Identification of Polygonum orientale constituents using high-performance liquid chromatography high-resolution tandem mass spectrometry

Abstract

Our primary focus in this research was to identify and characterize its bioactive compounds for potential therapeutic use. Twenty-seven metabolites of Polygonum orientale were identified using LC-QTOF tandem mass spectrometry. Interestingly, P. orientale extracts included several highly oxygenated flavonoids were isolated from P. orientale by column chromatography. ¹³C NMR data of highly oxygenated flavonoids (1–7) are reported here for the first time. In addition, nitric oxide, 1,1-diphenyl-2-picrylhydrazyl, and water-soluble tetrazolium salt assays were carried out on the isolated compounds to investigate their anti-inflammatory, anti-oxidant, and neuroprotective activities, respectively. Compounds 1, 2, 3, 5, 7, and 8 significantly attenuated lipopolysaccharide-stimulated NO production in BV2 cells without affecting cell viability. Compounds 9–12 exhibited significant antioxidant activity, while compounds 8, 9, and 12 exhibited protective effects against glutamate-induced neurotoxicity in HT22 cells. Our results indicate that P. orientale is a promising source of natural agents for the potential treatment of inflammation and neurodegenerative diseases.

Graphical abstract

Chemical profile of Polygonum orientale.

Keywords:

• Polygonum orientale
• LC-QTOF MS
• anti-inflammatory
• anti-oxidant
• neuroprotective

Plants produce a wide range of chemically and biologically diverse secondary metabolites, which continue to play a highly significant role in drug discovery and development [1,2]. However, the identification of potentially bioactive constituents of natural products is challenging because of limitations in quantifying the compounds present in these products [3]. The coupling of high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (MS), however, has produced a system that can identify the molecular weight and structure of unknown constituents of interesting mixtures [4–6].

Polygonum orientale (Polygonaceae), an annual plant, has a cosmopolitan distribution and is cultivated domestically as well as grows at roadsides and on wastelands. P. orientale is also a well-known traditional Chinese medicine [7,8], and the whole planth as been used in the treatment of conditions such as muscle injuries [9], rheumatoid arthritis [7,9], coronary heart disease [10], and hypertension [11]. Several studies have reported various pharmacological effects of P. orientale, including antimicrobial [12], antimutagenic [7], antithrombotic [8], anti-inflammatory [13], and anti-oxidative activities [8,11]. Liang et al. investigated the anti-inflammatory effect of P. orientale extract (POE) in vivo, and found that it attenuated xylene-induced ear edema in mice and egg white-induced paw edema in rats. Moreover, POE inhibited cotton pellet-induced granuloma in a rat model of chronic inflammation [13].

The main constituents of P. orientale are the flavonoids orientin, vitexin, cynaroside, and quercitrin, and lignan and saponin have also been reported as components [10]. However, little has been reported to date on any direct correlation between the chemical composition and pharmacological effects of P. orientale. Therefore, we performed chemical profiling of P. orientale using liquid chromatography-quadrupole time-of-flight (LC-QTOF) MS. Biological activities of isolated compounds were investigated. Anti-inflammatory effects were tested via the measurement of the produced nitric oxide (NO) levels in lipopolysaccharide (LPS)-stimulated BV2 cells. Anti-oxidant activity was investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay. DPPH assay is based on the principle that DPPH, free radical, is reduced and scavenged by accepting hydrogen atom from antioxidant. Neuroprotective activities against L-glutamate induced neurotoxicity in HT22 cells were evaluated with water-soluble tetrazolium salt (WST) assay. WST assay is used for measurement of cell viability by reductive reaction from succinate-tetrazolium reductase of mitochondria of live cells.

