Evidence-Based Complementary and Alternative Medicine
Volume 2015 (2015), Article ID 165457, 13 pages
http://dx.doi.org/10.1155/2015/165457

Research Article

Antioxidant and Anti-Inflammatory Activity Determination of One Hundred Kinds of Pure Chemical Compounds Using Offline and Online Screening HPLC Assay

Kwang Jin Lee, You Chang Oh, Won Kyung Cho, and Jin Yeul Ma

Korean Institute of Oriental Medicine (KIOM), KM Application Center, 70 Cheomdan-ro, Dong-gu, Daegu 701-300, Republic of Korea

Received 21 April 2015; Revised 3 July 2015; Accepted 30 July 2015

Academic Editor: Abbas A. Mahdi

Copyright © 2015 Kwang Jin Lee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

This study investigated the antioxidant activity of one hundred kinds of pure chemical compounds found within a number of natural substances and oriental medicinal herbs (OMH). Three different methods were used to evaluate the antioxidant activity of DPPH radical-scavenging activity, ABTS radical-scavenging activity, and online screening HPLC-ABTS assays. The results indicated that 17 compounds exhibited better inhibitory activity against ABTS radical than DPPH radical. The IC₅₀ rate of a more practical substance is determined, and the ABTS assay IC₅₀ values of gallic acid hydrate, (+)-catechin hydrate, caffeic acid, rutin hydrate, hyperoside, quercetin, and kaempferol compounds were 1.03 ± 0.25, 3.12 ± 0.51, 1.59 ± 0.06, 4.68 ± 1.24, 3.54 ± 0.39, 1.89 ± 0.33, and 3.70 ± 0.15 μg/mL, respectively. The ABTS assay is more sensitive to identifying the antioxidant activity since it has faster reaction kinetics and a heightened response to antioxidants. In addition, there was a very small margin of error between the results of the offline-ABTS assay and those of the online screening HPLC-ABTS assay. We also evaluated the effects of 17 compounds on the NO secretion in LPS-stimulated RAW 264.7 cells and also investigated the cytotoxicity of 17 compounds using a cell counting kit (CCK) in order to determine the optimal concentration that would provide an effective anti-inflammatory action with minimum toxicity. These results will be compiled into a database, and this method can be a powerful preselection tool for compounds intended to be studied for their potential bioactivity and antioxidant activity related to their radical-scavenging capacity.

1. Introduction

Natural substances and oriental medicinal herbs (OMH) have been traditionally administered to treat or prevent various diseases in Asia, including Korea, China, and Japan [1]. Generally OMH have very effective anticancer, anti-inflammatory, and antivirus properties [2], and researchers have reported that these natural substances also exhibit antioxidant activity. In addition, their long historical clinical practice and reliable therapeutic efficacy make them excellent sources to discover natural bioactive compounds [3]. OMH have received extensive attention for their use as drugs, functional foods, and cosmetic materials [1, 4]. An extraction solvent composed of water and ethanol is commonly used to extract the bioactive compounds from OMH with subsequent boiling and distillation to obtain useful components [5]. The chemical constituents of OMH have been shown to be composed of natural products, including triterpenes, steroids, alkaloids, flavonoids, and polysaccharides [3, 6]. During our investigation on the potential antioxidant activity of the commonly known phytochemical, one hundred kinds of pure compounds were identified. Reactive oxygen species (ROS), which originate from oxygen, are naturally produced by some enzymes as part of the metabolism within the cytoplasm [7, 8]. However, excess ROS have a fatal effect on oxygen toxicity and cellular dysfunction. In addition, excess ROS have also been linked to maladies such as cancer, stroke, Parkinson’s disease, heart disease, arteriosclerosis, infection, ageing, and autoimmune disease [8, 9]. Many studies have been carried out on the antioxidant activity that eliminates ROS to obtain more conclusive information [10, 11], and OMH have been reported to contain these kinds of antioxidants: ABTS, DPPH, and lipid peroxidation inhibition. The corresponding target compounds were used to identify the antioxidant activity, especially using DPPH or ABTS radical technique [12]. Recently, sensitive online HPLC methods (online HPLC-DPPH and online HPLC-ABTS assays) have been developed to analyse free radical-scavenging activity [5, 13]. An online system has been introduced to rapidly determine the antioxidant activity of each component in the given compounds, and online screening HPLC postcolumn assay involving DPPH or ABTS radical techniques has been developed to provide a new analysis screening technology method with which the bioactive compounds can be spectrophotometrically monitored due to the decrease in absorbance at 515 or 734 nm [14]. This new method was successfully applied to screen and identify the natural bioactivity of complex mixtures, especially for OMH [15]. In this study, we conduct DPPH and ABTS assays to screen for the antioxidant activity of one hundred kinds of pure chemical compounds, so the IC₅₀ rate of a more practical substance is determined. We also evaluated the cytotoxicity of 17 compounds, including (+)-catechin hydrate, calycosin, (3) caffeic acid, (4) curcumin, (5) eugenol, (6) ferulic acid, (7) gallic acid hydrate, (8) hyperoside, (9) kaempferol, (10) magnolol, (11) quercetin, (12) quercetin 3-beta-D-glucoside, (13) quercitrin hydrate, (14) rutin hydrate, (15) sinapic acid, (16) vanillylacetone, and (17) L-(+)-ascorbic acid, by using a CCK assay to determine the optimal concentration that would be effective for anti-inflammatory activity with a minimum toxicity [9, 16]. In addition, the results of an online HPLC-ABTS assay of some of the compounds were compared and analysed to find a more practical approach toward the use of online screening HPLC-ABTS assays to quickly pinpoint peaks in chromatograms that correspond to bioactive compounds.

