CHAPTER 5 5.0 RESULTS 5.1 PRELIMINARY …shodhganga.inflibnet.ac.in/bitstream/10603/32049/8/chapter...

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CHAPTER 5

5.0 RESULTS

5.1 PRELIMINARY PHYTOCHEMICAL INVESTIGATIONS

5.1.1 Percentage Yield

The quantitative estimation of percentage yield of various crude

extracts of Indigofera aspalathoides, Myristica andamanica, Adhatoda

vasica,Azadirachta indica, Saraca asoca and Aeglemarmeloswas calculated,

the results of the percentage yield is given in table 5.1

Table 5.1: Percentage yield of various extracts of six Indian medicinal

plants*

Extract Indigofera aspalathoides

Myristica andamanica

Adhatoda vasica

Azadirachta indica

Saraca asoca

Aegle marmelos

Methanol 19.8 % 19.6 % 18.4 % 19.6 % 18.5 % 18.2 %Ethanol 16.7 % 15.0 % 16.3 % 16.5 % 15.8 % 16.6 %Ethyl acetate

13.8 % 14.6 % 13.6 % 14.6 % 13.4 % 13.2 %

Chloroform 8.8 % 9.2 % 8.9 % 8.7 % 8.7 % 8.6 %Hexane 8.4 % 8.3 % 8.1 % 8.3 % 8.0 % 8.2 %

*The table shows thepercentage yield of methanolic extract, ethanol extract,

ethyl acetate extract, chloroform extract and hexane extract of Indigofera

aspalathoides, Myristica andamanica, Adhatoda vasica,Azadirachta indica,

Saraca asoca and Aeglemarmelos.

Comparing the percentage yield of the plant extracts it was observed

that the methanolic extract gave the maximum amount of yield and the hexane

extract gave the minimum amount of yield for all the six medicinal plants.

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Among the methanolic extracts of all the six plants Indigofera aspalathoides

shows the highest yield percentage in methanolic extract. Whereas the lowest

percentage of yield was observed in hexane extract of Adhatoda vasica among

the six plants studied. When the percentage of yield, is higher the quantity of

soluble compounds will also be higher. It was observed from our study that the

methanolic extract shows the higher percentage of yield for all the six plants

hence the methanolic extract was used.

Table 5.2: Percentage yield of various extracts of Indigofera aspalathoides*

S.No: Extracts Nature of

Extracts

Colour Yield(%

w/w)

1 Methanol Semisolid Dark green 19.8 %

2 Ethanol Semisolid Dark green 16.7 %

3 Ethyl acetate Semisolid Dark green 13.8 %

4 Chloroform Semisolid Dark green 8.8 %

5 Hexane Semisolid Dark green 8.4 %

*The table shows thenature, color and percentage yield of methanolic extract,

ethanol extract, ethyl acetate extract, chloroform extract and hexane extract of

Indigofera aspalathoides.

Table 5.3: Percentage yield of various extracts of Myristicaandamanica*


Extracts

Colour Yield(%

w/w)






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Myristica andamanica.

Table 5.4: Percentage yield of various extracts of Adhatoda vasica*


Extracts

Colour Yield(%

w/w)








Adhatoda vasica.

Table 5.5: Percentage yield of various extracts of Azadirachta indica*

S.No: Extracts Nature of Extracts

Colour Yield(% w/w)








Azadirachta indica.

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Table 5.6: Percentage yield of various extracts of Saraca asoca*


Colour Yield(% w/w)








Saraca asoca.

Table 5.7: Percentage yield of various extracts of Aegle marmelos*


Colour Yield(% w/w)








Aeglemarmelos.

Table 5.2 to 5.7 gives the nature, color and percentage yield of the

crude extracts from different solvents. All the crude extracts were semi solid

in nature and dark green in color.

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5.2 PRELIMINARY PHYTOCHEMICAL ANALYSIS OF SIX INDIAN MEDICINAL PLANTS

5.2.1 Phytochemical Screening Methods

Methanol extract was evaluated for presence of various

phytoconstituents by performing different qualitative chemical tests reported

[280,184]. It showed the presence of anthraquinone glycosides, saponins,

tannins and phytosterols.

Table 5.8: Phytochemical Screening of methanolic extract of six Indian

medicinal plants*

Phytochemical Indigofera aspalathoides

Myristica andamanica

Adhatoda vasica

Azadirachta indica

Saraca asoca

Aegle marmelos

Amino acids + + + + + +Carbohydrates + + + + + +Terpenoids + - + + + +Tannins + - - - - -Alkaloids + + + + + +Steroids - + + + - +Flavonoids + - + + + +Saponins + - - - - -Glycosides + + + - + -Lipids + + + + + +Key:+ Present,-Absent

*The table shows the phytochemical compounds of methanolic extract of

Indigofera aspalathoides, Myristica andamanica, Adhatoda

vasica,Azadirachta indica, Saraca asoca and Aeglemarmelos.

From the Table-5.8 it is clear that carbohydrates, amino acids and lipids

are present in all the medicinal plant studied. Saponins and tannins are present

only in Indigoferaaspalathoides. Myristicaandamanicashows negative result

for terpenoids, while AzadirachtaindicaandAeglemarmelosshows the absence

of glycosides. Saracaasoca shows the absence of steroids.From this we

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conclude that the methanolic extract of Indigofera aspalathoides primarily

contains amino acids, carbohydrates, terpenoids, tannins, alkaloids,

flavonoids, saponins, glycosides and lipids.

Preliminary phytochemical analysis revealed thepresence of alkaloids

and saponins. The other secondary metabolites liketannins, flavonoids,

steroids, glycosides, etc. were also present in Indigoferaaspalathoides. Since

most of the phytochemicals were present in Indigofera aspalathoides the

plant extract ofIndigofera aspalathoideswas used for the characterization of

the bio active components by UV visible spectroscopy, FTIR, 13C NMR and 1H NMR studies .

5.3 ANTIOXIDANT ASSAYS OF SIX INDIAN MEDICINAL PLANTS:

5.3.1 2,2- Diphenyl 1-picra Hydrazyl (DPPH)ASSAY

After Screening for phytochemical analysis, the antioxidant assay was

determined by 2,2- Diphenyl 1-picra Hydrazyl (DPPH), the percentage of

antioxidant by DPPH activity is highest in Myristica andamanicaas shown in

Table-5.9 & Figure 5.1.

