In Vitro Antimicrobial Activity Studiesshodhganga.inflibnet.ac.in/bitstream/10603/15922/11/11...Chapter – 1 : In vitro antimicrobial activity studies Introduction 28 Introduction

Chapter - 1

In Vitro Antimicrobial Activity Studies

Water is H2O, hydrogen two parts, oxygen one, but there is also a third thing, that makes it water

and nobody knows what that is.

- D.H. Lawrence

Chapter – 1 : In vitro antimicrobial activity studies Introduction

28

Introduction

The use of plants and plant products as medicine could be traced far back as

the beginning of human civilization. From ancient literature it is evident that various

parts of the plants were used in Ayurveda, Unani and Siddha medicines for the

treatment of disease of human beings (Das et al., 2009). Medicinal plants constitute

the main source of new pharmaceuticals and health care products (Ivanova et al.,

2005). Extraction and characterization of several active phytocompounds from these

green factories have given birth to some of the high activity profile drugs (Mandal et

al., 2007). It is believed that crude extracts from medicinal plants are more

biologically active than isolated compounds due to their synergistic effects (Jana and

Shekhawat, 2010). Phytochemical screening of plants has revealed the presence of

numerous chemicals including phenols, flavonoids, alkaloids, steroids, saponins and

glycosides. These secondary metabolites of plants serve as medicine in the treatment

of various infectious diseases (Cowan, 1999). Herbal medicines have become more

popular in the treatment of many diseases due to the belief that green medicine is safe,

easily available and with fewer side effects. Given the alarming incidence of

antibiotic resistance in bacteria of medical importance, there is a constant need for

new and effective therapeutic agents which are natural, stable, non toxic and

multifunctional.

A systematic review of literature on research work of A. paniculata,

T. cordifolia and T. foenum-graecum have revealed that there are very few studies

showing the antibacterial activity of these plants, especially the leaves. Surprisingly,

there are no reports on the antifungal activities of these plants on dermatophytes.

Hence, in this study we have screened for the antibacterial activity in different solvent

leaf extracts of these plants using clinical isolates causing urinary tract infections.

Antifungal activity studies were performed on fungi causing dermatomycosis.

Phytochemical screening of the leaf extracts were also carried out to know the active

compounds responsible for the antimicrobial activity. The details of the work carried

out are presented in this chapter.

Chapter – 1 : In vitro antimicrobial activity studies Material and Methods

29

Material and Methods

Plant material

Fresh and healthy leaves of A. paniculata and T. cordifolia were obtained from

local growers of Mysore. T. foenum-graecum leaves were obtained from the local

market, Mysore. The sample specimen was identified based on the taxonomical

characteristics. The leaves were washed thoroughly in distilled water and the surface

water was removed by air drying under shade. The leaves were subsequently dried in

a hot air oven at 40°C for 48 h, powdered to 100-120 mesh in an apex grinder [Apex

Constructions, London] and used for extraction.

Chemicals

Solvents viz., chloroform, hexane, methanol and ethanol were of AR grade

from Merck (Mumbai, India). Nutrient agar, nutrient broth, saborauds dextrose broth

and saborauds dextrose agar were from Himedia Laboratories (Mumbai, India). Ferric

chloride, phenol reagent, α-napthol, sodium carbonate, sodium hydroxide, copper

sulphate, acetic anyhydride, glacial acetic acid, iodine and potassium iodide were

from SD Fine Chemicals (Mumbai, India).

Test microorganisms

The test organisms used were clinical isolates viz., Escherichia coli, Proteus

vulgaris, Klebsiella sp., Staphylococcus aureus, Pseudomonas aeruginosa,

Enterobacter aerogens, Trichophyton rubrum and Epidermophyton floccosum which

were obtained from Department of Microbiology, JSS Medical College, Mysore. The

bacterial and the fungal cultures were maintained on nutrient agar medium and

saborauds dextrose agar (SDA) medium respectively.

Preparation of aqueous extract

Fifty grams each of powdered leaves of A. paniculata, T. cordifolia and

T. foenum-graecum were macerated with 100 ml sterile distilled water in a blender for

10 min. The macerate was first filtered through double layered muslin cloth and

centrifuged at 4000 rpm for 30 min. The supernatant was filtered through Whatman

No.1 filter paper and heat sterilized at 120°C for 30 min. The extracts were preserved

aseptically in brown bottles at 4°C until further use.


30

Preparation of solvent extract

Extraction was carried out according to the method of Okigbo et al. (2005).

Fifty grams each of the powdered material was extracted initially with 300 ml of

chloroform, hexane, methanol and ethanol separately for 24 h at 23 ± 2°C. The extract

was filtered with Whatman No. 1 filter paper into a clean conical flask. Second

extraction was carried out with same amount of solvent for another 24 h at 23 ± 2°C

and filtered. The extracts were later pooled and transferred into the sample holder of

the rotary flash evaporator [Buchi Rotavapor R-124, Switzerland] for the evaporation

of the solvents. The evaporated extracts so obtained were preserved at 4°C in airtight

bottles until further use.

Antibacterial screening

The antibacterial activity was carried out by disc diffusion method (Bauer

et al., 1966). Bacterial cultures (adjusted to 1 × 106 CFU/ml using spectrophotometer)

were used to lawn nutrient agar plates evenly using a sterile swab. The plates were

dried for 15 min and sterile discs (5 mm in diameter, Whatman No.1) impregnated

with 10 µl (1 mg/ml) of the plant extracts were placed on the nutrient agar surface.

10 µl of the respective solvent served as the negative control. Streptomycin standard

antibiotic disc served as the positive control (10 µg/disc). The plates were then

incubated at 37°C for 18-24 h. After overnight incubation the plates were examined

for the zone of inhibition.

