5.1. Extraction - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/40994/11/11... ·...

Chapter 5 RESULTS AND DISCUSSION

79

5.1. Extraction

The extraction of Andrographis paniculata and Silybum marianum aerial

part (leaves and stem) was done with 70% ethanol. The percentage yield

obtained was 16.83 and 15.04 for A. paniculata and S. marianum

respectively (Table 5.1; Fig 5.1).

Table 5.1: Percentage yield of hydroalcoholic extract of A. paniculata and S. marianum

Solvent Plant Dry wt.

(g)

Yield

(g)

Time

(h)

Temperature

(˚C) % yield

70%

ethanol

A. paniculata 150 25.25 96 68 16.83

S. marianum 150 22.57 72 68 15.04

Fig. 5.1: Percentage yield of hydroalcoholic extract of A. paniculata and S. marianum

In the present study, hydroalcoholic solvent system was used for

the extraction, which contains 70% ethanol and 30% water. Ethanol is

16.83

15.04

0

5

10

15

20

A. paniculata S. marianum

% yield


80

less polar in nature as compared to water, which is the most polar

solvent on earth. Therefore, both the polar as well as non-polar

compounds present in the plants are extracted out in the same solvent.

The yield of the extract depends, upon the type of solvent (low-polarity

solvents yield more lipophilic compounds, whereas alcoholic extract

yields both polar and non-polar compounds), the time given for

extraction, and the method used for the extraction. Soxhlet extraction

yields better amount of extract as compared to the extraction by flask

method in water bath. The main advantage of the Soxhlet technique is

that, it is an automatic and continuous method that does not require

much manipulation. It has also been shown to be very effective in terms

of extraction yield and therefore, often used as a reference methods.


81

5.2. Phytochemical Analysis

In the present investigation, the phytochemical screening showed the

presence of flavonoids, tannins, carbohydrate, cardiac glycosides,

terpenes and steroids in both the plant extracts. Apart from this,

alkaloids were present only in the extract of Andrographis paniculata

while, saponins were present only in Silybum marianum extract (Table

5.2). Adegoke et al., (2009) performed similar studies and showed the

presence of alkaloids, flavonoids, saponins, anthraquinones,

plobatannins, tannins, cyanogenetic glycosides and cardiac glycosides in

the extract of Lasienthera africanum. Similarly, Reuben et al., (2008)

showed the presence of alkaloids, carbohydrate, cardiac glycosides,

flavonoids, saponins, terpenes and steroids in the alcoholic extract of

Croton zambesicus. Phytochemical screening of Carica papaya, Psidium

guajava, Vernonia amygdalina, and Mangifera indica showed the

presence of flavonoids, terpenoids, saponins, tannins and reducing

sugars. Mangifera indica did not contain cardiac glycosides and alkaloids

while, Psidium guajava could also show the absence of alkaloids and

anthraquinones. Anthraquinone was absent in Vernonia amygdalina

(Ayoola et al., 2008). Methanolic extract of Murraya koenigii also showed

the presence of alkaloids, flavonoids, saponins, anthraquinones,

plobatannins and tannins (Shivkanya et al., 2009).


82

Table 5.2: Phytochemical analysis of hydroalcoholic extract of A. paniculata and S.

marianum extracts

Phytochemical/Test A. paniculata S. marianum

Alkaloids: Dragendorff’s test Mayer’s test Hager’s test Wagner’s test

+ -

+ -

+ -

+ -

Flavonoids: Shibata’s test + +

Tannins: Ferric chloride test + +

Terpenoids: Salkowski’s test + +

Steroids: Liebermann’s Burchard test + +

Glycosides: Fehling’s test + +

Carbohydrates: Molisch’s test + +

Saponins: Frothing test - +

+ : Present

─ : Absent


83

5.3. Thin Layer Chromatography (TLC)

Thin layer chromatography is a separation technique which is used to

separate out the components present in a given mixture. The

phytochemical test for both the extracts reveals the presence of

flavonoid, tannins, glycosides etc. which when subjected to TLC with

different solvent system gave different colored bands on separation. The

Rf value (Relative front) is calculated for each band as this value is

specific for each compound. Just like the Rf value for each amino acid

for a particular solvent is specific and comparable to the unknown

sample run on the same solvent. The result for TLC of both the extracts

showed maximum number of bands i.e., 9 for Andrographis paniculata

with Rf (Relative front) value 0.98, 0.95, 0.92, 0.89, 0.86, 0.72, 0.69,

0.67, and 0.14 respectively, and 8 for Silybum marianum with Rf value

0.97, 0.96, 0.87, 0.85, 0.76, 0.70, 0.46, 0.20, 0.09 respectively, in

Chloroform: Methanol: Ethyl acetate (8:1.5:1) solvent system (Fig. 5.2;

Table 5.3), while Chloroform: Toluene: Methanol (6:2.5:1.5) gave 7 colored

bands for A. paniculata with Rf value 0.98, 0.93, 0.90, 0.84, 0.78, 0.72

and 0.12 respectively and 8 colored bands for S. marianum with Rf value

0.97, 0.95, 0.89, 0.86, 0.75, 0.61, 0.38 and 0.19 respectively (Table 5.4).