Table 5.9: Percentage of Antioxidant activity of six Indian medicinal

plants*

List of Medicinal Plants Percentage of Antioxidant activity

Myristica andamanica 95.2

Indigofera aspalathoides, 74.4

Azadirachta indica, 22.8

Saraca asoca 46.2

Aegle marmelos 18.6

Adhatoda vasica 13.8*The table shows the percentage of antioxidant activity of Indigofera



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Figure 5.1: Antioxidant activity (DPPH Assay)

Figure 5.1:The figure shows the antioxidant activity (DPPH Assay) of

Indigofera aspalathoides, Myristica andamanica, Adhatoda

vasica,Azadirachta indica, Saraca asoca and Aeglemarmelos.

5.3.2 Determination of Total Phenolic Content

The total phenolic content for methanolic extract of all the six

medicinal plants were determined, and the results are given in the Table

5.10and Figure 5.2. The phenolic content of six methanolic plant extract was

similar to that of antioxidant activity by DPPH.

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Table 5.10: Phenolic content of six Indian medicinal plants*

List of Medicinal Plants Phenolic Content (µg)Myristica andamanica 2.5

Indigofera aspalathoides 1.9Azadirachta indica 0.75

Saraca asoca 0.90Aegle marmelos 0.55Adhatoda vasica 0.40

*The table shows the phenolic content of Indigofera aspalathoides, Myristica

andamanica, Adhatoda vasica,Azadirachta indica, Saraca asoca and

Aeglemarmelos.

Figure 5.2: Determination of total Phenolic content

Figure 5.2: The figure shows the total Phenolic content of Indigofera



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5.4 ANTIMICROBIAL ACTIVITY

The antimicrobial activity of the methanolic extracts was tested against

the pathogens by well diffusion method and they the antibacterial activity of

the six medicinal plants were compared with the standards. The antibacterial

activity of the methanolic extract of Indigofera aspalathoideswas also

compared with the commercially available kuli thailam (Cocus nucifera). The

zone of inhibition of the antimicrobial activity for all the six plants is given in

Plate 5. 1- Plate – 5.9 and Table 5.11.

Plate 5. 1 : Antimicrobial Activity of Indigofera aspalathoides againstBacillus Subtilis

Plate 5.2 : Antimicrobial Activity of Indigofera aspalathoides against Streptococcus pneumonia

Plate 5. 3 : Antimicrobial Activity of Indigofera aspalathoides againstStaphylococcus aureus

Plate 5.4 : Antimicrobial Activity of Indigofera aspalathoides against Salmonella typhimurium

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Plate 5.5 : Antimicrobial Activity of Indigofera aspalathoides against Klebsiella aerogenes

Plate 5.6 : Antimicrobial Activity of Indigofera aspalathoides against

Escherichia coliPlate 5. 1 to Plate 5.6 : Antimicrobial activity of Indigofera aspalathoides

(Ia), Cocos nucifera (Cn), Standards (S). Cocos nucifera (Cn) is the

commercially available thailam which was compared with the Indigofera

aspalathoides (Ia). The plates show antimicrobial activity of Indigofera

aspalathoides against the growth of Bacillus subtilis, Streptococcus

pneumoniae, Staphylococcus aureus, Salmonella typhimurium, Klebsiella

aerogenes and Escherichia coli.

Plate 5.7: Antimicrobial activity of Plant Extract against Bacillus

Subtilis

Plate 5. 8: Antimicrobial activity of Plant extracts against Staphylococcus aureus

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Plate 5. 9: Antimicrobial activity of Plant extracts against Salmonella

typhimurium

Plate 5.7 to Plate 5. 9:1. Myristica andamanica, 2. Adhatoda vasica, 3.

Azadirachta indica, 4. Saraca asoca and 5. Aeglemarmelos. The plates show

antimicrobial activity of 1. Myristica andamanica, 2. Adhatoda vasica, 3.

Azadirachta indica, 4. Saraca asoca and 5. Aeglemarmelos against the growth

of Bacillus subtilis, Staphylococcus aureus and Salmonella typhimurium.

Table 5.11: Antimicrobial activity of six Indian medicinal plants*

Methanolic Extract Diameter of inhibition zone (in mm)Bacillus subtilis

Staphylococcus aureus

Salmonella typhimurium

Escherichiacoli

Myristica andamanica 11 10 8 21

Adhatoda vasica 7.5 7 11 8

Azadirachta indica 9.5 15 6 15

Saraca asoca 5.0 13 6.5 17

Aeglemarmelos 4.5 9 5 13

Indigofera

aspalathoides4.0 12.5 8.0 17

*The table shows the antimicrobial activity of Indigofera aspalathoides,

Myristica andamanica, Adhatoda vasica,Azadirachta indica, Saraca asoca

and Aeglemarmelos.

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Myristica andamanica showed the highest antimicrobial activity

against Bacillus subtilis and Escherichia coli. Adhatoda vasica was effective

against Salmonella typhimurium, andAzadirachta indica showed the highest

inhibitory zone against Staphylococcus aureus.

The order of antimicrobial activity against the Gram positive, catalase-

positive (rod) bacterium Bacillus subtilis is Myristica andamanica>

Azadirachta indica>Adhatoda vasica >Saraca asoca

>Aeglemarmelos>Indigofera aspalathoides.

The inhibitory activity of facultative anaerobic Gram-positive coccal

bacterium Staphylococcus aureus is in the order ofAzadirachta indica >Saraca

asoca > Indigofera aspalathoides >Myristica andamanica > Aeglemarmelos

>Adhatoda vasica.

The growth of the organism Salmonellae typhimurium a Gram-

negative, facultative anaerobic bacteria was inhibited by all the six plant

extract and the order of inhibition is as follows : Adhatoda vasica >Indigofera

aspalathoides = Myristica andamanica > Saraca asoca > Azadirachta

indica > Aeglemarmelos(plate 5.9 and table 5.11).

The order of antimicrobial activity against Escherichia coli a Gram-

negative, rod-shaped bacterium is Myristica andamanica > Saraca asoca =

Indigofera aspalathoides > Azadirachta indica > Aeglemarmelos > Adhatoda

vasica(Plate 5.6)

Based on the results of antimicrobial studies all the methanolic plant

extracts were tested for the wound healing activity in Swiss albino mice by

excision wound model.

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5.5 HPTLC RESULTS

The HPTLC reports of all the six Indian medicinal plants are given below for

the following extracts in the figures 5.3 - figure 5.32

1. Methanol

2. Ethanol

3. Ethyl Acetate

4. Chloroform

5. Hexane

Figure 5.3 – figure 5.7 shows the HPTLC reports of methanolic extract,


Indigofera aspalathoides. Figure 5.8 – figure 5.12 shows the HPTLC reports

of methanolic extract, ethanol extract, ethyl acetate extract, chloroform extract

and hexane extract of Myristica andamanica. Figure 5.13 – figure 5.17 shows

the HPTLC reports of methanolic extract, ethanol extract, ethyl acetate extract,

chloroform extract and hexane extract of Adhatoda vasica. Figure 5.18 –

figure 5.22 shows the HPTLC reports of methanolic extract, ethanol extract,

ethyl acetate extract, chloroform extract and hexane extract of Azadirachta

indica. Figure 5.23 – figure 5.27 shows the HPTLC reports of methanolic

extract, ethanol extract, ethyl acetate extract, chloroform extract and hexane

extract of Saraca asoca. Figure 5.28 – figure 5.32 shows the HPTLC reports

of methanolic extract, ethanol extract, ethyl acetate extract, chloroform extract

and hexane extract of Aeglemarmelos.

