1

CHAPTER 1

BIOLOGICAL ACTIVITY OF EXTRACTS OF

TRIANTHEMA DECANDRA

1.1 INTRODUCTION

Plant materials have provided the models for 25-50% Western drugs

[1, 2]. Many commercially proven drugs used in modern medicine were initially

used in crude form in traditional or folk healing practices, or for other purposes that

suggested potentially useful biological activity. Plant derived medicines are

relatively safer than synthetic alternatives, offering profound therapeutic benefits

and more affordable treatment [3]. Modern clinicians are now inclined towards the

use of herbal medicines due to efficiency of a number of phytopharmaceuticals and

herbal drugs which is often enhanced by the presence of many minor constituents

producing synergistic action, which results in lower side effects in comparison to

many allopathic modern drugs [4].

Drug resistance in pathogenic bacteria and fungi has increasingly been

reported all over the world [5]. The increasing prevalence of multidrug resistant

strains of microorganisms creates an urgent need to search for new sources of

antimicrobial agents [6, 7] with other strategies such as regulated and rational use of

antibiotics [8] to combat this crisis. Many plant extracts have proven to be good

antimicrobial agents and the active components in those extracts could be a new

source of antimicrobial compounds [9]. Therefore it is necessary to evaluate the

potential use of folk medicine for the treatment of infectious diseases.

2

Isolation of active principles from medicinal plants and characterization

can be traced from the beginning of 19th century. Large number of drugs from

medicinal plants were discovered and introduced in modern pharmacopoeias during

1850-1950. During this period there was outstanding contribution of fine drugs from

higher plants like reserpine, deserpidine and rescinnamine from Rouvolfia

serpentina, vincristine and vinblastine from Catharanthus roseus [10]. Ephedrine

was isolated from Ephedra sinica in 1887 and later introduced as drug in 1925.

Likewise, from opium and morphine were isolated in 1820 and introduced as drug in

1825. In some cases, the crude extracts of medicinal plants may be used as

medicament. On the other hand, the isolation and identification of the active

principles and elucidation of the mechanism of action of a drug is of paramount

importance. More than 121 major plant drugs have been identified for which no

synthetic one is currently available [11].

In herbal medicine, crude plant extracts in the form of decoction, tincture

or herbal extract are traditionally used for the treatment of various diseases.

Although their efficacy and mechanisms of action have not been tested scientifically

in most cases, these simple medicinal preparations often mediate beneficial

responses due to their active chemical constituents [12]. Plant-derived products

contain a great diversity of phytochemicals that are known to possess antibacterial,

antioxidants, antimutagenic, anticarcinogenic, antithrombotic and vasodilatory

effects [13]. Plant-derived antioxidants such as tannins, lignans, stilbenes,

coumarins, quinones, xanthones, phenolic acids, flavones, flavonols, catechins,

anthocyanins and proanthocyanins could delay or prevent the onset of degenerative

diseases because of their redox properties, which allow them to act as hydrogen

donors, reducing agents, hydroxyl radicals (OH�) or superoxide radical (O2�)

scavengers [14]. In addition to antioxidant activity, several studies demonstrated the

antimicrobial activity of phenols and/or phenolic extracts making them a good

alternative or supplement to the existing antibiotics [15].

Baker et al. (1995) [16], suggested the following criteria for investigating

medicinal potential of plants i) evidence suggesting the traditional usage of the plant

3

by the native population, ii) the purpose for which it is being used,

iii) the abundance of the specific plant species in nature and iv) the sustainable

utilization of the plant. One of the plant families that measure up to the criteria set

by Baker et al. and needs special consideration, is Aizoaceae. Besides the role that

many species in this family play as soil stabilizers, their leaves are traditionally used

as fodder while extracts from these plants are used as preservatives and as a remedy

against throat infections [17].

The species Carpobrotus edulis, traditionally utilized for its medicinal

properties,the leaf juice from C. edulis is widely used as a traditional remedy for a

wide range of fungal and bacterial infections [17] and in the treatment of sinusitis,

diarrhea, infantile eczema, tuberculosis and other internal chest conditions [18].

According to Roberts (1995) [19] the leaf juice is also effective in soothing itching

caused by spider and tick bites. The leaves contain an astringent antiseptic juice

which can be taken orally for treating sore throat and mouth infections [20]. In the

coastal regions of South Africa it is best known for its use as a soothing remedy for

wounds and burns [19]. Several members of Aizoaceae are edible, including

Carpobrotus edulis and Mesembryanthemum crystallinum. Tetragonia

tetragonioides known as New Zealand spinach is grown as a garden plant in dry

climates and used as an alternative to spinach.

Trianthema decandra (Aizoaceae) is a prostrate herb distributed in the

Southern parts of India with opposite pairs of unequal leaves, flowers are borne in

dense clusters. Fruit is a capsule with black seeds. The habit of T. decandra is

shown in Plate 1.1. The plant grows as a weed commonly known as “Gadabani” in

Hindi and “Vellai sharunnai” in Tamil. The roots of the plant are well-known as an

aperient [21] and reported to be useful in hepatitis, asthma and in orchitis [22, 23].

The leaves are eaten during the times of scarcity [24], they contains huge amount of

vitamin C which is used in the treatment of edema [25]. A decoction of this herb is

used in the treatment of rheumatism and as an antidote to alcoholic poison [26].

