Introduction

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Herbal medicines are plant-derived remedies that are used for their therapeutic
properties and they have been an important tradition of many cultures and beliefs of
African people [1]. Sanitation and hygiene levels for the majority of people in Africa
are not comparable to those of the first world countries. This exposes African people
to a wider array of microbial pathogens, which increases their susceptibility to
bacterial, fungal and viral infections. Indigenous plants are often the only available
means of treating such infections [2]. Since there is an increasing resistance to
antibiotics by many pathogenic and opportunistic bacteria, plant extracts and plant-
derived compounds have emerged as potential and promising antimicrobial agents [3].

Empiriological and experimental evidence suggest that free radicals and reactive
oxygen species (ROS) are implicated in more than 100 diseases, including malaria,
acquired immunodeficiency syndrome (AIDS), heart disease, stroke, arteriosclerosis,
diabetes, cancer and gastric ulcer [4, 5]. Antioxidants can protect the human body
from free radicals, ROS effects and may retard progression of many chronic diseases
as well as lipid oxidative rancidity in foods [4, 6, 7]. However, butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), the most commonly
used antioxidants at present, are suspected of being responsible for liver damage and
carcinogenesis [8]. As a result scientists are trying to incorporate traditional medicine
within primary health care [1]. This renewed interest in traditional medicines means
that scientists are not only concerned in determining the scientific rationale of
traditional practice of medicine plant usages, but also aspire to discover novel and
safe plant compounds of pharmacological importance [9].

Plants have an almost limitless ability to synthesize aromatic substances, most of

which are phenols or their oxygen-substituted derivatives which are mainly secondary
metabolites [10]. Currently identified secondary plant metabolites exceed 100 000
substances, belonging to a variety of chemical classes, including terpenoids, phenolics
and alkaloids [11]. There is an interest in these secondary metabolites since they are
known to demonstrate various biological activities that encourage positive health
effects, such as antibacterial, anticancer, antifungal, antioxidant and antiviral activities
that can be used in the food, agricultural and pharmaceutical industries.

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The present study is intended to investigate nine indigenous medicinal plants for
the presence of antioxidant and antibacterial compounds. These indigenous plants
were mainly collected in the surrounds of the Andhra Pradesh Most of the selected
plants are used to treat different infections and also for blood purifying purposes by
traditional healers. For antibacterial activity, the plant extracts were tested against
four pathogenic microorganisms, i.e., Escherichia coli, Enterococcus faecalis,
Pseudomonas aeruginosa and Staphylococcus aureus using bioautography approach.
The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, TLC-
DPPH antioxidant screening and reducing power assays were used to evaluate the
potential antioxidant activity of these plant extracts.

1.2 Free radicals and their sources

Free radicals are organic molecules responsible for aging, tissue damage, and
a wide variety of diseases. These molecules are very unstable, and thus bond with other
molecules, destroying their strength and perpetuating the detrimental process. Oxidation
reactions are an essential part of normal metabolism as oxygen is the ultimate electron
acceptor in the electron flow system that produces ATP [35]. Problems may arise when
electron flow and energy production become uncoupled so that oxygen free radicals, that
is, reactive oxygen species (ROS), are produced [8, 36]. Actually, ROS are continuously
produced within the cell as a result of mitochondrial electron transfer processes or as by-
products of the enzymes xantine oxidase, lipoxygenases and cyclooxygenases [37].
Furthermore, ROS can be generated as a consequence of the intracellular metabolism of
foreign compounds, toxins or drugs by cytochrome P450, monoxygenases, or because of
exposure to environmental factors such as excessive iron salts or UV irradiation [38].
Other sources of ROS are macrophages and neutrophils that contain enzymes, such as
NADPH oxidase complex, able to generate superoxide radicals and hydrogen peroxide.
Reactive oxygen species thus play different positive roles in vivo, being involved in
energy production, phagocytosis, cell growth and intercellular signaling regulation.
Reactive oxygen species may also be highly damaging, as they can attack biological
macromolecules, namely, lipids, proteins and DNA, induce oxidation and cause
membrane damage, enzyme inactivation and DNA damage [39]. However, when the
level of ROS level exceeds the antioxidant capacity of the cell, the intracellular redox
homeostasis is altered and oxidative stress ensues [40].

