Chemical Profile, Anti‐Inflammatory, and Antioxidant Potential of Hydroalcoholic Extracts and Fractions From Baccharis glaziovii Baker (Asteraceae)

TL;DR

This study explores the chemical makeup and health benefits of a Brazilian plant, finding it has strong antioxidant and anti-inflammatory properties.

Contribution

The study identifies apigenin and quantifies high phenolic content in fractions of Baccharis glaziovii, highlighting its potential for phytotherapeutic use.

Findings

Hydroalcoholic extracts of Baccharis glaziovii showed 62-64% inhibition of BSA denaturation, indicating anti-inflammatory potential.

Dichloromethane fractions contained up to 1540.1 mg GAE/g of total phenolics, the highest among tested fractions.

Antioxidant activity measured via DPPH and phosphomolybdenum assays showed values up to 126 µg TE/mg and 183.8%, respectively.

Abstract

Baccharis glaziovii Baker, known as carqueja‐arbustinho, is a native Brazilian plant characterized by its three‐winged stems. This study investigated the chemical profile and biological potential of hydroalcoholic extracts and fractions from male and female specimens. Chemical characterization by nuclear magnetic resonance and mass spectrometry indicated a phenolic profile, with the flavonoid apigenin identified in the dichloromethane fraction of both extracts. Anti‐inflammatory activity was assessed by the inhibition of bovine serum albumin (BSA) denaturation, with the female and male extracts showing inhibition of 64.1% and 62.2%, respectively, compared to 84.3% for sodium diclofenac. The highest effect was observed at 400 µg mL−1. Regarding antioxidant potential, the DPPH assay indicated values near 126 µg TE/mg, while the phosphomolybdenum test showed capacities of up to 183.8%. The dichloromethane fractions stood out for their extremely high total phenolic content, reaching 1540.1 mg GAE/g in the male and 1474.2 mg GAE/g in the female. These results provide evidence that the species possesses robust antioxidant and anti‐inflammatory properties, without critical disparities between the plant genus. Such findings position B. glaziovii as a promising candidate for the development of phytotherapeutic formulations, reinforcing the need for additional studies to isolate its bioactive compounds.

Image adapted from Jasinski et al. [79], Revista Brasileira de Farmacognosia 24, no. 6: 609–616. Reproduced under the terms of the CC BY license. Copyright 2014 by the authors; published by Elsevier.

Figures

Molecular structure of apigenin identified in the hydroalcoholic extracts of male and female specimens of Baccharis glaziovii.

Anti‐inflammatory activity of hydroalcoholic extracts of Baccharis glaziovii. MS: crude extract male specimen. FS: crude extract female specimen. Values are presented as the mean ± SD compared to diclofenac as a standard. Different letters within the same group indicate statistical differences (p < 0.05) between the sample and controls using ANOVA followed by Tukey's test.

Antioxidant potential of hydroalcoholic extracts and fractions of Baccharis glaziovii through phosphomolybdenum complex reduction. Values are presented as the mean ± SD compared to AA, rutin, and BHT as standard. Different letters within the same group indicate statistical differences (p < 0.05) between the sample and controls using ANOVA followed by Tukey's test. AA, ascorbic acid; AF, aqueous fraction; BHT, butylhydroxytoluene; DF, dichloromethane fraction; EAF, ethyl acetate fraction; FS, crude extract of the female specimen; HF, hexane fraction; MS, crude extract of the male specimen; RAA, relative antioxidant activity.

Antioxidant potential of hydroalcoholic extracts and fractions of Baccharis glaziovii through DPPH radical scavenging expressed as Trolox equivalents. Values are presented as the mean ± SD expressed as the Trolox equivalent. Different letters in the same group indicate statistical differences (p < 0.05) between samples and controls according to ANOVA followed by Tukey's test. AF, aqueous fraction; DF, dichloromethane fraction; EAF, ethyl acetate fraction; FS, crude extract of the female specimen; HF, hexane fraction; MS, crude extract of the male specimen.

Total phenolic content in hydroalcoholic extracts and fractions of male and female Baccharis glaziovii. Values are presented as mean ± SD and expressed as gallic acid equivalents. Different letters in the same group indicate statistical differences (p < 0.05) between samples and controls according to ANOVA followed by Tukey's test. AF, aqueous fraction; DF, dichloromethane fraction; EAF, ethyl acetate fraction; FS, crude extract of the female specimen; HF, hexane fraction; MS, crude extract of the male specimen.

Tables

TABLE 1. NMR spectral data of apigenin in the hydroalcoholic extracts of male and female specimens of Baccharis glaziovii.