2. Experimental

2.1. Reagents and Materials

One hundred kinds of pure chemical compounds were purchased from KFDA (Korea), Daejung (Korea), Sigma (USA), Chem Faces (China), TCI (Japan), ChromaDex (USA), Fluka (USA), Wako (Japan), GlycoSyn (New Zealand, USA), Santa Cruz Biotech (USA), China Lang Chem Inc. (China), and RD Chemical (USA). The following reagents were used for radical-scavenging assays: ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), DPPH (2,2-diphenyl-1-picrylhydrazyl), potassium persulfate, and trifluoroacetic acid (TFA) were purchased from Sigma (USA). The HPLC-grade methanol and acetonitrile were purchased from J. T. Baker (USA). The triple distilled water was filtered with a 0.2 m membrane filter (Advantec, Tokyo, Japan) before analysis. Materials for cell culture were obtained from Lonza (Basel, Switzerland). LPS, bovine serum albumin (BSA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies for ELISA were obtained from eBioscience (San Diego, CA, USA). The chemical structures of potentially selected compounds are shown in Figure 1.

Figure 1: Chemical structure of the superior antioxidant activity compounds.

2.2. Standard Sample Preparation

The high purity standard sample (higher than >95%) was prepared by dissolving 2 mg of each standard chemical in 20 mL of methanol and adjusting the concentration to 100 μg/mL.

2.3. Offline DPPH Assay for Antioxidant Activity Evaluation

The DPPH radical cation method [17] was modified to evaluate the free radical-scavenging effect of one hundred pure chemical compounds. The DPPH reagent was DPPH (8 mg) dissolved in MeOH (100 mL) for a solution concentration of 80 μL/mL. To determine the scavenging activity, 100 μL DPPH reagent was mixed with 100 μL of sample in a 96-well microplate and was incubated at room temperature for 30 min. After incubation, the absorbance was measured 514 nm using an ELISA reader (TECAN, Gröding, Austria), and 100% methanol was used as a control. The DPPH scavenging effect was measured using the following formula:

The IC₅₀ DPPH values (the concentration of sample required for inhibition of 50% of DPPH radicals) were obtained through extrapolation from regression analysis. The antioxidant was evaluated based on this IC₅₀ value.

2.4. Offline-ABTS Assay for Antioxidant Activity Evaluation

The ABTS radical cation method [17] was modified to evaluate the free radical-scavenging effect of one hundred pure chemical compounds. The ABTS reagent was prepared by mixing 5 mL of 7 mM ABTS with 88 μL of 140 mM potassium persulfate. The mixture was then kept in the dark at room temperature for 16 h to allow free radical generation and was then diluted with water (1 : 44, v/v). To determine the scavenging activity, 100 μL ABTS reagent was mixed with 100 μL of sample in a 96-well microplate and was incubated at room temperature for 6 min. After incubation, the absorbance was measured 734 nm using an ELISA reader (TECAN, Gröding, Austria), and 100% methanol was used as a control. The ABTS scavenging effect was measured using the following formula:

The IC₅₀ ABTS values (the concentration of sample required for inhibition of 50% of ABTS radicals) were obtained through extrapolation from regression analysis. The antioxidant activity was evaluated based on this IC₅₀ value.

2.5. Online Screening HPLC-ABTS Analysis

The online radical-scavenging activity of one hundred kinds of pure standard compounds was determined using the ABTS assay modifying the methods used by Stewart et al. [18]. A 2 mM ABTS stock solution containing 3.5 mM potassium persulfate was prepared and was kept in the dark at room temperature for 16 h to allow the completion of radical generation and was then diluted with water (1 : 29, v/v). Each pure sample was injected into a Dionex Ultimate 3000 HPLC system (Thromo Scientific). The chromatographic columns used in this experiment are commercially available; this is obtained from RS-tech (0.46 × 25 cm, 5 μm, C₁₈, Daejeon, Korea). The injection volume was 10 μL, and the flow rate of the mobile phase was 1.0 mL/min. The wavelength of the UV detector was fixed at 203, 254, and 320 nm. The composition of the mobile phases was as follows: A, water/trifluoroacetic acid = 99.9/0.1, vol%, and B, acetonitrile 100%. The run time was 70 min and the solvent program was the linear gradient method (90 : 10–60 : 40, A : B vol%). Figure 2 is a schematic showing the online coupling of HPLC to a DAD (diode array detector) and the continuous flow ABTS assay. Online HPLC then arrived at a “T” piece, where ABTS was added. The ABTS flow rate was 0.5 mL/min, delivered by a Dionex Ultimate 3000 Pump. After mixing through a 1 mL loop which was maintained at 40°C, the absorbance was measured by a VIS detector at 734 nm. Data were analyzed using Chromeleon 7 software.