Determination of Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) was carried out by broth

dilution method (Brantner and Grein, 1994). The test organisms were grown in

nutrient broth medium to a concentration of 1 × 106 CFU/ml. Extract of about 0.5 ml

(0.25-2 mg/ml) was mixed with 4 ml of nutrient broth inoculated with 0.5 ml of

bacterial suspension. The tubes containing 4.5 ml of broth and 0.5 ml of bacterial

suspension served as bacterial control and 5 ml of un-inoculated broth served as

blank. The tubes were incubated at 37°C for 18 h. Inhibition of bacterial growth was

determined by measuring the absorbance at 600 nm in a colorimeter. The lowest

concentration of the compound that inhibits the growth of the organism was


31

determined as the MIC. The percentage of growth inhibition was calculated according

to the formula:

Per cent growth inhibition = [(Acontrol – Atest ) / Acontrol ] × 100

Antifungal activity assay

Mycelial dry weight method was carried out to determine the antifungal

activity of the ethanol extracts at concentrations ranging from 1 to 10 mg/ml

according to the method of Rasooli and Abyaneh (2004). The dermatophytes grown

on SDA medium for a week were flooded with 0.85% saline. After settling of the

larger particles, conidia were counted with a haemocytometer and diluted in

saborauds dextrose broth to a final spore concentration of 1×106

spores/ml. For

antidermatophytic assay in broth, 5 ml of sterile saborauds dextrose broth medium

taken in screw capped tubes were inoculated with 20 µl of fungal suspension and

1-10 mg/ml concentration of the extract. The tubes were incubated for a week at

30°C. The visible mycelial growth in the tubes expressed the degree of activity of the

extract. Fungal mycelia from the above tubes were separated by passing through

Whatman No. 1 filter paper. The filter paper was allowed to dry at 60°C to reach a

constant weight. Fungal growth inhibition was calculated by considering the control

and sample mycelial dry weights. Ketaconozole was used as a standard antifungal

agent. The percentage of growth inhibition was calculated according to the formula:

Per cent growth inhibition = [(Acontrol – Atest ) / Acontrol ] × 100

Phytochemical screening

Phytochemical analysis of ethanol leaf extracts of A. paniculata, T. cordifolia

and T. foenum-graecum was carried out qualitatively to test for the presence of

carbohydrates, proteins, tannins, phenols, flavonoids, cardiac glycosides, phytosterols,

saponins and alkaloids (Harborne, 1998).

Test for carbohydrates

Molisch’s test: Two drops of Molisch’s reagent was added to 2 - 5 ml of the extract in

a test tube to which 1 ml of concentrated sulphuric acid was allowed to flow down the

side of the tube. Appearance of a violet ring at the junction indicated the presence of

carbohydrates.


32

Test for proteins

Biuret test: To 2 ml of the extract, 2 ml of 10% sodium hydroxide was added and

mixed well. To this, 2 drops of 0.1% copper sulphate solution was added. Formation

of violet or pink colour indicated the presence of proteins.

Test for phenols

FC reagent method: A known volume of the extract was made up to 3 ml with

distilled water to which 0.5 ml of freshly prepared FC reagent was added. After 3 – 5

min, 2 ml of 20% sodium carbonate was added. Appearance of blue colour indicated

the presence of phenols.

Test for flavonoids

Ferric chloride test: To a small amount of extract, neutral ferric chloride solution

was added. Appearance of blackish red colour indicated the presence of flavonoids.

Test for tannins

Ferric chloride test: Extract was treated with alcoholic ferric chloride solution.

Formation of blue colour indicated the presence of tannins.

Test for terpenoids

Libermann Burchard test: Ten milligram of the extract was dissolved in 1 ml of

chloroform and 1 ml of acetic anhydride. Concentrated sulphuric acid (2 ml) was

added along the sides of the test tube. Formation of reddish violet colour ring at the

junction indicated the presence of terpenoids.

Test for steroids

Salkowski test: Extract was dissolved in chloroform and few drops of concentrated

sulphuric acid were added to it. Formation of red colour in chloroform layer suggested

the presence of steroids.

Test for cardiac glycosides

Keller Kiliani test: The extract was treated with 1 ml of ferric chloride reagent

(mixture of 1 volume of 5% ferric chloride and 99 volumes of glacial acetic acid). To

this solution a few drops of concentrated sulphuric acid was added. Appearance of

greenish blue colour indicated the presence of cardiac glycosides.


33

Test for alkaloids

Mayers test: To a small amount of the extract, potassium mercuric iodide solution

was added. Formation of cream precipitate indicated the presence of alkaloid.

Wagners test: To a small amount of extract, iodine-potassium solution was added.

Appearance of reddish brown precipitate indicated the presence of alkaloid.

Test for saponins

Frothing test: The dry extract was vigorously shaken with distilled water and was

allowed to stand for 10 min. Stable froth indicated the presence of saponins.

Statistical analysis

The experimental results are expressed as mean ± standard deviation (SD) of

triplicate measurements. The data was subjected to One Way Analysis of Variance

(ANOVA) and the significance of differences between the sample means was

calculated by Turkeys test. Data was considered statistically significant at P value ≤ 0.05.

Statistical analysis was performed using Graph Pad statistical software.

Chapter – 1 : In vitro antimicrobial activity studies Results and Discussion

34

Results and discussion

Antibacterial screening of solvent extracts

The antibacterial activity of different solvent extracts of A. paniculata leaf

against the pathogenic bacteria showed varied levels of inhibition. As shown in

Table 1.1, among the solvent extracts tested, ethanol extract had a broad spectrum of

activity against all the bacteria tested and showed the highest zones of inhibition

against P. aeruginosa (15.0 mm) and S. aureus (13.0 mm). The least zone of

inhibition was observed with the chloroform extract which showed an inhibition of

1.0 mm against E. coli, 2.2 mm against S. aureus and 3.0 mm against P. aeruginosa

and did not show any inhibition against E. aerogenes, P. vulgaris and Klebsiella sp.

