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Discussion

Bioactivity and Phytochemical analysis of Amorphophallus commutatus SCHOTT (Engl.) an Endemic Aroid of Western Ghats, South India

1

IINNTTRROODDUUCCTTIIOONN

Herbal medicine plays an important role in the healthcare of many developing

countries. The use of herbal products is increasing worldwide due to the distinct

advantages. Herbal medicines include herbs, herbal materials, herbal preparations and

finished herbal products that contains as active ingredients, parts of plants, or other

plant materials, or combinations (WHO, 2008). Herbal medicine remains one of the

common forms of therapy available to much of the world’s population. According to the

WHO, about three quarters of the world population currently uses herbs and other forms

of traditional medicine to treat diseases. Traditional medicine is widely used in India.

Even in USA, use of plants and phytomedicine has increased dramatically in the last

two decades. However, the scientific basis for the bioactivity and the underlying

molecular mechanism for most of these products is presently unknown or incomplete

(Rao et al., 2004).

There are two major ways of bioprospecting natural products for investigation:

� First, the classical method that relies on phytochemical factors, serendipity and

random screening approaches.

� Second, the use of traditional knowledge and practices as a drug discovery

engine - this is also called as an ethnopharmacology approach, which is time

and cost effective and could lead to better success than random routine

screening (Patwardhan et al., 2004).

Traditional methods, for example, Chinese medicine, Japanese Kampo and Indian

Ayurveda, are becoming important bioprospecting tools (Patwardhan, 2000). These

botanicals have an array of compounds with diverse activities directed at various targets

of the immune matrix, in cancer and in infection and inflammation (Patwardhan and

Gautam, 2005).

Botanicals produce diverse range of natural products with antimicrobial

and immunomodulating potential, including isoflavanoids, indoles, phytosterols,

1

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Discussion


2

polysaccharides, sesquiterpenes, alkaloids, glucans and tannins. Designer drugs, which

involve safer, curative and synergistic combinations, are needed. Botanicals are

chemically complex and diverse, and could therefore provide appropriate complex of

synergistic combinations useful in drug discovery (Patwardhan and Gautam, 2005).

Bioprospecting of traditional medicine play a vital role in natural product drug

discovery.

Medicinal plants contain physiologically active principles that over the years

have been exploited in traditional medicine for the treatment of various ailments

(Adebanjo et al., 1983) as they contain anti-microbial properties (Sokmen et al., 1999;

Kelmanson et al., 2000). These medicinal herbs constitute indespensible components of

the traditional medicine practiced worldwide due to the low cost, easy access and

ancestral experience (Marini-Bettolo, 1980).

Free radicals, Reactive Oxygen Species (ROS) and Reactive Nitrogen Species

(RNS) play a vital role in various biological processes especially in host defense

mechanisms. But over production of these species contribute to oxidative stress related

pathological phenomenon. Halliwell and Gutteridge (1990) have reported that the

excess free radicals are removed by various reactive oxygen species scavenging

enzymes and antioxidant chemical species. The involvement of active oxygen and free

radicals in the pathogenesis of human disease such as cancer, aging, artherosclerosis,

malaria, rheumatoid arthritis, neurodegenerative disorders has been recently understood

more and more (Vasconcelos et al., 2007). The mechanisms of action of antioxidants

include complexation of redox-catalytic metal ions, scavenging of free radicals and

decomposition of peroxides. The antioxidant extracts have been found to involve lot of

mechanism and mechanistic synergism. Therefore a multiple experimental approaches

are recommended for identification of complete putative chain- breaking capacity

(Mello and Kubota, 2007).

Growing knowledge about the health promoting impact of antioxidants and

its additives in everyday food, combined with the hazardous effect of synthetic additive

(Krishnakumar and Gordon, 1996) has led to increased interest in identification of


Discussion


3

natural antioxidants like flavonoids, anthocyanins, carotenoids, dietary glutathionine,

vitamins and endogenous metabolites (Moure et al., 2001).

It is therefore evident about the need for developing functional food from

phytochemicals of plant origin as they are viewed to be promising as therapeutic agent ,

nutrceutical and food preservative to improve the status of human health and disease

prevention (Kitts et al., 2000; Noguchi and Nikki, 2000).

Plants behold a defense mechanism to protect against these toxic reactive

oxygen intermediates (ROIs). The defense mechanism involve antioxidant and

intracellular enzymes such as superoxide dismutase (SOD), peroxidase (POD),

glutathione peroxidase (GSH-Px), catalase (CAT), and ascorbate peroxidase (APX)

(Kim et al., 2004). The non enzymatic antioxidants like glutathione, ascorbic acid

Sgherri et al., 2003; Liu et al., 2009) and phenols (Debnath et al., 2011). These

enzymatic and non enzymatic antioxidants can terminate or prevent the formation

of free radicals by donating hydrogen or electrons to reactive radicals or species

(Rice-Evans et al., 1997).

Cancer is a leading health problem around the globe. Natural products

have been used for a long time to prevent and treat many diseases, including cancer

(Smith-Warner et al., 2000). Tumorigenesis is manifested when the delicate balancing

act of cell cycle regulation is lost (Mathew and White, 2006). Apoptosis is a self

destructive metabolic mechanism occurring in normal cells by genetically encoded

death signals, in which the DNA or other components are irreversibly damaged

(Hooper et al., 1999). The, cancer cells, which are already irreversibly developed,

obtain the capability to evade apoptosis by various ways. Therefore the aim of

anticancer agents is to trigger apoptosis signaling system in the cancer cells there by

disturbing their proliferation.

The induction of apoptosis in tumor cells is considered very useful in the

management and therapy as well as in the prevention of cancer. A wide variety of

natural substances have been recognised to have the ability to induce apoptosis in


Discussion


4

various tumor cells (Amit et al., 2001). It is thus considered important to screen

apoptotic inducers from plants, especially those used in traditional medicine for cancer

treatment either in the form of crude extracts or as components isolated from them. A

lot of traditional herbs do not have any extensive scientific review of the effect on the

body system. To fill up the gap, researchers have started to study the biological

properties of the traditional herbs (Yeap et al., 2007).

Plant materials remain an important resource to combat serious diseases in the

world. Pharmacognostic investigations of plants are carried out to find novel drugs or

templates for the development of new therapeutic agents. Among the more than 250,000

species of higher plants, only about 5–10% is chemically investigated. Since many

drugs, e.g. quinine and artemisinin (Wright and Phillipson, 1990), taxol and

campothecin (Debernardis et al., 1996) were isolated from plants, and because of

increased resistance of many microorganisms, e.g. malaria parasites, towards

established drugs, investigation of the chemical compounds within traditional plants is

necessary (Phillipson, 1991).

Progress made in therapy of various ailments has not been sufficient to

significantly lower annual death rates from most diseases, and there is an urgent need

for new strategies. Thus, the identification, mechanistic investigation, validation and

utilization of dietary components, natural products, or their synthetic analogues as

potential bioactive agents has become an important issue in current public health-related

research, in the form of functional foods or nutraceuticals. Considering the complexity

and causes of various diseases, it will be important to provide a variety of bioactive

agents with different molecular and cellular targets, acting by multiple mechanisms.

India has two of the thirty four identified 'hot spots' for Biodiversity. These are:

Eastern Himalayas and Western Ghats. The Western Ghats region is considered as one

of the most important biogeographic zones of India, as it is one of the richest centers of

endemism (http://www.biodiversityhotspots.org/). Numerous drugs have entered the

international pharmacopoeia via the study of ethno pharmacology and traditional

medicine (Ayyanar and Ignacimuthu, 2011). In synergy, the country has research


Discussion


5

strengths in natural products chemistry, medical biotechnology, conventional

biochemistry, plant molecular biology and tissue culture. This has led to sustainable

exploitation of bioactivity of medicinal plants, exploring the possibility of application in

drug discovery (Patwardhan and Gautam, 2005).

Development of novel therapeutics for the treatment of diseases has become a

clinical imperative. In view of the emergence of drug resistance among clinical isolates

for infectious agents or for other functional disorders a search for novel molecules is on

the rise. A solution to this dilemma is to test a broad range of lead compounds or their

intermediates and secondary metabolites. Many of these compounds show impressive

in vitro activity against microorganisms resistant to conventional antibiotics and exhibit

other desirable properties. Development of such lead molecules by characterizing their

biological activity spectrum, potency, toxicity and safety is the need of the hour.

Several diseases in the recent times are becoming increasingly important, of

which infectious diseases caused by microbes and cancer are two areas that demand

immediate attention. Cancer is a disease that results from changes in cellular behavior

caused by underlying changes in genetic information of the cells. Progress made in

cancer therapy has not been sufficient to significantly lower annual death rates from

most epithelial tumor types, and there is an urgent need for new strategies in cancer

control.

Figure 1. Golden triangle approach


Discussion


6

The golden triangle approach promotes effective integration of traditional

wisdom, the power of contemporary science and technology, and the evidence base of

modern medicine, where the holistic strategies are reflected from the principles of

systems biology. This provides a holistic or whole-person healing viewpoint alongside a

reverse pharmacology-based platform for drug discovery (Patwardhan and Gautam,

2005).

Most literature reveals that these endemic plants and the climax communities

have therapeutic potentials. These potentials are obviously due to the presence of

bioactive compounds that are being exploited by practitioners of alternative system of

medicines like Siddha and Ayurveda. The latent scientific knowledge and related

research is very much abysmal. Therefore the scientific documentation of bioactivities

will have vital impacts like – identification of new compounds with antimicrobial, anti

inflammatory, anticancer, immunomodulatory, antiviral properties etc., A golden

triangle, with the integration of modern medicine, traditional knowledge and the robust

use of science and technologies with a systems biology approach, could open new

opportunities for drug discovery (Kim, 2004).

Amorphophallus commutatus (Schott.) Engl. (Araceae) is a rare cormous herb

endemic to Western Ghats. It is also reported to be found in the marginal forest in

Gujarat, Maharashtra, Goa, Karnataka and Kerala states of India (Ravikumar and Ved,

2004). It is found in the evergreen and semi-evergreen forests under the shade of trees,

rarely in the open forming small colonies at the altitude of 60m (Hetterscheid and

Ittenbach, 1996).

Plants of the genus Amorphophallus have a long history of use in tropical and

subtropical Asia as a food source and as a traditional Chinese medicine (TCM). They

are perennial plants with an underground stem in the form of a corm and a highly

dissected umbrella-shaped leaf blade. There are 170 species distributed mainly in the

tropics from West Africa eastward into Polynesia and around nine species is used for

food, fodder and medicine (Hetterscheid and Ittenbach, 1996).


Discussion


7

Tuberous corms of A.commutatus were used for treatment of piles, tumours and

cysts (Ravikumar and Ved, 2004). Tubers of Amorphophallus commutatus has also

been used as antidote for snake bite by tribal’s living in fifty villages of Sitamata

wildlife, sanctuary Rajasthan, India (Jain et al., 2005). There are no other reports on the

bioactivity of the plant Amorphophallus commutatus, an endemic aroid of Western

Ghats but the cormous herb has been reported to be used by the tribes for various

ailments. Hence, this work is done with the aim of validating the following bioactivities.

OBJECTIVE

• Preparation of the plant extracts using solvents of increasing polarity namely,

Petroleum ether, Chloroform, Ethyl acetate, Methanol and Hot water.

• Evaluation of in vitro free radical scavenging and metal chelating properties of the

five different fractions.

• Analysis of enzymatic and non enzymatic antioxidant content of the freshly

collected tuber and leaves.

• In vitro cytotoxicity analysis of different fractions against mitogen induced

human lymphocyte culture by Sulpho Rhodamine B (SRB) assay and DNA

Fragmentation analysis by diphenyl amine method.

• In vitro cytotoxicity analysis of different fractions against human adenocarcinoma

cell line Colo 205 and human gyneacological cell line SiHa by Sulpho Rhodamine

B (SRB) assay.

• Evaluation of antimicrobial activity of the five different fractions.

• Phytochemical analysis of different fractions and possible identification of the

lead molecule in one of the extract with good bioactivity.


Discussion


8

RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE

The topics related to the current research work are reviewed in this chapter

under the following titles.

2.1 Introduction

2.2 Medicinal plants in traditional systems

2.3 Binomial classification of Amorphophallus commutatus

2.4 Properties of Araceae

2.5 Properties of Amorphophallus.

2.6 Ethnopharmacological utilisation of Araceae

2.6.1 Ethnopharmacological utilisation of Amorphophallus sps.

2.7 Bioactivity

2.7.1 Emergence of free radicals in biological systems

2.7.2 Effect of free radicals on biological system

2.7.3 Antioxidant enzymes – Natural defense against free radicals

2.7.4 Non enzymic antioxidant systems as natural defense against free radicals

2.7.5 Antimicrobial agents – Methods and mechanism

2.7.5.1 Recommendations for developing ‘Proof-of-concept’ for anti-infective

agents.

2.7.5.2 Selection of the appropriate bioassay

2.7.5.3 Antimicrobial agents from araceae

2.7.5.4 Antimicrobial agents from Amorphophallus sps

2.7.6 Anticancer agents – Mode of action

2.7.6.1 Anticancer agents from Araceae

2.7.7 Identified bioactivities of Amorphophallus sps

2.8 Phytochemistry

2.8.1 Molecules identified in Araceae/Amorphophallus sps

2.9 Conclusion

2


Discussion


9

2.1 Introduction

India represented by rich culture, traditions, and natural biodiversity, offer

unique opportunity for the drug discovery researchers. The country is blessed with two

(Eastern Himalaya and Western Ghats) of the hotspots of plant biodiversity and is 7th

among the 16 Megadiverse countries where 70% of the world’s species occur

collectively. In India there are over 17,500 species of higher plants, 64 gymnosperms,

1200 pteridophytes, 2850 bryophytes, 2021 lichens, 15,500 fungi and 6500 algae

reported. India is rich in its own flora i.e. endemic plant species (5725 angiosperms,

10 gymnosperms, 193 pteridophytes, 678 bryophytes, 260 liverworts, 466 lichens,

3500 fungi, and 1924 algae). Over 7500 plant species have been reported to be used in

the Indian traditional systems including ethnomedicines (Sanjappa, 2005).

Plant and plant based medicines are the basis of many of the modern

pharmaceuticals we use today for various ailments (Kaur et al., 2008). The medicinal

value of plants lies in some chemical substances that produce a definite physiological

action on the human body. The most important of these bioactive compounds are

alkaloids, flavonoids, phenolics etc. The phytochemical research based on ethno

pharmacological information is generally considered to be an effective approach in the

discovery of new bioactive compounds from plants (Duraipandiyan et al., 2006).

Plant-derived substances have recently become a great interest owing to their

versatile applications. The development of pharmaceuticals begins with identification of

active principles, detailed biological assays and dosage formulations, followed by

clinical studies to establish safety, efficacy and pharmacokinetic profile of new drug

(Khan et al., 2010). It is well known that natural products from the extracts of medicinal

plants are used in the treatment of skin, respiratory, neuromuscular and mental health

disorders and also in obstetrics and gynecology (Ankli et al., 2002).

Indian Materia Medica includes about 2000 drugs of natural origin almost all of

which are derived from different traditional systems and folklore practices. Nature was

considered as a compendium for templates of new chemical entities (NCEs). The plant


Discussion


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species mentioned in the ancient texts of Ayurveda and other Indian systems of

medicines may be explored with the modern scientific approaches for better leads in the

health care (Mukherjee and Wahile, 2006).

2.2. Medicinal plants in traditional systems

It is difficult to get reliable figures for the total number of medicinal plants on

earth; according to some estimation, around 35,000–70,000 plant species are being used

worldwide in health care systems (Farnsworth and Soejarto, 1991). According to

WHO estimations the populations in developing countries like India (70%), Ruwanda

(70%), Uganda (60%), Tanzania (60%), Benin (80%) and Ethiopia (90%) extensively

use traditional and alternative medicines for health care. Plants and plant-based

products are an integrated part of most of the traditional and alternative systems of

medicines worldwide (Figure 2). In developed countries like Belgium (31%), USA

(42%), Australia (48%), France (49%), Canada (70%), a significant percentage of the

population has used traditional and alternative remedies at least once for health care

(WHO, 2002). The table 1 lists the government organisation involved in research and

development of traditional systems of medicine.

Figure 2

Number of plants used in different systems of medicines in India

(Mukherjee and Wahile, 2006).

Modern

2%Tibetan

5%

Siddha

13%Ayurveda

19%

Folk

43%

Homeopathy

8%Unani

10%


Discussion


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Table 1

Government Institutes dealing with the research and development of the

traditional systems of medicines in India

Name of the Council Different Centers of the

Council

Research activities

Central council for Research in

Ayurveda and Siddha • 8 regional research institute

• 12 research centers

• 60 units and dispensaries

Medico-botanical survey and

development of Ayurvedic and

Siddha medicine based on

folklore uses and their

scientific validation and

implication.


Unani Medicines • 1 central research institute

• 8 regional research institute

• 11 clinical research units

• 5 drug standardisation units

Developing independent and

multidimensional research in to

various fundamental and

applied aspects of Unani

system of medicines.

Central council for research in

Homeopathy • 51 research centers across

the country

Screening of homeopathic

medicine for treating different

ailments and development of

standardisation parameters


Yoga and Naturopathy • Head quaters and central

unit at New Delhi, India

Development and propagation

of natural cure, yoga and

related aspects of yoga and

naturopathy

Council for scientific and

industrial Research and Regional

research laboratories

• Regional research

laboratory, Jammu.

• Central Drug Research

Institute, Lucknow.

• Central Institute of

Medicinal and Aromatic

Plants, Lucknow

Cultivation of medicinal plants,

quality control and

investigation of medicinal

plants and pharmacology

including development of agro-

biotechnological aspects.

2.3 Binomial classification of Amorphophallus commutatus

Kingdom : Plantae

Order : Alismatales

Family : Araceae

Subfamily: Aroideae

Tribe : Thomsonieae

Genus : Amorphophallus

Speices : commutatus


Discussion


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2.4 Properties of Araceae

Araceae includes 107 genera and 3200 species (Croat, 1998); distributed

mostly in tropical and subtropical regions, rarely in temperate regions. Twenty

five genera including 163 spp. and 21 vars. occur in India. These include newly

described and newly recorded taxa also. The largest genera in this family are Arisaema (46

spp. and 10 vars.), Amorphophallus (19 spp. and 2 vars.), Rhaphidophora (14 spp. and

one var.), Typhonium (10 spp. and 2 vars.), Alocasia (10 spp.), Pothos (10 spp. and 3

vars.) and Cryptocoryne (7 spp.) (Sakuragui, 2000).

The Araceae are best known as ornamental plants, e.g. Swiss-Cheese plant,

Monstera deliciosa; many Philodendron and Anthurium species, Zantedeschia

aethiopica (Calla lily), among many others (Sakuragui, 2000). The value of aroids is not

limited to the ornamentals alone. In the tropics various aroids are cultivated for food;

400 million people include taro (Colocasia esculanta) in their diets and several others

including species of Amorphophallus have significant potential in diet. Their

importance is often overlooked because they are generally consumed where they are

grown and are rarely exported. Some species are of medicinal importance. The use of

aroids as food and medicine is particularly interesting in view of the fact that they are

invariably toxic and require careful preparation for safe consumption (Sivadasan, 1999).

Their corms are rich in vitamin A, B, C and starch and are consumed after

roasting or boiling along with tamarind pulp to get rid off the acrid principle. Corms are

easily digestible and hence recommended for use in infant foods. Corms of Colocasia

esculenta are used against rheumatism, piles and as an antidote to stings of wasps and

insects. Internally it acts as a laxative (Plowman, 1969).

2.5 Properties of Amorphophallus

Plants of the genus Amorphophallus have a long history of use in tropical and

subtropical Asia as a food source and as a traditional Chinese medicine (TCM). They

are perennial plants with an underground stem in the form of a corm and a highly

dissected umbrella-shaped leaf blade. Of the 170 species distributed mainly in the

tropics from West Africa eastward into Polynesia (Hetterscheid and Ittenbach, 1996),


Discussion


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nine Amorphophallus species, i.e. A. albus, A. corrugates, A. kachinensis, A. konjac,

A. krausei, A. nanus, A. paeoniifolius, A. yuloensis and A. yunnanensis have been used

as food, medicine, fodder and for wine production. One of the most widely utilised is

Amorphophallus konjac which has been used in China for thousands of years. Despite

the historical use in China of whole corm extracts as a TCM, the current usage of konjac

in the West are in the food and nutraceutical industries; where soluble fibre extracted

from the corms, commonly known as konjac glucomannan (KGM) is used as a food

additive and in the development of dietary supplements or nutraceuticals (Chua et al.,

2010).

Figure 3

Habit of Amorphophallus commutatus

Corms of Amorphophallus campanulatus are used in dysentery and piles. Fruits

and seeds of Amorphophallus sylvaticus are made into a paste and employed in

toothache and bruises (Muthu et al., 2006). The figure 3 illustrates the habit of

Amorphophallus commutatus with vegetative structure and corm with inflorescence.

There are no identified reports on the bioactivity of the plant Amorphophallus

commutatus except for the reports on its ethnomedicinal usage. This has in turn

developed intrest in choosing the plant for this research.

a. Habit with vegetative

structure

b. Corm with inflorescence c. Habit

Review of Literature


Discussion


14

2.6 Ethnopharmacological utilisation of Araceae

Tribal communities are mainly the forest dwellers who have accumulated a rich

knowledge on the uses of various forests and forest products over the centuries. India

possesses a total of 427 tribal communities, of these more than 130 major tribal

communities live in North East India, which is comprised of the 8 states Meghalaya,

Mizoram, Manipur, Tripura, Sikkim, Assam, Nagaland and Arunachal Pradesh

(Kala, 2005).

Acorus gramineus (Araceae), which is distributed throughout Korea, Japan, and

China, has been used as a Korean traditional medicine for learning and memory

improvement, sedation and analgesia. Moreover, this herb has long been used for the

treatment of stomach ache and swelling as well as for the extermination of insects

(Park et al., 2011).

Documentation and determination of consensus about phytotherapeutic

veterinary practices among the Tharu tribal community of Uttar Pradesh, India was

conducted by Kumar et al., 2012, according to their report two araceae species namely

Acorus calamus and Alocasia indica contain ethno veterinary practice. The leaf paste of

the wild herb Acorus calamus has acted as a wound healer when applied externally. One

leaf of Alocasia indica (Roxb), is finely chopped and given with in morning for

3-4 days for lack of estrus.

The apatani tribes in Eastern Himalayas use Acorus calamus L. (Araceae) root to

treat cut wounds, skin diseases and bone fracture. The roots of Alocasia forniculata

(Roxb.) Schott. (Araceae) to cure the crack of heels. Amorphophallus paeoniifolius

(Dennst.) Nicolson (Araceae) Corn is used to treat Piles. The leaf of Colocasia affinis

Schott (Araceae) used to treat fever and respiratory disorders. There are five genus

belonging to araceae among the 138 plant used by apatani tribe as medicine

(Kala, 2005). Calamus oil, an essential oil from Acorus calamus (Araceae) has been

used as a Carminative, bitter stimulant, vermifuge and insect repellent (Rastogi and

Mehrotra, 2002).


Discussion


15

Traditionally Acorus calamus L was used to treat Leprosy (Sharma, 1998);

bronchitis, expectorant, pain in chest, asthma, cough, sore throat (Kirtikar and Basu,

1935). In ayurveda the plant has been used to treat leprosy (Gautam et al., 2007).

Whole plant of Pothos scandens (Araceae) are ground and the paste is externally

applied by rubbing 2-3 times a day, for muscle catches and sprains (Bhandary et al.,

1995).

2.6.1 Ethnopharmacological utilization of Amorphophallus sps

Amorphophallus campanulatus was reported to be used along with A. indica

Ficus retusa, Hibiscus rosa-sinensis, Pedalium murex, Pergularia daemia, P.granatum

(poison bites, stomachache), Elephantopus scaber (scabies), Pungam oil (M. pinnata)

and Mustard oil by Kani traditional healers in Tirunelveli district of Western Ghats

(Ayyanar and Ignacimuthu, 2011).

Upadhyay et al. (2010) in their study entitled “Ethnomedicinal and

ethnopharmaco-statistical studies of Eastern Rajasthan, India,” has identified the utility

of Amorphophallus companultus tuber in the 844 villages in Eastern Rajasthan. The

tuber is rubbed on stone with water and given orally to treat pneumonia and asthma. The

roots are useful in ophthalmia, amenorrhea and boils. They have also reported that the

plant is used in auyrveda, folk lore, siddha and unani.

Two araceae member Amorphophallus companulatus and Acorus calamus L

were evaluated to be positive as antimycobacterial agent (Gautam et al., 2007).

Similarly Amorphophallus companulatus was also reported to be used to treat leprosy

according to ayurveda (Gautam et al., 2007). The ethnomedicinal information put forths

that the plant is used to treat leprosy, asthma and bronchitis (Kirtikar and Basu, 1935);

respiratory disease, various fever, pulmonary disorders (Gupta and Viswanathan, 1956;

Sharma, 1998).

The rhizome of Amorphophallus bulbifer was powdered with that of turmeric

(Curcuma longa L., Zingiberaceae) and made into a paste with lime juice which is

applied 2-3 times a day into the anus to treat haemorrhoids. Two seeds are powdered


Discussion


16

and taken orally with a cup of cow's milk, 2-3 times a day, to cure diarrhea (Bhandary

et al., 1995).

Giday et al. (2009) had conducted an ethnobotanical study of Medicinal plants

of the Meinit ethnic group of Ethiopia and has identified Amorphophallus gallaensis

(Engl.) root to be applied topically to cure wound in cattles. A review on medicinal

plants useful for treating chronic obstructive pulmonary disease (COPD) reports the

utility of Amorphophallus campanulatus Roxb (Araceae) in treating bronchitis and

asthma (Ram et al., 2011).

Amorphophallus companulatus has been identified to be used traditionally in

Bangladesh for piles, enlargement of spleen, constipation, tumor, asthma, bronchitis,

vomiting, abdominal pain, blood diseases, elephentiasis, acute rheumatism, boils,

opthalmia, insect bites, as apetite and taste promoter and stomach ache tonic.

According to the authors there are no previously identified phytoconstituent

(Haque et al., 2000).

