5.1 Pharmacognostical Evaluation 5.1.1...

60
Chapter-5 Results Bhagwant University, Ajmer 83 5.1 Pharmacognostical Evaluation 5.1.1 Pharmacognostic studies of Emblica officinalis a) Macroscopical characters Table 12: Morphological features of fresh fruit of Emblica officinalis Sl.No. Features Observation 1. Color light yellowish 2. Odour Slight 3. Taste Sour 4. Shape Obovate - Elliptical 5. Size 2.5-3.5 cm in diameter 6. Surface Smooth with six prominent lines b) Microscopical characters: Anatomical study: The epicarpic cells are rectangular in shape and their outer and radial walls are highly cuticularized. In surface view the epicarpic cells appear polygonal in outline with thick walls. Anomoytic type of stomata are found to be present, but rare. Collateral fibro vascular bundles are scattered throughout the inner mesocarp. Pitted and helical tracheids with tapering ends are seen. At places in the phloem, large cavities filled with crystal mass are present. Powder characteristics: Colour: Buff green; Odour: Indistinct; Taste: Bitter. Microscopical characters of the powder include paracytic stomata, uniseriate multicellular covering trichomes, (usually collapsed trichomes), reticulated wood elements and lignocellulose fibers.

Transcript of 5.1 Pharmacognostical Evaluation 5.1.1...

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5.1 Pharmacognostical Evaluation

5.1.1 Pharmacognostic studies of Emblica officinalis

a) Macroscopical characters

Table 12: Morphological features of fresh fruit of Emblica officinalis

Sl.No. Features Observation

1. Color light yellowish

2. Odour Slight

3. Taste Sour

4. Shape Obovate - Elliptical

5. Size 2.5-3.5 cm in diameter

6. Surface Smooth with six prominent lines

b) Microscopical characters:

Anatomical study:

The epicarpic cells are rectangular in shape and their outer and radial walls are highly

cuticularized. In surface view the epicarpic cells appear polygonal in outline with thick

walls. Anomoytic type of stomata are found to be present, but rare. Collateral fibro

vascular bundles are scattered throughout the inner mesocarp. Pitted and helical tracheids

with tapering ends are seen. At places in the phloem, large cavities filled with crystal

mass are present.

Powder characteristics:

Colour: Buff green; Odour: Indistinct; Taste: Bitter. Microscopical characters of the

powder include paracytic stomata, uniseriate multicellular covering trichomes, (usually

collapsed trichomes), reticulated wood elements and lignocellulose fibers.

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Plate 3: Anatomy of the Pericarp

T.S. of the pericarp under low magnification(1) and Enlarged pericarp tissues(2)

Ep: Epidermis; GT:Ground Tissue; VS: Vascular Strand

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Plate 4: Fragment of the Epicarp of Emblica officinalis

Cells under low magnification (2.1) and enlarged cells showing cell wall structure (2.2)

Ep: Epidermal cells

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Plate 5: Structure of the Vascular Strands in the mesocarp of Emblica officinalis

Vascular strand and the ground tissue (3.1) and enlarged vascular strand showing xylem

and phloem (3.2) GT: Ground Tissue; Ph: Phloem; VB: Vascular Bundle; X: Xylem

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Plate 6: Powder Microscopic Structure of the Pericarp of Emblica officinalis

Ground parenchyma cells stained with Sudan-III to show the lipid bodies (4.1) and ged

lipid bodies (4.2) Li: Lipid

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5.1.2 Pharmacognostical studies of Eucalyptus globulus

a) Macroscopical characters

Table 13: Morphological features of fresh leaves of Eucalyptus globulus

Sl.No. Features Observation

1 Color Upper surface shining

Lower surface buff green color

2 Odour Strong and characteristic

3 Taste Bitter

4 Size Length in cms Width in cms

Min Max. Avg. Min Max. Avg.

22.3 24.5 23.4 4.5 3.6 4.1

5 Shape More or less scimitar shaped

6 Apex Acute to acuminate

7 Margin Entire/Undulate

8 Venation Reticulate

9 Surface

Dorsal surface Smooth

Ventral surface Slightly rough

10 Leaf base Obtuse

b) Microscopical characters:

Anatomical study:

Leaf - T.S. shows typical isobilateral structures with two or three rows of palisade cells

on both upper and lower sides, surfaces show thick cuticle; numerous sunken stomata and

large ovoid schizogenous oil cavities.; idioblasts present with rosettes or prismatic

calcium oxalate crystals; vascular bundle of midrib are crescent shaped with one vascular

strand present on each side, all having interrupted patches of sclerenchyma; corky warts

comprising of 10 or more layers of cells; laminary bundles enclosed in bundle sheath, the

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cells of which extend to the epidermis on both sides; upper and lower epidermal cells

have straight walls; stomata anomocytic.

Powder characteristics:

Colour: Yellowish brown; Odour: Indistinct; Taste: Bitter. Microscopical characters of

the powder include the presence of cluster and prismatic crystals of calcium oxalate;

epidermis straight walled with sunken stomata; fibers present.

Plate 7: TS of leaf midrib through laimina (a) and TS of midrib (b) of Eucalyptus

globulus

Abh - Abaxial hump; Adh – Adaxial hump; Col – Collenchyma; Ep – Epidermis; GT –

Ground tissue; La – Lamina; Ph - Phloem; Tpa – Transcurrent palisade tissue; VB –

Vascular bundles.

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Plate 8: Powder microscopical characters of Eucalyptus globulus

a. Diacytic stomata b. Multicellular uniseriate covering trichomes, c. Xylem vessel,

segments

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5.2 Physico-chemical evaluation

A) Foreign matter

Table 14 shows foreign matter of selected plants. Eucalyptus globulus showed

highest foreign matter compared to Emblica officinalis.

Table 14: Foreign matter of the selected plants

B) Extractive value

As shown in Table 15 extractive value of Emblica officinalis, and the percentage

yield was more in alcoholic extract than the water extract obtained by both cold and hot

maceration methods. Whereas in Eucalyptus globulus percentage yield of water extract was

more than that of alcoholic extract obtained by both cold and hot maceration methods.

Table 15: Alcoholic and water extractive value of the selected plants by cold and hot

maceration method

Plant Emblica officinalis

Eucalyptus globulus

Hot Cold Hot Cold

Alcoholic 57±0.76 42±0.66 20±0.63 13±0.92

Aqueous 55±0.52 32±0.9 28±1.7 26±3.4

C) Ash values

Table 16 shows ash values of selected plants. Eucalyptus globulus showed highest

ash content (total ash, water soluble ash, acid insoluble ash and sulphated ash)

compared to Emblica officinalis.

Plants Emblica officinalis

Eucalyptus globulus

Foreign matter 2±0.76 3±0.76

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Table 16: Ash values of the selected plants

D) Moisture content

Table 17 shows moisture content of selected plants. Eucalyptus globulus found to

contain high moisture content (6.78±1.66). Emblica officinalis contains the least

moisture content among the selected plants (2.42±1.42) %w/w of moisture content.

Table 17: Moisture content of the selected plants

All values are % w/w mean±S.D.

