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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
Chapter-5 Results
Bhagwant University, Ajmer 113
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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Bhagwant University, Ajmer 114
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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Bhagwant University, Ajmer 117
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.
Chapter-5 Results
Bhagwant University, Ajmer 118
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
Chapter-5 Results
Bhagwant University, Ajmer 119
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.
Chapter-5 Results
Bhagwant University, Ajmer 120
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).
Chapter-5 Results
Bhagwant University, Ajmer 121
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.
Chapter-5 Results
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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 (%)
Chapter-5 Results
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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 (%)
Chapter-5 Results
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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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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
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t p
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eat
ed
mg/
cm2
Time (hrs)
Effect of lecithin concentration
RPL1
RPL2
RPL3
RPL4
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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
Chapter-5 Results
Bhagwant University, Ajmer 127
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
Chapter-5 Results
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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
Chapter-5 Results
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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
Chapter-5 Results
Bhagwant University, Ajmer 131
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
Chapter-5 Results
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Fig 43: IR Spectra of Phospholipid and Rutin-Phospholipid Complex (1:1, 1:2 &1:3)
Chapter-5 Results
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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
Chapter-5 Results
Bhagwant University, Ajmer 134
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
Chapter-5 Results
Bhagwant University, Ajmer 135
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
Chapter-5 Results
Bhagwant University, Ajmer 136
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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Bhagwant University, Ajmer 137
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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Bhagwant University, Ajmer 138
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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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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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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Bhagwant University, Ajmer 142
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.