CHAPTER - V
RESULTS & DISCUSSION
TABLE OF CONTENT
HNGU Ph.D Thesis
CHAPTER 5
Chapter 5 RESULT AND DISCUSSION………………………………… Page No.
5 Result and discussion…………………………………………………… 88-138
5.1 Pharmacognostic Evaluation…………………………………. 88
5.1.1 Collection and authentication of plants……………….. 89
5.1.2 Morphological evaluation of plants…………………… 90
5.1.2.1 Leaves……………………………………….. 90
5.1.2.2 Fruits………………………………………… 92
5.1.2.3 Seeds…………………………………………. 93
5.1.3 Microscopical evaluation………………………………. 94
5.1.3.1 Microscopy of leaf…………………………… 94
5.1.3.2 Microscopy of fruit…………………………... 98
5.1.3.3 Microscopy of C. arborea seed………………. 100
5.1.4 Powder Microscopy……………………………………. 101
5.1.4.1 Powder microscopy of leaves………………… 102
5.1.4.2 Powder microscopy of fruits…………………. 103
5.1.4.3 Powder characteristic of C. arborea Seed…… 104
5.1.5 Quantitative Microscopy of leaf……………………….. 105
5.2 Physicochemical Evaluation…………………………………... 106-07
5.2.1 Fluorescence Analysis…………………………………. 108
5.3 Phytochemical Evaluation…………………………………….. 109-14
5.3.1 Successive solvent extraction………………………….. 109
5.3.2 Qualitative chemical examination……………………... 111
5.3.3 TLC profile of the extracts …………………………….. 114
5.4 Estimation of secondary metabolites…………………………. 118
5.4.1 Total phenolics content………………………………… 118
5.4.2 Flavonoid content……………………………………… 120
5.4.3 Total alkaloid content…………………………………. 121
5.4.4 Total saponin content………………………………….. 122
5.5 Estimation of Gallic Acid by HPTLC Method ………………. 123-24
TABLE OF CONTENT
HNGU Ph.D Thesis
5.5.1 Development of HPTLC method for estimation of gallic
acid...................................................................................
123
5.5.2 Validation of developed HPTLC method for estimation
of gallic acid.....................................................................
124
5.5.3 Quantification of Gallic acid using HPTLC method in
extracts…………………………………………………. 124
5.6 Pharmacological Evaluation…………………………………… 128-35
5.6.1 Acute toxicity study……………………………………. 128
5.6.2 Antiallergic activity…………………………………….. 129
5.6.2.1 In vitro on isolated guinea pig ileum
preparation…………………………………….
129
5.6.2.2 In vitro studies on isolated rat ileum
preparation……………………………………
130
5.6.2.3 Antiallergic activity by passive paw
anaphylaxis in rats…………………………….
131
5.6.3 Anti oxidant activity in vitro…………………………… 133
5.6.3.1 DPPH free radical scavenging activity……….. 133
5.6.3.2 Reducing power by FeCl3…………………….. 135
CHAPTER: 5
HNGU
5.1 Pharmacognostic Evaluation
5.1.1 Collection and authentication of plants
Fresh plant of Gmelina arborea
month of May 2011. Fresh leaves and fruits of
Botanical garden, M S University of Baroda, Vadodara in the month of
photographs of both plants
Gmelina arborea
Plants were identified as
characters and authenticated by Dr. P. S. Nagar at Botany Department, M. S. University
of Baroda, Vadodara. A certificate of authentication is given in
Photographs of voucher specimens of
2) were stored as herbarium in Pharmacognosy laboratory, Pioneer
College, Vadodara. Photographs of the same are given in
respectively.
RESULTS AND DISCUSSION
Ph.D Thesis
5. RESULTS AND DISCUSSION
5.1 Pharmacognostic Evaluation
5.1.1 Collection and authentication of plants
Gmelina arborea was collected from Waghodia road, Vadodara in the
Fresh leaves and fruits of Careya arborea were collected from
Botanical garden, M S University of Baroda, Vadodara in the month of
plants are given in Figure 5.1.