E) Swelling index

Table 18: Swelling index of the selected plants

F) Foaming index

Table 19: Foaming index of the selected plants

Plant Emblica officinalis

Eucalyptus globulus

Total ash 02±0.58 9±0.76

Water soluble

ash

1.1±2 .0 4.0±0.068

Acid insoluble

ash

0.55±0.22 0.093±0.024

Sulfated ash 0.5±012 0.82±0.20

Plants Emblica officinalis

Eucalyptus globulus

Moisture

content

2.42±1.42

6.78±1.66

Plants Emblica officinalis

Eucalyptus globulus

Swelling index 1.5ml/gm 1.7ml/gm

Plants Emblica officinalis

Eucalyptus globulus

Foaming index 15 17

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5.3 Physico-chemical evaluation

5.3.1 Extraction and fractionation of plant material:

The plant material of the both herbs were extracted and fractionated, the highest yield

was obtained from the ethyl acetate fraction and water fraction. Least yield was obtained

by Chloroform fraction. Percentage yield (w/w) of ethyl acetate fraction of Emblica

officinalis and Eucalyptus globulus, were found to be 6.67, and 11.45 where as

Chloroform fractions were 0.081and 0.45 respectively.

Table 20: Yield of extracts and fractions of selected herbs

5.3.2 Qualitative Phytochemical Screening:

Results of preliminary phytochemical screening are given in Table 21-22. It was found

that Emblica officinalis contains mainly sterols and lactones in pet ether fraction;

flavonoids, glycosides, carbohydrates and lactones in butanol fraction, ethyl acetate,

water fraction and water extract.

Eucalyptus globulus was found to contain mainly, sterols, glycosides, flavonoids and

tannins in pet ether fraction; flavonoids, hydrolysable tannins, glycosides and

carbohydrates in butanol, ethyl acetate, water fraction and water extract.

Sl.

No

Plant Drug taken

in gms

Extract/Fractions Total yield

in gm

% Yield in

gm w/w

1

Emblica

officinalis

625

Pet. Ether 1.17 0.18

Chloroform 0.51 0.081

Ethyl acetate 41.24 6.76

Butanol 40.27 6.6

Water fractions 15.0 2.4

100 Aq. alcoholic extract 8.65 17.3

2 Eucalyptus

globulus

600

Pet. Ether 3.0 0.5

Chloroform 4.5 0.45

Ethyl acetate 68.7 11.45

Butanol 37.0 6.16

Water fractions 36.4 6.07

100 Aq. alcoholic extract 11.34 22.68

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Table 21: Qualitative Phytochemical screening of Emblica officinalis

S.

No. Chemical Tests

Aq.

Alc.

Pet.

Ether

Ethyl

acetate Butanol

Water

fraction Chloroform

1.

Tests for Sterols

A) Salkowski test

B) Liebermann-Burchard

+

+

+

+

-

-

-

-

-

-

-

-

2.

Tests for

Triterpenes

A) Salkowski test

B) Liebermann-

Burchard test

-

-

-

-

-

-

-

-

-

-

-

-

3.

Tests for

Saponins

A) Foam test

B) Haemolysis test

-

-

-

-

-

-

-

-

-

-

-

-

4.

Tests for

Alkaloids

A) Wagner’s test

B) Mayer’s test

C) Dragendorff’s

test

D) Hager’s test

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5.

Tests for

Carbohydrates

A) Fehling’s test

B) Molisch’s test

+

+

-

-

+

+

+

+

+

+

-

+

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6.

Tests for Tannins

A) Ferric chloride

test

B) Gelatin test

C) Vanillin-HCl

test

D) Match stick test

-

-

-

-

-

-

-

-

+

+

+

+

+

-

-

-

+

-

-

-

+

-

-

-

7.

Tests for

Flavanoids

A) Shinoda test

B) Ferric chloride

test

C) Lead acetate

test

D) Zinc-HCl test

E) NaOH test

F) NaOH- HCl test

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

+

+

-

+

+

+

-

+

+

+

+

+

-

+

+

+

-

+

8.

Tests for

Lactones

/Cardiac

Glycosides

A. Legal’s test

B. Baljet’s test

+

+

+

+

+

+

+

+

+

+

+

+

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Table 22: Qualitative Phytochemical screening of Eucalyptus globules

Sl.

No. Chemical Tests

Aq.

Alc.

Pet.

Ether

Ethyl

acetate

Butan

ol

Water

fraction

Chloro

form

1. Tests for Sterols

A) Salkowski test

B) Liebermann-Burchard

+

+

+

+

-

-

-

-

-

-

-

-

2. Tests for Triterpenes

A) Salkowski test

B) Liebermann-Burchard

test

-

-

-

-

-

-

-

-

-

-

-

-

3. Tests for Saponins

A) Foam test

B) Haemolysis test

+

-

+

-

-

-

+

-

-

-

-

-

4. Tests for Alkaloids

A) Wagner’s test

B) Mayer’s test

C) Dragendorff’s test

D) Hager’s test

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5. Tests for Carbohydrates

A) Fehling’s test

B) Molisch’s test

+

+

-

-

+

+

+

+

+

+

+

+

6. Tests for Tannins

A) Ferric chloride test

B) Gelatin test

C) Vanillin-HCl test

D) Match stick test

+

-

-

-

+

-

-

-

+

-

-

-

+

-

-

-

+

-

-

-

+

-

-

-

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7. Tests for Flavanoids

A) Shinoda test

B) Ferric chloride test

C) Lead acetate test

D) Zinc- HCl test

E) NaOH test

F) NaOH- HCl test

-

+

+

-

+

+

-

+

-

-

-

-

+

+

+

+

+

+

-

+

-

-

+

+

-

+

-

-

+

+

-

+

-

-

+

+

8. Tests for

Lactones/Cardiac

Glycosides

A. Legal’s test

B. Baljet test

-

-

-

-

-

-

-

-

-

-

-

-

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5.3.3 Qualitative Phytochemical Screening:

5.3.3.1 Estimation of tannins

Table 23: Tannins of the selected plants

5.3.3.2 Estimation of total phenolic content

The total phenolic content estimated in the Emblica officinalis showed the absorbance

0.635 at 765 nm. The calibration curve of standard gallic acid is shown in fig.9

Fig 9: Calibration curve of Gallic acid

calibration curve of gallic acid

R2 = 0.9968

y = 0.1035x + 0.041

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12

concentration (μg/ml)

ab

so

rban

ce

Plants Emblica officinalis

Eucalyptus globulus

Tannins 12-18%w/w 0.5-1.5%w/w

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Table 24: Results of Total phenolic contents of Emblica officinalis and Eucalyptus

globulus

Fig. 10: Total phenolic content of different fractions of selected plants

0

50

100

150

200

250

300

350

400

Tota

l ph

en

olic

co

nte

nt

mg/

gm

Total phenolic content of different fraction of selected plants

Emblica officinalis

Eucalyptus globulus

Fractions Emblica officinalis Eucalyptus globulus

Pet. Ether 61.45±5.04 238.36±7.03

Chloroform 27.56±3.41 51.40±4.47

Ethyl acetate 216.01±5.59 346.37±16.76

Butanol 93.58±2.79 267.23±5.81

Water fractions 32.59±2.79 46.55±2.13

Aq. alcoholic

extract 24.67±2.78 68.90±2.13

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5.3.3.3. Estimation of total flavonoid

The total flavonoids content from the methanol extract of Eucalyptus globulus showed

the absorbance 0.297 at 415 nm. The calibration curve of standard rutin is shown in

fig.11.

