Antioxidant Properties of Some Medicinal Lamiaceae (Labiatae) Species

Abstract

In this study, antioxidant properties of Thymus sipyleus. Boiss. subsp. sipyleus. var. sipyleus., Teucrium chamaedrys L.., Mentha longifolia. (L.) Hudson subsp. longifolia., Salvia limbata. C.A. Meyer, and Thymus fallax. Fisch. & Mey. were investigated. Antioxidant and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, reducing powers, and the amount of total phenolic compounds of the extracts were studied. The highest antioxidant activity was shown by T. chamaedrys. (decoction, IC₅₀: 9.2 µg/ml), and the lowest one was S. limbata. (decoction, IC₅₀: 619.5 µg/ml). The highest DPPH radical scavenging activity was shown by T. fallax. [decoction, IC₅₀S: 56 µg/ml (IC₅₀S is the extract concentration (µg/ml) required for 50% inhibition of the DPPH solution absorbance at 517 nm)] while the lowest one was S. limbata. (decoction, IC₅₀S: 335.8 µg/ml). The highest reducing power and amount of total phenolic compounds was shown by T. chamaedrys. (decoction, 29.9 µg/ml ascorbic acid equivalent, 27.9 µg/ml gallic acid equivalent, respectively), and the lowest one was S. limbata. (decoction, 5.1 µg/ml ascorbic acid equivalent, 9.9 µg/ml gallic acid equivalent, respectively) at 250 µg/ml extract concentration.

Keywords:

• Antioxidant activity
• Lamiaceae
• phenolic compound
• radical scavenging
• reducing power

Introduction

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are various forms of activated oxygen and nitrogen, which include free radicals such as superoxide ions (O₂^⋅−), hydroxyl (OH^⋅) and nitric oxide radicals (NO^⋅), as well as non-free-radical species such as hydrogen peroxide (H₂O₂), and nitrous acid (HNO₂) (Halliwell, 1995; Halliwell & Aruoma, 1997; Squadriato & Peyor, 1998). In living organisms. ROS and RNS can form in different ways. Normal aerobic respiration, stimulated polymorphonuclear leukocytes and macrophages, and peroxisomes appear to be the main endogenous sources of most of the oxidants produced by cells (Fridovich, 1986; Halliwell, 1994; Alho & Leinonen, 1999). Exogenous sources of free radicals include tobacco smoke, ionizing radiation, organic solvents, and pesticides (Halliwell & Gutteridge, 1989; Davies, 1995; Papas, 1996; Robinson et al., 1997). Free radicals can cause lipid peroxidation in foods that leads to their deterioration (Sasaki et al., 1996). ROS and RNS may cause DNA damage that could lead to mutation (Aruoma, 1998; Sawa et al., 1999). In addition, ROS and RNS have been implicated in more than 100 diseases, including malaria, acquired immunodeficiency syndrome, heart disease, stroke, arteriosclerosis, diabetes, and cancer (Tanizawa et al., 1992; Hertog et al., 1993; Duh, 1998; Alho & Leinonen, 1999). When produced in excess, ROS can cause tissue injury, while, tissue injury can itself cause ROS generation (Aruoma, 1998). Nevertheless, all aerobic organisms, including human beings, have antioxidant defenses that protect against oxidative damage and numerous damage removal and repair enzymes to remove or repair damaged molecules (Davies, 1995; Fridovich, 1995; Granelli et al., 1995; Sun et al., 1998). However, the natural antioxidant mechanisms can be inefficient, hence dietary intake of antioxidant compounds becomes important (Halliwell, 1994; Terao et al., 1994; Duh, 1998; Espin et al., 2000). Although there are some synthetic antioxidant compounds, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), that are commonly used in processed foods, it has been reported that these compounds may have side effects (Branien, 1975; Yamamoto et al., 1980; Witschi, 1981; Takahashi et al., 1985; Lindenschmidt et al., 1986; Patterson et al., 1987; Kehrer & DiGiovanni, 1990). In addition, it has been suggested that there is an inverse relationship between dietary intake of antioxidant-rich foods and the incidence of a number of human diseases (Rice-Evans et al., 1997; Lu & Foo, 2000). Therefore, research into the determination of natural antioxidant sources is important.

