Introduction Of Anxiety Disorder Biology Essay

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Anxiety is an uncomfortable feeling that responses to threatening situation. Anxiety can be caused by medical illness, such as pheochromocytoma, or medication use, such as sympathomimetic agents. However, if the anxiety is not due to a medical illness or a medication and it lasts for a long period of time, it may affect normal body function and may become an anxiety disorder (Bouayed et al., 2007).

Anxiety disorders are very common among all of the psychiatric illnesses. Statistics showed that one eighth of total population world wide suffered from anxiety disorder and lifetime prevalence rates of major anxiety disorders are approximately two times greater than among women than among men (Wittchen et al., 1994).

Until today, there is no well defined mechanism on the pathophysiology of the anxiety. Many studies has shown that normal and pathologic anxiety states are associated with abnormal function in several neurotransmitter systems, including noradrenaline, γ-aminobutyric acid (GABA), and serotonin (5-HT). Hence, a few hypotheses had been proposed, such as noradrenergic model, GABA receptor model and 5-HT model. Recently, oxidative stress has been shown to be associated with anxiety-related behaviour in animal model (Bouayed et al., 2007; Rammal et al., 2008). A study showed that overexpression of glyoxylase 1 and glutathione reductase 1, antioxidant enzyme in the cingulate cortex of the mice, are associated with the increase in anxiety-related behaviour while inhibition of both antioxidant enzymes expression is occurred with decreased level of anxiety-related behaviour of mice (Hovatta et al., 2005).

Nowadays, the main pharmacotherapeutic approaches for the treatment of anxiety disorders are benzodiazepines (BZDs) and selective serotonin reuptake inhibitors (SSRIs). They will exert anxiolytic effect through modulation of γ-aminobutyric acid (GABA) or serotonergic receptors (Uhlenhuth et al., 1999) respectively. However, benzodiazepines have narrow therapeutic index and may cause irritating side effects such as cognitive impairment, hyperlipidemia, altered regulation of blood glucose levels and physical dependance (Roerig, 1999; Wirshing et al., 2002) while fluoxetine, an example of SSRI, has delayed onset of action and has a lot of interaction with medication (Grundamn, 2007). Hence, the attempt to search for better alternatives has been carried out.

There are numerous animal studies show that introduction of N-acetylcysteine (NAC) are proven to be effective in alleviation of oxidative stress in neurodegenerative disease (Farr et al., 2003; Andreassen et al., 2000), prompt the researchers to introduce a more effective dosage form for NAC to be applied in the patients. In addition, there are studies suggest that extracts from herbal medicines such as Passiflora incarnata (Dhawan et al., 2001), Hypericum perforatum (Beijamini and Andreatini, 2003), and Ginkgo biloba (Woelk et al., 2007) show significant anxiolytic effects in various animal models and are proposed to be good alternatives in treatment of anxiety patients. It has been showed that Passiflora incarnata, Hypericum perforatum and Ginkgo biloba contain polyphenols which show atioxidant properties and able to alleviate the oxidative stress in anxiety mice. However, there is no studies show the comparison of relative antioxidant activity between the herbal medicines. In the project, the antioxidant profile of various herbal medicines will be measured and compared while optimisation and validation of the antioxidant assay will also be carried out.

2.0 Literature review

2.1 Introduction of anxiety disorder

Anxiety disorders are characterized as five major distinct conditions according to the DSM-IV-TR: Generalised Anxiety Disorder (GAD), Obsessive-Compulsive Disorder (OCD), panic disorder (PD), phobias [including social anxiety disorder (SAD)], and post-traumatic stress disorder (PTSD) (American Psychiatric Association, 2000). Patients with anxiety may show neuronal and motor systems such as feeling of stress, worry, fear, shortness of breath and trembling (Bouayed et al., 2007). Study shows that 12.5% of worlwide population suffered from anxiety disorder with women is the gender that tend to be predispose to anxiety disorders (Wittchen et al., 1994). Since such disorder has become the common psychiatric disorder, studies have been carried out to investigate the pathophysiological process of anxiety disorder.