Position	subfraction 3, DF MS a	DF FS b			Apigenin c
	1H (ppm) multiplicity; J in Hz	1H (ppm) multiplicity; J in Hz	13C (ppm)	HMBC	1H (ppm) multiplicity; J in Hz	13C (ppm)
2	—	—	166.4		—	163.0
3	6.59 1H, s	6.55 s	103.9	2, 4, 10	6.68 s	100.2
4	—	—	183.9		—	181.8
5	—	12.60 s	163.6		12.90	161.2
6	6.17 1H, d (1.8)	6.20 1H, d (2.1)	100.1	5, 8, 10	6.20 1H, d (2.0)	99.7
7	—	—	166.4		—	164.5
8	6.44 1H, d (1.8)	6.43 1H, d (2.1)	95.1	6, 7, 9, 10	6.44 1H, d (2.0)	94.8
9	—	—	159.5		—	157.0
10	—	—	105.5		—	103.2
1'	—	—	117.2		—	119.1
2'	7.84 1H, d (8.8)	7.82 1H, d (8.9)	129.4	2, 1’, 2’, 4’	7.92 1H, d (9.0)	121.5
3'	6.94 1H, d (8.8)	6.92 1H, d (8.9)	116.8	1’,4’,6’	6.90 1H, d (9.0)	116.0
4'	—	—	162.6		—	161.1
5'	6.94 1H, d (8.8)	6.92 1H, d (8.9)	116.8	1’, 4’, 6’	6.90 1H, d (9.0)	116.0
6'	7.84 1H, d (8.8)	7.82 1H, d (8.9)	129.4	2, 1’, 2’, 4’	7.92 1H, d (9.0)	121.5

Full text

Introduction

1

Phenolic compounds, widely distributed in plants and known for their antioxidant activities, have garnered significant attention due to their potential in preventing various diseases. These compounds play a vital role in neutralizing reactive oxygen species (ROS) and free radicals, preventing cellular damage caused during inflammatory processes and the development of various pathologies. Studies indicate that oxidative molecules are associated with inflammatory conditions and chronic diseases, such as hepatitis, ulcers, and various tumors, as they promote oxidative stress, damaging lipids, proteins, and nucleic acids in cells [1, 2]. Moreover, research emphasizes the contribution of natural antioxidants, particularly flavonoids and plant‐derived coumarins, which possess a high capacity to mitigate the impact of free radicals on tissues, aiding in the prevention and treatment of inflammatory processes. The identification and study of substances with antioxidant and anti‐inflammatory activities offer promising avenues for developing therapies to reduce the impact of ROS in inflammation‐related diseases [3].

Baccharis glaziovii (B. glaziovii) Baker, belonging to the family Asteraceae and popularly known as carqueja‐arbustinho, is a shrub that can grow up to 2.5 m tall, featuring trilobed branches typical of Baccharis species called “carquejas.” This species, like others in the genus, has attracted interest in ethnopharmacology due to its therapeutic potential, primarily linked to the presence of phenolic compounds [4]. The production of these in Baccharis includes flavonoids and phenolic acids, which are well known for their antioxidant, anti‐inflammatory, and gastroprotective properties. These characteristics have been widely documented in other species of the genus, such as B. dracunculifolia, B. grisebachii, and B. burchellii, whose extracts contain high levels of compounds like caffeic acid, ferulic acid, naringenin, and rutin—substances capable of neutralizing free radicals and protecting cellular tissues [5, 6]. Studies suggest that phenolic compounds present in Baccharis species exert biological activities that contribute to the body's antioxidant defense and the mitigation of inflammatory processes [7].

Although several species of the genus Baccharis have been extensively investigated for their antioxidant and anti‐inflammatory properties, Baccharis glaziovii remains poorly explored. To the best of our knowledge, no in vitro studies have evaluated hydroalcoholic extracts and fractions of this species using standardized antioxidant and anti‐inflammatory assays, nor examined the influence of plant sex on these activities. Therefore, this study investigated the chemical profile, in vitro antioxidant and anti‐inflammatory activities, and total phenolic content (TPC) of extracts and fractions obtained from male and female specimens of B. glaziovii. This work provides the first experimental evidence supporting the bioactive potential of this species.

Results and Discussion

2

Chemical Profile of the Hydroalcoholic Extracts by NMR

2.1

The aerial parts (cladodes) of Baccharis glaziovii from male (MS) and female (FS) specimens yielded 158.4 g and 119.4 g of dry crude extract, respectively, corresponding to extraction yields of 15.8% for MS and 11.9% for FS.

Analysis of the ^1^H NMR spectra for MS and FS extracts revealed highly consistent qualitative chemical profiles, particularly within the expanded aromatic region (8.0–6.0 ppm), as shown in Figures S1 and S2. While the presence of identical major metabolites in both sexes is evident, distinct variations in signal intensities suggest differences in their relative concentrations. This quantitative divergence aligns with literature reports indicating that phytochemical profiles can be modulated by sexual dimorphism or exposure to varying environmental conditions [8, 9, 10].

