Comparative Study of the Antioxidative Activity of Culinary Herbs and Spices, and Hepatoprotective Effects of Three Selected Lamiaceae Plants on Carbon Tetrachloride-Induced Oxidative Stress in Rats

Abstract

The purpose of this study was to compare the antioxidative activity of 68 culinary herbs and spices, and to evaluate the hepatoprotective effects of highly antioxidative plants (selected after an in vitro study) on carbon tetrachloride-induced oxidative stress in rats. Six Lamiaceae plants (oregano, common thyme, peppermint, lemon balm, sweet marjoram and rosemary) were found to be among the top 10 plants showing high total oxygen radical absorbance capacity (T-ORAC: sum of hydrophilic and lipophilic ORAC (H- and L-ORAC)). Their T-ORACs were composed of > 90% H-ORACs. Next, we prepared hydrophilic fractions of three selected Lamiaceae plants (peppermint, lemon balm and rosemary), in which bitterness and lipophilic odorants were reduced. Oral administration of these fractions in rats decreased aspartate aminotransferase and alanine aminotransferase activities in serum, inhibited lipid peroxidation, and restored superoxide dismutase and glutathione S-transferase activities in carbon tetrachloride-treated rats. Antioxidants in plants could exert a hepatoprotective effect by scavenging free radicals.

Introduction

A state of oxidative stress occurs when there is an excess of reactive species such as reactive oxygen and reactive nitrogen in relation to available antioxidants (Asimov, 2007). Oxidative stress is reported to lead to various diseases such as atherosclerosis, diabetes, inflammatory bowel disease and cancer (Schatz, 2007). Antioxidants synthesized in vivo and taken in from the diet play an important role in defending against reactive species (Reth, 2007).

Culinary herbs and spices are sources of dietary antioxidants. These natural materials contain components that differ in properties, e.g., hydrophilic and lipophilic compounds, and function as antioxidants at different reaction rates. Therefore, to evaluate the antioxidative activity of each culinary plant, it is necessary to assay both the hydrophilic and lipophilic fractions. Comparative studies on the antioxidative activities of culinary plants have been reported (Zheng and Wang, 2001; Wu et al., 2004; Ninfali et al., 2005; Shan et al., 2005). However, most of these have focused on hydrophilic compounds, such as polyphenolic compounds and ascorbic acid, and there are few studies on the oxidative activity of both the hydrophilic and lipophilic fractions of plants.

The oxygen radical absorbance capacity (ORAC) assay is based on the measurement of antioxidant scavenging activity against peroxyl-radical-induced-oxidation initiated by 2,2′-azobis(2-amidino-propane) dihydrochloride (AAPH) at physiological pH (Ou et al., 2001). In addition, the ORAC assay can be performed in both an aqueous solution and a lipophilic environment (Huang et al., 2002, 2005; Prior et al., 2003; Prior et al., 2005). Therefore, this assay is considered to be biologically relevant and suitable for both hydrophilic and lipophilic components in natural materials.

In vitro assays such as the ORAC do not measure bioavailability, in vivo stability, and reactivity in situ. Therefore, an in vivo study is useful in confirming whether the administration of potent antioxidants evaluated by the initial in vitro study improves oxidative stress levels.

Carbon tetrachloride (CCl₄)-induced liver injury is an oxidative-stress model used for screening hepatoprotective materials (McCay et al., 1984; Recknagel et al., 1989; Plaa, 2000; Weber et al., 2003). A toxic intermediate, trichloromethyl radical (CCl₃•), which is metabolized from CCl₄ by the cytochrome P450 enzyme system in liver microsomes, is reported to produce liver damage. CCl₃• might attack biological molecules directly, causing covalent modification and/or initiation of lipid peroxidation by abstracting H• from membrane lipids. In addition, a highly reactive trichloromethylperoxyl radical (CCl₃O₂•), which is a reactive intermediate arising from CCl₃• and oxygen, initiates lipid peroxidation by H• abstraction from membrane lipids. Some studies have reported the protective effects of commercially available culinary herbs and spices on CCl₄-induced hepatotoxicity (Deshpande et al., 1998; Sotelo-Félix et al., 2002; Botsoglou et al., 2008; Park, 2010; Farhoudi, 2011). However, in general, many of the phytonutrients in foods that possess biological activities are also unpalatable because of bitter tastes and odors (Drewnoski and Gomez-Carneros, 2000; Lesschaeve and Noble, 2005; Soares et al., 2013). For ease of consumption, improving unfavorable tastes, especially bitterness, and removing undesirable odors in healthy foods is desirable.

The objectives of this study were to compare the hydrophilic ORAC (H-ORAC), lipophilic ORAC (L-ORAC) and T-ORAC (sum of H-ORAC and L-ORAC) values of 68 culinary herbs and spices grouped into 25 families and assess the hepatoprotective effect of the hydrophilic fractions of three selected Lamiaceae plants (peppermint, lemon balm and rosemary), in which both the bitterness components and lipophilic odorants of each plant were reduced, on CCl₄-induced oxidative stress in rats.

Materials and Methods

Plant materials    Sixty-eight herbs and spices from a variety of countries/regions were purchased through import merchants (Table 1). These plants are distributed throughout 25 families (Apiaceae, Asteraceae, Brassicaceae, Cannabaceae, Fabaceae, Illiciaceae, Iridaceae, Lamiaceae, Lauraceae, Liliaceae, Myristicaceae, Myrtaceae, Orchidaceae, Papaveraceae, Pedaliaceae, Piperaceae, Poaceae, Ranunculaceae, Rosaceae, Rubiaceae, Rutaceae, Solanaceae, Tiliaceae, Verbenaceae and Zingiberaceae) (Table 2).