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5.5.1 HPTLC Results for Indigofera aspalathoides

Figure 5.3: HPTLC peaks formed for Methanolic extract of Indigofera aspalathoides

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Figure 5.4: HPTLC peaks formed for Ethanol extract of Indigofera aspalathoides

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Figure 5.5: HPTLC peaks formed for Ethyl acetate extract of Indigofera aspalathoides

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Figure 5.6: HPTLC peaks formed for Chloroform extract of Indigofera aspalathoides

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Figure 5.7: HPTLC peaks formed for Hexane extract of Indigofera aspalathoides

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5.5.2 HPTLC Results for Myristica andamanica

Figure 5.8: HPTLC peaks formed for Methanol extract of Myristicaandamanica

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Figure 5.9: HPTLC peaks formed for Ethanol extract of Myristicaandamanica

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Figure 5.10: HPTLC peaks formed for Ethyl acetate extract of Myristicaandamanica

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Figure 5.11: HPTLC peaks formed for Chloroform extract of Myristicaandamanica

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Figure 5.12: HPTLC peaks formed for Hexane extract of Myristicaandamanica

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5.5.3 Hptlc Results for Adhatoda Vasica

Figure 5.13: HPTLC peaks formed for Methanol extract of Adhatoda vasica

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Figure 5.14: HPTLC peaks formed for Ethanol extract of Adhatoda vasica

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Figure 5.15: HPTLC peaks formed for Ethyl acetate extract of Adhatoda vasica

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Figure 5.16: HPTLC peaks formed for Chloroform extract of Adhatoda vasica

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Figure 5.17: HPTLC peaks formed for Hexane extract of Adhatoda vasica

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5.5.4 HPTLC Results for Azadirachta indica

Figure 5.18: HPTLC peaks formed for Methanol extract of Azadirachta indica

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Figure 5.19: HPTLC peaks formed for Ethanol extract of Azadirachta indica

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Figure 5.20: HPTLC peaks formed for Ethyl acetate extract of Azadirachta indica

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Figure 5.21: HPTLC peaks formed for Chloroform extract of Azadirachta indica

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Figure 5.22: HPTLC peaks formed for Hexane extract of Azadirachta indica

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5.5.6 Hptlc Results for Saraca asoca

Figure 5.23: HPTLC peaks formed for Methanol extract of Saraca asoca

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Figure 5.24: HPTLC peaks formed for Ethanol extract of Saraca asoca

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Figure 5.25: HPTLC peaks formed for Ethyl acetate extract of Saraca asoca

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Figure 5.26: HPTLC peaks formed for Chloroform extract of Saraca asoca

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Figure 5.27: HPTLC peaks formed for Hexane extract of Saraca asoca

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5.5.7 HPTLC Results For Aegle marmelos

Figure 5.28: HPTLC peaks formed for Methanol extract of Aegle marmelos

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Figure 5.29: HPTLC peaks formed for Ethanol extract of Aegle marmelos

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Figure 5.30: HPTLC peaks formed for Ethyl acetate extract of Aegle marmelos

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Figure 5.31: HPTLC peaks formed for Chloroform extract of Aegle marmelos

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Figure 5.32: HPTLC peaks formed for Hexane extract of Aegle marmelos

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The above HPTLC figures 5.3 – 5.32 of all the six plants studied showed

that the polyphenolic compound peak is the major peak in the methanolic extract

while other solvents like ethanol, ethyl acetate, chloroform and hexane showed

the fat soluble and other non-polar soluble compounds.

Many different methods have been established and are being carried out to

isolate and extractantioxidant compounds from the plant material. When choosing

an extractionmethod, the major priority must be to preserve the bioactive

compounds and extractmost if not all the desired compounds from the plant

material.

Antioxidants can be physically classified by their solubility into two

groups(i) hydrophilic antioxidants, such as the majority of polyphenolic

compounds andvitamin C and (ii) lipophilic antioxidants, mainly carotenoids and

vitamin E.

Since the methanolic extract of the six selected Indian medicinal plants

contains the polyphenolic compounds necessary for the antioxidant activities,

wound healing activities and antigenotoxic activities the Methanolic extract of the

six plants were further characterized for its wound healing and antigenotoxic

activities.

This concept has been clearly explained from the HPTLC results of the six

selected Indian medicinal plants. So the methanolic extract containing the most

desired bio active compounds from the plant material was used for our further

pharmacological activity studies.

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5.5.8Reason for the Selection of the Methanol Extract for Gel Formulation

The methanolic extracts of Indigofera aspalathoides, Myristica

andamanica, Adhatoda vasica, Azadirachta indica, Saraca asoca and Aegle

marmelos were observed tocontain the maximum active ingredients as it is

evident from the figures 5.3, 5.8, 5.13, 5.18. 5.23. 5.28 of the HPTLC analysis.

Therefore, the bioactive compounds extracted by the methanolic extracts of

Indigofera aspalathoides, Myristica andamanica, Adhatoda vasica, Azadirachta

indica, Saraca asoca and Aegle marmelos were used in the treatment of wounds

on Swiss albino mice by excision wound models to verify the claim of traditional

medicine..

5.6 WOUND HEALING ACTIVITIES OF THE METHANOLIC EXTRACT

To study the effect of phytochemical activities of methanolic extracts from

the medicinal plants on wound healing was done bycreating wound on the dorsal

interscapular region of swiss albino mice by excising a predetermined area of

7mm x 7mm skin under ether anaesthesia (Suguna et al, 1996). Wounds were left

open and the medicine was applied topically twice a day (once in the morning and

once in the evening) to each mouse till the wound was completely healed for up

to twelve days. The progressive changes in wound area were monitored by a

camera every fourth day.

The wound area was measured and calculated on a daily basis . The

percentage of wound contractionwas monitored every day from the 0th day till

the 12th day. On the 12th day all the six mice under positivecontrol were healed,

whereas, in the mice under negativecontrol, the healing is delayed. The wound

healing activity is similar to the case of mice under treatment group(Figure 8).