Gmelina arborea Careya arborea
Figure 5.1 Photograph of plants
Plants were identified as G. arborea Roxb and C. arborea Roxb with
characters and authenticated by Dr. P. S. Nagar at Botany Department, M. S. University
, Vadodara. A certificate of authentication is given in Figure. 5.
specimens of G. arborea (DC-GM-1) and C. arborea
were stored as herbarium in Pharmacognosy laboratory, Pioneer Degree
. Photographs of the same are given in Figure 5.3
RESULTS AND DISCUSSION
88
from Waghodia road, Vadodara in the
were collected from
Botanical garden, M S University of Baroda, Vadodara in the month of may 2012. The
arborea
with morphological
characters and authenticated by Dr. P. S. Nagar at Botany Department, M. S. University
. 5.2.
C. arborea (DC-CA-
Degree Pharmacy
3 and Figure 5.4
CHAPTER: 5
HNGU
Figure 5.
Figure 5.
RESULTS AND DISCUSSION
Ph.D Thesis
Figure 5.2 Certificate of authentication of plants
Figure 5.3 Herbarium of Gmelina arborea
RESULTS AND DISCUSSION
89
CHAPTER: 5 RESULTS AND DISCUSSION
90
HNGU Ph.D Thesis
Figure 5.4 Herbarium of Careya arborea
5.1.2 Morphological evaluation of plants
5.1.2.1 Leaves
Fresh leaves of G. arborea and C. arborea were subjected for morphological character
like colour, shape, size, surface characteristics and appearance etc. Photographs of fresh
leaf of G. arborea and C. arborea are given in Figure 5.5. Morphological characters are
summarized in Table 5.1.
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91
HNGU Ph.D Thesis
Figure 5.5 Photographs of leaves
Table 5.1 Morphology of G. arborea and C. arborea leaves
Characters G. arborea C. arborea
Type Simple Simple
Phyllotaxy Opposite Alternate
Color Dark green Green
SizeLength
Width
7×21cm
7-13cm
13×27cm
6-13cm
Shape Broadly Ovate or cordate Broadly obovate
Apex acuminate or caudate acuminate or cuspidate
sometimes obtuse
Margin Entire in big leaf
Toothed or lobed in small leaf
Wavy
Venation Reticulate Pinnate
Surface Smooth Smooth
Base Symmetrical Symmetrical
Petiole Long , 5-15cm Short , 0.5-1cm
Midrib Lower
Upper
Prominent on lower surface ,
not prominent on upper surface
Prominent on lower surface
Flat on upper surface
Result showed that the leaves of both plants were green, simple with symmetrical base,
smooth surface, acuminate apex and prominent midrib on lower surface. A leaf of G.
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HNGU Ph.D Thesis
arborea was small, opposite, reticulate venation with long petiole whereas leaf of C.
arborea was broadly ovate, alternate, wavy margin; pinnate venation and short petiole.
5.1.2.2 Fruits
Fresh fruits of G .arborea and C. arborea were subjected for morphological character
like colour, shape, size, surface characteristics and appearance etc. Photographs of fresh
fruit of plants are given in Figure 5.6 and morphological characters in Table 5.2.
Figure 5.6 Photographs of fruits of the both plants
Table 5.2 Morphological character of C. arborea and G. arborea fruits
Characters G. arborea C. arborea Type Drupe Fleshy indehiscent Colour Unripe Ripe Dried
Dark green yellow Black
Green Greenish-brown/ yellow Brown
Size Length Diameter/Width
1.9- 2.5cm 1.2-1.5cm(W)
5-6cm 1.5-2cm(D)
Shape Obovoid or pyriform Globose Surface Smooth, glossy Smooth Odour Strong characteristic, disagreeable Characteristic, agreeable Exocarp Thin Leathery Mesocarp succulent and aromatic Pulpy and fibrous Extra feature Fruits with calyx Crowned with calyx limb
Result showed that the unripe fruits of the both plants were green while ripe was yellow
with smooth surface. A fruits of G. arborea was small, drupe, strong, disagreeable odour,
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HNGU Ph.D Thesis
obovoid or pyriform and with calyx whereas C. arborea was fleshy indehiscent, globose,
agreeable odour and crowned with calyx limb.