84

AP = Andrographis paniculata

SM = Silybum marianum

1 = Chloroform: Toluene: Methanol

2 = Chloroform: Methanol: Ethyl acetate

Fig. 5.2: TLC of hydoalcoholic extracts of A. paniculata and S. marianum in UV light


85

Table 5.3: TLC of hydroalcoholic extract of A. paniculata and S. marianum

Solvent system Extract Spots in decreasing order Colour Rf value

Ch

loro

form

: M

eth

an

ol: E

thyl aceta

te (8:1

.5:1

)

AP

1 Yellow 0.98

2 Blue 0.95

3 Dark red 0.92

4 Red 0.89

5 Magenta 0.86

6 Pale blue 0.72

7 Light green 0.69

8 Red 0.67

9 Blue 0.14

SM

1 Blue 0.97

2 Blue 0.96

3 Red 0.87

4 Blue 0.85

5 Red 0.76

6 Blue 0.70

7 Blue 0.46

8 Yellow 0.20

9 Blue

0.09




86

Table 5.4: TLC of hydroalcoholic extracts of A. paniculata and S. marianum

Solvent system Extract Spots in decreasing order Colour Rf value C

hlo

rofo

rm: Tolu

en

e: M

eth

an

ol (6

:2.5

:1.5

)

AP

1 Pale blue 0.98

2 Blue 0.93

3 Blue 0.90

4 Red 0.84

5 Yellow 0.78

6 Red 0.72

7 Pale yellow 0.12

SM

1 Blue 0.97

2 Blue 0.95

3 Bright blue 0.89

4 Pale yellow 0.86

5 Pale yellow 0.75

6 Yellow 0.61

7 Dark red 0.38

8 Red 0.19




87

5.4. Antimicrobial Activity

In the present study, different concentrations of Andrographis paniculata

and Silybum marianum extract (0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0

mg/ml) and their combination (1 mg/ml: 1 mg/ml) were assayed for

antimicrobial activity using agar well diffusion method against five

bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis,

Salmonella typhi and Pseudomonas aeruginosa) and two fungus

(Aspergillus niger and Cladosporium oxysporum). The results were

obtained as diameter of inhibition zone (mm).

Among the extracts, S. marianum was found most potent at a

concentration of 2 mg/ml against S. typhi (inhibition zone, 11 mm) and

B. subtilis (inhibition zone, 10 mm). At concentration of 1 mg/ml, it gave

7 mm zone of inhibition for S. typhi and B. subtilis. The combination of

both the extracts gave better result against S. typhi (inhibition zone, 4

mm) and B. subtilis (inhibition zone, 4 mm) as compared to that of A.

paniculata which gave 2 mm zone of inhibition for S. typhi and 3 mm for

B. subtilis. The combination of both the extracts (A. paniculata + S.

marianum) showed the best activity against E. coli (inhibition zone, 10

mm at 2 mg/ml and 4 mm at 1 mg/ml). At 2 mg/ml, S. marianum

extract gave 8 mm inhibition zone and A. paniculata extract gave 3 mm

zone of inhibition for E. coli. Against fungus A. niger, the combination

was more effective (inhibition zone, 2 mm) in comparison to A.

paniculata (inhibition zone, 1 mm) and S. marianum (inhibition zone, 1

mm). At concentration of 2 mg/ml, all the extracts showed zone of

inhibition against all five tested bacteria (E. coli, S. aureus, S. typhi, B.


88

subtilis and P. aeruginosa). Similar result was obtained for fungus A.

niger, while none of the extracts were found effective against C.

oxysporum (Fig. 5.3, 5.4; Table 5.5, 5.6). At low concentration (<

1mg/ml) none of the extracts showed significant results against all the

seven tested organisms. From the results obtained, it can be stated that

all the extracts showed a concentration dependent activity. The

antimicrobial effect was in the order of S. marianum > A. paniculata + S.

marianum > A. paniculata. The observations were compared with that of

standard drug Gentamycin (antibacterial) and Amphotericin (antifungal).


89

Table 5.5: Antibacterial activity of hydroalcoholic extracts of A. paniculata and S.

marianum

Extract Concentration

(mg/ml)

Zone of Inhibition (mm)

E. coli S. aureus B. subtilis P. aeruginosa S. typhi

AP

0.25 - - - - -

0.5 - - 1 - -

1.0 2 - 2 - -

2.0 3 1 3 3 2

SM

0.25 3 - 1 - 1

0.5 6 - 4 - 6

1.0 7 - 7 2 7

2.0 8 1 10 4 11

AP+SM

(1:1)

0.25 - - - - -

0.5 - - - - 1

1.0 4 - - - 2

2.0 10 1 4 2 4

Gentamycin

(Standard

drug)

0.025 16 10 6 - 2

0.05 20 15 8 - 3

0.1 22 16 12 1 12

0.25 25 20 14 03 16



- = No zone of inhibition


90



Gentamycin = Standard drug

Fig 5.3: Antibacterial activity of hydroalcoholic extract of A. paniculata and S. marianum

0

5

10

15

20

25

30

E. coli S. aureus B. subtilis P. aeruginosa S. typhi

Zon

e o

f In

hib

itio

n (

mm

)

Bacteria

AP

SM

AP + SM

Gentamycin

(St. drug)


91

Table 5.6: Antifungal activity of hydroalcoholic extract of A. paniculata and S. marianum

Extract Concentration

(mg/ml)

Zone of Inhibition (mm)

A. niger C. oxysporum

AP

0.25 - -

0.5 - -

1.0 - -

2.0 1 -

SM

0.25 - -

0.5 - -

1.0 1 -

2.0 1 -

AP+SM

(1:1)

