Differential Antioxidant Effects of Consuming Tea from Sideritis clandestina subsp. peloponnesiaca on Cerebral Regions of Adult Mice

Abstract

Oxidative stress is involved in the pathophysiology of neurodegenerative diseases and aging. Many species of the genus Sideritis (mountain tea) are widely consumed in the Mediterranean region as herbal tea. This study evaluated the effect of supplementation of mice with herbal tea from Sideritis clandestina subsp. peloponnesiaca on the antioxidant status of different brain regions. To select the most bioactive herbal tea, the polyphenolic content (Folin–Ciocalteu method) and the antioxidant properties (ferric reducing antioxidant power [FRAP] and 2,2-diphenyl-1-picrylhydrazyl assays) of several taxa and different populations of the S. clandestina infusions were measured in vitro. Male adult mice had ad libitum access to water (control) or the herbal tea (4% w/v) for 6 weeks. At the end of the treatment period we assessed the total antioxidant power (FRAP assay) and the levels of malondialdehyde (indicator of lipid peroxidation) and reduced glutathione in the cerebral cortex, cerebellum, and midbrain. These biochemical measures have also been determined in liver samples used as a comparative reference peripheral tissue. Consumption of 4% herbal tea increased the total antioxidant power of the midbrain by 72% (P<.05); a significant (P<.05) decrease in malondialdehyde levels and increase in reduced glutathione content of the cerebellum (78% and 27%, respectively) and midbrain (59% and 32%, respectively) were also observed. These findings indicate that mountain tea consumption enhances the antioxidant defense of the adult rodent brain in a region-specific manner.

Introduction

S ideritis L. (Lamiaceae), growing mainly in the Mediterranean region, comprises approximately 150 species worldwide. The dried aerial parts of Sideritis species are widely consumed in Greece as an herbal tea, popularly termed mountain tea.¹ It has been used extensively in folk medicine as a calmative agent and as a treatment for inflammation, cough, and gastrointestinal disorders. Recent research has demonstrated that Sideritis species have anti-inflammatory² and antimicrobial³ activities, which have been attributed to their chemical composition. Indeed, many reports indicated that these plants contain essential oils,⁴ polyphenols⁵ (especially flavonoids⁶), and diterpenoids,⁷ which are of particular pharmacologic and nutritional interest.

A moderate in vitro antioxidant action of extracts of Sideritis species has been described elsewhere,⁶ but to our knowledge no in vivo studies have examined their effect. Mammalian brain is particularly vulnerable to oxidative stress,⁸ a factor that is considered to play a pivotal role in the pathogenesis of several age-related neurodegenerative disorders.⁹ However, the brain areas exhibit differential vulnerability to oxidative damage, probably because of differences in their endogenous antioxidant defense system.¹⁰ Therefore, we sought to investigate the antioxidant effect of consumption of tea composed of 4% S. clandestina on the cerebral cortex, cerebellum, and midbrain of adult normal mice. We also assessed the effects on the mice's livers, a reference peripheral tissue. This plant was chosen after in vitro evaluation of the polyphenolic content and the antioxidant properties of infusions of several taxa and different populations of S. clandestina, grown in different regions of the Peloponnese.

Materials and Methods

Plant material and preparation of herb extracts

S. clandestina is a species endemic in Greece (specifically, the Peloponnese). It is taxonomically divided into 2 subspecies: S. clandestina subsp. clandestina prospers in the mountains of the southern Peloponnese, and S. clandestina subsp. peloponnesiaca prospers in the mountains of the northern Peloponnese. S. clandestina subsp. clandestina was collected between July and August 2008 from the Taygetos (southern Peloponnese), Parnon (eastern Peloponnese), and Mainalo (Central Peloponnese) mountains; S. clandestina subsp. peloponnesiaca was collected from the Kyllini Mountain (northern Peloponnese). Taxonomic identification was performed at the Division of Plant Biology, Department of Biology, University of Patras, where herbarium vouchers are deposited. Green tea (blended and packed in Sri Lanka under the direction of St. Dalfour, Freres & Cie, Cour Cheverny, France), in 2-g packs, was used as standard.

Infusions were prepared with addition of 4 g of equal amounts of leaves, flowers, and stems of Sideritis taxa to 100 mL boiling water for 5 minutes. After 5 minutes, the solution was filtered and the filtrate volume was adjusted to 100 mL. Green tea was also prepared in the same way. For the animal studies, the infusions were prepared daily. For the in vitro studies, deionized distilled water was used. The weight of the dry extract was recorded after lyophilization in a Labconco FreezeZone 6 freeze-dry system.

