CHAPTER – 5

RESULTS & DISCUSSION

Section 5.1

Preliminary antifungal, phytochemical and toxicological screening of plants

5.1.1. Introduction

Natural products, either as pure compounds or as standardized plant extracts, provide

unlimited opportunities for the development of novel drugs because of the great diversity in

their chemical structure. There is a continuous and urgent need to discover new antifungal

compounds with diverse chemical structures and novel mechanisms of action for new and re-

emerging infectious diseases (Rojas et al., 2004). Therefore, researchers are increasingly

turning their attention to ethno-medicine, looking for new leads to develop more effective

drugs against fungal infections (Bhaskarwar et al., 2008) and this has led to the screening of

several medicinal plants for potential antifungal activity. The majority of studies dedicated to

antifungal activity of Indian medicinal plants used in Ayurveda and traditional medicinal

system focus on extracts. This might be due to the fact that the traditional medicines contain a

wide range of substances that can be used to treat chronic as well as infectious diseases and

have always played a key role in the health system of many countries (Gupta et al., 2010).

In Haryana region (state of India) there is a great biodiversity of medicinal plants and

there is a long tradition of using herbal products for skin and other problems by healers and

old peoples. Besides, the plants have been used throughout the world as good reservoir of

bioactive compounds, drugs and remedies for various diseases since time immemorial. The

most important of these bioactive compounds of plants are alkaloids, flavanoids, tannins and

phenolic compounds which can prove to be an important source of lead compounds in the

development of new antifungal drugs. Therefore, nine plants (Achyranthes aspera, Aegle

marmelos, Argemona mexicana, Callistemon lanceolatus, Capparis aphylla, Catharanthus

roseus, Commelina bengalensis, Justicia adhatoda and Syzygium cumini) were selected as

per use in Ayurveda and traditional system of medicines. Only leaves (except C. aphylla)

were used of all plant species for conservation and sustainability purposes. Other important

parameters which were used for preliminary screening includes availability of species and

number of phytochemical compounds. The nine plants used in the study are summarized in

Table 5.1.1 with their Ayurvedic and traditional uses.

Table 5.1.1- Various medicinal plants selected on the bases of traditional and Ayurvedic

uses.

Sr.

No.

Name of Plants/

Family

Common

Name

Traditional & Ayurvedic Uses of Plants

1 Argemone

mexicana

(Papaveraceae)

Prickly

Poppy,

Shialkanta,

Satyanashi.

According to Ayurveda the plant is diuretic,

purgative and destroys worms. It cures leprosy, skin-

diseases, bilious fevers and inflammations. Juice is

used to cure ophthalmia and opacity of cornea.

Moreover roots are used as anthelmintic.

2 Catharanthus

roseus (Linn.)

G. Don

(Apocynaceae)

Madagascar

periwinkle,

Sadabahar

The hot water extract of dried entire plant is taken

orally by human for cancer, heart disease,

leishmaniasis and taken by pregnant women to cause

abortion. The root extract is taken orally for

menorrhagia. Hot water extract of dried leaves is

taken orally for menorrhagia, diabetes, Hodgkin’s

disease and extract of root bark is taken orally as

febrifuge.

3 Achyranthes aspera

(Amaranthaceae)

Ulta Kanta,

Prickly-

chaff flower

It is commonly used for the treatment of fever,

especially malarial fever, dysentery, asthma,

hypertension and diabetes. The dried herb is used to

treat children for colic and also as an astringent in

gonorrhoea treatment.

4

Syzygium cumini

(Linn.) Skeels.

(Myrtaceae)

Jambul tree,

Jamuna,

Jamun

Charaka used seeds, leaves and fruits in decoctions

for diarrhoea and the bark as an astringent, dysentery,

and menorrhagia. The decoction of the bark is an

efficacious mouth-wash and gargle for treating

spongy gums, stomatitis, relaxed throat and other

diseases of mouth. According to Ayurveda, its bark is

acrid, sweet, digestive, astringent to the bowels,

anthelmintic and good for sore throat, bronchitis,

asthma, thirst, biliousness, dysentery, blood

impurities and to cure ulcers. Leaf juice is taken

orally to treat diabetes.

5 Aegle marmelos

(linn.) Correa

(Rutaceae)

Stone apple,

Indian Bail,

Holy fruit,

Plant used for intermittent fever, intestinal ailments,

fertility control and treatment after childbirth and fish

poison. Fruits are used in diarrhoea and dysentery.

Decoction of the root has been used to treat

melancholia, intermittent fevers and palpitation

6 Capparis aphylla

(Capparaceae)

Caper plant,

kair, Karil,

Kurel

Plant pacifies vitiated pitta, kapha, boils, eruptions,

swelling, chronic and foul ulcers, cough, asthma,

vomiting, haemorrhoids, intermittent fevers, arthritis,

lumbago, dyspepsia, flatulence, constipation,

intestinal worms, cardiac debility, gout, amenorrhea,

dysmenorrhoeal and general debility

7 Commelina

benghalensis

(Commelinaceae)

Benghal

dayflower

The plant is used medicinally as a diuretic, febrifuge,

anti -inflammatory, laxative and to cure

inflammations of the skin as well as leprosy. It can be

used to treat burns and indigestion with a juice

produced from the roots.

8 Justicia adhatoda

(Acanthaceae)

Vasaka,

Bansa,

Adulsa.

The various plant parts are used for the treatment of

asthma, joint pain, lumber pain, sprains, cold, cough,

eczema, malaria, rheumatism, swelling and venereal

diseases. It is also used for the treatment of bleeding

piles, Impotence and sexual disorders.

9 Callistemon

lanceolatus

(Myrataceae)

Bottle brush The plant is used medicinally as an antifungal,

antibacterial and antiviral as well as anti –

inflammatory. It also shows strong antioxidant activity.

5.1.2. Material and methods

5.1.2.1. Plant material

Nine plants species were collected from Jhajjar and Rohtak district of Haryana as

shown in Figure 5.1.1. Plants were selected based on their use in traditional and Ayurvedic

system of medicines. Nine plants were selected and identified from botany department of

Maharshi Dayanand University, Rohtak (India).

Figure 5.1.1- The plants selected in the present study for evaluation of their biological

activity.

The identified plants were further authenticated with the help of flora of Haryana

(Jain et al., 2000) and the voucher specimens were deposited in the herbarium of Centre for

Biotechnology, M. D. University, Rohtak. The voucher numbers are given respectively as

Achyranthes aspera (CBT 002), Aegle marmelos (CBT 003), Argemona mexicana (CBT

004), Callistemon lanceolatus (CBT 008), Capparis aphylla (CBT 009), Catharanthus roseus

(CBT 012), Commelina bengalensis (CBT 013), Justicia adhatoda (CBT 017), and Syzygium

cumini (CBT 022).

5.1.2.2. Sample preparation and extraction of crude extracts

Eight plants leaves and stem of Capparis aphylla were collected from Haryana

(30.73°N 76.78°E), India. The plant material was washed with tap water, chopped into small

pieces and air dried under shade for two weeks and, then oven dried at 40 °C for 18-24h. The

dried plant material was grinded and powdered with a mortar and pestle. The powder was

weighed (50 g for each plant sample) and the Soxhlet’s method was used for extraction. The

five solvents (250 ml for each sample): petroleum ether, chloroform, acetone, methanol and

water were used in ascending order of polarity (Harborne, 1980; Rajesh and Sharma, 2002).

The combined suspensions were filtered twice, first under vacuum through a double layer of

Whatman filter paper and then by gravity through a single sheet of Whatman No. 1 filter

paper. The solvents were removed from the clear supernatant by means of vacuum distillation

at 30-35°C using a Buichi Rotary Evaporator. The remaining solid was referred to as the

crude extract.

5.1.2.3. Pathogens

Pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC

5192) and Aspergillus niger (ITCC 5405) were obtained from IARI, New Delhi and used in

the present study. All the Aspergillus strains were cultured in the laboratory on Sabouraud

dextrose agar plates.

5.1.2.4. Culture of Pathogens

The pathogenic strains of Aspergillus were cultured on Sabouraud Dextrose Agar (SDA)

plates. The plates were inoculated with stock cultures of A. fumigatus (ITCC 4517), A. flavus

(ITCC 5192) and A. niger (ITCC 5405) and incubated for 96 h in BOD incubator at 37 C

(Chhillar et al., 2008).

5.1.2.5. Antimycotic activity

5.1.2.5.1. Microbroth-dilution assay

The spores of Aspergillus were harvested from 96 h cultures and treated with various extracts

of different plants in a 96-well culture plate and examined macroscopically after 48 h for the

growth of Aspergillus mycelia (Annette et al., 1995; Yadav et al., 2005).

5.1.2.5.2. Disc-diffusion assay

This test was performed in radiation-sterilized Petri plates of 10.0 cm diameter

(Tarsons). Sterilized discs (5.0 mm of Whatman paper) impregnated with various extracts of

different plants were placed on the surface of agar plates already inoculated with Aspergillus

spores (1 x 106). The plates were incubated at 37

oC and examined at 48 h for the zone of

inhibition, if any, around the discs (Indian Pharmacopoeia, 1996; Yadav et al., 2005).

5.1.2.5.3. Spore-germination-inhibition assay

The spore-germination-inhibition activity of the preparations was represented as the

MIC90 which inhibit the germination of spores in the range of 90–100% (Surender and

Janaiah, 1987; Chhillar et al., 2006). Detail is given in material and methods section of the

thesis.

5.1.2.6. Qualitative phytochemical analysis

All the extracts obtained from nine plants were subjected to various phytochemical

analysis tests for the identification of various bioactive constituents present in these plants

(Harborne, 1980). Detail is given in material and methods section of the thesis.

5.1.2.7. Toxicity studies

5.1.2.7.1. Acute toxicity

All the bio- extracts at the range 100mg to 1000mg/kg were administered orally to the

groups of rats comprising six rats in each group. Mortality and general behaviour was

observed for 14 days.

5.1.2.7.2. Haemolytic assay

Human erythrocytes, collected from apparently healthy individuals, were washed

three times with PBS by centrifugation at 1500 r.p.m. for 10 min. A 2% erythrocyte

suspension was incubated at 37 oC for 1 h with different concentrations of extracts ranging

from 500.00 to 3.9 µg/ml plant extracts. After incubation, cells were pelleted at 5000 r.p.m.

for 10 min. The supernatant was collected and the A450 was determined using a

spectrophotometer (UV Vis Spect Lambda Bio 20, Perkin Elmer). In negative control sets,

only buffer was used for background lysis, whereas in positive controls, lysis buffer was used

for completely lysing the erythrocytes. For each sample the percentage of maximum

haemolytic activity was determined (Yadav et al., 2005).

5.1.2.7.3. Single cell gel electrophoresis assay (Comet assay)

Blood was taken from a healthy donor by venipuncture; comet assay and

classification of comet category and their tail measurements were carried out according to

Garcia et al., 2007. Detail is given in material and methods section of the thesis.

5.1.2.8. Gas Chromatography Mass Spectrometric (GCMS) analysis

The GC-MS investigation of plant extracts were carried out using Shimadzu QP-2010

plus with thermal desorption system TD-20 and identification of constituent of the extract

was achieved on the basis of their retention indices determined with a reference to a

homologous series of phytoconstituents and by comparison of their mass spectral

fragmentation patterns (NIST database/ chemstation data system) with data previously

reported in literature (Sathyaprabha et al., 2010). Detail is given in material and methods

section of the thesis.

5.1.3. Results and Discussion

A number of medicinal plants described in Ayurveda still need to be testified

according to the modern parameters to ensure their activity and efficacy. Therefore, nine

Plants were selected as per use in Ayurveda and traditional system of medicines. In most of

the cases the amount of residue extracted with water and acetone is high as compared to that

of other solvents. The highest percentage yield of the nine plant leaf extracts screened was

obtained from Aegle marmelos (26.46), with the lowest from Commelina bengalensis (0.19), as

shown in Figure 5.1.2.

Many traditional health practitioners believe that the whole plant extract is more

active than isolated compounds (Rodriguez-Fragoso et al., 2008). In cases where mature

trees or plants cannot be found, the younger ones suffice, which results in availability of

inconsistent plant material of the same species (Von Ahlefeldat et al., 2003). The amount of

the bioactive compound(s) from plants may vary with both the locality and the season in

which they are collected. Moreover, the plants harvested from the wild generally vary in

quality and consistency of active compounds (Bopana and Saxena, 2007). Also, bioactive

molecules of many plants are powerful poisons when taken in excess, and if the plant extract

contains a lower content of bioactive compound(s) than usual, suboptimal dosage may not be

effective (Navarro García et al., 2003).

V a r io u s P la n t E x tra c ts in D if fe r e n t S o lv e n ts

Pe

rc

en

t Y

ield

of

Pla

nt

Ex

tra

cts

Petr

ole

um

eth

er

Ch

loro

form

Aceto

ne

Meth

an

ol

Aq

ueo

us

0

1 0

2 0

3 0

A . m a r m e lo s

C . a p h y lla

C . la n c e o la te

C . b e n g a le n s is

J . a d h a to d a

A . m e x ic a n a

A . a s p e r a

C . r o s e u s

S . c u m in i

Figure 5.1.2- Percentage yield of crude extracts of selected nine plants in different

solvents.

Furthermore, crude extracts from many medicinal plants may contain, in addition to

the bioactive molecules, other constituents which have harmful effects. For example

aristolochic acids present in a Chinese plant, Aristolochia fangch are nephrotoxic and

carcinogenic compounds closely associated with renal failure (Loset et al., 2001). Medicinal

properties of many plants are also rapidly lost on storage, for example, foxglove leaf’s

bioactive molecules decompose on long storage, unless dried quickly after collection. The

preliminary phytochemical screening of all the extracts revealed the presence of alkaloids,

flavanoids, saponins, tannins, phenols, terpenoid and phytosteroids (Table 5.1.2 and Figure

5.1.3).

Table 5.1.2- Qualitative phytochemical analysis of selected medicinal plants extracts.

Sr.No Plants Name Presence of phytochemicals in plant extracts of various serial

solvents

Petroleum

ether

Chloroform Acetone Methanol Water

1 Aegle marmelos S/t S/t, T S/t, T S/t, Sp S/t, Sp

2 C. aphylla S/t, T S/t, A, Sp S/t, Sp S/t, A, T P, S/t, Sp

3 Callistemon S/t, A, T S/t, T S/t, T S/t, Sp T,

4 C .bengalensis S/t, Sp A, Sp S/t, Sp S/t, Sp S/t, Sp

5 J. adhatoda S/t A, F S/t A, F, Sp P, T, F

6 Argemone

mexicana

S/t, T, Sp A, F, P P, Sp P, S/t, A, T,

F, Sp

T, Sp

7 Catharanthus

roseus

S/t A, S/t S/t, A P, S/t, A, T,

F

P, T

8 Achyranthes

aspera

- A, F P, F, Sp P, A, T, F,

Sp

P, T, F, Sp

9 Syzygium cumini - T A, T, Sp A, T, F, Sp P, F, Sp

(P = Phenol; S/t = Sterol/terpene; A = Alkaloid; T = Tannin, F = Flavanoids; Sp = Saponins)

Figure 5.1.3- Qualitative phytochemical analysis of various extracts by preliminary

phytochemicals tests.

Forty five extracts of nine plants tested for their antifungal potential and two plants

were found to be reasonable active against three pathogenic species of Aspergillus.

Amphotericin B, the positive control used in this study shows MICs in the range 0.73-1.95

µg/ml against pathogenic species of Aspergillus. The initial screening of plants extracts for

antifungal activity showed that J. adhatoda and C. bengalensis had potential against

pathogenic species of Aspergillus (Table 5.1.3 and Figure 5.1.4).

