Metabolomic Profiling and Antioxidant Activities of Breonadia salicina Using 1H-NMR and UPLC-QTOF-MS Analysis

Breonadia salicina (Vahl) Hepper and J.R.I. Wood is widely used in South Africa and some other African countries for treatment of various infectious diseases such as diarrhea, fevers, cancer, diabetes and malaria. However, little is known about the active constituents associated with the biological activities. This study is aimed at exploring the metabolomics profile and antioxidant constituents of B. salicina. The chemical profiles of the leaf, stem bark and root of B. salicina were comprehensively characterized using proton nuclear magnetic resonance (1H-NMR) spectroscopy and ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The antioxidant activities of the crude extracts, fractions and pure compounds were determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging and reducing power assays. A total of 25 compounds were tentatively identified using the UPLC-QTOF-MS. Furthermore, the 1H-NMR fingerprint revealed that the different parts of plant had differences and similarities among the different crude extracts and fractions. The crude extracts and fractions of the root, stem bark and leaf showed the presence of α-glucose, β-glucose, glucose and fructose. However, catechin was not found in the stem bark crude extracts but was found in the fractions of the stem bark. Lupeol was present only in the root crude extract and fractions of the stem bark. Furthermore, 5-O-caffeoylquinic acid was identified in the methanol leaf extract and its respective fractions, while the crude extracts and fractions from the root and dichloromethane leaf revealed the presence of hexadecane. Column chromatography and preparative thin-layer chromatography were used to isolate kaempferol 3-O-(2″-O-galloyl)-glucuronide, lupeol, d-galactopyranose, bodinioside Q, 5-O-caffeoylquinic acid, sucrose, hexadecane and palmitic acid. The crude methanol stem bark showed the highest antioxidant activity in the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity with an IC50 value of 41.7263 ± 7.6401 μg/mL, whereas the root crude extract had the highest reducing power activity with an IC0.5 value of 0.1481 ± 0.1441 μg/mL. Furthermore, the 1H-NMR and UPLC-QTOF-MS profiles showed the presence of hydroxycinnamic acids, polyphenols and flavonoids. According to a literature survey, these phytochemicals have been reported to display antioxidant activities. Therefore, the identified hydroxycinnamic acid (caffeic acid), polyphenol (ellagic acid) and flavonoids (catechin and (epi) gallocatechin) significantly contribute to the antioxidant activity of the different parts of plant of B. salicina. The results obtained in this study provides information about the phytochemistry and phytochemical compositions of Breonadia salicina, confirming that the species is promising in obtaining constituents with medicinal potential primarily antioxidant potential.


