ANTIOXIDANT POTENTIAL OF DIFFERENT TYPES OF TEA

ABSTRACT

The present study evaluated the phytochemical constituents and in vitro antioxidant potential of different types of tea namely; black tea, un-caffeinated tea, green tea and herbal tea. Radical scavenging capacities of the tea extracts were determined using 2,2-diphenyl-2-picrylhydrazyl (DPPH) assay. Total antioxidant activity was determined using ferric reducing antioxidant power (FRAP) assay. The results showed that the total flavonoid content (TFC) of green tea (215.61±48.83 QE/mg) wassignificantly (p<0.05) higher than that of un-caffeinated tea (184.32±33.62 QE/mg) and herbal tea (167.25±31.25 QE/mg). There was no significant (p >

0.05) difference between the TFC of un-caffeinated and herbal tea samples. However, the TFC of un-caffeinated and herbal tea samples were found to be significantly (p<0.05) higher than that of black tea (142.32±22.73 QE/mg). There was no significant (p > 0.05) difference in the total tannin content (TTC) of un-caffeinated tea (411.55±9.21 GAEmg/ml), green tea (406.83±22.71

GAEmg/ml) and herbal tea (402.74±13.2 GAEmg/ml). However, their TTC were found to be significantly (p < 0.05) higher than that of black tea (325.14±108 GAEmg/ml). The total phenol content (TPC) of green tea (124.81±79.05 GAEmg/ml) was found to be significantly (p < 0.05) higher  than  that  of  un-caffeinated  tea  (63.87±35.76  GAEmg/ml),  black  tea  (51.81±8.90

GAEmg/ml) and herbal tea (15.78±13.02 GAEmg/ml). The antioxidant activity of black tea and herbal tea was found to be significantly (p < 0.05) higher than that of un-caffeinated tea. Green tea showed the least radical scavenging activity. A correlation between the antioxidant capacity and the phytochemical constituent of the teas was observed. A positive correlation (r = 0.060) was observed between the TTC and FRAP of the tea samples, however, a negative correlation (r

= -0.137) was observed between the TTC and DPPH radical reducing power of the tea samples.

A positive correlation (r = 0.448) was observed between the TFC and FRAP as well as between TFC and DPPH (r = 0.347) radical scavenging activities of the tea samples. These findings demonstrated that the green tea, black tea, un-caffeinated tea and herbal tea samples are rich in important phytochemicals such     as flavonoids and tannins), and possess antioxidant potentials. However, the tea types vary in their content of antioxidants and in their antioxidant potential. Based on the FRAP assay, black tea had the highest antioxidant potential while green tea had the least. Conversely, based on the DPPH assay, black tea, un-caffeinated tea and green tea had equal antioxidant potential while herbal tea had the highest antioxidant potential.

CHAPTER ONE

INTRODUCTION

Oxygen is an element indispensable for life. When cells use oxygen to generate energy, free radicals are created as a result of cellular redox process which leads to ATP production by the mitochondria (Kabel, 2014). These products are called reactive oxygen species (ROS).   Reactive oxygen species (ROS) is a collective term used for a group of oxygen-centred oxidants, which are either free radicals or molecular species capable of generating free radicals. Free radicals are generated from either endogenous or exogenous sources. Endogenous free radicals are generated from  immune  cell  activation,  inflammation,  mental  stress,  excessive  exercise,  ischemia, infection,  cancer  and  ageing.  Exogenous free  radicals  result  from air  and  water  pollution, cigarette smoking, alcohol, heavy metals, certain drugs (cyclosporine, tacrolimus), industrial solvents, cooking and radiation. After penetration into the body, these exogenous compounds are decomposed into free radicals (Valko et al., 2007). Under normal physiologic conditions, nearly