Table 1.1 : Zone of inhibitory activity (in millimeter) of different solvent extracts

of Andrographis paniculata leaves against the test bacteria

Solvent extract E. coli E. aerogenes Klebsiella sp. P. vulgaris S. aureus P. aeruginosa

Aqueous

Ethanol

Methanol

Hexane

Chloroform

Streptomycin

(10µg/disc)

2.0 ± 2.0a

11.0 ± 1.0bc

8.0 ± 2.64b

3.0 ± 1.0a

1.0 ± 0.0a

15.0 ± 2.0c

0.0 ± 0.0a

10.0 ± 1.73c

6.0 ± 1.73b

1.0 ± 1.0a

0.0 ± 0.0a

14.0 ± 1.73d

0.0 ± 0.0a

8.0 ± 3.0bc

5.0 ± 1.0b

0.0 ± 0.0a

0.0 ± 0.0a

11.0 ± 1.72c

1.0 ± 1.0a

9.0 ± 1.70bc

7.0 ± 2.0b

2.0 ± 1.0a

0.0 ± 0.0a

12.0 ± 1.73c

3.0 ± 1.73a

13.0 ± 2.64b

9.0 ± 1.0b

4.0 ± 1.73a

2.2 ± 0.26a

11.0 ± 2.0b

4.0 ± 1.72a

15.0 ± 1.74c

10.5 ± 1.80b

4.5 ± 1.32a

3.0 ± 1.0a

14.0 ± 1.70bc

1 mg/ml concentration of the extract used

Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)

Table 1.2 represents the antibacterial activity of T. cordifolia leaf extracts in

different solvent extracts against the pathogenic bacteria. The highest antibacterial

activity as indicated by the zone of inhibition was achieved with ethanol extract which

showed inhibition zones of 9.0 mm, 6.6 mm,

6.1 mm,

6.0 mm,

12.0 mm and

9.6 mm

against E. coli, E. aerogenes, Klebsiella sp., P. vulgaris, S. aureus and P. aeruginosa

respectively. Among the bacteria tested, S. aureus showed maximum susceptibility to

T. cordifolia ethanol extract. Chloroform and aqueous extracts showed the least

antibacterial activity.


35


of Tinospora cordifolia leaves against the test bacteria


Aqueous 1.0 ± 2.0ab

0.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

3.0 ± 1.0a

2.0 ± 0.0a

Ethanol 9.0 ± 1.0d

6.6 ± 0.57d

6.1 ± 0.9c

6.0 ± 1.0c

12.0 ± 2.0d

9.6 ± 0.5c

Methanol 5.6 ± 0.57c

4.4 ± 0.50c

4.3 ± 0.4bc

4.0 ± 1.0bc

10.0 ± 0.0cd

8.3 ± 0.5bc

Hexane 3.1 ± 0.17bc

2.2 ± 0.20b

2.6 ± 0.2b

2.3 ± 0.1ab

7.2 ± 1.3bc

6.8 ± 0.7b

Chloroform 0.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

4.1 ± 0.3ab

3.5 ± 0.9a

Streptomycin

(10µg/disc)

15.0 ± 2.0e

14.0 ± 1.73e

11.0 ± 1.72d

12.0 ± 1.73d

11.0 ± 2.0d

14.0 ± 1.70d



The efficacy of various solvent extracts of T. foenum-graecum leaf against the

pathogenic bacteria showed varied levels of inhibition (Table 1.3). Among the various

extracts, maximum in vitro inhibition of the tested bacteria E. coli, E. aerogenes,

Klebsiella sp., P. vulgaris., S. aureus and P. aeruginosa was achieved in ethanol

extract with zones of inhibition of 12 mm, 11 mm, 10 mm, 12 mm, 14 mm and

13 mm respectively. Methanol extract showed an inhibition of 12 mm against

S. aureus and 10 mm against P. aeruginosa. As with other solvent extracts tested, a

zone of inhibition greater than 10 mm could not be achieved with any of the bacteria.

The least inhibition was observed with the aqueous extract.


of Trigonella foenum-graecum leaves against the test bacteria


Aqueous 2.0 ± 0.2cd

1.0 ± 0.4c

1.0 ± 0.5d

2.0 ± 0.7d

3.0 ± 0.5ef

2.0 ± 0.0de

Ethanol 12.0 ± 0.8ab

11.0 ± 0.2a

10.0 ± 0.3a

12.0 ± 0.5a

14.0 ± 0.5a

13.0 ± 0.0a

Methanol 9.0 ± 0.4b

8.0 ± 0.4b

8.0 ± 0.2b

10.0 ± 0.4bc

12.0 ± 0.7b

10.0 ± 0.5b

Hexane 5.0 ± 0.7c

5.0 ± 0.4c

4.0 ± 0.4c

5.0 ± 0.5c

7.0 ± 0.2d

5.0 ± 0.3c

Chloroform 3.0 ± 0.4c

2.0 ± 0.7c

1.0 ± 0.2d

2.0 ± 0.0d

4.0 ± 0.7e

3.0 ± 0.5d

Streptomycin

(10µg/disc)

