Chapter 1

INTRODUCTION
 Chapter 1: Introduction

Strive constantly to serve the welfare of the world; by devotion to selfless

work one attains the supreme goal of life. Do your work with the welfare
of others always in mind.

- Bhagwat Gita

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 Chapter 1: Introduction

1.1 INTRODUCTION

Earth is a planet dominated by plants. The green plant is fundamental to all other life. The
oxygen we breathe, the nutrients we consume, the fuels we burn and many of the most
important materials we use are produced by plants. The association between plant and
man is an age-old process starting from human civilization. Since time immemorial,
nature has been a rich source of medicinal agents. Using the traditional knowledge there
are isolation of phytoconstituents carried out from natural sources.

The importance of plants as one of the natural sources of drugs cannot be over-
emphasized as about 25% of the drugs prescribed worldwide come from plants (Rates,
2001). Ethnobotany has developed as an important research field as plants continues to be
an integral part of human existence (Sumner, 2000; Okujagu et al., 2006). This has
prompted humanity to undertake a systematic study of the plants from different point of
view.

India, like all other countries, has made significant progress in the scientific study of
plant drugs from the pharmacological, chemical, pharmacognostical and clinical points of
view during the past 65 years (Krishnan, 1999; Subramani, 2001). The field of
phytochemistry which deals with natural products chemistry is related to the study of
products elaborated by plants and has become a distinct discipline having combination of
natural product organic chemistry and plant biochemistry. This branch of science deals
with the study of chemical structures of plant constituents, their biosynthesis,
metabolism, natural distribution and biological functions. The fact that only less than
10% of the total identified species of plants on earth have been investigated indicates the
opportunities and challenges which have been thrown open to phytochemists (Subramani,
2001).

Medicinal plants owe their therapeutic properties to their ability to synthesize various
complex phytochemical substances broadly grouped as phenolics, alkaloids and

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terpenoids. Medicinal uses of plants demonstrated by the pharmacological and clinical

studies carried out on the phytochemicals present in plants. This serves as the major
backbone of traditional medicine (Van Wyk and Wink, 2004). Extraction of bioactive
compounds from various plants permits the demonstration of their physiological activity
and facilitates various pharmacological studies (Babayi et al., 2004; Verma et al, 2013).

Extraction of the secondary plant metabolites is carried out to obtain the individual
compounds which can be further used as active ingredients in pharmaceuticals, cosmetics
or as food supplements etc. (Dai and Mumper, 2010). Moreover, to check the authenticity
of the raw material during manufacturing of drugs of prescribed quality, it becomes
necessary to identify and evaluate the raw materials used (Mahajan and Pai, 2010).
Figure 1.1 shows the different phytochemicals present in plants.

Figure 1.1: Different phytochemicals present in plants

Among the large number of secondary metabolites, phenolic constitute one of the most
common and widespread groups of substances in plants. These include a wide range of

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 Chapter 1: Introduction

compounds, containing an aromatic ring with one or more hydroxy substituents or their
ether or glycoside derivatives (Lattanzio, 2006). These compounds have great diversity in
their structure and their occurrence widely distributed among the secondary metabolites.

Among the several thousands of naturally occurring polyphenolic compounds, the

flavonoids are the largest and the most widespread. About 48 years ago, Geissman and
Hinereiner used the term "flavonoid to include all the compounds having a flavone (2-
phenyl chromone) basic ring with C6-C3-C6 skeleton (Krishnan, 1999). Flavonoids are
considered to be the most familiar and widespread class of compounds in plant kingdom.
In the plant kingdom, more than 4000 naturally occurring flavonoids are being estimated
to be identified (Wink, 2008; Tahrani, 2011). Flavonoids contribute to the flavor and
pigmentation of the fruits and vegetables commonly used in the human diet (Timberlake,
1986). They also have important roles in plant growth, reproduction and pathogen and
predator resistance (Harborne and Williams, 2000).

Flavonoids are present in plants either as aglycones or as glycoside conjugates. The

attached sugar moieties include D-glucose, L- rhamnose, glucorhamnose, galactose,
lignin and arabinose (Ross and Kasum, 2002, Peluso, 2006). Over 4000 different
flavonoids have been identified within the major flavonoid classes which include
flavonols, flavones, flavanones, catechins, anthocyanidins, isoflavones, dihydroflavonols,
and chalcones (Symonowicz and Kolanek, 2012). Flavonoids are known to possess
several therapeutic activities like antiinflammatory, antiallergic, hepatoprotective,
antithrombotic, antiviral, and anticarcinogenic activities (Tapas et al., 2008).

The beneficial effect of flavonoids is mainly associated with their antioxidant activity.
Antioxidants act as reducing agents by donating hydrogen, quenching singlet oxygen,
acting as chelators or by trapping free radicals. These highly reactive molecules, present
in biological systems, can reduce the risk of degenerative diseases such as cancer, heart
disease, dermal disorders and ageing (Bimakr et al., 2008).