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1.2.1 Free radicals associated diseases

There have been accumulating evidence that suggests that cellular damage arising
from ROS can be involved in the aetiology and pathophysiology of human diseases such
as neurodegenerative disorders (e.g., Alzheimer’s disease, Parkinson disease, Multiple
sclerosis, Down’s syndrome, etc.), inflammation, viral infections, autoimmune
pathologies and digestive system disorders such as gastrointestinal inflammation and
ulcer [41]. In living systems, free-radicals are generated as part of the body’s normal
metabolic process and the free radical chain reactions are usually produced in the
mitochondrial respiratory chain, liver mixed function oxidases, by bacterial leucocytes,
through xanthine oxidase activity, atmospheric pollutants, and from transitional metal
catalysts, drugs and xenobiotics. In addition, chemical mobilization of fat stores under
various conditions such as lactation, exercise, fever, infection and even fasting, can result
in increased radical activity and damage, in particular, to the immune and nervous
systems, while the stress hormones (adrenalin and noradrenalin) secreted by the adrenal
glands under conditions of continuing and excessive emotional stress, are metabolised
into simpler, although, free radical molecules [42].

Free radicals or oxidative injury appears to be the fundamental mechanism underlying a

number of human neurologic and other disorders. For instance in diabetes, increased
oxidative stress which co-exists with reduction in the antioxidant status has been
postulated: Oxygen free-radical can initiate peroxidation of lipids, which in turn
stimulates glycation of protein, inactivation of enzymes and alteration in the structure
and function of collagen basement and other membranes, and play a role in the long-term
complication of diabetes [43]. Similarly, in carcinogenesis, reactive oxygen species are
responsible for initiating the multistage carcinogenesis process starting with DNA
damage and accumulation of genetic events in one or few cell lines which leads to
progressively dysplastic cellular appearance, deregulated cell growth, and finally
carcinoma [44].

1.2.2 The mechanism of antioxidant free radical scavenging

There is currently much interest in phytochemicals as bioactive components of food.

The roles of fruit, vegetables and red wine in disease prevention have been attributed, in
part, to the antioxidant properties of their constituent polyphenols (vitamins E and C, and
the carotenoids). Recent studies have shown that many dietary polyphenolic constituents

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derived from plants are more effective antioxidants in vitro than vitamins E or C, and
thus might contribute significantly to the protective effects in vivo. It is now possible to
establish the antioxidant activities of plant derived flavonoids in the aqueous and
lipophilic phases, and to assess the extent to which the total antioxidant potentials of
wine and tea can be accounted for by the activities of individual polyphenols [45].

Antioxidant compounds reduce or prevent the action of reactive oxygen species in tissue
damage. The oxidation proceeds in lipids with polyunsaturated fatty acids, generating
ROS such as hydroxyl radicals. Natural products with antioxidant activity are used to aid
the endogenous protective system, increasing interest in the antioxidative role of
nutraceutic products [46]. Antioxidants may act by decreasing oxygen concentration,
intercepting singlet oxygen, or preventing first chain initiation by scavenging initial
radicals [47]. Plants such as fruits, vegetables and medicinal herbs may contain a wide
variety of free radical scavenging molecules, such as phenolic compounds, vitamins,
terpenoids and some other endogenous metabolites, which are rich in antioxidant
activity. Epidemiological studies have shown that many of these antioxidant compounds
possess anti-inflammatory, antitumour, antibacterial or antiviral activities to a greater or
lesser extent. The intake of natural antioxidants has been associated with reduced risks of
cancer, diabetes and other diseases associated with ageing [48, 49]. These protective
mechanisms either scavenge or detoxify ROS, block their production, or sequester
transition metals that are the source of free radicals, and include enzymatic and
nonenzymatic antioxidant defenses produced in the body, namely, endogenous [50], and
others supplied with the diet, namely, exogenous [51]. The two types of antioxidant
defenses, the endogenous and exogenous antioxidants can be classified as follows:

1.2.3 Exogenous antioxidants

Many compounds in plants and vegetables have the ability of reacting with free radicals
without generating further radicals, therefore, quenching chain reactions. Other
compounds scavenge ROS and in so doing they become oxidized and need to be
regenerated for further use. Antioxidant compounds react directly with radicals reducing
oxidative stress and exerting their protective effects against cellular damage [52].
Polyphenols comprise a wide variety of compounds, divided into several classes (i.e.,
hydroxybenzoic acids, hydroxycinnamic acids, anthocyanins, proanthocyanindins,
flavonols, flavones, flavanols, flavanones, isoflavones, stilbenes and lignans), that occur

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in fruits and vegetables, wine and tea, chocolate and other cocoa products.
Epidemiological studies showed that increased intake of polyphenols was associated with
reduced risk of cardiovascular diseases, cancer and neurodegenerative disorders [53].
The beneficial effects of polyphenols are mainly ascribed to their capacity to counteract
conditions of oxidative stress that accompany these pathologies. Several polyphenols
have been demonstrated to have clear antioxidant properties in vitro as they can act as
chain breakers or radicals scavengers depending on their chemical structures, which also
influence their antioxidant power. A hierarchy has been established for the different
polyphenolic compounds within each class on the basis of their capability to protect
lipids, proteins or DNA against oxidative injury [54].