15.0 ± 2.0e

14.0 ± 1.73e

11.0 ± 1.72d

12.0 ± 1.73d

11.0 ± 2.0d

14.0 ± 1.70d




36

Fig. 1.1 : Antibacterial activity of ethanol leaf extract of Andrographis paniculata

against the test bacteria (a) Escherichia coli (b) Enterobacter aerogenes

(c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus aureus and

(f) Pseudomonas aeruginosa


37

Fig. 1.2 : Antibacterial activity of ethanol leaf extract of Tinospora cordifolia

against the test bacteria (a) Escherichia coli (b) Enterobacter aerogenes

(c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus aureus

and (f) Pseudomonas aeruginosa


38

Fig. 1.3 : Antibacterial activity of ethanol leaf extract of Trigonella foenum-

graecum against the test bacteria (a) Escherichia coli (b) Enterobacter

aerogenes (c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus

aureus and (f) Pseudomonas aeruginosa


39

Fig. 1.4 : Antibacterial activity of the standard antibiotic streptomycin (10

µg/disc) against the test bacteria (a) Escherichia coli (b) Enterobacter

aerogenes (c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus

aureus and (f) Pseudomonas aeruginosa


40

The traditional medicinal methods, especially the use of medicinal plants, still

play a vital role to cover the basic health needs in the developing countries and

moreover, the use of herbal remedies has risen in the developed countries in the last

decade. Plants have provided a source of inspiration of novel drug compounds, as

plant derived medicines have made large contributions to human health and well

being. Their role is twofold namely; they provide key chemical structure for the

development of new antimicrobial drugs and also as a phytomedicine to be used for

the treatment of disease (Abukakar et al., 2008). It is anticipated that phytochemicals

with adequate antibacterial efficacy will be used for the treatment of bacterial

infections (Balandrin et al., 1985). The first step towards this goal is the in vitro

antibacterial activity assay (Tona et al., 1998). Diffusion method is extensively used

to investigate the antimicrobial activity of natural substances and plant extracts. These

assays are based on the use of discs or holes as reservoirs containing the solutions of

substances to be examined (Rauha et al., 2000).

In the present study, bacteria commonly causing urinary tract infection (UTI)

have been selected. Urinary tract infection represents one of the most common

diseases occurring from the neonates to the geriatric age groups (Raju and Tiwari,

2004). The increasing drug resistance among the bacteria causing UTI has made

therapy difficult and has led to greater use of expensive broad spectrum drugs. This

resistance problem needs a renewed effort resulting in searching of effective

antibacterial agents against pathogenic microorganisms resistant to current antibiotics

(Soulsby, 2005).

The chloroform, hexane, methanol, ethanol and water extracts of the leaves of

A. paniculata, T. cordifolia and T. foenum-graecum were subjected to a preliminary

screening for antimicrobial activity against six human pathogenic bacteria commonly

associated with UTI infections. It was clear from the present results that ethanol leaf

extract of all the three plants had a broad spectrum of activity against all the tested six

bacteria. This tends to show that the active ingredients of the leaves are better

extracted with ethanol than with any other solvent. Since, nearly all of the identified

compounds from plants active against microorganisms are aromatic or saturated

organic compounds, they are most often obtained through initial ethanol or methanol

extraction (Cowan, 1999). Earlier studies have also reported the greater activity of


41

ethanol extracts compared to other solvent extracts (Aqil and Ahmad 2003; Allero and

Afolayan, 2006; Parekh and Chanda, 2007b; Kannan et al., 2009). Least antibacterial

activity was observed in the aqueous extracts of A. paniculata, T. cordifolia and

T. foenum-graecum. Investigators in the past have also clearly shown that ethanol

extracts are more effective than water extracts (Koduru et al., 2006; Aiyegoro et al.,

2008; Ashrafa et al., 2008). Two possibilities that may account for the higher

antibacterial activity of alcoholic extracts are the nature of bioactive components

(polyphenols, flavonoids, alkaloids, essential oil and terpenoids) which may be

enhanced in the presence of ethanol; and the stronger extraction capacity of ethanol

that may have yielded a greater number of active constituents responsible for

antibacterial activity (Ming et al., 2005; Vaghasiya and Chanda, 2007; Ghosh et al.,

2008).

Prajjal et al. (2003) have shown the antibacterial activity of aqueous extract of

A. paniculata against E. coli, S. aureus and P. aeruginosa at a concentration of

10 mg/disc. But studies by Leelarasamee et al. (1990), Xu et al. (2006) and Anjana

et al. (2009) have shown no antibacterial activity of A. paniculata aqueous extract

against these organisms. The result of the antibacterial activity of A. paniculata

aqueous extract in the present study is in agreement with the earlier done studies.

Antibacterial activity was not observed with the ethanol extract of A. paniculata

against E. coli, S. aureus and P. aeruginosa in a study by Xu et al. (2006). But, the

present study showed significant antibacterial activity of A. paniculata ethanol extract

against these organisms which supports the earlier studies by Zaidan et al. (2005) and

Anjana et al. (2009).

Srinivasan et al. (2001) have shown the broad spectrum activity of

T. cordifolia root and stem extracts. Similarly in the present study, T. cordifolia leaf

extracts exhibited antibacterial activity against both Gram positive and Gram negative

bacteria. The antibacterial activity of aqueous, ethanol and chloroform stem extracts

of T. cordifolia (100 mg/ml) against E. coli, P. vulgaris and S. aureus were carried out

by Jeyachandran et al. (2003). They found that the ethanol extract displayed

significant antibacterial activity while the chloroform and aqueous extracts displayed

least antibacterial activity. Mahesh and Satish (2008) have shown the antibacterial

activity of methanol leaf extract of T. cordifolia against E. coli, P. fluorescens and


42

S. aureus at a concentration of 0.1 mg/ml. The findings from the present study also

indicate significant antibacterial activity in a polar solvent like ethanol. Aqueous

extract of T. cordifolia has shown the least antibacterial activity in the present study

which is contrary to the study by Upadhyay et al. (2011) which has shown higher

percentage of growth inhibition of K. pneumoniae, E. coli and S. aureus in

T. cordifolia stem aqueous extracts in comparison to other solvent extracts.

Ethanol leaf extract of T. foenum-graecum exhibited broad spectrum activity

against both Gram positive and Gram negative bacteria. A study by Aqil and Ahmad

(2003) has also shown the broad spectrum activity of fenugreek leaves against

S. aureus, E. coli and P. aeruginosa in the ethanol extract. El-Kamali and El-Karim

(2009) in their study on T. foenum-graecum seeds indicated pronounced antibacterial

activity of ethanol seed extract. Bonjar (2004) has shown the antibacterial activity of

fenugreek seed extract in a polar solvent like methanol at a concentration of 20 mg/ml

against E. coli. Similar to the earlier findings in fenugreek seed, the findings of the

present study also show significant antibacterial activity in a polar solvent like ethanol.