Animals

Adult (3–4 months old) male Balb-c mice were kept in the same room under a constant temperature (23–25°C) with alternating 12-hour light/dark cycles. The mice were allowed free access to food. Mice were managed according to the Greek National Laws (Animal Act, PD 160/91).

The animals were divided into 2 groups consisting of 8 animals each. Group 1 mice served as controls and received water ad libitum. Group 2 mice had ad libitum access to 4% (w/v) tea infusion of S. clandestina subsp. peloponnesiaca for 6 weeks (40 days), based on the protocol of Choudhary and Verma¹¹ with slight modifications. Body weight was measured weekly in both groups and did not significantly differ between them at the end of the treatment period.

On the completion of the 40-day treatment period, all animals were sacrificed by light ether anesthesia. Liver and brain were excised immediately, and cerebral cortex, cerebellum, and midbrain were separated from the whole brain. The tissue samples were kept at −75°C until use.

Preparation of tissue homogenates

Brain regions and liver samples were weighed and homogenized (10% w/v) with a glass-Teflon homogenizer in ice-cold 1.15% KCl.¹² Tissue homogenates were centrifuged for 5 minutes at 15000 g, and the supernatants were used to determine the antioxidant activity (by ferric reducing antioxidant power [FRAP] assay) and malondialdehyde (MDA) and reduced glutathione levels.

Methods

Total phenolics assay

Total polyphenols were determined spectrophotometrically by using Folin–Ciocalteu reagent.^13,14 Aqueous solutions of gallic acid (Sigma-Aldrich), in the range of 25–800 mg/L, were used as reference standards, and total phenol content was expressed as gallic acid equivalents.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical–scavenging capacity assay

The antioxidant properties of the infusions were evaluated by the DPPH free-radical method. This assay is based on the measurement of the reducing ability of antioxidants toward DPPH•^• by measuring the decrease of its absorbance at 515 nm.¹⁵ The radical-scavenging activity of the samples tested, expressed as percentage inhibition of DPPH^•, was calculated according to the following formula:¹⁶ Inhibition (%)=[(A_B − A_A)/A_B]×100, where A_B is the absorbance of the blank sample and A_A is the absorbance of the antioxidant tested. IC₅₀ values of the plant extracts, defined as the concentration of the sample that causes 50% inhibition of DPPH^•, were also calculated from the kinetic curve of the percentage inhibition of DPPH^• toward different concentrations of each sample and were expressed in mg/mL.

FRAP assay

The antioxidant activity of the plant extracts and the tissue samples was determined by FRAP assay.¹² Aqueous solutions of known Fe^II concentration (range, 5–1000 μM FeSO₄) were used as standard solutions. The data were expressed as μmol of Fe^II equivalents per mg dry extract for the tea infusions and as nmol of Fe^II equivalents per mg wet tissue weight for the brain and liver tissues (FRAP value).

Lipid peroxidation assay

Lipid peroxidation in the brain regions and liver was assessed fluorometrically at 553 nm (emission wavelength) and 515 nm (excitation wavelength) by measuring the formation of MDA.¹⁷ Solutions of known MDA concentration (0.05–10 μM) were used as standards. Results are reported as μmol MDA per g of protein.

Reduced glutathione determination

Reduced glutathione content of the tissue samples was estimated fluorometrically according to the procedure of Mokrasch and Teschke.¹⁸ Standard solutions of reduced glutathione were prepared in a range of 0.5–100 μM. The tissue reduced glutathione content was expressed as μmol reduced glutathione per g of protein.

Protein determination

Total protein concentration was determined by the Bradford method,¹⁹ using bovine serum albumin as standard.

Statistical analysis

The results are expressed as mean±standard error. Statistical analysis was performed with GraphPad Instat 3 software using the nonparametric Mann–Whitney test for evaluating statistically significant differences (P<.05) between the experimental groups.

Results and Discussion

To select the most bioactive population of all S. clandestina samples among those collected, their polyphenolic content and antioxidant activity were determined in vitro. Table 1 presents the total polyphenol content of all Sideritis taxa infusions tested and their solid residue contents. All tea infusions of S. clandestina taxa and populations had significantly (P<.05) lower polyphenolic composition than Camellia sinensis leaves, which were used as the standard. The polyphenolic content of 4 of 5 populations of S. clandestina subsp. clandestina was significantly lower than that of S. clandestina subsp. peloponnesiaca. The in vitro antioxidant properties of the plant extracts were measured by DPPH and FRAP assays (Table 1). The infusion of S. clandestina subsp. peloponnesiaca displayed significantly (P<.05) stronger in vitro antioxidant properties than the other taxa of S. clandestina. However, the in vitro antioxidant properties of S. clandestina subsp. peloponnesiaca were lower than those of green tea. Similarly, Triantaphyllou et al.²⁰ showed that the water extract of China black tea displayed higher total phenol content and antioxidant activity than that of S. reaseri collected from a mountainous region of northern Greece.

