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1. HERBS AND SPICES

1.1. DEFINITION

Dietary supplements are products taken by mouth that contain a “dietary ingredient”

intended to supplement the diet. Dietary ingredients may include vitamins, minerals,

herbs or other botanicals, amino acids, and substances such as enzymes, organ tissues,

and metabolites.

The botanical term herb refers to seed-producing plants with nonwoody stems that die

down at the end of the growing season. In herbal medicine the term herbs is used loosely

to refer not only to herbaceous plants but also to bark; roots; leaves; seeds; flowers and

fruit of trees, shrubs, and woody vines; and extracts of the same that are valued for their

savory, aromatic, or medicinal qualities (Craig, 1999).

Spices are vegetable substances used to season foods. Examples include clove, cinnamon,

nutmeg, pepper, garlic, onion, curry, ginger, etc. They are usually dried for use and have

distinctive flavors and aromas (Guralnik, 1986).

1.2. CONSUMPTION

Food, nutrition and health have always been in the attention of the public, but in the

1990s focus on dietary supplements and natural remedies have increased (Fisher and

Ward, 1994; Goldbeck-Wood et al., 1996; MacLennan et al., 1996; Ernst, 2000). The

increase has been noted among the general public as well as among those who are

suffering from diseases such as cancer (Burstein et al., 1999). Traditional medicine

(natural remedies) is used in the maintenance of health. Usually people search for the

solutions without side effects or lesser side effects, for problems such as arthritis,

allergies, insomnia, headaches, anxiety, and depression. Today the interest in the use of

herbal remedies is growing because they believe that the herbal medicine derived from

plants contain natural substances that can improve health and alleviate illness.

Two of the largest users of medicinal plants are China and India. Traditional Chinese

Medicines uses over 5000 plant species; India uses about 7000 (Verma and Singh, 2008).

For thousands of years plants have played an important role in maintaining human health

and improving the quality of human life as well as in the prevention, diagnosis,

improvement or treatment of physical and mental illnesses, and have been used as

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acomponents of seasonings, beverages, cosmetics, dyes, and medicines. According to

World Health Organization, about 80% of the earth’s population uses traditional

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

plant extracts or their active components. Furthermore, many Western drugs had their

origin in a plant extract (Craig, 1999). Reserpine, an antihypertensive drug used for the

control of high blood pressure, was originally extracted from the plant Rauwolfia

serpentina, while digitalis drugs are heart stimulant medicines which are derived from a

type of foxglove plant (Digitalis Purpurea) that has a stimulating effect on the heart,

Over-the-counter laxatives commonly contain psyllium, senna, or Cascara sagrada. The

laxative effect of the latter 2 herbs is due to the presence of anthraquinones, which

stimulate peristalsis, whereas the mucilages in psyllium provide a bulking effect

(Bruneton, 1995; Hauptman and Kelly, 1999). Today herbal preparations are widely used

for many conditions, such as anxiety, hyperlipidemia, hypertension, arthritis, depression,

colds, coughs, constipation, fever, headaches, infections, insomnia, premenstrual

syndrome, stress, ulcers, and weakness.

Garlic, ginseng, goldenseal, ginkgo, saw palmetto and aloe-vera are some of the more

popular herb used nowadays. Researchers showed the usefulness of ginger for motion

sickness; licorice for treatment of ulcers as well as treatment of hops, passionflower, and

valerian for treating insomnia; borage for anxiety and depression (Lien et al., 2003;

Morin et al., 2005; Sayyah and Kamalinejad, 2006; Martin et al., 2008). Culinary herbs

have also been used to flavor foods since ancient times. The aroma in most of the herbs is

provided by the aromatic ingredients in their oleoresins and essential oils. Some herbs

can also give color to the food, like saffron, paprika, and turmeric. The main sources of

natural antioxidants were reported as spices and herbs (Siddhuraju and Becker, 2003).

In plant foods thousands of chemical structures have been recognized (Lampe, 2003).

Many of them are available in spices. In general, spices are the dried aromatic parts of

plants—generally the seeds, berries, roots, pods, and sometimes leaves—that mainly, but

not invariably, grow in hot countries. Spices are derived from a wide range of plant parts

and botanical species which can provide significant variety and complexity to the human

diet. Moreover apart from the flavoring purposes, spices and herbs have also been

consumed for their medical or antiseptic properties (Kahkonen et al., 1999). In the past,

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the medicinal uses of spices were often impossible to differentiate from their culinary

uses and for centuries both the inherent value, and the potential toxicity, of

phytochemical in relation to human health was recognized. Plants have the ability to

synthesize a various array of chemicals, and understanding their phytochemical role in

plants may further our understanding of the mechanisms by which they profit humans. In

plants, these compounds function to attract beneficial and repel harmful organisms, serve

as photoprotectants, and react to environmental changes. In humans, they can have

complementary and overlapping actions, including antioxidant effects, modulation of

detoxification enzymes, stimulation of the immune system, reduction of inflammation,

modulation of steroid metabolism, and antibacterial and antiviral effects (Lampe, 2003).

For the first time Chipault et al (1952) examined 72 spices, their petroleum ether, and

alcohol soluble fractions and found 32 spices to retard the oxidation of lard. Rosemary

and sage were remarkably effective, and oregano, thyme, turmeric, and nutmeg also

exhibited high antioxidant activity in the ground form and as extracts. Several later

studies established that many leafy spices, especially those belonging to the Labiatae

family such as sage, rosemary, oregano, and thyme, show strong antioxidant activity

(Hirasa and Takemasa, 1998).

Proximate and nutrient analysis of edible fruit and vegetables plays an essential role in

assessing their nutritional significance. As various medicinal plant species are also used

as food along with their medicinal benefits, evaluating their nutritional significance can

help to understand the worth of these plant species (Pandey et al., 2006). As far herbal

drug’s standardization is concerned, WHO also highlights the need and importance of

determining proximate and micronutrients composition. Such herbal formulations must

pass through standardization processes (Rajani and Kanaki, 2008).

1.3. MEDICINAL PROPERTIES OF HERBS AND SPICES

Many herbs which are used regularly have been identified by the National Cancer

Institute as possessing cancer-preventive properties.

These herbs are members of the Allium sp. (garlic, onions, and chives); members of the

Labiatae (mint) family (basil, mints, oregano, rosemary, sage, and thyme); members of

the Zingiberaceae family (turmeric and ginger); licorice root; green tea; flax; members of

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the Umbelliferae (carrot) family (anise, caraway, celery, chervil, cilantro, coriander,

cumin, dill, fennel, and parsley); and tarragon (Caragay, 1992). Furthermore, many herbs

have a variety of phytosterols, triterpenes, flavonoids, saponins, and carotenoids, which

have been shown from studies of legumes, fruit, and vegetables to be cancer

chemoprotective (Steinmetz and Potter, 1991). These helpful substances perform as

antioxidants and electrophile scavengers, motivate the immune system, inhibit nitrosation

and the creation of DNA adducts with carcinogens, inhibit hormonal actions and

metabolic pathways associated with the development of cancer, and persuade phase I or

II detoxification enzymes (Swine, 1988; Steinmetz and Potter, 1991; Caragay, 1992;

Kikuzaki and Nakatani, 1993; Zheng et al., 1993; Cuvelier et al., 1994; Ho et al., 1994;

Lam et al., 1994; Robbers et al., 1994; Smith and Yang, 1994; Haraguchi et al., 1995)

1.4. POSSIBLE ADVERSE EFFECTS OF HERBS AND SPICES

Herbal products can also be unsafe to use. The Food and Drug Administration has

classified several herbs as unsafe, even in small amounts, so they should not be used in

either foods or beverages (Larkin, 1983; Saxe, 1987). There are also data suggesting that

some spices may increase cancer risk. Several case-control studies in India have observed

that gastrointestinal cancer risk was higher with use of spicy foods and chili (Mathew et

al., 2000; Phukan et al., 2001). The work of Jensen-Jarolim et al (1998) suggests that

spices may increase intestinal epithelial permeability through loosening cell contacts (eg,

paprika, cayenne pepper, chili pepper) or decrease permeability (eg, black pepper,

nutmeg), possibly by cell swelling. Solanaceae spices (eg, chili peppers) increase

permeability for macromolecules of 10–40 kDa (Lampe, 2003).

2. ANTIOXIDANT

2.1. DEFINITION AND IMPORTANCE

Free radical can be explained as any species able of independent survival that possess

one or more unpaired electrons, an unpaired electron being one that is alone in an orbital.

Biological molecules are usually non-radicals containing only paired electrons, but when

a radical gives one electron to take one electron from, or simply adds itself to a non-

radical, then it becomes a radical; the feature of the reactions is that they have a tendency

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to proceed as chain reactions (Halliwell and Gutteridge, 1999). Generally free radicals are

not stable and highly reactive. Some of the free radicals and oxidants found in living

organisms are shown in Table 1. Free radicals and various reactive oxygen species can be

derived either from normal, essential metabolic processes or from external sources.

Examples of both are shown in Table 2 (Fürst, 1996).

Chemical reactivity of free radicals can damage all types of cellular macromolecules,

including proteins, carbohydrates, lipids and nucleic acids, in case free radicals are not

inactivated, which can result in cell injury and atherosclerosis. Superoxide radical is able

to damage membrane proteins and cell membrane phospholipids. 02' can inhibit ATP

synthesis and generate breaks in DNA molecules (Cochrane, 1991; Ward, 1991).

Definitely, many of the free-radicals cause degenerative diseases.

Table 1: Selected reactive oxygen species in living organisms

Non-radicals Free radicals

Hydrogen peroxide H2O2 Superoxide radical O2·

Hypochloric acid HOCl Hydroxyl radical OH·

Ozone O3 Nitric oxide radical NO·

Singlet oxygen 1O2 Lipid peroxyl radical LOO'

Table 2: Selected sources of free radicals

Internally generated External sources

Mitochondria Cigarette smoke

Phagocytes Radiation

Xanthine oxidase U.V. light

Reactions with Fe and with other transition metals

Certain drugs, reagents and industrial solvents

Arachidonate pathways Pollution

Peroxisomes -

Exercise -

Inflammation -

Ischaemia and reperfusion -

(Fürst, 1996)

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Important mechanisms in cancer prevention are the processes which prevent free radical

formation, eliminate radicals before damage can occur, repair oxidative damage, remove

damaged molecules, or prevent mutations (Gordon, 1996).

2.2. SYNTHETIC ANTIOXIDANTS

Such as butylated hydroxytoluene, butylated hydroxyanisole, tert-butylhydroquinone and

propyl gallate are broadly used as antioxidants in the food industry. Their safety,

however, has been questioned. Butylated hydroxyanisole was shown to be carcinogenic

in experimental animals. At high doses, butylated hydroxytoluene may cause internal and

external hemorrhaging, which lead to death in some strain of mice and guinea pigs. It is

reported that this effect may be due to the ability of butylated hydroxytoluene to reduce

vitamin K-depending blood-clotting factor (Ito et al., 1986). Therefore, the importance of

replacing synthetic antioxidants by natural ingredients from oilseeds, herbs and spices

and other plant materials has increased due to health implications and increased

functionality which improves solubility in both, oil and water.

2.3. ANTIOXIDANT COMPONENTS

2.3.1. Polyphenols

These compounds depend on phenolic rings that they contain and of the structural

elements that bind these rings to one another, classify them into different groups.

Differences are made between the phenolic acids, flavonoids, stilbens, and lignans.

Classification of polyphenols is illustrated in Figure 1.

Studies suggest a possible hypocholesterolemic effect of polyphenols. When rats were

fed with green tea polyphenols, blood cholesterol concentrations declined in

hypercholesterolemic animals and blood pressure decreased in spontaneously

hypertensive animals. Some of these effects may be explained by the capacity of green

tea catechins and gallate esters to reduce intestinal cholesterol absorption, lower blood

coagulability, and inhibit proliferation of human aortic smooth muscle cells (Dreosti,

1996). Recently, low density lipoprotein (LDL)-cholesterol oxidation was inhibited by

exposure to tea flavonoids, specifically the catechins from green tea leaves or theaflavins

(catechin dimers) from black tea leaves (Ishikawa et al., 1997). Of the catechins,

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epigallocatechin gallate provided the most protection and was more protective than

vitamin E, whereas the theaflavins exerted even stronger inhibitory effects than the

catechins, also exhibited anticarcinogenic activity and inhibited atherosclerosis (Decker,

1995). Phenolics and polyphenols are responsible for most of the antioxidant activity in

plant-based foods (Prior and Cao, 1999). However, sulfur-containing compounds such as

diallylthiosulfinate (allicin) from garlic, 6-gingerol from ginger, and capsaicin from chili

pepper have also been shown to have considerable antioxidant capacity as isolated

compounds (Wong et al., 2006; Shukla and Singh, 2007; Dairam et al., 2008).

Figure 1: polyphenol classifications

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2.3.2. Phenolic acids

Phenolic acids classify to two different classes, derivatives of benzoic acid and cinnamic

acid. Generally in edible plants, the hydroxybenzoic acid content is very low, with the

exception of certain red fruits, black radish, and onions, which can have concentrations of

several tens of milligrams per kilogram fresh weight. Hydrolyzable tannins (gallotannins

in mangoes and ellagitannins in red fruit such as strawberries, raspberries, and

blackberries) are complex structure in which hydroxybenzoic acids is seen (Clifford,

2000). The types of fruit containing the highest hydroxycinnamic acids (blueberries,

kiwis, plums, cherries, apples) contain 0.5-2.0 g hydroxycinnamic acids/kg fresh weight

(Macheix et al., 1990).

2.3.3. Flavonoids

Flavonoids have widespread biological properties that support human health and help to

decrease the risk of disease. Flavonoids are helpful for the activity of vitamin C, act as

antioxidants, protect LDL cholesterol from oxidation, inhibit platelet aggregation, and act

as anti-inflammatory and antitumor agents (Smith and Yang, 1994; Cook and Samman,

1996; Manach et al., 1996). The Zutphen study of elderly men in the Netherlands found

that flavonoid intake from fruit, vegetables, and tea was inversely associated with heart

disease mortality and occurrence of heart attack and stroke for duration of 5 years.

Subjects who had the highest use of flavonoids had 60% lower mortality from heart

disease and showed 70% lower risk of stroke than those who consumed low amounts of

flavonoids (Hertog et al., 1993; Keli et al., 1996). In addition Hertog et al (1995) stated

that data from the 16 cohorts participating in the seven countries study showed an

opposite relation between the average flavonoid intake and age-adjusted mortality from

heart disease after 25 y of follow-up.

Additionally, flavonoids have been shown to have antiviral and carcinostatic effect.

Though, flavonoids are poorly absorbed from the lumen and are subject to degradation by

intestinal micro-organisms. The amount of quercetin that remains biologically available

may not be of sufficient concentration, theoretically, to elucidate the beneficial properties

seen with the Mediterranean diet. The role of flavonoids may transcend their presence in

food. The activity of flavonoids as inhibitors of reverse transcriptase suggests that these

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compounds may have role in controlling retrovirus infections, such as acquired

immunodeficiency syndrome. In addition to specific effects, the broad-modulating effects

of flavonoids as antioxidants, inhibitors of ubiquitous enzymes (ornithine carboxylase,

protein kinase, calmodulin), and promoters of vasodilatation and platelet disaggregation

can serve as starting material for drug development programmes (Formica and Regelson,

1995).

Since 1936 over 6000 flavonoids have been identified in plant (Harborne and Williams,

2000; Godevac et al., 2004) and many of flavonoids activity against several oxidants is

studied by many authors (Wojdylo et al., 2007; Fardet, 2010; Ghasemzadeh et al., 2010).

The flavonoids, has a common structure consisting of 2 aromatic rings (A and B) that are

bound together with 3 carbon atoms that form an oxygenated heterocycle (ring C), these

may themselves be divided into 6 subclasses as a function of the type of heterocycle

involved: flavonols, flavones, isoflavones, flavanones, anthocyanidins, and flavanols

(catechins and proanthocyanidins) (Manach et al., 2004).

The antioxidant efficacy of flavonoid compounds depends on structural features such as

the number and position of the hydroxyl moieties on the ring systems and the extent by

which unpaired electron in the oxidized phenolic intermediate can delocalize throughout

the molecules (Lugasi and Hóvári, 2003).