Table 5.1.3- Antifungal activity of selected plant extracts against pathogenic fungi.

{The abbreviated words are given respectively as Aegle marmelos (A m), Capparis aphylla (C a),

Callistemon lanceolatus (C l), Commelina bengalensis (C b), Justicia adhatoda (J a), Achyranthes

aspera (A a), Argemona mexicana (A m), Catharanthus roseus (C r), and Syzygium cumini (S c).}.

Moreover, the petroleum ether extract of all the nine plants inhibited the growth of

pathogenic Aspergilli (A. fumigatus, A. niger and A. flavus) in the range of 0.156 - 6.00 mg/ml by

microbroth dilution assay. Furthermore, it was observed that only four out of forty five plants

extracts were found to be endowed with anti-aspergilli activity at a preset concentration of 10 µg/

disc (Table 5.1.4 and Figure 5.1.4) by disc diffusion assay.

Pathogens

name

Solvent

Used

Minimum inhibitory concentration (MIC =mg/ml) of extracts

A m C a C l C b J a A a A m C r S c

Aspergillus

fumigatus

Petroleum

ether

0.625 1.25 0.625 0.625 0.156 5.00 2.50 0.75 5.00

Chloroform 2.50 1.25 0.625 1.25 0.156 2.50 - 2.50 1.25

Acetone 5.00 2.50 0.625 1.25 0.312 1.25 5.00 1.25 5.00

Methanol

2.50 5.00 0.312 1.25 2.50 - 1.25 1.25 1.25

Water

5.00 2.50 - 0.156 2.50 1.25 1.25 1.25 1.25

A. flavus

Petroleum

ether

1.25 1.25 2.50 0.312 0.312 2.50 - 2.50 5.00

Chloroform

0.625 1.25 1.25 0.625 0.625 1.25 - 2.50 5.00

Acetone

1.25 1.25 5.00 0.625 0.625 2.50 2.50 1.25 2.50

Methanol

1.25 1.25 2.50 0.625 1.25 2.50 2.50 2.50 5.00

Water

5.00 1.25 - 1.25 1.25 0. 62 2.50 1.25 2.50

A.niger

Petroleum

ether

1.25 0.625 0.625 0.312 0.156 5.00 2.50 1.25 1.25

Chloroform

2.50 1.25 2.50 0.625 0.312 2.50 - - 5.00

Acetone

1.25 1.25 - 1.25 0.156 2.50 1.25 2.50 2.50

Methanol

0. 625 2.50 5.00 0.625 5.00 2.50 1.25 0.62 1.25

Water

1.25 5.00 1.25 0.312 2.50 - 5.00 1.25 1.25

Figure 5.1.4- (i) Photoplate (A) and (C) showed antifungal activity ( Minimum inhibitory concentration) of

Justicia adhatoda (3-7) and Commelina bengalensis (8-12) against Aspergillus fumigatus and A. niger

respectively.Column- 1 & 2: Control [Row A, B & C- Positive control: Media + drug + fungal strain; Row D, E

& F- Negative control: Media + fungal strain; Row G & H- Negative control: Media]; Column- 3, 4, 5, 6 & 7:

Petroleum ether (3), Chloroform (4), Acetone (5), Methanol (6) and water (7) extract of J. adhatoda. Column- 8,

9, 10, 11 & 12: Petroleum ether (3), Chloroform (4), Acetone (5), Methanol (6) and water (7) extract of C.

bengalensis.(ii) Photoplate (B) and (D) showed zone of inhibition against A. fumigatus and A. niger

respectively. Number 1 showed the water extract of C. bengalensis. Number 2,3 and 4 showed the chloroform,

acetone and petroleum ether extract of J. adhatoda. Number 5 showed the standard drug: Amphotericin B.

In addition, the very low concentration of these four extracts inhibited the growth

(100%) of A. niger, A.fumigatus and A. flavus in range of 0.156 -0.625 mg/ml by spore-

germination-inhibition assay (Table 5.1.4).

Table 5.1.4- Activity of some evaluated best extracts against pathogenic Aspergilli by disc

diffusion assay and spore germination inhibition assay.

Sr. No. Name of crude

extracts/drug

Pathogens name

Aspergillus

fumigatus

A. flavus A.niger

1. Disc

diffusion

assay at the

concentration

(10µg/disc)

Commelina

bengalensis (water)

7.3±.40 _ _

Justicia adhatoda

(Petroleum ether)

7.2±.80 _ 6.2±.60

Justicia adhatoda

(Chloroform)

6.1±.20 _ _

Justicia adhatoda

(Acetone)

_ _ 6.2±.30

Amphotericin B

(2.5 µg/disc)

8.3±.30 8.2±.6 8.6 ±.20

2. Spore

germination

inhibition

assay

(mg/ml)

Commelina

bengalensis (water)

0.156 1.25 0.312

Justicia adhatoda

(Petroleum ether)

0.312 0.625 0.625

Justicia adhatoda

(Chloroform)

0.625 0.625 0.625

Justicia adhatoda

(Acetone)

0.312 0.625 0.156

Finally, with only the exception of few extracting fractions, all the plant species

which were screened had reasonable activity against all the tested fungal species. Both, J.

adhatoda and C. bengalensis had the most consistent MIC values with an overall average of

0.156 mg/ml to 0.312 mg/ml (Table 5.1.3). Therefore, the antifungal activity of J.

adhatoda and C. bengalensis might be attributed to either the individual class of compounds

present in each herb, as confirmed by the phytochemical screening, or to the synergistic

effect that each class of compounds exert to give the observed biological activity. Hence,

further in-depth investigations should be carried out to resolve this issue. The fact that C.

bengalensis contain saponins and J. adhatoda contain phytosteroids or terpenes, might

contribute to their antifungal activity. Previous studies reported the antimicrobial activity of

many saponins and phytosteroids/terpenes rich plants that have broad antimicrobial activity.

These compounds are known to disrupt the cell wall and cell membranes of

microorganisms causing lysis of the microbial cells. Although, the antifungal activities of

these herbal drugs are less likely to be due to polar compounds like saponins, the surface

active property of these compounds may still contribute to the activity by reducing surface

tension and facilitating the penetration of another active agent into the protoplasm.

Nevertheless, since polar solvent extracts were relatively less active as compared to non-

polar solvent extracts, the activity might be attributed to lipophilic phytochemicals.

Additionally phytosteroids/terpenes are characterized by low toxicity since they are widely

distributed in edible plants.

In the present study, increased attention was focused on whether naturally occurring

compounds present in biological active medicinal herb can produce acute and cytotoxic

effects. Therefore, the different groups of rats were treated by four extracts showed no

discernible behavioural changes up to 1000 mg/kg by oral route. No mortality was observed

at this dose during 14 days observation period. The toxicological effect in percent haemolysis

of the four extracts has been given in Figure 5.1.5.

Hemolytic assay

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00

Concn. in log

Perc

en

t h

em

oly

sis

AmpB

A

B

C

D

Figure

5.1.5- Cytotoxicity analysis of amphotericin B and four extracts by haemolytic method.{Amp B=

amphotericin B; A= acetonic extract fraction of J. adhatoda; B= chloroform extract fraction of J.

adhatoda; C= petroleum ether extract fraction of J. adhatoda; D= aqueous extract fraction of C.

bengalensis.}

The Petroleum ether, chloroform and acetonic fractions of J. adhatoda leaves did not

show 50% haemolysis even at 175 µg/ml and thus can be considered safe for use. These did

not reveal any toxicity to human erythrocytes as compared to standard drug (Amphotericin B)

which causes 100% lyses at concentration of 37.5 µg/ml (Figure 5.1.5). However, the water

extract fraction of C. bengalensis is highly toxic at even at low concentration (Figure 5.1.5).

Available data are insufficient to support the safety of J. adhatoda extract by above

experimented methods. Thus, considering the strong therapeutic use of J. adhatoda, it is

significant to investigate the genotoxicity of petroleum ether extract of J. adhatoda utilizing

the Comet assay. Single Cell Gel Electrophoresis or Comet assay is a highly sensitive method

for the estimation of DNA damage both at clinically significant and low doses. From the

Comet assay, it was clearly evident that the ionic liquid induced DNA damage responded in a

dose dependent manner. The results of the J. adhatoda extract by the comet assay, namely

data on the total number of cells with damage, and scores of various concentrations of

petroleum ether extracts with 10, 100, and 500 mg/ml, besides negative (2.5% DMSO; 10

mg/ml) and positive control (H2O2 ; 10 mg/ml) are presented in (Figure 5.1.6).

As expected, H2O2, the positive control, induced a significant increase in DNA mi-

gration in maximum observed leukocytes. Although there was some increase in damaged

cells in the group of cells treated with high doses of the extract, no significant differences

were found between treated vs untreated cells. When cells were exposed to three

concentrations of the extract, most of the cells examined on slides were undamaged, a few

cells showed minor damage (category 0- 1) and very few had a large amount of damage

(category 2- 4). There were also no major differences in DNA migration between the three

extract concentrations tested. Thus, the data obtained in the present study permit us to

conclude that, under the experimental conditions employed here, the J. adhatoda leaf extract

appears to be safe as a therapeutic agent.

Figure 5.1.6- Comets showing tails of different length induced by various concentrations of

extract of Justicia adhatoda in petroleum ether fraction: (A) control (-), 2.5% DMSO (10

mg/ml); (B) 10 mg/ml; (C) 100 mg/ ml; (D) 500 mg/ ml and (E) control (+), H2O2 (10

mg/ml). A total of 60 comets were examined for each treatment with two replicates.

Similarly, in a previous screening study of anti-fertility activity of J. adhatoda, after

administration of extract of leaves either in mice or in rats, no effects on the pregnancy were

recorded (Bhaduri et al., 1968). The effect of J. adhatoda leaf extract on early gestation was

studied. There was no effect on the maternal body weight or any other parameter recorded in

the form of statistically significant differences between the treated and control animals

(Bhaduri et al., 1968). Therefore, the highest total activity without any toxicity was obtained

with J. adhatoda extracts. For that reason, J. adhatoda was chosen for further investigation.

While, it is reported that crude ethanolic extract of the J. adhatoda leaves exhibited

antimicrobial activity against Staphylococcus epidermidis, Bacillus subtilis, Proteus vulgaris

and Candida albicans (Karthikeyan et al., 2009). Moreover the methanolic extract of J.

adhatoda exhibited positive antimicrobial activity for P. aeruginosa, S. aureus and B. subtilis

while E. coli was not effectively inhibited by extracts of tested plant (Shinwari et al., 2009).

While the extract of plant showed minimum inhibition in the growth of fungi, Microsporum

gypseum, Chrysosporium tropicum and Trichophyton terrestre (Quershi et al., 1997). The

present phytochemical study concludes that the antifungal activity of J. adhatoda was mainly

due to the presence of phenolic compounds, monoterpenes alcohols and sesquiterpenes. As in

earlier study, some important bioactive compounds have been reported in various part of J.

adhatoda are essential oil and quinazoline alkaloids which possesses activities like

antitussive, abortifacient, antimicrobial, cardiovascular protection, anticholinesterase, anti-

inflammatory and other important activities (Karthikeyan et al, 2009). Some plant extracts, as

per observations in this study, such as three extract of J. adhatoda were exhibited the broad

range activity, possibly due to presence of multiple antimicrobial compounds or synergic

effects of these compounds which were identified by GC-MS and given in Table 5.1.5.

A thorough analysis of the results indicated that among the three extracts of J. adhatoda

leaves, only the petroleum ether extract of leaves showed promising activity against tested

fungi. The l-(+)-Ascorbic acid 2, 6-dihexadecanoate comprising as the major

phytoconstituents in this extract, has been reported to have an antioxidant, anti-inflammatory

and antinociceptive properties which support the bioactive extract to be safe as a therapeutic

agent. It also enhances sperm quality and prevents sperm agglutination thus making them

more motile with forward progression (Ogunlesi et al, 2010).Therefore, petroleum ether

extract of J. adhatoda leaves can be considered a potential source of candidate drug in the

treatment of infectious diseases caused by the tested pathogenic fungi.

Table 5.1.5- Identification of phytoconstituents from various fractions isolated from leaves of

J. adhatoda.

Sr. No. Plant extracts Major phytochemical components identified by GC-MS

5.1.4. Conclusion

The results of the present study revealed the highest anti-Aspergilli activity without any

toxicity by three extract fractions of J. adhatoda. Therefore, the crude plant extracts may be

employed as a model to develop new antifungal drugs, or can be used directly after further

studies to reduce the severity of fungal infections. Furthermore, all of the plants extracts

tested in this study had potential antifungal activities against the Aspergillus spp. Our results

support the use of these plants as traditional medicine and suggest that some of the plant

extracts possess compounds with good antifungal properties that can be used as antimicrobial

agents in the search of new drugs.

1 Petroleum ether

fraction of crude

extract

l-(+)-Ascorbic acid 2,6 dihexadecanoate; Dotriacontane;

3,7,11,15- Tetramethyl-2- hexadecen-1-ol; Celidoniol;

9,12,15-Octadecatrien -1- ol (Z,Z,Z); Tetracontane;

Hexatriacontane; 9-Tricosene, (Z)-; Octadecanoic acid;

Caryophyllene oxide; Andrographolide; n-Hexadecanoic acid

methyl ester.

2 Chloroform

fraction of crude

extract

n-Hexadecanoic acid; Pentacosanoic acid, methyl ester;

3,7,11,15-Tetramethyl-2-hexadecen-1-ol; Octadecanoic acid;

9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- ; 1-

Octadecanol; But-3-enal, 2-methyl-4-(2,6,6-trimethyl-1-

cyclohexenyl)-; 2-Pentadecanone, 6,10,14-trimethyl-.

3 Acetone fraction

of crude extract

1,2,4-Butanetriol; 3-Hydroxy-4,5-dimethyl-2(5H)-furanone;

Neophytadiene; Ascorbic acid 2,6-dihexadecanoate; 1,2-

Benzenedicarboxylic acid, dibutyl ester; 3,7,11,15-

Tetramethyl-2- hexadecen-1-ol; 9,12,15-Octadecatrien-1-ol,

(Z,Z,Z)-; Hexatriacontane; Methyl pentacosanoate.

Section 5.2

Isolation and purification of antifungal compound from Justicia adhatoda

5.2.1. Introduction

Today, more pharmacognostic investigations of plants are carried out to find novel

drugs or templates for the development of new therapeutic agents. Many useful drugs that are

currently in use for different diseases were derived and developed from medicinal plants,

because of their use in traditional medicine (Gurib-Fakim, 2006). With the emergence of new

diseases and resistant to already available drugs, many medicinal plants will continue to be

the best source of new and active drugs. There are still a large number of higher plant species

that have never been investigated for their chemical or biologically active constituents.

One of medicinal plants is Justicia adhatoda (Vasaka), which is found in temperate and

tropical regions in the East, native mainly throughout India. Vasaka belongs to the plant

family Acanthaceae. This plant has a handful of medicinal uses, to name a few, mainly

antispasmodic, fever reducer, anti-inflammatory, anti-bleeding, bronchodilating, antidiabetic,

disinfectant, anti-jaundice, assistance in uterine contractions, and expectorant (Dhankhar et

al., 2011).

J. adhatoda is used by many cultures in folk medicine for the treatment of several

diseases and their established medicinal values prompted us to investigate the antifungal

potential of this plant. In many cases aqueous plant extracts have been used to combat

infections in traditional medicine. Because aqueous plant extracts available to poor rural

people do not contain the non-polar medicinal compounds. This motivated the investigation

of the antifungal activity of J. adhatoda leaf extracts extracted in different polarity solvents.

