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CHAPTER-I: INTRODUTION AND REVIEW OF LITERATURE 1

Chapter- I

INTRODUCTION AND REVIEW OF LITERATURE

1.1 INTRODUCTION

Advancement in technology has eased human life but these facilities have

increased competition and obscured life. The need to perform has resulted into increase in

stress as compared to early days.

Biologically, stress refers to consequences of failure of a human or animal body to

react appropriately to its emotional or physical threats. Stress in optimum quantum acts as

stimulator to achieve the best, but when it exceeds, it surely causes imbalance in the

biochemical parameters (Chandira et al, 2010). When stress is associated with oxygen it

is termed as oxidative stress. It plays a crucial role in the development of chronic and

degenerative diseases such as cancer, autoimmune disorders, rheumatoid arthritis,

cataract, aging, cardiovascular and neurodegenerative diseases (Willcox et al., 2004;

Pham-Huy et al., 2008). Chemically, oxidative stress is associated with increased

production of oxidizing species or a significant decrease in the effectiveness of

antioxidant defenses.

Oxygen is used by cells to generate energy, but paradoxically, as a by-product of

metabolism; it produces free radicals called reactive oxygen species (ROS) and reactive

nitrogen species (RNS) (Andersen, 2004). ROS and RNS exert beneficial effects at low

or moderate concentrations on cellular responses and immune function. But at high level,

these free radicals and oxidants generate oxidative stress, which is a deleterious process

that can damage cell structures, including lipids, proteins and DNA (Pham-Huy et al.,

2008).

To combat these free radicals, plants and animals maintain enzymatic antioxidant

system such as catalase, superoxide dismutase and various peroxidase and non enzymatic

complex systems such as glutathione, phenols, flavonoids and vitamins (Ganapaty et al.,

2007). Many synthetic antioxidants such as butylated hydroxyanisole (BHA) and

butylated hydroxytoluene (BHT) are also available in market. However, safety of these

synthetic antioxidants is now doubted (Moein et al., 2008). Thus, it is increasingly


emphasized to develop and utilize effective and non-toxic antioxidants of natural origin.

A great number of raw extracts or isolated pure compounds from medicinal plants

showed more effective antioxidant activity than BHA, BHT or vitamin E; hence they can

be potential sources of natural antioxidants (Pyo et al., 2004).

Arishtas and Asavas are complicated ayurvedic formulations, used as medicines

for over 3000 years to treat various disorders. They are taken as appetizers and

stimulants. They are alcoholic fermented preparations produced using microorganism in a

sugar/ jaggery/ honey or fruit base. It is a complicated biotechnological process

developed by ancient Indians. Charaka Samhita stated that, Arishta and Asava generally

promote courage, corpulence, satisfaction, imagination, strength of mind, body and

digestion. They alleviate sleeplessness, anxiety, and anorexia and are exhilarating.

Further, various Arishtas and Asavas available in market and claim their usefulness in

treatment of problems like backache, abdominal pain, irritation, improving strength and

stamina with rejuvenation as main claim (Govindarajan et al., 2008).

Though, traditional knowledge about preparation and applications of these

fermented polyherbal formulations exist in literature, standard parameters like alcohol

content, pH, acid value and other constituents are normally checked for validation.

However, bulk of knowledge about interesting antioxidant property of these fermented

medicines has remained unrecognized or not been studied in detail and validated.

1.2 AIMS AND OBJECTIVES

Considering above revealed views in mind, an investigation was carried out to study

Arishtas and Asavas with following aims and objectives:

1. To determine the antioxidant potential of Asavas and Arishtas.

2. To determine the biochemical parameters of Asavas and Arishtas.

1.3 REVIEW OF LITERATURE

1.3.1 Free Radicals

Excess stress leads to suppression in physical endurance as well as mental

capability for logical thinking and it also suppresses immunity leading to pathological

conditions (Kanoor et al., 2008). According to Hazra et al. (2008), oxidative stress is

most important reason behind today's modern diseases, which results from an imbalance


between formation and neutralization of pro-oxidants (Braca et al., 2002). This oxidative

stress leads to formation of free radicals, which seek stability through electron pairing

with biological macromolecules such as proteins, lipids and DNA. This ultimately cause

protein and DNA damage along with lipid peroxidation. These changes could lead to

cancer, atherosclerosis, cardiovascular diseases, ageing and inflammatory diseases

(Maxwell, 1995).

One major problem associated with inhaled oxygen is its pronounced tendency to

form free radicals. These free radicals are highly reactive molecules and hence

dangerous. Their role as culprits in causing disorders is being debated. These free radicals

are electrically charged molecules. So, to neutralize themselves, they attack our cells

tearing through cellular membranes to react and create havoc with the nucleic acid,

proteins, and enzymes present in our body. This attack by free radicals, collectively

known as oxidative stress, are capable of causing cells to lose their structure, function and

can eventually destroy them. They are continuously produced in our body, by continuous

use of oxygen such as in respiration and some cell-mediated immune functions. Free

radicals also generated due to environmental pollutions, cigarette smoke, automobile

exhaust, radiation, air pollution, pesticides etc. Stress is one of the oldest and chronic

disorders of mankind. Because of stress, patients often have lots of diseases that are

difficult to treat and manage or delay in treatment gives rise to other chronic conditions

and diseases (Chandira et al., 2010).

During oxidation, many free radicals are produced, which have an unpaired,

nascent electron. Free radicals are any atom (e.g. oxygen, nitrogen), molecule or

compound with at least one unpaired electron in their outermost orbit. Such a situation is

energetically unstable, making such species often highly reactive and short-lived. As a

result, they attempt to pair-up with other molecules, atoms or even individual electron to

create a stable compound. These free radicals achieve stability by removing or filching of

electron from surrounding molecule to produce an electron pair. They can also pinch

hydrogen atom from molecule which has bound to another molecule, or interact in

various ways with other free radicals to stabilize themselves (Wu and Cederbaum, 2003).

Though, by snatching an electron form surrounding atom, molecules or

compound these free radicals become stable, however, leftover of attacked atom,


molecules or compound then possess an unpaired electron and turned into a free radical.

These new born free radicals also want to stabilize themselves, so they fallow same path

of stabilization. In this way presence of a single radical may initiate a chain sequence of

electron transfer (redox) reactions. And until, subsequent free radicals are neutralized,

thousands of free radical reactions can occur within seconds of initial reaction. These

reactions leads to damages of important biomolecules such as proteins, lipids, nucleic

acids etc. This implicated in cause of several diseases like neurogenerative diseases,

cancer, arthritis, diabetes etc. Free radicals can have positive, negative or neutral charge.

They are generated as necessary intermediates in a variety of normal biochemical

reactions, but when generated in excess or uncontrolled, radicals can wreak havoc on a

broad range of macromolecules (Anonymous, 2012).

1.3.1.1 Reactive Oxygen Species

Various forms of activated oxygen are generally known as reactive oxygen

species (ROS). These lead to the oxidative cell damage and ends up in number of

pathological conditions like cancer, diabetes, arthrosclerosis and heart disease (Halliwell,

1998). ROS are classified as free radicals which includes superoxide anion (O2•),

hydroxyl radical (•OH), singlet oxygen (½O2), and non free radical i.e. Hydrogen

peroxide (H2O2) (Silva et al., 2010). These are produced from endogenous sources within

living organisms via various mechanisms like normal aerobic respiration, stimulated

poly-morpho-nuclear leukocytes, macrophages, and peroxisomes or from exogenous

sources like tobacco smoke, ionizing radiation, certain pollutants, organic solvents,

pesticides etc. (Zakaria, 2007). According to Ebrahimzadeh et al. (2010), these ROS are

responsible for cellular redox process.

The oxidation potential and reactivity of various ROS are given in the order

O2• < H2O2<

•OH (Fridovich, 1978).

Superoxide anions are formed when oxygen (O2) achieve an extra electron,

leaving the molecule with only one unpaired electron.