Flavonols are the most abundant flavonoids in foods, and the main representatives are

quercetin and kaempferol (Macheix et al., 1990). These flavonols are gathered in the

outer and aerial tissues (skin and leaves) since they need light to stimulate biosynthesis.

There is a remarkable difference in concentration between pieces of fruit on the same tree

and even between different sides of a single piece of fruit, which depends on exposure to

sunlight (Price et al., 1995). Flavones are much less common than flavonols in fruit and

vegetables. Flavones are made chiefly of glycosides of luteolin and apigenin. Parsley and

celery are the only important edible sources of flavones identified to date. Isoflavones are

flavonoids with structural similarities to estrogens. Isoflavones are found almost

exclusively in leguminous plants. Main source of isoflavones in the human diet is Soya

and its processed products. Flavanols exist in a monomer form (catechins) and the

polymer form (proanthocyanidins). Catechins are found in many types of fruit (apricots,

which contain 250 mg/kg fresh wt, are the richest source. They are also present in red

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wine (up to 300 mg/l), but green tea and chocolate are by far the richest sources (Manach

et al., 2004).

Anthocyanins are natural, nontoxic, belonging to the flavonoid family and water-soluble

pigments responsible for the red, purple, blue, and orange colors of fruits, vegetables, and

flowers. Anthocyanins exist in different chemical forms and according to pH they can be

colored or uncolored. In the aglycone form (anthocyanidins) they are highly unstable but

while they are in plants, they are resistant to light, pH, and oxidation conditions that are

likely to degrade them.

In food plants, anthocyanins are widespread occurring in at least 27 families, 73 genera

and a multitude of species. The most common anthocyanidin in foods is cyanidin. Food

contents are generally related to color intensity and values reach up to 2–4 g/kg fresh

weight in blackcurrants or blackberries (Clifford, 2000; Es-Safi et al., 2002). Figure 2

presents the structure of flavonoids.

Figure 2: Structure of flavonoids

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2.3.4. Lignans

Lignans are from 2 phenylpropane units. Linseed is the richest dietary source. Thompson

et al (1991) confirmed that the richest sources are oleaginous seeds (linseed) and minor

sources identified are algae, leguminous plants (lentils), cereals (triticale and wheat),

vegetables (garlic, asparagus, carrots), and fruit (pears, prunes).

2.3.5. Stilbenes

Only in low quantities stilbenes are found in the human diet. Resveratrol, one of stilbenes

for which anticarcinogenic effects have been shown during screening of medicinal plants

and which has been widely studied, is found in low quantities in wine (0.3–7 mg

aglycones/L and 15 mg glycosides/L in red wine) (Bertelli et al., 1998; Bhat and Pezzuto,

2002; Vitrac et al., 2002). However, because resveratrol is found in a very small

quantities in the diet, any protective effect of this molecule is not likely at normal

nutritional intakes (Manach et al., 2004).

2.4. WHAT IS OXIDATIVE STRESS?

Oxidative stress is defined as a state of imbalance between pro-oxidants and antioxidants.

Pro-oxidant elements include all factors which play an active role in the enhanced

formation of free radicals or other reactive oxygen species. In this instance cellular

mechanisms (fault in mitochondria1 respiration, specific enzymes) as well as exogenous

mechanisms (smoking, toxins, polyunsaturated fatty acids, air pollution, drugs, etc.) may

play role (Fürst, 1996).

2.5. ANTIOXIDANT PREVENTING ROLE IN DISEASE

2.5.1. Cardiovascular disease

The oxidative-modification theory of atherosclerosis (Berliner et al., 1995; Navab et al.,

1996; Epstein et al., 1997) has encouraged the study of antioxidant vitamins in the

prevention of the initiation and progression of cardiovascular disease. Preclinical studies

recommend that supplementation of the diet with various compounds that have

antioxidant properties before the development of vascular disease inhibited the

atherogenic process (Crawford et al., 1998; Praticò et al., 1998; Thomas et al., 2001).

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2.5.2. Hyperlipidemia

Hypercholesterolemia is an important risk factor for atherosclerosis (Sloop, 1999).

Though, there is great inter-individual difference in the progress of atherosclerotic lesions

at given cholesterol level. The reason for this phenomenon is not yet completely

understood. It has been suggested that further than the known risk markers for

cardiovascular disease, oxidation of LDL could play a significant role in the development

and progression of atherosclerosis (Esterbauer et al., 1992; Lynch and Frei, 1994;

Berliner et al., 1995). It may be clinically relevant whether an elevated blood cholesterol

concentration is associated with increased LDL oxidation, which would represent an

additional risk. Conflicting results are reported about the LDL oxidation, lipid

peroxidation and antioxidants in hypercholesterolemia and hypertension. This may be

due primarily to the different nutritional habits of the groups studied, as well as the

different analytical methodologies used to estimate these variables.

2.5.3. Diabetes and oxidative stress

Diabetes has reached epidemic problem all over the world. Since diabetes damages

arterial blood vessels, the leading causes of death are myocardial infarction, stroke, and

peripheral vascular disease, which are 2 to 4 times more common in diabetic patients.

Moreover, microvascular complications, as well as retinopathy, nephropathy, and

neuropathy, ultimately affect nearly all patients with diabetes (Fürst, 1996). Increasing

evidence in both experimental and clinical studies suggests that there is a close

relationship between hyperglycemia, oxidative stress and diabetic complications. High

blood glucose level determines overproduction of reactive oxygen species (ROS) by the

mitochondria electron transport chain (Piconi et al., 2003). During the past two decades,

significant evidence has implicated oxidative stress in diabetes and other diseases,

including atherosclerosis, neurodegenerative diseases, and end-stage kidney disease

(Brownlee, 1995; Degenhardt et al., 1998), as well as in aging.

Pennathur and Heinecke (2004) explained the oxidative stress pathway and it is presented

in Figure 3.

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Figure 3: Proposed oxidant-generating pathways in diabetes.

AGE: Advanced glycosylation end products; NOx: Nitric oxide derived oxidants; RNS:

Reactive nitrogen species; IIOC1: Hypochlorous acid.

2.6. Antioxidants for preventing diabetic complications

Since it is proved that oxidative stress is a major contributor to diabetic complications,

effective antioxidant regimens would be important therapies. More than the past decade,

there was a sudden increase of interest by scientists and the public in the possibility that

dietary and supplemental antioxidant vitamins, such as beta-carotene, vitamin E, and

vitamin A can prevent some human disease (Vivekananthan et al., 2003; Eidelman et al.,

2004; Sesso et al., 2008). However, trials of antioxidants and carbonyl trapping agents in

humans suffering from diabetes have yielded mixed results, though the results of animal

studies have been positive. Ziegler et al (1999) demonstrated that chronic treatment with

vitamin E failed to reduce chances of cardiovascular diseases in a large study that

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included a high percentage of diabetic patients. In some studies on lipoic acid, which is

known to be an antioxidant, was used to treat diabetic neuropathy, results have been

equivocal (Reljanovic et al., 1999; Ziegler et al., 1999). Pennathur and Heinecke (2004)

express the one possible reason for these discouraging results is that antioxidant therapy

might benefit only subjects who are in a condition with high oxidative stress.

Indeed, the renal failure patients who benefited from vitamin E therapy (Boaz et al.,

2000) might have been a subset with greatly increased carbonyl and oxidative stress

(Miyata et al., 1999). However Pennathur and Heinecke (2004) stated that there is little

evidence that compounds like vitamin E and vitamin C in fact inhibit oxidative reactions

in humans.

Degenerative diseases such as alzheimer’s disease, rheumatoid arthritis, coronary artery

disease, Parkinson’s disease, cataracts, inflammation, cancer, aging, pancreas damage in

diabetes are accompanied by the free radicals and reactive oxygen species oxidative

stress (Evans et al., 2002). Oxidative stress happens by the source of stimulated

polymorphonuclear leukocytes, macrophages, peroxysomes and aerobic respiration,

which cause damage of macromolecules like proteins, lipids and nucleic acid in

intracellular and extracellular regions (Vijayakumar et al., 2006). Superoxide dismutase,

glutathione peroxidase and catalase are the antioxidant enzymes. Hydroxyl radical

generated by metal catalyzing process by cleavage of water and hydrogen peroxide,

cause for peroxidation of the cell membrane lipids (lipid peroxides), converts

malondialdehyde into carcinogenic and mutagenic complex (Srinivasan et al., 2007).

Superoxide dismutase, glutathione peroxidase is an enzyme which contains selenium and

is active in the reduced form. These enzyme catalyses the oxidations of glutathione

(GSH) to glutathione disulfide (GSSG) at the expense of hydrogen peroxide (Maritim et

al., 2003). An in vivo study on the Streptozotocin induced diabetes on rats showed a

marked reduction in the hepatic GSH (Orhan et al., 2005).

Reduction in the level of insulin in diabetic patients impair the enzymatic antioxidant

system causing increased fatty acyl‐coA oxidase and initiates β‐oxidation of fatty acids

that helps the accumulation of free radicals which results in lipid peroxidation. Increased

lipid peroxidation damage membrane function by decreasing the fluidity and changing

the activity of the membrane bound enzymes and receptors (Babre et al., 2010).

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3.1. BOTANICAL INFORMATION

E. amoenum is a biennial or perennial herb original to the narrow region of northern part

of Iran and Caucasus, where it grows at an height ranging from 60-2200 m. Echium

genus has 4 species in Iran (Mozzaffarian, 1998) and only E. amoenum has medicinal

uses (Hooper et al., 1937; Delorme et al., 1977).

3.2. HISTORY OF USE

Dried violet petals of Echium amoenum are known as Gol-e-gavzaban in Iran. In

traditional medicine of Iran it has long been used as a tonic, tranquillizer, diaphoretic and

as a remedy for common cold, fever, pneumonia, demulcent, anxiolytic, sedative, anti-

inflammatory and analgesic effects (Hooper et al., 1937; Amin, 1991; Zargari, 1995;

Kapoor and Klimaszewski, 2000; Kast, 2001; Shafaghi et al., 2002; Abolhassani, 2004).

E. amoenum has anxiolytic effect in mice (Shafaghi et al., 2002; Rabbani et al., 2004) and

has the capacity to increase the cellular immune response (Amirghofran et al., 2000).

A large percentage of the world’s population uses folk medicine, but much scientific data

are not available their safety, purity, and standardization; therefore, it is required to

investigate on their efficacy and toxicity (Ernst, 1998).

3.3. NUTRITIONAL AND CHEMICAL COMPOSITION

USDA reported the nutritive value of borage leaves and it is reported in Table 3.

Table 3: Nutritive value of fresh leaves of borage (per 100 g)

Nutrient Value (g)

Value (% of RDA)

Minerals (mg)

Value (% of RDA)

Vitamin (mg) Value (% of RDA)

Carbohydrate 3.06 (1%) Sodium 80 (5%) Folates (µg) 13 (3%) Protein 1.8 (3%) Potassium 470 (10%) Niacin 0.9 (25.5%)

Total fat 0.70 (2%) Calcium 93 (9%) Pantotenic

acid 0.041(1%)

Cholesterol 0 (0%) Copper 0.13 (15%) Pyridoxine 0.084 (6.5%) Energy (Kcal)

21 (1%) Iron 3.3 (41%) Riboflavin 0.15 (12%)

- - Magnesium 52 (13%) thiamin 0.060 (5%)

- - Manganese 0.349 (15) Vitamin A

(IU) 4200 (140%)

- - Zinc 0.20 (2%) Vitamin C 35 (60%) (USDA 2011)

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Abbaszadeh et al (2009) analysed seed, leaf and stem of borage plant for its fatty acid

composition and it is presented in Table 4.

Table 4: Fatty acid composition of borage plant

Plant part

Total oil

S US MU. PU. L/α-

L α/γ- L

ω3/ω ratio

UN/S P/M

Seed 33.8 10.85 88.13 14.96 73.17 0.46 6.52 2 8.12 4.89

Leaf 1.9 14.39 67.66 11.06 59,21 0.40 14.11 2.5 5.7 6.42

Stem 0.84 27.72 62.18 8.15 54.03 1.25 5.2 0.78 2.24 6.63

S: saturated, US: Unsaturated, MU: Monounsaturated, PU: Polyunsaturated, L: Linoleic

Rosmarinic acid is an ester of caffeic acid and 3, 4-dihydroxyphenyl lactic acid and in

significant quantities is seen in the plants of Lamiaceae and Boraginaceae families

(Petersen and Simmonds, 2003; Toth et al., 2003) (Figure 4). The results of the

spectroscopic data in comparison with references (Kelley et al., 1976; Lu and Foo, 1999;

Wettasinghe et al., 2001), suggest that the major phenolic compound of the ethyl acetate

extract of petals of E. amoenum is rosmarinic acid.

Figure 4: Chemical structure of rosmarinic acid

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The callus culture is a technique of tissue culture, it is usually carried out on solidified gel

medium in the presence of growth regulators and initiated by inoculation of small

explants or sections from established organ or other cultures. This technique is one of the

ways for production of secondary metabolites in medicinal plants. Mehrabani et al (2005)

investigated the major secondary metabolite of E. amoenum with callus culture

technique. They initiated the callus culture of E. amoenum and established from seeds in

Murashige and Skoog media with three different ratios of plant growth regulatory:

kinetin, 2, 4-dichlorophenoxy acetic acid and 1-naphtalen acetic acid. They compared the

methanolic extracts of freeze-dried calluses by thin layer chromatography and high

performance liquid chromatography (HPLC). By preparative HPLC they separated the

major secondary metabolite and by UV IR, one and two dimensional 1H and 13C-

Nuclear magnetic resonance and Mass spectroscopy, they elucidated the structure of this

pure compound. They identified the rosmarinic acid with different spectroscopic methods

from callus culture of E. amoenum. Rosmarinic acid is common within the plant cell

tissue culture of the Lamiaceae and Boraginaceae families, although in insignificant

quantities. Rosmarinic acid has shown antimicrobial, antiviral, and anti-inflammatory

activity, which makes it a valuable product for the pharmaceutical and cosmetic

industries (Ghassemi et al., 2003).

3.4. IN VITRO ANTIOXIDANT STUDIES

The phytochemical studies on E. amoenum revealed that petals of this plant have

anthocyanidine (13%), flavonoid aglycons (0.15%), traces of alkaloids (Delorme et al.,

1977; Anon, 2002), volatile oils (0.05%) (Ghassemi et al., 2003) and rosmarinic acid

(Mehrabani et al., 2005b). Volatile compounds (0.05%) include octadecane, heptadecane,

viridiflorol, alpha cadinene and δ-cadinene. δ-cadinene (24.25%) is the major volatile

component (Ghassemi et al., 2003). Different investigators have isolated the plant

constituents; they include gamma-linolenic acid, alpha-linolenic acid, delta6-fatty acyl

desaturase, delta8-sphingolipid desaturase (Sperling et al., 2001), pyrrolizidine alkaloids,

mucilage, resin, potassium nitrate and calcium salt combined with mineral acids

(Abolhassani and Darabi, 2004).

24

Ghassemi et al (2003) studied volatile constituents of Echium amoenum. They isolated

the chemical composition of volatile fraction of dried petals of this plant by steam

distillation extraction with pentane (in yield of 0.05%) and examined that by GS-MS. By

mass spectra and Kovats indices the constituents were identified. The major components

except aliphatic alkanes which belong to sesquiterpenes were: ä-cadinene (24.25%),

viridiflorol (4.9%), á-muurolene (4.52%), ledene (3.8%), á-calacorene (3.04%), and ã-

cadinene (2.9%).

Ethanolic extracts of defatted borage (Borago offcinalis L.) seeds were studied for

antioxidant and free radical scavenging properties (Wettasinghe and Shahidi, 1999). The

resulting extract inhibited (p≤0.05) the coupled oxidation of β-carotene and linoleate in a

β-carotene-linoleate system. The system containing extract at a level providing 200 ppm

phenolics, retained 81% of the initial β-carotene after 2 h of assay whereas the control

retained only 11%. Inhibition (p≤0.05) of thiobarbituric acid reactive substances

(TBARS), hexanal and total volatile formation in a meat system containing 200 ppm

extract was reported to range from 18.9 to 88.3%, depending upon the concentration. The

extract inhibited (p≤0.05) conjugated diene, hexanal and total volatile formation in bulk

corn oil (8.3±49.6% inhibition) and corn-oil-in water emulsion (5.2±32.2% inhibition).

