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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

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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 world’s 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.

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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

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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

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(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).

166

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).

167

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

168

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

169

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; Dall’Agnol 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

170

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).

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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.

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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, H2O2 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).

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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

(O2•-)

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

174

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 H2O2 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 H2O2 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.

175

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 H2O2), 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.

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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.

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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.

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EFFECTS OF Couroupita guianensis ON DNA DAMAGE

The leaf, flower and fruit pulp extracts of Couroupita guianensis

reduced the damage to λ 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).

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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 (Glöckner et al., 2008).

The goat liver slices were exposed to H2O2 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.

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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 H2O2-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

181

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.

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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.

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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).

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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).

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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, a

polymethoxylated flavonoid commonly found in citrus fruit and an FDA-

approved 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 3β-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-β-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).

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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, H2O2 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

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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). Paixão 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

(Dall’Acqua 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.

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Many reports have indicated that bands were visualized on silica gel

TLC developed by spraying 10% H2SO4, followed by heating the plate.

β-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 (α-tocopherol), 0.65 (carotene isomer), 0.81 (α-carotene) and 0.94 (β-

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

Dragendroff’s spraying reagent, showed a major band with the Rf value 0.74.

These results reported the bands obtained earlier when sprayed 10% H2SO4

at spot 5 with Rf value 0.77. Hence, it confirmed the presence of alkaloids in

the methanolic extract of the flower sample.

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Similar studies have been reported that the alkaloids can be best

studied on TLC plate using Dragendroff’s reagent. Marino et al. (2008)

reported alkaloids from Croton lechleri. The studies in different parts of

Strychnos potatorum with Dragendroff’s reagent have reported seventeen

alkaloid spots in the stem bark, while the seeds showed thirteen alkaloid

spots (Mallikharjuna et al., 2007). A modified Dragendroff’s 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 Folin-

Ciocalteau 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

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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 n-

butanol: 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-H2SO4 reagent and heated at 105°C for

5 minutes (Ding et al., 2008). The studies in Lepidium sativum on HPTLC

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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 λmax 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 320–330nm, flavonols and flavanols at 340–350nm 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

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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

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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 Kiadó (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.

194

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

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(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

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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.

197

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

GC–MS 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, beta-

ketoacyl-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, 3-

geranylemodin 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 µM 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.