Identification, quantification, and characterization of the phenolic fraction of Brunfelsia grandiflora: In vitro antioxidant capacity

Citation: Mateos, R.; Ramos-Cevallos, N.; Castro-Luna, A.; Ramos-Gonzalez, M.; Clavo, Z.-M.; Quispe-Solano, M.; Goya, L.; Rodríguez, J.-L. Identification, Quantification, and Characterization of the Phenolic Fraction of Brunfelsia grandiflora: In Vitro Antioxidant Capacity. Molecules 2022,27, 6510. https://doi.org/10.3390/ molecules27196510 Academic Editors: María Isabel Calvo, Maria Luisa Kennedy and Francisco Leon Received: 15 September 2022 Accepted: 26 September 2022 Published: 2 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). molecules Article Identification, Quantification, and Characterization of the Phenolic Fraction of Brunfelsia grandiflora: In Vitro Antioxidant Capacity Raquel Mateos 1,* , Norma Ramos-Cevallos 2, Americo Castro-Luna 2, Mariella Ramos-Gonzalez 3, Zoyla-Mirella Clavo 4, Miguel Quispe-Solano 5, Luis Goya 1,* and José-Luis Rodríguez 6 1 Department of Metabolism and Nutrition, Institute of Food Science, Food Technology and Nutrition (ICTAN), Spanish National Research Council (CSIC), Jose Antonio Novais 10, 28040 Madrid, Spain 2Research Institute Juan de Dios Guevara, Faculty of Pharmacy and Biochemistry, Major National University of San Marcos, Lima 15021, Peru 3Zootecnia an Animal Production Laboratory, Faculty of Veterinary Medicine, Major National University of San Marcos, Lima 15021, Peru 4 Research Institute IVITA-Pucallpa, Faculty of Veterinary Medicine, Major National University of San Marcos, Lima 15021, Peru 5Functional Products Laboratory, Faculty of Engineering in Food Industries, National University of Central Peru, Huancayo 12006, Peru 6 Pharmacology and Toxicology Laboratory, Faculty of Veterinary Medicine, Major National University of San Marcos, Lima 15021, Peru *Correspondence: [email protected] (R.M.); [email protected] (L.G.); Tel.: +34-915492300 (R.M. & L.G.) Abstract: 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-MS n . 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. Keywords: Brunfelsia grandiflora; medicinal plant; polyphenols; hydroxycoumarins; antioxidant activity; LC-MS-QToF 1. 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 Molecules 2022,27, 6510. https://doi.org/10.3390/molecules27196510 https://www.mdpi.com/journal/molecules Molecules 2022,27, 6510 2 of 14 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 healthpromoting properties. Molecules 2022, 27, x FOR PEER REVIEW 2 of 14 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. Figure 1. Brunfelsia grandiflora from Perú. On the right is the plant in the flowering period (photo courtesy of Jana Horackova), and on the left is the bark used to obtain the extract used in the present study. 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 [7–9]. 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 Figure 1. Brunfelsia grandiflora from Perú. On the right is the plant in the flowering period (photo courtesy of Jana Horackova), and on the left is the bark used to obtain the extract used in the present study. 