Introduction
Breonadia salicina (Vahl) Hepper and J.R.I. Wood is a plant species belonging to the family Rubiaceae. The Rubiaceae family consists of~13,500 species in~620 genera and is one of the largest of the angiosperm family. It is divided into four subfamilies: Cinchonoideae, Ixoroideae, Antirheoideae and Rubioideae [1,2]. Breoanadia salicina is commonly known as "Transvaal teak" (in English), "mingerhout", "waterboekenhout" or "basterkiaat" (in Afrikaans), "mutulume" (in Venda) and "matumi" (in Pedi). It is a small to large tree up to 40 m in height [3]. B. salicina occurs in the tropical and subtropical regions of Africa and Saudi Arabia. In South Africa, it is widely distributed in the northeast, from KwaZulu-Natal to Mpumalanga and Limpopo near the banks or in the waters of permanent streams and rivers [4]. The secondary metabolite characteristic of the Rubiaceae family includes alkaloids, terpenes, iridoids, quinonic acid glycosides, flavonoids, coumarins, anthraquinones and other phenolic derivatives [5]. These secondary metabolites possess pharmacological activities such as antioxidant, anti-inflammatory, anti-diabetic, antimicrobial, antiplasmodial, antidiarrheal and antitumor [6].
Different parts of Breonadia salicina are commonly used by traditional healers in South Africa and other African countries for the treatment of many infectious diseases such as arthritis, pneumonia, tachycardia, vomiting, ulcers, stomach pains, gastrointestinal illness, headaches, inflamed wounds, and bacterial and fungal infections [7][8][9][10]. Despite B. salicina being used in traditional medicine, little is known about the phytochemistry and pharmacological activities. Moreover, many of the reports on the biological activities of this plant have been limited to crude extracts. Therefore, there is a need to isolate compounds from different parts of B. salicina and evaluate their pharmacological activities which can contribute to human health. Studies on the phytochemistry of Breonadia salicina has revealed very few isolated compounds, mainly pentacyclic triterpenoids (ursolic acid and α-amyrin [8,11]), hydroxycinnamic acid (2,4-dihydroxycinnamic acid [12]), phytosterol (stigmasterol [11]) and coumarins (7-(β-D-apiofuranosyl (1-6)-β-D-glucopyranosyl) umbelliferone, 7-hydroxy coumarin and 6-hydroxy-7-methoxy coumarin [11]). According to a literature survey, the biological activities of these compounds isolated from B. salicina have never been evaluated. In order to characterize the chemical fingerprints or profile completely; the crude extracts and fractions of the stem bark, root and leaf of Breonadia salicina were evaluated using metabolomics approach that coupled the use of proton nuclear magnetic resonance ( 1 H-NMR) and UPLC-QTOF-MS. The distribution of antioxidants in different parts of the plant were determined using the DPPH free radical scavenging and reducing power assays. Natural antioxidants play an important role as part of the human diet and for their potential health benefits [13]. Oxidative stress, caused by the accumulation of free radicals and reactive oxygen species (ROS), has been associated with the pathogenesis of many degenerative and chronic conditions such as atherosclerosis, cancer, inflammation, Alzheimer's, diabetes and inflammation [14]. Antioxidants protect biological molecules (DNA) from oxidation to reduce the risk of developing degenerative and chronic diseases [15]. Studies have revealed that medicinal plants are good sources of antioxidants, because they are rich in phenolic compounds [16]. These compounds can protect humans from high level of free radicals, inhibit lipid peroxidation, scavenge free radicals and chelate metal ions [17].
A previous study proved that the stem bark crude extracts of B. salicina has strong antioxidant activity against DPPH free radical scavenging assay [18]. However, the antioxidant activity of the crude root extract from B. salicina has never been assessed. Furthermore, there are no reports on the isolation and evaluation of the compounds responsible for the antioxidant activity of this plant. Therefore, this study aimed at determining the phytochemical of different parts of Breonadia salicina and linking these results with antioxidant activity using a metabolomics approach to isolate the major compounds and to evaluate their role in the antioxidant activity. Therefore, this study is the first study to detect significant antioxidant activity of different parts of Breonadia salicina.