Fig. 11: Calibration curve of Rutin

Table 25: Results of Total flavonoid content of Emblica officinalis and Eucalyptus

globulus

calibration curve of rutin

y = 0.0028x - 0.0154

R2 = 0.987

0

0.05

0.1

0.15

0.2

0.25

0.3

0 20 40 60 80 100 120

concentration (μg/ml)

ab

so

rban

ce

Fractions Emblica officinalis Eucalyptus globulus

Pet. Ether 28.86±4.27 35.38±3.51

Chloroform 64.89±6.46 20.11±2.23

Ethyl acetate 59.59±4.27 97.77±1.40

Butanol 54.93±5.81 96.83±2.13

Water fractions 24.21±4.27 18.62±2.13

Aq. alcoholic

extract 19.55±2.79 25.14±2.79

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Fig. 12: Total flavonoid content of different fractions of selected plants

0

20

40

60

80

100

120

Tota

l fla

von

oid

co

nte

nt

Total flavonoid content of different selected plant fractions

Emblica officinalis

Eucalyptus globulus

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5.3.3.4 Estimation of gallic acid in Emblica officinalis by RP-HPLC

Estimation of gallic acid in Emblica officinalis methanol extract was carried out

by RP-HPLC method using the optimized chromatographic conditions. The calibration

curve of standard gallic acid is shown in Fig. 14. A typical chromatogram of gallic acid is

shown in Fig 13. Detection was done at 255 nm. The retention time of gallic acid

standard was found to be 2.237 min. The retention time of gallic acid in the methanol

extract of Emblica officinalis was found to be 2.268 min. The peak area ratios of

standard and sample solutions were calculated. The assay procedure was repeated for six

times and the mean peak area and mean peak area ratios of standards were calculated.

The chromatogram of the methanol extract of Emblica officinalis is shown in Fig. 15.

The percentage of gallic acid in the methanol extract of Emblica officinalis was found

to be 0.0424% (w/w) as shown in table 11.

Fig. 13: Overlay chromatogram of standard (Gallic acid, quercetin and rutin)

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

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Fig. 14: Calibration curve of Gallic acid by RP-HPLC

Fig. 15: Chromatogram of gallic acid in the methanol extract of Emblica officinalis

GA

LLIC

AC

ID -

2.2

68

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

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Table 26: HPLC analysis of gallic acid in Emblica officinalis

Plant extract Constituents Amount found (% w/w)

Ethyl Acetate Fraction Gallic acid 1.542

Linearity and Range

The linearity of the method was determined at five concentration levels ranging from 0.1

to 1 μg/ml. The calibration curve was constructed by plotting peak area against

concentration of drugs. The slope and intercept value for calibration curve were Y =

3.77e+004X-1.43e+003 and R2

= 0.996. The results show an excellent correlation

between peak area and concentration of gallic acid within the concentration range

indicated above. The calibration curve is shown in Fig.14

5.3.3.5 Estimation of rutin in Eucalyptus globulus by RP-HPLC

Estimation of rutin in Eucalyptus globulus. methanol extract was carried out by RP-

HPLC method using the optimized chromatographic conditions. The calibration curve of

standard rutin is shown in Fig. 16. A typical chromatogram of rutin is shown in Fig 13.

Detection was done at 255 nm. The retention time of rutin standard was found to be 6.294

min. The retention time of rutin in Eucalyptus globulus the methanol extract was found to

6.458 min. The peak area ratios of standard and sample solutions were calculated. The

assay procedure was repeated for six times and the mean peak area and mean peak area

ratios of standards were calculated. The chromatogram of the methanol extract of

Eucalyptus globulus. is shown in Fig. 17. The percentage of gallic acid in the methanol

extract of Eucalyptus globulus was found to be 1.542% (w/w) as shown in table 26.

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Fig 16: Calibration curve of rutin by RP-HPLC

Fig 17: Chromatogram of rutin in the methanol extract of Eucalyptus globulus.

RU

TIN

- 6

.458

6.5

55

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

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Table 27: HPLC analysis of rutin in Eucalyptus globulus.

Plant extract Constituents Amount found (% w/w)

methanol Rutin 3.0425

Linearity and Range

The linearity of the method was determined at five concentration levels ranging from 0.1

to 1 μg/ml. The calibration curve was constructed by plotting peak area against

concentration of drugs. The slope and intercept value for calibration curve were Y =

4.26e+004X-1.15e+004 and R2

= 0.985. The results show an excellent correlation

between peak area and concentration of rutin within the concentration range indicated

above. The calibration curve is shown in fig. 16.

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5.4 Preparation and Characterization of Gallic acid Phytosomes:

Preparation:

In this study, we prepared the, gallic acid-phospholipids complex to improve the

lipophilic properties of gallic acid. We prepared the complex with different quantity

ratios of phospholipids and gallic acid such as 0.5, 1, 1.5, 2, 2.5 and 3. The results

showed that when the ratio was lower than 1, the stability of the gallic acid–

phospholipids complex was worse. To get the best complex and use the smallest quantity

of phospholipid, we finally prepared a gallic acid-phospholipids complex with a 1 ratio of

ingredients. The obtained complex was used for the subsequent structural analysis.

Process variables used for optimization

Table 28: Effect of types of alcohol on gallic acid phytosomes

Formulation

code

Vesicle size

(µm)

Entrapment

(%)

Cumulative amount of

drug permeated (mg/cm2)

GAPB 6.90 ± 0.184 86.02 ± 0.83 1.3052±0.086

GAPE 6.80 ± 0.237 89.12 ± 0.86 1.4184±0.068

GAPI 7.80 ± 0.267 85.00 ± 0.85 1.2506±0.065

Fig 18: Effect of type of alcohol on entrapment of gallic acid

828384858687888990

GAPB GAPE GAPI

% e

ntr

apm

en

t

Types of alcohol

Effect of types of alchol

Entrapment %

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Table 29: Effect of lecithin concentration on gallic acid phytosomes

Formulation

code

Vesicle size

(µm)

Entrapment (%) Cumulative amount of

drug permeated

(mg/cm2)

GAPL1 6.0 ± 0.131 78.49 ± 0.85 1.2801±0.031

GAPL2 6.8 ± 0.138 82.26 ± 0.60 1.4236±0.085

GAPL3 7.0 ± 0.057 85.03 ± 0.449 1.4236±0.026

GAPL4 7.0 ± 0.064 88.64 ± 0.56 1.3460±0.025

GAPL5 7.0 ± 0.09 92.26 ± 0.70 1.3452±0.048

Fig 19: Effect of lecithin concentration on entrapment of gallic acid

70

75

80

85

90

95

0.5 1 1.5 2 3

% E

ntr

apm

en

t

Lecithin concentration (%)

Entrapment efficeancy

Entrapment (%)

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Table 30: Effect of Drug concentration on gallic acid phytosomes

