ANTIOXIDENT, ANTIMICROBIAL AND PULICARIA INULOIDES AND OCIMUM FROSKOLEI: A REVIEW

973

AMERICAN RESEARCH THOUGHTS ISSN: 2392 – 876X

Volume 1 │ Issue 2 │ December 2014

Available online at: www.researchthoughts.us

ANTIOXIDENT, ANTIMICROBIAL AND

PULICARIA INULOIDES AND OCIMUM

FROSKOLEI: A REVIEW

Nabil Qaid M Al-Hajj1,2*, Hana Rashid2, Hong xin Wang1, Riyadh Thabit1

and Marwan M. A. Rashed1

1State Key Laboratory of Food Science and Technology, School of Food Science and

Technology, Jiangnan University, Wuxi 214122, P.R. China

2Marine Science and Biological Research Authority, Aden, Yemen

Abstract: The traditional medicinal plants still play a vital role to cover the basic health needs in the

developing countries. Herbal remedies used in the traditional folk medicine provide an

interesting and still largely an unexplored source for the creation and development of potential

new drugs. Plant extracts represent a continuous effort to find new compounds with the

potential to act against multi-resistant bacteria. The medicinal value of plants lies in some

chemical substances that have biological activities; the most important of these bioactive compounds

of plant are alkaloids, flavonoids, tannins, essential oils, glycosides, saponins, steroids,

phenylpropanoids and carbohydrates. In addition, interest in the antioxidant activity of plant

extracts has become larger and very important due to the fact that free radicals, e.g. reactive

oxygen species can be responsible for various diseases, including cancer, coronary heart diseases,

inflammatory conditions, gastric ulcer, cirrhosis, diabetes and aging are associated with

oxidative stress caused by an imbalance between oxidants and antioxidants in the body. So, the

need for new sources of antioxidants is escalating to make life easier and health better. Many

antioxidant compounds, naturally occurring from plant sources, have been identified as a free radical

or active oxygen scavengers and Beta carotene bleaching assay.

Key words: traditional medicinal plants, developing countries, plant extracts, bacteria,

alkaloids, flavonoids, tannins, essential oils, glycosides, saponins, steroids,

phenylpropanoids and carbohydrates

* Corresponding author: [email protected]

Nabil Qaid M Al-Hajj, Hana Rashid, Hong xin Wang, Riyadh Thabit, Marwan M. A. Rashed- ANTIOXIDENT,

ANTIMICROBIAL AND PULICARIA INULOIDES AND OCIMUM FROSKOLEI: A REVIEW

974 AMERICAN RESEARCH THOUGHTS- Volume 1 │ Issue 2 │2014

INTRODUCTION

The flora of Yemen, where is very rich and heterogeneous. Species diversity is a result

of considerable climatic variation, which enabled different species to survive in the

different ecological habitats. Over 3000 plant species are possibly found in the

mainland, and about 10% of them are endemic, comprised 467 plant species belonging

to 244 genera from 71 Families [1]. Socotra Island is undoubtedly a most precious

natural asset not only in the Republic of Yemen but also for the whole world and

humankind. This Island is unique in its flora and like many oceanic islands, have a high

level of endemism [2]. Medicinal plants have been identified and used throughout

human history. Plants have the ability to synthesize a wide variety of chemical

compounds that are used to perform important biological functions, and to defend

against attack from predators such as insects, fungi and herbivorous mammals. At

least 12,000 such compounds have been isolated so far; a number estimated to be less

than 10% of the total [3]. Chemical compounds in plants mediated their effects on the

human body through processes identical to those already well understood for the

chemical compounds in conventional drugs; thus herbal medicines do not differ greatly

from conventional drugs in terms of how they work. This enables herbal medicines to

be as effective as conventional medicines, but also gives them the same potential to

cause harmful side effects [3]. The use of plants as medicines predates written human

history. Ethnobotany (the study of traditional human uses of plants) is recognized as an

effective way to discover future medicines. In 2001, researchers identified 122

compounds used in modern medicine which were derived from "ethnomedical" plant

sources; 80% of these have had an ethnomedical use identical or related to the current

use of the active elements of the plant [4]. Many of the pharmaceuticals currently

available to physicians have a long history of use as herbal remedies,

including aspirin, digitalis, quinine, and opium [4]. The use of herbs to treat disease is

almost universal among non-industrialized societies, and is often more affordable than

purchasing expensive modern pharmaceuticals. The World Health

Organization (WHO) estimates that 80 percent of the population of some Asian and

African countries presently use herbal medicine for some aspect of primary health care.

