In Vitro Studies on The Antioxidant potential of Cyperus rotundus linn. 1. Introduction Antioxidants are compounds that inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions. There are two basic categories of antioxidants, namely, synthetic and natural. In general, synthetic antioxidants are compounds with phenolic structures of various degrees of alkyl substitution, whereas natural antioxidants can be phenolic compounds (tocopherols, flavonoids, and phenolic acids), nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids, and amines), or carotenoids as well as ascorbic acid (Larson, 1988; Hudson, 1990; Hall and Cuppett, 1997). Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been used as antioxidants since the beginning of this century. Restrictions on the use of these compounds, however, are being imposed because of their carcinogenicity (Branen, 1975; Ito et al., 1983). Thus, the interest in natural antioxidants has increased considerably (Lo¨liger, 1991). The ability of some phenolic compounds to act as antioxidants has been demonstrated in the literature. Several researchers have investigated the ant oxidative activity of flavonoid compounds and have attempted to define the structural characteristics of flavonoids that contribute to their activity (Nieto et al., 1993; Das and Pereira, 1990; Foti et al., 1996). O-Dihydroxy groups in the B ring, the presence of a C2-3 double bond in conjunction with 4-oxo in the C ring, and 3- and 5-hydroxy groups and the 4-oxo function in the A and C rings are associated with antioxidant activity. Phenolic acids, such as caffeic, chlorogenic, ferulic, sinapic, and pcoumaric acids, appear to be more active antioxidants than the hydroxy derivatives of benzoic acid such as p-hydroxybenzoic, vanillic, and syringic acids (Dziedzic and Hudson, 1983; Larson, 1988). Burton and Ingold (1981) have shown that R-tocopherol is one of the most active in vitro chain-breaking antioxidants. Carotenoids also have a protective function against oxidative damage, and singlet oxygen is very powerfully quenched by â-carotene (Foote et al., 1971). Many of the natural antioxidants, especially flavonoids, exhibit a wide range of
biological effects, including antibacterial, antiviral, anti-inflammatory, antiallergic, antithrombotic, and vasodilatory actions (Cook and Sammon, 1996). Antioxidant activity is a fundamental property important for life. Many of the Biological functions, such as antimutagenicity, ant carcinogenicity, and antiaging, among others, originate from this property (Cook and Samman, 1996; Huang et al., 1992). The antioxidant activity of several plant materials has recently been reported (AlSaikhan et al., 1995; Yen and Duh, 1995; Oomah and Mazza, 1996; Wang et al., 1996; Cao et al., 1996; Amarowicz et al., 1996); however, information on the relationship between antioxidant activity and phenolic content and composition of many food plants is not available. The objective of this study was to determine the contents of total phenolics in several plant products and to explore relationship(s) between phenolic content and antioxidant activity. In addition, the antioxidant activities of alcoholic extracts of herbal products, such as ginseng, echinacea, and sea buckthorn were compared. At the very beginning, prior to the footprint of modern civilization, health care system for the mankind was solely dependent on the plant diversity. Curing of diseases and caring of health, natural plants played a vital role all over the universe. Through the history of mankind, naturally occurring plant-derived substances with minimal or no industrial processing have been used to treat illness within local or regional healing practices. This traditional practice is now concerning the promising factor for the development of better health care system and getting significant attention in global health debates. In the last decade, there has been a global upsurge in the use of traditional medicine(TM) and complementary and alternative medicine (CAM) in both developed and developing countries. Today, therefore, certain forms of traditional, complementary and alternative medicines play an increasingly important role in health care reform globally.
The development of traditional medicines has been influenced by the different cultural and historic conditions in which they were first developed. Their common basic is a holistic approach to life, equilibrium between the mind, body and environment, and an emphasis on health rather than on disease. Generally, the treatment focuses on the overall condition of the individual patient, rather than on the ailment or disease. This more complex approach makes evaluation highly difficult, since so many factors must be taken into account. Geographical point of view, Bangladesh is enriched with diversified natural plants. Natural plants actually are God gifted asset and with proper utilization mankind can have the chance to develop its whole life through the appropriate collaboration of nature and life.
1.1. MEDICINAL PLANTS: A medicinal plant is any plant which, in one or more of its organ, contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs. This definition of Medicinal Plant has been formulated by WHO (World Health Organization). Actually, the plants that possess therapeutic properties or exert beneficial pharmacological effects on the animal body are generally designated as “Medicinal Plants�. Although there r no apparent morphological characteristics in the medicinal plants growing with them, yet they possess some special qualities or virtues that make them medicinally important. It has now been established that the plants which naturally synthesis and accumulate some secondary metabolites, like alkaloids, glycosides, tannins, volatiles oils and contain minerals and vitamins, possess medicinal properties. Medicinal plants constitute an important natural wealth of a country. They play a significant role in providing primary health care services to rural people. They serve
as therapeutic agent as well as important raw materials for the manufacture of traditional and modern medicine. Substantial amount of foreign exchange can be possible to earn by exporting medicinal plants to other countries. 1.2. DEVELOPMENT OF MODERN MEDICINE Genesis of modern medicine is actually the continuous innovative process of traditionally utilized plant medicine through its improvement and utilization. Traditional medicine is the synthesis of therapeutic experience of generations of practicing physicians of indigenous system of medicine. Throughout the history of mankind, many infectious diseases have been treated with herbals. The traditional medicine is increasingly solicited through tradipractitioners. 1.3 Table 1: List of Medicinal Plants and uses
Plant Image Common name Amla
Parts Used Fruit
Medicinal Use Vitamin – C, Cough , Diabetes, Cold, Laxative, hyperacidity. Menstrual Pain, Diabetes, Uterine disorder.
Ashok
Bark Flower
Aswagandha
Root, Leafs
Restorative tonic, stress, Nerves disorder, Aphrodisiac.
Bael / Bilva
Fruit, Bark
Diarrhea, Dysentery, Constipation.
Bhumi Amla
Whole Plant
Anaemic, Jaundice, Dropsy.
Brahmi
Whole plant
Nervous, Memory enhancer, Mental disorder.
Chiraita
Whole Plant
Skin Disease, Burning sensation, Fever.
Gudmar / madhunasini
Leaves
Diabetes, Hydrocil, Asthma.
Guggul
Gum rasine
Arthritis, Paralysis, Laxative.
Guluchi
Stem
Calihari
Seed
Gout, Piles, Fever, Jaundice. Skin Disease, Labour pain, Abortion, etc.
Kalmegh/ Bhui neem
Whole Plant
Fever, weekness, Release of gas.
Long peeper / Pippali
Fruit, Root
Makoi
Fruit/whole plant
Appetizers, Enlarged spleen, Bronchitis, Cold, Antidote. Dropsy, Diuretic, Anti dysenteric.
Pashan Bheda
Root
Sandal Wood
Heart oil
Satavari
Senna
Kidney stone, Calculus.
wood ,Skin disorder, Burning sensation, Jaundice, Cough. Root Enhance lactation, General weakness, Fatigue, Cough. Dry Tubers Rheumatism, Aphrodisiac.
Tulsi
Leaves/Seed
Cough, Cold, Bronchitis, Expectorant. Fruit,Skin disease, Snake Bite, Helminthiasis.
Vai Vidanka
Root, Leaves
Peppermint
Leaves, Flower,Digestive, Oil Pain killer.
Gritkumari
Leaves
Sada Bahar
Whole Plant
Vringraj
Seed/whole
Swetchitrak
Root, Root bar Appetizer, Antibacterial, Anti-cancer.
Rakta Chitrak
Root, Root bar Colic, Inflammation, Cough.
Kochila
Seed
Nervous, Paralysis, Healing wound.
Harida
Seed
Trifala, wound ulcer, leprosy, inflammation, Cough.
Bahada
Seed, Bark
Cough, Insomnia, Dropsy, Vomiting, Ulcer, Trifala.
Gokhur
Whole Plant
Sweet cooling, Aphrodisiac, appetizer, Digestive, Urinary.
Laxative, Wound healing, Skin burns & care, Ulcer. Leukemia, Hypotension, Antispasmodic , Antidote. Anti-inflammatory, Digestive, Hair-tonic.
Bach
Rhizome
Sedative, analgesic, antepilepsy, hypertensive.
Vasa
Whole Plant
Antispasmodic, respiratory, Stimulant.
Nag Champa
Bark, Flower
Benachar
Root
Burning, ulcer, Skin, Vomiting.
Mandukparni
Whole plant
Anti-inflammatory, Jaundice, Diuretic, Diarrhoea.
Leaf,Asthma, Skin, Burning, Vomiting, Dysentery, Piles.
Kaincha/CreeperBaidanka Root, Hair,Nervous, Disorder, Seed, Leaf Constipation, Dropsy. Dalchini
Bark, Oil
Bronchitis, Asthma, Cardiac,Disorder, Fever. Scabies, Antipyretic, Amoebic dysentery.
Kurai
Bark, Seed
Kantakari
Whole Plant,Diuretic, Fruit, Seed Anti-inflammatory, Appetizer, Stomachic.
2.1 Practice of Modern medicine Medicinal plant is pivotal for the development of new drugs. During 1950-1970 approximately 100 plants based new drugs were introduced in USA drug market including deserpidine, reseinnamine, reserpine, vinblastine and vincristine which are derived from higher plants. From 1971-1990 new drugs such as ectoposide, Eguggulsterone,
teniposide,
nabilone,
plaunotol,
Z-guggulsterone,
lactinan,
artemisinin and ginkgo ides were appeared all over the world.2% of drugs were introduced from 1991-1995 including paciltaxel, toptecan, gomishin, irinotecan etc.
Table 2: Chronicle of plant medicine in world market 1950-1970 Deserpidine Reseinnamine Reserpine Vinblastine Vincristine Serpentine Cynarin Danthron
1971-1990 Ectoposide Teniposides E-guggulsterone Z-guggulsterone Nabilone Plaunotol Lactinan Artemisinin
1991-1995 Paciitaxel Toptecan Gomishin Irinotecan Ginkgolides Tubocurarine Valapotriates Monocrotaline
1996-2008 Digoxin Taxol lupeol acetate Gossypol Hemsleyadin Qulsqualic acid Rescinnamine Yohimbine
Plant based drugs provide outstanding contribution to modern medicine. For example: Serpentine isolated from the root of Indian plant Rauwolfia serpentine in 1953 was a revolutionary event in the treatment of hypertension and lowering of blood pressure. Vinblastine isolated from Catharanthus rosesus (Farnsworth and Blowster, 1967) is used in the treatment of Hodgkin’s, choriocarcinoma, non-hodgkins lymphomas, leukemia in children, testicular and neck cancer. Table 3: Some of the important medicinal plants are used for major modern drugs for cancer. Plant Name Cathranthus rosesus
Drugs Treatment L. Vinblastine and vincristine Hodgkins,
(Apocynaceae)
Lymphosarcomas
Podophyllum emodi Wall. Podophyllotaxin,
children leukemia. Testicular cancer,
(Beriberidaceae)
cell
Taxus
lymphomas. Ovarian cancer,
brevifolius Paciltaxel, taxotere
(Taxaceae) Mappia foetida Miers.
lung
cancer
and
cancer
and small and lung
malignant
melanoma. Comptothecin, Irenoteccan Lung, ovarian and cervical
Comptotheca acuminata
and Topotecan Quinoline
cancer. and used in Japan for the
comptothecin alkaloids
treatment
of
cervical
cancer
Vincristine is recommended for acute lymphocytic leukemia in childhood advanced stages of Hodgkin’s, lymophosarcoma, small cell lung, cervical and breast cancer. (Farnsworth and Bingel, 1977). Phophyllotoxin is a constituent of Phodophyllum emodi currently used against testicular, small cell lung cancer and lymphomas. Indian indigenous tree of Nothapodytes nimmoniana (Magpie foetida) are mostly used in Japan for the treatment of cervical cancer. Plant derived drugs are used to cure mental illness, skin diseases, tuberculosis, diabetes, jaundice, hypertension and cancer. Medicinal plants play an important role in the development of potent therapeutic agents. Plant derived drugs came into use in the modern medicine through the uses of plant material as indigenous cure in folklore or traditional systems of medicine. More than 64 plants have been found to possess significant antibacterial properties; and more than 24 plants have been found to possess antidiabetic properties (Arcamone et al., 1980), antimicrobial studies of plants (Perumal Samy and Ignacimuthu, 1998; 1999 and Perumal Samy et al., 2006), plant for antidotes activity - Daboia russellii and Naja kaouthia venom neutralization by lupeol acetate isolated from the root extract of Indian sarsaparilla Hemidesmus indicus R.Br (Chatterjee, et al., 2006). Which effectively neutralized Daboia russellii venom induced path physiological changes (Alam et al., 1994). The present investigation explores the isolation and purification of another active compound from the methanolic root extract of Hemidesmus indicus, which was responsible for snake venom neutralization. Antagonism of both viper and cobra venom and antiserum action potentiating, antioxidant property of the active
compound was studied in experimental animals. Recently, Chatterjee et al. (2004) from this Nature preceding hdl:10101/npre.2007.1176.1: Posted 28 Sep 2007 Table 4: Plant derived ethno therapeutics and traditional modern medicine SLNo. Drugs 1 Codeine,
Basic Investigation Opium the latex of Papaver somniferum used by ancient
morphin
Sumarians. Egyptaians and Greeks for the treatment of
2
Atropine,
headaches, arthritis and inducing sleep. Atropa belladonna, Hyascyamus Niger etc., were important
3
Hyoscyamine Ephedrine
drugs in Babylonian folklore. Crude drug (astringent yellow) derived from Ephedra sinica had been used by Chinese for respiratory ailments
4 5
Quinine
since 2700 BC. Cinchona spp were used by Peruvian Indians for the
Emetine
treatment of fevers Brazilian Indians and several others South American tribes used root and rhizomes of Cephaelis spp to induce vomiting
6 7
Colchicine
and cure dysentery. Use of Colchicum in the treatment of gout has been known
Digoxin
in Europe since 78 AD. Digitalis leaves were being used in heart therapy in
Europe during the 18th century. Laboratory reported that an active compound from the Strychnus nux vomica seed extract, inhibited viper venom induced lipid per oxidation in experimental animals. The mechanism of action of the plant derived micromolecules induced venom neutralization need further attention, for the development of plant-derived therapeutic antagonist against snakebite for the community in need. However, the toxicity of plants has known for a long period of time, and the history of these toxic plants side by side with medicinal ones are very old and popular worldwide, they considered the major natural source of folk medication and toxication even after arising of recent chemical synthesis of the active constituents contained by these plants (Adailkan and Gauthaman, 2001; Heinrich, 2000; Pfister et al., 2002).
