5

REVIEW OF LITERATURE

Quercetin is a plant-based flavonoid widely present in leaves and flowers and

also has a unique ability to act as an antioxidant (Chen et al., 2012a). The evolving

commercial importance of quercetin has in recent years resulted in a great interest in

secondary metabolism, particularly in the possibility of altering the production of

quercetin by means of tissue culture technology. In vitro production of secondary

metabolites in plant cell suspension cultures has been reported from various medicinal

plants are the key step for their commercial production. Based on this lime light, the

present review is aimed to cover phytotherapeutic application, purification and

biotechnological methods adopted for the production of quercetin.

The literature relevant to present study on ―Isolation, Purification and

Structural Characterization of Quercetin and its Derivatives from In Vitro

Suspension Cultures of Selected Plants and Its Comparison to In Vivo Plant Parts”

is reviewed in this chapter under the following headings:

2.1. Plant Secondary metabolites

2.2. Flavonoids

2.3. Methods for separation, purification and quantification of flavonoids.

2.4. Quercetin and quercetin derivatives

2.5. In vitro culture techniques

2.6. Cell suspension cultures for secondary metabolite production

2.7. Evaluation and elicitation of flavonoids in cell suspension cultures

2.8. Importance of the plants selected

2.1. Plant secondary metabolites

Humans consume a wide range of foods, drugs, and dietary supplements that are

derived from plants. Two hundred years of modern chemistry and biology have

described the role of primary metabolites in basic life functions such as cell division and

growth, respiration, storage, and reproduction. Since Frankel (1959) recognized that

plant secondary metabolites were not simply plant waste products but served as

2

6

defensive, protective or offensive chemicals against microorganisms, insects, and higher

herbivorous predators (Udomsuk et al., 2011), several studies have been carried out

either to understand their biosynthesis or to exploit their potential bioactivity. The

absence of secondary metabolites generally does not immediately kill plants but can

have profound effects on a species ecology and evolution. Related to the latter,

particular secondary metabolites are often restricted to individual species or narrow sets

of species within a phylogenetic group, providing the potential for biochemically

mediated behaviour almost as diverse as the plant kingdom (Metlen et al., 2008). But

plants can behave dynamically in response to environmental stimuli by rapid induction

and reversal of secondary metabolite production. Thereby, a great array of molecules,

derived from plant secondary metabolism, is of extreme interest in human nutrition and

pharmacology, in addition to perfumery and cosmetic industries.

Phytochemicals could provide health benefits as: (1) substrates for biochemical

reactions; (2) cofactors of enzymatic reactions; (3) inhibitors of enzymatic reactions;

(4) absorbents/ sequestrants that bind to and eliminate undesirable constituents in the

intestine; (5) ligands that agonize or antagonize cell surface or intracellular receptors;

(6) scavengers of reactive or toxic chemicals; (7) compounds that enhance the

absorption and or stability of essential nutrients; (8) selective growth factors for

beneficial gastrointestinal bacteria; (9) fermentation substrates for beneficial oral, gastric

or intestinal bacteria; and (10) selective inhibitors of deleterious intestinal bacteria. Such

phytochemicals include terpenoids, phenolics, alkaloids and fiber (Dillard and German,

2000).

Polyphenols are common constituents of foods of plant origin and major

antioxidants of our diet. The main dietary sources of polyphenols are fruits and

beverages. Fruits like apple, grape, pear, cherry, and various berries contain up to

200-300mg polyphenols per 100g fresh weight. Several hundreds of different

polyphenols have been identified in foods: two main types of polyphenols are flavonoids

and phenolic acids. Phenolics synthesized primarily from products of the shikimic acid

pathway, have several important defensive role in the plants. A protective role of

7

polyphenols against degenerative diseases is supported today by many studies carried

out on animals, and different mechanisms of action have been proposed to explain such

protective effects. Much progress has also been made on the evaluation of their

bioavailability (Scalbert et al., 2005).

(Taiz and Zeiger, 2006)

2.2. Flavonoids

The term ―flavonoid‖ is generally used to describe a broad collection of natural

products that include a C6-C3-C6 carbon framework, or more specifically a

phenylbenzopyran functionality (Grotewold, 2006). Flavonoids are a class of plant

phenolics that comprise an astonishingly diverse group of more than 4500 compounds

and derived from the condensation of a cinnamic acid with three malonyl-CoA groups

(Figure.2.1). All flavonoids arise from the initial reaction, which is catalyzed by the

chalone synthase enzyme. The chalcone is usually converted rapidly into a

8

phenylbenzopyran, (Packer and Sies, 2001). Flavonoids have two aromatic rings

(A & B) enclosing a heterocyclic six membered ring, ring C with oxygen (Figure.2.2).

Figure.2.1.

Biosynthesis pathway of flavonoids

(Packer and Sies, 2001).

According to the modifications of the central C-ring, they can be divided into

different structural classes including flavonols (represented mainly by quercetin,

kaempferol, and myricetin), flavones (represented by apigenin and luteolin), flavan-3-ols

(ranging from the simple monomers (+)-catechin and its isomer (−)-epicatechin to the

oligomeric and polymeric proanthocyanidins), flavanones, isoflavones, and

anthocyanidins (Goncalves et al., 2012).

9

Figure.2.2.

Major classes of Flavonoids

(http://supplementscience.org/antioxidants.html)

The flavonoid content in plants is strongly influenced by extrinsic factors such as

variations in plant type and growth, season, climate, degree of ripeness, food preparation

and processing (Aherne & O´Brien, 2002). Flavonoids have a wide range of biological

activities, such as inhibition of cell- proliferation, inducing-apoptosis, enzyme inhibiting

activity, antibacterial, and antioxidant effects (Figure.2.3). Moreover, some findings

indicate that flavonoids possess various clinical properties, such as antiatherosclerotic,

antiinflammatory, antitumour, antithrombogenic, antiosteoporotic, and antiviral effects

(Buer et al., 2010). Flavonoids decrease the risk of coronary heart disease by three major

10

actions: improving coronary vasodilatation, decreasing the ability of platelets in the

blood to clot, and preventing low-density lipoproteins (LDLs) from oxidizing

(Benavente-Garcia and Castillo, 2008). These polyphenols interact with ABC drug

transporters involved in drug resistance and drug absorption, distribution and excretion.

This flavonoid-ABC-transporter interaction could be beneficial for poorly absorbed

drugs but could also result in severe drug intoxication, especially drugs with a narrow

therapeutic window (Alvarez et al., 2010).

Figure.2.3.

