5.

DISCUSSION
One of the ongoing problems scientists and medical workers face in
the fight against infectious diseases is the development of resistance to the
agents used to control them. The phenomenon of resistance has been known
since almost the beginning of antibiotic use (http://science.education.nih.gov
/supplements/nih1/diseases/guide/understanding1.htm).
There has been a remarkable progress in the prevention, control and
even eradication of infectious diseases with improved hygiene and
development of antimicrobials and vaccines. However, infectious diseases
still remain a leading cause of global disease burden with high morbidity and
mortality, especially in the developing world. Furthermore, there have been
threats of new diseases during the past three decades due to the evolution
and adaptation of microbes and the re-emergence of old diseases due to the
development of antimicrobial resistance and the capacity to spread to new
geographic areas. The impact of the emerging and re-emerging diseases in
India has been tremendous at the socioeconomic and public health levels.
Their control requires continuing surveillance, research and training, better
diagnostic facilities and improved public health system. Emerging and
re-emerging zoonotic diseases, foodborne and waterborne diseases and
diseases caused by multiresistant organisms constitute the major threats in
India (Chugh, 2008).
More than two-thirds of the antibiotics used to treat humans are
microbial natural products or semisynthetic derivatives of these molecules.
Advances in genetics, biochemistry and bioinformatics have transformed the
study of antibiotics and other natural products, not just by revealing how
they are synthesized but also by casting them as phenotypes encoded by gene

161

collectives that can be studied through an evolutionary lens (Fischbach,

2009).
Plant-derived herbal remedies have remained a vital part of traditional
medicine for thousands of years. It has been estimated by the World Health
Organization that approximately 80% of the worlds inhabitants rely mainly
on traditional medicines for their primary health care. Plant products also
play an important role in the health care systems of the remaining 20% of the
population, mainly residing in developed countries. The use of data on
traditional medicine can provide a very valuable short cut by indicating
plants with specific folkmedicinal uses, which might be likely sources of
biologically active compounds. Recent investigations on medicinal plants
used in traditional medicine have led to the discovery of many new drugs
and hundreds of pharmacologically active substances for synthetic
modifications (Wang, 2008).
One such medicinal plant is Couroupita guianensis, commonly called
as the cannon ball tree. The medicinal use of the parts of the cannon ball tree
are strongly implicated in traditional medical practices. The flowers, leaves,
bark and fruit flesh are used to treat various ailments. The tree parts are used
to cure colds and stomach aches. The juice made from the leaves is used to
cure skin diseases, and the Shamans of South America have even used the
tree parts for treating malaria. The fruit pulp can disinfect wounds and young
leaves ease toothache (Geetha et al., 2004).
Albeit the known uses of the plant parts in various disorders,
especially those against microbial infections, no systematic study on the
nature of the antimicrobial action and the phytochemical responsible for this
action has been reported. Hence, the present study was formulated to analyze
these aspects.

162

The study was formulated in 4 phases. In the first phase, the

antibacterial and antifungal activity of the various parts of the Couroupita
guianensis namely, leaves, bark, flowers and fruit pulp were evaluated. In
phase II, the levels of enzymic and non-enzymic antioxidants in the plant
parts were determined, followed by their radical scavenging ability and
biomolecular protective effects. In phase III, the phytochemical fractions
present in the plant were isolated and assessed for their antmicrobial activity
and free radical scavenging potential. The phytochemical fractions were also
subjected to spectral analyses. In the last phase, the efficacy of the identified
phytochemicals were checked by an in silico approach against the most
susceptible microorganisms.
The results obtained in the study are discussed in this chapter with
appropriate literature support.
PHASE I
In this phase, the antibacterial and antifungal activity of the leaves,
bark, flowers and fruit pulp of Couroupita guianensis were assessed against
seven clinical bacterial isolates namely Staphylococcus aureus, Proteus
vulgaris, Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhi,
Klebsiella pneumoniae and Escherichia coli and six clinical fungal isolates
namely Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
Candida albicans, Rhizopus oryzae and Mucor indicus.
ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY
The leaves, flowers and fruit pulp of Couroupita guianensis exhibited
considerable antibacterial activity, while the bark showed the least activity.
The methanolic extracts of all the parts exhibited a better antibacterial
activity than the aqueous and chloroform extracts. Klebsiella pneumoniae

163

and Staphylococcus aureus were the most susceptible bacteria, among the
seven tested.
The antifungal activity was found to be maximum in the methanolic
extract of leaves, flower and fruit pulp, with the bark showing the least
activity. Candida albicans and Aspergillus fumigatus were the most
susceptible organisms among the seven studied.
There are several reports in the literature indicating the antibacterial
and antifungal activity of the medicinal plants. Unal et al. (2008) have
reported the antimicrobial activities of chloroform, acetone, ethanol and
water extracts of 25 plants, used in Turkish traditional medicine, against ten
pathogenic bacteria and one fungus (Candida albicans) using the disk
diffusion method. The chloroform extract was found to exhibit the maximum
antimicrobial activity in most of the plants.
The results obtained from the study of Buwa and Afolayan (2009)
justified the use of Artemisia afra, Carpobrotus edulis and Tulbaghia
violacea in TB and their related symptoms due to the antibacterial activity of
by their aqueous, ethanol and dichloromethane extracts. The antibacterial
activity was found to be high in dichloromethane extracts.
The ethnobotanical efficacy of Indian medicinal plants, Achyranthes
aspera, Artemisia parviflora, Azadirachta indica, Calotropis gigantean,
Lawsonia

inermis,

Mimosa

pudica,

Ixora

coccinea,

Parthenium

hysterophorus and Chromolaena odorata were examined against clinical

bacteria (Escherichia coli and Staphylococcus aureus) and phytopathogenic
bacteria (Xanthomonas vesicatoria and Ralstonia solanacearum). The
solvents used were methanol, ethanol, ethyl acetate and chloroform. Among
all the extracts, maximum in vitro inhibition was scored in methanol extracts

164

(Sukanya et al., 2009). This reveals that the active components have been
extracted in methanol, which is similar to our results.
The antibacterial and antifungal activities of aqueous, ethanol and
ethyl acetate extract of Torilis anthriscus (L.) Gmel. (Apiaceae) were tested
in vitro against ten species of bacteria and five species of fungi, wherein the
ethanol extracts exerted the strong inhibition of microorganisms than the
other extracts (Stefanovic et al., 2009). Among the n-hexane, ethyl acetate,
n-butanol, methanol and water fractions of sorghum (Sorghum bicolor
Moench), the methanol extract elicited the maximum antimicrobial activity
against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium,
Klebsiella pneumoniae, Candida albicans and Bacillus subtilis (Kil et al.,
2009).
In our study, the methanolic extract of the flowers and fruit pulp
showed a better antimicrobial activity. Many reports are available in the
literature, wherein an alcoholic extract (ethanolic or methanolic) of various
plant parts has shown better antimicrobial activity than other solvent
extracts.
An ethanol extract from the fruits of Tribulus terrestris L., was more
active examined against both Gram-negative and Gram-positive bacteria
than the leaf and root extracts, when tested on 11 species of pathogenic and
non-pathogenic microorganisms (Al-Bayati and Al-mola, 2008). A
methanolic extract of Anthemis cotula flowers, showed good antimicrobial
activity against both Gram-negative and Gram-positive microorganisms
(Quarenghi et al., 2000). The antibacterial activity of ethanolic extracts of
the leaves, flowers and stems of Acacia aroma were found to be more potent
compared to aqueous extracts (Arias et al., 2004).

165

The maximum antibacterial activity among the ethanol, methanol,

acetone and aqueous extracts of dry flower and fresh flower of Cassia
auriculata was associated with the acetone extract of the fresh flower
(Maneemegalai and Naveen, 2010). The organic extracts (petroleum ether
and methanol) of Coccinia indica showed a better activity against the
pathogenic bacteria than the aqueous extract (Shaheen et al., 2009).
In another investigation on the antibacterial effects of root, stalk and
leaf extracts of Rheum ribes on a few common clinical isolates of gram
negative pathogens, the root and leaf extracts showed significant
antibacterial activities than the stalk extracts (Bazzaz et al., 2005). Analysis
of the antimicrobial potential of methanol extracts of root, stem, leaf, flower
and fruits of Lantana camara against ten bacteria and five fungi revealed
that the leaf extract rendered the maximum antimicrobial action followed by
the flower and root extracts (Pour et al., 2009).
Our results show that the flowers of Couroupita guianensis exhibited
maximum antibacterial effect followed by fruit pulp and leaves. The bark of
the plant showed the least activity.
The antimicrobial activities of Cynara scolymus L. leaf, head and stem
extracts were tested against 15 microbial species and the leaf extract was
found to be the most effective, followed by head and stem extracts (Zhu
et al., 2005). The n-hexane, ethyl acetate, n-butanol, methanol and water
fractions of sorghum (Sorghum bicolor Moench) have been tested for their
antimicrobial activity against Staphylococcus aureus, Escherichia coli,
Salmonella typhimurium, Klebsiella pneumoniae, Candida albicans and
Bacillus subtilis, wherein the methanol extract showed high levels of
antimicrobial activity than the other fractions (Kil et al., 2009).