Studies in the United States and Europe have shown that their use is less common in

clinical settings, but has become increasingly more in recent years as scientific evidence

about the effectiveness of herbal medicine has become more widely available. The

http://en.wikipedia.org/wiki/Predator

http://en.wikipedia.org/wiki/Insect

http://en.wikipedia.org/wiki/Fungi

http://en.wikipedia.org/wiki/Herbivorous

http://en.wikipedia.org/wiki/Mammal

http://en.wikipedia.org/wiki/Aspirin

http://en.wikipedia.org/wiki/Digitalis

http://en.wikipedia.org/wiki/Quinine

http://en.wikipedia.org/wiki/Opium

http://en.wikipedia.org/wiki/Herb

http://en.wikipedia.org/wiki/Disease

http://en.wikipedia.org/wiki/World_Health_Organization

http://en.wikipedia.org/wiki/World_Health_Organization




annual global export value of pharmaceutical plants in 2011 accounted for over US$2. 2

billion [4].

The genus Pulicaria which belongs to the Asteraceae family (Compositae, trible

Inuleae, subtrible Inulinae), comprises more than 77 species widespread all around the

world [5]. Pulicaria genus is an annual herb producing small bright yellow flowers [6].

Chemically, this genus is not homogenous. As pointed out previously some species

contain monoterpenes, diterpenes, sesquiterpene lactones [7] and caryophyllane

derivatives [6]. Also the literature reports that Pulicaria species afforded different

flavonoid profiles [4]. The Pulicaria species proved various activities such as

antiinflammatory, antilukemic [8], potential cancer chemopreventive and cytotoxic

agents [8]. The Pulicaria jaubertii indigenous to Yemen, locally known as Anssif, is

traditionally used as diuretic, pyritic conditions in urogenetic organs, and to cure fever.

The flowers of Pulicaria jaubertii was also used as spice and to make various delicious

foods. Some investigation reported that this species reveal antimicrobial, antifungal,

antimalarial and insecticides properties [9]. The leaves of Pulicaria inuloides had

antibacterial and antioxidant activities and was also used to flavor foods and to make

an herbal tea [5]. Essential oil of Pulicaria inuloides is volatile, natural, complex

compounds characterized by a strong odor and formed by aromatic plants as secondary

metabolites and it plays an important role in the protection of the plants as

antibacterials, antivirals, antifungals and insecticides [5].

Figure 1. Macrograph of Pulicaria inuloides

The genus Ocimum, which belongs to the Lamiaceae family, is a low-growing (30-100

cm), annual plant. It has a square, slightly hairy stem and ovate, entire to slightly




toothed leaves. Leaves vary in color: from bright green to dark purple. Flowers also

vary in color: white, pink or red. They appear along the leaf axils, during August and

September. Ocimum has many uses. It is considered to be antibacterial, antifungal,

antispasmodic, carminative, diaphoretic, digestive, emmenagogue, expectorant,

stimulant, stomachic, refrigerant etc. The plant is generally used in treatments of

problems concerning digestion and nervous system. Leaves are taken (fresh or dried) in

cases of fevers, abdominal cramps, gastro-enteritis, constipation, nausea and poor

digestion. Tea prepared from the leaves is considered to obviate mild nervous tension,

headaches and nausea. Water boiled with basil leaves is taken in case of sore throat.

Decoction of the leaves acts as a helpful remedy in treatment of respiratory disorders.

Juice of basil leaves promotes expulsion of kidney stones. Chewing on basil leaves on a

daily basis can act as a significant protection against stress, ulcer and mouth infections.

Plant is also useful in reduction of blood cholesterol. Ocimum plants are also called

basil with many widespread medicinal uses. Based on essential oil composition,

there has been many chemotypes reported from basil species, which fall either

under terpenoid or phenylpropanoid class. Moreover, monoterpenoids dominate

basil essential oils in various proportions. [10]. 1,8-cineole [11], linalool [12], terpinen-

4-ol [12], citral [13], anisole and methyl-(E)-cinnamate [13]. Similarly, Ocimum

forskolei has been reported with high Camphor, cineole, estragol, eugenol, linalool,

pinene [14]. P. arabica is used medicinally to treat painful swellings and boils. The

whole plant is strongly aromatic and sprigs were often plucked and rubbed between the

hands and then over the body as a deodorant. It grows abundantly in drier areas, where

the water levels lie close to the surface, and is a reliable indicator of the proximity of

water [11]. The biological activities of medicinal plants are mainly due to the presence

of their active constituents or secondary metabolites, which are present almost in higher

plants, usually in a high structural diversity. As a rule, a single group of secondary

metabolites dominates within a given taxon. A few major compounds are often

accompanied by several derivatives and minor components [15].




