) were identified as kaempherol-O-rutinoside and kaempherol-O-galactosiderhamnoside based on their different fragmentation pattern (Table 2). In addition, they eluted at 4.9 and 12.0 min, respectively, in agreement with their polar nature. Likewise happened with isorhamnetin-O-rutinoside and isorhamnetin-O-glucoside-O-rhamnoside, although they showed the same quasimolecular ion at m/z 623.1618 and fragment ion at m/z 315 corresponding to isorhamnetin, the greater polarity of isorhamnetin-O-rutinoside allowed it to be associated with the chromatographic peak that eluted at 10.6 min, and isorhamnetin-O-glucoside-O-rhamnoside with that which eluted at 12.2 min.
The phenolic fraction of Brunfelsia grandiflora was also constituted by lignans, such as pinoresinol and matairesinol. These phenolic compounds presented the same molecular formula and, therefore, equal quasimolecular ion at m/z 357.1344, and, unfortunately, MS/MS analysis showed no fragment ion. Nevertheless, their different polar nature allowed us to know that pinoresinol eluted at 8.8 min while matairesinol at 9.5 min. Two isomers of hydroxysecoisolariciresinol were identified at 9.6 min and 9.9 min based on their MS analysis ([M − H] − at m/z 377.1606 and fragment ion at m/z 329). Secoisolariciresinol, along with two isomers, were characterized thanks to their MS analysis, which eluted between 9.8 and 15.2 min (Table 2). Sesamol with a quasimolecular ion at m/z 137.0244 was associated with the chromatographic peak eluting at 11.  15 and fragment ion at m/z 313), and the molecular formula (C 20 H 24 O 6 ) compatible with cyclolariciresinol and isolariciresinol, which did not allow us to determine the identity of the lignin. Likewise, the chromatographic peak at 12.9 min showed MS spectra compatible with hydroxymatairesinol and nortrachelogenin.
Finally, simple phenolic acids were also characterized in this medicinal plant, most of them supported by commercial standards such as protocatechuic acid, 3-and 4-hydroxybenzoic acid, vanillic acid, homovanillic acid, 3,4-dihydroxyphenylpropionic acid, 3-and 4-hydroxyphenylpropionic acids and 3-methoxy-4-hydroxyphenylpropionic acid. Additionally, three isomers of dihydroxybenzoic acid, two isomers of methoxyhydroxybenzoic acid, two isomers hydroxyphenylacetic acid, and the other isomer of methoxy-hydroxyphenylpropionic acid were characterized based on their respective MS spectra ( Table 2). This group was completed with the characterization of glycosylated derivatives of dihydroxybenzoic acid and methoxy-hydroxybenzoic acid, thanks to the quasimolecular ion at m/z 315.0722 and 329.0878, respectively, and the presence of their precursor, benzoic acid, among the fragment ions.