Teniposide and etoposide isolated from Podophyllum species are used for testicular and lung cancer. Taxol isolated from Taxus brevifolius is used for the treatment of metastatic ovarian cancer and lung cancer. The above drugs came into use through the screening study of medicinal plants because they showed fewer side effects, were cost effective and possessed better compatibility. 2.2 STATUS OF MEDICINAL PLANTS IN BANGLADESH Medicinal plants are an accessible, affordable and culturally appropriate source of primary health care system in Bangladesh. Marginalized, rural and indigenous people, who can not afford or access formal health care systems, are especially dependent on these culturally familiar, technically simple, financially affordable and generally effective traditional medicines. As such, there is widespread interest in promoting traditional health systems to meet primary health care needs. This is especially true in this country, as prices of modern medicines spiral and governments find it increasingly difficult to meet the cost of pharmaceutical-based health care. Throughout the region, there is strong and sustained public support for the protection and promotion of the cultural and spiritual values of traditional medicines. The total number of plants with medicinal properties in the subcontinent at present stands at about 2000. About 450 to 500 of such medicinal plants have so far been enlisted as growing or available in Bangladesh. Table 5: Common name of some Medicinal plants with scientific name and uses found in Bangladesh.
Local name Ulatkambal
Scientific name Abroma augusta
Uses Emmenogogue; used in amenorrhoea
Muktajhuri
Acalypha indica
and dysmenorrhoea. Expectorant, emetic, diuretic; used in
Apang
Achyranthes aspera
bronchitis and asthma. Purgative, diuretic,
ecbolic,
hypoglycemic; used in renal dropsy, piles, anasarca, boils and other skin Basak
Adhatoda zeylanica
eruptions. Expectorant, bronchodilator, used in cough,
asthma,
bronchitis,
pneumonia, phthisis and respiratory Bel
Aegle marmelos
problems. Digestive,
stomachic,
laxative,
astringent; used in constipation and Rashun
Allium sativum
dysentery. Carminative,
diuretic,
hypotensive,
used in indigestion, hypertension and Chhatim
Alstonia scholaris
diabetes. Febrifuge, ant periodic, astringent, anthelmintic, hypotensive; used in fever, hypertension, diarrhoea and
Kalomegh
Andrographis
dysentery. Febrifuge,
paniculata
anthelmintic, cholagogue; used in
alterative,
stomachic,
liver diseases, colic, fever, diarrhea Shatamuli
Neem
Asparagus
and dyspepsia. Roots aphrodisiac, alterative, diuretic;
racemosus
promotes
Azadirachta indica
diabetes. Antiseptic; ulcers,
Brahmishak
Bacopa monniera
lactation; used
eczema
also
in and
used
in
fevers,
boils,
other
skin
diseases. Blood purifier, brain-, nerve- and cardiac tonic, diuretic; also used for
Nayantara
Catharanthus
epilepsy. Used in blood cancer, Hodgkin's
roseus
disease and diabetes.
Thankuni
Centella asiatica
Leaf juice is used in cataract and other eye
diseases;
dysentery, Babchi
Cullen corylifolia
plant
internal
is and
used
in
external
ulcers, and convulsive disorders. Seed extract is used in leucoderma, leprosy, psoriasis and inflammatory
Kalo Dhutra
Datura metel
skin diseases. Narcotic, anodyne and antispasmodic; leaves used in spasmodic asthma, colic,
Ayapan
sciatica
painful
tumours,
Eupatorium
glandular inflammations. Haemostatic and antiseptic; used in
triplinerve
ulcers and haemorrhages and also as cardiac stimulant, emetic, diaphoretic
Hemidesmus
and laxative. Alterative, sudurific,
indicus
blood purifier; used in abdominal
Holarrhena
tumours. Antidysenteric, astringent, stomachic
antidysenterica
and
Chalmoogra
Hydnocarpus
hypoglycaemic. Seed oil is used as a cure for leprosy
Mehedi
kurzii Lawsonia inermis
and other skin diseases. Paste of leaves and bark is used in
Anantamul
Kurchi
burns
anthelmintic.
and
scalds,
diuretic
Fruits
and
are
dandruff
and
various other skin diseases. Decoction Sarpagondha
Ashoke
Raulwolfia
in jaundice. Roots are used
serpentina
hypertension,
Saraca asoca
excitement and insanity. Strongly astringent and sedative;
Arjun
Terminalia arjuna
used
as
remedy
insomnia,
in
for
anxiety, uterine
menorrhagia,
haemorrhoids and ulcers. Bark is hypotensive, cardiac tonic,
astringent and febrifuge; has tonic Methi
Ashwagondha
Ada
Trigonella foenum-
effect on liver cirrhosis. Diuretic, carminative, emollient, tonic;
graecum
used in menstrual disorders, diabetes,
Withania
hypertension and sexual problems Roots are used in headache,
somniferum
convulsions, insomnia, hiccup, coughs
Zingiber officinale
and dropsy. Rhizome is carminative, stomachic, digestive;
used
in
dyspepsia,
vomiting, loss of voice, coughs, sore throat and fever 2.3 CHEMICAL CONSTITUENTS OF MEDICINAL PLANTS Plants have been serving the animal kingdom as its source of energy (food, fuel) as well as its means of shelter and sustenance since the very beginning of its existence on earth’s surface habitable for the animals. In addition to providing the animal kingdom its food, fuel and shelter, each of these plants has been synthesizing a large variety of chemical substances since their first day of life on earth. These substances include, in addition to the basic metabolites, phenolic compounds, terpenes, steroids, alkaloids, glycosides, tannins, volatile oils, contain minerals, vitamins and a host of other chemical substances referred to as secondary metabolites which are of no apparent importance to the plants own life. But many of these compounds have prominent effects on the animal system and some possess important therapeutic properties which can be and have been utilized in the treatment and cure of human and other animal diseases since time immemorial. These secondary metabolites differ from plant to plant. Thus, the plant kingdom provides a tremendous reservoir of various chemical substances with potential therapeutic properties3.
The chemical constituents, which are capable of influencing the physiological systems of the animal body by exerting some pharmalogical actions, are designated as the active chemical constituents or simply active constituents. In short, it may be said that the chemical constituents present in the medicinal plants constitute the most important aspect of all medicinal plants. Green leaves are the sites of a great deal of such chemical activity. The chemical substances with medicinal properties found in some plants are the products of such chemical processes. The occurrence of the active chemical substances in all parts of the plant body or they may be accumulated in higher concentrations in some specific part. The types of chemical constituents are as follows: A. Alkaloids and amides •
Pyridine group
•
Tropane group
•
Isoquinoline group
•
Quinoline group
•
Quinolizidine group
•
Indole group
•
Steroidal group
•
Imidazole group
•
Phenylthylamine group
•
Alkaloidal amines
B. Antibiotic and Anti-inflammatory principles C. Bitter and Pungent principles D. Volatile oils and Fixed oils E. Glycosides •
Anthraquinone glycosides
•
Cardiac glycosides
•
Saponin glycosides
•
Thiocyanate glycosides
•
Other glycosides
F. Gum-resins and Mucilage G. Vitamins and Minerals. 3. FREE RADICAL Like all matter, our bodies are composed entirely of tiny particles called molecules. Each molecule is made up of atoms, and each atom is made up of a center or nucleus and electrons which spin in orbits around it. Ordinarily, the electrons occur in balanced pairs. This keeps the atom and molecule stable. Sometimes a molecule loses one of its electrons or gains and extra one. This causes the molecule to become unbalanced and highly reactive. Such a molecule is called a 'Free Radical'. Actually a free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. Many radicals are highly reactive and can either donate an electron to or extract an electron from other molecules, therefore behaving as oxidants or reductants. As a result of this high reactivity, most radicals have a very short half life (10−6 seconds or less) in biological systems, although some species may survive for much longer. The most important free radicals in many disease states are oxygen derivatives, particularly superoxide and the hydroxyl radical.
Free radicals and other reactive oxygen species are derived either from normal essential metabolic processes in the human body or from external sources such as exposure to X-rays, ozone, cigarette smoking, air pollutants and industrial chemicals. Figure 1: Major sources of free radicals in the body and the consequence of free radical damage.
Free radical formation occurs continuously in the cells as a consequence of both enzymatic and non-enzymatic reactions. Enzymatic reactions which serve as sources of free radicals include those involved in the respiratory chain, in phagocytosis, in prostaglandin synthesis and in the cytochrome P450 system. Free radicals also arise in non-enzymatic reactions of oxygen with organic compounds as well as those initiated by ionizing radiations. With electrons unhinged, free radicals roam the body, wreaking havoc. The free radical, in an effort to achieve stability, it takes nearby molecules to obtain another electron and, in doing so, damages those molecules. This situation can be compared to letting a bachelor into a dance where people have come as couples. The bachelor begins cutting in, each time leaving another bachelor, so the breaking up of couples spreads through the dance floor. If free radicals are not inactivated, their chemical reactivity can damage all cellular macromolecules including proteins, carbohydrates, lipids and nucleic acids. Their destructive effects on proteins may play a role in the causation of cataracts. Free radical damage to DNA is also implicated in the causation of cancer and its effect on LDL cholesterol is very likely responsible for heart disease. In fact, the theory associating free radicals with the aging process has also gained widespread acceptance. 4. ANTI-OXIDANT Antioxidants are molecules that can neutralize free radicals by accepting or donating an electron to eliminate the unpaired condition. Typically this means that the antioxidant molecule becomes a free radical in the process of neutralizing a free radical molecule to a non-free-radical molecule. But the antioxidant molecule will usually be a much less reactive free radical than the free radical neutralized. The antioxidant molecule may be very large (allowing it to "dilute" the unpaired
electron), it may be readily neutralized by another antioxidant and/or it may have another mechanism for terminating its free radical condition. A free radical attack on a membrane usually damages a cell to the point that it must be removed by the immune system. If free radical formation and attack are not controlled within the muscle during exercise a large quantity of muscle could easily be damaged. Damaged muscle could in turn inhibit performance by the induction of fatigue. The roles of individual antioxidants have in inhibiting this damage. The major benefits of Antioxidant through the examination of different health benefits including: 1. Counteraction of the damaging oxidative action of low-density lipoproteins (LDLs), the so-called bad cholesterol, thereby protecting the arteries from worsening effects of atherosclerosis. 2. Protection of the endothelial cells of the arteries themselves from free radical damage, permitting them to be compliant and reactive rather than rigid and dysfunctional. 3. Decrease in platelet aggregation (clumping), protecting the vascular system from clot formation that has potentially damaging effects such as heart attacks and strokes. 4. Counteraction of the oxidation-promoting effects of stress hormones such as the catecholamine’s (epinephrine and nor epinephrine), often secreted in high amounts during chronic stress. 5. Counteraction of free radical damage to many cells of the body that could potentially trigger undesired proliferation in the form of cancer. 6. Protection from some of the damaging effects of aberrant metabolism that can contribute to the triggering of type II diabetes. 7. Protection of important connective tissues of the body to help counteract many age-related forms of deterioration.
8. Protection and enhancement of immune responses important in protective responses against viral infections and surveillance and protection from the formation or spread of many types of cancer. 9. Counteraction of damaging effects of inflammatory responses in diverse systems of the body, including joints (arthritis), and brain (Alzheimer's disease). 10. Protection against degenerative processes in the brain that can lead to specific neuronal damage associated with Parkinson's disease and Alzheimer's disease. 4.1 Classification of anti-oxidant Antioxidants are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants react with oxidants in the cell cytosol and the blood plasma, while lipidsoluble antioxidants protect cell membranes from peroxidation.These compounds may be synthesized in the body or obtained from the diet. The different antioxidants are present at a wide range of concentrations in body fluids and tissues, with some such as glutathione or ubiquinone mostly present within cells, while others such as uric acid are more evenly distributed. Some antioxidants are only found in a few organisms and these compounds can be important in pathogens and can be virulence factors.