Fig 2. Macrograph of Ocimum forskolei

ANTIOXIDANT NATURAL PLANT

An array of intra and extracellular antioxidant mechanisms are essential to scavenge

any oxidants ‘reactive intermediate’ which are continually generated in almost all

aerobic cells, otherwise tissue damage occurs .[61] The antioxidant is any substance

which, when present at low concentrations compared with those of an oxidizable

substrate, significantly delays or prevents oxidation of substrate. The term ’oxidizable

substrate’ includes almost everything found in the living cells, including proteins,

lipids, DNA and carbohydrates .[11] Biological antioxidants have been defined as

compounds that protect biological systems against the potentially harmful effects of

processes or reaction that can cause excessive oxidation [11] . Our body is rich in

endogenous antioxidants, the substances that have the ability to stop the free radical

formation or to limit the damage they cause [11] . The effectiveness of current used

exogenous antioxidants arises most probably from the increase of the endogenous free

radical scavengers as enzymes (superoxide dismutase and selenium-dependent

glutathione peroxidase), vitamins (alpha tocopherol and ascorbic acid). Many plants

have also been found to possess free radical scavenging activity (Polyphenols, alkaloids

and terpenoids). Low levels of one or more of the essential antioxidants have been

shown to be associated with many disorders, including cancer, inflammation,

atherosclerosis, coronary heart disease and diabetes. Thus, in such cases, the

administration of exogenous antioxidants seems to be salutary. Nowadays, a great deal




of effort being expended to find effective antioxidants for the treatment or prevention of

free radical-mediated deleterious effects [11] .

SOURCE OF ANTIOXIDANTS

There are several sources of antioxidant: those that we can get from food and food

supplements e.g. vitamin E, D and b carotene; and those that are produced within our

own bodies they are less well known but vital. The latter type includes molecules such

as glutathione and uric acid, which scavenge free radicals directly; and enzymes such as

superoxide dismutase, catalase and glutathione peroxides which can break free radical

into nontoxic products. There is also melatonin, which comes as a new member in

antioxidant systems besides macromolecules such as caeruloplasmin and transferring

and an array of small molecules including Methionine [02] , vitamins E & C. Plant

extracts containing low molecular mass compounds have been successively used in

phytotherapy since ancient times, as reactive oxygen species are involved in several

diseases. It has been demonstrated that many naturally occurring possess notable

activity as radical scavengers and lipid peroxidation inhibitors .[11] In addition to plant

extracts, numerous naturally occurring compounds are useful as antioxidant, ranging

from alpha tocopherol and beta carotene to plant antioxidants such phenolic

compounds (tannins, flavonoids, anthrocyanins, chalcones, xanthones, xanthones,

liganans, depsides, and depsidones ….etc), terpenes (sesquterpens and diterpineses),

alkaloids, organic sulfur compounds [01] ….etc.

PHENOLICS COMPOUNDS

Phenolic compounds are a key source of antioxidant activity in fruits. Flavonoids, the

fraction of phenolics comprised of such compounds as flavones, is flavones, flavonones

and anthocyanins are known to be potent antioxidants in vitro [00] . Polyphenols are

able to act as reducing agents, hydrogen donating antioxidants, as well as singlet

oxygen quenchers [02] . Phenolic compounds are known to terminate oxidation by

participating in the reactions through resonance stabilized free radical forms, as well as

acting as free radical scavengers .[02] As suggested by [02] , hydrogen donation may be a

key mechanism of action for the antioxidant activity of phenolic compounds.

Polyphenols compounds are an essential part of the defense mechanisms in plants.




These compounds protect plants against the attack of environmental stresses such as

ultraviolet light, microorganisms and insects.

Simple phenolic acids and flavonoids are the most common phenolic compounds

and they generally occur as soluble conjugated (glycosides) and insoluble forms .[02] In

nature, phenolic acids occur mostly in the insoluble or bound forms whereas flavonoids

present as glycosides with a single or multiple sugar moieties linked through an OH

group (O-glycosides) or through carbon–carbon bonds (C-glycosides). Polyphenols are

extensively studied and around 8000 are characterized although it is possible that over

one million molecules possessing protective functions may occur naturally in food

plants [06,27 [. In the early 1980, an accurate procedure for the estimation of free,

soluble conjugated and insoluble bound phenolics was developed and proved in

different foods [06]. the insoluble bound phenolics have demonstrated a significantly

higher antioxidant capacity compared to free and soluble conjugated phenolics

(Chandrasekara & Shahidi, 2010). Nutritionally important bioactive compounds can be

divided into more than ten different classes including: phenolic acids, benzoquinones,

hydroxycinnamic acids, phenylpropenes, coumarins, chromones, naphthoquinones,

xanthones, stilbenes, flavonoids, and lignans [28]. In fruits, polyphenols are commonly

observed as flavonoids, phenolic acids, and tannins (Figure 1).

Figure 1. Common polyphenols in fruits. (A) generic structure of the flavonoid

skeletion, (B) Phenolic acid(ellagic acid), (C) tannins (tannic acid).