Figure 2: Schematic of online screening HPLC-ABTS system.

2.6. Cell Culture and Drug Treatment

RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in RPMI 1640 medium containing 10% FBS and 100 U/mL of antibiotics sulfate. The cells were incubated in humidified 5% CO₂ atmosphere at 37°C. To stimulate the cells, the medium was changed with fresh RPMI 1640 medium and LPS (200 ng/mL) [18, 19] was added in the presence or absence of 17 compounds (1, 5, and 10 μg/mL) for 24 h.

2.7. Cell Viability Assay

Cytotoxicity was analyzed using CCK (Dojindo, Japan). 17 compounds were added to the cells and incubated for 24 hours at 37°C with 5% CO₂. 10 μL CCK solutions were added to each well and the cells were incubated for another 1 h. Then the optical density was read at 450 nm using an ELISA reader (Infinite M200, Tecan, Männedorf, Switzerland).

2.8. Measurement of NO Production

NO production was analyzed by measuring the nitrite in the supernatants of cultured macrophage cells. The cells were pretreated with 17 compounds and stimulated with LPS for 24 hours. The supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) and incubated at room temperature (RT) for 5 min [19]. The absorbance at 570 nm was read.

2.9. Inflammatory Cytokine Determination

Cells were seeded at a density of 5 × 10⁵ cells/mL in 24-well culture plates and pretreated with three concentrations of 17 compounds for 1 hour before LPS stimulation. ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with capture antibody diluted in coating buffer (0.1 M carbonate, pH 9.5) and then washed five times with phosphate-buffered saline (PBS) containing 0.05% Tween 20. The nonspecific protein-binding sites were blocked with assay diluent buffer (PBS containing 10% FBS, pH 7.0) for more than 1 hour. Promptly, samples and standards were added to the wells. After overnight of incubation at 4°C, the working detector solution (biotinylated detection antibody and streptavidin-HRP reagent) was added and incubated for 1 hour. Subsequently, substrate solution (tetramethylbenzidine) was added to the wells and incubated for 30 min in darkness before the reaction was stopped with stop solution (2 N H₃PO₄). The optical density was read at 450 nm [19].

2.10. Statistical Analysis

The results are expressed as mean ± SD values for the number ( times) of experiments. Statistical significance was compared for each treated group with the control and determined by Student’s -tests. Each experiment was repeated at least three times to yield comparable results. Values with and were considered significant.

3. Result and Discussion

Several researches have revealed that a variety of natural and chemical compounds from natural substance crops, fruits, vegetables, and oriental medicinal herbs (OMH) have shown high antioxidant activity after the extraction and purification processes [3]. In addition, various methods have been used to determine the antioxidant activity of natural substance crops, foods, and plant products [1, 4]. The present study used three different methods to evaluate the antioxidant activity consisting of DPPH radical-scavenging activity, ABTS radical-scavenging activity, and online screening HPLC-ABTS assays. Therefore, this work documented for the first time a comparison of the antioxidant activities of one hundred kinds of pure chemical compounds.