Hydrogen peroxide, hydroxyl radical and superoxide radical scavenging properties of the

extract were reported less than, but comparable to, those observed for trans-sinapic acid

at similar concentrations of phenolics. At 200 ppm, a 100% quenching of the hydroxyl

radical and superoxide radical was observed. The extract scavenged 29±75% of the

hydrogen peroxide in assay media after 10 min of assay as compared to 3% reduction in

the control.

3.5. IN VIVO ANTIOXIDANT STUDIES

Ranjbar et al (2006) studied the antioxidant activity of Iranian Echium amoenum Fisch &

C.A. Mey flower decoction in humans. In this study, they carried a cross-sectional

clinical trial to investigate the antioxidant properties of the decoction of the flowers of

Echium amoenum Fisch & C.A. Mey in human subjects. They invited a group of 38

healthy subjects to use the E. amoenum (7 mg/kg body weight) two times for 14 days.

Lipid peroxidation level, total antioxidant capacity and total thiol molecules were

25

measured in the blood before and after the study. They observed a significant reduction

of blood lipid peroxidation (24.65 ± 11.3 versus 19.05 ± 9.7, P= 0.029) after 14 days of

E. amoenum consumption. Blood total antioxidant capacity (1.46 ± 0.51 versus 1.70 ±

0.36, P= 0.018) and total thiol molecules (0.49 ± 0.11 versus 0.56 ± 0.12, P= 0.001)

improved after two weeks of intervention. They concluded that, this antioxidative stress

potential of E. amoenum may be due to its bioactive antioxidant components, especially

rosmarinic acid and flavonoids.

3.6. NUTRITIONAL AND MEDICINAL PROPERTIES

3.6.1. Antibacterial properties

Antibacterial effect of E. amoenum on Staphylococcus aureus was reported by

(Abolhassani, 2004).

Mahony et al. (2005) studied the bactericidal and anti-adhesive properties of 25 culinary

and medicinal plants against Helicobacter pylori. All plants were boiled in water (to

stimulate the effect of cooking) and aqueous extracts were prepared.

They assessed the bactericidal activity of samples by a standard-curve with seven strains

of H. pylori. Also anti-adhesive property was evaluated by the inhibition of binding of

four strains of FITC-labeled H. Pylori to stomach sections.

Among the plants that were able to kill H. Pylori, turmeric was the most efficient,

followed by cumin, ginger, chilli, borage, black caraway, oregano and liquorice. In

addition, extracts of turmeric, borage and parsley could inhibit the adhesion of H. Pylori

strains to the stomach sections.

In the other study (Jahandideh et al., 2011) studied the utilization of Echium amoenum

extract as a growth medium for the production of organic acids by selected lactic acid

bacteria. They examined the suitability of Echium extract as a raw material for production

of fermented juice by four strains of lactic acid bacteria (L. paracasei, L. acidophilus, L.

plantarum, and L. delbrueckii). Echium extract was inoculated with these bacteria and

incubated at 30 °C for 24 h. Changes in microbial population, pH, acidity, sugars, and

organic acids metabolism were observed during the fermentation process. Results

revealed that all of the selected bacteria were able to grow well in Echium extract without

any supplementation. They all metabolized sugars including glucose, fructose and

26

sucrose simultaneously. L. paracasei had more affinity to sugar consumption at almost

45.2%, 38%, and 21.6% of initial glucose, fructose, and sucrose concentration,

respectively. Lactic acid was formed immediately after the fermentation started. L.

plantarum, L. delbrueckii, and L. acidophilus produced 6.8, 6.6, and 6.4 g/l lactic acid,

respectively, which were significantly higher than that produced by L. paracasei (5 g/l).

On the other hand in the case of acetic acid, L. paracasei produced significantly larger

quantities in comparison with other strains (4.48 g/l). In this study researchers proved that

E. amoenum was a desirable media for the production of some organic acids by all these

bacterial strains.

In a study, anti-viral activity of aqueous extract of E. amoenum dried flower on

bacteriophage 3C and its specific host, Staphylococcus aureus 8327 was studied by agar

overlay method and the burst size was examined by one-step growth experiment. They

studied the antibacterial activity of flower by disc diffusion, agar-well diffusion and

minimum inhibitory concentration methods. They observed that the extract has

concentration-dependent antiviral activity against free bacteriophage 3C and the yield of

phage from the host Staphylococcus aureus 8327 was reduced. Also it has been shown

that antiviral activity of the extract is heat resistant. Autoclaving the extract at 110°C for

1 h did not eliminate its antiviral activity and the effect was similar to the extract that was

filter sterilized. However, the freeze-dried extract showed reduction in the activity during

90 days of storage at 4°C, also the activity of the working solution was reduced in the

period of one-week at 4°C storage. They explained results can justify for the traditional

use of the Iranian borage flower for infectious diseases and antifebrile activity

(Abolhassani, 2010).

One of the major public health problem causing significant morbidity and mortality in

Africa, Asia, and Latin America is Leishmaniasis (Croft and Coombs, 2003).

Leishmaniasis is caused by parasitic protozoa transmitted by the bite of a female sand fly,

and according to the world health organization (WHO), leishmaniasis is currently

endemic in 88 countries, affecting 12 million people worldwide and threatening 350

million more (Croft and Coombs, 2003).

BALB/c mice are highly susceptible to the infection with the parasite Leishmania major.

This susceptibility has been related, in part, to the expansion of Th2 cells and production

27

of their cytokines, interleukin (IL)-4 and IL-10, and down regulation of Th1 cytokine,

interferon gamma (IFN-γ) (Heinzel et al., 1991). The incapability of susceptible hosts to

increase the immune response necessary to activate macrophage and destroy the parasites

may be due to the parasite-specific proteins which can modulate the immune system

(Abolhassani and Darabi, 2004).

The treatment of leishmaniasis is based on antimonial compounds, pentamidine, and

liposomal amphotericin B. While antimonial compounds and pentamidine have more

severe side effects, these are less with AmBisome when compared with amphotericin B

deoxycholate (Croft and Coombs, 2003). The severe toxicity, high cost of chemotherapy,

emerging resistance of Leishmania spp. against most conventional drugs, and increased

rate of co-infection leishmaniasis–HIV demands the innovation of new, safe, more

efficient, and economically feasible drugs for the treatment of leishmaniasis and the

development of a successful vaccine. Although there are several researches for finding an

effective vaccine against leishmaniasis, there is no vaccine so far to prevent the disease.

For that reason, the interest goes toward traditional medicine to assess several plant

extracts for their anti-leishmanial effects. Medicinal plants hold a promise as sources of

chemical leads for improvement of novel therapeutic agents in the fight against many

diseases including parasite infections (Rates, 2001; Anthony et al., 2005).

Hosseini and Abolhassani (2011) studied immunomodulatory properties of Borage

(Echium amoenum) on BALB/c mice infected with Leishmania major. They evaluated

the in vivo anti-leishmanial and immunomodulatory activities of Iranian E. amoenum.

They used both aqueous and alcoholic extracts of Iranian borage (Echium amoenum Fisch

& C.A. Mey) for treatment of L. major infection in BALB/c mice. They found that both

extracts had immunomodulatory properties and increased the level of IFN-γ and lowered

the parasite burden in the proximal lymph nodes and prevented the necrosis of the

footpad as compared with the untreated infected mice.

3.6.2. Psychiatric disease

Iranian borage (E. amoenum) was studied for its analgesic and anticonvulsant effect in

mice (Heidari et al., 2006a; Heidari et al., 2006b). The effect of methanolic extract of

Echium amoenum Fisch & C.A. Mey. against Picrotoxin induced seizure in mice was

28

determined. Twenty min before administration of picrotoxin 10 mg kg-1body weight, the

extract with doses of 3.125, 6.25, 12.5 and 25 mg kg-1 were injected intraperitoneally to

mice and the latency of seizure, death time and percentage of mortality were studied in

animals. It was observed that the latency of seizure increased in groups pretreated with

different doses of extract and this effect was only significant at the dose of 6.25 mg/kg

body weight. In the other study the analgesic effect of the methanolic extract of the petals

of E. amoenum on male albino mice was evaluated by formalin and hot-plate test. The

methanolic percolated extract with different doses 5, 10, 20 and 30 mg/kg body weight

were injected intraperitoneally to mice. The results revealed that the dose of 10 mg/kg

body weight showed the highest analgesic effect in formalin (P < 0.05) and hot-plate test

(P < 0.01) in comparison to the control group.

3.6.3. Anxiolytic effect

It has been demonstrated that flavonoids possess mild sedative and anxiolytic effects.

The naturally occurring flavonoids and their synthetic derivatives, bind selectively to the

central benzodiazepine receptors and exhibit anxiolytic and other benzodiazepine like

effects in animals (Medina et al., 1997).

Gholamzadeh (2009) studied the anxiolytic effect of petals of Iranian borage. In this

study, an aqueous extract from petals of this plant was used (125 mg/kg) as compared to

diazepam (1 mg/kg), intraperitoneally, during two different treatment courses, 15 and 30

days. The aqueous extract was prepared by soxhlet apparatus with distilled water. A

sample size of 36 rats in 6 groups was selected for this experiment. Three groups were

treated by saline, diazepam and extract daily for 15 days and the other groups for 30 days.

Anxiolytic effect of extract was investigated in rat using the elevated plus-maze model of

anxiety. After finishing these courses and 30 min after the last injections, the test was

performed. The results revealed that in 30-day treatment course, time spent in open arms

was significantly higher than that of 15-day treatment in both diazepam and extract

groups and this effect was the highest in the diazepam group. So, the results of the study

indicated a significant duration dependent increase in time spent in open arms of plus-

maze.

29

In an in-vivo study, putative activity of hydroalcoholic and aqueous infusion extracts of

Echium amoenum was evaluated in mice using the rotarod model of motor coordination

and the elevated plus maze model of anxiety. The extracts were administered

intraperitoneally one time, one hour before performing the experiment. Preliminary

phytochemical analysis of sample showed that it contained saponins, flavonoids,

unsaturated terpenoids and sterols whereas there was no evidence of tannins, alkaloids

and cyanogenic glycosides. The hydroalcoholic extract of Echium amoenum at 125, 250

and 500 mg/kg did not show significant effect on motor coordination whereas the

aqueous extract at 62.5, 125, 250 and 500 mg/kg significantly disrupted motor

coordination. Intraperitoneal injection of aqueous extract at 5, 10, 20, 30, 62.5, 80 and

125 mg/kg showed a significant dose-dependent increase in time spent in open arm with

no significant change in open arm entries, closed arm entries and total arm entries. The

anxiolytic effect was most evident in 125 mg/kg group. It is almost observable that the

extract exhibited its anxiolytic effect in the doses in which no change in motor activity is

evident. Comparison of the dose response curve with the anxiolytic dose response of

diazepam at 0.25, 0.5, 1.0 and 2.0 mg/kg) in the same setting revealed that the maximal

efficacy of the extract is significantly lower than diazepam. It is concluded that single

administration of aqueous extract of Echium amoenum was found to be marginally

significant (Shafaghi et al., 2002).

Sayyah et al. (2009) investigated the efficacy and safety of an aqueous extract of E.

amoenum in treatment of obsessive–compulsive disorder. Obsessive–compulsive is an

anxiety disorder which is characterized by intrusive thoughts that feels uneasiness,

nervousness, fear, or worry. Symptoms of the disorder include unnecessary washing or

cleaning; repeated checking; excessive hoarding; preoccupation with sexual, violent or

religious thoughts; aversion to particular numbers; and nervous rituals, such as opening

and closing a door a certain number of times before entering or leaving a room. In

present study forty-four patients were randomly allocated to receive either E. amoenum

aqueous extract (500 mg/day) or placebo for duration of 6-week, double blind, and

parallel-group trial. Patients were assessed before the study and during weeks 1, 2, 4, and

6 by the yale-brown obsessive compulsive, the Hamilton Rating Scale for Anxiety

(HAM-A), and a score sheet on adverse effects. Researchers observed that in weeks 4 and

30

6, the extract has a significant positive effect in reducing obsessive and compulsive and

anxiety symptoms in comparison to placebo group. But there was no significant

difference between the two groups in terms of adverse effects. In this study they suggest

that E. amoenum aqueous extract has some anti obsessive and compulsive effects.

3.7. BORAGE TOXICITY STUDIES

The main source of pyrrolizidine alkaloids are found in plants of three families:

Boraginaceae, Compositae and Leguminosae. Two most important plants in the family

Boraginaceae are Ecbium plantagineum and HeIiotropium europaeum that cause

livestock toxicosis (Cheeke, 1988).

It is well established that plants containing pyrrolizidine alkaloids are hepatotoxic. Acute

toxicity is reproducible in animals and appears to be associated to biotransformation of

pyrrolizidine alkaloids by cytochrome P450s into pyrrole derivatives, which then act as

alkylating agents (Stickel and Seitz, 2000).

In a study the hepatotoxicity effects of aqueous extract of Echium amoenum in rats was

examined by Zamansoltani et al. (2008). Due to pyrrolizidine alkaloid constituents of

Borago officinalis, it is well accepted that this plant has Hepatotoxicity effects. Rats were

divided into 8 groups (n=7/group) and treated for 1 or 2 weeks with saline or E. amoenum

at doses of 100, 200 or 400 mg/kg/day. Serum biochemistry of analine aminotransferase,

aspartate aminotransferases and alkaline phosphatase was assessed to evaluate the rat

liver function. Histopathological assessment of liver sections was stained with H&E. In

both 1 and 2 weeks treated groups, serum levels of analine aminotransferase and alkaline

phosphatase reduced significantly (p<0.05) compared to controls. All treated groups had

shown a normal structure of liver tissue. No sign of evidence of characteristic

hepatotoxicity was found in rats treated with E. amoenum extract.

Mehrabani et al (2006) studied the toxic pyrolizidine alkaloids of Echium amoenum Fisch

& C.A. Mey. They separated and purified alkaloids by preparative TLC and characterized

by IR, one and two dimensional 1H and

13C-NMR and Mass spectroscopy. Results

showed four toxic alkaloids namely: echimidine I, echimidine isomer II, 7-angeloyl

retronecine III and 7-tigloyl retronecine IV are present in Echium amoenum Fisch & C.A.

Mey flower.

31

4.1. BOTANICAL INFORMATION

Valerian (Valeriana officinalis) plant root is a herb which is used worldwide over

centuries. It belongs to Valerianaceae family. There are 10 genera and about 300 species

in the family Valerianaceae (Simpson 2006), or the Valeriana genus is of the family

Caprifoliaceae and approximately contains 200 species (Judd et al., 1999). The

Valerianaceae are typically distributed worldwide and consist of herbs, rarely shrubs,

with opposite leaves, a sympetalous, spurred corolla, 1–4 stamens, and a tricarpellate,

poorer ovary with 1 functional locule and a single, apical ovule, the fruit is an achene,

with a pappuslike calyx in some members. The economic uses include some cultivated

ornamentals (e.g. Centranthus) and negligible edible, medicinal, or essential oil plants.

The plant of Valeriana officinalis is native to Europe and Asia and in addition has

naturalized in eastern North America. This tall perennial has a preference in moist

woodlands; it has been broadly cultivated in northern Europe. Most of the European

supply is grown in Holland. Low lying, damp sandy humus with lime fertilizer is the way

to cultivate Valerian. It harvests in the late fall and dries. V. officinalis is the species

which is used in Europe. This genus contains more than 250 species. In traditional

Chinese and Japanese medicine V. fauriei is used commonly (Hikino et al., 1971; Hikino

et al., 1972a; Hikino et al., 1972b; Huang and Williams, 1999). Valerian capensis is other

species which is used in African traditional medicine (Iwu, 1993), V. edulis is used in

Mexico and V. wallichii is used in India (Schulz et al., 1997).