The analysis of the expanded aromatic region (8.0–6.0 ppm) is consistent with hydrogens from substituted aromatic systems typically found in flavonoids, coumarins, and phenolic acid derivatives. In this analysis, the coupling patterns characteristic of flavones were observed, particularly doublets between 7.8 and 8.2 ppm with coupling constants around 8.5 Hz, attributed to ortho‐coupled protons on the B‐ring of flavonoids and indicative of para‐disubstituted aromatic systems [11, 12, 13, 14, 15]. Also, this region exhibits aromatic and olefinic proton signals between 6.0 and 9.0 ppm, being observed as singlets and doublets forming AB systems, typically appearing with coupling constants of 7–10 Hz in pyranocoumarins and 1–2.5 Hz in furanocoumarins [16]. The spectrum exhibited also characteristic signals that suggest the presence of both coumarin skeletons and conjugated derivatives. The doublets at δ 6.50 and 8.08 (J = 9.5 Hz) [17] are consistent with the H‐3 and H‐4 protons of non‐substituted coumarins. Additionally, the presence of signals at δ 6.47 and 7.76 with a larger coupling constant (J = 15.1 Hz) indicates the existence of trans‐olefinic protons, typical of conjugated systems within these derivatives [18, 19]. The spectrum also exhibited signals at 7.65 and 7.25 ppm attributed to phenylene protons, indicating the presence of phenolic acid derivatives [20, 21]. In general, the downfield region between 6.0 and 10.0 ppm is typical of phenolic constituents and frequently shows signal overlap due to the coexistence of multiple phenolic species [22].

Building upon these preliminary findings, which led to the identification of apigenin, further chromatographic studies are being conducted to purify and isolate additional constituents from the observed chemical classes by NMR. This systematic approach will ensure a more comprehensive chemical characterization of the extract profiles.

According to the literature, species of the Baccharis genus are rich in flavonoids, caffeoylquinic acids, clerodane diterpenes, and other phenolic compounds with known biological properties such as antioxidant and anti‐inflammatory activities [4]. Studies by Negri et al. [9] and Bernardes et al. [23] support the presence of such metabolites in extracts of species like B. trimera and B. dracunculifolia, reinforcing the attribution of the observed signals in this study to these classes of constituents.

Identification of Apigenin in the Hydroalcoholic Extracts and Fractions From Baccharis glaziovii

2.2

The flavonoid apigenin (Figure 1) was identified in both female (FS) and male (MS) specimens of B. glaziovii. It was isolated as subfraction 3 from the dichloromethane fraction (DF) of the MS extract and characterized through 1D and 2D NMR (Figures S1–S7) and ESI‐MS/MS (Figures S8–S9).

Molecular structure of apigenin identified in the hydroalcoholic extracts of male and female specimens of Baccharis glaziovii.

The ^1^H NMR spectra displayed characteristic signals for the flavone skeleton, notably a singlet between δ 6.55 and 6.59 (H‐3) and the meta‐coupled doublets at δ 6.17–6.20 (H‐6, J = 2 Hz) and δ 6.43–6.44 (H‐8). The AA'BB' spin system in ring B confirmed the 4’‐hydroxy substitution pattern. The structure was further validated by HSQC and HMBC correlations, showing 13 carbon signals consistent with the apigenin backbone, including the carbonyl at δ 183.9 (C‐4). Detailed spectroscopic assignments and comparisons with literature data are summarized in Table 1 [8]

The Mass Spectrometry (ESI, negative mode) analysis of subfraction 3 revealed a deprotonated molecular ion at m/z 269 [M – H]^−^. A subsequent ESI‐MS/MS experiment of this molecular ion yielded product ions at m/z 225, 149, and 117. These signals correspond to the characteristic loss of CO_2_ and Retro–Diels–Alder (RDA) cleavages, which allowed the identification of apigenin by comparison with literature [24, 25].

The spectroscopic data of the extract fractions from both specimens revealed the presence of apigenin, a flavonoid previously reported in species of the section Caulopterae, such as B. crispa, B. gaudichaniana, B. genistelloides, B. milleflora, B. notosergila, and B. trimera [26, 27, 28, 29, 30, 31], as well as in other species of the genus, including B. salzmannii, B. dracunculifolia, B. salicifolia, and B. dentata [32, 33, 34, 35]. These findings reinforce the importance of apigenin as a chemical marker and contribute to the understanding of the chemosystematics of the genus Baccharis.