Materials and methods

General experimental procedures

NMR spectra were recorded on Bruker Avance DPX 300 and Bruker Avance III 600 spectrometers (Bruker, Billerica, MA, USA). ESI-MS spectra were measured using an Agilent 6530 Q-TOF mass spectrometer (Agilent, Santa Clara, CA, USA). Dimethyl sulfoxide (DMSO) and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphoric acid was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone Laboratories Inc. (Logan, UT, USA). Fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were purchased from Gibco Industries Inc. (Auckland, NZ). HPLC-grade water and acetonitrile were purchased from Fisher Scientific Korea Ltd. (Seoul, Korea). Chloroform-d, DMSO-d6, and methanol-d4 were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). A Griess reagent for measurement of NO production was manufactured with 1% sulfanilamide (Sigma-Aldrich, St. Louis, MO, USA), 0.1% naphtylethylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA), and 2% phosphoric acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan). A WST kit (EZ-cytox) for cell viability assay was purchased from Daeil Lab Service Co., Ltd. (Seoul, Korea). Antioxidant activity was assessed by DPPH radical scavenging capacity. DPPH was purchased from Sigma-Aldrich and made with 15 μM DPPH solution in ethanol (EtOH). ESI-L Low Concentration Tuning Mix and Biopolymer Reference Mass Standard Kit for mass calibration were purchased from Agilent Technologies (Santa Clara, CA, USA).

Plant material

P. orientale was purchased from Kyungdong Oriental Herbal Market, Seoul, Korea and identified by Dr. Ki Yong Lee, a professor at the College of Pharmacy, Korea University (Sejong, Republic of Korea). The voucher specimen (KUP-HD053) has been deposited at the laboratory of pharmacognosy, College of Pharmacy, Korea University.

Extraction and isolation

Dried whole plants of P. orientale (1.6 kg) were cut into pieces and extracted three times (each for 1.5 h) with 8 L of 80% MeOH under ultrasonic conditions. After removing the solvent in vacuo, the obtained residue (46.6 g) was suspended in distilled water (1.5 L) and partitioned successively with n-hexane (1.5 L × 3), EtOAc (1.5 L × 3), and n-BuOH (1.5 L × 3) to yield ann-hexane fraction (2.6 g), an EtOAc fraction (4.6 g), and ann-BuOH fraction (18.4 g).

The n-hexane fraction was subjected to medium-pressure liquid chromatography (MPLC) using an Isolera One system (Biotage, Uppsala, Sweden) on a silica gel column (Biotage SNAPcartridge, KP-Sil 100 g) and eluted with an n-hexane/EtOAc gradient (95:5, 60:40) to yield 11 fractions (H1–H11). Compounds 1 (21.3 mg) and 2 (42.7 mg) were purified from H7 and H8 by recrystallization. Fraction H9 was re-chromatographed by MPLC using a Biotage SNAP cartridge, KP-Sil 10 g (CH₂Cl₂/MeOH, 100:0, 92:8), to afford 8 fractions (H9–1 to H9–8). Compound 3 (19.2 mg) was obtained from H9–3. The EtOAc fraction was subjected to silica gel (0.040–0.063 mm, 230–400 mesh)column chromatography (CC) and eluted with a gradient of n-hexane/EtOAc/MeOH (1:0:0, 20:1:0, 10:1:0, 5:1:0, 2:1:0, 1:1:0, 0:1:0, 0:20:1, 0:10:1, 0:5:1, 0:2:1,1 L of each solvent) to yield 20 fractions (EA1–EA20). Compounds 4 (6 mg), 5 (22 mg), 6 (5 mg), and 7 (7 mg) were purified by recrystallization from EA5, EA7, EA10, and EA11, respectively. EA15 was separated by Sephadex LH-20 (18–111 μm) CC (MeOH, isocratic, 2L) to yield 4 subfractions (EA15-1 to EA15-4). EA15-2 was purified by recrystallization to yield compound 8 (32 mg). EA16 was subjected to silica gel CC (CHCl₃/MeOH/H₂O, 50:4:0.1, 25:4:1, 15:4:0.4, 1 L of each solvent) to yield 9 fractions (EA16-1 to EA16-9). EA16-7 was further purified by recrystallization to yield compound 9 (57 mg). The n-BuOH fraction was subjected to adsorptive macroporous resin HP-20 (250–850 μm) CC and eluted successively with water, 20% aqueous MeOH, 40% aqueous MeOH, 60% aqueous MeOH, 80% aqueous MeOH, and 100% MeOH (2 L of each solvent) to yield 6 fractions (B1–B6). B3 and B4 were combined and further subjected to reversed-phase (RP)-C₁₈ (30–50 μm) CC (MeOH/H₂O, 1:4, 1:1.5, 2 L of each solvent) to afford 8 subfractions (B3-1 to B3-8). Compound 10 (47 mg) was obtained by the separation of B3-5 on a silica gel column (CH₂Cl₂/MeOH/H₂O, 5:1:0.1, 4:1:0.1, 1 L of each solvent). Fraction B3-6 was subjected to silica gel CC (CHCl₃/MeOH/H₂O, 3.5:1:0.1, 3:1:0.1, 1 L of each solvent) to yield two subfractions (B3-6-1 and B3-6-2). B3-6-2 was recrystallized to provide compound 11 (142 mg). B3-8 was separated by silica gel CC (CHCl₃/MeOH/H₂O, 4.5:1:0.1, 3.5:1:0.1, 1 L of each solvent) to yield compound 12 (27 mg).