4.2. HISTORY OF USE

The roots of V. officinalis known as valerian, since long is taken as sedative medicine in

Europe. Valerian is an agent with mild sedative and sleep-promoting properties that is

often used as a milder substitute or a possible alternate for stronger synthetic sedatives,

such as the benzodiazepines, in the treatment of nervous states and anxiety-induced sleep

disturbances (Miyasaka et al., 2006). Lesniewicz et al., (2006) reported that valerian is

tranquillizer for people with hyper-excitability and as a smooth-muscle relaxing agent to

treat stomach and intestine cramp. Valerian is also a component of many herbal mixtures,

which are widely used to treat sleeping disorders (Bent et al., 2006). Nowadays, valerian

extracts are available as dietary supplements, which primarily involve dried root or

32

extracts from the root, formulated into tablets or soft gelatin capsules. Usually each dose

contains approximately between 50 mg and 1 gram of dried root or extract. The use of

these dietary supplements is widespread, with an estimated 210 and 125 million doses

sold annually in the United States and in Europe respectively (Grunwald, 1995). Though

not supported by research, traditionally it has been recommended for epilepsy (Spinella,

2001). Restlessness, insomnia, nervousness, and tension are the present indications for

valerian as reported by Tariq and Pulisetty (2008). It is suggested that the large doses

when stopped, as most sleep aids, cause withdrawal symptoms (Garges et al., 1998).

Cohen and Toro (2008) expressed that patients with liver disease are advised not to take

valerian. Although it is shown to be a successful remedy for the reduction of anxiety, in

some individuals some side effects like headaches and night terrors were reported

(Dennehy et al., 2005). He explained the reason may be due to the fact that some people

lack a digestive conversion property required to effectively break down valerian. In these

individuals, valerian can cause agitation.

4.3. NUTRITIONAL AND CHEMICAL CONSTITUENT

Mineral content of valerian was studied by Adamczyk and Jankiewicz (2008) and they reported that

valerian root contains 13.1 ppm copper, 75.1 ppm zinc and 16.8 ppm manganese. More than 150

chemical constituents were found in valerian of which many are physiologically active (Jiang et al., 2007).

There is significant variation in the chemical constituents in plants from different sources and growing

conditions, processing methods and storage conditions (Wagner et al., 1972). To guarantee the quality of

the drug, producers have standardized production of the plant extracts (Gutierrez et al., 2004).

Alkaloids, terpenes, organic acids and its derivatives, valepotriates and flavones are the

known pharmacologically active compounds found in valerian extract. In general, it is

accepted that the valepotriates are the compounds responsible for the sedative activity of

the Valerianaceae (Patočka and Jakl, 2010).

Alkaloids (0.01–0.05%), notably terpene alkaloids are present in valerian (Duke, 1985).

The main valerian alkaloids are actinidine, chatinine, valerianine, valerine, alpha-methyl

pyrryl ketone and naphthyridin methyl ketone (Torssell and Wahlberg, 1967; Franck et

al., 1970; Janot et al., 1979). The structures of some valerian alkaloids are shown in

Figure 5.

33

Figure 5: The structures of principal compounds present in volatile essential oil of

Valeriana officinalis.

Valerian alkaloids actinidine (Ia) and valerianine (Ib), valerenic acid (IIa), its aldehyde

valerenal (IIb) and terpene valeranone (III).

4.3.1. Actinidine

Actinidine (Ia) is a steam-volatile monoterpenoid pyridine alkaloid with a cyclopenta [c]

pyridine skeleton present in the essential oil of valerian root (Johnson and Waller, 1971)

and Actinidia polygama (silver vine) (Sakan, 1967). Actinidine is compound in valerian,

which can attract cats (Patočka and Jakl, 2010). Biosynthesis of actinidine results from

lysine and quinolinic acid as precursors (Auda et al., 1967). Actinidine is an alkaloid

which is psychoactive which interferes with the gamma-aminobutyric acid (GABA)-ergic

metabolism; it is an agonist on benzodiazepine receptors and thus revealed an allosteric

modulation of the GABA-receptor-proteins (Patočka and Jakl, 2010).

34

Waliszewski was isolated Chatinine from valerian (Baby et al., 2005) but its biological

properties have not been studied. Alpha-methyl pyrryl ketone has been studied in

Germany as a central nervous system active compound on 1970 (Sándor et al., 1970).

Synthetic naphthyridinones similar in structure to natural naphthyridyl methyl ketone

were introduced as potential drugs for the treatment of schizophrenia (Clark et al., 2006;

Favor et al., 2006). Since the pharmacological properties of valerian alkaloids have been

studied separately only infrequently, it is difficult to say how these participate in the

medical effects of V. officinalis.

4.3.2. Organic Acids and Terpenes

Organic Acids and Terpenes are available in the volatile essential oil which is 0.2–2.8%

of the dry weight of the root. The essential oils are not only seen in the subterranean parts

of the plants but also in the aerial parts (Funke and Friedrich, 1975). Terpenes are

characterized chemically as monoterpenes and sesquiterpenes. Valeric, isovaleric,

valerenic, isovalerenic and acetoxyvalerenic acids, bornyl acetate, bornyl isovalerenate,

1-pinene, 1-comphene, 1-borneol, terpineol, valeranone and cryptofauronol are most

considerable valerian organic compound. It is suggested that some of the oil components

pose sedative properties. Isovaleric acid and bornyl isovalerate are two compounds which

are mainly responsible for the characteristic aroma of valerian.

Isovaleric acid and 3-methylbutanoic acid do not have significant pharmacological and

toxicological properties and only share the drug’s odor. However, it was found in 2007

that isovaleric acid decreases ATPase activity in the synaptic membranes of the cerebral

cortex and it may be necessary in the pathophysiology of the neurological dysfunction of

isovaleric acidemic patients (Ribeiro et al., 2007).

Valerenic acid (IIa) and its aldehyde valerenal (IIb) are monoterpenes which are

pharmacologically active compound. Cavadas et al (1995) recommended that valerian

acts via GABA mechanisms. Other studies have revealed binding of valerian extract to

GABA receptors, but the functional effect of the binding has not been demonstrated. Data

from the study of Yuan et al. (2004) and Trauner et al (2008) suggest that the

pharmacological effects of valerian extract and valerenic acid are mediated through

modulation of GABAA receptor function. By passive diffusion valerenic acid is known

35

to penetrate into the central nervous system trans cellulary (Neuhaus et al., 2008). Dietz

et al. (2005) showed that valerenic acid is a partial agonist of the 5HT receptor with the

strong binding affinity to the 5-HT (5a) receptor, but only weak binding affinity to the 5-

HT(2b) and the serotonin transporter. In a study valerenic acid, acetylvalerenolic acid and

valerenal served as a inhibitors of NF-κB at a concentration of 100 µg/ml.

Acetylvalerenolic acid reduced NF-κB activity to 4%, while valerenic acid reduced NF-

κB activity to 25% (Jacobo et al., 2006).

Valeranone (III) was tested as a medical drug in hyperkinetic behavior disorders (Gupta

and Virmani, 1968). In animal experiments its sedative, tranquilizing and

antihypertensive properties was pharmacologically investigated but the activity of

valeranone was found to be lesser than those of the standard substances used (Rucker et

al., 1978). Thus, valerian may carry the sedative effects of anaesthetics and other

medications that act on GABA receptors, and use of valerian before surgery may cause a

valerian-anaesthetic interaction.

4.3.3. Valepotriates

Valepotriates are esterified iridoid-monoterpenes. Their name is derived from the

valeriana-epoxy-triester, because these are triesters of polyhydroxycyclopenta-(c)-pyrans

with carboxylic acids: acetic, valeric, isovaleric, α-isovaleroxy-isovaleric, β-

methylvaleric, β acetoxy-isovaleric, β-hydroxyisovaleric and β-acetoxy-β-methylvaleric

acid (Thies, 1969). It is a major component consisting of 50–80% active compounds.

Valepotriates are divided into two classes: monoene and the diene derivatives. The

principal diene valepotriates are valtrate, isovaltrate, 7-desisovaleroyl-7-acetylvaltrate

and 7-homovaltrate, and the major monoene derivatives are didrovaltrate and

isovaleroxyhydroxydidrovaltrate. The amount of valepotriates varies widely between

species. In general the underground parts of plant contain higher amount of valepotriates

than the other parts of the plant (Violon et al., 1984). Valepotriates are unstable

compounds: they are thermolabile and decompose quickly under acidic or alkaline

conditions in water, as well as in alcoholic solutions. However in anhydrous methanol,

and stored at 20°C, the diene valepotriates were found to be relatively stable. Dissolved

in methanol or ethanol, with only a small amount of water and stored at room

36

temperature, gives 90% decomposition within a few weeks (Bos et al., 1996). The main

decomposition products of the valepotriates are the yellow-coloured baldrinals (Bos et

al., 2002). Baldrinals are chemically reactive and may subsequently form polymers (Bos

et al., 1996).

4.4. MECHANISM OF ACTION

Because of valerian's traditional use as a sedative, anti-convulsant, migraine treatment

and pain reliever, most basic science research has been directed at the interaction of

valerian constituents with the GABA neurotransmitter receptor system (Trauner et al.,

2008). The mechanism of action of valerian in general and as a mild sedative in particular

is not known (Wheatley, 2005). Valerian extracts and some of its constituents, mainly

valerenic acid, appear to have some affinity for the GABAA receptor, but the exact

mechanism of action is not clear. Benke et al. (2009) described a specific binding site on

GABAA receptors with nM affinity for two general constituents of valerian namely

valerenic acid and valerenol. Both valerenic acid and valerenol increased the response to

GABA at multiple types of recombinant GABAA receptors. A point mutation in the

beta2 or beta3 subunit of recombinant receptors strongly decreased the drug response. In

vivo, valerenic acid and valerenol have shown anxiolytic activity with high potencies in

the elevated plus maze and the light/dark choice test in wild type mice. In beta3 point-

mutated mice the anxiolytic activity of valerenic acid was found to be absent. Thus,

neurons expressing beta3 containing GABAA receptors are a main cellular substrate for

the anxiolytic action of valerian extracts (Benke et al., 2009). Substances such as

valerenic acid and its derivatives acetoxyvalerenic acid and hydroxyvalerenic acid have

to pass the blood-brain barrier and interact with this receptor in the brain. It was

hypothesized that the investigated terpenes from V. officinalis can probably only cross

through the blood-brain barrier by a still unknown transport system and not

transcellularly by passive diffusion (Neuhaus et al., 2008).

4.5. IN VITRO ANTIOXIDANT STUDIES

Zheng and Wang (2001) studied the antioxidant activity of selected herbs which were

grown in the same place with similar conditions to avoid variations of oxygen radical

37

absorbance capacity (ORAC) values because of ecological factors. Herbs (2.0 g) were

extracted with 15 ml of phosphate buffer (75 mM, pH 7.0) using a Polytron homogenizer

(Brinkmann Instruments, Inc., Westbury, NY) for 1 min and were then centrifuged at

20000g for 20 min. The supernatant was used for the ORAC and total phenolic

compound assay after suitable dilution with phosphate buffer (75 mM, pH 7.0). They

reported the total phenolic content of valerian as 1.78 mg of Gallic acid equivalent

(GAE)/g of fresh weight and ORAC as 15.82 µmol of TE/g of fresh weight.

4.6. NUTRITIONAL AND MEDICINAL PROPERTIES

The root and rhizome of the valerian plant (Valeriana officinalis L.) is used medicinally

for its sedative properties with indications including nervous tension, insomnia, anxiety

and stress (Houghton, 1999). The concentration of valerenic acid significantly diminished

over time and was reduced by temperature and moisture. The greatest loss was reported

at 30°C in low humidity (Wills and Shohet, 2009). In an in vivo and in vitro investigation

of valepotriates and valeranone on guinea-pig ileum smooth muscle preparations it was

found that dihydrovalerate and valeranone were able to relax stimulated smooth muscle

preparations with potency comparable to that of papaverine. Moreover, it was shown that

these valeriana compounds cause smooth muscle relaxation through a musculotropic

action, which is also known to be the case for papaverine (Hazelhoff, 1984).

Hazelhoff (1984), in his dissertation, showed that there is a significant reduction in the

locomotor activity of mice when the valerian and V. officinalis extract was administered.

The effect of a mixture of valepotriates on the elevated plus-maze performance of

diazepam withdrawn rats was evaluated by Andreatini and Leite (1994). The rats were

chronically (28 days) treated with diazepam (doses increased up to 5.0 mg/kg) and to

provide a withdrawal syndrome they were treated with a control solution for 3 days.

Chronically vehicle-treated rats were used as control. The abstinent animals treated with

the vehicle showed a significant reduction in the percentage of time spent in the open

arms when compared with the control animals. Diazepam and valerian 12.0 mg/kg

reversed this anxiogenic effect. They did found significant diference in zalerian 6.0

mg/kg other group.

38

5.1. BOTANICAL INFORMATION

Ginger (Zingiber officinale) described as herbaceous rhizomatous perennial, which can

reach up to 90 cm in height under cultivation. Ginger plant rhizomes of the plant are with

strong aromatic odor, sweet, warm, thick lobed, pale yellowish, bearing simple alternate

distichous narrow oblong lanceolate leaves. During the growing process the herb

develops several lateral shoots in clumps, which start drying when the plant matures.

Leaves are long being 2 - 3 cm in width with sheathing bases, the blade gradually

tapering to a point. Inflorescence is solitary, lateral radical pedunculate with oblong

cylindrical points. Flowers are rare, smaller in size, calyx superior, gamosepalous, three

toothed, open splitting on one side, corolla of three subequal oblong to lanceolate connate

greenish segments (Kawai et al., 1994; Burdock, 1996).

5.2. HISTORY OF USE

Food condiments or spices are strong smelling, sharp tasting substances. They are usually

of vegetable origin and used to improve the flavor of food. Mustard, nutmeg, ginger,

garlic, pepper, coriander, locust bean etc are common examples. Since ginger produces

many complex compounds that are useful in food similar to herbs and spices, as flavoring

and seasoning, in the cosmetics and medicinal industries, antioxidant and antimicrobial

agents, it has received much attention. For over 2000 years, ginger has been used as a

spice (Bartley and Jacobs, 2000b).

It is used as an essential spice in dishes like curry powder, gingerbread and in some kind

of beers and other drinks. When the plant matures, the taste and pungency of ginger

increases, therefore young rhizomes are juicy and fleshy with a very mild taste while

juice from old rhizomes are extremely potent and sharp and is often used as a spice in

Chinese cuisines (Burdock, 1996).

Ginger is used as ingredient in making soups, as a spice in ginger bread and other recipes

and can be stewed in boiling water to make ginger tea. It can also be made into candy or

used as flavoring for cookies, crackers and cake. It is used to relieve motion sickness and

especially in the Far East, it is used as a digestive aid and also a food preservative

(Macrae et al., 2002). In Nigeria, ginger is used to flavor a local drink named Kunnu.

39

It is also an important ingredient of many herbal formulations, is carminative, pungent,

stimulant, consumed widely for indigestion, stomach ache, malaria and fevers. Its

traditional medicinal uses are also listed for abdominal pain, chest congestion, chronic

bronchitis, colic and vomiting (Jatoi et al., 2007).

The characteristic odor and flavor of ginger is caused by a mixture of zingerone, shogaols

and gingerols, volatile oils that made one to three percent of fresh ginger weight. The

gingerols in laboratory animals, increase the motility of the gastrointestinal tract and

shown to have analgesic, sedative, antipyretic and antibacterial properties (Ahmed et al.,

2011). Ginger oil has been shown to prevent skin cancer in mice and a study at the

University of Michigan demonstrated that gingerols can kill ovarian cancer cells (Singh

et al., 2011). 6-gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone), the

major pungent principle of ginger, has anti-oxidant, anti-inflammation and anti-tumor

promoting activities (Surh et al., 1998; Bode et al., 2001; Jagtap et al., 2009). Ginger

contains up to three percent of a fragrant essential oil whose main constituents are

sesquiterpenoids, with (-)-zingiberene as the main component. Smaller amounts of other

sesquiterpenoids (β-sesquiphellandrene, bisabolene and farnesene) and a small monoterpenoid

fraction (β-phelladrene, cineol, and citral) have also been recognized (Ahmed et al., 2011).

It is said that the pungent taste of ginger is due to nonvolatile phenylpropanoid-derived

compounds, particularly gingerols and shogaols, which form from gingerols when ginger

is dried or cooked (Malu et al., 2009). Zingerone is also produced from gingerols during

this process; this compound is less pungent and has a spicy-sweet aroma (Riazur et al.,

2011). Ginger is also a minor chemical irritant. Ginger has a sialagogue action,

stimulating the production of saliva, which makes swallowing easier (O'Hara et al.,

1998).