In Vitro Anti‐Inflammatory Activity of Hydroalcoholic Extracts

2.3

The hydroalcoholic extracts of Baccharis glaziovii from male (MS) and female (FS) specimens showed significant anti‐inflammatory activity in the BSA denaturation assay (Figure 2). At 400 µg mL^−^ ^1^, MS and FS inhibited protein denaturation by 62.2% and 64.2%, respectively. Although lower than diclofenac sodium (84.4%), these values indicate relevant biological activity. All inhibition percentages obtained at the other tested concentrations are presented in Table S1.

Anti‐inflammatory activity of hydroalcoholic extracts of Baccharis glaziovii. MS: crude extract male specimen. FS: crude extract female specimen. Values are presented as the mean ± SD compared to diclofenac as a standard. Different letters within the same group indicate statistical differences (p < 0.05) between the sample and controls using ANOVA followed by Tukey's test.

Both extracts showed a clear concentration‐dependent response. These extracts exhibited slightly higher inhibition at 400 and 200 µg mL^−^ ^1^, suggesting higher levels or a more effective profile of anti‐inflammatory metabolites, possibly related to phenolic acids, flavonoids, and coumarins [36, 37, 38, 39, 40].

At lower concentrations, MS extract maintained greater residual activity, inhibiting denaturation by 4.7% at 12.5 µg mL^−^ ^1^, while FS extract showed minimal effect (0.6%). These differences reflect qualitative variations in secondary metabolites between sexes, reinforcing the role of sexual dimorphism in dioecious species [41]. Overall, Figure 2 confirms the anti‐inflammatory potential of B. glaziovii, consistent with reports on the genus Baccharis [42].

2.4 In Vitro Antioxidant Activity of Hydroalcoholic Extracts and Fractions

Phosphomolybdenum Complex Reduction

2.1

All samples showed antioxidant activity in the phosphomolybdenum assay (Figure 3). The male crude extract (MS) and its ethyl acetate fraction (EAF‐MS) exhibited the highest relative antioxidant activity, exceeding BHT and rutin but remaining below ascorbic acid. Using rutin as a reference, MS (183.8%) and DF‐MS (130.4%) showed superior performance compared to female samples. The complete set of relative antioxidant activity values for all extracts and fractions is provided in Table S2.

Antioxidant potential of hydroalcoholic extracts and fractions of Baccharis glaziovii through phosphomolybdenum complex reduction. Values are presented as the mean ± SD compared to AA, rutin, and BHT as standard. Different letters within the same group indicate statistical differences (p < 0.05) between the sample and controls using ANOVA followed by Tukey's test. AA, ascorbic acid; AF, aqueous fraction; BHT, butylhydroxytoluene; DF, dichloromethane fraction; EAF, ethyl acetate fraction; FS, crude extract of the female specimen; HF, hexane fraction; MS, crude extract of the male specimen; RAA, relative antioxidant activity.

Crude extracts and dichloromethane fractions from both specimens displayed pronounced antioxidant activity, whereas hexane fractions were weak. When compared with BHT, all samples surpassed this synthetic standard, with the MS and EAF‐MS showing the highest activities and no significant differences between them. Although less active than ascorbic acid, MS (40.1%) and EAF‐MS (39.5%) retained approximately half of its antioxidant capacity, indicating relevant bioactivity.

The use of rutin, ascorbic acid, and BHT as standards enables reliable benchmarking. Rutin represents phenolic electron‐donor activity, while BHT is a widely used synthetic antioxidant [43]. The superior performance of MS and its fractions relative to BHT highlights their potential as natural antioxidant alternatives. These differences are likely associated with sex‐related variations in phenolic composition, as previously reported for Baccharis species [43, 44].

DPPH Radical Scavenging

2.2

In the DPPH assay (Figure 4), the dichloromethane fractions (DF‐MS and DF‐FS) and the EAF‐MS fraction showed the highest radical scavenging activities, with values ranging from 117.0 ± 1.27 to 130.4 ± 1.06 µg TE mg^−1^. No significant statistical difference was observed between DF‐MS and DF‐FS, indicating equivalent potential in this system (letter “a,” p > 0.05). All DPPH scavenging values obtained for the remaining extracts and fractions are detailed in Table S3. In contrast, hexane fractions were largely inactive, confirming that the key antioxidants are concentrated in fractions of intermediate polarity.

Antioxidant potential of hydroalcoholic extracts and fractions of Baccharis glaziovii through DPPH radical scavenging expressed as Trolox equivalents. Values are presented as the mean ± SD expressed as the Trolox equivalent. Different letters in the same group indicate statistical differences (p < 0.05) between samples and controls according to ANOVA followed by Tukey's test. AF, aqueous fraction; DF, dichloromethane fraction; EAF, ethyl acetate fraction; FS, crude extract of the female specimen; HF, hexane fraction; MS, crude extract of the male specimen.