The above review describes the utilisation of members of Araceae as food,

fodder and medicine. The bioactivity of Amorphophallus commutatus has not been

reported in any of the studies so far except for the reports on ethnomedicinal values. The

reason might be due to the non availability of the plant in other regions due to its

endemism. Therefore it becomes inevitable to focus on the bioactivity of the plant and

bioprospecting it to identify the lead molecule responsible for the bioactivity.

2.7 Bioactivity

2.7.1 Emergence of free radicals in biological systems

Oxygen consumption inherent in cell growth leads to the generation of a series

of reactive oxygen species (ROS) (Barros et al., 2006). They are continuously produced

by the body’s normal use of oxygen such as respiration and some cell-mediated immune

functions (Figure 4). ROS include free radicals such as superoxide anion radicals

(O2 •−

), hydroxyl radicals (OH•) and non-free radical species such as hydrogen peroxide

(H2O2) and singlet oxygen (1O2) (Gulcin, 2006). ROS are continuously produced during


Discussion


17

normal physiologic events and can easily initiate the peroxidation of membrane lipids,

leading to the accumulation of lipid peroxides.

Figure 4

An unbalance between the procuction of prooxidants and antioxidants

in the cell lead to serious cellular damage

It was suggested that the electron donating capacity (Figure 5), reflecting

the reducing power of bioactive compounds, is associated with antioxidant activity

(Arabshahi-Delouee and Urooj, 2007). Antioxidants can be reductants, and inactivation

of oxidants by reductants can be described as redox reactions in which one reaction

species is reduced at the expense of the oxidation of the other (Chung et al., 2002).

Figure 5

Antioxidant neutralizing a free radical

Glutathinone reductase

Glutathinone peroxidase


Discussion


18

Lipid peroxidation consists of a series of free radical mediated chain

reaction processes (Figure 6) and is associated with several types of biological damage

(Ak and Gulcin, 2008).

Figure 6

Mechanism of Lipid peroxidation

Figure 7

The proposed reaction for chelating of ferrous ions by curcumin

(Ak and Gulcin, 2008)


Discussion


19

Among the transition metals, iron is known as the most important lipid oxidation

pro-oxidant due to its high reactivity. The effective ferrous ion chelators may also afford

protection against oxidative damage by removing iron that may otherwise participate in

HO• generating Fenton type reactions.

Fe2

+ + H2O2 Fe3

+ + OH

− + OH

•

Ferric ions also produce radicals from peroxides although the rate is 10-fold less

than that of ferrous ion (Kehrer, 2000). The model proposed for chelation of ferrous ion

by a well known antioxidant curcumin is illustrated in Figure 7.

Superoxide is an oxygen-centred radical with selective reactivity. Although a

relatively weak oxidant, superoxide exhibits limited chemical reactivity, but can

generate more dangerous species, including singlet oxygen and hydroxyl radicals, which

cause the peroxidation of lipids (Halliwell and Chirico, 1993). These species are

produced by a number of enzyme systems.

Superoxide can also reduce certain iron complexes such as cytochrome C.

Superoxide anions are thus precursors to active free radicals that have potential for

reacting with biological macromolecules and thereby inducing tissue damage (Halliwell

and Gutteridge, 1984). Also, superoxide has been observed to directly initiate lipid

peroxidation.

Nitric oxide (NO) is an important chemical mediator generated by endothelial

cells, macrophages, neurons, etc. and is involved in the regulation of various

physiological processes. Excess concentration of NO is associated with several diseases

(Forstermann, 2010). Oxygen reacts with the excess nitric oxide to generate nitrite and

peroxynitrite anions, which act as free radicals. In the present study the crude

hydroalcoholic extracts was checked for its inhibitory effect on nitric oxide production.

Nitric oxide radical generated from sodium nitroprusside at physiological pH was found

to be inhibited by plant extracts by a dose dependent way. Nitric oxide radical has been

involved in the cancer pathogenesis by increasing tumor vascularization and metastasis.


Discussion


20

Studies using nitric oxide inhibitors have shown decrease in tumor growth and play

important role in cancer therapy (Jayakumar and Kanthimathi, 2011).

The highly reactive hydroxyl radicals is formed in all biological systems and has

been implicated as a highly damaging species in free radical pathology, capable of

damaging almost every molecule found in living cells. This radical has the capacity to

join nucleotides in DNA and cause strand breakage which contributes to carcinogenesis,

mutagenesis and cytotoxicity. Hydroxyl radical scavenging capacity of an extract is

directly related to its antioxidant activity (Babu et al., 2001). There is no specific

enzyme to defense against them in human body. For this reason, the discovery of some

compounds with excellent hydroxyl radical scavenging ability would be significant for

some ailments induced by oxidative stress (Zhou et al., 2010).

2.7.2 Effect of free radicals on biological system

ROS are also capable of damaging crucial biomolecules such as nucleic acids,

lipids, proteins and carbohydrates and may cause DNA damage that can lead to

mutations. If ROS are not effectively scavenged by cellular constituents, they lead to

disease conditions. ROS have been implicated in more than 100 diseases (Halliwell

and Gutteridge, 1990). The Figure 8 illustrates how the free radicals can damage a cell.

Figure 8

Effect of free radical on cells

A B

A. Free radical attacking and weakening a cell

B. Cells with strengthened antioxidant defense


Discussion


21

The reduction of chronic diseases, DNA damage, mutagenesis, carcinogenesis

and inhibition of pathogenic bacterial growth is often associated with the termination of

free radical propagation in biological systems (Zhu et al., 2002). Lipid peroxidation

induced by hydroxyl radicals act as a vital step for degeneration of other intacellular

components. The benefits of compounds that possess lipid peroxidation inhibition

is shown in Figure 9.

Figure 9

Advantages of compounds inhibiting lipid peroxidation

2.7.3 Antioxidant enzymes - Natural defense against free radicals

All aerobic organisms have antioxidant defense (Figure 10), including

antioxidant enzymes and antioxidant food constituents, to remove or repair the damaged

molecules. Antioxidant compounds can scavenge free radicals and increase shelf life

by retarding the process of lipid peroxidation, which is one of the major reasons for

deterioration of food and pharmaceutical products during processing and storage

(Halliwell, 1997). Antioxidants can protect the human body from free radicals and

ROS effects by neutalising them (Figure 11). They retard the progress of many chronic

diseases as well as lipid peroxidation (Gulcin et al., 2002).


Discussion


22

Free radicals have been shown to be harmful as they react with important

cellular components such as proteins, DNA and cell membrane. The body on the other

hand, requires free radicals for immune system responses.

Figure 10 Figure 11

Antioxidant defense in cells Action of antioxidants on free radicals

However, an overload of these molecules has been linked to certain chronic

diseases of heart, liver and some form of cancers. All organisms contain anti-free

radical defence system, which includes antioxidant enzymes like catalase, peroxidase

and superoxide dismutase and antioxidants like ascorbic acid and tocopherol.

At present, there is special interest on natural antioxidants coming from the plant

resources (Kalaivani and Mathew, 2010). There are more evidences suggesting that

phytochemicals having antioxidant properties are associated with a lower risk of

mortality from many of the diseases (Rice- Evans, 2004; Dixon et al., 2005). The

Figure 12 describes the initiators and inhibitors of free radical formation.

Plants have defense systems that protect them against toxic ROIs. The resistance

of a plant to stress is correlated with its increased capacity to scavenge or detoxify

ROIs. The best well known antioxidant enzymes are intracellular enzymes such as

superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GSH-Px),

catalase (CAT) and ascorbate peroxidase (APX), which protect against the toxic effects

of oxidants generated within cells.

Catalase

Vitamin E

ββββ - Carotene

Cu / Zn SOD

Lipid Bilayer

Vitamin E+

ββββ - Carotene

Mn SOD + Glutathione peroxidase + GSH

Vitamin E

Vitamin C

Glutathione peroxidase

GSH

Vitamin C & E

ββββ - Carotene


Discussion


23

Figure 12

Initiators and Inhibitors of free radical formation

Recently, SOD and GSH-Px were characterized in the cell culture medium and

the extracellular space of mammalian cells (Ookawara et al., 2003). Ookawara et al.,

(2003) reported upregulated translocation of extracellular SOD (a Cu/Zn-containing

secretory glycoprotein) from the medium to the nucleus in 3T3-L1 cells under oxidative

stress, suggesting that SOD plays a role in protecting the nucleus against oxidative

damage to genomic DNA. In human BET1A cells, extracellular GSH-Px expression

was increased in response to ROI, providing clear evidence for the redox regulation of

expression (Comhair et al., 2001).

Plant cells are endowed with very important antioxidants such as glutathione

(GSH) and ascorbate (AsA), and antioxidative enzymes, such as superoxide dismutase

(SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) (Sgherri et al.,

2003).

Super oxide dismutase (SOD), the first enzyme in the detoxifying process,

converts O2•−

radicals to H2O2. APX reduces H2O2 using ascorbate as an electron donor

in the ascorbate-glutathione cycle. Oxidized ascorbate is then reduced by GSH, which is

generated from oxidized glutathione (GSSG) by glutathione reductase (GR). GR also

plays an important role in protecting against oxidative damage by maintaining a high

GSH/GSSG ratio (Foyer et al., 1997).


Discussion


24

To alleviate the damaging effects of ROS, plants have evolved enzymatic

antioxidants that include superoxide dismutase (SOD), catalase (CAT), guaiacol

peroxidase (GPX) (Foyer et al., 1994), glutathione peroxidase (GSH-Px) (Xue et al.,

2001) and ascorbate peroxidase (APX) and also possess non-enzymatic antioxidants

such as reduced glutathione (GSH) and ascorbate (AsA) (Liu et al., 2009).

The detoxification of O2 − is always accompanied by the production of H2O2,

which is toxic and must be eliminated (Foyer et al., 1997). In plants, enzymes such as

CAT, GPX, GSH-Px and APX are important for regulating intracellular H2O2 (Noctor

and Foyer, 1998). CAT acts in the microbody of cells, while GPX exists in the apoplast,

chloroplast and cytosol. It has been shown that chloroplasts contain the enzyme

GSH-Px. APX, a key enzyme in an ascorbate–glutathione cycle, exists in the

chloroplasts, cytosol, mitochondria and peroxisomes (Li et al., 2011). The figure 13

depicts the protection offered by antioxidants in regeneration of healthy skin cells.

Figure 13

Effect of free radicals and antioxidants on a skin cell

All living bodies have a complex antioxidant defence system that includes

various antioxidant enzymes, such as superoxide dismutase and catalase. Aerobic

metabolism produces superoxide anion as a byproduct and superoxide dismutase breaks

it up into H2O and H2O2 and then H2O2 is converted to H2O and O2 by catalase.

Therefore, the catalase activity of extracts is very important (Debnath et al., 2011).


Discussion


25

Guaicol peroxidase (G-POD) is involved in a large number of biochemical and

physiological processes. Glutathione peroxidase (GSH-POD) may be responsible for

scavenging H2O2, catalysing the peroxidation of reduced glutathione (GSH), and

forming the oxidized disulfide form of glutathione (GSSG) as a product. AsA-POD is

highly specific for ascorbate as the electron donor. Ascorbic acid serves as an excellent

antioxidant and plays a fundamental role in the removal of hydrogen peroxide and

produces DHAsA. DHAsA is reduced to ascorbic acid by MDAR or DHAR at the

expense of NADH and GSH (Halliwell, 1982). GSH has an important function in

maintaining cellular redox status (Wang and Ballington, 2007). GR is a ubiquitous

NADPH-dependent enzyme and may be a rate-limiting enzyme for defense against

active O2 toxicity (Gossett et al., 1996).

2.7.4 Non Enzymic antioxidant systems as natural defense against free radicals

The commonly known non-enzymatic antioxidants are GSH and AsA, which are

redox buffering in the apoplasts (Foyer et al., 2001). To mitigate stress conditions, AsA

directly scavenges ROS. GSH takes parts in the control of H2O2 levels. Chilling

increased the activities of antioxidant enzymes such as CAT, GSH-Px and APX and

elevated the contents of AsA and GSH (Li et al., 2011). The functions of non enzymatic

antioxidants are shown in Table 2.

Table 2

Role of non enzymatic antioxidants

Antioxidant Antioxidant Function

Vitamin E Chain-breaking antioxidant, prevent ion of ROS proliferation, binding ROS

Vitamin C Restriction of ROS propagation, Vitamin E recycling, ROS scavenging

Carotenoids Restriction of ROS propagation, immune function, moppinf up excess ROS

Glutathione Prevention of ROS formation by enzyme GSH-Px

Selenium Prevention of ROS formation by enzymes: GSH-Px, thioredoxin reductase

and others.

Zinc Prevention of free radical formation (superoxide dismutase enzyme)

Copper Prevention of free radical formation (superoxide dismutase enzyme). Must

be bound to protein to reduce oxidative potential.

Manganese Prevention of free radical formation (superoxide dismutase enzyme)

Iron Prevention of free radical formation (catalase enzyme). Must be bound to

protein to reduce oxidative potential.


Discussion


26

Glutathione, a sulphur-containing tripeptide, plays a prominent role in the

defence against the free radicals in plants under oxidative stress conditions and is

involved in the complex enzymatic machinery that controls the intracellular levels of

H2O2. It is also the precursor of phytochelatins that act as heavy metal-binding peptides

in plants. The level of GSH in plant tissues is known to change under metal stress

(Lomonte et al., 2010). The multilevel action of glutathione defense is showm in

Figure 14.

Figure 14

Glutathione dependent defense against ROS at multilevels

2.7.5 Antimicrobial agents – Methods and mechanism

Infectious diseases caused by bacteria, fungi, viruses and parasites are still a

major threat to public health, despite the tremendous progress in human medicine. Their

impact is particularly large in developing countries due to the relative unavailability of

medicines and the emergence of widespread drug resistance (Okeke et al., 2005).

Research on new antimicrobial substances must therefore be continued and all possible

strategies should be explored. Besides small molecules from medicinal chemistry,

natural products are still major sources of innovative therapeutic agents for various

conditions, including infectious diseases (Clardy and Walsh, 2004).


Discussion


27

Only a minute portion of the available diversity among fungi, marine fauna and

flora, bacteria and plants has yet been explored and ample opportunities lie theoretically

ahead. Current research on natural molecules and products primarily focuses on plants

since they can be sourced more easily and be selected on the basis of their

ethno-medicinal use (Verpoorte et al., 2005).

The chemical complexity of many natural products and the lack of assurance of

a renewable supply have created a diminishing interest by the pharmaceutical industry,

which in turn endorses the pivotal role of academia and public organisations in the

protracted exploration and evaluation of natural products. Use of ethnopharmacological

knowledge is one attractive way to reduce empiricism and enhance the probability of

success in new drug-finding efforts (Patwardhan, 2005).

2.7.5.1 Recommendations for developing ‘Proof-of-concept’ for anti-infective

agents

The recommendations that will help to define a more sound ‘proof-of-concept’

for antibacterial, antifungal, antiviral and antiparasitic potential in natural products and

their primary requirements include (Cos et al., 2006):

(1) use of reference strains or fully characterized clinical isolates,

(2) in vitro models on the whole organism and if possible cell-based,

(3) evaluation of selectivity by parallel cytotoxicity testing and/or integrated profiling

against unrelated micro-organisms,

(4) adequately broad dose range, enabling dose–response curves,

(5) stringent endpoint criteria with IC50-values generally below 100µg/ml for extracts

and below 25 µM for pure compounds,

(6) proper preparation, storage and in-test processing of extracts,

(7) inclusion of appropriate controls in each in vitro test replicate (blanks, infected and

reference controls) and

(8) follow-up of in vitro activity (‘hit’-status) in matching animal models

(‘lead’-status).


Discussion


28

2.7.5.2 Selection of the appropriate bioassay

Different screening approaches are available to identify the primary

pharmacological activity in chemical and/or natural products. The screening option will

largely depend on the specific nature of the disease being targeted and on the

availability of practical and biologically validated laboratory models.

As illustrated in Figure 15, four levels of screening can be identified and the

most rewarding strategy is to opt for models that remain as close as possible to the final

target, i.e. the patient. Whenever possible, activities discovered at one particular

screening level should be confirmed using a model in the next higher evaluation level

(Cos et al., 2006).

Figure 15

General approaches in anti-infective drug screening

For example, results obtained in a subcellular (enzymatic) screen should be

confirmed against the whole organism. A good in vitro activity against the whole

organism should then be linked to a confirmation test in an animal model. For most

infectious diseases, this can easily be achieved since validated in vitro and in vivo

laboratory models using the whole organism are available. The advantages and

disadvantage of various systems are shown in the Table 3 (Cos et al., 2006).


Discussion


29

Table 3

Advantages and Disadvantages of different drug screening approaches

2.7.5.3 Antimicrobial agents from araceae

Totally eight endophytic fungi were isolated from different parts of some plants

of araceae species. The isolated fungi were evaluated for their antibacterial and

cytotoxic activities and the endophytic fungi from araceae species was found to have

significant antibacterial activity against Pseudomonas aeroginosa (Hazalin et al., 2009).

Calamus oil, an essential oil from Acorus calamus (Araceae) has been screened

for in vitro anti bacterial activity and was identified to inhibit the growth of Bacillus

subtilis and Pseudomonas aeroginosa (Prabuseenivasan et al., 2006).

2.7.5.4 Antimicrobial agents from Amorphophallus sps.

The dichloromethane extract of Amorphophallus bequaertii was reported to

inhibit the growth of Mycobacterium tuberculosis with a MIC of 100µg/ml. The tuber

of Amorphophallus bequaertii was traditionally in the Republic of Congo for the

treatment of malaria, fever, abdominal pain and snake bite. The tuber was ground with

warm water and the filtrate was utilised for this purpose. The observed inhibition of


Discussion


30

growth of T. cruzi and T. rhodesiense by the aqueous extract of A. bequaertii was weak

when compared to the reference drugs melarsoprol and benznidazole, which have MIC

values of 0.011 and 0.4 µg/ml respectively. These compounds were around 103 and

102 times more active than the extract (Tshibangu et al., 2002).

Amorphophallus campanulatus, commonly known as 'OL', is a stout herbaceous

plant of India and neighbouring countries. The vegetative parts of this wild plant are

used in several Ayurvedic (traditional medicine) preparations by the tribal people. The

corm is also used as a condiment. Preliminary work with this plant showed that the

corm is by and large free of infection with mycotoxigenic fungi including

Aspergillusjavus. The aflatoxin has also not been found as a natural contaminant of the

plant and hence anti-aflatoxigenic property of leaf and corm extracts against the

aflatoxin-producing capacity of a toxigenic strain of Aspergillus javus (Prasad et al.,

1994).

The essential oil isolated from the rhizome of Amorphophallus companulatus

inhibited the growth of various strains of Mycobacterium tuberculosis - H37Rv

(human), B19-3 (bovine), and B19-1 (avian) strains at 10µg/ml, while that of H52RS

(streptomycin resistant) at 12.5µg/ml in broth dilution assay (Chopra et al., 1957).

The ether extract of the stem of Amorphophallus companulatus has been

reported to be active against Mycobacterium tuberculosis - MT B19-3 (bovine) strain at

MIC 1:5000 dilution (Gupta and Viswanathan, 1956).

2.7.6 Anticancer agents – Mode of action

Cancer is a growing health problem around the world. Natural products have

long been used to prevent and treat many diseases, including cancer and thus they are

good candidates for the development of anti-cancer drugs (Smith-Warner et al., 2000).

Phytochemicals, found in fruits and vegetables, have been proposed as the major

bioactive compounds providing the health benefits associated with diets rich in plant-

foods. Since the prevention of chronic diseases is a more effective strategy than their

treatment, reducing the risk of diseases such as cardiovascular disease and cancer is a


Discussion


31

Figure 18 DNA Fragmentation

Figure17 Electron micrograph of a normal cell

and an apoptotic cell

Figure 16 Process of Apoptosis

subject of great interest for doctors, scientists in general, consumers and the food

industry (Liu, 2003).

Apoptosis is a selective, controlled, and

genetically programmed cell death process that plays an

important role in the balance between cell replication

and cell death. In contrast to necrosis, this tightly

regulated and complex process exhibits some typical

morphological changes, such as chromatin condensation,

membrane blebbing, formation of apoptotic bodies (Figure 16 and 17), and in most

cases, DNA fragmentation (Figure 18). Natural products have been shown to be

excellent and reliable sources for the development of new drugs (Haddad et al., 2004).

Anticancer agents, on

the other hand, are mainly

related to their curative role

in a damaged system. Under

normal conditions, the cells

in which the DNA or other

components are irreversibly

damaged by various causes

undergo apoptotic cell death,


Discussion


32

which is a self-destructive metabolism according to the genetically encoded cell death-

signal (Hooper et al., 1999). However, cancer cells, which are already irreversibly

developed, obtain the capability to evade apoptosis by various ways. The aim of

anticancer agents is to trigger the apoptosis signaling system in these cancer cells whilst

disturbing their proliferation (Lee et al., 2004). Plants have many phytochemicals with

various bioactivities, including antioxidant, anti-inflammatory and anticancer activities.

For example, some studies have reported that extracts from natural products, such as

fruits, vegetables and medicinal herbs, have positive effects against cancer, compared

with chemotherapy or recent hormonal treatments (Wu et al., 2002). Therefore, many

plants have been examined to identify new and effective antioxidant and anticancer

compounds, as well as to elucidate the mechanisms of cancer prevention and apoptosis

(Swamy and Tan, 2000). There are various test systems available targeting different

cellular locations (Figure 19) and can be chosen based on the necessity.

Figure 19

Cytotoxicity test systems targeting different cellular locations

2.7.6.1 Anticancer agents from Araceae

Phenolic Constituents of Acorus gramineus (Araceae) was isolated and were

tested for cytotoxicity against four human tumor cell lines in vitro using a

Sulforhodamine B (SRB). Eighteen compounds were isolated and among them

CRYSTAL VIOLET


Discussion


33

kaempferol 3-methyl ether (compound 11) exhibited good cytotoxic activity against

A549, SK-OV-3, SK-MEL-2, and HCT15 cell lines (IC50: 11.37, 5.74, 7.19 and

9.06 µM, respectively). (2S,5S)-diveratryl- (3R,4S)-dimethyltetrahydrofuran (compound

15) showed moderate cytotoxic activity against A549, SK-OV-3, SK-MEL-2, and

HCT15 cells (IC50: 14.05, 19.27, 32.14 and 12.86 µM, respectively) (Park et al., 2011).

Two novel lectins were purified from rhizomes of two sweet flag species,

belonging to Araceae namely Acorus calamus and Acorus gramineus by affinity

chromatography. These lectins showed potent mitogenic activity towards mouse

splenocytes and human lymphocytes. Both the lectins also significantly inhibited the

growth of J774, a murine macrophage cancer cell-line and to lesser extent WEHI-279, a

B-cell lymphoma (Bains et al., 2005).

Amorphophallus companulatus have been screened for phytoconstituent and

identified the presence of terpenoids and phenols. The evaluation of antitumor activity

of the plant by potato disk bioassay had exhibited 25% inhibition of crown gall tumor

while vincristine showed 100% growth inhibition (Haque et al., 2000).

2.7.7 Identified bioactivities of Amorphophallus sps

Tinworth et al. (2010) has reviewed some of the potential species that can be

used as potential agents to treat insulin resistance in horse. They have reported that

Amorphophallus konjac as one potential species that contain glucomannan and

phytosterols. Glucomannan, being a water soluble fibre has glucose lowering and

insulin sensitizing activities and mediates antioxidant effects at dose of 2-4 g/ day in

humans (Eshun and He, 2004). As a soluble fibre glucomannan must absorb water to

form a viscous gel like mass that promotes the feeling of staitey while travelling through

the gastro intestinal tract (Keithley and Swanson, 2005).

The pharmacotherapeutic efficacy of Garcinia cambogia plus Amorphophallus

konjac for the treatment of obesity was evaluated by Vasques et al. (2008). The results

obtained by them suggest that joint daily administration of standardized extracts of

Garcinia cambogia (2.4 g) and Amorphophallus konjac (1.5 g) over a 12 week period


Discussion


34

had a significant hypocholesterolemic effect, without influencing the anthropometric or

colorimetric parameters tested. This effect took the form of a significant reduction in

total cholesterol, LDL-c and HDL-c levels; no effect was noted on triglyceride

levels. The drop in LDL-c values was proportionally greater than that observed in

HDL-c values, and thus contributed more significantly to the decrease in total

cholesterol levels. HCA), the main compound of Garcinia cambogia extract, is a

competitive blocker of ATP-citrate-lyase, presenting a potential inhibition of fatty acid

biosynthesis. Glucomannan fibers, abundant in Amorphophallus konjac, seem to reduce

the absorption kinetics of dietary fat. Hence this combination is a recommended

treatment for obesity (Vasques et al., 2008).

Figure 20

Role of antioxidants in obesity

A daily dosage of 1800 to 2004 mg of Amorphophallus koniac (Araceae) fibre

has been declared to have therapeutic indications on Obesity, lipid and glucose

metabolism alterations. The active principle of the plant is its mucilage and fibre. The

mucilage of Amorphophallus koniac at daily dosage of 2004 mg can act as an appetite


Discussion


35

modulator (Moro and Basile, 2000). The aromatic compounds isolated from

Amorphophallus konjac possess peroxynitrire scavenging activity (Niwa et al., 2002).

The importance of antioxidant balance to reduce obesity is illustrated in the Figure 20.

However the factor responsible for controlling obesity in Amorphophallus is its fibre.

Three oligosaccharide fractions from the root of Amorphophallus Konjac, which

was reported with hypoglycemic effects on diabetes subjects, were isolated and studied

using the STZ-treated diabetes model. Among them, one fraction named as KOS-A, was

found with nitric oxide (NOo) free radical regulation effect. At concentrations less than

1.5 mM, KOS-A positively decreased STZ-induced NOo level of islets, but normal NO

o

release for non-STZ-treated islets was not affected within the range. At 15 mM, KOS-A

played a contrary role and increased NOo level for islets both with and without

STZ-treatment. Islets insulin secretion changed corresponding to NOo level in the assay.

