Phytochemical Composition, Hepatoprotective, and Antioxidant Activities of Phyllodium pulchellum (L.) Desv

Phyllodiumpulchellum has been traditionally used as a medicinal herb because of its health-promoting effects, such as its hepatoprotective and antioxidant activities. In the present study, the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and aqueous fraction were successively obtained from the ethanol extract of P. pulchellum. Two fractions, ethyl acetate fraction and n-butanol fraction, were found to display hepatoprotective and antioxidant activities. Further chemical investigation of the active fractions led to the isolation of its main constituents, including 11 flavonoids (1–11) and 8 indole alkaloids (12–19). There were 9 flavonoids (1–9) that were obtained from the ethyl acetate fraction, and 2 flavonoids (10 and 11) and 8 alkaloids (12–19) from the n-butanol fraction. Compounds 1–11 and 16–19 were isolated for the first time from P. pulchellum, and 1, 2, 8, 11, and 18 were obtained from the genus Phyllodium initially. Subsequently, the isolated compounds were evaluated for their in vitro hepatoprotective effects on the human normal hepatocyte cell line L-O2 injured by d-galactosamine and radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH). The flavonoids (−)-epigallocatechin (5) and (−)-epicatechin (6) exhibited prominent hepatoprotective activities with higher cell viability values (65.53% and 62.40% at 10 μM·mL−1, respectively) than the positive control, silymarin (61.85% at 10 μM·mL−1). In addition, compared with the positive control of vitamin C (IC50: 5.14 μg·mL−1), (−)-gallocatechin (3) and (−)-epigallocatechin (5) exhibited stronger antioxidant activities with IC50 values of 3.80 and 3.97 μg·mL−1, respectively. Furthermore, the total flavonoids from P. pulchellum were characterized using a high-performance liquid chromatography-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC-LTQ-Orbitrap-MS). In total, 34 flavonoids were tentatively identified, which had not been previously reported from P. pulchellum. In addition, we performed a semi-quantitative analysis of the isolated flavonoids. The contents of compounds 1–11 were 3.88, 17.73, 140.35, 41.93, 27.80, 4.34, 0.01, 0.20, 9.67, 795.85, and 5.23 μg·g−1, respectively. In conclusion, this study revealed that the flavonoids that were isolated from P. pulchellum showed hepatoprotective and antioxidant activities, indicating that, besides alkaloids, the flavonoids should be the potential pharmacodynamic ingredients that are responsible for the hepatoprotective and antioxidant activities of P. pulchellum.

This study attempted to investigate the main hepatoprotective and antioxidant ingredients of P. pulchellum.Four fractions, namely, the petroleum ether fraction (PPP), ethyl acetate fraction (PPE), n-butanol fraction (PPB), and aqueous fraction (PPA), were successively obtained from the ethanol extract of P. pulchellum.These four fractions were screened for their hepatoprotective and antioxidant activities.As the PPE and PPB showed the hepatoprotective and antioxidant activities, we further investigated the active principles from the target fractions.Subsequently, the structure elucidation, biological activities, and structure-activity relationships of the isolated compounds were studied and discussed.It was indicated that, besides the alkaloids, the flavonoids could be another potential pharmacodynamic ingredient for driving the hepatoprotective and antioxidant activities of P. pulchellum.Because the flavonoids showed good activities, we further investigated the composition of the total flavonoids of P. pulchellum using high-performance liquid chromatography-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC-LTQ-Orbitrap-MS).It was the first report to characterize the flavonoid compositions of P. pulchellum by LC-MS.

Plant Material
The aerial parts of the P. pulchellum were collected from Xingning (GPS coordinates: N 23 • 50 , E 115 • 30 ), Guangdong, China, in July 2014, and were identified by Professor Fengqin Zhou, Shandong University of Chinese Medicine.A voucher specimen (No.PP-201407) was deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, Ocean University of China, Qingdao, China.

Extraction and Isolation
The dried aerial parts of the P. pulchellum (8.0 kg) were consecutively extracted three times by reflux, with 95% ethanol at 80 • C for 3 h.The combined ethanol layer was lyophilized in order to obtain the ethanol extract (950 g).This dried residue was mixed with distilled water (1000 mL) and successively partitioned three times with the same volume of petroleum ether (PE), EtOAc, and n-butanol (n-BuOH).The respective fractions were dried under reduced pressure and lyophilized to yield four fractions, namely, PPP (60.0 g), PPE (50.0 g), PPB (30.0 g), and PPA (20.0 g).The above fractions were stored at −20 • C.