Formulation

code

Vesicle size

(µm)

Entrapment

(%)

Cumulative amount of

drug permeated

(mg/cm2)

GAPD1 7.8 ± 0.048 90.10 ± 0.758 1.0530±0.076

GAPD2 7.0 ± 0.073 89.80 ± 0.65 1.3215±0.083

GAPD3 7.0 ± 0.085 89.58 ± 0.70 1.3913±0.079

GAPD4 6.0 ± 0.054 89.49 ± 0.54 1.5012±0.086

GAPD5 4.0 ± 0.061 86.20 ± 0.450 1.2721±0.049

Fig 20: Effect of drug concentration on entrapment

84

85

86

87

88

89

90

91

3 2.5 2 1.5 1

% E

ntr

apm

en

t

Drug concentration (%)

Effect of drug concentration

Entrapment (%)

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Table 31: Effect of Ethanol Concentration on gallic acid phytosomes

Formulation

code

Vesicle size

(µm)

Entrapment

(%)

Cumulative amount of

drug permeated

(mg/cm2)

GAPE1 6.0 ± 0.074 88.00 ± 0.873 1.3406±0.035

GAPE2 6.0 ± 0.097 89.49 ± 0.488 1.4890±0.093

GAPE3 6.8 ± 0.068 89.48 ± 0.657 1.4794±0.067

GAPE4 7.0 ± 0.083 88.20 ± 0.731 1.4779±0.052

Fig21: Effect of ethanol concentration on entrapment of gallic acid

87.8

88

88.2

88.4

88.6

88.8

89

89.2

89.4

89.6

0 5 10 15 20 25

% e

ntr

apm

en

t

Ethanol concentration (%)

Effect of ethanol concentration

Entrapment (%)

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Table 32: Effect of Types of alcohols on cumulative amount of drug permeated

Time(hrs) Cumulative Amount Of Drug Permeated (Mg / Cm2)

GAPB GAPE GAPI

1 0.1270±0.084 0.1592±0.074 0.0947±0.082

2 0.2926±0.095 0.2942±.035 0.2275±0.054

3 0.4661±0.096 0.5000±0.028 0.3343±0.061

4 0.5841±0.010 0.6508±0.036 0.4450±0.042

5 0.7069±0.056 0.7768±0.096 0.5614±0.019

6 0.8335±0.086 0.9076±0.069 0.6503±0.071

7 0.9658±0.037 1.0421±0.061 0.7747±0.066

8 1.0395±0.054 1.0867±0.078 0.8716±0.091

24 1.3052±0.086 1.4184±0.068 1.2506±0.065

Fig 22: Effect of types of alcohols on cumulative amount of drug permeated

0

0.5

1

1.5

1 2 3 4 5 6 7 8 24

cum

. am

ou

nt

pe

rme

ate

dm

g/cm

2

Time (hrs)

Types of alcohol

GAPB

GAPE

GAPI

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Table 33: Effect of Lecithin Concentration on cum. amount of drug permeated

Time(hrs) Cumulative amount of drug permeated (mg / cm2)

GAPL1 GAPL2 GAPL3 GAPL4 GAPL5

1 0.1270±0.032 0.1592±0.058 0.1592±0.048 0.1270±0.064 0.1210±0.067

2 0.2603±0.095 0.2619±0.094 0.2619±0.098 0.2603±0.049 0.2624±0.037

3 0.4010±0.074 0.4338±0.054 0.4338±0.064 0.4645±0.062 0.4541±0.046

4 0.4836±0.068 0.4545±0.061 0.4545±0.079 0.6137±0.054 0.6159±0.042

5 0.6016±0.046 0.5074±0.029 0.5074±0.094 0.7069±0.031 0.7105±1.004

6 0.6922±0.051 0.6567±0.082 0.6567±0.084 0.8782±0.094 0.8777±0.031

7 0.8181±0.083 0.8133±0.074 0.8133±0.046 1.0003±0.054 1.0003±0.085

8 0.9489±0.049 0.9753±0.091 0.9753±0.055 1.1390±0.082 1.1389±0.046

24 1.2801±0.031 1.4236±0.085 1.4236±0.026 1.3460±0.025 1.3452±0.048

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Fig 23: Effect of lecithin concentration on cumulative amount of drug permeated

Table 34 Effect of drug Concentration on cumulative amount of drug permeated

Time

(hrs)

cumulative amount of drug permeated (mg / cm2)

GAPD1 GAPD 2 GAPD 3 GAPD 4 GAPD 5

1 0.0791±0.03 0.1270±0.086 0.1592±0.082 0.1905±0.05 0.1270±0.08

2 0.1944±0.04 0.2926±0.045 0.2619±0.091 0.3915±0.08 0.2603±0.06

3 0.3319±0.09 0.4661±0.064 0.4338±0.052 0.6114±0.03 0.4322±0.02

4 0.5071±0.04 0.6153±0.091 0.4545±0.062 0.6947±0.04 0.4851±0.02

5 0.6580±0.06 0.7397±0.034 0.5074±0.046 0.8213±0.06 0.5397±0.03

6 0.7205±0.08 0.8689±0.065 0.6254±0.035 0.9536±0.04 0.6271±0.04

7 0.8470±0.06 1.0028±0.082 0.8117±0.095 1.0907±0.08 0.7499±0.08

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 2 3 4 5 6 7 8 24

cum

.am

ou

nt

pe

rme

ate

dm

g/cm

2

Time (hrs)

Effect of Lecithin concentration

GAPL1

GAPL2

GAPL3

GAPL4

GAPL5

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8 0.9794±0.03 1.1416±0.038 0.9425±0.034 1.1941±0.02 0.8764±0.08

24 1.0530±0.07 1.3215±0.083 1.3913±0.079 1.5012±0.08 1.2721±0.04

Fig24: Effect of different concentration of drug on cumulative amount of drug

permeated

Table 35: Effect of ethanol concentration on cumulative amount of drug permeated.

Time(hrs) Cumulative amount of drug permeated (mg / cm2)

GAPE1 GAPE 2 GAPE 3 GAPE 4

1 0.1592±0.068 0.1905±0.031 0.1592±0.031 0.1905±0.024

2 0.2942±0.033 0.3915±0.062 0.3577±0.061 0.3592±0.074

3 0.4677±0.084 0.5376±0.056 0.4709±0.041 0.4402±0.084

4 0.6170±0.031 0.6910±0.085 0.6524±0.072 0.6846±0.081

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 2 3 4 5 6 7 8 24

Cu

m. a

mo

un

t p

erm

eat

ed

mg/

cm2

Time (hrs)

Effect of drug concentration

GAPD1

GAPD 2

GAPD 3

GAPD 4

GAPD 5

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5 0.7413±0.066 0.8176±0.094 0.7784±0.046 0.8434±0.092

6 0.8705±0.018 0.9499±0.035 0.9092±0.085 0.8816±0.034

7 0.9722±0.035 1.0548±0.027 1.0124±0.067 1.0156±0.055

8 1.0458±0.042 1.1950±0.037 1.1189±0.085 1.1543±0.044

24 1.3406±0.035 1.4890±0.093 1.4794±0.067 1.4779±0.052

Fig 25: Effect of different concentration of ethanol on cumulative amount of drug

permeated

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 2 3 4 5 6 7 8 24

Cu

m. a

mo

un

t p

erm

eat

ed

mg/

cm2

Time (hrs)