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 [7–9]. 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. Molecules 2022,27, 6510 3 of 14 2. 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. 2.1. 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 µ g/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]. IC50 values determined by the ABTS assay showed significantly higher values (1678.00 ± 11.26 µ g/mL and 3441.00 ±36.05 µg/mL for HR and CHE, respectively) than that determined for Brunfelsia grandiflora bark (4.55 µ g/mL). Likewise occurred with the IC 50 determined by DPPH for HR and CHE, where the values of 37,698.00 ± 3437.00 µ g/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 µ g/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 µ g/mL, which were higher than that determined for ascorbic acid, BHT, and quercetin (11.5, 15.3, and 14.8 µ g/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. Molecules 2022,27, 6510 4 of 14 Table 1. Total polyphenolic content and antioxidant capacity of lyophilized extract obtained from Brunfelsia grandiflora. Brunfelsia grandiflora Total Phenolic content by Folin (g/100 g d.m.) 3.02 ±0.33 DPPH (mg Trolox/g d.m.) 11.86 ±1.45 ABTS (mg Trolox/g d.m.) 80.38 ±4.22 IC50 by DPPH (µg/mL) 2.55 ±0.12 IC50 by ABTS (µg/mL) 4.55 ±0.10 Values are the average ± standard deviation of three different samples and expressed on a dry matter (d.m.) basis. 2.2. 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 2shows 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. Table 2. LC-QToF identification of phenolic compounds of Brunfelsia grandiflora. Identified Compound RT (min) Molecular Formula Molecular Weight [M −H]−Fragment MS2 HYDROXYCOUMARINS Esculin 3.7 C15H16O9340.0794 339.0722 177; 133 Esculetin 5.7 C9H6O4178.0266 177.0193 133; 105; 149 Scopoletin 8.9 C10H8O4192.0423 191.0350 104; 120; 148 GALLATES Gallic acid 2.0 C7H6O5170.0215 169.0142 125 Methyl-gallate 3.6 C8H8O5184.0372 183.0299 168; 124 Galloyl-glucose 5.0 C13H16O10 332.0743 331.0671 169 Methyl-gallate 6.6 C8H8O5184.0372 183.0299 124 Ethyl-gallate 6.3 C9H10O5198.0528 197.0455 169; 124 Ethyl-gallate 7.2 C9H10O5198.0528 197.0455 169; 124 Methyl-gallate 9.4 C8H8O5184.0372 183.0299 168 Ethyl-gallate 11.4 C9H10O5198.0528 197.0455 169; 124 Ethyl-gallate 13.0 C9H10O5198.0528 197.0455 169; 124 HYDROXYCINNAMIC ACIDS AND HYDROXYCINNAMATES 5-Caffeoylquinic acid 4.7 C16H18O9354.0951 353.0878 191; 93; 173 Caffeoylquinic acid 5.0 C16H18O9354.0951 353.0878 191 Caffeoylquinic acid 5.1 C16H18O9354.0951 353.0878 191 Caffeic acid 5.9 C9H8O4180.0423 179.0350 135 Coumaric acid 8.0 C9H8O3164.0473 163.0401 119 Ferulic acid 9.0 C10H10O4194.0579 193.0506 134; 149 Sinapic acid 9.4 C11H12O5224.0685 223.0612 193, 149, 165 Dehydrodiferulic acid 10.2 C20H18O8386.1002 385.0929 193; 177; 149 Caffeic acid-O-glucoside 11.3 C15H18O9342.0951 341.0878 179; 161 Coumaric acid-O-glucoside 12.5 C15H18O8326.1002 325.0929 163; 119 Coumaroylquinic acid 12.8 C16H18O8338.1002 337.0929 191; 163 Ferulic acid-O-glucoside 13.0 C16H20O9356.1107 355.1035 193; 149; 134 Sinapic acid-O-glucoside 13.1 C17H22O10 386.1213 385.1140 223 Ferulic