4.1.1 Antioxidant Enzymes: Even though the production of antioxidant enzymes in the body is a complex process that is not yet totally understood, there are several processes that we are aware of and which seem to constitute a large part of the finished system. The antioxidant enzyme defense system consists of hundreds of different substances and mechanisms. This is why only an adequate combination of whole foods, such as
sprouted food concentrates, will contain all of the known and unknown nutritional factors that the body requires to enhance its antioxidant enzyme supply. 4.1.1.1 Superoxide- Dismutase & Catalase We are a species that cannot live without breathing. As a result of this circumstance, oxygen has turned into a synonym of life. A fact much less known is that not all oxygen atoms are life supporting. Some are actually quite destructive for our cells. These unhealthy oxygen atoms are unbalanced and constitute the most common "Free Radical" known. The "Oxygen Free Radical" is characterized by having and unpaired electron in its molecular structure. Called “Superoxide", it is quite capable of causing cell damage. The first line of defense that the body has against superoxide free radicals is the enzyme known as "Superoxide Dismutase" or (SOD), which is considered the most effective antioxidant. The importance of SOD is so paramount for the protection of our cells, that it represents a substantial proportion of the proteins manufactured by the body. In brief, SOD keeps oxygen under control. In the process of removing superoxide free radicals, SOD rarely operates alone. It requires the enzyme called “Catalase" or (CAT) to remove hydrogen peroxide molecules which are by-products of the reactions created by SOD. Similar to SOD, CAT is abundant in the body. Integrated in all red blood cells, CAT removes hydrogen peroxide from our tissues, preventing both cell damage and , more importantly, the formation of other more toxic free radicals. In nature, and in the body, SOD and CAT always co-exist. The natural interactions - synergy - between these two antioxidant enzymes constitute the most effective system of free radical control in our bodies. Superoxide free radicals initiate the breakdown of "Synovial Fluid" (the lubricating element in the joints of our bodies) causing friction and, eventually, inflammation.
For this reason, the attention of clinical SOD research has been focused primarily on inflammatory processes triggered by superoxide free radicals such as arthritis, bursitis and gout. Deficiency in SOD/CAT is the most notorious nutritional factor in most 'inflammatory' processes. Recent applications of SOD/CAT enhancing foods have also proven to be extremely useful as a (pre- & post) operative supplement which stimulates recovery and reduces convalescence periods remarkably. Considering the powerful link between free radicals and many of today's health problems, supplements that enhance SOD/CAT activity in the body offer tremendous potential in the field of preventative nutrition. 4.1.1.2 Glutathione Peroxidase Glutathione Peroxidase is another of the body's major protectors against free radicals. This antioxidant enzyme consists of the amino acid Glutathione and the trace mineral 'Selenium'. These two nutrients team up to combat a specific class of free radicals called peroxides. The main biological function of selenium in mammals is a component of the Glutathione Peroxidase enzyme. Many of the attributes of selenium and Glutathione are actually attributes of Glutathione Peroxidase. Cell membranes consist primarily of lipids (fats). These lipid membranes are very susceptible to damage by free radicals, especially peroxide radicals. This is why rancid fats (lipid peroxides) have proven to be highly toxic. Glutathione Peroxidase prevents destruction of cell membranes by removing several classes of these lipid peroxides. The main symptoms of excess peroxide free radicals include heart disease, liver disease, premature aging and skin disease such as skin cancer, eczema, wrinkling, age spots, dermatitis and psoriasis. Peroxide free radicals mediate much damage to
the body by impairing liver functions. Consisting of nearly 50% fatty tissue, the liver is very susceptible to lipid per oxidative damage. Although used primarily for skin related problems, many environmentally sensitive and chemically poisoned people report that Glutathione Peroxidase helps them control their allergies and build resistance to the effects of pollution. Generally speaking, all of the antioxidant enzymes are important where pollution is a concern due to their ability to remove free radicals generated by toxic substances. The list of protective effects of Glutathione Peroxidase is growing and is in no way limited to any single symptom such as age spots. The effects of excess per oxidation in our cells are diverse and dangerous and must be limited to maintain cellular health. 4.1.1.3 Methionine Reductase Methionine Reductase is a unique enzyme that has demonstrated an ability to remove an extremely toxic free radical called the "Hydroxyl Radical". The hydroxyl radical is commonly formed through reactions involving heavy metals and other less toxic free radicals, such as mercury reacting with hydrogen peroxide. The hydroxyl radical has the ability to damage any type of organic tissue and is considered to be the most dangerous free radical. Hydroxyl radicals are also the main toxins generated by exposure to excessive radiation. With their ability to damage any type of tissue, symptoms directly related to hydroxyl radical induced tissue damage are difficult to identify. One effective detoxifying application for Methionine Reductase is in the removal of free radical toxins generated by the mercury found in dental fillings. Another interesting application of Methionine Reductase is for the modern day athlete. It seems that hydroxyl radicals are also formed during exercise. This is especially true if we are exercising in oxygen starved closed rooms or in a auto exhaust filled polluted environments. It is amazing to see joggers running along the road during rush hour traffic. Quite possibly, they are doing more harm than good!
Most avid exercisers are always aware of the need to obtain extra nutrition to fuel their activities. What many miss is the importance of cleaning out the extra metabolic wastes that are a direct result of this exercise. Hydroxyl radicals can be created when we burn fat molecules to produce energy as in strenuous exercise or dieting. This is due to the lack of evacuation of chemicals and toxins stored in the fatty tissue which are released when these tissues are used for fuel. These toxins, when not properly evacuated, generate the formation of hydroxyl radicals. People supplementing their diets with "Methionine Reductase" have reported greater resistance to the ill effects of pollution as well as greater endurance, stamina, flexibility and the ability to recover from extensive exercise. Although it is generally important to exercise, our modern civilized environments force us to compensate for free radical by-products if we wish to gain health or longevity form our workout programs. 4.1.2 The Chain Breaking Antioxidants Whenever a free radical interacts with another molecule, secondary radicals may be generated that can then react with other targets to produce yet more radical species. The classic example of such a chain reaction is lipid per oxidation and the reaction will continue to propagate until two radicals combine to form a stable product or the radicals are neutralized by a chain breaking antioxidant. A simplified scheme of free radical initiated lipid per oxidation is summarized here. In the first reaction, hydrogen from an unsaturated fatty acid (RH) is abstracted by a free radical initiator to form a free radical (R•) (I). This is followed by the rearrangement of the double bond and acceptance of oxygen by the free radical to produce a fatty acid peroxyl radical (Roo•) (II). The peroxyl radical then reacts with another molecule of unsaturated fatty acid to form a semis table unsaturated hydro peroxide (ROOH) which also regenerates a molecule of free radical (III). These
reactions may propagate unless all the free radical is scavenged by an antioxidant (AH) (IV), or by self-quenching (V).
However, the hydro peroxide may undergo homolytic fission of covalent bonds to form more free radicals (VI, VII). Upon hemolytic fission of the hydro peroxide, a free radical chain reaction may again be initiated. The reaction can be catalyzed via one electron rcdox reaction in the presence of heme or certain heavy metal ions such as copper or iron.
Chain breaking antioxidants are small molecules that can receive an electron from a radical or donate an electron to a radical with the formation of stable byproducts. 46 In general, the charge associated with the presence of an unpaired electron becomes dissociated over the scavenger and the resulting product will not readily accept an electron from or donate an electron to another molecule, preventing the further propagation of the chain reaction. Such antioxidants can be conveniently divided into aqueous phase and lipid phase antioxidants. 4.1.2.1 Lipid phase chain breaking antioxidants These antioxidants scavenge radicals in membranes and lipoprotein particles and are crucial in preventing lipid per oxidation. The most important lipid phase antioxidant is probably vitamin E.47 Vitamin E occurs in nature in eight different forms, which differ greatly in their degree of biological activity. Both classes of compounds are lipid soluble and have pronounced antioxidant properties. They react more rapidly than polyunsaturated fatty acids with peroxyl radicals and hence act to break the chain reaction of lipid per oxidation.
In cell membranes and lipoproteins the essential antioxidant function of vitamin E is to trap peroxyl radicals and to break the chain reaction of lipid peroxidation. 53 Vitamin E will not prevent the initial formation of carbon centred radicals in a lipid rich environment, but does minimize the formation of secondary radicals. ΑTocopherol is the most potent antioxidant of the tocopherols and is also the most abundant in humans. It quickly reacts with a peroxyl radical to form a relatively stable tocopheroxyl radical, with the excess charge associated with the extra electron being dispersed across the chromanol ring. This resonance stabilized radical might subsequently react in one of several ways.
Figure 2: Forms of α – tocopheryl radical resonance
A-Tocopherol might be regenerated by reaction at the aqueous interface with ascorbate or another aqueous phase chain breaking antioxidant, such as reduced glutathione or urate.Alternatively, two A-tocopheroxyl radicals might combine to form a stable dimer, or the radical may be completely oxidized to form tocopherol quinone. The carotenoids are a group of lipid soluble antioxidants based around an isoprenoid carbon skeleton. The most important of these is β-carotene, although at least 20 others may be present in membranes and lipoproteins. They are particularly efficient scavengers of singlet oxygen, but can also trap peroxyl radicals at low oxygen pressure with efficiency at least as great as that of A-tocopherol. Because these conditions prevail in many biological tissues, the carotenoids might play a role
in preventing in vivo lipid peroxidation.The other important role of certain carotenoids is as precursors of vitamin A (retinol). Vitamin A also has antioxidant properties, which do not, however, show any dependency on oxygen concentration.
Figure 3: Radical trapping mechanism for carotenoids
Flavonoids are a large group of polyphenolic antioxidants found in many fruits, vegetables, and beverages such as tea and wine. Over 4000 flavonoids have been identified and they are divided into several groups according to their chemical structure, including flavonols (quercetin and kaempherol), flavanols (the catechins), flavones (apigenin), and isoflavones (genistein). There is some evidence that augmenting the intake of flavonoids might improve biochemical indices of oxidative damage.Apart from flavonoids, other dietary phenolic compounds might also make a small contribution to total antioxidant capacity.
A
B Figure 4:
Radical trapping mechanism for phenolic compound (A) and
Structure after oxidation of flavon-3-ols with 4’-hydroxyl groups (B)
Ubiquinol-10, the reduced form of coenzyme Q10, is also an effective lipid soluble chain breaking antioxidant. Although present in lower concentrations than αtocopherol, it can scavenge lipid peroxyl radicals with higher efficiency than either α-tocopherol or the carotenoids, and can also regenerate membrane bound αtocopherol from the tocopheryl radical. Indeed, whenever plasma or isolated low density lipoprotein (LDL) cholesterol is exposed to radicals generated in the lipid phase, ubiquinol-10 is the first antioxidant to be consumed, suggesting that it might be of particular importance in preventing the propagation of lipid per oxidation.
4.1.2.2 Aqueous phase chain breaking antioxidants These antioxidants will directly scavenge radicals present in the aqueous compartment. Qualitatively the most important antioxidant of this type is vitamin C (ascorbate).Ascorbate has been shown to scavenge superoxide, hydrogen peroxide, the hydroxyl radical, hypochlorous acid, aqueous peroxyl radicals, and singlet oxygen. During its antioxidant action, ascorbate undergoes a two electron reduction, initially to the semidehydroascorbyl radical and subsequently to dehydroascorbate. The semidehydroascorbyl radical is relatively stable owing to dispersion of the charge associated with the presence of a single electron over the three oxygen atoms, and it can be readily detected by electron spin resonance in body fluids in the presence of increased free radical production. Dehydroascorbate is relatively unstable and hydrolyses readily to diketogulonic acid, which is subsequently broken down to oxalic acid.
Two mechanisms have been described by which dehydroascorbate can be reduced back to ascorbate; one is mediated by the selenoenzyme thioredoxin reductase and the other is a non-enzyme mediated reaction that uses reduced glutathione. Dehydroascorbate in plasma is probably rapidly taken up by red blood cells before recycling, so that very little, if any, dehydroascorbate is present in plasma.
Uric acid efficiently scavenges radicals, being converted in the process to allantoin A-tocopherol. Urate might be particularly important in providing protection against certain oxidizing agents, such as ozone. Part of the antioxidant effect of urate might be attributable to the formation of stable non-reactive complexes with iron, but it is also a direct free radical scavenger. Albumin bound bilirubin is also an efficient radical scavenger, and it has been suggested that it might play a particularly crucial role in protecting the neonate from oxidative damage, because deficiency of other chain breaking antioxidants is common in the newborn. The other major chain breaking antioxidants in plasma are the protein bound thiol groups. The sulphydryl groups present on plasma proteins can function as chain breaking antioxidants by donating an electron to neutralise a free radical, with the resultant formation of a protein thiyl radical. Reduced glutathione (GSH) is a major source of thiol groups in the intracellular compartment but is of little importance in the extracellular space.GSH might function directly as an antioxidant, scavenging a variety of radical species, as well as acting as an essential factor for glutathione peroxidase. Thioredoxin might also function as a key intracellular antioxidant, particularly in redox induced activation of transcription factors
Table 10: Some nutrients & their concentration in the body
Antioxidant
Solubility
metabolite
Concentration in human Concentration
in
serum (μM)
liver tissue (μmol/kg)
Ascorbic acid (vitamin C)
Water
50 – 60
260 (human)
Glutathione
Water
325 – 650
6,400 (human)
Lipoic acid
Water
0.1 – 0.7
4 – 5 (rat)
Uric acid
Water
200 – 400
1,600 (human)
Carotenes
Lipid
α-Tocopherol (vitamin E) Ubiquinol (coenzyme Q)
β-carotene:
0.5
–
1, 5
retinol (vitamin A): 1 – 3
Lipid
10 – 40
Lipid
5
(human,
total
carotenoids) 50 (human) 200 (human)
4.1.2.3 The Transition Metal Binding Proteins As discussed above, transition metal binding proteins (ferritin, transferring, lactoferrin, and caeruloplasmin) act as a crucial component of the antioxidant defence system by sequestering iron and copper so that they are not available to drive the formation of the hydroxyl radical. The main copper binding protein, caeruloplasmin, might also function as an antioxidant enzyme that can catalyze the oxidation of divalent iron.94 4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2O
Fe2+ is the form of iron that drives the Fenton reaction and the rapid oxidation of Fe 2+ to the less reactive Fe3+ form is therefore an antioxidant effect.
