Protective effects of luteolin-7-O-β-D-glucuronide methyl ester from the ethyl acetate fraction of Lycopi Herba against pro-oxidant reactive species and low-density lipoprotein peroxidation

Figures & data

Table 1.  Extraction yields and contents of total phenolics in the extracts of Lycopi Herba.

Sample^1	Yield (%)^2	Total phenolics(μg GA eq/mg)^3
E	21.32	162.25 ± 1.24^c
H	4.06	112.83 ± 1.17^e
DCM	0.55	96.67 ± 0.98^f
EA	1	576.83 ± 2.35^a
B	3.15	227 ± 2.73^b
A	10.23	135.17 ± 3.65^d

1E, 70% ethanol extract; H, hexane fraction; DCM, dichloromethane fraction; EA, ethyl acetate fraction; B, butanol fraction; A, aqueous layer.

2Extraction yield is expressed as the percentage dry weight of Lycopi Herba.

3Each value represents the mean ± SE of triplicate measurements.

a∼f Values with different superscripts in the same column are significantly different at a p < 0.05 by the Tukey test.

Table 2.  Antioxidant activities of the extracts and 1 from Lycopi Herba as determined by the ABTS^·+ and DPPH assays.

Sample^1	TEAC^2(mM Trolox equivalent)	DPPH^2(IC50 = μg/mL)
E	0.207 ± 0.008^g	434.39 ± 13.93^d
H	0.046 ± 0.005^h	654.7 ± 15.57^b
DCM	0.425 ± 0.012^e	821.93 ± 19.5^a
EA	0.483 ± 0.025^d	129.78 ± 3.5^fg
B	0.364 ± 0.016^f	329.68 ± 5.54^e
A	0.056 ± 0.005^h	469.73 ± 14.69^c
1	1.99 ± 0.02^b	60.92 ± 1.67^h
AA	2.349 ± 0.01^a	123.58 ± 1.9^g
BHT	1.869 ± 0.03^c	156.6 ± 1.64^f

¹E, 70% ethanol extract; H, hexane fraction; DCM, dichloromethane fraction; EA, ethyl acetate fraction; B, butanol fraction; A, aqueous layer; 1, luteolin-7-O-β-D-glucuronide methyl ester.

²Each value represents mM Trolox equivalent and IC₅₀ respectively showing the mean ± SE of triplicate measurements.

^a∼hValues with different superscripts in the same column are significantly different at a p < 0.05 by the Tukey test.

Figure 1.  Structure of luteolin-7-O-β-D-glucuronide methyl ester from Lycopi Herba.

Table 3.  ROS (superoxide anion and hydroxyl radical) and RNS (nitric oxide radical and peroxynitrite) scavenging activities of the extracts and 1 from Lycopi Herba.

ROS
Sample^1	Superoxide anion^2 (IC50 = μg/mL)	Hydroxyl radical^2   (IC50 = μg/mL)
E	1,210.63 ± 113.51^bc	423.95 ± 5.27^c
H	1,818.77 ± 262.05^ab	315.08 ± 3.48^e
DCM	2,537.31 ± 790.02^a	368.51 ± 2.96^d
EA	509.29 ± 5.35^ef	16.76 ± 0.47^h
B	860.24 ± 70.95^cd	471.17 ± 7.14^b
A	1457.95 ± 129.20bc	777.97 ± 13.28^a
1	937.61 ± 14.63^cd	99.4 ± 1.49^f
AA	NA	61.34 ± 1.27^g
BHT	NA	NA
RNS
Sample	Nitric oxide radical^2  (IC50 = μg/mL)	Peroxynitrite^2(IC50 = μg/mL)
E	28.62 ± 3.3^c	31.18 ± 2.22^cd
H	65.46 ± 8.37^b	26.82 ± 1.73^e
DCM	96.77 ± 8.2^a	35.87 ± 1.17^c
EA	3.78 ± 0.15^d	4.79 ± 0.06^g
B	6.01 ± 0.58^d	13.68 ± 1.35^f
A	53.28 ± 6.36^b	77.41 ± 5.48^b
1	7.84 ± 0.16^d	7.04 ± 0.22^g
AA	9.13 ± 0.15^d	3.45 ± 0.06^g
BHT	NA	123.76 ± 2.34^a

¹E, 70% ethanol extract; H, hexane fraction; DCM, dichloromethane fraction; EA, ethyl acetate fraction; B, butanol fraction; A, aqueous layer; 1, luteolin-7-O-β-D-glucuronide methyl ester.

²Each value represents the IC₅₀, mean ± SE of triplicate measurements.

^a∼g Values with different superscripts in the same column are significantly different at a p < 0.05 by the Tukey test. NA is not active.

Table 4.  Inhibitory effect on Cu²⁺-induced LDL oxidation of the extracts and 1 from Lycopi Herba.

Sample^1	Inhibitory effect on Cu^2+-induced LDL oxidation^2
E	110.15 ± 11.25^b
H	129.67 ± 8.76^b
DCM	55.14 ± 4.51^c
EA	128.04 ± 10.35^b
B	52.44 ± 2.47^c
A	125.4 ± 13.04^b
1	71.13 ± 0.4^c
AA	167.68 ± 5.62^a
BHT	NA

¹E, 70% ethanol extract; H, hexane fraction; DCM, dichloromethane fraction; EA, ethyl acetate fraction; B, butanol fraction; A, aqueous layer; 1, luteolin-7-O-β-D-glucuronide methyl ester.

²Each value represents the IC₅₀, mean ± SE of triplicate measurements.

^a∼cValues with different superscripts in the same column are significantly different at a p < 0.05 by the Tukey test. NA is not active.

Figure 2.  The relative electrophoretic mobility (REM) of human LDL incubated with Cu²⁺, with or without extracts and one from Lycopi Herba. * LDL (120 μg/mL) was oxidized with 10 μM CuSO4 at 37°C in the presence of LH extracts for 12 h. (A): Lane 1: native LDL; Lane 2: LDL and Cu2+; Lane 3,4: LDL and Cu2+ and 5, 10 μg of E; Lanes 5,6: LDL and Cu2+ and 5, 10 μg of H; Lanes 7,8: LDL and Cu2+ and 5, 10 μg of DCM; Lanes 9,10: LDL and Cu2+ and 5, 10 μg of EA; Lanes 11,12: LDL and Cu2+ and 5, 10 μg of B; Lanes13,14: LDL and Cu2+ and 5, 10 μg of A; Lanes 15,16: LDL and Cu2+ and 5, 10 μg of luteolin-7-O-β-D-glucuronide methyl ester (#1); Lanes 17,18: LDL and Cu2+ and 5, 10 μg of AA. (B): Protection rate (%), (Each value represents the mean ± SE of triplicate measurements.).