acid-O-glucoside 13.4 C16H20O9356.1107 355.1035 193; 134 Feruloylquinic acid 13.4 C17H20O9368.1107 367.1035 191; 193 Sinapoylquinic acid 13.5 C18H22O10 398.1213 397.1140 223 Feruloylquinic acid 13.7 C17H20O9368.1107 367.1035 193; 191 Ferulic acid-O-glucoside 13.8 C16H20O9356.1107 355.1035 193; 134 Molecules 2022,27, 6510 5 of 14 Table 2. Cont. Identified Compound RT (min) Molecular Formula Molecular Weight [M −H]−Fragment MS2 Sinapic acid-O-glucoside 15.2 C17H22O10 386.1213 385.1140 223; 149 Coumaric acid-O-glucoside 15.6 C15H18O8326.1002 325.0929 163 Coumaric acid-O-glucoside 17.4 C15H18O8326.1002 325.0929 119 FLAVANOLS Gallocatechin 6.1 C15H14O7306.0740 305.0667 125; 137 Methyl-epigallocatechin 14.2 C16H16O7320.0896 319.0823 275; 137 FLAVANONES Eriodictyol 10.2 C15H12O6288.0634 287.0561 285; 283; 287; 255 Naringenin-O-glucoside 12.1 C21H22O10 434.1213 433.1140 271; 151 Eriodictyol-O-glucoside 12.3 C21H22O11 450.1162 449.1089 287; 255 Naringenin 15.9 C15H12O5272.0685 271.0612 151; 177 Eriodictyol-O-glucoside 16.2 C21H22O11 450.1162 449.1089 287 Hesperetin 16.9 C16H14O6302.0790 301.0718 286; 242 Naringenin-O-glucoside 18.0 C21H22O10 434.1213 433.1140 271 FLAVONOLS Kaempherol-O-rutinoside 4.9 C27H30O15 594.1585 593.1512 284, 285, 255 Isorhamnetin-O-rutinoside 10.6 C28H32O16 624.1690 623.1618 315 Kaempherol-O-galactoside-O-rhamnoside 12.0 C27H30O15 594.1585 593.1512 285; 257; 284 Isorhamnetin-O-glucoside-O-rhamnoside 12.2 C28H32O16 624.1690 623.1618 315 LIGNANS Pinoresinol 8.8 C20H22O6358.1416 357.1344 N.D. Matairesinol 9.5 C20H22O6358.1416 357.1344 N.D. Hydroxysecoisolariciresinol isomer 9.6 C20H26O7378.1679 377.1606 329 Secoisolariciresinol isomer 9.8 C20H26O6362.1729 361.1657 165 Hydroxysecoisolariciresinol isomer 9.9 C20H26O7378.1679 377.1606 329 Sesamol 11.3 C7H6O3138.0317 137.0244 N.D. Secoisolariciresinol 11.8 C20H26O6362.1729 361.1657 346; 165 Cyclolariciresinol or Isolariciresinol 12.7 C20H24O6360.1573 359.15 313 Hydroxymatairesinol/Nortrachelogenin 12.9 C20H22O7374.1366 373.1293 355 Sesamin 13.4 C20H18O6354.1103 353.1031 96 Secoisolariciresinol Isomer 15.2 C20H26O6362.1729 361.1657 165 Episesamin 19.4 C20H18O6354.1103 353.1031 96 OTHER PHENOLIC ACIDS Methoxy-hydroxybenzoic acid glucoside 2.3 C14H18O9330.0951 329.0878 167; 108 Dihydroxybenzoic acid glucose 2.4 C13H16O9316.0794 315.0722 153; 109 Dihydroxybenzoic acid glucose 2.6 C13H16O9316.0794 315.0722 153; 109 3,4-Dihydroxybenzoic acid (protocatechuic acid) 3.2 C7H6O4154.0266 153.0193 109 3-Hydroxybenzoic acid 4.8 C7H6O3138.0317 137.0244 93 3-Hydroxyphenylpropionic acid 4.9 C9H10O3166.0630 165.0557 121 Dihydroxybenzoic acid 5.1 C7H6O4154.0266 153.0193 109 3,4-Dihydroxyphenylpropionic acid 5.4 C9H10O4182.0579 181.0506 137; 109 Hydroxyphenylacetic acid 5.4 C8H8O3152.0473 151.0401 107 3-Methoxy-4-hydroxybenzoic acid (vanillic acid) 5.9 C8H8O4168.0423 167.0350 152; 108 4-Hydroxybenzoic acid 6.1 C7H6O3138.0317 137.0244 93 Dihydroxybenzoic acid 6.4 C7H6O4154.0266 153.0193 109 3-Methoxy-4-hydroxyphenylacetic acid (Homovanillic acid) 6.4 C9H10O4182.0579 181.0506 137; 122 Dihydroxybenzoic acid 6.7 C7H6O4154.0266 153.0193 109 Methoxy-hydroxybenzoic