4

Plate 1.1 Habit of Trianthema decandra

Systematic position of Trianthema decandra:

Kingdom Plantae Subkingdom Viridaeplantae Phylum Tracheophyta Subphylum SpermatophytinaInfraphylum Angiospermae Class Magnoliopsida Subclass Caryophyllidae Superorder Caryophyllanae Order Caryophyllales Family AizoaceaeGenus TrianthemaSpecies decandra L.

5

1.2 REVIEW OF LITERATURE

1.2.1 Importance of Medicinal Plants

Plants are not only important to the millions of people to whom

traditional medicine serves as the only opportunity for health care but also as a

source of new pharmaceuticals [27]. The earliest mention of the medicinal use of

plants has been found in “Rigveda” which were written between 4000 and 1600

B.C. The traditional Indian system of medicine, namely Ayurveda which involves

dispensing of herbal and plant products in various forms such as powders, extracts

and decoctions, dates back to the vedic period, around 2000 BC where the first

mention of diseases and drugs is found. The earliest comprehensive descriptions of

Ayurveda are available in the Atharvaveda (1600-1000BC) which contains interlaid

description of human anatomy, rudiments of classifications of diseases and

reference to herbal medicine [28].

India is one among the world’s 12 biodiversity centers with over 45,000

different plant species. Of these, about 15000-20000 plants have good medicinal

value. In India about 7300 plant species are used in traditional health care systems

[29]. Among ancient civilizations, India has been known to be rich source of

medicinal plants. The forests in India are the principal repository of large number of

medicinal and aromatic plants, collected as raw materials for manufacture of drugs

and perfumery products. About 8000 herbal remedies have been codified in

Ayurveda. The Rigveda has described properties and uses of 67 medicinal plants,

Yajurveda, 81, Atharvaveda, 290, Charak Samhita, 1100 and Sushrut Samhita, 1270

species, in compounding of drugs. These are still used in the classical formulations,

in the Ayurvedic system of medicine [30].

Today over three-quarters of the world population relies mainly on plants

and plant extracts for health care. The global trade in medicinal plants is of the order

of US$ 800 million per year. Export statistics available indicate that, India exports

more than 32,600 tonnes of crude drugs valued at $US 46 million [31]. It is

estimated that world market for plant derived drugs may account for about

6

Rs. 200000 crores [32]. All the major herbal-based pharmaceutical companies are

showing a constant annual growth of about 15 percent [33].

Plants constitute one of the major raw materials for drugs for treating

various ailments of human beings, although there has been significant development

in the field of synthetic drug chemistry and antibiotics [28]. The use of plant

extracts, as well as other alternative forms of medical treatments, is enjoying great

popularity in late 1990s [34].

1.2.2 Antimicrobial Activities of Medicinal Plants

During the last two decades considerable changes have taken place in the

medicinal system all over the world. The interest in antimicrobial plant extracts can

be attributed to three factors, first, it is very likely that these phytochemicals will

find their way into the arsenal of antimicrobial drugs prescribed by physicians, the

second, the public is becoming increasingly aware of problems with the over

prescription and misuse of traditional antibiotics, and third the increase in resistance

of microbes to the available antibiotics. A contemporary (2002-2003) national

collection of 2100 strains of Streptococcus pneumoniae obtained from 30 sites in the

United States (US) census regions were tested to determine the comparative

antimicrobial properties of amoxicillin/clavulanate and 15 other antimicrobials. The

rank order of antimicrobials with the lowest susceptibility rates was penicillin <

trimethoprim/sulphamethoxazole < macrolides < orally administred cephalosporins.

Blood stream infection isolates were more susceptible than strains isolated from

community-acquired respiratory tract infections; penicillin, erythromycin and

trimethoprim / sulphamethoxazole [35].

Antimicrobials are a chemical compound biosynthetically or

synthetically produced, which either destroys or usually suppress the growth or

metabolism of a variety of microscopic or submicroscopic forms of life [36]. The

basic mechanisms of antibiotic action against bacterial cells include i) inhibition of

cell wall synthesis (Penicillins); ii) inhibition of protein synthesis (Tetracyclines);

iii) alteration of cell membrane (Polymyxins); iv) inhibition of nucleic acid

7

synthesis (Quinolones); and v) antimetabolite activity (Sulfonamides) of which

inhibition of cell wall synthesis is the most common [37].

There are many reports on antimicrobial activity of plant extracts

(Table 1.1). Leaf extract of Ziziphus mauritiana showed highest activity against

E. coli and least activity were observed against S. aureus, P. fluorescens and

B. subtilis (ref). Bark extract of Tinospora cordifolia showed varied of antifungal

and antibacterial property reacording zone of inhibition from 10-14 mm. Root and

leaf extract of Withania somifera showed almost similar antibacterial activity

against all the tested bacteria [38]. Many authors concluded that plants containing

high concentrations of polyphenols, alkaloids, tannins, sterols/terpenes, saponins

and glycosides demonstrated very strong anti-microbial properties [39-41].

The Family Aizoaceae contains 135 genera and about 1900 species. They

are commonly known as “stone plants”, “carpet weeds” or “ice plant”. Carpobrotus

edulis, C. acinaciformis, C. mellei and Sesuvium portulacastrum are some of the

examples of this family that demonstrated appreciable activity against various

microorganisms [42-45]. Growth of Staphylococcus aureus, Escherichia coli,

Bacillus subtilis, Proteus vulgaris and Pseudomonas aeruginosa was inhibited by

methanolic extract of T. decandra [46]. Chloroform extract of T. decandra was

investigated for its anti-inflammatory activity using rats in carrageenan induced

edema and cotton pellet induced edema which showed significant activity when

compared to the control animals [47].

