Isolation and Identification of the Anti-Oxidant Constituents from Loropetalum chinense (R. Brown) Oliv. Based on UHPLC–Q-TOF-MS/MS

The aim of this study was to identify the chemical constituents of Loropetalum chinense (R. Brown) Oliv. (LCO) and determine which of these had antioxidant effects. The chemical composition of a 70% ethanol extract of LCO was analyzed systematically using UHPLC–Q-TOF-MS/MS. The chemical components of the 70% ethanol extract of LCO were then separated and purified using macroporous resin and chromatographic techniques. Antioxidant activity was evaluated using a DPPH assay. In total, 100 compounds were identified tentatively, including 42 gallic acid tannins, 49 flavones, and 9 phenolic compounds. Of these, 7 gallium gallate, 4 flavonoid and 8 quinic acid compounds were separated and purified from the 70% ethanol extract of LCO. The compounds identified for the first time in LCO and in the genus Loropetalum were 3,4,5-trimethoxyphenyl-(6′-O-galloyl)-O-β-d-glucopyranoside, protocatechuic acid, ethyl gallate, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 3,5-O-diocaffeoylquinic acid, 4,5-O-diocaffeoylquinic acid and 3,4-O-diocaffeoylquinic acid. The 50% inhibitory concentration (IC50) values of compounds 1,2,3,4,6-penta-O-galloyl-β-d-glucose, gallic acid, protocatechuic acid, and ethyl gallate were 1.88, 1.05, 1.18, and 1.05 μg/mL, respectively. Compared with the control group (VC) (2.08 μg/mL), these compounds exhibited stronger anti-oxidation activity. This study offered considerable insight into the chemical composition of LCO, with preliminary identification of the antioxidant ingredients.


Introduction
Reactive oxygen species (ROS) refers to oxygen-containing reactive species and includes superoxide anions (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (•OH) [1].Under normal conditions, ROS are in a constant dynamic state of production and elimination in vivo.They play an important role in physiological metabolic processes such as enhancing leukocyte phagocytosis and prostaglandin synthesis, and participating in enzymatic pathways that contribute to immunity [2].
A net excess of ROS can follow an imbalance in ROS production and elimination.This can lead to a series of peroxidation reactions, cross-linking or breakages with subsequent cellular structural damage and dysfunction.If this occurs chronically, a number of pathophysiological processes may ensue including arteriosclerosis, cardiovascular diseases, neurodegenerative diseases, cancers, and other disorders associated with aging [3][4][5][6].Therefore, elimination of excess ROS in the body is important for maintaining physiological health.Recently, there has been an increase in interest in antioxidants as substances that protect against oxidative damage.A particular focus has been secondary plant polyphenol or phenolic metabolites such as catechins, epigallocatechin gallate, and catechin-aldehyde polycondensates [7][8][9].These phenolic compounds also possess other bioactivities including antimicrobial and anti-inflammatory effects [10,11].Therefore, screening for antioxidants could be an effective strategy for sourcing new drugs or functional foods.
Loropetalum, as a member of the Hamamelidaceae family, contains three species (Loropetalum lanceum Hand-Mzt, Loropetalum subcapitatum Chun ex Chang, and Loropetalum chinense (R. Brown) Oliv.) as well as one variety (Loropetalum chinense var.rubrum) in China.Among them, Loropetalum chinense (R. Brown) Oliv.(LCO), an evergreen shrub or small arbor, was first recorded officially in the 1970 edition of the Chinese pharmacopoeia.LCO has antipyretic, hemostatic, and detoxificant effects, and is a traditional medicine widely used in the treatment of bleeding disorders, burns, skin infections, dysentery, and diarrhea [12].Modern pharmacological research has shown that LCO has bacteriostatic, anti-inflammatory, healing, and antioxidant effects [13].
The multiple biological activities of LCO are attributed to its diverse constituents.Phytochemical studies reported the presence of tannins, flavonoids, lignans, terpenoids, and steroids in LCO [14][15][16][17].Alkanes, aldehydes, and terpenoids have been identified as the dominant constituents of LCO essential oil [13].However, there is little information on the biological constituents that have antioxidant effects.In earlier work, we used DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity to evaluate the antioxidant effects of four fractions (water, and 10%, 70%, and 95% ethanol eluates) separated with HPD-400 macroporous resin.Our results showed that the four fractions exhibited various antioxidant effects.The 50% inhibitory concentrations (IC 50 ) were 22.53 µg/mL for the water eluate, 11.47 µg/mL for 10% ethanol eluate, 9.73 µg/mL for 70% ethanol eluate and 39.20 µg/mL for 95% ethanol eluate [18].However, this work only assessed the antioxidant effects of various LCO fractions.Details of the specific constituents responsible for the antioxidant activities remain unclear.
Therefore, in the present study, we used UHPLC-Q-TOF-MS/MS to systemically analyze the chemical constituents of the ethanol extract of LCO to identify the components responsible for its antioxidant effects.

