Correlation between Solid Content and Antioxidant Activities in Umbelliferae Salad Plants.

The aim of this study is to evaluate the antioxidant properties of 70% methanolic extracts and the correlation between several antioxidant activities in selected Umbelliferae plants, based on total phenolic content (TPC) and total flavonoid content (TFC). For Umbelliferae plants extracts, the IC50 of DPPH radical (100 μM) quenching activities for extract, TPC, and TFC were 39∼179 μg dry weight (DW)/mL, 14.08∼38.11 μg TPC/mL, and 0.36∼1.51 μg TFC/mL, respectively. The oxygen radical absorbance capacity (ORAC) of extracts ranged from 11.44 to 42.88 mg Trolox equivalent (TE)/g DW extract, whereas ORAC for TPC and TFC was 47.40∼240.19 mg TE/g and 0.72∼11.22 g TE/g, respectively. The TPC had a superior linear correlation (r2=0.817) with 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) values. Of the 14 Umbelliferae plant extracts, Sanicula rubiflora, Sanicula chinensis, Torilis japonica, Torilis scabra, and Angelica fallax showed the strongest antioxidant activity.

INTRODUCTION

Umbelliferae (Apiaceae) is widely distributed throughout the world, from polar regions to subtropical regions, and is particularly abundant in temperate zones of the northern hemisphere (Sayed-Ahmad et al., 2017). Approximately 3,780 species in Umbelliferae include caraway, carrot, celery, chervil, coriander, cumin, dill, fennel, hemlock, parsley, parsnip, and sea holly, which are important in the production and consumption in food industry (Cherng et al., 2008). Various Umbelliferae plants, including Angelica dahurica (Fisch.) Benth. & Kook.f., Angelica decursiva (Miq.) Franch. & Sav., Bupleurum chinense DC., Cnidium monnieri (L.) Cusson, and Oenanthe javanica (Blume) DC inhabit in Korea (Wiart, 2012).

Traditional Korean food is primarily prepared using a variety of vegetables; in particular, this includes the fermented food Kimchi and the non-fermented salad Namul. Angelica decursiva, Bupleurum longiradiatum, Coriandrum sativum, Cryptotaenia japonica, Daucus carota subsp. sativus, Ostericum koreanum, and Sanicula chinensis belong to the family Umbelliferae, and have been used in various Kimchis and Namuls. In addition, the several Umbelliferae plants contain the potent anti-oxidants carotenoids, flavonoids, and various polyphenols, which have several physiological activities (Lee et al., 2011a; Sayed-Ahmad et al., 2017).

Excessive amounts of extracellular and intracellular reactive oxygen species (ROS) are produced during metabolism and can modify DNA/RNA in the cell; these modifications can lead to mutations or cancer. ROSs have also been implicated in the early stages as well as in the progression of diseases (Pham-Huy et al., 2008). To maintain optimal ROS levels is considered an important factor for maintaining health and preventing diseases. Many investigations have been carried out to understand the antioxidant efficacy of several plant extracts for removing ROS (Mahdi-Pour et al., 2012; Fernandes et al., 2016). Most antioxidative studies are based on total phenolic content (TPC) and total flavonoid content (TFC) in plant extracts, and the scavenging activities of free radicals, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis (3-ethylbenzothiazoline 6-sulfonic acid) (ABTS), oxygen radical absorbance capacity (ORAC), and hydroxyl oxygen radical averting capacity (HORAC). However, there has been very few reports on the effects of TPC and TFC on the antioxidant activity of extracts (Mahdi-Pour et al., 2012; Fernandes et al., 2016).

Antioxidant activity of plants is mostly due to the amount and type of polyphenolic and flavonoid compounds contained in them (Prior et al., 1998; Di Majo et al., 2008). The specificity of antioxidant activity in plant resources is determined by the amount and type of polyphenolic and flavonoid compounds due to climate and soil conditions in certain habitats (Moore et al., 2006; Liu et al., 2016). Therefore, it is imperative to show that the antioxidant activity of a plant extract depends on the amount of polyphenols or flavonoids.

The purpose of this study is to measure the amount of TPC and TFC and to evaluate the antioxidant activities of Korean 14 Umbelliferae plants by a correlation between TPC (or TFC) and the antioxidant activities. These results could be applied to develop health enhancing foods and cosmetics containing antioxidative substances from Umbelliferae plants.

MATERIALS AND METHODS

Chemicals and reagents

Dimethyl sulfoxide (DMSO), Folin-Ciocalteu reagent, DPPH, ascorbic acid, pyrogallol, ABTS, 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and fluorescein disodium salt were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ethanol and other reagents were used as first grade reagents.

Plant materials

The extracts of 14 Umbelliferae plants were obtained from the Korea Plant Extract Bank, Daejeon, Korea. For preparation of extracts, plants were washed, freeze dried, and crushed. Crushed plants were extracted with 70% methanol (in water). Extracts were filtered through Whatman No. 1 filter paper and concentrated using an evaporator under reduced pressure. Extracts were re-dissolved in DMSO to a concentration of 100 mg/mL, stored at −20℃, and used as a stock solution.