4.2 Counteracting Free Radical Damage The body has developed several endogenous antioxidant systems to deal with the production of ROI. These systems can be divided into enzymatic and nonenzymatic groups. Figure summarizes the sites of action of the various antioxidants.
O2-
•
O
SOD
2
Catalase
H2O2 β-Carotene
GSH
1
O
2
OH
+ Vit E Vit C
RO
•
Vit E
•
ROO
•
Vit E Vit C
Fig 7: Generation sequence of reactive oxygen species following univalent reduction of oxygen and the various sites of action of the different antioxidants.
The enzymatic antioxidants include superoxide dismutase (SOD), which catalyses the conversion of O2·-to H2O2 and H2O; catalase, which then converts H2O2 to H2O and O2; and glutathione peroxidase, which reduces H2O2 to H2O. The nonenzymatic antioxidants include the lipid-soluble vitamins, vitamin E and vitamin A or provitamin a (beta-carotene), and the water-soluble vitamin C and GSH. Vitamin E has been described as the major chain-breaking antioxidant in humans25. Because of its lipid solubility, vitamin E is located within cell membranes, where it interrupts lipid per oxidation and may play a role in modulating intracellular signalling pathways that rely on ROI. Vitamin E can also directly
quench ROI, including O2·-, ·OH, and 1O2. The enzymatic and nonenzymatic antioxidant systems are intimately linked to one another and appear to interact with one another. Both vitamin C and GSH have been implicated in the recycling of alpha-tocopherol radicals. In addition, the trace elements selenium, manganese, copper, and zinc also play important roles as nutritional antioxidant cofactors. Selenium is a cofactor for the enzyme glutathione peroxidase, and manganese, copper, and zinc are cofactors for SOD. Zinc also acts to stabilize the cellular metallothionein pool, which has direct free radical quenching ability. The complex interactions of these different antioxidant systems may imply that therapeutic strategies will depend on combination therapy of various antioxidants rather than a single agent.
AA metabolism
NADPH oxidase ATP
Mitochondria
Hypoxanthine + O2 XO
x Allopurinol
O2- •
x
H2O2
x
Vitamin C
Vit E & β-Carotene
SOD
ROO• Lipid peroxidation
H2O
Fe++
Deferoxamine
x
Catalase GSH
OH•
DNA damage
x
DMS O
Fig 8: Sources of intracellular oxidative stress and sites of antioxidant activity.
Figure 9 describes the interactions among antioxidants. Reactive oxygen intermediates (ROI) induce membrane lipid per oxidation resulting in a chain reaction that can be interrupted by the direct scavenging of lipid peroxyl radicals by vitamin E (VE) and beta-carotene. Vitamin E can then be recycled by both vitamin C (VC) and glutathione (GSH).
Fig 9: Interactions among antioxidants
The reducing ability of GSH is catalyzed by the enzyme glutathione peroxidase (GSSG). Glutathione is then recycled by NADPH, which is facilitated by glutathione reductase (GSSG). LOO路 = active species of the lipid peroxyradical; LOOH-reduced lipid radical; VEO路 = active radical form of VE; VE-OH = the reduced form. The human body has several mechanisms to counteract damage by free radicals and other reactive oxygen species. These act on different oxidants as well as in different cellular compartments.
One important line of defense is a system of enzymes, including glutathione peroxidases, superoxide dismutases and catalase, which decrease concentrations of the most harmful oxidants in the tissues. Several essential minerals including selenium, copper, manganese and zinc are necessary for the formation or activity of these enzymes. Hence, if the nutritional supply of these minerals is inadequate, enzymatic defences against free radicals may be impaired. The second line of defence against free radical damage is the presence of antioxidants. Antioxidant is stable enough to donate an electron to a rampaging free radical and neutralize it, thus reducing its capacity to damage. Some such antioxidants, including glutathione, ubiquinol and uric acid, are produced during normal metabolism in the body. Other lighter antioxidants are found in the diet. Although about 4000 antioxidants have been identified, the best known are vitamin
E, vitamin C and the carotenoids. Many other non-nutrient food substances, generally phenolic or polyphenolic compounds, display antioxidant properties and, thus, may be important for health. Although a wide variety of antioxidants in foods contribute to disease prevention, the bulk of research has focused on three antioxidants which are essential nutrients or precursors of nutrients. These are vitamin E, vitamin C and the carotenoids. Each of these antioxidant nutrients has specific activities and they often work synergistically to enhance the overall antioxidant capability of the body. The balance between the production of free radicals and the antioxidant defenses in the body has important health implications. If there are too many free radicals produced and too few antioxidants, a condition of "oxidative stress" develops which may cause chronic damage. As mentioned above, free radicals have been implicated in several health problems. Cancer, atherosclerosis, cerebrovascular accidents, myocardial infarction, senile cataracts, acute respiratory distress syndrome and rheumatoid arthritis are just a few examples. Numerous studies have shown the protective effects of antioxidant nutrients on these health problems. Oxidative damage to DNA caused by free radicals generated as a damaging sideeffect of aerobic metabolism. The theory that free radicals are a major cause of human cancer and that the risk of disease can be reduced by increased consumption of food-borne antioxidants has prompted an enormous growth of interest in antioxidant nutrients and other antioxidant substances in food. Diet-related anticarcinogenesis can usefully be classified into blocking mechanisms, which operate during the initiation phase of carcinogenesis, and suppressing mechanisms, which delay or reverse tumor promotion at a later stage. A schematic illustration of these concepts and a summary of the mechanisms through which they may act are given in fig10:
Figure 10: Hypothetical sites of interaction between anti-carcinogenic substances in the diet and the progressive stages of carcinogenesis. Blocking agents are those acting to prevent initiation, whereas suppressing agents act to inhibit the development of tumors from initiated cells.
Some evidence also suggests that the reduced risk of lung cancer associated with the intake of fruits and vegetables may be due to some other micronutrients, such as vitamin C (Block et al., 1992), flavonoids (Knekt et al., 1997a), and selenium (Clark et al., 1996). Increasingly, research suggests that antioxidant nutrients, including vitamin C, vitamin E, and the carotenoids are of great importance in reducing the risk of cancer and heart disease. However, beyond these diseases, it is increasingly apparent that antioxidants may be important in most of the diseases of aging, including age-related eye diseases such as cataracts, and impaired immune function resulting in increased susceptibility to infection.
The third line of defense is Self repair The body also has systems to repair or replace damaged building blocks of cells. These systems are rapid and thorough. For example, the system for repairing damage to DNA and other nucleic acids is particularly elaborate and efficient, with various specialized enzymes that locate damaged areas, snip out ruined bits, sreplace them with the correct sequence of molecules, and seal up the strand once again. Every aspect of the cell receives similar attention. Most protein constituents in the cell, for example, are completely replaced every few days. Scavenger enzymes break used and damaged proteins into their component parts for reuse by the cell. Mechanism of action of Antioxidants: There are four routes •
Chain breaking reactions, e.g. alpha-tocopherol which acts in lipid phase to trap “ROD” radical.
•
Reducing the concentration of reactive oxygen species e.g. glutathione.
•
Scavanging initiating radicals e.g. superoxide dismustase which acts in aqueous phase to trap superoxide free radicals.
•
Chelating the transition metal catalysts: A group of compounds serves an antioxidant function by sequestration of transition metals that are well established pro-oxidants. In this way, transferring, lactoferrin, and ferritin function to keep iron induced oxidant stress in check and ceruloplasmin and albumin as copper sequestrants. Basic mechanism of some common Antioxidant Ascorbic Acid (Vitamin C)
Ascorbic Acid, the formula of which is C6H8O6, with antioxidant properties. Mechanism of action: The chemo preventive action of Vitamin C is attributed to two of its functions. It is a water soluble chain braking antioxidant (Ishwarial et at 1991). As an antioxidant, it scavenges free radicals and reactive oxygen molecules, which are produced during metabolic pathways of detoxification. It also prevents formation of carcinogens from precursor compounds (Bock and menkes, 1988). The structure of ascorbic acid is reminiscent of glucose, from which it is derived in the majority of mammals.
One important property is its ability to act as a reducing agent (electron donor). Ascorbic acid is a reducing agent with a hydrogen potential of +0.08 V, making it capable of reducing such compounds as a molecular oxygen, nitrate and cytochromes a and c. Donation of one electron by acrobat gives the semidehydroascorbate radical (DHA). Ascorbate reacts rapidly with superoxide and even more rapidly with hydroxyl ion to give DHA. DHA, itself can act as a source of Vitamin C Ascorbic acid + 2O2.- + 2H+ → H2O2 + DHA α-tocopherol (Vitamin E) Mechanism of Action: Vitamin E is more appropriately described as an antioxidant than a vitamin. This is because, unlike most vitamins, it does not act as a co-factor for enzymatic reactions. Also deficiency of vitamin E does not produce a disease with rapidly developing symptoms such as scurvy or beriberi. Overt symptoms due to vitamin E deficiency occur only in cases involving fat malabsorption syndromes, premature infants and patients on total parenteral nutrition. The effects of inadequate Vitamin E intake usually develop over a long time, typically decades and have been linked to chronic diseases such as cancer and atherosclerosis. Hence, its main function is to prevent the peroxidation of membrane phospholipids, and avoids cell membrane damage through its antioxidant action, the lipophilic character of tocopherol enables it to locate in the interior of the cell membrane bilayers (halliway and Getteridge,1992; borg 1993). Tocopherol-OH can transfer a hydrogen atom with a single electron to a free radical, thus removing the radical before it can interact with cell membrane proteins or generate lipid per oxidation. When tocopherol-OH combines with the free radical, it becomes tocopherol-O ., itself a radical. When ascorbic acid is available, tocopherol-O . Plus ascorbate (with its available hydrogen) yields semidehydroascorbate (a weak radical) plus tocopherolOH (halliway and Gutteridge, 1992). By this process, an aggressive ROI is eliminated and a weak ROI (dehydroascorbate) is formed and tocopherol-OH is regenerated. Despite this complex defence system, there are no known endogenous enzaymatic antioxidant systems for the hydroxyl radical. Alpha tocopherol + LOO·® Alpha tocopherol· +LOOH Alpha tocopherol·+ LOO· ® LOO-alpha tocopherol Vitamin E also stimulates the immune response. Some studies have shawn lower incidence of infections when vitamin E levels are high and vitamin E may inhibit cancer initiation through enhanced immunocompetence.
Vitamin E also has a direct chemical function. It inhibits the conversion of nitrites in smoked, pickled and cured foods to nitrosamines in the stomach. Nitrosamines are strong tumour promoters. Alpha tocopherol has been shown to be capable of reducing ferric iron to ferrous iron (i.e. to act as a pro-oxidant). Moreover, the ability of alphatocopherol to act as a pro-oxidant (reducing agent) or antioxidant depends on whether all of the alpha tocopherol becomes consumed in the conversion from ferric to ferrous ion or whether following this interaction residual alpha tocopherol is available to scavenge the resultant ROI (Yamamoto and Nike, 1988).
Beta Carotene The antioxidant function of beta-carotene is due to its ability to quench singlet oxygen, scavenge free radicals and protect the cell membrane lipids from the harmful effects of oxidative degradation (krinsky and deneke, 1982; Santamaria et al. 1991). The quenching involves a physical reaction in whch the energy of the excited oxygen is transferred to the carrotenoid, forming an excited state molecule (Krinsky, 1993). Quenching of singlet oxygen is the basis for beta carotenes well known therapeutic efficacy in erythropoietic protoporphyria ( a photosensitivity disorder) (Matthews-Roth,1993) . The ability of beta carotene and other carotenoid itself can be oxidized during the process (autooxidation). Burton and ingold (Burton and Ignold,1984) and others have shown that beta carotene autoxidation in vitro is dose dependent and dependent upon oxygen concentrations. At higher concentrations, it may function as a pro oxidant and can activate proteases. In addition to singlet oxygen, carotenoids are also thought to quench other oxygen free radicals. It is also suggested that beta carotene might react directly with the peroxyl radical at low oxygen tensions; this may provide some synergism to vitamin E which reacts with peroxyl radicals at higher oxygen tensions (Cotgreave et al 1988). Beta carotene (CAR) + LOO·® LOO-CAR· LOO-CAR· + LOO· ® LOO-CAR-OOL Carotenoids also have been reported to have a number of other biologic actions, including immuno-enhancement;inhibition of mutagenesis and transformation and regression of premalignant lesions. Lutein Lutein is a carotenoid, a natural antioxidant and a free radical scavenger found in highest levels in the eye. The eye is continually subjected to oxidative stress due to
light exposure and retinal metabolism and research indicates that Luteins chemical properties may retard the onset of degenerative and harmful effects in the eye. OTHER ANTIIOXIDANT Alpha-linolenic acid: Alpha-linolenic acid, an antioxidant that helps the body turn glucose into energy. Rutin: Rutin is a flavanoid glycoside also called rutoside, rectine-3-rutinoside and sophorin. Often refered to as a bioflavonoid, it is a powerful antioxidant found in both citrus and non-citrus sources. Retinoids: Retinol, retinoic acid but not retinyl palmitate or retinyl acetate all have antioxidant properties (Prasad, 1989). However, retinoids in general are not classified as antioxidants as they mainly function as antiproliferatives. Glutathione (GSH): GSH is synthesized intracellarly from cysteine, glycine and glutamate. In addition to its role as a substrate in GSH redox cycle, GSH is also a scavenger of hydroxyl radicals and singlet oxygen. It is capable of either directly scavenging ROI or enzymatically via glutathione peroxidase, as described previously. In addition, GSH is crucial to the maintenance of enzymes and other cellular componentsin as reduced state. GSH also has an important role in xenobiotic metabolism and leukotriene syntesis. It is found in milllimolar concentration in all human cells (Halliwell, 1994). The majority of GSH is synthesized in the liver, and approximately 40% is secreted in the bile. The biologic role of GSH in bile is believed to be defence against dietary xenobiotics and lipid peroxidation in the lumen of the gut and protection of the intestinal epitheilium from oxygen radical attack (AW,1994).