2.2 Pathophysiology of anxiety

Until today, there is no definitive pathophysiologic mechanisms have been determined, but biochemical and neuroimaging study shows that normal and pathologic anxiety states is associated with abnormal function in hormonal and neurotransmitter systems, including corticotrophic releasing hormone (CRH) (Strohle and Holsboer, 2003), noradrenaline (Berridge and Waterhouse, 2003) , γ-amino butyric acid (GABA), and serotonin (5-HT) (Tunnicliff et al., 1991), which give rise to some suggested receptor hypothesis for anxiety which is useful in development of anxiolytic currently used in treatment. Recently, there is an animal study suggests that oxidative stress in brain is related to the development of anxiety (Hovatta et al., 2005).

CRH is the hormone which will released upon activation of hypothalamic-pituitary-adrenal (HPA) axis. Upon exposure to stress, the HPA axis will be activated in order to release glucocorticoid hormone to increase the blood pressure, heart rate, and downregulate immune system, which is the symptom showed in anxiety patients (Strohle and Holsboer, 2003). Prolonged exposure to stress will cause sustained activation of the HPA which will increase the anxiety level associated with cardiovascular disease, memory impairment, susceptibility to infection and fatigue appearance (Kim and Gorman, 2005).

Noradrenaline is an important neurotransmitter that play role in modulation of

anxiety related behaviour. Study suggests that noradrenergic system is closely related to the HPA system via locus ceruleus (LC) which is located adjacent to fourth ventricle in the brain stem (Berridge and Waterhouse, 2003). It can be showed through the studies which suggest that increase in the corticotrophin-releasing factor (CRF) within the locus ceruleus will result in increased secretion of noradrenaline levels in the adrenal medulla (Kim and Gorman, 2005) which cause direct vascular effects such as increased in the blood pressure and heart rate, which are the symptoms shown in anxiety patients.

GABA is the major inhibitory neurotransmitter in the central nervous system that is able to activate GABAA and GABAB and GABAC receptor (Bormann et al., 2000; Chebib et al., 1999). Activation of GABAA, GABAB and GABAC receptor will causing neuronal membrane becomes hyperpolarized, resulting in inhibition of neuronal transmission (Weinberger et al., 2001; Bowery et al., 2002). There are findings suggest that impairment of GABAA receptor will lead to increased harm-avoidance behaviour and hypersensitivity to negative behaviour, hence predispose individual to GAD and PD (Crestani et al., 1999)

Serotonin is the inhibitory neurotransmitter which regulates sleep, appetite, memory, impulsitivity, sexual behaviour, and motor function. Importance of 5-HT1A receptors in anxiety was raised by evidence that the anxiolytic medication, buspirone, has shown 5-HT1A receptor agonist/partial agonist properties (Tunnicliff et al., 1991). Recent studies suggested that anxiety patients, who had reduced neurogenesis, may be reversed after treatment with SSRIs (Vermetten et al., 2003; Ressler and Nemeroff, 2000).

GABA system

Oxidative stress

Hypothalamic-pituitary-adrenal axis (HPA)


Serotonergic system

Noradrenergic system

Diagram 1.0 Pathophysiology of anxiety

2.3 Oxidative stress

Oxidative stress is defined as an imbalance between free radical or reactive oxygen species (ROS) production and opposing antioxidant defence system in the body (Cui et al., 2004). Numerous physiological and pathological processes such as ageing, excessive caloric intake, infections, inflammatory disorders, environmental toxins, pharmacological treatments, emotional or psychological stress, ionizing radiation, cigarette smoke and alcohol increase the bodily concentration of oxidizing substances, known as ROS (Emmanuel, 2005). ROS, highly reactive species that formed in vivo, can be divided into free radicals such as superoxide anion (O2.), hydroxyl radical (.OH) and non free radical such as hydrogen peroxide (H2O2) ( Huda-Faujan et al., 2007). ROS can be produced by enzymatic activation of oxygen, through lipoxygenase activity during prostaglandin synthesis (Cui, et al., 2004). Normally, ROS produced can be inactivated by the antioxidant defence system in the body. Antioxidant defence system in the body contains the enzymes which are able to deactivate the ROS, such as superoxide dismutase, glutathione peroxidase, and catalase (Ali et al., 2008). Superoxide dismutase able to convert the superoxide radical to H2O2, which can be converted further into the water and oxygen by catalase (Berk et al., 2008) while glutathione peroxidase able to reduce the H2O2 into water (Ursini et al., 1995).