1.2.4 Endogenous antioxidants

Several antioxidant enzymes exist that convert ROS into less noxious compounds, for
example, superoxide dismutase (SOD), catalase, thioredoxin reductase, peroxiredoxin
and glutathione peroxidase (GPx) [55]. Collectively, these enzymes provide a first line of
defense against superoxide and hydrogen peroxides. They are of enormous importance in
limiting ROS-mediated damages to biological macromolecules, but they are not able to
be 100% effective because certain compounds generated by the interaction of ROS with
macromolecules are highly reactive. It is then mandatory to detoxify these secondary
products in order to prevent further intracellular damage, degradation of cell components
and eventual cell death. This second line of defense against ROS is provided by enzymes
such as GPx, glutathione S-transferase (GST), aldo-keto reductase and aldehyde
dehydrogenase [56]. The detoxified metabolites produced by these enzymes are
eliminated from the cell by efflux pumps such as the glutathione S-conjugate transporter
[57].

1.3. Extraction and isolation of natural products

Extraction of natural products from crude material is one of the simple steps towards
isolation whilst on the other hand isolation process of natural products remains a tough,
extensive and a monotonous task [58]. Spectroscopic methods coupled with good
separation techniques like chromatography, have contributed to the phenomenal success
of natural product chemistry over the past 50 years. Sound strategies have helped in the
isolation and characterization of many bioactive molecules [59]. Nowadays, bioassay-

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guided fractionation of medicinal plants is a routine feature in the attempt to isolate

bioactive components from natural sources [60].

1.3.1. Extraction

In practice, as soon as the material is collected, in the case of plants, it needs to be

identified by a taxonomist so as to ascertain the correct identity of the material. Various
parts of the plant or the whole plant are collected (leaves, flowers, stem, wood, bark,
root, root bark, etc.) and dried quickly in drying cabinets because fresh material has
much water and this can lead to degradation of the components of the plants by
microbes. Good ventilation conditions or high speed fans can be used [60]. Once the
material has been dried to constant weight, it is ground up to smaller particles and
extracted usually using solvents of different polarities. The extraction process could
either use one solvent or be stepwise extraction in which the same material is extracted
using different solvents normally from non-polar solvents to polar solvents. There are
different techniques which can be applied to extract natural products from crude samples;
the following are some of the example:

1.3.2 Maceration

Here the plant material is extracted in solvents of differing polarity at room temperature,
by leaving the plant material soaked in the selected solvents and/or shaking at room
temperature and this allows for maximum extraction of most components.

1.3.3 Decoction

The plant material is boiled with the solvent usually under reflux. This method allows for
extraction of a large number of metabolites, from the most insoluble material like the
waxes to the lipophilic natural products.

1.3.4 Continuous extraction

Perhaps the most widely and commonly used technique for the extraction of natural
products. The polarity gradient of the solvent is applied. Although some components
may be destroyed in the process, it is still the best method of extraction used in natural
product chemistry.

1.3.5 Infusion

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In this technique hot liquid ‘solvent’ is poured on the plant material and this is different
from decoction where the plant material is boiled with the solvent.

1.4 Partial purification and fractionation

Once the extraction is complete, the extract is usually concentrated under vacuum. The
activity within the obtained extract can then be demonstrated by bioassay methods using
both the crude and the fractionated or semi-purified extracts. Fractionation has the added
advantage of getting to the biologically active material faster. There are techniques
which can be applied to partially purify the desired components by removing some of the
undesired components. The techniques which can be used are solvent/solvent partition
and precipitation. Solvent/solvent partition needs two immiscible liquids and separates
the components according to solubility which can be polarity or charge. This method
relies on the ability of the components to be either soluble in water or the organic phase
[60]. In precipitation/precipitation an excessive amount of the solvent e.g. ethanol,
ammonium sulphate, lead etc. is added to the crude material to and some components in
the crude material will precipitate.

1.5 Chromatographic techniques

For the separation of compounds within the extract, chromatographic techniques are
employed. Chromatographic techniques have been instrumental in the separation of
natural products. Chromatography is a process whereby a mixture of solutes may be
resolved into components by exploiting differences in affinity of the solutes for particles
of an insoluble matrix over which a solution of the components is passing. The insoluble
matrix is called the stationary phase, while the solution which passes through it is called
the mobile phase [60]. There are different types of chromatographic techniques which
can be utilised to separate compounds here three chromatographic techniques will be
discussed.

1.5.1 Thin layer chromatography

Thin layer chromatography (TLC) is one of the fastest and most widely used
chromatographic techniques in the separation of natural products. TLC mostly used for
phytochemical analysis of plant extracts and to check purity of isolated compounds. TLC
method employs glass or aluminium plates pre-coated with the sorbent (e.g., silica gel) to
varying thickness depending on the amount of the sample to be loaded. The compound

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mixture is loaded on plates at around 1-2 cm from the bottom of the plate and lowered in
a tank containing the solvent. The latter migrates up the plates and separates the
compound mixture according to the polarity of the components. Several reagents are
available for visualization of the separated materials. TLC has the advantage of being a
highly cost-effective qualitative technique since a large number of samples can be
analysed simultaneously.