Increased insulin secretion appeared parallel to the decrease of NOo, and normal insulin

release was not affected by KOS-A less than 1.5 mM. Structure determination of KOS-

A shows that it is a tetrasaccharide with Mw of 666 Da and reductive end of a-D-

mannose. These results indicate that low dosage of KOS-A, with its function on

attenuating STZ-induced NOo level, doesn’t alter normal NO

o and insulin secretion

pathways of isolated islets. The NOo attenuation function of KOS-A on the diabetes

model is mainly resulted from environmental free radical scavenging by the

oligosaccharide. Present results also imply the mechanism of clinical Amorphophallus

Konjac hypoglycemic function maybe related with free radical attenuation and lower

risks of islets damage from NOo radical (Lu et al., 2002).

2.8 Phytochemistry

Irrespective of the adopted plant collection strategy, a critical step is the

processing of the plant material that will be used in the panel of screens. Appropriate

measures must be taken to guarantee that potential active constituents are not lost,

altered or destroyed during the preparation of the extract. Plant extracts are prepared by

maceration or percolation of fresh green plants or dried powdered plant material in

water and/or organic solvents. For hydrophilic compounds, polar solvents such as

methanol, ethanol or ethyl-acetate are used. For extraction of more lipophilic


Discussion


36

compounds, dichloromethane or a mixture of dichloromethane/methanol 1:1 are used. In

some instances, extraction in hexane is used to remove chlorophyll.

Important to consider in the ethnomedical approach is the need to prepare the

extract as described by the traditional healer in order to mimic as closely as possible the

traditional ‘herbal’ drug. To detect active substances present in very small quantities in

the extracts, a concentration step is usually required and is based on evaporation of the

solvent in vacuo. It is advised to extract and evaporate at low temperature not to destroy

any thermolabile constituent. Unfortunately, this concentration step often results in

precipitation or co-precipitation thereby hampering proper performance and

interpretation of the bioassay. Introducing pH differences may further enhance

separation of acid, neutral and basic constituents.

In some instances and if logistics permit, a ‘primary’ fractionation of the total

extract can be carried out prior to testing to separate polar from less-polar constituents

by sequential use of solvents from high to low polarity (Vanden Berghe and Vlietinck,

1991). This permits better discrimination between fractions that exhibit aspecific

activity or cytotoxicity and fractions that show selective antimicrobial activity.

This ‘primary’ fractionation scheme may also contain dereplication steps to avoid

re-isolation of known compounds (Cordell et al., 1993).

2.8.1 Molecules identified in Araceae/Amorphophallus sps

Cyanogenic glucosides (cyanohydrins), which are derived from the five

proteinogenic amino acids phenylalanine, tyrosine, valine, isoleucine, leucine and the

non-proteinogenic amino acid cyclopentenyl-glycine. Despite their widespread

occurrence, these natural products are found predominantly in the families - Araceae,

Asteraceae, Euphorbiaceae, Fabaceae, Passifloraceae, Poaceae, and Rosaceae (Dewick,

2002). Reynolds 2005 has identified the presence of alkaloids in the aroids

Amorphophallus, Arisarum, Arum and Caladium but no convincing isolations were

made.

Water-soluble O-carboxymethyl glucomannan derivatives (O-CMG) with

different degrees of substitution were synthesized successfully by reaction of a konjac


Discussion


37

glucomannan (isolated from the tubers of Amorphophallus paeoniifolius, one of the

most abundant Amorphophallus species in Vietnam forest) directly with

monochloroacetic acid (MCA) without methanol. The structure of O-carboxymethyl

glucomannan derivatives was characterized by FTIR, 1H, 13C and 1H–13C NMR-

HSQC (Hetro nuclear single quantum coherance spectroscopy). The conditions for

synthesizing of O-CMG derivatives were also evaluated. The results showed that the

optimal conditions for carboxymethylation of glucomannan were pH 10, temperature

60◦C for 3 h. The degree of substitution (DS), of O-substitution increased from 0.363 to

0.697 since the mass ratio (w/w) of glucomannan/monochloroacetic acid changed from

1/1 to 1/5 (An et al., 2011).

N-p-Coumaroylserotonin was isolated from the powder of Amorphophallus

konjac. N-p-Coumaroylserotonin has been reported as having strong antioxiditant

activity and inhibiting the production of pro inflammatory cytokines by human

monocytes (Tanaka et al., 2003).

A series of homologous x-phenylalkanoic acids and x-phenylalkenoic acids

were isolated from seed lipids of various genera of the subfamily Aroideae of Araceae

(the Jack-in-the-Pulpit family) and characterized. Besides the major acids,

11-phenylundecanoic acid, 13-phenyltridecanoic acid and 15-phenylpentadecanoic

acid, all other homologous odd carbon number x-phenylalkanoic acids from C7 to C23

were detected in trace amounts. Additionally, one even carbon number acid,

12-phenyldodecanoic acid was found in several specimens in trace amounts. Similarly,

two series of homologous odd carbon number monounsaturated x-phenylalkenoic acids

were found and characterized using dimethyl disulfide derivatization to locate the

positions of their double bonds. In five acids from C11 to C19, the double bond is

located at the same distance, D7, from the phenyl ring. In the other two acids of

C13 and C15 chain length, the double bond is located at D5 from the phenyl ring (Meija

and Soukup, 2004).

Konjac glucomannans (KGM) have been isolated from Amorphophallus tubers

(used three Amorphophallus species: Amorphophallus panomensis, Amorphophallus


Discussion


38

paeoniifolius and Amorphophallus tonkinensis) by a simple method without using toxic

chemicals. The konjac glucomannan content was about 5–9% (w/w) of original

Amorphophallus tubers. The structure, moisture uptake, molecular weight of konjac

glucomannan was investigated by nuclear magnetic resonance spectroscopy (NMR),

differential scanning colorimetry (DSC) and viscosimetry. The results indicated that the

main component of konjac flour was glucomannan. The mannose/glucose molar ratio

and molecular weight (Mw) of glucomannan isolated from Amorphophallus

paeoniifolius, Amorphophallus panomensis and Amorphophallus tonkinensis were

1/0.13; (Mw = 1.115_106), 1/0.10; (Mw = 1.023_106) and 1/0.25; (Mw = 1.043_106),

respectively. The moisture uptake of konjac glucomannans was about 7.5 - 9.2%

(An et al., 2010).

2.9 Conclusion

All above reviews hardly have given any emphasis on Indian medicinal plants.

To establish the potentiality of traditional medicine, research needs to be conducted on

different disciplines to meet the requirement of the society based on the various aspects.

The most vital aspect include standardization of materials, methods and measures for

preparation, preservation, presentation and administration of plant based drugs. These

standardizations will provide proper scientific validation and significance to the

fundamental principles of the system to the extent possible, so that they can be accepted

within a scientific framework. Biodiversity of natural resources has served not only for

the primary human needs but also for health care, since time immemorial.

The Indian subcontinent, with the history of one of the oldest civilization,

harbors many traditional health care systems. Their development was supported by the

diverse biodiversity in flora and fauna due to variations in geographical landscaping.

The compounds like campothecin, vinca alkaloids, taxol etc., and the plants like alovera

have gained importance only after scientific validation. The plant Amorphophallus

commutatus has not been reported except for very few papers stating the ethnomedicinal

application of the plant. Therefore scientifical validation of the bioactivity of the plant

becomes a must and hence this research is an initiative to validate the biological activity

of the plant.


Discussion


39

MMAATTEERRIIAALLSS AANNDD MMEETTHHOODDSS

The current therapies are inadequate and have numerous adverse effects. There

is an acute need for potential alternative therapies. Medicinal plants are classical and

most widespread form of medication for treating various human ailments throughout the

world (Ram et al., 2011). Bioassay-directed fractionation is an important process in the

identification of active principle(s) in natural product extract(s). In a drug discovery

programme from natural products, two steps are generally followed, viz., development

of new/novel (phyto) chemical methods and a suitable bioassay in order to provide

valid guidance with respect to ultimate endpoint (Gautam et al., 2007).

The present study was initiated with an aim to identify the bioactivity of

Amorphophallus commutatus an endemic aroid of Western Ghats, S. India. In order to

achieve the objectives the study was divided in to various phases as follows and as

mentioned in the flow chart 1.

Phase I

Preparation of the plant extracts using solvents of increasing polarity namely,

Petroleum ether, Chloroform, Ethyl acetate, Methanol and Hot water.

Phase II

• Evaluation of in vitro free radical scavenging and metal chelating properties of

the five different fractions.

• Analysis of enzymatic and non enzymatic antioxidant content of the freshly

collected tuber and leaves.

Phase III

• In vitro cytotoxicity analysis of different fractions against mitogen induced

human lymphocyte culture by SRB assay and DNA Fragmentation analysis by

diphenyl amine method.

• In vitro cytotoxicity analysis of different fractions against human

adenocarcinoma cell line Colo 205 and human gyneacological cell line SiHa by

SRB assay.

3


Discussion


40

Bioactivity and Phytochemical analysis of Amorphophallus commutatus (Schott) Engl. an

Endemic Aroid of Western Ghats, South India

40


Discussion


41

Phase IV

• Evaluation of antimicrobial activity of the five different fractions

Phase V

• Phytochemical analysis of different fractions and possible identification of the

lead molecule in one of the extract with good bioactivity

PHASE I

3.1 Plant sample

The whole plants of Amorphophallus commutatus was obtained from All India

Coordinated Research Project on Medicinal and Aromatic plants (AICRP on M and A),

College of Horticulture, Kerala Agricultural University, Vellanikkara, Thrissur, Kerala.

The plant was authenticated by Dr. V.V.Radhakrishnan, Associate professor and Head,

AICRP on M and A, College of Horticulture, Kerala Agricultural University,

Vellanikkara, Thrissur, Kerala.

3.2 Extraction

Organic solvents in the increasing order of polarity (Petroleum ether,

chloroform, ethyl acetate, methanol) and aqueous extract (hot water) of the plant

materials were prepared according to the method described by Harbone, 1998. The

samples were sequentially extracted using a soxhelet apparatus. Each of the five extracts

was evaporated to dryness after extraction under reduced pressure using rotary

evaporator. The concentrated extracts were lyophilized and were solubilised with

DMSO for organic solvents and with sterile distilled water for hotwater extract. The

desired concentration was used for different experiments.

Phase II

3.3 In vitro Radical Scavenging Activity

3.3.1 Sample

All the five extracts were diluted and concentration ranging between 25µg to

100 µg was used for free radical scavenging activity studies. Standard BHT in the same

concentration was used as positive control. EDTA was used as a positive control for

metal ion chelation studies.


Discussion


42

3.3.2 Scavenging activity on synthetic radicals

3.3.2.1 DPPH radical scavenging activity

The free radical scavenging activity of different solvent fractions

of Amorphophallus commutatus was measured according to the method of

Mensor et al. (2001) using DPPH as free radical. The reaction mixture contained 0.1 ml

of different concentration of solvent fractions with 1.9 ml of 0.1 mM DPPH in

methanol. The control was devoid of the sample. BHT was used as a positive control.

The reaction mixture was shaken well and read at 517 nm after incubation at 25o C for

30 min. The percentage DPPH radical scavenging activity was calculated from the

following equation.

DPPH Scavenging activity (%) =

Where Ac was the absorbance of control reaction and As the absorbance in the

presence of A.commutatus tuber extracts.

3.3.2.2 ABTS radical cation decolorisation assay

The ABTS•+

scavenging activity was determined according to the method of

Re et al. (1999). The plant extract with antioxidant property was added to a pre-formed

ABTS radical solution and after a fixed time period the remaining ABTS•+

is quantified

spectrophotometrically at 734 nm (Gulcin et al., 2006). ABTS•+

was produced by

reacting 2 mM ABTS in H2O with 2.45mM potassium persulfate (K2S2O8), stored in the

dark at room temperature for 4 h. The ABTS•+

solution was diluted to give an

absorbance of 0.750 ± 0.025 at 734 nm in 0.1M sodium phosphate buffer (pH 7.4).

Then, 1 mL of ABTS•+

solution was added to different concentration of various solvent

fractions. The absorbance was recorded after 30 min. The percentage of radical

scavenging was calculated for each concentration relative to a control without extract.

The scavenging capabilities of test compounds were calculated using the following

equation.

ABTS•+

scavenging (%) =

Where Ac was the absorbance of control reaction and As the absorbance of

remaining ABTS•+

(Gulcin, 2006; Gulcin and Dastan, 2007) in the presence of

A.commutatus tuber extracts.


Discussion


43

3.3.2 Superoxide anion radical scavenging activity

The assay depends on the capacity of different solvent fractions of

A.commutatus tuber extracts to inhibit formazan formation by scavenging the

superoxide radicals generated in riboflavin-light-NBT system (Beauchamp and

Fridovich, 1971). The method adopted by Martinez et al. (2001) was followed with

slight modification. The reaction mixture contained 50 mM phosphate buffer, pH 7.6,

20 µg riboflavin, 12 mM EDTA, and NBT 0.1 mg/3 ml, added in sequence. Different

concentrations of A. commutatus tuber extracts were added to the reaction mixture and

the reaction was initiated by illuminating the tubes under fluorescent lamp (20 w) for

15minutes. Immediately after illumination, the absorbance was measured at 560 nm.

Decreased absorbance of the reaction mixture indicates increased superoxide anion

scavenging activity. Identical tubes, with reaction mixture, were kept in the dark and

served as blanks. The percentage inhibition of superoxide anion generation was

calculated using the following equation.

Super oxide radical scavenging capacity (%) =


presence of A.commutatus tuber extracts (Gulcin et al., 2003; Gulcin et al., 2004).

3.3.3 Nitric oxide radical scavenging activity

The method adopted by Marcocci et al., 1994 with slight modifications was

adopted. Sodium nitroprusside in aqueous solution at physiological pH spontaneously

generates nitric oxide (NO) which interacts with oxygen to produce nitric ions that can

be estimated by using Greiss reagent. Scavengers of nitric oxide compete with oxygen

leading to reduced production of nitric oxide. Various concentrations of different

solvent fractions of A. commutatus were mixed with 0.4 ml of Sodium nitroprusside

(10 mM) in phosphate buffer saline (PBS) pH 7.4. The volume was made up to 1.5 ml

with distilled water and incubated at room temperature for 30 min. After incubation

0.5 ml of Greiss reagent (1% sulfanilamide, 2% H3PO4 and 0.1% napthylethylenedi-

amine dihydrochloride) was added. The absorbance of the chromaphore formed during

the diazotization of nitrite with sulfanilamide and subsequent coupling with napthyl


Discussion


44

ethylene diamine was read at 540 nm. BHT treated in the same way as the samples and

served as a positive control. The same reaction mixture without extract served as the

control. The percentage inhibition of Nitric oxide formation by the extracts was

measured by the following formula.

Nitric oxide radical scavenging capacity (%) =



3.3.4 Prevention of deoxyribose degradation

3.3.4.1 Hydroxy radical scavenging activity

The ability of test compound to prevent Fe2+

/H2O2 induced decomposition of

deoxyribose was carried out using the method of Halliwell and Gutteridge (1981). Test

compounds of different fractions of Amorphophallus commutatus tuber was added to a

reaction mixture containing 120 µl of 20 mM deoxyribose, 400µl 0.1M phosphate

buffer, 40µl of 20mM hydrogen peroxide and 40µl of 500µM FeSo4, and the volume

was made up to 800µl with distilled water. The reaction mixture was incubated at 37ºC

for 30 min, and the reaction was then arrested by adding 0.5 ml of 2.8% TCA. This was

followed by the addition of 0.4ml of 0.6% TBA (Thiobarbuturic acid) solution. The

tubes were subsequently incubated in boiling water for 20 min. The absorbance was

measured at 532 nm in spectrophotometer. The percentage inhibition of hydroxyl

radical production by the extracts was calculated using the following equation.

Hydroxy radical scavenging capacity (%) =


presence of A. commutatus tuber extracts.

3.3.4.2 DNA nicking assay

The ability of different fractions of Amorphophallus commutatus tuber to protect

plasmid DNA from devastating effects of hydroxyl radicals generated by Fenton’s

reagent was assessed according to the DNA nicking assay described by Lee et al. (2002)

with slight modifications. The reaction mixture contained 0.3µl of plasmid DNA, 10 µl


Discussion


45

Fenton’s reagent (30 mM H2O2, 50 mM ascorbic acid, and 80 mM FeCl3) followed by

the addition of 10 µl of different concentration of extracts and the final volume of the

mixture was brought up to 25 µl using distilled water. The mixture was then incubated

for 30 min at 37oC and the DNA was analyzed on 1% agarose gel (prepared by

dissolving 0.5 g of agarose in 50 ml of 1× TBE buffer) with 2.5 µl of Ethidium bromide

solution added at bearable warmth. Vitamin E was used as a positive control.

3.3.5 Inhibition of lipid peroxidation

A modified thiobarbituric acid reactive species (TBARS) assay (Ohkowa et al.,

1979; Banerjee et al., 2005) was used to measure the lipid peroxide formed, using

egg yolk homogenates as lipid rich media (Ruberto et al., 2000). Egg homogenate

(0.5 ml 10% in distilled water, v/v) and different concentrations of Amorphophallus

commutatus tuber extracts were mixed in test tube and the volume was made up to 1ml,

by adding distilled water. Finally, 0.05 ml FeSO4 (0.07 M) was added to above

mixture and incubated for 30 min to induce lipid peroxidation. Thereafter, 1.5ml of 20%

acetic acid (pH adjusted to 3.5 with NaOH) and 1.5 ml of 0.8% TBA (W/V) (prepared in

1.1% sodium dodecyl sulphate) and 0.05ml 20% TCA were added, vortexed and then

heated in a boiling water bath for 60 min. After cooling, 5.0 ml of 1-butanol was added

to each tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic

upper layer was measured at 532 nm. The ability of the extract to inhibit lipid

peroxidation was calculated using the following equation:

% inhibition of Lipid Peroxidation =



3.3.6 Total antioxidant capacity by phosphomolybdenum assay

The method of Pietro et al. (1999) was adopted for the assay of total antioxidant

capacity (TAC) of different doses of Amorphophallus commutatus solvent fraction

from the tuber. Aliquot of 0.1 ml of sample solution was combined with 1 ml of reagent

solution (600 mM sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium

molybdate). The tubes were capped and incubated in a boiling water bath at 95oC for


Discussion


46

90 min. After the samples had cooled to room temperature, the absorbance was

measured at 695 nm against a blank. Total antioxidant activity was expressed in relation

of ascorbic acid and calculated by following formula

% TAC =

Where Ac was the absorbance of the control (blank, without extract), As was the

absorbance in the presence of the extract and Aaa was absorbance of ascorbic acid.

3.3.7 Total reducing power activity

The reducing power of different doses of A.commutatus tubers were determined

by the method of Yildrim et al., 2001. The sample 0.1ml was mixed with 2.5 ml of

0.2 M Phosphate buffer pH 6.6 and 2.5 ml of 1% potassium ferricyanide. The aliquot

was incubated at 50oc for 30 min. A volume of 2.5 ml of 10 % TCA was added to the

above mixture and centrifuged for 10 min at 3000 rpm. An aliquot of 2.5 ml of the

supernatant was mixed with 2.5 ml of distilled water and 0.5ml of 1% ferric chloride.

Absorbance was measured at 700nm. Higher the absorbance of reaction mixture, greater

is the reducing activity of the extract. The results were compared with BHT (positive

control). The percent reduction of the sample as compared to the standard, i.e. BHT was

calculated using the following equation.

Reducing Power (%) =

Where Ac was the absorbance of standard at maximum concentration tested and

As the absorbance in the presence of A.commutatus tuber extracts.

3.3.8 Ferrous ion chelating activity:

The ferrous ion (Fe2+)

- chelating potential of the extract was investigated according

to the method of Decker and Welch (1990), the Fe2+

- chelating ability of the extract was

monitored by measuring the ferrous iron - ferrozine complex at 562 nm. To 0.1 ml of

different solvent fractions of A.commutatus tuber, FeCl2 (2 mM), and ferrozine (5 mM),

was added and adjusted to a total volume of 0.8 ml with methanol, shaken well and

incubated for 10 min at room temperature. The absorbance of the mixture was measured


Discussion


47

at 562 nm against blank. EDTA along with BHT at the same concentration range as

extracts was used as positive control. The ability of the extract to chelate ferrous ion

was calculated using the following equation:

% inhibition =



3.3.9 Calculation of IC50

Various concentrations (25 -100 µg) of A.commutatus tuber extracts were taken for

the study and IC50 value (which shows 50% inhibition) was calculated using Regression

analysis in MS Excel.

3.4 ENZYMATIC ANTIOXIDANTS

3.4.1 Plant Sample

Fresh tuber and leaves was taken for the study. The samples were prepared by

grinding one gram of the tuber in 5 ml of 30% ethanol, in a pre-chilled mortar and pestle

and the extracts were centrifuged at 10,000 pm at 4ºC for 10 minutes. The supernatant

thus obtained were used with in four hours for various enzymatic antioxidants assays

(Rani et al., 2004).

3.4.2 Protein Content

Protein content of tissue homogenates was determined by a colorimetric method

of Bradford (1976), using Bovine serum albumin (BSA) as a standard. For the

estimation of protein 0.1 ml of sample were taken, along with sample 2 ml of Bradford’s

dye solution were added and mixed gently. After 5 minutes, absorbance was read at 595

nm using colorimeter. Protein concentration was determined by plotting graph, optical

density at x axis and the concentration at y axis.

3.4.3 Antioxidant enzyme measurements

3.4.3.1 Superoxide dismutase (SOD)

The assay of superoxide dismutase was done according to the procedure of

Das et al. (2000). In this method, 1.4ml aliquot of the reaction mixture (containing

1.11 ml of 50 mM phosphate buffer of pH 7.4, 0.075 ml of 20 nM L-Methionine,


Discussion


48

0.04 ml of 10 mM hydroxylamine hydrochloride and 0.1 ml of 50 mM EDTA) was

added to 100 µl of the sample extract and incubated at 30ºC for 5 minutes. 80 µl of

50 µM riboflavin was then added and the tubes were exposed for 10 min to 200 W -

Philips fluorescent lamps. After the exposure time, 1ml of greiss reagent (mixture of

equal volume of 1% sulphanilamide in 5% phosphoric acid) was added and absorbance

of the colour formed was measured at 543nm. One unit of enzyme activity was

measured as the amount of SOD capable of inhibiting 50% of nitrite formation under

assay conditions.

The SOD activity was calculated by the following formula,

% inhibition of nitrite formation =

Where As is the absorbance of the sample and Ac is the absorbance of

the control.

3.4.3.2 Catalase (CAT)

The method of Luck (1963), as mentioned in Sadasivam and Manikam (1992)

was adopted to measure the activity of catalase. The enzyme extract (0.1 ml) was added

to the reaction mixture containing 3 ml of H2O2 and 0.01 M phosphate buffer (pH 7.0)

and the OD change was measured at 240 nm, the time taken for decrease in the

absorbance from 0.45 to 0.4 is noted as ∆T. The activity of the enzyme is expressed in

the terms of µmole of H2O2 consumed/ min/ mg protein.

The activity of catalase was calculated by the following formula,

Unit in the assay mixture = 17 / ∆T

3.4.3.3 Guaiacol peroxidase (GPOD)

The assay was carried out by the method of Putter (1974), Malik and Singh

(1980). The reaction mixture consisted of 3ml of assay buffer (0.1 M phosphate buffer,

(pH 7.0), 20 mM guaiacol, and 0.03 ml of 30% H2O2). To this 0.1 ml of enzyme extract

added and O.D change was measured at 436 nm. The peroxidase activity was calculated

using an extinction coefficient of guaiacol dehydrogenase (liters /mol). The activity of

were calculated by the formula.


Discussion


49

Guaicol peroxidase activity units / litre =

Where 0.1 = volume of sample; 6.39 = extinction coefficient; ∆t = time in minutes

3.4.3.4 Ascorbic acid oxidase (AAO)

Assay of ascorbic acid oxidase activity was carried out according to the

procedure of Oberbacher and Vines (1963). To 3.0 ml of the substrate solution (8.8 mg

of ascorbic acid in 300 ml phosphate buffer, pH 5.6) 0.1 ml of the enzyme extract was

added and the absorbance change was measured at 265 nm for every 30 second for a

period of 5 minutes. One enzyme unit is equivalent to 0.01 OD change per minute.

3.4.3.5 Glucose- 6-phosphate dehydrogenase (G6PD)

The method of Balinsky and Bernstein (1963) was adopted to assay glucose

6- phosphate dehydrogenase. The reaction mixture containing 0.4 ml Tris- Hcl buffer,

0.2 ml of NADP, 0.2 ml of magnesium chloride, and 1.0 ml of water were added in a

cuvette. The reaction was started by adding 0.2 ml of glucose - 6- phosphate and the

increase in the absorbance was measured at 340 nm.

The activity of the enzyme is expressed in terms of units/g in which one unit is

equal to the amount of enzyme that brought about a change in optical density of

0.01/minute.

3.4.3.6 Glutathione peroxidase (GSH-Px)

Glutathione peroxidase was assayed by the procedure of Wendel (1980). To

0.1 ml of enzyme extract was added the reaction mixture containing 50 mM sodium

phosphate buffer with 40 mM EDTA pH 7.0, 1.0 mM sodiumazide solution 1 mg of

β-NADPH, 1 mM of DTT with sodium phosphate buffer, 200 mM reduced glutathione,

0.042% of H2O2. The decrease in absorbance was recorded at 340 nm for 5 minutes.

The enzyme activity is expressed in terms of µg of glutathione utilised / min /mg

protein. Glutathione activity was calculated by the formula,

(∆A340/test-∆A340/blank)(2)(3.1)(DF)

U/ml = —————————————————

(6.22)(0.05)


Discussion


50

2=2µmoles of GSH produced per µmole of β-NADPH oxidized; 3.1=total

volume (in millimeters) ofassay DF= Dilution Factor; 6.22=millimeter extinction co

efficient of β-NADPH at 340 nm; 0.05 = volume (in millimeter) of enzyme used.