Ethanol concemtration

GAPE1

GAPE 2

GAPE 3

GAPE 4

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Table 36: In vitro permeation study of optimized gallic acid phytosomes

Time(hrs) Cumulative amount of drug

permeated (mg / cm2) RPE3

1 0.1905±0.031

2 0.3915±0.062

3 0.5376±0.056

4 0.6910±0.085

5 0.8176±0.094

6 0.9499±0.035

7 1.0548±0.027

8 1.1950±0.037

24 1.4890±0.093

Fig 26: In vitro permeation study of optimized gallic acid phytosomes

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 2 3 4 5 6 7 8 24

Cu

m. a

mo

un

t p

erm

eat

ed

mg/

cm2

Time (hrs)

Optimized prepration GAPE 2

GAPE 2

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Characterization: The prepared Gallic acid-phospholipid complex (1:1) was subjected

to structural analysis by Scanning electron microscopy (SEM), transmission electron

microscopy (TEM), solubility studies, differential scanning calorimetry (DSC) of the

complex, UV spectroscopy and IR spectroscopy.

Scanning Electron Microscopy (SEM)

The surface morphology of gallic acid –phospholipids complex as shown in Figure.3,

indicate the presence of spherical shape of complex. The vesicles consisted of

phospholipids and gallic acid was intercalated in lipid layer.

Fig 27: Scanning electron micrographs of gallic acid phytosomes at ×200

magnification

Transmission Electron Microscopy (TEM)

The TEM of gallic acid phospholipids complex after shaking in distilled water are shown

in Figure.4. We could observed that there were many particles suspended in the distilled

water and infusible particles still exited in the solution. For phospholipids complex, the

drugs were combined with phospholipids by the polar part of phospholipids, when

swirled in distilled water many complex molecules arranged in order and formed the

structure of vesicles.

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Fig 28: Transmission electron micrographs of gallic acid phytosomes after slightly

shaking in distilled water at×4000 magnification

Solubility Studies

Determination of solubility characteristics of gallic acid, gallic acid–phospholipids

complex and physical mixture of gallic acid and phospholipids in water and n-octanol

were shown in table 37.

Table 37: Apparent solubility of Gallic acid, gallic acid–phospholipids complex and

physical mixture of gallic acid and phospholipids in water at 25 ◦C

Sample Solubility in water

(µg/ml)

Solubility in n-octanol

(µg/ml)

Gallic acid 10.86±2.73 6.63±1.86

Gallic acid -phospholipids

complex

26.35±1.78 32.31±3.76

Physical mixture of Gallic acid

and phospholipids

18.12±1.03 11.66±2.70

Differential Scanning Calorimetry (DSC) of the complex

The DSC thermo grams of phospholipids, gallic acid, their physical mixture and

phospholipids complex were shown in Figure 5. Phospholipids show two different kinds

of endothermal peaks, and the first (74.85°C) endothermal peak appears mild, it was

considered that the formation of this peak was duo to hot movements of phospholipids

molecule polarity parts. However, the second endothermal peak at 190.6◦C appears

sharp-pointed; it was considered that owing to the transition from gel state to liquid

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crystal state, the carbon–hydrogen chain in phospholipids perhaps happened to be melt,

isomerous or the crystal changes. Gallic acid is not pure, so it shows abroad endothermal

peak, and its beginning melting point at 136.5 ◦C. Physical mixture of gallic acid and

phospholipids shows that there are two endothermal peaks, and the former is 28.8 ◦C, the

same with the onset temperature of phospholipids complex; another is 136.5 ◦C, the same

with the onset temperature of gallic acid. It was considered that when the temperature

was increased, phospholipids were melt and drugs were dissolved in the phospholipids

and partly formed phospholipids complex, which could be explained through the theory

of preparation by melt-out method. DSC of phospholipids complex shows the

endothermal peaks of drug and phospholipid are disappeared and the phase transition

temperature is lower than the phase transition temperature of phospholipids After the

combination of gallic acid and the phospholipids molecule polarity parts, the carbon–

hydrogen chain in phospholipids could turn freely and enwrap the phospholipids

molecule polarity parts, which made the sequence decrease between phospholipids

aliphatic hydrocarbon chains, made the second endothermal peak of phospholipids

disappear and depressed the phase transition temperature.

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Fig 29: DSC thermo grams of Gallic acid (1), Phospholipids (2), Physical mixture of

Gallic acid - phospholipids (3), Gallic acid -phospholipids complex (4).

UV and IR Analysis

The UV and IR spectra of phospholipid, gallic acid, their physical mixture and the

complex are shown in Figure.6 and 7 respectively.

Fig 30: UV spectra of phospholipids (1), physical mixture of gallic acid and

phospholipids (2), Gallic acid-phospholipids complex (3) and gallic acid (4).

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Fig 31: IR spectra of phospholipid (1), physical mixture of gallic acid and

phospholipids (2), Gallic acid-phospholipid complex (3) and gallic acid (4).

5.5 Preparation of Rutin-phospholipids complex:

Preparation:

Rutin-phospholipid complex was prepared by anhydrous co-solvent lyophilization

method. In briefly, rutin powder was dissolve in methanol and phospholipid was dissolve

in methanol separately. Both are mix by gentle agitation until formation of a clear

mixture. The resultant homogeneous solution was then freeze-dried under vacuum and

stored in air tight container for further use . For preparation of complex in the ratio of 1:1,

100 mg of rutin was dissolved in 10 ml of methanol separately and 100 mg phospholipid

was dissolved in 10 ml chloroform, separately. Both the solution was mixed and stirred in

mechanical stirrer up to methanol was completely evaporated. The residue was ground

and the resultant powder was collected as rutin-phospholipid complex.

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Table 38: Effect of lecithin concentration on rutin phytosomes

Formulation

code

Vesicle size

(µm)

Entrapment (%) Cumulative amount of

drug permeated

(mg/cm2)

RPL1 6.0 ± 0.121 77.03±0.80 0.32±0.067

RPL2 6.5 ± 0.122 92.01±0.92 0.42±0.022

RPL3 7.0 ± 0.157 91.06±0.88 0.41±0.014

RPL4 7.0 ± 0.064 91.21±0.79 0.33±0.036

RPL5 7.0 ± 0.59 90.26 ± 0.70 0.34±0.048

Fig32: Effect of lecithin concentration on entrapment efficiency.