Chemicals and Reagents
DPPH and vitamin C (V C ) were purchased from Sigma Chemicals (Shanghai, China).Chemical standards of gallic acid, protocatechuic acid, quercetin, kaempferol, and chlorogenic acid were obtained from Chengdu Munster Biotechnology Co., Ltd.(Chengdu, China).LC-MS grade methanol and HPLC grade methanol for use as solvents, and acetic acid, formic acid, and acetonitrile were purchased from Fisher Scientific (Shanghai, China).HPD-400 macroporous resin was purchased from Cangzhou Bon Adsorber Technology Co., Ltd (Jiangsu, China).Various types of silica gel were obtained from Qingdao Haiyang Chemical Co., Ltd (Shandong, China).Double distilled water was used in the LC mobile phase.All other chemicals used were analytical grade.

Plant Material and Extraction
LCO was collected in May around the city of Jindezhen (29 Extraction parameters were as described in earlier work by our group [19].Dried branches and leaves of LCO (17 kg) were milled and reflux-extracted twice with an 8-fold weight of 60% ethanol solution for 2 h.Extracts were then combined and filtered with vacuum suction filtration.The combined filtrates were concentrated to dryness under vacuum with a rotary evaporator to yield a yellowish-brown residue (2.754 kg) that was stored at 4 • C until needed.The residue was re-dissolved with methanol to give a final concentration of 10 mg/mL and then filtered through a 0.22 µm filter membrane before UHPLC analysis.

DPPH Assay
DPPH, control, blank and sample solutions were prepared as follows.DPPH solution: 3 mg DPPH was dissolved in absolute ethanol and mixed with 0.03 g/L reaction solution protected from light.Control: 1 mL solvent and 3 mL DPPH solution were combined in a test tube, shaken to mix and left to react for 30 min in the dark at room temperature (25 • C).Absorbance (A 0 ) was then measured at a wavelength of 517 nm with a UV visible spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan).Blank: Various concentrations of sample solution (1 mL) were added to 3 mL absolute ethanol in a test tube, shaken to mix, and then stored in the dark at 25 • C.After 30 min, absorbance (A j ) was measured at 517 nm wavelength.Sample: Various concentrations of sample solution (1 mL) were mixed with 3 mL DPPH solution in a test tube, stored in the dark at 25 • C for 30 min before measuring the absorbance (A i ) at a wavelength of 517 nm [20][21][22].The scavenging rate was calculated as follows: scavenging activity (%) = [1 − (A i − A j )/A 0 ] × 100.

UHPLC-Q-TOF-MS/MS Analysis of the Crude Extract
Chromatographic analysis was carried out on a Prominence™ UHPLC system (Shimadzu, Japan) coupled with a triple TOF™ 5600+ MS/MS system (AB Sciex, Framingham, MA, USA).Separations were accomplished on a Welch C 18 column (2.1 mm × 100 mm, 1.8 µm, Shanghai, China) and 2 µL injected into UHPLC.The column oven was maintained at 40 The mass spectrometer was operated both in negative and positive ion modes.The following parameter settings were used: ion spray voltage 4500 V; turbo spray temperature 600 • C; curtain gas 25 psi, nebulizer gas (GS 1) 50 psi, heater gas (GS 2) 50 psi, declustering potential 100 V. TOF MS and TOF MS/MS were scanned with the mass range of m/z 50-1250 and 50-1250, respectively.In the IDA-MS/MS experiment, collision energy was set at 35 eV and collision energy spread was (±) 10 eV.Accurate mass and composition for the precursor and fragment ions were analyzed using Peakview software (Version 1.2, AB Sciex) integrated with the instrument.

Isolation of the Crude Extract
The crude extract solution was adsorbed by the HPD-400 macroporous resin and eluted with water, and 10%, 70%, and 95% ethanol-water.The 10% and 70% fractions were concentrated under vacuum to recover the organic solvent to dryness.They were then isolated using various chromatographic techniques including silica gel, Sephadex LH-20, ODS, and MCI columns, and re-crystallization methods.
The compounds were identified using UV, MS, 1 H-NMR, and 13 C-NMR experiments along with comparison of their spectroscopic properties from the literature.Their antioxidant activities were measured by DPPH assay.