Potential effects of flavonoids

(Grassi et al., 2010)

2.3. Methods for separation, purification and quantification of flavonoids

Although quercetin is generally recognized as a phytochemical agent with

biological activities, few of the published reports present details on the sample

preparation, separation and identification of quercetin from non-commercial plants. The

determination of flavonoids in biological fluids, drinks, plants and food follow

11

different types of strategies and procedures (de Rijke et al., 2006). The necessity of

standardization of natural products and their registration as functional nutraceuticals

demand easy, quick and inexpensive methods of analysis (Medic-Saric et al., 2009).

Figure.2.4. Scheme for studying flavonoids in biological samples

(de Rijke et al., 2006).

Extraction of flavonoids

Most of flavonoids are extracted readily from source material by alcohol or

alcohol/ water mixtures. Prior knowledge of the nature of the flavonoids is helpful in

determining the most efficient extraction techniques. The extraction method allows to

12

concentrate and to clean up the extracted sample before chromatographic injection

(Daniela et al., 2007) and during the extraction of plant material, it is important to

minimize interfering compounds that may co-extract with the target compounds, and to

avoid contamination of the extract, as well as to prevent decomposition of important

metabolites or solvent impurities (Jones and Kinghorn, 2012).

Method of Soxhlet extraction is not always acceptable for industrial applications

due to long extraction time, and large consumption of hazardous solvents

(Grigonis et al., 2005). This extraction method is also not suitable for the extraction of

thermo-sensitive compounds due to the probability of thermal decomposition of target

compounds as extraction usually occurs at the boiling point of used solvent for a long

time (Wang and Weller, 2006).

In general, a larger solvent volume can dissolve constituents more effectively

leading to an enhancement of the extraction yield (Yang and Zhang, 2008). An increase

in the volume of extraction solvent leads to an increase in the amount of extracted

flavonoids, since higher numbers of solvent molecules can interact with solid materials

and enhance the extraction (Hadjmohammadi and Sharifi, 2009).

Concerning to the extraction methods, maceration method uses limited volume

of solvent, which could lead to saturation of the extracting liquid due to the reduced

contact time of the solvent with the plant material (Nobre et al., 2005).

Trabelsi et al. (2010) proposed that the extraction with solvent system of varying

polarities, differ significantly in their extraction capacity and selectivity for quercetin

content in leaves. It was concluded from his studies that the extracts obtained using

solvents of higher polarity were more effective. Chen et al., (2012b) found that an

optimal condition for flavonoid extraction from lotus leaves was when 1g of leaf tissues

were extracted in 30mL methanol–water mixture (70:30) for 36h.

The first process can be a liquid-liquid extraction, which consists of a partition of

extract components into two immiscible solvents. The crude extracts obtained can be

used for purification of flavonoids. The primary water extract can then be successfully

13

extracted using ethylic ether (to extract flavonoid aglycones), ethyl acetate (to recover

flavone monoglycosides), and n-butanol (to extract flavone C-glycosides and poly-O-

glycosylated flavones). Hence, different flavonoids are extracted depending on their

polarity (Nollet and Toldra, 2012). Wu et al. (2012) described a novel ionic liquid-based

pressurized liquid extraction procedure coupled with high performance liquid

chromatography (HPLC) tandem chemiluminescence (CL) detection capable of

quantifying trace amounts of rutin and quercetin in four Chinese medicinal plants. Chen

and Xiao (2005) demonstrated that highest concentration of quercetin was better

extracted from the Marchantia convoluta inflorescences with the alcoholic solvent of

lower polarity.

Acid hydrolysis for separation of aglycones

Most methods for screening total amounts of different flavonoids have utilized

complicated sample preparation procedures in which the glycosidic compounds are

transformed into their aglycones by acid hydrolysis. Under the hydrolysis process,

optimum compromise is to be found to minimize degradation reactions of glycosides

and to achieve complete release of aglycones. Evaluating various hydrolysis conditions,

even in the case of the same matrix, aiming to release the same aglycone(s) reflect the

difficulty and importance of this preparation step.

Optimum acid concentration and hydrolysis time for hydrolysis of flavonol

glucuronides, flavonol glucosides and flavone glucosides, expressed in HCl

concentration (M)/h, were reported on the quercetin, kaempferol, myricetin, luteolin and

apigenin yields from cranberry, onion, leek, lettuce, endive and celery, in order of

listing, 1.2 M/0.5 h, 1.2 M/2 h, 1.6 M/4 h, 2.0 M/2 h, 1.6 M/4 h and 2.0 M/4 h,

respectively. These data proved the fact that unified optimum conditions could not be

suggested for purification of aglycones (Molnar-Perl and Fuzfai, 2005).

Quantification of flavonoids

The total flavonoid content in extractive solvents represents an important

parameter in evaluating the extractive process and is frequently based on the

14

complexation with aluminium chloride (Nobre et al., 2005) or based on the formation of

terbium (Tb3+)-flavonoids (quercetin as a reference standard) complex at pH 7.0, which

has fluorescence intensely with maximum emission at 545 nm when excited at 310 nm

(Shaghaghi et al., 2008).

The methods of determination of chemical composition of plant extracts include

preliminary chromatographic separation, thin layer chromatography (TLC), High

performance chromatography (HPLC) and preparative thin layer chromatography, and

hyphenated techniques such as Nuclear Magnetic Resonance spectrophotometry (NMR).

These first three methods were used for rapid initial screening of crude plant extract, and

provides initial information on the content and the nature of constitutions in the matrix.

Chromatographic methods (thin layer chromatography and high performance liquid

chromatography) were used for identification, quantification, and characterization of

individual flavonoid or phenolic acid (Medic-Saric et al., 2009; Menon et al., 2012).

Thin layer chromatography

Chromatography is a powerful analytical method suitable for the separation and

quantitative determination of a considerable number of compounds, even from

complicated matrix. Thin Layer Chromatography (TLC) has some advantages such as

rapidity, sensitivity, easiness, cheapness and this method does not require complex

instrumental equipment (Guliyev et al., 2004).

Silica gel is by far the most frequently used layer material for phytochemical

analysis by TLC and High performance TLC (HPTLC) methods. This phase has been

used for separation of all classes of compounds. Unmodified silica gel and silica gel

bonded phases with polar (hydrophilic bonded): cyano, amino, diol, and nonpolar

(hydrophobic bonded): n-octadecyl groups have been employed for analysis of

polyphenolic compounds (Tomczyk et al. 2012). The purpose of optimization of

chromatographic systems is to find the one that shows the greatest difference in

identification characteristics between substances e.g. retention factor (RF) value in thin

layer chromatography.

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Quercetin and rutin was determined in Tephrosia purpurea Pers leaves on TLC

plates using the solvent system methanol-water-formic acid (40:57:3, v/v/v), which

produced good separation with RF values of 0.07 (quercetin) and 0.17 (rutin) for the

ethanolic extract and reference compounds (Jain et al., 2009). Attarde et al. (2008)

estimated quercetin, quercetin-3-O-galactoside, quercetin-3-O-xyloside and quercetin-3-

O-rutinoside in leaflets of Soymida febrifuga with the help of HPTLC technique using

Toluene: ethyla acetate: formic acid (5:4:1) as mobile phase.