Table 1.

Total Polyphenol Content and Antioxidant Activity of Sideritis Taxa Water Extracts

			Antioxidant Activity
Sideritis samples	Solid residue content of extracts (mg/100 mL) ^a	Concentration of polyphenols (μg gallic acid/mg) ^b	DPPH assay—IC₅₀ (mg/mL) ^c	FRAP assay (μmol Fe^II/mg) ^d
S. clandestina subsp. clandestina (Taygetos I)	302	36.8±0.9^**	7.75±0.55^**	0.43±0.07^**
S. clandestina subsp. clandestina (Taygetos II)	332	34.6±1.8^**	7.31±0.39^**	0.49±0.12^**
S. clandestina subsp. clandestina (Parnon)	404	30.3±2.2^**	7.58±1.12^**	0.56±0.09^**
S. clandestina subsp. clandestina (Taygetos III)	262	44.7±0.8	16.18±1.69^**	0.27±0.07^**
S. clandestina subsp. clandestina (Mainalo)	500	23.7±0.5^**	8.90±1.55^**	0.33±0.03^**
Green tea (Camellia sinensis)	744	165.0±8.8	0.38±0.12	4.84±0.33
S. clandestina subsp. peloponnesiaca (Kyllini)	330	45.6±1.7	2.79±0.21	1.19±0.09

Values are the mean of 4 or 5 independent tea preparations±standard error.

Expressed as mg dry extract per 100 mL of infusion.

Expressed as μg gallic acid equivalent per mg of dry extract.

Expressed as IC₅₀ values corresponding to the aqueous extract concentration (mg/mL) causing 50% inhibition of DPPH radical.

Expressed as μmol Fe^II per mg dry extract.

P<.05 compared with S. clandestina subsp. peloponnesiaca.

DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric reducing antioxidant power; IC₅₀, 50% inhibitory concentration.

Another study showed that not only the species but also many environmental factors,²¹ such as the soil type, sun exposure, and rainfall, have a strong effect on polyphenol composition. This finding explains the variation observed among the different populations of S. clandestina subsp. clandestina. All S. clandestina infusions had significantly lower antioxidant activity than C. sinensis, in accordance with their lower polyphenolic content. Tunalier et al.,⁵ in their study of the antioxidant properties and phenolic composition of 27 Sideritis species grown in Turkey, indicated a linear relationship between these 2 measures. Thus, the infusion of S. clandestina subsp. peloponnesiaca displayed high polyphenolic content and strong antioxidant properties in vitro, rendering it suitable for the further in vivo study.

To this end, the biological effects of 4% (w/v) herbal tea from S. clandestina subsp. peloponnesiaca consumption for 6 weeks by adult control mice were further investigated. We assessed the total antioxidant activity, the extent of lipid peroxidation, and reduced glutathione content of 3 brain regions (cerebral cortex, cerebellum, and midbrain) and a peripheral tissue (liver) in control and treated mice. As shown in Table 2, there is a hierarchy in antioxidant/reducing capacity and MDA levels of the examined brain regions in control animals: cerebral cortex > cerebellum > midbrain; the differences reached significance (P<.05) between all brain areas. However, with respect to their reduced glutathione content, the cerebellum ranks higher than the cerebral cortex. In accordance with our observation, Feoli et al. also reported a similar hierarchy in total antioxidant reactivity levels, as estimated by total antioxidant reactivity assay.²² Other studies also reported greater lipid peroxidation in the cerebral cortex than in the cerebellum or midbrain of young control rats^23,24 and higher reduced glutathione content in rat cerebellum than in the cortex and midbrain.¹⁰ These regional differences could be attributed to the differences in structure and physiology of the brain areas, leading to differences in vulnerability to oxidative stress and activity of their endogenous antioxidant defense system.

Table 2.