2%  of  the  oxygen  consumed  by  the  body  is  converted  into  reactive  oxygen  through mitochondrial respiration, phagocytosis, etc. However, free radicals play a dual role as both toxic and beneficial compound (Kunwar and Priyadesh, 2011). At low or moderate level, ROS exert beneficial effects on cellular responses and immune function. At high concentration, they cause oxidative stress, a deleterious process that can damage all cellular structures (Halliwell, 2007). Oxidative stress results from an imbalance between formation and neutralization of free radicals. For example, hydroxyl radical and peroxynitrite in excess can damage cell membranes and lipoproteins by a process called  lipid peroxidation. This reaction leads to the formation of malondialdehyde (MDA) and conjugated diene compounds, which are cytotoxic and mutagenic. Lipid peroxidation occurs by a radical chain reaction, i.e once started; it spreads rapidly and affects a great number of lipid molecules (Halliwel, 2007; Valko et al., 2007). Oxidative stress plays a major part in the development of chronic and degenerative diseases such as cancer, arthritis,  aging,  autoimmune  disorders,  cardiovascular  and  neurodegenerative diseases.  The human body has several mechanisms to counteract oxidative stress by producing antioxidants, which are either naturally produced in situ, or externally supplied through foods and/or supplements. Endogenous and exogenous antioxidants act as free radical scavengers by preventing and repairing damages caused by ROS, and therefore can enhance the immune system

and lower the risk of cancer and degenerative diseases (Valko et al., 2007). An Antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Antioxidants do so by terminating chain reactions initiated by free radicals, removing radical intermediates and inhibiting other oxidation reactions by being oxidized themselves. So, antioxidants are often reducing agents such as thiols or polyphenols (Duarte and Lunec, 2005). Hence, plants and animals contain various antioxidants, such as glutathione, vitamin C, and vitamin E; polyphenols as well as enzymes such as catalase, superoxide dismutase and peroxidases. A number of clinical studies reported that the antioxidant in fruits, vegetables, teas and wine are the main factors for the observed efficacy of these foods in reducing the incidence of chronic diseases (Li et al.,

1999; Zaveri 2006). Processed tea, which is one of the most popular beverages, is manufactured from the young tender leaves of the plant Camellia sinensis (Cabrera et al., 2003). Two types of tea products are most widely consumed; green and black tea. In both cases, it is the chemical composition of the tea shoots and the reactions that occur during processing that determine the nature of the finished product and its quality. Though most of the tea produced in the world can be classified as non-fermented/aerated green tea, semi-fermented (oolong) tea and fermented black tea (Reeves et al., 1987), processing and emerging technologies have led to the production of special teas e.g. white tea, flavoured teas, organic teas, decaffeinated teas, herbal teas, scented teas,  un-caffeinated teas  and  various  other  blends.  The  tea  beverage  has  continued  to  be considered medicinal since ancient times because of its polyphenols and there is already growing evidence that tea polyphenols reduce the risk of heart diseases, neurological disease, cancer and obesity in humans (Vanessa and Williamson, 2004). In some studies, tea has been associated with anti-allergic (Yamamoto et al., 2004) and antimicrobial properties (Paola et al., 2005). Study has demonstrated that the co-administration of drugs with catechins (EC and EGCG) inhibits glucoronidation and sulfonation of orally administered drugs thereby increasing the bioavailability of such drugs (Hang et al., 2003). Moreover, some epidemiological studies have associated consumption of tea with a lower risk of several types of cancer including those of the stomach, oral cavity, oesophagus and lungs, therefore, tea appears to be an effective chemo- preventive agent for toxic chemicals and carcinogens (Cabrera et al., 2003; Hakim and Chow,