The size of the wound was also measured using a scale daily and the wound area

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was calculated. Wound contraction was calculated as percentage of the reduction

in wound area (Table 5.12 & 5.13).

The preliminary phytochemical investigation of the leafextract showed the

presence of tannins, triterpenes andalkaloids. Any one of the observed

phytochemical constituents present in the medicinal plant extractmay be

responsible for the wound healing activities. Studies have shown

thatphytochemical constituents like flavonoids [300](Tsuchiya etal., 1996) and

triterpenoids [301](Scortichini and Pia, 1991)are known to promote the wound

healing process mainlydue to their astringent and antimicrobial properties

whichappear to be responsible for the wound healing andincreased rate of

epithelialisation [300](Tsuchiya et al., 1996).

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Table 5.12:Effect of methanolic extract of six medicinal plants on reduction in wound area*

*The table shows the effect of methanolic extract of Indigofera aspalathoides, Myristica andamanica, Adhatoda vasica,

Azadirachta indica, Saraca asoca and Aegle marmeloson reduction in wound area.

Table- 5.13: Effect of methanolic extract of six medicinal plants on Percentage of wound contraction*

Days

Treatment Group

Reduction of wound area in mm2

0 2 4 6 8 10 12

Positive Control 49±0.24 39.08±1.47 26.69±1.32 18.96±1.32 6.82±1.69 1.00±0.14 0

Negative Control 49±0.05 47.49±1.01 44.34±1.99 38.16±2.58 26.85±0.76 16.46±0.59 10.12±0.49

Indigofera aspalathoides 49±0.05 40.07±4.47 19.21±1.07 8.98±1.525 2.60±.0113 0.46±0.18 0.018±0.02

Myristica andamanica 49±0.05 41.81±0.77 35.595±1.45 19.25±2.11 10.05±0.45 1.03±0.32 0.38±0.34

Adhatoda vasica 49±0.05 40.43±1.36 18.8±1.57 12.60±1.0.01 2±1.45 0.34±0.32 0.026±0.039

Azadirachta indica 49±0.05 40.21±1.82 27.65±1.59 18.29±1.50 9.58±1.23 2.59±0.89 0.021±0.02

Saraca asoca 49±0.05 40.33±1.74 25.04±1.28 12.16±1.31 2.21±0.33 0.54±0.35 0.04±0.035

Aegle marmelos 49±0.05 39.38±1.30 25.50±1.30 10.85±1.08 1.84±0.19 0.16±0.11 0.03±0.02

No of Days

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*The table shows the effect of methanolic extract of Indigofera aspalathoides, Myristica andamanica, Adhatoda vasica,

Azadirachta indica, Saraca asoca and Aegle marmelos on the percentage of wound contraction. The table shows the

reduction in the size of wound on daily basis. On the 12th day all the six mice under positive control were healed whereas

the mice under negative control the healing is delayed. The healing is intermediate in the case of mice under treatment

groups.

0 2 4 6 8 10 12

Positive 0 20.22±4.93 45.52±3.0 61.29±2.70 86.06±3.44 97.95±0.29 100

Negative 0 3.06±2.06 9.49±4.07 22.12±5.28 45.20±1.56 66.40±1.21 79.35±1.01

Indigoferaaspalathoides 0 20.77±3.3 60.78±2.20 81.66±3.11 94.67±0.23 99.05±.38 99.96±0.04

Myristicaandamanica 0 14.67±1.57 27.35±2.96 60.71±4.32 79.47±0.93 97.88±0.65 99.21±0.70

Adhatodavasica 0 17.49±2.77 61.63±3.20 74.27±2.08 95.91±2.96 99.32±0.67 99.94±0.07

Azadirachtaindica 0 17.93±3.72 43.57±3.25 62.58±2.94 80.43±2.51 94.70±1.81 99.95±0.051

Saracaasoca 0 17.69±3.55 48.88±2.62 75.16±2.67 95.48±0.68 98.89±0.72 99.92±0.07

Aeglemarmelos 0 19.63±2.67 47.94±2.65 77.85±2.21 96.23±0.40 99.66±0.241 99.93±0.05

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5.6.1.1Reduction In Wound Area For Mice Treated With Indigofera aspalathoides

Figure 5.33:Reduction in wound areafor mice treated withIndigofera

aspalathoides

Figure 5.33:The figure shows reduction in wound area for mice in treatment

group compared to mice in positive control group and mice in negative control

group. This graph has been calculated for a span of 12 days. The x – axis denotes

the number of days and the y – axis denotes the wound area in sq.mm.

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5.6.1.2Percentage of Wound Contraction in Mice Treated withIndigoferaaspalathoides

Figure 5.34:Percentage of wound contraction for mice treated with

Indigofera aspalathoides

Figure 5.34:The figure shows percentage of wound contraction for mice in

treatment group compared to mice in positive control group and mice in negative

control group. This graph has been calculated for a period of 12 days. The x - axis

denotes the number of days and the y - axis denotes the percentage of wound

contraction.

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5.6.2.1 Reduction in Wound Area for Mice Treated with Myristica andamanica

Figure 5.35:Reduction in wound areafor mice treated

withMyristicaandamanica





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5.6.2.2 Percentage of Wound Contraction in Mice Treated with Myristicaandamanica

Figure 5.36: Percentage of wound contractionfor mice treated

withMyristicaandamanica



control group. This graph has been calculated for a span of 12 days. The x – axis

denotes the number of days and the y – axis denotes the wound area in sq.mm.

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5.6.3.1Reduction in Wound Area for Mice Treated with Adhatoda vasica

Figure 5.37:Reduction in wound areafor mice treated withAdhatoda vasica





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5.6.3.2 Percentage of Wound Contraction in Mice Treated with Adhatoda vasica

.