Measurement of TPC and TFC

TPC was determined using the Folin-Ciocalteu method (Oh et al., 2004), with modifications. Each Umbelliferae extract was dissolved in 1 mL of 1 N Folin-Ciocalteu reagent and was incubated for 5 min. Then, 2 mL of 20% (w/v) Na2CO3 was added to the mixture. After 10 min at room temperature, the mixture was centrifuged at 15,000 g for 1 min, and the absorbance of the supernatant was measured on 765 nm using a spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). Gallic acid was used to plot a standard calibration curve. The TPC content of extracts was expressed as mg of gallic acid equivalents (GAE)/g dry weight (DW).

Analysis of TFC was performed using the colorimetric Dowd method (Zhishen et al., 1999), with modifications. Extracts were added to a test tube containing distilled water (1.25 mL) and 5% (w/v) NaNO2 (75 mL), and the mixture was incubated for 5 min. Then, 0.15 mL of 10% (w/v) AlCl3·6H2O was added to the mixture. After 6 min at room temperature, 0.5 mL of 1 M NaOH was added, and the mixture was diluted with 0.275 mL distilled water. The absorbance of the mixture was measured immediately at 510 nm using a UV-spectrophotometer (Libra S22, Biochrom Ltd.). The TFC content of extracts was expressed as mg of catechin equivalents (CE)/g DW.

Antioxidant assay

The DPPH radical scavenging activity of Umbelliferae extracts was measured by Lee and collegues’ method with modifications (Lee et al., 2005). Methanolic extracts (0.2 mL) were mixed with 4 mL of methanol, and then methanolic solution of DPPH (1 mM, 0.5 mL) was added. This mixture was vortexed for 15 s, incubated at room temperature for 30 min, and the absorbance was measured on 517 nm using UV-spectrophotometry (Libra S22, Biochrom Ltd.).

The reducing capacity of Umbelliferae extracts was determined by Fe3+ reduction (Oyaizu, 1986). Extracts (1 mg/mL) in distilled water were first mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) K3Fe(CN)6. The mixture were then incubated at 50℃ for 20 min. Next, 2.5 mL of 10% (w/v) trichloroacetic acid was added and the mixture was centrifuged at 2,090 g for 10 min. Supernatant solution (2.5 mL) was then added to 2.5 mL of distilled water and 0.5 mL of 0.1% (w/v) FeCl3. The absorbance of the mixture was measured at 700 nm using UV-spectrophotometry (Libra S22, Biochrom Ltd.).

The ABTS+· free radical scavenging activity of Umbelliferae extracts was measured using the methods of Thaipong et al. (2006), with some modifications. Assays were performed using the ABTS+· cation decolorization reaction (the blue-green color). To generate ABTS+· in phosphate-buffered saline (pH 7.4), the stock solution was prepared to have 7 mM ABTS and 2.45 mM potassium persulfate and was allowed to react for 24 h at room temperature in the dark. Then, the dark blue-green colored ABTS+· radical solution was diluted to obtain an absorbance of 0.70 (±0.02) on 732 nm using the spectrophotometer (Libra S22, Biochrom Ltd.). Fresh ABTS+· solution was prepared for each assay. Umbelliferae extracts (10 μL) were incubated with 190 μL of the ABTS+· solution for 30 min in the dark. The absorbance of the reactant was determined at 734 nm using a spectrophotometer. Trolox was used as a standard for the ABTS assay. Results were expressed as mg of Trolox equivalents (TE)/g DW by comparing the slope for ABTS+· scavenging to Trolox.

ORAC assays were performed based on a previous method (Číž et al., 2010), with a few modifications. The ORAC assay measures the peroxyl radical antioxidant scavenging activity induced by AAPH at 37℃. Fluorescein was used as a fluorescent probe. Loss of the fluorescence of fluorescein indicates the peroxyl radical production (Gomes et al., 2005). Fluorescein (70 nM) and other reagents were prepared in phosphate buffer (75 mM, pH 7.4). One-hundred and seventy microliters of fluorescein solution (60 nM final concentration) and 10 μL of sample were placed in wells of a microplate (clear bottom, black plate) and incubated at 37℃ for 30 min. AAPH (20 μL, 50 mM final concentration) was then added using a multichannel pipette to initiate the reaction. The fluorescence was recorded at 460 and 550 nm for excitation and emission, respectively, at every 5 min, and the microplate was automatically shaken prior to each reading in an enzyme-linked immunosorbent assay plate reader (SparkⓇ 10M, Tecan, Grödig, Austria) at 37℃. For the blank, phosphate buffer was used instead of sample, and Trolox was used as an antioxidant standard for plotting the calibration curve to quantitate oxygen radical absorbance capacity in each assay. The final ORAC values were calculated using a regression equation between the Trolox concentration and the net area under the curve (AUC). The net AUC corresponding to the sample was calculated by subtracting the AUC corresponding to the blank. ORAC values were expressed as mg TE/g DW of Umbelliferae extract (Lee and Lee, 2014).