The polarity of solvents is important when extracting plant material, in terms of targeting

specific compounds from crude extracts. In order to yield pure compounds, several steps need

to be followed and this includes: extraction, isolation, purification, separation, detection of

the active compounds and quantitative data analyses (Abidi, 2001). A well known isolation

procedure is the solvent extraction of the plant sample followed by column chromatography

on different sorbents. Column chromatography and TLC (Thin layer chromatography)

techniques are most affordable procedures and are suitable for sample purification, qualitative

assays and preliminary estimates of the compounds in plant extracts. In this study, we follow

column chromatography for isolating antifungal compound since it can purify larger samples

and also use normal phase systems, i.e. a polar stationary phase (silica) eluted with organic

solvents of increasing polarities. Petroleum ether extract fraction of J. adhatoda may contain

highly non-polar substances which can interfere with the separation of impure non-polar

compounds during isolation if they are present in a very high concentration. Therefore,

preliminary removal of inactive non-polar substances during isolation is useful since it

increases extract purity and allows more accurate determination of antifungal activity and

easier isolation of active compounds. The successful isolation of bioactive compounds from

indigenous medicinal plants will validate indigenous knowledge adding value to plants and

support plant conservation and knowledge preservation (Webster et al., 2008). It may also

contribute to research and development in the production of new pharmaceutical drugs for the

treatment of various infectious diseases caused by micro-organisms such as fungi and

bacteria.

5.2.2. Material and methods

5.2.2.1. Chromatographic Methods

Chromatography is the best method of choice in handling the problem of isolation of a

compound of interest from a complex natural mixture. Therefore, the chromatographic

methods used during the present work are briefly described.

5.2.2.1.1. Thin layer chromatography (TLC)

TLC involves the use of a particulate sorbent (silica) spread on an inert sheet of metal

(aluminium) as a stationary phase. The sample of plants extracts fraction were applied on the

aluminium silica plate with the help of capillaries as small streaks at about 2.0 cm above the

lower edge of the TLC plate (aluminium silica). Detail is given in material and methods

section of the thesis.

5.2.2.1.2. Column Chromatography

Column Chromatography (CC) consists of a column of particulate material such as

silica or alumina that has a solvent passed through it at atmospheric, medium or low pressure.

Detail is given in material and methods section of the thesis.

5.2.2.2. Antimycotic activity (Microbroth-dilution assay)

The spores of Aspergillus were harvested from 96 h cultures and treated with various

extracts of different plants in a 96-well culture plate. The plates were incubated at 37oC and

examined microscopically after 48 h for the growth of Aspergillus mycelia (Annette et al.,

1995; Yadav et al., 2005).

5.2.2.3. Gas Chromatography/Mass Spectrometry (GC/MS)

The GC-MS investigation of plant extracts were carried out using Shimadzu QP-2010

plus with thermal desorption system TD-20 and identification of constituent of the extract

was achieved on the basis of their retention indices determined with a reference to a

homologous series of phytoconstituents and by comparison of their mass spectral

fragmentation patterns (NIST database/ chemstation data system) with data previously

reported in literature (Sathyaprabha et al., 2010). Detail is given in material and methods

section of the thesis.

5.2.3. Results and Discussion

The extracts of herbal plants can be analyzed for the presence of desired constituents

and absence of impurities or compounds characteristic of common adulterants by various

qualitative techniques. Even if the precise identification of the constituents is difficult, the

pattern of zones may be used to characterize particular drugs (Tyler, 2002). The

chromatographic profiles of major components are used to evaluate herbs, by herbal growers

and suppliers, to standardize raw materials and to control formulation and tablet content

uniformity (Cai et al., 2002). Moreover, TLC has been widely used for the analysis of

medicinal plants and it is included as a method for identification in monographs of herbal

drugs in most pharmacopoeias throughout the world. For example, TLC has been the

most widely used classical method for fingerprinting analysis in Chinese medicines. To find

the antifungal compounds in present study, the plant extracts are first qualitatively analyzed

by thin layer chromatography and other chromatographic (GC-MS) methods to determine the

biological activity.

As described above, TLC is an ideal technique for screening of herbal drugs because

of its low cost, easy maintenance and selectivity of detection reagents. Therefore, the

extracts of the J. adhatoda was analyzed by TLC on silica gel in order to obtain

information on the active compounds from the components separated into a sequence of

discrete zones or the fingerprints distinctive for individual plant species. In such conditions

compounds can be characterized by the distance they travel in a particular TLC system

and the appearance of each zones after visualization. Thus, a data set of Rf values for the

separation of various constituents in the crude extract of petroleum ether fractions of J.

adhatoda and its corresponding appearance under daylight and UV illumination is shown

in Figure 5.2.1.

Figure 5.2.1- Chromatogram fingerprints of pure fractions isolated from column sub-

fractions of J. adhatoda (petroleum ether fraction).

{(A) J. adhatoda plant; (B) Leaves of J. adhatoda plant; (C) TLC chamber; (D) TLC profile

of crude extract J. adhatoda (petroleum ether fraction) in visible light; (E) TLC profile of

crude extract in UV light; (F) Column chromatography; (G),(H), (I) and (J) TLC profile of

purified fractions with respect to crude extract as a standard.}

5.2.3. 1. Isolation of bioactive compounds from petroleum ether fraction

5.2.3.1.1. Equilibration of Chromatographic Chamber

About 1.0 cm of height of solvent system was taken in a clean dry glass chamber. The

chamber was covered with air tight lid and allowed to soak with vapours of solvent. The

inner side of the back wall of chamber was lined with a piece of filter paper. The lower edge

of the filter paper was dipped into the solvent present at the bottom of the chamber to ensure

the even distribution of the solvent vapours throughout the volume of developing tank.

5.2.3.1.2. Application of Sample

Fifteen micro litres of neat plant extract at 100 mg/mL was applied 2 cm from the

base of aluminium-backed silica plates (Merck 60F254, Germany) cut to size (10x5 cm). The

plates were dried for 15 minutes at room temperature and separately developed in the various

solvent combinations. The plates were prepared in duplicates for each solvent combination

(‘A’ and ‘B’) and developed in glass tanks closed with aluminium foil. Plate ‘A’ was used as

a reference chromatogram to visualize the separated spots under visible light and UV

irradiation at 365 nm and sprayed with vanillin (Eloff, 2001). The plate was carefully heated

at 105 ºC for optimal colour development. The Rf values (Retention factor) of the spots on the

plate were computed and recorded.

5.2.3.1.3. Development of Chromatogram

The plates loaded with petroleum ether fraction of J. adhatoda were kept in the

chromatographic chamber containing the mixture of petroleum ether and ethyl acetate in ratio

of 80.0:20.0. The chamber was closed with an air tight lid to saturate it properly. The solvent

was allowed to rise up to a height of about 9.0 cm at room temperature. After developing the

chromatogram, the plates were removed from the tank, the solvent front was marked and they

were allowed to dry in the air. The various components in the fraction were detected with UV

light and by spraying the group specific reagents.

5.2.3.2. Sub-fractionation by Column Chromatography

The components of various bands having different Rf values were fragmented and

examined for antifungal activity. The Column Chromatography was performed to obtain

active component of our interest. The silica gel was suspended in petroleum ether and packed

in a glass of 5x 35 cm size. The column was equilibrated with petroleum ether for 16 h before

loading the sample.

Slurry of petroleum ether fraction was prepared in petroleum ether and loaded on pre-

equilibrated silica gel column carefully. The polarity was increased by addition of ethyl

acetate (EtAC) at an interval of 1% until 20% EtAC. The components of petroleum ether

fraction were eluted with 200 ml of petroleum ether at a flow rate of 1.0 ml/min followed by

different ratio of Petroleum ether: Ethyl acetate ranging from 100: 0 to 80: 20. Eighty

fractions collected in test tubes were allowed to concentrate under a stream of cold air.

Fractions containing similar constituents were combined (monitored by TLC fingerprinting).

Total 80 sub-fractions of 50 ml each were analysed by TLC using the solvent of Petroleum

ether: Ethyl acetate (90.0: 10.0). The sub-fractions showing similar profile of Rf values were

pooled and dried in vacuo-rotavapour. The pooling of same sub-fractions resulted into 12

column sub-fractions which are monitored by TLC fingerprinting (Figure 5.2.2).

Figure 5.2.2- Chromatogram fingerprints of column sub-fractions isolated from petroleum

ether fraction of J. adhatoda.

{ST= standard crude extract; 1,2,3,4,5,6,7,8,9,10,11,12,= various fraction’s fingerprint;

Upper fingerprints (B) are in daylight and Lower fingerprints (C) are in UV light.}

5.2.3.3. Antifungal Activity of Column Sub- fractions

The antifungal activity of each sub-fraction was tested against A. fumigatus, A. flavus

and A. niger by microbroth dilution assay as given in Table 5.2.1. The stock solution of all

the 12 column sub-fractions was prepared and their antifungal activity (A. fumigatus) was

examined in the range of 0.00 to 93.0 µg/ml by microbroth dilution assay (Table 5.2.1 and

Figure 5.2.3) and the further subjected to the thin layer chromatography for identifying and

separating out pure active component.

Table 5.2.1- Antifungal activity (MIC) of twelve fractions isolated from petroleum ether

extracts of Justicia adhatoda.

Pathogens

MIC of plant extract fractions (mg/ml)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12

A. fumigatus - - - - - - 1.5 0.750 0.187 0.093 0.375 1.5

A. niger - - - - - 1.5 1.5 0.750 0.187 0.093 0.375 1.5

A. flavus - - - - - - 1.5 0.750 0.187 0.093 0.375 0.750

{(-) means no activity at 3 mg/ml. Because initial concentrations of samples were 3 mg/ml; F1 to F12

= Fraction 1 to Fraction 12 respectively = twelve fractions isolated from petroleum ether extracts of

Justicia adhatoda respectively}

Figure 5.2.3- Antifungal activity of twelve fractions isolated from petroleum ether extracts of

Justicia adhatoda against A. fumigatus by microbroth dilution assay.

5.2.3.4. TLC of Active Column Sub-fraction

The TLC of active column sub-fraction was performed using solvent system of

Petroleum ether: Ethyl acetate (90.0:10.0). The distance travelled by component and solvent

front was measured and Rf values were calculated as 0.31, given in Figure 5.2.1. The

preparative TLC was run in the prescribed solvent system and the bands were detected under

UV light and single band was observed.

5.2.3.5. GC-MS of Active Column Sub-fraction

The plant crude extract was initially fractionated by silica gel column and thin layer

chromatography techniques. Subsequent fractionation and identification of the phyto-

constituents was achieved by gas chromatography and mass spectrometry (GC/MS) analysis.

The antifungal activity of the fractions and compounds was evaluated against three

pathogenic Aspergilli fungi using micro-broth dilution assay. Amphotericin (MIC = 0.73-1.95

µg/ml) was included in these experiments as a positive control antifungal drug. Seven of the

twelve fractions collected, demonstrated antifungal activity with minimum inhibitory

concentration (MIC 90) values ranging from 1500- 93.0 μg/ml.

The GC-MS of Active Column Sub-fraction was performed using solvent system of

chloroform and investigation was carried out using Shimadzu QP-2010 plus with thermal

desorption system TD-20 gas chromatography equipped with an Turbomolecular pump

(58l/Sec for He), Rotary pump 30L/min (60Hz) and Column (Inert Cap Pure-WAX) flow up

to 4mL/min which was operated in EI mode (1 pg octafluoronaphthalene m/z 272 S/N ˃200).

Helium was the carrier gas at a flow rate of 1ml/min. The injector was operated at 250°C and

the column temperature was programmed as follows; 35°C for 5min to 4°C/min, then

gradually increased to 250°C for 10min. Identification of constituent of the extract was

achieved on the basis of their retention indices determined with a reference to a homologous

series of phytoconstituents and by comparison of their mass spectral fragmentation patterns

(NIST database/ chemstation data system) with data previously reported in literature

(Sathyaprabha et al., 2010). The GC-MS spectra of purified fraction of leaves extract

(petroleum ether) of J. adhatoda which exhibited the best antifungal activity, has been given

in Figure 5.2.4.

The most abundant identified compound was 2-Hexadecen-1-ol, 3, 7, 11, 15-

tetramethyl-, (64.20%), followed by 1-Hexadecyne (28.55%) and 1-Octadecyne (7.24%). 2-

Hexadecen-1-ol, 3, 7, 11, 15-tetramethyl-, is present in a major quantity has been previously

isolated and identified from jute leaves, is a well known acyclic diterpene, present as the ester

side chain in the molecule of chlorophylls (Harborne, 1998). The compound is a constituent

of nettles, Leucas volkensii, alga, Perilla spp, Solidago virga-aurea, Tetragonia

tetragonoides (New Zealand spinach), Garcilaria andersoniana, Megaceros flagellaris and

other plants. It is one of the main compounds present in extracts of Mentha spicata as well as

Camella sinensis (Padmini et al., 2010) and reported for various biological activities such as

antimicrobial, anticancer, antiinflammatory and diuretic activity (Praveen et al., 2010). The

phenolic constituents of the extracts of M. spicata namely rosmarinic acid, luteolin and 2-

Hexadecen-1-ol, 3, 7, 11, 15-tetramethyl- are reported for their antimicrobial and antiviral

activities, strong antioxidant and antitumor action (Mckay and Blumberg, 2006).

Figure 5.2.4- GC-MS spectra of bio-active (antifungal) sub-fraction of leaves extract

(petroleum ether) of J. adhatoda.

The phytol (2-Hexadecen-1-ol, 3, 7, 11, 15-tetramethyl-) is also an anticancer agent

(colon and gastric cancer) and used in the preparation of vitamin E and K. It has potent

antitumor activity against induced Raji cells at concentration of 15µg/ml and 30µg/ml in the

medium (Furumoto et al, 2002). Moreover, Eicosane and 2-Hexadecen-1-ol, 3, 7, 11, 15-

tetramethyl- are reported to be the main constituents in the Aloe vera extract;

responsible for high antimicrobial activity against clinical pathogens (Arunkumar and

Muthuselvam, 2009). Octadene is also said to possess various activities such as anticancer,

antioxidant and antimicrobial activities (Vinay et al., 2011; Lee et al., 2007).

5.2.4. Conclusion

During isolation of compounds from crude extracts, a large proportion of plant

constituents are lost especially when using silica gel chromatography. The quantity of some

plant constituents is higher than others hence they can be easily isolated. Only, twelve

column sub-fractions were isolated from leaves extract (petroleum ether) of J. adhatoda. The

10th

sub-fraction (viscous yellowish green liquid; Rf = 0.31) indicated the presence of

antifungal constituents using GC-MS, and low MIC values in the microbroth dilution assay

against A. fumigatus, A. flavus and A. niger. These isolated compounds preliminary

recognized to be the terpene oils by GC-MS before further identification studies such as

NMR (1H,

13C, and COSY), FTIR and mass spectroscopy analysis which are necessary to

confirm this supposition. This facilitates the identification and isolation of active constituents,

and hence J. adhatoda was selected for isolation and characterization of plant compounds

active against J. adhatoda. In the following chapter the structures of the isolated compounds

are determined using NMR, FTIR, UV-spectra and MS spectroscopy techniques.