2.3.1 Oxidative stress in anxiety

Upon exposure to stress, the brain cell will utilise the glucose and oxygen extensively in meeting the 'fight and flight' response. Hence, the free radical will be generated through the metabolic process and it will be deactivated by antioxidant enzyme in the brain tissue. However, if the patient experience stress for a prolonged period of time, for example anxiety disorder, the production of the free radical exceed the scavenging capacity of the antioxidant enzyme in the brain, leading to oxidative stress in the brain (Cui et al., 2004). Furthermore, the antioxidant enzymes in the brain contain negligible amount of catalase, relatively less glutathione peroxidase and vitamin C compared to liver, plus high lipid content in the brain tissue, causing accumulation of the free radical in the brain cells and formation of oxidative stress in the brain (Reiter, 1995a; Olanow, 1993; Halliwell, 2001). There is study showed that patients with social phobia (Atmaca et al., 2004) and obsessive-compulsive disorder (Kuloglu et al., 2002) has less antioxidants enzyme activities in the brain. High level of oxidative stress is one of the factors which contribute to a loss of membrane integrity, protein damage, and neuronal dysfunction, leading to many degenerative illnesses in the central nervous system, as well as psychiatric disturbances (Atmaca et al., 2004). There are numerous studies indicating that free radical mediated neuronal damage plays a role in the pathophysiology of post traumatic stress disorder (Attari et al., 2002; Tezcan et al., 2003) and obsessive compulsive disorder ((Kuloglu et al., 2002). From the studies' results, the theory that oxidative injuries could be involved in the pathogenesis of psychiatric disorders has been suggested.

2.4 Management of anxiety disorder

There are two types of treatment for anxiety disorders, psychotherapy (primarily cognitive-behavioural) and pharmacotherapy (Devane et al., 2005). Psychotherapy has been proven effective for social anxiety disorder (Acarturk et al., 2009), whereas pharmacotherapy has been considered the standard treatment for most of anxiety disorders and it can be used alone or in combination with psychotherapy. The main pharmacotherapeutic approaches for the treatment of anxiety disorders are benzodiazepines (BZDs) and selective serotonin reuptake inhibitors (SSRIs), which act on the GABAA receptor or the serotonergic system, respectively (Uhlenhuth et al., 1999). Example of the benzodiazepines used in the treatment of anxiety is diazepam. The binding of benzodiazepines to the GABAA receptor increases the affinity of gamma amino butyric acid (GABA) to its receptor, thereby increasing the opening frequency of GABAA receptor. Hence, it cause increased conductance to chloride ions, which produces membrane hyperpolarisation, thus induces inhibition of neuronal impulse transmission (Nemeroff, 2003). The benzodiazepines, due to rapid onset of action, have been widely used for short-term relief of anxiety symptom in patients with GAD and PD (Uhlenhuth et al., 1999). High-potency BZDs will only use in patients who express a high level of anticipatory anxiety and a reduction in the severity of their panic attacks. It is because other anxiolytic drugs and cognitive behaviour therapy (CBT) often take weeks for therapeutic effect to be seen (Cloos and Ferreira, 2008). However, BZDs will produce less good outcome in the long term treatment and hence they should not be used beyond 2 to 4 weeks. Typical side-effects of benzodiazepine (BZD) include drowsiness and impairment of cognitive and motor function ( Roerig, 1999; Ninan et al., 1998). Besides that, relapse rates following even gradual taper and discontinuation of benzodiazepines are high. Statistics showed that 63% to 81% of patients with GAD experience relapse within weeks or months of drug discontinuation (Ballenger, 2001). BZDs may also produce deleterious metabolic effects such as hyperlipidemia and altered regulation of blood glucose levels (Wirshing et al., 2002). In summary, there are numerous study states that BZDs are effective treatments due to the advantage of a rapid onset of action, but that their use is limited by their potential for abuse and lack of antidepressant properties (Hidalgo et al., 2007).