O2 + e- → O2

•

Though, within mitochondria, O2• is continuously being formed; however rate of

formation depends upon the amount of oxygen flowing through mitochondria at any

given time. Superoxide is capable of damaging cellular membranes (through


peroxidation), proteins (through varieties of mechanisms) and other macromolecules.

Superoxide radicals also play very important role in formation of hydroxyl radicals which

are most damaging radical within the body.

In-vivo hydrogen peroxide (H2O2) is produced by many reactions, either being

converted to highly damaging hydroxyl radical or catalyzed and excreted harmlessly as

water (Goldfarb, 1999). It may form by conversion of O2•.

O2• + 2H

+ + e

– → H2O2

The principal problem with H2O2 is that, it easily crosses cellular membrane

while receiving one more electron, normally originating from iron or copper, gives rise to

hydroxyl radical (•OH). It has ability to damage DNA and can lead to cancer-causing

illnesses, Down syndrome and other genetic disorders.

Though •OH is short-lived, they are most dangerous free radicals. Formed when

O2• and H2O2 react together, which causes an extreme reaction; they will attack any

molecule present around them. They can cause damage to cells where they react with

DNA, lipids, and proteins.

H2O → •OH

+

H

•

H2O2 → 2•OH

This type of free radicals can be formed from O2•, O

• and H2O2 via Harber-Weiss

and Fenton reaction respectively (Knight, 1999).

O2• + H2O2 →

•OH +

•OH + O2 Haber-Weiss reaction

Fe2+

+ H2O2 → Fe3+

+ •OH +

•OH Fenton reaction

1.3.1.2 Reactive Nitrogen Species

Nitric oxide (NO) is an important bio-regulatory molecule, which has a number of

physiological effects including control of blood pressure, neural signal transduction,

platelet function, antimicrobial and antitumor activity (Jagetia et al., 2004). In most

cases, low concentrations of NO are sufficient to affect these beneficial functions.

However, during infections and inflammations, formation of NO is elevated and may

bring about some undesired deleterious effects.

As a free radical and depending on the microenvironment, NO is oxidized,

reduced or form complex with other biomolecules. Though, NO does not directly interact

with the bio-organic macromolecules like DNA or proteins, but under aerobic condition,


NO molecule become very unstable and reacts with oxygen to produce intermediates

such as NO2, N2O4, N3O4 (Marcocci et al., 1994). When it comes in contact with O2•, it

forms highly reactive peroxynitrite anion (ONOO•) which leads to oxidative injury as

well as lung damage (Levi, 1987; Wink et al., 1991).

According to Jagita et al. (2004), there are increasing evidences which suggest

that NO and its derivatives produced by activated phagocytes may have a geno-toxic

effect and may contribute in multistage carcinogenesis process (Wink et al., 1991).

1.3.1.3 Transitional- metal ions

Ions are species which can lose or gain single electron as they change from one

valence state to another and because of their excited energy state; they have long been

recognized as powerful initiator of free radical. According to Fenton reaction, in vivo,

majority of •OH comes from metal catalytic breakdown of H2O2 (Valko et al., 2006).

Devasagayam et al. (2004) report some other sources of free radicals include

redox cycling of xenobiotics, exposure to physicochemical agents like ionizing radiations

and an endogenous compound or a drug that acts as photosensitizer. Most of the damage

induced by ionizing radiations in biological systems is indirect and mediated by products

of radiolysis of water including hydrogen radical (H•),

•OH, hydrated electron (eaq

–),

H2O2, peroxyl radical (ROO•), O2

•, ½O2 etc. (Von Sonntag, 1987; Devasagayam and

Kesavan, 1996).

Cigarette smoke contains a large amount of reactive species (Devasagayam and

Kamat, 2002). Cigarette tar contains quinone-hydroquinone-semiquinone system which

reduces O2 to O2•, H2O2 and

•OH. Cigarette smoke contains small oxygen and carbon

centered radicals as well as active oxidants such as NO• and nitrogen dioxide (NO2).

Wentworth et al. (2003) stated that antibodies convert ½O2 into H2O2 via a process that

they have postulated to involve dihydrogen trioxide (H2O3). During ischemia-reperfusion,

oxidants like O2•,

•OH and H2O2 are produced. This occurs during non-fatal myocardial

infarction, surgeries, stroke, transplantation, blockage of arteries under pathological

conditions. During ischemia in heart (in myocyte mitochondria) conversion of ATP to

adenosine causes generation of O2•, while in the blood vessels (endothelium) pathway

involved is transition from xanthine to uric acid (Yoshikawa et al 2000). Vogiatzi et al.

(2009) found some evidence which suggests that, common risk factors for atherosclerosis


increase production of ROS, not only from endothelial cells, but also from the smooth

muscle cells and the adventitial cells (Gozin et al., 1998). Thus, hypercholesterolemia,

diabetes mellitus (DM), arterial hypertension, smoking, age, and nitrate intolerance

increase the production of free ROS.

1.3.1.4 Free radicals in biological system

Free radicals, mostly in the form of ROS/RNS are known to play a dual role in

biological systems. They can either harmful or beneficial to living systems (Valko et al.,

2004). Beneficial effects of ROS/RNS comprise physiological role in cellular response to

anoxia. They play vital role against infectious agents and in function of a number of

cellular signaling systems. Furthermore, lower concentration of free radical is helpful in

induction of mitogenic response. In contrast, at high concentration, free radical can be

important mediators of damage to cell structures including lipids and membranes,

proteins and nucleic acids.

1.3.1.4.1 Free radicals in diseases

According to Hemnani and Parihar (1998) ROS and RNS have been implicated in

the patho-physiology of various clinical disorders including ischemia, reperfusion injury,

myocardial infarction, rheumatoid arthritis, neurodegenerative, atherosclerosis, acute

hypertension, hemorrhagic shock and diabetes mellitus. Some tumor cells also produce

ROS and RNS (Szatrowski and Nathan, 1991). Source of these products and their

contribution to transformed phenotypes are not known. Formation of oxidative stress may

result in damage of critical cellular macromolecules such as DNA, lipid and proteins and

causes many chronic diseases (Poli et al., 2004). Vogiatzi et al. (2009) stated that during

last decade, several studies have examined potential role of oxidative stress in

atherogenesis (Stephens et al., 1996; Ohara et al., 1993). Further, according to theory of

oxidative stress, oxidative modifications of low density lipoproteins (LDL) in the arterial

wall by ROS resulted in atherosclerosis.

Oxidative stress occurs in brain not only because of physical damage but also due

to free radicals produced by viruses, bacteria, fungi or parasitic diseases (Floyd, 1999).

According to Lushchak and Semchyshyn (2012), different types of physical or anoxic

trauma stimulate the generation of ROS/RNS that can recombine with metals and


produce powerful oxidants and also create other forms of ROS and RNS. These forms of

free radicals are well known to create major toxicities to brain and which cause

Parkinson’s disease, amyotrophic lateral sclerosis and Huntington’s disease.

Diabetes mellitus (DM) is caused due to excessive production of RNS; produced

due to oxidative metabolism. Such free radicals damage pancreatic β-cells which

produce, store and release insulin. So, once β-cells die, body becomes deficient in insulin.

These β-beta cells are then replace by α- cells (which produce glucagon) causes an

increase in blood glucose in the body. Once diabetic state is established, there is a

deficiency of NO in the vascular system; which is necessary to maintain proper blood

pressure. This condition leads to damage endothelial cells; which line the blood vessels.

Therefore, blood pressure increases and hypertension occurs (Van Dyke et al., 2010). The

role of transition metal ions is of particular interest because malignant diseases like

chronic inflammation produced changes in distribution in the body. (Halliwel and

gtteridge, 1989; Yagi, 1982). Some other examples originating from free radicals are

Alzheimer’s, Parkinson’s, Down’s syndrome, etc. (Das et al., 1997; Koleva et al., 2002).