Quantitative determination of quercetin (RF 0.75) in flower of Acacia nilotica

using high performance thin layer chromatography (HPTLC) was performed on

aluminum plates precoated with silica gel 60 F254 and separation was achieved in the

mobile phase of chloroform: methanol: water (7: 3: 0.5 v/v) and densitometry

determination was carried out at 280 nm in absorbance mode (Leela et al., 2011).

Similarly, Sehgal et al., (2011) developed a rapid HPTLC method for separation of

quercetin from Mimusops elengi L. using Toluene: Ethyl acetate: Formic acid (5:4:1) as

mobile phase.

Quercetin 3 -O -α- d- glucuro pyranoside (miquelianin; QG), quercetin 3-O-α-d

glucopyranoside (isoquercitrin), quercetin 3-O-α-d-galactopyranoside (hyperoside) and

rutin in ethyl acetate fractions from aerial parts of selected Potentilla species (Tomczyk

et al., 2012) on a HPTLC plates using a mixture consisting of ethyl acetate/methyl ethyl

ketone/diisopropyl ether/formic acid (3:10:4:1, v/v/v/v). Rutinosides and quercetin were

also eluted using methanol-water-acetic acid (50/44/6, v/v/v) (Vila-Real et al., 2011) and

benzene: pyridine: formic acid (36:9:5) (Audu et al., 2007). The spot visualization was

evaluated under UV light at 254nm and Ferric chloride reagent.

Preparative thin layer chromatography

The inexpensive and disposable nature of individual TLC plates allow for it to be

used to identify impurities that may cause damage to HPLC columns and detectors. TLC

is widely used in organic synthesis for monitoring reactions and the identification of

product formation. TLC plates also allow for multiple samples to run in parallel to each

other and normal TLC run times are often shorter than HPLC. These advantages have

16

resulted in extensive development and applications of TLC for analytical and

preparative applications (Dillon et al., 2012). For the preparative separation of

compounds, TLC plates with a thicker silica gel layer than that on analytical plates are

regularly used. After the chromatographic run and nondestructive staining, the clearly

separated compounds obtained can be eluted with organic solvents from the TLC plate

(Christie, 2003). These isolated compounds can then be analyzed by conventional

spectroscopic or MS methods. This is a common technique that allows the isolation of

milligram amounts of clearly separated metabolite fractions in a very fast way

(Teuber et al., 2010).

Preparative TLC also acting as an additional clean-up step for separation of

flavonoids from natural products (Xiao et al., 2010), which can then be used for

different purposes – for example determination by spectroscopic methods for the

structure of the compounds isolated (Cunha et al., 2012), or investigation of their

biological activity. In preparative-layer chromatography UV light or UV–visible

densitometry are used for location of the separated bands.

High performance liquid chromatography (HPLC)

An important aspect of flavonoid analysis is whether to determine the target

analytes in their various conjugated forms or as the aglycones (de Rijke et al., 2006). In

that case, analyses becomes much more complicated, because the number of target

analytes increases significantly: much more selective and sensitive analytical methods

are now required. In general, separations of flavonoids by HPLC are followed mainly on

C18 RP columns, differences in identification and quantitation characteristics of

procedures are associated with the detection system. Quantification of flavonoids is

another forte of HPLC in combination with UV detection.

RP-HPLC is a powerful analytical technique for this purpose which has the

potential benefits of high sensitivity, accuracy, reproducibility, and time-saving

(Liu et al., 2011). Optimum solvent combination and wavelength (254-365nm)

selection for estimation of quercetin are also based on plant species used for the study.

Various solvent systems i.e. methanol and water (Marshall et al., 2012), methylcyanide

17

(Yang et al., 2012), orthophosphoric acid (Lin-Chin et al., 2000) and acetonitrile (Notas

et al., 2012) were also reported for estimation of quercetin from plant extracts. Quercetin

and rutin quantification from methanolic extract of Amarnthus viridis was carried out at

340nm using 0.5% Formic acid: Acetonitrile (70%:30%) by Kumar et al. (2009). HPLC

analysis of quercetin at 254nm was also reported in flower extracts (Chafer et al., 2005;

Sumathy et al., 2011), leaf extracts, honey samples (Michalkiewicz et al., 2008) and

whole plant extracts (Moustapha et al., 2011).

In another RP-HPLC method (Rajeswari and Andallu, 2011), Rutin, quercetin,

chlorogenic acid and caffeic acid separation was carried out from coriander extracts

(Coriandrum sativum L.) using formic acid and acetonitrile gradient as mobile phase.

Misan et al. (2011) developed rapid resolution HPLC/DAD method, on a 1.8 μm,

4.6×50 mm column, to enable a rapid separation of a mixture of 17 compounds, which

consisted of hydroxybenzoic acids, hydroxycinnamic acids, flavones, flavonols,

flavanone, flavonol-glycoside and antraquinone, in a single run, within 22 minutes.

Nuclear magnetic resonance spectrometry (NMR)

Nuclear magnetic resonance (NMR) is traditionally considered as a prime tool in

identification, characterization, and structure elucidation of molecules. NMR is

becoming increasingly popular for metabolomics studies. It is also used for

distinguishing the sites of methylation, glycosylation and acylation in flavonoid

glycoiides, and in some cases the nature and sites of specific sugars and acyl groups

(Markham et al., 1978). Structural assignments for glycosides spectroscopy can be used

to identify and quantify chemicals from complex mixtures. This can be done semi-

automatically by comparing the mixture of interest to a library of reference spectra

derived from pure compounds of known concentrations (Wishart, 2008). Recently,

NMR is widely used to identify different compounds including hydroxylated or

methoxylated flavonoids. Because the position and the number of substituted hydroxy

or/and methoxy groups will change the 1H and

13C chemical shifts, it is important to

understand these changes. So, the structures of newly isolated hydroxy/methoxy-

flavonoids can be easily identified (Yoon et al., 2011).

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2.4. Quercetin and quercetin derivatives

Quercetin (3,3‘,4‘,5,7-pentahydroxyflavone) is an important member of the class

of flavonoid food components (Figure 2.5). The average western daily diet contains

about 16 mg of quercetin (Hertog et al., 1993). The interest for the use of flavonoids like

quercetin as functional food ingredient and/or food supplement is increasing rapidly,

owing to the several health claims. Use of these supplements by consumers at the

indicated daily levels may increase the daily intake of quercetin 25- to 60-fold (van der

Woude et al., 2004).

Figure 2.5.

Chemical structure of quercetin.