UHPLC-Q-TOF-MS/MS Analysis of the Crude Extract
To obtain the abundant constituents from LCO, UHPLC conditions (type of column, mobile phase system, column temperature, and flow rate) were first optimized followed by the MS conditions (capillary voltage, declustering potential, and collision energy).UHPLC-Q-TOF-MS/MS in positive (A) and negative (B) ion modes was employed to characterize the corresponding signals.The base peak chromatogram under optimized chromatographic and MS conditions are presented in Figure 1.Retention times, observed molecular weight, and fragment ions for each metabolite, and their identities are presented in Table 1.A total of 100 compounds were identified or tentatively characterized including 42 gallic acid tannins, 49 flavones, and 9 phenolic compounds.The structures of these compounds were tentatively assigned by matching the MS/MS data with a reference or public database such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) or MassBank (http://www.massbank.jp/).2A2).Three fragment ion at m/z: 171.0289, 153.0185, and 125.0232 showed that peak 4 contained gallic acid units.Based on these MS/MS data, peak 4 was identified as theogallin (C 14 H 16 O 10 ).Under the negative ion mode, peak 11 had [M − H] − ions at m/z 331.0667, and fragment ions at m/z 169.0148, 151.0031, and 125.0257 indicating the presence of gallic acid units.[M − H] ions produce m/z 169.0148 by losing 162 Da (-C 6 H 10 O 5 ), which indicated the presence of hexose units and thus peak 11 peak was determined to be monogalloylglucose.

Fragmentation of Flavonoids Compounds
The Retro-Diels-Alder (RDA) cleavage reaction involves loss and rearrangement of flavonoid aglycone C rings in different ways.The main fragment was derived from cleavage of C-C and C-O bonds on the C ring, and neutral fragments such as CO, CO 2 , H 2 O, and C 2 H 2 O. Hexose (m/z: 162) and pentose (m/z: 132) fragments often appear in the MS/MS cracking spectrum of flavonoid glycosides [27][28][29].In this study, flavonoid glycosides produced gallic acid (m/z: 169) fragments.For example, peak 71 was identified as quercetin (     2B1).As peak 73 had the same fragments, it was presumed to be an isomer of peak 66.The [M − H] − ion at m/z: 593.1341 of peak 89 was found in the negative ion MS/MS spectrum.Fragments m/z: 285.0399 and 151.0038 were consistent with kaempferol cleavage.Combined with m/z: 307.0835, 163.0398, and 145.0296, we speculated that this was tribuloside (C30H26O13) (Figure 2B2), and that peaks 84 and 93 were its isomers.2B1).As peak 73 had the same fragments, it was presumed to be an isomer of peak 66.
The [M − H] − ion at m/z: 593.1341 of peak 89 was found in the negative ion MS/MS spectrum.Fragments m/z: 285.0399 and 151.0038 were consistent with kaempferol cleavage.Combined with m/z: 307.0835, 163.0398, and 145.0296, we speculated that this was tribuloside (C 30 H 26 O 13 ) (Figure 2B2), and that peaks 84 and 93 were its isomers.

Fragmentation of Quinine Acid Compounds
Nine caffeoylquinic acid compounds, which lost CO (m/z 28), CO 2 (m/z 44), and H 2 O (m/z 18) during MS/MS cleavage, were observed temporarily in this study [30][31][32].For example, [M + H] + ion at m/z: 193.0706 of peak 3 was matched with the molecular formula, C 7 H 12 O 6 .Compared with [M + H] + ions, m/z:175.0608ions were less than 18 Da and m/z: 147.0656 ions were less than 46 Da, which meant that peak 3 contained hydroxyl and carboxyl groups.After comparison with standard products, peak 3 was identified as quinic acid.
The negative MS/MS spectra of peaks 22, 28, 37, and 68 showed the presence of quinic and caffeic acids in the structure.For example, in the MS/MS spectra of peak 22, the [M − H] − ion at m/z: 353.0878 lost 162 Da and acquired m/z 191.0565, and lost 180 Da and acquired m/z 173.0445, which confirmed the presence of caffeic acid.In addition, [M − H] − ion lost 192 Da and acquired m/z 161.0242, and lost 174 Da and acquired m/z 179.0344, which indicated the presence of quinic acid.Therefore, peaks 22, 28, and 37 were identified as caffeoylquinic acid isomers (C 16 H 18 O 9 ) and peak 68 was dicaffeoylquinic acid (C 25 H 24 O 12 ).

Structural Identification of Purified Samples
The 10% ethanol elution (101.2 g) was dissolved in water and then adsorbed by MCI-gel pore resin to obtain five fractions (Fr1-Fr5) by gradient elution of methanol/water (1:0-0:1).Fr1 underwent repeated ODS column chromatography and preparative HPLC to give compound 1.Compound 2 was obtained by repeated crystallization of Fr3.Separation of Fr4 by ODS column chromatography gave compound 3.