2004). Research on the effects of tea on human health has been fuelled by the growing need to provide naturally healthy diets that include plant-derived polyphenols. In line with this, there is need to elucidate how known functional components in foods could expand the role of diet in

disease prevention and treatment (Mandel et al., 2006). The ability to scavenge free radicals by tea polyphenols is due to possession of a phenolic hydroxyl group attached to the flavan-3-ol structure has been associated with teas therapeutic actions against free radical mediated diseases thereby attracting tremendous research interest (Amie et al., 2003). Many plant phenolics have been reported to have antioxidant properties that are even much stronger than vitamins E and C. In addition, currently available synthetic antioxidant like butylated hydroxyl anisole (BHA), butylated hydroxytoluene (BHT) and gallic acid esters have been suspected to cause or prompt negative  health  effects  and  hence  the  need  to  substitute  them  with  naturally  occurring antioxidants (Aqil et al., 2006; Pourmorad et al., 2006). There is therefore an increased quest to obtain natural antioxidants with broad-spectrum action. The upsurge of free radical related diseases such as diabetes (type 2), cancer, neurodegeneration e.t.c have provoked the search for food sources with natural antioxidants. Few studies have been carried out using processed tea. Therefore, the  present  study evaluated  the  total  polyphenols,  total  flavonoids  and  in  vitro antioxidant activities of a set of twenty commercial Nigerian tea samples (black, green, un- caffeinated and  herbal tea)  using  DPPH  radical scavenging  ability and  the  ferric  reducing antioxidant power.

1.1 Antioxidants

An Antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Antioxidants do so by terminating chain reactions initiated by free radicals, removing radical intermediates and inhibiting other oxidation reactions by being oxidized themselves. So, antioxidants are often reducing agents such as thiols or polyphenols (Duarte and Lunec, 2005). Antioxidants are capable of stabilizing, or deactivating free radicals before they attack cells and are absolutely critical for  maintaining optimal cellular and  systemic  health and well-being. Antioxidants are the first line of defence against free radical damage in living organism and are critical for maintaining optimum health and wellbeing. The need for antioxidants becomes even more critical with increased exposure to free radicals. Pollution, cigarette smoke, drugs, illness, stress, and even exercise can increase the chances of exposure to free radicals. Because so many factors can contribute to oxidative stress, individual assessment of susceptibility becomes very important. Many experts believe that the Recommended Dietary Allowance (RDA) for specific antioxidants may be inadequate and in some instances, the need may be several times higher than

the RDA. Antioxidant supplementation is  now being recognized as an important means of improving free radical protection, in addition to healthy lifestyle and balanced diet (Mark, 1999).

1.1.1 Free Radical Production and Antioxidant Defence Mechanism

The ability to utilize oxygen has provided humans with the benefit of metabolizing fats, proteins, and carbohydrates for energy; however, it does not come without cost. Oxygen is a highly reactive atom that is capable of becoming part of potentially damaging molecules commonly called “free radicals.” Free radicals are capable of attacking the healthy cells of the body, causing them to lose their structure and function. Free radicals have been implicated in the pathogenesis of at least 50 diseases (Langseth, 1993; Halliwell, 1994). Fortunately, free radical formation is controlled naturally by various beneficial compounds known as antioxidants. It is when the availability of antioxidants is limited that this damage can become cumulative and debilitating. However, nature have made provisions for both endogenous and exogenous antioxidant, in such a way that if the ratio of free radicals to endogenous antioxidant is on the rise, exogenous antioxidant can boost up the antioxidant level of biological system.

1.1.2 Endogenous and Exogenous Antioxidants

Endogenous antioxidant compounds in cells can be classified as enzymatic antioxidants and non- enzymatic antioxidants. The major antioxidant enzymes directly involved in the neutralization of ROS and RNS are: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GRx) (Genestra, 2007; Pham-Huy et al., 2008). SOD, the first line of defence against free radicals, catalyses the dismutation of superoxide anion radical (O2•ˉ ) into hydrogen peroxide (H2O2) by reduction. The oxidant formed is transformed into water and oxygen by catalase (CAT) or glutathione peroxidase (GPx). The GPx, a selenoprotein, removes H2O2   by using  it  to  oxidize  reduced glutathione (GSH)  into  oxidized glutathione (GSSG). Glutathione reductase, a flavoprotein, regenerates GSH from GSSG, with NADPH as a source of reducing power. Besides hydrogen peroxide, GPx also reduces lipid or non-lipid hydroperoxides while oxidizing glutathione (GSH) (Young and Woodside 2001; Bahorun et al., 2006). The non- enzymatic antioxidants are also divided into metabolic antioxidants and nutrient antioxidants. Metabolic antioxidants, belonging to endogenous antioxidants, are produced by metabolism in the body. They include lipoic acid, glutathione, L-arginine, coenzyme Q10, melatonin, uric acid,