8

Tab

le 1

.1 A

ntim

icro

bial

act

ivity

of e

xtra

cts o

f diff

eren

t pla

nts

Mic

roor

gani

sms

Plan

t Fa

mily

E

xtra

ct

Zone

of i

nhib

ition

(m

m)

Ref

eren

ce

Stap

hylo

cocc

us a

ureu

sC

urcu

ma

long

a Zi

ngib

erac

eae

Etha

nol

1048

Salm

onel

la ty

phi

Alpi

nia

gala

nga

Zing

iber

acea

e Et

hano

l 11

48St

rept

ococ

cus f

aeca

lis

Paliu

rus s

pina

Chr

isti

Mill

R

ham

nace

ae

Etha

nol

1449

Can

dida

alb

ican

s Vi

smia

laur

entii

De

Wild

G

uttif

erae

M

etha

nol

1650

Prot

eus v

ulga

ris

Pseu

dogn

apha

lium

sp.

Ast

erac

eae

Dic

hlor

omet

hane

1251

Stap

hylo

cocc

us a

ureu

sC

roto

n ur

ucur

ana

Euph

orbi

acea

e Et

hano

l 21

52C

andi

da a

lbic

ans

Sant

olin

a ch

amae

cypa

rissu

sC

ompo

sitae

H

exan

e 20

53Es

cher

ichi

a co

li Lu

dwig

ia su

ffrut

icos

a O

nagr

acea

e Et

hano

l 19

54St

rept

ococ

cus

pyog

enes

Car

pobr

otus

edu

lis

Aiz

oace

aeEt

hyl a

ceta

te

1455

Baci

llus s

ubtil

is

Acac

ia n

ilotic

a Fa

bace

aeM

etha

nol

20

38

Pseu

dom

onas

ae

rogi

nosa

Ac

hyra

nthe

s asp

era

Am

aran

thac

eae

Met

hano

l

1456

Kle

bsie

lla p

neum

onia

eVi

tex

negu

ndo

Ver

bena

ceae

Et

hano

l 11

57Pr

oteu

s mir

abili

s C

assi

a oc

cide

ntal

is

Faba

ceae

Met

hano

l 15

58K

lebs

iella

sp.

Cym

odoc

ea se

rrul

ata

Cym

odoc

eace

ae

Hex

ane

1559

Esch

eric

hia

coli

Zing

iber

offi

cina

le

Zing

iber

acea

e Et

hano

lic

1260

Pseu

dom

onas

ae

rogi

nosa

C

aric

a pa

paya

C

aric

acea

e W

ater

15

.761

9

Tab

le 1

.1 (C

ontin

ued)

Mic

roor

gani

sms

Plan

t Fa

mily

E

xtra

ct

Zone

of i

nhib

ition

(m

m)

Ref

eren

ce

Salm

onel

la ty

phi

Andr

ogra

phis

serp

yllif

olia

A

cant

hace

ae

Leaf

ext

ract

8

62C

andi

da a

lbic

ans

Lobe

lia n

icot

iana

efol

ia

Com

posit

ae

Chl

orof

orm

11

.263

Stre

ptoc

occu

s fae

calis

Ec

lipta

alb

a A

ster

acea

e M

etha

nol

1864

Baci

llus s

ubtil

isC

urcu

ma

long

a Z

ingi

bera

ceae

M

etha

nol

2265

Stap

hylo

cocc

us a

ureu

s P

lant

ago

lanc

eola

ta

Plan

tagi

nace

ae

Ethy

lace

tate

14.3

66Ps

eudo

mon

as

aero

gino

sa

Alte

rnan

ther

a ph

iloxe

roid

esA

mar

anth

acea

e W

ater

17

.167

Stap

hylo

cocc

us a

ureu

sC

assi

a au

ricu

lata

L

Faba

ceae

Dic

hlor

omet

hane

12

.768

Vibr

io c

hole

rae

Lant

ana

cam

era

Ver

bena

ceae

W

ater

11

69Ba

cillu

s cer

eus

Mel

ia a

zeda

rach

M

elia

ceae

Met

hano

l 17

.670

Prot

eus v

ulga

ris

Pipe

r bet

el

Pipe

race

ae

Etha

nol

1071

Stre

ptoc

occu

s fae

calis

Al

lium

sativ

um

Lilia

ceae

Etha

nol

3072

Prot

eus v

ulga

ris

Andr

ogra

phis

nee

sian

a A

cant

hace

ae

Ace

tone

8

73St

rept

ococ

cus m

utan

s Te

rmin

alia

che

bula

C

ombr

etac

eae

Met

hano

l

2374

10

1.2.3 Objectives of the Study

The objectives of the study are

� To extract the phytochemical constituents from the leaves of

Trianthema decandra using solvents of increasing polarity.

� To study the antimicrobial activity of the extracts against selected

bacterial and fungal strains.

� To determine the Total Phenolic Content (TPC) and free radical

scavenging activity of the extracts.