The activity profiles indicate that dichloromethane fractions perform comparably to crude extracts. The order of activity—EAF‐MS > DF‐FS > AF‐MS > DF‐MS > AF‐FS > MS > FS—highlights the relevance of medium‐polarity fractions as sources of bioactive compounds.

Expression of DPPH activity as Trolox equivalents allows standardized comparison across studies [45, 46]. Except for hexane fractions, most samples showed activity close to Trolox, consistent with the presence of phenolic compounds and flavonoids capable of hydrogen donation and radical stabilization [47, 48, 49, 50].

Determination of TPC

2.4

The TPC, determined by the Folin–Ciocalteu method and expressed as mg of gallic acid equivalents per gram of extract (mg GAE g^−^ ^1^), was quantified in order to establish a direct relationship between chemical composition and the observed biological activities. The hydroalcoholic extracts and fractions of Baccharis glaziovii showed a wide variation in phenolic levels, highlighting the influence of extraction solvent polarity and plant sex on the concentration of these metabolites (Figure 5; Table S4).

Total phenolic content in hydroalcoholic extracts and fractions of male and female Baccharis glaziovii. Values are presented as mean ± SD and expressed as gallic acid equivalents. Different letters in the same group indicate statistical differences (p < 0.05) between samples and controls according to ANOVA followed by Tukey's test. AF, aqueous fraction; DF, dichloromethane fraction; EAF, ethyl acetate fraction; FS, crude extract of the female specimen; HF, hexane fraction; MS, crude extract of the male specimen.

Fractions of intermediate polarity exhibited the highest TPC, particularly the dichloromethane fractions obtained from male and female specimens (DF‐MS and DF‐FS), which reached 1540.1 ± 30.21 and 1474.2 ± 55.00 mg GAE g^−^ ^1^ dry sample, respectively. These values exceed those reported for other species of the genus, such as Baccharis macrantha (approximately 17 mg GAE g^−^ ^1^ of dry extract) [51] and B. dracunculifolia (values ranging from 20 to 37 mg GAE g^−^ ^1^ of dry plant material) [52], and are comparable to the highest levels described for B. trimera, including ethyl acetate extracts (1387.95 ± 42.45 mg GAE g^−^ ^1^ dry extract) and phenolic extracts (1482.00 ± 50.41 mg GAE g^−^ ^1^ dry extract) [53]. Given the lack of previous data for B. glaziovii, these findings reveal an unprecedented and remarkably enriched phenolic profile within the genus Baccharis.

The observed biological activity can be partially attributed to apigenin, a flavonoid with well‐documented bioactive properties [54]. When evaluated in isolation, apigenin shows strong antioxidant activity, including DPPH radical scavenging (IC_50_ 37.9 µg/mL, > 95% inhibition) [55] and high values in complementary assays, such as TEAC (2022.2 ± 154.8 µmol TE/mmol), ORAC (887.9 ± 5.8 µmol TE/mmol), and FRAP (113.2 ± 12.2 µmol Fe^2^ ^+^/mmol) [56]. However, the activity of complex fractions cannot be attributed to a single compound, as synergistic interactions among multiple metabolites often enhance antioxidant and anti‐inflammatory effects, as demonstrated in bioactivity‐guided fractionation studies [57].

The relevance of apigenin is closely linked to its chemical structure. The C2═C3 double bond conjugated with the C4‐carbonyl group enables electron delocalization and stabilization of phenoxyl radicals, while the 4′‐hydroxyl group on the B‐ring acts as an efficient hydrogen donor in the neutralization of ROS [58, 59, 60]. In addition, its planar structure favors hydrogen bonding and hydrophobic interactions with protein residues, contributing to the inhibition of protein denaturation [61]. These structural features provide a mechanistic basis for the anti‐inflammatory effects observed in phenolic‐enriched extracts.

In line with structure–activity relationships and potential synergistic effects, phenolic‐rich hydroalcoholic extracts of Baccharis glaziovii exhibited marked anti‐inflammatory activity in the bovine serum albumin (BSA) denaturation assay, with inhibition values of 64.15% for FS and 62.20% for MS (Figure 2; Table S1). Protein denaturation is closely associated with inflammatory processes, and phenolic compounds are known to counteract this phenomenon through protein stabilization and modulation of oxidative stress [57, 60]. Comparable inhibition levels have been reported for other Asteraceae species, including Atractylis aristata (70.84%), Tridax procumbens (approximately 80.0%), Launaea intybacea (81.0%), and Cynara cardunculus L. var. ferocissima (96.03%) [62, 63, 64, 65]. In this context, the values obtained for B. glaziovii represent the first evidence of anti‐inflammatory activity evaluated by this model within the genus Baccharis.