In this study, we have determined the antioxidant and DPPH radical scavenging activities (AA and DPPH-RS), reducing powers (RP), and amount of phenolic compounds (APC) of some medicinal Lamiaceae plants that have been used especially in Eastern Turkey.

Materials and Methods

Collection of plants and identification

Five Lamiaceae species were collected in July–August in villages of Ilιca District: Thymus sipyleus. subsp. sipyleus. var. sipyleus. (“kekik otu”), Teucrium chamaedrys. (“basur otu”), Mentha longifolia. subsp. longifolia. (“yarpuz”), Salvia limbata. (“kedi kuyruğu”), and Thymus fallax. (“kekik otu”). Their local names, parts used, uses/ailments treated, preparations, and extraction solvents are given in Tables 1 and 2. They were authenticated by Dr. Ufuk Özgen and Dr. Yusuf Kaya. Voucher specimens were deposited at Ankara Üniversitesi Eczacιlιk Fakültesi Herbaryumu (AEF) and Atatürk Üniversitesi Fen Fakültesi Herbaryumu (ATA): Mentha longifolia. (L.) Hudson subsp. longifolia. (AEF 21171), Salvia limbata. C.A. Meyer (AEF 21163), Teucrium chamaedrys. L. (AEF 21179), Thymus fallax. Fisch. & Mey. (AEF 21199), and Thymus sipyleus. Boiss. subsp. sipyleus. var. sipyleus. (ATA 9718).

Table 1 Local names, parts used, uses/ailments treated, and preparations of some Lamiaceae plants used by the villagers of Ilιca District, Erzurum, Turkey.

Botanical name and voucher specimen	Local names	Part used	Use/ailment treated	Preparation
Mentha longifolia. subsp. longifolia. (AEF 21171)	Yarpuz	Herb	Against dyspnea, cough, stomachache, hemorrhoids, and pains; for wound healing	Decoction
Salvia limbata. (AEF 21163)	Kedi otu	Herb	Against toothache	Decoction (gargle)
Teucrium chamaedrys. (AEF 21179)	Basur otu	Herb	For hemorrhoids	Powder (mixed with honey and eaten)
			Against gallbladder stones and kidney stones	Decoction
Thymus fallax. (AEF 21199) Thymus sipyleus. subsp. sipyleus. var. sipyleus. (ATA 9718)	Kekik	Herb	Against stomachache, gastric ulcer, tonsillitis, urinary system diseases, internal diseases, dyspnea; for eczema and hemorrhoids	Decoction

Table 2 Localities, parts, and extraction solvents of plants used in activity studies.

Species	Village	Collection date	Altitude (m)	Part used	Extraction solvent
Mentha longifolia. subsp. longifolia.	Başçakmak	27 Aug. 2000	1800	Herb	Water and methanol
Salvia limbata.	Rizekent	8 Aug. 2000	1800	Herb	Water and methanol
Teucrium chamaedrys.	Sorkunlu	15 Aug. 2000	1900	Herb	Water
Thymus fallax.	Sorkunlu	15 July 2000	1900	Herb	Water
Thymus sipyleus. subsp. sipyleus. var. sipyleus.	Sorkunlu	15 July 2000	1900	Herb	Water

Preparation of extract solutions

Taking into consideration traditional usage in general, the most suitable parts of plants and extraction solvents were chosen. Plants that have been used generally as decoctions by the public were extracted with water. Plants that have been employed generally for other usages (eating, powdering for treatment) were also extracted with methanol. All plants were dried and powdered using a mill before extraction.