166

The Chinese medicinal plants extracted with hot water, methanol and
acetone were evaluated for their antifungal activity, among which the
acetone extracts had the lowest MIC values indicating their effective
antifungal property (Lee et al., 2007). The aqueous extract of Fragaria
virginiana Duchesne, Epilobium angustifolium L. and Potentilla simplex
Michx. demonstrated strong antifungal potential among fourteen medicinal
plants analyzed (Webstera et al., 2007). The aqueous extracts of five of the
ten medicinal plants tested exhibited broad spectrum antibacterial activity
(Arora and Kaur, 2007). But in our study, the aqueous extracts showed lower
antimicrobial action.
Thobunluepop et al. (2008) have studied the antifungal activity of
crude extracts of Acorus calamus L., Stemona curtisii HK. f., Stemona
tuberose L., Memmea siamensis Kost., Eugenia caryophyllus and eugenol
against several fungal pathogens, where eugenol was found to have the
maximum antifungal activity followed by Eugenia caryophyllus.
Mutai

et

al.

(2009)

have

analysed

the

methanolic

and

methanol:dichloromethane (1:1) extracts of the stem bark of Acacia

mellifera for their antimicrobial action against some bacterial and fungal
strains. The results indicated the effectiveness of methanolic extracts of
Acacia mellifera.
The in vitro antibacterial activity of the ethanolic extracts of herbal
products were tested against both Gram positive (Bacillus subtilis,
Micrococcus luteus, and Staphylococcus aureus) and Gram negative bacteria
(Escherichia

coli

and

Pseudomonas

aeruginosa),

among

which

Staphylococcus aureus and Pseudomonas aeruginosa were found to be

sensitive (Gamboe et al., 2008). The ethanolic extract of Laetiporus
sulphureus was found to possess a narrow range of antibacterial activity

167

against the Gram negative organisms and a broad spectrum antibacterial

activity against the Gram positive organisms (Turkoglu et al., 2007).
In the present study, the methanolic extract of Couroupita guianensis
showed the maximum inhibitory effect against Staphylococcus aureus and
Shigella flexneri, which is in accordance with the results reported by
Ushimaru et al. (2007) that the methanolic extract of Caryophyllus
aromaticus presented the highest anti-S. aureus activity, though effective
against all bacterial strains tested.
The hot aqueous and methanolic extracts prepared from six plants
(Terminallia chebula, Terminallia bellerica, Phyllanthus emblica, Punica
granatum, Lawsonia alba and Mikania micrantha) used in traditional folk
medicines of India, when screened against five pathogenic bacteria
(Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus vulgaris
and Enterobacter aerogenes), showed potential antibacterial activity by the
methanolic extracts (Ghosh et al., 2008).
The crude methanolic extracts of the leaves, stem bark, stem heart
wood, root bark and root heart wood of Euroschinus papuanus and the
fractions obtained on partitioning with petrol, dichloromethane, ethyl acetate
and butanol exhibited a broad spectrum of antibacterial activity (Khan et al.,
2004). The methanol extract of the leaves of Lawsonia inermis showed
significant antibacterial activity, comparable to ciproflaxacin against the
Gram-negative microorganisms with special reference to E.coli, Vibrio
cholerae and Shigella species (Mazumdar et al., 2004).
Good antimicrobial activity was seen in the methanolic extract of
Byrsonima verbascifolia (Lopez et al., 2001). The crude methanolic extract
of Barringtonia asiatica (leaves, fruits, seeds, stem and root barks) and the
fractions (petroleum ether, dichloromethane, ethyl acetate, butanol) exhibited
168

a very good level of broad spectrum antibacterial and antifungal activity

(Khan and Omoloso, 2002). Ethanolic extract and ethyl acetate fractions of
the seeds of Garcinia kola showed good microbicidal activity against most
clinical isolates of bacteria and fungi (Akerele et al., 2008).
The studies on the antibacterial and antifungal activity of leaves and
stem of C. indica have shown significant activity of methanol and ethyl
acetate extracts against different bacteria and fungi and provided support to
our observation that methanol is a better solvent for extraction and isolation
of phytochemicals having antimicrobial activity (Dewanjee et al., 2007).
In past decade, a large number of studies dealing with antimicrobial
screening of the extracts of medicinal plants have been reported (Sokmen
et al., 1999; Keles et al., 2001; Erdogrul, 2002; Mukherjee et al., 2002;
Rabanal et al., 2002; DallAgnol et al., 2003; Sauza et al., 2004; Dulger and
Gonuz, 2005; Duraipandiyan et al., 2006; Arora and Kaur, 2007; Cho et al.,
2008; Hendry et al., 2009).
Our results indicate that the methanol extracts of Couroupita
guianensis flowers and fruit pulp exhibit broad spectrum antimicrobial
activity. The intensity of the antimicrobial action varied depending on the
species of microorganism. The flowers and fruit pulp of Couroupita
guianensis showed equal effectiveness against both Gram positive and Gram
negative bacteria and effective inhibitory effect against some fungal strains
followed by the leaves. Hence, our results support the therapeutic value of
the candidate plant.
PHASE II
In this phase, the enzymic and non-enzymic antioxidants were
analysed in the leaves, flowers and fruit pulp of Couroupita guianensis. Also

169

the ability of these plant parts to scavenge a battery of free radicals has also
been evaluated. The antioxidant property of Couroupita guianensis was also
checked using an in vitro model namely goat liver slices subjected to
oxidative stress using H2O2.
ENZYMIC AND NON-ENZYMIC ANTIOXIDANTS IN Couroupita
guianensis
The enzymic antioxidants analysed were superoxide dismutase,
catalase, peroxidase, glutathione S-transferase and polyphenol oxidases
(catechol oxidase and laccase). The non-enzymic antioxidants estimated
were ascorbate, tocopherol, total carotenoids, lycopene, reduced glutathione,
total phenols and flavonoids. The leaves, flowers and fruit pulp of
Couroupita guianensis were found to contain considerable activities of all
the enzymes analysed and moderate levels of non-enzymic antioxidants.
These results clearly indicate that the flowers of Couroupita
guianensis are a good source of antioxidants. There are several reports in the
literature suggesting that the antioxidant potential of plants contribute to
their medicinal properties.
Ksouri et al. (2009) have reported that the leaves and flowers of the
edible medicinal halophyte Tamarix gallica L. exhibited higher antioxidant
activity validating its traditional use in the treatment of liver disorders.
Sixteen methanol extracts from thirteen medicinal plants collected from
Western Ghats, India, were screened for in vitro antioxidant activity using
different models, among which the methanol extract of C. aromaticus was
found to be more active (Badami and Channabasavaraj, 2007). The
antioxidative potential of the ethanol extracts of Hypericum triquetrifolium
Turra and Hypericum scabroides both belonging to Hypericaceae were
found to possess high antioxidant activities (Kizil et al., 2008).
170

Bhaskar et al. (2009) have evaluated the antioxidant potential of

Clitoria ternatea L. and Eclipta prostrata L. by determining the levels of
enzymatic and non-enzymatic antioxidants. Kim et al. (2009) have reported
a comparative study on the antioxidant potential of leaves and roots of the
wild plant of Adenophora triphylla by relating their phenolic and flavonoid
constituents and found that the leaves are a better source of antioxidants.
The antioxidant potential of buntan pumelo (Citrus grandis Osbeck)
and its ethanolic and acetified fermentation products were analysed and
found that the essential oil had higher phenolic contents than the fruit pulp
extracts (Jang et al., 2010). The antioxidant potential in the herbal bark
extracts of five therapeutically important medicinal plants native to India,
i.e., Crataeva nurvala Buch.-Ham., Buchanania lanzan Spreng., Aegle
marmelos Corr., Dalbergia sissoo Roxb. ex DC., and Cedrela toona Roxb.
was suggested to be associated with their total phenolic and tannin contents
and a high SOD activity (Kumari and Kakkar, 2008).
Plants having vitamins, flavonoids and polyphenols have been
reported to possess remarkable antioxidant activity (Gupta and Sharma,
2006). Emami et al. (2007) have reported a strong antioxidant potential of
Cuprescus semiprevivenseven. The quantitative analysis of the total phenolic
content of the seaweeds indicated that Gelidella acerosa and Haligra species
had high phenolic contents, which correlated with their respective
antioxidant and antimicrobial activity (Devi et al., 2008).
In the present study, the leaves, flowers and fruit pulp of Couroupita
guianensis were found to exhibit considerable enzymic and non-enzymic
antioxidant levels, which supports their traditional uses in several disorders.