5,5′-dihydroxy-3,3′,4′,6,7,8-hexamethoxyflavone (digicitrin, 1):Yellow powder;C₂₁H₂₂O₁₀; ESI-MS m/z435 [M + H]⁺; ¹H and ¹³C NMR, see Table 2.

3,3′,5,6,7,8-hexamethoxy-4′,5′-methylenedioxyflavone (2): Pale yellow powder;C₂₂H₂₂O₁₀; ESI-MS m/z447[M + H]⁺; ¹H and ¹³C NMR, see Table 2.

3,3′,4′,5,5′,6,7,8-octamethoxyflavone (exoticin, 3):Yellow powder;C₂₃H₂₆O₁₀; ESI-MS m/z463 [M + H]⁺; ¹H and ¹³C NMR, see Table 2.

5-hydroxy-3,3′,6,7,8-pentamethoxy-4′,5′-methylenedioxyflavone(4): Yellow powder; C₂₁H₂₀O₁₀; ESI-MS m/z433 [M + H]⁺; ¹H and ¹³C NMR, see Table 2.

5,5′-dihydroxy-3,3′,4′,8-tetramethoxy-6,7-methylenedioxyflavone(5): Yellow powder; C₂₀H₁₈O₁₀; ESI-MS 419 [M + H]⁺; ¹H and ¹³C NMR, see Table 2.

3,3′,5,8-tetramethoxy-4′,5′,6,7-bis(methylenedioxy)flavone (6): Yellow powder; C₂₁H₁₈O₁₀; ESI-MS m/z431[M + H]⁺; ¹H and ¹³C NMR, see Table 2.

3,3′,4′,5,5′,8-hexamethoxy-6,7-methylenedioxyflavone (7): Yellow powder; C₂₂H₂₂O₁₀; ESI-MS m/z447 [M + H]⁺; ¹H and ¹³C NMR, see Table 2.

