Introduction

INTRODUCTION

The origin of disease is multifactorial in nature and is now being understood

as a violation in the basic hom(o)eostatic balance phenomenon in the body. It is
increasingly realized that the majority of the present day diseases are due to the shift
in the balance of the pro-oxidant and the antioxidant hom (o)eostatic phenomenon in
the body. Pro-oxidant condition dominates either due to the increased generation of
the free radicals caused by excessive oxidative stress of the current life, or due to the
poor scavenging/quenching in the body caused by depletion of the dietary
antioxidants (Rakesh and Rajesh, 2006).

So it becomes very essential to aim at the balancing of oxidants in the body.

During the last few decades, there has been a considerable growth in the field of
herbal medicine. It is becoming popularized in both developing and developed
countries due to its natural origin and lesser side effects (Khopde et al., 2001, Naik et
al., 2003). It is classified in the ancient Indian system of medicine (Ayurveda) as a
Rasayana. A group of plant derived drugs that improves overall physical and mental
health and puts off diseases by rejuvenating the body during the incapacitated
conditions of disease or disorder.

1.2 Free radicals

Free radicals are molecules containing unpaired electrons. The unpaired electron is
highly reactive. It either burns a molecule (causes oxidative damage) or is passed
from one molecule to another molecule. In that process it turns the recipient into a
free radical and neutralizes the donor. Most of the free radicals are produced in the
mitochondria and most of the free radical damage is done to mitochondrial
membranes and mitochondrial DNA (Rakesh and Rajesh, 2006).

Free radicals can also be produced by many cells as a protective mechanism.

Neutrophils produce free radicals to attack and destroy pathogens, while the liver uses
free radicals for detoxification (Lunec et al., 2002). However, the presence of free
radicals within the body can also have a significant role in the development and
progression of many disease processes like heart disease, congestive heart failure,

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hypertension, cerebrovascular accidents, and diabetic complications (Chen et al.,

2002). Excess free radicals can result from tissue damage and hypoxia, over exposure
to environmental factors (eg: smoking, ultraviolet radiation, and pollutants) and due
to lack of antioxidants or destruction of free radical scavengers. When the production
of damaging free radicals exceed the capacity of the body‟s antioxidant defenses to
detoxify them, a condition known as oxidative stress occurs (Kohen and Nyska,
2002).

As free radicals play an important role in the diseases scenario of an

individual, a thorough understanding of the various physiologically significant free
radicals is of paramount importance (Rakesh and Rajesh, 2006).

1.3 Processes of Free Radical Formation

- -.
Radical formation by electron transfer: A + e  A
. .
Radical formation by homolytic fission: X: YX + Y
- +
Ion formation by heterolytic fission: X: YX + Y

1.4 Types of free radicals

-
Reactive oxygen and nitrogen free radicals include superoxide (O 2 ), hydroxyl
(•OH), peroxyl (RO2•), alkoxyl (RO•), hydroperoxyl (HO2•), nitric oxide (NO) and
nitrogen dioxide (•NO2) radicals. Oxygen and nitrogen free radicals can be converted
to other non-radical reactive species, such as hydrogen peroxide (H 2O2),
-
hypochlorous acid (HOCl), and peroxynitrite (ONOO ). The reactive oxygen species
(ROS) are continuously produced during normal physiological events and are
removed by antioxidant defense mechanisms. There is a balance between the
generation of ROS and inactivation of ROS by the antioxidant system in organisms.

Under pathological conditions, the imbalance between ROS and antioxidant

defence mechanism leads to oxidative modification in cellular membrane or
intracellular molecules (Duh et al., 1999). Consequently, antioxidants that can
neutralize direct ROS attacks and terminate free radical-mediated oxidative reaction

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would have beneficial effect in protecting the human body from various diseases
(Havsteen, 2002, Hertog et al., 1995).

The most important free radicals in biological systems are radical derivatives
of oxygen. Reduction of oxygen by the transfer of single electron will produce the
superoxide free radical anion (super oxide).

- -
O2 + e  O2 .

A two electron reduction of oxygen would yield hydrogen peroxide:

- +
O2 + 2e + 2H  H2O2

1.4.1 Superoxide
Components as a precursor of more reactive oxidative species, such as single
oxygen and hydroxyl radicals (Aurand and Boonme, 1977). Furthermore, superoxide
radical is considered to play an important role in the peroxidation of lipids (Dahl and
Richardson, 1978). At low pH value superoxide will protonate to form the
.
perhydroxyl radical (HO2 ), a more reactive oxidizing species but at physiological pH
less than 1%will be in protonated form (Yun-zhong Fang et al., 2002).

1.4.2 Hydrogen peroxide

Although not a radical species, H2O2 is an important ROS contributing to

oxidative stress. The generation of even low levels of H 2O2 in biological systems
may be important, since naturally occurring iron complexes are believed to react with

H2O2 in vivo to generate the highly reactive hydroxyl radicals in a superoxide driven
Fenton reaction. Hydrogen peroxide is an oxidizing agent but not specially reactive.
Its main significance lies in it being a source of hydroxyl radicals in the presence of
reactive transition metal ions. Hydrogen peroxide is often generated in biological
systems via the production of superoxide, two superoxide molecules can react
together to form hydrogen peroxide and oxygen.