Figure 5.38: Percentage of wound contractionfor mice treated withAdhatoda

vasica





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5.6.4.1 Reduction in Wound Area For Mice Treated With Azadirachta indica

Figure 5.39: Reduction in wound areafor mice treated withAzadirachta

indica




the number of days and the y – axis denotes the wound area in sq.mm

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5.6.4.2 Percentage Of Wound Contraction In Mice Treated With Azadirachta indica

Figure 5.40: Percentage of wound contractionfor mice treated

withAzadirachta indica





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5.6.5.1 Reduction in Wound Area for Mice Treated withSaraca asoca

Figure 5.41: Reduction in wound areafor mice treated withSaraca asoca





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5.6.5.2Percentage of Wound Contraction in Mice Treated with Saraca asoca

Figure 5.42: Percentage of wound contractionfor mice treated withSaraca

asoca





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5.6.6.1 Reduction In Wound Area For Mice Treated With Aegle marmelos

Figure 5.43: Reduction in wound areafor mice treated withAegle marmelos





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5.6.6.2 Percentage of Wound Contraction in Mice Treated With Aegle marmelos

Figure 5.44: Percentage of wound contractionfor mice treated withAegle

marmelos





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Plate 5.10: Reduction in wound area in mice treated with methanolic extract of Indigofera aspalathoides

0th day: 5.10-A1)Treatment Group 5.10-A2) Positive Control Group 5.10-A3) Negative Control Group

4th day: 5.10-B1) Treatment Group 5.10-B2) Positive Control Group 5.10-B3) Negative Control Group

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8th day: 5.10-C1) Treatment Group 5.10-C2) Positive Control Group 5.10-C3) Negative Control Group

12th day: 5.10-D1) Treatment Group 5.10-D2) Positive Control Group 5.10-D3) Negative Control Group

Plate 5.10: The plates show reduction in wound area in mice treated with methanolic extract of Indigofera aspalathoides. The photos of mice were taken on the 0th day, 4th day, 8th day and 12th dayof the wound healing period of study.

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Plate 5.11: Reduction in wound area in mice treated with methanolic extract of Myristica andamanica

0th day: 5.11-A1) Treatment Group 5.11-A2) Positive Control Group 5.11-A3) Negative Control Group


174



Plate 5.11: The plates show reduction in wound area in mice treated with methanolic extract of Myristica andamanica.

The photos of mice were taken on the 0th day, 4th day, 8th day and 12th dayof the wound healing period of study.

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Plate 5.12: Reduction in wound area in mice treated with methanolic extract ofAdhatoda vasica



176



Plate 5.12: The plates show reduction in wound area in mice treated with methanolic extract of Adhatoda vasica.The photos of mice were taken on the 0th day, 4th day, 8th day and 12th dayof the wound healing period of study.

177

Plate 5.13: Reduction in wound area in mice treated with methanolic extract of Azadirachta indica



178



Plate 5.13: The plates show reduction in wound area in mice treated with methanolic extract of Azadirachta indica. The photos of mice were taken on the 0th day, 4th day, 8th day and 12th dayof the wound healing period of study.

179

Plate 5.14: Reduction in wound area in mice treated with methanolic extract of Saraca asoca

0th day: 5.14-A1)Treatment Group 5.14-A2) Positive Control Group 5.14-A3) Negative Control Group


180



Plate 5.14: The plates show reduction in wound area in mice treated with methanolic extract of Saraca asoca. The photos of mice were taken on the 0th day, 4th day, 8th day and 12th dayof the wound healing period of study.

181

Plate 5.15: Reduction in wound area in mice treated with methanolic extract of Aegle marmelos



182



Plate 5.15: The plates show reduction in wound area in mice treated with methanolic extract of Aegle marmelos.The photos of mice were taken on the 0th day, 4th day, 8th day and 12th dayof the wound healing period of study.

183

Compared to the day 1 of wound healing, to the day 12 of wound healing

showed complete wound contraction and reduction in wounded area as seen in

Table 5.12 for all the methanolic extracts of the selected plants. On the 12th day

the positive control mice treated with Povidine iodine showed 100% wound

reduction while the negative control showed 80% wound reduction. But the

methanolic extracts treated animal showed above 99% and was equivalent to the

positive control. The methanolic extract of all the six selected medicinal plants

showed similar wound healing activity in 1% w/w of methanolic extract (Plate

5.10 to Plate 5.15: A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2 and D3). This

wound healing activity was possible because of the presence of phenolics,

alkaloids, glycosides, flavonoids, tannins, anthraquinone and saponins present in

the extract which is shown in the phytochemical screening table (Table 5.8). The

presence of wound healing was also revealed from the table that the healing

process was slowly progressing from day 1 to day 12 which is shown as reduction

in wound area in Table 5.12 and in table 5.13 as percentage of wound

contraction. This was also well supported by our histopathological studies from

Plate 5.16 to Plate 5.21: A1, A2, A3, B1, B2, B3, C1, C2 and C3. The preliminary

phytochemical analysis of the crude extracts of the six selected Indian medicinal

plants indicated the presence of major phytochemical compounds,including

phenolics, alkaloids, glycosides, flavonoids, andtannins which may be responsible

forthe observed wound healing activities.

184

5.7 HISTOPATHOLOGICAL EXAMINATION

5.7.1 Histopathological Examination of Excised Tissues Treated withIndigofera aspalathoides

Plate 5.16:Histopathological examination of excised tissues treated with Indigofera aspalathoides

Plate 5.16-A:Tissue stained with Hematoxylin& Eosin Stain (HE)

5.16-A1) Treatment Group 5.16-A2) Positive Control Group 5.16-A3) Negative Control Group

Plate 5.16-A:Tissue stained with Hematoxylin & Eosin Stain (HE): Treatment Group: Collagen is seen in pink color. Muscle is seen in deep pink color. Basophilic cytoplasm is seen in purple color. Nuclei are seen in blue color. Positive Control Group: Collagen is seen in pink color. Muscle is seen in deep pink color. Basophilic cytoplasm is seen in purple color. Nuclei are seen in blue color. Negative Control Group: No deposition of collagen or muscles are seen which indicatesthat the tissue has not still developed. Proper nucleus has not still developed since no blue color is observed.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S- Scab,BC- Basophilic Cytoplasm.

185


Plate 5.16-B:Tissue stained with Van Gieson’s Stain (VG)

5.16-B1) Treatment Group 5.16-B2) Positive Control Group 5.16-B3) Negative Control Group

Plate 5.16-B: Tissue stained with Van Gieson’s Stain (VG): Treatment Group: Collagen is seen in bright red color. Cytoplasm, muscle, fibrin and RBC are seen in yellow color. Nucleus is seen in blue color. Positive Control Group: Collagen is seen in bright red color. Cytoplasm, muscle, fibrin and RBC are seen in yellow color. Nucleus is seen in blue color. Negative Control Group: No deposition of collagen is seen.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S- Scab, BC- Basophilic Cytoplasm.

186


Plate 5.16-C:Tissue stained with Toluidine blue Stain (TB)

5.16-C1) Treatment Group 5.16-C2) Positive Control Group 5.16-C3) Negative Control Group

Plate 5.16-C: Tissue stained with Toluidine blue Stain (TB): Treatment Group: Mast cells are seen in blue color. Positive Control Group: Mast cells are seen in blue color. Negative Control Group: No mast cells are observed which indicates thattissue development has not started.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S- Scab, BC- Basophilic Cytoplasm.