Chemical Fingerprint of the Crude Extracts and Fractions of the Stem Bark, Root and Leaf Using 1 H-NMR
The Breonadia salicina crude extracts and fractions were subjected to 1 H-NMR analysis, and the chemical shifts were compared to known standards or from literature [19][20][21][22][23][24]. Different classes of identified metabolites such as triterpenoids, fatty acids, sugars (monosaccharides), phenols and quinic acids were identified. This is the first study to identify these metabolites from different parts of B. salicina. The chemical shifts of the identified metabolites are presented in Table 1. In the aromatic region of fraction S 1 , proton signals belonging to catechin were detected at δ H 7.05 ppm, δ H 6.72-6.85 ppm, δ H 5.86 ppm and δ H 5.94 ppm, respectively, as shown in Figure S1A. However, fraction S 2 showed the aromatic proton signals for catechin at δ H 7.06 ppm, δ H 6.72-6.86 ppm, δ H 5.87 ppm and δ H 5.94 ppm, respectively, as presented in Figure S2. However, catechin was not found in the stem bark crude extract but was found in the fractions of the stem bark. This may be because the signals of catechin were not visible in the 1 H-NMR spectra of the stem bark crude extract or the concentration of catechin was low in the stem bark extract. The signals of lupeol, a pentacyclic triterpenoid, were identified in the root crude extract (R.crude, Figure S4), fraction S 1 ( Figure S1B) and fraction S 2 ( Figure S3). Moreover, signals belonging to 5-O-caffeoylquinic acid were detected in the methanol leaf crude extract (LM.crude, Figure S5), fraction LM 2 ( Figure S6) and fraction LM 3 ( Figure S7). The dichloromethane leaf crude extract (LD.crude, Figure S8), fraction R 1 ( Figure S9) and fraction LD 3 ( Figure S10) exhibited proton signals of hexadecane at δ H 0.90 (6H, t)-1.69 (28H, m) ppm, δ H 0.84 (6H, t)-1.27 (28H, m) ppm and δ H 0.87 (6H, t)-1.27 (28H, t) ppm, respectively. Monosaccharides (sugars) such as α-glucose, β-glucose and fructose were observed in the root crude extract (R.crude, Figure S4), stem bark crude extract (S.crude, Figure S11), methanol leaf crude extract (LM.crude, Figure S5) and fraction LM 3 ( Figure S7). The 1 H-NMR spectra revealed that the crude and fractions of the stem bark, root and leaf contained differences and similarities among the different crude extracts and fractions. The crude extracts and fractions of the root, stem bark and leaf showed the presence of α-glucose, β-glucose, glucose and fructose. However, catechin was not found in the stem bark crude extracts but was found in the fractions of the stem bark. Lupeol was present only in the root crude extract and fractions of the stem bark. Furthermore, 5-O-caffeoylquinic acid was identified in the methanol leaf extract and its respective fractions, while the crude extracts and fractions from the dichloromethane leaf revealed the presence of hexadecane.   [29][30][31][32][33]. Peak 11 (Rt = 1.876 min), peak 12 (Rt = 4.728 min) and peak 13 (Rt = 11.530 min) were assigned to be (epi) gallocatechin (Figure S22 Figure S36) were tentatively identified as deacetyl asperuloside acid (C 16 [26,[42][43][44][45], respectively. Finally, a data comparison with the literature confirmed the identification of these compounds. Therefore, metabolites including polyphenols, flavanoids, hydrolyzable tannin, triterpenoids, hydroxycinnamic acids and quinic acids were tentatively identified and characterized from Breonadia salicina. Many of these metabolites were mostly found in the stem bark than in the root or leaf samples. Furthermore, this is the first study to identify and report these metabolites from Breonadia salicina.   (5) showed the presence of 16 carbons, including two carbonyl groups at δ C 145.4 ppm and δ C 166.5 ppm, corresponding to carbons at positions 7 and 9 , respectively, on the 13 C-NMR spectrum. Furthermore, the 13 C-NMR spectrum presented two aromatic carbons bonded to hydroxyl groups at δ C 148.5 ppm and δ C 145.8 ppm corresponding to carbons at positions 4 and 3 , and two olefinic carbons at δ C 180.2 ppm and δ C 113.6 ppm corresponding to carbons at positions 7 and 8 . The 1 H-NMR spectrum showed two ortho-coupled doublets at δ H 6.79 ppm and δ H 6.95 ppm corresponding to protons at positions 5 and 6 . A broad singlet at δ H 7.06 ppm has been assigned to a proton at position 2 , confirming the presence of a tri-substituted aromatic ring. Moreover, the 1 H-NMR revealed two doublets at corresponding to protons at δ H 6.21 ppm and δ H 7.52 ppm positions 7 and 8 , indicating the presence of trans-di-substituted ethylene moiety in the compound. These assignments are in good agreement with the structure of 5-O-caffeoylquinic acid.