The variation in the antimicrobial activity of different solvents can be

rationalized in terms of the polarity of the solvents used, polarity of the compounds

being extracted by each solvent and, in addition to their extrinsic bioactivity and by

their ability to dissolve or diffuse in the media used in the assay (Anjana et al., 2009).

The other possibility may be due to loss of some active compounds during extraction

process of the sample, lack of solubility of active constituents in the solvent (Kumar

et al., 2008) or the active compounds may be present in insufficient quantities in the

crude extracts to show their activity with the dose levels employed (Taylor et al.,

2001). Alternatively, if the active principle is present in high enough quantities there

could be other constituents exerting antagonistic effects (Jager et al., 1996). Extracts

which have shown least antibacterial activity may be active against other bacterial

species which were not tested (Shale et al., 1999).

As shown in Table 1.8, leaves of A. paniculata, T. cordifolia and T. foenum-

graecum contain phenols, flavonoids, terpenoids and tannins which have biological

significance as antimicrobials. The antibacterial activity as seen in this study might be

due to the presence of these compounds.


43

Determination of MIC

Broth dilution method which was used to determine the MIC values of

A. paniculata ethanol leaf extract was carried out with concentrations ranging from

0.25-2.0 mg/ml (Table 1.4). Minimum inhibitory concentration values for

P. aeruginosa and S. aureus were found to be 0.75 mg/ml and 1.0 mg/ml respectively.

At 600 nm, P. aeruginosa and S. aureus showed an absorbance of 0.02 (83.3%

growth inhibition) and 0.03 (84.8% growth inhibition) as against a control absorbance

of 0.14 and 0.22 respectively. Minimum inhibitory concentration values were found to

exceed 2.0 mg/ml for other bacteria tested. The per cent growth inhibition of bacteria

is shown in Figure 1.5.

Table 1.4 : Minimum inhibitory concentration (MIC) of Andrographis paniculata

leaf ethanol extract using broth dilution method

Bacteria Control Concentration (mg/ml)

0.25 0.5 0.75 1.0 2.0

E. coli 0.27 ± 0.05c 0.27 ± 0.01

c 0.17 ± 0.02

b 0.14 ± 0.03

ab 0.13 ± 0.00

ab 0.11 ± 0.00

a

E. aerogenes 0.30 ± 0.02d

0.29 ± 0.01d

0.24 ± 0.01c

0.19 ± 0.02bc

0.15 ± 0.01ab

0.14 ± 0.01a

Klebsiella sp. 0.25 ± 0.00c

0.24 ± 0.01bc

0.20 ± 0.02ab

0.20 ± 0.02ab

0.18 ± 0.02a

0.16 ± 0.01a

P. vulgaris 0.25 ± 0.01c

0.24 ± 0.00c

0.20 ± 0.00b

0.19 ± 0.01b

0.16 ± 0.00a

0.15 ± 0.01a

S. aureus 0.22 ± 0.04e

0.21 ± 0.01de

0.15 ± 0.00cd

0.09 ± 0.02bc

0.03 ± 0.01ab

0.02 ± 0.00a

P. aeruginosa 0.14 ± 0.03b

0.13 ± 0.02b

0.06 ± 0.01a

0.02 ± 0.01a

0.02 ± 0.00a

0.01 ± 0.01a

Values are absorbance at 600 nm


Fig. 1.5 : Per cent growth inhibition of bacteria at different concentrations

(0.25- 2.0 mg/ml) of Andrographis paniculata ethanol leaf extract

Values are means of three independent replicates

Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)


44

The MIC values of ethanol leaf extract of T. cordifolia at different

concentrations (0.5-2.0 mg/ml) against the tested six bacteria are summarized in Table

1.5. The ethanol extract was more effective in inhibiting S. aureus. The MIC value of

S. aureus was found to be 1.75 mg/ml. Staphylococcus aureus showed an absorbance

of 0.07 at 1.75 mg/ml as against a control absorbance of 0.22 at 600 nm. For other

bacteria tested the MIC values exceeded 2 mg/ml. There was 68.18% growth

inhibition of S. aureus at 1.75 mg/ml (Fig. 1.6). Pseudomonas aeruginosa recorded

61.90% growth inhibition at 2 mg/ml.

Table 1.5 : Minimum inhibitory concentration (MIC) of Tinospora cordifolia

leaf ethanol extract using broth dilution method

Bacteria

Control

Concentration (mg/ml)

0.5 1.0 1.25 1.50 1.75 2.0

E. coli 0.27 ± 0.05d 0.28 ± 0.00d 0.23 ± 0.00c 0.20 ± 0.05b 0.19 ± 0.00b 0.17 ± 0.01a 0.16 ± 0.00a

E. aerogenes 0.30 ± 0.02b 0.30 ± 0.00b 0.30 ± 0.01b 0.28 ± 0.01ab 0.26 ± 0.01a 0.25 ± 0.00a 0.25 ± 0.01a

Klebsiella sp. 0.25 ± 0.00cd 0.26 ± 0.00d 0.25 ± 0.00cd 0.24 ± 0.00bc 0.23 ± 0.01ab 0.21 ± 0.00ab 0.21 ± 0.01a

P. vulgaris 0.25 ± 0.01b 0.25 ± 0.00b 0.25 ± 0.01ab 0.24 ± 0.01ab 0.24 ± 0.00ab 0.22 ± 0.00a 0.22 ± 0.01a

S. aureus 0.22 ± 0.04c 0.23 ± 0.01c 0.18 ± 0.01c 0.12 ± 0.00b 0.08 ± 0.00ab 0.07 ± 0.00a 0.07 ± 0.01a

P. aeruginosa 0.14 ± 0.03cd 0.14 ± 0.01d 0.13 ± 0.00cd 0.10 ± 0.01bc 0.09 ± 0.00ab 0.06 ± 0.01ab 0.05 ± 0.00a




(0.5- 2.0 mg/ml) of Tinospora cordifolia ethanol leaf extract




45

The MIC values of ethanol extract of T. foenum-graecum against the tested six

bacteria are represented in Table 1.6. Maximum antibacterial activity was observed

against S. aureus followed by P. aeruginosa with a MIC value of 1 mg/ml.