1.3 MATERIALS AND METHODS

1.3.1 Plant Material

The leaves of T. decandra L. were collected from Nagerkoil district,

Tamil Nadu, India during June 2008. The plant was taxonomically identified and

authenticated by the Botanical Survey of India, Coimbatore, Tamil Nadu, India and

voucher specimen No.BSI/SRC/5/23/10-11/Tech.975 (Plate 1.2) is deposited in

Plant Tissue Culture Laboratory, SRM University for future reference.

1.3.2 Extraction

Leaves of Trianthema decandra were washed with distilled water to

remove dirt and soil and were shade dried. The dried plant material was powdered

and sieved through a 40-mesh sieve. The coarse powder (500g) was extracted

initially with petroleum ether (35ºC) and then extracted three times each, with

chloroform (59.5-61.5�C), ethyl acetate (76.5-77.5�C), 100% ethanol (79�C), 70%

ethanol (78.4-100�C) and water (100�C) sequentially using a Soxhlet apparatus.

The solvents from various extracts were then concentrated in a rotary vacuum

evaporator at reduced pressure at temperature below 40�C.

11

Plate 1.2 Botanical Survey of India Certificate

12

1.3.3 Source of Microorganisms

Strains of bacteria including Staphylococcus aureus (MTCC 29213),

Streptococcus faecalis (MTCC 0459), Enterococcus faecalis (MTCC 2729),

Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 1035), Salmonella

typhi (MTCC 98), Vibrio cholerae (MTCC 3906), Proteus vulgaris (MTCC 1771)

and Bacillus subtilis (MTCC 121), Yersinia enterocolitica (MTCC 840), yeast,

Candida albicans (MTCC 183) and fungus Cryptococcus neoformans (MTCC 1346)

were obtained from Institute of Microbial Technology, Chandigarh.

1.3.4 Antimicrobial Susceptibility Tests

Antimicrobial susceptibility test was carried out using the disc diffusion

assay, followed by the determination of the minimal concentrations of the extracts

that could inhibit the growth of bacteria.

1.3.4.1 Disc Diffusion Assay

Antimicrobial susceptibility tests measure the ability of an antibiotic or

other antimicrobial agent to inhibit bacterial growth in vitro which can be estimated

by the diffusion method.

Materials required

1. Extracts of T. decandra

2. Nutrient Agar Medium

Glucose - 5g

Beef extract - 3g

Peptone - 5g

Sodium chloride (Nacl) - 5g

Agar -15g

Distilled water - 1L

pH -7.0

13

3. Sabourad’s Dextrose Agar Medium

Glucose - 20g

Peptone -10g

Agar -15g

Distilled water -1L

pH - 6.5

4. Sterile Whatman No.1 filter paper disc

5. 1% Dimethyl sulfoxide (DMSO)

6. Chloramphenicol

7. Nystatin

Procedure

All bacterial strains were cultured on nutrient agar plates and incubated at

37°C while fungal strains were cultured on Sabourad’s dextrose agar plates and

incubated at 25°C. Overnight grown cultures of the selected bacterial and fungal

strains were inoculated in respective media and incubated at specified temperature

for 24h and were used for the disc diffusion assay [75]. Sterile media plates were

swabbed with 100µL the overnight grown cultures of selected microorganims.

Sterile What-man No.1 filter paper discs (6mm diameter) were impregnated with

20�L (1mg/mL) each of six different extracts of T. decandra in dimethyl sulfoxide,

air dried and placed on the seeded agar plates. The plates containing bacterial swabs

were incubated at 37°C and those with fungal swabs were incubated at 25°C for 24h.

Chloramphenicol and nystatin (30µg) were used as positive control for bacterial and

fungal strains respectively while 1% DMSO was used as negative control.

Triplicates were maintained and diameters of inhibition zone (DIZ) were measured

for each of the organisms and expressed as mean ± S.D.

14

1.3.4.2 Determination of Minimal Inhibitory Concentration (MIC)

For quantitative estimates of antibiotic activity, antibiotics with different

dilutions are added to the broth and inoculated with the test organism. The least

concentration of the antibiotic that prevents growth after overnight incubation is

taken as the MIC of the extract. Resazurin is a redox indicator, a blue non-

fluorescent dye that turns pink and fluorescent on reduction to give resorufin.

Reduction to pink colour is brought about by oxidoreductases in the viable of cells

and retention of blue colour is indicative of non viablility.

Materials required

1. Extracts of T. decandra

2. Nutrient broth

3. Sabourad’s Dextrose broth

4. 1% Dimethyl sulfoxide (DMSO)

5. Chloramphenicol

6. Nystatin

7. Resazurin solution - 6.75 mg/mL of water

Procedure

Minimum inhibitory concentration was determined in nutrient broth

supplemented with resazurin according to the method described by Sarker [76]. All

the test samples including standard drugs were initially dissolved in DMSO and

added to nutrient broth to a final concentration of 1250 �g/100µL. This was serially

diluted by twofold, to obtain concentration ranging from 1250 �g to 9.762 �g/100 µL.

One hundred µL of each was then added to wells in 96-well microtitre plate

containing 95�L of respective broth and 5�L of standard inoculum at a density of

2x105 CFU/mL of the selected bacterial and fungal strains. To each well 10µL of

resazurin indicator solution was added. The plates were covered with a sterile plate-

15

sealer, agitated to mix the contents using a plate shaker and incubated at respective

temrature or 24h. Chloramphenicol and nystatin were serially diluted by twofold, to

obtain concentration ranging from 200 �g to 1.56�g/100µL and served as positive

controls for bacteria and fungi respectively. Wells without extract, with 1% DMSO

served as a negative control. The experiment was carried out in triplicates and

microbial growth was determined by observing the change of colour in the wells.