Isolation and Identification of Chemical Constituents
Sucrose (6)  , was evident as a multiplet that integrated for one proton. A single long peak multiplet at δ C 29.3-29.7 ppm appeared from C-4, C-5, C-6, C-7, C-8, C-9, C-10, C-11, C-12 and C-13 on the 13 C-NMR spectra. Furthermore, hexadecane (7) was isolated as white crystals with a melting point of 16-18 • C. Therefore, the spectroscopic data and physical property comparison with the literature confirmed the isolation of hexadecane.
The infrared (IR) spectrum of palmitic acid (8) revealed absorptions at 3436.00 cm −1 which is characteristic of O-H stretching, 2849.51 cm −1 which is due to aliphatics (CH 3 ) stretching, 1703.71 cm −1 due to carbonyl (C=O) stretching and 2917.27 cm −1 due to overtone of the long chain (CH 2 )n bending frequency. The 1 H-NMR spectrum showed a long peak of multiplet at δ H 1.23 ppm assigned to protons at position 4-H, 5-H, 6-H, 7-H, 8-H, 9-H, 10-H, 11-H, 12-H and 13-H of the long chain, was evident as a multiplet that integrated for one proton. Furthermore, the 1 H-NMR spectrum displayed a triplet at δ H 2.27 ppm (2H, t) and δ H 0.83 ppm (3H, t) corresponding to protons at positions 2 and 16, respectively. The 13 C-NMR has shown double bonds of the acidic group (-COOH) at δ C 178.4 ppm assigned to C-1 as singlet, the alkane carbon C-16 appeared at δ C 14.1 ppm as a triplet. A single long peak multiplet at δ C 29.0 ppm appeared from C-4 to C-13, whereas an alpha and beta carbon to the acidic group (-COOH) appeared at δ C 31.9 ppm and δ C 34.0 ppm corresponding to carbons at position 3 and 2 as doublets respectively. Furthermore, an alpha and beta carbon to the alkane carbon appeared as doublets at δ C 22.6 ppm and δ C 24.7 ppm corresponding to carbons at position 15 and 14.

Antioxidant Activity
The crude methanol stem bark (S.crude) showed the highest antioxidant activity in the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity with an IC 50 value of 41.7263 ± 7.6401 µg/mL, whereas the root crude extract (R.crude) had the highest reducing power activity with an IC 0.5 value of 0.1481 ± 0.1441 µg/mL, as shown in Table 3. The presence of the identified hydroxycinnamic acids, polyphenols and flavonoids might have contributed to the potent antioxidant activities in the stem bark and root extracts, as shown in Tables 1 and 2. Kilic et al. (2014) reported that ellagic acid, tentatively identified from the stem bark crude extract (S.crude, as shown in Table 2), exhibited a high DPPH radical scavenging activity of 85.6% at 30 µg/mL [47]. However, Grzesik et al. (2018) indicated that catechin, (epi) gallocatechin and caffeic acid found in the stem bark crude extract (S.crude, as presented in Table 2) have good antioxidant activities against DPPH radical scavenging and reducing power activities with IC 50 values of 3.965 ± 0.067 mol TE/mol, 2.939 ± 0.037 mol TE/mol and 0.965 ± 0.015 mol TE/mol, respectively, and IC 0.5 values of 0.793 ± 0.004 mol TE/mol, 1.032 ± 0.007 mol TE/mol and 1.018 ± 0.004 mol TE/mol, respectively [48]. Therefore, ellagic acid, catechin, (epi) gallocatechin and caffeic acid play an important role in the antioxidant activities of the stem bark crude extract (S.crude). Furthermore, D-galactopyranose (3) exhibited the highest antioxidant activity against DPPH free radical scavenging activity compared to the other isolated compounds, with an IC 50 value of 44.5613 ± 2.6772 µg/mL, whereas kaempferol 3-O-(2 -O-galloyl)-glucuronide (1) exhibited the highest reducing power activity compared to the other isolated compounds, with an IC 0.5 value of 3.3742 ± 1.7492 µg/mL, as shown in Table 3. Moreover, D-galactopyranose (3) was less active than the parent fraction S 4 , as shown in Table 3. This could be because the interaction of D-galactopyranose (3) with other constituents from the fraction it was isolated from could be responsible for the higher antioxidant activity observed in the DPPH free radical scavenging and reducing power. According to a literature survey, the antioxidant activities of D-galactopyranose (3) have never been evaluated. Therefore, our study is the first to detect important antioxidant activity of the crude extracts, fractions and pure compounds from Breonadia salicina. Notes: A different superscript letter indicates significant differences using one-way ANOVA at p < 0.05. Data (n = 3) expressed as mean ± standard deviation. For DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity: a -Stem bark crude extract (S.crude) was significantly different to all samples; a,b -Root crude extract (R.crude) was only significantly different to lupeol (2) and palmitic acid (7); a,c -Methanol leaf crude extract (LM.crude) was only significantly different to lupeol (2) and palmitic acid (7); a,d -Dichloromethane leaf crude extract (LD.crude) was only significantly different to lupeol (2) and palmitic acid (7), and a,b,c,d -lupeol (2) was only significantly different to palmitic acid (7). For reducing power activity: a -Stem bark crude extract was not significantly different to all samples.