acid 6.8 C8H8O4168.0423 167.0350 108 Hydroxyphenylacetic acid 8.0 C8H8O3152.0473 151.0401 107 Dihydroxybenzoic acid glucose 8.1 C13H16O9316.0794 315.0722 153 3-Methoxy-4-hydroxyphenylpropionic acid 8.4 C10H12O4196.0736 195.0663 136 4-Hydroxyphenylpropionic acid 8.7 C9H10O3166.0630 165.0557 121 Methoxy-hydroxyphenylpropionic acid 9.8 C10H12O4196.0736 195.0663 136 Methoxy-hydroxybenzoic acid 11.3 C8H8O4168.0423 167.0350 152; 108 Molecules 2022,27, 6510 6 of 14 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/z191.0350 and fragment ions at m/z148, 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/z177.0193 and fragment ions at m/z149, 133, and 105 compatible with its chemical structure, and the glycosylated derivative ([M − H] − at m/z339.0722) showed as the main fragment ion that corresponds to its free precursor, esculetin (m/z177) (Table 2). Some of the identified compounds showed a chemical structure belonging to gallates, such as methyland ethyl-gallate, as well as galloyl glucose. Three isomers of methylgallates at 3.6, 6.6, and 9.4 min showed a [M − H] − at m/z183.0299 and fragmented ions at m/z168 and 124. Likewise, four isomers of ethyl-gallate at 6.3, 7.2, 11.4, and 13.0 min were identified based on their compatible MS spectra ([M − H] − at m/z197.0455 and fragment ions at m/z169 and 124). Only one chromatographic peak showed MS spectra suited with galloyl-glucose at 5.0 min ([M − H] − at m/z331.0671 and fragment ion at m/z169). Finally, gallic acid was unambiguously identified at 2.0 min, thanks to its commercial standard and the MS spectra (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, 5.0, and 5.1 min due to their MS spectra ([M − H] − at m/z353.0878 and fragment ion at m/z191, characteristic of quinic acid). The earliest chromatographic peak was assigned to 5-caffeoylquinic acid (chlorogenic acid) thanks to the commercial standard. More hydroxycinnamic acids esterified with quinic acid were identified in this plant. Coumaroyland sinapoylquinic acids, along with two isomers of feruloylquinic acid were identified at 12.8, 13.5, 13.4, and 13.7 min, respectively, due to their respective quasimolecular ions at m/z 337.0929, 397.1140 and 367.1035, respectively. MS/MS allowed confirming their identity after determining their respective precursor at m/z163, 223, and 193, respectively. Related to the described hydroxycinnamates, it was identified both their free hydroxycinnamic acids and glycosylated forms. Thus, caffeic acid ([M − H] − at m/z179.0350 and fragment ion at m/z135) as well as caffeic acid-O-glucoside ([M − H] − at m/z341.0878 and fragment ions at m/z179 and 161) appeared at 5.9 and 11.3 min, respectively. Ferulic acid at 9.0 min ([M − H] − at m/z193.0506 and fragment ions at m/z134 and 149) and three isomers of ferulic acid-O-glucoside at 13.0, 13.4, and 13.8 min ([M − H] − at m/z355.1035 and fragment ions at m/z193, 149 and 134) were identified in Brunfelsia grandiflora. Coumaric acid at 8.0 min ([M − H] − at m/z163.0401 and fragment ion at m/z119), three isomers of coumaric acid-O-glucoside at 12.5, 15.6, and 17.4 min ([M − H] − at m/z325.0929 and