Quercetin derivatives

Quercetin O-glycosides are quercetin derivatives with at least one O-glycosidic

bond which are widely distributed in the plant kingdom (Materska, 2008). A quercetin

glycoside is formed by attaching a glycosyl group (a sugar such as glucose, rhamnose, or

rutinose) as a replacement for one of the OH groups (commonly at position 3). The

attached glycosyl group can change the solubility, absorption, and in vivo effects of

quercetin glycosides (Kelly, 2011). Quercetin 7-O- rhamnoside reduces porcine

epidemic diarrhea virus replication via independent pathway of viral induced reactive

oxygen species (Song et al., 2011). Isoquercetin (quercetin 3-O-glucoside) had a

regulative role in blood glucose level and lipids, which improved the function of

pancreatic islets, and may be useful in the treatment of type 2 diabetes mellitus

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(Zhang et al., 2011a). The chelating site of quercetin involves the deprotonated C5-OH

and the C4=O groups at ring C of quercetin (Ahmedova et al., 2012). Leishmanicidal

flavonol quercetin is a mixed inhibitor whereas quercitrin and isoquercitrin are

uncompetitive inhibitors of Leishmania (Leishmania) amazonensis arginase

(da Silva et al., 2012).

Initially, Quercetin methyl ether derivatives displayed cytotoxic properties,

which induced G2–M phase cell cycle arrest and apoptosis on human myeloid leukemia

cells (Rubio et al., 2007) and nowadays, these are used for treatment of inflammatory

diseases related with NO overproduction due to inducible nitric oxide synthase

(Gil-Ortega et al., 2010). Quercetin- 3-gallate showed comparable antiviral activity

against influenza virus and no cytotoxic effects on Caucasian hepatocyte or lung

carcinoma at concentrations as high as 200 µM (Katalinic et al., 2010). Nowadays,

Quercetin derivatives act as novel antibacterial agents by showing dual inhibition on

DNA gyrase and topoisomerase (Hossion et al., 2011).

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Structural modification of quercetin by methylation at the 5th

or 7th

position is the

most optimal to retain most of the antioxidant capacity of quercetin (Moalin et al., 2011)

and it protect brain from disorders associated with oxidative stress (Ishisaka et al. 2011).

They interact with lipid bilayers, and play a role of ―guarding‖ molecules, preventing

penetration of acyl chain regions by reactive oxygen species (Cieslik-Boczula et al.,

2012). Thapa et al. (2012) investigated the synthesis and antiviral activities of various

quercetin derivatives with substitution of C3, and C5 hydroxyl functions with various

phenolic ester, alkoxy, and amino alkoxy moieties.

Health effects of quercetin

For decades, quercetin has been widely investigated, mainly because of its

putative health-promoting effects, for example as a potential protection against coronary

heart disease (Pace-asciak et al., 1996) and hypertension (Larson et al., 2010). These

health claims are merely supported by in vitro evidence related to the cytoprotective

ability of quercetin to modulate the activity of numerous enzymes involved in energy

metabolism (An et al., 2010), signal transduction, cell growth (Nakamura et al., 2011)

and antioxidant activity (Zhang et al., 2011b). Quercetin is an emerging prospective

anticancer drug candidate and its prodrug QC12 has entered phase-I clinical studies

(Hirpara et al., 2009). Quercetin showed neuronal cell protective effect against

glutamate-induced neurotoxicity (Yang et al., 2012), and recovered the mitotic

index and chromosomal instability after treatment with hydrogen peroxide

(Boligon et al., 2012).

Bioavailability of quercetin

Bioavailability studies performed with quercetin have recently been reviewed

and showed that the rate of elimination of quercetin metabolites is relatively slow, with

half-lives ranging from 11 to 28 hours. This could favour accumulation in plasma with

repeated intakes (Manach et al., 2005). Quercetin, and possibly other related flavonoids,

given orally is rapidly metabolized in the intestine and liver into glucuronidated

derivatives which act as carriers of quercetin and deliver the free aglycone in situ by

deconjugation. Moreover, the intravenous administration of quercetin-3-O-glucuronide

21

resulted in a slow onset and sustained blood pressure lowering effect

(Menendez et al., 2011).

2.5. In vitro culture techniques

Plant cell/tissue culture, also referred to as in vitro, axenic, or sterile culture, is

an important tool in commercial application for production of whole plants (Thrope,

1990). The capacity for plant cell, callus, and organ cultures to produce and

accumulate many of the same valuable chemical compounds as the parent plant in nature

has been recognized almost since the inception of in vitro technology

(Karuppusamy, 2009; Hussain et al., 2012). Callus cultures, containing more or less

homogenous clumps of dedifferentiated cells, are used for secondary metabolite

production (Jedinak et al., 2004).

The production of compact callus at cut edge of explant may be due to the

wound during the process of cutting which resolved in a synchronous cell division

(Tahir et al., 2011). Changes in hormone composition provoke marked perturbations in

growth and biosynthetic characteristics of the cultures (Kiselev et al., 2007). The size of

callus clumps may determine the chemical gradients that influence chemical synthesis

(Wu et al., 2003). However, many factors such as genotype, composition of the nutrient

medium, and physical growth factors such as light, temperature, humidity, and

endogenous supply of growth regulators are important for callus induction

(Pierik, 1987).

Under in vitro conditions, explants have been removed from their original tissue

environment and transferred to synthetic media containing non-physiological

concentrations of growth regulators and organic and inorganic constituents, resulting in

exposure to significant stresses. Plant growth regulator can be defined as either natural

or synthetic compounds that modify the plant growth and development pattern exerting

profound influence on many physiological processes (Jaleel et al., 2009) and involved

in the regulation of these developmental switches under in vitro conditions

(Feher et al., 2003). Auxins, cytokinins, gibberellins, abscisic acid and ethylene are

commonly recognized as naturally occurring plant hormones (Rai et al., 2011).

22

Morphogenetic response of leaf discs- explants was greatly influenced by the

type of growth regulator used in the medium (Singh and Chaturvedi, 2012). Different

plant hormones like 2,4-D, kinetin, IAA, BAP influence callus induction from explants

(Roy et al., 2008). For continuity of callus induction, 2,4-D alone or in combination with

Kn are essential and auxin reduction may lead to organogenesis and embryo formation

(Valizadeh and Tabar, 2009). Changes in hormone composition provoke marked

perturbations in growth and biosynthetic characteristics of the cultures (Kiselev et al.,

2007). As callus growing in a nutrient-rich culture medium undoubtedly exposed to

more carbon influx than the field- grown plant parts, it may influence the metabolic flux

for the biosynthesis of elevated levels of phenols (Hakkim et al., 2007). The age of the

cells in the inoculum also had influence on metabolite production and determines the

way the culture grows (Palacio et al., 2010).