The HORAC assay (Lee and Lee, 2014) measures the metal-chelating activity of antioxidants under conditions of Fenton-like reactions using a Co(II) complex; hence, HORAC assays measure the ability to protect against hydroxyl radical formation. A hydrogen peroxide solution (0.55 M) was prepared in distilled water. Co(II) was prepared as follows: 15.7 mg of CoF2· 4H2O and 20 mg of picolinic acid were dissolved in 20 μL of distilled water. Fluorescein 170 μL (60 nM, final concentration) and 10 μL of sample were incubated for 10 min at 37℃ in the dark. Then, 10 μL of H2O2 (27.5 mM, final concentration) and 10 μL of Co(II) were added to each well. The fluorescence was recorded at 460 and 550 nm for excitation and emission, respectively, every 5 min for 1 h; the microplate was automatically shaken prior to each reading. Phosphate buffer solution was used as a blank. Gallic acid solutions were used to plot a standard curve. AUC values were calculated in the same manner as for the ORAC assay. Final HORAC values were calculated using regression analysis between the gallic acid concentration and the net area under the curve. The HORAC of the sample was expressed as mg of GAE/g DW of Umbelliferae plant extract.

Statistical analysis

SPSS 24.0 (IBM SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All results were expressed as mean ±standard deviation (SD) in at least triplicate and were analyzed using one way analysis of variance (ANOVA) and Duncan’s multiple comparison test for individual comparisons. The correlation between TPC, TFC, and antioxidant activity is presented by Pearson correlation coefficient. Results were considered statistically significant when P-values were below 0.05.

RESULTS AND DISCUSSION

TPC and TFC in Umbelliferae plant extracts

The TPC contents of the extracts of the selected 14 Umbelliferae plants cultivated in Korea, are tabulated in Table 1. Extracts of S. chinensis and Sanicula rubiflora had the highest TPC contents of the selected Umbelliferae extracts (587.5 and 401.3 mg GAE/g DW, respectively). The TPC content of S. chinensis, S. rubiflora, Angelica fallax, B. longiradiatum, C. japonica, Glehnia littoralis, Ostericum koreanum, Torilis japonica, and Torilis scabra extracts showed more than 300 μg GAE/g DW. However, C. sativum had the lowest TPC content (71.5 mg GAE/g DW). The TPC contents of A. decursiva, B. longiradiatum, C. japonica, D. carota, O. koreanum, Pimpinella brachycarpa, and T. japonica have been reported in several previous studies (Croft, 1998; Robbins, 2003; Tsao, 2010; Ghasemzadeh and Ghasemzadeh, 2011; Del Rio et al., 2013). However, the comparison of TPC contents between the present study and earlier studies is not possible since the methods used to prepare the plant extracts, extraction solvents, and equivalent compounds for quantification are different in each other.

Table 1

Source and optimum conditions of various proteasesTotal phenolic and flavonoid contents of Umbelliferae plant extracts

Umbelliferae	Korean name	Voucher no.1)	TPC (mg GAE/g DW)	TFC (mg CE/g DW)	Flavonoids ratio2)
Angelica decursiva	Badi-namul	PB4036.1	113.0±12.0de	2.3±0.5g	2.0
Angelica fallax	Sayakchae	PB4038A.1	325.3±11.4b	20.9±2.8d	6.4
Bupleurum longeradiatum	Gaesiho	PB3994.1	358.9±15.2b	14.2±1.6e	4.0
Coriandrum sativum	Gosu	PB4005.1	71.5±1.8e	10.0±4.3f	14.0
Cryptotaenia japonica	Paddeuk-namul	PB4006.2	364.8±73.0b	24.1±1.8bc	6.6
Daucus carota var. sativa	Carrot	PB4059.1	151.2±17.7de	4.5±0.0g	3.0
Glehnia littoralis	Gaekbangpung	PB4027.4	361.0±29.3b	9.2±0.3f	2.5
Ostericum koreanum	Gangwhal	PB4048.1	360.6±37.0b	3.5±0.2g	1.0
Ostericum sieboldii	Muecminari	PB4049.1	212.9±11.4cd	5.4±0.8g	2.5
Pimpinella brachycarpa	Cham-namul	PB4010.2	184.1±32.9d	8.9±0.5f	4.8
Sanicula chinensis	Chambandi	PB3996.1	587.5±12.5a	31.4±1.3a	5.3
Sanicula rubiflora	Red chambandi	PB3997.1	401.3±67.6b	26.6±0.6b	6.6
Torilis japonica	Sasangza	PB4003.2	361.0±39.2b	22.1±2.0cd	6.1
Torilis scabra	Gaesasangza	PB4004.2	322.0±5.3bc	20.0±1.2d	6.2

1)Korea Plant Extract Bank (KPEB), Daejeon, Korea.