0.25 - -

0.5 - -

1.0 1 -

2.0 2 -

Amphotericin

(Standard drug)

0.025 - -

0.05 - 2

0.1 2 4

0.25 3 6



- = No zone of inhibition


92



Amphotericin= Standard drug

Fig. 5.4: Antifungal effect of hydroalcoholic extracts of A. paniculata and S. marianum

Antibiotics like Gentamycin, Tetracyclin and Streptomycin inhibit

protein synthesis of the organism, by binding to the 30S ribosome and

freeze the 30S initiation complex (30S-mRNA-tRNA), so that no further

initiation can occur. Antibiotics like Chloramphenicol, Lincomycin and

Clindamycin binds to the 50S ribosome and inhibit peptidyl transferase

activity, while Rifamycin and Rifampicin binds to DNA-dependent RNA

polymerase and inhibit initiation of RNA synthesis. Also, such antibiotic

degrades the peptidoglycan cell wall of bacteria and chitinous cell wall of

the fungus. Similar mode of action may be performed by the plants

0

1

2

3

4

5

6

7

AP SM AP+SM Amphotericin

Zon

e o

f in

hib

itio

n (

mm

)

Extract/drug (mg/ml)

A. niger

C. oxysporum


93

secondary metabolites, against microbial infection, which are present in

both the plant extracts as well as in their combination.

The phenolic compounds (flavonoids and tannins) and terpenoids

present in both the extracts may have inhibited microbial growth by

disrupting the cell wall, binding to the adhesion complex with the cell

wall and also inactivating the enzyme for microbial synthesis (Brownlee

et al., 1990; Rojas et al., 1992; Perrett et al., 1995; Haslam, 1996;

Cichewicz and Thorpe, 1996), while alkaloid present in the extracts may

have intercalated into the cell wall and DNA of the microbes inhibiting

their growth and synthesis (Burdick, 1971; Rahman and Choudhary,

1995). The crude leaf and stem bark extract of Ficus capensis inhibited

the growth of Escherichia coli and Shigella sp. This was mainly due to the

presence of phytochemicals like alkaloids, tannins, carbohydrates,

flavonoids, sterols and terpenes present in the extract. The ethanolic

extract of Olax subscorpioidea could show considerable antibacterial

activity against S. aureus and E. coli due to the presence of alkaloids,

flavonoids and steroids (Ayandele and Adebiyi, 2007). The phytochemical

analysis revealed the presence of alkaloids, saponins, tannins, flavonoids

in the extract of Lasienthera africanum which was tested against

Escherichia coli, Salmonella typhi and Staphylococcus aureus and

inhibited the growth of the tested bacteria (Adegoke et al., 2009).

Combination therapy with two or more plant extracts can be used

in special cases like preventing the emergence of resistant strains, to

treat emergency cases during the period when an etiological diagnosis is

still in progress and also when to take advantage of extract synergism,


94

which occur when the effects of a combination of extract is greater than

the sum of the effects of the individual extract as showed by the

combination of A. paniculata and S. marianum in the case of E.coli and A.

niger. Extract antagonism occurs when one extract, usually the one with

the least effect, interferes with the effect of another extract as seen in the

case of B. subtilis and S. typhi. Further studies can be conducted to

attain a combinational therapy to achieve better synergistic effect.


95

Organism AP Gentamycin

(Standard drug)

E. coli

S. aureus

A. subtilis

P. aeruginosa

S. typhi


Fig. 5.5: Antibacterial effect of hydroalcoholic extract of A. paniculata and Gentamycin


96

Organism AP Amphotericin

(Standard drug)

A. niger

C. oxysporum


Fig. 5.6: Antifungal effect of hydroalcoholic extract of A. paniculata and Amphotericin


97

Organism SM Gentamycin

(Standard drug)

E. coli

S. aureus

B. subtilis

P. aeruginosa

S. typhi


Fig. 5.7: Antibacterial effect of hydroalcoholic extract of S. marianum and Gentamycin


98

Organism SM Amphotericin

(Standard drug)

A. niger

C. oxysporum


Fig. 5.8: Antifungal effect of hydroalcoholic extract of S. marianum and Amphotericin


99

Organism AP + SM (1:1) Gentamycin

(Standard drug)

E. coli

S. aureus

B. subtilis

P. aeruginosa

S. typhi

AP + SM = Andrographis paniculata + Silybum marianum (1:1 concentration)

Fig. 5.9: Antibacterial effect of hydroalcoholic extract of S. marianum and A. paniculata

(1:1) and Gentamycin


100

Organism AP + SM Amphotericin

(Standard drug)

A. niger

C. oxysporum

AP + SM = Andrographis paniculata + Silybum marianum (1:1 concentration)

Fig. 5.10: Antifungal effect of hydroalcoholic extract of S. marianum and A. paniculata

(1:1) and Amphotericin


101

5.5. Anthelmintic Activity

Andrographis paniculata and Silybum marianum and their combination

when analyzed for anthelmintic potential showed a concentration

dependent activity. A. paniculata extract showed better activity by way of

causing the paralysis of the worms at 3.33 min at 40 mg/ml and 5.33

min at 20 mg/ml and death in 5.16 min at 40 mg/ml and 7.50 min at

20 mg/ml. At 40 mg/ml, S. marianum extract caused paralysis at 3.83

min, while death at 7.5 min. Combination with A. paniculata and S.

marianum extract (1:1) was found most potent and caused paralysis of

the worm at 2.83 min and death at 6.33 min. The time taken by the

standard drug (20 mg/ml) for the paralysis and death of the worms was

7.0 and 14.83 min respectively (Table 5.7, Fig 5.11).