Antioxidant Activity Analysis of Purified Compounds
The antioxidant activity of the purified compounds was tested using DPPH methods (Table 2, Figure 3).With the exception of compound 11, all test compounds showed significant antioxidant activity.The IC 50 values of compounds 2, 3, 11, 12, and 14 ranged from 3.00 to 4.05 µg/mL, and all were slightly higher than in the V C control group (2.08 µg/mL).The IC 50 values of compounds 1, 4, 5, and 6 were 1.88, 1.05, 1.18, and 1.05 µg/mL, respectively, and were significantly lower than those of the V C control group.

Conclusions
In this study, UHPLC-Q-TOF-MS/MS and a variety of chromatographic separation techniques were used to systematically analyze and separate the active antioxidant components of LCO.A total of 100 compounds were identified from the 70% ethanol extract of LCO, and these were mainly gallic acid tannins and flavonoids.Of these, 18 pure compounds were isolated, with compounds 2, 5, 6, 12, 14, and 16-18 identified for the first time in LCO and the genus Loropetalum.DPPH results showed that compounds 1, 4, 5, and 6 had significant antioxidant activity.

Supplementary Materials:
The following are available online.

Conclusions
In this study, UHPLC-Q-TOF-MS/MS and a variety of chromatographic separation techniques were used to systematically analyze and separate the active antioxidant components of LCO.A total of 100 compounds were identified from the 70% ethanol extract of LCO, and these were mainly gallic acid tannins and flavonoids.Of these, 18 pure compounds were isolated, with compounds 2, 5, 6, 12, 14, and 16-18 identified for the first time in LCO and the genus Loropetalum.DPPH results showed that compounds 1, 4, 5, and 6 had significant antioxidant activity.
C 15 H 10 O 7 ) through comparison with the reference substance.[M + H] + ions at m/z: 303.0305, and RDA cleavage of C 1 -C 2 and C 3 -C 4 bonds in the C ring generated the fragment m/z: 153.0181.After breakage of the C 2 -O and C 10 -O bonds of the C ring, the B ring produced m/z: 137.0229 by losing two CO.[M + H] + ion lost one H 2 O (18 Da) to form m/z: 285.0037, and one CO (28 Da) to form m/z: 275.0176 (-C 14 H 10 O 6 ).Subsequently, m/z: 275.0176 (-C 14 H 10 O 6 ) was produced m/z: 257.0436 (C 14 H 8 O 5 ) and m/z: 229.0493 (-C 13 H 8 O 4 ) by losing -H 2 O and -CO.As peaks 57, 59, 62, and 85 had the same fragments, they were presumed to be quercetin isomers.

Figure 2 .
Figure 2. The MS/MS spectrum information of different compounds in LCO.A1: Peak 5; A2: Peak 4; B1: Peak 66; B2: Peak 89.3.1.3.Fragmentation of Quinine Acid CompoundsNine caffeoylquinic acid compounds, which lost CO (m/z 28), CO2 (m/z 44), and H2O (m/z 18) during MS/MS cleavage, were observed temporarily in this study[30][31][32].For example, [M + H] + ion at m/z: 193.0706 of peak 3 was matched with the molecular formula, C7H12O6.Compared with [M + H] + ions, m/z:175.0608ions were less than 18 Da and m/z: 147.0656 ions were less than 46 Da, which meant that peak 3 contained hydroxyl and carboxyl groups.After comparison with standard products, peak 3 was identified as quinic acid.The negative MS/MS spectra of peaks 22, 28, 37, and 68 showed the presence of quinic and caffeic acids in the structure.For example, in the MS/MS spectra of peak 22, the [M − H] − ion at m/z: 353.0878 lost 162 Da and acquired m/z 191.0565, and lost 180 Da and acquired m/z 173.0445, which confirmed the presence of caffeic acid.In addition, [M − H] − ion lost 192 Da and acquired m/z 161.0242, and lost 174 Da and acquired m/z 179.0344, which indicated the presence of quinic acid.Therefore, peaks 22, 28, and 37 were identified as caffeoylquinic acid isomers (C16H18O9) and peak 68 was dicaffeoylquinic acid (C25H24O12).

Figure 3 .
Figure 3.The IC50 values of purified compounds in LCO on antioxidant activity.

Figure 3 .
Figure 3.The IC 50 values of purified compounds in LCO on antioxidant activity.
• 25 N, 117 • 16 E) (Jiangxi province, China).Plants were authenticated by Prof. Shi-lin Yang, Jiangxi University of Traditional Chinese Medicine.A voucher specimen (No. 20100521) was stored in the National Pharmaceutical Engineering Center for Solid Preparation in Chinese Materia Medica, Jiangxi University of Traditional Chinese Medicine.
Fragment m/z: 447.0969 lost 162 Da to form m/z: 285.0414, and fragment m/z: 313.0582 in MS/MS spectrum showed the presence of hexose units.Fragments m/z: 285.0414, 151.0036, and 125.0245 were consistent with quercetin cleavage.Therefore, peak 66 was identified as astragalin-O-gallate (Figure