The antioxidant capacity of B. glaziovii extracts and fractions was consistently confirmed in both antioxidant models employed. In the phosphomolybdenum assay, samples with high phenolic content—particularly the male crude extract (MS) and the EAF‐MS fraction—exhibited elevated total antioxidant capacity, surpassing BHT and rutin, although remaining lower than ascorbic acid (Figure 3; Table S2). Similarly, the highest DPPH radical scavenging activities were observed in phenolic‐enriched fractions (DF‐MS, DF‐FS, and EAF‐MS), with values ranging from 126 to 130 µg TE·mg^−^ ^1^, reflecting hydrogen donation and radical stabilization mechanisms characteristic of flavonoids [66, 67, 68]. The weak activity of hexane fractions further reinforces the central role of phenolic compounds in these effects.

In comparative terms, the antioxidant potential of B. glaziovii is consistent with that reported for other species of the genus. Baccharis trimera exhibits DPPH IC_50_ values around 118.18 µg·mL^−^ ^1^ [69], whereas Baccharis dracunculifolia shows pronounced activity, with EC_50_ values of 15.75 µg·mL^−^ ^1^ in phenolic‐enriched extracts [70]. Although expressed in different units, these results converge toward a consistent antioxidant profile, positioning B. glaziovii among Baccharis species with recognized ethnopharmacological relevance [42, 71].

Finally, the determination of TPC provided a quantitative basis for correlating chemical composition with biological activities. The enrichment of phenolic substances in the dichloromethane and ethyl acetate fractions—compounds well known for their redox properties and ability to donate electrons or hydrogen atoms [13, 72]—directly explains the antioxidant and anti‐inflammatory responses observed. The identification of apigenin in the phenolic‐rich dichloromethane fractions supports its relevant contribution to these effects [54, 73]; however, the bioactivity reflects the behavior of a complex phytochemical matrix, in which synergistic interactions among co‐occurring metabolites may enhance the overall response [57]. Therefore, further studies involving bioactivity‐guided isolation and comparative assays with high‐purity standards are required to elucidate the individual and combined contributions of the metabolites present, positioning B. glaziovii as a promising source of bioactive phenolics within the genus Baccharis.

Conclusion

3

The results of this study demonstrate that the hydroalcoholic extracts of Baccharis glaziovii exhibit a characteristic chemical profile, with variations in metabolite classes, evidencing the presence of phenolic compounds such as flavonoids—particularly apigenin, which was identified in the dichloromethane fractions—as well as other phenolic classes, including coumarins and phenolic acid derivatives. These metabolites, derived from secondary metabolism, may be associated with the biological activities observed, such as protein denaturation inhibition, as well as the antioxidant and free radical scavenging effects detected in the extracts and their fractions. However, further phytochemical characterization and complementary biological assays are required to elucidate the mechanisms of action, investigate possible synergistic interactions among the compounds, and assess their viability as therapeutic alternatives, in addition to their potential for commercial or industrial applications.

Materials and Methods

4

Solvents and Reagents

4.1

Analytical grade solvents (hexane, dichloromethane, ethyl acetate, and methanol) used in the preparation and fractionation of extracts were pre‐distilled to remove impurities. Deuterated methanol from Sigma‐Aldrich (Brazil), dimethyl sulfoxide (DMSO) from Labsynth (Diadema, Brazil), bovine serum albumin (BSA) from Sigma‐Aldrich (Germany), sulfuric acid Biophar Anadiol (São José dos Pinhais, Brazil), anhydrous sodium phosphate Dinâmica Química Contemporânea (Brazil), ammonium molybdate Cromoline Química Fina (Diadema, Brazil), ascorbic acid Labimpex (Brazil), butylated hydroxytoluene (BHT) Sigma Fine Chemicals Limited (Uppsala, Sweden), 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) Calbiochem (Billerica, USA), and 6‐hydroxy‐2,5,7,8‐tetramethylchromane‐2‐carboxylic acid (Trolox) EMD Millipore Corp., Merck KGaA (Darmstadt, Germany).

Collection and Identification of Plant Material

4.2

Plant material (cladodes) of B. glaziovii was collected from Colônia Dúlcio, Municipality of Mallet [coordinates: 25°48“26.99” S, 50°52“54.91” W, altitude 881 m], Paraná, Brazil in Novembro de 2012. The material was identified by botanist Osmar dos Santos Ribas. The voucher specimens (#381048, female; #381049, male) were deposited in the Herbarium of the Botanical Museum of Curitiba. The study was authorized by the National Council for Scientific and Technological Development (CNPq) and the Genetic Heritage Management Council (CGEN), registered under number 010304/2013‐4.