2)Flavonoids ratio=(TFC/TPC)×100.

TPC, total phenolic content; TFC, total flavonoid content; GAE, gallic acid equivalent; DW, dry weight; CE, catechin equivalent.

Values followed by the different letters (a-h) in the same column are significantly different (P<0.05).

More than 8,000 phenolic plant compounds exist as secondary metabolites in plants and have various physiological activities (Tsao, 2010), many of which have been developed as pharmaceuticals. Phenolic compounds are considered to be natural substances with health-improving functions, which eliminate active oxygen free radicals and reduce the risk of inflammation and cancers (Ghasemzadeh and Ghasemzadeh, 2011). Phenolic compounds have at least one aromatic ring with one or more hydroxyl groups and are classified as flavonoids and non-flavonoids (Del Rio et al., 2013). Therefore, the TPC values observed in the present study indicate the total amount of flavonoids and non-flavonoids phenolic compounds in each Umbelliferae plant extract.

The TFC contents of the 14 Umbelliferae plant extracts are shown in Table 1. S. chinensis extract had the highest TPC (587.5 mg GAE/g DW) and TFC (31.4 mg CE/g DW) contents. The TFC values of A. fallax, C. japonica, S. chinensis, S. rubiflora, T. japonica, and T. scabra extracts were higher than 20 mg CE/g DW. However, the TFC levels were lowest in the A. decursiva and O. koreanum extracts (2.3 and 3.5 mg CE/g DW, respectively). Flavonoids are a group of phenolic compounds synthesized through the phenylpropanoid pathway from phenylalanine, and consist of approximately 4,000 compounds, such as quercetin, lutin, apigenin, and baicalein (Tsao, 2010). All flavonoids have a basic C6-C3-C6 structural skeleton, which is composed of two aromatic C6 rings and one heterocyclic ring with one oxygen atom (Tsao, 2010; Ghasemzadeh and Ghasemzadeh, 2011; Del Rio et al., 2013). The main subclasses of flavonoids are flavonols, flavones, isoflavones, flavanones, anthocyanidins, flavan-3-ols, and dihydrochalcones (Tsao, 2010; Ghasemzadeh and Ghasemzadeh, 2011; Del Rio et al., 2013).

C. sativum extracts had the highest TFC/TPC ratio (14.0%), and A. fallax, C. japonica, S. rubiflora, T. japonica, and T. scabra extracts all showed TFC/TPC ratios >6.0%. On the other hand, O. koreanum and A. decursiva had the lowest TFC/TPC ratios (1.0 and 2.0%, respectively). TFC /TPC ratios indicate comparative levels of flavonoids to total phenolic acid in the extracts. Therefore, TPC, excluding TFC, indicates only non-flavonoids, such as gallic acid, chlorogenic acid, and cinnamic acid. In 14 Umbelliferae plant extracts, the ratio of the maximum to minimum TPC was 8.21, and the ratio of the maximum to minimum TFC was 13.6. The difference for the amount of TPC and TFC in extracts results in variation in the ABTS radical scavenging activity, ORAC, and HORAC of Umbelliferae plant extracts.

Antioxidative activities of Umbelliferae plant extracts

To evaluate the antioxidant activity of the Umbelliferae plant extracts, DPPH radical scavenging activity was measured (Table 2). G. littoralis extracts (300 μg/mL) were able to quench DPPH radicals (100 mM) by 84.1%. Several of the extracts (300 μg/mL) were able to quench DPPH radical (100 mM) by over 80%; in decreasing order of magnitude: G. littoralis (84.1%)>A. fallax (82.7%)>B. longiradiatum, S. rubiflora, and T. scabra (82.4%)>T. japonica (82.0%)>O. koreanum (81.6%). In contrast, D. carota and C. sativum showed the weakest DPPH radical scavenging activity (46.4% and 37.6%, respectively). Umbelliferae plant extracts exhibited half-maximal inhibitory concentration (IC50) values for quenching DPPH radicals (100 mM) in the range of 39∼179 μg DW/mL. IC50 values for A. decursiva, C. sativum, and D. carota were not analyzed because their DPPH radical scavenging activities were <50%. T. japonica, S. chinensis, T. scabra, and S. rubiflora extracts showed the most potent DPPH radical quenching activities, exhibiting IC50 values of 39, 48, 49, and 50 μg DW/mL, respectively.