171

FREE RADICAL SCAVENGING EFFECTS OF Couroupita guianensis

Free radical-mediated oxidative stress is believed to be the primary
cause of many diseases and disorders. Hence, therapy using free-radical
scavengers (antioxidants) has a potential to prevent, delay or ameliorate
many of these disorders. Over the past two decades, an expanding body of
evidence from epidemiological and laboratory studies have demonstrated
that many plants as a whole, or their identified ingredients with antioxidant
properties have substantial protective effects on human diseases.
In the present study, the antioxidant potential of Couroupita
guianensis was assessed by subjecting the various plant parts to the battery
of radicals and oxidants namely DPPH, ABTS, H 2O2 and hydroxyl radicals.
The flowers of Couroupita guianensis exhibited strong radical scavenging
activity against DPPH, ABTS, H2O2 and hydroxyl radicals followed by the
fruit pulp. The leaves also exhibited a radical scavenging activity, but to a
lesser extent than the flowers and fruit pulp.
DPPH is a stable nitrogen-centered free radical the color of which
changes from violet to yellow upon reduction by either hydrogen or electron
donating. Substances that are able to perform this reaction can be considered
as antioxidants and therefore radical scavengers (Brand-Williams et al.,
1995).
Many studies have been reported on the use of medicinal plants as
radical scavengers. Aqueous leaf extracts of Chenopodium album and
methanolic fruit extract of Vitis trifolia exhibited significant reducing power
and free radical scavenging effect on DPPH, hydroxyl, superoxide, hydrogen
peroxide radicals in a study conducted by Kumar and Kumar (2009).

172

Siddhuraju and Becker (2007) have reported the antioxidant and free
radical scavenging activities of processed cowpea (Vigna unguiculata (L.)
Walp.) seed extracts, wherein the DPPH radical and ABTS cation radical
scavenging activities correlated with the ferric reducing antioxidant capacity
of the extracts. The antioxidant activity of the aqueous extracts of the leaves
of B. forficata and C. sicyoides were determined by using several different
assay systems, namely (ABTS) decolorization, superoxide anion radical
(O 2-) scavenging and myeloperoxidase activity (Khalil et al., 2008).
Among twenty two species of medicinal plants, the methanol extract
of Annona squamosa and Sapium macrocarpum showed two times more
DPPH scavenging activity than the commercial antioxidant butylated
hydroxyl anisole (Ruiz-Teran et al., 2008). An aqueous extract from
Choerospondias axillaries showed a potent scavenging effect on DPPH
(Wang et al., 2008).
Methanol extracts of bark, fruits and leaves of Ficus microcarpa
exhibited excellent ABTS+ scavenging activity (Ao et al., 2008). The
scavenging effect of Andrographis paniculata was demonstrated against
DPPH and ABTS showing its ability to convert unpaired electrons to paired
ones (Tripathi and Kamat, 2007). A crude aqueous extract of Chlorophytum
borivilianum has been shown to scavenge DPPH free radicals and decrease
TBARS, revealing that it is a promising anti-stress agent as well as a
potential antioxidant (Kenjale et al., 2007).
The scavenging antioxidant activities of the three plants namely
Daniella oliveri, Ficus capensis and Vitex doniana were reported by the
DPPH and ABTS assays. This study showed the relationship between the
antioxidant activities and the polyphenolic compounds (Muanda et al.,
2009). Desai et al. (2008) have examined the free radical scavenging

173

potential of the aqueous extract of roots of Baliospermum montanum

Muell-Arg., which was evaluated using DPPH assay and nitric oxide (NO)
scavenging assay and showed a remarkable and concentration-dependent
free radical scavenging activity.
H2O2 has two contradicting functions in cells, one as an oxidant
responsible for lipid peroxidation, membrane injury, pigment bleaching,
protein / enzyme inactivation, and the other as an inducer of antioxidant
enzymes (Sairam and Srivastava, 2000). Although H 2O2 may play an
important role in the development of cancer, it can also be an efficient
inducer of apoptosis in cancer cells, via mitochondrial and endoplasmic
reticulum stress pathway in oral cancer cells, such that increasing the cellular
levels of H 2O 2 sufficiently may lead to selective killing of oral cancer cells
and therefore be therapeutically useful (Seung-Ki et al., 2008). Leaf
metabolism produces H2O2 at high rates, but current concepts suggest that
the potent signaling effects of this oxidant require that the concentrations be
controlled by a battery of antioxidative enzymes (Queval et al., 2008).
Curcumin had an effective hydrogen peroxide scavenging activity due
to which it is found to be useful in the pharmacological and food industry
(Ak and Gulcin, 2008). L-tyrosine and L-DOPA had an ability to scavenge
hydrogen peroxide (Gulcin, 2007). Our results are also comparable with the
reports by Kumaran and Karunakaran (2007a) that the hydrogen peroxide
scavenging effect was strong in the methanol extracts of five plants from the
genus Phyllanthus.
The Couroupita guianensis parts rendered protection to DNA
molecules by exhibiting considerable hydroxyl radical scavenging activity,
thus protecting the deoxyribose moiety of DNA. This effect is a significant
one and is supported by a lot of available literature.

174

Hydroxyl radical is an extremely reactive free radical formed in

biological systems and has the capacity to cause DNA strand breakage,
which contributes to carcinogenesis, mutagenesis and cytotoxicity. In
addition, this radical species is considered as one of the quick initiators of
the LPO process, abstracting hydrogen atoms from unsaturated fatty acids
(Gordon, 1990). Strong hydroxyl radical inhibitory activity was shown by
different extracts obtained from Teucrium montanum and Teucrium
chamaedrys (Panovska et al., 2005).
The biological antioxidants extracted from many plants and fungi have
been reported to possess potential abilities to scavenge free radicals. An
aqueous extract of Corduceps militaris (L.) link fruit bodies showed
significant scavenging effect on eliminating hydroxyl radicals (Zhan et al.,
2006). Among the methanolic and 70% acetone extracts of Camellia sinensis
(L.) o. Kuntz, Ficus bengalensis L. and Ficus recemosa L., the methanol
extracts of all the samples were found to have more hydroxyl radical
scavenging activity (Manian et al., 2008).
In the present study, the oxidant-scavenging effects of Couroupita
guianensis plant parts were tested against a battery of radicals and oxidants,
which were either pre-formed (stable DPPH and unstable H 2O2), formed
during the reaction of the assay (ABTS+) or formed and exert their action on
biomolecular components (hydroxyl radicals on deoxyribose). In all the
cases, the methanolic extracts of the leaves, flowers and fruit pulp of
Couroupita guianensis showed strong scavenging effects. Thus, it can be
concluded that Couroupita guianensis can scavenge pre-formed radicals,
prevent new radical formation and protect biomolecular components from
oxidant damage.

175

EFFECT OF Couroupita guianensis ON LPO

The extent of LPO (lipid peroxidation) was tracked in three diverse
membrane preparations, namely goat RBC ghosts, goat liver homogenate
and goat liver slices. All the three extracts caused a substantial decline in the
extent of LPO in three different membrane preparations. The flower extract
caused a better protection to all the three lipid preparations, followed by the
fruit and leaf extracts.
LPO has been broadly defined as the oxidative deterioration of
polyunsaturated lipids. Initiation of a peroxidation sequence in a membrane
or polyunsaturated fatty acid is due to abstraction of a hydrogen atom from
the double bond in the fatty acid. The free radical tends to stabilize by a
molecular rearrangement to produce a conjugated diene, which then readily
reacts with oxygen molecule to give a peroxy radical. Peroxy radicals can
abstract a hydrogen atom from another molecule to give lipid hydroperoxide,
R-OOH (Singh et al., 2009).
LPO, measured as TBARS, is a reliable index that reflects the extent
of oxidative damage to membrane lipids, as reported by several researchers.
Karuna et al. (2009) have reported that the aqueous extract of Phyllanthus
amarus treated rats showed a significant decrease in plasma LPO and a
significant increase in plasma vitamin C, uric acid, GSH levels and GPx,
CAT and SOD activities.
In another study postulated by Verma et al. (2007) the ameliorative
effect of the aqueous extracts of three medicinal plants (Phyllanthus
fraternus, Terminalia arjuna, and Moringa oleifera) on arsenic trioxide
induced alteration of lipid peroxidation were evaluated, when the aqueous
extract of P. fraternus was observed to cause better inhibition of lipid
peroxidation.
176

The antioxidant activity of Acacia nilotica Willd. was evaluated by

checking the extent of inhibition of lipid peroxidation in rat tissue
homogenate, using solvents of different polarity, where the ethyl acetate and
water fractions were found to have better effects (Singh et al., 2009). The
methanol extract of the leaves of Iryanthera juruensis, and the hexane
extract from its seeds inhibited lipid peroxidation in in vitro assays, proving
their medicinal importance (Silva et al., 2007).
Azadirachta indica leaf extracts decreased the levels of LPO in the
hepatic tissue, confirming its antioxidative potential (Gangar and Koul,
2007). The protective effect of a butanolic extract of Paronychia argentea L.
against toxicity caused by the organophosphorus pesticide, chloropyriphos
ethyl (CE) was evaluated by the significant decrease in the lipid peroxidation
(Zama et al., 2007). In another investigation, G. sylvestre extract showed
significant hepatoprotective effect against irradiated mice by significantly
lowering

the

radiation-induced

lipid

peroxidation

in

terms

of

malondialdehyde (Bhatia et al., 2008).