3.1. Offline DPPH and ABTS Assay

Antioxidant activity reportedly has an effect on various different bioactivities (whitening, anti-inflammation, and high blood pressure). The antioxidant activity of natural substances and OMH has been widely studied, and, thus, this study identifies the antioxidant activity of standard substances that have originated from various OMH in terms of their DPPH radical-scavenging activity and ABTS radical-scavenging activity. The DPPH and ABTS radical-scavenging assays offer a redox-functioned proton ion for unstable free radicals and play a critical role in stabilizing detrimental free radicals in the human body. This is generally achieved by taking advantage of the fact that unstable violet DPPH and ABTS free radicals transform to stable yellow DPPH free radicals by accepting a hydrogen ion from antioxidants. In terms of the antioxidant activity, the ability to eliminate hydroxyl radicals or superoxide radicals through a physiologic action or through oxidation is evaluated, and a high index indicates a strong antioxidant activity. Table 1 provides the results of the DPPH and ABTS radical scavenging in 100 ppm for one hundred kinds of pure standard compounds used in this study. 17 compounds among the one hundred kinds of pure standard compounds ( (+)-catechin hydrate, calycosin, (3) caffeic acid, (4) curcumin, (5) eugenol, (6) ferulic acid, (7) gallic acid hydrate, (8) hyperoside, (9) kaempferol, (10) magnolol, (11) quercetin, (12) quercetin 3-beta-D-glucoside, (13) quercitrin, (14) rutin hydrate, (15) sinapic acid, (16) vanillylacetone, and (17) L-(+)-ascorbic acid) have an antioxidant activity of over 90%. Table 2 shows the IC₅₀ rate of compounds with a strong antioxidant activity. The ABTS radical-scavenging measurement method, which is commonly used to evaluate the antioxidant activity, takes advantage of the fact that ABTS free radicals become stable by accepting a hydrogen ion from the antioxidant, losing their blue colors. Moreover, in the ABTS assay as well as in the DPPH assay, when antioxidant activity occurs, the ability to eliminate hydroxyl radicals or superoxide radicals through physiologic action or oxidation is evaluated with a high index indicating a strong antioxidant activity. Each of the DPPH and ABTS are compounds that have a proton free radical, with a characteristic absorption that decreases significantly upon exposure to proton radical scavengers. DPPH and ABTS radical-scavenging through antioxidant activity are well known to be attributable to their hydrogen-donating ability (Tables 1 and 2). The concentration of these compounds required to inhibit 50% of the radical-scavenging effect (IC₅₀) has been determined by testing a series of concentrations. In particular, the sample with (+)-catechin hydrate, caffeic acid, eugenol, gallic acid hydrate, hyperoside, quercetin, vanillylacetone, and L-(+)-ascorbic acid compounds showed the strongest activity. In addition, the 17 compounds showed better inhibitory activity against ABTS radical than the DPPH radicals. That is, the ABTS assay is more sensitive in identifying antioxidant activity because of the faster reaction kinetics, and its response to antioxidants is higher. Consequently, this study shows that the ABTS assay IC₅₀ values of gallic acid hydrate, (+)-catechin hydrate, caffeic acid, rutin hydrate, hyperoside, quercetin, and kaempferol compounds were 1.03 ± 0.25, 3.12 ± 0.51, 1.59 ± 0.06, 4.68 ± 1.24, 3.54 ± 0.39, 1.89 ± 0.33, and 3.70 ± 0.15 μg/mL, respectively.

Table 1: Free radical-scavenging capacities of antioxidant activity available measured with DPPH and ABTS assay on microwell plate.

Number
Compounds names
Compounds
purchased
Concentration
M (mol/L)
Radical scavenging (%)

