discussion review of literature -...

Discussion

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

8

RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE

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

under the following titles.

2.1 Introduction

2.2 Medicinal plants in traditional systems

2.3 Binomial classification of Amorphophallus commutatus

2.4 Properties of Araceae

2.5 Properties of Amorphophallus.

2.6 Ethnopharmacological utilisation of Araceae

2.6.1 Ethnopharmacological utilisation of Amorphophallus sps.

2.7 Bioactivity

2.7.1 Emergence of free radicals in biological systems

2.7.2 Effect of free radicals on biological system

2.7.3 Antioxidant enzymes – Natural defense against free radicals

2.7.4 Non enzymic antioxidant systems as natural defense against free radicals

2.7.5 Antimicrobial agents – Methods and mechanism

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

agents.

2.7.5.2 Selection of the appropriate bioassay

2.7.5.3 Antimicrobial agents from araceae

2.7.5.4 Antimicrobial agents from Amorphophallus sps

2.7.6 Anticancer agents – Mode of action

2.7.6.1 Anticancer agents from Araceae

2.7.7 Identified bioactivities of Amorphophallus sps

2.8 Phytochemistry

2.8.1 Molecules identified in Araceae/Amorphophallus sps

2.9 Conclusion

2

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Discussion


9

2.1 Introduction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

versatile applications. The development of pharmaceuticals begins with identification of

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

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

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

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

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

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

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

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


Discussion


10

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

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

health care (Mukherjee and Wahile, 2006).

2.2. Medicinal plants in traditional systems

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

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

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

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

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

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

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

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

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

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

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

development of traditional systems of medicine.

Figure 2

Number of plants used in different systems of medicines in India

(Mukherjee and Wahile, 2006).

Modern

2%Tibetan

5%

Siddha

13%Ayurveda

19%

Folk

43%

Homeopathy

8%Unani

10%


Discussion


11

Table 1

Government Institutes dealing with the research and development of the

traditional systems of medicines in India

Name of the Council Different Centers of the

Council

Research activities

Central council for Research in

Ayurveda and Siddha • 8 regional research institute

• 12 research centers

• 60 units and dispensaries

Medico-botanical survey and

development of Ayurvedic and

Siddha medicine based on

folklore uses and their

scientific validation and

implication.


Unani Medicines • 1 central research institute

• 8 regional research institute

• 11 clinical research units

• 5 drug standardisation units

Developing independent and

multidimensional research in to

various fundamental and

applied aspects of Unani

system of medicines.

Central council for research in

Homeopathy • 51 research centers across

the country

Screening of homeopathic

medicine for treating different

ailments and development of

standardisation parameters


Yoga and Naturopathy • Head quaters and central

unit at New Delhi, India

Development and propagation

of natural cure, yoga and

related aspects of yoga and

naturopathy

Council for scientific and

industrial Research and Regional

research laboratories

• Regional research

laboratory, Jammu.

• Central Drug Research

Institute, Lucknow.

• Central Institute of

Medicinal and Aromatic

Plants, Lucknow

Cultivation of medicinal plants,

quality control and

investigation of medicinal

plants and pharmacology

including development of agro-

biotechnological aspects.

2.3 Binomial classification of Amorphophallus commutatus

Kingdom : Plantae

Order : Alismatales

Family : Araceae

Subfamily: Aroideae

Tribe : Thomsonieae

Genus : Amorphophallus

Speices : commutatus


Discussion


12

2.4 Properties of Araceae

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

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

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

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

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

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

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

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

Monstera deliciosa; many Philodendron and Anthurium species, Zantedeschia

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

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

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

including species of Amorphophallus have significant potential in diet. Their

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

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

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

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

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

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

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

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

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

2.5 Properties of Amorphophallus

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

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

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

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

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


Discussion


13

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

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

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

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

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

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

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

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

2010).

Figure 3

Habit of Amorphophallus commutatus

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

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

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

Amorphophallus commutatus with vegetative structure and corm with inflorescence.

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

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

developed intrest in choosing the plant for this research.

a. Habit with vegetative

structure

b. Corm with inflorescence c. Habit

Review of Literature


Discussion


14

2.6 Ethnopharmacological utilisation of Araceae

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

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

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

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

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

(Kala, 2005).

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

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

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

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

(Park et al., 2011).

Documentation and determination of consensus about phytotherapeutic

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

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

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

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

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

3-4 days for lack of estrus.

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

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

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

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

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

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

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

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

Mehrotra, 2002).


Discussion


15

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

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

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

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

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

1995).

2.6.1 Ethnopharmacological utilization of Amorphophallus sps

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

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

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

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

(Ayyanar and Ignacimuthu, 2011).