102

Table 5.7: Anthelmintic activity of hydroalcoholic extracts of A. paniculata and S. marianum

Extract/Drug Concentration

(mg/ml)

Paralysis time Death time

Min.

AP

20 5.33 ± 0.40 7.50 ± 0.20

40 3.33 ± 0.18 5.16 ± 0.14

SM

20 6 ± 0.23 10.16 ± 0.43

40 3.83 ± 0.14 7.5 ± 0.31

AP + SM

(1:1)

20 5 ± 0.23 8.5 ± 0.20

40 2.83 ± 0.14 6.33 ± 0.18

Piperazine citrate 20 7 ± 0.33 14.83 ± 0.36

Normal Saline (Control) - - -



Piperazine citrate = Standard drug

─ = No activity


103

AP= Andrographis paniculata

SM= Silybum marianum

Piperazine citrate = Standard drug

Fig. 5.11: Anthelmintic activity of hydroalcoholic extract of A. paniculata and S. marianum

Synthetic anthelmintic drug like piperazine citrate is known to

cause paralysis of worms so that they are expelled in the faeces of men

and animals. These drugs may reach the target site in worms either

orally or by diffusion and/or uptake through the cuticle, however the

major uptake of the drug is through cuticle (Geary et al., 1999). Some

synthetic phenolic anthelmintics, e.g. niclosamide, oxyclozanide and

bithionol are shown to interfere with energy generation in helminth

parasites by uncoupling oxidative phosphorylation (Martin, 1997).

5.33 6

5

7 7.5

10.1

8.5

14.8

0

2

4

6

8

10

12

14

16

18

AP SM AP + SM Piperazine citrate

Tim

e (

min

)

Extacts/Drug (mg/ml)

Paralysis time

Death time


104

A. paniculata S. marianum

A. paniculata + S. marianum Piperazine citrate

Fig. 5.12: Anthelmintic activity of hydroalcoholic extract of A. paniculata and S. marianum

In anthelmintic activity, most of the screenings reported are in vitro

studies using some worm samples like Pheretima posthuma, Ascardia galli

and Ascaris lumbricoids, etc (Kosalge and Fursule, 2009; Kane et al.,

2009). Adult Indian earthworm, Pheretima posthuma has been used as a

test worm in most of the anthelmintic screenings, as it shows anatomical

and physiological resemblance with the intestinal roundworm parasite of

human (Mali and Mehta, 2007). Because of easy availability, earthworms

and Ascardia galli worms are used as suitable models for screening of

anthelmintic studies (Dash et al., 2002; Mali et al., 2007). The extracts

in the present study caused paralysis as well as the death of the worms


105

may be due to the presence of phenolic compounds. Both the extracts

when taken in combination, showed synergistically better activity as

compared to individual effect. The variation in activity of the plant

extract might be due to the difference in the proportion of the active

compounds responsible for the anthelmintic property (Eguale et al.,

2006). The active constituents may be the phenolics, such as flavonoids

and tannins, present in both the plant extracts may be the reason for

causing paralysis and death of the worms. In the present investigation,

all the extracts were shown to possess tannins which were previously

reported to produce anthelmintic activities (Niezen et al., 1995). The

possible action of tannins is that they can bind to free proteins in the

gastrointestinal tract of the host animal (Athnasiadou et al., 2001) or

glycoprotein on the cuticle of the parasite (Thompson and Geary, 1995)

and may cause death. The results of the combinational study showed

synergistic effect and it may be suggested that both the plants when

consumed together can show better effect against intestinal worms

rather then taken individually.


106

5.6. Hepatoprotective activity

In the present study, the effect of different concentrations of

Andrographis paniculata and Silybum marianum (100, 200 and 400

mg/ml) and their different combinations (1:1, 1:2 and 2:1 to make final

concentration of 400 mg/ml) were assayed on CCl4 induced liver damage

in Wistar rats. After assessment of the biochemical parameters, CCl4

treated animals showed significant increase in the levels of AST (187.22

U/L) and ALT (90.66 U/L), while decrease in the level of total protein

(1.57 mg/dL) as compared to the normal control group (54.91 U/L,

31.65 U/L and 4.89 mg/dL for AST, ALT and total protein, respectively).

Whereas, animals treated with different extracts at a dose of 400 mg/kg

BW, showed significant decrease in the levels of serum marker enzymes

and significant increase in the total protein to the near normal value

which are comparable to that of standard drug Liv 52 (65.31 U/L, 42.31

U/L and 5.51 mg/dL for AST, ALT and total protein, respectively),

indicating the recovery of hepatic cells against the damage. At 200

mg/ml, S. marianum extract lowered the level of AST to 90.16 U/ml, ALT

to 51.16 U/ml and increased the total protein level to 4.27 U/mg of

protein), while, at a dose of 400 mg/kg it significantly reduced the AST

level to 64.35 U/ml and ALT level to 39.47, while increased the total

protein to 4.78 mg/dL and was found to be more potent among all the

extracts. S. marianum and its combination with A. paniculata in the

concentration ratio of 2:1 (S. marianum 266 mg and A. paniculata 133

mg) was found to be most effective among all the three combinations and

reduced the AST level to 76.24 U/ml, ALT to 45.44 and increased the


107

total protein to 4.67 mg/dL. This combination was also found better

when compared to that of A. paniculata at a dose of 400 mg/ml (AST

level 78.32 U/ml, ALT 46.49 and total protein 4.34 mg/dL). At a

concentration of 100 mg/ml for all the extracts, could not show

significant changes in the level of AST, ALT and total protein. Significant

results for all the three extracts were obtained at higher concentration

(400 mg/ml). The present finding showed that both the extracts and

their combinations were found effective against the CCl4 induced liver

damage in rats (Table 5.8, 5.9, 5.10; Fig. 5.13).