3.4.3.7 Glutathione reductase (GR)

The assay of glutathione reductase was done according to the procedure of

David and Richard (1983). To 0.1 ml of sample, 1 ml of Potassium buffer (0.12 M pH

7.2), 0.1ml of EDTA, 0.1 ml of Sodium azide and 0.1 ml of oxidized glutathione were

added and the volume was made up to 2 ml with water. The mixture was kept at room

temperature for three minutes and 0.1 ml of NADPH was added. The absorbance at

340nm was recorded at intervals of 15 seconds for 2 to 3 minutes. One unit of GR is

expressed as µM of NADPH oxidized/ minute/gram. The GR activity was calculated by

the formula,

∆A340nm/min×3×Df

U /ml= ————————

(6.22)(0.1)

Where 6.22-millimeter extinction coefficient of β-NADPH; 0.1- volume of

enzyme used for assay; 3-volume of reaction mixture; Df – dilution factor if samples are

diluted.

3.4.3.8 Polyphenol oxidase (PPO)

Polyphenol oxidases activity was assayed by the procedure of Esterbauer et al.

(1977). Into a cuvette, 0.2 ml of the sample extract was added to the reaction mixture

containing, 2.5 ml of phosphate buffer and 0.3 ml of catechol solution. The change in the

absorbance was recorded every 30 sec up to 5 minutes. One unit of either catechol

oxidase or laccase is defined as the amount of enzyme that transforms one µmole of

dihydrophenol to one µmole of quinine/minute. The enzyme activity is expressed as u/g

tissue. The activity of PPO can be calculated using the formula,

Enzyme unit = k × (A/minute)

k for catechol oxidase = 0.272

3.4.4 Non-enzymatic component measurement

Non- enzymic antioxidant contents such as ascorbic acid, glutathione reduced,

total phenol was estimated in different parts of Amorphophallus commutatus.


Discussion


51

3.4.4.1 Total phenols

The method proposed by Malick and Singh (1980) was used to determine the

total phenols in the different parts of the Amorphophallus commutatus.

Fresh tuber and the leaf sample were homogenized in 30% of ethanol and

centrifuged at 10,000 rpm for 10 minutes. To 0.5 ml of supernatant added 0.5 ml of

folin-ciocalteau reagent and after 5 minutes, 2.0 ml of 20% sodium carbonate was

added. The tubes were placed in the boiling water bath for 1 minute. Then the blue

colored complex was measured at 650 nm in a spectrophotometer. The values are

expressed as mg phenols/g tissue.

3.4.4.2 Ascorbic acid

For the estimation of ascorbic acid, 1 g of the different parts of sample were

homogenized with by 4% of TCA after centrifugation a pinch of activated charcoal was

added, mixed vigorously using cyclo mixer and stand for 5 minutes. The tubes were

centrifuged again to pellet the charcoal particles. Aliquots of supernatant were taken for

the estimation and as adopted by Roe and Keuther (1943).

To 0.5ml of charcoal treated supernatant 2.0 ml of 4% TCA, 0.5 ml of Di Nitro

Phenyl Hydrazine was added followed by 2 drops of thiourea solution and mixed well.

The tubes were incubated for 3 hours. Removed, placed in ice cold water and added

2.5 ml of 85% H2SO4 drop by drop and the absorbance were recorded at 540 nm.

Concentration of ascorbic acid in the samples were calculated and expressed as mg/g

tissue.

3.4.4.3 Reduced Glutathione (GSH)

The method of Moron et al. (1979) was followed to determine the amount of

reduced glutathione. For the estimation, 2 g of different part of the sample were

homogenized with 5% TCA and centrifuged at 10,000 rpm for 10 minutes at 4ºC. Then

supernatant was used for the estimation of GSH. To 0.1 ml of supernatant, 1.0 ml of

phosphate buffer then 2.0 ml of freshly prepared DTNB (Ellman’s reagent) solution was

added and the intensity of the yellow color formed was read at 412 nm in a

spectrophotometer after 10 minutes. The values are expressed as µmoles of GSH/g

tissue.


Discussion


52

PHASE III

3.5 ANTIPROLIFERATIVE ACTIVITY

3.5.1 Preparation of Sample

The condensed sample obtained from the rotary vaccum evaporator 1 mg was

dissolved in 1ml of DMSO and serially diluted to concentration ranging between

61.5 -500 µg/ml. From the concentration range 20µl of the extracts is used for the

assays. Querecetin was used as positive control.

3.5.2 In vitro cytotoxicity of mitogen induced blood lymphocytes

3.5.2.1 Isolation of Peripheral blood lymphocytes

Venous peripheral blood was collected aseptically from healthy donors using

EDTA as anticoagulant. The blood (3 ml) was diluted with equal volume of RPMI 1640

medium. Peripheral blood lymphocytes were separated by density gradient

centrifugation at 400 g using lymphocyte separation medium. The white layer formed

intermittently was taken and washed by using RPMI 1640. After centrifugation

at 160-200 g for 10 minutes, the pellet was collected. Isolated Peripheral blood

lymphocytes were suspended in RPMI medium with 10% sterile bovine serum (FBS),

2 mM L-glutamine-streptomycin solution (Suganthy et al., 2010).

3.5.2.2 Cell viability

Tryphan blue is a vital dye. The reactivity of tryphan blue is based on the fact

that the chromophore is negatively charged and does not interact with the cell unless the

membrane is damaged. Therefore, all the cells which exclude the dye are viable. The

cell suspension is mixed with equal volume of 0.4% tryphan blue and loaded in to a

hemocytometer for cell count (Freshney, 2006).

3.5.2.3 Lymphocyte culture

The lymphocytes were grown to log phase by overnight culture in RPMI 1640

containing 10% heat inactivated bovine serum and 1% antibacterial antimycotic solution

(Sigma). After checking the viability 1.0 x 106 cells /ml was seeded in six well plate and

made up to 3.0 ml with complete media (Freshney, 2006).


Discussion


53

3.5.2.4 Mitogen induction of isolated lymphocytes

Concanavalin A, a mitogen that encourages the proliferation of T lymphocytes

was used in this study. Concanavalin A, in the concentration of 10 µg / ml of media was

used for inducing cell proliferation. The lymphocytes after checking the viability

1.0 x 106 cells / ml were seeded in six well plates with Concanavalin A (Bains et al.,

2005).

3.5.2.5 In vitro cytotoxicity assay using Sulpho Rhodamine B (SRB)

The in vitro cytotoxic effect of various fractions of Amorphophallus commutatus

tuber was done by sulphorhodamine B assay following the method given by

Skehan et al. (1990). The cells at sub confluent stage were harvested from the flask

by centrifugation. The cells were checked for viability by tryphan blue dye exclusion.

Cells with viability of more than 98%, as determined by tryphan blue exclusion,

were used for the assay. The cell suspension of 1 x105 cells/ ml was prepared

in complete growth medium for determination of cytotoxicity.

To 96 well micro titre plates 100 µl of cell suspension was added followed by

addition of test material, after 24 h incubation. The plates were further incubated for

48 h, after addition of test material, and the cell growth was stopped by fixing with

50% trichloroacetic acid (50% TCA). The plates were then incubated at 4 oC for one

hour and then washed with distilled water to remove TCA, growth medium, low

molecular weight metabolites, serum proteins etc.

Cell growth was measured by staining the plates for 30 min, with 0.4%

sulforhodamine B dye in 1% acetic acid. Unbound dye was removed by rinsing 4 times

with 1% acetic acid. The plates were air dried and again washed with 10 mM

un buffered Tris base to extract the protein bound dye. The optical density (OD) was

recorded at 540 nm, on ELISA reader and percent growth inhibition in the presence of

test material was calculated. Each compound at a given concentration was tested in

triplicates in each experiment, which was repeated two times.


Discussion


54

3.5.2.6 Quantitation of DNA Fragmentation of mitogen induced lymphocytes by

Diphenylamine method (Squier and Cohen, 2001)

Transfer cells (1–5 million per point) to a 1.5 mL micro centrifuge tube labeled

B (for bottom). Pellet cells by centrifugation at 1600 rpm for 10 min. Aspirate and some

fragments may be present within this supernatant. If the supernatant may contain

significant amounts of DNA, one may elect to keep it in a separate tube, labeled S.

It should be treated in a manner similar to the T and B fractions, beginning with TCA

precipitation (12.5% TCA, overnight at 4°C, then centrifugation, as for the T and B

tubes). The constituents of tissue culture medium may interfere with the final OD

reading, a separate blank tube containing medium was also included.

Add 0.5 mL of TTE to pellet and vortex. Let stand at least 10 min to allow for

cell and nuclear lysis. Separate DNA fragments from intact chromatin by micro

centrifugation at 12800 rpm. Carefully remove most of the supernatant to a separate

micro centrifuge tube labeled T (for top). Add 0.5 mL of TE to the pellets in the B

tubes. Add 0.5 mL of 25% TCA to all (B and T) tubes and vortex. Place at 4°C

overnight to precipitate DNA. Pellet precipitated DNA in all tubes by centrifugation at

12800 rpm for 10 min. Aspirate and discard supernatants. To all the tubes (B, S and T)

add 80 µL 5% TCA (prepared by a 1:5 dilution of 25% TCA) and hydrolyze the DNA

by heating tubes to 90°C for 15 min in a heat block. Include a blank tube containing

only 80 µL of 5% TCA. Prepare Diphenylamine reagent.

The DPA protocol is a micro method (Sellins and Cohen, 1987) adapted from

the original protocol of Burton (Burton, 1956). Add 160 µL of DPA reagent to each

tube, including the blank, and vortex. Allow color to develop overnight at room

temperature. Transfer 200µL of the colored solution (ignore any dark particles) to wells

of a 96-well plate and read optical density (OD) at 600 nm, setting the blank to zero

(The OD wavelength used should be within the range of 560 to 620 nm. When

comparing results by fragmentation with morphology, the percent DNA fragmentation

will usually be lower than the percent apoptosis. This probably arises from incomplete

separation of fragments from intact DNA. A small amount of fragmented DNA can

typically be found in the B fraction.


Discussion


55

T

Percentage of DNA fragmentation = x 100

T+B

T stands for Top, B stands for Bottom

3.5.3 Cell lines and culture conditions

Human colo rectal carcinoma cell lines colo 205 and human cervical cancer cell

lines SiHa were obtained from National Centre for Cell Sciences (NCCS) Pune, India.

The cell line Colo 205 was anchorage independent and was grown as suspension

culture. The cell line SiHa was anchorage dependent and was grown as a monolayer.

The cell lines were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal

bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ L streptomycin) in a

humidified atmosphere of 5% CO2 at 37° C and were sub cultured twice a week.

3.5.3.1 In vitro cytotoxicity assay using Sulpho Rhodamine B (SRB)

The cell lines were subjected to SRB assay as mentioned in the section 3.5.2.5

3.5.3.2 Calculation of IC50

Various concentrations (62.5-500µg/ml) of Amorphophallus commutatus tuber

extracts were taken for the study and IC50 value (which shows 50% inhibition) was

calculated using MS Excel.

PHASE IV

3.6. Screening for anti-bacterial activity

3.6.1 Preparation of Sample

The condensed sample obtained from the rotary vaccum evaporator 1mg was

dissolved in 1ml of DMSO (dissolved with sterile distilled water for hot water extract)

and serially diluted to concentration ranging between 15.6 - 500 µg/ml. From the

concentration range 100µl of the extracts is used for the assays. Standard antibiotics was

used as positive control and DMSO was used as control.

3.6.2 Bacterial cultures

A total of six clinical isolates, three gram positive - Methicillin Sensitive

Staphylococcus aureus (MSSA); Methicillin Resistant Staphylococcus aureus (MRSA);


Discussion


56

and Enterococcus faecalis and three gram negative organisms namely; Klebsiella

pneumonia; Pseudomonas aeruginosa; and Escherichia coli were obtained from the

Department of Microbiology, Bharathidasan University, Trichy. All the strains were

multiple drug resistant clinical isolates. The bacterial strains were grown in Muller-

Hinton agar plates at 370C and maintained on nutrient agar slants.

3.6.3 Well-in agar method

Anti-bacterial activity of plant extracts was tested by a modified well-in agar

method using lawn culture technique (Parekh et al., 2005; Vuddhakul et al., 2007).

The inoculum was adjusted to final concentration of 0.8 (1.5 x 108 CFU/ml) on the Mac

Farland’s scale. The inoculum suspension was spread uniformly over the agar plates

using sterile glass rod spreader, to get uniform distribution of bacteria. Subsequently,

using a sterile borer, six wells of 0.5 cm diameter was made equidistant in the

inoculated media. The wells were aseptically filled with 0.1 ml of various

concentrations of five different extracts. Later the plates were placed at room

temperature for an hour to allow diffusion of extract into the agar. Then the plates

were incubated for 24 h at 37 oC. The results were recorded by measuring the diameter

of inhibition zone at the end of 24 h.

3.6.4 Minimum Inhibitory Concentration

Minimum inhibitory concentration values of the extracts were determined by

broth dilution method (Rahman et al., 2000). The five different extracts were serially

diluted in the concentration range of 7.78 µg/ml to 500 µg/ml. The bacterial strains were

grown over night on MHA plates at 37oC before being used. To 1.0 ml of the extract of

different concentration 4.0 ml of nutrient broth was added and each of the tubes was

inoculated with 100 µl (5.9 x 104 CFU) of bacterial suspension. The tubes were

incubated at 37oC in an orbital shaker at 150 rpm for 24 h. After 24 h, bacterial growth

was assayed by measuring absorbance at 625 nm. MIC was defined as the lowest

concentration of the extract that restricted the growth and it was equal to the absorbance

of 0.05 at 625 nm.


Discussion


57

PHASE V

3.7 PHYTOCHEMISTRY

3.7.1 Identification of biologically active compounds using preliminary qualitative

tests

Phytochemical screening was carried out by the methods used by Amarasingham

et al., 1964; Das and Bhattacharjee, 1970; Treas and Evans, 1978 and Harborne, 1984.

The tuber was washed with water, chopped into small fragments and shade dried. The

dried samples were powdered in a Wiley Mill to 60 –mesh size. The powdered samples

were stored in air tight container until further use.

3.7.1.1 Test for alkaloids

Two ml aliquot of the extract was treated with the following reagents to test the

presence of alkaloids.

Reagent Positive result

a) Dragendroff’s reagent orange or orange red precipitate

b) Mayer’s reagent white precipitate or turbidity

Dragendorff’s reagent is prepared by mixing 10.0 ml of Solution A (0.17 g of

Bismuth nitrate is dissolved in 2.0 ml 0f acetic acid and made up to 10.0 ml with

distilled water) and 20.0 ml of Solution B (4.0 g of potassium iodide is dissolved

in 10.0 ml of acetic acid and made up to 20.0 ml with distilled water) and diluted to

100.0 ml with distilled water.

Mayer’s reagent is prepared by mixing 60.0 ml of solution A (1.358 grams of

mercuric chloride is dissolved in 60 ml water), with 10.0 ml of solution B (5 g of

potassium iodide siddolved in 10 ml of water) and the volume was made uo to 100.0 ml

with distilled water.

3.7.1.2 Test for Steroids and Sterols

Salkowski’s test

The extracts were dissolved in 1.0 or 2.0 ml of chloroform and equal volume of

concentrated sulphuric acid was added by the sides of the test tubes. The upper layer

turns red and sulphuric acid layer showed yellow with green fluorescence. This

indicated the presence of steroid and sterol compound in the extract.


Discussion


58

3.7.1.3 Test for triterpenoids

a. Liebermann-Burchard’s test

The extracts were dissolved in 2.0 ml of chloroform followed by 10 drops of

acetic anhydride and 5 drops of concentrated sulphuric acid. Appearance of red to violet

colour indicated the presence of triterpenoids.

3.7.1.4 Test for flavonoids

a. Shinoda test: one ml of the extract was treated with magnesium turnings and

1-2 drops of concentrated hydrochloric acid. Formation of pink or red colour shows

the presence of flavonoids.

b. One ml of the extract was treated with 1.0 ml of ferric chloride. The formation of

brown colour confirms the presence of flavonoids.

3.7.1.5 Test for tannins and phenolic compounds

a. One ml of the extract was treated with few ml of 5% neutral ferric chloride, a dark

blue or bluish black colour product shows the presence of tannins.

b. One ml of the extract was treated with few ml of gelatin solution, a white precipitate

reveals the presence of tannins and phenolic compounds.

c. One ml of the extract was treated with lead tetra acetate solution, and a yellow

precipitate production indicates the presence of tannins and phenolic compounds.

3.7.1.6 Test for Anthraquinones

a. Borntrager’s test - To 5.0 ml of plant extract 10ml of benzene solution was added

and shaken well. The appearance of pink, red or violet colour in the ammoniacal

(lower) phase indicates the presence of free anthraquinones.

b. For combined anthraquinones, 5.0 ml each of plant extracts was boiled with 10.0 ml

of aqueous sulphuric acid and filtered while hot. The filtrate was shaken with 5.0 ml

of benzene, the benzene layer was separated and half its own volume of 10 percent

ammonia phase (lower layer) indicates the presence of anthraquinone derivatives in

the extract.


Discussion


59

3.7.1.7 Test for Cardiac glycosides

Salkowski test: - 0.5 ml of the extract was dissolved in 2.0 ml of chloroform.

Sulphuric acid was carefully added to form a lower layer. A reddish brown colour at the

interface indicates the presence of steroidal ring.

3.7.1.8 Test for Saponins

a. About 1.0 ml of alcoholic extract was diluted separately with 20 ml of distilled

water and shaken in a graduated cylinder for 15 minutes. A one cm layer of foam

indicates the presence of saponins.

b. To 1.0 ml of the extract, alcoholic vanillin solution and a few drops of concentrated

sulphuric acid were added. A deep violet colour confirms the presence of saponins.

3.7.1.9 Test for volatile oil

2.0 ml of extract was evaporated on a porcelain crucible. If the residue has an

aromatic smell, it indicates the presence of volatile oils.

3.7.1.10 Test for fatty acids

The extracts were evaporated on a filter paper. A translucent spot indicates the

presence of fatty acids.

3.7.1.11 Test for coumarins

The extracts were evaporated and dissolved in water. The presence of UV

florescence and the increase in intensity of fluorescence indicates the presence of

coumarins.

3.7.1.12 Test for emodins

The extracts were treated with 25% (w/v) ammonium hydroxide solution. The

appearance of red colour indicates the presence of emodins.

3.7.2 Isolation of active principle in Ethyl acetate fraction

Analysis of the results of antibacterial activity, free radical scavenging activity and

anti proliferative activity has lead to the identification of ethylacetate fraction to hold a

promising activity and hence the extract was choosen for isolation of the active

principle adopting methods described by Khan et al. (2008a) and Khan et al. (2008b).


Discussion


60

3.7.2.1 Column Chromatography

The ethyl acetate extract which exhibited significant activity was subjected to

column chromatography. Methanol soluble portion of ethyl acetate fraction 2.2 g was

activated with silica gel and packed on to a column for isolation of active principle.

The fractions were subjected to Silica gel column using same solvent (ethyl acetate) as

mobile phase and increasing the polarity with methanol. Fractions of 100 ml were

collected with a flow rate of 10 ml / minute. Each of the eluted column chromatography

fractions were subjected to preparative TLC and UV-visible spectrophotometry for

checking the presence of single compound.

3.7.2.2 TLC detection

Silica gel plates were prepared and activated at 120ºC for 1 h before being used.

Ten microliters of each fraction collected from column chromatography was loaded to

the marked points about 10 mm from the bottom of silica plate. The plates were

developed in ethyl acetate: chloroform (50:50, v/v) at room temperature and the

separated spots were visualized by iodine fume (Xia, 2003). Ingredients of each eluted

fraction were compared with ethyl acetate fraction for identification. The Rf values of

each of the fractions were noted.

3.7.2.3 UV-Visible spectrophotometry

The spectrums of each of the fractions obtained from column were recorded on a

Genesys 5 UV-Visible spectrophotometer at room temperature. About 0.5 ml of the

eluted fraction mixed with 1.5 ml of the corresponding mobile phase solvent was used

to record the spectrum (from 200 to 1100 nm) to obtain the absorption maxima (λmax)

of the eluted fractions.

3.7.3 Identification of the active principle

The eluted fractions that possessed similar Rf value and absorption maxima were

pooled and subjected to (Liquid Chromatography Mass Spectroscopy) LC-MS analysis

and NMR spectroscopy (Khan et al.,2008a; Khan et al.,2008b).

3.7.3.1 Liquid chromatography-mass spectrometry (LCMS):

The mass spectrum of the isolated compound was recorded on an Thermo

LCQ Deca XP Max instrument (m/z value range was 1 – 2000) by the electrospray


Discussion


61

ionization (ESI) technique with a flow rate of 0.2 ml/min on a C-18 column and a total

run time of 40 min. Diode array was used as a detector. About 1 mg of isolated

compound dissolved in 5 ml of DMSO was used to record the spectrum.

3.7.3.2 Nuclear magnetic resonance (NMR) spectra:

The 1H and

13C NMR spectra of the pooled fractions were recorded on 400MHz

Bruker advance spectrometer (Rheinstetten, Germany). Regions from 0 to 12 ppm were

employed for 1H and 0 – 200 ppm for

13C.

3.8 Statistical analysis

Experimental results concerning this study were represented as mean ± SD of

three parallel measurements. Analysis of variance was performed by two - way

ANOVA employing Agres stat version 7.01. The p< 0.05 were regarded as significant.


Discussion


62

RREESSUULLTTSS

Drug discovery strategies based on natural products and traditional medicines

are re-emerging as attractive options. Drug discovery and development need not always

be confined to new molecular entities. Rationally designed, carefully standardized,

synergistic traditional herbal formulations and botanical drug products with robust

scientific evidence can also be alternatives. A reverse pharmacology approach, inspired

by traditional medicine can offer a smart strategy for new drug candidates to facilitate

discovery process and also for the development of rational synergistic botanical

formulations (Patwardhan and Mashelkar 2009). Biodiversity of natural resources has

served not only for the primary human needs but also for health care, since time

immemorial. The Indian subcontinent, with the history of one of the oldest civilization,

harbors many traditional health care systems. Their development was supported by the

diverse biodiversity in flora and fauna due to variations in geographical landscaping

(Mukherjee and Wahile, 2006).

The results of the research findings entitled “Bioactivity and Phytochemical

analysis of Amorphophallus commutatus (Schott) Engl. an Endemic aroid of Western

Ghats, South India” is discussed in under the following headings:

4.1 IN VITRO RADICAL SCAVENGING ASSAYS



4.1.1.2 ABTS radical cation scavenging activity



4.1.4 Prevention of Deoxy Ribose degradation


4.1.4.2 DNA Nicking assay

4.1.5 Inhibition of Lipid peroxidation


4


Discussion


63

4.1.7 Total Reducing power activity

4.1.8 Ferrous Iron Chelating activity


4.2.1 Protein content




4.2.2.3 Guaicol peroxidase (GPOD)


4.2.2.5 Activity of Glucose-6-phospate- dehydrogenase (G6PD)

4.2.2.6 Activity of Glutothione peroxidase (GPx)

4.2.2.7 Activity of Glutathione reductase (GR)


4.2.3 Determination of non- enzymatic antioxidant

4.2.3.1 Total phenol


4.2.3.3 Reduced glutathione

4.3 ANTIPROLIFERATIVE STUDIES


4.3.1.1 Cell Viability

4.3.1.2 Comparison of cell quantity in normal and mitogen induced human

peripheral blood lymphocytes

4.3.1.3 In vitro Cytotoxicity by SRB assay

4.3.1.4 Quantitation of DNA Fragmentation by Diphenylamine method

4.3.2 In vitro cytotoxicity of Colo 205 by SRB assay

4.3.3 In vitro cytotoxicity of SiHa by SRB assay

4.4 ANTI BACTERIAL ACTIVITY

4.4.1 Well in Agar Method

4.4.1.1 Gram positive Bacteria

4.4.1.2 Gram negative bacteria

4.4.2 Minimum Inhibitory concentration (MIC)


Discussion


64

4.5 PHYTOCHEMISTRY

4.5.1 Identification of biologically active compounds using preliminary

qualitative tests


4.1 IN VITRO RADICAL SCAVENGING ASSAYS

Antioxidant capacity is widely used as a parameter for identification of

bioactive medicinal components. It is well understood that termination of free radical

chain propagation by antioxidants results in reduction of chronic diseases, DNA

damage, Mutagenesis, carcinogenesis and inhibition of pathogenic bacterial growth

(Zhu et al., 2002). The IC50 values of radical scavenging assays are denoted in table 4.


ABTSo+

or DPPHo

radical scavenging assays are common spectrophotometric

methods used to determine the antioxidant capacity of bioactive medicinal components.

Both the assays are easy, highly sensitive and can be employed for rapid analysis of

large number of samples (Awika et al., 2003). ABTSo+

assay which involve electron

transfer is comparatively sensitive than DPPHo

assay which involves H atom transfer

(Kaviarasan et al., 2007). In the current study these two assays were employed to assess

the radical scavenging activity of five different solvent fractions of A. Commutatus

tuber extracts.


The Figure 21 indicates the free radical scavenging ability of different

solvent fractions of Amorophophallu commutatus and standard BHT. Ethyl acetate

fraction exhibited significant radical scavenging effect of 80.10 ± 2.07 % at 100 µg

concentration. This value was significant (p<0.05) than standard BHT which

showed 76.79 ± 1.10 % at 100 µg concentration. The methanol fraction showed

60.68 ± 1.77 % followed by chloroform 38.46 ± 1.36 % and Petroleum ether

corresponding to 30.59 ± 1.40 %. IC50 values were as shown in Table 4. The IC50 value

of BHT was 53.9 ± 0.6 µg followed by ethyl acetate, methanol, chloroform and

petroleum ether corresponding to 61.32 ± 1.39 µg, 77.01 ± 2.4 µg, 120.80 ± 4.77 µg

and 209.62 ± 14.83 µg respectively.