-.
2O2 + 2H+ SOD H2O2 + O2 (dismutase reaction)

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Here hydrogen peroxide is the non-radical product. H 2O2 is not a free radical
but falls into the category of “reactive oxygen species” (ROS) that includes not only
oxygen free radicals but also non-radical oxygen derivatives that are involved in
oxygen radical production. Hydrogen peroxide is an important

compound in free radical biochemistry because it can rather easily break down,
particularly in the presence of transition metal ions, to produce the most reactive and
damaging of the oxygen free radicals, the hydroxyl radical (OH).

2+ 3+
H2O2 + Fe 
.
OH + OH + Fe (Fentons reaction)

O H O .OHOHO
2 2 2 2 (Haber-weiss reaction)

O2  Fe3  Fe2 O2

O2  Cu2  Cu  O2

2+ +
Ferrous (Fe ) iron and cuprous (Cu ) copper are much more reactive with
3+ 2+
H2O2 than their oxidized counterparts Ferric (Fe ) and cupric (Cu ), respectively. In
the absence of metal catalysts, superoxide and H 2O2 also readily removed and are
virtually harmless. (Yun-zhong Fang et al., 2002)

5.4.3 Hydroxyl radical

The reactive oxygen radicals are unstable and react readily with other groups
or substances in the body, resulting in cell damage and hence human diseases
(Halliwell and Gutteridge, 1989). Among the oxygen radicals specifically, the
hydroxyl radical is the most reactive.

It severely damages adjacent biomolecules such as all proteins, DNA, PUFA,

Nucleic acid, and almost any biological molecule it touches. This damage causes
ageing, cancer and several diseases (Aruoma, 1989). Therefore, the removal of

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hydroxyl radical is probably one of the most effective defenses of a living body
against various diseases.

5.4.4 DPPH
The DPPH radicals are widely used to investigate the scavenging activity of
some natural compounds. In the DPPH assay, the antioxidants are able to reduce the
stable radical DPPH to the yellow colored diphenyl-picrylhydrazine. The method is
based on the reduction of alcoholic DPPH solution in the presence of a hydrogen-
donating antioxidant due to the formation of the non-radical form DPPH-H by the
reaction. The method helps to determine the antiradical power of an antioxidant by
measuring of a decrease in the absorbance of DPPH at 517 nm.

Resulting 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. (Matthaus 2002)

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Calotropis gigantea
Synonyms

Asclepias gigantea L.

Periploca cochinchinensis Lour.

Streptocaulon cochinchinense (Lour.) [Link]

Common Name: Crown Flower

General information:
Crown flower is a fast-growing, attractive, evergreen flowering shrub or small tree
that grows about 5 metres tall, occasionally to 10 metres. It is much branched at the base,
with stems up to 20cm in diameter[299 ]. The plant is harvested from the wild for a wide
range of uses, but is especially valued as a medicinal plant and source of fibre. It is widely
cultivated as an ornamental in the tropics, and is also sometimes grown for the fibre obtained
from its stems, and for its medicinal uses[302 307 ]. Although the fibre is of very good
quality, the plant is unsuitable for commercial cultivation because individual fibres are too
short and the proportion of fibre in the stem is too low[ 454 ]

The flowers of Calotropis gigantea

Pollination:

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This is plant plays host to a variety of in Tamil Nadu and butterflies. It is the host plant for
Hawaii's non-migratory monarch butterflies.[2] Calotropis is an example of entomophily
pollination (pollination by insects) and pollination is achieved with the help of bees. In
Calotropis, gynostegium is present (formed by the fusion of stigma and androecium). The pollen
are in a structure named pollinia which is attached to a glandular, adhesive disc at the stigmatic
angle (translator mechanism). These sticky discs get attached to the legs of visiting bees that pull
out pollinia when a bee moves away. When such a bee visits another flower, this flower might be
pollinated by the pollinium.

Calotropis gigantea (Ankhade) plant with full bloom at Haridwar

Uses

The flowers are long lasting, and in Thailand they are used in floral arrangements. The extract of
flowers and leaves has shown hypoglycemic effect in preclinical studies.[3] They were favored
by the Hawaiian Queen Liliuokalani, who considered them a symbol of royalty and wore them
strung into leis. In Cambodia, they are used in funerals to decorate the urn or sarcophagus and
the interior of the house holding the funeral. The fruit is a follicle and when dry, seed dispersal is
by wind. In Indonesia its flowers are called widuri. According to Shivpuran (Hindu religion) the
madar flower/crown flower is very much liked by Lord Shiva; therefore the crown flower and its
garland are offered to Lord Shiva for peace, prosperity and stability in society.[4] The Crown
flower is also one of the major parts of the nine astrological trees (Navagrah tree).