DPPH
ABTS

1
Albiflorin
Wako
208.13
−0.15 ± 0.39
0.14 ± 5.27

2
Alisol A
Wako
203.79
−0.47 ± 0.71
12.92 ± 0.86

3
Alisol B
Wako
211.55
−1.66 ± 0.23
12.78 ± 1.35

4
Amygdalin
KFDA
218.61
−0.85 ± 0.67
12.31 ± 0.03

5
Anthraquinone
Wako
480.28
0.12 ± 0.70
0.86 ± 5.12

6
Atractylenolide iii
Chem Faces
402.71
−2.07 ± 0.53
1.34 ± 8.29

7
Aucubin
Wako
288.74
−1.64 ± 0.79
1.22 ± 9.74

8
Baicalein
KFDA
370.04
95.84 ± 0.15
99.37 ± 0.12

9
Benzoic acid
Sigma
818.87
1.88 ± 0.42
3.80 ± 0.26

10
Berberine
Chem Faces
297.30
0.64 ± 0.43
84.68 ± 2.55

11
Berberine HCl
KFDA
268.95
−0.30 ± 0.30
8.74 ± 8.38

12
Caffeic acid
Sigma
555.06
95.91 ± 0.16
99.66 ± 0.24

13
Calycosin
Chem Faces
351.79
64.51 ± 0.59
99.19 ± 0.05

14
Catalpol
Wako
275.99
−2.55 ± 0.47
1.12 ± 9.62

15
Chrysin
Sigma
393.33
0.45 ± 0.53
99.13 ± 0.06

16
Cimifugin
Chem Faces
326.46
−0.89 ± 0.21
14.39 ± 1.81

17
Cinnamyl alcohol
Sigma
745.27
−0.03 ± 0.37
15.18 ± 0.75

18
cis-Inositol
Sigma
555.06
0.15 ± 0.26
0.54 ± 1.22

19
Costunolide
Sigma
430.44
2.86 ± 0.17
20.13 ± 8.74

20
Crocin
Sigma
102.36
24.43 ± 1.28
47.73 ± 0.67

21
Curcumin
Sigma
271.46
97.50 ± 0.63
99.97 ± 0.16

22
(+)-Catechin hydrate
TCI
344.51
94.50 ± 0.16
99.15 ± 0.06

23
1,8-Dihydroxy-3-methylanthraquinone
Sigma
393.33
−0.94 ± 0.54
2.09 ± 2.60

24
D-(−)-Lactic acid
Sigma
1110.12
2.51 ± 2.40
28.29 ± 0.74

25
D-(+)-Chiro-inositol
Sigma
555.06
0.49 ± 0.33
0.21 ± 0.45

26
Daidzein
Wako
393.33
0.52 ± 0.61
99.49 ± 0.49

27
Decursin
KFDA
304.54
−3.01 ± 0.91
0.97 ± 2.75

28
Decursinol
Chem Faces
406.07
−1.59 ± 1.03
15.12 ± 1.81

29
Dioscin
Sigma
115.07
−2.93 ± 1.30
0.43 ± 2.75

30
Diosgenin
Sigma
241.18
−1.38 ± 0.70
1.96 ± 9.24

31
D-Pinitol
Sigma
514.99
−2.27 ± 0.33
12.72 ± 1.85

32
6,7-Dimethylesculetin
RD Chemical
484.99
−2.74 ± 0.54
1.31 ± 0.21

33
(−)-Epicatechin
Sigma
344.51
94.51 ± 0.41
99.61 ± 0.16

34
(−)-Epigallocatechin gallate
Sigma
218.16
95.69 ± 0.14
99.51 ± 0.24

35
Eleutheroside B
Wako
268.55
−2.04 ± 1.02
1.97 ± 2.85

36
Emodin
TCI
370.04
2.20 ± 1.16
91.27 ± 1.39

37
Ephedrine-HCl
KFDA
495.81
−2.11 ± 1.52
0.00 ± 0.12

38
Ergosterol
Chem Faces
252.11
−1.65 ± 1.34
0.55 ± 9.46

39
Eugenol
Sigma
609.01
93.72 ± 0.12
99.91 ± 0.55

40
Evodiamine
KFDA
329.64
4.21 ± 1.55
40.76 ± 2.09

41
Ferulic acid
Sigma
514.99
95.12 ± 0.24
98.96 ± 0.27

42
Gallic acid hydrate
TCI
587.82
95.56 ± 0.03
101.30 ± 2.12

43
Geniposide
Chem Faces
257.49
−1.45 ± 0.90
1.22 ± 9.49

44
Genistein
TCI
370.04
−1.52 ± 0.30
98.50 ± 0.48

45
Genistin
Wako
231.28
2.23 ± 0.64
100.65 ± 0.03

46
Geraniol
Sigma
112.49
−2.43 ± 1.67
14.94 ± 0.95

47
Glabridin
Wako
308.29
42.20 ± 0.88
100.58 ± 0.04

48
Glimepiride
Sigma
203.82
−1.33 ± 0.63
7.84 ± 2.30

49
Glycyrrhetic acid
TCI
212.46
0.66 ± 0.65
10.23 ± 0.62

50
Glycyrrhizin
TCI
121.52
1.59 ± 1.96
12.36 ± 1.15

51
Gomisin A
KFDA
240.12
1.80 ± 0.28
1.74 ± 0.22

52
Gomisin N
KFDA
249.71
0.45 ± 0.51
3.80 ± 0.21

53
Hesperidin