5.1.2.3 Seeds
Morphology of G. arborea and C. arborea seeds were subjected for morphological
character like colour, shape, size, surface characteristics and appearance etc. Photographs
of seed of plants are given in Figure 5.7 and morphological characters in Table 5.3.
Figure 5.7 Photographs of both plants seed
Table 5.3 Morphological character of C. arborea and G. arborea seed
Characters G. arborea C. arborea
Type Exalbuminous Exalbuminous
Shape Oblong, oval ellipsoid or oblong
SizeLength
Thickness
Width
0.7-0.9cm
0.1- 0.2cm
0.2-0.5cm
1.5-2cm,
1-1.2cm
Colour External
Internal
Light brown
Creamish white
Dark brown,
Creamish white
Taste Oily Oily, astringent
Odour Characteristic, unpleasant Characteristic pleasant
Result indicated that the seeds of the both plants were exalbuminous, oily, internally
creamish white and externally brown. A seeds of G. arborea was small, obovoid or
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HNGU Ph.D Thesis
pyriform and with calyx having strong disagreeable odour whereas C. arborea was oval
ellipsoid or oblong with agreeable odour. Thus the morphological characters help in
distinguishing G. arborea and C. arborea from each other and other plants.
5.1.3 Microscopical evaluation
The transverse section of leaves, fruits and seeds of G. arborea and C. arborea were
taken.
5.1.3.1 Microscopy of leaf
A. Microscopy of G. arborea leaf
Microscopical characters of G. arborea leaf are reported in Figure 5.8-5.9.
Figure 5.8 T.S. of G. arborea leaf
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HNGU Ph.D Thesis
Figure 5.9 Microscopical characteristic of G. arborea leaf
Transverse section of leaf showed following characters.
Lamina: It was a dorsiventral. Upper epidermis was single layer of polygonal
cells, covering trichome and anomocytic stomata. Cell wall was covered with
thick cuticle. Mesophyll was discriminated into palisade and spongy parenchyma.
Single layered palisade was present below the upper epidermis. Vascular strand
was present in mesophyll. Spongy parenchyma was a thin, 3 to 6 layers, loosely
arranged with intercellular space. Lower epidermis was similar to upper epidermis
but having more number of anomocytic stomata. Uni-multicellular (2-3 celled)
trichomes were present on the lower epidermis.
Midrib: The dorsal surface and ventral surface were bulged. A 2 to 4 layered
collenchyma were present below the upper epidermis and above the lower
epidermis. Two small vascular bundles were present below the upper
collenchymatous layer of midrib. The rest of midrib was occupied by the cortical
parenchyma with the collateral vascular bundle embedded in the middle. Xylem
was towards the centre and phloem towards the periphery. Parenchymatous tissue
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96
HNGU Ph.D Thesis
was thin walled with prominent intercellular spaces. The vascular bundles were
surrounded by incomplete sheath of pericycle. Ground tissue was present in the
centre of vascular bundle.
Petiole: A microscopical character of G. arborea leaf petiole was reported in
Figure 5.10.
Figure 5.10 T.S. of petiole of G. arborea leaf
Transverse section of petiole was more or less concave to convex, having single
layer epidermal cell with cuticle. Multicellular uniserriate trichomes were present
on epidermis. Dorsal surface was convex and grooved. Below the each groove
vascular bundle was present. Outer 3-5 layer of cortex was collenchymatous while
inner 3-4 layers were parenchymatous contain chlorophyll. Endodermis was
indistinct. Vascular bundles were collateral arranged in ring, grouped of lignified
pericycle fibre crown the phloem. Ground tissue was parenchymatous with
intercellular space.