65

70

75

80

85

90

95

RPL1 RPL2 RPL3 RPL4 RPL5

Entr

apm

en

t e

ffic

ean

cy (

%)

Formulation code

Entrapment efficeancy

Entrapment (%)

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Table 39: Effect of Drug concentration on rutin phytosomes

Formulation

code

Vesicle size

(µm)

Entrapment

(%)

Cumulative amount of

drug permeated

(mg/cm2)

RPD1 7.4 ± 0.042 88.10 ± 0.758 0.45±0.054

RPD2 7.0 ± 0.013 90.11±0.1.01 0.65±0.049

RPD3 7.0 ± 0.025 89.52 ± 0.70 0.32±0.073

RPD4 6.2 ± 0.014 89.86±0.85 0.27±0.080

RPD5 5.0 ± 0.061 89.28±0.98 0.49±0.032

Fig 33: Effect of drug concentration on entrapment efficiency

87

87.5

88

88.5

89

89.5

90

90.5

RPD1 RPD2 RPD3 RPD4 RPD5

Entr

apm

en

t e

ffic

ean

cy (

%)

Formulation code

Effect of drug concentration

Entrapment (%)

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Table 40: Effect of Ethanol Concentration on rutin phytosomes

Formulation

code

Vesicle size

(µm)

Entrapment

(%)

Cumulative amount of

drug permeated

(mg/cm2)

RPE1 7.0 ± 0.075 84.59±0.86 0.30±0.010

RPE2 6.9 ± 0.027 87.45±0.81 0.38±0.012

RPE3 6.2 ± 0.061 91.06±0.79 0.41±0.080

RPE4 7.0 ± 0.081 62.31±0.84 0.35±0.022

Fig 34: Effect of ethanol concentration on entrapment efficiency

0

10

20

30

40

50

60

70

80

90

100

RPE1 RPE2 RPE3 RPE4

% e

ntr

apm

en

t

Formulation code

Effect of etanol concentration

Entrapment (%)

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Chapter-5 Results

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Table 41: Effect of lecithin concentration on cumulative amount of drug permeated

Time (hrs) Cumulative amount of drug permeated (mg/cm

2)

RPL1 RPL2 RPL3 RPL4

1 0.02±0.004 0.03±0.001 0.03±0.005 0.02±0.084

2 0.03±0.003 0.05±0.004 0.05±0.084 0.04±0.084

3 0.05±0.001 0.10±0.022 0.10±0.001 0.07±0.022

4 0.07±0.003 0.14±0.012 0.14±0.014 0.10±0.021

5 0.09±0.001 0.20±0.001 0.18±0.013 0.14±0.001

6 0.14±0.084 0.25±0.034 0.24±0.009 0.18±0.004

7 0.20±0.057 0.30±0.067 0.29±0.084 0.23±0.013

8 0.26±0.043 0.35±0.023 0.34±0.031 0.27±0.069

24 0.32±0.067 0.42±0.022 0.41±0.014 0.33±0.036

Fig 35: Effect of lecithin concentration on cumulative amount of drug permeated

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

1 2 3 4 5 6 7 8 24

Cu

m. a

mo

un

t p

erm

eat

ed

mg/

cm2

Time (hrs)

Effect of lecithin concentration

RPL1

RPL2

RPL3

RPL4

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Chapter-5 Results

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Table 42: Effect of drug concentration on cumulative amount of drug permeated

Time(hrs) cumulative amount of drug permeated (mg / cm2)

RPD1 RPD 2 RPD 3 RPD 4 RPD 5

1 0.03±0.003

0.070±0.021 0.01±0.005 0.01±0.002

0.06±0.009

2 0.06±0.007

0.093±0.062 0.03±0.003 0.02±0.003

0.08±0.008

3 0.10±0.009

0.14±0.028 0.05±0.004 0.04±0.004

0.13±0.013

4 0.15±0.016

0.19±0.027 0.08±0.005 0.06±0.007

0.18±0.016

5 0.20±0.012

0.33±0.021 0.11±0.004 0.09±0.002

0.22±0.014

6 0.25±0.062

0.37±0.046 0.15±0.011 0.12±0.013

0.28±0.022

7 0.31±0.024

0.44±0.015 0.19±0.030 0.16±0.010

0.33±0.054

8 0.37±0.072

0.57±0.087 0.24±0.037 0.21±0.071

0.39±0.063

24 0.45±0.054

0.77±0.029 0.32±0.073 0.27±0.080

0.49±0.032

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Fig 36: Effect of lecithin concentration on cumulative amount of drug permeated

Table 43: Effect of ethanol concentration on cumulative amount of drug permeated.

Time(hrs) Cumulative amount of drug permeated (mg / cm2)

RPE1 RPE2 RPE3 RPE4

1 0.01±0.004 0.02±0.002 0.03±0.001 0.02±0.005

2 0.02±0.003 0.04±0.004 0.05±0.005 0.05±0.002

3 0.05±0.007 0.08±0.004 0.10±0.009 0.08±0.003

4 0.07±0.006 0.11±0.003 0.14±0.008 0.11±0.031

5 0.09±0.001 0.17±0.002 0.18±0.005 0.15±0.054

6 0.14±0.009 0.21±0.004 0.24±0.004 0.20±0.023

7 0.17±0.006 0.26±0.007 0.29±0.002 0.24±0.067

8 0.22±0.004 0.31±0.013 0.34±0.084 0.29±0.071

24 0.30±0.010 0.38±0.012 0.41±0.080 0.35±0.022

0

0.2

0.4

0.6

0.8

1

1 2 3 4 5 6 7 8 24

Cu

m a

mo

un

t p

erm

eat

ed

mg/

cm2

Time (hrs)

Effect of drug concentration

RPD1

RPD 2

RPD 3

RPD 4

RPD 5

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Fig37: Effect of ethanol concentration on cumulative amount of drug permeated

Table 44: In vitro permeation study of optimized rutin phytosomes

Time(hrs) Cumulative amount of drug

permeated (mg / cm2) RPE3

1 0.03±0.001

2 0.05±0.005

3 0.10±0.009

4 0.14±0.008

5 0.18±0.005

6 0.24±0.004

7 0.29±0.002

8 0.34±0.084

24 0.41±0.080

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

1 2 3 4 5 6 7 8 24

Cu

m a

mo

un

t p

erm

eat

ed

mg/

cm2

Time (hrs)

Effect of ethanol concentration

RPE1

RPE2

RPE3

RPE4

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Fig 38: In vitro permeation study of optimized rutin phytosomes

Characterization:

The prepared Rutin-phospholipid complex was subjected to structural analysis by

Scanning electron microscopy (SEM), transmission electron microscopy (TEM),

solubility studies, differential scanning calorimetry (DSC) of the complex, UV

spectroscopy and IR spectroscopy.

Scanning Electron Microscopy (SEM)

The surface morphology of Rutin –phospholipids complex as shown in Figure.3, indicate

the presence of spherical shape of complex. The vesicles consisted of phospholipids and

gallic acid was intercalated in lipid layer.

Fig 39: Scanning electron micrographs of rutin phytosomes at ×200 magnification

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

1 2 3 4 5 6 7 8 24

Cu

m. a

mo

un

t p

erm

eat

ed

mg/

cm2

Time (hrs)

Optimized prepration RPE3

RPE3

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Transmission Electron Microscopy (TEM)

The TEM of Rutin acid phospholipids complex after shaking in distilled water are shown

in Figure.4. We could observed that there were many particles suspended in the distilled

water and infusible particles still exited in the solution. For phospholipids complex, the

drugs were combined with phospholipids by the polar part of phospholipids, when

swirled in distilled water many complex molecules arranged in order and formed the

structure of vesicles.