Selective serotonin reuptake inhibitors (SSRIs) are also the alternatives in treatment of anxiety disorder. Anxiety is associated with reduced levels of the monoamines in the brain, such as serotonin (5-HT). The SSRIs are thought to restore the levels of serotonin in the synaptic cleft by binding at the 5-HT reuptake transporter preventing the reuptake and subsequent degradation of 5-HT (Vaswani et al., 2003). This action leads to the accumulation of 5-HT in the synaptic cleft and the concentration of 5-HT returns to within the normal range. Use of SSRIs has increased over the last decade due to lesser side effects and a higher effectiveness especially for obsessive-compulsive disorders compared to the long-established benzodiazepines (Sheehan et al., 2007). One disadvantage of SSRIs and many other anti-anxiety drugs are the delayed onset of action and several drug related interaction (Sheehan et al., 2007). CYP2D6 is inhibited by SSRIs, in order of decreasing potency paroxetine, norfluoxetine, fluoxetine, sertraline, citalopram and fluvoxamine and hence will interact with the monoamine oxidase inhibitor and NSAIDs that metabolised by this enzyme (Baumann, 1996a). Hence, usage of SSRIs is limited and is given with precautions to patients. Another treatment option are duloxetine, an SNRI, which shows an anxiolytic effect based on reduction in animal anxiety behaviours and is approved by FDA for treatment of GAD in 2007 (Khan and Macaluso, 2009).

2.5 Alternative treatment in anxiety disorder

Studies using herbal remedies to treat mild to moderate anxiety disorders have emerged and their use has dramatically increased over the past decade (Bystritsky et al., 2005). The clinical effects of herbal medicines are usually moderate and often appear only after prolonged periods of administration. However, research study also shows that the herbal medicines rarely have adverse effects (Ernst et al., 2006). Herbal medicine, compared to western medicine, contain active ingredients which are often unknown and do not have pure compounds. Besides, the herbal medicines have variable quality (Garrard et al, 2003) due to a range of factors such as soil, climate, storage, extraction method and have wide therapeutic range. Although there is limited clinical data on the efficacy of herbal medicines for the treatment of anxiety disorders (Ernst et al., 2006), there is strong evidence from traditional uses, case reports, and numerous animal behavioural experiments that showed promising results (Zhang et al., 2004). Some studies suggest that certain herbal plants that have antioxidant properties are effective in treatment of anxiety. These plants are reported to contain phenolic compound which able to extinguish oxygen derived free radicals by donating a hydrogen atom or an electron to the free radical (Wanasundara and Shahidi, 1998). Extracts from herbs such as Hypericum perforatum (Hunt et al., 2001), passionflower (Bergner et al., 1995), and Gingko biloba (Ramassamy et al., 1992) contain phenolic compound which show significant anxiolytic activity in various animal models.

2.5.1 Ginkgo Biloba

Ginkgo biloba is belonging to Ginkgoaceae family and is indigenous to China and Japan. Ginkgo biloba leaves has been used for medicinal purposes for several years in China. It has been consumed in order to maintain their health as it has been reported that it possess anticancer activities (Youdim and Joseph, 2001). Recently, it has been used in the patients with vascular cognitive impairment, cerebral and vascular insufficiency (Helen et al., 1996). Gingko biloba extracts is obtained from the dried leaves in an acetone/ water (60:40) solution and is found containing a series of flavonoids. Flavonoid is a large group of chemical structure contain containing a series of carbon rings. Flavonoids that found in the Ginkgo biloba include isohamnetin, anacardic acid and kaempterol-3. Ginkgo extracts also contain terpenes, which can be further divide into diterpenes and sesquiterpenes (Gold et al., 2002). Examples of diterpenes in the ginkgo extracts are ginkgolide A, ginkgolide B, while the example of sesquiterpenes is bilobalide. Ginkgo biloba has been shown in a study that it can modulate the function of serotonin neurotransmitter in the brain system during exposure to stress in anxious mice (Ramassamy et al., 1992). Studies also showed that flavonoids component of Ginkgo biloba extracts may have greater effects on the serotonin neurotransmitter system compared to the terpenes (Bolanos-Jimenez et al., 1995; Huguet, Drieu, and Piriou, 1994).