Section 5.3

Structure elucidation and Antifungal potential of purified compound

5.3.1. Introduction

Over the lifetime of the modern pharmaceutical industry, natural products have been

established as an excellent supply source for the discovery of novel compounds with

therapeutic potential (Svetaz et al., 2010). Obviously natural products will continue to be

extremely important as sources of medicinal agents (Singh et al., 2004). In addition to the

natural products which have found direct medicinal application as drug entities, many others

can serve as chemical models or templates for the design, synthesis, and semi synthesis of

novel substances for treating humankind’s diseases (Sunita and Mahendra, 2008). Therefore,

the ultimate goal of surveying plants for biological activity is thus to isolate one or

more biologically active compounds that may be potentially useful in treating certain

disease conditions or serve as a structural analogue (template) from which better

synthetic modifications can be derived. Chemical characterization and compositional

analysis of traditional medicines provide the necessary scientific basis for the discovery

and development of new drugs of natural origin (Shrestha and Dhillion, 2003). Compounds

derived from natural products are principally identified using techniques such as nuclear

magnetic resonance (NMR) and mass spectroscopy (MS) that provides structural information

leading to the complete structure determination of natural products (Silva-Elipe, 2003). MS is

used in conjunction with NMR spectroscopy to allow determination of the molecular formula

of a compound. Use of molecular weight and UV absorbance data with Bioactive Natural

Products Database allows rapid identification of compounds. The identification is then

confirmed by FT-IR yields information about functional groups (Duraipandiyan and

Ignacimuthu, 2009).

A number or methods may be used to elucidate the structure. These include: Gas

chromatography, mass spectrometry (MS), nuclear magnetic resonance (NMR), Fourier

transform infrared (FTIR) spectroscopy and ultra-violet (UV) absorption (Van der Watt and

Pretorius 2001). Gas chromatography and MS are used to study crude extracts and

multicomponent fractions for the presence of specific bioactive compounds or compound

types. These methods are used as part of the de-replication process to help identify known

classes of compounds when looking for novel bioactive compounds. Structural elucidation

based on these techniques has been the most successful for determining both simple and

complex structures. In this chapter, we used NMR, FT-IR, GC-MS, MS and ultra-violet (UV)

absorption spectrometry to determine the structure of bioactive compound isolated from

leaves of J. adhatoda.

5.3.2. Materials and methods

5.3.2.1. Spectroscopic Techniques

5.3.2.1.1. Nuclear Magnetic Resonance

Spectroscopy is the study of the interaction of electromagnetic radiation (EMR) with

matter. NMR spectroscopy is the study of interaction of radio frequency (RF) of the EMR

with unpaired nuclear spins in an external magnetic field to extract structural information

about a given sample. NMR spectroscopy is routinely used by chemists to study chemical

structure of simple molecules using simple one dimensional technique (1D-NMR). NMR is

the best method to use for non-crystalline compounds. Detail was given in material and

methods section of the thesis.

5.3.2.1.2. Mass Spectrometry (MS)

MS is an analytical technique that involves generating charged particles (ions) from

molecules of the analyte. The generated ions are analyzed to provide information about the

molecular weight of the compound and its chemical structure. There are many types of mass

spectrometers and different sample introduction techniques which allow a wide range of

samples to be analyzed. Detail was given in material and methods section of the thesis.

5.3.2.1.3. Other Spectroscopic methods

These include the Fourier transform infrared (FTIR) spectroscopy which offers

information relating to the functional groups, and the ultraviolet (UV) spectroscopy which

reveals information relating to the presence of sites of unsaturations in the structure. These

two methods are becoming less important in structure elucidation of natural products due to

the superiority of information obtained from the NMR experiments with much less sample

amounts. Detail was given in material and methods section of the thesis.

5.3.2.2. Antimycotic activity (Microbroth-dilution assay)

The spores of Aspergillus were harvested from 96 h cultures and treated with various

extracts of different plants in a 96-well culture plate. The plates were incubated at 37oC and

examined macroscopically after 48 h for the growth of Aspergillus mycelia (Annette et al.,

1995; Rajesh and Sharma, 2002).

5.3.3. Results and Discussion

NMR is the best method to use for non-crystalline compounds. It allows fragments of

compounds to be combined into complete molecules and may be used to definitively identify

metabolites. Before undertaking NMR analysis of a complex mixture, separation of the

individual compounds by chromatography is required (Silva-Elipe 2003). Nuclear magnetic

resonance is the best method for complete structure elucidation of non-crystalline samples.

When elucidating the structure of secondary natural products, 1H NMR,

13C NMR and 2D

NMR spectroscopy are important since hydrogen and carbon are the most abundant atoms in

natural products (Van der Watt and Pretorius, 2001). However, there are some difficulties

encountered when using NMR because it has a very low sensitivity compared to MS and it

therefore requires much larger samples for analysis. The machine can detect proton (1H)

sensitivity, high isotopic natural abundance and its ubiquitous presence in the organic

compounds. When using NMR, all samples require signal averaging to reach an acceptable

signal-to-noise level. The NMR analysis depends entirely on the size of the sample, and can

range anywhere from several minutes to several days. For example, in the case of metabolites

with a mass of 1-10 µg, an overnight experiment with a very powerful apparatus is required

(Silva-Elipe, 2003). MS does not always provide conclusive structural information, especially

when isomers of bioactive compounds are studied. It can be used to determine the molecular

weight and confirm the structure of the isolated compounds or natural products.

5.3.3.1. Structure elucidation of purified bioactive compound

An analytical Varian-NMR-vnmrs 600 instrument operating at proton frequency of 600

MHz was used for 1H and

13C. The compound isolated from leaves of J. adhatoda

was weighed (10-30 mg) and dissolved in deuterated CDCl3 since the compound was

soluble in CHCl3. Each sample were dissolved in 0.7 ml CHCl3 and

transferred into NMR tubes (5 mm). Spectral data were collected on a FTIR spectrometer

(Model FTS 7000; Varian Inc., Palo Alto, CA, USA) coupled to an infrared microscope

(model 600 UMA; Varian) using a 15 ×Varian objective and fitted with a liquid N2 cooled

MCT 64× 64 element array Stingray (Varian) focal plane array detector. The Varian system

was controlled by IBM compatible PC running WIN IR PRO 3.0 software (Varian). The

absorbance spectra were acquired in reflectance mode at a spectral resolution of 8 cm – 1

with

64 scans coadded. Apodization was performed using a triangular function. The system

enabled 4096 spectra to be acquired from a sample area of approx. 350 μm 2

in approx. 2 min.

PC (Purified Compound) was obtained as yellowish green viscous liquid and showed

a molecular ion peak in EI-MS (Mass spectroscopy) at m/z =296 (Figure 5.3.4) which

together with 1H and

13C NMR spectral data (Table 5.3.1), suggested a molecular formula of

C20H40O which indicates 1 degree of unsaturation that can be deduced to be a double bond by

examining FT-IR (ν max 1660 cm-1

) absorption spectrum, 1H NMR one olefinic proton at δ

5.40 ppm and 13

C NMR two signals at δ 123.02 and 140.38 ppm. The DEPT

(Distortionless Enhancement by Polarization Transfer-spectra) spectra of 13

C NMR showed

that compound contains five methyl, ten methylene, four methine and one quaternary carbon.

The 13

C NMR spectra (Figure 5.3.5) together with DEPT spectrum exhibited 20 carbon

signals: δ 140.1 (s), 123.1 (d), 59.3 (t), 39.8 (t), 39.3 (t), 37.4 (t), 37.2 (t), 37.0 (t), 36.6 (t),

32.9 (d), 32.6 (d), 27.9 (q), 25.1 (t), 24.7 (t), 24.2 (t), 22.6 (q), 22.5 (q), 19.7 (q), 19.6 (q),

16.1 (q).

The 1H NMR spectra (Figure 5.3.6) exhibited the signal of an olefine with protons δ

5.40, td, 1H, and oxide methylene group δ 4.14, d, J=6.9, 2H, methylene group δ 1.95, d, J=

7.7, 2H, 5 methyl group δ 1.67, 0.87, 0.84, 0.85, 0.86. The nature of oxygen atom was found

to be primary alcoholic from both 1H NMR δ 3.53 (t, J= 6.5) ppm and 13

C NMR δ 59.44

ppm. The UV spectra (Figure 5.3.8) and FTIR spectra (Figure 5.3.9) data (3100-3400 cm-1

: -

OH, 1500 cm-1

: double bond) also confirmed these results. We can observe a difference in the

position of the double bond. Localization of the hydroxyl group at C-1, methyl groups and

the double bond at C3-C4 were deduced from correlations and COSY experiment (Figure

5.3.7). Positions of the methyl groups also can be explained by the obtained spectral data

(Table 5.3.1) which is in agreement with the biogenetic rule of terpenoids.

Table 5.3.1- List of the chemical shift values for purified bioactive (antifungal) compound (Phytol) in

13C NMR (125 MHz) and

1H NMR (600 MHz) spectra in CDCl3.

C No. Purified compound

( δ13C ) ppm

Purified compound

( δ H)

ppm (m, J in Hz)

Proton Position

(H)

1 59.3 4.14 (d, J= 6.8 ) B

2 123.2 5.40 (d-q, d= 6.8, 1.4) A

3 140.0 - -

4 39.9 1.95 (t, J= 7) C

5 25.1 1.33 (m) G

6 36.7 1.25 (m) J

7 32.8 1.66 (m) E

8 37.4 1.29 (m) J

9 24.5 1.25 (m) J

10 37.4 1.25 (m) J

11 32.7 1.59 (m) F

12 37.3 1.25 (m) J

13 24.8 1.25 (m) J

14 39.4 1.15 (m) K

15 28.0 1.72 (m) F

16 22.6 0.87 (d, J= 6.3) L

17 22.7 0.86 (d, J= 6.3) M

18 19.7 0.85 (d, J= 6.1) N

19 19.7 0.84 (d, J= 6.3) N

20 16.1 1.67 (s) D

Therefore, the molecular formula of this compound was determined as C20H40O.

Comparing 1H,

13C NMR and GC-MS spectra of this compound with that of phytol (3, 7, 11,

15 tetramethyl-2-hexadecen-1-ol), both data were closely coincided. Therefore, this

compound was determined as (2E)-3, 7, 11, 15-tetramethyl- 2-hexadecen-1-ol (Figure 5.3.1-

5.3.3).

According to the data above and the degree of unsaturation in the molecule, it should

be a long chain compound containing a double bond. These data of allowed to be assigned

that it should be acyclic diterpene alcohol which can be deduced to be phytol following

structure, 3, 7, 11, 15 tetramethyl-3-hexadec-en-1-ol (Figure 5.3.1-5.3.3). This compound is

known as phytol, which is generated as the result of the decomposition of chlorophyll. Its

activity against Mycobacterium tuberculosis was reported (Rajab et al., 1998). Phytol and its

metabolites have been reported to bind and activate the transcription factors PPAR-alpha

(Gloerich et al., 2005). Peroxisome proliferator-activated receptor alpha (PPAR-alpha) is a

transcription factor and a major regulator of lipid metabolism in the liver.

5.3.3.2. Antifungal Activity of Column Sub- fractions

The stock solution of purified compound was prepared. The antifungal activity of

purified compound was tested against A. fumigatus, A. flavus and A. niger by microbroth

dilution assay. Its antifungal activity was examined as an average MIC value of 93.0 µg/ml

against A. fumigatus, A. flavus and A niger. Then, bioactive purified compound further

subjected to the toxicity test and biochemical analysis.

5.3.4. Conclusion

Medicinal plant, J. adhatoda provide leads to find therapeutically useful antifungal

compound (3, 7, 11, 15-tetramethyl- 2-hexadecen-1-ol), thus more efforts should be made

towards isolation and characterization of other active principles and elucidation of the

relationship between structure and activity. Therefore, a combination of traditional and

modern knowledge can produce better drugs for infectious diseases with fewer side effects.

Figure 5.3.1- The molecular structure of antifungal molecule (purified) named 3, 7, 11, 15-

tetramethyl- 2-hexadecen-1-ol.

Figure 5.3.2- Ball stick model of antifungal molecule (purified) named 3, 7, 11, 15-

tetramethyl- 2-hexadecen-1-ol.

Figure 5.3.3-The molecular structure of antifungal molecule (purified) named 3, 7, 11, 15-

tetramethyl- 2-hexadecen-1-ol.

Figure 5.3.4- Mass spectra of active (antifungal) molecule isolated from leaves extract (petroleum

ether) of J. adhatoda.

Figure 5.3.5- 13

C NMR spectra of active (antifungal) molecule isolated from leaves extract

(petroleum ether) of J. adhatoda.

Figure 5.3.6- 1H NMR spectra of active (antifungal) molecule isolated from leaves extract

(petroleum ether) of J. adhatoda.

Figure 5.3.7- 2D - NMR (COSY) spectra of active (antifungal) molecule isolated from leaves

extract (petroleum ether) of J. adhatoda.

W a v e le n g th (n m )

Ab

so

rba

nc

e

2 0 0 3 0 0 4 0 0 5 0 0 6 0 0

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Figure 5.3.8- UV absorbance spectra of active (antifungal) molecule isolated

from leaves extract (petroleum ether) of J. adhatoda.

Figure 5.3.9- FT-IR spectra of bio-active (antifungal) molecule isolated from leaves

extract (petroleum ether) of J. adhatoda.

Section 5.4

Toxicological and Biochemical characterization of Identified compound

5.4.1. Introduction

Toxicology is an aspect of pharmacology that deals with the adverse effect of

bioactive substance on living organisms. In order to establish the safety and efficiency of a

new drug, toxicological studies are very essential experiments in animals like mice, rats etc.

Toxicological studies help to make a decision whether a new drug should be adopted for

clinical use or not (Alam et al. 2006). From a scientific point of view, research has shown

that many substances, including natural products, are potentially toxic and therefore should

be used with care, respecting their toxicological risks (Veiga-Junior et al., 2005). In most

cases, the adverse effects of commonly used plants are not well documented in the literature

and their long term use by humans is usually correlated with low toxicity. However, studies

have shown that many foods and traditional plants used for their medicinal properties have

mutagenic effects (Elgorashi et al., 2003). The potential toxicity of the traditional medicines

is an important consideration when studying their biological activities (McGaw et al.,

2007). Plant extracts might be very toxic as they contain many different compounds;

therefore it is very important to investigate toxicity of both crude extracts and isolated

compounds.

Justicia adhatoda plants have attracted considerable attention from pharmacologists

due to a wide range of biological properties. J. adhatoda, a medicinal plant of the

Acanthaceae family, is widely distributed in several temperate and tropical regions in the

East, native mainly throughout India. It has long been used in traditional folk medicine to

treat diseases, such as chronic bronchitis, antidiabetic, disinfectant, anti-jaundice, anti-

inflammatory and so on (Dhankhar et al., 2011). Present study show that the extracts of this

plant exhibit excellent antifungal and antioxidant activities. The plant can be considered as a

multipotent herbal medicine. However, to our best knowledge, no references about its

antifungal constituents have been published. Considering the undesirable attributes of

synthetic fungicides, there is an urgent need to develop alternative treatments that are less

hazardous to humans and animals. Therefore, increased attention has focused on whether

naturally occurring compounds present in biological active medicinal herb, Justicia adhatoda

can produce toxicity. For that reason, the toxicity test of the isolated compound from

petroleum ether extract was performed by acute, cytotoxicity (Hemolytic assay) and

genotoxicity assays (Comets assay).

5.4.2. Material and Methods

5.4.2.1. Toxicity studies

5.4.2.1.1. Acute toxicity

The bioactive petroleum ether fraction of J. adhatoda, at the range 100mg to

1000mg/kg was administered orally to the groups of rats comprising six rats in each group.

Mortality and general behaviour was observed for 14 days.

5.4.2.1.2. Haemolytic assay

Human erythrocytes, collected from apparently healthy individuals, were washed

three times with PBS (phosphate buffer saline) by centrifugation at 1500 rpm for 10 min. A

2% erythrocyte suspension was incubated at 37.8 oC for 1 h with different concentrations of

purified compound (phytol) ranging from 500.00 to 3.9 µg/ml. After incubation, cells were

pelleted at 5000 rpm for 10 min. The supernatant was collected and the absorbance A450 was

determined using a spectrophotometer (UV Vis Spect Lambda Bio 20, Perkin Elmer). In

negative control sets, only buffer was used for background lysis, whereas in positive controls,

lysis buffer was used for completely lysing the erythrocytes. For each sample the percentage

of maximum haemolytic activity was determined (Yadav et al., 2005).