Fedoreyev et al. (2000) established callus cultures from the different parts of

Maackia amurensis and analyzed for isoflavonoids. The isoflavones daidzein, retuzin,

genistein, formononetin, and the pterocarpans maakiain were found to be produced by

these cultures. Shashikala et al. (2009), studied callus induction from leaf explants of

Centella asiatica and leaf callus showed high accumulation of asiaticoside compared to

parent plant. Quercetin production also observed in callus cultures, biosynthesis of

apigenin, kaempferol, quercetin were identified from Indigofera cordifolia callus

cultures (Upman and Sarin, 2011).

Sucrose

Sucrose is the most important carbon and energy source that influences plant cell

growth and metabolism. The growth and production of secondary compounds from cells

in culture depends greatly on the source of carbon employed, its concentration and on

the biosynthetic pathway or process studied. The optimum concentration varies

according to the requirement of different plant species (Malik et al., 2011). Meanwhile,

as an osmotic pressure agent, sucrose could maintain a stable osmotic pressure

environment for culture and support vigorous growth of in vitro tissue culture of most

plants (Ondo Ovono et al., 2009).

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Addition of sucrose increases survival of explants and callusing, where high

sucrose concentrations induce organogenesis (EI Tahchy et al., 2011). Sucrose

concentration along with plant growth regulators might be more responsible for the

secondary metabolite biosynthesis in cell suspension cultures (Ram et al., 2011) and the

extra carbohydrates accumulated in plants might be channeled for the production of

secondary metabolites (total phenols and flavonoids), thus explaining the reason why the

up regulation of plant secondary metabolites production. Stable and optimized callus

cultures are a logical step in the first phase of the cell culture production of plant

secondary metabolites, i.e. preparing the inoculum for liquid suspension cultures.

2.6. Cell suspension cultures for secondary metabolite production

Plant cell suspension cultures are potential sources of secondary metabolites. In

contrast to traditionally grown ―whole wild plants‖, their production in in vitro

guarantees defined controlled process conditions and therefore minimizes or even

prevents variations in product yield and quality, which simplifies process validation and

product registration (Eibl and Eibl, 2008). Secondary compounds from plant have been

incorporated into a wide range of both commercial and industrial applications, and in

many cases, rigorously controlled plant in vitro culture can generate the same valuable

natural products (Park et al., 2008). For commercial utilization of flavonoids produced

from cell suspension cultures requires large scale production along with increase in

specific metabolite accumulation. This criteria is fulfilled by the addition of precursor to

the medium to increase target metabolite accumulation.

Precursor feeding

Another successful strategy used in influencing the biosynthetic pathways to

activate and increase the production of secondary metabolites is by feeding cell cultures

with precursors (Baldi and Dixit, 2008). Precursors such as amino acids have been

successfully used when they are cheaper than the desired end products

(Tumova et al., 2004). Nonetheless, the induction of exogenous precursors to a plant in

vitro culture causes an increase in the production of secondary metabolites

(Namdeo, 2007). Phenylalanine is a precursor for most phenolic compounds in plants

24

and has been successfully used to induce metabolite production in vitro in many

different plant systems. Phenylalanine at 2mM concentration led to 1.3 fold higher

isoflavonoid production from hairy root cultures of Psoralea corylifolia (Shinde et al.,

2009). The content of p-coumaric acid in Larrea divaricata cell cultures increased to 100

fold in the medium supplemented with 3mM l-phenylalanine (Palacio et al., 2011).

Similarly, exogenously added phenylalanine, however, had little effect on resveratrol

production in V. amurensis callus cultures (Kiselev et al., 2007) and anthocyanin

production in cell suspension cultures of Vitis vinifera (Qu et al., 2011).

2.7. Evaluation and elicitation of flavonoids in cell suspension cultures

Production of secondary metabolites reflects the adaptations of plants to

abiotic and biotic stress, which was enhanced by the addition of elicitors

(Wongwicha et al., 2011). In general, the secondary metabolites that are involved in

plant defense functions undergo significant elicitation as a response to external physical,

chemical and biological stimuli (Savitha et al., 2006).

Physical Elicitors

Recently, some physical elicitors which include UV-ray, high or low

temperature, electromagnetic wave, mechanical wound, etc. are reported to have

remarkable effects on induced resistance in plants and cell suspension against pathogens

(Zhao et al., 2005) and these treated plant cells showed increase in phenylamine

ammonia lyase (PAL), peroxidase (POD) and polyphenoloxidase (PPO), which are

strongly associated with the biosynthesis of flavonoids in cells.

A wide array of external stimuli are capable of triggering changes in the plant

cell, leading to a cascade of reactions that ultimately result in the formation and

accumulation of secondary metabolites, which help plants to overcome the stress factors.

Amongst these, visible light and ultraviolet (UV) light which are parts of the solar

spectrum, represents an important ecological factor that influences the organisms and

ecosystems, and it is related to the occurrence of some adaptive changes in organisms

throughout the development of life on Earth. Increasing evidence indicates that light is

25

important for the proper induction of plant defense and for resistance to pathogens

(Jeong et al., 2010).

UV radiation is divided into three regions: UV-C (wavelengths below 280 nm),

UV-B (280- 315 nm) and UV-A (315-400 nm). UV-C is the most damaging, but it is

almost completely absorbed by the stratosphere. By contrast, UV-B radiation is only

partially absorbed by the stratospheric ozone layer, and UV-A is not absorbed at all.

Therefore, a fraction of UV-B and all UV-A reaches the earth's surface, where they

cause various biological effects. Moreover, the effectiveness of biological responses to

UV radiation increases with decreasing wavelength, so these responses are normally

dominated by the UV-B (Almagro et al., 2011).

In plant cell cultures, UV light acts as an abiotic factor which stimulates the

biosynthesis of secondary metabolites (Broeckling et al., 2005). Thus, it has been shown

that UV-B light induces both the formation of dimeric terpenoid indole alkaloids and

tryptophan decarboxylase and strictosidine synthase mRNA accumulation in

Catharanthus roseus (Ouwerkek et al., 1999). Ramani and Jayabaskaran (2008) also

observed the enhanced production of catharanthine and vindoline from C. roseus cell

cultures, when cells were irradiated with UV-B for 5 min.

Visible light primarily induces biosynthesis of phenolic acids and affects their

composition, whereas UV light specifically induces biosynthesis of flavonols

(Koyama et al., 2012). A significant increase in boswellic acid accumulation by UV

light in callus cultures of B.serrata has been observed by Ghorpade et al. (2011).

Pharmacological results confirmed that haem oxygenase-1 (HO-1) is involved in

protection of cells against ultraviolet (UV)-induced oxidative damage and Transcription

factors involved in the synthesis of flavonoid (Xie et al., 2012). UV-C light also is a

good inducer of salicylic acid biosynthesis (Nawrath et al., 2002) which in turn

increased accumulation of flavonoids through chemical signals.

