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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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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
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Bioactivity and Phytochemical analysis of Amorphophallus commutatus SCHOTT (Engl.) an Endemic Aroid of Western Ghats, South India
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
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Bioactivity and Phytochemical analysis of Amorphophallus commutatus SCHOTT (Engl.) an Endemic Aroid of Western Ghats, South India
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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
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Bioactivity and Phytochemical analysis of Amorphophallus commutatus SCHOTT (Engl.) an Endemic Aroid of Western Ghats, South India
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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
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Bioactivity and Phytochemical analysis of Amorphophallus commutatus SCHOTT (Engl.) an Endemic Aroid of Western Ghats, South India
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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).
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Bioactivity and Phytochemical analysis of Amorphophallus commutatus SCHOTT (Engl.) an Endemic Aroid of Western Ghats, South India
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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.
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Bioactivity and Phytochemical analysis of Amorphophallus commutatus SCHOTT (Engl.) an Endemic Aroid of Western Ghats, South India
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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
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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
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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%
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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.
Central council for Research in
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
Central council for Research in
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
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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),
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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
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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).
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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
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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
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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
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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)
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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.
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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
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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).
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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
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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).
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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).
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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.
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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).
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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).
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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).
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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
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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
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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,
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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
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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
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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
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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
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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
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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
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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.
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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
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Bioactivity and Phytochemical analysis of Amorphophallus commutatus (Schott) Engl. an
Endemic Aroid of Western Ghats, South India
40
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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.
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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.
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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 (%) =
Where Ac was the absorbance of control reaction and As the absorbance in the
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
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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 (%) =
Where Ac was the absorbance of control reaction and As the absorbance in the
presence of A.commutatus tuber extracts.
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 (%) =
Where Ac was the absorbance of control reaction and As the absorbance in the
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
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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 =
Where Ac was the absorbance of control reaction and As the absorbance in the
presence of A.commutatus tuber extracts.
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
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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
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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 =
Where Ac was the absorbance of control reaction and As the absorbance in the
presence of A.commutatus tuber extracts.
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,
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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.
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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)
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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.
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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.
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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).
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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.
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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.
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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);
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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.
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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.
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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.
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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).
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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
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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.
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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 Scavenging activity on synthetic radicals
4.1.1.1 DPPH radical scavenging activity
4.1.1.2 ABTS radical cation scavenging activity
4.1.2 Superoxide anion radical scavenging activity
4.1.3 Nitric oxide radical scavenging activity
4.1.4 Prevention of Deoxy Ribose degradation
4.1.4.1 Hydroxy radical scavenging activity
4.1.4.2 DNA Nicking assay
4.1.5 Inhibition of Lipid peroxidation
4.1.6 Total antioxidant capacity by phosphomolybdenum assay
4
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63
4.1.7 Total Reducing power activity
4.1.8 Ferrous Iron Chelating activity
4.2 ENZYMATIC ANTIOXIDANTS
4.2.1 Protein content
4.2.2 Antioxidant enzyme measurements
4.2.2.1 Superoxide dismutase (SOD)
4.2.2.2 Catalase (CAT)
4.2.2.3 Guaicol peroxidase (GPOD)
4.2.2.4 Ascorbic acid oxidase (AAO)
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)
4.2.2.8 Polyphenol oxidase (PPO)
4.2.3 Determination of non- enzymatic antioxidant
4.2.3.1 Total phenol
4.2.3.2 Ascorbic acid
4.2.3.3 Reduced glutathione
4.3 ANTIPROLIFERATIVE STUDIES
4.3.1 In vitro cytotoxicity of mitogen induced blood lymphocytes
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)
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4.5 PHYTOCHEMISTRY
4.5.1 Identification of biologically active compounds using preliminary
qualitative tests
4.5.2 Isolation of active principle in Ethyl acetate fraction
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.
4.1.1 Scavenging activity on synthetic radicals
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.
4.1.1.3 DPPH radical scavenging activity
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.
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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
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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.
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Figure 22
ABTS radical scavenging activity of Amorphophallus commutatus extracts
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
4.1.2 Superoxide anion radical scavenging activity
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.
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Figure 23
Super oxide radical scavenging activity of Amorphophallus commutatus extracts
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
4.1.3 Nitric oxide radical scavenging activity
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.
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Figure 24
Nitric oxide radical scavenging activity of Amorphophallus commutatus extracts
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
4.1.4 Prevention of Deoxy Ribose degradation
4.1.4.1 Hydroxy radical scavenging activity
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.