Hepatoprotective Activity Assay
The hepatoprotective effects were tested on human normal hepatocyte cell line L-O2 injured by D-GalN using MTT method, as reported previously [20].In this assay, the cell viability was measured so as to reflect the hepatoprotective activity.The cells were prepared into a 6.0 × 10 4 •mL −1 cell suspension with a DMEM medium containing 10% fetal bovine serum, penicillin (100 U•mL −1 ), and streptomycin (100 mg•mL −1 ), which were seeded in 96-well culture medium.After incubation for 24 h in an incubator with 5% CO 2 at 37 • C, 100 µL test samples were added.After incubation for 1 h, 37 mM D-GalN was added and incubated for 24 h.The incubation solution was discarded, and 100 µL MTT solution (0.5 mg•mL −1 ) was added to each well, followed by incubation at 37 • C for 4 h.The supernatant was discarded and 150 µL DMSO was added to each well so as to dissolve the fomazan particles.After a mild shaking, the optic density (OD) value was measured at the detection wavelength of 490 nm.Silymarin (purity > 98%) was used as a positive control.The blank control group was established at the same time, with 37 mM D-GalN.

DPPH Radical-Scavenging Activity Assay
Radical reducing effects against DPPH were tested according to a previously described method [21].The DPPH radical-scavenging was evaluated by comparing the percentage inhibition of the DPPH radicals.Specifically, for the fractions from P. pulchellum, each fraction of PPP, PPE, PPB, and PPA was dissolved in DMSO, and diluted into 156.25,78.13, 39.06, 19.5, and 9.75 µg•mL -1 , respectively.For the isolated compounds, each compound was dissolved in DMSO, and were diluted into 200, 100, 50, 25, and 12.5 µg•mL -1 .The DPPH solution was prepared with anhydrous ethanol to get the concentration of 0.05 mg•mL -1 .Each of the test samples were added to the DPPH solution 100 µL.After 30 min of dark reaction, the absorbance was measured at 517 nm.DPPH radical reducing activity of the test sample was expressed as I = [(A 0 /A X )/A 0 ] × 100%, where A 0 was the absorbance of the blank control reaction and A X was the absorbance in the presence of the sample.The sample concentration, providing 50% inhibition (IC 50 ), was calculated from the regression equation that was prepared from the concentration of the samples and the inhibition percentage.Vitamin C (purity > 99.7%) was used as a positive control.
An Agilent series 1290 Infinity HPLC instrument coupled with an LTQ/Orbitrap mass spectrometer equipped with an electrospray (ESI) interface was performed to analyze the total flavonoids extract.The chromatography conditions were as follows: a Kromasil C-18 analytical HPLC column (250 mm × 4.6 mm, I.D., 5 µm) was used; the flow rate was 1.0 mL•min −1 ; the sample injection volume was 10 µL; the column temperature was 30 • C; and the diode-array detector (DAD) scanned from 190 to 400 nm, and the samples were detected at 254 nm.The gradient profile of mobile phase A (acetonitrile) and mobile phase B (0.1% formic acid-water) was as follows: 0-10 min, 5-10% A; 10-20 min, 10-20% A; 20-30 min, 20-50% A; 30-50 min, 50-70% A; 50-60 min, 70-80% A; and 60-65 min, 80-100% A. The LTQ-Orbitrap-MS operating parameters were as follows: drying gas, high-purity nitrogen (N 2 ); capillary temperature, 350 • C; source voltage, 3000 V; sheath gas flow, 40 arb; aux gas flow, 10 arb; and tube lens, −100 V.Each sample was analyzed in both positive and negative modes so as to provide abundant information for structural identification.The mass spectra were recorded across the range of m/z 100-1500, with accurate mass measurement of all of the mass peaks.Accurate mass measurements of each peak from the total ion chromatogram (TIC) were obtained by means of ESI source.All of the data were processed using Xcalibur software version 3.0 (Thermo Fisher Scientific, San Jose, CA, USA).
The quantification of the isolated flavonoids was carried out by high performance liquid chromatography using a semi-quantitative analysis method.Rutin (purity > 92.6%) was used as a standard.The samples and standard were dissolved in ethanol (HPLC grade) and filtered using 0.22 µm sterile Millex filters before injection.Aliquots of 10 µL were injected into the HPLC system.