Discussion


65

Table 4

IC50 values of radical scavenging activity of Amorphophallus commutatus extracts (µg)

Assays Petroleum

ether Chloroform Ethyl acetate Methanol Hot water BHT

DPPH radical

scavenging activity 209.62 ± 14.83 120.80 ± 4.77 61.32 ± 1.39 77.0 ± 2.40 488.8 ± 15.8 53.9 ± 0.6

ABTS radical

scavenging activity 219.1 ± 6.80 83.3 ± 0.90 69.6 ± 1.10 104.8 ± 0.50 124.4 ± 0.8 80.8 ± 1.1

SO radical scavenging

activity 95.5 ± 2.70 115.4 ± 3.40 92.5 ± 1.10 198.1 ± 14.80 204.0 ± 15.8 66.1 ± 1.1

NO radical scavenging

aactivity 142.4 ± 5.20 176.4 ± 4.90 139.6 ± 2.20 158.6 ± 1.90 236.2 ± 10.4 82.4 ± 1.0

OH radical scavenging

activity 98.2 ± 0.90 65.0 ± 2.60 62.4 ± 1.10 111.1 ± 5.40 167.2 ± 11.2 49.6 ± 0.8

Inhibition of LPO 109.4 ± 0.8 126.7 ± 3.10 66.3 ± 0.90 67.6 ± 0.70 88.0 ± 1.5 65.2 ± 1.6

Reducing power 72.0 ± 1.60 62.1 ± 1.20 59.2 ± 0.10 69.9 ± 1.0 64.9 ± 0.8 -

FIC 170.02 ± 6.18 179.9 ± 9.0 132.8 ± 7.0 64.2 ± 1.30 42.3 ± 0.4

73.6 ± 1.8

EDTA

48.2 ± 0.8

65


Discussion


66

Figure 21

DPPH radical scavenging activity of Amorphophallus commutatus extracts

PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;

HW – Hot water BHT - Butylated hydroxyl toluene

4.1.1.4 ABTS radical cation scavenging activity

Bleaching of blue – green ABTSo+

at 734 nm can be monitored by decrease in

absorbance similar to DPPH assay. Among the tested solvent fractions ethyl acetate

fraction exhibited effective cation scavenging activity. As shown in Figure 22 ethyl

acetate fraction was identified as an effective ABTS•+

radical scavenger with % radical

scavenging corresponding to 76.33 ± 1.50 (p≤ 0.05) at 100µg and the corresponding

IC50 value was 69.6 ± 1.1 µg.

The values of BHT the positive standard at 100 µg was 69.53±0.49% (p≤ 0.05)

with an IC50 value (Table 4) of 80.8 ± 1.1 µg. The next better activity was observed in

chloroform extract with an IC50 value of 83.3±0.9 µg. Moderate radical scavenging

activity was produced by methanol and hot water with their IC50 values corresponding

to 104.8 ± 0.5 µg and 124.4 ± 0.8 µg respectively. Comparatively less significant

(p<0.05) activity was observed in petroleum ether extract with the IC50 value of 219.1 ±

6.8 µg. ABTSo+

scavenging activity was in correlation with DPPHo scavenging activity.


Discussion


67

Figure 22

ABTS radical scavenging activity of Amorphophallus commutatus extracts




The superoxide generated by photochemical reduction of riboflavin reduces

NBT (Nitro Blue Tetrazolium). The presence of super oxide radical scavenging capacity

in the extract will lead to reduced reduction of NBT as the superoxide being scavenged

by the extract (Beauchamp and Fridovich, 1971). The reduction of NBT was measured

as an indication of consumption of super oxide anion by the antioxidants (Shukla et al.,

2009) present in the extract.

The results are shown in Figure 23, the percentage scavenging effect of

superoxide anion was significant (p<0.05) in BHT at 100 µg corresponding to

72.51 ±0.89 %. The effect was similar between ethyl acetate and petroleum ether

fractions with their % scavenging at 100 µg corresponding to 62.88 ± 0.99 % and

61.65 ± 0.88 % respectively. The order of scavenging of other fractions at 100 µg

were as follows chloroform 45.39 ± 1.08 %, Hot water 31.20 ± 1.77 % and methanol

26.06 ± 1.88 %. The IC50 values are as shown in the Table 4.


Discussion


68

Figure 23

Super oxide radical scavenging activity of Amorphophallus commutatus extracts




In the present study nitric oxide radical (NO) generated from sodium nitro

prusside at physiological pH

was found to be inhibited by different fractions in a dose

dependent manner as shown in Figure 24. Standard BHT exhibited significant (p<0.05)

nitric oxide scavenging activity corresponding to 51.68 ± 1.13% at 100 µg. The %

scavenging of solvent fractions was in the following order Ethylacetate > Petroleum

ether > Methanol > Chloroform > Hotwater.

The corresponding values were 39.87 ± 0.51%, 34.46 ± 1.27%, 31.56 ± 0.92,

24.56 ± 1.18% and 23.26±1.51% respectively. Their corresponding IC50 (Table 4)

values were 139.6 ± 2.2 µg, 142.4 ± 5.2 µg, 158.6 ± 1.9 µg, 176.4 ± 4.9 µg and

236.2 ± 10.4 µg. Standard BHT exhibited IC50 value of 82.4 ± 1.0 µg which was

nearly 40% greater than ethylacetate fraction which showed significant (p<0.05)

activity among the extracts.


Discussion


69

Figure 24

Nitric oxide radical scavenging activity of Amorphophallus commutatus extracts



4.1.4 Prevention of Deoxy Ribose degradation


The OHo is known to cause DNA damage by degradation of the deoxyribose

moiety. The effect of different solvent fractions on degradation of deoxy ribose by

generation of hydroxyl radicals through fenton’s reaction is shown in Figure 25.

Ethyl acetate fraction exhibited significant (p<0.05) percentage of OH° scavenging next

to standard BHT. Their values at 100 µg were 64.8 ± 1.86 % and 87.42 ± 1.99%

respectively. The order of inhibition of hydroxyl radical by the other four extracts were

as follows chloroform > Petroleum ether > Methanol > Hot water with their values at

100 µg corresponding to 62.46 ± 3.04 %, 56.07 ± 1.77 %, 38.74 ± 4.10 % and 33.43 ±

2.19 % respectively.

The IC50 (Table 4) value of BHT was 49.6 ± 0.8 µg followed by ethyl

acetate 62.4 ± 1.1 µg, chloroform 65.0 ± 2.6 µg, petroleum ether 98.2 ± 0.9 µg,

Methanol 111.1 ± 5.4 µg and Hot water 167.2 ± 11.2 µg.


Discussion


70

Figure 25

Hydroxy radical scavenging activity of Amorphophallus commutatus extracts



4.1.8.2 DNA Nicking assay

The ability of different fractions of Amorphophallus commutatus tuber extracts

to protect supercoiled pUC18 DNA from devastating effect of hydroxyl radicals

generated by Fenton’s reagent are depicted in Plate 1.

The results shown in Plate 1 (pUC18) are in confirmation with the hydroxyl

radical scavenging ability of various fractions analysed by deoxy ribose degradation

method described in the previous section. Fenton’s reactions are known to cause

oxidatively induced breaks in DNA strand to yield its open circular or relaxed forms

(Prakash et al., 2007). The different concentrations of extracts exhibited more or less

protective effect. The observation from the plate-1c illustrates that ethyl acetate extract

exhibited significant presence of supercoiled (Form I) DNA. The petroleum ether

extract had least protection against degradation of deoxyribose. Plate 1.a reveals the

presence of linear (Form II) DNA alone in petroleum ether extract. Followed by ethyl

acetate extract comparatively significant retention of supercoiled (Form I) DNA could

be identified in Chloroform followed by Methanol and Hot water extracts.


Discussion


71

Plate 1

Effect of Different extracts of Amorphophallus commutatus tuber on pUC 18

Lane 1 - pUC18

Lane 2 - pUC18 with Fenton’s reagent

Lane 3, 4, 5 and 6 - pUC18 with Fenton’s reagent+ 500, 250,125 and 62.5µg

concentrations of Amorphophallus commutatus tuber extracts.

Lane 7 - pUC18 with Fenton’s reagent+ Vitamin E as Positive control.

4.1.9 Inhibition of Lipid peroxidation

A modified thio barbituric acid (TBA) reactive species was used to measure the

lipid peroxide formed, using egg-yolk homogenates as lipid rich media (Chang et al.,

2002; Banerjee et al., 2005). Malondialdehyde (MDA), a secondary end product of the

oxidation of polyunsaturated fatty acids, reacts with two molecules of TBA yielding a

pinkish red chromogen with an absorbance maximum at 532 nm.

Lane 1 2 3 4 5 6 7

Form III

Form II

Form I

Lane 1 2 3 4 5 6 7

Form III

Form II

Form I

Lane 1 2 3 4 5 6 7

Lane 1 2 3 4 5 6 7

8

Lane 1 2 3 4 5 6 7

Form III

Form II

Form I

Form III

Form II

Form I

Form III

Form II

Form I

A. Petroleum ether B. Chloroform C. Ethyl acetate

D. Methanol E. Hotwater


Discussion


72

Figure 26

Inhibition of Lipid peroxidation by Amorphophallus commutatus extracts



The inhibition of lipid peroxidation induced by Fenton reaction is shown in

Figure 26. The analysis of the results reveal that hot water extract exhibited significant

(p≤ 0.05) activity of 65.33 ± 1.16 % at 100 µg compared to 73.25 ± 1.25 % by

BHT at the same concentration. Methanol and ethylacetate fractions at 100 µg

concentration exhibited comparitively less significant (p<0.05) activity corresponding to

59.35 ± 1.92 % and 60.68 ± 1.38 respectively. Interestingly even at lowest concentration

they exhibited comparable activity of 49.29 ± 2.6 % and 47.49 ± 2.3 % respectively.

Petroleum ether had 40.08 ± 2.54 % scavenging while chloroform exhibited

33.28 ± 1.54% scavenging at 100 µg concentation. Comparing the IC50 values (Table 4)

of all the fraction revealed that ethyl acetate and methanol are comparable with BHT

with their respective values corresponding to 66.3 ± 0.9 µg ; 67.6 ± 0.7 µg and 65.2 ±

1.8 µg. The IC50 value of hot water, petroleum ether and chloroform were as

follows 88.0 ± 1.5 µg 109.4 ± 1.0 µg and 126.7 ± 3.1 µg.


Discussion


73


The spectrophotometric measurement of Total antioxidant capacity (TAC) is

based on the reduction of Mo (VI) to Mo (V) by antioxidant compound and the

formation of green phosphate / Mo (v) complex at acidic pH (Pietro et al, 1999).

Increase in absorbance indicates increase in total antioxidant capacity. All the fractions

exhibited significant activity and it increased with increase in concentration. The results

are shown in Figure 27 and the values are in comparison with ascorbic acid. At 100 µg

concentration ethyl acetate fraction exhibited significant (p≤ 0.05) activity of 66.2 ±

1.61 % followed by petroleum ether extract corresponding to 47.8 ± 1.7 %. The other

three extracts had no significant (p<0.05) total antioxidant capacity.

Figure 27

Total antioxidant capacity of Amorphophallus commutatus extracts



4.1.11 Total Reducing power activity

The principle of reducing power assay is based on the electron donating ability

(Yildrim et al., 2001). All the extract exhibited high reducing power activity with a dose

dependent effect and the values represent % ascorbic acid equivalent. The result


Discussion


74

(Figure 28) illustrates that at 100 µg ethyl acetate fraction exhibited 87.36 ± 0.94 % with

IC50 of 59.2 ± 0.10 µg. Hot water extract exhibited next higher value of 80.36 ± 0.67 %

with IC50 value of 64.9 ± 0.8 µg. The other three extracts at 100 µg exhibited following

values 72.28 ± 1.08 %. Chloroform 75.31 ± 1.40 %, Methanol 72.28 ± 1.06 % and

petroleum ether 71.37 ± 1.39 %. Their IC50 values (Table 4) were 62.1 ± 1.2 µg,

69.9 ± 1.0 µg and 72.0 ± 1.6 µg respectively. All extracts exhibited significant (p<0.05)

reducing power activity in a dose dependent effect.

Figure 28

Reducing power of Amorphophallus commutatus extracts



4.1.12 Ferrous Iron Chelating activity

The chelating agents present in the plant extract form σ bond with the metal

thereby decreasing the redox potential and stabilizing the oxidized form of metal ion

(Gordon, 1990).

The Ferrous ion chelating activity of the extracts along with standard BHT and

EDTA are shown in Figure 29.


Discussion


75

Figure 29

Ferrous ion chelating activity of Amorphophallus commutatus extracts



Hot water extract at 100 µg exhibited significant (p<0.05) activity of 93.19 ± 1.25

% while compared to 60.50 ± 1.52 % for BHT and 84.19 ± 1.02 for EDTA. At the same

concentration methanol fraction possessed 75.56 ± 0.94 % scavenging while the other

three fractions showed a nominal activity. IC50 value of hot water extract was 42.3 µg

followed by EDTA with 48.2 ± 0.8 µg. The IC50 values of methanol and BHT were

64.2 ± 1.3 and 73.6 ± 1.8 respectively. The IC50 values of ethyl acetate were three times

higher than that of EDTA corresponding to 132.8 ± 7.0 µg. The IC50 values of

Petroleum ether and chloroform were not significant (p<0.05) and were 170.02 ± 6.18

and 179.9 ± 9.0 µg respectively.


Redox reactions that occur naturally are very vital for controlling the metabolic

process occurring in the living system. Free radicals or reactive oxygen species (ROS)

are introduced in to the living system as a product of normal metabolic function or from


Discussion


76

the environment. Plants have evolved a well regulated mechanism for scavenging

ROS, generally through the production of various antioxidative enzymes such as

Superoxide dismutase, Peroxidase, Glutathione peroxidase, Ascorbate oxidase, Glucose

6-Phospate-dehydrogenase and Glutathione reductase. These enzymes are usually

considered to be the most predominant ROS scavenging in plant systems (Bowler et al.,

1992; Foyer et al., 1994; Allen, 1995; Rao et al., 1996; Liu et al., 2009). The result of

this report reveals for the first time the enzymatic and non enzymatic antioxidant

content present in different parts of Amorphophallus commutatus.

4.2.3 Protein content

Proteins are Macromolecules that act as alternate energy source when other

energy sources are in short supply. They are the building block of any organism. The

tuber, young leaves and mature leaves of Amorphophallus commutatus were analyzed

for its protein content and the results obtained are represented in Table 5. The tuber has

been identified to contain significant quantity of protein corresponding to 80±1.31 µg in

one gram tissue followed by young leaf and mature leaf. The enzyme activity is also

expressed in terms of activity per gram of protein.

Table 5

Protein content of different parts of Amorphophallus commutatus

Sample Concentration (µg/g of tissue)

Tuber 80 ± 1.31

Young leaves 60 ± 0.97

Mature leaves 46 ± 1.69



The photo induced reduction of riboflavin generates superoxide radical

which is detected by the formation of nitrite while reacting with hydroxylamine


Discussion


77

hydrochloride (Das et al., 2000). SOD, an important metal containing primary defense

enzyme, catalyzes the dismutation of superoxide radical anions into H2O2 and molecular

oxygen (Scandalios, 2001). SOD is classified in to three types based on the metal

cofactor present – Cu/Zn –SOD, Mn- SOD and Fe-SOD (McKersie et al., 1993).

However in this study the total SOD activity is only determined.

The SOD activity of different parts of the plant is represented in Table 6, which

shows that significant activity is observed in the tuber (47.7 ± 5.50 U/g tissue) followed

by matured leaves (21.1 ± 4.20 U/g tissue) and young leaves (17.3 ± 3.05 U/g tissue).

Since the protein content was identified to be higher in the young leaf, the specific

activity in terms of protein is higher in young leaf compared to mature leaf. The

transition metal present in the enzyme reacts with O2 that is superoxide taking its

electron and superoxide is the only substrate for superoxide dismutase (Oberley and

Oberley, 1997).

Table 6

Super oxide dismutase in different parts of Amorphophallus commutatus

Enzyme activity Sample

U/g of tissuea

U/g of protein

Tuber 47.7 ± 5.50 11.67 ± 1.55

Young leaf 17.3 ± 3.05 5.9 ± 0.70

Mature leaf 21.1 ± 4.20 5.7 ± 1.10

Values are mean ± SD; n=3

a1 unit = activity of enzyme that exhibits 50% inhibition of NBT reduction/minute


Catalase, a heme protein, is considered biologically essential in the reduction of

hydrogen peroxide. CAT appears to be the most effective defense against high

concentration on H2O2 (Atalay and Laksonen, 2002). Catalase has a double function as

it catalyses the, decomposition of hydrogen peroxide to water and oxygen and oxidation

of proton donors.


Discussion


78

Catalase activity, of different parts of Amorphophallus commutatus are

presented in Table 7, which shows that significant activity is observed in young leaf

(64.3 ± 6.02 U/g tissue) followed by mature leaves (57.3 ± 5.03 U/g tissue) and the

tuber (2.7 ± 0.40 U /g tissue). Similar pattern of activity was observed in terms of

protein content also. Hydrogen peroxide is generated by the dismutation of superoxide

radical by the enzyme superoxide dismutase. The H2O2 causes cell membrane damage

leading to release of arachidonic acid, a long acting cell damaging molecule

(Park et al., 2003).

Table 7

Catalase activity in different parts of Amorphophallus commutatus


U/g tissue a

U/g protein

Tuber 2.7 ± 0.40 0.66 ± 0.001

Young leaf 64.3 ± 6.02 19.8 ± 2.02

Mature leaf 57.3 ± 5.03 17.4 ± 2.80

Values are mean±SD; n=3

a1unit = µmoles of H2O2 utilised/ minute

4.2.3.4 Guaiacol peroxidase (GPOD)

Peroxidase (POD) is a group of specific and non specific enzymes from different

sources. POD catalyses the dehydrogenation of a large number of organic compounds

such as phenols, aromatic amines, hydroquinones etc., Guiacol peroxiase catalyzes

oxido reduction between hydrogen peroxide and various reductants (Hiraga et al.,

2001). Guaicol peroxidases are involved in large number of biochemical and

physiological process (Halliwell, 1982).

The activity of GPOD are represented in Table 8, it illustrates that the significant

activity is observed in mature leaves (1.9 ± 0.65 U/g tissue) followed by young leaves

(1.77 ± 0.25 U/g) and tuber (0.38 ± 0.12 U/g tissue). The specific activity was identified

to be significant in the young leaves with 1.2 ± 0.01 U/g protein.


Discussion


79

Table 8

Guaicol Peroxidase in different parts of Amorphophallus commutatus


U/g tissue a

U/g protein

Tuber 0.38 ± 0.12 0.07 ± 0.001

Young leaf 1.77 ± 0.25 1.2 ± 0.01

Mature leaf 1.9 ± 0.65 0.46 ± 0.02


a 1unit= µmoles of guaicol oxidized/minute


Ascorbic acid oxidase is a peroxide enzyme involved in the detoxification of

hydrogen peroxide utilizing two molecules of ascorbic acid and reduces it to water. It is

highly specific for ascorbic acid as electron donor (Noctor and Foyer, 1998).

The activity of ascorbate oxidase is represented in Table 9 which reveals that

significant activity is observed in tuber (0.38 ± 0.12 U/g tissue), followed by young

leaves (0.01 ± 0.004 U/g tissue) and matured leaves (0.005 ± 0.001 U/g tissue ). The

predominant ascorbate activity in tuber is associated with high SOD activity and

catalase, peroxidase and glutathione peroxidase, emphasizing the importance of

ascorbate system in tuber part. The specific avtivity also exhibited similar order of

activity.

Table 9

Ascorbic acid oxidase Activity in Amorphophallus commutatus


U/g tissue a

U/g protein

Tuber 0.38 ± 0.12 0.443 ± 0.016

Young leaf 0.01 ± 0.004 0.01 ± 0.002

Mature leaf 0.005 ± 0.001 0.003 ± 0.001


a I unit (AAO) = equivalent to 0.01change in OD /min


Discussion


80

4.2.2.11 Glucose-6-phospate- dehydrogenase (G6PD)

Glucose 6-phosphate dehydrogenase is an important enzyme for the generation

of NADPH, which is utilised for the regeneration of various antioxidant molecules.

G6PD was assayed by measuring the increase in the absorbance due to NADP being

reduced to NADPH. This reaction takes place when two electrons are transferred from

G-6-P to NADP in the reaction catalyzed by the enzyme Glucose 6-phospate

dehydrogenase (Balinsky and Bernstein, 1963).

Table 10

Glucose -6-phosphate dehydrogenase activity in Amorphophallus commutatus


U/g tissue a

U/g protein

Tuber 0.077 ± 0.002 0.057 ± 0.004

Young leaf 2.5 ± 0.568 0.5 ± 0.03

Mature leaf 9.97 ± 2.0 3.74 ± 0.015


a 1unit=change in OD of 0.01/minute

The activity of G6PD in different parts of Amorphophallus commutatus extracts

is represented in Table 10. It reveals the presence of significant activity in mature

leaves (9.97 ± 2.0 U/g tissue) followed by young leaves (2.5 ± 0.568 U/g tissue) and

tuber (0.077 ± 0.002 U/g tissue) respectively. Similar order of activity profile was

identified in the specific activity. The main function of the enzyme is to maintain GSH

in reduced state (Sultana et al., 1995).

4.2.2.12 Glutothione peroxidase (GPx)

Glutathione peroxidase is a major antioxidant enzyme counteracting the action

of oxidative molecules. It is responsible for scavenging H2O2 by peroxidation of

reduced glutathione (GSH) and forming oxidized glutathione (G-S-S-G) as a product

(Halliwell, 1982).


Discussion


81

The activity of GPx in different parts of plant extracts is represented in

Table 11 which shows the significant activity is observed in mature leaves (3.8±0.33

U/g tissue) followed by young leaves (2.3±0.066 U/g tissue) and tuber (0.46 ± 0.021U/

g tissue). The specific activity values for mature and young leaves were more or less

similar.

Table 11

Glutathione Peroxidase Activity in Amorphophallus commutatus


U/g tissue a

U/g protein

Tuber 0.46 ± 0.021 0.073 ± 0.020

Young leaf 2.3 ± 0.066 0.35 ± 0.06

Mature leaf 3.8 ± 0.33 0.34 ± 0.015


a 1unit = µmoles of GSH utilised/ minute

4.2.2.13 Glutathione reductase (GR)

Glutathione reductase is a flavo protein that regenerates Glutathione (GSH)

which has been oxidized to G-S-S-G by oxidation and thiol transfer reaction (Rana

et al., 2002). Glutothine reductase (GR) catalyses the reduction of oxidized glutathione

(GSSG) to reduced glutathione (GSH) employing NADPH as a substrate (David and

Richard, 1983)

Table 12

Glutathione Reductase Activity in Amorphophallus commutatus


U/g tissue a

U/g protein

Tuber 0.65± 0.025 0.057± 0.004

Young leaf 1.3±0.017 0.3± 0.009

Mature leaf 0.323± 0.075 0.11± 0.045


a 1unit = µmoles of NADPH oxidized / minute


Discussion


82

The activity of GR was assessed and the results obtained are shown in Table 12.

The young leaves (1.3±0.017 U/ g tissue) had a significant activity compared to the

tuber (0.65 ± 0.025 U/ g tissue) and the mature leaves (0.323 ± 0.075 U/g tissue). The

specific activity was significant in young leaves. GR is a ubiquitous NADPH dependent

enzyme and may be a rate limiting enzyme for defense against active oxygen toxicity

(Gossett et al., 1996).


Polyphenol oxidases are copper containing proteins, which catalyses the aerobic

oxidation of certain phenolic substrates to quinines, which are auto oxidized to dark

brown pigments known as melanins (Esterbauer et al., 1977). The most abundant in

terms of occurance amongst the polyphenol oxidases (PPOs), is the enzyme catechol

oxidase, while other enzymes falling under this class include tyrosinase, laccase and

oxygen reductase.

The activity of PPO was assessed and the results obtained are shown in

Table 13. The result illustrates that tuber possess significant activity (0.8 ± 0.014 U/g

tissue) followed by young leaves (0.453 ± 0.161 U/g tissue) and mature leaves (0.23 ±

0.014 U/g tissue). The specific activity was significant in the tuber among the parts

analysed.

Table 13

Poly Phenol Oxidase Activity in Amorphophallus commutatus


U/g tissue a

U/g protein

Tuber 0.8 ± 0.014 0.057 ± 0.004

Young leaf 0.453 ± 0.161 0.12 ± 0.063

Mature leaf 0.23 ± 0.014 0.15 ± 0.02


a1 unit = Activity of catechol oxidase/ laccase that transforms 1 unit of

dihydrophenol to quinine/minute


Discussion


83

4.2.4 Determination of non- enzymatic antioxidant

The antioxidants belonging to second line of defense include glutathione,

ascorbic acid and Phenols. The commonly known non enzymatic antioxidants

are glutathione and ascorbic acid which are essential for redox buffering

(Foyer et al., 2001). The concentration of different non- enzymatic antioxidants in

different parts of Amorphophallus commutatus was also assessed and the results are

represented in Table 14.

Table 14

Non - enzymatic antioxidant content of Amorphophallus commutatus

Non - enzymatic antioxidant (mg / g tissue) Parts of plant

Reduced glutathione Vitamin C Total phenol

Tuber 2.46 ± 0.75 2.6 ± 0.5 0.2 ± 0.007

Young leaves 6.21 ± 0.6 1.9 ± 0.9 0.02 ± 0.003

Matured leaves 3.83 ± 0.70 1.3 ± 0.7 0.019 ± 0.002


4.2.3.1 Total phenol

Phenolic compounds posses a wide spectrum of biological effects as antioxidant

and free radical scavenger (Pellati et al., 2004). They are classified into two groups such

as polyphenols and simple phenols (Marinova et al., 2005) and commonly found in both

edible and non edible plants. The phenolics act as reducing agents, hydrogen donors and

singlet oxygen quenchers, and also possesses a metal chelting potential (Oboh

and Rocha, 2007). Phenols are the aromatic compounds with hydroxyl group and it

forms an array of compounds like tannins, flavonols, etc.

The level of total phenol content in different parts of Amorphophallus

commutatus are as follows tuber possessed significant activity of 0.2 ± 0.007 mg/g

tissue while the young leaves contained 0.02 ± 0.003 mg/g tissue and matured leaves

0.019 ± 0.002 mg/g tissue.


Discussion


84


Ascorbic acid or Vitamin C is a natural water soluble antioxidant defense that

protects cells against lipid peroxidation (Maneesh et al., 2005). The vitamin C content

in different parts of Amorphophallus commutatus, exhibited that significant quantity is

identified in young leaves (1.9 ± 0.9 mg/g tissue) followed by tuber (2.6 ± 0.5 mg/g

tissue) and matured leaves (1.3 ± 0.7 mg/g tissue) respectively.