187

5.7.2 Histopathological Examination of Excised Tissues Treated with Myristica andamanica

Plate 5.17:Histopathological examination of excised tissues treated withMyristica andamanica



Plate 5.17-A: Tissue stained with Hematoxylin & Eosin Stain (HE): Treatment Group: Collagen is seen in pink color. Muscle is seen in deep pink color. Basophilic cytoplasm is seen in purple color. Nuclei are seen in blue color. Positive Control Group: Collagen is seen in pink color. Muscle is seen in deep pink color. Basophilic cytoplasm is seen in purple color. Nuclei are seen in blue color. Negative Control Group: No deposition of collagen or muscles are seen which indicatesthat the tissue has not still developed. Proper nucleus has not still developed since no blue color is observed.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S-Scab, BC- Basophilic Cytoplasm.

188

Plate 5.17: Histopathological examination of excised tissues treated with Myristica andamanica

Plate 5.17-B: Tissue stained with Van Gieson’s Stain (VG)


Plate 5.17-B: Tissue stained with Van Gieson’s Stain (VG): Treatment Group: Collagen is seen in bright red color. Cytoplasm, muscle, fibrin and RBC are seen in yellow color. Nucleus is seen in blue color. Positive Control Group: Collagen is seen in bright red color. Cytoplasm, muscle, fibrin and RBC are seen in yellow color. Nucleus is seen in blue color. Negative Control Group: No deposition of collagen is seen.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S-Scab, BC- Basophilic Cytoplasm.

189

Plate 5.17: Histopathological examination of excised tissues treated with Myristica andamanica

Plate 5.17-C: Tissue stained with Toluidine blue Stain (TB)


Plate 5.17-C: Tissue stained with Toluidine blue Stain (TB): Treatment Group: Mast cells are seen in blue color. Positive Control Group: Mast cells are seen in blue color. Negative Control Group: No mast cells are observed which indicates that tissue development has not started.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S- Scab, BC- Basophilic Cytoplasm.

190

5.7.3 Histopathological Examination of Excised Tissues Treated with Adhatoda vasica

Plate 5.18:Histopathological examination of excised tissues treated withAdhatoda vasica



Plate 5.18-A: Tissue stained with Hematoxylin & Eosin Stain (HE): Treatment Group: Collagen is seen in pink color. Muscle is seen in deep pink color. Basophilic cytoplasm is seen in purple color. Nuclei are seen in blue color. Positive Control Group: Collagen is seen in pink color. Muscle is seen in deep pink color. Basophilic cytoplasm is seen in purple color. Nuclei are seen in blue color. Negative Control Group: No deposition of collagen or muscles are seen which indicates that the tissue has not still developed. Proper nucleus has not still developed since no blue color is observed.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S- Scab,BC- Basophilic Cytoplasm.

191

Plate 5.18: Histopathological examination of excised tissues treated with Adhatoda vasica




192

Plate 5.18: Histopathological examination of excised tissues treated with Adhatoda vasica




193

5.7.4 Histopathological Examination of Excised Tissues Treated With Azadirachta indica

Plate 5.19: Histopathological examination of excised tissues treated with Azadirachta indica

Plate 5.19-A: Tissue stained with Hematoxylin& Eosin Stain (HE)



194





195




Plate 5.19-C: Tissue stained with Toluidine blue Stain (TB): Treatment Group: Mast cells are seen in blue color. Positive Control Group: Mast cells are seen in blue color. Negative Control Group: No mast cells are observed which indicates thattissue development has not started.C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S- Scab, BC- Basophilic Cytoplasm.

196

5.7.5 Histopathological Examination of Excised Tissues Treated With Saraca asoca

Plate 5.20:Histopathological examination of excised tissues treated withSaraca asoca




197

Plate 5.20: Histopathological examination of excised tissues treated with Saraca asoca




198

Plate 5.20: Histopathological examination of excised tissues treated with Saraca asoca



Plate 5.20-C: Tissue stained with Toluidine blue Stain (TB): Treatment Group: Mast cells are seen in blue color. Positive Control Group: Mast cells are seen in blue color. Negative Control Group: No mast cells are observed which indicates that tissue development has not started. C- Collagen, F- Fibroblast, PMC- Poly Morpho nuclear Cells, M- Mast Cells, N- Nuclei, RE- Re- Epithelialization, S- Scab, BC- Basophilic Cytoplasm.

199

5.7.6 Histopathological Examination of Excised Tissues Treated With Aegle marmelos

Plate 5.21:Histopathological examination of excised tissues treated withAegle marmelos




200

Plate 5.21: Histopathological examination of excised tissues treated with Aegle marmelos




201

Plate 5.21: Histopathological examination of excised tissues treated with Aegle marmelos




202

The tissues were stained for nucleus and collagen with Hematoxylin &

Eosin Stain in the treatment group as shown in plate 5.16-A1, 5.17-A1, 5.18-A1,

5.19-A1, 5.20-A1 and 5.21-A1 and positive control group as shown in plate 5.16-

A2, 5.17-A2, 5.18-A2, 5.19-A2, 5.20-A2, and 5.21-A2 which shows collagen in

pink color. Muscle fibres can be observed in deep pink color. Basophilic

cytoplasm is seen in purple color. The nucleus can be seen in blue color. The

formation of collagen and muscle fibres cannot be observed in the negative

control groupas shown in plate 5.16-A3, 5.17-A3, 5.18-A3, 5.19-A3, 5.20-A3 and

5.21-A3. The nucleus is also not formed properly.

The tissues were stained fornucleus, muscle fibres and collagen with Van

Gieson’s Stain in the treatment group as shown in plate 5.16-B1, 5.17-B1, 5.18-

B1, 5.19-B1, 5.20-B1 and 5.21-B1 and positive control group as shown in plate

5.16-B2, 5.17-B2, 5.18-B2, 5.19-B2, 5.20-B2 and 5.21-B2 which shows collagen

in bright red color. The cytoplasm, muscle fibres and fibrin is observed as yellow

color. The nucleus can be seen in blue color. But the formation of collagen and

nucleus cannot be observed in the negative control groupas shown in plate 5.16-

B3, 5.17-B3, 5.18-B3, 5.19-B3, 5.20-B3 and 5.21-B3.

The tissues were stained for mast cells with Toluidine blue Stain in the

treatment group as shown in plate 5.16-C1, 5.17-C1, 5.18-C1, 5.19-C1, 5.20-C1

and 5.21-C1 and as shown in plate 5.16-C2, 5.17-C2, 5.18-C2, 5.19-C2, 5.20-C2

and 5.21-C2 positive control group which shows mast cells in blue color.

Whereas, the mast cells cannot be observed in negative control groupas shown in

plate 5.16-C3, 5.17-C3, 5.18-C3, 5.19-C3, 5.20-C3 and 5.21-C3.