FLAVONOIDS

Flavonoids are the most abundant polyphenols in our diets. The basic flavonoid

structure is the flavan nucleus, containing 15 carbon atoms arranged in three rings (C6–




C3–C6). Flavonoids are themselves divided into six subgroups: flavones, flavonols,

flavanols, flavanones, isoflavones, and anthocyanins, according to the degree of

oxidation (oxidation state) of the oxygen heterocycle, central third ring. Their structural

variation in each subgroup is partly due to the degree and pattern of hydroxylation,

methoxylation, or glycosylation [29]. Flavonoids are the most abundant phenolic

compounds in fruits and vegetables with more than 5000 compounds identified to date

[30]. The fiavonoids, widely distributed compounds, are classified into 13 subclasses:

chalcones, dihydrochalcones, aurones, flavones, flavonols, dihydroflavonol, flavanones,

flavanols, flavandiols, anthocyanidins, isoflavonoids, biflavonoids, and

proanthocyanidins [31]. Fiavonoids have been associated with many physiological

properties, including antioxidant, anti-inflammatory, antimicrobial, anti-

hyperlipidemic, anticancer, anti-viral, and anti-allergenic, all of which are thought to

play a role in reducing the risk of degenerative diseases.

Figure 2. The Most Abundant Flavonoids in Fruits. (A) querectin(flavonol), (B) apigenin

(flavone), (C) naringenin (flvanone), (D) (+)- catachin ( flavan – 3 – ol), (E) cyanidin

(anthocyandins).

FLAVONOIDS AS ANTIOXIDANT

Flavonoids exerts their antioxidant effects by neutralizing all types of oxidizing radicals

including the superoxide and hydroxyl radicals and by chelation. Flavonoid can also act

as powerful chain breaking antioxidant due to the electron-donating capacity of their

phenolic groups. The potant antioxidant activity of flavonoids; their ability to scavenge




hydroxyl radicals. May be the most important function of flavonoids and underlies

many of their actions in the body [32]. Flavonoids by acting as free radical scavengers

were shown to exert a protective effect in perfusion ischemic tissue damage, and by

acting as antioxidants exhibited several beneficial effects as anti-inflammatory,

antiallergic, antiviral as well as anticancer activity.

TANNINS

Tannins known as the group of phenolic compounds are the significant plant secondary

metabolites. Tannins in vascular plants occur as two types, the condensed and the

hydrolysable [33]. Condensed tannins are also known as proanthocyanidins (PAs), the

oligomeric and polymeric flavan- 3-ols, which are linked through C4-C8 or C4-C6

linkages. The diversity of condensed tannins is given by the structural variability of the

monomer units. The size of PA molecules can be described by their degrees of

polymerization (DPs). The molecules are water-soluble and can form complexes with

proteins and polysaccharides [34]. PAs are of great interest in nutrition and medicine

because of their potent antioxidant capacity and possible protective effects on human

health [35]. They have antioxidant properties related to their radical scavenging

capacity [36], and these properties have been used against heart disease through

reducing lipid oxidation. It was hypothesized that the free radical scavenging

properties of PAs may reduce the risk of cardiovascular diseases, cancer [37] and blood

clotting, and certain types of trimeric PAs may protect against urinary tract infections

[34]. However, tannins are diverse compounds with great variation in structure and

concentration within and among plant species. Therefore, biomedical researches on the

health benefits and risks of increased tannins consumption are severely limited by lack

of methods for rapid characterization and standardization.

ANTHOCYANINS

Anthocyanins have some of the strongest medicinal effects of any plant compounds.

Physiologically, they are powerful antioxidants used as viable therapies that support

eye and heart health. Some anthocyanins have been shown to be four times as powerful

as vitamin E. The berry nectars including grapes (vitis vinifera var), bilberries, and

blueberries (vaccinium myrtillus), elderberries (sambucus cerulean), cranberries (vaccinium




macrocarpon) and prunes (prunus domestica) are some of the richest sources of

anthocyanins. Anthocyanins are most stable in low, acid Ph’s. However, these berries

have a powerful alkalinizing effect from their minerals and polyphenols. The ultimate

test of a nutrient’s effect of body pH is the pH of its ash, and when the nectars of these

anthocyanin-rich foods are heated to ash, the pH is quite alkaline. Red cabbage,

eggplant and apples (malva pumila) are some common foods that contain anthocyanins.

An easy way to identify them in your refrigerator is to notice which fruits and

vegetables do not spoil quickly [37]. Bilberry nectar is a rich source of anthocyanins. It is

also a rich botanical source of iron, magnesium, potassium and copper. It was used as

early as the Middle Ages to induce menstruation and as recently as World War II to

improve pilots’ night vision. One study showed that anthocyanins have the strongest

antioxidant power in the polyphenol family. The study found that the darker a berry’s

color, the greater its antioxidant power. Oligomeric proanthocyanidins (OPCs) can

prevent damage caused by atherosclerosis and unhealthy lifestyles. They inhibit platelet

aggregation four times better than aspirin in smokers. They also prevent damage from

blood clots, or ischemic reperfusion injury, as well as from venous insufficiency.