The least concentration which did not show growth of organisms in the wells was

considered to be MIC.

1.3.5 EVALUATION OF ANTIOXIDANT ACTIVITY

1.3.5.1 Estimation of Total phenolic content (TPC)

Principle

Total phenolic content of an extract is evaluated spectrophotometrically

using Phosphotungstat-phosphomolibdenum complex commonly referred to as Folin -

Ciocalteu reagent. Oxidation and reduction reaction of phenolat ion takes place in

alkaline conditions. The reduction of Folin-Ciocalteu reagent by phenolat ion causes

color change from yellow to blue which is measured spectrophotometrically at 765 nm.

Materials required

1. Extracts of T. decandra

2. Folin - Ciocalteau reagent - Diluted 10-fold with distilled water

3. 7.5% Sodium carbonate

7.5 g of sodium carbonate was dissolved in 100ml of distilled water

4. Ethanol

5. Gallic acid

10 mg of gallic acid was dissolved in 100mL of distilled water

16

Procedure

The total phenolic content of the extracts was determined according to

Singleton & Rossi [77]. An aliquot of 400 µL of each extract of T. decandra

dissolved in ethanol were taken in test tubes. To the test tubes 1.0 mL of Folin-

Ciocalteu reagent and 0.8 ml of 7.5% sodium carbonate were added, mixed

thoroughly and allowed to stand for 30 min at room temperature. The absorption

was measured at 765 nm against an ethanol blank. Triplicates were maintained. The

total phenolic content was expressed as mg/g gallic acid equivalents in of extract

calculated from a standard graph of gallic acid. Values are expressed as mean ± S.D.

1.3.5.2 Free radical scavenging activity

Principle

Free radical scavenging activity was studied using 2, 2-diphenyl-1-

picrylhydrazyl (DPPH). 2, 2-diphenyl-1-picrylhydrazyl is a stable free radical which

is reduced to DPPH-H on reaction with antioxidants. The scavenging reaction

between DPPH and an antioxidant (H-A) can be written as:

(DPPH) + (H-A) DPPH-H + (A)

DPPH is purple in colour and shows a characteristic absorption at 517 nm. On

scavenging of the free radical by hydrogen donation, changes colour to light yellow

resulting in a decrease in absorbance at 517 nm. The degree of discoloration is

directly proportional to the scavenging potential of the antioxidant.

Materials Required

1. Extracts of T. decandra

2. 2,2-diphenyl-1-picrylhydrazyl (DPPH) (100 µM)

3.9mg of DPPH was dissolved in 100mL of methanol

17

Procedure

The antioxidant activity of the plant extract was estimated according to

Chen [78]. To 2 mL of DPPH solution, 2 mL of extracts of T. decandra were added.

The reaction mixture was incubated in the dark for 15 min and optical density was

recorded at 517 nm against the methanol blank. The decrease in optical density of

DPPH on addition of test samples in relation to the control was used to calculate the

antioxidant activity, using the formula

control sample

control

(A A )Radical scavenging (%) 100

A�

� �

Triplicates were maintained and values are expressed as mean ± S.D.

1.4 RESULTS AND DISCUSSION

1.4.1 Extraction

The leaves of T. decandra were subjected to extraction using solvents of

increasing polarity namely petroleum ether (PE), chloroform (CHCl3), ethyl acetate

(EtOAc), ethanol (EtOH) and water. The yield of each extract was 2.56, 1.44, 1.36,

0.16, 21.08 and 3.24% respectively

1.4.2 Antimicrobial activity

In the past few decades the research for new anti-infectious agents has

occupied the field of ethnopharmacology. Diffusion methods and dilution methods

have been the most commonly used ones for determining the antimicrobial activity

of an extract. In the present study both disc diffusion assay and broth micro dilution

assay were performed to understand the activity of various extracts of leaves of T.

decandra against the selected microorganism listed in 1.3.3. The results obtained in

these methods are discussed below.

18

1.4.2.1 Disc diffusion assay

All the six extracts mentioned above were tested for antimicrobial

activity against Staphylococcus aureus, Streptococcus faecalis, Enterococcus

faecalis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Vibrio

cholerae (MTCC 3906), Proteus vulgaris (MTCC 1771) and Bacillus subtilis

(MTCC 121), Yersinia enterocolitica, the yeast Candida albicans and the fungus

Cryptococcus neoformans (Table 1.2). Figure 1.1 (a & b) show the zones of

inhibition (mm) recorded for different microorganisms in different extracts of T.

decandra using disc diffusion method. Among the six extracts studied, the

chloroform and ethyl acetate extracts from T. decandra inhibited the growth of all

the organisms tested, but their efficiency in inhibition varied between different

organisms. Streptococcus faecalis, Candida albicans and Cryptococcus neoformans

were inhibited to the maximum extent by the chloroform extract of leaves of T.

decandra, recording values greater than 23 mm diameter of inhibition zone (DIZ)

while Staphylococcus aureus and Candida albicans were inhibited to the same

extent by ethyl aceatae extract. Chloroform extract showed higher diameters of

inhibition zone (DIZ) ranging from 18.5 ± 0.05 to 23.2 ± 0.04 mm, whereas ethyl

acetate extract showed inhibition in the range of 16.5 ± 0.04 to 23.4 ± 0.05 mm at a

concentration of 20µg/mL. For chloramphenicol and nystatin, DIZ ranged from 18 ±

0.05 to 23.6 ± 0.02 mm at a concentration of 30µg/mL, these were comparable to

DIZ of chloroform and ethyl acetate extracts. Ethanol extracts, 70% ethanol extracts

and aqueous extracts showed relatively less antimicrobial activity. Plate 1.3 and 1.4

show the zones of inhibition of chloroform and ethyl acetate extracts of T. decandra.