5.4.2.1.1. Single cell gel electrophoresis assay (Comet assay)

Blood was taken from a healthy donor by venipuncture; comet assay and

classification of comet category and their tail measurements were carried out according to

Garcia et al., (2007). Detail is given in material and methods section of the thesis.

5.4.2.2. Study of physical/biochemical properties

The colour, boiling point, solubility behaviour and other properties of the purified

compound were determined as described by Furniss et al., (1989).

5.4.3. Results and Discussion

Medicinal plants continue to play a central role in the healthcare systems of a

large proportion of the world’s population. In developing countries, a substantial part of the

population uses folk medicine for its daily health care. Despite widespread use, few scientific

studies have been undertaken to ascertain the safety and efficacy of traditional remedies

(Veerappan et al., 2007). Although many medicinal plant products are used as relief for many

ailments in humans, very little is known about their toxicity. Safety should be the overriding

criterion in the selection of medicinal plants for use in healthcare systems (Cuzzolin et al.,

2006).

5.4.3.1. Toxicity studies

Some of the most common practices involve the use of crude plant extracts, which

may contain a broad diversity of molecules with often unknown biological effects (Konan et

al., 2007). The evaluation of the toxic action of plant extracts/purified compound is

indispensable in order to consider a treatment safe; it enables the definition of the intrinsic

toxicity of the plant and the effects of acute overdose. The administration of the

extracts/purified compound in increasing amounts enables the evaluation of the toxicity

limits, and the test should be carried out in three doses, taking into account such factors as

weight, sex, species, diet, and environmental conditions (Lagarto Parra et al., 2001).

5.4.3.1.1. Acute toxicity

The acute toxic class (ATC) method has been developed for hazard assessment, for

hazard classification purposes, and for risk assessment. The method enables the toxicologist

to allocate chemical substances to all classification systems currently in use. The LD50 (Lethal

dose) test was designed to give a numerical index of acute toxicity. However, substantial

experience has shown that data from such tests are highly variable between differing

experiments and laboratories. It will attempt to describe the statistical strengths and

limitations of the various methods for accurately determining a point estimate of the LD50.

It is the principle of the test that based on a stepwise procedure with the use of

a minimum number of animals per step; sufficient information is obtained on the acute

toxicity of the test substance to enable its classification. The substance is administered

orally to a group of experimental animals (Wistar rats) at one of the defined doses. Absence

or presence of compound-related morality of the animals dosed at one step will determine the

next step. The method enables a judgement with respect to classifying the test substances to one

of the series of toxicity classes defined by fixed LD50 cut-off values.

In the present study, increased attention has focused on whether naturally occurring

compounds present in biological active medicinal herb (J. adhatoda) can produce acute toxic

effects. Therefore, the different groups of rats (wistar) were treated by five different

concentrations of petroleum ether extract fractions; showed no discernible behavioural

changes up to 1000 mg/kg by oral route. No mortality was observed at this dose during 14

days observation period. The toxicological effect on wistar rats has been given in form of

outcome on body weight (Figure 5.4.1). The minor effect has been reported on the body

weights of rats after oral administration of purified fraction (petroleum ether) of J. adhatoda.

Furthermore, it has already mentioned in database (Dictionary of Natural Products, 2006) that

the lethal dose 50 (LD50) of 2-Hexadecen-1-ol, 3, 7, 11, 15-tetramethyl (rat, oral) is >

5000 mg/kg.

D a y s

Bo

dy

we

igh

t (m

g)

0 5 1 0 1 5 2 0

1 5 0

1 6 0

1 7 0

1 8 0

1 9 0

I (1 0 0 m g /K g )

II (2 0 0 m g /K g )

II I (4 0 0 m g /K g )

IV (7 0 0 m g /K g )

V (1 0 0 0 m g /K g )

N o rm a l C o n tro l

Figure 5.4.1- Effect on body weights of wistar rats due to different concentrations

administration (oral) of purified fraction (petroleum ether) of J. adhatoda.

As a result, currently available purified fraction (petroleum ether) of J. adhatoda after

the treatment of different concentrations is relatively safe and essentially effective, and the

negligible toxicity associated with this fraction may increase its utility.

5.4.3.1.2. Cytotoxic test (Haemolytic assay)

Since many people in developing countries depend on traditional medicinal plants for

their primary health care; it is very important to study the cytotoxic effects of the plant in use.

In vitro cytotoxicity is necessary to define basal cytotoxicity such as the intrinsic ability of a

compound to cause cell death as a result of damage to several cellular functions (Bouaziz

et al., 2006).

In the previous chapter, the structure of purified compound was elucidated as a

diterpenoid named 3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol. The crude extracts and pure

compounds of medicinal plants are important in drug discovery; however their toxicity

requires extensive attention since this can cause various side effects (biological implications)

to human and animals. In general, cell type cytotoxic specificity of plant extracts is likely due

to the presence of different classes of compounds (such as terpenes or terpenoids, and

alkaloids) in the extracts. There are several types of cytotoxicity assays that can be used to

determine the level of toxicity in the plant extracts. However, cytotoxicity (Haemolytic assay)

with red blood cell cultures is highly preferred because it is very common, rapid and

inexpensive. The purified compound of

J. adhatoda leaves was not showing 50% haemolysis even at 175 µg/ml and are safe for use

as these did not reveal any toxicity to human erythrocytes as compared to standard drug

(amphotericin B) which causes 100% lyses at concentration of 37.5 µg/ml (Figure 5.4.2).

Figure 5.4.2- Cytotoxicity analysis of amphotericin B and purified compound (3, 7, 11, 15

tetramethyl-2-hexadecen-1-ol) by haemolytic method.

5.4.3.1.3. Genotoxicity test (Comet assay)

Available data are insufficient to support the safety of purified compound isolated

from petroleum ether extract of J. adhatoda by above experimented methods. Thus,

considering the strong therapeutic use of J. adhatoda, it is significant to investigate the

genotoxic activity of purified compound utilizing the Comet assay. Single Cell Gel

Electrophoresis or Comet assay is a highly sensitive method for the estimation of DNA

damage both at clinically significant and low doses (Ferguson, 2001). From the Comet assay,

it was clearly evident that the ionic liquid induced DNA damage responded in a dose

dependent manner. The results of the purified compound (3, 7, 11, 15 tetramethyl-2-

hexadecen-1-ol) by the comet assay, namely data on the total number of cells with damage,

and scores of various concentrations of purified compound with 10, 100, and 500 mg/ml,

besides negative (2.5% DMSO; 10 mg/ml) and positive control (H2O2; 10 mg/ml) are

presented in (Figure 5.4.3).

As expected, H2O2, the positive control, induced a significant increase in DNA

migration in maximum observed leukocytes. Although there was some increase in damaged

cells in the group of cells treated with high doses of purified compound, no significant

differences were found between treated vs untreated cells. When cells were exposed to three

concentrations of purified compound (3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol), most of the

cells examined on slides were undamaged, a few cells showed minor damage (category 0- 1)

and very few had a large amount of damage (category 2- 4). There were also no major

differences in DNA migration between the three extract concentrations tested. Thus, the data

obtained in the present study permit us to conclude that, under the experimental conditions

employed here, the 3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol purified from J. adhatoda leaf

extract appears to be safe as a therapeutic agent. Therefore, the highest total activity without

any toxicity was obtained with purified compound. For that reason, J. adhatoda was chosen

for further investigation and drug development.

Figure 5.4.3- Comets showing tails of different length induced by various concentrations of

purified compound (3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol) of Justicia adhatoda: (a)

control (-), 2.5% DMSO; (b) 10 mg/ml; (c) 100 mg/ ml; (d) 500 mg/ ml and (e) control (+),

H2O2. A total of 60 comets were examined for each treatment with two replicates.

5.4.3.2. Therapeutic index of the extract fraction and isolated compound

The therapeutic index for the purified antifungal compound was calculated using the

cytotoxic concentrations of the compound. The therapeutic index against each fungus was

calculated as follows:

Therapeutic index (TI) = LC50 against red blood cells in µg/ml divided by the MIC in µg/ml.

The higher the therapeutic index the better the compounds can be considered for use in drug

discovery.

The MIC of purified compound (3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol) and

petroleum ether extract fraction of J. adhatoda are 98 µg/ml and 156 µg/ml respectively. While

both were not demonstrating 50% haemolysis even at 175 µg/ml concentration. Therefore, the

therapeutic index value of purified compound (3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol) and

petroleum ether extract fraction of J. adhatoda were calculated as 1.79 and 1.12 respectively.

The result concludes that both were harmless antifungal agent with higher therapeutic index

value and can be use, further for antifungal drug development.

5.4.3.3. Biochemical characterization of purified molecule

Phytol (3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol) is a branched-chain fatty alcohol

that is a naturally occurring precursor of phytanic acid, a fatty acid involved in the

pathogenesis of Refsum disease. A number of reports have appeared indicating the isoprenoid

phytyl moiety is effective in the treatment of several disorders. For example, phytol and

phytanic acid were found to be as effective as vitamin E (D-a-tocopherol) in alleviating

symptoms of nutritional muscular dystrophy in chicks. Vitamin E is ineffective in the

treatment of progressive muscular dystrophy in mammals. Phytol, like cholesterol,

intercalates between the fatty acid chains of a lecithin bilayer, but the resultant effect on the

lipid packing is completely different. Because of the disruptive effect of the phytol branched

chain, a general destabilization of the membrane occurs. Due to membrane disruptive effect

of the phytol branched chain, it might exhibit the strong antifungal activity against A.

fumigatus, A. flavus and A. niger. Some other biochemical and physico-chemical parameters

of 3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol have been given in Table 5.4.1.

Table 5.4.1-Biochemical/ physico-chemical properties of isolated and purified compound of

Justicia adhatoda.

Name of Purified compound: Phytol (3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol).

5.4.4. Conclusion

The correct use of plants by the general population requires the use of medicinal

plants selected for their efficacy and safety, based on folk tradition or validated scientifically.

Therefore, an assessment of their acute, cytotoxic and genotoxic potential is necessary to

ensure a relatively safe use of plant-derived medicines. As a result, currently available

purified compound (3, 7, 11, 15 tetramethyl-2-hexadecen-1-ol) for the treatment of various

fungal infections is relatively safe and essentially effective and the negligible toxicity

associated with this molecule may increase its utility. Some biochemical characters also

support its antifungal potential. The cytotoxicity (haemolytic assay) and genotoxicity (Comet

assay) of purified compound are reported for first time in this study.

S. No. Observation Parameter Observation

1 Molecular Weight & Formula 296 g mol-1

& C20H40O

2 Physical Test

(a) Nature

(b) Odour

Yellowish green viscous liquid

Odourless

3 Biochemistry Terpenes (diterpenes)

4 Solubility Soluble in non-polar solvents

5 Density 0.850 g cm-3

6 pH Value 6.7

7 Surface Tension 28.47 mN/m (20 °C)

8 Refractive index 1.461- 1.469

9 Boiling Point 202-204 0C

10 U.V max 400

Section 5.5

Analysis toward innovative herbal antibacterial drugs

5.5.1. Introduction

Infectious diseases caused by bacteria remain a leading cause of death worldwide.

Many of the antibiotics developed to combat bacterial infections have been rendered almost

impotent due to the rapid evolution and spread of antibiotic resistance. A common and major

resistance mechanism, the efflux system, enables bacteria to extrude structurally diverse

antimicrobials, facilitating survival in toxic environments. The pumps also have important

physiological functions, play major roles in bacterial pathogenesis and are distributed widely

across diverse bacterial species. In addition a single species may harbour several different

types of efflux systems: of these, active efflux has proven to be one of the most successful

detoxification mechanisms used by both Gram-positive and -negative pathogens. Unravelling

the intricacies of the microbial efflux systems is essential for the development of new

strategies to overcome antimicrobial resistance. This has inspired a plethora of

multidisciplinary research projects that have focused on the biochemistry, bioinformatics,

structural and molecular biology of this fascinating field. Therefore, multidrug resistant strain

of many microorganisms has revealed exploration of alternative antimicrobial agent. The

success story of chemotherapy lies in the continuous search for new drugs to counter the

challenge posed by these resistant strains of microorganisms. For example, antibiotic resistant

strain Staphylococcus aureus is virulent organism that causes a broad array of health

conditions including pneumonia, osteomyelitis, endocarditis and bacteraemia (Henry, 2000)

likewise Escherichia coli cause UTI, diarrhea and septicemia. These microorganisms develop

resistance to all the available drugs so they are called multidrug resistant (MDR) strain.

Hence there is urgent need to find alternative antimicrobial agent to treat these virulent

bacterial infection. Nature has been a source of medicinal agents for thousands of

years and an impressive number of modern drugs have been isolated from natural sources;

many of these isolations were based on the uses of the agents in traditional medicine. This

plant-based, traditional medicine system continues to play an essential role in health care,

with about 80% of the world’s inhabitants relying mainly on traditional medicines for their

primary health care (Owolabi et al., 2007). According to World Health Organization,

medicinal plants would be the best source to obtain a variety of drugs (Nascimento et al.,

2000). Medicinal plants have become the focus of intense study in terms of validation of their

traditional uses through the determination of their actual pharmacological effects. Synthetic

drugs are not only expensive and inadequate for the treatment of diseases but also often with

adulterations and side effects. Therefore, investigation of certain indigenous plants for their

antimicrobial properties may yield useful results. Many studies indicate that in some plants

there are many substances such as peptides, unsaturated long chain aldehydes, alkaloidal

constituents, some essential oils, phenols and water, ethanol, chloroform, methanol and

butanol soluble compounds. These plants then emerged as compounds with potentially

significant therapeutic application against human pathogens, including bacteria, fungi or virus

(Elastal et al., 2005). Therefore, such plants should be investigated to better understand their

properties, safety and efficacy.

In recent times, focus on plant research has increased all over the world and a large

body of evidence has been collected to show immense potential of medicinal plants used in

treatment of various infectious/microbes borne disease. Therefore, the present investigation

was under taken to find in vitro antibacterial activity of nine plants viz, Aegle marmelos,

Capparis aphylla, Callistemon lanceolatus, Commelina bengalensis, Justicia adhatoda,

Argemona mexicana, Achyranthes aspera, Catharanthus roseus, and Syzygium cumini.

5.5.2. Material and methods

5.5.2.1. Plant Material

Nine plants were selected based on their use in traditional system of medicines for

their antibacterial potential evaluation and identified from Botany Department of Maharshi

Dayanand University, Rohtak (India). The identified plants were further authenticated with

the help of flora of Haryana and the voucher specimens were deposited in the herbarium of

Centre for Biotechnology, M. D. University (Rohtak). The voucher numbers are given

respectively as Aegle marmelos (CBT 003), Capparis aphylla (CBT 009), Callistemon

lanceolatus (CBT 008), Commelina bengalensis (CBT 013), Justicia adhatoda (CBT 017),

Argemona mexicana (CBT 004), Achyranthes aspera (CBT 002), Catharanthus roseus (CBT

012) and Syzygium cumini (CBT 022).

5.5.2.2. Preparation of crude extract

The nine plants leaves were collected from Haryana (30.73°N76.78°E), India. The

plant material was air dried under shade for two weeks and then oven dried at 40 °C for 18-

24h. The dried plant material was grinded to a powdered with a mortar and pestle. The

powder was weighed (50 g for each plant sample) and the Soxhlet’s method was used for

extraction. The five solvents (250 ml for each sample): petroleum ether, chloroform, acetone,

methanol and water were used in ascending order of polarity. The combined suspensions

were filtered twice, first under vacuum through a double layer of Whatman filter paper and

then by gravity through a single sheet of Whatman filter paper. The solvents were removed

from the clear supernatant by means of vacuum distillation at 30-35° C using a Buichi Rotary

Evaporator. The remaining solid was referred to as the crude extract.