Anthocyanins are powerful atherosclerosis fighters, they prevent the damaging

oxidation of low density lipoproteins or LDL (bad) cholesterol, which is often the source of

inflammation, thickening of arteries and clotting mechanisms, all of which lead to heart

disease.

They help maintain healthy cholesterol levels and reduce the risk factors from

heart disease that can lead to death. Prune nectar has been shown to promote healthy

cholesterol levels, particularly high density lipoproteins or HDL (good) cholesterol in both

menopausal and post-menopausal women, compensating in part for the reduction of

estrogen levels and helping to maintain a healthy cardiovascular system [38].

PHENOLIC ACIDS

Phenolic acids are one such group of aromatic secondary plant metabolites widely

spread throughout the plant kingdom [37]. Phenolic acids are a subclass of a larger

category of metabolites commonly referred to as “phenolics”. The term phenolics

encompasses approximately 8000 naturally occurring compounds, all of which possess

one common structural feature, a phenol (an aromatic ring bearing at least one hydroxyl

substituent) [39]. Current classification divides the broad category of phenolics into




polyphenols and simple phenols, based solely on the number of phenol subunits

present (3). Polyphenols possessing at least two phenol subunits include the flavonoids,

and those compounds possessing three or more phenol subunits are referred to as the

tannins (hydrolyzable and non-hydrolyzable). The name “phenolic acids”, in general,

describes phenols that possess one carboxylic acid functionality. However, when

describing plant metabolites, it refers to a distinct group of organic acids (Figure 3).

Figure 3. Structures of sage phenolic acids (caffeic acid and oligomers).

ESSENTIAL OILS

The chemical components of essential oils can be divided into two main categories, the

hydrocarbon monoterpenes, diterpenes and sesquiterpenes, as well as some oxides,

phenolices and sulphur and nitrogen containing material. Common terpenes include




limonene which occurs in most citrus oils, and the antiseptic pine, found in pine and

terpene oils. Important sesquterpenes include chamzulene and farnesene which occur

in chamomile oil and which have been widely studied for anti-inflammatory and

bactericidal properties. Essential oils are volatile, natural, complex mixtures of

compounds characterized by a strong odour and are formed by aromatic plants as

secondary metabolites. They are usually obtained by steam or hydro-distillation.

Known for their antiseptic, i.e. bactericidal, virucidal, fungicidal, and medicinal

properties and their fragrance, they are used in preservation of foods and as

antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic and locally anesthesic

remedies. Up to the present day, these characteristics have not changed much except

that more is now known about some of their mechanisms of action, particularly at the

antimicrobial level [40]. In nature, essential oils play an important role in the protection

of the plants as antibacterial, antiviral, antifungal, insecticides and also against

herbivores by reducing their appetite for such plants. They also may attract some

insects to favour the dispersion of pollens and seeds, or repel undesirable others [40].

Essential oils are very complex natural mixtures which can may contain about 20–60

components at quite different concentrations. They are characterized by two or three

major components at fairly high concentrations (20–70%) compared to other

components present in trace amounts [41]. These essential oils are usually formed by

two modes namely; first, by hydrolysis of some glycosides; and secondly, by the

protoplasm directly. It has been observed that the essential oils are present in different

parts of a plant such as glands or glandular hairs [41].

USE OF ESSENTIAL OIL




WHAT ARE ANTIOXIDANTS?

From a biological point of view, antioxidants have been defined as substances

that at concentrations lower compared to the substrate susceptible to oxidation are

capable of delaying or inhibiting oxidative processes [46]. In this definition, a

“substrate” refers to any oxidisable molecule in vivo; such as lipids, proteins,

carbohydrates and DNA. As antioxidants react to protect biological targets from

oxidation, they are themselves oxidized. The stability of an oxidized antioxidant

molecule is essential, as to prevent oxidation from progressing; oxidized

antioxidants must retain low reactivity towards biomolecules [47].

ANTIOXIDANTS ACTIVITY

Oxidation can be defined as the loss of electrons by an atom or a molecule and the

adverse reaction is reduction which is the gain of electrons by an atom or a molecule

[42]. Antioxidants are of growing interest in recent years. More and more research is

focusing on natural food antioxidants as the public is becoming aware of the

importance antioxidants play in a healthy diet. Historically, antioxidants have been

broadly described as “all substances that inhibited oxidation reactions regardless of the

mechanism, and narrowly as “those compounds that interrupt the free-radical chain

reaction involved in lipid oxidation and those that scavenge singlet oxygen [23].