Calotropis yields a durable fiber (commercially known as bowstring of India) useful for ropes,
carpets, fishing nets, and sewing thread. Floss, obtained from seeds, is used as stuffing. Crown
flower cotton can also be used to make a pillow. A fermented mixture of Calotropis and salt is
used to remove the hair from goat skins for production of nari leather and from sheep skins to
make leather which is much used for inexpensive book binding.[5][full citation needed]
Fungicidal and insecticidal properties of Calotropis have been reported.[6][full citation needed]

In India, the plant is common in the compounds of temples and is known as madar in Hindi:
मदार. Its leaf (Marathi: rui) is one of the five leaves used in the Panch Pallava, a ritual
assortment of five different leaves used as a totem by the Maratha culture in India.[7]

It is known as වරා (waraa) in Sinhala, আকন (akon) in Assamese, aakonda in Bengali, erukku
(എരുക്ക്) in Malayalam, yakka (ಯಕ್ಕ) in Kannada, and ark (अर्क) in Sanskrit.

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Allelopathic effects

Allelopathic effects of Calotropis on different agricultural crops have been well studied.[8]
Extracts of plant parts such as root, stem, and leaf affect germination and seedling vigor of many
agricultural crops.[9][full citation needed][10][full citation needed] However, extracts of
Calotropis failed to produce any detrimental effects on weeds such as Chenopodium album,
Melilotus alba, Melilotus indica, Sphaeranthus indicus, and Phalaris minor.[11][full citation
needed]

Use as an Arrow Poison

Many plant and animal extracts have been used as arrow poisons all over the world. In many
cases, the poison was applied to the arrow or spear to aid the hunting of prey. Alkaloids are
among the most powerful plant poisons, and extracts of Strychnos species are commonly used.
Other arrow poisons are commonly cardiac glycosides, which can be found in digitalis, but most
of these arrow poisons are derived from plants in the Apocynaceae family.[12] This family
includes Calotropis gigantea and the more potent Calotropis procera. The latex of these plants
has been used in Africa as an arrow poison. Apocynaceae species often contain a mixture of
cardiac glycosides, including calactin, uscharin, calotoxin, and calotropin.[13] These poisons
work by inhibiting the sodium-potassium pump, and this effect is especially potent in the cardiac
tissues.[14] The cardiac effects can be applied for heart medication, and digitalis has been used
as such. However, excessive doses can cause arrhythmia, which can lead to death.[15]

Medical Uses

Given the potent bioactivity of calotropin, calotropis gigantea has been used as a folk medicine
in India for many years, and has been reported to have a variety of uses. In Ayurveda, Indian
practitioners have used the root and leaf of C. procera in asthma and shortness of breath and the
bark in liver and spleen diseases. The plant is reported as effective in treating skin, digestive,
respiratory, circulatory and neurological disorders and was used to treat fevers, elephantiasis,
nausea, vomiting, and diarrhea. The milky juice of Calotropis procera was used against arthritis,
cancer, and as an antidote for snake bite.[16] However, these reports are of folk uses and more
research is needed to confirm the clinical usefulness of the leaves, latex, and bark. Recent studies
have displayed use of calotropin as a contraceptive[17] and as a promising cancer medication.
[18] In one study of the cancer-fighting properties of Calotropis gigantea, DCM extracts were
demonstrated to be strongly cytotoxic against non-small cell lung carcinoma (A549), colon
carcinoma (HCT 116), and hepatocellular carcinoma (Hep G2). These extracts show promise as
cancer medications and warrant further clinical research.[19]

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Poisoning

Calotropis is a poisonous plant. The active principles are uscharin, calotoxin, calactin, and
calotropin.[citation needed] The leaves and stem when incised yield thick milky juice. It is used
as an arrow poison, cattle poison (see also Sutari), rarely for suicide and homicide and mostly an
accidental poison.

The milky latex sap of Calotropis gigantea is a known cause of toxic keratoconjunctivitis and
reversible vision loss. Crownflower keratitis is a rare condition and is usually the result of
accidental ocular exposure to the sap. During the process of making a Hawaiian lei flower
necklace, touching the sap and then touching the ocular surface may result in crownflower
keratitis. Damage (poisoning) of the cornea endothelium results in corneal stromal edema and
decreased visual acuity. Although there is some permanent damage to the corneal endothelium
with decreased endothelial cell count and irregular shape, the remaining corneal endothelial cells
usually recover with complete resolution of the corneal edema and a return to normal visual
acuity. The condition is usually self-limited and resolves faster with topical steroids. The clinical
course of this condition suggests that Calotropis is paradoxically relatively nontoxic to corneal
epithelium and highly toxic to corneal endothelium. The painless clinical course may be related
to anesthetic properties of Calotropis latex and relatively minor epithelial injury.[20][21][22]

Signs and symptoms

Applied to the skin, it causes redness and vesication. When taken orally, the juice produces an
acrid, bitter taste and burning pain in throat and stomach, salivation, stomatitis, vomiting,
diarrhea, dilated pupils, tetanic convulsions, collapse and death. The fatal period is 6 to 12 hours.
[citation needed] Treatment includes stomach wash, demulcents, and symptomatic treatment.
[medical citation needed]

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