1.3.1.4.2 Free radicals and Lipid

Lipids are major target for attack by free radical because oxygen is foremost

soluble in hydrophobic membrane. These free radicals easily break double bond present

in polyunsaturated fatty acid (PUFA). It leads to the peroxidation of PUFA in lipid

membrane; which severely damage cell membrane and thereby produces loss of fluidity

and breakdown of cell membrane. Such conditions make us to suggest possible role of

lipid peroxide in various pathological conditions and aging (Halliwell and Gtteridge,

1989). Significance of lipid peroxide on aging process is based on the fact that lipofuscin

age pigments accumulate almost linearly with advance age and these pigments

presumably results from polymerization of oxidized unsaturated lipids (Ohyashiki et al.,

1984).

It has been considered that, damage to lipid results in aging and pathological

disorders. Some phases of neuronal ceroid lipofuscinosis, intermittent claudication,

oxygen toxicity and liver injury caused by orotic acid, ethanol, phosphorus or chlorinated

hydrocarbon have been reported in relation to lipid peroxidation. Free radical mediated

lipid peroxidation has been proposed to be critically evolved in several diseases including


cancer, rheumatoid arthritis, drug associated toxicity and post-ischemic re-oxygenation

injury as well as in the degenerative diseases associated with aging (Halliwel and

gtteridge, 1989; Yagi, 1982).

1.3.1.4.3 Free radicals and Protein

Radical mediated damage to proteins may be initiated by electron leakage, metal-

ion dependant reactions and auto-oxidation of lipids and sugars. These reactive species

can interact with proteins directly, especially with their sulfhydryl groups (Herington,

1986). Among amino acids, histidine, tryptophan, methionine and tyrosine are more

reactive towards ROS resulting in sulphoxides and short-lived endo-peroxides that may

toxic to other cells. The consequent protein oxidation is O2 dependent and involves

several propagating radicals, especially alkoxyl radicals. This will results in modification

of enzyme activity (Bellomo et al., 1983). Damage to proteins lead to alterations in

intracellular calcium and potassium salts that will trigger a series of changes in cells

(Kerr et al., 1992). In selective cases, alteration of protein structure and the consequent

unfolding is associated with enhanced susceptibility to proteinases.

1.3.1.4.4 Free radicals and DNA

DNA damage is a result of extrinsic and intrinsic process including oxygen

derived free radicals which are normal consequences of cellular metabolism of oxygen.

Several types of damage including base lesions, sugar lesions, protein and DNA cross

link, breaking of single and double strand are produced by free radical induced reactions

(Kaneko et al., 1996). When these free radicals react with sugar moiety of DNA, some

sugar product and interacting bases are released; leads to DNA damage (Dizdaroglu et

al., 1975). Blake et al., (1987) stated that, when DNA is exposed to high fluxes of radical,

strand scission has been observed but changes in bases and deoxyribose sugars may be

more frequent. Hydroxyl radicals are thought to mediate these reactions. DNA damage

including fragmentation has been reported to occur during the respiratory burst and may

be a cause of neutrophil cell death and induction of 'autoimmune' processes (Birnboim

and Kanabus-Kaminska, 1985).


1.3.2 Defense against oxidative stress

To protect body cells and organs, from ill effect of free radicals, humans have

evolved a highly sophisticated and complex antioxidant defensive mechanism. This

defense system either naturally produced in-situ or externally supplied through foods

and/or medicines. These antioxidants act as free radical scavengers by preventing and

repairing damages caused by free radicals. Therefore they can enhance immune defense

and lower risk of cancer and degenerative diseases (Pham-Huy et al., 2008).

According to free radical theory, as a result of accumulation of oxidatively

damaged macromolecules and consequently cells or tissues due to aerobic metabolism to

which individuals are continuously exposed, ageing is initiated in human beings

(Harman, 1965). Thus, antioxidant defense may be one of major mechanism to combat

ageing and age-related problems (Mukherjee et al., 2011). They may be classified as-

Primary defense against free radicals

Secondary defense against free radicals

Primary defense is group of antioxidant enzymes, which are capable of

catalytically removing free radicals (Fridovich, 1978). On the other hand, the secondary

defense is group of macromolecules which act as free radical scavengers.

Now, antioxidants are substance, which when present in foods or body, markedly

delay or prevent oxidation of that substrate. Antioxidants may help body to protect itself

against various types of oxidative damage caused by ROS/RNS, which is linked to a

variety of diseases including cancer, diabetes, shock, arthritis, and acceleration of aging

process (Sanchez-Moreno et al., 1999). Thus, antioxidant defense systems along with

aerobic metabolism counteract oxidative damage rooted by free radicals (Gulcin et al.,

2002).

By definition antioxidants means “Against oxidation”; which has been broadly

explained by Halliwell (1998) as: “any substance when present at low concentrations

with those of an oxidisable substrate significantly delays or prevents oxidation of that

substrate”. Variety of biological activities are possessed by antioxidant compounds

including induction of drug metabolizing enzymes, inhibition of prostaglandin synthesis,

inhibition of carcinogen induced mutagenesis and scavenging of free radicals (Hirose et

al., 1994). In late 19th

and early 20th

century, extensive study was devoted to uses of


antioxidants in important industrial processes like prevention of metal corrosion,

vulcanization of rubber and polymerization of fuels (Matill, 1947).

Antioxidant compounds in food play vital role as a health-protecting factor.

Scientific evidence suggests that antioxidants reduce risk for chronic diseases including

cancer and heart disease. Whole grains, fruits and vegetables are primary basis of

naturally occurring antioxidants. Plant sourced food antioxidants like vitamin C and E,

carotenes, phenolic acids, phytate and phytoestrogens have been recognized as having

potential to diminish such diseases. Most of antioxidants, in a typical diet are derived

from plant sources and belong to various classes of compounds with a wide variety of

physical and chemical properties. Antioxidant compounds like polyphenols and

flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and

thus prevent body from oxidative damage (Miller et al., 2000). Numbers of clinical

studies conclude that antioxidants from fruits, vegetables, tea and red wine are major

source to overcome present problem of chronic diseases including cancer and heart

diseases.

Antioxidants are powerful electron donors; act as chain-breaker and also react

with free radicals before more important target molecules are damaged due to reaction of

free radicals. For example, antioxidant N-acetyl cysteine shows antimutagenic and

chemo-preventive activities in a variety of organs, such as lung, liver, skin and colon

(Flora et al., 1986; Izzotti et al., 1994). Antioxidant can alter gene expression induced by

ROS rather than their direct effect on transcription through Antioxidant Response

Element (ARE) present in promoters of many genes including phase II enzyme

glutathione stransferase (Primiano et al., 1997). Antioxidants show potent effect on

ability of several transcription factors to bind DNA (Winyard and Blake, 1997). They

have been shown to trigger apoptosis in smooth muscle cells independent of oxidative

reactions (Liu et al., 1998 and Tsai et al., 1996). Antioxidants can inhibit tumor initiation,

tumor promotion and cell transformation (Steele et al., 1990). Changes in antioxidant

defense enzymes such as SOD, GPx, CAT and Glutathione S-Tansferase (GST) have

been widely described in cancerous cells (Cerutti et al., 1994). Evidence suggests that

low molecular weight antioxidant enzymes and anti-inflammatory agents that inhibit

arachidonic acid metabolism are anti-carcinogenic (Kensler et al., 1983).


Specific type of free radical can be reduced by specific antioxidant compound or

enzyme. For example Super oxide dismutase (SOD) reduces O2• to H2O2, which can

further reduced by Glutathione Peroxidase (GPx) and Catalase (CAT). Vitamin E

prevents the propagation of free radical damage in biological membrane through its

ability to peroxyl radical (Babber and Harris, 1994). Table 1.1 indicates the mechanism

of antioxidant activity with respect to antioxidant compounds.

Table 1.1: Mechanisms of antioxidant activity.