Preparation of Extracts

4.3

The fresh plant material (cladodes) from male and female specimens of B. glaziovii was dried in a circulating air oven at 40°C, fragmented, and pulverized using a knife mill. Extraction was carried out with 1 kg of the pulverized material by maceration using 7.5 L of ethanol (90% v/v), in three repetitions at 3‐day intervals, at room temperature. The crude extracts from the male (MS) and female (FS) specimens were combined, concentrated using a rotary evaporator, and stored in a refrigerator for subsequent fractionation.

Fractionation of Hydroalcoholic Extracts

4.4

The crude extracts (80 g) of both specimens were suspended in ethanol/water (1:2, v/v), as described by Zampieri et al. [74] with modifications, and subjected to liquid–liquid partitioning using solvents of increasing polarity to obtain the hexane (HF), dichloromethane (DF), ethyl acetate (EAF), and residual aqueous (AF) fractions. For each extract, 3.5 L of hexane, 2.0 L of dichloromethane, and 2.0 L of ethyl acetate were used. The fractions were concentrated under reduced pressure using a rotary evaporator and lyophilized for further analyses.

The DF fraction of the extract from the MS specimen was purified by preparative TLC, using 90 mg of the sample dissolved in 2 mL of methanol, which was applied by capillarity onto a silica plate (UV254 fluorescent indicator). The plate was eluted with 250 mL of a dichloromethane (DCM) and ethyl acetate (EtOAc) solution in an 80:20 ratio and examined under UV light at 254 and 366 nm. The fractionation yielded six subfractions, and apigenin was identified in subfraction 3 after ^1^H NMR analysis.

NMR Analysis of Extracts and Fractions From Male and Female Specimens of Baccharis glaziovii

4.5

The MS and FS extracts of B. glaziovii were analyzed by ^1^H NMR (600 MHz). The DF fraction of the FS extract was analyzed by 1D and 2D NMR (600 MHz), and subfraction 3, derived from the DF fraction of the MS extract, was analyzed by ^1^H NMR (200 MHz). The spectra were acquired on a Bruker spectrometer operating at 14.1 and 4.7 Tesla (T). The analysis was performed using approximately 10–20 mg of the sample dissolved in 600 µL of deuterated methanol or chloroform, with tetramethylsilane (TMS) used as the internal reference standard (δ = 0 ppm). Spectral processing was carried out using TopSpin 3.1 software, and chemical shifts (δ) were reported in parts per million (ppm).

Low‐Resolution Mass Spectrometry Analysis of the Dichloromethane Fraction From the Male Specimen

4.6

The flavonoid apigenin, found in the dichloromethane fraction (DF) of the male specimen, was identified via direct infusion mass spectrometry (DIMS). Analysis was performed on a Waters Xevo TQD triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ionization mode.

The experiments were full scan mode acquisition (ESI‐MS) (mass range: 100–1000 Da) and tandem mass spectrometry analysis (ESI‐MS/MS) (mass range: 60–300 Da), using argon as the collision gas and a collision energy of 24 eV. The instrumental parameters were as follows: capillary voltage (IS), −3 kV; desolvation temperature of 300°C; desolvation gas flow rate of 400 L/h; cone gas flow rate of 10 L/h; injection volume of 20 µL; sample concentration of 5000 ng mL^−^ ^1^, and diluent composed of methanol/water/formic acid (90:10:0.1, v/v/v). Data acquisition and analysis were performed using the MassLynx software for low resolution mass spectrometry.

In Vitro Anti‐Inflammatory Activity of Hydroalcoholic Extracts

4.7

The anti‐inflammatory activity of the extracts was investigated in vitro using the thermal denaturation assay of bovine serum albumin (BSA), based on the method described by Mizushima and Kabayashi [75], with adaptations. For sample preparation, 1 mg of each extract was dissolved in 20 µL of DMSO and diluted with 980 µL of phosphate buffer (pH 7.0), resulting in a stock solution with a concentration of 1 mg mL^−^ ^1^. A 10% BSA solution was prepared by dissolving 10 mg of albumin in 100 mL of phosphate buffer. The assay was performed in 96‐well microplates, with the test samples evaluated at concentrations ranging from 400 to 12.5 µg mL^−^ ^1^. The negative control consisted of distilled water combined with the BSA solution, while the positive control used sodium diclofenac (1 mg mL^−^ ^1^ in phosphate buffer), tested at the same concentration range. All samples were analyzed in triplicate to ensure result reproducibility.