Table 2

DPPH radical scavenging activity of Umbelliferae plant extracts

Umbelliferae	DPPH radical scavenging activity1)
	% Control2)	IC50
		Extract (μg DW/mL)	TPC3) (μg TPC/mL)	TFC3) (μg TFC/mL)
Angelica decursiva	49.5±3.2f	NT	NT	NT
Angelica fallax	17.3±0.3ab	65.8	21.40	1.38
Bupleurum longeradiatum	17.4±0.4ab	79	28.35	1.12
Coriandrum sativum	62.4±3.4h	NT	NT	NT
Cryptotaenia japonica	23.0±0.6c	61	22.25	1.47
Daucus carota var. sativa	53.6±1.1g	NT	NT	NT
Glehnia littoralis	15.9±1.0a	94	33.93	0.86
Ostericum koreanum	18.4±0.3ab	102	36.78	0.36
Ostericum sieboldii	35.2±0.6e	179	38.11	0.97
Pimpinella brachycarpa	20.1±1.0b	122	22.46	1.09
Sanicula chinensis	26.4±1.7d	48	28.20	1.51
Sanicula rubiflora	17.6±0.9ab	50	20.07	1.33
Torilis japonica	18.0±0.0ab	39	14.08	0.86
Torilis scabra	17.6±1.0ab	49	15.78	0.98

1)IC50 for DPPH radical scavenging activity were measured in 0∼300 μg/mL of extract.

2)DPPH radical (100 mM) was quenched by extracts (300 μg/mL).

3)IC50 of TPC and TFC were derived from IC50 of extract.

IC50, the half maximal inhibitory concentration; DW, dry weight; TPC, total phenolic content; TFC, total flavonoid content; NT, not tested.

Values followed by the different letters (a-h) in the same column are significantly different (P<0.05).

The antioxidant activities of plant extracts are dependent on various phenolic compounds, including flavonoids. The IC50 values observed for the DPPH radical scavenging activity are estimated differently by the levels of TPC and TFC in the Umbelliferae plant extracts; these contents were calculated to be 14.08∼38.11 μg TPC/mL and 0.36∼1.51 μg TFC/mL, respectively (excluding A. decursiva, C. sativum, and D. carota) (Table 2). T. japonica showed the most potent DPPH radical scavenging activity with an IC50 of 14.08 μg TPC/mL. On the other hand, G. littoralis, O. koreanum, and Ostericum sieboldii extracts showed relatively low anti-oxidative activities, with IC50 values of 33.93, 36.78, and 38.11 μg TPC/mL, respectively. In addition, O. koreanum had the lowest IC50 (0.36 μg TFC/ mL), which suggests that the flavonoids in O. koreanum have potent DPPH radical scavenging activity. Thus, the IC50 expressed as TPC or TFC, is much lower than that expressed as whole extract contents and indicates that total polyphenol or total flavonoid in the extract is the major substance with potent anti-oxidative DPPH radical scavenging activity. Since many flavonoids and phenolic compounds in plant extracts mediate antioxidant activity, the DPPH radical scavenging activity might be more appropriately expressed as IC50 of TPC or TFC rather than that of total extract content.

Several earlier studies have reported the DPPH radical scavenging activity of Umbelliferae plant extracts. In a previous study, B. longiradiatum, C. sativum, O. koreanum, P. brachycarpa, and T. japonica have been shown to have DPPH radical scavenging activities (IC50) of 96.5, 161, 70, 257, and 58 μg/mL, respectively (Lee et al., 2011a; Lee et al., 2011b). Considering the total DPPH radical scavenging activities and IC50 values in the previous and present studies (Table 2), S. chinensis, S. rubiflora, T. japonica, and T. scabra have comparatively more potent antioxidant activities.

: The reducing capacity of Umbelliferae plant extracts is shown in Table 3; the pattern of reducing capacity was similar to that of DPPH radical scavenging activity. In 14 Umbelliferae plant extracts, S. rubiflora, S. chinensis, and A. fallax showed the highest reducing capacity (>0.5 on A700). In contrast, C. sativum, C. japonica, and D. carota showed the lowest reducing capacity (0.11 on A700).

Table 3

Reducing capacity and ABTS radical scavenging activity of Umbelliferae plant extracts

Umbelliferae	Reducing capacity1) (A700)	ABTS radical scavenging activity
		Extract (mg TE/g DW)	TPC (mg TE/g)	TFC (g TE/g)
Angelica decursiva	0.16±0.00hi	517.5±10.3e	4,579.94±90.82b	1,991.28±39.49a
Angelica fallax	0.52±0.06c	850.9±43.9b	2,615.64±135.03d	125.15±6.46h
Bupleurum longeradiatum	0.32±0.03f	914.2±55.2ab	2,547.23±153.93de	179.38±10.84g
Coriandrum sativum	0.11±0.00i	381.5±15.0f	5,336.13±209.95a	533.61±20.99c
Cryptotaenia japonica	0.11±0.00i	900.2±98.2b	2,467.65±269.31de	102.39±11.17h
Daucus carota var. sativa	0.11±0.01i	367.5±12.9f	2,430.78±85.04de	540.17±18.90c
Glehnia littoralis	0.35±0.01ef	746.9±51.6c	2,068.88±143.01fg	224.88±15.54f
Ostericum koreanum	0.35±0.03ef	865.5±11.6b	2,400.26±32.02de	685.79±9.15b
Ostericum sieboldii	0.19±0.00h	520.9±71.1e	2,446.53±333.77de	453.06±61.81d
Pimpinella brachycarpa	0.26±0.02g	536.9±20.4e	2,916.17±110.97c	327.66±12.47e
Sanicula chinensis	0.63±0.03b	986.2±58.4a	1,678.64±99.43h	53.46±3.17i
Sanicula rubiflora	0.78±0.05a	924.9±40.9ab	2,304.68±101.81ef	86.64±3.83hi
Torilis japonica	0.45±0.03d	863.5±35.0b	2,392.06±96.81de	108.24±4.38h
Torilis scabra	0.38±0.03e	619.5±22.7d	1,924.02±70.64gh	96.20±3.53h