Antioxidant class Mechanism of antioxidant

activity Example of antioxidant

Proper antioxidants Inactivating lipid free radicals Phenolic compounds

Hydroperoxide stabilizers Preventing decomposition of

hydroperoxides into free radicals Phenolic compounds

Synergists Promoting activity of proper

antioxidants Citric acid, ascorbic acid

Metal chelators Binding heavy metals into

inactive compounds

Phosphoric acid, Millard

compounds, citric acid

Singlet oxygen quenchers Transforming singlet oxygen into

triple oxygen Carotenes

Substances reducing Hydro-

peroxides

Reducing hydroperoxides in a

non-radical way Proteins, amino acids

Source: Pokorny et al. (2001)

All antioxidants are either endogenous, produced in the body e.g. enzymes like

super oxide dismutase (SOD), catalase (CAT), polyphenol oxidase (PPO) etc. or

exogenous antioxidants obtain from food like vitamins, phenols, flavonoids etc.

1.3.2.1 Endogenous antioxidants

Human body relies on several endogenous defense mechanisms to help protect

against free radical-induced cell damage. Body mainly have five types of endogenous

antioxidants like superoxide dismutase (SOD), α-lipoic acid (ALA), catalase (CAT) and

glutathione peroxidase (GPX). Out of which SOD, CAT and GPX are three most

important of these because body can produce more of them when certain free radicals are

present. Antioxidant enzymes catalyze breakdown of radical species usually in


intracellular environment (Fig. 1.1). Preventive antioxidants bind transition metal ions

such as iron and copper; avoid their interaction with H2O2 and O2• to produce highly

reactive •OH.

Fig. 1.1: Free Radicals and Antioxidant Enzymes

1.3.2.1.1 Superoxide Dismutase

In 1969, the work of Mc Cord and Fridovich in USA showed that erythrocyte

protein was able to remove O2• catalytically and thus identified as a superoxide dismutase

(SOD) enzyme. They include Mn++

enzyme in mitochondria and Cu++

/Zn++

enzyme

present in cytosol. Copper-zinc containing Superoxide Dismutase (SOD) having two

protein subunits, each of which bears one copper ion and one zinc ion for stability of

apoenzyme.

Superoxide Dismutase is a metallo-enzyme acting as primary preventive inhibitor

by catalyzing the conversion of O2• to H2O2 (Symons and Gutteridge 1998). Yagi (1982)

proposed two possible mechanisms for action of SOD that inhibit transformation-

1. It is likely to act at level of cell membrane and remove or prevent radical formation

that would cause membrane lipid peroxidation and leads to chain of extra-nuclear and

nuclear events, ultimately expressed as transformation.

2. It has been suggested that, oxygen radicals could be formed in growth medium. These

radicals may act as promoters in transformation process that would be removed by

SOD.

1.3.2.1.2 Catalase

Most aerobic cells contain Catalase activity (CAT) and are largely located in

peroxisomes. Catalase activity of tissue varies greatly; highest in liver and kidney and


lowest in connective tissue. Catalase activity is a metal-containing enzyme and has most

efficient enzyme that promotes redox reaction:

2H2O2 → 2H2O + O2

It also acts as hydrogen donors, for example methanol, ethanol, formic acid,

phenol with consumption of peroxide.

ROOH + AH2 → H2O + ROH + A

Hydrogen peroxide itself is not particularly reactive with most biologically

important molecules; however, it may acts as a precursor for more reactive oxidants such

as •OH. When concentration of H2O2 is high, it is removed by CAT (Bragadottir, 2001).

1.3.2.1.3 Glutathione system

Glutathione, a major non-protein thiol in living organism, plays role in

coordinating body’s antioxidant defense system. Glutathione peroxidase (GPX) is a

selenium-containing enzyme (Symons and Gutteridge, 1998) that catalyses the reduction

of hydrogen of lipid peroxides (LOOH) with reduced glutathione (GSH).

2GSH + H2O2 → 2H2O + GSSG

GSH-PX is capable of reacting with both lipid and hydrogen peroxides (Halliwell and

Gtterridge, 1989). Selenium, which is required for GPX activity, is obtained from the diet

and glutathione peroxidase activity can be enhanced in muscle by increasing the level of

selenium in the diet (Devore et al. 1983). GPX is only antioxidant enzyme, which has

been reported to be influenced by animal diet (Decker and Xu, 1998). Antioxidative

enzymes have little significance in food application and they are currently not of

commercial significance in the food industry (Meyer and Isaksen, 1995).

1.3.2.1.4 Lipoic acid

Lipoic acid, yet another important endogenous antioxidant, categorized as a

“thiol” or “biothiol”. It is a sulfur-containing molecule that known for its involvement in

reaction that catalyzes oxidative decarboxylation of α-keto acids and α-keto-glutarate in

Krebs Cycle. Lipoic acid and its reduced form i.e. dihydrolipoic acid (DHLA), are

capable of quenching free radicals in both lipid and aqueous domains and thus called as

“universal antioxidant”. It may also exert its antioxidant effect by chelating with pro-


oxidant metals. Research further suggests that lipoic acid has a sparing effect on other

antioxidants (Percival, 1998; Packer, 1995; Kagen, 1992).

1.3.2.2 Exogenous antioxidants

1.3.2.2.1 Antioxidant vitamins

Antioxidant vitamins show number of biological activities such as immune

stimulation, inhibition of nitrosamine and an alteration of metabolic activations of

carcinogens (Poppel and Berg, 1997). Experimental evidence suggests that, carotenoids

and other retinoids act in earlier and later stages respectively; that inhibit mutagenesis

and gene expression (Sies, 1993).

Vitamin ‘E’: Vitamin ‘E’ (α-tocopherol) is an important chain-breaking antioxidant

which can directly scavenge ROS. It is a major lipid-soluble antioxidant present in all

cellular membranes, which protects against lipid peroxidation. It also protects membrane

of Poly-Unsaturated Fatty Acid (PUFA) and Low Density Lipoprotein (LDL) (McCay,

1985). Vitamin ‘E’ can directly react with variety of oxygen radicals like peroxyl radical

ROO•, CCl3

•,

•OH, O2

• and singlet oxygen (Fukuzawa and Gebicki, 1983). Vitamin ‘E’

donates hydrogen from chromane ring to free radical. The toco-peroxyl radical formed

can be reduced to tocopherol by interaction with reductants serving as hydrogen donors.

ROO• + Vit. E-OH ROOH + Vit. E-O

•

Vit. E-O• + AH Vit. E-OH + A

•

Vitamin ‘E’ induces growth inhibition of cancer cell by scavenging free radicals,

thereby stabilizing cell membrane (Blot et al., 1993). Because of the antioxidant

properties, vitamin ‘E’ neutralizes ROS and RNS and reduces oxidative DNA damage

and mutation (Frei, 1994).

Vitamin ‘C’: Vitamin ‘C’ (ascorbic acid) is an important water-soluble antioxidant in

biological fluids and an essential micronutrient required for normal metabolic functioning

of body. Vitamin ‘C’ readily scavenges ROS, Ozone, ONOO•, NO2, NO

• and

hypochlorous acid (Noroozi et al., 1998) and reduces oxidative damage and mutations

(Frei, 1994). The most striking chemical activity of ascorbate is its ability to act as a

reducing agent (electron donor). Donation of one electron by ascorbate gives the semi-

dehydro-ascorbate radical, which can be further oxidized to give dehydroascorbate.

Epidemiological studies have indicated an inverse association between intake of vitamin


‘C’ and risk of cancers. Vitamin ‘C’ can act as a co-antioxidant by regenerating

tocopheroxyl radical produced during scavenging of ROS. Reports suggest that, Vitamin

‘C’ may prevent formation of ONOO• by reaction with O2

• and may help to release NO

•

from endothelial cell. Some studies have shown that, Vitamin ‘C’ restores all parameters

to normal by reducing oxidative stress and may help to reduce the risk of developing

diabetic complications. Ascorbic acid supplementation has also been shown to improve

glycemic control and decrease fasting blood glucose, triglycerides and cholesterol levels

in non-insulin dependent diabetes (Eriksson, 1995).

Antioxidants like ascorbic acid and various forms of vitamin E (tocopherols and

tocotrienols) inhibit the auto-oxidation of glucose and glycation of blood proteins e.g.