Borchers et al., 2004 [79] reported that crude extracts of plants are more

beneficial than isolated constituents, since a bioactive individual component can

change its properties in the presence of other compounds present in the extracts.

Artemisia douglasiana leaf extract exhibited antimicrobial activities against B.

cereus, S. aureus, E. coli, P. aeruginosa, C. albicans and A. niger [80]. The extracts

or compounds that can either inhibit the growth of pathogens or kill them with no or

least toxicity to host cells are considered as good candidates for developing new

antimicrobial drugs [81]. Pseudomonas aeruginosa (ATCC12462) was the most

19

sensitive strain to the methanol bark extract of Treculia africana and exhibited the

maximum zone of inhibition diameter of 21 mm at 250 mg/mL with ofloxacine

exhibiting 26 mm at 30µg/mL [82].

The diameter of zone of inhibition is based upon various factors

including the pathogen susceptibility, antibiotic diffusion effects, agar depth, pH,

size of the inoculated organism and presence of other metals. The rate of diffusion

of an antibiotic through the agar is not always same. The rate of diffusion of the

antimicrobial through the agar is dependent on the concentration of antibiotic,

molecular weight of antibiotic, solubility properties of antibiotic, pH and ionization,

binding upon agar. Larger molecules diffuse at a slower rate than lower molecular

weight compounds. These factors, in combination, result in each antimicrobial

having a unique breakpoint zone size indicating susceptibility to that antimicrobial

compounds [83].

In the present study, the DIZ varied for different extracts against the

selected microorganism which might be attributed to the rate of diffusion through

the agar. Since the compositions of the extracts are not known, the precise

component and/or its concentration inhibiting the growth of the microorganism

cannot be ascertained. Hence in the next step we have used the broth micro dilution

method to determine the minimum concentration of the extract that could inhibit the

growth of selected microorganisms as a measure to test the susceptibility of the

microbes to the extracts.

20

Tab

le 1

.2 D

iam

eter

of z

ones

of i

nhib

ition

rec

orde

d fo

r di

ffer

ent m

icro

orga

nism

s in

vari

ous e

xtra

cts o

f Tri

anth

ema

dean

dra

Zone

of i

nhib

ition

(Dia

met

er in

mm

)

S.

No

Nam

e of

the

mic

roor

gani

sms

PE

extr

act

CH

Cl 3

extr

act

EtO

Ac

ex

trac

t E

tOH

extr

act

70%

EtO

H

extr

act

Wat

er e

xtra

ct

DM

SO

Con

trol

Stan

dard

dr

ug1

Stap

hylo

cocc

us a

ureu

s20

±0.0

4b20

±0.0

4b 23

.4±0

.04a

20±0

.04b

5±0.

06d

--

19±0

.07c

2St

rept

ococ

cus f

aeca

lis15

±0.0

5e 23

.2±0

.05a

21.5

±0.0

7c 18

±0.0

6d12

±0.0

4f-

-23

±0.0

5b

3En

tero

cocc

us fa

ecal

is13

±0.0

4b21

±0.0

5a 21

±0.0

5a 3±

0.02

d 11

±0.0

5c 1±

0.02

e-

21±0

.05a

4Es

cher

ichi

a co

li13

±0.0

7f 20

.5±0

.07c

21.5

±0.0

5b 19

±0.0

5d15

±0.0

4e 3±

0.07

g-

23±0

.05a

5Vi

brio

cho

lera

e15

±0.0

6d18

±0.0

4c 18

.5±0

.04b

3±0.

03f

13±0

.05e

3±0.

01f

-19

±0.0

4a

6Ps

eudo

mon

as a

erug

inos

a23

±0.0

5a19

±0.0

5c 16

.5±0

.05d

10±0

.04e

6±0.

03f

3±0.

02g

-20

±0.0

4b

7Sa

lmon

ella

typh

i20

±0.0

5a18

.5±0

.05b

20±0

.05a

11±0

.05d

10±0

.04e

2±0.

05f

-17

.0±0

.05c

8Ba

cillu

s sub

tilis

15±0

.06d

18±0

.05b

17±0

.04c

12±0

.05e

11±0

.05f

2±0.

02g

-21

±0.0

5a

9Pr

oteu

s vul

gari

s13

±0.0

4e 21

±0.0

7b 18

±0.0

7c 14

±0.0

5d6±

0.04

f 3±

0.04

g-

23.6

±0.0

7a

10Ye

rsin

ia e

nter

ocol

itica

16±0

.03d

20±0

.06b

20±0

.05b

17±0

.04c

14±0

.03e

3±0.

04f

-23

±0.0

4a

11C

andi

daal

bica

ns17

±0.0

5c23

±0.0

4a 23

±0.0

4a 10

±0.0

4d10

±0.0

5d 2±

0.04

e-

20±0

.05b

12C

rypt

ococ

cus n

eofo

rman

s15

±0.0

4d23

±0.0

5a 21

±0.0

5b 10

±0.0

5f 11

±0.0

2e-

-19

±0.0

6c

Val

ues

are

expr

esse

d as

Mea

n ±

S.E.