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

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

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

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

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

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

Two araceae member Amorphophallus companulatus and Acorus calamus L

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

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

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

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

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

Sharma, 1998).

The rhizome of Amorphophallus bulbifer was powdered with that of turmeric

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

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


Discussion


16

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

et al., 1995).

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

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

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

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

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

asthma (Ram et al., 2011).

Amorphophallus companulatus has been identified to be used traditionally in

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

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

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

According to the authors there are no previously identified phytoconstituent

(Haque et al., 2000).

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

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

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

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

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

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

2.7 Bioactivity

2.7.1 Emergence of free radicals in biological systems

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

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

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

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

(O2 •−

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

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


Discussion


17

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

leading to the accumulation of lipid peroxides.

Figure 4

An unbalance between the procuction of prooxidants and antioxidants

in the cell lead to serious cellular damage

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

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

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

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

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

Figure 5

Antioxidant neutralizing a free radical

Glutathinone reductase

Glutathinone peroxidase


Discussion


18

Lipid peroxidation consists of a series of free radical mediated chain

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

(Ak and Gulcin, 2008).

Figure 6

Mechanism of Lipid peroxidation

Figure 7

The proposed reaction for chelating of ferrous ions by curcumin

(Ak and Gulcin, 2008)


Discussion


19

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

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

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

HO• generating Fenton type reactions.

Fe2

+ + H2O2 Fe3

+ + OH

− + OH

•

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

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

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

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

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

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

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

produced by a number of enzyme systems.

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

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

reacting with biological macromolecules and thereby inducing tissue damage (Halliwell

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

peroxidation.

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

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

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

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

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

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

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

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

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


Discussion


20

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

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

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

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

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

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

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

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

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

compounds with excellent hydroxyl radical scavenging ability would be significant for

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

2.7.2 Effect of free radicals on biological system

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

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

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

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

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

Figure 8

Effect of free radical on cells

A B

A. Free radical attacking and weakening a cell

B. Cells with strengthened antioxidant defense


Discussion


21

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

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

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

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

components. The benefits of compounds that possess lipid peroxidation inhibition

is shown in Figure 9.

Figure 9

Advantages of compounds inhibiting lipid peroxidation

2.7.3 Antioxidant enzymes - Natural defense against free radicals

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

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

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

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

deterioration of food and pharmaceutical products during processing and storage

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

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

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


Discussion


22

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

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

hand, requires free radicals for immune system responses.

Figure 10 Figure 11

Antioxidant defense in cells Action of antioxidants on free radicals

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

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

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

and superoxide dismutase and antioxidants like ascorbic acid and tocopherol.

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

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

phytochemicals having antioxidant properties are associated with a lower risk of

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

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

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

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

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

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

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

of oxidants generated within cells.

Catalase

Vitamin E

ββββ - Carotene

Cu / Zn SOD

Lipid Bilayer

Vitamin E+

ββββ - Carotene

Mn SOD + Glutathione peroxidase + GSH

Vitamin E

Vitamin C

Glutathione peroxidase

GSH

Vitamin C & E

ββββ - Carotene


Discussion


23

Figure 12

Initiators and Inhibitors of free radical formation

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

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

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

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

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

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

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

expression (Comhair et al., 2001).

Plant cells are endowed with very important antioxidants such as glutathione

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

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

2003).

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

converts O2•−

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

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

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

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

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


Discussion


24

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 13

Effect of free radicals and antioxidants on a skin cell

All living bodies have a complex antioxidant defence system that includes

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

metabolism produces superoxide anion as a byproduct and superoxide dismutase breaks

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

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


Discussion


25

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

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

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

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

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

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

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

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

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

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

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

2.7.4 Non Enzymic antioxidant systems as natural defense against free radicals

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

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

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

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

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

antioxidants are shown in Table 2.

Table 2

Role of non enzymatic antioxidants

Antioxidant Antioxidant Function

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

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

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

Glutathione Prevention of ROS formation by enzyme GSH-Px

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

and others.

Zinc Prevention of free radical formation (superoxide dismutase enzyme)

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

be bound to protein to reduce oxidative potential.

Manganese Prevention of free radical formation (superoxide dismutase enzyme)

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

protein to reduce oxidative potential.


Discussion


26

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

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

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

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

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

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

Figure 14.

Figure 14

Glutathione dependent defense against ROS at multilevels

2.7.5 Antimicrobial agents – Methods and mechanism

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

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

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

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

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

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

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

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


Discussion


27

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

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

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

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

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

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

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

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

protracted exploration and evaluation of natural products. Use of ethnopharmacological

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

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

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

agents

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

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

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

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

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

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

against unrelated micro-organisms,

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

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

and below 25 µM for pure compounds,

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

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

reference controls) and

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

(‘lead’-status).