Hemoglobin. Therefore, antioxidant therapy can ease toxic state which occurs in diabetes

Type I or II (Van Dyke et al., 2010).

1.3.2.2.2 Phenols and Flavonoids

In recent years, there is an increasing interest in finding antioxidant

phytochemicals, because they can inhibit propagation of free radical reactions, protect

human body from diseases. The most effective components seem to be flavonoids and

phenolics found in almost all plants. Their metal-chelating capabilities and radical-

scavenging properties have enabled phenolic compounds to be thought of as effective

free radical scavengers and inhibitors of lipid peroxidation (Terao and Piskula, 1997). It

has been found that phytochemicals like polyphenols and flavonoids are good

antioxidants and exert protective effects against development of cancer and

cardiovascular diseases (Cao et al., 1996).

Cheniany et al., (2010) noted that, phenolic compounds occur in abundance, as

secondary metabolites, in all plant (Kefeli et al., 2003). These compounds belong to a

large and heterogeneous group of biologically active and non-nutrient compound (Jay-

Allemand et al., 2001). The polyphenolic antioxidants either trap initiating radical,

propagating lipid peroxyl radicals, recycling α-tocopherol or deactivating excited

photosentizers, etc. (Tiwari, 2001). The electron and H+ donating capacity of flavonoids

seem to contribute to termination of lipid peroxidation chain reaction based on their

reducing power (Tiwari, 2001). It is well known that all aerobic organisms possess such


antioxidant mechanism to protect against oxidative damages and various types of

enzymes responsible for removal or repair of damaged molecules (Auroma, 1998).

1.3.2.2.3 Synthetic antioxidants

Many synthetic antioxidants have been used for stabilization of foods. Synthetic

antioxidants includes N-acetyl (NAC), butylated hydroxyanisole (BHA) and butylated

hydroxytoluene (BHT), which are added to fatty and oil foods to prevent oxidative

deterioration. NAC has anti-mutagenic and chemo-protective activity in the verity of

organs such as lung, liver skin etc. NAC regulate gene expression and modify cellular

signaling induced by ROS (Lin et al., 2012). BHA inhibits carcinogenicity and

mutagenicity of many chemicals in various experimental animals. BHT has been shown

to inhibit carcinogenic effect of DMBA, 4-DMBA and azoxymethane. (Loliger, 1991).

However, use of these chemical compounds has begun to be restricted because of their

induction of DNA damage and their toxicity (Ito et al., 1986; Sasaki et al., 1986).

Moreover, Zamani et al. (2009) noted that making process of such synthetic

antioxidants is very expensive and some of them like BHA and BHT have confirmed

undesirable effects. These synthetic antioxidants though acts as free radical scavengers by

breaking chain reaction, still some scientist reported that safety of such synthetic

antioxidants are now in doubt (Moein, 2008). Watts (1975) affirmed that, at high dose,

synthetic antioxidants such as BHA and BHT suffer from some disadvantage due to their

toxicity. According to Wettasinghe and Shahidi (1999) potential health hazards caused by

use of synthetic antioxidants in food products has led to scrutiny of natural antioxidants.

Human metabolism counts on antioxidant defensive system involving enzymes

and proteins to prevent adverse effects of oxidative stress. However, defenses can be

overwhelmed in certain circumstances, so that harmful effects occur. It is accepted that

intake of antioxidant substances reinforces defenses against free radicals. Use of synthetic

antioxidants has been limited because of their toxicity (Valentao et al., 2002). Botterweck

et al. (2000) reported that, synthetic antioxidants appear to be involved in tumor

promotion and hence use of a natural antioxidant source become crucial.

So, it is of great significant and necessity that research focuses on discovering

potential natural, effective antioxidants to replace synthetic ones. Therefore, researchers

turn their point of view to find naturally occurring antioxidants for use in foods to swap


such synthetic antioxidants, which are being restricted due to their carcinogenicity

(Velioglu et al., 1998; Singh et al., 2002; Gulcin et al., 2004; Rezaeizadeh et al., 2011).

Thus finding of natural anti-oxidant from new sources is very important.

All human cells protect themselves against free radical damage by antioxidant

enzymes such as superoxide dismutase (SOD) and catalase, or compounds such as

ascorbic acid, tocopherol and glutathione (Niki et al., 1994). Sometimes these protective

mechanisms are disrupted by various pathological processes. When ROS are produced in

huge quantities, aforesaid natural mechanism can be ineffective. In such cases dietary

intake of antioxidants becomes essential. So, recently much attention has been directed

towards the development of ethno medicines with strong antioxidant properties but low

cytotoxicities (Hazra et al., 2008).

1.3.3 Ayurveda

Ayurveda is a traditional Indian medicinal system being practiced for thousands of

years. Along with traditional Chinese System of Medicine, Ayurveda is regarded as one

of two living great traditions of the world (Sekar, 2007). Atharveda (around 1200 BC),

Charak Samhita and Sushrut Samhita (100 - 500 BC) (Dash and Sharma, 2001) are main

classics that given detailed descriptions of over 700 herbs (Shrikumar and Ravi, 2007).

Ayurvedic Pharmacopoeia comprise with more than 1,200 species of plants,

nearly 100 minerals and over 100 animal products. Numerous drugs have entered into the

international pharmacopoeial through considerable research on Ayurveda in respect with

pharmacognosy, chemistry, pharmacology and clinical therapeutics. World Health

Organization (WHO) specifies that, primary health needs of countries in Africa, Asia and

Latin America are met by traditional medicines. Such traditional medicines are adapted to

industrialized countries as Complementary or Alternative Medicines (CAM). Ayurvedic

system of treatment has been estimated to meet 70-80% of the healthcare needs of India

(Viswanathan et al., 2003).

Ayurvedic system of treatment is designed to attain svasthya (to establish one’s

own natural state on perfect health) and advocates samadosa (structural and physiological

equilibrium), samagni (equilibrium of metabolic processes), samadhatu (equilibrium of

body tissues), samamalakriya (equilibrium of eliminative systems), prasannendriya

(equilibrium of senses), prasannamana (equilibrium of mind) and prasannatma (state of

pure awareness or contended self). Ayurveda contains 8 branches of sciences and 10


different diagnostic tools based on tridosha theory (three humours of body). Ayurveda

also advocates a system of prevention of diseases by stipulating a set of practices as daily

routine (Dinacharya) and seasonal routine (Ritucharya). Ayurvedic treatment system also

takes into account individual variations. Herbal drugs have been used by mankind since

time immemorial to treat various disorders and offer an alternative to synthetic

compounds, as they have been considered either non-toxic or less toxic (Tiwari and

Upadhyay, 2009).

Ayurvedic medicines are of various types, so as to meet diverse requirements in

treatment of human illness. They are Swarasa (expressed juice), Kalka (paste), Hima

(cold infusion), Phanta (hot infusion), Kwatha (decoction), Churna (powder), etc. along

with Arishtas (fermented decoctions) and Asavas (fermented infusions) (Dhiman, 2004).

1.3.3.1 Research on Ayurvedic Formulations

Ayurveda is becoming one of the best alternatives for modern medicines. Many

medical practitioners started integrating modern and ayurvedic systems for the benefit of

both systems. In short, Ayurveda not only deals with diseases and health problems but it

also deals with an integrated concept of health which include psychology, sociology,

anthropology, spirituality, tradition, custom, ritual, profession, food, family, social

relation and so on. According to Parekh et al. (2005) mainly in developing countries,

herbal medicine is still mainstay of about 75-80% of whole population for primary health

care because of better cultural acceptability, better compatibility with human body and

fewer side effects. Herbal medicines are claimed to be an oldest system of medicines in

the world and used for curing various diseases. Search for new chemical entities obtained

by screening natural sources such as plant extracts and microbial fermentation had led to

discovery of many clinically useful drugs that play major role in the treatment of human

diseases (Anonymous, 1994; Chaudhari, 1996).