General Experimental Procedure
All chemicals used were analytical grade purchased from Sigma-Aldrich (Darmstadt, Germany). Silica gel (Kieselgel 60 (0.063-0.2 mm), Merck) was used as stationary phase and solvent mixtures described below were used as mobile phase in the chromatographic separations. Thin-layer chromatography (TLC) plates packed with silica gel (normal phase) were used to locate the major constituents of the fractions.

High-Resolution Mass Spectrometry
A Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) (Thermo Fisher Scientific, Waltham, MA, USA) connected to a Waters Acquity ultraperformance liquid chromatograph (UPLC) (Waters, Milford, MA, USA) was used for direct injection high-resolution mass spectrometric analysis. A volume of one µL of sample was injected into a stream of 60% acetonitrile and 40% dilute (0.1%) aqueous formic acid. This conveyed the sample directly to the QTOF mass spectrometer where data were acquired using both positive and negative electrospray ionization. The following MS settings were used: cone voltage of 15 V, desolvation temperature of 275 • C, desolvation gas at 650 L/h, and the rest of the MS settings optimized for best resolution and sensitivity.

UPLC Analysis
The ultra-performance liquid chromatography MS (UPLC-MS) was carried out using a Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) (Thermo Fisher Scientific, Waltham, MA, USA) connected to a Waters Acquity ultra-performance liquid chromatograph (UPLC) (Waters, Milford, MA, USA) and a photodiode array detector (PDA) (Waters, Milford, MA, USA). An injection volume of 2.0 µL (full-loop injection) was used. Separation was achieved on an Acquity UPLC BEH C18 column (150 mm X 2.1 mm i.d., 1.7 µm particle size; Waters, Milford, MA, USA), maintained at 40 • C. The mobile phase consisted of 0.1% formic acid (Solvent A) and HPLC grade (Merck, Darmstadt, Germany) acetonitrile (Solvent B) at a flow rate of 0.3 mL/min. Gradient elution was executed as follows: the initial ratio was 10% B for 4 min, changed to 50% B for 6 min, then 95% B in 2.5 min, maintaining for 0.5 min, before returning to the initial ratio in 0.5 min. The system was equilibrated for 2 min prior to the subsequent analysis. Both positive and negative electrospray ionization (ESI) modes were evaluated for further analysis.

FTIR Spectral Analysis of the Isolated Compounds
Attenuated total reflection (ATR) infrared (IR) spectra were recorded on an Alpha Fourier transform infrared (FTIR) spectrometer (Bruker, Fällanden, Switzerland).