108

Table 5.8: Hepatoprotective activity of hydroalcoholic extract of A. paniculata

Groups* Treatment

(mg/kg BW)

(U/L) (mg/dL)

AST ALT Total protein

I Normal Saline

(Control) 54.91 ± 6.30 31.65 ± 4.30 4.89 ± 0.41

II CCl4, 2ml

(Toxic control) 187.22 ± 12.50 90.66 ± 8.60 1.57 ± 0.26

III CCl4 + Liv 52 (150) 65.31 ± 7.30***

42.31 ± 4.50***

5.51 ± 0.47***

IV A CCl4 + AP (100) 134.62 ± 12.20ns

71.82 ± 6.70 ns

3.36 ± 0.43 ns

IV B CCl4 + AP (200) 107.21 ± 11.11**

59.88 ± 6.18 ns

3.98 ± 0.49*

IV C CCl4 + AP (400) 78.32 ± 9.18***

46.49 ± 7.81 **

4.34 ± 0.48**

p value; ***p<0.001; **p<0.01; *p<0.05.

ns = non significant.


CCl4 = Carbon tetrachloride

Liv 52 = Standard drug

AST= Aspartate amino transferase

ALT= Alanine amino transferase

* = 6 animals in each group


109

Table 5.9: Hepatoprotective activity of hydroalcoholic extract of S. marianum

Groups* Treatment

(mg/kg BW)

(U/L) (mg/dL)

AST ALT Total protein

I Normal Saline

(Control) 54.91 ± 6.30 31.65 ± 4.30 4.89 ± 0.41

II CCl4, 2ml

(Toxic control) 187.22 ± 12.50 90.66 ± 8.60 1.57 ± 0.26

III CCl4 + Liv 52 (150) 65.31 ± 7.30***

42.31 ± 4.50***

5.51 ± 0.47***

V A CCl4 + SM (100) 118.95 ± 14.43**

65.35 ± 6.21 ns

3.67 ± 0.47 ns

V B CCl4 + SM (200) 90.16 ± 9.70***

51.16 ± 9.21 **

4.27 ± 0.49**

V C CCl4 + SM (400) 64.35 ± 8.17***

39.47 ± 5.61***

4.78 ± 0.41***

p value; ***p<0.001; **p<0.01; *p<0.05.









110

Table 5.10: Hepatoprotective activity of hydroalcoholic extract of A. paniculata and S.

marianum

Groups* Treatment

(mg/kg BW)

(U/L) (mg/dL)

AST ALT Total

protein

I Normal Saline

(Control) 54.91 ± 6.30 31.65 ± 4.30 4.89 ± 0.41

II CCl4, 2ml

(Toxic control) 187.22 ± 12.50 90.66 ± 8.60 1.57 ± 0.26

III CCl4 + Liv 52 (150) 65.31 ± 7.30***

42.31 ± 4.50***

5.51 ± 0.47***

VI (CCl4) + AP + SM (1:1) 91.98 ± 10.55ns

57.09±6.56ns

3.89±0.22 ns

VII (CCl4) + AP + SM (1:2) 76.24 ± 10.21***

45.44±6.69ns

4.67±0.27 ns

VIII (CCl4) + AP + SM (2:1) 109.64 ± 10.19* 61.09±6.54

ns 3.01±0.19

ns

p value; ***p<0.001; **p<0.01; *p<0.05.








1:1= 200 + 200 mg

1:2=133 + 266 mg



111



CCl4= Carbon tetrachloride

AST = Aspartate amino transferase

ALT = Alanine amino transferase

Control = Normal saline

1:1= 200 + 200 mg, 1:2=133 + 266 mg

Fig. 5.13: Hepatoprotective activity of hydroalcoholic extract of A. paniculata and S.

marianum

One of the most commonly used chemical agents for liver damage

in hepatoprotective study is CCl4 (Johnston and Kroening, 1998). The

active radical of this compound is CCl3 which binds to the

macromolecules and induce peroxidative degradation of membrane lipids

of endoplasmic reticulum. This result in the formation of lipid peroxides

whose product malondialdehyde (MDA) causes severe membrane damage

0

50

100

150

200

250

Control CCl4 Liv 52 AP SM AP + SM(1:1)

AP + SM(1:2)

AP + SM(2:1)

U/l

Drug/Extract (mg/kg BW)

AST

ALT

Total protein


112

(Cotran et al., 1994; Kaplowitz et al., 1986; Deleve and Kaplowitz, 1995;

Farrel 1998). The extent of hepatic damage is assessed by the elevated

levels of serum marker enzyme AST and ALT which is significantly

lowered by the administration of A. paniculata, S. marianum and their

combinational extracts in the tested groups showing their hepato

protective potential. Apart from liver, AST and ALT are also concentrated

in heart muscles, brain, gastric mucosa, adipose tissue, skeletal muscle

and kidneys. When these organs are damaged or destroyed by free

radicals or any other carcinogenic substances, these enzymes are

released from the damaged cells and their concentration is increased in

the blood. Elevated levels of the enzymes can signal myocardial

infarction, hepatic disease, muscular dystrophy, and organ damage. The

total protein estimation is also useful in hepatoprotective study as its

decreased level indicates severe non viral liver cell damage (Shenoy et al.,

2001).