fragment ions at m/z163, 119), sinapic acid at 9.4 min ([M − H] − at m/z223.0612 and fragment ions at m/z193, 149 and 165) and two isomers of sinapic acid-O-glucoside at 13.1 and 15.2 min ([M − H] − at m/z385.1140 and fragment ions at m/z223 and 149) were characterized. Regarding ferulic acid, dehydrodiferulic acid was also identified in this extract based on its quasimolecular ion at m/z385.0929 and fragment ions at m/z193, 177, and 149, and its chromatographic peak eluted at 10.2 min (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/z449.1089 and fragment ions at m/z287 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. Molecules 2022,27, 6510 7 of 14 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/z623.1618 and fragment ion at m/z315 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/z357.1344, 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/z377.1606 and fragment ion at m/z329). 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/z137.0244 was associated with the chromatographic peak eluting at 11.3 min. The isomers sesamin and episesamin eluted at 13.4 and 19.4 min, respectively, and showed a [M − H] − at m/z353.1031 and fragment ion at m/z96, compatible with their chemical structure. The chromatographic peak at 12.7 min showed MS spectra ([M − H] − at m/z359.15 and fragment ion at m/z313), 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, 3and 4-hydroxybenzoic acid, vanillic acid, homovanillic acid, 3,4-dihydroxyphenylpropionic acid, 3and 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/z315.0722 and 329.0878, respectively, and the presence of their precursor, benzoic acid, among the fragment ions. 2.3. 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, coumaroyland 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). Molecules 2022,27, 6510 8 of 14 Table 3. Content of individual phenolic compounds present in Brunfelsia grandiflora. Results represent the mean ±standard deviation (n= 4). N.D.: not detected; d.w.: dry weight. RT (min) Proposed Compound Brunfelsia grandiflora (mg/100 g d.w.) HYDROXYCINNAMIC ACIDS and HYDROXYCINNAMATES 4.7 5-Chlorogenic acid 3.13 ±0.18 5.0 Caffeoylquinic acid 1.89 ±0.07 5.1 Caffeoylquinic acid 0.70 ±0.05 5.9 Caffeic acid 0.21 ±0.01 8.0 p-Coumaric acid 0.10 ±0.01 9.0 Ferulic acid 3.99 ±0.10 9.4 Sinapic acid 0.31 ±0.02 10.2 Dehydrodiferulic acid 7.62 ±0.11 11.3 Caffeic acid-O-glucoside 533.86 ±8.29 12.5 Coumaric acid-O-glucoside 57.57 ±0.50 12.8 Coumaroylquinic acid 2.26 ±0.10 13.0 Ferulic acid-O-glucoside 391.46 ±17.08 13.1 Sinapic acid-O-glucoside 81.55 ±1.66 13.4 Ferulic acid-O-glucoside 19.33 ±1.00 13.4 Feruloylquinic acid 151.04 ±4.07 13.5 Sinapoylquinic acid 6.54 ±0.09 13.7 Feruloylquinic acid 7.51 ±0.22 13.8 Ferulic acid-O-glucoside 64.22 ±2.11 15.2 Sinapic acid-O-glucoside 8.76 ±0.30 15.6 Coumaric acid-O-glucoside 1.61 ±0.14 17.4 Coumaric acid-O-glucoside 1.48 ±0.04 TOTAL HYDROXYCINNAMIC ACIDS (mg/100 g) (%) 1345.13 ±36.16 (66.77%) HYDROXYCOUMARINS 3.7 Esculin 4.71 ±1.16 5.7 Esculetin 21.49 ±0.66 8.9 Scopoletin 286.77 ±21.28 TOTAL HYDROXYCOUMARINS (mg/100 g) (%) 312.97 ±23.11 (15.13%) LIGNANS 8.8 Pinoresinol 