Ginkgo biloba extracts contain the components that can be act as free radical scavenger (Seif-El-Nasr and El-Fattah, 1995). There are studies shows that the ginkgo extracts are able to protect neurons from consequences of oxidative stress such as apoptosis (Oyama et al., 1996) by neutralising the harmful effects of free radicals. The diterpenes and sesquiterpenes, instead of flavoniods, appear to be the main component responsible for the protection of brain tissue against brain injury due to reduced blood flow or oxygen, such as cerebral ischemia (Gold et al., 2002). It is because flavoniods are not able to pass through the blood-brain barrier in sufficient quantity to produce such effects in the brain. However, there are no studies shows that terpenes in gingko extract can alleviate the oxidative stress in anxiety patients.

Kaempferol Isorhamnetin




Ginkgolide A




Ginkgolide B




Diagram 2.0 The chemical structures showed above are the example of flavonoid compound (Kaempferol, Isohamnetin, and ginkgolide) that are found in the Ginkgo biloba.

2.5.2 Passionflower

The genus Passiflora is the largest group in family Passifloraceae. The species of this genus are distributed in the warm temperate and tropical regionsof the world. The genus Passiflora is a good source of bioflavonoids, i.e., chrysin, apigenin, kaempferol, quercetin, apigenin, and genistein, which have tremendous therapeutic potentials as anti-oxidants, immuno-modulators, antianxiety agents and anti-carcinogens (Murcia et al., 2001). Passiflora incarnata is a popular traditional European remedy as well as a homoeopathic medicine for insomnia, anxiety (Dhawan et al., 2004) and has been used as a sedative tea in North America (Bergner, 1995). P. incarnata contain flavonoids which are reported to be the major phyto-constituents. These include apigenin, luteolin, quercetin, kaempferol (Murcia et al., 2001). On the other hand, Passiflora edulis has been used as a sedative, diuretic, anthelmintic, anti-diarrheal, stimulant, tonic and also in the treatment of hypertension (Patel, 2009). The leaves of P. edulis, is used traditionally to treat both anxiety and nervousness by folk medicine, are rich in polyphenols, which have been reported as natural antioxidant (Dhawan et al., 2004). The antioxidant activity of P. edulis leaves extract was significantly correlated with polyphenol contents (Patel, 2009). However, there is no study to investigate the antioxidant activity of polyphenol in the Passiflower extract to attenuate the oxidative stress in anxiety.

2.6 Antioxidant assays

Antioxidant is the substance that can deactivate free radicals induced by metabolic system in the body. Antioxidant ,which can exist in the body enzymes and the natural herbs (Ali et al., 2008), possesses free radical scavenging effects on body cells. Antioxidant capacity of the food and herbal extracts can be measured through two types of assay methods, Hydrogen Atom Transfer (HAT) based and Single Electron Transfer (SET) based methods. HAT based methods are used to measure the ability of antioxidants to quench free radicals through donation of hydrogen while SET based methods are used to detect the ability of antioxidants to transfer one electron to reduce the reactive compound (Wright et al., 2001). Example of the HAT based methods are oxygen radical absorbance capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) assay. Ferric reducing antioxidant power (FRAP), trolox equivalent antioxidant capacity (TEAC) and 1,1-diphenyl-2-picrylhyrazyl radical (DPPH) assay are belong to the SET method.