LC-Q-TOF MS/MS conditions

HPLC analysis was performed on an Agilent 1260 series system (Agilent, Santa Clara, CA, USA) with an auto-sampler, binary pump, degasser, and diode array detector. The P. orientale extracts were separated using a Kinetex C18 column (150 × 4.6 mm, 5 μm) with a C18 guard column (4.00 × 3.00 mm; Phenomenex, USA). The mobile phase consisted of water (solvent A) and acetonitrile (solvent B), each containing 0.1% formic acid. The elution gradient system was as follows: 15% B (0–5 min), and 90% B (5–30 min). The flow rate of the mobile phase was 0.6 mL/min and the injection volume was 10.0 μL. An Agilent 6530 Q-TOF mass spectrometer (Agilent, Santa Clara, CA, USA) was used for mass chromatography analysis with electrospray ionization (ESI) in the positive and negative mode. Before analysis, positive and negative ion mass calibration was performed using the tuning solution. Tuning solution was prepared by ESI-L Low Concentration Tuning Mix diluted with HPLC-grade water and acetonitrile. 0.1 mM HP-0321 which is included in the Biopolymer Reference Mass Standard Kit was mixed at positive tuning solution, additionally. The mass parameters were as follows: nebulizer pressure, 40 psi; capillary voltage, 4000 V; fragmentor voltage, 175 V; skimmer voltage, 65 V; drying gas temperature, 325 °C; flow rate of drying gas, 12.0 L/min. The MS/MS chromatogram of the EtOAc fraction and compounds from P. orientale was obtained using a collision energy of 10, 20, and 30 V. The mass scan range was set at 50–1700 m/z. Mass Hunter Workstation software LC/MS Data Acquisition for 6530 series Q-TOF (version B.05.00) was used to set all acquisition parameters for obtaining data.

NO production inhibition and cell viability assay

BV2 mouse microglia cells were obtained from the College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul, Korea. BV2 cells were grown in DMEM containing 10% FBS and 100 U/mL penicillin-streptomycin at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. For NO production assay, cells were seeded at 2 × 10⁵ cells/mL into 48-well plates for 24 h. Medium wasreplaced with DMEM without FBS and containing the experimental compounds for 1 h before exposure to 50 ng/mL of LPS. After 24 h, 100 μL of Griess reagent and 100 μL of supernatants in each well were reacted for 10 min at room temperature. Optical density (OD) was measured at 550 nm using ELISA reader, and the NO inhibition rate was calculated from the following: % NO inhibition = 100 × (OD of LPS treated cultures − OD of LPS-sample treated cultures)/(OD of LPS treated cultures − OD of control cultures).

100 μL of the remaining supernatants from the NO inhibition assay was used to determine cell viability by WST assay. 20 μL of WST solution was added to supernatants in a 96-well plate and incubated for 2 h. OD values were obtained at 450 nm. Percent cell viability was calculated as 100×(OD of LPS-sample treated cultures)/(OD of control cultures).

DPPH radical-scavenging assay

A DPPH assay was used to measurement of stable radical DPPH scavenging ability. Both a DPPH assay of the extract and compounds isolated from P. orientale and a time-based DPPH assay were performed as previously reported [6].

Neuroprotective assay

Mouse hippocampal HT22 cells were grown in DMEM (Hyclone) containing 10% FBS and 100 U/mL penicillin-streptomycin at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. HT22 cells were seeded at a density of 2 × 10⁴ cells/mL in 96-well plates and cultured for 24 h. Various concentrations of compounds were used to pretreat cells for 1 h before the addition of 6 mM glutamate. Trolox (Sigma) was used as a positive control. After 24 h, cell viability was measured by WST assay. 10 μL of WST solution was added to each well, and cells were incubated for 2 h. OD values were obtained at 450 nm, and cell viability was calculated from the following equation: % protection = 100 × (OD of glutamate-sample treated cultures or OD of glutamate treated cultures)/(OD of control cultures).

Results and discussion

Chemical profile of P. orientale

We identified the metabolites contained in P. orientale extract using LC-QTOF-MS. The structures of the major mass chromatogram peaks were predicted using the UV spectrum, molecular weight, MS/MS fragmentation, and previously reported data (Figure 1, Table 1). Twenty-seven peaks were identified from the mass chromatograms using electrospray ionization (ESI) in the positive and negative mode. Almost all peaks had maximum UV absorption (λmax) at 260 and 330 nm, indicating that the structures potentially were flavonoids. Peaks m, n, and q (vanicosides) were previously identified in other members of the Polygonum genus such as P. lapathifolium [14], P. hydropiper [15], and P. pensylvanicum [16]. However, the present study is the first to identify vanicoside in P. orientale. Peaks p and s~z were found to be highly oxygenated flavonoids. Although flavonoids having eight oxygen functional groups (at C-3, 3′, 4′, 5, 5′, 6, 7 and 8) have previously been reported in P. orientale [17], it has not previously been shown to contain highly oxygenated flavonoids.