ANTIOXIDANT ACTIVITY USING (DPPH) RADICAL SCAVENGING

The principle of this assay is based on the measurement of scavenging ability of the

antioxidants towards the stable radical. The free radical DPPH is reduced to the

corresponding hydrazine, when it reacts with hydrogen donors, this stability is

evaluated by decolorizing assay which evaluates the decrease in absorbance at 517 nm

produced by the addition of antioxidant to DPPH solution in ethanol. The scavenging

reaction between (DPPH.) and an antioxidant (H-A) can be written as:




Figure 3: DPPH radical scavenging activity

The color turns from purple to yellow as the molar absorptivity of the DPPH radical at

517 nm. The degree of discoloration indicates the scavenging potential of the

antioxidant compounds or extracts in terms of hydrogen donating ability [43].

Antioxidants react with DPPH, which is a stable free radical and is reduced to the

DPPH H and as consequence the absorbance’s decreased from the DPPH radical to the

DPPH-H form.

ANTIOXIDANT ACTIVITY USING Β-CAROTENE BLEACHING ASSAY.

Beta-carotene, like all carotenoids, is an antioxidant. An antioxidant is a substance that

inhibits the oxidation of other molecules; it protects the body from free radicals. Free

radicals damage cells through oxidation. Eventually, the damage caused by free radicals

can cause several chronic illnesses. Several studies have shown that antioxidants

through diet help people's immune systems, protect against free radicals, and lower the

risk of developing cancer and heart disease. Some studies have suggested that those

who consume at least four daily servings of beta-carotene rich fruits and/or vegetables

have a lower risk of developing cancer or heart disease.




Fig.4. Anti-oxidant interaction (BCB)

ANTIOXIDANT IMPORTANCE TO HUMAN HEALTH

The adverse effects of oxidative stress on human health have become a serious issue.

The WHO has estimated that 80% of the earth’s inhabitants rely on traditional medicine

for their primary health care needs, and most of this therapy involves the use of plant

extracts and their active components [44]. Under stress, our bodies produce more ROS

(e.g., superoxide anion radicals, hydroxyl radicals and hydrogen peroxide) than

enzymatic antioxidants (e.g., superoxide dismutase, glutathione peroxidase, and

catalase) and non-enzymatic antioxidants (e.g., ascorbic acid (vitamin C), α-tocopherol

(vitamin E), glutathione, carotenoids, and flavonoids); this imbalance leads to cell

damage [45]. The main characteristic of an antioxidant is its ability to trap free radicals;

antioxidant compounds in food play an important role as a health-protecting factor.

Scientific evidence suggests that antioxidants reduce risk for chronic diseases including

cancer and heart diseases.

Primary sources of naturally occurring antioxidants include whole grains, fruits

and vegetables. Plant sourced food antioxidants like vitamin C have been recognized as

having the potential to reduce disease risk. Most of the antioxidant compounds 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. Some compounds, such as

gallates, have a strong antioxidant activity, while others, such as the mono-phenols are




weak antioxidants. Highly reactive free radicals and oxygen species are present in

biological systems from a wide variety of sources [45].

MECHANISM OF ACTION OF ANTIOXIDANTS

Free radicals, such as superoxide, hydroxyl ions and nitric oxide all contain an unpaired

electron. These radicals can have a negative effect on cells causing oxidative damage

that leads to cell death. Antioxidants, such as vitamin E, prevent cell damage by binding

to the free radical and neutralising its unpaired electron. For example, when vitamin E

binds to OO· or O2· they form an intermediate structure that is converted to a-

tocopherylquinone. A recent population based study of antioxidants concluded that a

diet rich in foods containing vitamin E might help protect some people against

Alzheimer’s disease (AD). Vitamin E in the form of supplements was not associated

with a reduction in the risk of AD.




OXIDATION DETERIORATION OF QUALITY FISH OIL

Fish oils such as cod liver oil and AlaskaPollack liver oil are rich sources of DHA

(docosahexaenoic acid) and EPA (eicosapentaenoic acid) called highly or poly

unsaturated fatty acids (HUFA orPUFA). During the last several decades, interest in the

dietary effects of n-3 PUFA has increased because of their ability to lower serum

triacylglycerols and cholesterol and in their conversion to eicosanoids, which are known

to reduce thrombosis. In addition, these fatty acids play an important role in the

prevention and possible treatment of coronary heart disease, hypertension, arthritis,

and other inflammatory and autoimmune disorders, and DHA is particularly important

for brain development.

As in other common oils that contain a high concentration of triglycerides, the

most important cause of deterioration in the quality of fish oil, from a flavour and

odour standpoint, is oxidation by atmospheric oxygen.The sites of attack by oxygen are

the unsaturated portions of the fattyacid moieties of triglycerides [48]. Due to its high

content of polyunsaturated fatty acids, including EPA and DHA, fish oils are highly

susceptible to oxidative spoilage and the rate of fish oil oxidation is significantly

different from that of other oils [49]. Normally with polyunsaturated oils, initiation of

rancidity begins slowly, with the polyenoic ester (LH) giving the free-radical (Reaction

1) [50, 51]. This reacts withoxygen (Reaction 2) to give a peroxy species immediately,

which in turn, reacts with a fresh alkyl-proton to give a new free-radical (Reaction 3).