Sampling and Extraction
The leaves, stem bark and root samples of Breonadia salicina were collected at Fondwe, located at latitude 22 •  bark, root and leaves were air-dried for four weeks and then ground to a fine powder using an industrial grinding mill (NETZSCH, Selb, Germany). Approximately 1.74 kg ground stem bark and 1.01 kg root of Breonadia salicina were each soaked with 2 L of methanol for 48 h at room temperature, respectively. The extracts were filtered and then concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 45 • C to obtain 95.57 g and 68.82 g of dried extracts, respectively (as shown in Scheme 1). ground stem bark and 1.01 kg root of Breonadia salicina were each soaked with 2 L of methanol for 48 hours at room temperature, respectively. The extracts were filtered and then concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 45 °C to obtain 95.57 g and 68.82 g of dried extracts, respectively (as shown in Scheme 1).
Moreover, ~1.12 kg ground leaves of Breonadia salicina were extracted successively with 2 L of dichloromethane and methanol for 48 hours at room temperature, respectively. The leaf extracts were filtered and then concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 45 °C to obtain 20.52 g and 66.43 g of dried extracts, respectively (as shown in Scheme 2). Each dried extract (stem bark, root and leaves) was subjected to column chromatography over silica gel (Kieselgel 60 (0.063-0.2 mm), Merck) [49]. The column was eluted initially with hexane and the polarity was gradually increased with ethyl acetate and finally methanol. Moreover,~1.12 kg ground leaves of Breonadia salicina were extracted successively with 2 L of dichloromethane and methanol for 48 hours at room temperature, respectively. The leaf extracts were filtered and then concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 45 • C to obtain 20.52 g and 66.43 g of dried extracts, respectively (as shown in Scheme 2). Each dried extract (stem bark, root and leaves) was subjected to column chromatography over silica gel (Kieselgel 60 (0.063-0.2 mm), Merck) [49]. The column was eluted initially with hexane and the polarity was gradually increased with ethyl acetate and finally methanol. ground stem bark and 1.01 kg root of Breonadia salicina were each soaked with 2 L of methanol for 48 hours at room temperature, respectively. The extracts were filtered and then concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 45 °C to obtain 95.57 g and 68.82 g of dried extracts, respectively (as shown in Scheme 1). Moreover, ~1.12 kg ground leaves of Breonadia salicina were extracted successively with 2 L of dichloromethane and methanol for 48 hours at room temperature, respectively. The leaf extracts were filtered and then concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 45 °C to obtain 20.52 g and 66.43 g of dried extracts, respectively (as shown in Scheme 2). Each dried extract (stem bark, root and leaves) was subjected to column chromatography over silica gel (Kieselgel 60 (0.063-0.2 mm), Merck) [49]. The column was eluted initially with hexane and the polarity was gradually increased with ethyl acetate and finally methanol.