The results of the present study reveal that the antioxidants of the plant
Couroupita guianensis are very effective in protecting the internal
membranes and the plasma membranes from oxidative damage. The extent
of protection rendered by the plant extract was much better in the liver
homogenate than in the other two models. It seems possible that some, as yet
unidentified, endogenous factor in the cells, interacts synergistically with the
plant components to elucidate a better protection against lipid damage. The
observation that the extent of protection being lower in the intact cells (tissue
slices) suggests that these components may not be delivered efficiently into
the cells within the time interval employed in the present study.

177

EFFECTS OF Couroupita guianensis ON DNA DAMAGE

The leaf, flower and fruit pulp extracts of Couroupita guianensis
reduced the damage to DNA and herring sperm DNA that were exposed to
H2O2. Among the three parts, the flowers caused a better protection of DNA.
Russo et al. (2003) have revealed that Bacopa monniera L. is capable
of reducing the hydrogen peroxide-induced cytotoxicity and DNA damage in
human non-immortalized fibroblasts and showed a dose-dependent free
radical scavenging capacity and a protective effect on DNA cleavage. Jeong
et al. (2009) have revealed the protective effect of the extracts from Cnidium
officinale against oxidative DNA damage induced by hydrogen peroxide in
the human skin fibroblast cell showing its antioxidant effect. Cao et al.
(2008) have shown the protective effects of ethanolic extracts of four
buckwheat groats on DNA damage caused by hydroxyl radicals to
understand their antioxidant property.
GSH could significantly inhibit the UV induction of lambda prophage,
and this effect was correlated to its capacity to scavenge free radicals
generated after UV irradiation (Liu et al., 2005). Ingestion of antioxidant
containing foods or tables of antioxidants indicated a protective effect of
oxidative damage to DNA in white blood cells or a decreased urinary
excretion of 8-oxo dG (Loft et al., 2008).
Green tea consumption has been suggested to be effective in
decreasing DNA damage (Hakim et al., 2008). Oxidative DNA damage and
increased levels of DNA repair, both associated with diet-induced
hypercholesterolemia, are strongly reduced during dietary lipid lowering
(Martinet et al., 2001).

178

Hydroxylated 4-thiaflavan showed effective protection against the

oxidation damage induced in herring sperm DNA by cumene hydroperoxide
(CumOOH) or by the glutathione / ferric ion (GSH / Fe3+) system (Lodovici
et al., 2006). Kiwi fruit decreased H2O2 sensitivity of lymphocyte DNA
ex vivo and enhanced DNA repair (Freese, 2006).
Thus, the present work determined the strong antioxidant effect of
Couroupita guianensis flowers, fruits and leaves. This is evident from the
strong scavenging effects exerted on a battery of radicals and oxidants
(DPPH, H2O2, ABTS and OH) as well as the strong protective effect on
cellular biomolecules (lipids and DNA) subjected to oxidative stress.
ANTIOXIDANT POTENTIAL OF Couroupita guianensis EXTRACTS
IN GOAT LIVER SLICES EXPOSED TO H2O2
Precision-cut liver slices are one of the powerful tools used in
metabolic studies. It has the combined advantage of retaining the complex
organ structure as well as rapid and simple preparation. Changes in the gene
expression profile during in vitro incubation are smaller for liver slices than
for isolated cells. Thus, this in vitro tool functions in a relatively similar way
to the situation in vivo (Glckner et al., 2008).
The goat liver slices were exposed to H2O 2 in the presence and the
absence of the plant extracts and the protective role of Couroupita
guianensis were assessed by determining the enzymic and nonenzymic
antioxidants in the homogenate of the slices prepared after the incubation.
All the plant extracts rendered protection by reverting the decreased
antioxidant status in the oxidatively stressed in vitro systems. The flower
extracts rendered maximum protection followed by the fruit pulp and leaf
extracts.

179

The literature is studded with similar studies. The study of Cadirci

et al. (2007) revealed that Ososma armeniacum root extract showed some
antioxidant mechanism by increasing SOD level and inhibited some oxidant
mechanism in ethanol induced rat stomach tissue. The work of
Khan et al. (2009) suggested that green tea extract ameliorates gentamycin
elicited nephrotoxicity and oxidative damage in rat renal tissue by improving
antioxidant like SOD and CAT.
Mathivadhani et al. (2005) have revealed that Semecarpus anacardum
Linn. nut extract administration in liver and breast tissue of Sprague-Dawley
rats increased the level of SOD and POD. Our results also prove that the
administration of Couroupita guianensis extracts increased the level of CAT,
POD and SOD activities significantly in H 2O2-exposed goat liver slices.
Dal et al. (2009) have reported that saffron, a plant derivative,
suppresses oxidative stess induced by 7,12-dimethyl benz(a)anthrazene
(DMBA) induced skin carcinoma in mice by elevating the activities of
enzymic antioxidants like CAT and SOD. The results of the study of
Tung et al. (2009) suggested that Acacia confuse bark extract and its active
component gallic acid exhibited potent hepatoprotection against carbon
tetrachloride induced liver damage in rat, which may be due to the
modulation of antioxidant enzymes, namely catalase.
Another study postulated by Singh and Kakkar (2009) revealed that
berberine, berbamine and palmatine, which are constituents of Berberis
aristata root, have anti-hyperglycemic and antioxidant effect and increases
catalase

activity

significantly

in

the

liver

of

diabetic

rats.

Kapoor et al. (2008) have reported that the extract of Bocopa monneri
modulates enzymic antioxidants like peroxidase and catalase by increasing

180

their activity and enhancing the defense against ROS generated damage in
the brain and kidney of diabetic rats.
An earlier study has reported that that epigallocatechin gallate exerts a
cardioprotective effect against isoproterenol-induced stress in rats due to its
free radical scavenging effects and enzymic antioxidants like glutathione
peroxidase and glutathione S-transferase, which maintain tissue defense
system against myocardial damage (Devika and Stanely, 2007). Juan-xu
et al. (2009) showed that the powder of selenium enriched green tea was able
to enhance peroxidase and superoxide dismutase activity in blood, serum and
liver of mice.
Plant extracts have been reported to stimulate the glutathione
peroxidase activities to counteract oxidative stress. The Ginkgo biloba
extract retrieved the decreased activities of glutathione peroxidase towards
near-normalcy in the plasma and liver of rats intoxicated with CCl4 (He
et al., 2008).
It is also reported that the ethanol extract and the flavonoid isolated
from the leaves and stems of Aquilegia vulgaris ameliorated the antioxidant
enzymes such as SOD, GPx and GR to normal levels after having been
enhanced by hydrogen peroxide (Adamska et al., 2009).
Yu-Hua et al. (2008) have reported that the Amaranthus spinosus
ethanolic and whole extract significantly increased the GPx activities upon
carbon tetrachloride intoxication in the hepatic damaged rats. The study of
Ming et al. (2008) revealed that the administration of Hycium barbbarum
polysaccharide, significantly increased the level of vitamin A in mice fed
with high fat diet.