For extraction, 20 g of powdered sample was extracted with 400 ml water or methanol by reflux for about one-half hour and then filtered. Extracts were evaporated and then lyophilized. Water extracts of M. longifolia., T. chamaedrys., T. fallax., T. sipyleus., and also methanol extracts of M. longifolia. and S. limbata. were tested. In the experiments, different concentrations of all extracts were studied and results of 50, 100, 250, and 500 µg/ml concentrations are given in Table 3.

Table 3 Antioxidant activities of plant extracts.

	Concentration (µg/ml)
	AA				DPPH-RSC				RP				APC
	50	100	250	500	50	100	250	500	50	100	250	500	50	100	250	500
T. sipyleus.	15.0	85.1	86.5	89.3	19.5	45.7	83.8	83.3	5.8	10.5	19.8	27.0	6.2	11.2	21.9	35.1
T. chamaedrys.	65.0	65.7	76.3	81.5	27.3	64.2	89.4	89.3	7.9	14.8	29.9	41.5	7.6	13.8	27.9	45.7
M. longifolia.	41.0	57.2	79.2	94.8	32.8	59.5	89.4	88.8	5.2	9.6	18.5	27.0	6.4	10.8	22.0	35.3
M. longifolia.^a.	55.4	74.2	81.3	83.5	28.6	57.8	90.1	88.9	7.0	12.4	26.4	33.3	6.4	12.0	25.1	38.2
S. limbata.	4.6	9.1	20.8	38.4	7.0	15.4	36.6	74.7	1.3	2.4	5.1	9.0	3.2	4.6	9.9	17.0
S. limbata.^a.	57.3	79.4	82.7	85.3	10.3	21.7	56.5	90.9	4.4	6.7	14.1	23.0	3.6	6.8	13.5	23.8
T. fallax.	18.8	73.7	82.2	83.2	44.7	80.7	89.3	87.5	6.0	11.5	22.1	29.9	7.4	13.1	26.2	40.3

AA, antioxidant activity; DPPH-RSC, DPPH radical scavenging activity; RP, reducing power; APC, amount of phenolic compounds.

^a.Methanol extract.

Antioxidant activity (thiobarbituric acid test)

The in vitro. antioxidant activity tests were carried out using the lipid peroxidation of liposomes assay where the thiobarbitoric acid (TBA) test has been applied to assess the efficacy of the compounds to protect liposomes from lipid peroxidation (Fernàndez et al., 1997). The TBA reaction is based on the fact that peroxidation of most membrane systems leads to formation of small amounts of free malonaldehyde (MDA). One molecule of MDA reacts with two molecules of TBA to yield a colored product, which in an acid environment absorbs light at 532 nm, and it is readily extractable into organic solvents. It can, thus, be measured and quantified spectrophotometrically. The intensity of color is a measure of MDA concentration. Absorbance at 532 nm was determined on a Helios β. UV/Vis spectrophotometer (England).

The presence of any antioxidant compound in the lipid peroxidation assay reaction mixture will lead to a reduction of the extent of peroxidation. The methanolic and/or aqueous extracts from investigated plants were tested for their antioxidant activity against liposomes that were prepared from bovine brain extract in phosphate-buffered saline (5 mg/ml) (Jacobi et al., 1999). Peroxidation was started by adding FeCl₃ and ascorbic acid followed by incubation at 37°C for 20 min. Ascorbic acid is a well-known antioxidant but also exhibits pro-oxidant properties in the presence of certain transition metal ions, such as Fe or Cu. BHT in ethanol was added to prevent lipid peroxidation during the TBA test itself. Propyl gallate was used as a positive control. Data are given as % peroxidation inhibition-concentration graphic and IC₅₀ (mg/l extract concentration required for 50% peroxidation inhibition).