LH →L· (1)

L· + O2 →LOO· (2)

LOO· + LH →LOOH + L· (3)

Various factors govern the oxidative reactions that occur at centres of unsaturation [48].

In addition to being affected by temperature and the degree of unsaturation, oxidation

may be accelerated or retarded by various catalytic agents. Certain metals, visible light

and light of shorter wavelengths, some oxidative enzymes, and other biological

substances, such as hemoglobin, markedly accelerate this type of oxidative

deterioration. During the autoxidation of fish oils, undesirable flavours and odours

develop at very low peroxide values atan early stage of oxidation, even during the

induction period [49]. The hydro peroxides do not themselves contribute appreciably to




the deteriorated flavours and odours of oxidised oils [48]. In most cases, the

organoleptically detectablematerials appear to have low molecular weights and are

formed by the decomposition of peroxides and by further oxidation of the peroxides

and their breakdown products. A large number of saturated and unsaturated

aldehydes, ketones, acids, and other products have been isolated from oxidised oils,

and have been shown to contribute to the undesirable flavours and odours.

FISH SPOILAGE AND FOODBORNE PATHOGENIC BACTERIA

Marine fish products deteriorate rapidly post mortem as a consequence of various

microbial and biochemical breakdown processes. The rate of quality loss depends

directly on the nature of the fish species in question, as well as the handling and

storage conditions. The quality of wild caught cod may vary considerably, due to

seasonal variations, different handling, fishing gear and fishing ground. The time

between the catch and processing will in addition strongly influence the quality.

Compared to wild caught fish, farmed fish have several advantages as a raw

material. Wild caught cod is known to have a different body composition than

farmed cod, with a higher condition factor, smaller head and larger liver [52]. There

is also observed a higher carbohydrate level and lower pH in the muscle [53]. Shelf

life studies on the MA-packaged wild fish have shown the importance of




temperature, production hygiene and gas composition on the development of

specific spoilage organisms (SSO) [54]. The bacterial flora of wild cod stored

aerobically on ice is well studied and dominated by Pseudomonas sp. [54],

Shewanella baltica, Shewanella hafniensis and Shewanella morhuae [55], rather than

Shewanellaputrefaciens, which has previously been considered, in many studies, as

the main spoilage organism (Gram and Dalgaard, 2002). In a modified atmosphere

(MA) packaging with high CO2 concentration, the CO2 tolerant bacterium

Photobacterium phosphorus has been identified as the main organism responsible

for spoilage [56].

Listeria monocetogenes

L. monocytogenes was first described in 1924 by [57]. The organism was isolated from

rabbits and guinea pigs and observed to cause monocytogenes in the infected

animals. The bacterium was originally named Bacterium monocytogenes [58]. Listeria

monocytogenes is a Gram-positive, foodborne pathogen. It is widely distributed in the

environment and occurs naturally in many raw foods. Listeria monocytogenes is

psychotropic and halotolerant and can, under otherwise optimal conditions, grow in

the range of 1 to 45 °C (34 to 113 °F) and between 0 and 10% NaCl. As a

consequence, it may grow in many food products with extended shelf lives [59].

Listeria monocytogenes can grow in a wider temperature range, from -1.5 to 45ºC

[60]. It has already long been established as an important food borne pathogen.

However, the incidence in food related listeriosis outbreaks has increased

dramatically in the last few years and L. Monocytogenes is now considered a

pathogen of major concern. A diverse range of food has been associated with outbreaks

of listeriosis [61]. Various food products are consumed without further cooking

and are also capsule of supporting growth of L. monocytogenes. A range of

seafoods, particularly the lightly preserved products (6% waterphase salt, pH 5) such as

smoked fish products, lightly salted products (e.g. Brined cooked shrimp) or marinated

products fall within this category. Outbreaks of listeriosis associated with smoked

mussels, smoked trout and raw oysters, have been reported [61] Therefore, some

degree of poisoning risk from seafood is evident.




Escherichia coli

E. coli O157:H7 is a member of the enterohemorrhagic group of pathogenic E. coli that

has emerged as a foodborne and waterborne pathogen of major public health

concern. A wide variety of foods have been implicated as vehicles of E. coli

O157:H7 infection, including meat, milk, fruit juices, and vegetables [62]. Unlike

most foodborne pathogens, E. coli O157:H7 is tolerant of acidic environments. Survival

in apple cider (pH 3.6–4.0) and mayonnaise (pH 3.6–3.9) has been reported and E.