181

The protective effect of selenium polysaccharide from the Coprinus

comatus on alloxan induced oxidative stress in mice was reported to be
associated with increased levels of vitamin A in the liver and kidney of
diabetic mice (Yu et al., 2009). Studies by Xu et al. (2007) suggested that
the administration of green tea polyphenols elevated the levels of vitamin A
and reduced glutathione in liver tissue of mice. The administration of
phenol-rich wild blue berry extract to mice increased the ascorbic acid level
in the brain (Papandreou et al., 2008). From the study of Jia et al. (2008), it
is clear that Ganoderma lucidum polysaccharides improved the vitamin C
levels in the plasma and liver of diabetic rat.
Priya et al. (2007) clearly proved that the level of vitamin C
significantly increased after the administration of Kalpaamruthaa, a modified
indigenous preparation in adjuvant induced arthritis in the blood of rats. The
study of Priscilla et al. (2008) revealed that gallic acid protects the
myocardium against isoprotenanol induced oxidative stress in Wistar rats by
increasing the level of vitamin E in the heart issue.
Semecarpus anacardium Linn-nut extract significantly decreased the
arthritic score on administration to the spleen and thymus of arthritic rats by
increasing the level of nonenzymic antioxidants like vitamin E and reduced
glutathione (Ramprasad et al., 2005).
Anbuselvam et al. (2007) showed that the oral administration of
methanolic extract of Operculina turpethum stem increased the antioxidant
level especially non enzymic antioxidants like vitamin E, vitamin C and
reduced glutathione in serum, liver and breast of DMBA induced oxidative
stress in rats. Zheng et al. (2008) have reported that the administration of
Amaranthus spinosus increased the level of reduced glutathione, which
counteracted the CCl4 treatment in rat liver.

182

Another study postulated the administration of Kalpamrutha to DMBA

induced rat mitochondria, which increased the reduced gluthathione levels in
mitochondria (Arulkumaran et al., 2007).

Devi

et

al.

(2007)

have

reported that the administration of methanolic extract of Terminalia arjuna

caused an increase in reduced glutathione level in gastric mucosa of
experimental

rats.

In

another

experiment,

the

antioxidant

and

hepatoprotective activities of the methanol extract of Careya arborea bark in

Ehrlich ascites carcinoma-bearing mice was studied, wherein the plant
extract could significantly increase the SOD and CAT activities and decrease
the TBARS formation, proving its antioxidant potential (Senthilkumar et al.,
2008).
With the support of these reports, our study confirmed that the
Couroupita guianensis plant parts can improve the antioxidant status in
oxidatively stressed tissues, which is attributable to the antioxidant potential
of the plant.
PHASE III
The results obtained so far proved the therapeutic value of Couroupita
guianensis, with its strong antimicrobial and antioxidant potential. In this
phase, to identify the phytochemicals responsible for their medicinal value,
qualitative phytochemical analysis was performed. The results indicated the
presence of alkaloids, phenolics and flavonoids.
The literature is rich with the reports of the analysis of
phyotchemicals, especially in various medicinal plants. The analysis of
phytochemicals in the ethylacetate and methanol extracts of Syzygium cumini
showed the presence of alkaloids, anthraquinones, flavonoids, saponins,
steroids, tannins and triterpenoids (Kumar et al., 2009).

183

The successive extracts of root, stem bark and seeds of Strychnos

potatorum have revealed the presence of alkaloids, flavonoids, glycosides,
lignins, phenols, saponins, sterols and tannins (Mallikharjuna et al., 2007).
Euphorbia heterophylla contained high amount of tannins and alkaloids and
is used as a purgative (Edeoga et al., 2005).
In our study, the presence of alkaloids, phenolics and flavonoids
revealed the nature of the active principles responsible for the antimicrobial
and antioxidant activity of Couroupita guianensis. In order to further
confirm the role of the secondary metabolites, the alkaloid, phenolic and
flavonoid fractions were isolated and checked for their antimicrobial activity
and antioxidant potential.
ANTIMICROBIAL ACTIVITY OF THE ISOLATED FRACTIONS
The antimicrobial activity of the alkaloid, phenolics and flavonoid
fractions of the leaves, flowers and fruit pulp of Couroupita guianensis were
determined. Only the alkaloid fractions exhibited antimicrobial activity,
while the phenolic and flavonoid fractions did not show good antimicrobial
action. There are several reports about the antimicrobial activity of the
phytochemicals present in the medicinal plant.
The ethanolic extract of Boehmeria rugulosa and its isolated
components showed potent antimicrobial activity against two bacterial
species Staphylococcus aureus and Streptococcus mutans and three fungus
pathogens Microsporum gypseum, Microsporum canis and Trichophyton
rubrum (Semwal et al., 2009). The alkaloid berberine isolated from Coptidis
rhizome (Ranunculacea) showed bactericidal activity against oral bacteria,
with the greatest activity against Actinobacillus actinomycetemcomitans and
Porphyromonas gingivalis, and lesser activity against Lactobacillus and
Streptococcus species (Hu et al., 2000).
184

Seven isoflavonoids isolated from the roots of the plant Erythrina

variegata (Leguminosae) were tested for their ability to inhibit the growth of
cariogenic oral bacteria, among which erycristagallin was found to be have
potential

antibacterial

activity

(Sato

et

al.,

2002).

Naringin,

polymethoxylated flavonoid commonly found in citrus fruit and an FDAapproved health supplement was shown to inhibit the growth of periodontal
pathogens and other common oral microorganisms (Tsui et al., 2008).
The methanol extract from the stem bark of Terminalia superba
fractions

and

two

compounds

isolated

were

evaluated

for

their

antimycobacterial, antibacterial and antifungal activities, among which only

compound 2 was found to have the effective antimicrobial potential (Kuete
et al., 2010). Another study investigated the antimicrobial activity of the
methanolic extract as well as the compounds isolated from the stem bark of
Morus mesozygia, namely 3-acetoxyurs-12-en-11-one, moracin Q, moracin
T, artocarpesin, cycloartocarpesin, moracin R, moracin U, moracin C and
moracin M, among which four compounds did not allow the growth of the
tested bacterial species (Kuete et al., 2009).
Yang et al. (2008) have demonstrated the antifungal activity of the
extracts and fractions of six chenopodiaceaeous plant species. In another
study, N-alkyl--D-glucosylamines were evaluated for both antifungal and
antibacterial activity against Fusarium proliferatum, Listeria innocua and
Salmonella typhimurium (Muhizi et al., 2009).
A proteobacterium isolated from the coastal region, produced
appreciable secondary metabolite and partial purification of the obtained
secondary metabolite demonstrated antimicrobial activity against both
Gram-positive and negative organisms including MRSA (methicillin
resistant Staphylococcus aureus) ( Madhava et al., 2009).

185

Rahman et al. (2008) have reported that the salicylic acid derivative, a
cinnamaldehyde and six isoflavones obtained from the stem bark of
Flemingia paniculata Wall. (Leguminosae) exhibited antibacterial (both
Gram-positive and Gram-negative) and antifungal activities. The methanol
extract of Leucas aspera flowers, its fractions, the alkaloidal residue and the
expressed flower juice were tested for their antimicrobial activity and good
antibacterial activity was seen for the methanol extract and the alkaloidal
residue (Mangathayaru et al., 2005).
In tune with these reports, our study also revealed the antimicrobial
activity of the alkaloid fractions of Couroupita guianensis. The phenolic and
flavonoid fractions did not show any inhibitory effect against the tested
bacteria and fungi.
FREE RADICAL SCAVENGING ACTIVITY OF THE ISOLATED
FRACTIONS
The isolated phytochemical fractions namely alkaloids, phenolics and
flavonoids were assessed for their radical scavenging ability against DPPH,
ABTS, H 2O 2 and hydroxyl radicals. All the three fractions could readily
scavenge the free radicals, which confirmed their antioxidant potential.
Several scientific evidences are available in the literature indicating
the antioxidant role of phytochemicals. Chu et al. (2000) and Czeczot (2000)
have demonstrated that the flavonoids present in the plants are known to
possess strong antioxidant properties. Hypericum perforatum extract is
reported to contain flavonoids such as rutin, quercetin, and quercetrin, which
demonstrated free radical scavenging activity in a model of autooxidation of
rat cerebral membranes (Saija et al., 1995). Demiray et al. (2009) have
showed that Turkish species of Hawthorn leaves and T. argentea were rich
in phenolic constituents and demonstrated good antioxidant activity, whereas
186