CoQ10: CoQ10 (Coenzyme Q10) is also known as ubiquinone. It is found in almost every living cell (hence the name ubiquitous) and is essential to energy production by the
mitochondria. Far beyond producing energy, CoQ10 can protect the body from destructive free radicals and enhance immune defense. Uric acid: Uric acid acts as an endogenous radical scavenger and antioxidant. It is present in about 0.5 mmol/L in bodys fluids and is the end product of purine metabolism. Uric acid is a powerful scavenger of singlet oxygen, peroxyl radical (ROO .) and OH radical (Halliwell, 1994). Albumin: Depending on the fact that albumin has one sulfhydryl group per molecule, it itself scavenges several free radicals (Halliwell, 1994) and thus can be considered as one of the primary extracellular defense systems. Albumin is an additional sacrificial antioxidant that can bind copper tightly and iron weakly to its surface. The bound metals would still be on its surface. The bound metals would still be available for participation in Haber-Weiss reaction but any generated .OH would immediately react with and be scavenged by albumin. The resultant protein damage is biologically insignificant because of the large amount of available albumin and free radicals would be inactivated before reacting with other more vital protein structures. Other plasma proteins namely ceruloplasmin and transferring have also shown antioxidant activity. Drugs: Several pharmaceutical agents have been found to exert an antioxidant effect (Reilly et al. 1991): • Xanthine oxidase inhibitors: e.g. allopurinol, folic acid. • NADPH inhibitors: e.g. adenosine, calcium channel blockers. • Superoxide dismutase’s • Catalases • Albumin • Inhibits of iron redox cycling: Deferoxamine, apotransferrin and ceruloplasmin Morphology Cyperus rotundus is described as a perennial sedge grass with slender, scaly creeping rhizomes, bulbous at from the tubers which are about 1-3 cm long, externally blackish in color and reddish characteristics odour. It has a thin stem that is of dark green in color. The stem is up to 25 cm tall. Leaves are long having 1/6 inch to 1/3 inch broad and sharp, linear also dark green surface. The nodes on the stem are thick that bears ½ inch diameter, oval shape rhizomes. Rhizomes are aromatic & white in color from inside and brown from
outside. Inflorecsences are small with 2-4 bracts, consisting of tiny flowers with a red three angled, Oblong-ovate, yellow in color and black when ripe. The plant flowers in summers & fruits in winter.
Fig: Cyperus rotundus
Fig: Flower
stems triangular cross section
Habitat: Cyperus rotundus indigenous to India but now found all over the world, it is considerable to be among the world as weeds and is especially prevalent in lamp places, agricultural areas, coastland, natural forest, riparian zones and water courses.
Parts used: Tubers or Rhizomes.
Botanical Classification: Kingdom Sub-kingdom Superdivision Division Class Sub-class Order
Plantae Tracheobionta Spermatophyta Magnoliophyta Liliopsida Commelinidae Poales
Family Genus Species
Cyperaceae Cyperus Rotundus
Nomenclature: Binomial Name: Cyperus rotundus L. Botanical Name: Cyperus rotundus linn Biological Name: Cyperus rotundus English Name: Nut grass Hindi Name: Motha, Nagormotha Sanskrit Name: Mustak Gujarati Name: Motha Synonyms: Chlorocyperus rotundus (L) Palla, Cyperus purpuro-variegatus Boeckeler, Cyperus Stoloniferum pallidus Boeckeler. Chemical Constituents: Steam distillation of the tubers and bulbous rhizome yields about 0.5-0.9% of an essential oil. There is also present an aromatic oil that is 0.5-0.6%. Besides this it contains followings • • • • • •
Alkaloids Minerals Vitamins Triterpenes Flavonoids Sugar
Ash contains followings • • • •
Calcium Phosphorous Sodium Carbonate
Action: Cyperus rotundus is active against kapha and pitta suppressant. It is a good skin disorder healer and also helps in early healing of wounds, provides strength to the
body and gears up the nervous system. Improves breast and its functioning, relieves from in and & work as anti inflammatory agent. It improves digestion and curbs infection in body. Also helps in uterine contraction. There is an emerging interest in the use of naturally occurring anti-oxidant for the preservation of food and in the management of number of patho-physiological condition, most of which involve free radical damage. Recent reports indicated that there was an inverse relationship between the dietary intake of anti-oxidant rich food and incidence of human diseases. Although the protective effect has been primarily attributed to the well known anti-oxidant such as Vitamin-C, Vitamin-E and β-Carotene but plants phenolic also play a vital role. Moreover, restrictions over the use of synthetic antioxidant like BHT and BHA in food further strengthen the concept of using naturally occurring compounds as anti-oxidant. Keeping this in view coupled with the fact that Cyperus rotundus has been investigated in the laboratory to study the mechanistic aspect i.e. antioxidant activity employing different free radicals scavenging method. The principal constituents of Cyperus rotundus are Gallic acid, ellagic acid, ethyl gallate, galloyl glucose, chebulagic acid, glucose, galactose, fructose & raminose. Other constituents are Ellagic acid;trimethyl ether of Gallic acid; trimethyl of chebulic acid; chebulagic acid; chebulinic acid; chebulic acid; terchubin acid;shikimic acid;dehydroshikimic acid; quince acid;triacontanoic acid; palmitic acid; betasitosterol; daucoterol; 1,3,6-trigallylglucose; 1,2,3,4,6-pentagallylglucose;corilagin; glu-cogallin;sennoside oxide;arabinose;
A;
fructose;
tannase; glucose;
polyphenol sucrose;
oxidase;
rhamnose;
ascorbic vitamin
acid
C;2alfa-
hydroxymicromeric acid; maslinic acid; 2alfa-hydro-xyursolic acid; terminoic acid;arjugenin; arjunolic acid;chebupentol; punicalagin; terflavinA; terchebulin Cyperus rotundus is Astringent, Tonic, and Expectorant & Laxative. It has lithotriptic, rejuvenative & antibacterial properties. Further the use of T. Billerica in herbal eye
drops helps in diseases like myopia, corneal opacity, and immature cataract, chronic & acute infective conditions. The fruit possesses antibacterial properties. It is employed in dropsy, piles and diarrhea. While using herbal eye drops containing Cyperus rotundus, encouraging results have been obtained in cases of myopia, corneal opacity, pterigium, and immature cataract, chronic and acute infective conditions. The fruit possesses myocardial depressive activity. Sore throat, Pharyngitis, laryngitis, cough, catarrh, bronchitis, gastric ulcers, hemorrhoids, chronic diarrhea, dysentery, parasites, cholelithiasis, ophthalmia, headache, alopecia and premature graying, edema, rheumatism (topical), wounds (topical) (Dash 1991, 9-10; Frawley and Lad 1986, 164; Kirtikar and Basu 1993, 1018-1019; Nadkarni 1976, 1203-04; Varrier 1996, 258) Due to growing interest to explore the presence of anti-oxidant in different medicinal plants, a number of research and investigation has been undertaken throughout the world. Compiling Literature search, I came to find out a wealth of information regarding anti-oxidant properties of different medicinal plants. The following list compiles medicinal plants of different countries of the word which showed high antioxidant properties in different experiments. Table 1: List of medicinal plants with prominent antioxidant properties
Sl.No . 1 2 3 4 5 6 7 7 9 10 11
Plant name
Country
Plant part
Croton celtidifolius Baill Cassia auriculata Tagetes lucida Rhus succedanea Tachigalia paniculata. Sphacele salviae Hamamelis virginiana L. Sophora japonica Tournefortia sarmentosa Thonningia sanguinea Burkea Africana
Brazil India Italy China Italy Chile Korea China Taiwan Africa Sub-Saharan
Barks Flowers Leaves Sap Leaves Aerial parts Bark Pericarps Stems Herbs Bark
12 13 14 15
Ilex paraguensis Rosa sp. (Rosa de Castillo) Chinchona sp(Copalquin) Rumex hymenosepalus(Cana
16
Agria) Marrubium vulgare (Mastranzo)
17
Helichrysum italicum
18
Inula viscosa
19
Forsythia suspensa
20
Poria cocos
21 22 23
C. chinensis Nymphaea lotus L. Acacia nilotica Linn.
24
Terminalia belerica Roxb.
25 26 27 28 29 30 31 32 33 34 35
Terminalia chebula Retz. Terminalia chebula Retz. Terminalia chebula Retz. Ricinus communis L. Smilax china Kalopanax pictus Nakai Catharanthus roseus, Thymus vulgaris Hypericum perforatum Artemisia annua Terminalia arjuna
36
Daphne gnidium
37
Eucalyptus globulus
38
Calycotome villosa
39
Artemisia arborescens
Africa New Mexico New Mexico New Mexico
Leaves Flowers Bark
New Mexico
Stems
New Mexico Mediterranean
Leaves
and China Mediterranean and China Mediterranean and China Mediterranean and China Mediterranean Pakistan Pakistan Pakistan, Bangladesh Pakistan Pakistan Pakistan Pakistan China Korea USA USA USA USA Srilanka Mediterranean area (Sardinia) Mediterranean area (Sardinia) Mediterranean area (Sardinia) Mediterranean area (Sardinia)
Herbs Herbs Herbs Herbs Herbs Flowers Delile beans Fruits Fruits, black Fruits, brown Fruit coat Leaves Root Stem barks Herbs Herbs Herbs Herbs Bark Methanol extract Essential oil Essential oil Essential oil & methanol extract
Mediterranean
Methanol
area (Sardinia)
extract Leaves
40
Calycotome villosa.
41
Rumex crispus L
Turkey
42 43 44 45 46 47 48 49 50 51
Copaifera reticulate Ducke. Bauhinia tarapotensis Pteleopsis hylodendron Stachitarpheta jamaicensis (L.) Tinospora cordifolia Andrographis paniculata Acanthus ilicifolius Camellia sinensis (L) Artemisia afra Jacq Artemisia abyssinica Schultz-Bip Juniperus procera Hoechst ex
South American Italy Pakistan Cuba India India India India Austria Austria
seeds Barks Leaves Stem bark Aerial parts Root Whole plant Leaves Root Essential oils Essential oils
Austria
Essential oils
China Mongolia Mongolia Mongolia Mongolia Mongolia Mongolia Mongolia Mongolia Mongolia Europe and North
Leaves Herbs Herbs Herbs Herbs Herbs Herbs Herbs Herbs Herbs
52 53 54 55 56 57 58 59 60 61 62
Endl Eucommia ulmoides Oliv Chamenerion angustifolium Equisetum arvense Gentiana decumbens Geranium pretense Lomatogonium carinthiacum Nonea poulla Phodococcum vitis-idaea Sphallerocarpus gracilis Stellera chamaejasme
63
Crataegus oxyacantha
64
Hamamelis virginiana
65
Hydrastis Canadensis
66 67 68 69
Artemisia douglasiana Besser Tilia argentea Desf ex DC Salvia triloba Camellia sinensis (Rize tea ) Camellia sinensis (Young shoot
70 71 72 73 74
tea) Rhazya stricta Decne Brandisia hancei Artemisia campestris Feijoa sellowiana Berg
America Europe and North America Europe and North America Argentina Turkey Turkey Turkey
Plant extract Plant extract Plant extract H2O extract H2O extract H2O extract H2O extract
Turkey
H2O extract
Saudi Arabia China Okinawa, Japan Tropical
Leaves Herbs Herbs Fruit
and
75 76 77 78 79
Coptis chinensis Paeonia suffruticosa Prunella vulgaris Senecio scandens Hypericum perforatum Linn
China China China China India
80
Areca catechu
China, Japan
81
Dendrobium plicatile
China, Japan
82
Juglans regia
China, Japan
83
Paeonia lactiflora
China, Japan
84
Psychotria serpens
Herbs Herbs Herbs Herbs Shoot Methanol extract Methanol extract H2O extract Methanol extract Water
China, Japan
and
methanol extracts Water and
85
Rhodiola sacra
China, Japan
methanol
86
Uncaria rhynchophylla
China, Japan
extracts H2O extract Water and
87
J. regia
China, Japan
methanol extracts Water extract Water extract Water extract Ethanolic
88 89 90
Morus alba Schisandra chinensis Cassia tora L
China, Japan China, Japan China
91
Ephemerantha lonchophylla
China
92 93 94 95 96 97
Lespedeza homoloba Mutisia friesiana Sanicula graveolens Solanum melongena Tinospora cordifolia Thonningia sanguinea
Japan Argentina Argentina India India Ghana: Argentina,
98 99 100 101
Achyrocline satureioides Lam. DC. Heterotheca inuloides Cass Ulmus davidiana Heterotheca inuloides
Uruguay,
extract Stems Water extract Water extract Flavanoids Roots Herbs Brazil Herbs
and Paraguay USA Korea Mexico
Flower Root bark Terpenoids
102 103 104
Pluchea sagittalis Lam.Cabr Cudrania cochinchinensis Curcuma longa
South America Taiwan India
Herbs Root bark Aqueous extract
The following chemical compound has been identified in Cyperus rotundus.