Figure 1. MS and UV chromatograms of P. orientale extracts. (A) negative ionization mode; (B) positive ionization mode; (C) 330 nm.

Table 1. Peak identification of P. orientale extracts on chromatographic profiles in Figure 1.

Peak No.	Compounds identification	tR (mins)	Observed m/z	Calculated m/z	Molecular formula [M–H]^−	MS/MS fragments (m/z)	UV (λmax, nm)	Compounds
a	Isoorientin	10.076	447.0926	447.0933	C21H19O11	429 [M–H2O-H]^−	268, 350	11
b	Orientin	10.486	447.0924	447.0933	C21H19O11	327 [M–C4H8O4–H]^−	268, 350	10
c	Vitexin	11.407	431.0976	431.0978	C21H19O10	311 [M–C4H8O4–H]^−	268, 350
d	Isovitexin	11.612	431.0978	431.0978	C21H19O10	311 [M–C4H8O4–H]^−	269, 340
e	Cynaroside	12.021	447.0929	447.0933	C21H19O11	285 [M–C6H10O5–H]^−	266, 350	12
f	Catechin gallate	12.175	441.0811	441.0827	C22H18O10	289 [M–C7H4O4–H]^−	276
g	Rhamnetin-3-O-glucoside	12.482	477.1022	477.1038	C22H21O12	315 [M–C6H10O5–H]^−	270, 350
h	Flavonoid glycoside	12.943	447.0925	447.0933	C21H19O11	284 [M–C6H11O5–H]^−	266, 350
i	Quercitrin	13.097	447.0916	447.0933	C21H19O11	300 [M–C6H11O4–H]^−	258, 350	9
j	Afzelin	14.120	431.0974	431.0978	C21H19O10	284 [M–C6H11O4–H]^−	263, 350	8
k	Luteolin	16.270	285.0384	285.0405	C15H9O6	175 [M–C3O2–CH2CO–H]^−	276, 333
l	Unknown compound	16.782	329.0292	329.0303	C9H13O13	314 [M–CH3–H]^−	246, 367
m	Vanicoside C	17.857	821.2300	821.2298	C41H41O18	675 [M–C9H6O2–H]^−	315
n	Vanicoside B	18.881	955.2673	955.2666	C49H47O20	809 [M–C9H6O2–H]^−	315
o	Flavonoid	18.936	455.0976	455.0973	C23H19O10^a	440 [M–CH3+H]^−	280, 322
p	5,3′,4′-Trihydroxy-3-methoxy-6,7-methylenedioxyflavone	19.547	343.0442	343.0459	C17H11O8	328 [M–CH3–H]^−	328
q	Vanicoside A	19.956	997.2783	997.2772	C51H49O21	851 [M–C9H6O2–H]^−	315
r	Methoxyeugenol	20.314	193.0845	193.0870	C11H13O3	178 [M–CH3–H]^−	315
s	3,3′,4′,5,5′,8-Hexamethoxy-6,7-methylenedioxyflavone	21.496	447.1315	447.1286	C22H23O10^a	417 [M–CH2O+H]^+	280, 330	7
t	Gossypetin tetramethyl ether	21.901	373.0908	373.0929	C19H17O8	343 [M–CH2O–H]^−	280, 340
u	3,3′,5-Trimethoxy-4′,5,6,7′-bis(methylenedioxy)flavone	21.906	423.0711	423.0687	C20H17O9^a	408 [M–CH3+Na]^+	280, 340
v	5,5′-Dihydroxy-3,3′,4′,8-tetramethoxy-6,7-methylenedioxyflavone	22.008	419.1004	419.0973	C20H19O10^a	403 [M–O+H]^+	280, 350	5
w	3,3′,5,8-Tetramethoxy-4′,5′,6,7-bis(methylenedioxy)flavone	22.264	431.1007	431.0973	C21H19O10^a	401 [M–CH2O+H]^+	280, 340	6
x-1	Digicitrin	22.315	435.1323	435.1286	C21H23O10^a	405 [M–CH2O+H]^+	275, 330	1
x-2	Exoticin	22.418	463.1627	463.1599	C23H27O10^a	433 [M–CH2O+H]^+	275, 330	3
y	3,3′,5,6,7,8,-Hexamethoxy-4′,5′-methylenedioxyflavone	23.135	447.1315	447.1286	C22H23O10^a	417 [M–CH2O+H]^+	256, 345	2
z	5-Hydroxy-3,3′,6,7,8-pentamethoxy-4′,5′-methylenedioxyflavone	25.695	433.1161	433.1129	C21H21O10^a	403 [M–CH2O+H]^+	280, 350	4