Radix bistorta root extracts were found to be poor in antioxidant capacity

measured by ABTS assay.
Among the 20 compounds isolated from Nymphaea caerulea flowers,
9 compounds showed a powerful antioxidant activity (Agnihotri et al.,
2008). Paixo et al. (2007) have revealed the relationship between the
phenolic contents of red, rose and white wines with the antioxidant acitivity
by the DPPH and ABTS scavenging assays. Four of the five prenylated
arylbenzofurans isolated from Morus mesozygia (Moraceae) displayed
potent antioxidant activity (Kapche et al., 2009). Similarly, the phenolics
isolated from Rubus ulmifolius also exhibited antioxidant activity
(DallAcqua et al., 2008).
Our results indicated the antioxidant potential of alkaloid, phenolics
and flavonoids of the leaves, flower and fruit pulp of Couroupita guianensis.
However, the antimicrobial activity was strongly associated only with the
alkaloid fraction, with the phenolic and flavonoid fractions exhibiting
negligible activities. Thus, it is perceivable from our study that the
antimicrobial component in the plant is an alkaloid.
SPECTRAL ANALYSIS
The flowers of Couroupita guianensis showed the maximum
antimicrobial activity and better antioxidant potential compared to the leaves
and fruit pulp. Hence the flowers were subjected to various spectral analysis
by TLC, HPTLC, UV absorption, HPLC, IR and GC-MS techniques.
TLC OF METHANOLIC EXTRACT OF Couroupita guianensis
TLC analysis of the methanolic extract of Couroupita guianensis
revealed the presence of seven components in the flowers of Couroupita
guianensis.
187

Many reports have indicated that bands were visualized on silica gel
TLC developed by spraying 10% H2SO4, followed by heating the plate.
-diketones in Eucalyptus globules have been reported to give spots at
Rf 0.7-0.8 with n-hexane : ethyl acetate (3:1) under UV radiation and
chloroform and ethanol extracts of the dried leaves have been shown to
exhibit spots at Rf 0.8 under UV light and with the spray reagent of
dilute H2SO4 followed by heating (Amakura et al., 2009).
Singh and Arora (2009) observed that TLC analysis of the Acacia
nilotica (L) wild extract showed two spots at Rf value 0.48 and 0.64, when
run in the solvent system toluene: ethyl acetate: formic acid (45:55:15). The
characterization of nutraceutical compounds in the blue green algae
Spirulina maxima exhibited spots at Rf value 0.04 (phenolic compounds),
0.11 (zeaxanthin), 0.2 (violexathin), 0.32 (chlorophyll a), 0.42 (chlorophyll
b), 0.5 (-tocopherol), 0.65 (carotene isomer), 0.81 (-carotene) and 0.94 (carotene) (El-Baky et al., 2008).
DETECTION OF ALKALOIDS
Alkaloids are low-molecular weight, nitrogen containing compounds
that are found in approximately 20% of the plant species. Most alkaloids are
derived from amines produced by the decarboxylation of amino acids, such
as histidine, lysine, ornithine, tryptophan and tyrosine. Alkaloids are also
very important because of their high biological activites (Minami et al.,
2008).
The

TLC

plate,

when

detected

with

the

alkaloid-specific

Dragendroffs spraying reagent, showed a major band with the Rf value 0.74.
These results reported the bands obtained earlier when sprayed 10% H 2SO4
at spot 5 with Rf value 0.77. Hence, it confirmed the presence of alkaloids in
the methanolic extract of the flower sample.
188

Similar studies have been reported that the alkaloids can be best
studied on TLC plate using Dragendroffs reagent. Marino et al. (2008)
reported alkaloids from Croton lechleri. The studies in different parts of
Strychnos potatorum with Dragendroffs reagent have reported seventeen
alkaloid spots in the stem bark, while the seeds showed thirteen alkaloid
spots (Mallikharjuna et al., 2007). A modified Dragendroffs reagent has
been used to detect furocoumarin alkaloid and quinolone alkaloid rutamine
in aqueous ethanolic extract of Ruta chalepensis leaves (Emam et al., 2009).
In the present study, the presence of alkaloids is clearly evidenced,
which might be responsible for the biotherapeutic value of Couroupita
guianensis.
DETECTION OF PHENOLICS
Phenolic compounds in plants provide an array of natural sources of
antioxidants for use in foods and nutraceuticals (Unver et al., 2009).
Phenols, a major group of antioxidant phytochemicals, have profound
importance due to their biological and free radical scavenging activities
(Prakash et al., 2007).
The results for the presence of phenolics in TLC plate showed 3 major
spots with the Rf values of 0.57, 0.67 and 0.75 respectively. Our results are
supported by several scientific reports.
Naz et al. (2008) showed that phenolics from chick pea in vitro
cultures were seen as dark blue spots on light background with FolinCiocalteau reagent. The presence of quercetin, isoquercetin, hyperoside and
rutin has been indicated in the TLC separation of flavonoids and phenolic
acids from methanolic extracts of the Arbutus unedo leaves with the solvent

189

system ethyl acetate: formic acid: acetic acid: water (100:11:11:26) (Males
et al., 2006).
The results of the present study emphasized the presence of the major
plant secondary metabolites, phenolics, in the flowers of Couroupita
guianensis, which validates the antioxidant potential of the candidate plant.
DETECTION OF FLAVONOIDS
Flavonoids are a widely distributed group of polyphenolic compounds
characterized by a common benzopyrone ring structure, and have been
reported to act as antioxidants in various biological systems. The biological
functions of flavonoids, apart from their antioxidant properties, include
protection against allergies, inflammation, platelet aggregation, microbes,
ulcers, hepatotoxins, viruses and tumors (Poornima and Rai, 2009).
A pink coloured spot in the TLC plate with the Rf value of 0.81 after
detection with 10% vanillin sulphuric acid indicated the presence of one
major group of components belonging to flavonoids.
The phytochemical investigation and identification of medicinal plants
have become the recent focus in scientific research. Recent research has
shown that quercetin, a flavonoid, when run in the solvent mixture of nbutanol: acetic acid: water (4:1:5) and developed in UV light showed a spot
at 0.82 and ammonia vapours showed deep yellow colour and iodine vapours
as yellow brown spots (Meena and Patni, 2008) which is in comparable with
the Rf value, obtained in the present study.
2-methoxy-1,4-naphthoquinone has been identified in TLC with
chloroform: petroleum ether: methanol: formic acid (8:4:0.5:0.1) mobile
phase when stained with 5% vanillin-H 2SO4 reagent and heated at 105C for
5 minutes (Ding et al., 2008). The studies in Lepidium sativum on HPTLC
190

with butanol: acetic acid: water (4: 1: 5) have been reported to result in the
best separation of sinapic acid with Rf value 0.68 (Nayak et al., 2009).
Our results clearly indicate the presence of flavonoids in the flowers
of Couroupita guianensis, which can be attributed to their antioxidant
activity.
UV ABSORPTION SPECTRUM
The absorption spectra of the methanolic extract and the alkaloid,
flavonoid and phenolic fractions in the UV range gave specific absorption
patterns. The absorption pattern of the alkaloids, phenolics and flavonoids, in
comparison with that of the methanolic extract, confirmed that alkaloids,
phenolics and flavonoids are the major active principles responsible for the
therapeutic potential of Couroupita guianensis flowers, and are extracted
into methanol.
Researchers have described the UV spectrum of the Acacia nilotica
extract, which gave max at 273nm and 200nm, a small structureless
absorption above 300nm indicating low polyphenolics in the extract (Singh
and Arora, 2009). Arfan et al. (2009) have reported that the UV spectra of
phenolic fractions of Mallotus phillippinensis bark extract showed maxima
at 320330nm, flavonols and flavanols at 340350nm and strong absorption
at 278 and 282nm that was attributed to catechins or procyanidins. Wang
et al. (2007) have showed 330nm as the set wavelength of Erigeron
breviscapus for HPLC analysis, based on the UV absorption.
The UV absorbance maximum for the petals of Echium amoenum was
obtained at 330nm and a shoulder at 290nm was suggested to be due to the
presence of the phenolics acid with two aromatic rings (Mehrabani et al.,
2005). The UV spectrum of the flowers of Abelmoschus manihot showed

191

peaks at 204, 210, 228, 262, and 277nm (Lai et al., 2007). Ding et al. (2008)
have shown a characteristic UV absorption at 215, 241, 246, 275 and 329nm
for 2-methoxy-1, 4-naphthoquinone.
Another study was done in the methanolic extract of the seeds of Ilex
aquifolium, which resulted in the isolation of two antioxidant phenyl acetic
acid derivatives, 2,4-dihydroxy phenyl acetic acid and 2,4-dihydroxy phenyl
acetic acid methyl ester. The UV absorption spectra of these compounds
showed maxima at 278nm indicating the presence of aromatic nucleus in
these molecules (Nahar et al., 2005).
HPTLC ANALYSIS OF THE FLOWERS OF Couroupita guianensis
HPTLC method offers high degree of selectivity, sensitivity and
rapidity combined with single-step sample preparation. Simultaneously a
large number of samples, along with the standard can be analyzed in one
TLC plate and solvent requirement is also very negligible, thus making it
inexpensive compared to HPLC. In addition, it requires very small amount
of sample and can detect active principle concentration in nanograms level
(Tripathi et al., 2006).
The HPTLC analysis of flowers of Couroupita guianensis was
performed to confirm the presence of alkaloids, phenolics and flavonoids.
The results obtained confirmed the presence of alkaloids, phenolics and
flavonoids in Couroupita guianensis.
Raina et al. (2007) have reported the HPTLC analysis of
hepatoprotective diterpenoid andrographolide from Andrographis paniculata
nees (kalmegh). Abou-Donia et al. (2006) demonstrated the use of HPTLC to
quantify rutin in the alcoholic extract of Amaryllis belladonna L. flowers and