1)Reducing capacity was measured using 1 mg/mL of extract.

TPC, total phenolic content; TFC, total flavonoid content; TE, Trolox equivalent; DW, dry weight.

Values followed by the different letters (a-i) in the same column are significantly different (P<0.05).

The reducing capacity of plant extracts indicate the presence of reductones, which have an antioxidant effect through donating hydrogen atoms and breaking free radical chains (Duh, 1998). Reductones are involved in a reaction with peroxide precursors, thereby preventing formation of peroxides (Guo and Wang, 2007). In a previous study, the essential oil of A. decursiva has been reported to have a reducing capacity of 0.35 on A700 (Lim and Shin, 2012), which is higher than the reducing capacity observed in this study. One of the reasons for the difference may be the variation between the composition of the essential oil and extract used in the antioxidant experiments.

The ABTS radical scavenging activities of the Umbelliferae plant extracts were calculated using plots on a straight line derived from the Trolox standard (Table 3). Umbelliferae plant extracts had ABTS radical scavenging activities of 367.5∼986.2 mg TE/g DW. In this study, the Umbelliferae plant extracts with >900 mg TE/g DW for ABTS radical scavenging activity were (in decreasing order of magnitude): S. chinensis>S. rubiflora>B. longiradiatum>C. japonica. With respect to the TPC and TFC, the Umbelliferae plant extracts had ABTS radical scavenging activity in the range of 1,678.64∼5,336.13 mg TE/g and 53.46∼1,991.28 g TE/g, respectively.

ABTS scavenging activity of Umbelliferae plant extracts have been estimated in diverse units in previous studies. In one study, the ABTS scavenging activity for A. decursiva, B. longadiatum, and D. carota were expressed in percent (36%, 60%, and 83%, respectively) (Lee et al., 2011a), whereas in another study the ABTS scavenging activity of C. japonica was expressed in μM TE/100 g DW (82 μM TE/100 g DW) (Yao et al., 2010). The ABTS radical scavenging activities of the Umbelliferae plants in this study (Table 3) were observed to be higher than those reported previously.

The peroxyl radical scavenging activities of the Umbelliferae plant extracts were measured using ORAC and HORAC assays. The ORAC values ranged from 11.44 to 42.88 mg TE/g DW extract (Table 4), as follows; S. rubiflora (42.88 mg TE/g DW)>S. chinensis (41.64 mg TE/g DW)>D. carota (36.32 mg TE/g DW)>T. japonica (35.12 mg TE/g DW)>B. longiradiatum (32.66 mg TE/g DW)>T. scabra (30.86 mg TE/g DW)>O. sieboldii (30.15 mg TE/g DW). With respect to the TPC and TFC contents, the Umbelliferae plant extracts had ORAC values in the range of 47.40∼240.19 mg TE/g and 0.72∼11.22 g TE/g, respectively.

Table 4

Oxygen radical absorbance capacity (ORAC) and hydroxyl oxygen radical absorbance capacity (HORAC) of Umbelliferae plant extracts

	ORAC			HORAC
	Extract (mg TE/g DW)	TPC (mg TE/g)	TFC (g TE/g)	Extract (mg GAE/g DW)	TPC (mg GAE/g)	TFC (g GAE/g)
Angelica decursiva	25.81±1.23d	228.37±10.92a	11.22±0.54a	1.55±0.50h	13.70±4.43h	0.67±0.22f
Angelica fallax	29.54±3.90cd	90.82±11.99e	1.41±0.19f	26.93±1.06c	79.78±6.54c	1.24±0.10de
Bupleurum longeradiatum	32.66±3.48bc	91.01±9.69e	2.30±0.24e	19.09±1.59d	53.20±4.43e	1.34±0.11cd
Coriandrum sativum	11.44±1.36e	159.99±19.04b	1.14±0.14fg	ND	ND	ND
Cryptotaenia japonica	17.29±1.31e	47.40±3.60g	0.72±0.05g	3.26±1.31h	8.93±3.59h	0.09±0.05g
Daucus carota var. sativa	36.32±2.72b	240.19±18.01a	8.07±0.61b	25.99±2.29c	171.91±15.13a	5.78±0.51a
Glehnia littoralis	25.53±2.32d	70.73±6.42f	2.78±0.25d	12.28±1.53f	34.02±4.25fg	1.34±0.17cd
Ostericum koreanum	20.35±1.68e	56.43±4.67fg	5.81±0.48c	9.70±0.76f	26.89±2.10g	2.77±0.22b
Ostericum sieboldii	30.15±2.89c	141.61±13.55c	5.58±0.53c	15.78±1.25e	74.13±5.88c	2.92±0.23b
Pimpinella brachycarpa	20.08±0.62e	109.09±3.38d	2.26±0.07e	7.33±0.71g	39.81±3.88f	0.82±0.08f
Sanicula chinensis	41.64±2.79a	70.87±4.76f	1.33±0.09f	37.90±2.30a	64.51±3.92d	1.21±0.07de
Sanicula rubiflora	42.88±3.75a	106.84±9.34de	1.61±0.14f	38.05±1.13a	94.83±2.81b	1.43±0.04cd
Torilis japonica	35.15±2.23b	97.37±6.17de	1.59±0.10f	34.80±2.49b	96.41±6.90b	1.57±0.11c
Torilis scabra	30.86±4.57c	95.84±14.18de	1.54±0.23f	18.36±2.23d	57.01±6.92de	0.97±0.06ef