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Researchers have also been carrying out studies on antioxidants and their role in
mitigating / preventing cancer and heart disease as well as in boosting the immune system
and slowing the ageing process. There is considerable attention focused on polyphenolic
flavonoids because of their health alimenting properties. Researchers today are
employing different experimental models for evaluation of efficacy of polyphenols as
antioxidants. They employ different models starting from the simplest chemical
antioxidant assays to the most accurate animal models, and ultimately clinical studies in
humans (Vladimir-Knezevic et al., 2012).

There are several investigations done by several researchers not only for confirmation of
ethnomedicinal use of plant but also to identify, isolate and characterize the active
compounds (Fennell et al., 2004). However, the presence of numerous inactive
components makes the screening and isolation of the target component(s) extremely
cumbersome (Sticher, 2008). The general approaches in extraction, isolation and
characterization of bioactive compound from plants extract is illustrated in Figure 1.2.
(Sasidharan et al., 2011).

Figure 1.2: Schematic representation of general approaches in extraction, isolation

and characterization of bioactive compound from plants extract
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 Chapter 1: Introduction

Extraction is the crucial first step in the analysis of medicinal plants, because it is
necessary to extract the desired chemical components from the plant materials for further
separation and characterization (Sasidharan et al., 2011). There are various extraction
methods such as maceration, percolation, decoction, Soxhlet extraction, counter current
extraction, sonication, supercritical fluid extraction, steam distillation, etc. that are
employed for the extraction of phytochemicals (Kumar et al., 2011). However, the
concentration of active compounds in herbal plants being usually low, so a vast amount
of research has been undertaken to develop effective and selective extraction methods.
One such method, Supercritical Fluid Extraction (SCFE) has been recently employed to
extract these compounds from the herbal matrices (Lang and Wai, 2001).

The supercritical fluid extraction region of a pure compound is defined as the region
where the temperature and pressure are higher than their critical values. Advantages of
supercritical fluids (SCFs) have been discussed in the literature and these include: rapid,
simple and good analyte selectivity, its efficiency, and its suitability for thermally labile
compounds, near solvent free character and reduced environmental hazards (Goncalves et
al., 2006). Of all the gases and liquids studied, CO2 remains the most commonly used
fluid for SCFE applications on account of its low critical constants (Tc = 304.04 K, Pc =
7.38 MPa), its non toxic and non flammable properties and its availability in high purity
with low cost (Farshad et al., 2005). It is these properties which make supercritical
carbon dioxide (SC-CO2) an attractive green or environmentally friendly solvent. CO2
is a non-polar solvent, so extraction of polar compounds such as flavonoids is difficult
with supercritical carbon dioxide (SC-CO2). Supercritical Carbon dioxide extraction has
the ability to ensure minimal changes of the active ingredients and the curative properties
that can be preserved (Cavero et al., 2006).

After extraction the second step in phytochemical analysis involves isolation and
identification of phytoconstituents. Due to the fact that plant extracts usually occur as a
combination of various types of bioactive compounds or phytochemicals with different
polarities, their separation still remains a big challenge for the process of identification
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and characterization of bioactive compounds. Plant extracts are subjected to

phytochemical screening involving various chromatographic and spectroscopic methods
(Sasidharan et al., 2011).

The conventional chromatographic methods like Thin Layer Chromatography (TLC) and
Preparative TLC are still in use for separation and purification of the flavonoid
compounds. Recent developments have indicated that LC can be used to improve
conventional methods for the analysis of phenolics (De Villiers et al., 2010, Bleeder,
2011). Increase in the speed and efficiency in the separation of phytochemicals is
achieved by high pressure liquid chromatography (HPLC). This technique is selected as
the prime most method for fingerprint studies involving quality control of plant samples
(Fan et al., 2006; Sasidharan et al., 2011). For phenolic compounds, separation and
characterization is achieved using analytical technique such as reversed-phase liquid
chromatography (RP-LC) coupled with Photo diode array (PDA) detector and/or Mass
Spectrometry (MS) Tandem mass spectrometry, (MS-MS) provides additional structural
information about the target compounds in its further structural elucidation (Stalikas,
2007).

Against this background, the current study was undertaken for extraction and
identification of flavonoids from a plant source. The plant selected for the present study
was Nyctanthes arbor-tris Lin. Nyctanthes arbor-tristis, commonly known as parijat,
belongs to the family Oleaceae. Different phytochemical and pharmacological studies
reported for N. arbor-tristis are discussed in the following chapter. To the best of our
knowledge, there are no reports on the supercritical fluid extraction of flavonoid
compounds from N. arbor-tristis; therefore it was felt that it would prove to be of interest
to undertake such a study.

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1.2 SCOPE OF THE THESIS

The present thesis broadly comprises two main parts; the first part contains background
information about the project work and literature survey while the second part deals with
the experimental data (including methodology, results and discussions). The entire thesis
is divided into eight chapters

Chapter 1 summarizes the importance of phytochemical studies involving extraction,

isolation and identification of phytoconstituents from plant sources.

Chapter 2 describes the theory and literature review of all the techniques used in the
present study.

Chapter 3 describes the rationale of the work followed by aim and objectives of the study.

Chapter 4, Chapter 5, Chapter 6 and Chapter 7 describe the experimental work along with
its results and discussion.

Chapter 8 gives a brief summary of the present study highlighting the important
conclusions followed by elaborate list of references used for the study.

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