Gallic acid: 3, 4, 5-Trihydroxybenzoic acid: Formula: C7H6O5 Biological Activities: Analgesic, Antiallergenic, Antibronchitic, Anti-inflammatory, Antioxidant, Antiperoxidant, Antiviral, Astringent, Bacteristat, Bronchodilator, Immunosuppressant,
Structure:
Ellagic acid: Ellagic acid is a phenolic compound present in fruits and nuts including raspberries, strawberries and walnuts. It is known to inhibit certain carcinogen-induced cancers and may have other chemopreventive properties. The effects of ellagic acid on cell cycle events and apoptosis were studied in cervical carcinoma (CaSki) cells. Ellagic acid at a concentration of 10(-5) M induced G arrest within 48 h, inhibited overall cell growth and induced apoptosis in CaSki cells after 72 h of treatment. Activation of the cdk inhibitory protein p21 by ellagic acid suggests a role for ellagic acid in cell cycle regulation of cancer cells.
Ellagic acid Ethyl gallate: Formula: C6H2 (OH) 3COOC2H5; WT. 198.17 Synonyms: Gallic acid, ethyl ester; Ethyl 3, 4, 5-trihydroxybenzoate; 3,4,5Trihydroxybenzoic acid, ethyl ester
Other Constituents: Ellagic acid; trimethyl ether of gallic acid; trimethyl ethyl of chebulic acid; chebulagic acid; chebulinic acid; chebulic acid; terchubin acid;shikimic acid;dehydroshikimic acid; quinic acid;triacontanoic acid; palmitic acid; beta-sitosterol; daucoterol; 1,3,6trigallylglucose; 1,2,3,4,6-pentagallylglucose;corilagin; glu-cogallin;sennoside A; tannase; polyphenol oxidase; ascorbic acid oxide;arabinose; fructase; glucose; sucrose; rhamnose;vitamin C; 2alfa-hydroxymicromeric acid; maslinic acid; 2alfahydro-xyursolic
acid;
terminoic
acid;arjugenin;
arjunolic
acid;chebupentol;
punicalagin; terflavinA; terchebulin.
Constituents in Volatile Oil: Hexadecanoic
acid;
linoleic
acid;
9,12-octa-deeadienoic
acid;
heptadecane;octadecane; cis-alfa-santalol; 2,6-dimethyl hep-tadecane; 2,6-bis(1,1dimethylethyl)-4-methyl
phenol;
eicosane;
benzoic
acid;
pentadecane
etc.
4. Remedies and Uses Guide: Bohera is a stimulating astringent, and has a wide application in any condition of atony, prolapsed, and relaxation of the mucosa. For coughs, sore throats, laryngitis and dyspepsia the churna may be taken with honey. In the treatment of dry, irritative coughs Nadkarni recommends a linctus of equal parts Bohera, Saindhava (rock salt), Pippali (Piper longum), and Maricha (Piper nigrum) (1976, 1204).
The mature rhizome of Cyperus rotundus is effective in the treatment of diarrhea, dysentery and parasites, but in the latter case should be taken along with purgatives such as Senna (Cassia angustifolia) as Bohera can be constipate (Varier 1996, 258). A decoction of the fruit may be taken internally and can be used externally as eyewash in the treatment of ophthalmological disorders (Nadkarni 1976, 1204). Vaidya Mana Bhajracharya indicates that the fresh fruit pulp is used as a collyrium in the treatment of nontraumatic corneal ulcer (avranashukla) (1997, 85). Varier mentions that the oil from the seeds is trichogenous, and can be used topically for leucoderma and skin diseases (avranashukla) (1996, 258). Action & Uses in Ayurveda and Siddha: Rasam, Kashayam, Mathura vipakam, ushna veeryam, pitta kapha hararm, good for vision, hair. Internally for kasam, krimi, swarbhangam. Externally antiseptic, lotion. Paste for pitta swellings, eye diseases. Action & Uses in Unani: Tonic to brain and stomach liquefies matter, acts as astringent, expells touda and safra, dries ruthoobath, headache, piles, chronic diarrhea.
8. Modern Medical research: Scientific studies have shown that Cyperus rotundus lowers lipid levels in the liver and heart, and works well in preventing heart and liver fat congestion, which lowers the risk of disease associated with those organs. Preliminary studies have shown its effectiveness in inhibiting viral growth in leukemia patients, and other studies are showing promise in Cyperus rotundus ability to hinder the spread of the HIV virus.
Antimalarial: Four
lignans
(termilignan,
thannilignan,
hydroxy-3',4'-
[methylenedioxy] flavan, and anolignan B) possessed demonstrable antimalarial activity in vitro (Valsaraj et al 1997). Antidiarrhoeal: Methanolic extract of Cyperus rotundus shown antidiarrhoeal activity. (journal of Ethnopharmacology et al 2005, S.J Uddin, J.A Shilpi, M Alamgir, M.T Rahman, S.D Sarker. Antifungal: Four lignans (termilignan, thannilignan, hydroxy-3', 4’-[methylenedioxy] flavan, and anolignan B) possessed demonstrable antifungal activity in vitro (Valsaraj et al 1997).
1. PREPARATION OF THE PLANT SAMPLE FOR EXPERIMENTS 1.1 Collection and identification The plants Cyperus rotundus was collected from Jahangirnagar University, Savar, Dhaka area and identified taxonomically at the Department of Botany, Jahangirnagar University, Savar, Dhaka. 1.2 Drying of the plant samples The collected and identified plant samples were dried (after cutting and slicing where necessary) in the sun and finally in a mechanical dryer at 60 – 700C. 1.3 Grinding of the dried samples The dried samples were ground to coarse powder with a mechanical grinder (Grinding Mill) and powdered samples were kept in clean closed glass containers pending extraction. During grinding of sample, the grinder was thoroughly cleaned to avoid contamination with any remnant of previously ground material or other foreign matter deposited on the grinder. 1.4 Extraction of the dried powdered samples
Duration of extraction: 3 days Solvent used: Ethanol Volume of ethanol used: 800 ml Apparatus: •
A glass jar with plastic cover.
•
Solvent (ethanol 800 ml)
•
Aluminium foil.
Procedure: •
A glass made jar with plastic cover was taken and washed thoroughly The jar was rinsed with ethanol and dried
•
Then the dried sample of Cyperus rotundus was taken in the jar.
•
After that ethanol (800 ml) was poured into the jar up to 1-inch height above the sample surface as it can sufficiently cover the sample surface.
•
The plastic cover with aluminium foil was closed properly to resist the entrance of air into the jar.
•
This process was performed for 3 days. The jar was shaked in several times during the process to get better extraction.
The above procedure was applied similarly for the above mentioned all about dried powdered plant samples. 1.5 Filtration of the extracts Apparatus: •
Clean cotton
•
Beaker
Procedure: •
After the extraction process the Cyperus rotundus plant extract was filtered with sterilized cotton filter.
•
The cotton was rinsed with ethanol and fitted in a funnel.
•
The filtrate was collected in a beaker.
The above procedure was applied similarly for the above mentioned all about dried powdered plant samples. 1.6 Concentration of the plant extract The Cyperus rotundus plant extract was concentrated by evaporating the solvent using a water bath at a temperature of 60 0C. This procedure was applied similarly for the above mentioned all about dried powdered plant samples.
2. Determination of Antioxidant Compound 2.1.1 Determination of Total Phenol Principle: The content of total phenolic compounds in plant methanolic extracts was determined by Folin–Ciocalteu Reagent (FCR). The FCR actually measures a sample’s reducing capacity. The exact chemical nature of the FC reagent is not known, but it is believed to contain heteropolyphosphotunstates - molybdates. Sequences of reversible one- or two-electron reduction reactions lead to blue species, possibly (PMoW11O40)4-. In essence, it is believed that the molybdenum is easier to be reduced in the complex and electron-transfer reaction occurs between reluctant and Mo(VI): Mo(VI) + e → Mo (V)
Reagent: Folin – ciocalteu reagent Sodium carbonate (Na2CO3) Ethanol or Methanol Galic acid (Analytical or Reagent grade) Experimental procedure:
1. Take 1.0 ml of plant extract or standard of different concentration solution in a test tube. 2.
Add 5 ml of Folin – ciocalteu (Diluted 10 fold) reagent solution into the test tube.
3. Add 4 ml of Sodium carbonate solution into the test tube. 4. Incubate the test tube for 30 minutes at 20 0C to complete the reaction. (only applicable for standard) **Incubate the test tube for 1 hour at 20 0C to complete the reaction (Applicable for extract). 5. Then the absorbance of the solution was measured at 765 nm using a spectrophotometer against blank. 6. A typical blank solution contained ethanol or methanol. 7. The Total content of phenolic compounds in plant methanol extracts in gallic acid equivalents (GAE) was calculated by the following formula equation C = (c x V)/m Where: C = total content of phenolic compounds, mg/g plant extract, in GAE; c = the concentration of gallic acid established from the calibration curve, mg/ml; V = the volume of extract, ml; m = the weight of pure plant methanolic extract, g. 2.1.2 Determination of Flavonoid Content230 Reagent: Aluminium Chloride (AlCl3) Potassium Acetate Ethanol or Methanol Quercetin (Analytical or Reagent grade) Experimental procedure: 1. Take 1.0 ml of plant extract or standard of different concentration solution in a test tube. 2.
Add 3 ml of methanol into the test tube.
3. Add 200µl of 10% aluminium chloride solution into the test tube.
4. Add 200µl of 1M potassium acetate solution into the test tube. 5. Add 5.6 ml of distilled water into the test tube. 6. Incubate the test tube for 30 minutes at room temperature to complete the reaction. 7. Then the absorbance of the solution was measured at 415 nm using a spectrophotometer against blank. 8. A typical blank solution contained methanol. 9. The Total content of flavonoid compounds in plant methanol extracts in quercetin equivalents was calculated by the following formula equation C = (c x V)/m Where: C = total content of flavonoid compounds, mg/g plant extract, in quercetin; c = the concentration of quercetin established from the calibration curve, mg/ml; V = the volume of extract, ml; m = the weight of pure plant methanolic extract, g. 3. In Vitro Antioxidant Assay 3.1 Determination of Total Antioxidant Capacity237 Principle: The phosphomolybdenum method usually detects antioxidants such as ascorbic acid, some phenolics, α-tocopherol, and carotenoids. The phosphomolybdenum method was based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound and subsequent formation of a green phosphate/Mo (V) complex at acid pH. In essence, it is believed that the molybdenum is easier to be reduced in the complex and electron-transfer reaction occurs between reluctant and Mo (VI) and the formation of a green phosphate/Mo (V) complex with a maximal absorption at 695 nm. Mo(VI) + e → Mo (V)
Reagent: Concentrated H2SO4 (98%) Sodium Phosphate (Na3PO4) Ammonium Molybdate Ascorbic acid (Analytical or Reagent grade)
Experimental procedure: 1. Take 300Âľl of plant extract or standard of different concentration solution in a test tube. 2. Add 3 ml of reagent solution into the test tube. 3. Incubate the test tube at 950C for 90 minutes to complete the reaction. 4. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer against blank after cooling to room temperature. 5. A typical blank solution contained 3 ml of reagent solution and the appropriate volume (300Âľl) of the same solvent used for the sample, and it was incubated under the same conditions as the rest of the samples solution. 6. The antioxidant activity is expressed as the number of equivalents of ascorbic acid .and was calculated by the following formula equation A = (c x V)/m Where: A = total content of Antioxidant compounds, mg/g plant extract, in Ascorbic acid; c = the concentration of Ascorbic acid established from the calibration curve, mg/ml; V = the volume of extract, ml; m = the weight of pure plant methanolic extract, g. 3.2 DPPH (1, 1-diphenyl-2-picrylhydrazyl) Scavenging capacity Assay233 Principle: The 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) has been widely used to evaluate the free radical scavenging capacity of antioxidants. DPPH free radical is reduced to the corresponding hydrazine when it reacts with hydrogen donors. DPPH can make stable free radicals in aqueous or methanol solution. With this method it was possible to determine the antiradical power of an antioxidant activity by measurement of the decrease in the absorbance of DPPH at 517 nm. Resulting from a color change from purple to yellow the absorbance decreased when the DPPH was scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH molecule. In the radical form this molecule had an absorbance at 517 nm which disappeared after acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule.
Reagent: DPPH (1,1-diphenyl-2-picrylhydrazyl) Ethanol or Methanol Ascorbic acid (Analytical or Reagent grade) Experimental procedure: 1. Take 200µl of plant extract or standard of different concentration solution in a test tube. 2.
Add 2 ml of reagent solution into the test tube.
3. Incubate the test tube for 30 minutes to complete the reaction. 4. Then the absorbance of the solution was measured at 515 nm using a spectrophotometer against blank. 5. A typical blank solution contained ethanol or methanol. 6. The percentage (%) inhibition activity was calculated from the following equation {(Ao – A1)/Ao} X 100 Where, A0 is the absorbance of the control, and A1 is the absorbance of the extract/standard. 7. Then % inhibitions were plotted against log concentration and from the graph IC50 was calculated. 3.3 Reducing Power Capacity Assessment240 Principle: In this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of antioxidant samples. The reducing capacity of a compound may serve as a significant indicator of its potential
antioxidant activity. The presence of reductants such as antioxidant substances in the antioxidant samples causes the reduction of the Fe 3+/ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Reagent: Potassium ferricyanide [K3Fe(CN)6] Trichloro Acetic acid Ferric Chloride (Fe3Cl) Ascorbic acid (Analytical or Reagent grade) Experimental procedure: 1. Take 2.0 ml of plant extract or standard of different concentration solution in a test tube. 2. Add 2.5 ml of Potassium ferricyanide [K3Fe (CN)6], 1% solution into the test tube. 3. Incubate the test tube for 10 minutes at 500C to complete the reaction. 4. Add 2.5 ml of Trichloro Acetic acid, 10% solution into the test tube. 5. Centrifuged the total mixture at 3000 rpm for 10 min. 6.