LC-QToF Quantification of the Phenolic Content of Brunfelsia grandiflora
The quantification of these phytochemicals by LC-QToF showed that Brunfelsia grandiflora contained 2014.71 mg of phenolic compounds in 100 g dry matter. This amount is lower than that determined by the Folin-Ciocalteu assay (3017 mg/100 g dry matter) but coherent because it is well-known that the Folin-Ciocalteu assay could over-estimate the real polyphenol content. It is impossible to compare with data reported in the literature because this is the first time that the phenolic fraction of Brunfelsia grandiflora is characterized.
The most abundant group of polyphenols present in Brunfelsia grandiflora was composed of hydroxycinnamic acids, which amounted to 66,8% of the total phenols quantified. These compounds were preferentially present as hydroxycinnamates, either esterified with glucose to form glycosidic derivatives of caffeic, ferulic, coumaric, and sinapic acids (86.2% of the total hydroxycinnamic acids) or with quinic acid to form hydroxycinnamoyl derivatives such as caffeoyl-, feruloyl, coumaroyl-and sinapoylquinic acids (12.9% of the total hydroxycinnamic group). The free precursors, along with dehydrodiferulic acid, barely represented 0.9% of the total hydroxycinnamic acids (Table 3).  The following compound group characterized in Brunfelsia grandiflora by order of abundance was that corresponding to hydroxycoumarins (15.5% of the total phenolic content), led by scopoletin (91.6% of the total of this group) and followed by esculetin and esculin ( Table 3).
The following more abundant compound group was lignans (6.1% of the total phenolic content), led by sesamol and sesamin/episesamin, with 44.8% and 17.5%, respectively, of the total of this group. Secoisolariciresinol and hydroxysecoisolariciresinol were also predominant, amounting to 14.9% of the total lignans (Table 3).
The following group was that corresponding to phenolic acids, widely distributed in vegetable sources, reaching 3.1% of the total phenolic content. Although these com-pounds were preferentially in their free forms, glycosylated forms of dihydroxybenzoic and methoxy-hydroxybenzoic acids represented 13.8% of phenolic acids characterized. The top five most abundant compounds were two isomers of hydroxyphenylacetic acid, protocatechuic acid, methoxy-hydroxybenzoic acid, and 3-hydroxybenzoic acid (Table 3).
Gallates group represented 2.3% of the total phenolic fraction. This group was headed by ethyl-gallate followed by methyl-gallate, accounting for 85.1% and 8.9% of the total gallates, respectively. The remaining 6.0% was composed of galloyl glucose and free gallic acid (Table 3).
Preferentially glycosidic forms of flavanones eriodictyol, naringenin, along with their free precursors, and hesperetin represented 0.21% of the total phenolic content of Brunfelsia grandiflora. Approximately half was comprised of eriodictyol and derivatives, and the other half of naringenin and derivatives, while hesperetin barely reached 2% of the total of this specific fraction.
Regarding the correlation between chemical composition and antioxidant capacity, it is worth noting that the massive content of hydroxycinnamic acids/hydroxycinnamates in B. grandiflora is enough to grant a remarkable antioxidant power, as we have previously reported in vitro and cell culture [17][18][19][20][21][22][23]. It is well known the correlation of the antioxidant activity of polyphenols with the number and position of -OH groups or the presence of a double bond in the position 2-3 of C ring in flavonoids. Likewise, the antioxidant activity of polyphenolic acids depends on the number of -OH groups in their molecule. Thus, gallic acid, caffeic acid, catechin, and eriodictyol and their derivatives will strongly contribute to the antioxidant potency of this plant. Likewise, dihydroxybenzoic, dihydroxyphenylacetic, and dihydroxyphenylpropionic acids are also key antioxidants present in this medicinal plant. Further, this antioxidant capability has translated into beneficial biological effects in experimental animal models [24]. We are currently investigating the effect of B. grandiflora extracts on cultured endothelial EA.hy926 and neuronal SH-SY5Y cells submitted to oxidative stress to confirm the chemo-protective potential of extracts from the bark of this plant to explain and sustain its traditional medicinal utilization. Thus, this research should be considered as a starting point for a series of studies devoted to proving the cellular and molecular basis that supports the medicinal use of this plant.

Chemical Reagents
Bark of Brunfelsia grandiflora was collected from the native community of Canaán de Cachiyacú, Contamana district, Ucayali province, Loreto region (Peru). All solvents and reagents were of analytical grade unless otherwise stated.