Values followed by the different letters (a-h) in the same column are significantly different (P<0.05).

ORAC (or HORAC) of TPC and TFC were derived from ORAC (or HORAC) value of extract.

TPC, total phenolic content; TFC, total flavonoid content; TE, Trolox equivalent; GAE, gallic acid equivalent; DW, dry weight; ND, not detected.

The HORAC values of the Umbelliferae plant extracts (excluding C. sativum, which was not detected in the assay) ranged from 1.55 to 38.05 mg GAE/g DW extract (Table 4). S. chinensis, S. rubiflora, and T. japonica had potent HORAC values of 38.05, 37.90, and 34.80 mg GAE/g DW extract, respectively. With respect to the TPC and TFC content, the HORAC values ranged from 8.93∼171.91 mg GAE/g TPC and 0.09∼5.78 g GAE/g TFC, respectively. Judging by the ORAC and HORAC values observed, S. chinensis and S. rubiflora had the highest antioxidant activity of the Umbelliferae plant extracts tested in this study. From the studies on the antioxidant activity of plant foods, the ABTS activity and ORAC for 27 kinds of vegetables was reported as 4.1∼49.7 μmol TE/g (Gorinstein et al., 2009); for Peucedanum japonicum Thunberg roots were reported as 42.24∼50.55 μmol TE/g and 58.10∼133.37 μmol/g, respectively (Lim et al., 2019); and for Chrysanthemum boreale extract were reported as 0.47 μmol/mg and 94.34 μmol TE/g, respectively (Kim et al., 2014). In this study, the ABTS activity of S. chinensis was 3.94 μmol TE/g, indicating a higher antioxidant activity the vegetables and plants mentioned above. However, the ORAC of S. rubiflora was higher (0.22 μmol GAE/g), but it should not be directly compared due to the use of different standards.

Generally, the ABTS assay has been widely used for analysis of the total antioxidant capacity of plant extracts, but it is inadequate for assessing antioxidant activity in vivo (Scalzo et al., 2005; Lee and Kim, 2015). ORAC and HORAC methods can measure antioxidant activity according to the concentration of the antioxidant and the reaction time, are more sensitive than other antioxidant assays, and thus more accurately reflecting antioxidant activity in vivo (Prior and Cao, 1999; Číž et al., 2010). Therefore, the ORAC method has been extensively applied to and approved for use to evaluate the antioxidant activity of food ingredients, medicines, and plant extracts. There are numerous reports of free radical scavenging activities of food and plant-derived substances. Comparisons with antioxidant activities are reported regularly in the same study, but the antioxidant activities are rarely compared with those from other studies. This is because although the principles and basic methods of antioxidant analysis, such as ABTS and ORAC assays, are similar, the reagent concentrations and reaction times vary between studies. In addition, it is not meaningful to numerically compare antioxidant results because the conditions of the samples and the standards used as quantitative criteria vary from study to study. For example, Floegel et al. (2011) reported the ABTS activity and ORAC of onion as 29.6 mg vitamin C equivalents/100 g and 1,034 μmol TE/100 g, respectively; however, in another study, the ABTS activity and ORAC of onion were reported as 3.55 μmol TE/g wet weight and 0.50 μmol TE/g wet weight, respectively (Sun and Tanumihardjo, 2007).

The relative antioxidant potentials of Umbelliferae plant extracts varied depending on how the antioxidants are analyzed. In 14 Umbelliferae plant extracts, the highest antioxidant activities (as determined by DPPH, reducing capacity, ABTS, ORAC, and HORAC assays) were shown for (in order of magnitude) S. rubiflora, S. chinensis, T. japonica, T. scabra, A. fallax, and G. littoralis.