Staphylococcus aureus and P. aeruginosa recorded an absorbance of 0.03 and 0.05 as

against a control absorbance of 0.22 and 0.14 respectively at 600 nm. In this study,

S. aureus recorded a growth inhibition of around 86% and P. aeruginosa showed a

growth inhibition of nearly 65% at a concentration of 1 mg/ml (Fig. 1.7). The MIC

values exceeded 2 mg/ml as seen by the absorbance values in case of other bacteria.

Table 1.6 : Minimum inhibitory concentration (MIC) of Trigonella foenum-

graecum leaf ethanol extract using broth dilution method

Bacteria Control Concentration (mg/ml)

0.25 0.5 1 2

E. coli 0.27 ± 0.0a

0.26 ± 0.0a

0.17 ± 0.0b

0.19 ± 0.0b

0.25 ± 0.0a

E. aerogenes 0.30 ± 0.0a

0.3 ± 0.01a

0.28 ± 0.0a

0.27 ± 0.0a

0.3 ± 0.0a

Klebsiella sp. 0.25 ± 0.0a

0.23 ± 0.01ab

0.19 ± 0.0b

0.19 ± 0.0b

0.23 ± 0.0ab

P. vulgaris 0.25 ± 0.0a

0.23 ± 0.01ab

0.23 ± 0.0ab

0.2 ± 0.0b

0.22 ± 0.0ab

S. aureus 0.22 ± 0.0ab

0.21 ± 0.01ab

0.17 ± 0.01b

0.03 ± 0.0c

0.09 ± 0.0c

P. aeruginosa 0.14 ± 0.01b

0.14 ± 0.01b

0.06 ± 0.01c

0.05 ± 0.0c

0.07 ± 0.01c





(0.25- 2.0 mg/ml) of Trigonella foenum-graecum ethanol leaf extract



d

c

c

c e

dc

d

d

cb

abc

c

ca

a

a

b

bc

b

ab

a

d

0

10

20

30

40

50

60

70

80

90

100

0.25 0.5 1 2

Concentrations (mg/ml)

% g

row

th I

nh

ibit

ion

of

Bacte

ria

E.coli E.aerogenes Klebsiella sp.

P.vulgaris S.aureus P.aeruginosa

Concentration (mg/ml)

E. coli

P. vulgaris

E. aerogenes

S. aureus

Klebsiella sp.

P. aeruginosa


46

As ethanol extracts of A. paniculata, T. cordifolia and T. foenum-graecum

showed potent antibacterial activity against both Gram positive and Gram negative

organisms, the determination of MIC values was carried out only with the ethanol

extract. Broth dilution method which was used for the determination of MIC values of

ethanol extract of all the three plants indicated that S. aureus and P. aeruginosa were

more susceptible than any other bacteria at lower concentrations. Earlier findings have

reported that most medicinal plants attack Gram positive bacteria while few are active

against Gram negative bacteria (Srinivasan et al., 2001; Priscilla et al., 2007; Pavithra

et al., 2010). The reason for the different sensitivity between Gram positive and Gram

negative bacteria could be ascribed to the morphological differences between these

microorganisms. The Gram negative bacteria have an outer phospholipidic membrane

carrying the structural lipopolysaccharidic components which makes the cell wall

impermeable to lipophilic solutes, while porins constitute a selective barrier to the

hydrophilic solutes with an exclusion limit of about 600 Da (Nikaido and Vaara,

1985). The Gram positive bacteria are more susceptible having only an outer

peptidoglycon layer which is not an effective permeability barrier (Scherrer and

Gerhardt, 1971). The results of the present study are contrary with the earlier reports

which have shown greater activity of medicinal plants against Gram positive bacteria.

In spite of this permeability difference between Gram positive and Gram negative

bacteria, the ethanol extract of all the three plants had a broader spectrum of

inhibitory activity. This tends to show the involvement of more than one active

principle of biological significance (Ming et al., 2005). The possible mechanism for

their broad spectrum activity against both Gram positive and Gram negative bacteria

may be due to their ability to complex with the cell wall (Cowan, 1999).

The findings of the present study are in fair correlation with the study carried

out by Calabrese et al. (2000) who reported the antibacterial activity of A. paniculata

against both Gram positive and Gram negative organisms. Upadhyay et al. (2011)

have shown the MIC value of methanol extract of T. cordifolia stem against S. aureus

to be 0.06 mg/ml. But, the MIC value of T. cordifolia leaf ethanol extract in the

present study was found to be 1.75 mg/ml. The reason for this variation may be

attributed to the presence of different biological substances in the stem and leaf (Shan

et al., 2005). Earlier studies by Aqil and Ahmad (2003) and Bonjar (2004) have

shown the broad spectrum activity of fenugreek seeds against both Gram positive and

Gram negative bacteria. Similar to the earlier findings in fenugreek seeds, we have

also shown the wide spectrum activity of fenugreek leaves.