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Figure 25
Hydroxy radical scavenging activity of Amorphophallus commutatus extracts
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
4.1.8.2 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.
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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
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Figure 26
Inhibition of Lipid peroxidation by Amorphophallus commutatus extracts
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
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.
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4.1.10 Total antioxidant capacity by phosphomolybdenum assay
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
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
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
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(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
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
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.
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Figure 29
Ferrous ion chelating activity of Amorphophallus commutatus extracts
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water BHT - Butylated hydroxyl toluene
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.
4.2 ENZYMATIC ANTIOXIDANTS
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
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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
4.2.4 Antioxidant enzyme measurements
4.2.2.1 Superoxide dismutase (SOD)
The photo induced reduction of riboflavin generates superoxide radical
which is detected by the formation of nitrite while reacting with hydroxylamine
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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
4.2.2.9 Catalase (CAT)
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.
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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
Enzyme activity Sample
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.
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Table 8
Guaicol Peroxidase in different parts of Amorphophallus commutatus
Enzyme activity Sample
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
Values are mean±SD; n=3
a 1unit= µmoles of guaicol oxidized/minute
4.2.2.10 Ascorbic acid oxidase (AAO)
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
Enzyme activity Sample
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
Values are mean±SD; n=3
a I unit (AAO) = equivalent to 0.01change in OD /min
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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
Enzyme activity Sample
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
Values are mean±SD; n=3
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).
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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
Enzyme activity Sample
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
Values are mean±SD; n=3
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
Enzyme activity Sample
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
Values are mean±SD; n=3
a 1unit = µmoles of NADPH oxidized / minute
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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).
4.2.2.14 Polyphenol oxidase (PPO)
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
Enzyme activity Sample
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
Values are mean ± SD; n=3
a1 unit = Activity of catechol oxidase/ laccase that transforms 1 unit of
dihydrophenol to quinine/minute
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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
Values are mean ± SD; n=3
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.
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84
4.2.3.2 Ascorbic acid
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 In vitro cytotoxicity of mitogen induced blood lymphocytes
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
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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
peripheral blood lymphocytes
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
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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
peripheral blood lymphocytes
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water
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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
Values are mean ± SD (n=3)
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.
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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
Values are mean ± SD (n=3)
88
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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
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
HW – Hot water
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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
S.No. Extracts IC50 (mg/ml)
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
Values are mean ± SD (n=3)
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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
PE – Petroleum ether; Ch – Chloroform; EA – Ethyl acetate; M – Methanol;
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.
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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
S.No. Extracts IC50 (mg/ml)
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
Values are mean ± SD (n=3)
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.
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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.
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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
acetate Methanol Hotwater
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
Values are mean ± SD of three samples
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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
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PLATE 9
Antibacterial activity of Methycillin resistant Staphylococcus aureus (MRSA)
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 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
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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
Values are mean ± SD of three samples
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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
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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
Values are mean ± SD of three samples
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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
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Table 25
Antibacterial activity of Pseudomonas aeruginosa
Zone of inhibition (mm)
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
Values are mean ± SD of three samples
Table 26
Antibacterial activity of Escherichia coli
Zone of inhibition (mm)
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
Values are mean ± SD of three samples
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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
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PLATE 13
Antibacterial activity of Escheichia coli
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 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
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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
acetate Methanol Hotwater
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
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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
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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.
4.5.3 Isolation of active principle in Ethyl acetate fraction
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
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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.
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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.
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Figure 36
1H –NMR spectrum of ethyl acetate fraction collected from column chromatography
109
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Figure 37
1H –NMR spectrum of ethyl acetate fraction collected from column chromatography
110
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Figure 38
1H –NMR spectrum of ethyl acetate fraction collected from column chromatography
111
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Figure 39
1H –NMR spectrum of ethyl acetate fraction collected from column chromatography
112
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Figure 40
13C - NMR spectrum of ethyl acetate fraction collected from column chromatography
113
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Figure 41
13C - NMR spectrum of ethyl acetate fraction collected from column chromatography
114
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Figure 42
13
C - NMR spectrum of ethyl acetate fraction collected from column chromatography
115
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Figure 43
13C - NMR spectrum of ethyl acetate fraction collected from column chromatography
116
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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.
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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
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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
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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
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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
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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,
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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.
5.2.1 Antioxidant enzyme measurements
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).
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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.
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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
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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).
5.3.1 In vitro cytotoxicity of mitogen induced blood lymphocytes
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.
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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
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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
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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
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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
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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,
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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.
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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.
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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.
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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
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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
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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.
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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
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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.
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140
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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).
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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).
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