Correlation between TPC and various antioxidant activities of Umbelliferae plant extracts

The relationship between antioxidant activity and TPC is shown in Fig. 1. TPC had a superior linear correlation (r2=0.817) with ABTS values (Fig. 1A). Additionally, there was a strong linear correlation between ORAC and HORAC, r2=0.820 (Fig. 1B). This relationship is consistent with the Pearson correlation between TPC, TFC and various antioxidant activities (Table 5). Since total phenolic compounds contain flavonoids and non-flavonoid polyphenols, the TPC and TFC contents of 14 Umbelliferae plant extracts showed a strong positive correlation (r=0.747, P<0.01), indicating that TFC contributes a significant amount of TPC. TPC (and TFC) showed a strong positive correlation to ABTS radical scavenging activity (r=0.904, P<0.01), reducing capacity (r=0.737, P<0.01) and HORAC (r=0.630, P<0.05). The DPPH radical scavenging activity also showed a strong negative correlation with TPC (r=−0.725, P<0.01). DPPH radical quenching activity is indicated by a negative correlation since the radical content decreases as activity increases. Therefore, TPC in Umbelliferae plant extracts may play a major role in the increase in DPPH radical scavenging activity.

Fig. 1

Correlations between TPC content and various antioxidant activities of Umbelliferae plant extracts. (A) TPC versus ABTS: y=288.7+1.426x, r2=0.817; (B) ORAC versus HORAC: y=−23.40+1.430x, r2=0.820. TPC, total phenolic content; GAE, gallic acid equivalent; DW, dry weight; ABTS, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); TE, Trolox equivalent; ORAC, oxygen radical absorbance capacity; HORAC, hydroxyl oxygen radical absorbance capacity.

Table 5

Pearson’s correlations between antioxidant activities measured using different assays and total phenolic/flavonoid contents

	TPC	TFC	DPPH	ABTS	Reducing capacity	ORAC	HORAC
TPC	1.000
TFC	0.747**	1.000
DPPH	−0.725**	−0.473	1.000
ABTS	0.904**	0.696**	−0.770**	1.000
Reducing capacity	0.737**	0.685**	−0.608*	0.695**	1.000
ORAC	0.532	0.465	−0.275	0.375	0.683**	1.000
HORAC	0.545	0.630*	−0.172	0.384	0.764**	0.905**	1.000

P<0.05, **P<0.01.

TPC, total phenolic content; TFC, total flavonoid content; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); ORAC, oxygen radical absorbance capacity; HORAC, hydroxyl oxygen radical absorbance capacity.

The TPC (or TFC) of Umbelliferae plant extracts showed a stronger correlation with ABTS radical scavenging activity (r=0.904, P<0.01) than with DPPH radical scavenging activity (r=−0.725, P<0.01). Methanol, a polar solvent used to prepare Umbelliferae plant extracts, removes hydrophilic, lipophilic, and highly pigmented compounds from samples (Sasidharan et al., 2011). The antioxidant activities of the hydrophilic and lipophilic compounds are sensitively analyzed by the ABTS assay, while the DPPH radical scavenging assay is appropriate for analysis of hydrophobic compounds (Floegel et al., 2011; Kim, 2015). Therefore, ABTS activity have a stronger correlation with TPC content than DPPH activity. The reducing capacity of Umbelliferae plant extracts was positively correlated with the ABTS radical scavenging activity and ORAC (r=0.695, P<0.01 and r=0.683, P<0.01, respectively). ORAC and HORAC assays measure peroxyl radical scavenging activity and are therefore strongly correlated (r=0.905, P<0.01). DPPH radical scavenging activity showed a strong correlation with ABTS radical scavenging activity and reducing capacity (r=−0.770, P<0.01 and r=−0.608, P<0.05, respectively). In addition, the reducing capacity showed a strong positive correlation with HORAC (r=0.764, P<0.01). The reliability for the antioxidant activity results are confirmed by strong correlations between results from different assays.

The different types of polyphenols present in plant extracts exert several antioxidant effects and properties through numerous mechanisms, including free radical scavenging and transition metal chelation and singlet oxygen quenching (Prior and Cao, 1999). Using one type of detection method provides only limited information about the antioxidant activity of plants. Therefore, it is more appropriate to carry out analyses of the antioxidant activity of plant extracts using a variety of methods. Using multiple analytical methods is a more comprehensive approach to determine the antioxidant activity of plant extracts (Prior and Cao, 1999).

To conclude, the results of this study indicate that the TPC contents and antioxidant activities of Umbelliferae plant extracts are closely correlated, and that there is a significant correlation between the antioxidant activities measured by different assays. Therefore, the TPC contents and potent antioxidant activities of 14 Umbelliferae plant extracts calculated in this study are considered reliable. Umbelliferae plant extracts with potent antioxidant activities (including S. rubiflora, S. chinensis, T. japonica, T. scabra, A. fallax, and G. littoralis) are expected to play a role in adding antioxidant functions to foods and cosmetics.