2.6.1 Trolox equivalent antioxidant capacity (TEAC)

TEAC assay is based on the inhibition by antioxidants of the absorbance of the radical cation of 2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS), which has a characteristic long-wavelength absorption spectrum showing maxima at 660, 734 and 820 nm (Ali et al., 2008; Prior et al., 2005). It have been applied to the measurement of the total antioxidant capacity of biological matrices,such as plasma, as well as single compounds, food components or food extracts (Gil, 2000). TEAC assay held some advantages where it tend to be operationally simple, able to determine both hydrophilic and lipophilic antioxidant capacity of food extracts and physiological fluids (Awika et al., 2003), since the reagent is soluble in both aqueous and organic solvent media. However, many phenolic compounds that have low reduction potential than ABTS radical are able to to react with the radical, hence affect the final results. The TEAC reaction may not be the same for slow reaction and may need a long time to reach the endpoint (Prior et al., 2005).

2.6.2 Oxygen Radical Absorbance Capacity (ORAC)

In the ORAC assay, fluorescein is used as a fluorescence probe and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is used as a peroxyl radical generator (Ali et al., 2008).

It is the only method that takes free radical action to completion and uses an area-under-curve (AUC) technique for quantitation and thus, combines both inhibition percentage and the length of inhibition time of the free radical action by antioxidants into a single quantity.

The principle of ORAC assay is begun with AAPH, peroxyl radical (ROO•) generator, will breakdown to form peroxyl radical. The peroxyl radical will oxidise the fluorescein to produce a substance without fluorescence. Hence, less fluorescence will be detected and the fluorescence reading is low. However, in the presence of the antioxidants, the oxidation reaction of the fluorescein will be suppressed by transfering hydorgen atom to the peroxyl radical to form a stable product. Hence, the fluorescence readings will be higher compared to that without antioxidants. The concentration of antioxidants in the test sample is proportional to the fluorescein intensity in this assay and it will be assessed by comparing the area under the curve (AUC) to the vitamin E analogue, Trolox, that has known antioxidant profile.


Fluorescein Non-fluorescence product




This method offer advantage of applicable to both food samples and biological fluids (Apak et al., 2007), and therefore may be useful in detecting and therapeutical monitoring of diseases(Ou et al., 2001). This assay is also able to measure hydrophilic and hydrophobic antioxidants by altering the radical source and solvent (Ou et al., 2005). However, If the probe used in ORAC assay isβ-PE, it shows inconsistent reactivity toward ROO• and it is photobleached due to peroxyl radical attack (Ou et al., 2001).

2.6.3 Ferric reducing ability of plasma (FRAP) assay

Ferric reducing ability of plasma (FRAP) assay is a technique to determine the total antioxidant power interpreted as the reducing capability (Ali et al., 2008). In this assay reductants (antioxidants) in the sample reduce Fe (III)/tripyridyltriazine complex, present in stoichiometric excess, to the blue ferrous form, with an increase in absorbance at 593 nm. This assay is carried out at acidic pH 3.6 to maintain iron solubility (Prior et al., 2005). The advantage of this assay is simple, speedy, inexpensive, and do not require specialised eqiupment (Benzie and Strain, 1999). However, FRAP assay is just able to measure only the hydrophilic antioxidants (e.g. ascorbic acid) (Apak et al., 2007). This assay also unable to detect compounds that act by hydrogen transfer, especially thiols and proteins (Ou et al., 2002)

3.0 Aim and Objectives

3.1 Aim

To investigate the antioxidant capacity of the complementary medicines that are useful in anxiety treatment.

3.2 Objectives

To optimize and validate the antioxidant assays that are used in determination of antioxidant capacities of selected materials.

To determine and compare the antioxidant potential of selected complementary medicines.

4.0 Methodology

4.1 Oxygen Radical Absorbance Capacity (ORAC) assay

4.1.1 Materials

St John's wort tablets, Passionflower tablets, Ginkgo tablets, vitamin A tablets, vitamin B tablets of different brands will be obtained from community pharmacy in Kuala Lumpur. Fluorescein, Trolox, ascorbic acid, disodium fluorescein, potassium phosphate buffer and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) will be purchased from Sigma-Aldrich, USA. Fluorescent microplate reader is purchased from Eppendorf, Hamburg. Fluorescent microplate is purchased from Eppendorf, Hamburg. Fluorescence filter with an excitation wavelength of 485nm and an emission wavelength of 520 nm is used in the microplate reader.