B. Microscopy of C. arborea leaf: Microscopical characters of C. arborea leaf are
reported in Figure 5.11-5.12.
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HNGU Ph.D Thesis
Figure 5.11 TS of C. arborea leaf
Lamina
Midrib
Figure 5.12 Microscopical character of C. arborea leaf
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HNGU Ph.D Thesis
Transverse section of leaf showed following characters.
Lamina: It was a dorsiventral. Upper epidermis was single layer of rectangular
cells, covered with thick walled cuticle. Mesophyll was divided into palisade and
spongy parenchyma. Single layered palisade was present below the upper
epidermis. Vascular strands were observed in mesophyll. Spongy parenchyma
was loosely arranged with intercellular space. Lower epidermis was similar to
upper epidermis but having more number of anisocytic stomata. Plenty of calcium
oxalate crystals were present in spongy parenchyma.
Midrib: It showed slight round notch at upper surface and large notch at lower
surface. Collenchyma was present below the upper notch and above the lower
notch. A bunch of sclerenchymatous cells was present below the upper
collenchyma. Major portion of midrib was occupied with large vascular bundles.
Median vascular bundle was largest, lateral vascular bundles are getting gradually
smaller towards lamina region. Vascular bundle was covered with fibre bundle
sheath which was broad at lower side (5-7 layers) and narrow (1-2 layers) at upper
side. Xylems were arranged in arc shape which was surrounded by phloem toward
lower surface. Abundant calcium oxalate clusters were present in entire tissues
except epidermis.
Result showed that the G. arborea and C. arborea leaves were dorsiventral. G.
arborea leaf in transverse section showed covering trichome and anomocytic
stomata while calcium oxalate crystals were absent. In transverse section of C.
arborea leaf showed anisocytic stomata and cluster of calcium oxalate crystals
while trichomes were absent.
5.1.3.2 Microscopy of fruit
A. Microscopy of G. arborea fruits: Microscopical characters of G. arborea fruits
are reported in Figure 5.13.
Transverse section of fruits of G. arborea showed following characters;
Pericarp of G. arborea fruit was differentiated in to epicarp, mesocarp and endocarp.
Epicarp was made up of single layered cells. Mesocarp was made up of fleshy and
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HNGU Ph.D Thesis
multilayered parenchymatous cells. Outer portion of mesocarp cells were isodiametric
or oblong while inner portion of mesocarp made up of thin walled elongated cells,
vascular strand and sclerides. Hard endocarp was made up of Sclerenchymatous
tissue.
Figure 5.13 TS of G. arborea fruits pericarp (Excluding endocarp)
B. Microscopy of C. arborea fruits
Microscopical characters of C. arborea fruits are reported in Figure 5.14-5.15.
Figure 5.14 Transverse section of C. arborea fruits
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HNGU Ph.D Thesis
Figure 5.15 Microscopic characters of C. arborea fruits
Transverse section of the fruit showed epicarp having single epidermal cell layered
covered by a cuticle; followed by few layers of compactly arranged hypodermal cells.
Major portion of fruit was occupied by fleshy mesocarp. Mesocarp was composed of
thin walled, oval to polygonal parenchymatous cells. Lignified fibers and thick walled
sclereids were scattered in mesocarp. Vascular bundles were embedded throughout
parenchymatous tissue.
Cells and tissues arrangement in G. arborea and C. arborea fruits helps in
distinguishing each other and other plants.
5.1.3.3 Microscopy of C. arborea seed
Microscopical characters of C. arborea seeds are reported in Figure 5.16-5.17.
Figure 5.16 Transverse section of seed of C. arborea
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HNGU Ph.D Thesis
Figure 5.17 Microscopical characters of seed of C. arborea
The Testa was sclerenchymatous followed by a zone of collapsed cells of outer and
inner integument lined by cuticle on both sides. Outer layers of both integuments
filled with dark brown material. Cotyledons made up of thin walled polygonal
parenchymatous cells. Cotyledon consists of single epidermal cells, followed by
palisade cell and parenchymatous cells. It was contains abundant starch grains and oil
globules.