KFDA
202.23
31.84 ± 0.37
100.21 ± 0.01

54
Hyperoside
Chem Faces
215.34
93.16 ± 0.25
99.62 ± 0.20

55
2′-Hydroxy-4′-methoxy-acetophenone
Sigma
601.76
−2.81 ± 0.28
99.73 ± 1.04

56
Icariin
TCI
147.78
3.82 ± 1.08
14.60 ± 0.12

57
Imperatorin
Chem Faces
369.99
−0.71 ± 2.31
0.74 ± 9.51

58
Isoimperatorin
Santa Cruz Biotech
369.99
0.27 ± 0.22
3.06 ± 2.17

59
Jujuboside A
Chem Faces
82.83
−1.63 ± 0.35
0.26 ± 9.74

60
Kaempferol
Chem Faces
349.36
95.02 ± 0.22
99.84 ± 0.41

61
Liquiritigenin
Chem Faces
390.24
12.26 ± 0.86
0.12 ± 0.68

62
Loganin
KFDA
256.16
2.62 ± 1.28
5.80 ± 0.62

63
Magnolol
KFDA
375.47
54.50 ± 0.12
77.74 ± 1.06

64
Mevinolin
Sigma
247.19
−0.26 ± 0.07
2.78 ± 0.69

65
Morroniside
China Lang Chem Inc.
246.08
7.11 ± 0.58
19.62 ± 1.83

66
Naringin
Sigma
172.26
3.05 ± 0.37
100.36 ± 0.05

67
Nodakenin
Chem Faces
244.86
0.68 ± 0.45
15.70 ± 1.92

68
Oleanolic acid
Wako
218.96
−0.37 ± 0.54
0.00 ± 0.12

69
Ononin
Sigma
232.34
2.23 ± 1.58
22.91 ± 1.89

70
Oxymatrine
Chem Faces
378.26
−1.63 ± 0.17
0.72 ± 8.85

71
Oxypeucedanin
Chem Faces
349.31
−0.11 ± 0.27
19.10 ± 2.38

72
Paeoniflorin
TCI
208.13
4.29 ± 1.43
22.03 ± 0.91

73
Paeonol
Sigma
601.79
0.99 ± 2.21
19.79 ± 2.35

74
Palmatine chloride hydrate
Sigma
257.82
2.57 ± 2.42
84.15 ± 2.43

75
Palmatine
Chem Faces
292.05
0.12 ± 0.23
68.05 ± 3.63

76
p-Coumaric acid
Sigma
609.16
8.89 ± 0.04
38.52 ± 0.84

77
Poncirin
KFDA
168.19
−1.44 ± 1.27
84.20 ± 3.66

78
Puerarin
Wako
240.17
9.89 ± 0.16
100.62 ± 0.06

79
Quercetin
Sigma
330.86
96.02 ± 0.08
100.18 ± 0.06

80
Quercetin 3--D-glucoside
Sigma
215.34
94.43 ± 0.02
99.94 ± 0.06

81
Quercitrin hydrate
Sigma
223.03
93.32 ± 0.04
99.12 ± 0.14

82
Rutaecarpine
KFDA
348.04
−1.41 ± 0.60
97.23 ± 0.63

83
Rutin hydrate
Sigma
163.79
93.57 ± 0.13
100.10 ± 0.02

84
Saikosaponin a
KFDA
128.04
−0.67 ± 0.79
16.17 ± 1.62

85
Salicylaldehyde
Sigma
818.87
2.07 ± 0.04
100.36 ± 0.05

86
Schisandrin
KFDA
231.21
−2.39 ± 0.89
10.29 ± 2.43

87
Sennoside A
KFDA
115.91
−3.09 ± 0.64
87.86 ± 3.21

88
Sequoyitol
GlycoSyn
514.99
−3.20 ± 1.37
10.28 ± 2.07

89
Sinapic acid
Fluka
445.99
94.84 ± 0.33
99.99 ± 0.23

90
Spinosin
Chem Faces
164.33
−0.96 ± 2.26
80.16 ± 2.20

91
Tetrandrine
Fluka
160.58
60.83 ± 2.11
100.28 ± 0.06

92
trans-Cinnamaldehyde
Sigma
756.66
2.83 ± 0.69
19.00 ± 1.37

93
trans-Cinnamic acid
Sigma
674.95
0.19 ± 0.15
0.02 ± 9.45

94
Uric acid
Sigma
594.85
40.37 ± 1.97
98.33 ± 0.31

95
Vanillylacetone
Sigma
514.85
93.78 ± 0.06
99.92 ± 0.24

96
Wogonin
KFDA
351.79
1.02 ± 0.51
100.57 ± 0.13

97
Wogonoside
Chem Faces
217.21
10.84 ± 0.10
98.62 ± 0.32

98
Ziyuglycoside I
Chem Faces
130.38
−1.71 ± 1.00
0.01 ± 9.85

99
Z-Ligustilide
Chem Faces
531.29
1.92 ± 0.76
68.69 ± 2.14

100
Ascorbic acid (Vitamin C)
Daejung
567.79
99.56 ± 0.89
99.89 ± 0.04

Table 2: Antioxidant activity of 17 compounds with offline DPPH and ABTS IC₅₀ assay.

Number
Name
Radical- scavenging IC₅₀

(g/mL)