4.2.3.3 Reduced glutathione

Glutathione is a sulphur containing tripeptide and plays a predominant role in

defense against free radicals. Glutathione takes part in the control of H2O2 level and has

an important function in maintaining the cellular redox status. It is an important

antioxidant that is found to detoxify toxic substances by conjugation. In plants,

glutathione act as radical scavenger, membrane stabilizer and precursor of heavy metal

binding peptides (Peklak-scott et al., 2005)

The reduced glutathione content was observed to be significant in young leaves

(6.21 ± 0.6 mg/g tissue) followed by matured leaves (3.83 ± 0.70 mg/g tissue) and tuber

(2.46 ± 0.75 mg/g tissue) respectively.

4.3 ANTIPROLIFERATIVE STUDIES

Plants are exploited for new and novel chemotherapeutics (Reed and Pellechia

2005). The continuing search for new anticancer compounds in plant medicines and

traditional foods is a realistic and promising strategy for its prevention (Wei et al.,

2009). Numerous groups with antitumor properties are plant derived natural products

including alkaloids, phenyl propanoids and terpenoids (Park et al., 2008). The present

study was conducted to evaluate the antiproliferative activity of fractionated extracts

from Amorphophallus commutatus tuber.


4.3.1.1 Cell Viability

The quantity of lymphocytes obtained by density gradient centrifugation was

equivalent to 1.15 x 106 cells per ml. The viability of the isolated lymphocytes was

analysed by tryphan blue dye exclusion method and the cell viability was highly


Discussion


85

significant ranging to 98.2%. The percentage of viable cells was good and therefore

used as such for further analysis.

4.3.1.2 Comparison of cell quantity in normal and mitogen induced human


The cells were cultured overnight in two different plates one with Concanavalin

A (mitogen induced) and another one without Concanavalin A (normal). It shows that

there is 70.86% increase in the cell number in mitogen induced culture. The results

of Normal and mitogen induced cells in the culture are shown in Plate 2 and Plate 3

respectively. The number of cells obtained after overnight culture are depicted in

Table 15.

Plate 2

Normal healthy humanperipheral blood lymphocytes

Plate 3

The mitogen induced human peripheral blood lymphocytes


Discussion


86

Table 15

Comparison of the viability of normal lymphocytes and

mitogen induced lymphocytes

S.No. Lymphocyte culture cells/ml

1 Normal lymphocytes 5.4 x 105

2 Mitogen induced lymphocytes 7.62 x 105

4.3.1.3 In vitro Cytotoxicity of mitogen induced lymphocytes by SRB assay

The percentage control of growth (cytotoxicity) offered by the extract was

found to be significant in petroleum ether extract at 500µg concentration with the

percentage cytotoxicity corresponding to 80.54 ± 0.704, followed by methanol extract

61.62 ± 1.147 and hot water 45.6 ± 0.754 at the same concentration. Less significant or

negligible activity was observed in chloroform extract, followed by ethyl acetate

fraction. The results of percentage cytotoxicity are depicted in Figure 30.

Figure 30

Cytotoxic effect of different extracts on mitogen induced human



HW – Hot water


Discussion


87

The values were used iteratively to calculate the concentration of plant extracts

required to cause a 50% reduction (IC50) in growth (cell number). The Table 16 shows

the IC50 value of different extracts. The IC50 value indicates that petroleum ether extract

has significant cytotoxicity 0.257±0.003mg/ml. Quercetin, the positive control exhibited

significant cytotoxicity (Table 17) and almost destroyed 96.80 ± 0.58% of cells at 80µg

concentration.. The IC50 value of quercetin was 1.811±0.901µg/ml. The result shows

that the extracts have less significant activity compared to quercetin.

Table 16

IC50 value of cytotoxicity of different extracts on mitogen induced

human peripheral blood lymphocytes

S.No. Extracts IC50 (mg/ml)

1 Petroleum Ether 0.257±0.003

2 Chloroform 1.536±0.121

3 Ethyl Acetate 3.782±0.420

4 Methanol 0.366±0.007

5 Hot water 0.431±0.007

Values are mean ± SD (n=3)

Table 17

Cytotoxicity exhibited by Quercetin positive control on

mitogen induced lymphocytes.

Concentration (µg/ml) Percentage of cytotoxicity

20 52.55±0.71

40 63.80±0.68

60 78.17±0.62

80 96.80±0.58

IC50 (µg/ml) 1.811±0.901


4.3.1.4 Quantitation of DNA Fragmentation by Diphenylamine method

DNA fragmentation of mitogen induced human lymphocytes for development of

apoptotic induction by the different fractions of Amorphophallus commutatus tuber is

shown in Table 18.


Discussion


88

Table 18

Quantitation of DNA Fragmentation by Diphenylamine method

Concentration

(µg/ml)

Petroleum

ether Chloroform Ethyl acetate Methanol Hot water Quercetin

500 66.68 ± 0.80 53.3 ± 0.65 41.11 ± 1.01 62.42 ± 0.53 55.56 ± 0.62 78.80 ± 0.66

250 56.86 ± 0.82 52.28 ± 0.58 37.35 ± 0.63 54.25 ± 0.63 49.22 ± 0.94 50.84 ± 0.70

125 53.39 ± 0.70 51.64 ± 0.61 31.70 ± 0.67 43.22 ± 0.81 38.38 ± 0.58 49.71 ± 0.64

62.5 52.93 ± 0.72 47.88 ± 0.61 23.28 ± 0.54 39.54 ± 0.92 31.22 ± 0.82 31.49 ± 0.60


88


Discussion


89

Analysis of the results of percentage DNA fragmentation obtained by treating

the mitogen induced lymphocytes with the extracts reveals that all the extracts showed

increased percentage of DNA fragmentation as the concentration increases. The positive

control Quercetin showed significant fragmentation percentage of 78.8 ± 0.66. Among

the extracts Petroleum ether exhibited significant activity followed by Methanol,

Hot water and Chloroform. The percentage of DNA fragmentation of Ethyl acetate

fraction was less than 50%. The results are in coordination with the SRB assay.

4.3.2 In vitro cytotoxicity of Colo 205 by SRB assay

The growth of adenocarcinoma cell line colo 205 (Plate 4 & 5) was controlled

by different fractions of Amorphophallus commutatus tuber extracts. The Petroleum

ether extract (Figure 31) exhibited least significant activity among the extracts. It

inhibited 33.33 ± 0.72% growth at 500 µg. The chloroform extract showed activity

better than petroleum ether but less significant among the extracts. At 500 µg it

exhibited 32.57 ± 1.15% control of growth.

Figure 31

Cytotoxic effect of different extracts on Colo 205 cell line


HW – Hot water


Discussion


90

Plate 4 Plate 5

Colo 205 cell line (10x 1) Colo 205 cell line (20x 1)

The Methanol fraction with significant activity among the extracts was found

to inhibit 94.7 ± 0.90% of growth at 500 µg concentration. The ethylacetate fraction

was observed to have significant control of growth at 500 µg corresponding to

89.63 ± 0.81%. The Hot water fraction showed moderate inhibition of growth with its

activity corresponding to 60.70 ± 0.75% at 500 µg.

Table 19

IC50 value of cytotoxicity of different extracts on Colo 205


1 Petroleum Ether 0.79 ± 0.014

2 Chloroform 0.69 ± 0.02

3 Ethyl Acetate 0.23 ± 0.01

4 Methanol 0.23 ± 0.01

5 Hot water 0.21 ± 0.02



Discussion


91

The IC50 values are as shown in Table 19. It clearly indicates that methanol

followed by ethyl acetate fraction contains significant activity. The Hot water extract

exhibited moderate significance. The other two petroleum ether and chloroform extracts

had no significant activity comparitively.

4.3.3 In vitro cytotoxicity of SiHa by SRB assay

The percentage control of growth exhibited by the Amorphophallus commutatus

extract against human gynecological cell line SiHa (Plate 6 & 7) are as shown below.

The % control of growth exhibited by petroleum ether extract (Figure 32) was not

significant. The next polar solvent chloroform also exhibited least activity and therefore

not significant.

Figure 32

Cytotoxic effect of different extracts on SiHa cell line


HW – Hot water

Ethylacetate fraction was observed to possess significant activity and

inhibited 57.74 ± 1.06% growth at 500 µg. The next significant activity was observed

in the methanol fraction with % of growth inhibition at 500µg corresponding to

41.23 ± 0.67%, the activity exhibited by hot water fraction was not significant.


Discussion


92

Plate 6 Plate 7

SiHa cell line (10x 1) SiHa cell line (20x 1)

Table 20

IC50 value of cytotoxicity of different extracts on SiHa


1 Petroleum Ether 2.05 ± 0.48

2 Chloroform 2.21 ± 0.19

3 Ethyl Acetate 0.39 ± 0.04

4 Methanol 0.52 ± 0.05

5 Hot water 2.43 ± 0.18


The IC50 values as shown in Table 20 puts forth the significant cytotoxic property

of ethyl acetate fraction with an IC50 value of 0.39 ± 0.04 mg/ml. The next considerable

activity was identified in methanol fraction and the other three fractions exhibited less

significant activity with IC50 value ranging between 2.05 to 2.43 mg.


Discussion


93

4.4 ANTI BACTERIAL ACTIVITY

The development of resistance among the bacterial strains has increased the need

for new antibiotics. Bioassay guided fractionation of plant species may lead to the

discovery of new antibacterial agents. Staphylococcus aureus, Escherichia coli and

Enterococcus faecalis are the three important clinically significant strains that

are developing resistance (Langfield et al., 2004). The five different extracts of

Amorphophallus commutatus were screened for presence of antibacterial activity

against six different strains of bacteria – methicillin resistant and sensitive

Staphylococcus aureus a gram positive coci, Enterococcus faecalis a gram positive

spherical bacteris and gram negative rods – Escherichia coli, Pseudomonas aeruginosa

and Klebsiella pneumoniae. The control DMSO and the antibiotics did not produce any

zone of inhibition against the multiple drug resistant strains used in this study.

Therefore, the results are not discussed.

4.4.1 Well in Agar Method

4.4.1.1 Gram positive Bacteria

The methicillin sensitive Staphylococcus aureus (MSSA) was inhibited by all

the extracts in a dose dependent manner (Table 21). The ethyl acetate fraction showed

significant activity among the extracts (Plate 8). It was found to lyse the organism

completely and the zone of inhibition was found to be more than 30mm in the observed

concentration range. Petroleum ether extract was found to have a minimum zone of

23 ± 1.15 mm at 15.6 µg and maximum zone of inhibition was identified at 500 µg

concentration corresponding to 31 ± 0.58 mm. The next significant activity was

exhibited by chloroform and hot water extract with zone of inhibition ranging between

16 ± 1.15mm at lower concentration and 28 ± 1.73 mm at the higher concentration.

Methanol exhibited least significant activity with 9 ± 0.58 mm at 15.6 µg and 21 ± 0.58

at 500 µg.

Methicillin resistant Staphylococcus aureus (MRSA) was found to be inhibited

significantly by ethylacetate extract similar to MSSA. The maximum zone of inhibition

was observed (Table 22 and Plate 9) in ethylacetate extract at 500 µg corresponding

to 28 ± 1.15 mm.


Discussion


94

Table 21

Antibacterial activity of Methycillin sensitive Staphylococcus aureus (MSSA)

Zone of inhibition (mm) Concentration

(µg) Petroleum

ether Chloroform

Ethyl

acetate Methanol Hotwater

15.6 23±1.15 16±1.15 >30 9±0.58 17±1.53

31.3 25±1.00 19±1.53 >30 12±1.53 19±1.53

62.5 26±1.15 21±1.00 >30 16±2.08 22±1.73

125 28±0.58 23±0.58 >30 17±1.53 23±1.53

250 29±1.00 25±1.00 >30 20±0.58 25±2.08

500 31±0.58 28±1.73 >30 21±0.58 27±0.58

Values are mean ± SD of three samples

Table 22

Antibacterial activity of Methycillin resistant Staphylococcus aureus (MRSA)

Zone of inhibition (mm) Concentration

(µg) Petroleum

ether Chloroform

Ethyl


15.6 11±1.13 12±1.00 21±2.08 12±0.58 13±1.15

31.3 13±1.00 13±0.00 22±0.58 13±1.00 15±1.53

62.5 14±0.58 14±0.58 24±1.15 14±1.00 16±1.00

125 15±1.00 15±1.00 25±1.73 16±1.15 18±1.53

250 18±1.15 17±1.53 26±2.08 18±1.15 18±1.53

500 21±1.73 20±2.08 28±1.15 19±1.53 20±1.53



Discussion


95

Plate 8

Antibacterial activity of Methycillin sensitive Staphylococcus aureus (MSSA)

Petroleum ether Chloroform

Ethyl acetate Methanol

Hot water

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µg

31.3 31.3 31.3 31.3 µµµµg 62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg 500 500 500 500 µµµµg


Discussion


96

PLATE 9

Antibacterial activity of Methycillin resistant Staphylococcus aureus (MRSA)



Hot water

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg 125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg 15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg 125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg 15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg


Discussion


97

The next significant activity at the same concentration was identified in all the

other extracts with minor variation in the zone of inhibition. Petroleum ether with

21 ± 1.73 mm, chloroform with 20 ± 2.08mm, hot water with 20 ± 1.53 mm and

methanol with 19±1.53 mm. Similar pattern of inhibition zone was found at the lower

concentration range also with ethylacetate extract exhibiting maximum inhibition zone

of 21 ± 2.08 mm and the other four extracts possessed inhibition zone in the range

of 11 ± 1.13 to 13 ± 1.15 mm.

The zone of inhibition of the extracts against Enterococcus faecalis is elicited in

Table 23. The results reveal that only ethyl acetate fraction exhibited activity and other

extracts did not inhibit the growth of the organisms (Plate 10). The ethyl acetate extract

showed a maximum zone of inhibition corresponding to 25 ± 1.15 mm at 500 µg, while

minimum zone of 17 ± 1.53 mm was observed at 15.6 µg.

Table 23

Antibacterial activity of Enterococcus faecalis

Zone of inhibition (mm) Concentration (µg)

Ethyl acetate

15.6 17±1.53

31.3 18±0.58

62.5 19±1.00

125 20±0.00

250 23±0.58

500 25±1.15



Discussion


98

PLATE 10

Antibacterial activity of Enterococcus faecalis

Ethyl acetate

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg


Discussion


99

4.4.1.2 Gram negative bacteria

Klebsiella pneumoniae was not inhibited by methanol and hot water extract. The

results are depicted in Table 24 and Plate 11. The zone of inhibition at 500 µg was

identified to be 22 ± 1.73 mm. At the same concentration petroleum ether extract

showed 16±1.53 mm and chloroform extract exhibited 13 ± 1.53 mm. The petroleum

ether extract though exhibited next significant activity to ethyl acetate extract but at

lower concentration (15.6 µg) it did not possess any zone of inhibition while the

chloroform extract possessed a zone of 6 ± 1.73 mm.

Table 25 brings forth the results of antibacterial activity of the extracts against

Pseudomonas aeruginosa. To our surprise only ethyl acetate fraction inhibited the

organisms growth by zone formation and the other extracts did not show any activity.

Ethyl acetate fraction possessed a maximum activity of 20 ± 1.15 mm at 500µg

concentration (Plate 12).

Escherichia coli was inhibited by all the extracts in a dose dependent manner

(Table 26; plate 13). The zone of inhibition exhibited by ethyl acetate fraction was

identified to be > 30 mm at all concentration range and the organisms were lysed

completely similar to MSSA.

Table 24

Antibacterial activity of Klebsiella pneumoniae

Zone of inhibition (mm)

Concentration

(µg)

Petroleum

ether Chloroform Ethyl acetate

15.6 0 6±1.73 14±1.53

31.3 9±0.58 8±0.58 18±1.00

62.5 11±1.00 9±0.58 19±0.00

125 12±1.15 11±0.58 21±0.58

250 14±1.00 12±0.58 21±1.15

500 16±1.53 13±1.53 22±1.73



Discussion


100

PLATE 11

Antibacterial activity of Klebsiella pneumoniae

Petroleum ether

Chloroform

Ethyl acetate

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg


Discussion


101

Table 25

Antibacterial activity of Pseudomonas aeruginosa


Concentration (µg) Ethyl acetate

15.6 14±1.00

31.3 16±1.53

62.5 17±1.00

125 18±1.15

250 18±1.53

500 20±1.15


Table 26

Antibacterial activity of Escherichia coli


Concentration

(µg)

Petroleum

ether Chloroform

Ethyl

acetate Methanol Hot water

15.6 15±1.00 16±1.53 >30 14±1.00 17±1.15

31.3 18±0.58 18±0.58 >30 16±0.58 20 ±1.00

62.5 21±1.53 19±1.53 >30 18±1.00 21 ± 1.00

125 23±1.15 21±1.15 >30 19±1.15 22

250 24±1.00 22±1.73 >30 21±1.53 23 ±0.58

500 27±1.53 23±1.53 >30 22±1.53 26 ±1.15



Discussion


102

PLATE 12

Antibacterial activity of Pseudomonas aeruginosa

Ethyl acetate

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg


Discussion


103

PLATE 13

Antibacterial activity of Escheichia coli



Hot water

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg 15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg 125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg 125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg

15.6 µµµµg

31.3 31.3 31.3 31.3 µµµµg

62.5 62.5 62.5 62.5 µµµµg

125 125 125 125 µµµµg

250 250 250 250 µµµµg

500 500 500 500 µµµµg


Discussion


104

The next significant activity in E. coli (Table 26) was observed in petroleum

ether and hot water extract with values at 500 µg corresponding to 27 ± 1.53 mm and

26 ± 1.15 mm respectively. Chloroform and methanol extracts possessed activity

corresponding to 23 ± 1.53 mm and 22 ± 1.53 mm respectively at 500 µg concentration.

Similar pattern of inhibition was identified even at the lower concentration range and

the values ranged between 14 ± 1.00 mm to 17 ± 1.15 mm.

4.4.2 Minimum Inhibitory concentration (MIC)

The results of minimum inhibitory concentration of the organisms tested are

shown in Table 27. Among the extracts ethylacetate was identified to have a significant

MIC value corresponding to 15.6µg/ml against all tested organisms. Petroleum ether

and ethyl acetate fraction had a significant MIC value of 15.6 µg/ml against MSSA

followed by chloroform with MIC value of 125µg/ml, while methanol and hot water

had an MIC value of 250 µg/ml. The results of MIC value of extracts against

MRSA reveals that followed by ethyl acetate fraction petroleum ether and chloroform

had 31.25 µg/ml while methanol and hot water had an MIC value of 250 µg/ml. The

extracts exhibited an MIC value of > 500 µg /ml against Enterococcus faecalis which

was not significant and only ethylacetate had a significant MIC value of 15.6 µg/ml.

Table 27

Minimum Inhibitory concentration (MIC) of the organisms

MIC VALUES (µg/ml)

Organisms Petroleum

ether Chloroform

Ethyl


MSSA 15.6 125 15.6 250 250

MRSA 31.25 31.25 15.6 250 250

Klebsiella 15.6 62.5 15.6 >500 >500

Pseudomonas >500 >500 15.6 >500 >500

Enterococcus >500 >500 15.6 >500 >500

E.coli 62.5 62.5 15.6 250 250


Discussion


105

The petroleum ether and ethyl acetate fraction possessed an MIC value

of 15.6 µg/ml followed by chloroform extract corresponding to MIC value of

62.5 µg/ml against Klebsiella pneumoniae. The other two extracts showed negligible

values. In the case of Pseudomonas aeruginosa only ethyl acetate fraction behold an

MIC values and the other four extracts possessed an MIC value > 500 µg/ml. The MIC

values against E.coli revealed that followed by ethylacetate fraction petroleum ether and

chloroform extract exhibited 62.5 µg/ml and methanol and hot water extract possessed

250 µg/ml.

4.2 Phytochemistry

4.5.1 Identification of biologically active compounds using preliminary qualitative

tests

All the five fractions of Amorphophallus commutatus tuber were analysed

qualitatively for the existence of various phytoconstituents. The results are depicted in

Table 28. The results reveal the presence of cardiac glycosides in all the extracts,

followed by saponins which are not identified in petroleum ether extract alone.

Table 28

Qualitative tests for phytochemical constituent of the extracts

Extracts

Alk

alo

id

Ste

roid

s

& s

terols

Trit

erp

en

oid

s

Fla

va

noid

Ta

nn

ins

&

Ph

en

ols

Ca

rd

iac

Gly

co

sid

es

Sa

po

nin

s

Fa

tty

acid

s

Petroleum ether + - + - - + - -

Chloroform + - - + + + + -

Ethyl acetate - - - - - + + -

Methanol + + - + - + + +

Hotwater + - + - - + + +

+ - Presence of phyto constituents – - Absence


Discussion


106

Apart from the above mentioned phyto constituents petroleum ether fraction

possessed alkaloid and triterpenoid. Chloroform fraction contained alkaloid, flavanoid,

tannins and phenols. Methanol extract was identified to possess maximum constituents

namely alkaloid, steroids, flavonoid, and fatty acids. Next to methanol extract

significant constituents like alkaloids, triterpenoids, and fatty acids were identified in

hot water extract. Ethyl acetate fraction which has kindled the interest by its bioactivity

possessed positive results for saponins and cardiac glycosides.


The column chromatography of ethyl acetate fraction was done with silica gel

(100 -200 mesh). The mobile phase was 100% ethyl acetate and the polarity was

increased with methanol. The fractions were checked for Rf values in a preparative TLC

and using a UV-visible spectrophotometer. When eluted with mobile phase of 5%

methanol in ethyl acetate to 14% methanol in ethyl acetate the chromatography fractions

exhibited Rf value of 0.68 in TLC and absorption maxima (λmax) of 263 nm (Figure 33).

These fractions were pooled and labeled as compound – x.

Figure 33

UV visible spectrum of ethylacetate fraction


Discussion


107

The compound x was subjected to LC-MS analysis and the results are shown

in Figure 34.

Figure 34

LC-MS analysis of ethyl acetate fraction collected from column chromatography

The LC results discloses that the compound x hold a retention time

14.61 minute. The m/z ratio of the molecules present in the compound x is also depicted

in the Figure 34. The LC-MS spectrum suggests the following groups as shown in

Figure 35 to be present in the compound.


Discussion


108

Figure 35

Suggested groups to be present according to LC-MS spectra

+

m/z 99

+.

m/z 128

OH

m/z 128

+.

O

m/z 240

The results of NMR spectroscopy are exhibited as follows 1H –NMR spectrum

(Figure 36 - 39) and 13

C-NMR spectrum (Figure 40 - 43). In its 1H –NMR spectrum it

exhibits signals at δ 0.82 indicating the presence of methyl groups, a strong singlet at

δ 1.29 and bunch of signals around δ 2.00 indicates the presence of long chain of

methylene groups. The two strong signals at δ 2.00 are due to methyl groups attached to

carbonyl groups. The group of multiplet signals at δ 3.7, 4.01, 4.8 and 5.3 suggests the

presence of protons under oxygen function and probably a glycosidic function.

In its 13

C-NMR spectrum it exhibits a signal at δ 14.00 is attributed to a methyl

group, 21.00, 22,05, 24.45 and a bunch of signals centred at δ 29.00 are due to long

chain methylene carbons. The signals at δ 72.45, 69.22, 65.67, 63.06 (for 2 carbon

atoms), 62.59, 61.00 and 59.71 are due to carbon atoms under oxygen function. The

anomeric carbon atom appears as a small signal at δ 101.00. The signal at δ 170.38 and

171.95 indicates the presence of two carbonyl functional groups.


Discussion


109

Figure 36

1H –NMR spectrum of ethyl acetate fraction collected from column chromatography

109


Discussion


110

Figure 37


110


Discussion


111

Figure 38


111


Discussion


112

Figure 39


112


Discussion


113

Figure 40

13C - NMR spectrum of ethyl acetate fraction collected from column chromatography

113


Discussion


114

Figure 41


114


Discussion


115

Figure 42

13

C - NMR spectrum of ethyl acetate fraction collected from column chromatography

115


Discussion


116

Figure 43


116


Discussion


117

Figure 44

Partial structure elucidated based on NMR & LCMS spectra of compound - x

O

O

OHHO

O

OO

OO

m/z 504

Based on the above data arrived from LC-MS and NMR spectrum the partial

structure of the compound may be formulated as shown in Figure 44. But however,

many things are to be confirmed by further spectral data.


Discussion


118

DDIISSCCUUSSSSIIOONN

The development of pharmaceuticals begins with identification of active

principles, detailed biological assays and dosage formulations, followed by clinical

studies to establish safety, efficacy and pharmacokinetic profile of new drug (Iwu et al.,

1999). Plants will continue to serve for the exploration of new chemical entities and

also as raw material for semi-synthetic chemical compounds used in health care systems

(Mukherjee and Whaile, 2006). This chapter discusses the identified biological activities

and phytochemistry of Amorphophallus commutatus.

5.1 In vitro Radical Scavenging Activity

ABTSo+

or DPPHo

radical scavenging assays are common spectrophotometric

methods used to determine the antioxidant capacity of bioactive medicinal components.

Both the assays are easy, highly sensitive and can be employed for rapid analysis of

large number of samples (Awika et al., 2003). ABTSo+

assay which involve electron

transfer is comparatively sensitive than DPPHo

assay which involves H atom transfer

(Kaviarasan et al., 2007). In the current study these two assays were employed to assess

the radical scavenging activity of five different solvent fractions of A.Commutatus tuber

extracts.

DPPH is a proton free radical and possesses a characteristic absorption, which

decreases significantly on exposure to proton radical scavengers (Yamaguchi et al.,

1998). The decrease in absorbance of DPPHo at 517nm was due to the scavenging

activity of antioxidant present in the extract through donation of hydrogen atom to form

a stable diamagnetic molecule (Mattaus, 2002).

The results of DPPH radical scavenging activity indicate that ethyl acetate

fraction at 100 µg concentration exhibited significant (p<0.05) radical scavenging

ability when compared to standard BHT. But BHT even at lower concentration has

shown 59.35 ± 2.89% that was almost 46% higher than the ethyl acetate fraction and

that is the reason that the IC50 value of BHT is better than ethyl acetate fraction.