^a [M + H]^+.

Isolation of compounds from P. orientale

Twelve flavonoids (1–12) were isolated from P. orientale extracts (Figure 2), and were identified as 5,5′-dihydroxy-3,3′,4′,6,7,8-hexamethoxyflavone (digicitrin,1) [17], 3,3′,5,6,7,8-hexamethoxy-4′,5′-methylenedioxyflavone (2) [17], 3,3′,4′,5,5′,6,7,8-octamethoxyflavone (exoticin, 3) [17], 5-hydroxy-3,3′,6,7,8-pentamethoxy-4′,5′-methylenedioxyflavone (4) [17], 5,5′-dihydroxy-3,3′,4′,8-tetramethoxy-6,7-methylenedioxyflavone (5) [17], 3,3′,5,8-tetramethoxy-4′,5′,6,7-bis(methylenedioxy)flavone (6) [17], 3,3′,4′,5,5′,8-hexamethoxy-6,7-methylenedioxyflavone (7) [17], kaempferol 3-O-α-L-rhamnopyranoside (afzelin, 8) [18], quercetin 3-O-α-L-rhamnopyranoside (quercitrin, 9) [19], luteolin 8-C-β-D-glucopyranoside (orientin, 10) [20], luteolin 6-C-β-D-glucopyranoside (isoorientin, 11) [20], and luteolin 7-O-β-D-glucopyranoside (cynaroside, 12) [21] by comparing their physical and spectroscopic data with those reported previously. Interestingly, all proton positions of compounds 1–7 structures are substituted oxygen molecules, producing the unusual compounds that are highly oxygenated flavonoids. The present study is the first to report ¹³C- NMR data for these compounds. ¹H- and ¹³C NMR data of compounds 1–7 were displayed in Table 2.

Figure 2. Chemical structures of isolated compounds 1–12 from P. orientale extracts.

Table 2. ¹H- and ¹³C NMR spectroscopic data for compounds 1–7 (δ in ppm).