4.1.2 Sample Preparation

Valerian tablets, St John's worts tablets, Passionflower tablets and Ginkgo tablets, melatonin tablets, vitamin A tablets and vitamin B tablets will be initially solubilised with 1:10 w/v in potassium phosphate buffer (pH7.4). The solutions formed are then homogenised in an homogenizer for 5 seconds. This process is then repeated again in order to obtain absolute homogenized solutions. The homogenates will be centrifuged at 3000g for 10 minutes to remove the insoluble residue. The supernatant retained will be collected and used as the sample solution for antioxidant analysis.

4.1.3 Reagent and Standard Preparation

AAPH (0.414 g) will completely dissolved in 10 mL of 75 mM potassium phosphate buffer (pH 7.4) to a final concentration of 153 mM and was kept in an ice bath. Fluorescein stock solution (4.19 x 10-3 mM) will be made in 75 mM phosphate buffer (pH 7.4) and will keep at 4 °C in dark condition. The fluorescein stock solution at such condition can last several months. The 48 nM fresh fluorescein working solution will be made daily by further diluting the stock solution in 75 mM potassium phosphate buffer (pH 7.4). Trolox standard will be prepared as follows: 0.250 g of Trolox was dissolved in 50 mL of 75 mM potassium phosphate buffer (pH 7.4) to give a 0.02 M stock solution. The stock solution will be diluted with the potassium phosphate buffer to 50, 25, 12.5, and 6.25 µM working solutions.

4.1.4 Assay protocol

Optimization of ORAC assay can be done by adding different concentration of Trolox solution and fixed amount of AAPH into each wells of microplate. Then run the assay to obtain the fluorescence reading. According to Ou et al.(2001), ORAC assay is initiated by incubating the microplate reader at 370C for 15 minutes. After that, 150µL of fluorescein working solution will be added to each well of the fluorescent microplate. The wells of the microplate can be divided into 3 parts where the first part of the wells is allocated to the samples. The second and third part of wells of microplate are designated to the Trolox solution and phosphate buffer respectively. 25µL of the samples will be added into the designated wells of microplate while 25µL of Trolox solution will be added into the allocated wells of assay plate and act as positive control. Finally, 25µL of the phosphate buffer will be added into individual wells of microplate and act as negative control. After the samples, Trolox solution and phosphate buffer have been added, 25µL of the AAPH working solution will be added to each well of the microplate. Immediately after the AAPH working solution is added, the microplate will be placed in the microplate reader and take the fluorescence reading, f0. Then, the subsequent fluoresence reading will be taken every 2 minutes for 90 minutes. The assay is repeated 3 times. The results obtained are plotted into the graph and measure the AUC of samples, Trolox solution and phosphate buffer.

The AUC of the samples, trolox solution and phosphate buffer can be calculated using the following formula:

AUCA= 1+ f1/ f0 + f2/ f0+ f3/ f0+ f4/ f0+.....+ f34/ f0+ f35/ f0

A= sample/ Trolox/ phosphate buffer, f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i.

ORAC values of the samples will be calculated by using a regression equation between the Trolox concentration and the net area under the fluorescein decay curve. ORAC value is expressed as Trolox equivalents(TE) and the unit is micromole of TE per gram of sample. It also can be calculated using the following equation:

ORAC value = [(AUCSample - AUCBlank) / (AUCTrolox - AUCBlank)] x (dilution factor x concentration of trolox)

Add 150 µL of fluorescein into each well of the microplate.

Add 25µL of samples into individual wells of microplate

Add 25µL of potassium phosphate buffer into individual wells of microplate

Add 25µL of Trolox solution into individual wells of microplate

Add 25µL of AAPH working solution into each wells of microplate containing samples, Trolox solution and potassium phosphate buffer respectively.

Run the assay and take the fluorescence readings every 2 minutes for 90 minutes. Plot the graph and measure AUC



AUCPhosphate buffer

Diagram 3.0 Flow chart of the ORAC assay procedures