5


Discussion


119

Singh and Rajini (2004) has reported the DPPHo scavenging activity of aqueous

extract of potato peel which exhibited 90% scavenging at 5mg concentration. Kaur

et al. (2008) revealed the DPPH radical scavenging ability of different solvent fractions

of Chukrasia tabularis and similar to the current report the ethyl acetate fraction

exhibited maximum scavenging activity among the fractions corresponding to

93.14% at 100 µg concentration. The methanolic extract of the tuber of Amorphophallus

companulatus the species phylogenetically closer to A.Commutatus was examined for

the DPPH radical scavenging activity and was reported to have an IC50 value of

3.39 mg/ml (Ramesh et al., 2011).

ABTS also forms a relatively stable free radical, which decolorizes in its

non-radical form (Shirwaikar et al., 2006). ABTSo+

, a nitrogen centered cation

radical generated by oxidation of ABTS in the presence of potassium per sulphate

prior to reaction with putative antioxidants (MacDonald-Wicks et al., 2006). Debnath et

al., 2011 characterised ethanolic and aqueous extracts of Gardenia Jasminoides

for their ABTSo+

scavenging activity and reported the IC50 value to be 0.21 mg and

0.39 mg respectively. Also Prabakar et al. (2006) has fractionated the whole plant

of Coronopus didymus on the basis of polarity to evaluate the radical scavenging ability

and reported the presence of ABTSo+

scavenging activity in the non polar fractions.

Super oxide anion is oxygen centered weak oxidant play a vital role in the

formation of other reactive oxygen species like singlet oxygen and hydroxyl radicals

leading to lipid peroxidation (Halliwell and Gutteridge, 2000). SOo-

is known to involve

in the accumulation of ROS/ RNS in cells leading to redox imbalance and associated

physiological consequences (Pervaiz and Clement, 2007). It also reduces iron

complexes like Cytochrome c (Halliwell and Gutteridge, 1984) and can directly induce

lipid peroxidation (Yen and Duh, 1994). The potato peel extract was identified to

scavenge SOo effectively in a dose dependent manner (Singh and Rajini, 2004).

Nitric oxide radical (NO) act as a chemical mediator and is involved in the

regulation of physiological activities. It is found to be generated by endothelial cells,

macrophages and neurons (Fostermann, 2010). Despite of its biological effects excess


Discussion


120

NO can react with oxygen and superoxide anion to produce peroxynitrite anion and

nitrite a potential oxidants which in turn can lead to the production of free radicals like

OHo

and NO2 (Pacher et al., 2007). The ethyl acetate extract of Stachytarpheta

jamaicensis has also been shown to possess antioxidant effects, inhibiting super oxide

radical and NO radical in peritoneal macrophages (Alvarez et al., 2004). Nitric oxide

radicals play a vital role in vascularisation and metastasis of tumor (Jayakumar and

Gandhimathi, 2011) and extracts with higher NO scavenging activity may be utilised in

reducing the pathogenesis caused by cancer.

Hydroxy radical is an extremely reactive free radical produced by the biological

system (Yen and Duh, 1994). It quickly initiates the process of free radical chain

reaction by abstracting hydrogen atom from unsaturated fatty acids (Gordon, 1990).

Thereby, leading to membrane damage, DNA strand breakage, and finally inducing

cytotoxicity, carcinogenesis or mutagenesis (Yen and Duh, 1994; Babu et al., 2001).

The prevention of deoxyribose degradation by OHo

was reported in different parts of

pomegranate and their IC50 values ranged between 85 to 107 µg (Zhang et al., 2011).

The hydroxyl radical scavenging activity of the peanut skin extract was noted to be

higher than BHT by Wang et al. (2007). The potato peel extract exhibited a strong

concentration dependent hydroxyl radical scavenging activity in deoxy ribose

degradation system (Singh and Rajini, 2004). There are no specific enzymes to defend

against OHo

radicals (Zhou et al., 2010) and discovery of some compounds with

hydroxyl radical scavenging ability would gain significant role by providing ailment to

the damage caused by OHo.

DNA damage is related to conversion of the supercoiled form of plasmid DNA

to open-circular and further linear forms (Jung and Surh, 2001). The damage of plasmid

DNA results in a cleavage of one of the phosphodiester chains and produces a relaxed

open circular form. Further cleavage near the first breakage results in linear double

stranded DNA molecules. The formation of circular form of DNA is indicative of

single-strand breaks and the formation of linear form of DNA is indicative of double-

strand breaks (Singh et al., 2009). The plasmid DNA was mainly of the supercoiled

form in the absence of Fe2+

and H2O2. During the addition of Fe2+

and H2O2, the


Discussion


121

supercoiled form of DNA is converted into the open circular and linear forms indicating

that OHo

generated from iron-mediated decomposition of H2O2 produced both single-

strand and double-strand DNA breaks.

The protective effect of Gelidiella acerosa red algae against pBR322 is reported

by Suganthy et al., 2010. They have identified that the non polar fraction protective

better than the polar fraction. The DNA damage protective activity of Chukrasia

tabularis was in correlation with hydroxyl radical activity and the extracts

was identified to minimize the formation of Form II and Form III DNA in pBR322

(Kaur et al., 2008). Polyphenolics from various extracts/fractions of Allium cepa peel

was found to protect DNA by maintaining supercoiled nicked circular form in pUC18

(Singh et al., 2009).

The oxidation of unsaturated lipids by free radicals (Kaur and Perkins, 1991) is

counter acted by antioxidants. They donate hydrogen from their functional group which

breaks the free radical mediated oxidative chain, there by forming a stable end product

(Sherwin, 1978). The inhibition of Lipid peroxidation in egg yolk by Stachytarpheta

angustifolia has been identified by Awah et al., 2010. Singh and Rajini, 2004 has

identified the prevention of lipid peroxidation by potato peel extract and was reported

to give 80% inhibition at 5 mg concentration.

Total antioxidant capacity of aqueous and alcoholic extracts of Coronopus

didymus was evaluated by Prabhakaran et al., 2006 and identified the total antioxidant

capacity of some of their alcoholic extract was equivalent to 115 µg of ascorbic acid.

da Silva et al. (2011) has compared the total antioxidant capacity (TAC) of hydro

alcoholic extracts of Anadenanthera colubrine, Libidibia ferrea and Pityrocarpa

moniliformis and reported the TAC ranged between 24% and 18% of ascorbic acid. The

total antioxidant capacity of Amorphophallus companulatus was reported to be 19.5 µg

ascorbic acid equivalent by Ramesh et al., 2011.

The electron donating capacity associated with reducing power of bioactive

compound reflects on the antioxidant capacity of extracts (MacDonald-Wicks et al.,

2006; Ak and Gulcin, 2008). Reducing power is associated with presence of reductones


Discussion


122

(antioxidants) which have been shown to exert antioxidant action by breaking free

radical chain by donating by hydrogen atom (Sawant et al., 2009). The existence of

direct correlation between antioxidant potential and reducing power of certain plant

extracts has been reported by Kumaran and Joel Karunakaran (2006) against Coleus

aromaticus. The presence of reductones attributing to the reducing power activity in

Amorphophallus companulatus was reported by Ramesh et al. (2011). Similarly the

Reducing power capacity of potato peel extract is reported by Singh and Rajini (2004).

Iron is an important proxidant involved in lipid peroxidation, if not chelated, the

Fe2+

will produce OHo

leading to fenton reaction. Fe2+

state is ten times more powerful

that Fe3+

state (Liu et al, 2007; Kehrer, 2000). The results of this assay is indicative that

the extract, EDTA and BHT interfered with the formation of ferrous and ferrozine

complex, suggesting that it has chelating activity and captures ferrous ion before

ferrozine. The Percentage of chelation increased with increasing concentration. The

presence of Fe2+

chelating activity is reported in Peanut extract (Wang et al., 2007),

Gardenia jasminoides fruit extract (Debnath et al., 2011), Rhus coriaria extracts (Bursal

and Koksal 2010), Polysaccharide of Astragalus membranaceus (Niu et al., 2011) peel

of potato (Singh and Rajini, 2004).

5.2 ENZYMATIC ANTIOXIDANT

To elevate the damaging effects of ROS, plants have evolved intracellular

enzymatic antioxidants that include superoxide dismutase (SOD), catalase (CAT),

guiacol peroxidase (G-Px), ascorbic acid peroxidase (AAO) (Kim et al., 2004;

Xue et al., 2001), glutathione peroxidsae (GSH-Px) and glutathione reductase (GR)

(Sgherri et al., 2003). They also possess non-enzymatic antioxidants such as Phenol,

reduced glutathione (GSH) and ascorbate (Liu et al., 2009). Plants are still a large

source of natural antioxidants that might serve as leads for the development of novel

drugs (Linn and Huang, 2002).

Therefore the present investigation evaluate the enzymatic and non- enzymatic

capacity of Amorphophallus commutatus tubers and leaves adapting eight enzymatic

methods such as superoxide dismutase, catalase, peroxidase, ascorbate oxidase,


Discussion


123

glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione reductase,

polyphenol oxidase and three non- enzymatic methods such as reduced glutathione, total

poly phenols and ascorbic acid. Analysis of the results indicated that the extracts

exhibited differential antioxidant profile.


The Amorphophallus commutatus tuber exhibited significantly greater activities

of superoxide dismutase, ascorbic acid oxidase and polyphenol oxidase. It is also found

to have higher non-enzymatic content of ascorbic acid and total phenol. The young

leaves of Amorphophallus commutatus exhibited significant catalase and glutathione

reductase activity and found to have significant glutathione content among the plant

parts. The matured leaves of Amorphophallus commutatus exhibited significant activity

of glucose-6-phospate-dehydrogenase and peroxidase.

The tuber with significant phenol content exhibits poly phenol oxidase

whereas the leaves have no significant poly phenol oxidase activity and phenol content.

In the young leaves the activity of glutathione reductase and glutathione content are in

significant quantity revealing the conversion of oxidized glutathione (G-S-S-G) to

reduced (GSH) by glutathione reductase. In mature leaves the activity of glucose-6-

phosphate dehydrogenase activity might be high for it might require high amount

of NADPH for other activities with its hexose monophosphate (HMP) shunt functioning

effectively. Glucose-6-phosphate dehydrogenase is the first enzyme in the HMP shunt.

Super oxide radicals are inactivated by the enzyme superoxide dismutase (SOD),

the only enzyme known to use a free radical as a substrate. The radical scavenging

activity of SOD is effective only when it is followed by increase in activity of catalase

and other peroxidases (Ramasarma, 1990). SOD generates H2O2 as a product which is

in turn more toxic to the cells and requires catalase or peroxidases to scavange. Thus a

concomitant increase in catalase and or peroxidase is essential for the beneficial

effect from increase superoxide dismutase activity (Harman, 1991). Catalase act in the

microbody of cells, while guaiacol peroxidise exist in the apoplast, chloroplast and

cytosol (Shigeoka et al., 2002).


Discussion


124

H2O2 can penetrate the cell membrane and then react with metal ions through the

fenton reaction to produce extremely highly toxic hydroxyl radicals, which cause DNA

damage and cell injury. These harmful free radicals can be scavenged by intracellular

antioxidant enzymes like guaiacol peroxidase and glutathione reductase that minimize

or remove cellular reactive radical cascades and decrease cytotoxic oxidative damage in

cells. Guaiacol peroxidases are able to catalyze the reduction of lipid hydroperoxides to

hydroxides during the oxidation of reduced glutathione (GSH). Subsequently,

glutathione reductase regenerates GSH and provides reducing power for various

coupled thiol transferase and peroxidase. Moreover, compounds inducing antioxidative

enzymes or decreasing free radicals levels could decrease mutation production and

cancer initiation because they might reduce intracellular oxidative stress and DNA

damage (Yen and Chen, 1998).

5.2.2 Non-enzymatic component measurement

It is understood that defense against oxidative stress is primarily dependent upon

orchestere synergism between exogenous and endogenous antioxidants. The exogenous

antioxidants like vitamin E and vitamin C are recycled continuously by thiols like GSH

and dihydrolipoate. Thus vitamin C and glutathione react cooperatively in vivo leading

to greater protection against radical damage which could not be provided by any single

antioxidant (Gul et al., 2000).

Berries are the best known for their antioxidant properties. Rani et al. (2004) has

observed the activity of superoxide dismutase in orange (13.424 U/ mg protein) and in

grapes (2.62 U/ mg protein). The presence of significant levels of catalase, peroxidase,

superoxide dismutase, glutathione peroxidase, ascorbate oxidase, glucose 6-phospate

dehydrogenase in the enzymatic antioxidants and the presence of reduced glutathione ,

vitamin C, total phenol among the non enzymatic antioxidants in berries like goose

berry, grapes , orange and tomato are reported by Rani et al., 2004.

Shyles and Padikala (1999) have reported the presence of SOD activity of

alcoholic extracts of Emilia-sonchofoila. The peroxidase and catalase activity was

reported to be higher in Coleus forskohlii tuber and their activities were highly

significant and almost 70% higher activity is reported in tubers than leaves and stems.


Discussion


125

Li et al. (2011) has studied the importance of active antioxidant enzyme system

which will help in treating the chilling stress of cucumber. They have reported the

enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase

and ascorbate oxidase. Chilling increased the activities of antioxidant enzyme such as

CAT, GSH - Px and AAO elevated the contents of Ascorbic acid and GSH.

The results obtained by Duh et al. (2009) by analyzing the three biological thiols

on antimutagenic and antioxidant enzyme activities indicate that GSH may have

inhibitory effect on indirect and acting mutagen. In addition cellular glutathione

dependent enzymes were induced in BNL cell by the addition of GSH. Therefore, GSH

has potential for the exploration of wider application as anti mutagenic and chemo

preventive agent. The higher antioxidant capacity and antioxidant enzyme activity has

been reported by Chanjirakul et al., 2006 in raspberries.

The reports of Suriyavathana and Indhupriya (2011) are relevant to the current

report. They have screened the antioxidant potential of Dioscorea bulbifera tuber, they

have reported the presence of glutathione peroxidase, catalase, superoxide dismutase,

glucose 6- phosphate dehydrogenase and glutathione s- transferase. The non-enzymatic

antioxidants like vitamin E, vitamin C and reduced glutathione also has been reported in

Dioscorea bulbifera tuber.

Tubers tend to be starchy and typically rich in vitamins and minerals. The main

nutritional value of roots and tubers lie in the potential ability to provide one of the

cheapest source of dietary energy in the form of carbohydrate this make them an

excellent addition to human diet. Antioxidant enzymes have the capacity to lower the

free radical burden and neutralize the excess free radicals created by the stress and

normal metabolic conditions. The present study has been initiated with the view and

objective to explore the antioxidants store in Amorphophallus commutatus.

5.3 ANTIPROLIFERATIVE ACTIVITY

Plants have many phytochemical with various bioactivities, including

antioxidant, anti-inflammatory and anticancer activities. For example, some studies

have reported that extracts from natural products, such as fruits, vegetables and


Discussion


126

medicinal herbs, have positive effects against cancer, compared with chemotherapy or

recent hormonal treatments (Wu et al., 2002). Therefore, many plants have been

examined to identify new and effective antioxidant and anticancer compounds, as well

as to elucidate the mechanisms of cancer prevention and apoptosis (Swamy and Tan,

2000). Several antioxidants in plants have been suggested to contribute to the anti

carcinogenic effects and other such as flavanols have been able to inhibit cancer cell

proliferation in vitro (Scalbet et al., 2005).

Cancer remains one of the leading causes of death worldwide even today.

Various cancer therapies have currently been tried, including the use of natural products

from higher plants. Therefore, the need to discover an effective, novel and scientifically

reliable natural compound is urgent. Natural products provide a fertile ground

for seeking out treatments with fewer side effects and equal or better results (Wicaksono

et al., 2009). The antitumor area has the greatest impact of plant derived drugs,

where drugs like vinblastine, vincristine, taxol and camptothecin have improved

the chemotherapy of some cancers (Newman et al., 2003).


The SRB assay provided a rapid and sensitive method for measuring the drug-

induced cytotoxicity in both attached and suspension cultures in 96-well microtiter

plates. SRB binds to protein basic amino acid residues in TCA-fixed cells to provide a

sensitive index of cellular protein content that is linear over a cell density range of at

least 2 orders of magnitude. The SRB assay provides a colorimetric end point that is

nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive

measure of drug-induced cytotoxicity. Color development in the SRB assay is rapid,

stable, and visible (Skehan et al., 1990).

The following discusses the antiproliferative properties of petroleum ether,

chloroform, ethyl acetate, methanol and hot water extracts of Amorphophallus

commutatus tuber. The in vitro cytotoxic activity against mitogen induced human

peripheral blood lymphocytes showed that significant activity was exhibited by

petroleum ether extract with IC50 value corresponding to 0.257 ± 0.003 mg/ml. All the

crude extracts inhibited mitogen induced T lymphocytes in a dose dependent manner.


Discussion


127

To identify a compound to possess antiproliferative property, identification of

compounds which triggers apoptosis need to be done. The preliminary methods of

identifying apoptosis inducing properties of a compound are DNA fragmentation by gel

electrophoresis or quantitation of DNA fragmentation by Diphenylamine method or

radiolabel method. When comparing the results of fragment with morphological

observation the percent DNA fragmentation usually is lower than the percent apoptosis.

This is due to incomplete separation of fragments from intact DNA (Squier and Cohen,

2001). The DNA fragmentation assay was in coordination with SRB assay with

petroleum ether fraction exhibiting significant fragmentation and ethyl acetate showing

least fragmentation among the extracts.

Artyukho et al. (2011) has reported on DNA fragmentation of human

lymphocytes in dynamics of development of apoptosis induced by action of UV

radiation and reactive oxygen species. The degradation of DNA in to fragments of

18 - 200 nucleotide pairs during apoptosis can be electrophoretically visualized or

quantified spectrophotometrically.

5.3.2 In vitro cytotoxicity of Colo 205 cell line

The presence of polyps and bleeding stools are common for piles and colon

cancer. The report of Ravikumar and Ved, 2004 authenticates the usage of

Amorphophallus commutatus tuber against piles and cyst by the tribes. This study was

taken up with the suspicion that the plant might possess antiproliferative activity against

colon cancer and hence colo 205 the adenocarcinoma cell line was used. The results

clearly indicate that methanol and ethyl acetate fraction contains significant cytotoxicity

that has to be elucidated for the responsible compound. Hsu et al. (2008) has reported

that the crude extracts of Solanum lyratum induced cytotoxicity and apoptosis in human

adenocarcinoma cell line Colo 205. Zhou et al. (2006) demonstrated that the volatile oil

of ginger down regulated the T lymphocytes proliferatioin in vitro.

5.3.3 In vitro cytotoxicity of SiHa Cell line

The mortality rate of cervical cancer has been gradually reduced after the

introduction of PAP-smear programme. It is still one of the leading female malignancies


Discussion


128

worldwide (Ng et al., 2011). The reports of Ravikumar and Ved (2004) indicate that

tribes have used the tuber to cure cyst and tumor. The results of SRB assay against

human gyneacological cell line SiHa reveals that among the extracts ethylacetate

fraction contains significant activity against human cervical carcinoma cell line SiHa.

The methanol extract contained moderate activity and the others were not significant.

Cytotoxicity of extracts and compounds from Commiphora myrrha resin against human

gynecologic cancer cells were analysed by Su et al. (2011) and results reveal that the

fat-soluble extracts and chemical components exhibited significant activities on four

human gynecological cancer cell lines.

The intresting result that needs to be highlighted is that the ethylacetate fraction

which has been identified to possess DNA damage protecting activity has exhibited

significant cytotoxicity against Colo 205 and SiHa cell lines but has not shown

cytotoxicity on mitogen induced lymphocytes. The extract might possess compound that

specifically target the altered self cells i.e cancerous cells which have altered protein

and glycoproteins on their surface. The mitogen induced lymphocytes does not

transform in to a cancerous cell and would have not lost its normal cell properties upon

addition of a mitogen. The proliferation could be a temporary response to the mitogen.

Therefore special studies on the target group for the ethyl acetate fraction becomes vital

and if it possess such specificity towards the cancerous cells it will lead to new arena in

cancer chemotherapy.

It has been reported that the isolated compounds of Rhaphidophora decursiva

(Araceae) possess antimalarial and cytotoxic activity (Zhang et al., 2001).

Rhaphidophora korthalsii (Araceae) is widely known with the common name “Dragon

tail” due to the morphology of the plant. Traditionally, this plant is used as anticancer

drug and to treat skin disease. Wong and Tan (1996) who studied this plant found that

the ether fraction of Rhaphidophora korthalsii (Araceae) showed cytotoxicity in P388,

Molt 4, KB and SW 620 cancer cell line at the concentration above 50 µg/mL. The

cytotoxicity of the plant may be due to the presence of 5, 6-dihydroxyindole. The above

reports indicate the existence of antiproliferative activity among the members of


Discussion


129

Araceae family, belonging to the same family the presence of cytotoxicity in

Amorphophallus commutatus is evident from our reports.

Many compounds purified from plants, such as taxol (Bhalla et al., 1993) and

camptothecin (Kaufman, 1989), have revealed anticancer activity and induce cancer

cells to differentiate and undergo apoptosis. Curcumin, the active portion of turmeric

(Curcuma longa L.) has been shown to inhibit proliferation of human adeno carcinoma

cell line colo 205 by induction of apoptosis, production of Reactive oxygen species and

activation of Caspase-3 (Su et al., 2006).

Many more screening studies are necessary using plant extracts and compounds

isolated from them. Potential apoptotic inducers should not be cytotoxic to normal

tissues and the immune cell system. Naturally occurring compounds that are included in

the diet are non-toxic and may partially regulate programmed cell death in several

tissues and organs. Elaborate studies with such compounds with respect to their abilities

to induce apoptosis and understanding their mechanism of action may provide

valuable information for their possible application in cancer therapy and prevention

(Amit, 2001).

The identification, mechanistic, investigation, validation and utilization of

dietary components, natural products, or their synthetic analogous as potential cancer

chemo preventive agents has become an important issue in current public health related

research, in the form of functional foods or nutraceuticals. Considering the complexity

of cancer causes and development, it will be important to provide a variety of cancer

chemo preventive with different molecular and cellular targets, acting by multiple

mechanisms. Thus enhanced apoptosis may be responsible for reduction of many of the

adverse effects of chemotherapy and for tumor regression (Mathew and White, 2006).

5.4 SCREENING FOR ANTI-BACTERIAL ACTIVITY

The Frequency of life threatening infections caused by pathogenic

microorganism has increased the mortality rate of immunologically suppressed patients

worldwide, especially in the developing countries (Al – Bari et al., 2006). A very large

number of antimicrobial agents are being released but, the pathogenic microorganisms


Discussion


130

equally develop resistance against these agents. The reason being irrational use of

antimicrobial agent by the third world countries (Al - Bari et al., 2007). In recent years,

attempts have been made to investigate the indigenous drugs against infectious diseases.

Research in the field of indigenous plant is a significant aspect to develop a safer

antimicrobial principle through isolation, characterization, identification and biological

studies (Rahman et al., 2001).

The five different extracts of Amorphophallus commutatus were screened for

antibacterial activity by agar well diffusion and the MIC values were determined by

broth dilution method. Three gram positive and three gram negative clinical isolates

were used for the analysis. Relevant to the above discussed points the organisms chosen

for this study were those that are rapidly developing resistance and therefore becoming

potential threats.

The ethyl acetate fraction exhibited significant activity against all the six tested

clinical isolates. The zone of inhibition observed against all the organisms was above

20 mm. interestingly ethyl acetate fraction has inhibited MSSA and E.coli with good

significance. The MIC values were identified to be 15.6 µg against all the tested

organisms. Pseudomonas aeruginosa was inhibited only by ethyl acetate fraction.

The next significant activity was exhibited by both petroleum ether and

methanol extracts. Along with ethyl acetate extract methanol also inhibited the growth

of the spherical gram positive bacteria Enterococcus faecalis. But the zone produces by

petroleum ether extracts were only 8 to 15% less than the ethylacetate fraction which

stood first among the fractions. Methanol fraction was identified to be equivalent to

chloroform extract except in the case of MSSA, were chloroform extract behold

significant activity. Hot water extract did not inhibit the growth of two gram negative

bacterias Pseudomonas and Klebsiella. MSSA, MRSA and E.coli were inhibited by all

the extracts.

The MIC measurement to determine antimicrobial activity is a quantitative

method based on the principle of contact of a test organism to a series of dilutions of

test substance. Assays involving MIC methodology are widely used and an accepted


Discussion


131

criterion for measuring the susceptibility of organisms to inhibitors (Lambert and

Pearson, 2000). There exists a good correlation in the range of sensitivity among the

extracts measured by agar well diffusion and MIC.

The MIC values of all the fractions ranged between 15.6 µg/ml to 250 µg/ml and

those extracts that did not exhibit any zone of inhibition were expected to have an MIC

value greater than 500 µg/ml. the results of MIC were relevant to agar well diffusion

method and in both ethyl acetate fraction possessed notable significance followed by

petroleum ether, chloroform, methanol and hot water. Extracts having activities where

MIC values are below 8 mg/ml (Fabry et al., 1998) are considered to possess some

antimicrobial activity and natural products with MIC values below 1 mg/ml are

considered noteworthy (Gibbons, 2004; Rios and Recio, 2005).

Diacetyltambulin isolated from Amorphophallus companulatus exhibited

significant antibacterial activity against four gram-positive bacteria (Bacillus subtilis,

Bacillus megaterium, Staphylococcus aureus, Streptococcus β-haemolyticus) and six

Gram-negative bacteria (Escheichia coli, Shigella dysenteriae, Shigella sonnei, Shigella

flexneri, Pseudomonus aeruginosa, Salmonella typhi). The MIC values against these

bacteria ranged from 8 to 64 µg /ml but had weak antifungal activity against a number

of fungi (Khan et al., 2008a). Similarly antibacterial activity of ambylone isolated

from Amorphophallus companulatus has exhibited large zones of inhibition against

four gram-positive bacteria (Bacillus subtilis, Bacillus megaterium, Staphylococcus

aureus, Streptococcus pyogenes) and six Gram-negative bacteria (Escheichia coli,

Shigella dysenteriae, Shigella sonnei, Shigella flexneri, Pseudomonus aeruginosa,

Salmonella typhi). The MIC values against these bacteria ranged from 8 to 64 µg/ml.