Effect of Herbal Tea Intake on Biochemical Measures on Oxidant/Antioxidant Status of Adult Mouse Brain and Liver

	FRAP (nmol Fe^II/mg wet tissue)		MDA (μmol/g protein)		GSH (μmol/g protein)
Mouse tissue	Control mice	Treated mice (4% herbal tea)	Control mice	Treated mice (4% herbal tea)	Control mice	Treated mice (4% herbal tea)
Cerebral cortex	6.018±0.408	6.047±0.172	3.273±0.273	2.802±0.144	12.472±0.430	12.560±0.683
Cerebellum	3.404±0.378^**	3.744±0.389	1.957±0.203^**	0.426±0.011^* (↓ 78%)^a	16.265±1.085^**	20.612±1.109^* (↑ 27%)^a
Midbrain	2.176±0.281^,*	3.743±0.391^* (↑ 72%)^a	1.381±0.112^,*	0.555±0.047^* (↓ 59%)^a	14.145±0.493	18.737±0.919^* (↑ 32%)^a
Liver	17.222±0.512^****	18.717±1.976	1.659±0.136^**	1.524±0.077	7.271±0.144^****	6.836±0.159

Values are expressed as mean±standard error of 8 values corresponding to 8 animals.

Percentage change (↑ increase, ↓ decrease) compared with control mice.

P<.05 compared with control mice.

P<.05 compared with cerebral cortex of the control mice.

***

P<.05 compared with cerebellum of the control mice.

****

P<.05 compared with each brain region of the control mice.

FRAP, ferric reducing antioxidant power; GSH, reduced glutathione; MDA, malondialdehyde.

It is known that the brain regions vary with respect to their fatty-acid composition because white matter rich in myelin contains fewer polyunsaturated fatty acids (the major targets of free radicals) than the grey matter.²⁵ In this context, the midbrain, a heavily myelinated region, is expected to be more resistant to lipid peroxidation than cerebral cortex and cerebellum^10,26 and thus demanding the development of lower degree of endogenous antioxidant defense. As shown in Table 2, comparison of the above biochemical measures among liver and the brain regions of the controls revealed significantly higher total antioxidant capacity, significantly lower reduced glutathione content, and a moderate level of lipid peroxidation in liver, in accordance with findings from a previous study in normal rats.²⁷

Consumption of the tea significantly increased (P<.05) the antioxidant capacity of midbrain by 72% but did not significantly influence the antioxidant power of the cerebral cortex, cerebellum, and liver (Table 2). Furthermore, herbal tea intake led to a significant reduction in MDA levels (Table 2) in the cerebellum (78%) and midbrain (59%), with simultaneous significant elevation in reduced glutathione content of these regions (Table 2) by 27% and 32%, respectively, compared with the control mice. Conversely, in accordance with their total antioxidant power, the MDA and reduced glutathione levels of the cerebral cortex and liver remained unchanged after tea consumption. These findings clearly show that consumption of the tea infusion reinforces the antioxidant defense system of the mouse brain in a region-specific manner.

The diverse pattern of antioxidant effects of tea consumption obtained in different brain areas may be attributed to differences in regional antioxidant defense. This region specificity of antioxidant reinforcement in the brain by polyphenol-rich plant extracts has been also observed in previous studies.^{24, 28} Ultimately, the cerebral cortex seems to have a stronger antioxidant defense system, resulting in resistance to changes in its oxidant/antioxidant status. The comparison between the brain regions and liver reveals that the cerebral cortex and liver share the same antioxidant response pattern in response to the herbal tea consumption. As in the cortex, the high antioxidant power of liver, shown in the present and previous studies,²⁷ might render this tissue insensitive to changes in its oxidant/antioxidant status. This finding is somewhat expected because liver executes several vital functions of the organism.

Our findings conclusively indicate that the consumption of the herbal tea from Sideritis species induces a significant region-specific antioxidant reinforcement in the brain, with the cerebellum and midbrain the most affected brain areas. Although further studies should address the specific bioactive ingredients of Sideritis infusions, their absorption and bioavailability, and their mechanism of action, the present data strongly suggest that this herbal tea could be a good source of natural antioxidants. The tea might help prevent the age-related deficits and neurodegenerative disorders related to oxidative damage of the specific brain regions.

Footnotes

Acknowledgment

This research was partially supported by BIOFLORA, a network of the University of Patras.

Author Disclosure Statement

No competing financial interests exist.

References

Baden

, Sideritis

. Mountain Flora of Greece, Vol.2. Strid

, Tan

. Edinburgh, Scotland: Edinburgh University Press, 1991; 84–91.

Hernandez-Perez

, Gallego

RMR

. Analgesic and antiinflammatory properties of Sideritis lotsyi var. mascaensis. Phytother Res, 2002; 16:264–266.

Logoglu

, Arslan

, Oktemer

, Sakıyan

. Biological activities of some natural compounds from Sideritis sipylea Boiss. Phytother Res, 2006; 20:294–297.