Discussion


28

2.7.5.2 Selection of the appropriate bioassay

Different screening approaches are available to identify the primary

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

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

availability of practical and biologically validated laboratory models.

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

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

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

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

(Cos et al., 2006).

Figure 15

General approaches in anti-infective drug screening

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

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

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

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

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

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


Discussion


29

Table 3

Advantages and Disadvantages of different drug screening approaches

2.7.5.3 Antimicrobial agents from araceae

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

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

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

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

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

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

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

2.7.5.4 Antimicrobial agents from Amorphophallus sps.

The dichloromethane extract of Amorphophallus bequaertii was reported to

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

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

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

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


Discussion


30

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

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

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

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

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

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

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

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

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

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

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

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

1994).

The essential oil isolated from the rhizome of Amorphophallus companulatus

inhibited the growth of various strains of Mycobacterium tuberculosis - H37Rv

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

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

The ether extract of the stem of Amorphophallus companulatus has been

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

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

2.7.6 Anticancer agents – Mode of action

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

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

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

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

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

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

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


Discussion


31

Figure 18 DNA Fragmentation

Figure17 Electron micrograph of a normal cell

and an apoptotic cell

Figure 16 Process of Apoptosis

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

industry (Liu, 2003).

Apoptosis is a selective, controlled, and

genetically programmed cell death process that plays an

important role in the balance between cell replication

and cell death. In contrast to necrosis, this tightly

regulated and complex process exhibits some typical

morphological changes, such as chromatin condensation,

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

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

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

Anticancer agents, on

the other hand, are mainly

related to their curative role

in a damaged system. Under

normal conditions, the cells

in which the DNA or other

components are irreversibly

damaged by various causes

undergo apoptotic cell death,


Discussion


32

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 19

Cytotoxicity test systems targeting different cellular locations

2.7.6.1 Anticancer agents from Araceae

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

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

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

CRYSTAL VIOLET


Discussion


33

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

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

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

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

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

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

belonging to Araceae namely Acorus calamus and Acorus gramineus by affinity

chromatography. These lectins showed potent mitogenic activity towards mouse

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

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

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

Amorphophallus companulatus have been screened for phytoconstituent and

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

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

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

2.7.7 Identified bioactivities of Amorphophallus sps

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

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

Amorphophallus konjac as one potential species that contain glucomannan and

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

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

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

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

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

The pharmacotherapeutic efficacy of Garcinia cambogia plus Amorphophallus

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

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

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


Discussion


34

had a significant hypocholesterolemic effect, without influencing the anthropometric or

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

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

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

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

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

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

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

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

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

Figure 20

Role of antioxidants in obesity

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

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

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

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


Discussion


35

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

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

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

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

Three oligosaccharide fractions from the root of Amorphophallus Konjac, which

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

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

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

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

o

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

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

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

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

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

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

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

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

o and insulin secretion

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

model is mainly resulted from environmental free radical scavenging by the

oligosaccharide. Present results also imply the mechanism of clinical Amorphophallus

Konjac hypoglycemic function maybe related with free radical attenuation and lower

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

2.8 Phytochemistry

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

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

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

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

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

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

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


Discussion


36

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

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

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

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

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

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

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

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

precipitation or co-precipitation thereby hampering proper performance and

interpretation of the bioassay. Introducing pH differences may further enhance

separation of acid, neutral and basic constituents.

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

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

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

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

activity or cytotoxicity and fractions that show selective antimicrobial activity.

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

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

2.8.1 Molecules identified in Araceae/Amorphophallus sps

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

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

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

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

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

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

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

made.

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

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


Discussion


37

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

most abundant Amorphophallus species in Vietnam forest) directly with

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

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

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

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

optimal conditions for carboxymethylation of glucomannan were pH 10, temperature

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

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

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

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

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

activity and inhibiting the production of pro inflammatory cytokines by human

monocytes (Tanaka et al., 2003).

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

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

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

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

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

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

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

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

were found and characterized using dimethyl disulfide derivatization to locate the

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

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

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

and Soukup, 2004).

Konjac glucomannans (KGM) have been isolated from Amorphophallus tubers

(used three Amorphophallus species: Amorphophallus panomensis, Amorphophallus


Discussion


38

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

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

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

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

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

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

and molecular weight (Mw) of glucomannan isolated from Amorphophallus

paeoniifolius, Amorphophallus panomensis and Amorphophallus tonkinensis were

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

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

(An et al., 2010).

2.9 Conclusion

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

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

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

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

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

standardizations will provide proper scientific validation and significance to the

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

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

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

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

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

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

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

have gained importance only after scientific validation. The plant Amorphophallus

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

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

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

of the plant.


discussion review of literature -...

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