Table 1. General information regarding 68 culinary herbs and spices.

no	common name	scientific name	country/region
1	clove	Syzygium aromaticum (L.) Merrill et Perry	Madagascar
2	oregano	Origanum vulgare L.	Albania
3	common thyme	Thymus vulgaris L.	Morocco
4	peppermint	Mentha x piperita L.	Egypt
5	lemon balm	Melissa officinalis L.	Bulgaria
6	Sichuan pepper	Zanthoxylum bungeanum Maxim.	China
7	sweet marjoram	Origanum majorana L.	Egypt
8	tarragon	Artemisia dracunculus L.	East Europe
9	damask rose	Rosa damascena Mill.	Pakistan
10	rosemary	Rosmarinus officinalis L.	Albania
11	winter savory	Satureja montana L.	Albania
12	Japanese pepper	Zanthoxylum piperitum DC.	Japan
13	cinnamon	Cinnamomum verum Presl	Sri Lanka
14	common linden	Tilia x europaea L.	France
15	bay laurel	Laurus nobilis L.	Turkey
16	perilla	Perilla frutescens Britton var. acuta Kubo	China
17	true lavender	Lavandula angustifolia (L.) Mill.	France
18	Japanese mugwort	Artemisia princeps Pamp.	Japan
19	sage	Salvia officinalis L.	Greece
20	Roman chamomile	Chamaemelum nobile (L.) All.	Egypt
21	star anise	Illicium verum Hook., f.	China
22	nigella	Nigella sativa L.	India
23	curry leaf tree	Murray a koenigii (L.) Spreng.	India
24	turmeric	Curcuma longa L.	India
25	hyssop	Hyssopus officinalis L.	Hungary
26	allspice	Pimenta dioica (L.) Merrill	Mexico
27	anise	Pimpinella anisum L.	Spain
28	brown mustard	Brassica juncea (L.) Czern.	India
29	nutmeg	Myristica fragrans Houtt.	Indonesia
30	vanilla	Vanilla planifolia Andr.	Madagascar
31	Japanese mint	Mentha arvensis L. var. piperascens Malinv.	China
32	German chamomile	Matricaria chamomilla L.	Egypt
33	cape jasmine	Gardenia jasminoides Ellis	China
34	cumin	Cuminum cyminum L.	Iran
35	lemongrass	Cymbopogon citratus (DC. Ex Nees) Stapf	Thailand
36	parsley	Petroselinum crispum (Mill.) Nym. ex A.W. Hill.	USA
37	sweet basil*	Ocimum basilicum L.	Egypt
38	saffron	Crocus sativus L.	Spain
39	licorice	Glycyrrhiza glabra L.	China
40	garden chervil	Anthriscus cerefolium (L.) Hoffm.	Germany
41	common juniper	Juniperus communis L.	Mediterranean
42	celery	Apium graveolens L.	India
43	ginger	Zingiber officinale Rosc.	China
44	ajwain	Carum ajowan BENITH.	India
45	pepper	Piper nigrum L.	Vietnam
46	sweet orange	Citrus sinensis (L) Osbeck	Spain
47	red pepper	Capsicum annuum L.	China
48	caraway	Carum carvi L.	Netherlands
49	cardamon	Elettaria cardamomum (L.) Maton	Guatemala
50	fenugreek	Trigonella foenum-graecum L.	India
51	dill**	Anethum graveolens L.	India
52	fennel	Foeniculum vulgare Mill.	China
53	coriander***	Coriandrum sativum L.	Morocco
54	angelica	Angelica archangelica L.	Poland
55	sweet basil*	Ocimum basilicum L.	Egypt
56	chives	Allium schoenoprasum L.	China
57	common wormwood	Artemisia absinthium L.	China
58	dill**	Anethum graveolens L.	India
59	hemp	Cannabis sativa L.	China
60	sesame	Sesamum indicum L	Myanmar
61	asafoetida	Ferula assafoetida L.	India
62	coriander***	Coriandrum sativum L.	Morocco
63	watercress	Nasturtium officinale R. Br.	France
64	poppy	Papaver somniferum L.	Turkey
65	horseradish	Armoracia rusticana (Lam) Gaertn., Mey. et Scherb.	China
66	garlic	Allium sativum L.	China
67	onion	Allium cepa L.	USA
68	tamarind	Tamarindus indica L.	India

*  sweet basil (no. 37: leaf; no. 55: seed),

**  dill (no. 51: leaf; no. 58: seed),

***  coriander (no. 53: leaf; no. 62: seed).

Table 2. ORAC values of 68 culinary herbs and spices.