5.5.2.3. Pathogens and reference strain

Antimicrobial activity of leaves extract was investigated against six registered

Microbial Type Culture Collection (MTCC) bacterial isolates viz. Escherichia coli

(MTCC433), Vibrio cholerae (MTCC3904), Klebsiella pneumoniae (MTCC3384), Proteus

vulgaris (MTCC426), Bacillus subtilis (MTCC441), Salmonella typhi (MTCC531) which

were obtained from the from Institute of Microbial Technology, Chandigarh.

5.5.2.4. Culture of pathogens

5.5.2.4.1. Bacteria

Luria broth was used to culture bacteria. The method ensures that a uniform number

of bacteria were always used; a set of graphs of killing/viability curves for each strain of

bacterial species was prepared. A final concentration of 5 × 106 CFU (Colony Forming Unit)

/ml was adopted for this assay. Thus different strains and different bacterial species could be

compared (Sarkar et al., 2007).

5.5.2.4.2. Preparation of bacterial culture

Using aseptic techniques, a single colony was transferred into a 100 ml bottle of Luria

broth capped and placed in incubator overnight at 35°C. After 12–18 h of incubation, using

aseptic preparation and the aid of a centrifuge, a clean sample of bacteria was prepared. The

culture was centrifuged at 4000 rpm for 5 min with appropriate aseptic precautions. The

supernatant was discarded. The pellet was resuspended using 20 ml of sterile normal saline

and centrifuged again at 4000 rpm for 5 min. This step was repeated until the supernatant was

clear. The pellet was then suspended in 20 ml of sterile normal saline and was labelled as

bacteria. The optical density of the bacteria was recorded at 500 nm and serial dilutions were

carried out with appropriate aseptic techniques until the optical density was in the range of

0.5-1.0 .The actual number of colony forming units was calculated from the viability graph.

The dilution factor needed was calculated and the dilution was carried out to obtain a

concentration of 5 × 106 CFU/ml (Sarkar et al., 2007).

5.5.2.5. Antibacterial evaluation of various plant extracts

The antibacterial activity was studied by Resazurin based Microtitre Dilution Assay

and Disc Diffusion assay. Brief description of these assays is given below:

5.5.2.5.1. Resazurin based Microtitre Dilution Assay

The resazurin solution was prepared by dissolving 300 mg resazurin powder in 50 ml

of sterile distilled water. A vortex mixer was used to ensure that it was a well-dissolved and

homogenous solution prepared. Resazurin based MDA was performed in 96 well plates under

aseptic conditions. A volume of 100 μl of test materials in 10% (v/v) DMSO or sterile water

(usually a stock concentration 12 mg/ml for crude extracts) added into the first row of the

plate. To all wells of plate 50 μl of nutrient broth and 50 μl of normal saline was added. Serial

dilutions were performed using a multichannel pipette such that each well had 100 μl of the

test material in serially descending concentrations. Tips were discarded after use. 10 μl of

resazurin indicator solution was added in each well. Finally 10 μl of bacterial suspension was

added to each well to achieve a concentration of 5 × 106

CFU/ml. Each plate was wrapped

loosely with cling film to ensure that bacteria did not become dehydrated. Each plate had a

set of controls: a column with tetracycline as positive control. The plates were prepared in

triplicate and placed in an incubator set at 37°C for 18–24 h. The colour change was then

assessed visually. Any colour change from purple to pink or colourless was recorded as

positive. The lowest concentration at which colour change occurred was taken as the MIC

(minimum inhibitory concentration) value. The average of three values was calculated and

was considered as the MIC for the test material against bacterial strain (Sarkar et al., 2007).

5.5.2.5.2. Disc Diffusion Assay

The disc diffusion test performed in radiation sterilized Petri plates of 10.0 cm

diameter (Tarson). The disc of the sample placed on the surface of the agar plates already

inoculated with bacterial culture. The plates incubated at 37°C and examined at 48 h for zone

of inhibition, if any, around the discs (Chhillar et al., 2006).

5.5.3. Results and Discussion

The Leaves and stem extracts in five different solvent viz. Petroleum ether, acetone,

chloroform, methanol and water were evaluated for antimicrobial activity against the

reference strains of E. coli, V. cholerae, K. pneumoniae, P. vulgaris, B. Subtilis and S. typhi.

The test organisms used in this study are associated with various forms of human infections.

From a clinical point of view, K. pneumoniae is the most important member of the Klebsiella

genus of Entero-bacteriaceae and it is emerging as an important cause of neonatal nosocomial

infection (Gupta et al., 1993). E. Coli causes septicemias and can infect the gall bladder,

meninges, surgical wounds, skin lesions and the lungs, especially in debilitate and

immunodeficient patients (Black, 1996). Infection caused by S. typhi is a serious public

health problem in developing countries and represents a constant concern for the food

industry (Mastroeni, 2002). The demonstration of activity against both gram-negative and

gram-positive bacteria is an indication that the plant can be a source of bioactive substances

that could be of broad spectrum of activity.

Therefore, forty five extracts of nine plants were tested for their antibacterial potential

against six microorganisms. The initial screening of plants extracts for antibacterial activity

showed that C. roseus and A. aspera had potential against some pathogenic bacterial strains

as given in Figure 5.5.1 and Figure 5.5.2. Tetracycline, the positive control used in this

study shows MICs in the range 0.004-0.078 mg/ml against different bacterial strains (Table

5.5.1). The MIC of the extracts ranged from 0.37 to 6.00 mg/ml, with the aqueous extracts of

C. roseus demonstrating the lowest values (MIC 0.37 mg/ml) against E. coli and B. Subtilis

followed by the petroleum ether extract of S. cumini against E. coli (Table 5.5.2). The

chloroform extract of A. aspera showed best activity against S. typhi and V. cholerae (MIC

0.375mg/ml); while acetone, methanol and water extracts showed mild activity against S.

typhi and V. cholerae (MIC 1.50 mg/ml). In addition, the petroleum ether extract prepared of

A. mexicana showed growth inhibition at 0.75 mg/ml against S. typhi and did not show

activity against K. pneumoniae. While, the S. cumini water extract was found to be reasonable

active against all six bacterial strains in the range of 0.750-6.00 mg/ml.

Furthermore, it was observed that only seventeen out of forty five plants extracts were

found to be endowed with antibacterial activity at a preset concentration of 25 µg/ disc

(Table 5.5.3) by disc diffusion assay. Maximum zone of inhibition at this concentration was

7.7±0.1 mm against S. typhi by petroleum ether extract of A. aspera. Most of the extracts

were active against two or three bacteria in disc diffusion assay at the preset concentration of

25 µg/ disc, while C. roseus aqueous extract shows activity against all six bacteria.

The strongest antibacterial activities against tested microorganisms were obtained for

extracts of C. roseus and A. aspera. These plant extract exhibited moderate antimicrobial

activity towards the others investigated bacteria especially S. typhi. Different solvents have

various degrees of solubility for different phytoconstituents (Majorie, 1999), these results

show that there are differences in the antimicrobial effect of plant groups; which may be due

to phytochemical differences among the various plant species.

Table 5.5.1- Activity (MIC) of standard drug tetracycline against various bacteria.

Name of bacteria MIC of Tetracycline (mg/ml)

against various bacteria

Escherichia coli 0.078

Salmonella typhi 0.0156

Vibrio cholerae 0.0172

Bacillus subtilis 0.0172

Figure 5.5.1. (i) Photoplate (A) and (C) showed antibacterial activity ( Minimum inhibitory

concentration) of C. roseus (3-7) and A. aspera (8-12) against E. coli and V. cholerae

respectively.

Column- 1 & 2: Control [Row A & B- Negative control: Media + resazurin; Row C, D & E- -

Positive control: Media + drug + bacteria+ resazurin; Row F, G & H - Negative control:

Media + bacteria+ resazurin];

Column- 3, 4, 5, 6 & 7: Petroleum ether (3), Chloroform (4), Acetone (5), Methanol (6) and

water (7) extract of C. roseus.

Column- 8, 9, 10, 11 & 12: Petroleum ether (3), Chloroform (4), Acetone (5), Methanol (6)

and water (7) extract of A. aspera.

Proteus vulgaris 0.004

Klebsiella pneumoniae 0.005

(ii) Photoplate (B) and (D) showed zone of inhibition against E. coli and V. cholerae

respectively. Number 1 showed the water extract of C. roseus. Number 2,3,4 and 5 showed

the chloroform, acetone, petroleum ether and methanol extract of C. roseus. Number 6

showed the standard drug: Tetracycline.

Figure 5.5.2. (i) Photoplate (A) and (C) showed antibacterial activity ( Minimum inhibitory

concentration) of C. roseus (3-7) and A. aspera (8-12) against B. subtilis and S. typhi respectively.

Column- 1 & 2: Control [Row A & B- Negative control: Media + resazurin; Row C, D & E- - Positive

control: Media + drug + bacteria+ resazurin; Row F, G & H - Negative control: Media + bacteria+

resazurin]; Column- 3, 4, 5, 6 & 7: Petroleum ether (3), Chloroform (4), Acetone (5), Methanol (6)

and water (7) extract of C. roseus;Column- 8, 9, 10, 11 & 12: Petroleum ether (3), Chloroform (4),

Acetone (5), Methanol (6) and water (7) extract of A. aspera;

(ii) Photoplate (B) and (D) showed zone of inhibition against B. subtilis and S. typhi respectively.

Number 1 showed the water extract of C. roseus. Number 2,3,4 and 5 showed the petroleum ether,

chloroform, acetone and methanol extract of C. roseus. Number 6 showed the standard drug:

Tetracycline.

Plants name

/Voucher No.

Solvent/ Fraction MIC (mg/ml) of various Plants extracts used

Table 5.5.2- Antibacterial Potential Exposed as Minimum Inhibitory Concentration of Various Fractions

Extracted from Various Plants.

{The abbreviated words are given respectively as Aegle marmelos (A m), Capparis aphylla (C a), Callistemon

lanceolatus (C l), Commelina bengalensis (C b), Justicia adhatoda (J a), Achyranthes aspera (A s), Argemona

mexicana (A m), Catharanthus roseus (C r), and Syzygium cumini (S c).

Table 5.5.3. Antibacterial Potential / Zone of Inhibition of Various Plants Extracts Shown by

Disc Diffusion Assay (25 µg/ disc).

Am1 C a C l C b J a A as Am2 C r S c

Escherichia coli

(MTCC433)

Petroleum ether 3.00 3.00 6.00 1.50 1.50 3.00 3.00 1.50 0.375

Chloroform 1.50 1.50 6.00 0.75 1.50 6.00 3.00 0.75 1.50

Acetone 1.50 1.50 1.50 1.50 1.50 6.00 3.00 3.00 6.00

Methanol 1.50 1.50 3.00 1.50 1.50 6.00 - 3.00 3.00

Water - 1.50 6.00 0.75 0.75 6.00 - 0.37 1.50

Vibrio cholerae

(MTCC 3904)

Petroleum ether - 3.00 3.00 1.50 3.00 3.00 1.50 1.50 3.00

Chloroform 3.00 3.00 6.00 3.00 - 0.37 3.00 1.50 1.50

Acetone 1.50 3.00 1.50 1.50 1.50 1.50 3.00 6.00 6.00

Methanol 1.50 3.00 1.50 1.50 3.00 1.50 - 3.00 6.00

Water 3.00 3.00 1.50 1.50 0.75 1.50 - 0.75 3.00

Klebsiella

pneumoniae

(MTCC3384)

Petroleum ether 3.00 3.00 6.00 1.50 1.50 3.00 - 1.50 6.00

Chloroform 1.50 1.50 6.00 0.75 1.50 3.00 - 1.50 6.00

Acetone 3.00 3.00 3.00 1.50 0.75 3.00 3.00 1.50 6.00

Methanol 1.50 3.00 1.50 1.50 3.00 6.00 3.00 0.75 1.50

Water - 3.00 6.00 1.50 1.50 6.00 1.50 1.50 0.75

Proteus vulgaris

(MTCC426)

Petroleum ether 6.00 - 3.00 - 3.00 1.50 1.50 3.00 3.00

Chloroform 3.00 - 3.00 - 3.00 3.00 1.50 3.00 1.50

Acetone 1.50 - 6.00 1.50 3.00 3.00 1.50 6.00 3.00

Methanol 3.00 6.00 6.00 1.50 - 6.00 - 3.00 6.00

Water 3.00 1.50 3.00 3.00 3.00 3.00 - 0.75 6.00

Bacillus subtilis

(MTCC 441)

Petroleum ether - 3.00 6.00 3.00 3.00 3.00 3.00 0.75 1.50

Chloroform 6.00 6.00 3.00 3.00 6.00 6.00 3.00 0.75 1.50

Acetone 3.00 3.00 1.50 1.50 1.50 3.00 3.00 1.50 -

Methanol 1.50 3.00 1.50 1.50 3.00 3.00 - 3.00 6.00

Water 3.00 6.00 6.00 0.75 3.00 6.00 - 0.37 6.00

Salmonella typhi

(MTCC-531)

Petroleum ether 6.00 - 3.00 3.00 3.00 1.50 0.75 3.00 1.50

Chloroform 6.00 3.00 3.00 3.00 - 0.37 1.50 0.75 6.00

Acetone 3.00 0.75 1.50 1.50 3.00 1.50 1.50 6.00 -

Methanol 1.50 1.50 3.00 1.50 1.50 3.00 - 3.00 -

Water 3.00 1.50 3.00 1.50 3.00 3.00 - 0.75 3.00

Plant Name

Solvent

Name

Zone of inhibition (mm)

S. typhi V. B. E. coli P K.

{It is considering that, (-) means no activity at 25 µg/ disc}

Some plant extracts, as per observations in this study, such as aqueous extract of C.

roseus were exhibited the broad range activity, possibly due to presence of multiple

antimicrobial compounds or synergic effects of these compounds which were identified by

GC-MS and given in Table 5.5.4 .