5.2.1. In Traditional Chinese Medicine

Ginger is very widely used in Traditional Chinese Medicine. It is said to be a spleen

remedy and is supportive to the spleen, stomach and kidneys (Perez, 2005). Ginger also

has antiemetic activity and is used to prevent motion sickness (Vishwakarma et al., 2002;

Sontakke et al., 2003; Manusirivithaya et al., 2004).

40

5.2.2. In Arabic Medicine

Ginger is said to be hot in the second degree and moist in the first. It is warming and has

a softening effect on the belly; it is beneficial to the body against digestive ailments such

as flatulence, food toxins and constipation. The use of ginger has also been mentioned in

western medicine. It has been used as such or as an ingredient in specific herbal formula

and also consumed as a ‘corrective remedy’ against the unwanted effects of other plants

(Perez, 2005).

5.3. NUTRITIONAL AND CHEMICAL COMPOSITION

Many authors have reported the chemical composition of ginger (Sakamura and Hayashi,

1978; Smith and Robinson, 1981; Nishimura, 1995; Bartley and Jacobs, 2000), but toxic

effects have not been reported. Different authors have reported the nutritional

composition of ginger rhizome and it is presented in Table 5.

Table 5: Reported Nutritional composition of ginger rhizome (g/100 g), minerals

(mg/100g)

Adanlawo (2007)

Bhowmik (2008)

Nwinuka et al (2005)

Odebunmi et al (2010)

Meadows (1988)

Kirk and Othme (1982)

Dry matter 92.7 96.2 93.3 23.14 - 93.1 Crude protein 5.25 11.88 8.58 8.75 8.39 8.6 Ether extract 5.54 7.65 - - - - Crud fiber 9.74 9.88 2.93 - - Fat - - 5.53 15.21 6.4 Ash 5.97 11.6 6.40 2.54 5.7 Total carbohydrate

66.26 - 72.84 - 67.21% 72.4

Oxalate 4.55 - 0.23 - - - Phytate 28.83 - - - - - Tannin 0.26 - 0.01 - - - Phosphorous 25.7 1.16 - - - - Sodium 40.96 17 - - - - Potassium 37.34 563 - - - - Calcium 35.66 0.004 - - - - Manganese 19.6 78 - - - - Zinc 4.06 5.62 - - - - Iron 1.44 - - - - - Copper 0.76 0.50 - - - -

41

5.4. ANTIOXIDANTS PROPERTIES OF GINGER

The theory of oxidative-stress on aging and age-degenerated diseases shows the importance

of daily use of natural phytochemicals and compounds (Harman, 1992). In a variety of

spice extracts, the systematic evaluation of total antioxidant concentration has been

conducted using in vitro assays, including common Indian spices, which also have been

shown to inhibit lipid peroxidation. In one study, relative antioxidant activities from highest

to lowest were found in cloves, cinnamon, pepper, ginger, and garlic (Shobana and Naidu,

2000). In vitro studies also show that ginger extract has antioxidative properties and

scavenges superoxide anion and hydroxyl radicals (Cao et al., 1993; Krishnakantha and

Lokesh, 1993), and several studies have identified ginger with high antioxidant content

(Shobana and Naidu, 2000; Halvorsen et al., 2002). Several in vivo studies also have

determined the antioxidant capacity of spices or their constituents. The antioxidant capacity

of ginger has been reported in relation to LDL cholesterol oxidation in apolipoprotein E-

deficient mice (Fuhrman et al., 2000). The efficiency of endogenous antioxidant systems

in different organism is not enough to combat all oxidative damages, therefore the interest

for natural exogenous antioxidants like flavonoids and polyphenols in the human diet has

become greater. Natural antioxidants influence the safety and acceptability of the food

system. They can keep the food stable against oxidation and also be effective in controlling

microbial growth. Dietary supplementation can enhance the inherent levels of the

antioxidants in animal products and offer a more consumer acceptable product. However,

the traditional practice of adding antioxidants during processing can still play a very

important role since the added compounds have the potential for enhancing the activity of

the inherent antioxidants systems (Stoilova et al., 2007).

5.5. ANTIOXIDANT COMPONENTS

Ginger roots and its extracts have polyphenol compounds (6-gingerol, gingerol,

gingerdiol, gingerdione) and other compounds which could be responsible for antioxidant

activities of ginger (Chen et al., 1986; Kikuzaki and Nakatani, 1996). Ginger contains

11% of gingerols including 5% of 6-gingerol (Nazemieh et al., 2002). Though various

extracts are obtained from ginger, the CO2 extracts are richest in polyphenol compounds

and have a composition that is closest to that of the roots (Chen et al., 1986; Bartley and

42

Jacobs, 2000). At high concentration the active component of ginger, -gingerol- inhibits

ascorbate– ferrous complex which in turn induces lipid peroxidation (Reddy and Lokesh,

1992). Several studies have shown that consumption of foods rich in polyphenolic

antioxidants such as tea, garlic, olive oil, ginger, tomato and others reduce diabetic

complications and progressed the antioxidant system of the body (Aviram and Eias,

1993; Serafini et al., 1994; Fuhrman et al., 2000; George et al., 2004). Nwinuka et al

(2005) reported the saponin content of ginger as 3.99g/100g. Antioxidant active

chemicals isolated from ginger (Zingiber officinale) (USDA 2011) are listed in Table 6.

Table 6: Antioxidant active chemicals isolated from ginger (Zingiber officinale)

Antioxidant Quantity Antioxidant Quantity From rhizome

6-gingerol 130–7138 gamma-terpinene 0.4–25

6-shogaol 40–330 lauric-acid 390–3630 alanine 310–1793 methionine 130–737 ascorbic-acid 0–317 myristic-acid 180–1650 beta-carotene 0–4 p-coumaric-acid 0–19 caffeic-acid - palmitic-acid 1200–11,220

camphene 28–6300 Selenium 10 sucrose - tryptophan 120–693 terpinen-4-ol - - -

From plant

myricetin - vanillic-acid - p-hydroxy-benzoic-acid

- vanillin

-

quercetin - kaempferol -

delphinidin - capsaicin -

ferulic-acid - chlorogenic-acid -

beta-sitosterol - - -

Others

shikimic-acid - myrcene 2–950

curcumin plant - - -

5.6. IN VITRO ANTIOXIDANT STUDIES

The antioxidant activity of each plant enhances in proportion to the concentration of

extract. It could be related to the presence of antioxidants compounds, especially phenols

(Matsufuji et al., 1998; Chu et al., 2002) and in particular flavanoids, catechin and

43

isocatechin (Materska and Perucka, 2005; Gramza et al., 2006; Dubost et al., 2007). Kaur

and Kapoor (2002) studied the antioxidant activity and total phenolic content of some

Asian vegetables and examined ginger (Zingiber officinale) for these parameters. The

antioxidant activity of ethanolic extract was reported to be 71.8 %, water extract 65.0%,

and total phenolic compound of 80% ethanolic extract, 221.3 mg/100g.

Stoilova et al (2007) studied the total phenols and antioxidant effect of ginger extract.

The total phenols of the alcohol extract were found to be 870.1 mg/g dry extract. Free

radical scavenging activity (FRSA) by 2,2-diphenyl-1-picrylhydrazyl (DPPH), reached

90.1% and exceeded that of butylated hydroxytoluene (BHT), the IC50 concentration for

inhibition of DPPH was found to be 0.64 µg/ml. The antioxidant activity in a linoleic

acid/water emulsion system evaluated by means of TBARS was highest at 37ºC, 73.2%,

and 71.6% when the formation of conjugated dienes was inhibited. At 80ºC the

antioxidant activity at the highest concentration of a ginger extract was less efficient:

65.7% for conjugated dienes formation and 68.2% for TBARS. The ginger extract

inhibited the hydroxyl radicals 79.6% at 37ºC and 74.8% at 80ºC, which showed a higher

antioxidant activity than quercetin. The IC50 concentration for inhibiting OHº at 37ºC was

slower than that at 80ºC – 1.90 and 2.78 µg/ml respectively. The ginger extract chelated

Fe3+ in the solution (Stoilova et al., 2007). Ahmed and Rocha (2009) analyzed the total

phenol content of ginger plant originating from Iraq and reported the content as 266.3 ±

1.2 mg gallic acid equivalent/100g. In vivo antioxidant activity of Zingiber officinalis

determined against lipid peroxidation in brain homogenate TBARS in rat showed an

activity of 32.9 – 64.8% at concentrations of 3.5 – 16.9 mg/ml.

They also determined the Fé+2 chelating ability of the water extractable phytochemicals

of Zingiber officinalis and reported an activity of 54.7- 64.1 % at the concentration tested

3.5-16.9 mg/ml. The use of iron chelation is a popular therapy for the management of

Fé+2-associated oxidative stress in brain. The iron chelating ability of the Zingiber

officinalis was an indicator of the neuroprotective property because iron is involved in the

pathogenesis of Alzeimer’s and other diseases by multiple mechanisms (Malecki and

Connor, 2002). In this study they found a good correlation between % antioxidant

activity and phenolic content. Jitoe et al. (1992) studied the antioxidant activity of

tropical ginger extracts and analyzed the curcuminoids. In this study antioxidant activity

44

of the rhizomes of nine tropical gingers (Curcuma aeruginosa, Curcuma domestica,

Curcuma heyneana, Curcuma mangga, Curcuma xanthorrhiza, Zingiber cassumunar,

Phaeomeria speciosa, Alpinia galanga, and Amomum kepulaga) was determined by

thiocyanate and TBA methods in a water/alcohol system. The antioxidant activity of the

extracts of the gingers was greater than that evaluated from the actual quantity of three

known curcuminoids in the extracts. In a research conducted by Chen et al (2008a), the

antioxidant activity of 18 different species of ginger from Taiwan was analyzed. The

methanolic extracts of the plants were analyzed for their total phenol compounds, FRSA

and reducing power. As shown in Table 7, total phenol compounds of the Alpinia genus

averaged 17.30 mg/g for Curcumas, and the highest, 36.5 mg/g for Vanoverberghia

sasakiana. The best antioxidant performances were seen in Vanoverberghia and

Hedychium, both 89%, and FRSA followed similar trends. Particularly, Zingiber

oligophyllum, considered as a traditional medicinal plant used in Taiwan exhibited low

DPPH scavenging activity and reducing power.

Ghasemzadeh (2010) evaluated the antioxidant activities of methanol extracts from the

leaves, stems and rhizomes of two Zingiber officinale varieties (Halia Bentong and Halia

Bara) to explore the potential medicinal properties of different parts of plant. The free

radical scavenging activity was higher in plant leaves, which had more phenolic and

flavonoids content than rhizomes. In contrast, the ferric reducing/antioxidant potential

(FRAP) activity was more in rhizomes than that of the leaves. At low concentration, the

inhibition activity in leaves of both varieties was significantly higher than or comparable

to those of the young rhizomes. Between the two variety, Halia Bara had higher

antioxidant activities as well as total contents of phenolic and flavonoid than Halia

Bentong. The author authenticated the medicinal potential of the leaves and young

rhizome of Zingiber officinale (Halia Bara) and the positive relationship between total

phenolics content and antioxidant activities.

Hinneburg (2006) studied the antioxidant activities of extracts from selected culinary

herbs and spices. In this study they found the total polyphenols of ginger to be 23.5 ±

1.26 (mg GAE/g).

45

Table 7: Extraction yield & contents of total phenolic & antioxidant capacity of

ginger

Species Extraction

yield (mg/g)

Total phenols (mg/g)

Antioxidantcapacity (%)

Free radical scavenging activity (%)

Reducing power

Alpinia japonica (Thunb.)

Mig 58.8±7.7 18.3±6.3 69.5a±6.7b 71.8±6.4 0.8±0.2

Alpinia kawakamii Hayata 79.3±15.6 18.7±3.4 73.5±4.6 76.7±5.3 0.9±0.1 Alpinia kusshakuensis

Hayata 64.3±6.4 19.5±1.45 70.9±5.67 76.7±6.3 0.8±0.1

Alpinia mesanthera 82.6±5.8 19.5±4.3 62.3±7.6 64.6±7.4 0.8±0.3 Alpinia officinarum 52.2±6.3 19.3±2.5 75.4±9.6 80.5±9.3 1.4±0.2 Alpinia pricei Hayata 41.1±11.7 19.2±5.1 70.6±7.9 74.9±5.4 0.7±0.15 Alpinia shimadai 107.5±19.4 20.2±5.3 72.2±1.3 69.3±7.3 1.0±0.3 Alpinia uraiensis Hayata 89.5±13.9 17.6±1.5 62.5±5.8 68.6±4.3 0.8±0.4 Alpinia zerumbet (Pers.)

Burtt & Smith 127.5±3.8 15.3±2.2 53.8±8.6 59.4±3.5 0.47±0.2

Costus speciosus (Koenig)

Smith 93.2±9.7 22.9±2.3 81.3±6.7 78.5±4.4 1.3±0.3

Curcuma domestica 87.1±6.3 35.6±5.5 89±7.4 8 1.3±6.3 1.6±0.4 Curcuma longa L. 94.2±7.5 21.4±1.7 76.1±5.1 72.1±5.2 1.2±0.5 Curcuma viridiflora 91.4±5.8 29.4±3.7 53±5.3 76.4±5.0 1.1±0.2 Curcuma zedoaria 92.5±7.6 33.4±5.7 76±6.4 65.4±6.1 0.9±0.2 Hedychium coronarium

Koenig 32.6±5.8 25.8±4.4 89.6±11.6 90.1±7.2 1.6±0.3

Vanoverberghia sasakiana 117±12.6 36.5±8.9 89±6. 9 89.5±7.1 1.01±0.2 Zingiber kawagoii Hayata 95.6±7.4 28±2.9 79±4.8 42±7.8 0.9±0.08 Zingiber oligophyllum K.

Schumann 74±4.3 20±4.5 88±5.9 32±9.3 0.34±0.08

a Concentration of each methanolic extract was adjusted to 100 mg/ml

b All values in this table represent the mean ± SD (n=3)

5.7. IN VIVO ANTIOXIDANT STUDIES

One of the factors which induce oxidative stress is the exposure to pesticide chemicals

which lead to generation of free radicals and alterations in antioxidants or oxygen free

radical scavenging enzymes. Hence, in a research study, the effect of sub chronic

malathion (O,O-dimethyl-S-1,2, bis ethoxy carbonyl ethyl phosphorodithioate) exposure

was determined on lipid peroxidation, glutathione and related enzymes and oxygen free

radical scavenging enzymes in albino rats. Malathion (20 ppm) was administered for 4

46

weeks and it was observed that the malondialdehyde levels in serum, activities of

superoxide dismutase, catalase and glutathione peroxidase in erythrocytes and glutathione

reductase and glutathione S-transferase in serum increased. However, the glutathione

level in whole blood was reduced. Concomitant dietary feeding of Zingiber officinales

Rosc (ginger 1%, w/w) significantly reduced malathion induced lipid peroxidation and

oxidative stress in these rats. These results indicate the possible involvement of free

radicals in organophosphate-induced toxicity and highlight the protective action of

ginger, against these in biological systems (Ahmed et al., 2000).

Manju and Nalini (2005) studied chemopreventive efficacy of ginger in animal models. A

weekly subcutaneous injection of 1,2 dimethylhydrazine (20 mg/kg body weight) in the

groin was given to rats for duration of 15 weeks. Ginger (50 mg/kg body

weight/everyday) was given to the rats at the beginning and after initiation stages of

carcinogenesis. The activity of lipid peroxidation was studied by measuring the formation

of thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes,

and the antioxidant status by measuring superoxide dismutase, catalase, glutathione

peroxidase, glutathione-S-transferase, glutathione reductase, reduced glutathione,

vitamins C, E, and A concentrations in the circulation of 1,2-dimethylhydrazine-induced

experimental colon cancer. They observed that in the presence of a known colon

carcinogen, 1,2 dimethylhydrazine, plasma lipid peroxidation (thiobarbituric acid reactive

substances, lipid hydroperoxides and conjugated dienes) and cancer incidence were

significantly increased whereas enzymic (glutathione peroxidase, glutathione-S-

transferase, glutathione reductase, superoxide dismutase and catalase) and non-enzymic

antioxidant concentrations (reduced glutathione, vitamins C, E, and A) were diminished

as compared to control rats. The number of tumors as well as the incidence of cancer was

significantly reduced on treatment with ginger. In addition, ginger supplementation at the

initiation stage and also at the post-initiation stages of carcinogenesis significantly

decreased circulating lipid peroxidation and significantly increased the enzymic and non-

enzymic antioxidants as compared to unsupplemented 1,2 dimethylhydrazine -treated

rats. Authors concluded that ginger supplementation can suppresses colon carcinogenesis

in the presence of the procarcinogen 1,2 dimethylhydrazine.

47

5.8. NUTRITIONAL AND MEDICINAL PROPERTIES

5.8.1. Hypolipidemic and hypoglycemic effect

Metabolic syndrome, including obesity, dyslipidaemia, hyperglycaemia and insulin

resistance that predisposes type 2 diabetes are major disease problem all around the

world and herbal medicines can effectively control these disorders. The root of ginger is

commonly used as a spice in various foods and beverages. Apart from its other traditional

medical uses, Z. officinale has been used to control diabetes and dyslipidaemia.

Ginger can increase the pancreatic lipase (Platel and Srinivasan, 2000). Ginger extract is

also known as functional food because it posses both nutritional and medicinal benefits.

Consumption of ginger extract is said to be beneficial in attenuation of atherosclerosis

development, it is associated with down-regulating the macrophage-mediated oxidation

of LDL, as well as reduction in (i) uptake of oxidized LDL by macrophages, (ii)

oxidative state of LDL and (iii) LDL aggregation (Fuhrman et al., 2000). All these effects

lead to a reduction in accumulation of cellular cholesterol and foam cell formation, the

hallmark of early atherosclerosis. Ginger has hypolipidemic effect in rabbits and rat fed

foods containing cholesterol (Bhandari et al., 1998; Bhandari, 2005).

Incorporating ginger in rat’s diet significantly increased the activity of hepatic

cholesterol 7a-hydroxylase. This is a rate-limiting enzyme in the biosynthesis of bile

acids and stimulates the conversion of cholesterol to bile acids leading to the excretion of

cholesterol from the body (Srinivasan, 1991). The effect of ginger (Zingiber officinale

Rosc.) on blood lipids, blood sugar and platelet aggregation in patients with coronary

artery disease was studied by Bordia and verma (1997). Ginger was administered in the

capsule form in two different doses, 4 g daily for 3 months and 10g as a single dose. The

placebo was given to control group in the same dosage for the same duration. Initial two

blood samples were collected at an interval of 2 weeks. Patients were then administered

the spice or placebo for 3 months. They examined the lipid profile, fibrinogen,

fibrinolytic activity, platelet aggregation and blood sugar at 1.5 and 3 months. They

observed that powdered ginger given in a dose of 4 g daily did not affect Adenosine di

phosphate (ADP)- and epinephrine-induced platelet aggregation measured at 1.5 and 3

months of administration. In addition, no change in the fibrinolytic activity and

fibrinogen level was observed. A significant reduction in platelet aggregation was

48

observed after 4 h from administration of single ginger powder (Table 8). Also it was

reported that ginger did not affect the blood lipids and blood sugar when administered in

4 g daily dose for 3 months.

Table 8: Effect of single dose of powdered ginger on platelet aggregation in patients

with coronary artery disease

ADP Epinephrine

Initial After 4 h Initial After 4 h Placebo group (n = 10)

55.0 ± 3.4 57.8 ± 4.7 56.1± 7.6 58.6±7.5 P value NS NS

Ginger group (n = 10) 52.5± 5.7 38.7 ± 5.1 52.0± 9.5 40.0 ± 9.0

P < 0.05 < 0.05 Values (Mean± S.D.) are % platelet aggregation. Dose: 10 g.

Another study was undertaken to investigate an in-vivo effect of standardized ginger

extract on the development of atherosclerosis in apolipoprotein E-deficient (E0) mice, in

relation to plasma cholesterol levels and the resistance of their LDL to oxidation and

aggregation. Sixty, 6 weeks old E0 mice were divided into three groups and for 10 weeks

they were fed via drinking water with 1.1% alcohol. Placebo group I had no ginger

extracts, whereas experimental group II & III had 25 and 250 mg of ginger extract added

to alcohol and water base. There was a reduction in aortic atherosclerotic lesion areas by

44% (P < 0.01) in mice that consumed 250 µg of ginger extract/day. Reductions (P <

0.01) in plasma triglycerides and cholesterol (by 27 and 29%, respectively), in VLDL (by

36 and 53%, respectively) and in LDL (by 58 and 33%, respectively) was observed in

third group. These results were associated with a 76% reduction in cellular cholesterol

biosynthesis rate in peritoneal macrophages derived from the E0 mice that consumed the

high dose of ginger extract for 10 wk (P < 0.01). In addition, peritoneal macrophages

produced from E0 mice in group 2 and 3 had shown a lower (P < 0.01) capacity to

oxidize LDL (by 45 and by 60%, respectively), and to take up and degrade oxidized LDL

(by 43 and 47%, respectively). Daily consumption of 250 µg of ginger extract could

reduce the basal level of LDL-associated lipid peroxides by 62% (P < 0.01). In parallel,

49

there was a 33% inhibition (P < 0.01) in LDL aggregation (induced by vortexing) in mice

fed ginger extract. They concluded that dietary consumption of ginger extract by E0 mice

significantly attenuate the development of atherosclerotic lesions. This antiatherogenic

effect is related to a significant reduction in plasma and LDL cholesterol levels and a

significant reduction in the LDL basal oxidative state, as well as their susceptibility to

oxidation and aggregation (Fuhrman et al., 2000).

Bhandari et al (2005) evaluated the lipid lowering and antioxidant potential of ethanolic

extract of Zingiber officinale Roscoe in streptozotocin-induced rats. They fed 200 mg/kg

ethanolic extract of Z. officinale for 20 days to diabetic rats. They observed a significant

antihyperglycaemic effect (P < 0.01) in diabetic rats. The total cholesterol and

triglycerides were decreased and the high density lipoprotein (HDL)-cholesterol level

was increased in treatment group when compared with pathogenic diabetic rats

(P < 0.01). As compared to normal healthy control rats, streptozotocin-treatment also

exhibited a significant increase in liver and pancreas lipid peroxide levels (P < 0.01). The

liver and pancreas TBARS values (P < 0.01) were lower in treatment group as compared

to pathogenic diabetic rats. The results of test drug were comparable to a standard

antihyperglycaemic agent, gliclazide (25 mg/kg, orally). The results specify that ethanolic

extract of Zingiber officinale Roscoe can protect the tissues from lipid peroxidation. The

extract also showed a significant lipid lowering activity in diabetic rats.

Nammi et al (2008) treated rats with an ethanol extract of Zingiber officinale (400 mg⁄

kg) extract along with a high-fat diet. The extract of Zingiber officinale administered over

6 weeks to the rats fed a high-fat diet significantly decreased hepatic triglyceride and

tended to decrease hepatic cholesterol levels. They observed that in parallel, the extract

increased both LDL receptor mRNA and protein level and decreased HMG-CoA

reductase protein expression in the liver of these rats. The metabolic control of body lipid

homeostasis was said to be in part due to enhanced cholesterol biosynthesis and reduced

expression of LDL receptor sites following long-term consumption of high-fat diets. The

results showed restoration of transcriptional and post-transcriptional changes in low-

density lipoprotein and HMG CoA reductase by Zingiber officinale administration with a

high-fat diet and provide a rational explanation for the effect of ginger in the treatment of

hyperlipidaemia.

50

Lam et al. (2007) studied antioxidant actions of phenolic compounds found in dietary

plants on low-density lipoprotein and erythrocytes in vitro. LDL, erythrocytes and

erythrocyte membranes were subjected to several in vitro oxidative systems. They

assessed the antioxidant effect of the phenolic compounds and their abilities to inhibit

hemolysis and lipid peroxidation of LDL and erythrocyte membranes, and in protecting

adenine tri phosphatase activities (ATPase) and protein sulfhydryl groups of erythrocyte

membranes. Results revealed that 6-Gingerol and rhapontin showed strong inhibition

against lipid peroxidation in LDL induced by 2, 2'-azobis (2-amidinopropane)

hydrochloride and hemin whereas barbaloin showed weaker effects. On the lipid

peroxidation of erythrocyte membranes in a tert-butylhydroperoxide (tBHP)/hemin

oxidation system a similar order of antioxidant potencies among the three compounds

was observed. In contrast, barbaloin and rhapontin were comparatively stronger

antioxidants than 6-gingerol in preventing 2, 2'-azobis (2-amidinopropane)

hydrochloride-induced hemolysis of erythrocytes. Among the three compounds, only

barbaloin protected Ca2+-ATPase and protein sulfhydryl groups on erythrocyte

membranes against oxidative attack by tBHP/hemin. They observed that, under the same

experimental conditions, rhapontin showed protective actions on Na+/K+-ATPase in a

sulfhydryl group-independent manner. These studies results provide scientific evidence to

substantiate the traditional use of Z. officinale in preventing metabolic disorders.

5.8.2. Antibacterial activity

According to Azu et al. (2007), flavonoids are a second class of health enhancing

compound produced by onions, an example is quercetin. Flavonoids are chemical

compounds active against microorganisms. They have been found to be effective

antimicrobial substance against a wide array of microorganisms in-vitro (Ekwenye and

Elegalam, 2005). In vitro studies support that apart from antiradical activity, ginger

extract also posses antibacterial activity (Jirovetz et al., 2005).

Chen et al (2008a) studied antioxidant and antimicrobial activity of Zingiberaceae plants

in Taiwan. Eighteen species of five genuses of Zingiberaceae plants from Taiwan area

were collected and analyzed for their antibacterial activity. As presented in Table 9, most

of the Zingiberaceae plant extracts exhibited antimicrobial activity against all tested food

51

microorganisms. Only Hedychium and Vanoverberghia, did not show antimicrobial

activities on Escherichia coli and Vibrio parahaemolyticus.

Table 9: Antimicrobial activities of gingers, and amoxicillin–clavulanic acid,

ofloxacin

Species Escherichia

coli

Salmonella

enterica

Staphylococcus

aureus

Vibrio

parahaemolyticus

Alpinia japonica

(Thunb.) Mig 13a (0.05) 8 (0.05) 8 (0.08) 15 (0.05)

Alpinia kawakamii

Hayata 12.5 (0.05) 9 (0.05) 7 (0.05) 12 (0.05)

Alpinia

kusshakuensis Hayata 7 (0.05) 10 (0.08) 9 (0.05) 8 (0.05)

Alpinia mesanthera 9 (0.05) 10 (0.05) 13 (0.08) 11 (0.05) Alpinia officinarum 11 (0.05) – 5 (0.05) – Alpinia pricei Hayata 4 (0.05) 9 (0.05) 10 (0.05) – Alpinia shimadai 9 (0.08) 11 (0.05) 9 (0.05) – Alpinia uraiensis

Hayata 12 (0.05) 12 (0.05) 14 (0.08) –

Alpinia zerumbet

(Pers.) Burtt & Smith 6 (0.05) 7 (0.08) 9 (0.05) 8 (0.05)

Costus speciosus

(Koenig) Smith 11 (0.05) 8 (0.05) 11 (0.05) –

Curcuma domestica 15 (0.05) 17 (0.05) 20 (0.05) 18 (0.05) Curcuma longa L. 17 (0.05) 6 (0.05) – 13 (1.0) Curcuma viridiflora 15.6 (0.05) 19 (1.0) 18 (0.05) 20 (0.05) Curcuma zedoaria 16 (0.05) 13.5 (0.05) 11 (0.05) – Hedychium

coronarium Koenig - 9 (0.05) 14 (0.05) –

Vanoverberghia

sasakiana - 8 (0.05) 9 (0.05) –

Zingiber kawagoii

Hayata 11 (0.05) 9 (0.02) 11 (0.05) 8 (0.05)

Zingiber

oligophyllum K. 9 (0.08) – 6.5 (0.05) 8 (0.08)

Schumann 20 18 15 15 Ofloxacinc 18 (parenthesis indicates minimum inhibitory concentration (mg/ml) for antimicrobial activity of Zingiberaceae extracts) a Diameter of zone (mm) b Amoxicillin-clavulanic acid (20 µg /1 ml per disc) c Ofloxacin (20 µg /1 ml per disc)

52

Malu et al. (2009) illustrated the antibacterial activity of ginger root in different extracts.

Results revealed that ginger roots extracts, viz. hexane, ethyl acetate and soxhlet extracts

had antibacterial activities on coliform bacillus, staphylococcus epidermidis and

streptococcus viridians but water extract did not show antibacterial activity. The results

may suggest that hexane; ethyl acetate and soxhlet extract of ginger root could be potent

against bacterial infections while the water extract of ginger roots could be ineffective.

They also studied the zone of inhibition of ginger on these bacteria. Results are presented

in Table 10.

Table 10: Inhibition of bacterial growth by the ginger extracts

Test organism

Dilution (%)

Zone of inhibition (mm)

n-hexane Ethyl

acetate Soxhlet Water

Coliform bacillus

1.00 4.0 5.0 5.5 - 0.50 1.5 2.5 3.0 - 0.25 - - - -

0.125 - - - -

Straphylococcus

Epidermidis

1.00 4.5 5.5 6.5 - 0.50 2.5 3.5 4.0 - 0.25 - 1.0 2.5 -

0.125 - - - -

Streptococcus

viridans

1.00 5.0 5.6 7.0 -

0.50 3.0 4.0 4.5 - 0.25 - - - -

0.125 - - - -

Azu and Onyeagba (2007) studied the antimicrobial properties of various extracts of

Allium cepa (onions) and Zingiber officinale (ginger) against Escherichia coli,

Salmonella typhi and Bacillus subtilis. These bacterias are common cause of

gastrointestinal tract infections so the researchers investigated the antibacterial properties

of these 2 commonly used spices, with the use of cup-plate diffusion method. They

observed that ethanolic extract of ginger gave the widest zone of inhibition against two

out of the three test organisms at the concentration of 0.8 gml-1. Result revealed that the

extraction solvent and its different concentrations affected the sensitivity of two of the

test organisms to the plant materials. The minimum inhibitory concentration of ginger

53

extracts on the test organisms ranged from 0.1gml-1 - 0.2 gml-1, showing that ginger was

more effective and produced significant inhibitory effect on the two out of the three test

organisms in comparison to the onion extracts. This investigation indicates that, though

both plants had antimicrobial activities on the two gram negative test organisms but not

effective on the gram positive test organism, ginger had more inhibitory effect thus

confirming their use in folk medicine.

The effect of ginger on diabetic nephropathy, plasma antioxidant capacity and lipid

peroxidation in rats was examined by Afshari et al. (2007). Wistar rats were divided into

3 groups (n =8) of non-diabetic, diabetic non-treated and diabetic rats treated with ginger

powder. Rats weighing 250±20 g were treated with streptozotocin 60 mg/kg. The diabetic

treated with ginger group was fed with ginger at 5% of their daily consumed food. The

rats were anaesthetized after 8 weeks with 10% chloral hydrate.

Blood samples were collected from the heart of each rat and kidneys were removed and

kept in 10% formalin buffer. Plasma and red blood cells were separated. They evaluated

the plasma antioxidant capacity by the FRAP method and red blood cells

malondialdehyde (MDA) as an indicator of lipids peroxidation. For renal samples

sections were taken, stained and were studied for focal cell proliferation and glomerular

and tubular structural changes. The MDA levels in diabetic rats treated with ginger were

significantly lower than in the other groups (P < 0.01). Plasma antioxidant capacity in

ginger treated rats was more compared to the first two groups. Diabetes induced

nephropathies were also lower in the ginger treated group.

This study demonstrated that ginger causes a reduction in lipid peroxidation, a rise of

plasma antioxidant capacity and a decrease in renal nephropathy.

5.8.3. Weight gain

Although, since a long time ago the digestion stimulating effect of this spice became

known, the stimulating effect on peptic juices, such as gastric juice, bile, pancreatic and

intestinal juices, was discovered later. Bile acids is an enzyme which play a major role in

the uptake of fats and each upset in the metabolism of fats would slow down food

digestion as a whole, because the fatty particles cover the other food elements and make

them inaccessible for the action of the digestive enzymes. Lipase is the other key factor

54

which plays a very important role in fat digestion. It was found that when ginger was

included in animal diets, there was a considerable increase in the pancreatic and intestine

lipase (Platel and Srinivasan, 2000).

David et al. (2007) studied the effect of a Chinese herbal extract on body weight of rats.

The Chinese herbal extract Number Ten (NT) is a dietary herbal formulation prepared

from rhubarb, ginger, astragalus, red sage and turmeric. This study tested the

effectiveness of NT in reducing body weight gain in rats. In the study, sixty female wistar

rats were fed a high fat diet and adapted to gavage feeding. Animals were divided into

five treatment groups: (1) Control (n = 15); (2) NT-H (high) (n = 15), 1.5 g/day; (3) NT-L

(low) (n = 10), 0.75 g/day; (4) Pr-fed (n = 10), pair fed to NT-H; (5) d-FF (n = 10), d-

fenfluramine 2 mg/kg. In each group ten rats were sacrificed on day 56. Weight, food

intake, clinical chemistry and body composition were evaluated. In the control five

animals and 1.5 g/day NT groups were left untreated during a two week recovery period.

They demonstrated that the 0.75 g/day NT, 1.5 g/day NT, d-fenfluramine and pair fed

groups gained 24.6%, 33.3%, 12.3% and 33.3% less than the control respectively (P <

0.0006). Leptin hormone reduced 27.5% to 46.2% in the treatment groups vs. control (P

< 0.009). Parametrical fat reduced 14.1% to 55.5% in the NT and pair fed groups vs.

control (P < 0.006). It is also observed that NT groups had soft stools, loss of hair around

the mouth and coloration to the urine and stool without evidence of blood or bilirubin

(attributed to chromogens in NT). There were no differences between groups in the

clinical chemistry. This study concluded the efficacy of NT in reducing weight gain in

rodents.

5.8.4. Anti-inflammatory action

It is hypothesized that Prostaglandins and other eicosanoids influence carcinogenesis

through action on nuclear transcription sites and downstream gene products important in

the control of cell proliferation. Non-steroidal anti-inflammatory drugs, potent inhibitors

of cyclooxygenase (COX), the enzyme responsible for prostaglandin synthesis, are

associated with reduced risk of several cancers (Wargovich et al., 2001). Thus, natural

products, including spices, have been examined for their capacity to inhibit COX or other

55

parts of the inflammation pathway. Ginger has been reported to interfere with

inflammatory processes (Ozaki Y et al., 1991).

Thomson et al. (2002) studied the use of ginger (Zingiber officinale Rosc.) as a potential

anti-inflammatory and antithrombotic agent. The effect of an aqueous extract of ginger

(Zingiber officinale) on serum cholesterol and triglyceride levels as well as platelet

thromboxane-B2 and prostaglandin-E2 production was studied. For a duration of 4 weeks

a raw aqueous extract of ginger was administered daily, either orally or intraperitoneally

(IP) to rats. Fasting blood serum was investigated for thromboxane-B2, prostaglandin-E2,

cholesterol and triglycerides. A low dose of ginger (50 mg/kg) administered either orally

or IP did not cause any significant reduction in the serum thromboxane-B2 levels when

compared to saline-treated animals. However, ginger administered orally caused

significant changes in the serum PGE2 at this dose. High doses of ginger (500 mg/kg)

were significantly effective in lowering serum PGE2 when given either orally or IP.

However, TXB2 levels were significantly lower in rats given 500 mg/kg ginger orally but

not IP. A significant reduction in serum cholesterol was observed when a higher dose of

ginger (500 mg/kg) was given. At a low dose of ginger (50 mg/kg), only when ginger was

administered IP, a significant reduction in the serum cholesterol was observed. There

were no significant changes in serum triglyceride levels upon administration of either the

low or high dose of ginger. These results suggest that ginger could be used as a

cholesterol-lowering, antithrombotic and anti-inflammatory agent.

Tripathi et al. (2007) studied the inhibition of ginger extract on Lipopolysaccharide

induced macrophage activation and function. Production of proinflammatory cytokines

and chemokines were observed after stimulation of murine peritoneal macrophages by

Lipopolysaccharidein presence and absence of ginger extract. They also observed the

effect of ginger extract on the Lipopolysaccharide induced expression of MHC II, B7.1,

B7.2 and CD40 molecules. They also studied the antigen presenting function of ginger

extract treated macrophages by primary mixed lymphocyte reaction. It was observed that

in Lipopolysaccharide stimulated macrophages ginger extract inhibited IL-12, TNF-α, IL-

1β (pro inflammatory cytokines) and RANTES, MCP-1 (pro inflammatory chemokines)

production. Ginger extract also down regulated the expression of B7.1, B7.2 and MHC

class II molecules. Additionally ginger extract negatively affected the antigen presenting

56

function of macrophages and when ginger extract treated macrophages were used as

antigen presenting cells, they observed a significant reduction in T cell proliferation in

response to allostimulation. A significant decrease in IFN-γ and IL-2 production by T

cells in response to allostimulation was also seen. In conclusion they reported that, ginger

extract inhibits macrophage activation and antigen presenting cell function and indirectly

inhibits T cell activation.

5.9. TOXICITY STUDY

Ginger (Zingiber officinale Roscoe, Zingiberacae) is one of the most commonly used

spices around the world and a traditional medicinal plant that has been widely used in

Chinese, Ayurvedic and Unani-Tibb medicines for several thousand years. This spice is

listed as GRAS (generally recognized as safe) by food and drug administration (FDA)

(FDA, 2011).

In a research conducted by Rong et al. (2009) a 35-day toxicity of ginger in rats was

evaluated. Ginger powder at the dosages of 500, 1000 and 2000 mg/kg body weight by a

gavage method for 35 days was given daily to both male and female rats. The results

revealed that this chronic administration of ginger was not associated with any mortalities

and abnormalities in general conditions, behavior, growth, and food and water

consumption. Except for dose-related decrease in serum lactate dehydrogenase activity in

males, there was no significant difference in hematological and blood biochemical

parameters between ginger treatment group and controlled animals. In general, ginger

treatment did not cause evident organ abnormality. Only at a very high dose

(2000 mg/kg), ginger led to slightly reduced absolute and relative weights of testes (by

14.4% and 11.5%, respectively).

57

6.1. BOTANICAL INFORMATION

Lime (C. aurantifolia) is a fruit belonging to citrus sp. It is greenish yellow color, 5 - 10

cm in size, round shaped, with sour taste and fibrous texture (Ghafar et al., 2010).

6.2. HISTORY OF USE

Traditionally Citrus aurantifolia is used against menstrual disorder, diarrhea and

dysentery. It has been reported that lime lowers cholesterol level of the blood and can

cure ulcer (Okwu and Emenike, 2006).

6.3. NUTRITIONAL COMPOSITION

Though citrus fruits are well known as source of ascorbic acid and folic acid, they are

good sources of fiber also. Citrus are fat free, low in sodium and without cholesterol

(Economos and Clay, 1999).

Nutritional composition of lime is reported by many researchers. It has been reported that

lime fruit contain alkaloids- 0.33, flavonoids- 0.29, tannin- 0.04, phenols- 0.02, saponins-

0.22, vitamin C, 22.88 and thiamin, 0.11 mg/100g (Okwu and Emenike, 2006) . Lime is

composed of dry matter, 92.31; crude protein, 12.25; ether extract, 3.56; crude fiber, 3.14

and ash, 3.93% (Belewu et al., 2009). In a phytochemical screening, Belewu et al (2009)

found alkaloids and tannin in lime. Patil et al. (2009b) reported 22 volatile compounds

which represent more than 89.5% of the volatile oil in Citrus aurantifolia. These volatile

oil were reported as a-Pinene, 2,3-Dehydro-1,8-cineole, Camphene, p-Cymene, m-

cymene, m-Mentha-6,8-diene R(+), D-Limonene, b-cis-Ocimene,Trans-p-mentha-2,

8dienol, b-Linalool, Fenchol, P-Menth-8-en-1-ol, D-Verbenone, a-Terpineol , D-

Dihydrocarvone, Myrtenol, c-Terpineol, Verbenone, d-Elemene, Neryl acetate,

Caryophyllene, trans-a-Bergamotene. D-limonene (30.13%) and D-dihydrocarvone

(30.47%) are two major compounds in the lime volatile oil.

Rich dietary fiber powders from Persian lime peels were prepared and their dietary fiber

composition and antioxidant abilities were assessed. The total dietary fiber contents was

high (70.4%), with a suitable ratio of soluble/insoluble fractions. The water holding

capacities of dietary fiber concentrate was found to be high (6.96g of water/g of dietary

fiber). The water holding capacities was related to the soluble dietary fiber. Three

58

methods (azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ABTS radical-scavenging

activity, a,a-diphenyl–picrylhydrazyl (DPPH) and β-carotene-linoleic acid) was used for

antioxidant activity and total extractable polyphenols. Polyphenol content of dietary fiber

concentrates of Persian lime peel was found to be 19.90 mg/g. The polyphenols

associated with the lime dietary fiber peel showed a good antioxidant activity. From a

nutritional standpoint, dietary fiber lime concentrates may be appropriate to use as natural

food additives (Ubando-Rivera et al., 2005).

6.4. ANTIOXIDANT COMPONENTS

It has been reported that the solid parts of citrus fruit, particularly the albedo (the white

spongy portion) and the membranes separating the segments, are very rich in flavanone,

so that the whole fruit may contain up to 5 times as much flavanone as a glass of orange

juice (Manach et al., 2004).

6.5. IN VITRO ANTIOXIDANT STUDIES

Total phenol content and antioxidant activity by 3 different methods (TBARS, DPPH

scavenger free radical activity and the iron chelation capacity) was studied in boiling

water extract of lime. The total phenolic compound of Citrus aurantifolia was reported

as 404.24 mg GA/100g, percent antioxidant activity 42.6 – 88.3% at concentration of 3.5,

6.9, 10,13.5 and 16.9 mg/ml, Fe+2 chelating ability 61-83% at the same concentration and

DPPH 15.4-73.8% at 2.5, 5,12.5, 25 and 50 µg/ml (Ahmed and Rocha, 2009).

Calliste et al. (2001) reported that phenolic acids and their glycosides, aglycones, and

monoglycosyl or diglycosyl flavonoids can be solubilized in different solvents as a

function of polarity and water extracts contain the most polar compounds. Hence, water

extracts of Phoenix dactylifera, Citrus aurantifolia have strong scavenging and

antioxidant activity.

The total polyphenol, flavonoids and hisperidine contents of lime juice was reported as

211.70 mgGAE/100 ml of juice, 10.67 mg of hesperidine equivalent/ 100 ml of juice and

16.67 mg/100ml of juice respectively (Ghafar et al., 2010).

Patil et al (2009a) studied bioactive compounds from Mexican lime (Citrus aurantifolia)

juice with induced apoptosis in human pancreatic cells. Freeze-dried lime juice was

59

extracted with different extracting media, such as chloroform, acetone, methanol, and

methanol/water (8:2). It was observed that the chloroform extract showed the highest

(85.4 and 90%) radical-scavenging activity by DPPH and 2,20-azino-bis (3-

ethylbenzthiazoline-6-sulfonic acid) methods at 624 µg/ml, whereas the methanol/water

extract showed the lowest (<20%) activity. The active components were identified with

the help of HPLC using a C-18 column as rutin, neohesperidin, hesperidin, and

hesperitin. In addition, the limonoids identified were limonexic acid, isolimonexic acid,

and limonin. All of the lime juice extracts inhibited Panc-28 cancer cell growth. The

methanol extract exhibited the maximum activity, with an IC50 value of 81.20 µg/ml

after 72 h. The inhibition of Panc-28 cells was found to be in the range of 73-89%, at 100

µg/ml at 96 h. The involvement of apoptosis in initiation of cytotoxicity was established

by expression of Bax, Bcl-2, casapase-3, and p53. The results of the present study

evidently specify that antioxidant activity is proportionate to the content of flavonoids

and proliferation inhibition ability is proportionate to the content of both flavonoids and

limonoids.

6.6. NUTRITIONAL AND MEDICINAL PROPERTIES

6.6.1. Antibacterial activity

Onyeagba et al. (2004) studied antibacterial activity of fresh lime (Citrus aurantifolia

Linn.) juice against Staphylococcus aureus; Bacillus spp., E. coli and Salmonella spp.

Results revealed that lime has antibacterial properties against all these tested bacteria. It

was observed that the zone of inhibition in Staphylococcus aureus and Bacillus spp. is 17,

E. coli 11 and Salmonella spp. 13mm. It is demonstrated that the effect of lime juice on

the gram-positive organisms is higher than gram-negative organisms. Oboh et al. (1992)

and Oboh and Abulu (1997) investigated antibacterial activity of lime leaves and found

similar results.

An antimicrobial activity of essential oils and crude extracts from tropical Citrus spp.

against food-related microorganisms was studied by Chanthaphon et al. (2008). In

present research to achieve essential oil, citrus peels (500 g) were subjected to

hydrodistillation for duration of 4 hours. It was dried over anhydrous sodium sulfate and

stored under N2 in sealed vials at 4°C. Ethyl acetate extracts were attained by grinding

60

500 g of citrus peels to fine powder, then it was soaked in 2 liter of ethyl acetate and

shaken at the speed of 130 rpm for 8 h, the extract was filtered (Whatman No. 4) and

dried over anhydrous sodium sulfate. With the help of rotary-vacuum evaporator, ethyl

acetate was eliminated totally by nitrogen evaporator to yield dry ethyl acetate extracts,

which were stored under N2 in sealed vials at 4°C. The antibacterial activity in different

extracting media and MIC, minimum bacterial concentration (MBC) and minimum

fungicidal concentration (MFC) are presented in Table 11.

Table 11: MIC, MBC and MFC (mg/ml) of crude extracts from lime prepared by

ethyl acetate extraction and hydrodistillation against Gram-positive bacteria, mold

and yeast

Extraction methods

Microorganisms MIC & MBC

Con. (mg/ml)

Mold & yeast MIC & MFC

Con. (mg/ml)

Ethyl acetate B. cereus MIC MBC

0.56 0.56

Sac.

Cerevisiae

var. sake

MIC MFC

0.56 0.56

Hydrodistillation MIC MBC

>2.25 >2.25

MIC MFC

>2.25 >2.25

Ethyl acetate S. aureus MIC MBC

1.13 1.13

A. fumigates

TISTR 3180 MIC MFC

2.25 2.25

Hydrodistillation MIC MBC

>2.25 >2.25

MIC MFC

>2.25 >2.25

Ethyl acetate L.

monocytogenes MIC MBC

1.13 2.25

Salmonella

sp. MIC MFC

>2.25 >2.25

Hydrodistillation MIC MBC

>2.25 >2.25

MIC MFC

>2.25 >2.25

Ethyl acetate -

- - - E. coli O157: H7 DMST 12743

MIC MFC

>2.25 >2.25

Hydrodistillation - - - MIC MFC

>2.25 >2.25

MIC- Minimum inhibitory concentration MFC- Minimum fungicidal concentration MBC- Minimum bacterial concentration

Antimicrobial activity of ethyl acetate extracts at 55°C for 48 hours was assessed by disk

diffusion assay and zone of inhibition were measured and expressed as mm (Table 12).

61

Table 12: Antimicrobial activity of ethyl acetate extracts at 55°C

Organism

Fresh peel Dried peel

Concentration (µg) Concentration (µg)

25 50 100 200 25 50 100 200

Staphylococcus aureus 10.0 10.5 9.5 12.0 6.0 7.5 7.5 8.5

Escherichia coli Nil 5.0 9.0 10.5 Nil Nil Nil Nil

(Chanthaphon et al., 2008)

In a study conducted by Camacho-Corona et al (2008) the activity of lime (a traditional

plant in Mexico used for respiratory track disease and as antiseptic), against drug

resistant-tuberculosis was studied. The peel of Citrus aurantifolia was extracted in

hexane, chloroform, methanol, aqueous and antibacterial activity of extract was studied

(in-vitro). It has been shown that only hexane extracts of lime were active against the

pan-sensitive strain M. tuberculosis H37Rv. Results revealed that RIF, INH, STR and

EMB resistance strain showed 200, >200, 25, 50 and 25 µg/ml inhibition respectively.

Evaluation of the antimicrobial properties of different parts of Citrus Aurantifolia (lime

fruit) as used was reported by Aibinu et al. (2007). The lime fruits (unripe) were dried at

50°C for whole week until whole fruits were completely dried and burnt. This is another

traditional way of using lime fruit and traditionally it is believed that it is a healing herbal

medicine. Dry lime powder, lime fruit oil and fresh lime juice were tested for its

antibacterial properties. Dry lime powder extract was taken in 5 different extract (distilled

water, ethanol, palm-wine, Seamann’s Schnapps and fermented water. All extracts were

examined for purity by plating them on nutrient agar and incubated for 24 hours at 37 oC

and 25 oC. MIC of isolates to Citrus aurantifolia (Dry lime fruit) extracts is shown in

Table 13 and zones of inhibition (mm) of organisms to Citrus aurantifolia extracts at neat

and stock concentration of 512 mg/ml is shown in Table 14.

62

Table 13: Minimum inhibitory concentration (MIC) of isolates to Citrus aurantifolia

(Dry lime fruit) extracts (mg/ml)

Test Organisms PWE SE EE AQE JUICE OOE Gram-Positive Organisms Staphylococcus aureus ATCC 25213 32 64 64 256 256 128 Staphylococcus aureus (1) 32 32 64 128 64 128 Enterococcus faecalis 128 64 256 256 256 128 Gram-Negative Organisms Escherichia coli ATCC 25922 128 64 256 256 256 256 Salmonella paratyphi 64 256 256 256 256 256 Citrobacter spp 512 128 256 256 256 256 Serratia spp 64 512 64 256 128 256 Shigella flexnerii 256 256 256 256 64 128 Klebsiella pneumoniae 256 256 256 256 128 128 Pseudomonas aeruginosa 128 128 256 256 256 128 Escherichia coli 128 64 128 256 256 256 Fungi Candida albicans 256 256 256 512 256 512 Aspergilus niger - - - - - - Anaerobes Bacteroides spp 64 128 128 128 128 128 Clostridium spp 32 32 64 64 128 128 Porphyromonas spp 32 32 64 64 128 128 SE=Schnapps extract; Juice=Lime juice; PWE=Palm wine extract; EE=Ethanol extract AQE=Aqueous extract; OOE=Omi-ogi extract

Melendez and Capriles (2006) evaluated the antibacterial activity of Citrus aurantifolia

fruit against several bacteria. Zone of inhibition for different bacteria were reported as

follow, E.coli 20, S. aureus 30, A. faecalis 25, E. aerogenes 16, E. cloacae 45, P.

fluorescens 43, P. vulgaris 16, S. marcescens 17, A. globiformis 35, B. cereus 23, B.

coagulans 24, B. subtilis 20, M. luteus 37, M. roseus 0, M. phlei 24, M. rodochrus 25, M.

smegmatis 21mm.

63

Table 14: Zones of inhibition (mm) of organisms to Citrus aurantifolia extracts at

neat and stock concentration of 512mg/ml

Test Organisms CIP OIL.E (Neat)

PWE SE EE AQE JUICE (Neat)

OOE

Gram-Positive Organisms Staphylococcus aureus

ATCC 25921 20 30 26 24 22 22 24 24

Staphylococcus aureus

(1) 20 40 30 28 35 33 22 25

Enterococcus faecalis 16 25 26 22 20 24 20 20 Gram-Negative Organisms Escherichia coli ATCC

25922 27 30 19 25 14 16 17 19

Escherichia coli 27 25 20 20 15 20 14 15 Shigella flexnerii 33 30 19 19 17 21 21 20 Salmonella paratyphi 30 40 17 19 12 21 9 18 Citrobacter spp 30 19 17 18 16 22 12 18 Serratia spp 25 17 24 21 24 17 13 23 Pseudomonas

aeruginosa 17 13 18 24 20 19 14 15

Klebsiella pneumoniae 30 19 16 18 16 15 17 17 Fungi Aspergillus niger - 48 40 0 0 0 0 0 Candida albicans - 40 30 30 30 15 32 18 Anaerobes Porphyromonas spp 26 27 23 22 18 18 21 15 Clostridium spp 27 31 28 25 23 20 22 15 Bacteroides spp 24 30 21 21 20 18 20 18 Oil .E=Oil extract; SE=Schnapps extract; PWE=Palm wine extract; EE=Ethanol-extract; AQE=Aqueous extract; OOE=Omi-ogi extract Juice=Lime juice CIP=Ciprofloxacin

Patil et al (2009a) reported that D-dihydrocarvone volatile oil of lime at 100 µg/ml

concentration for 48 h showed 78% inhibition of human colon cancer cells (SW-480). It

was observed that after 24 h and 48 h lime volatile oil showed DNA fragmentation and

induction of caspase-3 up to 1.8 and 2- folds respectively, which may be due to the

involvement of apoptosis. Analysis of apoptosis-related protein expression further

confirmed apoptosis induction by lime volatile oil. These results suggest that lime

volatile oil have potential benefits in prevention of colon cancer.

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7.1. BOTANICAL INFORMATION

Shallots (Allium ascalonicum) possibly originated in Central or Southeast Asia, traveled

from there to India and the eastern Mediterranean. The name "shallot" comes from

Ashkelon, an ancient Philistine city (McKean, 2005). Shallot leafs are long and hollow

and flowers are red or violet colored. Often the upper parts of leaf head are thrown out.

But these parts are vitamin rich and we can be used for salad (Moghaddasi, 2011).

7.2. HISTORY OF USE

Shallot (Allium ascalonicum L.), is a major component of many Asian diets and is

believed to be beneficial to health. This bulb is darker than garlic and has a stronger odor

that correlates with its sulfide content (Mubarak and Kulatilleke, 1990).

7.3. NUTRITIONAL COMPOSITION

Organosulfur compounds are the main constituents of allium vegetables which are

present in all allium vegetables and flavonoids are abundantly present in onion. Thus, it

can be considered that the medicinal properties of the allium families may be due to the

presence of these chemical groups which would be of great importance in relation to

cancer (Sengupta et al., 2004).

7.4. ANTIOXIDANT COMPONENTS

Analysis of shallot extracts has confirmed the existence of flavone and polyphenolic

derivatives for instance quercetin, quercetin 4'-glucoside, quercetin 7,4'-diglucoside,

quercetin 3,4'-diglucoside, and quercetin mono-D-glucose, suggesting that it also may

have antioxidant properties (Kiviranta et al., 1988; Leighton et al., 1992; Fattorusso et al.,

2002). Antioxidant activities are directly linked to the contents of phenolic compounds

and it is observed that it is significantly more potent in fresh freeze-dried extracts than

commercial preparations. Hexane extracted shallot and garlic is reported to have higher

antioxidant activity, followed by water extracts, bulb pressings, and commercial products.

Fresh shallot and garlic preparations inhibited lipid oxidation and accelerated their

decomposition but did not exhibit, any effect on protein oxidation. Organic solvent and

aqueous extracts of garlic and shallot bulbs have shown significant antioxidant potential,

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as measured by decreases in free radicals and an ability to inhibit lipid oxidation

(Leelarungrayub et al., 2006).

Flavonoids are effective antioxidants due to their capability to scavenge free radicals of

fatty acids and oxygen. One of the richest sources of flavonoids in human diet is reported

to be common onion (Allium cepa L.) and shallot (Allium ascaloni L.) (Kopec and

Minarova, 1985). Onion and shallot’s flavonol content noticeably reduces atherosclerotic

processes, inhibits accumulation of cholesterol in the blood serum and increases vascular

walls resistance. It is known that flavonoids reduce risk of coronary heart disease.

In onion quercetin di- and triglycosides: 3,4´-O-β-D-diglucoside, 7,4´-O-β-D-diglucoside,

3,7-O-β-D-diglucoside, 3-O-sophoroside-7-O-β-D-glucuronide, 3,7,4´-O-β-D-

triglucoside and rutin were found (Fossen et al., 1998). Quercetin-monoglycosides

spiraeoside (4´-O-β-D-glucoside), 3-O-β-D-glucoside, 3´-O-β-D-glucoside, and 7-O-β-D-

glucoside are very highly evidented (Tsushida and Suzuki, 1995; Ioku et al., 2001). In a

minor amounts kaempferol-glycosides are also present (3,4´-O-β-D-diglucoside, 7,4´-O-

β-D-diglucoside, 3-O-sophoroside-7-O-β-D-glucuronide, 4´-O-β-D-glucoside). Only in

yellow and red cultivars of onion, another type of flavonols – isorhamnetin – is present in

the free form and in the bound form in glycosides like; 3,4´-O-β-D-diglucoside, 4´-O-β-

D-glucoside and 3-O-β-D-glucoside (Park and Lee, 1996). β-D-glucopyranoside of

dihydroflavonol type is a minor compound. Red onion cultivars contain the highest

contents of flavonoids, posses other Anthocyanins like peonidin-3-O-β-D-arabinoside

and peonidin-3-β-D-glucoside, pelargonidin-3-O-β-D-glucoside and three cyanidin-

glycosides like; 3-O-β-D-glucoside, 3-O-β-D-glycoside and 3-O-β-D-laminariobioside

(3-O-β-D-glucopyranosyl-D-glucoside). Malonylesters of some of these glycosides, such

as cyanidin-3-O-malβ-D-glucoside and 3-O-malonyllaminariobioside and peonidin-3-O-

malonyl-β-D-glucoside are compounds which were reported in last decade. As for this

rich flavonoid content, the proportions of quercetin-4´-O-β-D-glucoside and spiraeoside

are highest. Yellow and red onion cultivars were reported to have 60–1,000 mg/kg

flavonoids. Primary flavonoids identified in shallots are quercetin-4´-O-β-D-glucoside

and free quercetin. The other groups of polyphenolic compounds in onion are

phenolcarboxylic acids, such as protocatechuic, caffeic, ferulic, p-coumaric, p-

hydroxybenzoic and vanillic acid. These acids are represented both in the free and bound

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form as 1-O-β-D-glucosides, methyl esters or bound to phloroglucinol units. Among

other polyphenols catechol and phloroglucinol are stated. Onion and shallot also contain

significant amount of amino acid (Lachman et al., 2003). Major polyphenols in onions is

presented in Table 15.

Table 15: Content of major polyphenols in onion

Compound

Content (mg/kg DM)

Proportion in total polyphenol content

(%) Quercetin 48,000 63.36 Protocatechuic acid 11,020 14.52 Spiraeoside (quercetin-4´-O-β-D-glucoside) 10,650 14.03 Quercetin-3,4´-O-β-D-diglucoside 3,650 4.81 Tyrosine 1,725 2.27 Vanillic acid 228 0.34 Quercetin-7,4´-O-β-D-diglucoside 160 0.21 p-Hydroxybenzoic acid 258 0.14 Total amount of major polyphenols 75,910 99.68

Quercetin and other flavonoids have been shown to affect the biosynthesis of eicosanoid

(antiprostanoid and anti-inflammatory responses), protect low-density lipoprotein from

oxidation (prevent atherosclerotic and formation of plaque), prevent platelet aggregation

(antithrombic effects), and encourage relaxation of cardiovascular smooth muscle

(antihypertensive, antiarrhythmic effects).

In addition, flavonoids have been shown to have antiviral and carcinostatic properties.

However, flavonoids are poorly absorbed from the gut and are subject to degradation by

intestinal micro-organisms. The quantity of quercetin that remains biologically available

may not be of adequate concentration, theoretically, to explain the beneficial effects seen

with the Mediterranean diet. The function of flavonoids may transcend their presence in

food. The activity of flavonoids as inhibitors of reverse transcriptase recommends a place

for these compounds in the managing of retrovirus infections, such as acquired

immunodeficiency syndrome. In addition to specific effects, the broad-modulating effects

of flavonoids as antioxidants, inhibitors of many enzymes like ornithine carboxylase,

protein kinase and calmodulin, and promoters of vasodilatation and platelet

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disaggregation can be used as starting material for development drugs (Formica and

Regelson, 1995).

7.5. IN VITRO ANTIOXIDANT STUDIES

Ismail et al. (2004) studied total antioxidant activity and phenolic content in selected

vegetables. They examined the effect of thermal treatment on total antioxidant activity

and total phenolic content of some vegetables. They found that there is a significant

reduction of total phenolic content in shallot after thermal treatment whereas total

antioxidant activity did not show significant difference.

Yin and cheng (1998) studied the antioxidant activity of several members of the Allium

family with liposome model. Allicin formation of these plants, the effect of heat, pH, and

salt on the antioxidant activity of these foods was studied.

He reported the water content and pH value of shallot bulb to be 77.8% and 6.44

respectively and allicin yield from shallot to be 2.87 mg/g of fresh weight. Effect of heat-

treated allium foods on lipid oxidation (TBA-No.) after 36 h incubation was reported to

be 0.053 at 25°C, 0.093 at 65°C and 0.175 at 100°C. Effect of acid or base-treated allium

foods on lipid oxidation at pH 2, 4, 6 and 8 after 36 h of incubation was reported to be

0.172, 0.086, 0.063 and 0.076. Effect of Heat pH-treated shallot bulb on lipid oxidation

after 36 h incubation at 65°C in pH 2,4 and 6 was determined as 0.197, 0.137, 0.089

respectively and at 100°C in pH 2,4 and 6 was 0.293, 0.209, 0.182 respectively. Effect of

salt-treated shallot bulb on lipid oxidation after 36 h incubation was 0.053, 0.052 and

0.049 at 0, 0.2 and 0.4 M solution of NaCl respectively.

7.6. NUTRITIONAL AND MEDICINAL PROPERTIES

7.6.1. Antibacterial activity

Antimicrobial properties of extracts of Allium cepa (onions) and Zingiber officinale

(ginger) on E.coli, Salmonella typhi and Bacillus subtilis was studied by Azu and

Onyeagba (Azu and Onyeagba, 2007). The antimicrobial properties of various extracts of

Allium cepa (onions) and Zingiber officinale (ginger) against Escherichia coli,

Salmonella typhi and Bacillus subtilis that are common cause of gastrointestinal tract

infections were examined with the cup-plate diffusion method. E. coli and Salmonella

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typhi were found to be more sensitive to the extract of onion bulbs compared to Bacillus

subtilis which was predominantly resistant. It was also observed that the solvent of

extraction and its varying concentrations affected the sensitivity of two of the test

organisms to the plant materials.

The antifungal effects of fresh crude juice of shallot were determined against 11 isolates

of Candida albicans, three species of dermatophytes (Microsporum gypseum,

Trichophyton mentagrophytes and Epidermophyton floccosum) and Syncephalastrum,

Aspergillus niger, Penicillium sp., Paecilomyces sp., Scopulariopsis sp., Cladosporium

sp., Alternaria sp., Drechslera sp. by agar well diffusion method (Mahmoudabadi and

Nasery, 2009). MIC of the fresh extract of A. ascalonicum was reported to be 0.25% for

most tested fungi; however extract showed remarkable activity against saprophytic fungi

followed by Candida species and dermatophytes. It is concluded that fresh extract of A.

ascalonicum has more anti-saprophytes effect at 0.25% with a mean diameter of

inhibition zone 21.83 mm.

In a research, scientists focused on beneficial effect of shallot (Allium ascalonicum L.)

extract on cyclosporine nephrotoxicity in rats. Male Wistar rats were treated orally with

vehicle, immunosuppressive cyclosporine A (CsA) (25 mg/kg), shallot extracts (1 g/kg),

and CsA plus shallot extract for 21 days. Renal function, histopathology, tissue MDA and

GSH levels 24 h after the last treatment were estimated. CsA-induced nephrotoxicity was

evidenced by increased blood urea nitrogen and serum creatinine, but reduced urea and

creatinine clearance. The kidney of CsA treated rats exhibited severe vacuolations and

tubular necrosis. CsA also induced oxidative stress, as indicated by increased renal MDA

and reduced GSH concentrations. Administration of shallot extract along with CsA

counteracted the deleterious effects of CsA on renal dysfunction, oxidative stress

markers, and morphological changes. These data specify the protective potential of

shallot extract against CsA nephrotoxicity and suggest a significant contribution of its

antioxidant property to this beneficial effect (Wongmekiat et al., 2008).