The microplates were incubated at 37°C for 15 min in a BOD incubator and subsequently heated at 60°C for 10 min in a water bath to induce protein denaturation. After a 5‐min cooling period at room temperature, absorbance readings were taken at 660 nm using a microplate reader (Bio‐Tek). The percentage inhibition of BSA denaturation was calculated using the following Equation (1):

$$[eqn]$$

This method enabled the assessment of the ability of the extracts to prevent heat‐induced conformational changes in the protein, indicating potential anti‐inflammatory activity.

In Vitro Antioxidant Activity of Hydroalcoholic Extracts and Fractions

4.8

Phosphomolybdenum Complex Formation

4.8.1

The antioxidant activity of the samples was assessed based on the formation of the phosphomolybdenum complex. A reagent solution was prepared containing ammonium molybdate (4 mmol L^−^ ^1^), sodium phosphate (28 mmol L^−^ ^1^), and sulfuric acid (0.6 mol L^−^ ^1^), following the procedure described by Pietro et al. [76]. Standard solutions of rutin, ascorbic acid, and butylated hydroxytoluene (BHT), as well as hydroalcoholic extracts from male (MS) and female (FS) specimens and their respective fractions (hexane, dichloromethane, ethyl acetate, and aqueous), were prepared in methanol at a concentration of 200 µg.mL^−^ ^1^.

In test tubes, 0.3 mL of each sample or standard was combined with 3 mL of the reagent solution. For the blank control, 0.3 mL of methanol was mixed with 3 mL of the reagent. The mixtures were incubated at 95°C for 90 min. After cooling to room temperature, the contents were transferred to a 96‐well microplate, and absorbance readings were taken at 695 nm using a microplate spectrophotometer (Multiskan FC, Thermo Scientific). The relative antioxidant activity (RAA%) of each sample was calculated in comparison to the standards (considered as 100% antioxidant activity) using the following Equation (2):

$$[eqn]$$

All analyses were performed in triplicate to ensure the reliability of the results.

Evaluation by DPPH Radical Scavenging

4.9

The antioxidant activity against the DPPH free radical was assessed following the procedures described by Mensor et al. [77]. Methanolic solutions of the DPPH reagent (0.03 µg mL^−^ ^1^) and stock solutions of the samples (1 µg mL^−^ ^1^) were prepared. The assays were performed in quintuplicate using 96‐well microplates, where 71 µL of each extract (MS and FS) or their respective fractions (hexane, dichloromethane, ethyl acetate, and aqueous) were mixed with 29 µL of the DPPH solution.

Control conditions included, for the blank, a mixture of 71 µL of the sample (or standard solution) with 29 µL of methanol; and for the negative control, 71 µL of methanol mixed with 29 µL of the DPPH solution. After incubating the microplates in the dark for 30 min, absorbance was measured at 517 nm using a Multiskan FC spectrophotometer (Thermo Scientific). The percentage of DPPH radical scavenging activity was estimated using Equation (3), where A sample is the absorbance of the sample, A blank is the absorbance of the blank solution, and A negative is the absorbance of the negative control:

$$[eqn]$$

A solution of the Trolox standard was prepared at different concentrations to obtain a calibration curve (y = −0.0108x + 0.2664; r ^2^ = 0.9978), and the antioxidant capacity was expressed in µg of Trolox equivalents per mg of sample (µg TE mg^−^ ^1^).

Determination of TPC

4.10

The TPC of the hydroalcoholic extracts and fractions of Baccharis glaziovii (male and female specimens) was determined using the Folin–Ciocalteu method, as described by Woisky and Salatino [78], with modifications. In 96‐well microplates, aliquots (20 µL) of methanolic sample solutions (200 µg mL^−^ ^1^) were mixed with diluted Folin–Ciocalteu reagent (1:20, v/v) and 7.5% (w/v) sodium carbonate, reaching a final volume of 200 µL per well. After incubation for 60 min at room temperature in the dark, absorbance was measured at 690 nm using a Multiskan FC spectrophotometer (Thermo Scientific), with methanol as the blank. Quantification was performed using a gallic acid calibration curve (20–150 µg mL^−^ ^1^), and results were expressed as mg gallic acid equivalents per g of dry extract.

4.11

The results obtained in the in vitro assays were evaluated using ANOVA for variance analysis and Tukey's test for statistical differences between results, with p values < 0.05 considered significantly different. These analyses were performed using the Sisvar software, version 5.8.92.

Author Contributions

Fabio Vidal Tananta: conceptualization, methodology, results, data analysis, and review – original writing. Vanessa Cristina Godoy Jasinski: methodology, results, and data analysis. Marcos José Salvador: anti‐inflammatory assay methodology. Josiane de Fátima Gáspari Dias: antioxidant assays methodology. Francinete Ramos Campos: review and supervision of original writing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting file 1: cbdv71103‐sup‐0001‐SuppMat.pdf