no	common name	family name	parts used	H-ORAC (mmol of TE/g of dry plant)	L-ORAC (mmol of TE/g of dry plant)	T-ORAC (mmol of TE/g of dry plant)	H-ORAC/T-ORAC (%)	L-ORAC/T-ORAC (%)
				means ± SEM	means ± SEM	means ± SEM	ratio of means	ratio of means
1	clove	Myrtaceae	bud	1.19 ± 0.05	1.70 ± 0.12	2.89 ± 0.17	41.2	58.8
2	oregano	Lamiaceae	leaf	2.15 ± 0.08	0.14 ± 0.03	2.29 ± 0.11	93.9	6.1
3	common thyme	Lamiaceae	leaf	1.55 ± 0.03	0.03	1.58 ± 0.03	98.1	1.9
4	peppermint	Lamiaceae	leaf	1.45 ± 0.04	0.03 ± 0.01	1.48 ± 0.05	98.0	2.0
5	lemon balm	Lamiaceae	leaf	1.46 ± 0.05	0.02	1.48 ± 0.05	98.6	1.4
6	Sichuan pepper	Rutaceae	fruit	1.10 ± 0.07	0.29 ± 0.05	1.39 ± 0.12	79.1	20.9
7	sweet marjoram	Lamiaceae	leaf	1.36 ± 0.09	0.02 ± 0.01	1.38 ± 0.10	98.6	1.4
8	tarragon	Asteraceae	leaf, stem	1.37 ± 0.05	0.01	1.38 ± 0.05	99.3	0.7
9	damask rose	Rosaceae	flower	1.23 ± 0.08	0.01	1.24 ± 0.08	99.2	0.8
10	rosemary	Lamiaceae	leaf	1.14 ± 0.06	0.06 ± 0.01	1.20 ± 0.07	95.0	5.0
11	winter savory	Lamiaceae	leaf	1.12 ± 0.09	0.04 ± 0.01	1.16 ± 0.10	96.6	3.4
12	Japanese pepper	Rutaceae	peel	0.99 ± 0.04	0.11 ± 0.01	1.10 ± 0.05	90.0	10.0
13	cinnamon	Lauraceae	bark	1.05 ± 0.05	0.01	1.06 ± 0.05	99.1	0.9
14	common linden	Tiliaceae	leaf, flower	0.98 ± 0.03	0.02	1.00 ± 0.03	98.0	2.0
15	bay laurel	Lauraceae	leaf	0.91 ± 0.08	0.08	0.99 ± 0.08	91.9	8.1
16	perilla	Lamiaceae	leaf	0.91 ± 0.04	0.08 ± 0.01	0.99 ± 0.05	91.9	8.1
17	true lavender	Lamiaceae	flower	0.92 ± 0.06	0.06	0.98 ± 0.06	93.9	6.1
18	Japanese mugwort	Asteraceae	leaf	0.78 ± 0.09	0.01	0.79 ± 0.09	98.7	1.3
19	sage	Lamiaceae	leaf	0.58 ± 0.04	0.20 ± 0.01	0.78 ± 0.05	74.4	25.6
20	Roman chamomile	Asteraceae	flower	0.75 ± 0.01	0.01	0.76 ± 0.01	98.7	1.3
21	star anise	Illiciaceae	fruit	0.29	0.42 ± 0.02	0.71 ± 0.02	40.8	59.2
22	nigella	Ranunculaceae	seed	0.15 ± 0.06	0.52 ± 0.03	0.67 ± 0.09	22.4	77.6
23	curry leaf tree	Rutaceae	leaf	0.40 ± 0.06	0.11 ± 0.01	0.51 ± 0.07	78.4	21.6
24	turmeric	Zingiberaceae	rhizome	0.24	0.26 ± 0.04	0.50 ± 0.04	48.0	52.0
25	hyssop	Lamiaceae	leaf, flower	0.48 ± 0.02	0.02	0.50 ± 0.02	96.0	4.0
26	allspice	Myrtaceae	fruit	0.31 ± 0.01	0.18 ± 0.02	0.49 ± 0.03	63.3	36.7
27	anise	Apiaceae	seed	0.16 ± 0.01	0.30 ± 0.09	0.46 ± 0.10	34.8	65.2
28	brown mustard	Brassicaceae	seed	0.45	0.01	0.46	97.8	2.2
29	nutmeg	Myristicaceae	kernel	0.12 ± 0.01	0.33 ± 0.07	0.45 ± 0.08	26.7	73.3
30	vanilla	Orchidaceae	fruit	0.13 ± 0.01	0.32 ± 0.03	0.45 ± 0.04	28.9	71.1
31	Japanese mint	Lamiaceae	leaf, stem	0.39 ± 0.01	0.05	0.44 ± 0.01	88.6	11.4
32	German chamomile	Asteraceae	flower	0.38 ± 0.01	0.06	0.44 ± 0.01	86.4	13.6
33	cape jasmine	Rubiaceae	fruit	0.21 ± 0.01	0.20 ± 0.03	0.42 ± 0.04	50.0	47.6
34	cumin	Apiaceae	seed	0.34 ± 0.01	0.08 ± 0.03	0.42 ± 0.01	81.0	19.0
35	lemongrass	Poaceae	leaf	0.25 ± 0.01	0.15 ± 0.02	0.40 ± 0.03	62.5	37.5
36	parsley	Apiaceae	leaf, stem	0.39 ± 0.01	0.01 ± 0.01	0.40 ± 0.02	97.5	2.5
37	sweet basil	Lamiaceae	leaf	0.37 ± 0.01	0.02	0.39 ± 0.01	94.9	5.1
38	saffron	Iridaceae	pistil	0.35 ± 0.01	0.03	0.38 ± 0.01	92.1	7.9
39	licorice	Fabaceae	rhizome	0.36 ± 0.01	0.01	0.37 ± 0.01	97.3	2.7
40	garden chervil	Apiaceae	leaf	0.36 ± 0.01	0.01	0.37 ± 0.01	97.3	2.7
41	common juniper	Verbenaceae	fruit	0.19 ± 0.01	0.17 ± 0.05	0.36 ± 0.06	52.8	47.2
42	celery	Apiaceae	seed	0.19 ± 0.01	0.16 ± 0.03	0.35 ± 0.04	54.3	45.7
43	ginger	Zingiberaceae	rhizome	0.07 ± 0.01	0.26 ± 0.05	0.33 ± 0.06	21.2	78.8
44	ajwain	Apiaceae	seed	0.22 ± 0.01	0.12 ± 0.01	0.33 ± 0.03	66.7	36.4
45	pepper	Piperaceae	fruit	0.17 ± 0.01	0.15 ± 0.01	0.32 ± 0.02	53.1	46.9
46	sweet orange	Rutaceae	peel	0.30 ± 0.01	0.01	0.31 ± 0.01	96.8	3.2
47	red pepper	Solanaceae	fruit	0.14 ± 0.01	0.16 ± 0.05	0.30 ± 0.06	46.7	53.3
48	caraway	Apiaceae	seed	0.11	0.14 ± 0.04	0.25 ± 0.04	44.0	56.0
49	cardamon	Zingiberaceae	fruit	0.04 ± 0.01	0.21 ± 0.03	0.25 ± 0.04	16.0	84.0
50	fenugreek	Fabaceae	seed	0.16 ± 0.01	0.09 ± 0.01	0.25 ± 0.02	64.0	36.0
51	dill	Apiaceae	leaf	0.23 ± 0.01	0.02 ± 0.01	0.25 ± 0.02	92.0	8.0
52	fennel	Apiaceae	seed	0.11 ± 0.01	0.11	0.22 ± 0.01	50.0	50.0
53	coriander	Apiaceae	leaf	0.19 ± 0.01	0.01	0.20 ± 0.01	95.0	5.0
54	angelica	Apiaceae	root	0.13	0.06 ± 0.01	0.19 ± 0.01	68.4	31.6
55	sweet basil	Lamiaceae	seed	0.08 ± 0.01	0.10 ± 0.01	0.18 ± 0.02	44.4	55.6
56	chives	Liliaceae	leaf	0.17 ± 0.01	0.00	0.17 ± 0.01	>99.9	<0.1
57	common wormwood	Asteraceae	leaf	0.16	0.01	0.17	94.1	5.9
58	dill	Apiaceae	seed	0.14	0.02 ± 0.01	0.16 ± 0.01	87.5	12.5
59	hemp	Cannabaceae	seed	0.03	0.12 ± 0.01	0.15 ± 0.01	20.0	80.0
60	sesame	Pedaliaceae	seed	0.03	0.11 ± 0.01	0.14 ± 0.01	21.4	78.6
61	asafoetida	Apiaceae	rhizome	0.09	0.04 ± 0.01	0.13 ± 0.01	69.2	30.8
62	coriander	Apiaceae	seed	0.05	0.08 ± 0.03	0.13 ± 0.03	38.5	61.5
63	watercress	Brassicaceae	leaf	0.08 ± 0.01	0.03	0.11 ± 0.01	72.7	27.3
64	poppy	Papaveraceae	seed	0.01	0.07 ± 0.01	0.08 ± 0.01	12.5	87.5
65	horseradish	Brassicaceae	rhizome	0.04	0.01	0.05	80.0	20.0
66	garlic	Liliaceae	bulb	0.03 ± 0.01	0.01	0.04 ± 0.01	75.0	25.0
67	onion	Liliaceae	bulb	0.04	0.00	0.04	>99.9	<0.1
68	tamarind	Fabaceae	fruit	0.03	0.00	0.03	>99.9	<0.1