192

the results obtained by HPTLC showed excellent accuracy and precision

when compared to those obtained by spectrophotometric methods.
HPTLC fingerprinting of ethyl acetate extract of Vitex agnus-castus
revealed eight spots and showed the presence of flavonoids, terpenoids,
steroids, and carbohydrates (Arokiyaraj et al., 2009). An efficient HPTLC
method for the analysis of alkaloids in harding grass (Phalaris aquatica L.)
has been reported by Zhou et al. (2006). The HPTLC method for the
determination of curcumin, demethoxycurcumin and bisdemethoxycurcumin
from turmeric powder have been reported by Paramasivam et al. (2008).
HPTLC analysis of myristicin and safrole in the seed powder of
Myristica fragrans Houtt was performed by Kiad (2009). Tripathi et al.
(2006) have demonstrated the quantitative determination of phyllanthin and
hypophyllanthin in Phyllanthus species by HPTLC. HPTLC analysis showed
that Semecarpus anacardium seeds contained tetrahydroamentoflavone
(Aravind et al., 2008).
Our HPTLC fingerprinting also revealed the presence of alkaloids,
phenolics and flavonoids in the flowers of Couroupita guianensis, indicating
the type of active principles present in the plant.
HPLC ANALYSIS OF THE METHANOLIC EXTRACT OF THE
FLOWERS OF Couroupita guianensis
The isolation of pure plant constituents started many years ago, and
the term phytochemistry was coined. Since then, we have come a long way
and many new techniques have been introduced, most noticeably HPLC and
various powerful spectroscopic techniques, making the identification of new
compounds ever faster.

193

HPLC analysis of secondary metabolites represents an efficient tool

for the study of plant chemical diversity under different aspects. Statistical
analysis of HPLC databases, like correlation analysis between HPLC peaks,
can reliably provide information on the similarity/dissimilarity degrees
between the chemical compounds. The similarities, corresponding to positive
correlations, can be interpreted in terms of analogies between chemical
structures, synchronic metabolisms or co-evolution of two compounds under
certain environment conditions (Semmar et al., 2007).
The analysis of RP-HPLC spectrum of the methanolic extract of
Couroupita guianensis flowers carried out in the UV range, revealed the
presence of 5 major peaks and several minor peaks.
Meena and Patni (2008) identified and isolated quercetin, a flavonoid
from Citrullus colocynthus, which showed a characteristic peak at Rt 3.475
minutes. Rapeseed meal showed sinapine and sinapic acid as the main
phenolic acids at 325 nm (Cai and Arntfield, 2001). The HPLC analysis of
the extracts of B. racemosa belonging to family Lecythdaceae (leaves, sticks
and barks) showed two different phenolic acids (gallic acid and ferulic acid)
and four different flavonoids (naringin, rutin, luteolin and kaempferol)
(Hussin et al., 2009). HPLC identification and quantification of the isolated
compounds from Coleus aromaticus showed that rosmarinic acid was the
major component and principally responsible for the radical-scavenging
activity of the plant (Kumaran and Karunakaran, 2007b).
An RP-HPLC method was developed for the determination of five
major compounds in Polygonum cuspidatum namely resveratrol, polydatin,
anthraglycoside B, emodin and physcion at UV detection at 306 nm
(Zhang et al., 2007b). The RP-HPLC analysis was done in the methanolic
extract of Citrullus colocynthis fruits reporting their antioxidant properties

194

(Delazar et al., 2006a). A study on the leaves of Ilex paraguariens using

reverse-phase HPLC separation combined with UV detection for saponin
assay, showed sapogenins were present in the saponin fraction of the leaf
extract and the retention time was found to be 15 minutes (Gnoatto et al.,
2005).
Preliminary identification of the major phenolic compounds from 83
selected medicinal plants by reverse-phase HPLC revealed phenolic acids,
tannins, flavonoids, curcuminoids, coumarins, lignans, and quinines
(Surveswaran et al., 2007). In another study, Delazar et al. (2006b) have
reported that the Chrozophora tinctoria afforded four flavonoid glycosides
by RP-HPLC analysis. da Silva et al. (2008) have demonstrated the
separation of the ethanol extract from the leaves of Casearia sylvestris by
RP-HPLC and reported eight major peaks in the plant.
In the present study, the methanolic extract of the flowers of
Couroupita guianensis showed five major peaks indicating the presence of
five major components that are probably responsible for the biological
activity of the plant.
INFRA RED SPECTRUM OF THE FLOWERS OF Couroupita
guianensis
FT-IR is one of the most widely used methods to identify the chemical
constituents and elucidate the structures, and has been used as a requisite
method to identify medicinal compounds. Owing to the fingerprint
characters and extensive applicability to the samples, FT-IR has played an
important role in pharmaceutical analysis in recent years.
The IR spectrum of the methanolic extract of Couroupita guianensis
indicated the presence of polyphenolics (-OH) and presence of alkaloid type

195

compounds in the methanolic extract of the flowers of Couroupita

guianensis.
Zhang et al. (2007c) have reported the identification of medicinal
plants namely Tripterygium wilfordii and T. hypoglaucum by pattern
recognition within the infrared spectra of crude extracts. Fourier transform
infrared spectroscopy (FT-IR) associated with second derivative infrared
spectroscopy and two-dimensional correlation infrared spectroscopy (2D-IR)
have been applied to study the main constituents in traditional Chinese
medicine Angelica and its different extracts (Druy, 2004).
The IR spectroscopic analysis of hexane, ethyl acetate and ethanol
crude extracts of Plectranthus glandulosus suggested the presence of
different functional groups ranging from O-H stretching, hydroxyl, C-H
stretching, alkyl, C=C stretching aromatic ring , C-O bending, and alcohols,
ethers, esters, carboxylic acid and anhydrides, C=O stretching, carboxylic,
carbonyl and C-N bending, alkaloids (Egwaikhide and Gimba, 2007).
The infra-red spectra of the crude extract of Plumeria rubra (flower
and leaf) and Eucalyptus globules (leaf) revealed the presence of different
functional groups, ranging from hydroxyl group (OH) (3406-3338.6 cm-1),
C-H stretching for alkyl group (2926.6 cm-1), C=O stretching for carbonyls
(2162.1 cm-1), C-O bending for alcohols, ethers, esters, carboxylic acid and
anhydrides (1310.6 - 1059.6 cm-1), C H bending alkyl (1453.4 1376.2
cm-1) and C H bending for methyl group (864.1 668.4 cm-1)
(Egwaikhide et al., 2009).
In the present study, the IR spectrum of Couroupita guianensis
confirmed the presence of polyphenolics and alkaloids in the flower sample.

196

GC-MS ANALYSIS OF THE FLOWERS OF Couroupita guianensis

The GC-MS spectrum of the flowers of Couroupita guianensis
revealed the presence of five peaks at 9.69, 9.98, 10.88, 11.03 and 11.13.
The interpretation of this spectrum confirmed the presence of polyphenolic
type of compounds and nitrogen containing alkaloid type of compounds.
Extraction of the dried fruits from the cannon ball tree, Couroupita
guianensis Aubl., yielded 6,12-dihydro-6, 12-dioxoindolo[2,1-b]quinazoline
(tryptanthrin), indigo, indirubin and isatin (Bergman et al., 2008).
da Silva et al. (2001) have reported the presence of isatins in Couroupita
guianensis.
The structure of indirubin contains two NH and two -CO groups and
the molecular weight of indirubin is 262. Since the GC-MS spectrum of
Couroupita guianensis plant extract reveals the presence of N and CO
group, in the fraction at 10.88, with higher molecular ion peak at m/e 300, it
may be concluded that this extract contains a higher derivative of indirubin.
Roy et al. (2009) have done the phytochemical analysis of
Andrographis paniculata extract and subjected it to GC-MS. GC-MS results
revealed phenols, aromatic carboxylic acids and esters in the chloroform
extract to be the molecules responsible for the antimicrobial activity of A.
paniculata. Phytochemical screening of three different oil fractions, obtained
from n-hexane extract of Prunus domestica shoots analyzed by GC and
GCMS resulted in the identification of 9, 16 and 24 compounds, which
represented 92.56%, 90.6% and 90.69% of these oil fractions, respectively
(Mahmood et al., 2009).
In another study, the essential oil from the fresh leaves of Schinus
terebinthifolius was extracted and subjected to GC-MS analysis. This