The result of the present study on wound healing activity indicated that the

methanolic extract of the six selected medicinal plants promotes wound healing in

experimental mice. This was demonstrated by a significant decrease in the

wound area and increase in the percentage of wound contraction. The increased

percentage of wound contraction in the methanolic extract treated mice may be

203

due to enhanced activity of fibroblasts in the regenerated wound tissues.

Myofibroblasts are believed to play a major role in the wound contraction by

exerting tension on the surrounding extracellular matrix and secreting

extracellular matrix proteins such as collagen to stabilize the contraction of

wound. Collagen is a major protein of the extracellular matrix and an important

component that ultimately contributes to the wound strength [281].

Angiogenesis is a critical component of wound healing process. Delayed

or aberrant revascularization at the site of wounds contributes to the etiology of

chronic wounds and prolonged and delayed wound healing process [282]. The

methanolic extract treatment on the wounds promoted angiogenesis in the mice as

is evident in the histopathological studies. Free radicals are generated at the site

of injury which impairs the healing process by causing damage to cellular

membranes, nucleotides, proteins and lipids [283].

The use of antioxidants has been shown to increase the wound healing

activity [115]. Flavonoids are efficient antioxidants that is capable of scavenging

the free radicals and has also been reported to play an important role on the

wound healing process in many animal models [284,285,286].

The present study on wound healing activities clearly indicates that the

methanolic extract of the selected medicinal plants promotes the wound healing

process in experimental mice. The treatment of the wounds with the plant extract

augmented the endogenous antioxidants and prevented the free radical mediated

tissue injury. The methanolic extract of the medicinal plants also played a vital

role in the angiogenesis, formation of the extracellular matrix and remodeling

phase of wound healing process in mice.

Wound healing is a very complex, multifactor sequence of events

involving several cellular and biochemical processes. The aim in these processes

is to regenerate and reconstruct the disrupted anatomical continuity and functional

204

status of the skin. Healing process, a natural body reaction to injury, initiates

immediately after wounding and occurs in four stages.

The first phase is coagulation which controls excessive blood loss from the

damaged vessels. The next stage of the healing process is inflammation and

debridement of wound followed by re-epithelialization which includes

proliferation, migration and differentiation of squamous epithelial cells of the

epidermis. In the final stage of the healing process collagen deposition and

remodeling occurs within the dermis.

The results showed wound healing and repair, accelerated by applying gel

of medicinal plant extract which was highlighted by the full thickness coverage of

the wound area by an organized epidermis.Study on animal models showed

enhanced rate of wound contraction and drastic reduction in healingtime than

control, which might be due to enhanced epitheliasation.

The treated woundafter 4 days itself exhibit marked dryness of wound

margins with tissue regeneration.However, histological evaluation showed that,

increased cellular infiltration from hematoxylin and eosin staining in treated cases

may be due to chemo tactic effect enhanced by the crude extract which mighthave

attracted inflammatory cells towards the wound site.

Increased cellular proliferation may be due to the mitogenic activity of the

plant extract, which might have significantly contributed to healing process. Early

dermal and epidermal regeneration in treated mice also confirmed that the extract

had a positive effect towards cellular proliferation, granular tissue formationand

epitheliasation.

The histopathological study revealed increased collagen deposition in the

treatment group as compared to the positive and the negative control group, as

confirmed by the Hematoxylin & Eosin Stain and also by Van Gieson’s Stain

205

5.8 ANTIGENOTOXIC ACTIVITY

Plate 5.22: Fish Erythrocytes of Control CellsPlate 5.22: The plate showsfish erythrocytes of control cells

Plate 5.23: Fish Erythrocytes showing the Micro nucleated and

Multinucleated Cells

Plate 5.23: The plate shows fish erythrocytes showing the micro nucleated and

multinucleated cells

1000 X

206

Plate 5.24: Fish Erythrocyte showing the Control Cells

Plate 5. 25: Fish Erythrocyte Showing Micronucleus Cells

Plate 5. 26 Fish Erythrocyte Showing Multinucleated Cells

207

5.8.1Micronucleus Assay 5.8.1.1 Micronucleus Assay for Pungasius pungasius treated with aqueousextract of Indigofera aspalathoides leaves

Table 5.14: Micronucleus assay for Pungasius pungasius treated with

aqueous extract of Indigofera aspalathoides leaves*

Sample 13.5 Gy 27 Gy% of

Micronucleated cells

% of Binucleated

cells

% of Multinucleated

cells

% of Micronucleated

cells

% of Binucleated

cells

% of Multinucleated

cells+ ve

control0.8 0.2 0.4 0.5 0.2 0.3

- ve

control11.5 4.7 1.2 15.1 7.4 5.3

10 ppm 3.1 1.5 1.3 10.1 3.3 2.1

*The table shows micronucleus assay for Pungasius pungasius treated with

aqueous extract of Indigofera aspalathoides leaves.

5.8.1.2 Micronucleus Assay for Pungasius pungasius treated with aqueousextract of Myristica Andamanica leaves

Table 5.15:Micronucleus assay for Pungasius pungasius treated with

aqueous extract of Myristica andamanica leaves*

Sample13.5 Gy 27 Gy

% of Micronucleated

cells

% of Binucleated

cells

% of Multinucleated

cells

% of Micronucleated

cells

% of Binucleated

cells

% of Multinucleated

cells+ ve control

1.2 0.3 0.4 1.5 0.2 0.6- ve control

11.5 2.8 1.7 15.5 4.2 3.210 ppm 3.7 2.2 0.6 3.1 1.8 0.2*The table shows the micronucleus assay for Pungasius pungasius treated with

aqueous extract of Myristica andamanica leaves.

208

5.8.1.3 Micronucleus Assay for Pungasius pungasius treated with aqueousextract of Adhatoda Vasica leaves


aqueous extract of Adhatoda vasica leaves*



% of Binucleated

cells

% of Multinucleated

cells

% of Micronucleated

cells

% of Binucleated

cells

% of Multinucleated

cells+ ve

control0.5 0.1 0.2 0.1 0.1 0.2

- ve

control14.1 5.5 1.9 8.9 6.2 2.1

10 ppm 2.2 1.7 0.2 0.8 2.4 0.8


aqueous extract of Adhatoda vasica leaves.

5.8.1.4 Micronucleus Assay for Pungasius pungasius treated with aqueousextract of Azadirachta indica leaves


aqueous extract of Azadirachta indica leaves*



% of Binucleated

cells

% of Multinucleated

cells

% of Micronucleated

cells

% of Binucleated

cells

% of Multinucleated

cells+ ve

control1.7 0.5 0.7 1.7 0.7 0.6

- ve

control12.4 5 1.5 16 7.7 5.6

10 ppm 3.9 1.8 1.6 10.9 3.6 2.4*The table shows micronucleus assay for Pungasius pungasius treated with

aqueous extract of Azadirachta indica leaves.