Chemical elicitors

The exposure of cultures to the chemical mediators, such as jasmonates,

salicylates, and ethylene (often referred as ‗‗stress hormones‘‘) is also employed to

26

reproduce the stimulus that triggers secondary metabolites accumulation

(Vazquez-Flota et al., 2009).

Salicylic acid

Salicylic acid (SA) is a well-known signal transducer for wound and microbial

defense responses in plants (Klessig and Malamy, 1994), and has been used as elicitors

or potentiators of secondary metabolic pathways in cell culture systems. Different

response of callus cultures to salicylic acid and conditions of cultivation (light, darkness)

is suggested to be associated with the antioxidant defense system, which is, in particular,

characterized by the hydrogen peroxide content in the callus.

Salicylic acid increased the H2O2 content in non-morphogenic calluses more

strongly than in morphogenic calluses, and the difference was more significant for the

calluses cultivated in the light (Maksyutova et al., 2005), whereas increased H2O2 level

induce flavonoid accumulation in Glycyrriza inflata Batal cultures (Yang et al., 2007).

Satdive et al. (2007) suggested that the azadirachtin production could be increased by

27

decreasing the concentration of signal compounds (SA and JA) in the culture medium

and similar results were observed by Goyal and Ramawat, (2008) in cell suspension

cultures of Pueraria tuberosa.

The examination of growth and production parameters of the callus cultures

growing in the presence of SA showed that SA inhibited growth of all cultures at 100µM

and stimulated anthraquinone production (Bulgakov et al., 2002). Subsequent increase

of SA concentrations in the media strongly suppressed the growth of cultures. The

reason behind this phenomenon is suitable concentration of SA activated oxidative

enzymes activities, however higher concentration of SA inversely depressed oxidative

enzymes activities (Jing-Hua et al., 2008).

Exogenous salicylic acid coupled with an increase in beta-1,3-glucanase,

maintains cellular integrity and prevents damage to cell wall and plasma membrane

(Zhen and Li, 2004). It is feasible that elicitation with salicylic acid regulates the

jasmonate pathway, which in turn mediates the elicitor-induced accumulation of

silymarin in hairy root cultures of S.marianum (Khalili et al., 2009).

Abscisic acid (ABA)

Abscisic acid (ABA) plays a significant role in the regulation of many

physiological processes of plants. It is often used in tissue culture systems to promote

somatic embryogenesis and enhance somatic embryo quality by increasing desiccation

tolerance and preventing precocious germination. ABA is also employed to induce

somatic embryos to enter a quiescent state in plant tissue culture systems and during

synthetic seed research. Application of ABA and a related analog, (+)- 80 acetylene

ABA, mediate induction of lipid accumulation in the L. fendleri suspension cultures

(Kharenko et al., 2011).

Exogenous ABA could elevate thermo tolerance in calluses by alleviating ion

leakage, lipid peroxidation and growth suppression induced by heat stress, respectively.

Employment of exogenous ABA remarkably activated NOS activity and increased NO

release (Song et al., 2008) and NO has been reported to play important roles in elicitor-

induced secondary metabolite production (Lu et al., 2011).

28

Increase in cell ABA content induced both structural and regulatory genes

involved in anthocyanin production in grape culture (Gagne et al., 2011). Furthermore,

ABA induced the important genes in the artemisinin biosynthetic pathway

(Jing et al., 2009) and Hao et al., (2010) suggested that a causal relationship present

between ABA release and both PAL activity and ABA is involved in fungal endophytes-

induced flavonoids accumulation in Ginkgo biloba.

Gibberellic acid (GA3)

GA3 is also a member of plant hormone called gibberellins, which regulate the

growth rate of plants (Abbas et al., 2010). Gibberellic acid (GA3) is widely used in

agriculture, to end dormancy in seeds, promote flowering or accelerate germination in

the brewery industry (Balakrishnan and Pandey, 1996). Gibberellins are also involved in

several plant development processes and promote a number of desirable effects

including stem elongation, uniform flowering, reduced time to flowering, and increased

flower number and size (Srivastava and Srivastava, 2007).

GA3 can increase the antioxidant metabolism and alkaloid production in

Catharanthus roseus (Jaleel et al., 2007), and exogenously supplied gibberellic acid at

the 0.5 mg/l level enhanced growth and coumarin content in hairy root cultures of

Cichorium intybus L (Bais et al., 2001). Gibberellic acid at lower concentrations

(0.01mg/l) showed 91% increase artemisinin production in Artemisia dubia hairy root

cultures (Ali et al., 2012). In combination with dry yeast (6g/L) gibberellic acid

enhanced essential oil yield in Lemon balm plants (Sharaf El-din et al., 2009) and a

combined treatment of GA3 with paclobutrazol to the in vitro plants generally increased

the concentration of caftaric acid in the roots of Echinacea purpurea L. (Jones et al.,

2009).

Heavy metals

Heavy metals are included in the main category of environmental pollutants as

they can remain in the environment for long periods; their accumulation is potentially

hazardous to humans, animals and plants (Benavides et al., 2005; Gratao et al., 2005). It

is well known that any pollutant, heavy metals in particular, has the potential to cause

29

damage to plants, which, in turn, respond to the stress to survive. Heavy metals caused

the change in the plant defense system and induced antioxidants accumulation

(Azevedo and Azevedo, 2006).

Heavy metals such as Cadmium, Lead, Arsenic, Zinc and Copper

(Raeymaekers et al., 2003) induce rapid generation of Reactive Oxygen Species (ROS)

and oxidative stress (Pompeu et al., 2008) in cell suspension cultures. Presence of ROS

and other stress increase PAL activity in plants (Pawlak-Sprada et al., 2011a) and

activate phenyl propanoid pathway which is involved in flavonoid biosynthesis (Pawlak-

Sprada et al., 2011b). Silver and Cadmium showed stimulating effect on tanshinone

accumulation in Salvia miltiorrhiza cell cultures (Zhao et al., 2010). Copper and zinc are

essential micronutrients and components of several enzymatic systems, participating

primarily in electron flow and catalysis of redox reactions, but excess of these metals

become toxic (Sharma and Dietz, 2009).

Copper

Copper at high concentrations decreased both growth and chlorophyll content to

a greater extent, the decrease in growth under all treatments was also accompanied by

leaf necrosis, a possible indicator of cell death. Copper increases calcium, Nitric oxide,

and H2O2 levels and there is a cross talk between these intracellular signals, leading to a

calcium-dependent activation of gene expression in Ulva compressa

(Gonzalez et al., 2012). It also increased Nitric oxide release and NADPH-diaphorase

activity in root tips of Vicia faba (Zou et al., 2012). Interestingly, the copper-alone and

UV -alone treatments caused accumulation of virtually the same population of

phenylpropanoid compounds (Babu et al., 2003). Copper ion (0.1mM) enhanced growth

and accumulation of decursinol angelate production up to 3.22 fold than control in

Angelica gigas root cultures (Rhee et al., 2010).