bilirubin  and  metal-chelating  proteins  such  as  ferritin,  lactoferrin,  transferrin  e.t.c.,  While nutrient antioxidants, belonging to exogenous antioxidants, are compounds which cannot be produced in the body and must be provided through foods or supplements. They include vitamins C and E, carotenoids, flavonoids, omega-3 and omega-6 fatty acids, and trace metals such as selenium, manganese, zinc etc. (Droge, 2002; Willcox et al., 2004).

1.1.3 Diet, Antioxidant and Health

There has been much interest in the mechanisms of actions of antioxidants which might explain the relationships between dietary quality and health status (Siex, 1997; Aruoma, 1999; Halliwell,

2006). For example, mixtures of fruit and vegetables can increase the antioxidant capacity of blood (Kawashima et al., 2007). Antioxidants from our diet play an important role in helping endogenous antioxidants in the neutralization of oxidative stress. The nutrient antioxidant deficiency  is  one  of  the  causes  of  numerous  chronic  and  degenerative  pathologies  (zinc deficiency hypogonadism, selenium deficiency keshan disease cardiac myoparty). Each of the antioxidant nutrients is unique in terms of its structure and antioxidant function (Wilcox et al., 2004).

1.1.3.1 Flavonoids

Flavonoids are polyphenolic compounds which are present in most plants. Based on chemical structure, over 4000 flavonoids have been identified and classified into flavanols, flavanones, flavones, isoflavones, catechins, anthocyanins and proanthocyanidins. Beneficial effects of flavonoids on human health mainly reside in their potent antioxidant activity (Miller, 1996). They have been reported to prevent or delay a number of chronic and degenerative ailments such as cancer, cardiovascular diseases, arthritis, ageing, cataract, memory loss, stroke, Alzheimer’s disease, inflammation and infection. Every plant contains a unique combination of flavonoids, which is why different herbs rich in these substances have very different effects on the body (Hanneken et al., 2006). The main natural sources of flavonoids include green tea, grapes (red wine), apple, cocoa (chocolate), ginkgo biloba, soybean, curcuma, berries, onion, broccoli, etc. For example, green tea  is a rich source of flavonoids, especially flavonols (catechins) and quercetin. Catechin levels are 4-6 times greater in green tea than in black tea. Many health

benefits of green tea reside in its antioxidant, anticarcinogenic, anti-hypercholesterolemic, antibacterial (dental caries) and anti-inflammatory activities (Pham-Huy et al., 2008).

Fig. 1: General structures for various classes of flavonoid

Source: (Andrea et al., 2003)