Purification of Fractions
Fraction S 1 (0.5 g) was subjected to preparative TLC (normal phase) to obtain compound 1 (0.28 g). Fraction S 2 (0.34 g) was not purified due to having less amount of material. Fraction S 3 (0.5 g) was also subjected to preparative TLC (normal phase) to obtain compound 2 (0.36 g). Fraction S 4 (4 g) was subjected to silica gel column chromatography; the column was eluted using CH 2 Cl 2 /MeOH (50:50) followed by an increasing gradient of CH 2 Cl 2 /MeOH (up to 90:10) to obtain compounds 3 (0.27 g). Fraction R 2 (4.89 g) was a pure fraction, yielded compound 4. Fraction LM 1 (4.46 g) was a pure fraction, yielded compound 5. Fraction LM 3 (5 g) was subjected to silica gel column chromatography; the column was eluted using CH 2 Cl 2 /EtOAc (50:50) followed by an increasing gradient elution with a mixture of dicholoromethane, ethyl acetate and methanol to yield compound 6 (0.96 g). Fraction LD 1 was a pure fraction, yielded compound 7 (1.74 g). Fraction LD 3 (2 g) was subjected to silica gel chromatography; the column was eluted using CH 2 Cl 2 /EtOAc (50:50) followed by an increasing gradient elution with a mixture of dicholoromethane, ethyl acetate and methanol to obtain compound 8 (0.13 g). Moreover, fractions S 5 (17.10 g), R 1 (4.44 g), LM 2 (5.29 g) and LD 2 (1.69 g) could not be purified due to the complexity of the fractions. Compounds 1 [50], 2 [23], 3 [46], 4 [51], 5 [24], 6 [20], 7 [52] and 8 [53] are known compounds, as shown in Figure 1.  The DPPH free radical scavenging activity of the crude extracts, fractions and pure compounds were determined according to the modified spectrophotometric method of Motamed and Naghibi (2010) [58]. A solution of 125 mM DPPH/methanol was prepared by dissolving 10 mg DPPH (2,2-diphenyl-1-picrylhydrazyl) in 200 mL methanol. A 100 µL volume of distilled water was added in each 96-well plate. Therefore, a 100 µL volume of the crude extracts, fractions and pure compounds was added in triplicate into the first three wells followed by serial dilution using a multi-channel micropipette. Finally, a volume of 200 µL of DPPH (2,2-diphenyl-1-picrylhydrazyl) was added to each well containing the mixtures and the 96-well plate was kept in the dark for not more than 30 min. The absorbance was evaluated using a VersaMax TM tuneable microplate reader at 517 nm.
The percentage radical scavenging was determined by using the following formula:

Reducing Power
The reducing power was determined according to the modified method of Pereira et al. (2013) [59]. A volume of 100 µL of the samples (crude extracts, fractions and pure compounds) and standards (ascorbic acid and gallic acid) was added in triplicate in the first three wells of a 96-well plate, each containing 100 µL of deionized water, followed by serial dilution. A volume of 0.2 M (pH 6.6) sodium phosphate buffer (50 µL) was added into all 96-well plates and 50 µL volume of a 1% aqueous potassium hexacyanoferrate(III) [K 3 Fe (CN) 6 ] solution was added in each well. The mixture was incubated for 20 minutes at 45 • C. After incubation, a volume of 50 µL of 10% trichloroacetic acid solution was added to each well. An 80 µL volume of each mixture was transferred to another 96-well plate containing a volume of 80 µL of distilled water and 16 µL ferric chloride (0.1% w/v). Absorbance was determined using a VersaMax™ tuneable microplate reader at 700 nm.

Statistical Analysis
Statistical analysis was undertaken using the SPSS package (Chicago, IL, USA). The data was shown as mean ± standard deviation (SD). Mean differences of the crude extracts, fractions and pure compounds were assessed by one-way analysis of variance (ANOVA, Graph pad prism 6) in the antioxidant tests; p < 0.05 was considered statistically significant.

Conclusions
The main objective of this study was to determine the phytochemistry, phytochemical compositions and antioxidant activity of Breonadia salicina. Eight compounds (kaempferol 3-O-(2 -O-galloyl)-glucuronide, lupeol, D-galactopyranose, bodinioside Q, 5-O-caffeoylquinic acid, sucrose, hexadecane and palmitic acid) were isolated from the stem bark, root and leaf extracts of B. salicina. This is the first study to report the isolation of these compounds from the genus Breonadia Ridsdale and B. salicina species. Consequently, a total of 25 metabolites were tentatively identified from different parts of B. salicina using UPLC-QTOF-MS. This is the first study to identify and report these metabolites from the genus Breonadia Ridsdale and B. salicina species. Furthermore, the study showed that the stem bark crude extract contained a significantly higher antioxidant capacity compared to the root and leaf samples. The findings in this work comprehensively indicate that polyphenols, hydroxycinnamic acids and flavonoids contribute to the biological activities evaluated.