Ayurvedic literatures are very rich in therapeutic formulations for all sorts of

disorders. Likewise references are also available on standardization of formulations and

their bioactivity. Naik et al. (2005) made phytochemical analysis and antioxidant

potential of ayurvedic formulation Triphala. Further, they examined Triphala extract for

its radio protective activity and confirmed that free radical reactions, antioxidant and

radio protecting activity of Triphala arise from polyphenols, which reduce oxidative

stress by converting reactive oxygen species and free radicals to non reactive products.


Recently, Patel et al. (2010) made detailed study and developed HPLC method for the

identification of such phenolic compounds e.g. Ellagic acid and Gallic acid in Triphala.

More recently, Samarakoon et al. (2011) evaluated antioxidant potential of

Amakajayas by using different antioxidant modules and some preliminary phytochemical

like phenols. Amalakayas Rasayana is a polyherbal composition in which Phyllanthus

emblica is the principal ingredient. Borde et al. (2011) analyzed some ayurvedic

formulations for their gallic acid contain.

Mukherjee et al. (2011) reported that, Rasayana therapy is one of the major

methods for preservation of health and delaying the process of ageing as described in

Ayurvedic system of medicine. Mukherjee et al. (2011) check the free radical quenching

property of Vayasthapana Rasayana and concluded that it possess good antioxidant

activity to fight age-related problems. In last decade Jagetia et al. (2004) evaluate NO

scavenging activity of certain herbal formulations like abana, chyavanaprasha, triphala,

geriforte, septilin and mentat. Velioglu et al. (1998) examined 28 plant products and

found a significant relationship between antioxidant activity and phenolics.

Various scientists have actively participated to validate Ayurvedic formulations

(Yadav and Dixit, 2008; Shenoy and Yoganarasimhan, 2008; Dubey et al., 2008). Kumar

et al. (2011) intended a method for evaluation of Nisamalaki Churna tablet which is a

Ayurvedic formulation used for anti-diabetic and consists of Curcuma longa and Emblica

officinalis (Anonymous, 2000). Waghmare et al. (2011) standardized parameters such as

physicochemical, chemo profiles as preliminary analysis, TLC fingerprint profiles and

safety evaluation as microbial contamination. Heavy metal determination was also

evaluated with marketed formulations of Varunakwatha Churna. Parameswaran and

Mandar (2010) standardized Ayurvedic formulation Sanjivani Vati through RP-HPLC.

Jaiprakash et al. (2012) worked on polyherbal formulation Vyaghriharitaki Avaleha with

a classical point of view. Somanathan et al. (1989) reported a chemical method for

standardization of Dasamulam Kasayam, which can be of general applicability to other

formulations.

Origin of Ayurveda indeed difficult to pinpoint, it have been placed by scholars of

Ayurveda and ancient Indian literature at around 6000 B.C. (Sayyad et al., 2012; Thatte

and Dahanukar, 1986). Ayurvedic remedy considered as cost effective, inherently safe

and with lesser side effects (Bouldin et al., 1999). This traditional system comprises of


various types of medicines including fermented forms, namely, Arishtas and Asavas.

(Vyas et al., 2010).

1.3.3.2 Arishta and Asava

Arishta and Asava are liquid preparations containing self generated alcohol, thus

contain water soluble as well as alcohol soluble substances of drugs. They are alcoholic

medicaments prepared by allowing herbal juices or their decoctions to undergo

fermentation with the addition of sugars, jaggery or honey (Sekar and Mariappan, 2008).

Due to their medicinal value, sweet taste, and easy availability, people are prone to

consume higher doses of these drugs for longer periods (Narayana and Subhose, 2005).

Arishtas and Asava differ from each other owing to their difference in method of

preparation. Arishtas are preparations which are subjected to fermentation for a specific

time after adding main decoction of herbs and adding other ingredients. While, in Asavas

infusion of main herbs are subjected to fermentation with other ingredients for allotted

period (Shastri, 1968; Nadkarni, 1976; Srikantha, 1989; Dash and Hashyap, 2002;

Viswanathan et al., 2003; Patwardhan et al., 2004; Dhiman, 2004). Fermentation of both

preparations is brought about by addition of sugar or jagerry with Dhataki (Woodfordia

fruticosa) flowers and honey. Many contain additional spices for improving their

assimilation. They are moderately alcoholic (up to 12% by volume) and mostly sweetish

with slight acidity and agreeable aroma. These medicinal wines have several advantages,

like better keeping quality, enhanced therapeutic properties, improvement in efficiency of

extraction of drug molecules from herbs and improvement in drug delivery into human

body sites (Sekar and Mariappan, 2008).

Literature survey on Arishtas and Asavas

Weerasooriya et al. (2006) studied quantitative parameters of Arishta and Asava

to guarantee quality and safety of product to consumer and established quality and

standard parameters like alcohol level, pH, acid value and other constituents of these

preparations. Weerasooriya et al. (2006) determined level of alcohol, acidity and pH in

commercially available Ashwagandharishta and Aravindasava to establish a routine

procedure for standardisation of these Ayurvedic preparations. They further stated that

these medicinal wines have several advantages like better keeping quality, enhanced

therapeutic properties, improvement in efficiency of extraction of drug molecules from

herbs and improvement in drug delivery into the human body sites. Govindarajan et al.

(2008) studied chemical constituents of polyherbal formulation (Ashokarishta) and


validated this by RP–LC method. Tatiya et al. (2008) prepared Arishta and Asava by

fermentation method and standardized these formulations based upon physicochemical

and phytochemical parameters. Kushwaha and Karanjekar (2011) prepared

Ashwagandharishta by traditional method and standardized by TLC method. They also

studied physicochemical and phytochemical parameters to confirm chemical constituents

from Ashwagandharishta. Sayyad et al. (2012) formulated Arjunarishta by traditional

method and evaluated its quality assessment. Mishra et al. (2012) assessed different

marketed brands of Ashokarishta and analyzed gallic acid concentration in different

brands by using HPTLC. Kumar et al. (2009) studied effect of Ashokarishta on

endocrine, bone, blood and biochemical profile of postmenopausal women.

Gharate et al. (2011) believed that establishing quality and standard parameters

like alcohol level, pH, acid value, total viable count and boiling point of such fermented

medicaments are highly significant. So they determined level of alcohol, acidity and pH

in commercially available Kanakasava and established routine procedure for

standardization of this Ayurvedic preparation.

Jain et al. (2009) standardized Dashamularishta, while, Singh et al. (2010a)

standardized Arjunarishta by using TLC method. Kalaiselvan et al. (2010) assessed

different marketed brands of Dashamularishta. They further declared that herbal

formulations, available in the market are usually not properly standardized and are not

assessed for their quality. As use of herbal formulations by patients is increasing, there is

an urgent need for pharmacists and physicians to have knowledge about safety and

efficacy of these preparations. Rajalakshmy and Sindhu (2011) screened preliminary

phytochemical and evaluated antioxidant property of Balarishtam. Kulkarni et al., (2012)

evaluated physicochemical characters and compared efficacy of Saratswatarishta by

process variations. Asava and Arishta were screened for antimicrobial activity by Farooq

and Pathak (1998). Hepatoprotective effect of Kumariasava on carbon tetrachloride

induced hepatic damage in rats was studied by Kataria and Singh (1997).

Though traditional knowledge about preparation and applications of such

medicaments exists in literature, as well as standard parameters like alcohol level, pH,

acid value and other constituents are normally practiced for validation. However, bulk of

knowledge about interesting antioxidant properties on these fermented medicines has

remained unrecognized or not been studied in detail and validated.


Earlier research on role of antioxidants in biology, focused on their use in

preventing oxidation of unsaturated fats, which is the cause of rancidity (German, 1999).

A wide range of antioxidants both natural and synthetic has been proposed for use in

treatment of human diseases caused due to ROS. Interest in role of antioxidants in human

health has prompted research in fields of food science and medicinal plants, to assess role

of plants as antioxidants (Beris, 1991). Plants are rich source of chemical compounds,

which protect organism from free radical injury and disease (Hogman, 1989). Following

table indicate detail about antioxidant survey of plants used in the preparation of Arishtas

and Asavas with their main chemical compositions.