Each value represents the mean ± SEM (n = 3).

In vitro assay of ORAC values    The ORAC for each plant material was evaluated according to the methods of Prior et al. (2003). Hexane (10 mL × 2) was added to the dried plant (1 g), followed by vortexing (30 sec) and sonication (37°C, 5 min). After centrifugation (2500 g, 15 min), the hexane layer was collected.

The solution for the L-ORAC assay was prepared as follows. The hexane solution was concentrated. The dried hexane extract was dissolved in acetone (10 mL) and then diluted with 7% (w/v) randomly methylated β-cyclodextrin (RMCD) solution (50% acetone/50% water, v/v). The 7% RMCD solution was used as a blank.

The solution for the H-ORAC assay was prepared as follows. The residue, which was separated by hexane extraction, was extracted with acetone/water/acetic acid (70:29.5:0.5, v/v/v, 10 mL). The extract was vortexed (30 sec), followed by sonication (37°C, 5 min). After standing for 10 min with occasional shaking, the extract was filtered. The filtrate was diluted to a total volume of 25 mL with acetone/water/acetic acid (70:29.5:0.5, v/v/v).

The H-ORAC and L-ORAC assays were carried out as follows. As the standard, trolox (6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid; Sigma-Aldrich, St. Louis, MO, USA) calibration solutions (0 – 200 µM) were pipetted into a 96-well microplate. Each sample (15 µL) and 150 µL of 0.2 µM fluorescein sodium salt (Sigma-Aldrich) solution diluted with 75 mM phosphate buffer (pH 7.0) were pipetted into the 96-well microplate, and incubated at 37°C for 10 min. After the addition of 60 µL of 30 mM AAPH (Wako Pure Chemical Industries, Ltd., Osaka, Japan) solution diluted with 75 mM phosphate buffer (pH 7.0), the microplate was placed immediately in a microplate reader (Wallac 1420 Victor 2, Perkin-Elmer Inc., Minneapolis, MN, USA) preheated to 37°C. Fluorescence was recorded at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The calculation of net area under the curve (AUC) was obtained from the difference of AUC between the sample and the blank (phosphate buffer only). ORAC was expressed as mmol/Trolox equivalents (TE)/g of dried plant, which was calculated using the calibration curve generated in each assay.

Sensory testing of 10 Lamiaceae plants possessing high T-ORAC values    Hot water (100°C, 50 mL) was added to each dry material (0.5 g) of 10 plants (clove, oregano, common thyme, peppermint, lemon balm, Sichuan pepper, sweet marjoram, tarragon, damask rose and rosemary). The extract was obtained after immersing samples in boiling water for 3 min. Lemon balm extract was used as the standard of bitterness intensity because it had the lowest bitterness among the 10 plants. The bitterness intensity of each extract was evaluated by four evaluators using a two-fold dilution method. The evaluation was carried out with nose clips to exclude the effect of odor.

Preparation of hydrophilic fractions of three Lamiaceae plants    Lamiaceae plants (peppermint, lemon balm, rosemary; 10 g each) were crushed and extracted with 50% aqueous ethanol (100 g) for 10 min at room temperature. After filtration of the mixture, distilled water (DW) was added to the filtrate to adjust the concentration to 30% ethanol. The 30% aqueous ethanol solution was passed through SEPABEADS^® SP70 (Mitsubishi Chemical Co., Ltd., Tokyo, Japan), a synthetic absorbent, and subjected to the H-ORAC assay. Then, the eluent was evaporated and freeze-dried under vacuum. The yields of hydrophilic fractions of peppermint (PE), lemon balm (LB) and rosemary (RM) were 2.0 g, 2.3 g and 1.4 g, respectively.