0.77 ±0.03 9.5 Matairesinol 2.45 ±0.29 9.6 Hydroxysecoisolariciresinol isomer 5.19 ±0.19 9.8 Secoisolariciresinol isomer 3.35 ±0.20 9.9 Hydroxysecoisolariciresinol isomer 4.43 ±0.10 11.3 Sesamol 55.36 ±2.46 11.8 Secoisolariciresinol 2.42 ±0.09 12.7 Cyclolariciresinol or Isolariciresinol 9.70 ±0.57 12.9 Hydroxymatairesinol/Nortrachelogenin 15.23 ±0.75 13.4 Sesamin 16.00 ±0.47 15.2 Secoisolariciresinol isomer 2.95 ±0.20 19.4 Episesamin 5.51 ±0.13 TOTAL LIGNANS (mg/100 g) (%) 123.36 ±5.48 (6.12%) FLAVONOLS 4.9 Kaempherol-O-rutinoside 3.97 ±2.21 10.6 Isorhamnetin-O-rutinoside 8.09 ±0.09 12.0 Kaempherol-O-galactoside-O-rhamnoside 7.62 ±0.29 12.2 Isorhamnetin-O-glucoside-O-rhamnoside 94.51 ±2.47 TOTAL FLAVONOLS (mg/100 g) (%) 114.18 ±5.06 (5.67%) PHENOLIC ACIDS 2.3 Methoxy-hydroxybenzoic acid glucoside 2.86 ±0.22 2.4 Dihydroxybenzoic acid glucose 2.16 ±0.10 2.6 Dihydroxybenzoic acid glucose 2.80 ±0.20 3.2 3,4-Dihydroxybenzoic acid (protocatechuic acid) 5.23 ±0.25 4.8 3-Hydroxybenzoic acid 6.97 ±0.15 4.9 3-Hydroxyphenylpropionic acid 3.99 ±0.24 5.1 Dihydroxybenzoic acid 1.03 ±0.04 Molecules 2022,27, 6510 9 of 14 Table 3. Cont. RT (min) Proposed Compound Brunfelsia grandiflora (mg/100 g d.w.) 5.4 3,4-Dihydroxyphenylpropionic acid 3.81 ±0.15 5.9 3-Methoxy-4-hydroxybenzoic acid (vanillic acid) 2.73 ±0.16 6.1 4-Hydroxybenzoic acid 1.50 ±0.02 6.4 Dihydroxybenzoic acid 1.22 ±0.06 6.4 3-Methoxy-4-hydroxyphenylacetic acid (Homovanillic acid) 0.17 ±0.01 6.5 4-Hydroxyphenylacetic acid 7.61 ±0.09 6.7 Dihydroxybenzoic acid 0.59 ±0.06 6.8 Methoxy-hydroxybenzoic acid 2.11 ±0.06 8.0 Hydroxyphenylacetic acid 3.98 ±0.07 8.1 Dihydroxybenzoic acid glucose 0.81 ±0.02 8.4 3-Methoxy-4-hydroxyphenylpropionic acid 1.08 ±0.05 8.7 4-Hydroxyphenylpropionic acid 2.73 ±0.10 9.8 Methoxy-hydroxyphenylpropionic acid 1.85 ±0.07 11.3 Methoxy-hydroxybenzoic acid 7.23 ±0.24 TOTAL PHENOLIC ACIDS (mg/100 g) (%) 62.46 ±2.38 (3.10%) GALLATES 2.0 Gallic acid 0.97 ±0.13 3.6 Methyl-gallate 0.66 ±0.06 5.0 Galloyl-glucose 1.82 ±0.13 6.3 Ethyl-gallate 3.99 ±0.09 6.6 Methyl-gallate 2.89 ±0.10 7.2 Ethyl-gallate 12.38 ±0.38 9.4 Methyl-gallate 0.57 ±0.06 11.4 Ethyl-gallate 19.92 ±0.56 13.0 Ethyl-gallate 3.28 ±0.10 TOTAL GALLATES (mg/100 g) (%) 46.48 ±1.62 (2.31%) FLAVANOLS 6.1 Gallocatechin 5.54 ±0.12 14.2 Methyl-epigallocatechin 0.29 ±0.02 TOTAL FLAVANOLS (mg/100 g) (%) 5.83 ±0.14 (0.29%) FLAVANONES 10.2 Eriodictyol 0.54 ±0.05 12.1 Naringenin-O-glucoside 1.59 ±0.06 12.3 Eriodictyol-O-glucoside 0.65 ±0.02 15.9 Naringenin 0.06 ±0.01 16.2 Eriodictyol-O-glucoside 1.00 ±0.07 16.9 Hesperetin 0.09 ±0.01 18.0 Naringenin-O-glucoside 0.37 ±0.05 TOTAL FLAVANONES (mg/100 g) (%) 4.30 ±0.27 (0.21%) TOTAL PHENOLIC COMPOUNDS 2014.71 ±74.23 (100%) 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). Flavonols were the next most abundant compound group (5.7% of the total phenolic content), headed by isorhamnetin-O-glucoside-O-rhamnoside and followed by isorhamnetinO-rutinoside, kampherol-O-galactoside-O-rhamnoside, and kampherol-O-rutinoside (82.7, 7.1, 6.7 and 3.5%, respectively, of the total flavonols) (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-