Table 5.5.4- Chemical Components Identified by Gas Chromatography Mass Spectroscopy

(GC-MS) from Water Extract Fraction of C. roseus.

cholerae subtilis vulgaris pneumoniae

A. marmelos Acetone _ _ _ 6.1±0.4 _ _

A. marmelos methanol 6.0±0.3 6.1±0.8 _ 6.2±0.6 _ _

C. aphylla Acetone 7.1±0.5 _ _ 6.1±0.8 _ _

C. lanceolatus Acetone 6.0±0.8 _ 6.0±0.5 6.2±0.3 _ _

C. lanceolatus methanol _ 6.2±0.5 _ _ _ 6.1±0.7

C.bengalensis chloroform _ _ _ 7.2±0.5 _ 7.0±0.4

C.bengalensis water _ _ 7.1±0.3 7.3±0.4 _ _

J. adhatoda Acetone _ _ 6.7±0.4 _ _ 7.0±0.8

J. adhatoda water _ 6.8±0.3 _ 6.7±0.7 _ _

A. aspera chloroform 7.7±0.1 7.4±0.4 _ _ _ _

A. aspera water _ _ _ _ _ _

A. mexicana Petroleum

ether 7.0±0.8 6.2±0.6 _ _ 6.2±0.8 _

C. roseus methanol _ _ _ _ _ 6.6±0.7

C. roseus water 6.7±.2 6.6±0.5 7.1±0.8 7.3±0.2 6.7±0.4 6.0±0.2

C. roseus chloroform 6.2±0.0 _ 6.4±0.3 6.5±0.7 _ _

C. roseus Petroleum

ether _ _ 6.9±0.6 _ _ _

S. cumini water _ _ _ 6.3±0.4 _ 6.0±0.6

Peak# R. Time

Area% Match

quality

(%)

Name of chemical compounds

1 10.946 4.51 77 2-Bornanone

2 11.379 14.80 94 2,6-Bis(1,1-dimethylethyl)-4-methyl- phenol,

3 13.432 4.04 76 1,2-Oxathiane, 6-dodecyl-, 2,2-dioxide

One of the major compound present in C. roseus aqueous extract is 1,2-

Benzenedicarboxylic acid, dibutyl ester (17.33%) which may contribute for its antibacterial

activity as it previously accounted antimicrobial compound (Jia et al., 2010). Fatty acid

esters namely, dibutyl ester is responsible for anti inflammatory activity (Li et al., 2004) and

antibacterial activity (Modape et al., 2010). Moreover, the appreciable presence of various

phytoconstituents 2-Bornanone (Bayoub et al., 2010), Hexadecanoic acid, methyl ester

(Carballeira et al., 1998) and Hexatriacontane (Guerrini et al., 2006) in this extract could

explain its antibacterial activity against the tested bacterial strains. While, it is reported about

the antimicrobial potential of chloroform extract of C. roseus (Saravanan et al.,2012), we also

found chloroform extract to be active against S. typhi, B. Subtilis and E. coli, but the aqueous

extracts were more promising and active in broad range. In this regards, the above-mentioned

active compounds may act as potential antimicrobial agents against drug resistant microbes.

Some plant extracts, as per observations in this study, were exhibited the broad range

activity possibly due to presence of multiple antimicrobial compounds or synergic effects of

these compounds. The potential synergism between active plants constituents present in

extracts supports the use of crude plant extracts by people in developing countries who rely

heavily on medicinal plants. The use in antimicrobial therapy of crude extracts, which contain

4 13.825 1 2.20 79 (3e)-2-Methyl-4-(1,3,3-trimethyl-7-

oxabicyclo[4.1.0]hept-2-yl)-3-buten-2-ol

5 17.345 5.75 89 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl)

ester

6 17.866 2.63 89 9-Octadecenoic acid (z)-, methyl ester

7 18.229 9.23 94 Hexadecanoic acid, methyl ester

8 19.199 17.33 96 1,2-Benzenedicarboxylic acid, dibutyl ester

9 19.836 2.03 87 Cyclopropaneoctanoic acid, 2-hexyl-, methyl ester

10 21.258 7.37 92 Methyl (9z)-9-octadecenoate

11 21.504 10.46 93 Stearic acid methyl ester

12 25.918 3.84 89 1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl)

ester

13 28.725 2.39 80 Hexatriacontane

14 30.943 3.42 86 Tetratetracontane

100.00

a vast number of compounds, may act to reduce the occurrence or development of resistant

microbes. It is far easier for a pathogenic microorganism to develop resistance against a

single purified compound rather than against a suite of compounds acting simultaneously

possibly with different targets to result in antimicrobial activity. This supports the

development of efficacious herbal products derived from biologically active plant extracts,

rather than pharmaceuticals comprising a single active constituent isolated from the plant

extract.

5.5.4. Conclusion

The present study reveals that highest antibacterial potential was observed with

aqueous extract fraction of C. roseus against all pathogenic microbes studied. Owing to the

development of multi drug resistant bacterial strains, it will be interesting to purify the active

sub fractions for potential lead antibacterial compounds. Indeed; C. roseus plant presents a

versatile reservoir of the various bioactive metabolites and can be of potential use to modern

medicine. Expectantly, our study could contribute to the development of newer and highly

active antimicrobial drugs which would be rapidly progress to various stages of formulation

development and enter the pharmaceutical market, further contributing to a reduction in the

incidence of infections.

Section 5.6

Investigation of antioxidant potential of plants extracts

5.6.1. Introduction

Antioxidants are compounds that can delay or inhibit the oxidation of lipids or

other molecules by inhibiting the initiation or propagation of oxidative chain

reactions. The beneficial effects of antioxidants on promoting health is believed to be

achieved through several possible mechanisms, direct reaction with and quenching of free

radicals, chelation of transition metals, reduction of peroxides and stimulation of the

antioxidative enzyme defence system (Osman et al., 2009). Free radicals, formed as a result

of various metabolic reactions within the body and are the unstable species that react rapidly

and destructively with biomolecules such as protein, lipid, DNA, RNA in the body.

Uncontrolled generation of free radicals is associated with lipid and protein peroxidation,

resulting in cell structural damage, tissue injury or gene mutation and ultimately led to the

development of various health disorders such as Alzheimer’s disease, cancer, atherosclerosis,

diabetes, hypertension and ageing (Mantle et al., 2000). Antioxidants are therefore critical

for maintaining optimal cellular and systemic health. Recently, and mainly due to

undesirable side effects such as toxicity and carcinogenicity of synthetic additives, interest

has considerably increased for finding naturally occurring antioxidant compounds suitable for

use in food and/or medicine.

Plant phenolic compounds have attracted considerable attention for being the main

sources of antioxidant activity. Phenolic compounds such as flavonoids, phenolic acids,

diterpenes and tannins have received attention for their high antioxidative activity (Rice-

Evans et al., 1996). The antioxidant activity of phenolics is mainly due to their redox

properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen

quenchers. Hence most of the antioxidant compounds in a typical diet are derived from plant

sources and belong to various classes of compounds with a wide variety of physical and

chemical properties. Consequently, the natural food antioxidants like vitamin C, Vitamin

E, carotenes, phytate and phytoestrogens have been recognized as having the potential to

reduce disease risk.

Even as, many synthetic antioxidants such as BHA (Butylated Hydroxy Anisole),

BHT (Butylated Hydroxy Toluene) are very effective but they possess certain health risks

and toxic properties to human health. Therefore, the search for natural antioxidants of plant

origin has gained momentum in recent years (Mathew and Abraham, 2006). Hence, studies

on antioxidants present in plants and foods have come to be one of the most popular topics of

phytochemistry research. The plants selected; such as Aegle marmelos, Capparis aphylla,

Callistemon lanceolatus, Commelina bengalensis, Justicia adhatoda, Argemona mexicana,

Achyranthes aspera, Catharanthus roseus, and Syzygium cumini; for the present study have

been reported to encompass many medicinal properties and exploring the plant for

antioxidant activity can be of immense value as potential metabolites as natural antioxidants.

5.6.2. Material and methods

5.6.2.1. Plant material

Nine plants species were collected from Jhajjar and Rohtak district of Haryana. Plants

were selected based on their use in traditional and Ayurvedic system of medicines. Nine

plants were identified from botany department of Maharshi Dayanand University, Rohtak

(India). The voucher numbers are given respectively as Aegle marmelos (CBT 003), Capparis

aphylla (CBT 009), Callistemon lanceolatus (CBT 008), Commelina bengalensis (CBT 013),

Justicia adhatoda (CBT 017), Argemona mexicana (CBT 004), Achyranthes aspera (CBT

002), Catharanthus roseus (CBT 012), and Syzygium cumini (CBT 022).

5.6.2.2. Sample preparation and extraction of crude extracts

Eight plants leaves and stem of C. aphylla were collected from Haryana

(30.73°N76.78°E), India. The plant material was washed with tap water, chopped into small

pieces and air dried under shade for two weeks and, then oven dried at 40 °C for 18-24h. The

dried plant material was grinded to a powdered with a mortar and pestle. The powder was

weighed (50 g for each plant sample) and the Soxhlet’s method was used for extraction. The

five solvents (250 ml for each sample): petroleum ether, chloroform, acetone, methanol and

water were used in ascending order of polarity (Harborne, 1980; Rajesh and Sharma, 2002).

The combined suspensions were filtered twice, first under vacuum through a double layer of

Whatman filter paper and then by gravity through a single sheet of Whatman No. 1 filter

paper. The solvents were removed from the clear supernatant by means of vacuum distillation

at 30-35° C using a Buichi Rotary Evaporator. The remaining solid was referred to as the

crude extract.

5.6.2.3. Antioxidant Potential Assays

5.6.2.3.1. Total Phenolic content

The total phenolic content of the leaf extracts was determined by using method of

McDonald et al. (2001), with some modifications. The total phenolic content of the leaf

extracts was calculated in Gallic acid equivalents (% of GAE).

5.6.2.3.2. Hydroxyl Radical Assay

The hydroxyl radical scavenging activity was measured by the deoxyribose method

(Mathew and Abrahem, 2006; Halliwal et al., 1987). The reaction mixture which contained

extract in different concentrations (0.2 mg/ml to 1.0 mg/ml), deoxyribose (3.75 mM), H2O2 (1

mM), Potassium phosphate buffer (20 mM, pH 7.4), FeCl3 (0.1 mM), EDTA (0.1 mM) and

ascorbic acid (0.1 mM), was incubated in a water bath at 37±0.50C for 1 h. The extent of

deoxyribose degradation was measured by the TBA (Thio-barbituric acid) method (Ohkawa

et al., 1979). Then, 1 ml of TBA (0.1% w/v) and 1 ml of TCA (2.8% w/v) were added to the

mixture and heated in a water bath at 1000C for 20 min. The absorbance of the resulting

solution was measured at 532 nm. All the analysis was done in triplicates and the Percent

Inhibition (PI) of deoxyribose degradation was calculated according to the equation.

PI = A (Control) – A (Sample or Standard)/ A (Control) × 100.

Where A (Control) = Absorbance of control reaction

A (Sample or Standard) = Absorbance of sample extract or standard

5.6.2.3.1. β-Carotene -linoleic acid (linoleate) Assay

The antioxidant activity of extract was evaluated using a β-carotene-linoleic acid

model system (Miller, 1971). In briefly, 1 ml of β-carotene (0.2 mg/ml) dissolved in the

chloroform was took in to a small round-bottom flask. After removing the chloroform by

using a rotary evaporator, 20 mg of linoleic acid, 200 mg of Tween-40 and 50 ml of aerated

distilled water were added to the flask with vigorous stirring. Then, 5 ml aliquot of the

prepared emulsion were transferred to a series of tubes containing (0.2-1.0 mg/ml) the sample

extract. Here, ascorbic acid was used as a positive control. The test systems were placed in

the water bath at 500C for 2 h. The reaction was performed in triplicates and absorbance was

measured using the spectrophotometer at 470 nm, immediately after sample preparation (t=0

min) and then at the end of experiment (t=120 min). The antioxidant activity of the extracts

under investigation was expressed as:

% AA = 100[1 – (A1 (t=0) – A1 (t=120)) / (A0 (t=0) – A0 (t=120))]

Where, % AA = Antioxidant activity of the sample extract

A1 (t=0) = Absorbance of the test sample/standard at zero time.

A1 (t=120) = Absorbance of the test sample/standard after 120 min.

A0 (t=0) = Absorbance of the aqueous control sample at zero time.

A0 (t=120) = Absorbance of the aqueous control sample after 120 min

5.6.2.4. Chromatographic Methods

Chromatography is the best method of choice in handling the problem of isolation of a

compound of interest from a complex natural mixture. The chromatographic methods used

during the present work are already described in material and method section. 5.6.2.5.

Spectroscopic Techniques

Spectroscopy is the study of the interaction of electromagnetic radiation (EMR) with

matter. NMR spectroscopy is the study of interaction of radio frequency (RF) of the EMR

with unpaired nuclear spins in an external magnetic field to extract structural information

about a given sample. NMR spectroscopy is routinely used by chemists to study chemical

structure of simple molecules using simple one dimensional technique (1D-NMR). NMR is

the best method to use for non-crystalline compounds. Detail was given in material and

methods section of the thesis.

5.6.3. Results and Discussion

5.6.3.1. Total phenolic content (TPC)

Phenolics or polyphenols are the plant secondary metabolites and are very important

by virtue of their antioxidant activity by chelating redox active metal ions, inactivating lipid

free radical chains and preventing hydroperoxide conversions into reactive oxyradicals. The

Folin-Ciocalteu procedure has been proposed to rapidly estimate the level of total phenolics

in food and supplements (Prior et al., 2005). The total phenolic content of all the nine plants

was given in Table 5.6.1 and Figure 5.6.1. The TPC of various solvent extracts have been

expressed as gallic acid equivalent i.e., mg gallic acid/g dry wt. A high phenolic content

24.03±0.50 was found in acetonic extract of C. aphylla and decreased in the order of A.

marmelos > J. adhatoda > A. aspera (Table 5.6.1). Results showed that the levels of

phenolic compounds in different plant extracts were variable from each other. The TPC of the

acetonic extract in terms of GAE (Gallic acid equivalent) is indicative of high antioxidant

potential of the extract, because the phenolic constituents can react with active oxygen

radicals such as lipid peroxy radical, hydroxyl radical, and superoxide anion radical. In our

study, C. aphylla showed the highest phenolic content indicating good antioxidant potential.

All the nine plant extracts were screened on the basis of their total phenolic content and the

plants having maximum phenolic content were further tested by other potential antioxidant

assays. This is because the earlier studies reported that there is direct correlation between the

antioxidant activities and their phenolic content.

To

ta

l P

he

no

l C

on

te

nt

Petr

ole

um

eth

er

Chlo

rofo

rm

Aceto

ne

Meth

anol

Aqueous

0

1 0

2 0

3 0

A . M a rm e lo s

C .a p h y lla

C . la n c e o la te

C . b e n g a le n s is

J . a d h a to d a

A .m e x ic a n a

C . ro s e u s

A . a s p e ra

S .c u m in i

V a r io u s P la n t E x tra c ts Figure

5.6.1- Total Phenolic Content of various plant extracts

Table 5.6.1 - Total phenolic content (TPC) of extracts isolated from nine plants.

Name of

plants

Plant

part

Used

Total phenolic content (% dw Gallic acid equivalent)

PE CE AC ME AE

A. marmelos Leaves 14.19±0.70 9.06±0.50 22.17±0.30 23.56±0.70 20.30±0.30

C. aphylla Stem 11.15±0.10 15.34±0.50 24.03±0.50 23.94±0.89 21.10±0.45

C. lanceolatus Leaves 4.40±0.70 3.08±0.20 15.45±0.04 10.09±0.40 7.67±0.37

C.bengalensis Whole 3.45±0.77 3.05±0.50 11.40±0.24 8.09±0.50 5.60±0.30

J. adhatoda Leaves 20.10±0.70 16.05±0.54 18.47±0.36 14.36±0.73 10.50±0.40

A. mexicana Leaves 4.67±0.46 7.67±0.27 9.30±0.90 16.40±0.26 10.23±0.45

C. roseus Leaves 3.04±0.20 6.07±0.68 12.10±0.23 19.07±0.06 8.60±0.30

A. aspera Leaves 10.45±0.11 12.35±0.04 17.77±0.96 17.40±0.36 16.77±0.66

S. cumini Leaves 16.25±0.50 14.40±0.34 10.43±0.21 7.39±0.53 4.63±0.34

(Note = PE = Petroleum ether; CE = Chloroform extract; AC=Acetone extract; ME =

Methanol extract and AE = Aqueous extract)

5.6.3.2. Antioxidant Potential of three selected plants

5.6.3.2.1. Hydroxyl radical scavenging activity

After screening of the all plant extracts, three plants J. adhatoda, C. aphylla and A.

marmelos having maximum total phenolic contents are further tested by other antioxidant

assays for the antioxidant activities. Hydroxyl radical scavenging capacity of extracts is

directly related to its antioxidant activity (Babu et al., 2001). Generally molecules that inhibit

deoxyribose degradation are those that can chelate the iron ions and thereby prevent them

from complexing with deoxyribose and render them inactive in a Fenton reaction (Smith et

al., 1992). In case of leaves extracts of J. adhatoda, hydroxyl radical scavenging potential

was found in the following order: petroleum ether > acetone > methanol > chloroform> water

extracts. While petroleum ether extract of J. adhatoda, was most effective in inhibiting the

hydroxyl radicals among all the plant extracts tested (Table 5.6.2).

