results - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/13464/11/11_chapter 3.p… ·...

1

4. RESULTS

Free radicals are constantly generated in vivo for physiological purposes. They can be

over produced in pathological conditions, causing oxidative stress. A large number of

diseases such as autoimmune diseases, inflammation, cardiovascular, neurological diseases

and cancer are attributed to oxidative stress. An adequate intake of natural antioxidants is

believed to protect the macromolecules against this oxidative damage in cells (Riaz et al.,

2011).

The body is endowed with both endogenous (catalase, superoxide dismutase,

glutathione peroxidase / reductase) and exogenous (vitamins C and E, carotene, uric acid)

defense systems against free radicals generated within it. These systems are, however, not

sufficient in certain situations in which the production of free radicals significantly increases.

The beneficial effects of phytochemicals in this direction are associated with a number of

their biological activities including antioxidant and free radical scavenging properties

(Oyebanji and Saba, 2011).

There is currently a strong interest in plants as pharmaceuticals, especially from

edible plant parts, because these compounds play an important role in preventing free radical

induced diseases such as cancer. This interest focused not only on the discovery of new

biologically active molecules by the pharmaceutical industry, but also on the adoption of the

crude extract of the plants, such as infusions for self medication by the public (Haripyaree

et al., 2010).

The free radical neutralizing property of the extracts from a number of medicinal

plants is gaining a lot of importance. They are known to have some biologically active

principles and are used in Ayurvedic preparations (Mandade et al., 2011). Many synthetic

drugs protect against oxidative damage but they have adverse side effects. An alternative

solution to the problem is to consume natural antioxidants from food supplements and

traditional medicines. Recently, many natural antioxidants have been isolated from different

plant materials (Hazra et al., 2008). In the present study, we have studied the antioxidant

activity of M. hortensis leaves. The results obtained are presented below.

2

PHASE I

The antioxidant contents present in the leaves of M. hortensis were analyzed. Both

enzymic and non-enzymic antioxidants were quantified and the values obtained are presented

below.

4.1. ENZYMIC ANTIOXIDANT ACTIVITIES IN M. hortensis LEAVES

The enzymic antioxidants analysed in the leaves of M. hortensis were superoxide

dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR),

glutathione S-transferase (GST) and polyphenol oxidases (PPO). The activities obtained are

presented in Table 4.1.

The results revealed that the leaves of M. hortensis possess considerable activities of

all the enzymic antioxidants studied. It is evident from the above tabulated values that the

leaf of M. hortensis is a good source of enzymic antioxidants.

4.2. NON-ENZYMIC ANTIOXIDANT LEVELS IN M. hortensis LEAVES

The levels of non-enzymic antioxidants, namely ascorbic acid, tocopherol, reduced

glutathione, total phenols, total flavonoids, total chlorophyll, total carotenoids and lycopene

are presented in Table 4.2.

The results revealed that the leaves of M. hortensis exhibited appreciable amounts of

all the non-enzymic antioxidants analysed. Therefore, it is evident that the leaves of the

candidate plant are a rich source of antioxidants, both enzymic and non-enzymic.

PHASE II

Knowing that the M. hortensis leaves (Plate 4.1) are rich in antioxidants, further

analysis was carried out to assess the free radical scavenging activity of the same. In order to

identify the active principle and the solvents into which the maximum amount of antioxidants

got extracted, the leaves of M. hortensis were serially extracted into solvents of increasing

polarity (petroleum ether, benzene, chloroform, ethyl acetate and methanol) using a Soxhlet

apparatus. An aqueous extract was also prepared (as mentioned in the methodology chapter).

These extracts were then tested for their radical scavenging effects.

3

TABLE 4.1

ENZYMIC ANTIOXIDANT ACTIVITIES IN M. hortensis LEAVES

ENZYMES ACTIVITIES

Superoxide dismutase (U/g leaf)# 43.33 ± 0.86

Catalase (U/g leaf)$ 103.72 ± 3.50

Peroxidase (U/g leaf)* 19.40 ± 0.02

Glutathione reductase (U/g leaf)+ 2.50 ± 0.39

Glutathione S-transferase (U/g leaf)@ 0.16 ± 0.04

Catechol oxidase (Units X 10-3/ g leaf)¢ 0.54 ± 0.19

Laccase (Units X 10-3 / g leaf ) ¢ 0.50 ± 0.05

The values are mean ± S.D of triplicates. # 1 Unit = Amount of enzyme that causes 50% reduction in NBT oxidation

$ 1 Unit = Amount of enzyme required to decrease the absorbance at 240nm by 0.05 units/minute * 1 Unit = Change in absorbance at 430 nm/minute

+ 1 Unit = mmoles of NADPH oxidized/minute @ 1 Unit = nmoles of CDNB conjugated/minute

¢ 1 Unit = Amount of catechol oxidase/laccase enzyme which transforms 1 unit of dihydrophenol to quinine /minute

TABLE 4.2

NON-ENZYMIC ANTIOXIDANT LEVELS IN M. hortensis LEAVES

PARAMETERS LEVELS

Ascorbic acid (mg/g leaf) 1.70 ± 0.01

Tocopherol (µg/g leaf) 3.59 ± 0.25

Reduced glutathione (nmoles/g leaf) 256.19 ± 15.1

Total phenols (mg*/g leaf) 11.81 ± 0.14

Total flavonoids (mg^/g leaf) 5.08 ± 0.51

Total carotenoids (mg/g leaf) 24.47 ± 0.39

Lycopene (mg/g leaf) 4.26 ± 0.01

Total chlorophyll (mg/g leaf) 3.96 ± 0.22

The values are mean ± S.D. of triplicates * = catechin equivalents ^= catechol equivalents

4

PLATE 4.1

Majorana hortensis LEAVES

4.2.1. Radical Scavenging Effects of M. hortensis Leaf Extracts

The extracts were tested for their radical scavenging effects against a battery of

oxidant moieties that included the radicals DPPH, ABTS, H2O2 (non-radical), OH●, SO● and

NO. The ability of the different leaf extracts to scavenge DPPH was tested in a rapid dot blot

screening and quantified using a spectrophotometric assay. The picture obtained in the dot

blot screening is shown in Plate 4.2, where all the extracts showed significant free radical

scavenging ability. The maximum activity was observed in the methanolic extract.

1 – Petroleum ether 4 – Ethyl Acetate 2 – Benzene 5 – Methanol 3 – Chloroform 6 – Water

PLATE 4.2

DPPH DOT BLOT Assay

4.2.2. DPPH and ABTS Radical Scavenging Activity of M. hortensis Leaf Extracts

The per cent extent of DPPH and ABTS scavenging by the M. hortensis leaf extracts

were carried out spectrophotometrically and the results are presented in Figure 4.1. It was

observed that M. hortensis leaf extracts effectively reduced the stable radical DPPH to the

yellow-coloured compound diphenylpicryl hydrazine. The maximum extent of both DPPH

5

and ABTS radical scavenging was elicited by the methanolic extract, followed by the

aqueous extract. DPPH and ABTS scavenging effects of the other solvent extracts were

found to be moderate. The minimum radical scavenging activity was exhibited by the

petroleum ether extract.

4.2.3. Hydrogen Peroxide Scavenging Activity of M. hortensis Leaf Extracts

The ability of M. hortensis leaf extracts to scavenge H2O2 in an in vitro system was

studied and the results are also expressed in Figure 4.1. All the different solvent extracts of

M. hortensis leaves exhibited strong H2O2-scavenging effects. Though the extents of

scavenging varied, the methanolic extract showed the maximum scavenging activity,

followed by the aqueous extract. The least scavenging activity was observed in the petroleum

ether extract.

4.2.4. Hydroxyl Radical Scavenging Activity of M. hortensis Leaf Extracts

The hydroxyl radical has high reactivity and is short-lived. The extent of TBARS

produced in the reaction is taken as a measure of hydroxyl radical production. The inhibition

of TBARS production is, thus, considered as a measure of hydroxyl radical scavenging

efficiency. The exposure to H2O2 caused the maximum damage, which was very effectively

reduced by the presence of the leaf extracts. The methanolic extract exhibited the maximum

extent of radical scavenging (Figure 4.2). The other solvent extracts also showed a varied

percent of free radical scavenging activity though not as much as the methanolic extract of

the M. hortensis leaf.

4.2.5. Effect of M. hortensis Leaf Extracts on the in vitro Generation of Superoxide and

Nitric Oxide Radicals

The per cent inhibition of SO● and NO generation in the presence of the leaf extracts

was calculated and the values are depicted in Figure 4.3. All the different solvent extracts of

the leaves were found to be very good scavengers of superoxide in vitro, with the maximal

inhibitory effect found in the methanolic extract followed closely by the aqueous, chloroform

and ethyl acetate extracts. A reduction in NO generation was also observed with all the

different extracts of M. hortensis leaves. The methanolic extract showed the maximum

inhibition of nitric oxide generation, closely followed by the aqueous extract.

6

The values are Mean ± S.D. of triplicates

FIGURE 4.1 : DPPH, ABTS AND H2O2 SCAVENGING EFFECTS OF

M. hortensis LEAVES


The value of H2O2-treated group was fixed as 100 per cent and the relative values in percentage were

calculated for the other groups

FIGURE 4.2 : HYDROXYL RADICAL SCAVENGING EFFECT OF

M. hortensis LEAVES


The extent of inhibition of nitric oxide generation in vitro was found to be almost similar to that of the

extent of inhibition of SO● generation

FIGURE 4.3 : SUPEROXIDE AND NITRIC OXIDE SCAVENGING EFFECTS OF M. hortensis LEAVES

0

20

40

60

80

100

Petroleum

ether

Benzene Chloroform Ethyl

acetate

Methanol Water

Pe

rce

nt

Ra

dic

al

Sca

ve

ng

ed

DPPH ABTS H2O2

0

20

40

60

80

100

120

No Extract Petroleum

ether

Benzene Chloroform Ethyl

acetate

Methanol Water

Pe

rce

nt

TB

AR

S F

orm

ed

Without With H2O2 H2O2

0

20

40

60

80

Superoxide Nitric OxidePe

rce

nt

Inh

ibit

ion

of

SO

an

d N

O g

en

era

tio

n

Petroleum ether Benzene Chloroform Ethyl acetate Methanol Water

7

The results of all the above revealed that the methanolic extract exhibited the

maximum scavenging activity of all the radicals tested, compared to all the other extracts.

Therefore, only this extract was taken forward for the further studies. Once the extract with

the maximum scavenging activity was identified, the minimum concentration at which this

extract would evoke the maximum antioxidant response was analyzed in order to decide on

the dose to be used in the further experiments. For this purpose, a set of dose-response

experiments were conducted. Different concentrations of the methanolic extract of the leaves,

ranging from 0.1 to 0.4 mg were subjected to a battery of radical quenching assays (DPPH,

ABTS, H2O2, SO●, NO and OH● scavenging). The results obtained are depicted in Tables 4.3

and 4.4.

TABLE 4.3

M. hortensis LEAF EXTRACT DOSE OPTIMIZATION

Leaf

Extract

(mg)

Percent Radical Scavenging Percent Inhibition of in vitro

Generation

DPPH ABTS H2O2 SO● NO

0.1 76.30 ± 1.20 83.11 ± 2.01 72.50 ± 0.25 63.19 ± 0.75 58.19 ± 1.97

0.2 80.04 ± 0.58 85.12 ± 0.73 77.11 ± 0.95 69.63 ± 0.53 63.28 ± 0.64

0.3 78.88 ± 1.25 85.92 ± 1.17 75.00 ± 2.55 69.62 ± 1.65 59.50 ± 2.11

0.4 78.08 ± 0.66 84.46 ± 2.53 76.50 ± 3.96 68.81 ± 1.37 61.06 ± 2.28


TABLE 4.4

HYDROXYL RADICAL SCAVENGING EFFECTS OF M. hortensis LEAF

EXTRACT FOR DOSE OPTIMIZATION

Dose of Extract Percent TBARS formed

Without H2O2 With H2O2

0 21.14 ± 1.06 100

0.1 mg 36.47 ± 1.58 40.98 ± 1.36

0.2 mg 36.13 ± 0.85 40.43 ± 0.93

0.3 mg 36.94 ± 0.70 41.71 ± 1.47

0.4 mg 37.24 ± 1.08 41.11 ± 0.57

The values are Mean ± S.D. of triplicates The value of H2O2-treated group was fixed as 100 per cent and the relative values in percentage were calculated for the other groups

8

The values obtained showed that the extent of scavenging increased upto the dose of

200 µg, and thereafter exhibited a plateau. This clearly indicated that 200 µg was the optimal

dose that could be employed for further study. Therefore, only this dose level was used in

subsequent experiments.

4.3. EFFECT OF M. hortensis LEAVES ON OXIDATIVE DAMAGE TO

BIOMOLECULES

Normal aerobic metabolism is associated with ROS that can damage cellular

macromolecules (Breimer and Mikhailidis, 2011). Free radicals are by-products of

metabolism, which, in regard to their chemical structure, readily react with biomolecules

namely, DNA, lipids, proteins and carbohydrates, and cause changes in their structure and

function (Kupczyk et al., 2010). Hence, it is very crucial to study the extent of oxidative

damage to biomolecules by standard oxidants in the presence of the component under study

for antioxidant activity. For this, the methanolic extract (0.2 mg) of M. hortensis was studied

on the extent of oxidative damage to lipids, DNA and proteins.

4.3.1. Extent of Inhibition of in vitro Lipid Peroxidation

The damage to lipids and the extent to which the leaf extract inhibited this process

was quantified by measuring the extent of lipid peroxidation (LPO). To ascertain the damage

to lipids, three different membrane models were studied. They were RBC ghosts (plasma

membrane devoid of intracellular membranes), liver homogenate (a mixture of plasma

membrane and internal membranes) and precision-cut liver slices (intact cells). The extent of

inhibition of this LPO was studied in the presence of the leaf extract.

The per cent inhibition of in vitro lipid peroxidation by the leaf extract in all the three

membrane systems is presented in Table 4.4. The maximum inhibition of LPO was observed

in the goat liver homogenate, followed by the liver slices and then the RBC ghosts. These

results indicated that the lipid components of the liver homogenate, which constitute both the

plasma and internal membranes, can be protected from oxidative damage by the leaf extract

to a higher magnitude compared to the other lipid preparations in the presence of the leaf

extract.

9


FIGURE 4.4 : INHIBITION OF LIPID PEROXIDATION IN DIFFERENT

MEMBRANE PREPARATIONS BY M. hortensis LEAVES

4.3.2. Protective Effects of the M. hortensis Leaves on Oxidative Damage to DNA

The ultimate biomolecular target of the oxidative assault is DNA. The extent of

protection rendered by the leaf extract to DNA exposed to oxidants was studied. Here,

different sources of DNA, belonging to various evolutionary hierarchical levels, were used

for the analysis. Both the commercially available DNA preparations and DNA from intact

cells were used. They were,

� Lambda DNA (linear, viral phage)

� pUC18 DNA (plasmid, circular, bacterial)

� Herring sperm DNA (genomic, haploid, fish)

� Calf thymus DNA (genomic, diploid, mammal )

� Human peripheral blood lymphocytes (intact human cells)

i) Protective Effects of the Leaf Extract to λ DNA and pUC18 DNA

The extent of damage induced by H2O2 to DNA from these sources and the protective

effects of the extract were studied by viewing the migration pattern of the DNA in agarose

gels. The results are presented in Plate 4.3. H2O2 caused a significant extent of damage to

0

10

20

30

40

50

60

70

80

90

RBC ghosts Liver homogenate Liver slices

Pe

rce

nt

Inh

ibit

ion

10

both λ and pUC18 DNA. This was evident by the absence of the specific bands in lane 2,

wherein the DNA was treated with oxidant alone. The weakening of the bands in lane 2

suggested that the DNA was severely damaged resulting in very small fragments that cannot

be visualized sharply on the gel. M. hortensis leaf extract reversed this damage, which could

be seen in lane 4, as indicated by the intact bands. The leaf extract, by itself, did not cause

any DNA damage. This observation was reiterated by the Integrated Density Values (IDV) of

the bands, recorded using a digital gel documentation software (Alpha Ease FC of Alpha

Digidoc 1201), the values of which are presented in Table 4.5.

(a) Lambda DNA (b) pUC18 DNA

Lane 1: Control; Lane 2: H2O2; Lane 3: Leaf extract; Lane 4: Leaf extract + H2O2

PLATE 4.3

MIGRATION PATTERNS OF λ DNA AND pUC18 DNA TREATED WITH H2O2

WITH AND WITHOUT M. hortensis LEAF EXTRACT

Among the two DNA preparations from the lower organisms, the bacterial plasmid

DNA was more susceptible to oxidative damage and was also more receptive to the

protective effect by the leaf extract. The extent of damage by H2O2 in the DNA from the viral

source was lower; however, the extent of protection was also lower in λ DNA. The IDV

recorded clearly proved this observation.

11

TABLE 4.5

IDV OF THE BANDS IN THE AGAROSE GEL OF DNA DAMAGE

IN λ DNA and pUC18 DNA

Sample

IDV of the bands

of λ DNA

IDV of the bands

pUC18 DNA

Without H2O2 With H2O2 Without H2O2 With H2O2

No Extract 39046 20572 128440 10313

Leaf Extract 38968 30492 126578 122569

ii) Protective Effect of M. hortensis Leaf Extract on H2O2 Induced Damage to

Herring Sperm and Calf Thymus DNA

The results of the quantification of oxidative damage to herring sperm DNA is

schematically presented in Figure 4.5. It was found that H2O2 caused an increased extent of

damage to herring sperm DNA. The extent of damage decreased markedly in the presence of

the leaf extract. This indicated the protective effect rendered by the leaf extract against the

oxidant. Similar results were observed with calf thymus DNA as well (Figure 4.6). This

proved that M. hortensis leaf extract possess good protective effect against oxidative damage

to DNA.

iii) Effect of M. hortensis Leaf Extract on the Damage Induced by H2O2 to DNA in

Intact Cells

The DNA damaging effect of H2O2 was studied by following the formation of comets

in human peripheral blood cells exposed to the oxidant in vitro. The effect of the leaf extract

on this process was followed, and the results are presented in Table 4.6.

The photographic record of the comets in each of the treatment groups is depicted in

Plate 4.4.

12

The values are Mean ± S.D. of triplicates. The value of H2O2-treated group was fixed as 100 per cent and the relative values in percentage were calculated for the other groups

FIGURE 4.5 : INHIBITION OF OXIDANT-INDUCED DAMAGE TO HERRING

SPERM DNA BY M. hortensis LEAF EXTRACT

The values are Mean ± S.D. of triplicates The value of H2O2-treated group was fixed as 100 per cent and

the relative values in percentage were calculated for the other groups

FIGURE 4.6 : INHIBITION OF OXIDANT-INDUCED DAMAGE TO CALF

THYMUS DNA BY M. hortensis LEAF EXTRACT

0

20

40

60

80

100

120

No Extract Leaf Extract

PE

RC

EN

T T

BA

RS

PR

OD

UC

ED


0

20

40

60

80

100

120


PE

RC

EN

T T

BA

RS

PR

OD

UC

ED


13

TABLE 4.6

EFFECT OF M. hortensis LEAF EXTRACT ON DNA DAMAGE

INDUCED BY H2O2 IN HUMAN PERIPHERAL BLOOD CELLS

Treatment Groups No. of cells with comets/100 cells

Without H2O2 With H2O2

No Extract 5 ± 1 29 ± 1a

Leaf Extract 11 ± 1a 18 ± 2a,b,c

The values are Means ± SD of triplicates a – Statistically significant (P

Control

Methanolic Extract

COMET BEARING PERIPHERAL BLOOD LYMPHOCYTES

ii) Effect of M. hortensis

In an attempt to study the effect of the leaf extract on a mixture of proteins subjected

to oxidative stress in vitro, a mixture of bovine serum albumin and ovalbumin was prepared

in PBS and incubated in the presence and/or absence of H

the reaction mixture was loaded onto polyacrylamide gel and electrophoresed in the presence

of SDS.

The results of this SDS

in the H2O2-exposed group (lane 2) showed a clear dec

control (lane 1). The leaf extract was able to prevent this decrease to a large extent (lane 4)

(Plate 4.5).

The results thus far obtained, clearly indicated the strong free radical scavenging and

biomolecule-protecting effects of the leaf extract. Further to this, an extensive study was

formulated to analyze the effects of the leaf extract on live cells and tissues, in the presence

and the absence of induced oxidative stress.

14

Control Hydrogen peroxide

ethanolic Extract Methanolic Extract + H2O

PLATE 4.4


ortensis Leaf Extract on Protein Migration on 1D G


, a mixture of bovine serum albumin and ovalbumin was prepared

in PBS and incubated in the presence and/or absence of H2O2 and lesaf extrac


The results of this SDS-PAGE showed five distinct bands. The intensity of the bands

exposed group (lane 2) showed a clear decrease when compared to that of the



effects of the leaf extract. Further to this, an extensive study was


and the absence of induced oxidative stress.

O2


1D Gel


, a mixture of bovine serum albumin and ovalbumin was prepared

af extract. An aliquot of


PAGE showed five distinct bands. The intensity of the bands

rease when compared to that of the



effects of the leaf extract. Further to this, an extensive study was


The in vitro models adapted were goat liver slices,

primary chick embryo fibroblasts and cancer cell lines. The goat liver slices were taken as a

model that simulates in vivo

research group (Sumathi, 2007; Vidya, 2007; Nirmalade

Padma, 2010).

4.4. STUDIES ON THE ANTIOXIDANT STATUS IN LIVER SLICES EXPOSED

TO OXIDANT AND LEAF EXTRACT

The liver is a key metabolic organ and it plays a

(http://nst.berkeley.edu/faculty/stahl.html). Precision

model representing the liver under

Hence, the precision-

both in the presence and in the absence of the extracts of

(SOD, CAT, Px, GST and GR) and non

reduced glutathione) antioxidants were analyzed in the homogenate of the liver slices.

EFFECT OF M. hortensis

0

5

10

15

20

25

30

35

40

45

50

No ExtractPro

tein

ca

rbo

ny

l (n

mo

l/m

g)

Without

15

models adapted were goat liver slices, Saccharomyces cerevisiae


in vivo conditions. The model was validated in earlier studies in

research group (Sumathi, 2007; Vidya, 2007; Nirmaladevi, 2008; Radha 2010; Sreelatha

STUDIES ON THE ANTIOXIDANT STATUS IN LIVER SLICES EXPOSED

TO OXIDANT AND LEAF EXTRACT in vitro

The liver is a key metabolic organ and it plays an important role in the homeostasis

(http://nst.berkeley.edu/faculty/stahl.html). Precision-cut liver slices constitute an

model representing the liver under in vivo conditions.

-cut goat liver slices were challenged with an ox

both in the presence and in the absence of the extracts of M. hortensis leaves. The enzymic

(SOD, CAT, Px, GST and GR) and non-enzymic (vitamin C, vitamin E, vitamin A and


FIGURE 4.7

ortensis LEAF EXTRACT ON PROTEIN CARBONY

FORMATION



Saccharomyces cerevisiae cells,


conditions. The model was validated in earlier studies in our

vi, 2008; Radha 2010; Sreelatha and

STUDIES ON THE ANTIOXIDANT STATUS IN LIVER SLICES EXPOSED

n important role in the homeostasis

cut liver slices constitute an in vitro

cut goat liver slices were challenged with an oxidant (H2O2)

leaves. The enzymic

enzymic (vitamin C, vitamin E, vitamin A and


LEAF EXTRACT ON PROTEIN CARBONYL

Control Band 1 300126 Band 2 204869

Band 3 209050 Band 4 155375 Band 5 518448


SUBJECTED TO OXIDATIVE STRESS

4.4.1. Enzymic Antioxidant Status

Extract in vitro

The activities of the major enzymic antioxidants were assayed in the liver slice

homogenate prepared after exposure to H

enzymes assayed were SOD, CAT, Px, GST and GR (Table 4.7). Upon exposure to the

oxidant (H2O2), all the enzymic antioxidants showed significantly (P

17

TABLE 4.7

EFFECT OF M. hortensis LEAF EXTRACT ON ENZYMIC ANTIOXIDANTS

ACTIVITIES IN GOAT LIVER SLICES EXPOSED in vitro TO H2O2

Enzymic

Antioxidants

Groups

Untreated

control

H2O2

treated

Leaf extract

treated

H2O2 + leaf

extract treated

SOD (units#/g tissue)

9.09 ± 1.18 7.32 ± 0.44 a 13.84 ± 0.55 a 13.15 ± 0.79 a,b,c

CAT (units$/g tissue)

95.82 ± 1.87 82.98 ± 2.86 a 123.67 ± 3.19 a 101.52 ± 2.14 a,b,c

Px (units*/g tissue)

6.69 ± 0.50 4.61 ± 1.40 a 14.50 ± 0.84 a 12.38 ± 0.62 a,b,c

GST (units@/ g tissue)

0.27 ± 0.02 0.16 ± 0.01 a 0.56 ± 0.05 a 0.50 ± 0.05 a,b,c

GR (units+/g tissue)

2.48 ± 0.35 1.83 ± 0.10 a 2.60 ± 0.08 a 2.26 ± 0.50 a,b,c

Values are mean ± S.D. of triplicates # 1 Unit = 50% inhibition of NBT reduction in one minute

$ 1 Unit = Amount of enzyme required to decrease the absorbance at 240nm * 1 Unit = Changes in absorbance at 430 nm/minute

+ 1 Unit = mmoles of NADPH oxidized/minute @ 1 Unit = nmoles of CDNB conjugated/minute

a – statistically significant (p

18

TABLE 4.8

EFFECT OF M. hortensis LEAF EXTRACT ON NON-ENZYMIC ANTIOXIDANT

LEVELS IN GOAT LIVER SLICES EXPOSED in vitro TO H2O2

Non-enzymic

Antioxidant

Group

Untreated

control

H2O2

treated

Leaf extract

treated

H2O2 + leaf

extract treated

Vitamin C (mg/g tissue)

36.90 ± 0.87 33.62 ± 0.55 a 54.89 ± 1.01 a 45.49 ± 1.30 a,b,c

Vitamin E (µg/g tissue)

7.28 ± 0.29 5.42 ± 0.15 a 9.91 ± 0.12 a 8.81 ± 0.11 a,b,c

Vitamin A (µg/g tissue)

250.13 ±9.97 221.94 ± 0.01 a 288.89 ± 4.98 a 274.72 ± 4.98 a,b,c

Reduced Glutathione (nmoles/g tissue)

14.42 + 0.52 13.88 + 0.85 18.38 + 0.55 a 17.88 + 0.90 a,b

The values are mean ± S.D. of triplicates a – Statistically significant (P

19

Control Hydrogen Peroxide

Methanolic Extract Methanolic Extract+H2O2

PLATE 4.6

HISTOPATHOLOGICAL ARCHITECTURE OF THE GOAT LIVER SLICES

When the oxidant was co-administered along with the leaf extract, notable damage was

observed in certain areas, but some parts of the liver tissue showed recovered areas with

normal architecture. The periportal areas showed some edema but the peripheral areas

showed preserved architecture. This indicated that the methanolic extract of the M. horntesis

leaves is effective in protecting the liver tissue from oxidative damage.

PHASE III

The results of the first two phases of the study clearly indicated the antioxidant

potential of the M. hortensis leaf extract. Therefore, the next phase was initiated to validate

the effect of the leaf extract on oxidative cell death. For this, the apoptosis–modulating

effects of the leaf extract were studied under unstressed and oxidatively stressed conditions.

20

This effect was analyzed on both untransformed Saccharomyces cerevisiae and primary

chick embryo fibroblasts (non-cancerous) and transformed Hep2 (cancerous) cells. For

Saccharomyces cerevisiae cells, H2O2 was used to induce oxidative stress. In the primary

cells and in the cancer cell line, etoposide (a standard cancer chemotherapeutic agent that

induces cell death via oxidative stress) was used as the oxidant.

4.6. EFFECT OF M. hortensis LEAF EXTRACT ON H2O2-INDUCED

APOPTOSIS IN S. cerevisiae CELLS

Yeast cells were used to study the effects of the plant extract on the apoptotic events

associated in the presence of the oxidant, H2O2. In order to understand the nature of the

cellular death process and the molecular events involved in the process, studies were

conducted on morphological and nuclear changes that occur during apoptotic death.

4.6.1. Morphological Changes of Apoptosis Observed in S. cerevisiae Cells

The characteristic morphological changes in apoptotic cells were analysed by Giemsa

staining in the presence and the absence of the leaf extract and/or H2O2. The number of

apoptotic and non-apoptotic cells was counted under a phase contrast microscope (Table 4.9).

TABLE 4.9

EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL

CHANGES IN S. cerevisiae CELLS SUBJECTED TO OXIDATIVE STRESS

AS DETERMINED BY GIEMSA STAINING

TREATMENT

GROUPS

Number of Apoptotic

Cells /100 Cells Apoptotic Ratio

Control H2O2 treated Control H2O2 treated

No Extract 8 ± 2 75 ± 1a 0.09 3.00

Leaf Extract 16 ± 1a 26 ± 1 a,b,c 0.19 0.35

The values are mean ± S.D of triplicates a – Statistically significant (P

21

significantly, indicating the anti-apoptotic activity rendered by the leaf extract. The apoptotic

ratios in the treated and untreated cells were calculated and the values obtained are

represented in Table 4.9.

4.6.2. Nuclear Changes of Apoptosis Observed in S. cerevisiae Cells

The apoptotic nuclei stain strongly with the fluorescent dyes, which allow the non-

apoptotic cells to be discriminated from the apoptotic ones. The nuclear changes associated

with apoptosis were followed after staining the treated cells with the fluorescent dyes,

namely EtBr (Plate 4.7b), PI (Plate 4.7c) and DAPI (Plate 4.7d) the number of apoptotic cells

counted in the various staining experiments are presented in Tables 4.10 to 4.12 respectively.

The apoptotic ratios were also calculated for each group and are listed alongside the number

of apoptotic cells in Tables 4.10 to 4.12 respectively.

The results of EtBr, PI and DAPI staining indicated that the number of apoptotic cells

was highest in the oxidant treated group, which was significantly (P

Control

(a) S.

Control

(b)

Control

(c)

Control

(d)


NUCLEAR CHANGES INDUCED BY H

22

H2O2 Methanol Extract Methanol+H

S. cerevisiae cells stained with Giemsa


(b) S. cerevisiae cells stained with EtBr


(c) S. cerevisiae cells stained with PI


(d) S. cerevisiae cells stained with DAPI

PLATE 4.7

M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL AND

NUCLEAR CHANGES INDUCED BY H2O2 IN S.cerevisiae CELLS

Methanol+H2O2

Methanol+H2O2

Methanol+H2O2

Methanol+H2O2

LEAF EXTRACT ON THE MORPHOLOGICAL AND

CELLS

23

TABLE 4.11

EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN

S. cerevisiae CELLS SUBJECTED TO OXIDATIVE STRESS

AS DETERMINED BY PI STAINING

Treatment

Groups

Number of Apoptotic

Cells / 100 Cells Apoptotic Ratio

Control H2O2

treated Control H2O2 treated

No Extract 11 ± 2 74 ± 1a 0.12 2.85

Leaf Extract 20 ± 1a 30 ± 1a,b,c 0.25 0.43


24

Sulphorhodamine B assay was used as an additional parameter to calculate the cell

viability and proliferative potential of S. cerevisiae cells in the presence and the absence of

H2O2 and/or the leaf extract of M. hortensis. Figure 4.9 illustrates the results obtained for the

cell viability study in yeast using SRB. The viability of the cells decreased in the H2O2

treated group due to the effect of the oxidant. The groups that were simultaneously treated

with the leaf extract showed improved cell viability indicating the protective effect of the leaf

extract.

4.6.4. Effect of M. hortensis Leaves on the Viability of S. cerevisiae Cells Subjected to

Oxidative Stress (LDH Assay)

LDH release has been considered as a very reliable marker of membrane damage due

to cell lysis, indicating cytotoxicity. This is indicative of the protection rendered by the leaf

extract towards the cytotoxicity. The cytotoxicity in the cells treated with or without H2O2, in

the presence or absence of M. hortensis leaf extract was also determined by the LDH release.

The results are depicted in Figure 4.10. H2O2 exposure caused a steep rise in the extent of

apoptosis in yeast cells. When administered along with H2O2, the leaf extract resulted in a

significant decrease in the LDH release.

4.6.5. DNA Fragmentation in Yeast

DNA fragmentation in apoptotic S. cerevisiae cells were assayed using

diphenylamine in a spectrophotometric assay and the per cent extent of fragmentation

obtained is shown in Figure 4.11. The exposure of H2O2 to the S. cerevisiae cells caused

significant DNA damage. The co-administration of the methanolic extract of M. hortensis

leaves reduced the extent of DNA damage indicating the anti-apoptotic effect of M. hortensis

leaf extract.

25

FIGURE 4.8

Effect of M. hortensis leaf extract

on the viability of S. cerevisiae cells

subjected to oxidative stress as

determined by MTT assay

The values are mean ± SD of triplicates

The values of the untreated (negative) control group were fixed as 100% and

the per cent viabilities in the other groups were calculated relative to this

FIGURE 4.9

Effect of M. hortensis leaf extract on

the viability of S. cerevisiae cells

subjected to oxidative stress as

determined by SRB assay

The values are mean ± SD of triplicates The values of the untreated (negative) control

group were fixed as 100% and the per cent viabilities in the other groups

were calculated relative to this

FIGURE 4.10

Effect of M. hortensis leaf extract on

percent cytotoxicity in S. cerevisiae

cells as determined by LDH release

The values are means ± S.D. of triplicates

FIGURE 4.11

Effect of M. hortensis leaf extract

on DNA damage in S. cerevisiae

cells subjected to oxidative stress

The values are means ± S.D. of triplicates

0

20

40

60

80

100

120


Pe

rce

nt

Ce

ll V

iab

ilit

y

Without With H202 H202

0

20

40

60

80

100

120


Pe

rce

nt

Ce

ll V

iab

ilit

y


0

10

20

30

40

50


Perc

ent C

yto

toxic

ity


0

20

40

60

80

100

No Extract Leaf ExtractPe

rce

nt

DN

A

Da

ma

ge


26

4.7. EFFECT OF M. hortensis LEAF EXTRACT IN ETOPOSIDE INDUCED

STRESS IN PRIMARY CHICK EMBRYO FIBROBLASTS AND Hep2 CELLS

It is evident from the results obtained from the S. cerevisiae cells that M. hortensis

leaves can render protection to these cells against oxidative stress. As the next step of the

study, it was felt necessary to study the effect of the leaf extract on cancer cells. This was

done because cancer is recognized to be a result of oxidative genotoxicity. As a control to the

cancerous cells, non-cancerous primary cultured chick embryo fibroblasts were used.

Etoposide was employed as the oxidant to induce the oxidative stress. The influence of the

etoposide in the presence and the absence of the M. hortensis leaf extract in both chick

embryo fibroblasts (Plate 4.8a) and Hep2 cells (Plate 4.8b) were evaluated by various

(membrane and nuclear) staining techniques and the cytotoxicity assays.

4.7.1. Effect of M. hortensis Leaf Extract on the Morphological Changes in Etoposide

Induced Stress in Primary Chick Embryo Fibroblasts and Hep2 Cells

The morphological changes observed in primary chick embryo fibroblasts and Hep2

cells stained with Giemsa are depicted in Table 4.13 and Table 4.14 respectively.

Etoposide caused a steep increase in the number of cells (cancerous) showing

apoptotic morphology in both chick embryo fibroblasts and Hep2 cells (Plates 4.9a and

4.10a). However, the effect of M. hortensis leaf extract in the two types of cells, showed a

markedly differential response. In the chick embryo fibroblasts, the presence of the extract,

along with the oxidant showed a recovery in survival, with a decrease in the apoptotic cells

(Plate 4.9a). Whereas, in the case of Hep2 cells, the administration of the leaf extract alone

increased the number of apoptotic cells compared to control, indicating anticancer activity of

the leaf extract. Co-exposure of the Hep2 cells with leaf extract and etoposide caused a

further increase in the number of apoptotic cells. This observation indicates that the

M.hortensis leaf extract augments the cytotoxicity of the chemotherapeutic agent (etoposide)

only in the cancer cells, while protecting the non-cancerous cells from its cytotoxicity.

a) Primary Chick Embryo Fibroblasts

4.7.2. Effect of M. hortensis

Observed Primary Chick Embryo Fibroblasts

The nuclear changes in the cancerous and non

modulation in the presence/absence of leaf extract

namely EtBr, PI and DAPI.

27

Primary Chick Embryo Fibroblasts

b) Hep2 cells

PLATE 4.8

ortensis Leaf Extract on the Nuclear Changes

Observed Primary Chick Embryo Fibroblasts and Hep2 Cells

The nuclear changes in the cancerous and non-cancerous cells by etoposide and its

modulation in the presence/absence of leaf extract were studied using the nuclear stains,

Leaf Extract on the Nuclear Changes of Apoptosis

cancerous cells by etoposide and its

studied using the nuclear stains,

28

TABLE 4.13

EFFECT OF M. hortensis LEAF EXTRACT ON THE MORPHOLOGICAL

CHANGES IN CHICK EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE

STRESS AS DETERMINED BY GIEMSA STAINING

Treatment

Groups

Number of Apoptotic Cells

/ 100 Cells Apoptotic Ratio

Without

Etoposide

With

Etoposide

Without

Etoposide

With

Etoposide

No Extract 5 ± 1 71 ± 3a 0.05 2.45



29

significant, as the M. hortensis leaf extract protects noncancerous cells from oxidative death,

at the same time rendering the cancerous cells more susceptible to the chemotherapeutic

agent-induced oxidative death.

The photographic evidence of the noncancerous and cancerous cells showing nuclear

changes is presented in Plates 4.9 and 4.10 respectively. The individual cell numbers are the

calculated apoptotic ratios of both types of cells are listed in Tables 4.15 to 4.20 respectively.

TABLE 4.15

EFFECT OF M. hortensis LEAF EXTRACT ON THE NUCLEAR CHANGES IN

CHICK EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE STRESS

AS DETERMINED BY EtBr STAINING

Treatment

Groups

Number of Apoptotic

Cells / 100 Cells Apoptotic Ratio

Without

Etoposide

With

Etoposide

Without

Etoposide

With

Etoposide

No Extract 3 ± 1 78 ± 1a 0.03 3.54


The values are mean ± S.D. of triplicates a – Statistically significant (P

30

TABLE 4.17

EFFECT OF M. hortensis LEAF EXTRACT ON NUCLEAR CHANGES IN CHICK

EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE STRESS

AS DETERMINED BY PI STAINING

Treatment Groups

Number of Apoptotic

Cells/100 Cells Apoptotic Ratio

Without

Etoposide

With

Etoposide

Without

Etoposide

With

Etoposide

No Extract 5 ± 1 78 ± 2a 0.05 3.54

Leaf Extract 12 ± 2a 23 ± 4 a,b,c 0.14 0.30

The values are mean ± S.D of triplicates a –Statistically significant (P

Control

(a) Primary Chick Embyro Fibroblasts stained with

Control

(b) Primary

Control

(c) Primary

Control

(d) Primary Chick Embyro Fibroblasts stained with DAPI


NUCLEAR CHANGES IN PRIMARY CHICK EMBRYO FIBROBLASTS

31

Etoposide Leaf Extract Leaf Extract +

Chick Embyro Fibroblasts stained with Giemsa


Chick Embyro Fibroblasts stained with EtBr


Primary Chick Embyro Fibroblasts stained with PI


Chick Embyro Fibroblasts stained with DAPI

PLATE 4.9



Leaf Extract +

Etoposide

Giemsa

Leaf Extract +

Etoposide

Chick Embyro Fibroblasts stained with EtBr

Leaf Extract +

Etoposide

Leaf Extract +

Etoposide

Chick Embyro Fibroblasts stained with DAPI



Control

(a) Hep2 Cells Stained With

Control

(b)

Control

Control


NUCLEAR CHANGES IN Hep2 CELLS

32


(a) Hep2 Cells Stained With Giemsa


(b) Hep2 Cells Stained With Etbr


(c) Hep2 Cells Stained With PI


(d) Hep2 cells stained with DAPI

PLATE 4.10


NUCLEAR CHANGES IN Hep2 CELLS

Leaf Extract +

Etoposide

Leaf Extract +

Etoposide

Leaf Extract +

Etoposide

Leaf Extract +

Etoposide


33

TABLE 4.19

EFFECT OF M. hortensis LEAF EXTRACT ON NUCLEAR CHANGES IN CHICK

EMBRYO FIBROBLASTS SUBJECTED TO OXIDATIVE STRESS

AS DETERMINED BY DAPI STAINING

Treatment

Groups

Number of Apoptotic

Cells/100 cells Apoptotic Ratio

Without

Etoposide

With

Etoposide

Without

Etoposide

With

Etoposide

No Extract 4 ± 1 68 ± 4a 0.04 2.13



34

4.7.3. Effect of M. hortensis Leaf Extract on the Cell Viability of Primary Chick

Embryo Fibroblasts and Hep2 Cells

Cell viability was deduced by the MTT and SRB assays. In the chick embryo cells,

the cytotoxicity of etoposide was reduced by the presence of the leaf extract of M. hortensis.

The leaf extract, by itself, also caused a decrease in the viability of Hep2 cells, which was

decreased further in the presence of etoposide (Figure 4.12). These observations suggest that

the plant extract enhances the action of etoposide on Hep2 cells, while inhibiting the toxicity

of etoposide to the primary cells (Figure 4.13).

Similar results were obtained for the SRB assay, which was done to confirm the

results of MTT. The figures 4.14 and 4.15 show the cell viability results.

4.7.4. Effect of M. hortensis Leaf Extract on the LDH Release in Primary Chick

Embryo Fibroblasts and Hep2 Cells

In the present study, the extent of cell death due to oxidative stress and the effect of

the leaf extract on this process was confirmed by another assay, namely the release of LDH.

This enzyme is a very reliable marker of membrane damage, which, in turn, is indicative of

cell death. Hence, the extent of release of LDH from the primary as well as cancer cells,

subjected to various treatments, was analyzed.

The cytotoxicity in the untransformed cells, i.e., primary chick embryo fibroblasts,

when treated with etoposide, showed a higher extent of LDH release due to the cytotoxic

effect of the standard chemotherapeutic drug, etoposide. This value was drastically reduced

in the presence of the plant extract due to the protection rendered by the M.hortensis leaf

extract (Figure 4.16).

In the case of the cancerous cells, Hep2, the cytotoxicity was increased in the group

of cells that were treated with etoposide. Additionally, in the cells treated with both the drug

and the leaf extract, the extent of LDH release increased further. This observation showed the

anticancer activity rendered by the leaf extract (Figure 4.17).

Thus, the LDH release assay also reiterated the differential effect of the M. hortensis

leaf extract on the different types of cells.

Figure 4.12

Effect of M.hortensis Leaf Extract on the

Viability of Chick Embryo Fibroblasts Subjected

to Oxidative Stress as Determined by MTT Assay

Figure 4.14

Effect of M.hortensis Leaf Extract on the Viability

of Primary Chick Embryo Fibroblasts Subjected

to Oxidative Stress as Determined by SRB Assay

Figure 4.16

Effect of M. hortensis leaf extract on percent

cytotoxicity in primary chick embryo fibroblasts

as determined by LDH release

The values of the untreated (negative) control group were fixed as 100% andthe per cent viabilities in the other groups were calculated relative to this.

0

20

40

60

80

100

120

No Extract Leaf ExtractPe

rce

nt

Ce

ll V

iab

ilit

y

Without Etoposide With Etoposide

0

20

40

60

80

100

120

No Extract

Pe

rce

nt

Ce

ll V

iab

ilit

y

Without Etoposide

0

20

40

60

80


Pe

rce

nt

Cy

toto

xic

ity


35

Leaf Extract on the

Viability of Chick Embryo Fibroblasts Subjected

to Oxidative Stress as Determined by MTT Assay

Figure 4.13

Effect of M.hortensis Leaf Extract on the Viability of

Hep2 Cells Subjected to Oxidative Stress as

Determined by MTT assay

Leaf Extract on the Viability

of Primary Chick Embryo Fibroblasts Subjected

to Oxidative Stress as Determined by SRB Assay

Figure 4.15

Effect of M.hortensis Leaf Extract on the Viability of

Hep2 Cells Subjected to Oxidat

Determined by SRB Assay

leaf extract on percent

cytotoxicity in primary chick embryo fibroblasts

as determined by LDH release

Figure 4.17

Effect of M. hortensis leaf extract on percent

cytotoxicity in Hep2 cells as determined by LDH

release

The values are mean ± SD of triplicates The values of the untreated (negative) control group were fixed as 100% and

the per cent viabilities in the other groups were calculated relative to this.

Leaf Extract

With Etoposide

0

20

40

60

80

100

120

No Extract

Pe

rce

nt

Ce

ll V

iab

ilit

y

Without Etoposide

Leaf Extract

With Etoposide

0

20

40

60

80

100

120

No ExtractPe

rce

nt

Ce

ll V

iab

ilit

y

Without Etoposide

Leaf Extract

With Etoposide

0

20

40

60

80

100


Pe

rce

nt

Cy

toto

xic

ity


Leaf Extract on the Viability of


Determined by MTT assay

Leaf Extract on the Viability of


Assay

leaf extract on percent

Hep2 cells as determined by LDH

The values of the untreated (negative) control group were fixed as 100% and the per cent viabilities in the other groups were calculated relative to this.

Leaf Extract

With Etoposide

Leaf Extract

With Etoposide

Leaf Extract

With Etoposide

4.7.5. Effect of M. hortensis

Embryo Fibroblasts

The phenomenon of apoptosis is well characterized by DNA fragmentation. In the

present study, the extent of DNA fragmentation in the chick embryo fibroblasts and Hep2

cells subjected to the various treatments was analyzed by agarose gel electrophoresis.

Exposure of the primary chick embryo fibroblasts and Hep2 cells to the oxidant,

etoposide, caused DNA damage as evidenced by a faint band (Lane 2).

extract by itself, did not cause a major damage (Lane 3). When the leaf extract was co

administered along with the oxidant, it decreased the extent of DNA damage caused by

etoposide (lane 4) in the primary cells, (Plate 4.11a) whereas, in Hep2 cells,

administration of the plant extract augmented the extent of damage caused by etoposide

4) (Plate 4.11b). The IDV quantifying the intensities of the DNA bands are tabulated (Table

4.21). This clearly depicts the differential role played by

untransformed cells, it exhibited anti

augmented the activity of the anticancer agent.

a) Chick Embryo Fibroblasts


PATTERN OF PRIMARY CHICK EMBRYO FIBROBLASTS

36

ortensis Leaf Extract on the DNA Fragmentation in Primary Chick

Embryo Fibroblasts and Hep2 Cells against Induced Oxidative Stress




xposure of the primary chick embryo fibroblasts and Hep2 cells to the oxidant,

etoposide, caused DNA damage as evidenced by a faint band (Lane 2). M. hortensis

extract by itself, did not cause a major damage (Lane 3). When the leaf extract was co


(lane 4) in the primary cells, (Plate 4.11a) whereas, in Hep2 cells,

administration of the plant extract augmented the extent of damage caused by etoposide


4.21). This clearly depicts the differential role played by M. hortensis leaf extract; in the

untransformed cells, it exhibited anti-apoptotic activity and in transformed cells, it

augmented the activity of the anticancer agent.

a) Chick Embryo Fibroblasts b) Hep2 Cells

Lane 1 – Untreated group Lane 2 – Etoposide treated group Lane 3 – Majorana hortensis leaf extract treated group Lane 4 – Majorana hortensis leaf extract + Etoposide

PLATE 4.11

M. hortensis LEAF EXTRACT ON THE DNA FRAGMENTATION


AND Hep2 CELLS

n Primary Chick

gainst Induced Oxidative Stress




xposure of the primary chick embryo fibroblasts and Hep2 cells to the oxidant,

M. hortensis leaf

extract by itself, did not cause a major damage (Lane 3). When the leaf extract was co-


(lane 4) in the primary cells, (Plate 4.11a) whereas, in Hep2 cells, the

administration of the plant extract augmented the extent of damage caused by etoposide (lane


leaf extract; in the

transformed cells, it

ON THE DNA FRAGMENTATION


37

TABLE 4.21

IDV OF THE BANDS IN THE AGAROSE GEL OF DNA FRAGMENTATION Assay

OF PRIMARY CHICK EMBRYO FIBROBLASTS AND Hep2 CELLS

PHASE IV

The first three phases of the study evidenced that M. hortensis leaves are a good

source of antioxidants, and show very good anti-apoptotic activity in non-cancerous cells and

pro-apoptotic activity in cancer cells. These properties are presumably rendered by the

chemical substances or the secondary metabolites present in the leaves that get extracted into

methanol. Therefore, it becomes highly essential to identify the active principle(s) rendering

the protective effects of M. hortensis leaves. Hence, the fourth phase of this study

emphasized on the qualitative identification of the chemical nature of the active component

present in the candidate plant. This was followed by spectral studies such as UV absorption

spectrum, TLC, HPTLC, HPLC, IR and GC-MS to identify the major components present in

the leaves of M. hortensis.

4.8. PRELIMINARY QUALITATIVE PHYTOCHEMICAL ANALYSIS

The fresh leaves of M. hortensis were subjected to phytochemical analysis to identify

the presence of the major phytochemicals. The qualitative test showed the presence of

alkaloids, phenols, flavonoids, saponins, sterols and tannins (Table 4.22).

From these results, it can be inferred that the active components in M. hortensis

leaves may be alkaloids, phenols, flavonoids, sterols, saponins or tannins. Hence, these

phytochemical fractions were isolated and subjected to UV absorption.

Sample

IDV OF THE BANDS

Primary Chick Embryo Fibroblasts Hep2 Cells

Without

Etoposide

With

Etoposide

Without

Etoposide

With

Etoposide

No Extract 83050 77003 53485 28100

Methanolic Extract 83965 79492 39540 30500

38

TABLE 4. 22

QUALITATIVE PHYTOCHEMICAL ANALYSIS OF M. hortensis LEAVES

S. No. COMPONENTS RESULT

1. ALKALOIDS

Mayer’s test +

Dragondroff’s test +

Wagner’s test +

2. PHENOLS

Ferric chlride +

Lead acetate +

3. FLAVONOIDS

Aqueous NaOH test +

Concentrated sulfuric acid test +

Schinado’s test +

4. STEROIDS

Leibermann-Buchard test +

Salkowski test +

5. SAPONINS

Froth test +

Haemolytic test +

6. TANNINS

Braemer’s test +

4.9. UV ABSORPTION SPECTRUM OF THE PHYTOCHEMICAL FRACTIONS

OF M. hortensis LEAVES

The absorption spectrum of the different fractions namely, alkaloids, phenols,

flavonoids, sterols and tannins of the M. hortensis leaves were evaluated in the UV range

which gave specific absorption spectrum.

39

The alkaloid fraction of M. hortensis leaves (Figure 4.18) showed several major and

minor peaks, beginning with a sharp peak at 225 nm, followed by another major peak at 250

nm. A few more well-defined peaks were noted at 300, 320, 330 and 360 nm respectively.

Figure 4.19 shows the UV absorption spectrum of the phenolic fraction, which

revealed a minor peak at 200 nm, a major peak at 220 nm. Also, 350, 380 and 395 nm

exhibited sharp major peaks, indicating the presence of major active principles present in the

leaf extract. A few other minor peaks were also noted.

The UV absorption spectrum of the flavonoid fraction indicated well defined major

peaks at 230, 300 and 325 nm. At 310 nm also a peak was observed, though not well defined.

A few minor peaks were also noticed beginning with 205 nm as indicated in Figure 4.20.

The UV absorption spectrum of the saponin fraction was determined and the

spectrum is presented in Figure 4.21. The peaks observed here were similar to the flavonoids

and a few peaks coincided. At 230, 310 and 325 nm saponins also exhibited peaks as in

flavonoids.

Figure 4.22 showed the peaks obtained from the steroid fraction by UV absorption.

Several major peaks at 195, 240, 250, 275, 290 and 305 nm were observed and a minor peak

at 230 nm.

The UV absorption pattern of tannins (Figure 4.23) showed well-defined peaks at

200, 225, 235, 255-260 nm. Major peaks were noted at 340-350 nm and 370 nm. Other well

defined peaks were observed at 360, 380 and 395 nm.

4.10. TLC OF THE PHYTOCHEMICAL FRACTIONS OF M. hortensis LEAVES

The TLC plate, when detected with the alkaloid-specific Dragendroff’s spraying

reagent, showed six major bands with Rf vales 0.83, 0.74, 0.65, 0.53,0.40 and 0.26 (Plate

4.12a). The presence of phenolics was analysed using Folin-Ciocalteau reagent as the

spraying reagent. The results are shown in Plate 4.12b, wherein five major spots with Rf

values 0.68, 0.66, 0.53, 0.50 and 0.48 were visualized. The investigation of flavonoids

separated by TLC, sprayed with 10% vanillin in sulphuric acid, showed four major bands

with Rf values 0.81, 0.73, 0.62 and 0.50 as seen in

three major bands as indicated in P

were also subjected to TLC analysis and the chromatogram was sprayed with 10% sulphuric

acid which showed five distinct bands with R

4.12e). The number of tannin bands was found to be five, with R

0.48 and 0.19 respectively, which were developed by spraying 10% sulphuric acid (Plate

4.12f).

UV ABSORPTION SPECTRUM OF THE ALKALOID FRACTION OF

UV ABSORPTION SPECTRUM OF THE PHENOLIC FRACTION OF

40

values 0.81, 0.73, 0.62 and 0.50 as seen in Plate 4.12c. The saponin fraction showed

three major bands as indicated in Plate 4.12d with Rf values 0.75, 0.71 and 0.50. The sterols


acid which showed five distinct bands with Rf values 0.80, 0.78, 0.63, 0.49, and 0.44 (Plate

ber of tannin bands was found to be five, with Rf values 0.76, 0.73, 0.67,


FIGURE 4.18


M. hortensis LEAVES

FIGURE 4.19


M. hortensis LEAVES

. The saponin fraction showed

values 0.75, 0.71 and 0.50. The sterols


values 0.80, 0.78, 0.63, 0.49, and 0.44 (Plate

values 0.76, 0.73, 0.67,




UV ABSORPTION SPECTRUM OF THE FLAVONOID FRACTION OF

UV ABSORPTION SPECTRUM OF THE SAPONIN FRACTION OF

41

FIGURE 4.20

SPECTRUM OF THE FLAVONOID FRACTION OF

M. hortensis LEAVES

FIGURE 4.21


M. hortensis LEAVES

SPECTRUM OF THE FLAVONOID FRACTION OF


UV ABSORPTION SPECTRUM OF THE STEROID FRACTION OF

UV ABSORPTION SPECTRUM OF THE TANNIN FRACTION OF

42

FIGURE 4.22


M. hortensis LEAVES

FIGURE 4.23


M. hortensis LEAVES



a) Alkaloids b) Phenolics

d) Saponins

TLC OF THE PHYTOCHEMICAL FRACTIONS OF

4.11. HPTLC OF THE PHYTOCHEMICAL

The methanolic extract of

the presence of alkaloids, phenols, flavonoids, saponins, steroids and tannins.

The alkaloid profile of the methanolic extract was done with the refernce standard

colchicine and the developed plate was sprayed with Dragendroff’s reagent.

coloured zone at day light mode present in the given standard and sample tracks obser

0.83

0.74

0.65

0.53

0.40

0.26

0.75

0.71

0.50

43

a) Alkaloids b) Phenolics c) Flavonoids

d) Saponins e) Sterols f) Tannins

PLATE 4.12

TLC OF THE PHYTOCHEMICAL FRACTIONS OF M. hortensis

HPTLC OF THE PHYTOCHEMICAL FRACTIONS OF M. hortensis

The methanolic extract of M. hortensis leaves was subjected to HPTLC analysis for



colchicine and the developed plate was sprayed with Dragendroff’s reagent.

coloured zone at day light mode present in the given standard and sample tracks obser

0.48

0.81

0.50 0.50

0.53

0.66

0.68

0.62

0.73

0.80

0.78

0.63

0.49

0.44

0.76

0.67

0.48

0.19

0.73

c) Flavonoids

f) Tannins

M. hortensis LEAVES

hortensis LEAVES

leaves was subjected to HPTLC analysis for



colchicine and the developed plate was sprayed with Dragendroff’s reagent. Orange-brown

coloured zone at day light mode present in the given standard and sample tracks observed in

44

the chromatogram after derivatization confirmed the presence of six alkaloids in the leaves

(Plate 4.13). The peak table (Table 4.23) and peak densitogram (Figure 4.24) were recorded.

The phenolics present in the methanolic extract of M. hortensis leaves were analysed

using quercetin as the reference standard. Orange-brown colored zones at visible light mode

were present in the track, it was observed from the chromatogram after derivatization, which

confirmed the presence of phenolics in M. hortensis leaves (Plate 4.14). The peak table

(Table 4.24) and peak densitogram (Figure 4.25) showed the presence of four phenols.

The flavonoid profile of the methanolic extract of M. hortensis leaves was analysed

using rutin as the standard. Yellow and yellow green fluoresecenc zone at UV 366 nm was

seen from the chromatogram, which confirmed the presence of flavonoids (Plate 4.15). There

were six different flavonoids identified in the methanolic extract of M. hortensis leaves as

shown in the peak table (Table 4.25) and peak densitogram (Figure 4.26).

Plate 4.16 confirmed the presence of saponins in the methanolic extract of

M.hortensis leaves where saponin standard was used. Blue, yellowish brown coloured zones

in the visible light mode were present in the track observed from the chromatogram after

derivatization, which confirmed the presence of saponins in the given samples. The peak

table (Table 4.26) and the peak densitogram (Figure 4.27) represented 9 different saponins.

The steroid profile of the methanolic extract of M. hortensis leaves was analyzed

using solasodine as the standard. Blue-violet coloured zones in the day light mode present in

the given standard and sample tracks observed in the chromatogram after derivatization

confirmed the presence of sterols in the M. hortensis leaves (Plate 4.17). The peak table

(Table 4.27) and the peak densitogram (Figure 4.28) confirmed the presence of 5 different

steroids.

Using tannic acid as the standard, the tannin profile of the methanolic extract of M.

hortensis leaves was analyzed by spraying with 5% ferric chloride. Bluish brown coloured

zones in the day-light mode confirmed the presence of two tannins (Plate 4.18). The peak

table (Table 4.38) and peak densitogram (Figure 4.29) showed the second tannin to be tannic

acid.