The hepatoprotective potential of a drug depends upon its ability

in reducing the harmful effects caused by a hepatotoxin (Manjunatha et

al., 2008). In the present study, these phytoconstituents play a vital role

in inducing microsomal enzymes thereby accelerating the excretion of

CCl4, or inhibiting the lipid peroxidation induced by CCl4 (Mehta et al.,

1999). Phytoconstituents such as alkaloids (Vijyan et al., 2003) and

flavonoids (Baek et al., 1996) have been found effective in the

hepatoprotection against CCl4 induced liver damage. The hydroalcoholic

extract of both the plants showed the presence of alkaloids and

flavonoids, which may be responsible for their hepatoprotective


113

efficiency. Also, silymarin present in S. marianum extract increases

superoxide dismutase activity in erythrocytes and lympocytes thus

showing antioxidant activity (Feher et al., 1988). It stabilizes the

membrane structure of hepatocytes and thus prevents the toxins to

enter the cell through enterohepatic recirculation. It promotes liver

regeneration by increasing ribosomal protein synthesis. Similarly,

andographolide present in A. paniculata protect liver against the

hepatotoxins by reducing the levels of the lipid oxidation product,

malondialdehyde (MDA), and by maintaining high levels of the reduced

form of glutathione (GSH) (Kapil et al., 1993). Similar studies have been

done using six polyherbal liquid formulations (Liv 52, Livergen, Livokin,

Octogen, Stimuliv and Tefroliv), on CC14-induced liver injury on Swiss

albino mice. The formulation was effective in recovery of liver damage

(Girish et al., 2009). The ethanolic extracts of Launaea pinnatifida was

administered against CCl4 induced hepatic injury in rats. The extract

was effective in lowering the level of serum marker enzymes ALT and

AST (Pokharkar et al., 2007). The methanolic extract of Diospyros

malabarica is reported to have hepatoprotective activity due to the

presence of flavonoids (Mondal et al., 2005).


114

5.7. Antioxidant activity

In the present study, the hydroalcoholic extract of Andrographis

paniculata and Silybum marianum at different concentrations (100, 200

and 400 mg/ml) and their different combinations (1:1, 1:2 and 2:1 to

make final concentration of 400 mg/ml) were assayed for antioxidant

activity, analyzing SOD (Superoxide dismutase), CAT (Catalase) and GPx

(Glutathione peroxidase). After assessing of the biochemical parameters,

CCl4 treated animals showed significant decrease in the levels of SOD,

CAT and GPx (11.21, 27.57 and 25.34 U/mg of protein, respectively) as

compared to the normal control group (19.30 for SOD, 52.43 for CAT

and 25.34 for GPx). Whereas, animals treated with different extracts at

the dose of 400 mg/kg BW showed significant increase in the levels of

the enzymes to the near normal value which are comparable to the

values observed for standard drug, Liv 52 (18.71, 48.19 and 23.14 U/mg

of protein for SOD, CAT and GPx, respectively). At a dose of 200 mg/ml,

S. marianum increased the activity of SOD to 16.35 U/mg protein, CAT

to 38.58 and GPx to 20.10. However, at a dose of 400 mg/kg BW, it

significantly increased the level of SOD to 17.42 U/mg protein, CAT to

45.24 and GPx to 21.96 U/mg protein, while, the combination of S.

marianum with A. paniculata in the concentration ratio of 2:1 was found

most effective among all the three combinations, which increased the

SOD level to 16.26 U/mg protein, CAT level to 48.88 and GPx 21.41

U/mg protein. This combination was also better when compared to that

of A. paniculata at a dose of 400 mg/ml (16.64, 40.19 and 20.34 for

SOD, CAT and GPx level, respectively). At a concentration of 100 mg/ml


115

for all the extracts, no significant changes were observed in the levels of

SOD, CAT and GPx. Significant results for all the three extracts were

obtained only at higher concentration of 400 mg/ml. (Table 5.11, 5.12,

5.13 and Fig. 5.14).

Table 5.11: Antioxidant activity of hydroalcoholic extract of A. paniculata

Groups* Treatment

(mg/kg BW)

(U/mg protein)

SOD CAT GPx

I Normal Saline

(Control) 19.30 ± 1.30 52.43 ± 4.38 25.34 ± 0.48

II CCl4, 2ml

(Toxic control) 11.21 ± 0.94 27.57 ± 1.43 17.14 ± 0.26

III CCl4 + Liv 52 (150) 18.71 ± 1.12***

48.19 ± 3.87***

23.14 ± 0.46***

IV A CCl4 + AP (100) 11.34 ± 0.90ns

25.12 ± 1.33ns

12.98 ± 0.38ns

IV B CCl4 + AP (200) 15.67 ± 0.78* 35.01 ± 1.25

ns 18.23 ± 0.38

ns

IV C CCl4 + AP (400) 16.64 ± 0.65**

40.19 ± 2.87**

20.34 ± 0.39***

p value; ***p<0.001; **p<0.01; *p<0.05.