Fig 40: Transmission electron micrographs of rutin phytosomes after slightly

shaking in distilled water at×4000 magnification

Solubility Studies

Determination of solubility characteristics of Rutin, Rutin–phospholipids complex and

physical mixture of Rutin and phospholipids in water and n-octanol were shown in table

Table 45: Apparent solubility of Rutin, Rutin–phospholipids complex and physical

mixture of Rutin and phospholipids in water at 25 ◦C

Sample Solubility in water

(µg/ml)

Solubility in n-octanol

(µg/ml)

Rutin 14.26±2.13 6.63±1.86

Rutin -phospholipids

complex

22.15±1.18 31.35±3.16

Physical mixture of Rutin

and phospholipids

17.11±1.01 18.26±1.70

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Chapter-5 Results

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Differential Scanning Calorimetry (DSC) of the Complex

Fig 41: DSC thermo grams of rutin phytosomes

UV and IR Analysis

The UV spectra of rutin and their phospholipid complex are shown in Figure 4. The

characteristic absorption peaks of rutin (271 nm) were still present. The infrared spectra

of phospholipid, rutin and complexes are shown in Figure 5.

Fig 42: Overlay UV Spectra of Rutin and Rutin-Phospholipid Complex

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Chapter-5 Results

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Fig 43: IR Spectra of Phospholipid and Rutin-Phospholipid Complex (1:1, 1:2 &1:3)

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5.6 In vitro free radical scavenging and antioxidant screening

5.6.1 Free radical scavenging activity by DPPH Method

The high DPPH radical scavenging activity of BHT and gallic acid-phospholipids

complex was observed in a concentration dependent manner. The results are shown in

Figure 8. The high DPPH radical scavenging activity of the complex is due the presence

of higher amount of phenolic content.

Table 46: Free radical scavenging activity of different extracts and gallic acid

phytosomes

Conc. in µg/ml GAP EOE EOB

10 6.34±1.60 4.36±1.05 4.11±1.55

20 14.13±1.57 7.50±1.30 6.19±1.80

30 22.97±2.63 11.66±1.50 9.36±1.45

40 31.87±1.03 15.97±2.51 13.94±1.46

50 38.29±1.18 21.59±2.71 16.40±1.52

60 45.08±1.30 29.19±1.58 25.39±2.59

70 51.85±1.60 38.33±1.22 33.30±2.51

80 63.49±1.11 50.31±1.85 39.41±2.56

IC50 in

µg/ml 123.1±3 181.8±.0.5 230±10

Table 47: Free radical scavenging activity of BHT and Ascorbic acid

Conc. in µg/ml

Ascorbic acid Conc. in µg/ml

BHT

4.54 42.96 ±1.06 4.54 31.66 ±0.46

9.09 49.32 ±0.37 9.09 52.18 ±1.56

13.63 75.93 ±0.86 13.63 77.08 ±1.12

18.18 94.70 ±0.33 18.18 83.07 ±1.20

22.72 95.41 ±0.39 22.72 88.39 ±0.93

27.27 90.67 ±0.44

IC50 in µg/ml

4.91±0.36 21.88±2.1

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Chapter-5 Results

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Fig 44: DPPH radical scavenging activity of gallic acid-phospholipids complex

5.6.2 Reduction of ferric ions

Table 48: Ferric ion reduction activity of different fractions and phytosomes of

Emblica officinalis

Conc. in

µg/ml

EOE EOB GAP

10 21.40±2.45 10.23±1.31 22.26±1.94

20 28.23±1.18 12.54±0.68 33.49±2.10

40 40.24±1.32 19.91±2.09 57.73±2.92

60 55.49±4.01 25.08±1.36 85.62±7.25

80 69.89±5.44 33.68±1.21

IC50 in

µg/ml 44±1.86 132±13.76 32±±0.4

Table 49: Ferric ion reduction activity of standards

Conc. in µg/ml

BHT Conc. in µg/ml

ASC

10 7.51 ±1.05 2 18.87 ±0.64

20 11.55 ±0.74 4 26.63 ±2.06

40 12.11 ±1.06 6 38.72 ±5.62

60 14.25 ±0.33 8 48.66 ±5.09

80 16.67 ±0.35 10 60.95 ±1.43

IC50 in µg/ml

ND 7.83 ±0.73

0

10

20

30

40

50

60

70

10 20 30 40 50 60 70 80

% r

ed

ical

sca

ven

gin

g

Concentration in ug/ml

DPPH radical scavenging activity

GAP

EOE

EOB

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Fig 45: Ferric ion reduction activity of gallic acid-phospholipids complex

5.7 Pharmacological evaluation

5.7.1 Acute Toxicity Study

The LD50 value by the oral route could not be determined as no mortality was observed

until a dose of 8 g/kg of gallic acid Phytosomes.

5.7.2 Evaluation of hepatoprotective activity of gallic acid Phytosomes in vivo.

In this experiment, on the basis of biochemical evaluation shows in table no. 50, we find

that CCl4 induced toxicity has increased the serum Bilirubin, GOT,GPT level to

significantly higher level when compared to normal (P<0.001) as the table no. 1 show .

The selected gallic acid phytosmes were able to reduce the increased bilirubin, SGOT and

SGPT to highly significant level (P<0.001). Silymarin, GAP 60mg and GAP 40 mg when

compared to normal, found to be non significant. This shows that bilirubin, SGOT and

SGPT level of normal Silymarin, GAP 60mg and GAP 40 mg were similar indicating

reversal of liver injury caused by CCl4.

Lipid peroxidation, measured in terms of Malondialdehyde (MDA) in rat liver

homogenate was significantly increased (P<0.001) in CCl4 group (Control) as compared

to Normal group. MDA level of groups treated with gallic acid, gallic acid Phytosomes

and Silymarin significantly decreased the MDA content as compared to Control. when

compared to Normal, Silymarin, GA 100mg, GA 200mg, GAP40mg and GAP60mg were

0

10

20

30

40

50

60

70

80

90

10 20 40 60 80

% f

err

ic io

n r

ed

uct

ion

Concentration in ug/ml

Ferric ion reduction activity

EOE

EOB

GAP

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Chapter-5 Results

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found to be insignificant (P>0.05). This indicates that liver injury caused by CCl4 was

almost reversed by Silymarin, GA 100mg, GA 200mg, GAP 40mg and GAP 60mg.

SOD activity in CCl4 treated group (Control - 4.78 U/mg protein) was found significantly

low when compared with the Normal group (22.89 U/mg protein, P<0.001). SOD levels

of GA 200mg, GAP 40mg and GAP 60mg were significant to the level of P<0.00,

whereas SOD levels of GA 100mg was found less significant with( P<0.01 ) when

compared to Control. Silymarin at 50 mg/kg completely restored the enzyme activity

(22.69 U/mg proteins) to the normal level. GAP 60 mg restored the normal enzyme level

equally significant to the Silymarin. i.e when compared to the Normal level of SOD, both

Silymarin and GAP 60 mg were found to be insignificant (P<0.05). This shows that

Normal group and groups treated with Silymarin and GAP 60 mg are close to each other.