HPLC analysis of PE, LB and RM    The HPLC/UV chromatograms of PE, LB and RM were obtained using an Agilent 1200 Series HPLC system equipped with a diode array detector (Agilent Technologies, Santa Clara, CA, USA) and a CAPCELL PAK C18MG column (250 mm × 4.6 mm i.d.; particle size, 5 µm; Shiseido, Tokyo, Japan). The operating conditions were as follows: column oven temperature, 40°C; mobile phase, a linear gradient from 100% solvent A (10% acetonitrile adjusted to pH 2.5 by phosphoric acid) to 100% solvent B (acetonitrile) in 30 min; flow rate, 1 mL/min; and injection volume, 20 µL, detection, 280 nm (eriocitrin) and 310 nm (rosmarinic acid). All samples were filtered through a 0.45 µm PTFE membrane-filter (DISMIC-13HP, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) prior to injection.

Animals and experimental design    All procedures were performed in accordance with the Guidelines for Animal Experiments of the College of Bioresource Sciences of Nihon University (approval number, AP11B013). Six-week-old male Sprague-Dawley SPF rats (weighing 230 – 250 g at the onset of experiments) (Japan SLC, Inc., Shizuoka, Japan) were used in this study. Rats were housed individually at 22 ± 2°C under a controlled 12 h light/darkness cycle with free access to chow (CE-2, CLEA Japan, Inc., Tokyo, Japan) and tap water for one week. The rats were randomly allocated to 8 groups of 6 animals each (group I (DW, normal control), group II (PE), group III (LB), group IV (RM), group V (DW + CCl₄, CCl₄ model control), group VI (PE + CCl₄), group VII (LB + CCl₄) and group VIII (RM + CCl₄)). PE, LB or RM was dissolved in water at a final concentration of 10% (w/v) and intragastrically administered (1 g/kg of body weight) for 5 days under unanesthetized conditions. The same volume of water was administered to groups I and V. On the sixth day, 50% CCl₄ in olive oil (1 mL/kg) was intraperitoneally injected into the rats belonging to groups V – VIII. After fasting for 20 hours, blood and liver samples of all rats (groups I – VIII) were collected under anesthesia. Blood samples were drawn from the abdominal aorta. Liver samples were homogenized with 0.1 M potassium phosphate buffer containing 0.25 M sucrose solution (pH 7.4, 4 times the liver weight) and centrifuged (10000 g, 4°C, 15 min). The supernatant was further centrifuged (105000 g, 4°C, 60 min) to separate the cytosolic (supernatant) and microsomal (precipitate) fractions. The mixture of 0.1 M potassium phosphate buffer containing 0.25 M sucrose solution (pH 7.4, 5 mL) was added to the precipitate.

Biochemical assays    After centrifugation (1500 g, 4°C, 15 min) of the blood samples, the supernatants were obtained as serum samples. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in each serum sample were measured using the dry clinical chemical analyzer SpotChem™ EZ SP-4403 (Arkray Inc., Kyoto, Japan).

Liver lipid peroxidation was determined by measuring the formation of thiobarbituric acid reactive substances (TBARS) (Uchiyama and Mihara, 1978). A mixture of 1% phosphoric acid (0.3 mL) and 0.67% thiobarbituric acid (TBA, 1.0 mL) was added to the liver homogenate solution (0.5 mL), the reference solution of malondialdehyde (MDA) (0.5 mL) for a calibration curve and saline (0.5 mL) as a control, respectively. Each mixture was heated at 95°C for 45 min with continuous stirring. After cooling to room temperature, n-butanol (4 mL) was added to the mixture, followed by vigorous shaking. The n-butanol layer was separated by centrifugation (2650 g, 10 min). The value of TBARS was calculated from the difference in absorbance at 535 nm and 520 nm using a spectrophotometer (Cary 50; Varian Medical Systems, Inc., Palo Alto, CA, USA). The value of TBARS was expressed as nmol of MDA equivalents per mg of protein.

Superoxide dismutase (SOD) activity was obtained by measuring the inhibition rate of the formation of water insoluble blue formazan produced from the reaction of nitro blue tetrazolium (NBT) and O₂⁻• (Beauchamp and Fridovich, 1971). A mixture of 50 mM sodium carbonate buffer (pH 10.2, 960 µL) containing 0.1 mM xanthine, 0.025 mM NBT and 0.1 mM sodium-EDTA was preincubated at 25°C. The absorbance of the mixture at 560 nm was defined as the initial value. The liver cytosolic fraction (20 µL) and xanthine oxidase (20 µL) were added to the mixture to form formazan. The absorbance of formazan was measured at 560 nm for 5 min using a spectrophotometer (Cary 50). The value of the blank was measured by the addition of 0.1 M potassium phosphate buffer containing 0.25 M sucrose (pH 7.4) instead of the liver cytosolic fraction. The activity of SOD was expressed as units/min/mg of protein.

The activity of glutathione S-transferase (GST) was obtained by measuring the formation rate of S-(2,4-dinitrophenyl)glutathione produced by the conjugation of glutathione with 1-chloro-2,4-dinitrobenzene (Habig et al., 1974). A mixture of 30 mM glutathione (60 µL) and 0.1 M potassium phosphate buffer (pH 7.4, 1.48 mL) was prepared and maintained at 37°C. The absorbance of the mixture at 340 nm was defined as the initial value. The liver cytosolic fraction (300 µL) and 30 mM 1-chloro-2,4-dinitrobenzene (60 µL) were added to the mixture. The absorbance of S-(2,4-dinitrophenyl)-glutathione was measured at 340 nm for 5 min using a spectrophotometer (Cary 50). The value of the blank was obtained by measurement of 0.1 M potassium phosphate buffer containing 0.25 M sucrose (pH 7.4) instead of the cytosolic fraction. The activity of GST was expressed as µmol/min/mg of protein.