Table 1.2: A brief description of antioxidant plants.

Plant name

(Family): Local

Name

Part Used Chemical composition Antioxidant Reference

Adhatoda vasica

Nees

(Acanthaceae):

Vasa

Root

Vasicinolone, vasicol,

Peganine and 2' -hydroxy - 4

- glucosyl –oxychalcone ,

Maiontone, apigenin,

Astragalin, Kaempferol

(Sampath et al., 2010)

Padmaja et al., 2011;

Kaur et al., 2012;

Jahangir et al., 2006;

Gupta et al., 2010;

Khandelwal et al., 2011.

Aloe barbadensis

Miller (Liliaceae):

Kumari

Leaf

Anthraquinone glycosides,

Acemannan , Aloe-Emodin,

Aloins, Enzymes,

Mucopolysaccharides,

Galactomannans,

latexcontains (Adesuyi et al,

2012; Waller et al, 1978)

Lee et al., 2000;

Hu et al., 2003; Nwanjo,

2006.

Berberis aristata DC

(Berberidaceae):

Daruhadrika,

Daruhald

Stem

Alkaloids, Berberine,

Berbamine, Aromoline,

Palmatine oxyacanthine and

Oxyberberine (Shahid et al.,

2009; Mazumder et al.,

2010;)

Singh and Kakkar, 2009;

Mazumder et al., 2010;

Gupta et al., 2010;

Tiwari and Khosa, 2010.

Chlorophytum

tuberosum (Roxb.)

Baker (Liliaceae):

Musali

Root

Proteins, carbohydrates and

saponins (Patil and Deokule,

2010)

Narasimhan et al., 2006;

Chanda and Dave, 2009.


Plant name

(Family): Local

Name


Cinnamomum

tamala Nees &

Eberm. (Liliaceae):

Tejpatra

Leaf

Carbohydrates, Glycoside,

Alkaloid, Amino Acids,

Flavanoids, Fixed Oil,

Tannins, Mucilage,

Saponins, Terpenoids,

Phytosterols (Palanisamy et

al., 2011;Jain et al., 2011a;

Mishra et al., 2010)

Chakraborty and Das,

2010; Eswaran et al.,

2010; Mathew and

Abraham, 2006; Chanda

and Dave, 2009; Smerq

and Sharma, 2011;

Khandelwal et al.,2011.

Cinnamomum

zeylanicum Blume

(Liliaceae): Tvak

Stem Bark

Ascorbic Acid, α-

Tocopherol, Total

Carotenoids, Lycopene,

Reduced Glutathione, Total

Phenols and Flavonoids

(Varalakshmi et al., 2012)

Varalakshmi et al., 2012;

Jayaprakasha et al.,

2007; Pandey et al.,

2010;

Cuminum cyminum

L. (Apiaceae):

Ajaji, Sweta Jiraka,

Jire

Fruit

Terpenoids (Iacobellis et al.,

2005; Aina et al. 2012),

Carbohydrates, Thiamin

(Vit. B1), Riboflavin (Vit.

B2), Niacin (Vit. B3),

Vitamin B6, Vit. C, Vi. E, α-

Pinene, Pmentha- 1, 3-dien-

7-ol, D-terpinene, Cuminic

aldehyde, Cuminyl Alcohol

(Muhammad and Riaz,2012)

Koppula and Choi,

2011; Jagtap and Patil,

2010; Khandelwal et al.,

2011; Muhammad and

Riaz, 2012.

Curcuma longa L.

(Zingiberaceae):

Haridra, Haladi.

Rhizomes.

Curcumin (Jain et al., 2011);

Curcuminoids (Curcumin,

Bisdemethoxycurcumin,

Demethoxycurcumin),

Phenolic volatile oils (Cai et

al., 2003)

Cho et al., 2011; Kim et

al., 2005; Selvam et al.,

1995; Nishinaka et al.,

2007; Yu et al., 2002;

Khandelwal et al., 2011;

Sanhita et al., 2012.


Plant name

(Family): Local

Name


Cyperus rotundus

Linn. (Cyperaceae):

Mustaka, Musta

Rhizome

Carbohydrate, Tannins,

Saponins, Flavonoids,

Alkaloids, β-Cyanins,

Quinones, Terpenoids,

Phenols, Coumarins,

Proteins, Steroids (Lydia

and Sundarsanam, 2012;

Sharma and Singh, 2011)

Lydia and Sundarsanam,

2012;

Nagulendran et al.,,

2007.

Elettaria

cardamomum (L.)

Maton

(Zingiberaceae):

Ela

Seeds Total phenols (Abbas, 2011)

Jamal et al., 2006; Verma

et al., 2009; Jain et al.,

2011b; Khalaf et al.,

2008.

Glycyrrhiza glabra

L. (Leguminaceae):

Madhuka

Root

Triterpene, Saponins,

Flavonoids,

Polysaccharides, Pectins,

simple sugars, Amino acids,

Mineral salts (Meena et al.,

2010; Obolentseva et al.

(1999); Liquiritin,

Isoliquiritin (Yamamura et

al., 1992); Glabridin,

Glabrene (Tamir et al.,

2001); Glycyrrhetic acid

(Ploeger et al., 2001)

Naik and Satav, 2003;

Rafi, 2004; Wittschier et

al., 2009; Siracusa et al.,


2011; Vaya et al., 1997.

Jasminum officinale

L. (Oleaceae): Jati Flower

Secoiridoid glucoside,

Oleuropein (Teerarak et al.,

2010)

Tsai et al., 2006

Mangifera indica L.

(Anacardiaceae):

Amra, Amba

Ripe and

unripe

fruits

Alkaloids, Steroids,

Carbohydrates, Flavonoids,

Saponins, Amino acids,

Proteins, Phenols and tanins

(Latha, 2011)

Ribeiro et al., 2008;

Ghosal, 1996;

Maisuthisakul and

Gordon, 2009; Chanda

and Dave, 2009; Sanhita

et al., 2012.


Plant name

(Family): Local

Name


Nymphaea stellata

Burm. f.

(Nymphaeaceae)

Utpala

Flower

Nymphayol (Subhash et al.,

2009); Gallic acid (Rakesh

et al., 2009);

Shajeela et al., 2012;

Rajagopal and Sasikala,

2008; Raja et al.,

2011;Rakesh et al., 2010;

Alam et al., 2012.

Ocimum sanctum L.

(Lamiaceae) Tulsi Leaves

Rosmarinic acid,

Lithospermic acid, Eugenol,

Methyleugenol, Urosolic

acid, Phenolics and

Flavonoids such as Orientin,

Vicenin ( Kelm et al., 2000).

Kelm et al., 2000;

Yanpallewar et al., 2004;

Adhvaryu et al., 2007.

Phyllanthus emblica

L.

(Euphorbiaceae):

Amla, Amaliki.

Fruit

Vitamin C, Flavanoids,

Kaempferol, Ellagic Acid

and Gallic Acid (Singh,

2008)

Ghosal et al., 1996; Liu

et al., 2008; Chanda and

Dave, 2009; Scartezzini

et al., 2006; Yokozawa et

al., 2007; Mahesh et al.,

2009; Srikumar et al.,

2005; Rasool and Sabina,

2007; Chanda and Dave,

2009; Sanhita et al.,

2012.

Piper nigrum L

(Piperaceae):

Marika

Seeds,

Fruits

Alkaloid, Glycosides,

Terpenoid, Steroid,

Flavonoid, Tannins,

Reducing Sugar and

Anthraquinones (Nahak and

Sahu, 2011).

Gulcin, 2005; Singh et

al., 2008; Chanda and

Dave, 2009; Khalaf et

al., 2008; Nahak and

Sahu, 2011; Madhu et

al., 2012.