47

Antifungal activity assay

Mycelial dry weight method was used to determine the MIC values of A. paniculata,

T. cordifolia and T. foenum-graecum leaf ethanol extracts against two dermatophytes,

T. rubrum and E. floccosum. Table 1.7 represents the MIC values and the per cent

inhibition of ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum

against the dermatophytes as compared to the antifungal drug, ketaconozole. Among

the two dermatophytes, E. floccosum was found to be more susceptible to the ethanol

leaf extract of all the three plants. Andrographis paniculata extract had the MIC

values of 1.75 mg/ml (74.6% growth inhibition) and 3.0 mg/ml (70.9% growth

inhibition) against E. floccosum and T. rubrum respectively which was lower when

compared to other plant extracts. The MIC value of T. cordifolia extract exceeded

more than the used concentration range of 10 mg/ml against T. rubrum. The MIC

values obtained against T. rubrum and E. floccosum with all the three plant extracts

were higher than the standard antifungal drug ketaconazole.

Table 1.7 : Minimum Inhibitory Concentration (MIC) of Andrographis paniculata,

Tinospora cordifolia and Trigonella foenum-graecum leaf ethanol extracts

against dermatophytes using mycelial dry weight method



Dermatomycoses is the disease caused by a group of fungi known as

dermatophytes. Skin disease such as tinea is the most frequent mycoses which

involves superficial infections of keratinized tissue of the skin which occur in most

classes of patients, especially in immunosuppressed patients (Chuang et al., 2007).

Clinical surveys carried out in India have shown ringworm as one of the most

common dermatomycoses caused by species of Trichophyton, Epidermophyton and

Microsporum. The disease is predominant in tropical and subtropical countries due to

T. rubrum E. floccosum

MIC

( mg/ ml)

% growth

inhibition

MIC

( mg/ ml)

% growth

inhibition

Ketaconazole 0.4 ± 0.01a

90.3 0.25 ± 0.02a

93.0

A. paniculata 3.0 ± 0.04b

70.9 1.75 ± 0.05b

74.6

T. cordifolia > 10 ± 0.06d

- 8.0 ± 0.03d

56.3

T. foenum-graecum 6.5 ± 0.02c

62.3 4.0 ± 0.04c

65.0


48

their prevailing moisture and temperature regimes, and poses a therapeutic problem

despite several antimycotic drugs available in the market. These drugs are mostly

fungistatic. Besides, these drugs have been found to possess various side effects

(Shahi et al., 1998). Recently, products of higher plant origin have been shown as

effective sources of chemotherapeutic agents without undesirable side effects and

with strong fungicidal activity (Igbinosa et al., 2009; Mathur et al., 2011). Although

advances have been made in pharmacology and synthetic organic chemistry, the

reliance on natural products, particularly on plants, remains largely unchanged. It is

well established that some plants contain compounds that inhibits microbial growth

(Evarando et al., 2005). These plant compounds have different structures and

different actions when compared with antimicrobials conventionally used to control

microbial growth (Nascimento et al., 2000). These findings prompted us to explore

other plant products which could be exploited as effective antifungal agents.

A study by Wanchaitanawong et al. (2005) has shown the in vitro antifungal

activity of ethanol extract of A. paniculata plant against Aspergillus niger, Aspergillus oryzae

and Penicillium. Antifungal activity of hydroalcoholic extract of A. paniculata against

A. niger and Candida albicans has been shown by Mathur et al. (2011) with a MIC

value of 0.5 mg/ml. Similar to the earlier reports on antifungal activity of A. paniculata

ethanol extract, the result of the present study also indicates its antifungal activity

against dermatophytes. Based on the review of current literature, there are no previous

reports showing the antifungal activity of A. paniculata leaf ethanol extract on

dermatophytes.

Satish et al. (2007) have shown the antifungal activity of T. cordifolia stem

extract against eight species of Aspergillus. Methanol extract of T. cordifolia root and

stem exhibited antifungal activity against Helminthosporium sp. at a concentration of

5 mg/ml (Singh et al., 2010). Antifungal activity of T. cordifolia methanol leaf extract

against Aspergillus flavus, Dreschlera turcica and Fusarium verticilloides has been

shown by Mahesh and Satish (2008) at a concentration of 0.1 mg/ml. Hydroalcoholic

extract of T. cordifolia stem was found to have maximum antifungal activity against

A. niger and Candida albicans with a MIC value of 0.5 mg/ml (Mathur et al., 2011).

Eventhough, T. cordifolia stem and root extracts have shown antifungal activity

against the field fungi with lower MIC values, the MIC values in the present study for


49

T. cordifolia ethanol leaf extract against E. flocossum was quite high and with

T. rubrum it could not be achieved even at a higher concentration of 10 mg/ml. This

could be due to the presence of lower concentration of antifungal substances in the

extract.

In the present study, T. foenum-graecum ethanol leaf extract showed antifungal

activity against both fungi with MIC values ranging between 4.0 and 7.0 mg/ml. There are

no previous reports showing the antifungal activity of T. foenum-graecum leaf extract on

dermatophytes. But, a study by Aqil and Ahmad (2003) has shown less antifungal

activity of T. foenum-graecum leaf extract against the field fungi viz., A. niger,

Alternaria alternata, Fusarium chlamydosporum and Rhizoctonia bataticola.

It is well established that the potential antimicrobial properties of plants are

related to their ability to synthesize by secondary metabolism several chemical

compounds of relatively complex structures including tannins, alkaloids, cardiac

glycosides, saponins, terpenes, phenolics, flavonoids and coumarins (Evarando et al.,

2005; Matasyoh et al., 2009). The antifungal activity of plant extracts in the present

study may be due to the presence of any one or more of these active compounds

which might be acting synergistically.

Phytochemical screening

Ethanol extract which had shown good antibacterial and antifungal activities

was used for screening the phytochemical compounds. Investigations on the

phytochemical screening of A. paniculata, T. cordifolia and T. foenum-graecum leaf

ethanol extracts are summarized in Table 1.8. Results from the current study indicate

that ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum

contain varied types of pharmacologically active compounds. The commonly

identified components of pharmacological importance in the three plant extracts

included phenols, flavonoids and cardiac glycosides. In addition, tannins and

terpenoids were present in A. paniculata and T. cordifolia, polysterols were present in

T. cordifolia and T. foenum-graecum, saponins were present in T. foenum-graecum

and carbohydrates and proteins were present in all the three plant extracts. Alkaloids

were found to be absent in the plant extracts.