1.1.3.2 Tannins

The term tannin refers to the use of tannins in tanning animal hides into leather; however, the term tannins is widely applied to any large polyphenolic compound containing sufficient hydroxyls and other suitable groups (such as carboxyls) to form strong complexes with proteins and other macromolecules. They have molecular weights ranging from 500 to over 3000 (Bate- Smith, 1962). Tannins are astringent, bitter plant polyphenols, the astringency from the tannins is what causes the dry and pucker feeling in the mouth following the consumption of red wine, strong tea, or an unripe fruit; the sensation apparently results from the interaction between tannin constituents and proteins of the saliva  and/or the  mucous tissue of the  mouth (Ashok and Upadhyaya, 2012). Several groups of phenolic compounds, having the general properties of tannins as defined by Bate-Smith, are quite distinct from one another in terms of their chemical structure (Hagerman et al., 2005). Phytochemists have classified tannins into three main classes: condensed tannins (i.e., proanthocyanidins) are flavanol-based compounds that release anthocyanidins at high temperatures in alcohol solutions or a strong mineral acid; gallotannins and ellagitannins belong to the family of hydrolysable tannins. Gallotaninns are comprised of galloyl esters of glucose or quinic acid whereas ellagitannins are derivatives of hexahydroxydiphenic acid (HHDP). Phloroglucinols are subunits of phlorotannins, which are present only in marine brown algae (Amarowicz 2007). The tea plant (Camellia sinensis) is a good example of a plant that has high tannin content. When any type of tea leaf is steeped in hot water it brews a “tart” (astringent) flavor that is characteristic of tannins. This is due to the catechins and other flavonoids contained in the tea. Tea “tannins” are chemically distinct from other types of plant tannins such as tannic acid (Hamilton-Miller, 1995) and tea extracts have been reported to contain no tannic acid (Ashok and Upadhyaya, 2012). Black tea and peppermint tea are inhibitors of iron than herb teas like chamomile, vercain, lime flower and pennyroyal (Hurrell et al., 1999). Tannins have also been considered a bioactive compound with health promoting potential in plant  derived foods and  beverages. For instance, tannins  have  been reported to possess anticarcinogenic and antimutagenic potentials as well as antimicrobial properties. Several studies have reported on the antioxidant and antiradical activities of tannins (Amarowicz, 2007). Tannins do not function solely as primary antioxidants (i.e., they donate hydrogen atom or electrons), they also function as secondary antioxidants. Tannins have the ability to chelate metal ions such as Fe(II) and interfere with one of the reaction steps in the Fenton reaction and thereby retard oxidation (Karamac et al., 2006). The inhibition of lipid peroxidation by tannin constituents can act via the inhibition of cyclooxygenase (Zhang et al., 2004).

1.1.3.3 Ascorbic Acid

Ascorbic acid (vitamin C) is widely known for its antioxidant activity and is therefore used in cosmetics and degenerative disease treatments. Vitamin C has many physiological functions, among them a highly antioxidant power to recycle vitamin E in membrane and lipoprotein/lipid peroxidation. Paradoxically, however, it should also be noted that, in vitro, vitamin C is also capable of pro-oxidant activity. It has long been known that the combination of ascorbate and ferrous ions generates hydroxyl radicals, which induces lipid peroxidation (Shekelle, 2003). Vitamin C is a potent antioxidant for hydrophilic radicals, but poor against lipophilic radicals.

1.1.3.4 Carotenoids

Carotenoids are group of tetraterpenoids. The basic carotenoid structural backbone consists of isoprenoid units formed either by head-to-tail or by tail-to-tail biosynthesis. There are primarily 2 classes of carotenoids: carotenes and xanthophylls. Carotenes are hydrocarbon carotenoids and xanthophylls contain oxygen in the form of hydroxyl, methoxyl, carboxyl, keto, or epoxy groups. Lycopene and β–carotenes are typical carotenes whereas lutein in green leaves and zeaxanthin in corn are typical xanthophylls. The structures of carotenoids are acyclic, monocyclic, or bicyclic. For example, lycopene is acyclic, ϒ–carotene is monocyclic, and α– and β–carotenes are bicyclic carotenoids (Rumsey et al., 1999). Double bonds in carotenoids are conjugated and trans forms of carotenoids are found in plant tissues. Epidemiological studies have revealed that an increased consumption of a diet rich in carotenoids is correlated with a lower risk of age-related diseases (Glaser et al., 2015; Indaser et al., 2015). Carotenoids (Fig. 3) contain conjugated double bonds and their antioxidant activity arises due to/ their ability to delocalize unpaired electrons (DeMan,1999). This is also responsible for the ability of carotenoids to physically quench singlet oxygen without  degradation and  for  the  chemical reactivity of carotenoids with  free  radicals.  The efficacy of carotenoids for physical quenching is related to the number of conjugated double bonds present in the molecule.

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