The activity of CYP2E1 was obtained by measuring the formation rate of p-nitrocatechol produced by the hydroxylation of p-nitrophenol (Chang et al., 1998). A mixture containing 440 µL of 50 mM potassium phosphate buffer (pH 7.4), 10 µL of 5 mM p-nitrophenol in potassium buffer (pH 7.4), 25 µL of solution containing 26 mM NADP⁺, 66 mM D-glucose-6-phosphate and 66 mM magnesium chloride, and 5 µL of 5 mM sodium citrate solution containing glucose-6-phospate dehydrogenase (40 U/mL) was warmed at 37°C. The liver microsomal fraction (equivalent to 5 mg/mL of protein) was added to the mixture, followed by incubation at 37°C for 30 min in the dark. Then, 20% trichloroacetic acid (100 µL) was added to stop the reaction. After cooling under ice-cold conditions and centrifugation (13000 g, 5 min), the supernatant (500 µL) was separated. A solution of 2 M sodium hydroxide (250 µL) was added to the supernatant. The absorbance of p-nitrocatechol at 535 nm was measured using a spectrophotometer (UV-mini 1240, Shimadzu Corp., Kyoto, Japan). The value of the blank was obtained from the microsomal fraction boiled for 5 min to inactivate the enzyme. The activity of CYP2E1 was expressed as nmol/min/mg of protein.

Protein contents were determined by a BCA™ protein assay kit (Wako Pure Chemical Industries, Ltd.) using bovine serum albumin as a standard.

Statistical analysis    Results are presented as means ± SEM. Statistical analysis was conducted by one-way ANOVA with Tukey's multiple-comparison post-hoc test using the commercially available software package GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA). A probability value of p < 0.05 was regarded as significant.

Results

Antioxidant activities of 68 culinary herbs and spices    H-, L- and T-ORAC values of 68 culinary herbs and spices are shown in Table 2. T-ORAC values ranged from 0.03 ± 0.00 to 2.89 ± 0.17 mmol of TE/g of dry plant. Clove possessed the highest T-ORAC (2.89 ± 0.17 mmol of TE/g of dry plant) of all plants, with L-ORAC (1.70 ± 0.12 mmol of TE/g of dry plant) being higher than H-ORAC (1.19 ± 0.05 mmol of TE/g of dry plant). Six Lamiaceae leaves (oregano, common thyme, peppermint, lemon balm, sweet marjoram and rosemary; T-ORAC from 1.20 ± 0.07 to 2.29 ± 0.11 mmol of TE/g of dry plant) were included among the top 10 plants exhibiting high T-ORAC (from 1.20 ± 0.07 to 2.89 ± 0.17 mmol of TE/g of dry plant). Their H-ORACs made up 93.9 – 98.6% of T-ORAC. In addition, even for other Lamiaceae leaves or flowers (winter savory, perilla, true lavender, sage, hyssop, Japanese mint and sweet basil leaf), H-ORAC was higher than L-ORAC. In contrast, sixteen plants (clove, star anise, nigella, turmeric, anise, nutmeg, vanilla, ginger, red pepper, caraway, cardamom, sweet basil seed, hemp, sesame, coriander seed and poppy) showed higher L-ORAC compared to H-ORAC. As for the plant parts assessed, eight were seeds (nigella, anise, caraway, sweet basil seed, hemp, sesame, coriander seed and poppy), four were fruits (star anise, vanilla, red pepper and cardamom) and two were rhizomes (turmeric and ginger).

Bitterness of extracts of top 10 plant extracts possessing high T-ORAC values    As shown in Fig. 1, the bitterness intensity of the hot water extracts decreased in the order: clove > rosemary > oregano > Sichuan pepper > damask rose > sweet marjoram > common thyme > peppermint, tarragon > lemon balm.

Fig. 1.

Bitterness intensity of the extracts of 10 culinary herbs and spices.

Each value represents the mean ± SEM (n = 4).

Content and dose of eriocitrin and rosmarinic acid    The content of eriocitrin, a main component of PE, was 296 mg/g in PE, and that of rosmarinic acid, a main component of LB and RM, was 157 mg/g in LB and 119 mg/g in RM, respectively. Therefore, the dose of eriocitrin was 296 mg/kg of body weight for the PE administered group, and that of rosmarinic acid was 157 mg/kg of body weight for the LB administered group and 119 mg/kg of body weight for the RM administered group.

Effects on hepatotoxicity indices    AST and ALT activities in serum were used for the evaluation of hepatic disorder. As shown in Fig. 2A and 2B, the administration of LB, PE or RM did not affect the enzyme activities of either normal control. However, after CCl₄ administration, the activities of AST and ALT significantly increased compared to that of the normal controls. In contrast, the administration of PE or RM significantly reduced elevated AST and ALT activities induced by CCl₄ administration. As for LB administration, a similar tendency was observed.

Fig. 2.

Effects of PE, LB and RM on the serum (A) AST and (B) ALT activities in rats.

I: normal control; II: PE; III: LB; IV: RM; V: CCl₄ model control; VI: PE + CCl₄; VII: LB + CCl₄; VIII: RM + CCl₄. Each value represents the mean ± SEM for 4 – 6 rats per group. Values with different superscript letters are significant difference at p < 0.05.

Effects on hepatic oxidative indices    As shown in Fig. 3, CCl₄ administration significantly increased the TBARS level compared to the normal control. In contrast, the administration of PE, LB or RM significantly suppressed the elevation of TBARS induced by CCl₄ administration.

Fig. 3.

Effects of PE, LB and RM on the levels of TBARS in rats.

I: normal control; II: PE; III: LB; IV: RM; V: CCl₄ model control; VI: PE + CCl₄; VII: LB + CCl₄; VIII: RM + CCl₄. Each value represents the mean ± SEM for 4 – 6 rats per group. Values with different superscript letters are significant difference at p < 0.05.

Effects on antioxidant enzyme    As shown in Fig. 4, the administration of LB, PE or RM did not induce SOD, an endogenous antioxidant enzyme. After CCl₄ administration, SOD activity significantly decreased compared to that of the normal control, and was significantly recovered by the administration of PE, LB or RM.