50

Table 1.8 : Phytochemical constituents of ethanol leaf extracts of Andrographis

paniculata, Tinospora cordifolia and Trigonella foenum-graecum

Phytochemical

constituents

Ethanol leaf extracts

A. paniculata T. cordifolia T. foenum-graecum

Phenols + + +

Flavonoids + + +

Tannins + + _

Terpenoids + + _

Alkaloids _ _ _

Polysterols _ + +

Cardiac glycosides + + +

Saponins _ _ +

Carbohydrates + + +

Protiens + + +

+ Represents presence of the phytoconstituent; - represents absence of the phytoconstituent

Bioactive secondary metabolites have been utilized as natural medicines and

plants containing these compounds have been used as medicinal plants and prescribed

in many recipes as forms of crude drugs (Zuin and Vilegas, 2002; Rios and Recio,

2005). 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 flavonoids, tannins, alkaloids and phenolic compounds

(Weimann and Heinrich, 1997; Atindehou et al., 2002; Edeoga et al., 2005).

Phytochemical compounds are heterogeneous mixtures of substances acting in a

synergistic or antagonistic manner (Musyimi et al., 2008). Mixtures of active

constituents show a broad spectrum of biological and pharmacological activity

(Coelho-de-Souza et al., 2002). The need of the hour is to screen plants that are

traditionally used and also to evaluate their probable phytoconstituents (Parekh and

Chanda, 2007a; Parekh and Chanda, 2007c). Knowledge of the chemical constituents

of plants is desirable because such information will be of great value for the synthesis

of complex chemical substances. Many studies have been undertaken with the aim of

determining the different antimicrobial and phytochemical constituents of medicinal


51

plants and using them for the treatment of both topical and systemic microbial

infections as possible alternatives to chemical synthetic drugs to which many

infectious microorganisms have become resistant (Akinpelu and Onakoya, 2006).

Phytochemical analysis of the ethanol leaf extracts of A. paniculata,

T. cordifolia and T. foenum-graecum has revealed the presence of biologically active

compounds such as phenols, flavonoids, saponins, tannins, terpenoids, cardiac

glycosides and polysterols. The antimicrobial (antibacterial and antifungal) activity of

the plant extracts is due to the presence of these active compounds which may be

acting independently or synergistically with other compounds.

The phenolic compounds are one of the largest and most ubiquitous groups of

plant metabolites (Singh et al., 2007). Baravalia et al. (2009) have shown the

importance of polyphenols as antimicrobial compounds. Polyphenols and flavonoids

exhibit wide range of biological effects and antibacterial activity is one of them

(Hartung, 1990; Edenharder and Grunhage, 2003; Wang et al., 2003; Kathad et al.,

2010). According to Fatope (1995) polyphenols are important fungitoxic

phytochemical compounds.

Flavonoids have shown a wide range of biological activities like antimicrobial,

antiinflammmatory, antiallergic and antioxidant properties (Hodek et al., 2002). They

are found to be effective antimicrobial substances against a wide array of infectious

agents (Mendosa et al., 1997; Erdogrul, 2002; Jamine et al., 2007; Mostahar et al.,

2007). Flavonoids have been shown to be effective antimicrobial agents by

complexing with extracellular and soluble proteins and also with bacterial cells

(Tsuchiya et al., 1996). They also act by inhibiting enzymes that regulate cell

proliferation (Fatope, 1995).

Tannins are known for their antimicrobial activity (Stern et al., 1996; Rios et

al., 2000). Prasad et al. (2008) have reported the growth inhibition of many fungi,

bacteria and viruses by tannins. Tannins have been reported to prevent the

development of microorganisms by precipitating microbial proteins. They have been

found to form irreversible complexes with proline rich proteins resulting in the

inhibition of cell protein synthesis (Shimada, 2006).


52

Quinlan et al. (2000) have shown the presence of antibacterial activities of

steroidal extracts from medicinal plants. Terpenoids have been demonstrated to be

active against bacteria, fungi, viruses and protozoa (Aurelli et al. 1992; Tassou et al.,

1995). The mechanism of action of terpenes is by lipophilic membrane disruption

(Cowan, 1999). Saponins are known for their medicinal properties as a natural blood

cleanser, expectorant and antibiotic (Kalanithi and Lester, 2001).

Earlier studies on aerial parts of A. paniculata have shown the presence of

diterpenoids, flavonoids and polyphenols (Reddy et al., 2003; Rao et al., 2004; Chao

and Lin, 2010). Sule et al. (2010) in their study have shown the presence of

flavonoids, glycosides and saponins in methanol extract of A. paniculata. Similar to

the earlier findings, ethanol extract of A. paniculata leaves in the present study has

shown the presence of phenols, flavonoids, terpenoids and cardiac glycosides.

Reports on T. cordifolia stem extracts have shown the presence of phenols,

tannins, cardiac glycosides and steroids (Sivakumar et al., 2010; Vaghasiya et al.,

2011). Rose et al. (2010) have reported the presence of alkaloids, glycosides, steroids,

saponins, tannins, phenols and flavonoids in root extract of T. cordifolia. The result of

the present study is in agreement with an earlier study by Murthy et al. (2010) which

has shown the presence of tannins, flavonoids, phytosterols, glycosides in ethanol leaf

extract of T. cordifolia.

Trigonella foenum-graecum seeds have been shown to contain alkaloids,

saponins and flavonoids by Dheeraj et al. (2010). But in the present study, T. foenum-

graecum leaf extracts showed absence of alkaloids which may be due to the early

harvesting of the leaves (Srinivasan, 2006).

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