Zinc

Zn stress enhance energy metabolism in Arbis paniculata plants, including auxin

biosynthesis and protein metabolism to maintain plant growth and correct misfolded

proteins (Zeng et al., 2011). Kim et al. (2010) indicated that H2O2 levels increased in

30

response to heavy metal (Cu and Zn) stress and they were closely linked to an improved

antioxidant defense capability mediated by peroxidases. The phenomenon behind

increase in flavonoids was that peroxidases catalyze the reduction of H2O2 by

transferring electrons to various donor molecules such as phenolic compounds (Passardi

et al., 2005). Zn also induced phytochelatins and glutamylcysteine synthesis and

decreased Glutathione content in red spruce cells (Thangavel et al., 2007).

Elicitors may interfere with plant phenyl-propanoid compounds catalyzing

lignification of plant cells (Sreedhar et al., 2009), success of an elicitor to trigger a

particular metabolic pathway does not necessarily establish its total in-efficacy. An

elicitor may be ineffective under a combination of inappropriate conditions as well as

unsuitable concentration of an elicitor. Apart from elicitors, production of secondary

metabolites also influenced by the medium components such as casein hydrolysate,

chitosan, yeast extract, etc.

Organic supplements

The success of plant tissue culture as a means of plant propagation is greatly

influenced by nature of the culture medium used. In media, ‗undefined‘ components

such as fruit juices, yeast extracts and protein hydrolysates, coconut water were

frequently used in place of defined vitamins or amino acids, or even as further

supplements.

Coconut water

Coconut water (CW) is the colorless liquid endosperm of green coconuts (Cocos

nucifera), which is used in tissue culture media as organic supplement. It contain

complex combination of compounds such as amino acids, organic acids, nucleic acids,

vitamins, sugars, and sugar alcohols, plant hormones (auxins, cytokinins) and other

unidentified substances. When added to a medium containing auxin, the liquid CW can

induce plant cells to induce to divide and grow rapidly (Molnar et al., 2011) and often

used to maximize callus growth in various plant cultures like Rudgea jasminoides

(Oliveira et al., 2007) and Nopalea cochenillifera (Adki et al., 2012). The addition of

31

coconut water to the culture media resulted in the doubling of sub-culturing

time from four weeks to eight weeks in Cyamopsis tetragonolobust cultures

(Mohammad and Ali, 2010).

Yeast extract

Yeast extract (YE), which is one of the most frequently used elicitors, is

composed of poly- or oligo-saccharide mixtures, such as glucan, ergosterol, and

glycopeptide, derived from cell walls (Boller, 1995). Apart from amino acids, vitamins

and minerals (Ertola and Hours, 1998), and it is also possible that elicitation effects

might be due to the contents of cations like Zinc, Calcium and Cobalt in the yeast extract

(Samet et al., 2012). Reports suggested that yeast extract is used as a supplement in

order to promote plant growth, due to its high amino acid content (George et al., 2008).

The use of yeast extract not only induces the production of the metabolites, but

also reinforces the preeminence of specific metabolite and stimulates its release after

only 24 h of exposure (Pitta–Alvarez et al., 2000) and transiently up-regulated, mRNA

levels of polyketide reductase, an enzyme for flavonoid biosynthesis

(Hayashi et al., 2003), and initiated the jasmonic acid and ethylene pathways.

Enhancement of artemisinin levels in suspension cultures of Artemisia annua was also

observed by addition of yeast extract (Baldi and Dixit, 2008).

The effect of YE on secondary metabolism is proving to be very complicated,

probably because it is a mixture of numerous compounds. Other studies also found that

yeast extract at low concentrations did not favour cell growth but did enhance

scopolamine production in root cultures of Hyoscyamus niger (Hong et al., 2012). YE

also induced the formation of aucuparin as the major phytoalexin in Sorbus aucuparia

cell cultures (Huttner et al., 2010) and hyocyamine production in root cultures of Atropa

belladonna (Samet et al., 2012). Cell suspension cultures supplemented with yeast

extract showed increase in total isoflavonoids production in Pueraria tuberosa

(150 mg/L) (Goyal and Ramawat, 2008); whereas it also diminished rosmaric acid

production in Coleus blumei hairy roots (Bauer et al., 2009).

32

Jasmonic acid acts as a main mediator of yeast extract signal transduction which

independently induces secondary metabolite production in cell suspension cultures

(Zhao et al., 2004) and reactive oxygen species may mediate elicitor signals to the

jasmonate pathway that lead to the production of metabolites (Hasanloo et al., 2009).

Sasaki et al., (2008) detected induction of phenyl transferase activity in S.flavescens by

yeast extract, which mimics insect and pathogen attack.

2.8. Importance of the plants selected

Abutilon indicum

A. indicum (L.) Sweet is an erect, branched shrub of 0.5–1 m height, having the

Tamil name as ―thuthi‖. A. indicum L. is in the family Malvaceae, is easily identified by

its cog-like fruits, and is found abundantly in wastelands. This plant has a long history of

being used medicinally as an antidiabetic remedy, and phytochemical screening of the

plant revealed that it contained alkaloids, flavonoids, tannins, saponins and glycosides

(Seetharam et al., 2002; Krisanapun et al., 2009). This medicinal plant plays an

important role in folk medicine; as a blood tonic, carminative, antipyretic, anti-cough,

diuretic, anti-inflammatory, laxative and antidiabetic (Chuakul et al., 1997). It has been

used for the treatment of urinary diseases, gonorrhea, jaundice, rheumatism, high fever,

mumps, pulmonary tuberculosis, bronchitis, lack of urination and some nervous and ear

problems (Deokule and Patale, 2002; Abdul Rahuman et al., 2008).

According to many scientific reports, the leaf extracts possess hypoglycemic,

hepatoprotective, antibacterial and larvicidal properties (Porchezhian and Ansari, 2005;

Parekh and Chanda, 2007). Analgesic principles from this plant were also isolated

(Ahmed et al., 2000) and its polyherbal formulations have been reported as being

effective in treating diabetes and hyperlipidemia, and effective as free radical scavengers

(Ahmed et al., 2000). Krisanapun et al., (2011) suggested that A. indicum L. maybe also

beneficial for reducing insulin resistance through its potency in regulating adipocyte

differentiation through PPARγ agonist activity, and increasing glucose utilization via

GLUT1.