Fig. 4.

Effects of PE, LB and RM on the SOD activities in rats.

I: normal control; II: PE; III: LB; IV: RM; V: CCl₄ model control; VI: PE + CCl₄; VII: LB + CCl₄; VIII: RM + CCl₄. Each value represents the mean ± SEM for 4 – 6 rats per group. Values with different superscript letters are significant difference at p < 0.05.

Effects on detoxification enzymes    As shown in Fig. 5, the administration of LB, PE or RM did not induce GST, a phase II detoxification enzyme. After CCl₄ administration, the GST activity significantly decreased compared to that of normal control, and was recovered by the administration of PE, LB or RM, although not significantly so.

Fig. 5.

Effects of PE, LB and RM on the GST activities in rats.

I: normal control; II: PE; III: LB; IV: RM; V: CCl₄ model control; VI: PE + CCl₄; VII: LB + CCl₄; VIII: RM + CCl₄. Each value represents the mean ± SEM for 4 – 6 rats per group. Values with different superscript letters are significant difference at p < 0.05.

As shown in Fig. 6, the administration of LB, PE or RM did not affect the activity of CYP2E1, a phase I detoxification enzyme. After CCl₄ administration, CYP2E1 activity significantly decreased compared to that of the normal control. PE, LB or RM administration did not restore CYP2E1 activity.

Fig. 6.

Effects of PE, LB and RM on the CYP2E1 activities in rats.

I: normal control; II: PE; III: LB; IV: RM; V: CCl₄ model control; VI: PE + CCl₄; VII: LB + CCl₄; VIII: RM + CCl₄. Each value represents the mean ± SEM for 4 – 6 rats per group. Values with different superscript letters are significant difference at p < 0.05.

Discussion

In identifying antioxidants from culinary plants effective against oxidative stress in vivo, initial comparative study on their activities in vitro represents a good starting point. However, to date, reports on culinary herbs and spices remain limited (Zheng and Wang, 2001; Wu et al., 2004; Shan et al., 2005). In general, it is difficult to make comparisons between the antioxidative activity of plant extracts from these reports because of differences in assay conditions and the form of plant materials (fresh or dried). The aim of this study was to compare the data on 68 culinary herbs and spices, which comprised 25 families, under the same experimental conditions. Cultivating conditions such as production area, weather and harvest time can affect the content of secondary metabolites in plants. However, this study did not evaluate factors that influence antioxidative activity.

In this study, Lamiaceae leaves or flowers were found to have higher antioxidative activity compared to plants belonging to the other 24 families. In addition, the activities of the hydrophilic fractions were significantly higher than those of the lipophilic fractions. The ORAC assay is based on a hydrogen atom transfer (HAT) mechanism; therefore, phenolic compounds might predominantly contribute towards transferring one hydrogen atom to the peroxyl radical (ROO•). While the relation between total polyphenolic components and antioxidative capacity is not strong (Kähkönen et al., 1999), any hydrophilic polyphenols could predominantly contribute to the high H-ORAC values in Lamiaceae plants (Carnat et al., 1998; Dorman et al., 2003, 2004; Koşar et al., 2004; Fecka and Turek, 2007; Torras-Claveria et al., 2007; Dastmalchi et al., 2008; Barros et al., 2013).

Furthermore, the H-ORAC to L-ORAC ratio was associated not only with the plant family but also with the plant part assessed. In general, the seeds of many plants show higher L-ORAC compared to H-ORAC. For sweet basil (Fam. Lamiaceae), the seeds provided higher L-ORAC than H-ORAC; in contrast, the leaf showed higher H-ORAC than L-ORAC. Coriander seed and leaf also showed a similar tendency. Seed oil, obtained from herbs, spices and fruits, contains a variety of lipophilic compounds such as tocopherols, carotenoids, lignans and unsaturated fatty acids, which act differently in vivo compared to hydrophilic compounds (Yu et al., 2005; Parry et al., 2006). Therefore, it is necessary to properly select the plant species and parts for in vivo study.

In the context of high antioxidative activity and low bitterness, we selected peppermint and lemon balm from the top 10 plants possessing high T-ORAC values for an in vivo study of oxidative stress. Rosmarinic acid is commonly the main component in both rosemary and lemon balm. In addition, rosemary is well-known and widely used as an antioxidant in foods (Shahidi, 2000; Etter, 2004). Therefore, although the bitterness intensity of the hot water extract of rosemary was not weak, rosemary was included in vivo study.

Furthermore, to improve the flavor of foods, any odorants enhancing bitterness through odor-taste interactions should be eliminated (Caporale et al., 2004; Salles, 2006). Therefore, we removed the lipophilic odorants (essential oils) from the extracts of peppermint, lemon balm and rosemary, and prepared the hydrophilic fractions, PE, LB and RM, respectively. Through this process, carnosol and carnosic acid, which are reported to be highly bitter lipophilic-diterpenoids in rosemary (Charles, 2012), were also removed. Consequently, the bitterness intensity of PE, LB and RM decreased compared to peppermint, lemon balm and rosemary extracts, respectively (data not shown).

Lipid peroxidation by CCl₄ administration plays a significant role in hepatic injury, such as inactivation of the endogenous antioxidative enzymes and damage to cell membranes followed by the release of marker enzymes into the serum (Lee and Park, 1995; Koh et al., 1999; Weber et al., 2003; Masuda, 2006).

The TBARS assay can measure hydroperoxides and aldehydes produced during lipid peroxidation, and is usually expressed as MDA equivalents. TBARS is used as an index of oxidative damage (Botsoglou et al., 1994; Devasagayam, et al., 2003). SOD, an endogenous antioxidant enzyme that defends against reactive oxygen species formed by oxidative stress, is reported to transform O₂⁻• into H₂O₂, which is finally converted to H₂O by the action of catalase and glutathione peroxidase (Halliwell et al., 1992). In addition, the leakage of marker enzymes such as AST and ALT in serum after CCl₄ administration is reported to be strongly related to histological changes in hepatic cells (Nagase and Tanaka, 1980; Sallie et al., 1991).