2.5 ml supernatant solution was withdrawn from the mixture and mix with 2.5 ml of distilled water.
7. Add 0.5 ml of Ferric chloride (Fe3Cl), 0.1% solution. 8. Then the absorbance of the solution was measured at 700 nm using a spectrophotometer against blank. 9. A typical blank solution contained the same solution mixture without plant extract or standard and it was incubated under the same conditions as the rest of the samples solution. 10. Also take the absorbance of the blank solution was measured at 700 nm against the solvent used in solution preparation. 11. Increased absorbance of the reaction mixture indicated increase reducing power. The percentage (%) Reducing capacity was calculated from the following equation. {(Am – Ab)/Ab} X 100 Where, Am is the absorbance of the reaction mixture, and Ab is the absorbance of the blank.
3.4 Cupric Reducing Antioxidant Capacity (CUPRAC) Principle: The chromogenic oxidizing reagent of the developed CUPRAC method, that is, bis (neocuproine) copper (II) chloride [Cu (II)-NC], reacts with polyphenols [Ar (OH) n] in the manner 2n Cu(Nc)22+ + Ar(OH)n = 2n Cu(Nc)2+ + Ar(=O)n + 2n H+ Where the liberated protons may be buffered with the relatively concentrated ammonium acetate buffer solution. In this reaction, the reactive Ar-OH groups of polyphenols are oxidized to the corresponding quinones and Cu (II)-Nc is reduced to the highly colored Cu (I)-Nc chelate showing maximum absorption at 450 nm. In this reaction, each flavonoid (in the aglycon form) having n phenolic -OH groups theoretically acts as a 2n-e donor. Reagent: Cupric Chloride (CuCl2.2H2O) Neocaproin Ammonium acetate buffer, pH 7.0 Ascorbic acid (Analytical or Reagent grade)
Preparation of 0.01 M solution of Cupric Chloride (CuCl2.2H2O) Accurately weighing 0.4262 gm of CuCl2.2H2O and take place a 250 ml volumetric flask and adjust the volume with distilled water. Preparation of ammonium acetate buffer, pH 7.0 Accurately weighing 19.27 gm of ammonium acetate and take place a 250 ml volumetric flask and adjust the volume with distilled water. Preparation of 0.0075 M solution of Neocaproin
Accurately weighing 0.039 gm of Neocaproin and take place a 25 ml volumetric flask and adjust the volume with 96% of ethanol.
Preparation of Standard solution: Take 0.025 gm ascorbic acid and dissolved it into 5 ml of water. The concentration of this solution is 5µg/µl of ascorbic acid. This solution is called stock solution. Then prepared the experimental concentration from this stock solution by the following manner: Concentration
Solution taken Solution taken Adjust
the Final
(µg/ml)
from
by volume
stock from others
solution
volume
distilled water
200
80µl
-
100
-
1
1.92ml
2.0 ml
ml 1 ml
2.0 ml
ml 1 ml
2.0 ml
ml 1 ml
2.0 ml
(200µg/ml) 50
-
1 (100µg/ml)
25
--
1 (50µg/ml)
5
-
200µl
800µl
1.0 ml
(25µg/ml) Preparation of Extract solution: Take 0.025 gm of plant extract and dissolved it into 5 ml of methanol. The concentration of this solution is 5µg/µl of plant extract. This solution is called stock solution. Then prepared the experimental concentration from this stock solution by the following manner: Concentration
Solution taken Solution taken Adjust
the Final
(µg/ml)
from
by volume
stock from others
solution
volume distilled water
200
80µl
-
100
-
1
1.92ml ml 1 ml
2.0 ml 2.0 ml
(200µg/ml) 50
-
1
ml 1 ml
2.0 ml
ml 1 ml
2.0 ml
(100µg/ml) 25
-
1 (50µg/ml)
5
-
200µl
800µl
1.0 ml
(25µg/ml)
Experimental procedure: 1. Take 500µl of plant extract or standard of different concentration solution in a test tube. 2. Add 1.0 ml of 0.01M CuCl2.2H2O solution into the test tube. 3. Add 1.0 ml of ammonium acetate buffer, pH 7.0 into the test tube. 4. Add 1.0 ml of 0.0075 ml of neocaproin solution into the test tube. 5. Finally add 600 µl of distilled water and adjust the final volume of the mixture is 4.1 ml. 6. Incubate the total mixture for 1 hour at room temperature. 7. Then the absorbance of the solution was measured at 450 nm using a spectrophotometer against blank. 8. A typical blank solution contained the reagent mixture without extract or standard and treated as same. 9. The molar absorptivitiy of the CUPRAC method for each antioxidant was found from the slope of the calibration line concerned. Ref: RESAT. A., KUBILAY. G., MUSTAFA. O. AND SALIHA. E. K. Novel Total Antioxidant Capacity Index for Dietery Polyphenols and Vitamin C and E, Using their Cupric Ion Reducing Capability in the presence of Neocuproine: CUPRAC method, J. Agric. Food Chem. 2004, 52, 79707981. 1. RESULTS AND DISCUSSIONS In the present work Cyperus rotundus were screened for their free radical scavenging and in vitro antioxidant properties. The used antioxidant assay methods are serially: Total antioxidant capacity, reducing power assessment. In addition, the
total phenolic and flavonoid contents of these plants were determined by comparing with Galic acid and quercetin respectively flowing Folin–Ciocalteu Reagent (FCR) and Flavonoid content determination method. The observed results are summarized in the subsequent paragraphs. 1.1 In vitro Antioxidant Component Assay 1.1.1 Total Phenolic Compound and Flavonoid Assay The systematic literature collection, pertaining to this investigation indicates that the plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators. Therefore, it is worthwhile to determine their total amount in the plants chosen for the study. Flavonoids as one of the most diverse and widespread group of natural compounds, are likely to be the most important natural phenolics. These compounds possess a broad spectrum of chemical and biological activities including radical scavenging properties. Therefore, the content of both groups of phenolics is also determined in the extracts. The content of total phenolics in the methanolic plant extracts is determined using the Folin–Ciocalteu assay, calculated from regression equation of calibration curve (y = 0.013x + 0.127, r2 = 0.988) and is expressed as Gallic acid equivalents (GAE). It can be observed that the content of phenolics in the extracts correlates with the antioxidant activity in Cyperus rotundus 40.90 mg/g GAE) and the content of flavonoids (mg/g) in quercetin equivalents and showed 14.55 Table 1: Total Phenol Determination Standard: Galic Acid Conc 200 150 100 50 0
Abs 3.130 2.020 1.540 1.021 0.000
Abs 2.610 1.990 1.590 1.040 0.000
Abs 2.950 2.350 1.450 0.950 0.000
AVG 2.897 2.120 1.527 1.004 0.000
Calibration Curve of Galic acid y = 0.0138x + 0.1275 R2 = 0.9881
Absorbance at 695 nm
3.000
2.000
1.000
0.000 0
50
100
150
200
Concentration microgram/ml
Fig: Galic Acid Calibration curve Total Phenolic compound in Cyperous rotundus Conc 0.0002
Abs 0.221
Abs 0.239
Abs 0.240
AVG 0.233
ST. DE 0.011
Total Antioxidant Equivalent to Galic Acid Formula: A = (c x V)/m Calculation y = 0.013 + 0.127; Y= 0.233, M=0.013, C=0.127 A = Total content of Antioxidant compounds, mg/g plant extract, in Ascorbic acid c = The concentration of Ascorbic acid established from the calibration curve, mg/ml, 0.0082 V = The volume of extract, ml; 1.00 m = The weight of pure plant methanolic extract, g 0.0002
A= 40.90 So Cyperus rotundus 40.90 mg/g Galic Acid Total Flavonoid Content Determination standard: Quercetin Conc 100 50 25 12.5 0
Abs 1.060 0.342 0.210 0.117 0.000
Abs 0.939 0.352 0.183 0.120 0.000
Abs 0.985 0.339 0.195 0.112 0.000
AVG 0.995 0.344 0.196 0.116 0.000
Calibration Curve of Quercetin
Absorbance at 695 nm
1.000
y = 0.0098x - 0.0364 R2 = 0.9724
0.750
0.500
0.250
0.000 0
25
50
75
100
Concentration microgram/ml
Fig: Quercetin Calibration curve Total Flavonoid content assay Cyperus rotundus Conc 0.005
Abs 0.616
Abs 0.635
Abs 0.605
AVG 0.619
ST. DE 0.015
Calculation y = 0.009 - 0.0 3 6
Y=
0.619, m=0.009, c=0.036
Total Antioxidant Equivalent to Quercetin Formula: A = (c x V)/m A = Total content of Antioxidant compounds, mg/g plant extract, in Ascorbic acid c = The concentration of Ascorbic acid established from the calibration curve, mg/ml, 0.0727 V = The volume of extract, ml; 1.00 m = The weight of pure plant methanolic extract, g, 0.005
A=
Cyperous rotundus
Plant
14.5 5
14.54 8
mg/g Quercetin
name Local name
(Family) Cyperus rotundus Mutha
Plant
Total phenol mg/g plant parts extract (in GAE) Rhizome 40.90 Âą 0.10
Total Flavonoid mg/g plant extract (in Quercetin) 14.55 Âą 0.02
(Cyperaceae) Each value is expressed as mean Âą SD (n = 3). Phenolic compounds are commonly found in both edible and inedible plants, and they have been reported to have multiple biological effects, including antioxidant activity. The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. Crude extracts of fruits, herbs, vegetables, cereals, nuts, and other plant materials rich in phenolics are increasingly of interest in the food industry. The importance of the antioxidant constituents of plant materials in the maintenance of health and protection from coronary heart disease and cancer is also raising interest among scientists, food manufacturers, and consumers. It is suggested that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans, when up to 1.0 g was daily ingested from a diet rich in fruits and vegetables. Phenolic compounds form one of the main classes of secondary metabolites. They display a large range of structures and are responsible for the major organoleptic characteristics of plant-derived foods and beverages, particularly color and taste properties. They also contribute to the nutritional qualities of fruits and vegetables. Among these compounds, flavonoids constitute one of the most ubiquitous groups of plant phenolics. Owing to their importance in food organoleptic properties and human health, a better understanding of their structures and biological activities indicates their potentials as therapeutic agents and also for predicting and controlling food quality. Due to the variety of pharmacological activities in the mammalian body, flavonoids are more correctly referred as “nutraceuticalsâ€?. 1.2 In vitro Antioxidant Assay Antioxidants in foods are important for our health and eating five to seven serves each day of fresh fruit and vegetables has been show to help protect against heart disease, cancers and other diseases. (Ref: CSIRO) The consumption of fruits, vegetables, red wines, juices, etc (rich in antioxidant compounds) provides protection cancer and cardiovascular and cerebrovascular diseases. Recent research
shows the risk of cancer and heart disease is considerably lower in people who consume 5-7 serves of antioxidant-rich fruit and vegetables. (Ref: CSIRO) This protection can be explained by the capacity of these antioxidants to scavenge free radicals, which are responsible for the oxidative damage of lipids, proteins and nucleic acids. Therefore, in recent years, there has been a worldwide trend towards the use of the natural phytochemicals present in berry crops, teas, herbs, oilseeds, beans, fruits and vegetables since they contain plenty of antioxidant. It is well known that free radicals cause autoxidation of unsaturated lipids in food. In addition, antioxidants are known to interrupt the free-radical chain of oxidation and to donate hydrogen from phenolic hydroxyl groups, thereby, forming stable free radicals, which do not initiate or propagate further oxidation of lipids.
1.2.1 Total antioxidant activity Total Antioxidant capacity assay of Standard: Ascorbic acid Conc. 500 200 100 50 5 0
Abs 4.000 1.237 0.429 0.205 0.045 0.000
Abs 3.913 0.936 0.414 0.215 0.043 0.000
Abs 3.925 1.135 0.425 0.210 0.033 0.000
AVG 3.946 1.103 0.423 0.210 0.040 0.000
Calibration Curve of Ascorbic acid
Absorbance at 695 nm
1.250
y = 0.0054x - 0.028 R2 = 0.9826
1.000 0.750 0.500 0.250 0.000 0
50
100
150
200
250
Concentration microgram/ml
Fig: Ascorbic Acid Calibration Curve Total Antioxidant capacity assay of Cyperus rotundus: Conc. 200
Abs 0.366
Abs 0.389
Abs 0.374
AVG 0.376
ST. DE 0.007
Calculation: y = 0.005x - 0.028 Y=0.376, m=0.005, C=0.028 Total Antioxidant Equivalent to Ascorbic Acid Formula: A = (c x V)/m A = Total content of Antioxidant compounds, mg/g plant extract, in Ascorbic acid c = The concentration of Ascorbic acid established from the calibration curve, mg/ml, 0.0809 V = The volume of extract, ml; 0.3 m = The weight of pure plant methanolic extract, g, 0.0002 Cyperous rotundus 121.3 mg/g Ascorbic Acid
Plant
name Plant parts
(Family) Cyperus rotundus
Rhizome
Total antioxidant capacity Equiv. to ascorbic acid mg/g plant extract 121.3 ± 0.12
(Cyperaceae) Each value is expressed as mean ± SD (n = 3). The phosphomolybdenum method usually detects antioxidants such as ascorbic acid, some phenolics, α-tocopherol, and carotenoids. The phosphomolybdenum method was based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound. In essence, it is believed that the molybdenum is easier to be reduced in
the complex and electron-transfer reaction occurs between reductants and Mo(VI) and the formation of a green phosphate/Mo(V) complex with a maximal absorption at 695 nm. The assay is successfully used to quantify vitamin E in seeds and, being simple and independent of other antioxidant measurements commonly employed, it was decided to extend its application to plant extracts. Moreover, it is a quantitative one, since the antioxidant activity is expressed as the number of equivalents of ascorbic acid. The study reveals that the antioxidant activity of the extract is in the increasing trend with the increasing concentration of the plant extract.