33

Albizia julibrisin

The species Albizia julibrissin, commonly named mimosa, powder-puff tree, silk

tree, are widely distributed in Asia, Africa, Australia, and tropical and subtropical

America (Zheng et al., 2004; Kim et al., 2007). The genus Albizia (also Albizzia)

belonging to Fabaceae/ Leguminosae family (Mimosoideae subfamily), consists of

approximately 150 species (Wang et al., 2006). Most species are deciduous woody trees

and shrubs. They are easily identified by their bipinnately compound leaves. Its wood

can be used for building and furniture-making. The young leaves are edible

(Zheng et al., 2004). A.julibrissin is an umbrella-shaped tree growing to 6m tall

(Lau et al., 2007), with a broad crown of level or arching branches.

The bark and flowers of the A. julibrissin tree are used in China as medicine

(Lau et al., 2007). Bark extract is a sedative drug and an anti-inflammatory for treating

swelling and pain of the lungs, skin ulcers, wounds, bruises, abscesses, boils,

haemorrhoids and fractures, and has displayed cytotoxic activity (Higuchi et al., 1992;

Ikeda et al., 1997; Pharmacopoeia, 2005). Asians administered A.julibrissin bark extract

to patients to treat insomnia, diuresis, and confusion (Zhu, 1998). The flowers have been

commonly used to treat anxiety, depression and insomnia (Kang et al., 2007) and seeds

are a source of oil (Wang et al., 2006). The phytochemical study of this plant allowed

the isolation of two flavonol glycosides, quercitrin and isoquercitrin. The sedative

activity of these compounds was evaluated, and both compounds increased

pentobarbital-induced sleeping time in dose-dependent manner in mice

(Kang et al., 2000). Linoleic acid, palmitic acid and oleic acid were the dominant fatty

acids in the A.julibrissin seed oil (Nehdi, 2011).

Caesalpinia pulcherrima

Caesalpinia pulcherrima, is an evergreen, low-branching and fast growing shrub.

Leaves, flowers, bark and seeds are largely used in Indian medicine. Literature study

revealed that C.pulcherrima bark contain terpenoids is considered as antibacterial and

antifungal agent (Nasimul Islam et al., 2003). In Indo china, the plant is used as a tonic,

stimulant, and emmengogue. Some compounds of C. pulcherrima which possess

34

antiviral activities against herpesviruses (HSV-1, HSV-2) and adenoviruses (ADV-3,

ADV-8, ADV-11) may be derived from the flavonoid of quercetin (Chiang et al., 2003).

The leaves and flowers of this plant is considered as an antioxidant, cytotoxic agent

(Pawar et al., 2009), analgesic (Chakraborthy et al., 2009), antiulcer agent, and anti-

inflammatory agent (Sharma and Rajani, 2011).

Clitoria ternatea

Clitoria ternatea L. (Family: Fabaceae) a perennial twing herb, steams are terete,

more or less pubescent. The roots have a sharp bitter taste and cooling, laxative, diuretic,

anthelmintic, anti-inflammatory properties; they are useful in severe bronchitis, asthma

and hectic fever (Kirtikar and Basu, 1985; Nadkarni, 1992). The seeds also contain a

water-soluble mucilage, delphinidin 3,3‘,5‘-triglucoside useful as a food dye (Macedo

and Xavier-Filho, 1992). C. ternatea possesses number of pharmacological activities

such as nootropic, anxiolytic, antidepressant, anticonvulsant (Jain et al., 2003), sedative

(Kulkarni et al., 1988), antipyretic, anti-inflammatory and analgesic activities

(Devi et al., 2003). It enhance the memory, and increase acetylcholine content and

acetylcholinesterase activity in rats (Taranalli and Cheeramkuzhy, 2000; Rai et al.,

2001; Rai et al., 2002). In vitro multiplication of C.ternatea has been reported using

mature or seedling explants (Rout, 2004; Barik et al., 2007; Singh and Tiwari, 2010;

Ismail et al., 2011).

Euphorbia hirta

Euphorbia hirta is also commonly known as cats‘ hair, asthma weed, basri

dudhi, malnommee, and fei yang cao. The plant has been used widely in traditional

medicine as a treatment for skin problems, gastrointestinal disorders, particularly

intestinal parasitotosis, amoebic dysentery, diarrhoea, and ulcer. The plant is also used in

bronchial and respiratory disorders including asthma, bronchitis, and hay fever. E.hirta

is well documented for its biological activities such as anxiolytic (Lanhers, et al., 1990),

antifungal (Masood and Ranjan, 1991), diuretic (Johnson, et al., 1999), antihypertensive,

antidiarrhoeial, antimalarial (Tona, et al., 2004), anthelmintic (Adedapo, et al., 2005),

antibacterial (Sudhakar et al., 2006), anti-inflammatory (Singh, et al., 2006) and

35

antioxidant, anticancer/antiproliferative (Mothana, et al., 2009). Two novel butanol-

rhamnopyranosides have been isolated from various non-polar and polar extracts of an

Indian traditional herb, E.hirta (Mallavadhani and Narasimhan, 2009). E.hirta plants

and respective callus obtained in vitro showed that these materials accumulate phenolic

compounds and sterols in high quantity (Pioro-Jabruka et al., 2011).

Psidium guajava

Psidium guajava L. is a broad-spectrum folkloric herbal medicine. Its fruits and

leaves exhibit a diversity of bioactivities acting as antiparasitic, antibacterial, anti-

inflammatory, febrifuges, cicatrizants, antispasmodics, emmenagogues, central nervous

system depressants, hemostatics, antihemorrhoidals, antirheumatics and anticarcinoma

agents (Peng et al., 2008). In the last few decades, guava leaf teas (GLT) have been

heavily commercialized in Taiwan, Japan, China and Korea. The main constituents of

GLT are polyphenolics, flavonoids and triterpenoids (Peng et al., 2008).

Guava (Psidium guajava L.) is also a very nutritious and tasty tropical fruit,

which is characterised by a high content of pectin, dietary fibber, minerals, essential

amino acids, and vitamin C (Li, 2008), as well as, different pharmacological activities

(Gutierrez et al., 2008). Extracts from leaves of P. guajava contain mainly essential oils

(da Silva et al., 2003), tannins (Tanaka et al., 1992), triterpenoids (Begum et al., 2002),

carotenoids and flavonoids (Arima and Danno, 2002; Begum et al., 2004), and phenolic

compounds (Castro-Vargas et al., 2010). Other important chemical constituents of guava

leaves are phenolic compounds, isoflavonoids, gallic acid, catechin, epicathechin, rutin,

naringenin, kaempferol (Barbalho et al., 2012). Huang et al., (2011) reported that

P. guajava has a significant antihyperglycemic effect, and that this effect is associated

with its antioxidative activity.

In view of these reported information, modern biotechnological intervention

could be an effective means to exploit these plants for their biological activity. The

layout of the study, the materials used and the methodology adopted are explained, with

appropriate references, in the following chapter.