Sample Preparation
The bark was wholly collected and washed. It was dried in the open air to constant weight and reduced to a fine powder. The decoction was developed by placing distilled water and powdered plant material (10:1) in a beaker, heating to boiling, and holding for twenty minutes. The plant material exhausted by the extraction was separated by filtration, and the aqueous extract was concentrated and lyophilized for preservation.
To determine total phenolic content by Folin-Ciacalteu and antioxidant activity by DPPH and ABTS assays, a methanolic solution from the lyophilized extract obtained from the Brunfelsia grandiflora bark was prepared at 0.02 g/mL and diluting later with water up to 0.8 mg/mL.
To determine the antioxidant activity by DPPH and ABTS assays, the sample was dissolved in ethanol 96% (v/v) to obtain concentrations from 4 to 16 µg/mL and from 2 to 8 µg/mL, respectively.
The procedure of Perez-Jimenez et al. [25] was followed with minor modifications to isolate polyphenols from the bark of Brunfelsia grandiflora in order to characterize them by LC-ESI-QTOF. Briefly, 0.25 g by quadruplicate of the lyophilized extract was extracted in aqueous methanol (50:50, v/v, with HCl 2 N, 1 h) by constant shaking and centrifuged at 3000× g. Supernatants were separated, and the pellets were washed with acetone/water (70:30, v/v) by constant shaking and centrifuged at 3000× g. Supernatants from each extraction step were combined at 50 mL. An aliquot of 1 mL was concentrated under reduced pressure using a vacuum concentrator system (Speed-Vac, Thermo Fisher Scientific Inc., Waltham, MA, USA) and then resuspended in 0.5 mL of 1% formic acid in deionized water (v/v), filtered through a cellulose-acetate membrane filter of 0.45 µM pore-size, dispensed in chromatographic vials and stored at −80 • C until analysis.

Polyphenolic Content by Folin-Ciocalteu
The total phenolic content of Brunfelsia grandiflora bark was quantified spectrophotometrically at 765 nm using the Folin-Ciocalteu reagent following ISO 14502-1 procedure [26]. Then, 100 µL of the methanolic extract of the Brunfelsia grandiflora was prepared as described above in Section 3.2. The section was mixed with 500 µL of Folin-Ciocalteu diluted with water (1:9, v/v) and let stand for 5 min. Then, 400 µL of Na 2 CO 3 7.5% w/v was added and shaken vigorously. After 1 h incubation at room temperature (25 • C), the absorbance was measured in a spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 765 nm. Gallic acid was used as standard, and results were expressed as mg gallic acid equivalents (GAE) per 100 g of dry matter.

Determination of Antioxidant Capacity
The antioxidant capacity of Brunfelsia grandiflora extracts prepared as described in Section 3.2. was determined by two different methods. DPPH• radical scavenging assay: the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used to evaluate the radical scavenging activity of the samples, following the method reported by Brand-Williams et al. [27] and Thaipong et al. [28]. The stable free radical DPPH• is purple and is discolored to yellow in the presence of a free radicalcapturing substance whose measurement at 517 nm (spectrophotometer GENESYS TM 10S UV-VIS) is related to the antioxidant capacity of the substance. Trolox was taken as a reference, and the results were expressed as mg Trolox Equivalent Antioxidant Capacity (TEAC) per gram of dry matter. IC 50 was also determined and expressed as µg/mL. ABTS assay: the free radical cation ABTS+, which was prepared by reaction of ABTS with 2.45 mM potassium persulfate during 12-16 h at room temperature in the dark, was used to evaluate the free radical scavenging capacity of the samples. This radical decreases absorbance at 734 nm in the presence of an antioxidant [28,29]. The absorbance was monitored for 30 min at 37 • C in a spectrophotometer GENESYSTM 10S UV-VIS. Results were expressed as mg TEAC per gram of dry matter. IC50 was also determined and expressed as µg/mL.

Phenolic Characterization of Brunfelsia grandiflora by LC-ESI-QTOF Analysis
Phenolic compounds from Brunfelsia grandiflora were characterized by HPLC-ESI-QToF [30] in an Agilent 1200 series LC system coupled to an Agilent 6530A Accurate-Mass Quadrupole Time-of-Flight (Q-ToF) with ESI-Jet Stream Technology (Agilent Technologies). Compounds were separated on a reverse-phase InfinityLab Poroshell 120 EC-C18