Aligiannis

, Kalpoutzakis

, Chinou

, Mitakou

. Composition and antimicrobial activity of the essential oils of five taxa of Sideritis from Greece. J Agric Food Chem, 2001; 49:811–815.

Tunalier

, Kosar

, Ozturk

et al. Antioxidant properties and phenolic composition of Sideritis species. Chem Nat Comp, 2004; 40:206–210.

Gabrieli

, Kefalas

, Kokkalou

. Antioxidant activity of flavonoids from Sideritis raeseri. J Ethnopharmacol, 2005; 96:423–428.

Gomez-Serranillos

, El-Naggar

, Villar

, Carretero

. Analysis and retention behaviour in high-performance liquid chromatography of terpenic plant constituents (Sideritis spp.) with pharmacological interest. J Chromatogr B Analyt Technol Biomed Life Sci, 2004; 812:379–383.

Choi

. Oxygen, antioxidants and brain dysfunction. Yonsei Med J, 1993; 34:1–10.

Emerit

, Edeas

, Bricaire

. Neurodegenerative diseases and oxidative stress. Biomed Pharmacother, 2004; 58:39–46.

10.

Srivastava

, Shivanandappa

. Hexachlorocyclohexane differentially alters the antioxidant status of the brain regions in rat. Toxicology, 2005; 214:123–130.

11.

Choudhary

, Verma

. Ameliorative effects of black tea extract on aflatoxin-induced lipid peroxidation in the liver of mice. Food Chem Toxicol, 2005; 43:99–104.

12.

Katalinic

, Modun

, Music

, Boban

. Gender differences in antioxidant capacity of rat tissues determined by 2,2′-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays. Comp Biochem Physiol C Toxicol Pharmacol, 2005; 140:47–52.

13.

Singleton

, Rossi

Jr . Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic, 1965; 16:144–158.

14.

Prior

, Wu

, Schaich

. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem, 2005; 53:4290–4302.

15.

Huang

, Ou

, Prior

. The chemistry behind antioxidant capacity assays. J Agric Food Chem, 2005; 53:1841–1856.

16.

Koleva

, Linssen

JPH

, A van Beek

et al. Antioxidant activity screening of extracts from Sideritis species (Labiatae) grown in Bulgaria. J Sci Food Agric, 2003; 83:809–819.

17.

Papandreou

, Dimakopoulou

, Linardaki

et al. Effect of a polyphenol-rich wild blueberry extract on cognitive performance of mice, brain antioxidant markers and acetylcholinesterase activity. Behav Brain Res, 2009; 198:352–358.

18.

Mokrasch

, Teschke

. Glutathione content of cultured cells and rodent brain regions: a specific fluorometric assay. Anal Biochem, 1984; 140:506–509.

19.

Bradford

. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976; 72:248–254.

20.

Triantaphyllou

, Blekas

, Boskou

. Antioxidative properties of water extracts obtained from herbs of the species Lamiaceae. Int J Food Sci Nutr, 2001; 52:313–317.

21.

Manach

, Scalbert

, Morand

, Remesy

, Jimenez

. Polyphenols: food sources and bioavailability. Am J Clin Nutr, 2004; 79:727–747.

22.

Feoli

, Siqueira

, Almeida

et al. Effects of protein malnutrition on oxidative status in rat brain. Nutrition, 2006; 22:160–165.

23.

Manikandan

, Srikumar

, Jeya Parthasarathy

, Sheela Devi

. Protective effect of Acorus calamus LINN on free radical scavengers and lipid peroxidation in discrete regions of brain against noise stress exposed rat. Biol Pharm Bull, 2005; 28:2327–2330.

24.

Subathra

, Shila

, Devi

, Panneerselvam

. Emerging role of Centella asiatica in improving age-related neurological antioxidant status. Exp Gerontol, 2005; 40:707–715.

25.

Svennerholm

. Distribution and fatty acid composition of phosphoglycerides in normal human brain. J Lipid Res, 1968; 9:570–579.

26.

MacEvilly

, Muller

DPR

. Lipid peroxidation in neural tissues and fractions from vitamin E-deficient rats. Free Radic Biol Med, 1996; 20:639–648.

27.

Zafir

, Banu

. Antioxidant potential of fluoxetine in comparison to Curcuma longa in restraint-stressed rats. Eur J Pharmacol, 2007; 572:23–31.

28.

Balu

, Sangeetha

, Murali

, Panneerselvam

. Age-related oxidative protein damages in central nervous system of rats: modulatory role of grape seed extract. Int J Dev Neurosci, 2005; 23:501–507.