M. (

n =

3) a

nd v

alue

s fo

llow

ed b

y sa

me

lette

r do

not

diff

er s

igni

fican

tly a

t th

e p

< 0.

05 a

s

dete

rmin

ed b

y D

unca

n’s M

ultip

le R

ange

Tes

t.

- in

dica

tes

no in

hibi

tion.

PE

- Pe

trole

um e

ther

ext

ract

, CH

Cl 3

- C

hlor

ofor

m e

xtra

ct,

EtO

Ac

- Et

hyl a

ceta

te e

xtra

ct, E

tOH

- E

than

ol

extra

ct, N

egat

ive

cont

rol -

DM

SO, P

ositi

ve c

ontro

l - C

hlor

amph

enic

ol /N

ysta

tin

21

Figure 1.1(a) Diameter of Zones of inhibition (mm) recorded for different

microorganisms in different solvent extracts of leaves of

T. decandra

22

Figure 1.1(b) Diameter of Zones of inhibition (mm) recorded for different

microorganisms in different solvent extracts of leaves of

T. decandra

23

Plate 1.3 Activity of chloroform and ethyl acetate extracts against different microorganisms depicting zones of inhibition of a) chloroform extract b) ethyl acetate extract c) DMSO control d) Positive control (Chloramphenicol)

24

Plate 1.4 Activity of chloroform and ethyl acetate extracts against different microorganisms depicting zones of inhibition of a) chloroform extract b) ethyl acetate extract c) DMSO control d) Positive control (Chloramphenicol/Nystatin)

25

1.4.2.2 Minimal Inhibitory Concentration (MIC)

Table 1.3 shows the MIC values of the chloroform and ethyl acetate

extracts of leaves of Trianthema decandra for different test organisms. Plate 1.5,

1.6, 1.7 and 1.8 show the microtitre plates indicating MIC of chloroform and ethyl

acetate extracts of T. decandra, negative and positive controls respectively. All the

extracts revealed varying degrees of antimicrobial activity recording different MIC

values for each microorganism. The dose response however varied between different

microorganisms tested.

Table 1.3 Minimum Concentrations of chloroform and ethyl acetate extracts of

T. decandra inhibiting the growth of different microorganisms

MIC (µg/mL)

S.NoName of the

microorganisms Chloroform extract Ethyl acetate extract Standard

1 Staphylococcus aureus 312.5 312.5 50

2 Streptococcus faecalis 312.5 625 50

3 Enterococcus faecalis 156.2 312.5 25

4 Escherichia coli 78.1 312.5 50

5 Vibrio cholerae 78.1 156.2 50

6 Pseudomonas aeruginosa 156.2 312.5 25

7 Salmonella typhi 156.2 156.2 12.5

8 Bacillus subtilis 156.2 312.5 25

9 Proteus vulgaris 156.2 312.5 25

10 Yersinia enterocolitica 312.5 625 6.25

11 Candida albicans 39.05 156.2 3.13

12 Cryptococcus neoformans 156.2 312.5 6.25

Standard - Chloramphenicol /Nystatin

26

From the results it is clear that Candida albicans (MIC: 39µg/mL) was

the most sensitive organism to chloroform extract followed by Escherichia coli

(MIC: 78.1µg/mL) and Streptococcus faecalis (MIC: 312.5µg/mL) while S. typhi, V.

cholerae and C. albicans were sensitive to ethyl acetate extract at 156.2µg/mL. MIC

values for Chloramphenicol and Nystatin ranged from 3.13 to 50µg/mL. The

optimal effectiveness of a medicinal plant may not be due to one main active

constituent, but to the combined action of different compounds originally in the

plant [84].

Plate 1.5 Microtitre plate showing minimum inhibitory concentration of

chloroform extract of T. decandra for the selected

microorganisms

27

Plate 1.6 Microtitre plate showing minimum inhibitory concentration of

ethyl acetate extract of T. decandra for the selected

microorganisms

Plate 1.7 Microtitre plate showing minimum inhibitory concentration of

DMSO control for selected microorganisms

28

Plate 1.8 Microtitre plate showing minimum inhibitory concentration of

positive control (Chloramphenicol/Nystatin) for selected

microorganisms

Methanol extract and solvent soluble fractions from the date palm

showed 50µg/mL as MIC for P. aeruginosa [85]. Plant species used to treat

infections have been tested for antimicrobial activity, from Asteraceae,

Cucurbitaceae, Lamiaceae and Rosaceae and the most used plants were Artemisia

absinthium, Equisetum telmateia, Lavandula stoechas, Melissa officinalis, Tussilago

farfara and Urtica dioica. Extracts were tested in vitro for antimicrobial activity

against S. aueus, S. epidemidis, E. coli, K. pneumoniae, P. aeruginosa, S. typhi, S.

flexneri, P. mirabilis and C. albicans using microbroth dilution technique. This

research showed that Arum maculatum, Datura stramonium, Geranium

asphodeloides and Equisetum telmateia petroleum ether extracts had MIC values of

39.1µg/mL, 78.1µg/mL, 78.1µg/mL and 39.1µg/mL, respectively against S.

epidermidis. Datura stramonium petroleum ether extract had a MIC value of

39.1µg/mL against E. coli and Trachystemon orientalis ethanol extract had a MIC

value of 39.1µg/mL against E. coli [86]. The MIC value for ethanolic extract of

29

Glycyrrhiza glabra was observed at 500µg/mL against Mycobacterium tuberculosis

[87]. The results obtained in the present study are in accordance with the

mentioned research.