coli O157:H7 survived fermentation of buttermilk (pH 4.4) and drying and storage of

fermented sausage (pH 4.5) [62]. These organisms cause a spectrum of disease

increasing in severity from a mild diarrheal illness to hemorrhagic colitis, hemolytic

uremic syndrome, and, in some cases, death [63]. Several years ago, hurdle

technology was developed as a new concept for the realization of safe, stable,

nutritious, tasty, and economical foods. This approach uses a combination of

suboptimal growth factors, e.g. heating, chilling, drying, salting, conserving,

acidification, oxygen-removal, fermenting, adding various preservatives, to establish

growth inhibition of microorganisms in foods [61]. Escherichia is a member of the

Enterobacteriaceae family and is the most common organism in the intestinal tract

of man and warm blooded animals. Most of the E. coli strains are harmless. E.

coli colonizes the intestinal tract and probably plays important roles in maintaining

intestinal physiology. However, some strains of E. coli are pathogenic and can

cause diarrhoeal disease. E. coli strains are differentiated based on a serotyping the

scheme involving O (somatic), H (flagellar) and K (capsular) antigens. Pathogenic

E. coli are divided into specific groups depending on virulence, clinical symptoms

and distinct O: H antigens [64].

Staphelococcus auerus

Staphylococcus aureus is an important food-borne pathogen due to the ability of

enterotoxigenic strains to produce staphylococcal enterotoxins (SEs) preformed in food.

Up to present, 22 SEs have been described, designated SEA to SElV, in the chronological

order of their discovery [65]. Staphylococcal food poisoning is characterized by a

sudden onset of symptoms, with vomiting, abdomnal pain, and stomach cramps being

the most common [66]. Occasionally it can be severe enough to warrant hospitalization,

particularly among the group of YOPIs (young, old, pregnant, immuno suppressed

persons) [67]. Individual susceptibility to SE and the amount of SE ingested influence




the onset and severity of the symptoms [65, 66]. As less as 100–200 ng of enterotoxin A

(SEA) can lead to a disease [68]. Typically, a staphylococcal food poisoning (SFP) occurs

after ingestion of foods that are contaminated with S. aureus by improper handling and

subsequent storage at elevated temperatures. People colonized with S. aureus

asymptomatically, who handle food can introduce the bacteria into the food chain [69].

Approximately 20- 30% of humans persistently carry S. aureus as a commensal of the

skin and mucosal membranes, respectively [70]. One-half of the isolates found among

humans proved to be enterotoxigenic [71].

ADDITIVES USED IN FISH PRESERVATION

Chemical and natural substance added to food during preparation or achieving a

particular a part of the food or affects its characteristics for the purpose of

appearance, texture, or keeping quality of a food or serve as essential aids in the

processing of food are all considered to be food additives. The essential oil has

been used for decades in food preservation, protecting feed from microbial and

fungal hazards. In general, the preservative action of these compounds is mainly

due to their inhibitory effect not only on the metabolism of microorganism but also on

their growth. None of these chemicals have a complete spectrum of action against

the entire spoilage microorganism likely to occur in fish and fish products.

Plants protect themselves against microorganisms and other predators by

synthesising a wide range of compounds. Such compounds include essential oils,

phytoalexins, phenolics and related compounds. Herbs and spices are often rich in such

compounds. It is recognised that such compounds can be cidal, sporostatic or sporicidal

in respect to bacterial spores, as well as inhibitors of mycotoxin production [72, 73 and

74]. In recent years there has been considerable interest in the use of essential oils in

food preservation, although their fi rst use dates back to prehistoric times. Reports as

long as 5000 years ago can be traced to 1550 BC , when the ancient Egyptians used

cinnamon, cumin and thyme both for food preservation and mummifi cation [74]. The

antimicrobial compounds present in plant materials are commonly found in the

essential oil fractions obtained by steam or supercritical distillation, pressing, or

extraction by liquid or volatile solvents. Screening and isolation of antimicrobial phyto-

phenols requires a multidisciplinary approach [74]. When choosing a solvent for

extraction, its ability to extract components of a solute has to be considered. The more




effi cient the extraction, the greater is the range of compounds present in the extract.

The need to use pure solvents is essential, as is the requirement to carry out extraction

under mild conditions utilising, whenever possible, solvents of low reactivity. The

possibility of generating artefacts should never be discounted. Techniques to isolate

bioactive phytochemicals have been reviewed by [75]. Studies have been carried out on

the mode of action of spices or their essential oils to inhibit microorganisms. Since it is

apparent that the terpenes in essential oils are the primary antimicrobials, the

mechanism most likely involves phenols. The mode of action of phenolic compounds is

generally related to other phenolic compounds and is thought to involve interference

with functions of the cytoplasmic membrane [76, 77 and 78]. In addition terpenes may

have other antimicrobial mechanisms. It is suggested that essential oils may inhibit

yeast enzymes resulting in energy depletion and interference with synthesis of

structural components [72].

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ANTIOXIDENT, ANTIMICROBIAL AND PULICARIA INULOIDES AND OCIMUM FROSKOLEI: A REVIEW