1.5.2 Preparative thin layer chromatography

Preparative thin layer chromatography is a technique which is usually employed to

isolate bioactive natural compounds after column chromatography. Preparative thin layer
chromatography uses the same principles to those of thin layer chromatography the
difference is only that preparative thin layer chromatography has a thick stationary phase
compared to TLC. This gives preparative TLC the advantage in that large quantity of
sample is loaded on plates as a band and the developed in the chosen solvent system.
After developing the plates they can be analysed using non-destructive detection e.g.,
UV and/or destructive chromogenic spray by exposing only a small portion of the plate.
The band(s) with the compound(s) of interest now can be removed using a spatula or cut
out with scissors. The compound can be cleaned by filtration, size exclusion in column
chromatography, centrifugation, crystallisation etc. The purity of the compound(s) is
checked using TLC or High Performance Liquid Chromatography (HPLC).

1.5.3 Column chromatography

Column chromatography (CC) is a popular technique which is used for fractionation

and isolation of bioactive natural compounds. This technique is usually employed after
solvent/solvent partition. To fractionate or isolate bioactive compounds the stationary
phase normally used is silica gel with the mobile being the solvent(s) of choice. There
are eluting techniques which can be used which are isocratic elution and gradient elution.
Isocratic elution employs only one mobile phase while gradient elution employs a
sequence of mobile phases usually in order of polarity, increasing for normal phase
chromatography and decreasing polarity for reverse phase chromatography. Gradient
elution is normally employed when isolating and/or fractionating natural bioactive
compounds from crude samples. After elution fractions collected are analysed using e.g.,
chemical tests, TLC, bioassays etc. to identify fractions of interest, similar fractions are
grouped together for future work [61].

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Biological Source & Family:

Cassia auriculata (Leguminosae)26-30

SCIENTIFIC CLASSIFICATION

Kingdom : Plantae

Sub Division : Spermatophyta

Division : Magnoliophyta

Class : Magnoliopsida

Sub Class : Rosidae

Order : Fabales

Family : Fabaceae

Genus : Cassia

Species : auriculata

VERNACULAR NAMES

English : Tanner’s Cassia.

Ayurvedic: Aaavartaki, Aaadaari.

Unani : Tarwar.

Siddha/ Tamil : Aavaarai.

Folk : Tarwar.

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Figure 3.1. Habit of Cassia auriculata Linn.(Leguminosae)

Habitat: Wild in dry regions of Madhya Pradesh, Tamil Nadu and Rajasthan.

Cultivated in other parts of India.

Botanical description: Avaram (Cassia auriculata Linn), family Leguminosae, is also

known as Avaram tree, The leaves are alternate, stipulate, paripinnate compound, very
numerous, closely placed, rachis 8.8-12.5 cm long, narrowly furrowed, slender, pubescent,
with an erect linear gland between the leaflets of each pair, leaflets 16-24, very shortly
stalked 2-2.5 cm long 1-1.3 cm broad, slightly overlapping, oval oblong, obtuse, at both
ends, mucronate, glabrous or minutely downy, dull green, paler beneath, stipules very large,
reniform-rotund, produced at base on side of next petiole into a filliform point and
persistent.

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Its flowers are irregular, bisexual, bright yellow and large (nearly 5 cm across), the pedicels
glabrous and 2.5 cm long. The racemes are few-flowered, short, erect, crowded in axils of
upper leaves so as to form a large terminal inflorescence (leaves except stipules are
suppressed at the upper nodes). The 5 sepals are distinct, imbricate, glabrous, concave,
membranous and unequal, with the two outer ones much larger than the inner ones. The
petals also number 5, are free, imbricate and crisped along the margin, bright yellow veined
with orange. The anthers number 10 and are separate, with the three upper stamens barren;
the ovary is superior, unilocular, with marginal ovules.

The fruit is a short legume, 7.5–11 cm long, 1.5 cm broad, oblong, obtuse, tipped
with long style base, flat, thin, papery, undulately crimpled, pilose, pale brown. 12-20 seeds
per fruit are carried each in its separate cavity.

Chemical constituents: Pod husk contains nonacosane and nonacosan-6-one, chrysophanol,

emodin and rubiadin.

Medicinal uses: Roots- used in skin diseases and asthma.

Flowers used in diabetes, urinary disorders and nocturnal emissions.
Its Bark is used as astringent.
Leaves and Flowers – Anti-diabetic activity.

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