1.4.3 Evaluation of Antioxidant Activity

It has been reported that the phenolic compounds possess antioxidant

activity that allows them to scavenge both active oxygen species and electrophiles,

to inhibit nitrosation and to chelate metal ions, to have the potential for auto-

oxidation and the capability to modulate certain cellular enzyme activities [88].

Several studies showed a correlation between antioxidant activity and phenolic

content. Phenolic compounds are an important group of secondary metabolites,

which are synthesized by plants due to plant adaptation in response to biotic and

abiotic stresses [89].

1.4.3.1 Estimation of Total phenolic content (TPC)

Total phenolic content assayed in the extracts of Trianthema decandra

showed the presence of phenols in chloroform and ethyl aceatate extracts of leaves

of T. decandra. The total phenolic contents of petroleum ether, chloroform, ethyl

acetate, ethanol, 70% ethanol and water extracts have shown the values of 19.5 ±

0.5, 74.6 ± 1.26, 58.4 ± 0.7, 20.6 ± 0.75, 32.52 ± 0.86 and 63.7 ± 1.1 mg/g dry

extract respectively.The total phenolic content of different extracts of T. decandra

are depicted in Fig. 1.2. The total phenolic content of methanolic extract of Ocimum

selloi and Ocimum lamiifolium recorded were 42.1 ± 2.6 and 54.6 ± 2.1 mg/g dry

extract respectively [90]. These results on the total phenolic content recorded in the

present study indicate that the extract of leaves of T. decandra, has antioxidant

properties.

30

Figure 1.2 Total Phenolic Content of different extracts of T. decandra

Figure 1.3 Percentage inhibition of DPPH by different extracts of T. decandra

31

1.4.3.2 Free radical scavenging Activity

In order to continue investigating the antioxidant properties of the leaves

of T. decandra, the petroleum ether, chloroform, ethyl acetate, ethanol, 70% ethanol

and water extracts were tested for free radical scavenging activity. The DPPH test is

a very convenient method for screening small antioxidant molecules because the

reaction can be observed visually using common TLC and dot-blot techniques and

also its intensity can be analysed by simple spectrophotometric assays [91, 92].

The results of the free radical scavenging activity of Trianthema

decandra are depicted in Fig. 1.3. Inhibition Percentage (IP) values are considered

to be a good measure of the antioxidant efficiency of extracts. IP values recorded for

petroleum ether, chloroform, ethyl acetate, ethanol, 70% ethanol and water extracts

of T. decandra and butylated hydroxyl anisole are 2.99 ± 0.08, 46.94 ± 0.05, 12.73 ±

0.1, 15.34 ± 0.05, 13.52 ± 0.08, 33.55 ± 0.13 and 85.32 ± 0.24% respectively.

The results of the DPPH free radical scavenging activity assay suggests

that components within the extracts are capable of scavenging free radicals via

electron or hydrogen- donating mechanisms and thus should be potent enough to

prevent the initiation of deleterious free radical mediated chain reactions in

susceptible matrices e.g., biological membranes. The scavenging activity of Ocimum

selloi, Ocimum citriodorum and Ocimum lamiifolium showed 32.3 ± 2.1, 57.3 ± 1.1

and 38.9 ± 2.2% [90]. Typical IP values for plant materials which have

acknowledged potent antioxidant activities are 75% of Cassia fistula, a traditional

Indian medicine [93] and 52-80% for the edible seeds of Rosa rubiginosa [94]. Less

potent materials show IP values in the range 9-14%, as for example the seeds of

Gevuina avelana [94]. In the present study potent free radical scavenging activity

was recorded for water extract followed by moderate activity for chloroform extract.

32

1.5 CONCLUSION

In the present investigation, the phytochemical constituents of

T. decandra were extracted using different solvents namely petroleum ether,

chloroform, ethyl acetate, ethanol, 70% ethanol and water. All the extracts were

screened for antimicrobial activity against Staphylococcus aureus (MTCC 29213),

Streptococcus faecalis (MTCC 0459), Enterococcus faecalis (MTCC 2729),

Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 1035), Salmonella

typhi (MTCC 98), Vibrio cholerae (MTCC 3906), Proteus vulgaris (MTCC 1771),

Bacillus subtilis (MTCC 121), Yersinia enterocolitica (MTCC 840) and fungi such

as Candida albicans (MTCC 183) and Cryptococcus neoformans (MTCC 1346) by

disc diffusion assay and Minimum Inhibitory Concentration (MIC). Among the

extracts analyzed for antimicrobial activity, chloroform and ethyl acetate extracts

possessed good inhibition against all microorganisms tested. Further total phenolic

content and free radical scavenging activity was determined in all the six extracts.

Water and chloroform extracts showed higher phenolic content and free radical

scavenging activity among all the extracts. The effectiveness of a medicinal plant or

a formulation may not be due to one active ingredient, but a combination of all the

ingredients present there in. However to understand the mechanisms underlying the

effectiveness, it is necessary to separate the ingredients and study their activities

using in vitro and in vivo models.