Table 5.6.2 - Hydroxyl radical scavenging activity of leaf extracts of three plants.

Solvent/

Fraction

Con.

(mg/ml)

Percent Inhibition of deoxyribose degradation (Mean ± S.D.)

5.6.3.2.2. β-carotene-linoleic acid assay

In β-carotene-linoleic acid model, β-carotene undergoes rapid discolouration in the

absence of an antioxidant. During oxidation, an atom of hydrogen is abstracted from the

active bis-allylic methylene group of linoleic acid located on carbon-11 between two double

bonds (Frankel, 1998). The pentadienyl free radical so formed then attacks highly unsaturated

β-carotene molecule in an effort to reacquire an H-atom. As the β- carotene molecule loses

Ascorbic acid A. marmelos C. aphylla J. adhatoda

Petroleum

ether

0.2 62.40 ± 0.40 16.27 ± 0.44 24.40 ± 0.84 27.43 ± 0.18

0.4 63.03 ± 0.37 20.01 ± 0.10 27.30 ± 0.37 31.23± 0.45

0.6 66.66 ± 0.69 24.30 ± 0.10 32.16 ± 0.30 34.30 ± 0.09

0.8 70.40± 0.38 28.11 ± 0.11 36.75 ± 0.40 40.10 ± 0.18

1.0 72.56 ± 0.10 30.45 ± 0.60 40.12 ± 0.29 44.26 ± 0.34

Chloroform 0.2 62.40 ± 0.40 11.47 ± 0.40 26.03 ± 0.47 17.36 ± 0.90

0.4 63.03 ± 0.37 15.03 ± 0.17 31.13 ± 0.17 19.05 ± 0.31

0.6 66.66 ± 0.69 20.60 ± 0.40 34.16 ± 0.40 23.30 ± 0.11

0.8 70.40± 0.38 24.41 ± 0.18 40.20 ± 0.40 27.11 ± 0.46

1.0 72.56 ± 0.10 26.06 ± 0.69 43.12 ± 0.19 29.30 ± 0.01

Acetone 0.2 62.40 ± 0.40 28.40 ± 0.28 31.47 ± 0.40 26.33 ± 0.56

0.4 63.03 ± 0.37 32.03 ± 0.97 34.73 ± 0.37 30.20± 0.56

0.6 66.66 ± 0.69 37.09 ± 0.56 43.66 ± 0.49 35.36 ± 0.89

0.8 70.40± 0.38 43.19 ± 0.08 49.49 ± 0.38 39.10 ± 0.48

1.0 72.56 ± 0.10 47.76 ± 0.39 51.56 ± 0.19 42.06 ± 0.39

Methanol 0.2 62.40 ± 0.40 30.32 ± 0.20 30.46 ± 0.48 23.30 ± 0.16

0.4 63.03 ± 0.37 34.33 ± 0.34 34.73 ± 0.77 27.20± 0.50

0.6 66.66 ± 0.69 42.60 ± 0.19 41.09 ± 0.79 31.30 ± 0.80

0.8 70.40± 0.38 47.29 ± 0.18 48.19 ± 0.68 34.11 ± 0.08

1.0 72.56 ± 0.10 50.16 ± 0.10 50.76 ± 0.59 37.67 ± 0.39

Water 0.2 62.40 ± 0.40 24.20 ± 0.08 26.43 ± 0.20 13.46 ± 0.30

0.4 63.03 ± 0.37 28.03 ± 0.23 30.33 ± 0.27 18.65 ± 0.31

0.6 66.66 ± 0.69 33.02 ± 0.56 33.26 ± 0.40 19.30 ± 0.41

0.8 70.40± 0.38 37.09 ± 0.28 39.25 ± 0.30 21.41 ± 0.46

1.0 72.56 ± 0.10 41.31 ± 0.30 43.52 ± 0.59 25.33 ± 0.11

their conjugation, the carotenoids lose their characteristic orange colour. The presence of a

phenolic antioxidant can hinder the extent of β-carotene destruction by neutralizing the

linoleate free radical and any other free radicals formed within the system.

The antioxidant potential of all the tested three plant extracts was in a dose dependent

manner. In case of J. adhatoda, petroleum ether extract was found to be most effective

whereas methanolic extract of C. aphylla and A. marmelos were found to be most effective.

In this assay, the efficacies of this plant extract possess inhibition in a range 10.56 % to 52.16

% from concentration range 0.2 mg/ml to 1.0 mg/ml as shown in Table 5.6.3.

5.6.3.3. Isolation of bioactive compound from petroleum ether fraction of J. adhatoda

Fifteen micro litres of neat plant extract at 100 mg/ml was applied 2 cm from the base of

aluminium-backed silica plates (Merck 60F254, Germany) cut to size (10x5 cm). The plates

loaded with petroleum ether fraction of J. adhatoda were kept in the chromatographic

chamber containing the mixture of petroleum ether and ethyl acetate in ratio of 80.0:20.0.

The solvent was allowed to rise up to a height of about 9.0 cm at room temperature. After

developing the chromatogram, the plates were removed from the tank, the solvent front was

marked and they were allowed to dry in the air for 15 minutes at room temperature. The

components of various bands having different Rf values were fragmented by Column

Chromatography and examined for antioxidant activity. The sub-fractions showing similar

profile of Rf values were pooled and resulted into 12 column sub-fractions which are

monitored by TLC fingerprinting (Figure 5.6.2).

Table 5.6.3- Percent Antioxidant activity of leaf extracts of three plants by linoleate Assay

Solvent/

Fraction

Con.

(mg/ml)

Percent Antioxidant activity (Mean ± S.D.)

Ascorbic acid A. marmelos C. aphylla J. adhatoda

Petroleum

ether

0.2 44.39 ± 0.55 10.56 ± 0.05 26.44 ± 0.78 29.40 ± 0.28

0.4 58.30 ± 0.30 13.01 ± 0.10 28.90 ± 0.05 33.45± 0.50

0.6 63.44 ± 0.46 14.34 ± 0.13 31.46 ± 0.80 38.50 ± 0.48

0.8 69.27 ± 0.46 18.41 ± 0.10 36.05 ± 0.48 42.10 ± 0.18

1.0 74.11 ± 0.50 23.40 ± 0.30 41.78 ± 0.20 47.23 ± 0.30

Chloroform 0.2 44.39 ± 0.55 14.40 ± 0.30 20.78 ± 0.07 17.34 ± 0.48

0.4 58.30 ± 0.30 16.03 ± 0.12 21.10 ± 0.10 20.45 ± 0.11

0.6 63.44 ± 0.46 19.65 ± 0.49 25.56 ± 0.40 24.33 ± 0.51

0.8 69.27 ± 0.46 21.11 ± 0.10 27.20 ± 0.80 27.10 ± 0.06

1.0 74.11 ± 0.50 21.06 ± 0.60 31.12 ± 0.42 30.50 ± 0.41

Acetone 0.2 44.39 ± 0.55 30.43 ± 0.20 27.40 ± 0.40 25.53 ± 0.06

0.4 58.30 ± 0.30 34.06 ± 0.90 32.45 ± 0.76 29.28± 0.50

0.6 63.44 ± 0.46 39.09 ± 0.06 35.65 ± 0.45 34.56 ± 0.23

0.8 69.27 ± 0.46 45.19 ± 0.28 41.45 ± 0.30 38.90 ± 0.40

1.0 74.11 ± 0.50 49.26 ± 0.19 47.50 ± 0.19 41.02 ± 0.01

Methanol 0.2 44.39 ± 0.55 30.02 ± 0.50 30.40 ± 0.40 17.40 ± 0.11

0.4 58.30 ± 0.30 35.30 ± 0.30 34.56 ± 0.89 23.30± 0.23

0.6 63.44 ± 0.46 43.60 ± 0.10 40.34 ± 0.29 26 .30 ± 0.80

0.8 69.27 ± 0.46 48.29 ± 0.78 47.59 ± 0.60 30.14 ± 0.08

1.0 74.11 ± 0.50 52.16 ± 0.30 51.06 ± 0.09 35.87 ± 0.30

Water 0.2 44.39 ± 0.55 20.24 ± 0.38 25.40 ± 0.20 17.44± 0.70

0.4 58.30 ± 0.30 28.45 ± 0.20 28.83 ± 0.20 20.60 ± 0.91

0.6 63.44 ± 0.46 32.72 ± 0.66 32.56 ± 0.40 24.30 ± 0.40

0.8 69.27 ± 0.46 34.34 ± 0.20 37.40 ± 0.50 27.40 ± 0.60

1.0 74.11 ± 0.50 37.30 ± 0.36 40.32 ± 0.50 29.03 ± 0.01

F

igure 5.6.2- Chromatogram fingerprints of column sub-fractions isolated from petroleum

ether fraction of J. adhatoda.

5.6.3.4. Antioxidant Activity of Column Sub- fractions

The antioxidant activity of each sub-fraction was tested by hydroxyl radical assay as

given in Table 5.6.4. The stock solution of all the 12 column sub-fractions was prepared and

their antioxidant activity was examined in the range of 01 to 58% by hydroxyl radical assay

(Table 5.6.4) and the further subjected to the gas-chromatography mass spectroscopy (GC-

MS) for identifying and separating out pure active component.

Table 5.6.4- Antioxidant activity of twelve fractions isolated from petroleum ether extracts of

J. adhatoda.

Con.

(mg/ml)

Hydroxyl radical scavenging activity (%)

Asc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12

0.2 62 01 29 12 03 12 17 05 05 03 16 04 03

0.6 66 03 36 17 04 23 25 07 08 04 25 06 03

1.0 72 05 58 19 06 27 30 07 07 04 29 07 05

{Asc= Ascorbic acid (Standard); F1 to F12 = 1 to 12 Fractions respectively}

5.6.3.5. GC-MS of Active Column Sub-fraction

The GC-MS of Active Column Sub-fraction was performed using solvent system of

chloroform and investigation was carried out using Shimadzu QP-2010 plus with thermal

desorption system TD-20 gas chromatography equipped with an Turbomolecular pump (58

L/Sec for He), Rotary pump 30L/min (60Hz) and Column (Inert Cap Pure-WAX) flow up to

4ml/min which was operated in EI mode (1 pg octafluoronaphthalene m/z 272 S/N ˃200).

Helium was the carrier gas at a flow rate of 1ml/min. The injector was operated at 250°C and

the column temperature was programmed as follows; 35°C for 5 min to 4°C/min, then

gradually increased to 250°C for 10 min. Identification of constituent of the extract was

achieved on the basis of their retention indices determined with a reference to a homologous

series of phytoconstituents and by comparison of their mass spectral fragmentation patterns

(NIST database/ chemstation data system) with data previously reported in literature

(Sathyaprabha et al., 2010).

The pure compound was identified as 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-

hexamethyl- (100%) by GC-MS whose retention time was 21.289 (Figure 5.6.3).

Figure 5.6.3- GC-MS spectra of active (antioxidant) sub-fraction of leaves extract (petroleum

ether) of J. adhatoda.

5.6.3.6. Structure elucidation of compound isolated as an antioxidant

The active compound (40%) was isolated as yellow colored oil. Rf value: 0.725

(Petroleum ether: ethyl acetate 90:10). The mass spectra (Figure 5.6.4) showed the molecular

ion M+ peak at m/z = 410 corresponding to the molecular formula C30H50. The spectra

(Figure 5.6.5) of 1H NMR showed δ H: 1.67 (s), 1.55 (bs), 5.10 (m), 2.07 (m), 5.12 (m), 1.59

(bs), 5.10 (m), 2.04 (m), 2.05 (m), 1.56 (bs), 5.35 (m), 2.09 (m) peaks The 13

C NMR spectra

(Figure 5.6.6) data are presented as δ C: 135.08 (C-10, C-15), 134.88 (C-6, C-19), 131.22

(C-2, C-23), [methine carbons (CH)]: δ 124.40 (C-3, C-22), 124.34 (C-11, C-14), 124.22 (C-

7, C-18), [methylene carbons (CH2)]: 39.75 (C-5, C-20), 39.72 (C-9, C-16), 28.27 (C-12, C-

13I, 26.77 (C-4, C-21), 26.66 (C-8, C-17), [methyl carbons (CH3)]: 25.65 (C-24, C-1), 17.63

(C-30, C-25), 16.02 (C-29, C-26), 15.96 (C-27, C-28).

The 13

C-NMR spectra of compound displayed 15 distinct resonances represents 30

carbons, while the DEPT experiment showed the presence of eight methyls, ten methylenes,

six methines and six trisubstituted quarternary carbons. The 1H-NMR spectral data indicated

that the compound was an acyclic triterpenoids, thus the spectra showed six olefinic proton

(m, δ 5.10; H-3, H-7, H-11, H-14, H-18 and H-22). This was further substantiated by the

presence of three methine carbons resonating at δ 124.22, δ 124.34 and δ 124.40, ten

methylene proton (m δ 2.05; H-4, H-5, H-8, H-9, H-12, H-13, H-16 H-17, H-20 and H-21)

and finally a singlet at δ 1.67 (6H, s, H-1 and 24 Me) together with a broad singlet at δ 1.59

(9H, bs) which corresponded respectively, to an in-chain allylic methyl group and three out-

chain allylic groups of a polyprenoid system. In the 13

C-NMR spectrum, the out of chain

methyl groups resonating at δ 17.63, δ 16.02 and δ 15.96 indicated the geometry of the six

trisubstitutional double bonds, while signal appearing at δ 25.65, confirmed its in-chain

position. On the basis of these 'H and '3C-NMR spectral features and by comparison with the

authentic data compound was identified as 2,6,10,14,18,22-Tetracosahexaene,

2,6,10,15,19,23-hexamethyl (Figure 5.6.7 and 5.6.8) . It is also known as squalene. This is

the first report of its occurrence from the J. adhatoda.

It has suggested that squalene and its peroxidized derivatives occurring by UV

irradiation have an important role in the occurrence of sunburn and protection from sunburn

skin damage (Ohsawa et al., 1984). Furthermore, it has been suggested that squalene

peroxides may play an important part in the pathology of acne, pityriasis versicolor, and skin

aging. There is some evidence that squalene reduces colon cancer (Rao et al., 1998) and skin

cancer (Owen et al., 2000). These activities are likely related to its antioxidant effect.

5.6.4. Conclusion J.

adhatoda and A. aspera are rich in phenolics and may be responsible for the observed

antioxidant capacities of different extracts. The antioxidant activity of the extracts, however,

varied according to different solvents. These studies clearly establish the traditional usage of

these plants by villagers. These results imply that the extracts or the derived phytochemical

(2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl) from J. adhatoda have

great potential to prevent diseases caused by the overproduction of radicals, and they may be

suitable for the treatment of degenerative diseases.

Figure 5.6.4- Mass spectra of active (antioxidant) molecule isolated from leaves extract

(petroleum ether) of J. adhatoda.

Figure 5.6.5- 1H NMR spectra of active (antioxidant) molecule isolated from leaves extract

(petroleum ether) of J. adhatoda.

Figure 5.6.6- 13

C NMR spectra of active (antioxidant) molecule isolated from leaves extract

(petroleum ether) of J. adhatoda.

Figure 5.6.7- Ball stick model of antioxidant molecule (purified)

Figure 5.6.8- The molecular structure of antioxidant molecule (purified) named

2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl.