RESULTSAND DISCUSSION - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29166/4/rpappaammal... · antioxidant and cytotoxic properties of Murraya koenigii and Spinacia oleracea

Introduction

Evaluation of Thrombolytic and Antioxidant Potential of Murraya koenigii and Spinacia oleracea 39

RREESSUULLTTSS AANNDD DDIISSCCUUSSSSIIOONN

Green leafy vegetables contain various pharmacologically active compounds

and have been used as medicine since ancient times (Bhat and Al-Daihan, 2014).

Phytochemical constituents are playing a significant role in the identification of

crude drugs. There is a widespread interest in evaluating drugs derived from plant

sources. The interest mainly arises from the belief that green medicine is safe and

dependable, compared to costly synthetic drugs which are invariably associated

with adverse effects (Maobe et al., 2013). In recent times some evidence for the

role of specific plant food and phytochemicals in protecting against the onset of

diseases such as cancers and heart diseases has been put forward (Tiwari et al.,

2013). The present study has been formulated to evaluate the thrombolytic,

antioxidant and cytotoxic properties of Murraya koenigii and Spinacia oleracea. It

was carried out in five different phases.

Phase I involved the screening of various plants for thrombolytic activity and

analysis of phytoconstituents of the selected plants. Phase II included the

determination of the thrombolytic activity of the selected plants at various

concentrations and its correlation with serum cholesterol of the blood samples used

for thrombolysis. Phase III studied the establishment of antioxidant potential,

biosafety screening and spectral properties of the two different plants. Phase IV

involved in vivo experiments to ensure clot lysis by the plants. Phase V comprised

of in silico characterization of active components in the selected plants.

4.1 PHASE I

4.1.1 Screening of plants for thrombolytic activity

The thrombolytic activity of the aqueous extracts of plants, namely Murraya

koenigii, Spinacia oleracea, Basella alba, Talinum portulacifolium, Trigonella

foenum-graecum and Mentha piperita was determined in vitro using human blood.

The values are indicated in Table 1 and Figure I.

4

Introduction


Table 1Percentage Clot Lysis by the Selected Green Leafy Vegetables

Concentration(mg / ml)

Percentage Clot Lysis

Murrayakoenigii

Spinaciaoleracea

Basellaalba

Talinumportulacifolium

Trigonellafoenum-graecum

Menthapiperita

5 9.34±0.43 13.93±0.40 5.4±0.13 5.26±0.32 3.92±0.20 6.72±0.29

10 23.65±0.60 27.15±0.71 7.92±0.20 6.98±0.14 6.32±0.14 9.23±0.07

20 7.76±0.36 40.90±0.21 4.24±0.13 3.82±0.16 12.62±0.21 11.00±0.04

30 6.83±0.07 28.62±0.55 3.92±0.20 2.78±0.10 4.26±0.09 7.36±0.07

Values are mean ± SD of triplicates

Figure 1Clot Lysis of Green Leafy Vegetables

All the six plants tested showed clot-lysing abilities, albeit to varying extent.

Such diverse variations in the thrombolytic efficiency of different plants have been

reported in the literature. For instance, Prasad et al. (2007) have reported that

ability of various herbs namely Tinospora cordifolia, Rubia cordifolia, Hemidesmus

indicus, Glycyrrhiza glabra Linn, Fagonia arabica and Bacopa monnieri (Linn) to

lyse blood clots varied widely. A very high clot–lysing ability was reported for

0

5

10

15

20

25

30

35

40

45

5 10 20 30

Concentration (mg / ml)

Perc

enta

ge C

lot L

ysis

Murraya koenigii Spinacia oleracea Basella albaTalinum portulacifolium Trigonella foenum- graecum Mentha piperita

Introduction


Zanthoxylum budrunga (Khanb et al., 2011), which is in close agreement to our

results.

Another striking observation that can be made from our results is that the

thrombolytic activity of all the plants tested did not follow a dose-dependent

increase. On the other hand, the extent of clot lysis peaked at a particular optimum

concentration, on either side of which, a lower activity was observed. This peak

activity was attained at 10mg/ml concentration for Murraya koenigii, Basella alba

and Talinum portulacifolium, while it was at 20mg/ml for Spinacia oleracea,

Trigonella foenum-graecum and Mentha piperita. Such an optimal dose effect was

also reported by Islam et al. (2013) for Tinospora crispa.

The comparision of the thrombolytic activity of the six different plants

indicated that Murraya koenigii and Spinacia oleracea exhibited a higher extent of

clot lysis than the other plants. Hence these two plants were selected for further

analysis.

4.1.2 Phytochemical Analysis

Qualitative Analysis of Phytoconstituents of Murraya koenigii and Spinaciaoleracea

Analysing the phytochemicals in medicinal plants provides scientists with

insight to know how plants are medicinally effective and understanding the

chemical composition leads to the development of new medicines (Nithya et al.,

2011). Hence a qualitative phytochemical screening of the selected plants to

determine the presence or absence of bioactive compounds was performed and

the results are given in Table 2.

Phytochemicals such as carbohydrates, amino acids, proteins, phenols,

glycosides, saponins, quinones, flavonoids, tannins, volatile oils, terpenoids and

alkaloids were found to be present in the ethanolic extracts of Murraya koenigi,

whereas, glycosides and quinones were absent in the aqueous extract. In the case

of Spinacia oleracea it was found to be rich in all the above phytoconstituents

except volatile oils and tannins, which were absent in both aqueous and ethanolic

extracts. Saponins were absent only in the ethanolic extract.

Introduction


Table 2Phytochemical Constituents of Murraya koenigii and Spinacia oleracea

+ Present, - Absent

Parekh et al. (2005) have suggested that the beneficial medicinal effects of

plant materials typically result from the secondary products present in the plantalthough, it is usually not attributed to a single compound but a combination of the

metabolites. Subhash et al. (2010) have reported that secondary metabolites like

flavonoids, carotenoids and phenolic compounds were present in Spinacia

oleracea.

The results are supported by Bonde et al. (2011), who reported thatMurraya koenigii leaves are aromatic and contain proteins, carbohydrates, fiber,

minerals, carotene, nicotinic acid and vitaminC. Raghu et al. (2011) analysed ten

aqueous vegetable extracts and showed the presence of carbohydrates, proteins,amino acids, glycosides, flavonoids, tannins and polyphenols. Shanthi et al. (2011)

have shown the presence of carbohydrates, proteins, amino acids, sterols,

alkaloids, flavonoids, phlobatinins and terpenoids in the aqueous extracts of

Nerium oleander and Momordica charantia leaves.

S.No. PhytochemicalsMurraya koenigii Spinacia oleracea

Aqueousextract

Ethanolicextract

Aqueousextract

Ethanolicextract

1. Carbohydrates + + + +

2. Amino acids and Proteins + + + +

3. Phenols + + + +

4. Glycosides - + + +

5. Saponins + + + -

6. Quinones - + + +

7. Flavonoids + + + +

8. Tannins + + - -

9. Volatile Oils + + - -

10. Terpenoids + + + +

11. Alkaloids + + + +

Introduction


166

31

0

20

40

60

80

100

120

140

160

180

M. koenigii S. oleracea

mg

/ 100

g

7.31

1.64

0

1

2

3

4

5

6

7

8

M. koenigii S. oleracea

g / 1

00g

Quantitative estimation of phytochemical constituents of Murraya koenigiiand Spinacia oleracea

Phytochemicals namely proteins and alkaloids were quantitatively estimated

and the values are given in Figure 2.

Figure 2

Phytoconstituents in the Selected Plants

It is evident from the table that the alkaloid and protein contents were

greater in Murraya koenigii compared to Spinacia oleracea and the difference was

statistically significant (p<0.01). The alkaloid content was drastically increased in

Murraya koenigii suggesting it as a rich source of alkaloids.

Preliminary phytochemical screening of Murraya koenigii by Darvekar

et al. (2011) indicated the presence of mucilage, proteins, sterols and triterpenoids,

alkaloids, flavonoids and phenolic compounds. An intense research on literature

revealed that the stems, leaves, roots and seeds of Murraya koenigii are potential

sources of carbazole alkaloids (Nayak et al., 2010). Murraya koenigii possessed

potent antioxidant properties, which may be due to the presence of biological active

ingredients such as carbazole alkaloids, glycoside, triterpenoids and phenolic

Alkaloids Proteins

Introduction


compounds (Tembhurne and Sakarkar, 2010). Wight and Arn (2012) reported that

plants have thrombolytic activity that could be due to the wide range of

phytoconstituents including alkaloids, flavonoids, tannins and terpenoids. The

higher quantity of alkaloids in Murraya koenigii reported in the present study might

be due to the presence of carbazole alkaloids.

Ningappa et al. (2010) have isolated antioxidant protein from curry leaves,

which exhibited broad spectrum of antibacterial activity and suggested it as a

promising candidate for drug of an effective antioxidant antibiotic. Kavitha and

Ramadas (2013) have indicated that the protein content of spinach in raw and

powder form has the advantage as a rich source of vegetable protein over other

lesser known vegetables.

Many drugs are derived from alkaloids that are responsible for the

therapeutic effect of many medicinal plants. Earlier studies have indicated that

alkaloids possess antihyperglycemic and antilipidemic effects suggesting their

beneficial effect in the management of diabetes associated with abnormal lipid

profile and related cardiovascular diseases. From the results of the present study it

can be seen that alkaloids might be responsible for the medicinal properties of the

selected plants.

HPTLC Profiling of Murraya koenigii and Spinacia oleracea

Table 3, Plates 1, 2 and figure 3 represent the alkaloid profile of the

aqueous extracts of Murraya koenigii and Spinacia oleracea respectively. The

alkaloid standard exhibited Rf value of 0.51. Alkaloid profile by HPTLC analysis

revealed the presence of alkaloids in the aqueous extracts of Murraya koenigii and

Spinacia oleracea. Several bands appeared when the developed chromatogram

was sprayed with an alkaloid- specific reagent. However, only one band in each

leaf showed the characteristic colour associated with alkaloids. The Rf values of

these two bands differed widely from each other, and also from the standard

(Colchicine). This observation suggests that the leaves contain very different

phytochemicals.

Introduction


Table 3

Alkaloid Profile of Murraya koenigii and Spinacia oleracea

Track Peak Rf Height Area Assigned substance

Standard 1 0.51 514.4 14843.0 Colchicine

Murraya koenigii 1 0.01 428.0 3705.0 Unknown


Murraya koenigii 3 0.12 35.9 568.4 Alkaloid





Spinacia oleracea 1 0.01 373.4 3433.1 Unknown



Spinacia oleracea 4 0.29 31.3 699.7 Alkaloid





Introduction


Plate 1

Chromatogram of Murraya koenigii

Before derivatization

Daylight UV 366nm UV 254nm

After derivatization

Daylight UV 366nm

Introduction


Plate 1





Daylight UV 366nm

Introduction


Plate 1





Daylight UV 366nm

Introduction


Plate 2

Chromatogram of Spinacia oleracea




Daylight UV 366nm

Introduction


Plate 2





Daylight UV 366nm

Introduction


Plate 2





Daylight UV 366nm

Introduction


Figure 3

Peak densitogram of alkaloids

Alkaloid standard

Aqueous extract of Murraya koenigii Aqueous extract ofSpinacia oleracea

Introduction


Dineshkumar et al. (2010) have reported that mahanimbine, a carbazole

alkaloid in curry leaves, has a potential role to prevent atherosclerosis and coronary

heart disease. Nayak et al., (2010) stated that Murraya koenigii is a rich source of

biologically active carbazole alkaloids that attracts the attention of chemists and

pharmacologists and play a significant role in future research in medical science.

Kamba and Hassan (2010) supported that alkaloids were present in crude water

extract whereas they were absent in ethanolic extract of root bark of Securidaca

longepedunculata. They also mentioned that water was the best solvent to be used

in the extraction of sample. Beal and Lewis (2006) have also indicated that

alkaloids themselves are quite insoluble in water and soluble in organic solvents,

while their salts are soluble in water and insoluble in the organic solvents.

Thus the findings of HPTLC and the reports available in the literature

indicate the presence of alkaloids in plants and also suggest that they might be

present in the form of salts

4.2 PHASE II

4.2.1 Thrombolytic activity of Murraya koenigii and Spinacia oleracea

Thrombolytic activity of various concentrations (5-25mg) of aqueous extracts

of Murraya koenigii, Spinacia oleracea and combination of both the plants was

determined using human blood and the values are represented in Table 4 and

Figure 4.

As shown in the below table, all the five different concentrations of the plant

extracts induced significant clot lysis when compared to negative control. The

positive control streptokinase (30,000 IU) evoked a huge and significant clot lysis.

Murraya koenigii exhibited maximum clot lysis at a concentration of 10mg/ml while

Spinacia oleracea showed the maximal value at a concentration of 20mg/ml.

This finding has been supported by Chowdhury et al. (2011) who have indicated

that Aponogeton undulatus Roxb exhibited maximum percentage of clot lysis

46.13±3.87% at a dose of 10mg/ml. They have also indicated that the

phytochemicals such as tannins, alkaloids and saponins present in the crude

extract might have participated in clot lysis. It is evident from the table that

Introduction


percentage clot lysis of the extracts at the highest dose was lesser than that

produced by the penultimate dose. Similar finding has been reported by

Ratnasooriya et al. (2008) who have indicated that the thrombolytic activity of the

highest dose (20mg/ml) was lower than that produced by the penultimate dose

(10mg/ml).

Table 4

Thrombolytic Activity of Murraya koenigii and Spinacia oleracea

GroupsPercentage Clot Lysis

Concentration(mg/ml)

Murrayakoenigii

Spinaciaoleracea

M.koenigii andS.oleracea

G1 Distilled Water(Negative Control) 3.42 ± 0.50

G2 Streptokinase(Positive Control) 56.72 ± 3.78a**

G3 5 5.88 ± 0.64b** 13.58 ± 0.67 b** 15.76 ± 0.28 b**G4 10 22.14 ± 1.79b** 27.15 ± 0.26 b** 29.34 ± 1.21 b**G5 15 18.21 ± 1.59b** 35.91 ± 1.51 b** 31.65 ± 1.07 b**G6 20 7.23 ± 0.39b** 40.9 ± 0.25 b** 32.22 ± 1.80 b**G7 25 6.15 ± 0.35b** 23.85 ± 0.39 b** 20.22 ± 0.35 b**


a – G1 vs G2; b – G2 vs G3,G4,G5,G6,G7 Significant at **p<0.01

Determinations of the thrombolytic activity of Murraya koenigii and Spinacia

oleracea revealed that Spinacia oleracea exhibited higher percentage of clot lysis.

When compared to the positive control streptokinase both plants exhibited

moderate to good percentage of clot lysis. When both plant extracts were used in

combination, maximum clot lysis was observed at a concentration of 20 mg/ml but

the percentage lysis was lesser than the value shown by Spinacia oleracea alone.

This might be due to the interactive effect of Murraya koenigii, which registered

lower percentage of lysis than Spinacia oleracea. Comparison among the extracts

of Nigella sativa, Capsicum frutescens, Brassica oleracea, honey, combination of

honey and Nigella sativa and honey and Capsicum frutescens revealed that

Brassica oleracea, Capsicum frutescens and combination of honey and Nigella

sativa showed significant thrombolytic activity (Anwar et al., 2011).

Introduction


Figure 4

Thrombolytic Activity of the Selected Plants

Khan et al. (2011) reported that the aqueous extracts of Ocimum sanctum,

Curcuma longa, Azadirachta indica and Anacardium occidentale showed moderate

to good clot lysis activity. Al- Mamun et al. (2012) have observed 43.25% of clot

lysis for Coriandrum sativum. Mannan et al. (2011) have indicated that the Cassia

alata seed oil extract has moderate thrombolytic activity compared to negative

control (water). Anjum et al. (2013) reported that significant thrombolytic activity

was demonstrated by the aqueous soluble fraction of the stem bark of Bridelia

tomentosa (37.04%).

When the two different plants Murraya koenigii and Spinacia olearcea were

used individually, Spinacia oleracea registered greater thrombolytic activity (40.9%)

than Murraya koenigii at a concentration of 20mg/ml. When they were used in

combination maximum clot lysis (32.22%) was observed at the same concentration

(20mg/ml) but percentage was lesser than the value recorded by Spinacia oleracea

when used individually indicating that some compounds in Murraya koenigii would

have interfered in lysing the clot. Thus the results revealed that Murraya koenigii

and Spinacia oleracea exhibited considerable percentage of clot lysis whether used

0

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20

30

40

50

60

G1 G2 G3 G4 G5 G6 G7

Groups

Perc

enta

ge C

lot L

ysis

Murraya koenigii

Spinacia oleracea

M.koenigii and S.oleracea

Introduction


individually or in combination suggesting that both plants can be exploited for

thrombolytic therapy.

4.2.2 Comparison of Serum Cholesterol Level and Thrombolytic Activity ofMurraya koenigii and Spinacia oleracea

Total cholesterol was estimated in the serum obtained from an aliquots of

the blood samples used for clot lysis. A comparison was made between the

serum cholesterol level and percent clot lysis. The values are indicated in

Figures 5, 6 and 7.

The scattergram of paired values of cholesterol levels and the corresponding

clot lysis values in each sample varied widely. While a mild positive correlation

was observed with Murraya koenigii and the mixture of the two leaf extracts, there

was a slight negative correlation in the samples treated with Spinacia oleracea.

This lack of correlation indicates that both the leaves can render good protection

against clot induced blocks in blood vessels, irrespective of the cholesterol levels of

the individual. It also suggests that both the leaves can act as sources of potential

thrombolytic agents, irrespective of the cause of clot formation.

Figure 5

Correlation between Serum Cholesterol and ThrombolyticActivity of Murraya koenigii

rs = 0.1169p = 0.406ns

y = 0.002x + 21.69

20.50

21.00

21.50

22.00

22.50

23.00

23.50

24.00

0 100 200 300 400

Clot

lysi

s (%

)

Level of serum cholesterol (mg/dl)

Introduction


Figure 6

Correlation between Serum Cholesterol and ThrombolyticActivity of Spinacia oleracea

rs = -0.0654 p = 0.698ns

Figure 7

Correlation between Serum Cholesterol and Thrombolytic Activityof Murraya koenigii and Spinacia oleracea

rs = 0.147p = 0.429ns

y = -0.003x + 41.48

37.00

38.00

39.00

40.00

41.00

42.00

43.00

44.00

45.00

0 100 200 300 400

Clot

lysi

s (%

)


y = 0.006x + 31.01

29.00

30.00

31.00

32.00

33.00

34.00

35.00

36.00

0 100 200 300 400

Clot

lysi

s (%

)


Introduction


4.2.3 Membrane Stabilizing Potential of Murraya koenigii and Spinaciaoleracea

Figure 8 reflects the effect of plant extracts and standard acetyl salicylic

acid on percent haemolysis inhibition.

Figure 8Percentage Inhibition of Haemolysis by Murraya koenigii

and Spinacia oleracea

*- p<0.05 ns - Not Significant

The results suggest that the standard exhibited 59.36% and 64.75%

inhibition for heat induced and hypotonic solution induced haemolysis respectively.

The plants Spinacia oleracea and Murraya koenigii also showed inhibiting potential.

However, the magnitude of inhibition for Murraya was comparatively low (46.17%

for heat induced and 40.30% for hypotonic solution induced) when compared

to Spinacia oleracea which showed 57.82% for heat induced and 74.82% for

hypotonic solution induced haemolysis. The percentage inhibition of the latter was

significant when compared to the standard.

Kawsar et al. (2011) who reported that Vernonia cenerea a medicinal plant

of Bangladesh, showed 53.13% of haemolysis inhibition. Khan et al. (2013) also

reported that the different extracts of Vitex negundo Bark moderately protected the

a*46.17 a*

40.3

bns

57.82

b*74.82

c*64.75c*

59.36

0

10

20

30

40

50

60

70

80

90

Heat Induced Hypotonic Solution Induced

Perc

enta

ge in

hibi

tion

of H

aem

olys

is

M urraya koenigii Spinacia oeracea Acetyl salicylic acid

Introduction


lysis of human erythrocyte membrane induced by hypotonic solutions which

confirms that the plant has potent membrane stabilizing activity as it stabilized the

membrane of RBCs.

Shahriar et al. (2012) showed that in heat induced and hypotonic conditions

the methanol extract of Withania somnifera inhibited 45.91% and 63.95% of

haemolysis of RBCs respectively as compared to 42.12% and 72.9% inhibition by

acetyl salicylic acid respectively (0.1 mg/ml). The findings of the study are in

accordance with the present report.

Percentage of haemolysis by the plants and the standard acetyl salicylic

acid demonstrate that both plants moderately protected the membrane of RBC

suggesting the membrane stabilizing potential of the plant extracts which is an

essential quality for thrombolysis.

4.3 Phase III

Phase III comprised of the evaluation of antioxidant potential, biosafety

screening of Murraya koenigii and Spinacia oleracea and identification of functional

groups of the active components in the plants.

4.3.1 Antioxidant status of the selected plants

Antioxidants benefit our wellness by cleaning toxins out of our blood vessels

(Khan et al., 2013). The search for new antioxidant compounds is an ongoing area

of drug discovery and the plant kingdom has been generous in providing hundreds

of diverse natural products with such activity. In the present study, both the

enzymatic and nonenzymatic antioxidants were analysed in the plant samples and

the values are indicated below.

Enzymatic antioxidants

The activities of various antioxidative enzymes namely catalase, peroxidase

and superoxide dismutase were determined in the fresh leaves of the two different

plants and depicted in Table 5.

Introduction


Table 5Activity of Enzymatic Antioxidants in Murraya koenigii


Plant Catalase (U/g) Peroxidase(U/g)

Superoxidedismutase U/g)

M. koenigii 137.00 ± 0.72 6.51 ± 0.52 15.37 ± 0.15

S. oleracea 97.20 ± 0.40* 1.85 ± 0.08* 9.03 ± 0.14*

Values are mean ± SD of three samples in each plant

Significant at * - p<0.05

Catalase : Amount of enzyme required to decrease the optical density by 0.05 units

Peroxidase : Change in absorbance / min/ g of sample

SOD : The amount that causes 50% reduction in the extent of NBT oxidation

The activity of all the enzymatic antioxidants was found to be greater in

Murraya koenigii than Spinacia oleracea, suggesting Murraya koenigii to be a richer

source of enzymatic antioxidants. Catalase is one of the principal antioxidant

enzymes, it eliminates H2O2 by transforming it into H2O and O2. Beulah and

Ramana (2013) screened the leaves of different medicinal plants for catalase

activity and indicated that considerable activity has been noticed in the leaf extract

of Murraya koenigii. The stimulation of SOD activity along with catalase seemed to

play a protective role against membrane damage as Cu is particularly toxic to

membranes (Ahmed et al., 2010). Xu et al (2011) indicated that protection

of human body against both cellular oxidation and pathogens are due to

ROS-scavenging enzymes such as superoxide dismutase, catalase and

peroxidase.

Non enzymatic antioxidants

Apart from the enzymatic antioxidants, a spectrum of nonenzymatic

antioxidants namely vitamin C, vitamin E, reduced glutathione, polyphenols,

carotenoids and flavonoids are important in cellular system in curtailing reactive

oxygen species. Table 6 depicts the levels of nonenzymatic antioxidants in

Murraya koenigii and Spinacia oleracea.

Introduction


Table 6

The Levels of Non Enzymatic Antioxidants in Murraya koenigiiand Spinacia oleracea

Plant M. koenigii S. oleracea ‘t’ valueTotal Carotenoiods (mg/100g)

12.79 ± 0.33 13.12 ± 0.23 1.16ns

Flavonoids (mg/100) 151.80 ± 2.30 114.40 ± 1.98 16.91*

Vitamin C (mg/100g) 24.75 ± 0.34 68.20 ± 0.44 110.51*

Vitamin E (mg/100g) 26.52 ± 0.54 5.30 ± 0.39 45.05*

Reduced Glutathione(µmol/100g) 79.95 ± 0.22 60.75 ± 0.81 32.35*

Polyphenols (mg/100g) 165.00 ± 2.63 138.00 ± 1.95 9.61*


Significant at * - p<0.05 ns – Not significant

The level of nonenzymatic antioxidants such as flavonoids, vitamin E,

reduced glutathione and polyphenols content was significantly higher in Murraya

koenigii. Spinacia oleracea recorded significantly higher value of vitamin C.

There was no significant difference in the carotenoid content of the two different

plants.

Das and Guha (2008) reported that spinach contains different carotenoids

like lutein, β-carotene, violaxanthin and 9-(z)- neoxanthin and high concentration of

vitamins like A,E,C,K and folic acid. Bhatia and Jain (2004) have reported that

consumption of carotenoid-rich foods like spinach, even for a short period of time,

gives protection against oxidative stress. Chandrika et al. (2010) analysed the

carotenoid content of selected Srilankan green leafy vegetables and reported that

they can be exploited as rich sources of beta-carotene.

Saliu and Oboh (2013) analysed some tropical green leafy vegetables for

antioxidative properties and indicated that all the green leafy vegetables

demonstrated strong free radical scavenging abilities. Flavonoids are the most

Introduction


abundant polyphenols reported to possess antioxidant activity in plant foods. Rao

et al. (2011) have also indicated that flavonoids present in plants have the ability

to scavenge peroxyl, alkylperoxy radicals, superoxide hydroxyl radicals and

peroxynitrile in aqueous and organic environment. Bergman et al. (2001)

demonstrated for the first time the presence of both flavonoids and p-coumaric acid

derivatives as antioxidant components of the aqueous extract of spinach leaves.

Aehle et al. (2004) have observed that spinach (Spinacia oleracea) leaves contain

antioxidant flavonoids, in particular, spinacetin and patuletin, and also indicated that

spinach flavonoids, as well as the crude aqueous or resin-purified extracts,

exihibited high antioxidant activities

Ascorbic acid is an important antioxidant, which reacts not only with H2O2

but also with O2-, OH and lipid hydroperoxides. It has an additional role in

protecting or regenerating oxidized carotenoids or tocopherols. It occurs in all plant

tissues, usually being higher in photosynthetic cells and meristems. It reacts non

enzymatically with superoxide, hydrogen peroxide and singlet oxygen (Shao et al.,

2008).

High intake of vitamin E may slow down the development and progression

of atherosclerosis. Clinical trials also reported beneficial effects of vitamin E

supplementation in the secondary prevention of cardiovascular events. Reduced

glutathione acts as an antioxidant and is involved directly in the reduction of most

active oxygen radicals generated due to stress (Selvi et al., 2007).

Murraya koenigii L. Spreng, a member of the family Rutaceae is used

as a spice in India for its characteristic flavour and aroma (Ningappa et al., 2008).

A wide variety of phenolic compounds present in spices that are extensively used

as food adjuncts possess potent antioxidant, anti- inflammatory, antimutagenic

and cancer preventive activities (Srinivasan, 2014). It has been shown that Murraya

koenigii is a rich source of polyphenols, which inhibit the proteolytic activity of the

cancer cell proteasome, and cause cell death (Noolu et al., 2013). Andjelkovic et al.

(2008) have also indicated the presence of polyphenols namely para-coumaric

acid, ferulic acid, ortho-coumaric acid in Spinacia oleracea.

Introduction


2000

1700

1500

1550

1600

1650

1700

1750

1800

1850

1900

1950

2000

2050

M . koenigii S. oleracea

µmol

/ gFigure 9. Total Antioxidant Activity

Shabir et al. (2013) have suggested that the protective potential of the

Maytenus royleanus leaf extract may be attributed to the high concentration of

phenolics, flavonoids, tannins and terpenoids. The secondary metabolites such as

phenolics and flavonoids from plants have been reported to be potent free radical

scavengers. They are found in all parts of plants such as leaves, fruits, seeds, roots

and bark (Mathew and Abraham, 2006).

Enzymatic and nonenzymatic antioxidants of Murraya koenigii and Spinacia

oleracea revealed that both plants are the rich source of antioxidants which can

play an important role in scavenging the free radicals generated and the tissue

injury caused during thrombus formation.

Total Antioxidant Activity of Murraya koenigii and Spinacia oleracea

Total antioxidant potential was

determined in the selected plants and

indicated in Figure 9.

Murraya koenigii recorded significantly

greater antioxidant potential than Spinacia

oleracea. This finding has been supported

by Bhandari (2012) who reported that

amongst some green leafy vegetables the

total antioxidant activity was the highest in

Murraya koenigii (2691µmol of ascorbic

acid/g sample) as compared to that of

methanol extracts of Amaranthus sp.,

Centella asiatica and Trigonella foenum- graecum. The results are also in good

agreement with those of Gomes et al. (2013) who reported that plant extracts

present a positive relationship between total phenol content, flavonoid content and

antioxidant capacity, with higher phenol and flavonoid levels reflecting greater

antioxidant capacity. Rao et al. (2010) also reported that total antioxidant activity

showed a significant correlation with polyphenolic contents suggesting the

importance of polyphenolics as potential antioxidant biomolecules.

Introduction


Bhatia and Jain (2004) have indicated Spinacia as a promising rich source

of antioxidants because its use is cost effective, especially for people in adverse

and hazardous circumstances who are living in poverty. Aqueous extracts of ten

different vegetables showed very potent antioxidant capacity (Raghu et al., 2011).

Adedapo et al. (2008) also observed that the leaves and stem extracts of

Calpurnia aurea possess antioxidant properties and could serve as free radical

inhibitors or presence of chemical constituents including carbohydrates, alkaloids,

saponins, glycosides and flavonoids scavenger or, acting possibly, as primary

antioxidants.

4.3.2 Biosafety Screening

Brine shrimp lethality assay was carried out using brine shrimp larvae

(Artemia salina) to test the cytotoxicity of the plant extracts (Murraya koenigii

and Spinacia oleracea). Percentage lethality of brine shrimp was determined

after 24 hours of exposure to the plant extracts. Potassium dichromate was used

as a positive control. The findings are represented in Tables 7,8 and Figures 10

and 11.

Table 7Brine Shrimp Mortality on Exposure to Aqueous Extract of Murraya koenigii

S.No. Concentrationmg/ml

%Mortality

Corrected%

Mortality

Log 10Concentration

ProbitValue

ConcentrationK2Cr2O7(µg/ml)

%Mortality

1. Control 5 - - - 100 40

2. 5 45 42.11 0.699 4.80 200 45

3. 10 50 47.37 1.000 4.92 300 50

4. 15 65 63.16 1.176 5.33 400 60

5. 20 70 68.42 1.301 5.47 500 75

LC 50 - 11.25mg

The percentage mortality increased with increase in concentration of the

plant extracts. The maximum percentage mortality (68.4% and 47.4%) was

obtained at a concentration of 20mg/ml for Murraya koenigii and Spinacia oleracea

respectively and thus the degree of lethality was found to be directly proportional

Introduction


0

10

20

30

40

50

60

70

80

Control 5 10 15 20


% M

orta

lity

to the concentration of the extracts. The concentration-mortality relationship of plant

product is usually expressed as a median lethal concentration (LC 50).

Table 8Brine Shrimp Mortality on Exposure to Aqueous Extract of Spinacia oleracea

S.No. Concentrationmg/ml

%Mortality

Corrected%

MortalityLog10

ConcentrationProbitvalue

ConcentrationK2Cr2O7(µg/ml)

%Mortality

1. Control 5 - - - 100 40

2. 5 10 5.26 0.699 3.36 200 45

3. 10 20 15.79 1.000 3.96 300 50

4. 15 30 26.32 1.176 4.36 400 60

5. 20 50 47.37 1.301 4.92 500 75

LC 50 - 21.00mg

Figure 10

Brine Shrimp Mortality on Exposure to Murraya koenigii

Ramachandran et al. (2011) have shown aqueous and alcoholic

extracts of Agava cantula leaves exhibited potent brine shrimp lethality (LC50 as

15 and 25 mg respectively). Ved et al. (2010) denoted that majority of the extracts

tested showed good brine shrimp larvicidal activity. According to Deciga-campos

et al. (2007), criterion of toxicity for fractions is; LC50 values>1000 µg/ml (non toxic);

≥ 500 ≤1000 µg/ml (weak toxicity)and <500 µg/ml (toxic). These findings suggest

Introduction


that the plant extracts of Spinacia oleracea and Murraya koenigii are non toxic with

an LC50 values of 21mg/ml and 11.25 mg/ml respectively.

Figure 11Brine Shrimp Mortality on Exposure to Spinacia oleracea

4.3.3 Spectral analysis of selected plants

The IR spectrum of the aqueous extracts of selected plants was recorded in

a shimadzu FT-IR spectrophotometer using KBr pellet method. The IR spectrum

obtained is shown in figures 12 and 13.

Figure 12

FT-IR Spectrum of Murraya koenigii

05

101520253035404550

Control 5 10 15 20


% M

orta

lity

Wave numbers (cm-1)

% T

rans

mitt

ance

Introduction









Figure 12


05

101520253035404550

Control 5 10 15 20


% M

orta

lity

Wave numbers (cm-1)

% T

rans

mitt

ance

Introduction









Figure 12


Wave numbers (cm-1)

% T

rans

mitt

ance

Introduction


The IR spectrum of Murraya koenigii showed charecteristic peaks

at 3379.29 cm-1, 2137.13cm-1 , 1643.35cm-1, 1550.77cm-1 , 1373.32cm-1 and

1219.01 cm-1. The peak at 3379.29 cm-1 may be due to the presence of NH and

OH (hydroxyl) functional groups. The peak at 2137.13cm-1 may be due to the

presence of C ≡ C terminal alkyne. The peak at 1643.35 cm-1 showed the presence

of –C =O (carbonyl group). The peaks at 1550.77 cm-1 and 1373.32 cm-1 showed

the presence of OH bend. The peak at 1219.01 cm-1 indicated the presence of

CN stretch. The presence of the above functional groups indicated the presence of

polyphenolics and alkaloids in Murraya koenigii.

Figure 13

FT-IR Spectrum of Spinacia oleracea

The IR spectrum of Spinacia oleracea showed charecteristic peaks at

3379.29 cm-1, 1643.35 cm-1, 1550.77cm-1, 1381.03 cm-1 and 1219.01 cm-1. The

peak at 3379.29 cm-1 may be due to the presence of NH and OH functional groups.

The peak at 1643.35 cm-1 showed the presence of carbonyl group. The peak at

1550.77 cm-1 indicated the presence of NH bend and 1381.03 cm-1 showed the

presence of OH bend. The peak at 1219.01 cm-1 indicated the presence of

Wave numbers (cm-1)

% T

rans

mitt

ance

Introduction











Figure 13








Wave numbers (cm-1)

% T

rans

mitt

ance

Introduction











Figure 13








Wave numbers (cm-1)

% T

rans

mitt

ance

Introduction


CN stretch. The presence of above functional groups indicated the presence of

polyphenolics and alkaloids in Spinacia oleracea.

Similar results were observed by Dineshkumar et al. (2010) who reported

the presence of mahanimbine, a carbazole alkaloid from Murraya koenigii, which

showed IR peaks at 3440 (N-H), 2920,1642 (C=C), 1456, 1378, 1312 (C-N), 1211

(C-O),1164 and 741 cm-1.

Tachibana et al. (2003) suggested that an aryl hydroxyl substituent on the

carbazole rings plays a role in stabilizing the thermal oxidation and rate of reaction

against DPPH radical. Sukari et al. (2001) reported the absorption of NH group at

3420 cm-1, C-O stretching at 1232 and 1137 cm-1 for the carbazole alkaloid from the

roots of Murraya koenigii.

GC-MS Spectrum of selected plants

Spectral study of Murraya koenigii

The GC – MS Spectrum of Murraya koenigii leaves showed six major peaks

at retention times (RT) 7.20, 9.35, 11.78, 14.71, 18.91 and 20.05 in GC. The

GC–MS spectrum and peak fragmentation are shown in figure 14.

Figure 14

MS spectrum of Murraya koenigii

Introduction


Retention Time (7.20)- Murraya koenigii

The MS spectrum of GC peak at retention time 7.20 showed M+ ion at m/z

341.1 and base peak was observed at m/z 59. The other significant m/z peaks

were observed at m/z 73.1, 147.1, 207.1, 251 and 325. One (M-27) peak was

observed at m/z 207.1, indicating the presence of nitrogen. One (M-28) peak was

also observed at m/z 147.1 indicating the presence of carbonyl group. This

spectrum also showed the presence of one (M-44) peak at m/z 251 indicates the

presence of carboxyl group.


RT 7.20

Introduction


The MS spectrum of the GC peak at retention time 9.35 showed M+ ion at

m/z 342 and base peak was observed at m/z 57. The other significant m/z peaks

were at 60, 84.1, 147.1, 221.1, 281.1 and 327. This spectrum showed three (M-17)

peaks at m/z 268, 298 and 400 and two (M-18) peaks at m/z 118 and 147.1

indicating the presence of polyphenolic compounds in the extract. Two (M-27)

peaks were also observed at m/z 369 and 84.1 indicating the presence of nitrogen.

Two (M-44) peaks were observed at m/z 342 and 191 indicating the presence of

carboxyl group in the compounds.


The spectrum of GC peak at retention time 11.78 showed M+ ion at

m/z 401.1 and base peak was observed at m/z 59. The other significant m/z

peaks were observed at 73.1, 147.1, 221.1, 281.1 and 355.1. This spectrum

showed one (M-17) peak at m/z 340 and one (M-18) peak at m/z 401.1, which

indicated the presence of phenolic compound in the extract. Two (M-28) peaks

were observed at m/z 340 and 383, which indicated the presence of carbonyl

group. Three (M-44) peaks were observed at m/z 191, 252 and 312 and one (M-45)

peak was also observed at m/z 118, indicating the presence of carboxyl group in

the compounds.

Introduction





were at 68.1, 82, 95.1, 110, 123.2, 208.3, 278.3 and 340.9. This spectrum showed

three (M-27) peaks at m/z 95.1, 137 and 180 indicating the presence of nitrogen.

Five (M-28) peaks were observed at m/z 110, 123.2, 165, 193 and 208.3 which

indicated the presence of carbonyl group in the compounds.


Introduction


The MS Spectrum of GC peak at retention time 20.05 showed M+ ion at m/z

355.1 and the base peak was observed at m/z 60. The other significant m/z peaks

were at 73.1, 102.1, 147.1, 221.1 and 281.1. This spectrum showed two (M-17)

peaks at m/z 102.1 and 238 and one (M-18) peak at m/z 208, which indicated the

presence of phenolic compound in the extract. One (M-27) peak was observed at

m/z 295, indicating the presence of nitrogen. One (M-28) peak was also observed

at m/z 268, confirming the presence of carbonyl group in the compound. Two

(M-44) peaks were observed at m/z 325 and 369. Two (M-45) peaks were also

observed at m/z 147.1 and 340, which confirmed the presence of carboxyl group

in the compounds.

Spectral study of Spinacia oleracea

The GC-MS spectrum of Spinacia oleracea leaves showed five peaks at

retention times 9.34, 11.78, 18.91, 22.88 and 25.41 in GC. The GC-MS spectrum

and peak fragmentation are shown in figure 15.

Figure 15

MS spectrum of Spinacia oleracea

Introduction


Retention time (9.34)- Spinacia oleracea


400 and base peak was observed at m/z 59. The other significant m/z peaks were

observed at 73.1, 147.1, 221.1, 281.1, 327 and 342. This spectrum showed three

(M-17) peaks at m/z 252, 298 and 400 and one (M-18) peak at m/z 103 which

indicated the presence of polyphenolic compounds in the extract. One (M-28) peak

was observed at m/z 249, confirming the presence of carbonyl group. Three (M-44)

peaks at m/z 103, 265 and 342 and two (M-45) peaks were also observed at m/z

118 and 192, indicating the presence of carbonyl group in the compounds.


Introduction




observed at 73.1, 90.1, 117.1, 147.1, 221.2, 281.1, 327 and 355.1. This spectrum

showed four (M-17) peaks at m/z 190.1, 267, 312 and 355.1 and two (M-18) peaks

at m/z 178 and 268, indicating the presence of polyphenolic compound in the

extract. Five (M-27) peaks were observed at m/z 117.1, 125, 152, 160 and 295

confirming the presence of nitrogen. Six (M-28) peaks were observed at m/z 125,

188, 221.2, 295, 340 and 355.1, which confirmed the presence of carbonyl group in

the compound. Four (M-44) peaks were observed at m/z 117.1, 183, 312 and 325

and four (M-45) peaks were observed at m/z 178, 238, 295 and 340 indicating the

presence of carbonyl group in the compounds.




were observed at 68.1, 77, 80, 92, 109, 123.2, 138, 152, 179.2, 278.3, 315.1 and

355.3. This spectrum showed one (M-17) peak at m/z 109 indicating the presence

of phenolic compound in the extract. Two (M-27) peaks were observed at m/z 223

and one (M-44) peak was observed at m/z 179.2 indicating the presence of

nitrogen. One (M-44) peak was observed at m/z 223 and one (M-45) was observed

at m/z 209, all confirming the presence of carboxyl group in the compounds.

Introduction





observed at 58, 73.1, 97.1, 129.1, 157.2, 213.2, 256.3 and 284.1. This spectrum

showed two (M-17) peaks at m/z 73.1 and 75 and one (M-18) peak at m/z 75indicating the presence of phenolic compound in the extract. Two (M-27) peaks

were observed at m/z 102 and 129.1 which indicated the presence of nitrogen. Five

(M-28) peaks were observed at m/z 97.1, 157.2, 171, 227 and 284.1 indicating the

presence of carbonyl group. Two (M-44) peaks were observed at m/z 102 and 227and one (M-45) peak was observed at m/z 102 indicating the presence of carboxyl

group in the compounds.


Introduction


The MS spectrum of the GC peak at retention time 25.41 showed M+ ion at

m/z 405.3 and base peak was observed at m/z 55. The other significant m/z peaks

were observed at 71.2, 83, 92, 123.2, 196.3, 235.6, 279.3 and 313.1. This

spectrum showed one (M-18) peak at m/z 141 indicating the presence of phenolic

compound in the extract. Two (M-28) peaks were observed at m/z 83 and 177

indicating the presence of carbonyl group in the compound. One (M-44) peak at

m/z 279.3 and one (M-45) peak at m/z 257 were observed indicating the presence

of carboxyl group in the compounds.

Holzer et al. (2013) reported that the antioxidant activity of Cotoneaster

melanocarpus Lodd was based on the presence of polyphenolic compounds such

as flavonoids as well as various plant acids. Bunea et al. (2008) reported that the

LC-MS analysis of Spinacia oleracea showed the presence of three phenolic acids,

namely ortho-coumaric acid, ferulic acid and para coumaric acid. Bergman et al.

(2001) reported the presence of both flavonoids and p- coumaric acid derivatives

as antioxidant components of the aqueous extract of Spinach leaves.

The results of FT-IR and GC-MS confirms the presence of various

phytochemicals Identified by qualitative analysis of Murraya koenigii and Spinacia

oleracea.

4.4 PHASE IV

This phase was designed to explore the effect of selected plant extracts on

ferric chloride (FeCl3) - induced thrombus in experimental rats. An acute toxicity

study was carried out to find out the effective dose of plant extracts to be

administered. Appearance of thrombus in the tail region of experimental animals is

shown in Plate 3.

After the experimental period haematological parameters such as WBC

count, RBC count, platelet count, haemoglobin, bleeding time and activated partial

thromboplastin time were analysed in the control and experimental animals.

Biochemical parameters namely fibrinogen, D-dimer and creatine phosphokinase

were analysed in the serum sample of experimental animals. Total protein, SOD

and catalase were assessed in the liver sample of experimental animals.

Introduction


Histopathological examination was carried out to study the effect of plant extracts

on heart, liver and kidney.

Plate 3

Appearance of Thrombus in the Tail of Experimental Animals

Group I (Control) Group II (FeCl3)

Group III (FeCl3 + Streptokinase) Group IV (FeCl3 + Murraya koenigii)

Group V (FeCl3 + Spinacia oleracea)

)

Group VI (FeCl3 + Murraya koenigii +Spinacia oleracea)

)

Introduction




Plate 3





)


)

Introduction




Plate 3





)


)

Introduction


Figure 16RBC Count in Experimental Animals

0

1

2

3

4

5

6

7

8

9

10

G1 G2 G3 G4 G5 G6

Treatments

RB

C c

ount

(x 1

06/µ

l)

Haematological ParametersRBC, WBC, Platelet count and Haemoglobin Level in Experimental Animals

WBC, RBC, Haemoglobin level and Platelet count and in experimental rats

are depicted in Table 9 and Figures 16, 17, 18 and 19.

Table 9WBC, RBC, Platelet Count and Haemoglobin Level

in Experimental Animals

Column means followed by common superscript are not significant at 5% by DMRT

The control groups showed

7.73×106 cells/µl of RBC. When a

thrombus was induced, RBC

count increase to 9.06×106

cells/µl, which is similar to the

findings of Cadroy and Stephen

(1990) who have described that

thrombus formation increasds

when RBC count increased. On

the treatment of the thrombus

with streptokinase, the levels

decreased but were well within the control range, featuring the initiation of lysis.

Groups TreatmentsRBC count

X 106/µl

WBC countx 10

3/µl

Platelet countX 10

3/µl

Haemoglobing/dl

I Control 7.73 ± 0.95b

12.30 ± 1.05b

484.00 ± 0.57d

15.00 ± 0.35b

II FeCl3 9.06 ± 0.74

a14.00 ± 0.98

a450.00 ± 1.72

e16.30 ± 0.56

a

III FeCl3+ Streptokinase 8.31 ± 0.67

a10.89 ± 0.86

c707.00 ± 9.39

a12.40 ± 0.48

e

IV FeCl3+M. koenigii 8.62 ± 0.57

a12.00 ± 0.56

b649.00 ± 9.12

b14.50 ± 0.51

bc

V FeCl3+ S. oleracea 8.46 ± 0.64

a11.80 ± 0.53

bc660.00 ± 0.45

b14.10 ± 0.50

c

VIFeCl

3+M. koenigii and

S. oleracea 8.10 ± 0.55a

11.30 ± 0.59bc

610.00 ± 9.58c

13.40 ± 0.49d

Introduction


0

2

4

6

8

10

12

14

16

G1 G2 G3 G4 G5 G6

Treatments

Tota

l WB

C c

ount

(x 1

03/µ

l)

Figure 17WBC Count in Experimental Animals

Figure 18Haemoglobin Level in Experimental Animals

0

2

4

6

8

10

12

14

16

18

G1 G2 G3 G4 G5 G6

Treatments

Hae

mog

lobi

n (g

/dl)

A similar trend was followed

by the plant extracts when

administered individually. When the

extracts were given in

combination, their levels were

further decreased but not very

significantly. Both the WBC and

haemoglobin level increased to

14×103 cells/µl and 16.3 g/dl when

thrombus was formed and

subsequently on thrombolysis with streptokinase, the levels decreased to

10.89×103 cells/µl and 12.40 g/dl respectively.

Haemoglobin level and

WBC counts were lower in the

animals treated with plant

extracts than control groups

(15g/dl and 12.3×103 cells/µl)

but lay within the normal r

ange. The results are similar to

the findings of Zhou et al.

(2011), who have suggested

that excessive extracellular

haemoglobin may also

contribute to platelet activation and thrombosis. Reiter et al. (2003) have also

reported that thrombus formation significantly increased WBC levels. Barron et al.

(2000) have pointed out that patients treated with aspirin had a significantly lower

WBC levels

The platelet count in the control group was 484×103 cells/µl, which

decreased to 450×103 cells/µl, when a thrombus was formed. This is in agreement

with Monreal et al. (1991) who have described that patients with thromboembolism

Introduction


0

100

200

300

400

500

600

700

800

G1 G2 G3 G4 G5 G6

Treatments

Plat

elet

cou

nt (x

10

3 /µl)

Figure 19Platelet Count in Experimental Animals

may have lower platelet count.

The number of cells increased

to 707×103 cells/µl, when treated

with streptokinase, posing a

possible reocclusion that

remains a drawback for

thrombolytic therapies as

described by Montrucchio et al.

(1993). In contrast, when the

plant extracts were

administrated, platelets count

decreased to 649×103 cells /µl for Murraya koenigii and 660×103 cells /µl for

Spinacia oleracea. The plant extracts when g iven in combination, decreased the

platelet count more than when given individually. This effect could possibly

complement the negative effect of streptokinase in thrombolytic therapies.

Bleeding and Activated Partial Thromboplastin Time in Experimental Animals

Figures 20 and 21 represent the bleeding and activated partial

thromboplastin time in experimental rats

Figure 20

Bleeding Time in Experimental Animals

605

1205

608

1095

320

345

0

200

400

600

800

1000

1200

1400

Group I Group II Group III Group IV Group v Group VI

Ble

edin

g tim

e (s

)

a

Introduction


Figure 21

Activated Partial Thromboplastin Time in Experimental Animals

Bleeding time and Activated Partial ThromboplastinTime (APTT), the key

influential factors of thrombolysis were noted and depicted in figures. Both

the values were reduced on initiation of a clot. Lysis of the clot led to an increase in

bleeding time and APTT. A similar trend was seen in plant extracts also, with

Spinacia oleracea recording the least value of 608 s for bleeding time and 51 s for

APTT, which was comparable with the values of streptokinase. Though the

bleeding time and activated partial thromboplastin time have increased after

treatment, they lie within the normal range depicting that there is a minimal chance

of bleeding, a major complication in thrombolysis treatment.

Harrison (2005) reported that normal bleeding time is usually between 2

and 10 minutes, whereas severe platelet defects can result in bleeding time more

than 30 minutes. Prezoto et al. (2002) evaluated the possible antithrombotic and

thrombolytic activities of Lonomia obliqua caterpillar bristle extract (LOCB) in the

experimental animals and reported that siginificant increase in bleeding time seen

in LOCBE treated rats might be induced by the antithrombotic components or by

other substances present in the crude extract.

53

51

60

5450

36

0

10

20

30

40

50

60

70

Group I Group II Group III Group IV Group v Group VI

Act

ivat

ed P

artia

l Thr

ombo

plas

tin ti

me

(s)

Introduction


0

100

200

300

400

500

600

700

800

G1 G2 G3 G4 G5 G6

Treatments

Fibr

inog

en (m

g/dl

)

Figure 22Fibrinogen Level in Experimental Animals

Alterations observed in the haematological parameters due to thrombus

formation were reverted back to near normal values by administration of

streptokinase and plant extracts suggesting clot lysis activity of Murraya koenigii


Biochemical ParametersFibrinogen, D-dimer and Creatine phosphokinase Levels in ExperimentalAnimals

Table 10 and figures 22, 23 and 24 represent the fibrinogen, D-dimer and

Creatine phosphokinase in experimental rats.

Table 10

Fibrinogen, D-dimer and Creatine phosphokinase Levels in Experimental Animals

Groups Treatments Fibrinogenmg/dl

D-dimerng/ml

Creatinephosphokinase

U/LG1 Control 700.0± 6.78a 349.0±2.67f 129.00±1.94d

G2 FeCl3 650.0±6.18b 372±4.56e 84.00±1.45e

G3 FeCl3+ Streptokinase 425.0±5.39e 629.00±4.82a 164.00±1.23a

G4 FeCl3+M. Koenigii 569.0±4.89c 549.00±4.13c 146.00±1.55b

G5 FeCl3+ Spinaciaoleracea 465.0±6.15c 512.00±3.98d 146.00±2.05b

G6 FeCl3+M. Koenigii andS. oleracea 529.0±5.71d 592.004.52b 141.00±1.79c

CD (p<0.05) 8.237 6.452 2.489


The concentrations of fibri

nogen and D-dimer are inversely

proportional to each other, i.e.,

when a thrombus is formed,

fibrinogen is converted to fibrin,

which is then degraded to D-dimer

on thrombolysis. The same pattern

was observed in the present study,

where the levels of fibrinogen

Introduction


Figure 23D-dimer Level in Experimental Animals

0

100

200

300

400

500

600

700

G1 G2 G3 G4 G5 G6

Treatments

D-d

imer

(ng/

ml)

0

20

40

60

80

100

120

140

160

180

G1 G2 G3 G4 G5 G6

Treatments

Cre

atin

e ph

osph

okin

ase

(U/L

)

Figure 24Creatine phospokinase in Experimental Animals

decreased on the incidence of clot, and on clot lysis, the level of fibrinogen

decreased further, while the D-dimer concentration increased.

The D-dimer concentration

did not differ significantly when

compared to control and FeCl3induced group. But when

streptokinase was administered,

the D-dimer level increased

to 629 ng/ml confirming clot lysis.

Similarly, when the plant extracts

were administrated, there was an

increase in the D-dimer level (549

ng/ml for Murraya koenigii and 512

ng/ml for Spinacia oleracea). D-dimer concentration was higher when the plant

extract was given in combination (592ng/ml). These results collectively suggest that

the plant extract has clot lysing ability which is comparable to that of streptokinase.

Creatine phosphokinase

followed a similar pattern like

fibrinogen, where the level is

reduced when a thrombus is

formed, from 129 U/L in control

to 84 U/L in ferric chloride

induced grou p. Subsequently,

when the clot is lysed either with

streptokinase or with plant

extracts, the activity of creatine

phosphokinase increased

significantly. Streptokinase recorded the highest level (164 U/L) of creatine

phosphokinase. Both the plant extracts showed 146 U/L each. When they were

given in combination, the level was 141 U/L.

Introduction


Ratnasooriya (2008) reported that tea impairs the coagulation of blood of

animals and in man, both in vitro and in vivo and is claimed to be cardioprotective,

lowers fibrinogen and impairs platelet aggregation. Kadam et al. (2011) reported

that a crude aqueous leaf extract of Murraya koenigii showed a dose dependent

negative chronotropic effect on cardiovascular system of frog, which might be due

to its direct action on heart and blood vessels. They have also indicated that

Murraya koenigii has vasodilatory effect.

Catalase, Superoxide dismutase and Total Protein

Table 11 and figures 25, 26 and 27 represent the level of catalase,

superoxide dismutase (SOD) and total protein in experimental rats.

Table 11

Activity of Catalase, SOD and Total Protein Content in theLiver of Experimental Animals

Groups treatments Catalase(U/g)

Superoxidedismutase

(U/g)Total Protein

(U/g)

G1 Control 0.493 ± 0.005b 0.133 ± 0.004d 1.39 ± 0.17b

G2 FeCl3 0.276 ± 0.004f 0.066 ± 0.004e 0.62 ± 0.72c

G3 FeCl3+ Streptokinase 0.413 ± 0.003e 0.127 ± 0.005c 0.78 ± 0.71c

G4 FeCl3+M. Koenigii 0.424 ± 0.004d 0.124 ± 0.004c 1.51 ± 0.21b

G5 FeCl3+ Spinaciaoleracea 0.483 ± 0.003c 0.191 ± 0.007b 1.53 ± 0.13b

G6 FeCl3+M. Koenigiiand S. oleracea 0.528 ± 0.003a 0.213 ± 0.006a 2.11 ± 0.01a

CD (p<0.05) 0.0062 0.0091 0.578


Catalase : Amount of enzyme required to decrease the optical density by 0.05 units

SOD : The amount that causes 50% reduction in the extent of NBT oxidation

Introduction


Figure 25Catalase Activity in the Liver of Experimental Animals

Figure 26SOD Activity in the Liver of Experimental Animals

It can be seen from the values in the table, that the administration of FeCl3 to

the experimental animals decreased the activity of catalase, superoxide dismutase

and the concentration of total protein. After thrombus formation, when the animals

were treated with the standard drug streptokinase and the plant extracts, the

enzymatic activity and the protein concentration were found to be enhanced.

Among the three groups which received the plant extracts individually and

0

0.1

0.2

0.3

0.4

0.5

0.6

G1 G2 G3 G4 G5 G6

U /

g

Treatments

0

0.05

0.1

0.15

0.2

0.25

G1 G2 G3 G4 G5 G6

U /

g

Treatments

Introduction


in combination group VI (Murraya koenigii and Spinacia oleracea) showed

significantly higher activity of catalase and superoxide dismutase than the groups

which received either Murraya koenigii or Spinacia oleracea. In the case of protein,

though the value was found to be increased in the group that received the

combined plant extracts, the difference was not statistically significant. Comparison

of the levels of enzymes and protein in groups IV and V revealed that the animals

that received the extract of Spinacia oleracea recorded higher values than the other

group treated with Murraya koenigii.

Figure 27

Total Protein Content in the Liver of Experimental Animals

Studies conducted by Mitra et al. (2012) indicated that the aqueous extracts

of Murraya koenigii leaf conferred significant protection to rat cardiac tissue against

cadmium-induced oxidative stress, probably due to its antioxidant activity. Joseph

and Peter (2008) reported that the activities of SOD, catalase and glutathione

transferase were increased in the heart and liver of rats supplemented with curry

leaves. According to Xia et al. (2013) Smilax glabra Roxb significantly increased

the activities of catalase, superoxide dismutase and glutathione peroxidase in CCl4induced intoxicated liver.

0

0.5

1

1.5

2

2.5

3

G1 G2 G3 G4 G5 G6

U /

g

Treatments

Introduction


Bergman et al., (2001) elucidated the presence of powerful, natural

antioxidants (NAO) in the water extracts of Spinach leaves and demonstrated their

biological activity in both in vitro and in vivo systems. Gomati et al., (2012) also

supported that some of the antioxidant enzymes and non-enzymatic molecules

widely distributed in the biological system are capable of scavenging free radicals.

Rao et al. (2011) reported that the consumption of strawberries, spinach, red wine

or vitamin C increased the antioxidant capacity of serum in elderly women. Shetty

et al. (2007) reported that rats treated with the aqueous extracts of Ocimum

sanctum showed significant increase in the activity of SOD, catalase and GST

compared to control. These enzymes are known to quench the superoxide radical

and thus prevent the damage of cells caused by free radicals.

Histopathological Examination of Experimental Animals

Histopathological examination of sections of liver, heart and kidney of control

and experimental rats of the various groups were carried out to test thrombolytic

effect of the aqueous extract of Murraya koenigii and Spinacia oleracea.

The cellular changes in liver, heart and kidney are indicated in plates 4, 5 and 6

respectively and the findings of histopathological examination are discussed below.

The histology of the liver sections of control animals showed intact

hepatocytes and there was no inflammation, fibrosis and toxic change. Liver

sections of FeCl3 treated animals revealed mild lobular inflammation. The sinusoids

showed dilatation and central vein showed congestion. Portal traiditis was noticed

in the portal tract. In the case of animals treated with streptokinase showed normal

hepatocytes without any inflammation. Liver section of animals treated with

Murraya koenigii, Spinacia olereacea and combined extracts of Murraya koenigii

and Spinacia oleracea revealed moderate periportal inflammation.

Heart section of control animals showed normal myocardium and mild

congestion was observed in blood vessels. There was no inflammation, edema or

necrosis. Heart sections of FeCl3 treated animals revealed mild scattered

lymphocytic infiltrates in the myocardium. Myocytes showed mild degeneration and

areas of congested vessels and haemorrhage were also observed.

Introduction


Plate 4

Histophathological observations of Liver of Control and Experimental Animals

Control FeCl3 Induced

M.koenigii treated Streptokinase treated

S.oleracea treated Combined extract treated

Introduction


Plate 5

Histophathological observations of Heart of Control andExperimental Animals




Introduction


Plate 5





Introduction


Plate 5





Introduction


Plate 6

Histophathological observations of Kidney of Control andExperimental Animals




Introduction


In the case of streptokinase treated animal showed normal cardiac muscle

and the blood vessels showed mild congestion. No inflammation, edema or

necrosis was observed. Heart sections of animals treated with individual and

combined administration showed mild congestion.

The histolopathological examination of the kidney sections of control animals

showed normal cortex, medulla and pelvicalyceal system. The blood vessels

showed mild congestion and thickening. There was no inflammation or tubular

necrosis. Kidney sections of FeCl3 treated animals revealed focal lymphocytic

infiltration. Tubules showed focal nuclear loss. The blood vessels showed

thickening and congestion. The kidney sections of streptokinase treated animals

showed normal cortex and medulla without any inflammation. The animals treated

with Murraya koenigii indicated normal tubules. Treatment with Spinacia oleracea

animals showed nuclear loss in the tubules. Kidney sections of animals treated with

combined extracts revealed normal glomerui and tubules.

Jadhav et al. (2013) have indicated that the altered histoarchitecture of heart

and kidney tissue due to doxorubicin treatment was improved with Luffa acutangula

Roxb. Potential cardioprotective effect of Saraca Indica supported by

histospathological examination was demonstrated by Swamy et al. (2013). They

have also reported that the cardioprotective effect of Saraca Indica could be

attributed to antioxidant activity. Histopathological examination revealed that CCl4induced hepatic damage was markedly reversed by Smilax glabra Roxb (Xia et al.,

2013).

The results of histopathological examination showed that the changes

observed in the histoarchitecture of heart, liver and kidney due to thrombus

formation were improved by administration of the standard drug and the plant

extracts.

Introduction


4.5 PHASE V

In silico Characterization by Docking Studies

In silico screening of the ligands and/or of the receptors has become an

essential tool to facilitate the drug discovery process. Alkaloids namely

mahanimbine, mahanine and murrayanol from Murraya koenigii were subjected to

in silico studies using Maestero Molecular Modeling Environment, Schrodinger

software version 9.4.

Selection of Target Protein

Plasminogen activator inhibitor 1 is a regulator of plasminogen activators.

There are two types of plasminogen activator (tPA) and urokinase-type

plasminogen activator (uPA). In the blood vessel, tPA binds to fibrin and converts

plasminogen into plasmin for proteolytic degradation of the clot. uPA is the major

plasminogen activator for migrating cells, and it is activated by binding with uPA

receptor (uPAR) and subsequently intiates a proteinase cascade. Plasmin is a

proteinase with a wide range of substrates. It is able to degrade fibrin and other

extracellular matrix (ECM) components, to cleave and activate other proteinases

such as matrix metalloproteinases, and to activate latent transforming growth factor

β1 (TGF- β1), basic fibroblast growth factor (bFGF) and vascular endothelial growth

factor (VEGF). Plasminogen Activator Inhibitor-1 (PAI-1) is a primary inhibitor to

inhibit the activation of both tPA and uPA (Lee and Huang, 2005).

The 3D structure of the plasminogen activator inhibitor 1 was obtained from

the Protein Data Bank (4AQH) and the structure was refined using the protein

preparation wizard module of Schrödinger package. The molecular docking was

performed using Glide 5.9 and ADME studies were performed using QikProp 3.6 to

characterise the active components.

ADME Studies

ADME (Absorption, Distribution, Metabolism and Excretion) profile of

mahanimbine (CID 167963), mahanine (CID 56674845) and murrayanol (CID

44258048) are represented in Table 12. All the pharmacological parameters

Introduction


calculated for selected ligands were within the permissible limit thereby proving

their drug likeliness.

Table 12ADME Properties of the Selected Ligands

Descriptors Standardvalues

Ligand valuesMahanimbine Mahanine Murrayanol

Molecular weight (Da) 130.0 – 725.0 331.457 333.47 404

Number of hydrogen bondacceptors

2.0 - 20.0 1.0 2.0 1.0

Number of hydrogen bonddonors

0.0 / 6.0 0.750 1.5 6.75

QP log P for octanol/ water -2.0/6.5 6.5 5.4 3.152

Apparent CaCOPermeability (nm/sec)

<25 poor, >500great 6692 2029 786

Apparent MDCKPermeability (nm/sec)

<25 poor, >500great 3861 1063 381

Lipinski Rule of 5 Violations (maximum is 4 ) 1 1 0

% Human Oral Absorptionin Gl (±20%) (<25% is poor) 100 100 100

Molecular docking using Glide

The ligands, namely mahanimbine, mahanine and murrayanol were docked

with plasminogen activator inhibitor 1 using Glide, in Standard Precision (SP)

mode. The glide score, glide energy, good contacts and hydrogen bonds

parameters were considered, which indicates the binding affinity of the ligands with

the target protein. The top ranked poses generated by Glide XP docking are

presented in Table 13.

It is evident from the table that mahanimbine, mahanine and murrayanol

showed a glide score of -6.07, -4.89 and -4.51 respectively with the target protein

PAI 1. Mahanimbine showed the maximum glide score compared to mahanine and

murrayanol.

Introduction


Table 13

Glide XP Docking of the Ligands with the Target ProteinPlasminogen Activator Inhibitor 1

S.No DescriptorsLigand values

Mahanimbine Mahanine Murrayanol

1. Glide score -6.07 -4.89 -4.51

2. Energy (kcal/mol) -27.13 -27.88 -38.87

3. Good contacts 436 379 379

4. Bad contacts 40 55 30

5. Ugly contacts 4 07 02

6. Pose number 159 279 320

7. Number of H- bonds 0 1 2

All the three ligands showed a high number of good contacts and very low

bad and ugly contacts. This indicates that they bind with plasminogen activator

inhibitor 1 firmly. Additionally, the nature of binding was analysed, it was found that

all the three ligands bind to the same active pocket as the inhibitor (tert-butyl 3-[(4-

oxo-3h-pyrido[2,3-d]pyrimidin- 2-yl)amino]azetidine-1-carboxylate) of PAI 1. It is

known that tert-butyl 3-[(4-oxo-3h-pyrido[2,3-d]pyrimidin- 2-yl)amino]azetidine-1-

carboxylate inhibits PAI 1 in such a way that clot lysis occurs without interference

(Lee and Huang, 2005). This implies that the three ligands used in the present

study will be able to mimic the action of tert-butyl 3-[(4-oxo-3h-pyrido[2,3-

d]pyrimidin- 2-yl)amino]azetidine-1-carboxylate, thereby enhancing clot lysis in vivo.

The docking efficiency and the molecular interactions showing good contacts of

mahanimbine, mahanine and murrayanol with the PAI 1 are represented in

figure 28 and plates 7, 8 and 9.

Mahanimbine (C23H25NO), mahanine (C23H25NO2) and murrayanol

(C24H29NO2) were chosen as ligands because of their abundance in the leaves of

Murraya koenigii. It can be suggested that all the three alkaloids have good docking

Introduction


scores and high binding affinity to the plasminogen activator inhibitor 1. The

bioavailability of the compound was supported by the ADME profile. The in silico

studies reveal the interaction of mahanimbine, mahanine and murrayanol with

PAI1 thereby preventing the action of PAI 1 and favouring the action of tPA.

In silico study further supports the thrombolytic effect of Murraya koenigii.

Figure 28

Ligand Interaction Diagram with Target Protein PAI 1

Ligand Interaction Diagram of PAI 1(4AQH ) with co-crystalized

ligand AZ3976

Ligand Interaction Diagram of PAI 1(4AQH ) with Mahanimbine

ligand 167963

Ligand Interaction Diagram of PAI 1(4AQH ) with Mahanine

ligand 56674845

Ligand Interaction Diagram of PAI 1(4AQH ) with Murrayanol

ligand 44258048

Introduction







Figure 28



ligand AZ3976


ligand 167963


ligand 56674845


ligand 44258048

Introduction







Figure 28



ligand AZ3976


ligand 167963


ligand 56674845


ligand 44258048

Introduction


Plate 7

Docking of PAI 1 with Mahanimbine

Molecular Interaction of PAI 1 with Mahanimbineshowing good contacts

Introduction


Plate 7



Introduction


Plate 7



Introduction


Plate 8

Docking of PAI 1 with Mahanine

Molecular Interaction of PAI 1 with Mahanine showing goodcontacts and H- bond

Introduction


Plate 8



Introduction


Plate 8



Introduction


Plate 9

Docking of PAI 1 with Murrayanol

Molecular Interaction of PAI 1 with Murrayanol showinggood contacts and H- bonds

Introduction


Plate 9



Introduction


Plate 9



Introduction


The findings of five different phases of the present study collectively suggest

that Murraya koenigii and Spinacia oleracea have clot lysing ability which is

comparable to that of streptokinase.

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