Pluchea lanceolata

(DC.) Oliv. & Hiern

(Asteraceae):

Rasana

Root, Leaf

Terpenoids, Anthroquinone

Glycoside, Coumarin,

(Arora et al., 2011)

Arora et al., 2011


Plant name

(Family): Local

Name


Pueraria tuberosa

DC. (Fabaceae):

Vidari

Root

Tuberosin (Pandey and

Tripathi, 2010); Isoflavone,

Coumestan (Ramakrishna et

al., 1998); Puetuberosanol,

epoxychalcanol (Pawan et

al., 1996); Pterocarponoids

anhydrotuberosin 3-O

methylanhydrotuberosin,tub

erostan (Prasad et al., 1985)

Santosh et al., 2010;

Pandey and Tripathi,

2010; Bharti et al.,

2012.

Ricinus communis L.

(Euphorbiaceae):

Eranda

Root

Alkaloids and flavonoids

(Kang et al., 1985); Phenols

(Khogali et al., 1992); (Jena

and Gupta, 2012).

Singh and Chauhan

200); Kadri et al., 201);

Gupta et al., 2006;

Ilavarasan et al., 2006;

Jena and Gupta, 2012.

Rubia cordifolia L.

(Rubiaceae):

Manjishtha

Root

Anthraquinones (Purpurin,

Alizarin, Munjistin, and

their Glycosides) (Cai et al.,

2003); Triterpenoids,

Anthraquinone (Prajapati

and Parmar, 2011) ;

Rubiadin (Mohana et al.,

2006; Deoda et al., 2011)

Cai et al., 2003; Prajapati

and Parmar, 2011; Son

et al., 2008.

Santalum album L.

(Santalaceae):

Candana

Heard

wood

Santene, Nortricyclo-

Ekasantalene, Alcohols-

Santenol, Teresantalol,

(Kirtikar and Basu, 1933)

(Shankaranaryana, 1980;

Brunke et al., 1995) and the

acids α- santalic acids, β-

Santalic acids and

Teresantalic acids

(Kaur, 2005)

Banerjee et al. 1993;

Sindhu et al., 2010;


Plant name,

(Family): Local

Name

Part

Used Chemical composition Antioxidant Reference

Saraca asoca (Roxb.)

Wilde (Fabaceae):

Ashoka

Stem

bark

Tannins, Proteins, Steroids,

Glycosides, Carbohydrates,

Saponins, Flavonoids (Saha

et al., 2012;

Pradhan et al., 2010)

Parihar et al., 2010;

Panchawat and Sisodia,

2010.

Syzygium aromaticum

(L.) Merrill & Perry

(Myrtaceae):

Lavanga

Flower

Bud

Total Phenols and

Flavonoids (Kim et al.,

2011).

Banerjee and Das, 2005;

Shyamala et al., 2003;

Khandelwal et al., 2011;

Wojdyło et al., 2007;

Kim et al.,2011.

Terminalia arjuna

(Roxb.) Wight & Arn.

(Combretaceae):

Arjuna

Stem

Bark

Sugars, Amino Acids,

Proteins, Phenols,

Terpenoids, Alkaloids,

Flavonoids, Quinones,

Steroids (Doorika and

Ananthi, 2012);Oxalic acid,

Inorganic Acid,

Carbohydrate (Nema et al.,

2012); Vitamin C, Vitamin

E, Carbohydrates, Tannins

Ellagic acid (Raj et al.

2012);

Doorika and Ananthi ,

2012; Singh et al., 2011;

Raj et al., 2012.

Terminalia belerica

(Gaertn.) Roxb.

(Combretaceae):

Bibhitaka

Plant

Gallic acid, Tannic acid and

Ascorbic acid (Singh et al.,

2008)

Yokozawa et al., 2007;

Mahesh et al., 2009;

Srikumar et al., 2005.

Terminalia chebula

Retz.

(Combretaceae):

Haritki

Plant,

Fruits

Chebulagic acid, Chebulinic

acid, Tannic acid, Ellagic

acid, Gallic acid, Ascorbic

acid (Singh, 2008); Tannins

(Ellagitannins), Phenolic

acids (Cai et al., 2003)

Yokozawa et al., 2007;

Mahesh et al., 2009;

Srikumar et al., 2005;

Yadav et al., 2011;

Chanda and Dave, 2009.


Plant name,

(Family): Local

Name

Part

Used Chemical composition Antioxidant Reference

Vitis vinifera L.

(Vitaceae): Draksha

Fruits,

Seeds

Lipids, Proteins, Pectin,

Carbohydrate, Vit B2, Vit C,

Phenols, Oxalic Acids,

Alkaloids (Khan et al.,

2008; Lutz et al., 2011).

Baydar et al., 2007;

Chanda and Dave, 2009;

Fauconneau et al., 1997;

Lutz et al., 2011,

Jayaprakasha et al.,

2001.

Withania somnifera

(L.) Dunal

(Solanaceae):

Ashvagandha

Root,

Leaf,

Seed

Alkaloids (Isopellertierine,

anferine), Steroidal Lactones

(Withanolides, Withaferins),

Saponins containing an

additional acyl group

(Sitoindoside VII and VIII),

Withaferin A (Singh et al.,

2010b)

Ichikawa et al., 2006;

Maitra et al., 2009;

Udayakumar et al., 2010;

Bhattacharya et al.,


2011; Sanhita et al.,

2012.

Woodfordia fruticosa

(L.) Kurz

(Lythraceae): Dhatki

Flowers

Tannins, Cardiac

Glycosides, Steroids,

Saponins, (Vaghasiya et al.

2011; Finose and K.Devaki,

2011), Anthraquinonoes,

Flavons, Flavonols and

Chalcones, Terpenoids,

Phlobatanins and Cardiac

Glycosides (Khan et al.,

2011a).

Nitha et al., 2012;

Khan et al., 2011.

Zingiber officinale

(Zingiberaceae):

Sunthi

Rhizome

Phenolic, Volatile oils

(Gingerol Analogues:

Gingerols, Shogaol) (Cai et

al., 2003)

Kabuto et al., 2005;

Jiang et al., 2005; Rhode

et al., 2007; Shukla and

Singh, 2007; Ahmed et

al., 2008.

Many researchers, who have carried out work to prove antioxidant action of

drugs, have used either single in-vitro or in-vivo model. However, it is not advisable to

follow only one model and in many cases results are not reproducible. Various in-vitro

antioxidant models, which have proved efficient in confirming anti-stress properties of

extracts and formulations, would be of more helpful when used as a set of models at a

time for screening antioxidant potential. Such screening with various models would


enlighten anti-stress prospective of any drug or extract under various stress conditions.

Therefore, it is very important to use as much possible antioxidant models at a time to

generate more reliable results, to prove claims of polyherbal drugs which are already in

market and to screen new formulations or products.

Arishtas and Asavas are considered as unique and valuable therapeutics in

Ayurveda. Though traditional knowledge in literature as well as in practice exists about

Arishtas and Asavas, there was little effort to document, preserve and improve this

knowledge for betterment of mankind. A Literature survey revealed that although,

Arishta and Asava have been studied for ability to cure number of diseases, information

on quantitative parameters of Arishta and Asava to guarantee quality of the product to

consumer is less. Carrying out in detail studies on antioxidant potential and antioxidant

related biochemical parameters of Arishta and Asava may find applications of these

fermented polyherbal drugs in the management of diseases caused due to oxidative stress.

Therefore, it was intended to investigate the antioxidant potential of Arishtas and

Asavas by way of different in-vitro models to trough light on their possible exploitation

in stress management, cancer and heart diseases as well as general health problems.

1.4. SCOPE OF THE PRESENT STUDY

Antioxidants are micro-constituents of diet that are involved in structural

maintenance of DNA and cell and their repair. They protect DNA and cell membranes

against oxidative damage, including that induced by carcinogenic agents. It is therefore,

biologically believable that diets rich in antioxidants protect against cancer and other

chronic diseases. Individual and population requirements for antioxidants are determined

by the level of exposure to oxidative stress. Not only diets rich in vegetables and fruits

but regular consumption of fermented polyherbal medicine also give protection against

oxidative damage. Present study is the major attempt on antioxidant activity of Arishta

and Asava related with their biochemicals.

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