SOD = Superoxide dismutase

CAT = Catalase

GPx = Glutathione peroxidase



116

Table 5.12: Antioxidant activity of hydroalcoholic extract of S. marianum

Groups* Treatment

(mg/kg BW)

(U/mg protein)

SOD CAT GPx

I Normal Saline

(Control) 19.30 ± 1.30 52.43 ± 4.38 25.34 ± 0.48

II CCl4, 2ml

(Toxic control) 11.21 ± 0.94 27.57 ± 1.43 17.14 ± 0.26

III CCl4 + Liv 52 (150) 18.71 ± 1.12***

48.19 ± 3.87***

23.14 ± 0.46***

V A CCl4 + SM (100) 12.10 ± 0.79ns

28.11 ± 1.59ns

16.32 ± 0.38**

V B CCl4 + SM (200) 16.35 ± 0.71**

38.58 ± 1.64**

20.10 ± 0.41***

V C CCl4 + SM (400) 17.42 ± 0.63***

45.24 ± 1.84***

21.96 ± 0.39***

p value; ***p<0.001; **p<0.01; *p<0.05.






CAT = Catalase




117

Table 5.13: Antioxidant activity of hydroalcoholic extract of A. paniculata and S.

marianum

Groups* Treatment

(mg/kg BW)

(U/mg protein)

SOD CAT GPx

I Normal Saline

(Control) 19.30 ± 1.30 52.43 ± 4.38 25.34 ± 0.48

II CCl4, 2ml

(Toxic control) 11.21 ± 0.94 27.57 ± 1.43 17.14 ± 0.26

III CCl4 + Liv 52 (150) 18.71 ± 1.12***

48.19 ± 3.87***

23.14 ± 0.46***

VI (CCl4) + AP + SM (1:1) 14.52±0.91ns

37.91±1.67ns

19.21±0.28ns

VII (CCl4) + AP + SM (1:2) 16.26±0.77 ns

48.88± 2.09***

21.41±0.31ns

VIII (CCl4) + AP + SM (2:1) 13.33±0.67 ns

32.17±1.28ns

17.21±0.33 ns

p value; ***p<0.001; **p<0.01; *p<0.05.







CAT = Catalase


1:1= 200 + 200 mg

1:2=133 + 266 mg



118



Normal control= Normal saline

Liv. 52= Standard drug

CCl4= Carbon tetrachloride

CAT = Catalase

SOD= Superoxide dismutase

GPx= Glutathione peroxidase

1:1= 200 + 200 mg

1:2=133 + 266 mg

Fig. 5.14: Antioxidant activity of hydroalcoholic extract of A. paniculata and S. marianum

Antioxidants are intimately involved in the prevention of cellular

damage which is the common pathway for cancer, aging, and a variety of

diseases. Free radicals are atoms or groups of atoms with an odd

(unpaired) number of electrons which can be formed when oxygen

interacts with certain molecules. Once formed these highly reactive

0

10

20

30

40

50

60

NormalControl

CCl4 Liv 52 AP SM AP + SM(1:1)

AP + SM(1:2)

AP + SM(2:1)

U/m

g p

rote

in

Drug/Extract (mg/kg BW)

SOD

CAT

GPx


119

radicals can start a chain reaction. Their chief danger comes from the

damage they can do when they react with important cellular components

such as DNA, or the cell membrane. The body has a defense system of

antioxidants to prevent free radical damage. They are the molecules

which can safely interact with free radicals and terminate the chain

reaction before vital molecules are damaged. Catalase (CAT) an

antioxidant enzyme, like Superoxide dismutase (SOD) and Glutathione

peroxidase (GPx) is produced naturally within the body. It helps the body

to convert hydrogen peroxide into water and oxygen. It also uses

hydrogen peroxide to break down potentially harmful toxins in the body,

including alcohol, phenol, and formaldehyde. When our body uses

oxygen it produces free radicals that damage cell membranes, proteins

and DNA. Catalase works closely with superoxide dismutase to prevent

free radical damage to the body. SOD converts the dangerous superoxide

radical to hydrogen peroxide which is converted to harmless water and

oxygen by CAT and GPx. When the level of these enzymes decreases in

the body, the antioxidant system cannot function properly.

The results obtained in the present study showed that both the

extracts and their combinations were found effective in increasing SOD,

CAT and GPx activity. It also indicates that these extracts may be

associated with decreased oxidative stress, free radical-mediated tissue

damage and prevent the accumulation of excessive free radicals and

protects the liver from CCl4 induced liver damage in rats. Flavonoids and

other phenolic compounds of plant origin have been reported as

scavengers of free radicals (Formica and Regelson, 1995; Rice et al.,


120

1997). The results obtained in the present investigation of phytochemical

studies show that both A. paniculata and S. marianum are rich in

flavonoids and phenolic compounds. Varga et al., (2001) reported that

silymarin increases superoxide dismutase activity in erythrocytes. It’s

mechanism of action for hepatoprotection appears from its antioxidant

effect to scavenge free radicals and inhibit lipid peroxidation, which is

similar with the case of andrographolide present in A. paniculata (Flora

et al., 1998). Similar study was performed for evaluating antioxidant

effect of the methanolic extract of Eupatorium ayapana leaves in CCl4

induced liver damage in Wistar albino rats. SOD, CAT, GSH and protein

analysis revealed that the extract contains antioxidant activity (Bose et

al., 2007). Carica papaya, Psidium guajava, Vernonia amygdalina, and

Mangifera indica also possess potent antioxidant activity due to the

presence of various phytochemicals, like flavonoids, terpenoids and

tannins (Ayoola et al., 2008). The methanolic extract of Bauhinia

racemosa stem bark was investigated for the antioxidant effect in

paracetamol induced liver damage in Wistar albino rats. The extract

showed antioxidant effects on FeCl2-ascorbate-induced lipid peroxidation

in rat liver homogenate and on superoxide scavenging activity (Gupta et

al., 2004). The methanolic extract of Momordica cymbalaria was used

against CCl4 treated rats for antioxidant activity. The extract restored the

altered catalase level in CCl4 intoxicated rats (Rajshekhar et al., 2009).