DPPH radical scavenging

DPPH radical scavenging was carried out according to the Blois method (Blois, 1958) with a slight modification. Briefly, a 1 mM solution of DPPH radical solution in methanol was prepared and then 1 ml of this solution was mixed with 3 ml of extract solution in ethanol. Final concentrations of extracts were 50, 100, 250, and 500 µg/ml. After 30 min incubation in the dark, absorbance was measured at 517 nm. This activity is given as IC₅₀S. IC₅₀S is the extract concentration (μg/ml) required for 50% inhibition of the DPPH radical absorbance at 517 nm). The % DPPH radical scavenging that is calculated in the equation:

The control was the test solution without extract.

Reducing power

Measurement of the reducing power was carried out as described previously (Yιldιrιm et al., 2001). Briefly, the lyophilized extracts in 1 ml of corresponding solvents were mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide [K₃Fe(CN)₆] (1%), then the mixture was incubated at 50°C for 30 min. Afterward, 2.5 ml of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 ml of the supernatant was mixed with 2.5 ml distilled water and 0.5 ml FeCl₃ (0.1%), and the absorbance was measured at 700 nm. Final concentrations of extracts were 50, 100, 250, and 500 µg/ml. Data were presented as ascorbic acid equivalents (Asc. Acid Eq.) using a calibration curve.

Amount of total phenolic compounds

The determination of total phenolic compounds was carried out as described previously (Singleton et al., 1999). Briefly, extract solutions were transferred into test tubes, then final volumes were adjusted to 4 ml by addition of distilled water. Afterward, 0.25 ml of Folin-Ciocalteu reactive (FCR) (Fluka, Switzerland) was added to the mixture, and after 3 min, 0.75 ml of Na₂CO₃ (20%) was added. Final concentrations of extracts were 50, 100, 250, and 500 µg/ml. Subsequently, the mixtures were shaken on a shaker for 2 h at room temperature, and then the absorbance was measured at 760 nm. Gallic acid was used as a standard. The phenolic compound content was determined as the gallic acid equivalent using a calibration curve.

Statistical analysis

Each of the experiments were performed in triplicate. The results shown are the means of these measurements (Table 3). Data were analyzed with SPSS software to determine whether there was any correlation between antioxidant properties in an extract. Pearson parametric correlation analysis was carried out using SPSS software.

Results and Discussion

All of the extracts examined exhibited antioxidant properties. The most effective antioxidant potential was shown by T. chamaedrys., and the least effective was the aqueous extract of S. limbata. (Table 3 and Fig. 1).

Figure 1 IC₅₀ and IC₅₀S values of extracts (*methanol extract).

Data were analyzed with SPSS software to determine whether a correlation between antioxidant properties in an extract exist (Table 4). From the table, we can suggest that AA may be affected by different parameters, such as DPPH-RS, RP, and APC. Effects of these parameters are changeable. For example, there is a correlation between AA and DPPH-RS in some extracts but not others. However, there are statistically significant correlations between reducing powers and amount of phenolic compounds in all of the extracts. We found similar results in a previous study (Özgen et al., 2004).

Table 4 p values of correlation analyses.

		DPPH-RS	RP	APC	DPPH-RS	RP	APC
T. sipyleus.	AA	0.159	0.266	0.318	0.046	0.008	0.022	T. chamaedrys.
	DPPH-RS		0.060	0.109		0.091	0.127
	RP			0.008			0.003
M. longifolia.	AA	0.046	0.008	0.022	0.029	0.114	0.151	M. longifolia.
	DPPH-RS		0.091	0.127		0.057	0.108
	RP			0.003			0.012
S. limbata.	AA	0.001	0.000	0.001	0.234	0.255	0.244	S. limbata.
	DPPH-RS		0.002	0.002		0.001	0.005
	RP			0.001			0.002
T. fallax.	AA	0.002	0.206	0.259
	DPPH-RS		0.202	0.259
	RP			0.006

AA, antioxidant activity; DPPH-RSC, DPPH radical scavenging activity; RP, reducing power; APC, amount of phenolic compounds.

*. Methanol extract.