Position	1^a		2^a		3^a		4^a		5^b		6^c		7^c
	^1H- NMR	^13C NMR	^1H- NMR	^13C NMR	^1H- NMR	^13C NMR	^1H- NMR	^13C NMR	^1H- NMR	^13C NMR	^1H- NMR	^13C NMR	^1H- NMR	^13C NMR
2		155.4		152.7		152.7		155.4		155.1		151.4		151.6
3		139.3		143.9		143.9		138.9		138.6		140.2		139.5
4		179.4		173.8		173.9		179.3		178.7		172.3		172.4
5		149.1		148.2		148.2		149.2		136.0		134.9		135.0
6		136.2		140.9		141.2		135.4		129.8		136.8		136.8
7		153.0		151.4		151.4		152.3		145.3		145.5		145.7
8		132.9		137.8		137.8		132.8		124.4		127.8		127.8
9		144.9		146.6		146.7		144.8		144.3		143.0		143.2
10		107.5		115.0		115.0		109.1		107.1		113.2		113.2
11									6.20 (2H, s)	103.4	6.20 (2H, s)	103.1	6.20 (2H, s)	103.2
1′		126.0		124.9		126.0		124.5		124.9		124.2		125.5
2′	7.40 (1H, d, 2.1)	104.9	7.52 (1H, d, 1.5)	108.7	7.51 (1H, s)	105.7	7.53(1H, d, 1.5)	107.4	7.22 (1H, d, 2.0)	103.6	7.37 (1H, d, 1.5)	108.3	7.37 (1H, s)	105.3
3′		152.1		143.5		153.1		143.5		153.0		143.0		152.8
4′		138.0		137.4		140.2		137.4		139.0		137.0		140.5
5′		149.3		149.1		153.1		149.2		150.7		148.7		152.8
6′	7.46 (1H, d, 2.1)	108.8	7.40 (1H, d, 1.5)	102.7	7.51 (1H, s)	105.7	7.41 (1H, d, 1.5)	102.9	7.29 (1H, d, 2.0)	109.7	7.23 (1H, d, 1.5)	102.1	7.37 (1H, s)	105.3
7′	6.07 (2H, s)		6.07 (2H, s)	102.1			6.10 (2H, s)	102.2			6.13 (2H, s)	101.7
OMe	3.89 (3H, s)	56.0	3.88 (3H, s)	56.6	3.90 (3H, s)	56.2 (2C)	3.88 (3H, s)	56.6	3.77 (3H, s)	55.8	3.78 (3H, s)	56.3	3.76 (3H, s)	55.9
	3.94 (9H, s)	60.3	3.94 (3H, s)	59.9	3.94 (12H, s)	60.0	3.96 (6H, s)	60.2	3.83 (3H, s)	59.9	3.85 (3H, s)	59.3	3.81 (3H, s)	55.9
	4.01 (3H, s)	61.1	3.97 (3H, s)	61.7	3.97 (3H, s)	61.0	3.98 (3H, s)	61.2	3.84 (3H, s)	60.0	3.91 (3H, s)	61.0	3.86 (9H, s)	59.5
	4.10 (3H, s)	61.2	3.98 (3H, s)	61.9	4.00 (3H, s)	61.7	4.11 (3H, s)	61.7	3.96 (3H, s)	61.3	4.00 (3H, s)	61.1	4.00 (3H, s)	60.2
		61.7	3.99 (3H, s)	62.0	4.09 (3H, s)	61.8		62.1						61.1
		62.1	4.09 (3H, s)	62.3		61.9								61.2
						62.3

^a Recorded in chloroform-d, 300, 75 MHz;

^b Recorded in DMSO-d_6, 600, 125 MHz;

^c Recorded in DMSO-d_6, 600, 75 MHz;.

Biological activities of isolated compounds

We evaluated the inhibitory effect on NO production of an 80% MeOH extract of P. orientale, as well as of each fraction, using lipopolysaccharide (LPS)-stimulated BV2 cells. The ethyl acetate (EtOAc) and n-butanol (BuOH) fractions significantly inhibited NO production and did not influence cell viability at a concentration range of 10–100 μg/ml (S1). Compounds 1–12 of P. orientale were subjected to a Griess assay at concentrations of 1, 5, 15, 30, and 50 μM (Table 3), and the NO production inhibitory effect was demonstrated by the relative nitric oxide inhibition rate. Among the compounds, 1, 2, 3, 5, 7, and 8 exhibited the highest NO scavenging effect on LPS-induced BV2 cells (Table 3). Cell viability was not affected by the compounds at a concentration of 50 μM (S2). Compound 8 (afzelin) was shown in a previous study to inhibit NO production in LPS-stimulated RAW 264.7 cells with an IC₅₀ value of 42.8 μg/mL [22]. Compounds 1, 2, 3, 5, and 7 thus showed potential as novel candidates with NO inhibitory activity. The structure of these five compounds is related to their anti-inflammatory effect, as flavonoids having a 3′,4′-hydroxyl substituent on the B ring or with additional methoxy substituent have been shown to possess high anti-inflammatory activity [23] (see Figure 2).