Catalase activity in CCl4 group (Control - 1.78 U/mg protein) was observed to be

strikingly lower than the Normal group (5.15 U/mg protein, P<0.001). In case of

Silymarin, GAP 60 mg and GAP 40 mg CAT activity when compared to Control was

found to be highly significant (P<0.001). GA 100mg and GA 200mg also increased the

CAT level when compared to Control but less significantly (P<0.01). Silymarin at 50

mg/kg completely restored the enzyme activity (5.00 U/mg protein) to the normal level.

GAP 60 mg also restored the normal enzyme level equally significant to the Silymarin.

When compared to the Normal group Silymarin and GAP 60 mg showed no significant

difference indicating no difference between Normal, GAP 60 mg and Silymarin.

GSH level in the liver homogenate of Normal and Control group were found to be

11.41 and 3.16 nmol/mg of protein. GAP 60 mg, GAP 40 mg and GA 200 mg were

highly significant (P<0.001), Ga 100mg was less significant (P<0.01) when compared to

Control. But when compared to Normal GAP 60 mg GAP 40 mg ,GA 200 mg and GA

100mg were found to be insignificant indicating that the results obtained were very close

to Normal. Also, Silymarin almost completely restored the glutathione level in CCl4

treated groups to the normal level. Over all the plant extracts showed hepatoprotective

activity in CCl4 induced liver toxicity. But among the five plant extracts GAP 60 mg and

GAP 40 mg were found to be very potent.

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Chapter-5 Results

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Table 50: Effect of GA and GAP on various biochemical parameters in toxicity

induced rat liver

Bilirubin

in mg/dl

SGOT

in U/l

SGPT

in U/l

TBARS

in mol/mg

Catalase

in U/mg

SOD

in U/mg

GSH

in mol/mg

Normal 1.00 ±0.31*** 71.58 ±4.82*** 74.38 ±4.18*** 139.16 ±27.90*** 5.15 ±0.86*** 22.89 ±5.49*** 11.41 ±2.46***

Control 2.78 ±0.44 168.31 ±4.62 182.11 ±6.72 446.94 ±55.84 1.78 ±1.05 4.78 ±4.51 3.16 ±2.05

CCl4 +

sily (50

mg/kg) 0.90 ±0.13a 71.88 ±4.93***a 72.616 ±7.79 a 141.48 ±31.29***a 5.00 ±1.21***a 22.69 ±6.88***a 11.08 ±2.23***a

CCl4

+GA

(100

mg/kg) 1.72 ±0.35*** 126.16 ±7.78*** 156.83 ±8.11*** 297.13 ±47.00*** 4.33 ±1.66** a 16.91 ±5.04** a 7.81 ±1.38** a

CCl4

+GA

(200

mg/kg) 1.43 ±0.18** 124.2 ±2.71*** 135.33 ±10.8*** 189.30 ±18.23***a 4.57 ±0.90** a 20.69 ±6.69***a 8.76 ±2.59***a

CCl4

+GAP

(40

mg/kg) 0.97 ±0.14*** 72.96 ±3.19***a 119.16 ±9.59*** 172.97 ±76.87***a 4.89 ±1.06***a 22.21 ±4.54***a 10.17 ±1.17***a

CCl4

+GAP

(60

mg/kg) 1.07 ±0.29***

72.7 ±4.50***a 75.733 ±5.85 a 147.67 ±74.10***a 4.93 ±0.35***a 22.39 ±3.43***a 10.77 ±2.06***a

*** - p<0.001 Highly significant when compared to Control

** - p<0.01 Significant when compared to Control

* - p<0.05 Significant when compared to Control

a - Non significant when compared to Normal

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Chapter-5 Results

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Fig 46: Effect of phytosomes on serum bilirubin in CCl4 intoxicated rats

Fig 47: Effect of phytosomes on serum GOT levels in CCl4 intoxicated rats

0

0.5

1

1.5

2

2.5

3

Seru

m b

iliru

bin

in m

g/d

l

Effect of phytosomes on serum bilirubin

Bilirubin in mg/dl

0

20

40

60

80

100

120

140

160

180

Normal Control sily GA 100 GA 200 GAP 40 GAP 60

SGO

T in

U/l

Effect of phytosomes on serum GOT levels

SGOT in U/l

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Chapter-5 Results

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Fig 48: Effect of phytosomes on serum GPT levels in CCl4 intoxicated rats

Fig 49: Effect of phytosomes on TBARS levels in CCl4 intoxicated rats

0

20

40

60

80

100

120

140

160

180

200

Normal Control sily GA 100 GA 200 GAP 40 GAP 60

SGP

T in

U/l

Effect of phytosomes on serum GPT levels

SGPT in U/l

0

50

100

150

200

250

300

350

400

450

500

TBA

RS

in m

ol/

mg

Effect of phytosomes on TBARS level

TBARS in mol/mg

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Chapter-5 Results

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Fig 50: Effect of phytosomes on catalase of protein in CCl4 intoxicated rats

Fig 51: Effect of phytosomes on SOD of protein in CCl4 intoxicated rats

0

1

2

3

4

5

6

Cat

alag

e in

U/m

g o

f p

rote

in

Effect of phytosomes on catalage

Catalase in U/mg

0

5

10

15

20

25

Normal Control sily GA 100 GA 200 GAP 40 GAP 60

SOD

in U

/mg

of

pro

tein

SOD activity following the treatment wtih phytosoms

SODin U/mg

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Fig 52: Effect of phytosomes on GSH of protein in CCl4 intoxicated rats

Plate 9: Histopathology of rat livers

0

2

4

6

8

10

12

GSH

in m

ol/

mg

of

pro

tein

GSH activity following treatement with phytosomes

GSH in mol/mg

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Plate 15a. Microphotograph of section of the Normal untreated rat liver. Photo shows

Normal hepatocellular architecture like, normal liver parenchymal cells, small uniform

nuclei radially arranged around the central vein.

Plate 15b. Microphotograph of section of the rat live showing abnormal architecture after

treatment with CCl4 – Control. Photo shows gross structural alterations in Control group

like dilated portal triad with fibrosis, inflammation and necrosis of hepatic cells, fatty

degeneration of higher degree.

Plate 15c. Photo showing protection of liver treated with GA (200 mg/kg b.w.). GA

protected the liver moderately. Shows characters like, lesser degree of inflammation and

necrosis of hepatic cells, less dilation of central vein, fatty degeneration of slightly higher

degree.

Plate 15d. Photo showing protection of liver treated with GAP (40 mg/kg b.w.). GAP

protected the liver significantly. Photo shows almost normal gross anatomical characters

like normal hepatic cell with clear nuclei, cytoplasm. Normal central vein and portal vein.

Plate 15e. Photo showing protection of liver treated with GA (200 mg/kg b.w.). GA

protected the liver moderately. Shows characters like reduced inflammation and dilated

portal triad with fibrosis and fatty degeneration of lesser degree. But necrosis of hepatic

cells not reduced significantly.

Plate 15f. Photo showing protection of liver treated with GAP (60 mg/kg b.w.). GAP

protected the liver significantly. Photo shows almost normal gross anatomical characters

like normal hepatic cell with clear nuclei, cytoplasm. Normal central vein and portal vein.