The Lamiaceae family consists of approximately 200 genera of cosmopolitan distribution, many of them of economic importance due to essential oil production. Most genera of the Lamiaceae are thus rich sources of terpenoids and also they contain a considerable amount of various iridoid glycosides, flavonoids, and phenolic compounds. Within this family, the flavonoids are predominantly 6- and/or 8-substituted flavone derivatives, and flavonols are rarely encountered (Valant-Vetschera, 2003).

It has been shown that the aerial parts of M. longifolia. contain flavones and their glycosides (luteolin 7-glucoside, luteolin 7-rutinoside, luteolin 7-glucuronide, apigenin 7-glucuronide, acacetin 7-rutinoside, diosmetin 7-rutinoside, hesperetin 7-rutinoside, and eriodictyol 7-rutinoside) and phenolic acids (Bourwieg & Pohl, 1973; Mimica-Dukic et al., 19961999; Sharaf et al., 1999; Ghoulami et al., 2001; Jahan et al., 2001; Ali et al., 2002).

Salvia. species are widely used in traditional medicine and are a rich source of flavonoids and phenolic acids. Flavones, flavonols, and their glycosides that are present in Salvia. constitute the majority of flavonoids. Anthocyanins are abundant in red to blue Salvia. flowers, whereas the presence of proanthocyanidins (condensed tannins) in Salvia. has not been demonstrated conclusively (Lu & Foo, 2002). S. limbata. also contains norsesterter-penes and diterpenes (Ulubelen et al., 1996).

T. chamaedrys. contains neoclerodane diterpenoids, steroidal compounds, phenylpropanoid glycosides (teucrioside and poliumoside), 3,4-dihydroxyphenylethanoid glucoside (acteoside), flavonoids (cirsimaritin-5,4-OH-6,7-OMe-flavone-, cirsiliol-5,3′,4′-OH-6,7-OMe-flavone-, and luteolin-5,7,3′,4′-OH-flavone-) and phenolic acid (6-OH caffeic acid) (Kouzi et al., 1994; Ulubelen et al., 1994; Calis et al., 1996; Bedir & Calis, 1997; Pedersen, 2000; Valant-Vetschera, 2003).

Thymus. genus contains flavonoids (luteolin, apigenin, eryodictiol, naringenin, diosmetin, and their glycosides) and phenolic acids (rosmarinic acid, 6-hydroxyrosmarinic acid, caffeic acid, 6-hydroxycaffeic acid, protocatechuic acid, chlorogenic acid, syringic acid, p.-coumaric acid, 3,5-dicaffeoylquinic acid, gentisic acid, p.-hydroxybenzoic acid, vanillic acid, and p.-coumaric acid) (Voirin et al., 1985; Miura & Nakatani, 1989; Blazquez et al., 1994; Pedersen, 2000; Zgorka & Glowniak, 2001). There are so far no reports on flavonoids and phenolic acids associated with T. sipyleus. and T. fallax..

As mentioned above, these plants contain phenolic compounds, especially flavonoids. It is well-known that flavonoids possess a wide range of antioxidant activities. These antioxidant properties are based on their phenolic structures (Hall & Cuppett, 1997). Phenolic compounds are also thought to be capable of regenerating endogenous α -tocopherol, in the phospholipid bilayer of lipoprotein particles, back to its active antioxidant form. They are also known to inhibit various types of oxidizing enzymes. These potential mechanisms of antioxidant action make the diverse group of phenolic compounds an interesting target in the search for health-beneficial phytochemicals (Halliwell & Gutteridge, 1989; Hall & Cuppett, 1997). It has been reported that rosmarinic acid is the major component present in Lamiaceae plants, and it possesses a potent antioxidant activity. Thus, the observed antioxidant properties of Lamiaceae plants are presumably strongly depend on rosmarinic acid (Hall & Cuppett, 1997).

Acknowledgment

The authors would like to thank Atatürk University Rectorate for financial support (grant 2001/137 University Research Fund).