The antifungal screening exhibited weak zones of inhibition against Aspergillus niger,

Aspergillus flavus and Rhizopus aryzae. Candida albicans was resistant against the

compound (Khan et al., 2008b).

Several methods are currently available to detect their antimicrobial activity and

since not all of them are based on the same principles, the results obtained are

influenced not only by the method selected, but also by the microorganisms used,


Discussion


132

and by the extraction method or the degree of solubility of each test-compound

(Valgas et al., 2007; Tripoli et al., 2007).

5.5 PHYTOCHEMISTRY

The scientific validation of bioactivity of different fractions of Amorphophallus

commutatus could be achieved only if the active principle responsible for the bioactivity

could be identified. According to Mukherjee and Wahile, 2006 the finger printing and

marker compound analyses are nowadays getting momentum for the standardization of

traditional medicinal formulations. Only identification of the bioactive lead by isolation

and structural elucidation by bioactivity guided fractionation will provide valued

scientific standardization procedures. This technique helps not only in establishing the

correct botanical identity but also helps in regulating the chemical sanctity of the herbs.

Investigation of the tuber qualitatively for the secondary metabolite has revealed

that all the fractions were positive to cardiac glycosides. The tuber crops are known for

the starch and polysaccharide contents. Both saponins and cardiac glycosides are

classified under terpenes. Saponins are glycosides of terpenes or sterols. Cardiac

glycosides possess special sugar substituents that are usually not found in the plant

kingdom. Cardiac glycosides are toxic and are reported to have pharmacological activity

especially to heart (Harbone, 1998).

The petroleum ether soluble fraction of Amorphophallus companulatus tuber

was subjected to column chromatography, and various spectrophotometric methods and

the compound was identified to be amblyone a triterpenoid (Khan et al., 2008b).

Similarly chloroform soluble fraction of Amorphophallus companulatus was subjected

to various spectroscopic methods and the compound was identified to be a flavonoid

2, 3 – diacetyl amblyone (Khan et al., 2008a). Analysis of the results of the current

study indicates the presence of triterpenoid in petroleum ether extract and flavanoid in

chloroform extract. The activity exhibited by these two fractions might be due to the

presence of the above discussed compounds in their respective fractions. Both the

reports have revealed the presence of antibacterial activity and absence of antifungal

activity similar to the reports of the current study.


Discussion


133

Investigation of the structure of compound x reveals the presence of

carbohydrate molecules with long carbon chains. The Amorphophallus species is known

for its polysaccharide contents with lot of medicinal properties. Glucomannan (GM) is a

polysaccharide of the mannan family, very abundant in nature, specifically in softwoods

(hemicellulose), roots, tubers and many plants bulbs. Despite the variety of sources,

the most common used type of GM is named konjac glucomannan (KGM), which

is extracted from tubers of Amorphophallus plants (An et al., 2011). Konjac

glucomannans (KGM) has been isolated from other Amorphophallus species like

Amorphophallus panomensis, Amorphophallus paeoniifolius and Amorphophallus

tonkinensis (An et al., 2010).

Dietary fiber polysaccharides are of considerable physiological importance.

They influence the digestion of food in general and in particular reduce the insulin

needs of people with diabetes, influence bile acid metabolism, alter lipid digestion,

cholesterol absorption and protect against colonic cancer (Kok et al., 2009). KGM

and other polysaccharides were isolated from Amorphophallus species employing

chromatography, Raman spectroscopy, FT/IR spectroscopy, LC-MS, NMR and X – ray

crystallography in the following references Das et al. (2009); An et al. (2010);

An et al (2011). Other than polysaccharides, flavonoids and triterpenoids reported in

Amorphophallus species the aroid was identified to be a source of phenyl terminated

fatty acids (Meija and Soukup, 2004).

In order to further elucidate the structure and functional groups in compound x,

the compound need to be subjected to IR spectra for identification of the functional

group. Two dimensional NMR spectroscopy like dept 135, HSQC, 1H –

1H COSY

NMR. Further purification of the column fraction and identifying the above mentioned

NMR and IR spectrum might provide detailed information to elucidate the structure of

the isolated compound x. But the currently elucidated structure of x is in correlation

with the literature survey which indicates the presence of carbohydrate moiety. Apart

from the NMR spectrum the qualitative phytochemical screening also has showed

positive results for saponins and cardiac glycosides which are in turn glycosides.


Discussion


134

An essential element of any strategy for non-targeted metabolomics analysis of

complex biological extracts is the capacity to perform comparisons between large

numbers of samples. As the most widely used technologies are all based on mass

spectrometry (e.g. GCMS, LCMS), this entails that we must be able to compare

reliably and (semi) automatically large series of chromatographic mass spectra from

which compositional differences are to be extracted in a statistically justifiable manner

(Vorsta et al., 2005).

The presence of bioactivity in the plant Amorphophallus commutatus is evident

from the results. The ethyl acetate extract has significant bioactivity among the extracts,

though each extract possess its own potential activity. Validation of these properties lies

in identification of the lead molecule. Therefore the major efforts in this direction may

be augmented by introducing the element of research via the application of scientific

tools and techniques for new understanding about the bioactive principles. Medicinal

herbs as potential source of therapeutic aids have attained a significant position in health

systems all over the world for both humans and animals not only in the diseased

condition but also as potential material for prevention.


Discussion


135

SSUUMMMMAARRYY

Amorphophallus commutatus (Araceae) a rare cormous herb endemic to Western

Ghats has been used by the tribes for various ailments. The plant is used by the tribes of

sitamata wild life sanctuary, Rajasthan, India and tribes living in the Western Ghats,

India for various ailments. The current study was carried out with an objective to

investigate the radical scavenging properties, enzymatic and non enzymatic content, In

vitro cytotoxicity on mitogen induced lymphocytes, SiHa and Colo 205 cell lines,

antibacterial activity and isolation and identification of the phytochemical lead

responsible for the bioactivity.

In vitro, antioxidant potential and metal chelating properties of five solvent

fractions of Amorphophallus commutatus tuber fractions were evaluated using multiple

assays. The synthetic radicals DPPHo and ABTS

o+ were scavenged effectively by the

ethyl acetate fraction with IC50 value of 61.32 ± 1.39µg and 69.6 ± 1.1 µg respectively.

Ethyl acetate fraction exhibited significant (p<0.05) total antioxidant capacity of

66.2 ± 1.6% of ascorbic acid. Total Reducing Power activity was also significant in

ethyl acetate with an IC50 of 59.2 ± 0.1. The radical scavenging activity of ethyl acetate

fraction was observed to be significant (p<0.05) against radicals like super oxide, nitric

oxide and hydroxy radical with the IC50 values of 92.5 ± 1.1 µg, 139.6 ± 2.2 µg and

62.4 ± 1.1 µg respectively.

The DNA damage induced by hydroxyl radicals on pUC 18 was protected by

ethyl acetate extract significantly. The protecting effect of the extracts on pUC18 was

in correlation with hydroxyl radical scavenging activity. The inhibition of Lipid

peroxidation was also significant (p<0.05) in ethyl acetate fraction with IC50 of

66.3 ± 0.9 µg. The ferrous ion chelating potential of hot water extract was significant

with an IC50 value of 42.3 ± 0.4 µg.

Some extracts exhibited activities that were higher than that of the positive

standard used. ABTSo+

scavenging activity of ethyl acetate was higher than BHT and

ferrous Ion chelation of Hot water extract was higher than EDTA and BHT. The results

6


Discussion


136

assure the presence of antioxidant principles in various fractions and among the

fractions ethyl acetate exhibited significant activity.

The enzymatic and non enzymatic contents of the tuber and leaves of the plant

was investigated adopting eight enzymatic methods (Superoxide dismutase, Catalase,

Guaicol peroxidase, Ascorbic acid oxidase, Glutathione peroxidase, Glutathione

reductase, glucose-6- phosphate dehydrogenase and polyphenol oxidase) and three

non enzymatic methods (Phenol, Ascorbic acid, Glutathine reduced). The tuber

exhibited significant SOD (47.7 ± 5.5 U/g tissue), AAO (0.38 ± 0.12U/g tissue), PPO

(0.8 ± 0.014 U/g tissue), Ascorbic acid (2.6±0.5mg/g tissue) and total phenol

(0.2 ± 0.007 mg/g tissue). Young leaves contained significant CAT (64.3 ± 6.02 U/g

tissue), GR (1.3 ± 0.017 U/g tissue) and Glutathione (6.21 ± 0.6 mg/g tissue) content.

The mature leaf exhibited significant G6PD activity (9.97 ± 2.0 U/g tissue). Our results

reveal the innate antioxidant potential of Amorphophallus commutatus and therefore can

be utilised for supplementing the antioxidant needs in the diet.

The antiproliferative property of different fractions of Amorphophallus

commutatus tuber was anlysed by SRB assay against mitogen induced peripheral blood

lymphocytes. Peripheral blood lymphocytes were isolated for this study using density

gradient centrifugation with lymphocyte separation medium. The amount of cells

obtained was 1.15 x 106

cells / ml by density gradient centrifugation with 98.3%

viability. The cells obtained from density gradient centrifugation were cultured with and

without mitogen and the quantity of mitogen induced cells were nearly 73.3% more

compared to the normal cells in culture. Querecetin in different concentration range was

used as a positive control. The results revealed that petroleum ether exhibited significant

cytotoxic properties with IC50 value of 0.257 ± 0.003 mg/ml followed by methanol and

hot water extract. The chloroform and ethyl acetate extract showed negligible or less

significant activity.

The DNA fragmentation assay for apoptosis induction was carried out in

mitogen induced lymphocytes and the results were in coherence with SRB assay. The

petroleum ether extract at 500 µg/ml concentration exhibited 66.68±0.8% of DNA


Discussion


137

fragmentation. The ethyl acetate fraction was identified to contain least percentage of

fragmentation.

The Colo205 adeno carcinoma cell line was taken for anti proliferative

studies. The Methanol fraction possessed significant activity among the extracts with

94.7 ± 0.90% of growth inhibition at 500 µg concentration. The ethyl acetate fraction at

the same concentration was observed to have next significant control of growth

corresponding to 89.63 ± 0.81%.The Hot water fraction showed moderate inhibition and

the other two fractions were not significant.

In vitro cytotoxicity induced by the extracts on human gynecological cell line

SiHa was investigated by SRB assay. The investigation revealed ethyl acetate fraction

to possess significant activity of 57.74 ± 1.06% control of growth at 500 µg. The next

significant activity was observed in the methanol fraction with the percentage of growth

inhibition at 500µg corresponding to 41.23 ± 0.67%. The other three extract did not

produce any significant control on the growth of SiHa cells.

The antibacterial activity was carried out using six multiple drug resistant

bacterial strains three gram positive and three gram negative. The ethyl acetate fraction

exhibited significant activity against all the six tested clinical isolates. The Zone of

inhibition observed against all the organisms was above 20mm. The MIC values were

identified to be 15.6 µg against all the tested organisms. Pseudomonas aeruginosa was

inhibited only by ethyl acetate fraction.

The MIC values of all the fractions ranged between 15.6 µg/ml to 250 µg/ml and

those extracts that did not exhibit any zone of inhibition were expected to have an MIC

value greater than 500 µg/ml. The results of MIC were relevant to agar well diffusion

method and in both ethyl acetate fraction possessed notable significance followed by

petroleum ether, chloroform, methanol and hot water extracts respectively.

Qualitative phytochemical screening of all the five extract showed the presence

of cardiac glycosides and saponins. Since ethylacetate fraction had significant free

radical scavenging activity, antibacterial activity, anti proliferative activity against SiHa

and Colo 205 cell lines it was subjected to identification of lead molecule.


Discussion


138

Column chromatography fractions that exhibited Rf value of 0.68 and λmax of

263 nm were pooled and subjected to LC-MS and NMR spectroscopy. LC showed the

compound to possess a retention time of 14.61 minute and mass spectrum reported the

presence of molecules with m/z of 99, 128 and 240. NMR spectra and LCMS data were

analysed and the compound x was elucidated. As the tubers are known for their rich

carbohydrate content the compound x also contain carbohydrate moiety and the

qualitative tests confirm the presence of saponins and cardiac glycosides both belong to

the group of tri terpenoid.

Amorphophallus commutatus was found to be an effective antioxidant in

different in vitro assays. Ethyl acetate fraction was an effective free radical scavenger

and hot water demonstrated effective metal ion chelation. The presence of innate

antioxidant enzymes and non enzymatic components in different parts of the plant was

demonstrated. The in vitro cytotoxicity against - mitogen induced lymphocyte was

effective in petroleum ether extract, adeno carcinoma cell line colo 205 and human

gyneacological cell line SiHa were inhibited by ethyl acetate fraction. The antibacterial

studies suggest that among the extracts only ethyl acetate fraction exhibited potential

activity against all six tested organisms. The prelimnary phytochemical analysis reveals

the presence of tri terpenoids, cardiac glycoside and saponin in ethyl acetate fraction.

The structure of the compound isolated from ethyl acetate fraction was elucidated

partially by LC-MS and NMR.

THE FUTURE STUDIES PROPOSED INCLUDE

Elucidating the structure of compound x employing the following 2d NMR

spectroscopy.

• DEPT 135 - DEPT stands for Distortionless Enhancement by Polarization

Transfer, It is a very useful method for determining the presence of primary,

secondary and tertiary carbon atoms. The DEPT experiment differentiates

between CH, CH2 and CH3 groups by variation of the selection angle parameter.

The 135° angle gives all CH and CH3 in a phase opposite to CH2.

• HSQC – Hetronuclear Single Quantum Coherence Spectroscopy, the resulting

spectrum is two-dimensional with one axis for 1H and the other for a


Discussion


139

heteronucleus other than a proton. The spectrum contains a peak for each unique

proton attached to the heteronucleus being considered.

• 1H-

1H COSY is a two dimensional correlation spectroscopy indicationg the

relationship between the coupled protons. The identified compound should be

subjected to data mining and structure activity relationships for the target assays

which would greatly enhance predictability of the specific changes in the

identified compound either to enhance its activities or reduce its toxicity.

Determination of mechanism of action of antibacterial moiety by testing for

• Binding to endotoxins – A/B

• Disruption of bacterial membranes

• Haemolytic activity

• Bacterial D-ala-D- ala transpeptidase inhibition

• Formylase inhibitors

Identification of the mode of cytotoxicity against cell lines employing caspase assay

ans Annexin V binding assay.

To conclude, the potential activity exhibited by the extracts can be linked to the

presence of phytochemicals indicated in the preliminary screening tests. Further studies

on the extracts and their fractions should be carried out to have an insight in to the

mechanism involved in the observed effects. There by leading to scientific validation of

the lead molecule identified from the plant Amorphophallus commutatus which has not

been studied and reported previously.


Discussion


140

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http://www.biodiversityhotspots.org


Journal of Pharmacy Research Vol.4.Issue 3. March 2011

R. Kavitha Krishna et al. / Journal of Pharmacy Research 2011,4(3),710-711

710-711

Research Article

ISSN: 0974-6943 Available online through

www.jpronline.info

*Corresponding author.R. Kavitha Krishna

Research Department of Biotechnology,NGM

College,Pollachi – 642001,India

Tel.: + 91-9970372112

E-mail:[email protected]

Antibacterial activity of Amorphophallus commutatus, an endemic plant of Western Ghats,

South India.R. Kavitha Krishnaa*, S. Karthikeyanib, S. Krishna kumari c

a Research Department of Biotechnology,NGM College,Pollachi – 642001,Indiac Research Department of Botany,NGM College,Pollachi – 642001,India

c Department of Botany,Salem government arts and science College for women Salem, India.

Received on: 05-10-2010; Revised on: 14-12-2010; Accepted on:09-02-2011

ABSTRACTAmorphophallus commutatus (Schott) Engl (Araceae), is a rare cormous herb. Aqueous and organic solvent extracts of the tubers were investigated for anti-bacterial activity properties by usingdisc diffusion method, against pathogenic strains of gram negative bacteria (Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa and Salmonella typhii). The different extracts differed

significantly in their anti-bacterial properties with the benzene extract being very effective followed by petroleum ether, chloroform and ethyl acetate extracts. Aqueous and methanol extract

showed very least activity. The results of this study support the use of this plant in traditional medicine.

Key words: Amorphophallus commutatus;; anti-bacterial activity; human pathogens.

Table 1. Table.1. Antibacterial activity of different extracts of A.commutatus

S.No Organism Parts Zone of inhibition (in mm)

Disc diffusion method

used used S1 S2 S3 S4 S5 S61 E.coli Tuber 8 9 8 10 - -2 Proteus vulgaris Tuber - - 4 - - -3 Pseudomonas Tuber 22 19 14 8 15 94 Salmonella typhi Tuber 14 17 13 8 7 -

S1; Petroleum ether, S2; Benzene, S3; Chloroform, S4; Ethyl acetate, S5; Methanol, S6; Hotwater.

1. INTRODUCTIONPlants still continue to be almost the exclusive source of drugs for the majority of world’s 2.4. Screening of anti-bacterial activity

2.4.1. Disc diffusion methodThe disc diffusion assay methods of lennette (1985) as described by Rosoanaivo and Ratsimanaga-Urverg (1993) and Rabe and Van Staden (1997) were used with some modifications to

determine the rate of inhibition in growth of bacteria by plant extracts. The diluted bacterialcultures were spread over nutrient agar plates using sterile glass L-rod about 0.2ml of the eachextract was applied in filter paper disc 0.5mm diameter and allowed to dry before being placed

on the top layer of the agar plate. The plates were incubated at 37ºC for 24h and the growth ofinhibition zones were recorded.

3. RESULTS AND DISCUSSIONThe result of the anti-bacterial activity tests of A.commutatus tuberous extracts are presented inTable 1. Petroleum ether extracts of A.commutatus showed better growth inhibition in all

tested pathogens excluding Proteus vulgaris. The Benzene extracts of A.commutatus showedbetter growth inhibition in Pseudomonas followed by Salmonella. Chloroform extracts ofA.commutatus showed better growth inhibition in E.coli, Salmonella and Pseudomonas.

Ethyl acetate showed moderate growth inhibition in E.coli, Salmonella and Pseudomonas, butno inhibition in Proteus. The methanol extracts showed better growth inhibition in Pseudomonascompared to inhibition showed against Salmonella, but no inhibition in E.coli and Proteus.

The hot water extracts showed moderate inhibition in Pseudomonas, but no inhibition against

E.coli, Proteus and Salmonella.

There are reports on other Araceae species extracts that exhibit antibacterial activity. Althoughthese tested plant extracts may contain antibacterial constituents further phytochemical and

pharmacological studies by bioassay guided fractionation will be necessary to isolate the activeconstituent and evaluate the antibacterial activity against a wide range of microbial population.

Khana et al., (2008) have reported the antibacterial activity of 3,5- diacetyltambulin, aflavonoid isolated from the chloroform fraction of Amorphophallus campanulatus. Anotherreport indicates the antibacterial activity of ambylone, a triterpenoid isolated from the petro-

leum ether soluble fraction of Amorphophallus campanulatus. (Khanb et al., 2008).

Analyses of our reports indicate that both petroleum ether chloroform and benzene has estab-

lished good values on antibacterial activity compared to the other extracts. Therefore our resultsare in coherence with the above said reference. The antibacterial activity of the petroleum etherextract might be due to the flavonoid ambylone. Similarly the chloroform fraction might

contain 3,5-diacetyl ambulin as a lead molecule exhibiting antibacterial activity. This is thefirst step in Bioassay guided fractionation. The petroleum ether, chloroform and benzeneextracts require further fractionation and analysis to identify the active principle.

The development of resistant strains of bacteria has increased the need for new antibiotics(Eloff., 1998). Bioassay guided fractionation of plant species may lead to the discovery of new

antibacterial agents and better understanding of how ethanomedicine can treat infections.However, there is no report regarding bioactivity of this plant. Therefore, the aim of the presentwork was to evaluate the anti-bacterial potentiality of the tuberous extracts of the A.commutatus

against the growth of human pathogenic bacteria followed by identification of the compoundresponsible for the bioactivity.

2. MATERIALS AND METHODS

2.1. Plant materials

The whole plants of A.commutatus were collected from AICRPS on Medicinal and Aromaticplants, Kerala Agricultural University, Vellanikkara, Thrissur, Kerala.

2.2. Preparation of extractsOrganic solvents in the increasing order of polarity (Petroleum ether, benzene, chloroform,Ethyl acetate, methanol) and aqueous extract (hot water) of the plant materials were prepared

according to the method described by (Harbone,1998) with little modifications. Seventy fivegrams of plant material were air-dried, crushed and blended in to powder using an electricblender for each solvent. The blended material was transferred to a beaker and soaked separately

in room temperature. The mixture was extracted by agitation on a rotary shaker. The extractobtained was vaccum- dried and used for further test.

2.3. Microorganisms testedA total of four bacterial cultures (Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosaand Salmonella typhii) were used in this study. The cultures were procured from MTCC,

Chandigarh. The bacterial strains were grown in Muller Hinton plates at 37ºC and maintainedon Nutrient agar slants.

population (Sokmen et al., 1999). Substances derived from higher plants constitute 25% ofprescribed medicine and 74% of the 121 bioactive plant –derived compounds currently inworldwide use were identified via research based on leads from ethnomedicine (Farnsworth et

al., 1985; Sokmen et al., 1998). Medicinal plants contain physiologically active principlesthat over the years have been exploited in traditional medicine for the treatment of variousailments (Adebanjo et al.,1999; Natarajan et al., 2005) as they contain anti-microbial proper-

ties (Sokmen et al., 1999; Kelmanson et al., 2000; Srinivasan et al., 2001). Amorphophalluscommutatus (Schott) Engl, a member of the Araceae, is a tuber depressed globose, is a rarecormous herb that is found in evergreen and semi-evergreen forest of south western India

(endemic to Western Ghats). Tuberous corms of A.commutatus were used for treatment of piles,tumours and cysts (Ravikumar and Ved, 2004). Tubers of Amorphophallus commutatus has

also been used as antidote for snake bite by tribal’s living in fifty villages of Sitamata wildlife,sanctuary, Rajasthan, India (Jain et al.,2005).


Journal of Pharmacy Research Vol.4.Issue 3. March 2011

R. Kavitha Krishna et al. / Journal of Pharmacy Research 2011,4(3),710-711

710-711

Source of support: DST,India; Conflict of interest: None Declared

In fine the tuberous extracts of plant had potential antibacterial properties with significantgrowth inhibition against Pseudomonas and Salmonella, exhibited by petroleum ether, chlo-

roform and benzene extracts.

ACKNOWLEDGEMENT

The authors wish to thank the Department of Science and Tecnology, government of India forhaving funded the project under the women scientist scheme (WOS-A).

6. REFERENCE

1. Adebanjo, A.O., Adewama,C.O., Essein,E.E., 1983. Anti- infective agents of higher plants. IntInternational symposium of Medicinal plants. Fifth edn. University of lfe Niggeria, ,152-158.

2. Atalay Sokmen., Brian M. Jones., Murat Erturk., 1998. The in vitro antibacterial activity of Turkishmedicinal plants. Journal of Ethnopharmacology 67, 79-86.

3. Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitoryconcentration of plant extracts for bacteria. Planta Medica. 64, 711-713

4. Fransworth, N.R., Akerele, O., Bingel, A.S., Soejarto, D.D., Guo, Z.G., 1985. Medicinal, plants intherpy. Bull. Who.63, 965-9871.

5. Hamburger, M., Hostettmann, K., 1991. 7. Bioactivity in plants: The link between phytochemistry30 (12), 3864-3874.

6. Harbone, J.B. Phtochemicals methods: Aguide to modern technique of plant analysis. Londone:Chapman and Hall ltd. 1998. 52.

7. Jain, A., Katewa,S.S., Galav, P.K., Sharma,P.,2005. Medicinal plant diversity of Sitamata wildlifesanctuary, Rajasthan, India. Journal of ethnopharmacology. 102. 143–157.

8. Kelmanson, J.E., Jager,A.K., Van Staden, J., 2000. Zulu medicinal plants with antibacterial activity.Journal of ethnopharmacology. 69,241-246.

9 . aKhan, A., Rhaman, M., Islam, M.S. 2008. Antibacterial, antifungal and cytotoxic activities ofamblyone isolated from Amorphophallus campanulatus. Indian J Pharmacol. 40, 41-44.

10 . bKhan, A., Rahman, M., Islam, M.S. 2008. Antibacterial , Antifungal and cytotoxic activities of 3,5-diacetylambulin isolated from Amorphophallus campanula us. Blume ex. Decne. 16, 239-244.

11. Martin-Bettolo, G.B., 1980. Present aspects of the use of medicinal plants in traditional medicine.Jounal of ethanopharmacology. 2. 5-7.

12. Martin-Bettolo, G.B.,1980. Present aspects of the use of medicinal plnts in traditional medicine.Journal of ethnopharmacology 2,5-7.

13. Natarajan, D., John Britto, S., Srinivasan. K., Nagamurugan, N., Mohanasundhari, C., Perumal, G.,2005 Antibacterial activity of Euphorbia fusiforms A rare medicinal herb. Journal ofethnopharmacology. 102, 123-126.

14. Rabe,T., van Staden,J., 1997. Isolation of an antibacterial sesqui-terpenoid from Warburgia- salutaris.Journal of ethnopharmacology 73,171-174.

15. Ravikumar,K., and Ved,D,K., 2004. Illustrated Field Guide – 100 Red listed medicinal plants ofconservation concern in S.India, FRLTH, Bangalore.

16. Richard, D. Langfield., Frank, J. Scarona., Mary, E. Heitzman., Miwako., Gerald, B. Hammond.,Catherine, C. Neto. 2004. Use of a modified microplate bioassay method to investigate antibacterial activity in the Peruvian medicinal plant Peperomia galioides. Journal ofethanopharmacology. 94 ,279-281.

17. Rosqanaivo, P., Ratsimanaga-Urverg, S ., 1993. Biological Evaluation of Plants with Reference tothe Malagasy Flora Monograph for the IFS-NAPPECA Workshop on Bio-assays. Antananavivo,Madagascar,.72-79.

18. Sokmen,A.,Jones,B.M., Erturk,M., 1999. The in vitro antibacterial activity of Turkish medicinalplants. Journal of ethnopharmacology 67,79-86.


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