Hepatoprotective Activity of the Isolated Compounds
All of the isolated compounds 1−19 were tested for their hepatoprotective activities against the human normal hepatocyte cell line L-O2 injured by D-GalN.The results indicated that flavonoids 5 and 6 exhibited observably hepatoprotective activity, with higher cell viability values (65.53% and 62.40% at 10 µM•mL −1 , respectively) than the positive control, silymarin (61.85% at 10 µM•mL −1 ).Interestingly, alkaloid 18 was also found to show hepatoprotective activity, with the cell viability value of 60.72% at 10 µM•mL −1 , which was close to the positive control.According to the above results, it could be presumed that P. pulchellum displaying hepatoprotective activity might have been as a result of the presence of active compounds, mainly in the PPE and PPB fractions, such as compounds 5, 6, and 18.
A literature survey revealed that the flavonoids that were isolated from P. pulchellum, such as yukovanol and 8-prenylated 5,7,3 ,4 -tetrahydroxy flavanone, could have inhibited the proliferation of the activated hepatic stellate cells (HSC-T6 cells), in vitro at 10 µM•L −1 , with cell viability values of 54% and 42%, respectively [14].In addition, flavonoid pulcheloid B was reported to exhibit potent inhibitory activity in vitro against the proliferation of acetaldehyde-stimulated HSC-T6 cells, with the IC 50 value of 7.6 mM [16].Our study further proved that flavonoids had the hepatoprotective activity, which indicated that, besides the alkaloids, the flavonoids might have also been the potential pharmacodynamic ingredients that were responsible for the hepatoprotective activity of P. pulchellum.

Antioxidant Activity of the Isolated Compounds
A literature survey revealed that the hepatoprotective activity might have been related to antioxidant activity [38,39].In the present study, the isolated compounds 1−19 were further tested for their antioxidant activities against DPPH radicals.Only the flavonoids (−)-gallocatechin (compound 3) and (−)-epigallocatechin (compound 5) displayed strong antioxidant activities with the IC 50 values of 3.8 and 4.0 µg•mL −1 , respectively, which were more potent than the positive control, vitamin C (IC 50 : 5.1 µg•mL −1 ) (Table 1).Our results showed that flavonoids not only exhibited prominent hepatoprotective activity, but that they also displayed strong antioxidant activity.By comparison of the activities of flavonoids 3-6, we found that flavonoids 3 and 5 showed stronger activity than flavonoids 4 and 6, respectively.Furthermore, compound 7 (IC 50 : 47.5 µg•mL −1 ) displayed a stronger activity than that of compound 8 (IC 50 : >300 µg•mL −1 ).These results could have demonstrated that hydroxyl at C-5 might have played an important role in antioxidant activity, which was consistent with previous reports [40,41].Of flavonoids 1, 2, and 5, compound 5 with a hydroxyl substitution at C-3 demonstrated higher activity, which indicated that the hydroxyl group at C-3 had a positive contribution to improving the activity rather than the p-hydroxy-cinnamic acid substitution.Additionally, compared with compound 9, the glycoside substitutions of compounds 10 and 11 reduced the activity.Based on the above results, it could have been inferred that the extracts with better antioxidant activity might have been as a result of the active compounds contained in PPE fraction, such as flavonoids 1-7 and 9.
There was no report regarding the antioxidant activity of the flavonoids from P. pulchellum.Interestingly, the flavonoids that were isolated from the ethanol extract of the Distylium racemosum branches, including compounds 3−6 and 9, which were the same as our study, were also evaluated for their antioxidant activities [42].Particularly, compounds 3, 5, and 9 were reported for their radical scavenging activities toward DPPH, with the IC 50 values of 6.7, 62, and 65.3 µg•mL −1 , respectively [42], which were weaker than our test.While compounds 4 and 6 were found to display DPPH radical scavenging activities with the IC 50 values of 7.2 and 6.3 µg•mL −1 , respectively [42], which were stronger than our study.Thus, it was necessary to carry out the in vivo antioxidant experiment of the isolated compounds.

HPLC-LTQ-Orbitrap-MS Analysis of Total Flavonoids
To characterize the flavonoids in P. pulchellum, a HPLC-LTQ-Orbitrap-MS method was established.The total ion chromatograms (TIC) in negative ion mode (A) and positive ion mode (B) are displayed in Figure 2. Most of the constituents were well separated under the gradient elution condition, with high resolution and good sensitivity.