209

5.8.1.5 Micronucleus Assay for Pungasius pungasius treated with aqueousextract of Saraca asoca leaves


aqueous extract of Saraca asoca leaves*



% of Binucleated

cells

% of Multinucleated

cells

% of Micronucleated

cells

% of Binucleated

cells

% of Multinucleated

cells+ ve

control0.5 0.3 0.4 0.7 0.2 0.6

- ve

control5.6 4.2 1.7 9.5 2.8 3.2

10 ppm 0.9 2.2 0.6 1.1 1.8 2.0*The table shows micronucleus assay for Pungasius pungasius treated with

aqueous extract of Saraca asoca leaves.

5.8.1.6 Micronucleus Assay for Pungasiuspungasius treated with aqueousextract of Aegle marmelos leaves

Table: 5.19: Micronucleus assay for Pungasiuspungasius treated with

aqueous extract of Aegle marmelos leaves*



% of Binucleated

cells

% of Multinucleated

cells

% of Micronucleated

cells

% of Binucleated

cells

% of Multinucleated

cells+ ve

control0.9 0.4 0.2 1.0 0.2 0.3

- ve control

13 5.8 3.0 17.2 6.6 1.810 ppm 3.2 1.9 0.6 4.0 2.0 0.8


aqueous extract of Aegle marmelos leaves.

210

5.8.2 Enzymatic Assays 5.8.2.1 Oxidase Enzyme Test

The test batch solution of fish produced blue color immediately after the

addition of them to oxidase reagent discs. The control batch solution did not

produce any color change when added with the oxidase reagent discs (Plate 5.27).

Plate 5.27:Oxidase enzyme test

Plate 5.27: The plate shows Oxidase enzyme test for test and control.

5.8.2.2 Catalase Enzyme Test

The test batch smears of fish produced air bubbles immediately after the addition of them to catalase reagent. The control batch solution did not produce any air bubble when added with the catalase reagent (Plate 5.28).

Plate 5.28: Catalase enzyme test

Plate 5.28: The plate shows Catalase enzyme test for test and control.

211

5.9 ISOLATION AND PURIFICATION OF BIOACTIVE INGREDIENT FROM INDIGOFERA ASPALATHOIDES

5.9.1 Purification of Active Ingredient From Indigofera aspalathoides

• Column Chromatography Fractionation ratio:

Ethyl acetate: Hexane = 15:35

• TLC solvent ratio:

Ethyl acetate: Hexane = 2:8

• Rf : 9.2

5.9.2 Results for The Purity Of The Active Ingredient Isolated From Indigofera aspalathoides

The purity of the isolated and purified bioactive compound was confirmed

after several HPTLC analysis was done and checked to obtain a single peak

without any contamination.

212

5.9.2.1 HPTLC Analysis for Crude Extract

Figure 5.45: HPTLC chromatogram of the Crude Methanol extract of Indigofera aspalathoides

213

5.9.2.2 HPTLC Analysis for Purified Compound

Figure 5.46: HPTLC chromatogram of the Purified Compound isolated from the Methanol extract of Indigofera

aspalathoides

214

5.9.3 Identification of the Isolated and Purified Bioactive Compound

Identification of the isolated and purified bioactive ingredient from the

first peak was done using UV visible spectroscopy, FTIR spectroscopy, Mass

spectroscopy, 1H Nuclear Magnetic Resonance and 13C Nuclear Magnetic

Resonance.

5.9.4 Structural Elucidation Of The Isolated And Purified Bioactive Compound From Indigofera aspalathoides

5.9.4.1 UV visible spectroscopy report for the isolated and purified bioactive compound from Indigofera aspalathoides

Figure 5.47: UV visible spectroscopy report for the isolated and purified

bioactive compound from Indigofera aspalathoides

Figure 5.47: The figure shows UV visible spectroscopy report for the isolated and

purified bioactive compound from Indigofera aspalathoides.

215

5.9.4.2 FTIR Spectroscopy Report for The Isolated and Purified Bioactive Compound from Indigofera aspalathoides

Figure 5.48: FTIR spectroscopy report for the isolated and purified bioactive compound from Indigofera

aspalathoides

Figure 5.48: The figure shows FTIR spectroscopy report for the isolated and purified bioactive compound from Indigofera aspalathoides.

216

Table 5.20: FTIR Spectroscopy*

VALUES FUNCTIONAL GROUP

719 AROMATIC/ALKYNE1461 CH BENDING1717 CARBONYL2848 CH ALIPHATIC STRECH2916 CH ALIPHATIC STRECH3384 OH

*The table shows the functional group corresponding to the peak values from

FTIR Spectroscopy

217

5.9.4.3 1H NMR Report for The Isolated And Purified Bioactive Compound From Indigofera aspalathoides

Figure: 5.49: 1H NMR report for the isolated and purified bioactive compound from Indigofera aspalathoides

Figure: 5.49: The figure shows 1H NMR report for the isolated and purified bioactive compound from Indigofera

aspalathoides.

218

Table 5.21:1H NMR Spectroscopy*

CHEMICAL SHIFT PROTON

1.914 ALLYL OR TER CARBON

2.4-2.6 BENZYLIC PROTON

7.3-7.7 AROMATIC

8.4-8.6 UNSATURATED

*The table shows the proton corresponding to the chemical shift from 1H NMR

Spectroscopy

219

5.9.4.4 13C NMR Report for the Isolated And Purified Bioactive Compound From Indigofera aspalathoides

Figure 5.50: 13C NMR report for the isolated and purified bioactive compound from Indigofera aspalathoides

Figure 5.50: The figure shows 13C NMR report for the isolated and purified bioactive compound from Indigofera

aspalathoides.

220

Table 5.21:13C NMR Spectroscopy*

CHEMICAL SHIFT 13CARBON

76.816 ALKYNE OR CO

77.070 ALKYNE OR CO

77.324 ALKYNE OR CO

115.556 CH2

117.434 CH2

118.510 CH2

127.003 CH3

128.267 CH3

128.481 CH3

130.031 CH3

131.827 CH3

133.560 CH3

135.955 CH3

136.906 CH3

142.625 C

153.735 C*The table shows the 13C corresponding to the chemical shift from 13C NMR Spectroscopy

221

5.9.5 Structure identified

Cinnamyl alcohol

The structure identified using UV visible spectroscopy, FTIR spectroscopy,

Mass spectroscopy, 1H Nuclear Magnetic Resonance and 13C Nuclear Magnetic

Resonanceis Cinnamyl alcohol.

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