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showed that the major constituents of the essential oil were sabinene, pinene,
phellandrene, pinene, terpinene-4-ol, trans ocimene and myrcene (Gundidza
et al., 2009).
Henophyton deserti was characterized with respect to its chemical
composition, antioxidant potential and antimicrobial activity of its
methanolic extract. Fourteen compounds were identified by LC/MS,
GC/MS, and GC in leaf and seed extracts (Derbel et al., 2009). The essential
oils from the flowers, leaves, barks, roots and fruits of A. brachypus were
individually extracted by hydrodistillation, and their chemical constituents
were isolated and characterized by means of GC and GC-MS (Haobin et al.,
2009).
In our study, the combined analysis of the spectra of the flowers of
Couroupita guianensis, unequivocally confirmed the presence of alkaloids,
phenolics and flavonoids. The analysis of the biological activities of these
fractions revealed the antimicrobial activity to rest in the alkaloid fraction
and a strong antioxidant potential in the phenolic and flavonoid fractions.
Thus, our results strengthen the candidature of the plant Couroupita
guianensis against not only the microbial pathogenesis, but also the myriad
disorders resulting from oxidative stress.
PHASE IV
Computer-aided drug design (CADD) and discovery methods have put
much emphasis on the identification of novel active compounds and the
optimization of their potency. An important task in CADD is the
identification of small molecules that are selective against target families,
subfamilies or individual targets and can be used as molecular probes for
specific functions (Stumpfe et al., 2007).

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Lack of efficacy and toxicity are considered to be major reasons for

drug failures and pharmacokinetics governs them to a large extent.
Compounds with favorable pharmacokinetics is more likely to be efficacious
and safe. Therefore, the preclinical pharmacokinetic evaluation should be
comprehensive enough to ensure that compounds do not fail in the clinic.
Preclinical ADME screening facilitates the early elimination of weak
candidates and directs the entire focus of the drug development program
towards fewer potential lead candidates (Sonu, 2006). The ADME profile of
both isatin and indirubin showed that both the compounds exhibit good
bioavailability. This observation potentiates the drug candidature of the
compounds.
The two active components identified in the present study, isatin and
indirubin, were subjected to further in silico studies to analyse their efficacy
against the target proteins of the most susceptible organisms namely the
bacterial strains Shigella flexneri and Staphylococcus aureus and the fungal
strain of Candida albicans. The structures of the target proteins namely,
VirA (Shigella flexneri), MgrA (Staphylococcus aureus), SAP2 and SAP5
(Candida albicans) were obtained from the Protein Data Bank and refined
using the protein preparation wizard. The molecular docking studies were
performed to characterize the active components.
Both the ligands (isatin and indirubin) formed hydrogen bonds with
the target proteins. Among the two, isatin bound with greater affinity to the
targets, as indicated by more number of hydrogen bonds formed between the
ligand (isatin) and the targets. Eventhough the number of H-bonds formed
with indirubin were lesser, there were high affinity contacts between the
targets and indirubin. This observation indicates that indirubin probably
binds to the target molecules via weak bonds other than H-bonds.

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The docking between (2S,6S)-diaminopimelic acid (l,l-DAP) with its

target enzyme DAP-epimerase which is a key step in the lysine biosynthetic
pathway of bacteria showed that the molecular interactions occurred between
the functional groups bonded to the C2 and some residues of the binding
cavity immobilized the ligand in a position appropriate for the epimerization
reaction (Brunetti et al., 2008). The docking simulation to FabH, betaketoacyl-acyl carrier protein (ACP) synthase III, a particularly attractive
target in Gram positive and negative bacteria identified potent inhibitors of
E. coli FabH (Lv et al., 2009). The best thymidylate synthase inhibitors have
been tested against a number of clinical isolates of Gram-positive-resistant
bacterial strains by docking with new 1,2-naphthalein derivatives (Costi
et al., 2006).
The docking studies of philipimycin suggested that a part of the
molecule interacts with the ribosome and another part with Pro23, Pro22,
and Pro26 of L11 protein of Staphylococcus aureus, indicating the efficiency
of philipimycin against the target proteins (Zhang et al., 2008). To discover
non-nucleoside inhibitors of HIV-1 reverse transcriptase that are effective
against both wild-type (WT) virus and variants, virtual screening by docking
was carried out using three RT structures and more than 2 million
commercially available compounds. It was found that two of the structures
are for WT-virus with different conformations of Tyr181, while the third
structure incorporates the Y181C modification (Nichols et al., 2009).
A multistage molecular docking approach was performed to screen a
library of more than 5 million commercially available compounds against the
two binding sites of the enzyme, viral NS5 RNA methyltransferase. Among
the 263 compounds only 10 inhibitors were found to hit the target protein
efficiently (Podvinec et al., 2010).

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The molecular docking for the virtual screening of 1990 compounds

from the National Cancer Institute 'Diversity Set' on MurD and MurF, the
bacterial peptidoglycan biosynthesis enzymes, identified 50 top-scoring
compounds (Turk et al., 2009). Antitrypanosomal natural products with
different structural motifs previously shown to have growth inhibitory
activity against Trypanosoma brucei were docked into validated drug targets
of the parasite. The in silico calculations predicted the lowest energy docked
poses of a number of the compounds, among which cissampeloflavone, 3geranylemodin and ningpogenin offered profound promise (Ogungbe and
Setzer, 2009).
For antitubercular therapy, molecular docking was performed in order
to select the compounds to form favorable interactions with the thymidine
monophosphate kinase active site in M.tuberculosis. Eight compounds gave
a better docking score and all the identified hits highlighted a key hydrogen
bonding interaction with Arg74 (Kumar et al., 2009).
Chiu et al. (2009) have screened approximately 35 million
non redundant compounds for potential activity against a lethal factor
enzyme in Bacillus anthracis and comprised topomeric searching, docking
and scoring, and drug-like filtering. Among 5 hit compounds, none of which
has previously been identified as a LF inhibitor, three exhibited experimental
IC (50) values less than 100 M have been reported.
The molecular basis of IKKbeta inhibition by staurosporine and
quercetin as ATP-competitive inhibitors were studied by homology
modeling and molecular docking studies (Avila et al., 2009). Law et al.
(2010) have shown that, by computational virtual docking analysis, the
molecular target of the antitumor compound alisol B obtained from the

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rhizome of Alisma orientale is the sarcoplasmic/endoplasmic reticulum

Ca(2 +) ATPase.
Halofantrine, an antimalarial drug of unknown mechanism was
identified against plasmepsins target by docking (Friedman and Caflisch,
2009). In another study, docking experiments for fluconazole, voriconazole
and UR-9825 into the catalytic site of MT-CYP51 as template showed that
the molecules bind to the catalytic site adopting the similar bioactive
conformation as observed in the crystallized complex of fluconazole with the
enzyme (Nowaczyk and Banachiewicz, 2008).
Docking of the inhibitors to the active site of Candida albicans
lumazine synthase enzyme was performed with AutoDock 3.0 and found
compound TS44 to be effective than the other compounds (Morgunova et al.,
2007). Glucosamine-6-phosphate synthase, a novel target for antifungal
agents was docked with ADMP and ADGP, among which ADMP was found
to have better results (Wojciechowski et al., 2005).
Our study revealed that the two compounds possessed good docking
scores and reasonable stability. The ADME profile supports the
bioavailability of the compounds. Thus, our results in the fourth phase
provide evidence for the interaction of isatin and indirubin from Couroupita
guianensis with the bacterial and fungal virulence-causing target proteins.
This interaction is presumably vital in exerting the antimicrobial activity.
The results of the present investigation clearly demonstrate the
antimicrobial and antioxidant activity of Couroupita guianensis. The
antimicrobial effects rest with the alkaloid fraction, while the antioxidant
potential is exhibited predominantly by phenols and flavonoids. The study
provides further evidence of the antimicrobial activity of the alkaloids by

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demonstrating their in silico interaction with the target molecules involved in

the virulence of the organisms.
Thus, the present study strongly iterates the medicinal value of the
plant, Couroupita guianensis, and scientifically validates it for use as a
component of medicinal preparations.
The findings of the study are summarized and the conclusions drawn
therein are presented in the next chapter.

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