DPPH
ABTS

1
(+)-Catechin hydrate
5.25 ± 0.31
3.12 ± 0.51

2
Calycosin
61.88 ± 1.19
33.21 ± 3.59

3
Caffeic acid
4.50 ± 0.30
1.59 ± 0.06

4
Curcumin
8.89 ± 0.24
4.99 ± 0.45

5
Eugenol
5.22 ± 0.25
3.22 ± 0.45

6
Ferulic acid
9.49 ± 0.21
1.99 ± 0.12

7
Gallic acid hydrate
1.56 ± 0.38
1.03 ± 0.25

8
Hyperoside
5.44 ± 0.36
3.54 ± 0.39

9
Kaempferol
7.78 ± 0.30
3.70 ± 0.15

10
Magnolol
85.57 ± 1.40
8.37 ± 0.56

11
Quercetin
2.66 ± 0.24
1.89 ± 0.33

12
Quercetin 3--D-glucoside
7.05 ± 0.59
3.59 ± 0.89

13
Quercitrin hydrate
7.55 ± 0.77
4.23 ± 0.84

14
Rutin hydrate
9.72 ± 1.06
4.68 ± 1.24

15
Sinapic acid
8.26 ± 0.41
5.36 ± 0.85

16
Vanillylacetone
5.69 ± 0.00
3.45 ± 0.05

17
Ascorbic acid (Vitamin C)
3.65 ± 0.23
2.65 ± 0.46

3.2. Online HPLC-ABTS Assay Analysis

The most popular approach utilises a relatively stable, coloured radical solution of DPPH or ABTS, which is added postcolumn to the HPLC flow. Drug, food, functional material, and plant and OMH samples are evaluated for their antioxidant capacities according to a variety of antioxidant activity test methods, such as those for ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20]. The HPLC analyses react postcolumn with the ABTS, and the reduction is detected as a negative peak by a VIS absorbance detector at 734 nm. The ABTS radical is much more water soluble than DPPH [13], so ABTS better shows the details of an online HPLC-ABTS assay system that analysed the 17 given compounds (Figures 3(a) and 3(b)). Combined UV (positive signals) and ABTS quenching (negative signals) chromatograms of the 17 compounds ( gallic acid hydrate : 5.46 min, (+)-catechin hydrate : 9.26 min, (3) caffeic acid : 11.12 min, (4) ferulic acid : 15.87 min, (5) rutin hydrate : 15.99 min, (6) sinapic acid : 16.15 min, (7) hyperoside : 16.49 min, (8) quercetin 3-beta-D-glucoside : 16.82 min, (9) vanillylacetone : 17.92 min, (10) quercitrin : 18.90 min, (11) calycosin : 23.81 min, (12) quercetin : 24.31 min, (13) kaempferol : 28.37 min, (14) eugenol : 28.98 min, (15) curcumin : 40.20 min, (16) magnolol : 43.98 min, and (17) L-(+)-ascorbic acid) (not detected; L-(+)-ascorbic acid) (each concentration 100 ppm) are presented in Figure 3(a). Of these, seven compounds that showed excellent activity were further analysed. Several eluted substances were detected in the 7 compounds, including gallic acid hydrate (210 nm), (+)-catechin hydrate (210 nm), (3) caffeic acid (320 nm), (4) rutin hydrate (210 nm), (5) hyperoside (210 nm), (6) quercetin (210 nm), and (7) kaempferol (254 nm), which are observed as a positive signal on the UV detector (210, 254, and 320 nm). The retention times () of gallic acid hydrate ( 5.62 min), (+)-catechin hydrate ( 9.46 min), (3) caffeic acid ( 11.12 min), (4) rutin hydrate ( 15.86 min), (5) hyperoside ( 16.26 min), (6) quercetin ( 23.58 min), and (7) kaempferol ( 28.30 min) are reported in Figure 3(b). The other compounds exhibited a hydrogen-donating capacity (negative peak) towards the ABTS radical at the applied concentration. These results therefore reveal that this method can be applied for quick screening of antioxidant activity or, more precisely, of radical-scavenging activity (Table 3). This work confirms the feasibility of assessing the bioactivity of specific phytochemicals by using an online screening HPLC-ABTS assay. This method was successfully applied to screen and identify the antioxidant activity of natural substances and OMH complex mixtures [5, 15]. The results show the shape of the chromatogram by the competitive adsorption and desorption. In addition, the screening methods for the rapid activity can provide useful information in basic research on natural products chemistry and isolation analysis. It is considered that the data will only be valuable in engineering and also very useful as functional materials and pharmaceutical materials in commercial processes.

Table 3: Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay.

Compounds
UV wavelength (nm)
Retention time (min)
Peak area (mAu)
Positive
S.D.
Negative
S.D.

Positive (average)
Negative (average)

Gallic acid hydrate
210
5.623
72.116
51.624
0.054
0.405

(+)-Catechin hydrate
210
9.463
75.974
57.981
0.076
0.328

Caffeic acid
320
11.123
108.475
57.808
0.048
0.433

Rutin hydrate
210
15.860
51.185
13.241
0.393
0.023

Hyperoside
210
16.263
80.346
15.631
0.017
0.213

Quercetin
210
23.583
109.672
22.155
0.101
0.067

Kaempferol
254
28.303
56.806
30.651
0.143
0.071

(a)

(b)

Figure 3: Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds, gallic acid hydrate, (+)-catechin hydrate, (3) caffeic acid, (4) ferulic acid, (5) rutin hydrate, (6) sinapic acid, (7) hyperoside, (8) quercetin 3-β-D-glucoside, (9) vanillylacetone, (10) quercitrin hydrate, (11) calycosin, (12) quercetin, (13) kaempferol, (14) eugenol, (15) curcumin, (16) magnolol, and (17) ascorbic acid (not detected); (b) simultaneous analysis of 7 compounds).

3.3. Anti-Inflammatory Activity Screening

3.3.1. Effect of 17 Compounds on RAW 264.7 Cell Viability

We evaluated the cytotoxicity of the 17 compounds by using CCK to determine the optimal concentration that would be effective in providing anti-inflammatory activity with a minimum toxicity. As shown in Figure 4(a), kaempferol, quercetin, and curcumin show toxicity at a concentration of 10 μg/mL. Also, quercetin 3-beta-D-glucoside has a strong toxicity on macrophage viability at 5 μg/mL or more. Vanillylacetone, hyperoside, gallic acid hydrate, sinapic acid, rutin hydrate, ferulic acid, (+)-catechin hydrate, ascorbic acid, calycosin, caffeic acid, magnolol, quercitrin hydrate, and eugenol did not affect cell viability up to 10 μg/mL, indicating that these 13 compounds are not toxic to cells.