Table 3. Effect of isolated compounds on NO production in BV2 cells, DPPH assay, and glutamate-induced toxicity in HT22 cells.

Compounds	IC50, μM^a
	NO	DPPH	Glutamate
1	9.7 ± 0.7	>100	>100
2	34.9 ± 4.4	>100	>100
3	14.3 ± 0.5	>100	>100
4	>100	>100	>100
5	35.8 ± 0.5	>100	>100
6	>100	>100	>100
7	11.4 ± 2.1	>100	>100
8	41.4 ± 1.4	>100	2.4 ± 2.1
9	>100	11.5 ± 0.4	40.4 ± 9.9
10	>100	9.7 ± 0.9	>100
11	>100	8.6 ± 0.1	>100
12	>100	14.5 ± 0.6	1.7 ± 0.6
Positive control^b	4.6 ± 0.1	26.8 ± 0.2	68.0 ± 2.0

^a IC50 value of each compound was defined as the concentration (μM) that caused 50% inhibition.

^b NO dexamethasone; DPPH curcumin; glutamate Trolox.

The 80% MeOH extract and fractions of P. orientale were screened to identify free radical scavenging activity using a DPPH radical-scavenging assay at concentrations of 5, 25, 50, and 250 μg/mL. The EtOAc, n-BuOH, and water fractions showed a significant antioxidant effect (S3). In the DPPH radical-scavenging assay, compounds 9–12 showed strong antioxidant activity with IC₅₀values of 11.5 ± 0.4, 9.7 ± 0.9, 8.6 ± 0.1, and 14.5 ± 0.6 μm, respectively, compared with that of curcumin. DPPH radical-scavenging activity is dependent on the position of hydroxyl groups in the aromatic ring [24]. Compounds 9–12, with hydroxyl groups in the ortho position in the aromatic ring, exhibited a strong antioxidant activity.

The neuroprotective activity of the 80% MeOH extract, each fraction, and isolated compounds 1–12 of P. orientale was evaluated in glutamate-induced HT22 cells. Neither the extract nor the fractions showed a neuroprotective effect or influenced cell viability at 50 and 100 μg/mL (S4). However, compounds 8, 9, and 12 significantly attenuated the induced glutamate neurotoxicity (Table 3). The result exhibited that O-glycoside flavonoids have more neuroprotective activity than C-glycoside flavonoids. Compound 9 has previously been shown to exert a significant neuroprotective effect in HT22 cells induced by glutamate [25].

In conclusion, our present study was certified that LC-QTOF-MS analysis is a useful technology to identify the metabolites of plants. The twelve compounds were isolated from whole plant of P. orientale. All of them were flavonoids, some of them had a highly oxygenated structure uncommonly. The isolated constituents of P. orientale exhibited anti-inflammatory, anti-oxidant, and neuroprotective activities. Taken together, our findings indicate a potential role for isolated compounds of P. orientale as novel candidates for the prevention and/or treatment of inflammatory and neurodegenerative diseases.

Author contributions

H. Shin, Y. Park, Y.H. Jeon, X. Yan, and K. Y. Lee conceived and designed the experiments. H. Shin, Y. Park, and X. Yan performed all experiments and analyzed all the data. H. Shin, Y.H. Jeon, and K. Y. Lee wrote the manuscript. All the authors reviewed and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by a National Research Foundation of Korea grant funded by the Korean Government [NRF-2014R1A1A1003605], [NRF-2014R1A4A1007304], [NRF-2017R1A2B4003403].

Supplemental data

Supplemental material for this paper is available at https://doi.org/10.1080/09168451.2017.1415124.