121

4.8. Anticancer study

In the present study, an attempt was made to investigate the anticancer

potential of the extracts of A. paniculata and S. marianum and their

combination (1:1) at a concentration of 100 µg/ml against five human

cancer cell lines the human breast adenocarcinoma (MCF-7), the human

cervix (SiHa), colon (HT-29), liver (HepG2) and ovary cancer cell line

(ovcar-5). The result of in vitro, anticancer study suggest that S.

marianum extract showed the best cytotoxic activity against all given cell

lines (percentage inhibition was 21.34, 32.30, 46.56, 59.58, 36.20 for

MCF 7, SiHa, HT, Ovcar and HepG2, respectively), while significant one

was against HT and Ovcar. Andrographis paniculata extract was found

most effective against Ovcar cell line (51.12%). Again, the combination

(1:1) of both the plants showed an intermediate result for most of the cell

line but, the combination was most effective against the liver cell line

(HepG2) which showed better activity (42.76%), as compared with the

activity of both the plant extracts which gave individually (28.22 and

36.20% for A. paniculata and S. marianum respectively) (Table 5.14, Fig.

5.15).


122

Table 5.14: Anticancer activity of hydroalcoholic extract of A. paniculata and S.

marianum

Type of cell

line

Concentration of

Extract (µg/ml)

% inhibition

AP SM AP+SM

MCF 7 100 16.04 21.34 39.22

SiHa 100 24.08 32.30 33.46

HT 100 12.42 46.56 37.10

Ovcar 100 51.12 59.58 52.38

HepG2 100 28.22 36.20 42.76



MCF 7= breast cancer cell line

SiHa= cervix cancer cell line

HT= colon cancer cell line

Ovcar= Ovary cancer cell line

HepG2= Liver cancer cell line


123








Fig. 5.15: Anticancer activity of hydroalcoholic extract of A. paniculata and S. marianum

0

10

20

30

40

50

60

MCF 7 SiHa HT Ovcar HepG2

% in

hib

itio

n

Cell line

% inhibition AP

% inhibition SM

% inhibition AP+SM


124

Cell Line AP SM

MCF7

SiHa

HT

Ovcar

HepG2








Fig. 5.16: Anticancer activity of hydroalcoholic extract of A. paniculata and S. marianum


125

Cell line AP+SM Control (media)

MCF 7

SiHa

HT

Ovcar

HepG2








Fig. 5.17: Anticancer activity of hydroalcoholic extract of combination of A. paniculata and S. marianum


126

Medicinal plants maintain the health and vitality of individuals, and also

cure various diseases, including cancer without causing toxicity. These

medicinal plants possess good immunomodulatory and antioxidant

properties, leading to anticancer activities. The antioxidant

phytochemicals protect the cells from oxidative damage. Thus,

consuming a diet rich in antioxidant plant material can provide health-

protective effects. These natural products are supposed to minimize DNA

damage by reacting with free radicals and in this way they can prevent

cancer.

Phytochemicals, such as flavonols and flavonoids were

investigated to determine chemoprevention activity against cancer

(Conese and Blasi, 1995). Phenols, polyphenols, flavonoids and their

derivatives, are ubiquitous in plants and have been found associated

with the inhibition of atherosclerosis and cancer (Cirla and Mann, 2003).

Recent studies have reported antitumor effects of the flavonoids,

quercetin, genistein, and baicalein obtained from plant extracts

(Trangnos et al., 1992; Descher et al., 1991; Yoshida et al., 1990).

Similarly, alkaloids like schischkinnin and montamine have been

isolated from the seeds of Centaurea schischkinii and Centaurea montana

which showed anticancer property (Shoeb et al., 2006). Yang and Wang

(1993) reported the anticancer effect of polyphenols against tumor

formation and growth. Flavonoids isolated from Plantago species were

able to strongly inhibit the proliferation of human cancer cell lines

(Galvez et al., 2003). Aqueous extracts of Larrea tridentata and Juniperus

communis significantly decreased the growth of MCF-7 breast cancer


127

cells (Slambrouck et al., 2007). Flavopiridol is a synthetic flavone,

derived from the plant alkaloid rohitukine, which was isolated from

Dysoxylum binectariferum (Kellard et al., 2000). It is currently in phase I

and phase II clinical trials against a broad range of tumors, including

leukemia, lymphomas and solid tumors (Christian et al., 1997).

Synthetic agent roscovitine derived from natural product olomucine,

originally isolated from Raphanus sativus, is in Phase II clinical trials in

Europe (Cragg and Newman, 2005; Meijer et al., 2003).

Date post:	26-Mar-2020
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

5.1. Extraction - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/40994/11/11... ·...

Documents