45

Before derivatization After derivatization

Day light UV 366 nm UV 254 nm Day light

PLATE 4.13

HPTLC OF ALKALOIDS

FIGURE 4.24

HPTLC PEAK DENSITOGRAM OF ALKALOIDS IN M. hortensis LEAVES

TABLE 4.23

HPTLC PEAK TABLE FOR THE ALKALOIDS IN THE M. hortensis LEAVES

Track Peak Rf Height Area Assigned substance

A 1 0.04 12.0 166.2 Unknown

A 2 0.11 45.8 556.7 Alkaloid 1

A 3 0.20 133.1 3493.8 Alkaloid 2

A 4 0.26 142.9 4123.6 Alkaloid 3

A 5 0.31 342.4 9079.3 Alkaloid 4

A 6 0.34 277.1 5702.6 Unknown

A 7 0.40 534.2 35242.0 Alkaloid 5

A 8 0.53 13.7 124.6 Unknown

A 9 0.62 41.0 1257.9 Unknown

A 10 0.73 266.1 8645.6 Alkaloid 6

A 11 0.90 12.4 86.9 Unknown

COL 1 0.41 118.3 4333.6 Colchicine standard

Before derivatization

Day light UV 366 nm

HPTLC PEAK DENSITOGRAM OF PHENOLICS IN

HPTLC PEAK TABLE FOR THE PHENOLICS IN

Z Peak

QUER 1

MH 1

MH 2

MH 3

MH 4

MH 5

MH 6

MH 7

MH 8

MH 9

MH 10

46


UV 366 nm UV 254 nm Day light

PLATE 4.14

HPTLC OF PHENOLICS

FIGURE 4.25

PTLC PEAK DENSITOGRAM OF PHENOLICS IN M. hortensis

TABLE 4. 24

HPTLC PEAK TABLE FOR THE PHENOLICS IN M. hortensis

Peak Rf Height Area Assigned substance

0.56 525.6 11620.1 Quercetin standard

0.02 46.0 574.0 Unknown

0.06 21.5 488.3 Unknown

0.18 153.1 5510.8 Phenolics 1

0.29 92.1 2950.6 Phenolics 2

0.36 13.3 395.1 Unknown

0.46 193.0 6445.0 Phenolics 3

0.53 38.8 773.4 Unknown

0.57 118.4 6128.4 Phenolics 4

0.80 39.1 1348.4 Unknown

0.84 43.0 1066.0 Unknown

Day light

M. hortensis LEAVES

M. hortensis LEAVES

Assigned substance

Quercetin standard

47


Day light 366 nm 254 nm Day light 366 nm

PLATE 4.15

HPTLC OF FLAVONOIDS

FIGURE 4.26

HPTLC PEAK DENSITOGRAM OF FLAVONOIDS IN M. hortensis LEAVES

TABLE 4.25

HPTLC PEAK TABLE FOR THE FLAVONOIDS IN M. hortensis LEAVES


RUT 1 0.32 377.4 10448.2 Rutin standard

MH 1 0.13 77.6 2672.6 Flavonoid 1

MH 2 0.24 28.5 685.9 Unknown

MH 3 0.25 28.2 633.0 Unknown

MH 4 0.37 47.8 3241.8 Flavonoid 2

MH 5 0.47 181.4 9256.2 Flavonoid 3

MH 6 0.54 73.5 2801.7 Flavonoid 4

MH 7 0.59 67.8 3030.5 Flavonoid 5

MH 8 0.70 79.4 3747.4 Unknown

MH 9 0.88 286.2 16395.6 Flavonoid 6

MH 10 0.95 200.6 7346.6 Unknown


Day light UV 366 nm

HPTLC PEAK DENSITOGRAM OF SAPONINS

HPTLC PEAK TABLE FOR THE SAPONINS OF THE

Track Peak

SAP 1

MH 1

MH 2

MH 3

MH 4

MH 5

MH 6

MH 7

MH 8

MH 9

MH 10

MH 11

48


UV 366 nm UV 254 nm Day light UV 366 nm

PLATE 4.16

HPTLC OF SAPONINS

FIGURE 4.27

HPTLC PEAK DENSITOGRAM OF SAPONINS IN M. hortensis

TABLE 4.26

HPTLC PEAK TABLE FOR THE SAPONINS OF THE M. hortensis

Peak Rf Height Area Assigned substance

0.12 250.2 6815.2 Saponin standard

0.05 39.7 568.3 Saponin 1

0.10 104.1 2628.8 Saponin 2

0.16 71.6 2006.9 Saponin 3

0.20 84.7 2404.9 Saponin 4

0.30 545.7 20031.9 Saponin 5

0.34 84.8 1346.3 Unknown

0.39 139.4 5350.6 Saponin 6

0.46 91.8 3130.4 Saponin 7

0.52 68.4 2165.4 Saponin 8

0.60 162.2 5420.1 Saponin 9

0.65 33.2 1298.0 Unknown

After derivatization

UV 366 nm

M. hortensis LEAVES

M. hortensis LEAVES

Assigned substance

Saponin standard

49


Day light UV 366 nm UV 254 nm Day light UV 366 nm

PLATE 4.17

HPTLC OF STEROIDS

FIGURE 4.28

HPTLC PEAK DENSITOGRAM OF STEROIDS IN M. hortensis LEAVES

TABLE 4.27

HPTLC PEAK TABLE OF STEROIDS IN M. hortensis LEAVES


SOL 1 0.63 318.8 15382.6 Solasodine standard

A 1 0.17 30.1 1416.2 Unknown

A 2 0.20 31.7 497.8 Unknown

A 3 0.45 162.2 6142.5 Unknown

A 4 0.48 166.5 2713.4 Sterol 1

A 5 0.50 163.2 2971.0 Sterol 2

A 6 0.60 337.3 26544.9 Unknown

A 7 0.70 86.8 3971.8 Sterol 3

A 8 0.80 33.8 1746.0 Sterol 4

A 9 0.92 132.5 2670.9 Sterol 5

A 10 0.94 168.3 7638.5 Unknown


Day light UV 366 nm

HPTLC PEAK DENSITOGRAM OF TANNINS IN

HPTLC PEAK TABLE FOR THE TANNINS IN

Track Peak

A 1

A 2

A 3

A 4

A 5

A 6

TAN 1

50


UV 366 nm UV 254 nm Day light

PLATE 4.18

HPTLC OF TANNINS

FIGURE 4.29

HPTLC PEAK DENSITOGRAM OF TANNINS IN M. hortensis LEAVES

TABLE 4.28

HPTLC PEAK TABLE FOR THE TANNINS IN M. hortensis LEAVES

Rf Height Area Assigned substance

0.17 84.9 1029.9 Unknown

0.64 167.9 5900.6 Unknown

0.68 177.2 4344.5 Unknown

0.72 297.7 15878.5 Tannin 1

0.85 305.1 11634.7 Tannin 2 (Tannic acid)

0.92 384.4 25670.3 Unknown

0.86 179.2 5895.1 Tannic acid standard

After derivatization

LEAVES

LEAVES

Assigned substance

Tannin 2 (Tannic acid)

Tannic acid standard

51

4.12. HPLC ANALYSIS OF THE METHANOLIC EXTRACT OF M. hortensis

LEAVES

The HPLC analysis of the methanolic extract of M. hortensis leaves was carried out

using C18 reverse phase column (Shimadzu equipped with UV detector). The results

obtained are presented in Figure 4.30. The HPLC spectrum showed 5 peaks (2 major and 3

minor) in the methanolic extract of M. hortensis leaves. The retention time of the major and

minor peaks along with the peak area of all the 5 peaks are represented in Table 4.29.

FIGURE 4.30

HPLC ANALYSIS OF THE METHANOLIC EXTRACT OF M. hortensis LEAVES

TABLE 4. 29

PEAK TABLE OF THE METHANOLIC EXTRACT OF M. hortensis LEAVES

SUBJECTED TO HPLC

Scanning at 366nm (UV Long range)

Retention time Peak area

4.288 198311.1

4.958 1102922.8

41.607 195652.0

43.804 14563.6

44.387 40041.5

0 10 20 30 40 50 min

-10

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

65

mAU360nm,4nm (1.00)

335

194

446

441

441

52

4.13. IR ANALYSIS OF THE METHANOLIC EXTRACT OF M. hortensis LEAVES

The methanolic extract of M. hortensis leaves were analysed for the IR spectrum

using FT-IR spectrophotometer using KBr pellet method (Figure 4.31). It exhibited bands at

2910cm-1 along with bands at 1217cm-1 and 1012cm-1 which are characteristic of –OH

stretching, C-O-H bending and C-O stretching vibrations. This indicates the presence of –OH

group. A band at 1725cm-1 indicates the presence of –CO group. Hence, the extract may

contain saponins.

FIGURE 4.31

IR SPECTRUM OF THE METHANOLIC EXTRACT OF M. hortensis LEAVES

4.14. GC-MS ANALYSIS OF THE METHANOLIC EXTRACT OF M. hortensis

LEAVES

The GC-MS analysis of the methanolic extract of Majorana hortensis leaves was

carried out to identify the nature of the components present. The GC results showed the

presence of eight major components at retention times 3.120, 15.683, 18.565, 22.438, 23.486,

25.833, 31.924 and 33.375 respectively (Figure 4.32). In the mass spectrum of the GC peak

at retention time 33.375, molecular ion peak was observed at m/z 250, 239.7, 206.9, 165,

148, 111, 91, 86, 85 and 55 (Figure 4.33). Characteristic M-17 and M-18 peak were observed

at m/z 298 and m/z 148, indicating the presence of hydroxyl groups. The fragmentation

pattern of this compound was studied using the WILEY database.

53

The mass spectrum of the peak at retention time 31.924 registered M+ peaks at m/z

252.9. Other M+ peaks were observed at m/z 213.1, 199.0, 159.0, 130.0, 129.0, 112.0, 87,

69, and 63.8 (Figure 4.34). It also displayed a characteristic M-17 at m/z 112 indicating the

presence of hydroxyl group.

The mass spectrum of peak at retention time 23.486, showed M+ peak at m/z 147.1,

other characteristic peaks were observed at m/z 130.0, 129.0, 87, 86, 79 and 64.5 (Figure

4.36). (M-18) peak at m/z 129 was observed suggesting the presence of hydroxyl group.

The mass fragmentation pattern of peak at retention time 22.436, displayed M+ peaks

at m/z 282, 254, 216.2, 202.0, 196.1, 150, 133.0, 104.9, 95, 70 and 54 (Figure 4.37). A

characteristic M-17 peak was also observed at m/z 133, indicating the presence of hydroxyl

group. The presence of M-28 peak at m/z 254 in the spectrum indicted that the compound

may contain carbonyl group.

The mass spectrum of peak at retention time 18.565 registered M+ peaks at m/z

394.5, 380.3, 365, 326.8, 298.2, 281.1, 255.2, 222.0, 213.1, 185.1, 157.1, 143.0, 128.9, 119.0,

87, 74 and 55 (Figure 4.38). M-18 peak was observed at m/z 111, which is characteristic of

the presence of hydroxyl group. Also, three characteristic M-28 peaks were observed at m/z

298.2, 185.1 and 157.1 suggesting the presence of a carbonyl group in the compound.

The mass fragmentation pattern of peak at retention time 15.683 registered a

molecular peak at m/z 391. The other peaks were observed at m/z 373.4, 315.6, 278.3, 270.2,

239.1, 227.2, 185.1, 171.1, 143.1, 111.98, 87, 74, 71.1 and 54.9 (Figure 4.39). The spectrum

also displayed M-18 peak at m/z 373.4, M-45 peak at m/z 270.2 and M-28 at m/z 143, which

are characteristic for hydroxyl and an ester function. Hence the compound may contain

hydroxyl group along with an ester moiety.

The mass spectrum of peak at retention time 3.120 displayed M+ peaks at m/z 237,

208.1, 206.9, 192.4, 156.1, 131.9, 129.0, 104.9, 85, 71.8, 57 and 54.1(Figure 4.40). The

fragmentation pattern showed characteristic M-28 and M-27 peaks at m/z 129 and m/z 57.1

suggesting the presence of carbonyl group and nitrogen on the compound.

54

FIGURE 4.32

GC-MS PROFILE OF THE METHANOLIC EXTRACT OF M. hortensis LEAVES

FIGURE 4.33

PEAK FRAGMENTATION OF GC-MS SPECTRUM (33.375)

FIGURE 4.34


55

FIGURE 4.35


FIGURE 4.36


FIGURE 4.37


56

FIGURE 4.38


FIGURE 4.39


FIGURE 4.40


57

Thus, the phytochemical analyses of methanolic extract of M. hortensis leaves

revealed the presence of alkaloids, phenolics, flavonoids, saponins, sterols and tannins. These

compounds may be responsible for the antioxidant and apoptosis-modulating effects of M.

hortensis leaves.

The present study, thus, validates the M. hortensis leaves with strong antioxidant

effect. It also showed protection against the biomolecules like lipids, DNA and proteins. The

study was extrapolated into in vitro systems which simulate in the in vivo (goat liver slices,

yeast, primary cells and Hep2 cells) to study the antioxidant effect, apoptosis modulating

effect and anti-cancer effect. The phytochemical analysis shows supporting evidence for the

presence of few plant phytochemicals (alkaloids, phenols, flavonoids, saponins, tannins, and

steroids) which contribute to the antioxidant activity of the leaf extract. The spectral studies

are indicative of the presence of saponins as the major component.

The outcome of the research work is discussed in the next chapter with the support of

relevant published articles.

results - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/13464/11/11_chapter 3.p… ·...

Documents