1.2.2. Reducing power Assessment For the measurements of the reductive ability, it has been investigated from the Fe3+→Fe2+ transformation in the presence of extract samples using the method followed by. In this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of antioxidant samples. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The presence of reductants such as antioxidant substances in the antioxidant samples causes the reduction of the Fe 3+/ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the activity of antioxidants has been assigned to various mechanisms such as prevention of chain initiation, binding of transition-metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging depicts the reductive effect of plant extracts Similar to the antioxidant activity, the reducing power of plant extracts increased with increasing dosage. All the doses showed significantly higher activities than the control exhibited greater reducing power,
indicating that all plant extracts consist of hydrophilic polyphenolic compounds that cause the greater reducing power. Reducing power assessment of Cyperous rotundus Conc . Blank 0 5 25 50 100 200
Abs 1 0.213 0.000 0.178 0.198 0.217 0.264 0.371
Abs 2 0.156 0.000 0.182 0.215 0.235 0.289 0.378
Abs 3 0.168 0.000 0.185 0.205 0.254 0.279 0.398
Avg 0.179 0.000 0.182 0.206 0.235 0.277 0.382
Std dv 0.030 0.000 0.004 0.009 0.019 0.013 0.014
% Reducing Power 0.00 0.00 1.49 15.08 31.47 54.93 113.59
Reducing power assessment of Cyperous rotundus
% Reducing power
300.00 250.00 200.00 150.00 100.00 50.00 0.00 0
50
100
150
200
250
Concentration (microgram/ml)
Fig 5: % Reducing power assessment of plant extract Reducing power assessment of Ascorbic Acid: Conc Blank 0 5 50 100 200 500
Abs 1 0.250 0.000 0.285 0.421 0.883 1.424 3.010
Abs 2 0.235 0.000 0.287 0.425 0.887 1.427 3.017
Abs 3 0.245 0.000 0.285 0.422 0.885 1.425 3.015
Avg 0.243 0.000 0.286 0.423 0.885 1.425 3.014
Std dv 0.008 0.000 0.001 0.002 0.002 0.002 0.004
% Reducing Power 0.000 0.000 17.397 73.699 263.699 485.753 1138.630
Reducing power assessment of Ascorbic acid
% Reducing power
1000.000 750.000 500.000 250.000 0.000 0
50
100
150
200
250
Concentration (microgram/ml)
Fig 6: % Reducing power assessment of Ascorbic acid Reducing power assessment of Galic acid Conc. Blank 0 5 50 100 200 500
Abs 1 0.250 0.000 0.290 0.775 1.245 2.050 2.395
Abs 2 0.235 0.000 0.295 0.772 1.230 2.030 2.450
Abs 3 0.245 0.000 0.287 0.775 1.235 2.025 2.420
Avg 0.243 0.000 0.291 0.774 1.237 2.035 2.422
Std dv 0.008 0.000 0.004 0.002 0.008 0.013 0.028
% Reducing Power 0.000 0.000 19.452 218.082 408.219 736.301 895.205
Reducing power assessment of Galic acid
% Reducing power
1000.000 750.000 500.000 250.000 0.000 0
50
100
150
200
250
Concentration (microgram/ml)
Fig 7: % Reducing power assessment of Gallic acid Reducing power assessment of Quercetin Conc Blank 0 5 50
Abs 1 0.250 0.000 0.284 0.953
Abs 2 0.235 0.000 0.280 0.945
Abs 3 0.245 0.000 0.285 0.950
Avg 0.243 0.000 0.283 0.949
Std dv 0.008 0.000 0.003 0.004
% Reducing Power 0.00 0.00 16.30 290.14
100 200 500
1.300 2.100 3.200
1.250 2.150 3.050
1.350 2.050 3.150
1.300 2.100 3.133
0.050 0.050 0.076
434.25 763.01 1187.67
Reducing power assessment of Quercetin
% Reducing power
1000.00 750.00 500.00 250.00 0.00 0
50
100
150
200
250
Concentration (m icrogram/m l)
Fig 8: % Reducing power assessment of Quercetin
Reducing power assessment of Cyperous rotundus vs Starndards
% Reducing power
1000.00
750.00
500.00
250.00
0.00 0
Quercetin
50
100
As corbic acid
150
G a lic a cid
200
250
C ype rous rotundus
Concentration (microgram/ml)
Fig 9: % Reducing power assessment of plant extract and standards Earlier authors have observed a direct correlation between antioxidant activity and reducing power of certain plant extracts. The reducing properties are generally associated with the presence of reductones, which have been shown to exert
antioxidant action by breaking the free radical chain by donating a hydrogen atom. Reductones are also reported to react with certain precursors of peroxide, thus preventing peroxide formation. Our data on the reducing power of the tested extracts suggest that it is likely to contribute significantly towards the observed antioxidant effect. However, the antioxidant activity of antioxidants has been attributed by various mechanisms, among which some of them are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging. Like the antioxidant activity, the reducing power of ethanol extracts increases with increasing sample.
1.2.3 DPPH radical scavenging activity DPPH free radical is reduced to the corresponding hydrazine when it reacts with hydrogen donors. Free radicals are known to be a major factor in biological damages, and DPPH has been used to evaluate the free radical scavenging activity of natural antioxidants. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) has been widely used to evaluate the free radical scavenging capacity of antioxidants 233,234,235. It is well known that free radicals are able to induce lipid per oxidation. DPPH can make stable free radicals in aqueous or methanol solution. The determination of scavenging stable DPPH was a very fast method to evaluate the antioxidant activity of the extracts. With this method it was possible to determine the antiradical power of an antioxidant activity by measurement of the decrease in the absorbance of DPPH at 517 nm. Resulting from a color change from purple to yellow the absorbance decreased when the DPPH was scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH molecule. In the radical form this molecule had an absorbance at 517 nm which disappeared after acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule236. The scavenging effect of methanol extracts and standards with the DPPH radical is in the following order (according to IC50 value):
The reduction capability of the DPPH radical is determined by the decrease in its absorbance at 517 nm, induced by antioxidants. In order to evaluate the antioxidant potency through free radical scavenging by the test samples, the change of optical density of DPPH radicals was monitored. Figure10 and 11 showed the decrease in absorbance of DPPH radical due to the scavenging ability of soluble solids in different concentrations of plant extract and standard ascorbic acid. DPPH Scavenging activity of Cyperus rotundus: Conc. 0.000 0.699 1.000 1.699 2.000 2.699 IC 50
Abs 1 1.219 1.163 1.143 1.132 1.105 0.940
Abs 2 1.230 1.162 1.139 1.098 1.001 0.853
Abs 3 1.250 1.165 1.141 1.009 0.899 0.798
Avg 1.233 1.163 1.141 1.080 1.002 0.864
Std dv 0.009 0.001 0.001 0.037 0.059 0.041
Absorbance at 517 nm
DPPH scavenging activity of Cyperous rotundus 1.500 1.000 0.500 0.000 0.000
0.500
1.000
1.500
2.000
2.500
Concentration (microgram/ml)
Fig: Absorbance of DPPH of Cyperus rotundus DPPH Scavenging activity of Ascorbic Acid
3.000
% Inhibition 0.000 5.650 7.461 12.436 18.762 29.954 > 2.69
Conk 0.000 0.699 1.000 1.699 2.000 2.699 IC 50
Abs 1 1.830 1.314 1.145 0.229 0.056 0.052
Abs 2 1.892 1.413 1.154 0.230 0.065 0.045
Abs 3 1.883 1.434 1.135 0.227 0.075 0.053
Avg 1.868 1.387 1.145 0.229 0.065 0.050
Std dv 0.034 0.064 0.010 0.002 0.010 0.004
% Inhibition 0.000 25.763 38.733 87.761 96.503 97.324 1.16
DPPH scavenging activity of Ascorbic acid Absorbance at 517 nm
2.000 1.500 1.000 0.500 0.000 0.000
0.500
1.000
1.500
2.000
2.500
3.000
Concentration (microgram/ml)
Fig13: Absorbance ofFig12: DPPHAbsorbance of standardof DPPH of plant extract At the concentration of 0.699, 1.000, 1.699, 2.000 and 2.699µg/ml, methanolic extract of Cyperus rotundus scavenged 5.650%, 7.461%, 12.436%, 18.762% and 29.954%; DPPH free radical. Whereas ascorbic acid exhibited 25.76%, 38.73%, 87.76%, 96.50% and 97.32% respectively at the same concentration. IC50 is the concentration of methanolic extracts from Cyperus rotundus to quench 50% DPPH under the chosen experimental conditions. The IC50 of Cyperus rotundus extracts to quench DPPH was 2.69 µg/ml, where as for ascorbic acid was 1.16 µg/ml. This activity is increased by increasing the concentration of the sample extract.
DPPH scavenging activity of Cyperous rotundus vs Standard
% Inhibition
100.000
50.000
0.000 0.000
0.500
1.000
As corbic a cid
1.500
2.000
2.500
3.000
C yperous rotundus
Concentration (microgram/ml)
Fig 14: % inhibition of DPPH scavenging effects of plant extract From Fig.14 we observe that a dose–response relationship is found in the DPPH radical scavenging activity; the activity increased as the concentration increased for each individual plant species. The involvement of free radicals, especially their increased production, appears to be a feature of most, if not all human diseases, including cardiovascular disease and cancer. It has been found that cysteine, glutathione, ascorbic acid, tocopherol, flavonoids, tannins, and aromatic amines (pphenylene diamine, p-aminophenol, etc.), reduce and decolourise DPPH by their hydrogen donating ability. Phenolic compounds of the plant extracts are probably involved in their antiradical activity.
1.2.4 Cupric reducing antioxidant capacity (CUPRAC): The chromogenic oxidizing reagent of the developed CUPRAC method, that is, bis (neocuproine) copper (II) chloride [Cu (II)-Nc], reacts with polyphenols [Ar (OH) n] in the manner 2n Cu(Nc)22+ + Ar(OH)n = 2n Cu(Nc)2+ + Ar(=O)n + 2n H+
Where the liberated protons may be buffered with the relatively concentrated ammonium acetate buffer solution. In this reaction, the reactive Ar-OH groups of polyphenols are oxidized to the corresponding quinones and Cu (II)-NC is reduced to the highly colored Cu (I)-NC chelate showing maximum absorption at 450 nm. In this reaction, each flavonoid (in the aglycon form) having n phenolic -OH groups theoretically acts as a 2n-e donor. Table 7: CUPRAC assessment of Cyperus rotundus and standards methanolic extract at different concentration CUPRAC assessment of Cyperus rotundus Conc. 0 5 25 50 100 200
Abs 1 0.000 0.069 0.119 0.236 0.414
Abs 2 0.000 0.075 0.135 0.245 0.485
Abs 3 0.000 0.082 0.125 0.253 0.462
Avg 0.000 0.075 0.126 0.245 0.454
Std dv 0.000 0.007 0.008 0.009 0.036
0.777
0.784
0.791
0.784
0.007
CUPRAC assessment of Ascorbic Acid Conc.
Abs 1
Abs 2
Abs 3
Avg
Std dv
0 5 25 50 100 200
0.000 0.049 0.115 0.220 0.335 0.709
0.000 0.065 0.145 0.256 0.396 0.725
0.000 0.052 0.132 0.245 0.254 0.742
0.000 0.055 0.131 0.240 0.328 0.725
0.000 0.009 0.015 0.018 0.071 0.017
CUPRAC Assessment of Galic Acid Conc.
Abs 1
Abs 2
Abs 3
Avg
Std dv
0 5 25 50 100 200 Conc
0.000 0.525 1.020 2.080 2.790 3.100 Abs 1
0.000 0.515 1.010 1.997 2.590 2.980 Abs 2
0.000 0.509 1.001 1.972 2.300 2.790 Abs 3
0.000 0.516 1.010 2.016 2.560 2.957 Avg
0.000 0.008 0.010 0.057 0.246 0.156 Std dv
0.000 0.401 0.800 1.433 2.343 2.950
0.000 0.027 0.015 0.112 0.150 0.170
CUPRAC Assessment of Quercetin 0 5 25 50 100 200
0.000 0.430 0.815 1.560 2.490 3.120
0.000 0.398 0.785 1.350 2.190 2.950
0.000 0.376 0.801 1.390 2.350 2.780
3.500
Absorbance at 450 nm
3.000 2.500 Querc etin
2.000
Galic ac id
1.500
Cyperous rotundus
1.000
A s c orbic ac id
0.500 0.000 0
50
100
150
200
Concentration (microgram/ml)
Figure 10: Absorbance of Cyperus rotundus and standards at 450 nm Reduction of Cu2+ ions was found to rise with increasing concentrations of the extraction of rhizome parts of Cyperus rotundus as evident from a rise in absorbance. All extracts produced a dose dependent reduction of Cu2+ in a way similar to the standard antioxidant ascorbic acid. The fractions showed weak to moderate Cu 2+ ion reducing capacity. However, chloroform fraction (CFCB) showed highest Cu2+ ion reducing capacity (Figure 12).