In the present study, hepatic injury was evidenced by a significant elevation in TBARS, decrease in the activity of SOD, and increase in AST and ALT activities in CCl₄-treated rats. Notably, the administration of PE, LB or RM decreased TBARS levels and restored SOD activity, leading to decreases in the activities of AST and ALT in serum. As the H-ORAC values of peppermint, lemon balm and rosemary were high in the in vitro study, any antioxidative components in PE, LB and RM could have significantly contributed to the inhibition of lipid peroxidation by scavenging free radical species.

Peppermint extract contains a variety of antioxidants (Duband et al., 1992; Guédon et al., 1994; Fecka and Turek, 2007; Koşar et al., 2004). Among them, eriocitrin was identified as the main polyphenolic component. Eriocitrin is reported to have high antioxidative activities such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, superoxide anion radical scavenging activity, inhibition of linoleic acid peroxidation and H₂O₂ scavenging activity (Miyake et al., 1997; Miyake et al., 1998; Sroka et al., 2005; Miyake et al., 2007). In addition, Minato et al. (2003) reported that eriocitrin in lemon peel might control changes in glutathione redox status, followed by the prevention of oxidative damage induced by acute exercise. Miyake et al. (2000) suggested that eriocitrin metabolites such as eriodictyol and 3,4-dihydroxyhydrocinnamic acid formed by the action of intestinal bacteria would be absorbed into plasma followed by an inhibition of lipid peroxidation. Therefore, eriocitrin in PE and its metabolites would prevent oxidative stress by scavenging free radicals produced from CCl₄ and controlling redox system. Further studies are needed to assess the hepatoprotective effects of eriocitrin administration on CCl₄-induced oxidative stress.

As for the rosemary extract, the hepatoprotective effect on CCl₄-induced oxidative damage in rats has been reported (Sotelo-Félix et al., 2002; Gutiérrez et al., 2010). In addition, Aruoma et al. (1992) reported that carnosol and carnosic acid, powerful antioxidative components in rosemary extract, strongly contributed to the antioxidative activity of rosemary extract and effectively inhibited lipid peroxidation. However, in this study, carnosol and carnosic acid were not detected in RM. Therefore, components other than carnosol and carnosic acid might contribute to the hepatoprotective effect of RM. Li et al. (2010) and Domitrović et al. (2013) reported that rosmarinic acid exerted hepatoprotective effects against CCl₄-induced oxidative stress. The DPPH and H₂O₂ scavenging activities of rosmarinic acid were reported to be almost equal to those of eriocitrin (Sroka et al., 2005). Rosmarinic acid was absorbed and metabolized as conjugated and/or methylated forms, and the majority of rosmarinic acid absorbed was also degraded into conjugated and/or methylated forms of caffeic acid, ferulic acid and m-coumaric acid (Baba et al., 2004). Therefore, rosmarinic acid and its metabolites would contribute to the suppression of oxidative stress by scavenging free radicals and H₂O₂. Rosmarinic acid is a main polyphenolic component not only in RM but also in LB. The content of rosmarinic acid in LB was higher than that in RM, but the activities of AST and ALT with LB administration were lower compared to those with RM administration. Therefore, further study of the relation between the rosmarinic acid dose and its hepatoprotective effect is needed.

GST, a phase II detoxification enzyme, plays an important role in detoxification by catalyzing the nucleophilic attack of glutathione on electrophilic substances (glutathione conjugation), altering xenobiotics to less toxic compounds (Armstrong, 1997). Furthermore, GST exerts protective effects not only by detoxification in a cellular environment but also through antioxidative activities in cooperation with antioxidative enzymes such as glutathione peroxidase, glutathione reductase, catalase and SOD (Singh et al., 2008; Srivastava and Shivanandappa, 2010). The decreased activity of GST after CCl₄ treatment was likely because of the release of GST from liver cells into the serum, resulting from free radical damage to the cell membrane (Lee et al., 1991; Sheweita et al., 2001). On the other hand, the administration of PE, LB and RM suppressed the decrease in GST activity caused by CCl₄ administration. As for rosemary, its potent antioxidant, carnosol, is reported to ameliorate the decrease in GST activity by CCl₄ administration in rats (Singletary, 1996; Sotelo-Felix et al., 2002). However, as RM did not contain carnosol, some other antioxidants such as orthophenolic compounds (Fiander and Schneider, 2000) might have captured reactive radicals and suppressed the inactivation of GST. Further studies are necessary to confirm the in vivo effect of rosmarinic acid, a main component in RM and LB, and eriocitrin, a main component in PE, on suppression of enzyme inactivation caused by CCl₄ administration.

In conclusion, the comparative study of 68 culinary herbs and spices clarified that H-ORAC and L-ORAC values are related not only to the plant species, but also to the plant part assessed. Six Lamiaceae plants were contained among the top 10 plants possessing high T-ORAC, with H-ORAC contributing > 90% of T-ORAC. Taking into account the ORAC assay results and the sensory evaluation of bitterness, we then compared the hepatoprotective effects of the hydrophilic fractions of three selected Lamiaceae plants (peppermint, lemon balm and rosemary) on CCl₄-induced oxidative stress in rats. The antioxidative components in these plant extracts might play an important role in hepatoprotective effects through anti-peroxidation. The hydrophilic fractions of these three Lamiaceae plants, in which bitterness and lipophilic odorants were reduced, may be useful as foodstuffs for liver protection. In this report, the hepatoprotective effect was confirmed using one dose for each sample; therefore, further investigation of the relations between sample dose and effects are needed.

Acknowledgments    We are grateful to Ms. Satsuki Inagaki (Maihama Research Center, Ogawa & Company, Ltd.) for her technical assistance.

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