(a)

(b)

Figure 4: Effect of 17 compounds on (a) cell viability and LPS-induced (b) NO production in RAW 264.7 cells. RAW 264.7 cells were pretreated with 17 compounds for 1 hour before incubation with LPS for 24 hours. (a) Cytotoxicity was evaluated by a CCK. (b) The culture supernatant was analyzed for nitrite production. As a control, the cells were incubated with vehicle alone. Data shows mean ± SE values of triplicate determination from independent experiments. and were calculated from comparing with LPS-stimulation value.

3.3.2. Effect of the 17 Compounds on NO Production in LPS-Stimulated RAW 264.7 Macrophages

We evaluated the effects of 17 compounds on NO secretion in LPS-stimulated RAW 264.7 cells. The cells were pretreated with 17 compounds at concentrations of 1, 5, and 10 μg/mL prior to LPS stimulation, and NO production was also measured. We employed 10 μM dexamethasone as a positive control, since it is widely used as an anti-inflammatory agent. As shown in Figure 4(b), vanillylacetone, gallic acid hydrate, kaempferol, quercetin, magnolol, and curcumin exhibit a strong inhibitory effect on NO secretion upon LPS stimulation. The inhibitory effects of 10 μg/mL kaempferol, quercetin, and curcumin on NO production were a result of their cytotoxicity. However, kaempferol, quercetin, and curcumin exert effective inhibition at concentrations of 1 and 5 μg/mL. In particular, magnolol strongly inhibited NO production in a dose-dependent manner without toxicity. Hyperoside, sinapic acid, rutin hydrate, ferulic acid, (+)-catechin hydrate, ascorbic acid, calycosin, caffeic acid, quercitrin hydrate, eugenol, and quercetin 3-beta-D-glucoside do not show remarkable suppressive effects.

3.3.3. Effect of the 17 Compounds on LPS-Induced Inflammatory Cytokines Production

Next, we investigated the inhibitory effect of the 17 compounds on the production of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, which are the other parameters of the inflammation. Gallic acid hydrate exerts an inhibitory effect on the TNF-α cytokine production at all concentrations in a dose-dependent manner. In addition, 5 μg/mL kaempferol and 10 μg/mL magnolol show a strong inhibitory effect (Figure 5(a)). As shown in Figure 5(b), vanillylacetone, gallic acid hydrate, kaempferol, and quercetin significantly inhibited IL-6 cytokine secretion in a statistically significant, dose-dependent manner. In addition, all of the compounds showed an inhibitory effect on IL-1β cytokine production. Gallic acid hydrate, kaempferol, quercetin, and magnolol were especially strong inhibitors of IL-1β cytokine production in a dose-dependent manner (Figure 5(c)).

Figure 5: Effect of 17 compounds on the production of (a) TNF-α, (b) IL-6, and (c) IL-1β cytokine in macrophages. Cells were pretreated with 17 compounds for 1 hour before being incubated with LPS for 24 hours. Production of cytokines was measured by ELISA. Data shows mean ± SE values of triplicate determinations from three independent experiments. and were calculated from comparing with LPS-stimulation value.

4. Conclusions

This study provides a comparison of the free radical scavengers in the one hundred kinds of pure chemical compounds through an offline DPPH radical-scavenging activity assay, ABTS radical-scavenging activity assay, and an online screening HPLC-ABTS assay. Here, the IC₅₀ rate of a more practical substance is determined. The results indicate that the ABTS assay IC₅₀ values of gallic acid hydrate, (+)-catechin hydrate, caffeic acid, rutin hydrate, hyperoside, quercetin, and kaempferol compounds were 1.03 ± 0.25, 3.12 ± 0.51, 1.59 ± 0.06, 4.68 ± 1.24, 3.54 ± 0.39, 1.89 ± 0.33, and 3.70 ± 0.15 μg/mL, respectively. This testing methodology provided a useful tool to focus efforts on chemically active radical-scavenging compounds with high kinetic rates and allowed quick gathering of useful information related to the molecular compounds in terms of their antioxidant activity potential. In addition, there was a very small margin of error between the results of the offline-ABTS assay and those of the online screening HPLC-ABTS assay. We also evaluated the effects of 17 compounds on NO secretion in LPS-stimulated RAW 264.7 cells and the cytotoxicity of the 17 compounds using CCK to determine the optimal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicity. These results will be compiled into a database, and this method can therefore be a powerful preselection tool for compounds intended to be studied for their potential bioactivity and antioxidant activity related to their radical-scavenging capacity.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This study was achieved at KM Application Center, KIOM. The authors also acknowledge the support from the Study on Drug Efficacy Enhancement Using Bioconversion for Herbal Medicines (K15280) project.

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