5.1.4 Powder Microscopy
Powder microscopy of leaves and fruits of G. arborea and C. arborea were carried out.
The photographs of G. arborea and C. arborea leaf powder given in Figure 5.18.
C. arborea G. arborea
Figure 5.18 Photographs of C. arborea and G. arborea leaf powder
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HNGU Ph.D Thesis
G. arborea leaf powder was dark greenish colour, bitter taste and characteristic odour
while C. arborea leaf powder was greenish, astringent with characteristic odour.
5.1.4.1 Powder microscopy of leaves
A. G. arborea leaf: Powder microscopy of G. arborea is shown in Figure 5.19.
Figure 5.19 Powder microscopy of leaf of G. arborea
Microscopy of leaf powder of G. arborea showed presence of anomocytic
stomata, covering trichome, spiral xylem vessels and lamina fragments.
B. C. arborea leaf: Powder microscopy of leaf was reported in Figure 5.20.
Figure 5.20 Powder characteristic of C. arborea leaf
CHAPTER: 5
HNGU
Microscopy of leaf
oxalate cluster, epidermal cells
Result showed that
present while C. arborea
observed whereas In
were observed whereas
was observed.
5.1.4.2 Powder microscopy of
The photographs of powder
Figure 5.21 Photographs of
Fruit powder of G. arborea
unpleasant smell while
pleasant odour.
A. Powder microscopy of
reported in Figure 5.2
Figure 5.
A
RESULTS AND DISCUSSION
Ph.D Thesis
Microscopy of leaf powder showed presence of anisocytic stomata, calcium
oxalate cluster, epidermal cells, spiral xylem vessels and lamina fragments
Result showed that G. arborea was dark green and fine vascular
C. arborea powder was light green, coarse vascular strands were
observed whereas In G. arborea leaf, anomocytic stomata and covering trichome
were observed whereas C. arborea leaf anisocytic stomata and calcium oxalate
Powder microscopy of fruits
powder of both the plants fruits are given in Figure 5.21.
Photographs of fruits powder of (A) G. arborea and (B)
G. arborea was dark brown, acidic with characteristic, strong
while C. arborea was brownish, astringent with characteristic,
Powder microscopy of G. arborea fruits: G. arborea fruit powder microscopy is
reported in Figure 5.22.
Figure 5.22 Microscopy of fruits powder of G. arborea
B
RESULTS AND DISCUSSION
103
powder showed presence of anisocytic stomata, calcium
spiral xylem vessels and lamina fragments.
vascular strands were
powder was light green, coarse vascular strands were
leaf, anomocytic stomata and covering trichome
cytic stomata and calcium oxalate
in Figure 5.21.
(B) C. arborea
characteristic, strong
astringent with characteristic,
fruit powder microscopy is
G. arborea
CHAPTER: 5
HNGU
Microscopy of fruit powder of
with parenchyma, pitted parenchyma, vascular strand and sclerides.
B. C. arborea fruits :
5.23.
Figure 5.
Microscopy of fruit
fragments and epicarp cells,
Result showed that the
odour while G. arborea fruit powder is dark brown in
5.1.4.3 Powder characteristic of
The photographs of C. arborea
Figure 5.
Seed powder of C. arborea
RESULTS AND DISCUSSION
Ph.D Thesis
powder of G. arborea showed presence of epidermal cells,
parenchyma, vascular strand and sclerides.
: Microscopy of C. arborea fruit powder is reported in Figure
Figure 5.23 Powder characteristic of C. arborea fruits
of fruit powder of C. arborea showed fibers, sclereids, mesocarp
fragments and epicarp cells,
Result showed that the C. arborea fruit powder was brown coloured having pleasant
fruit powder is dark brown in colour and unpleasant
wder characteristic of C. arborea Seed
C. arborea seed powder is given in Figure 5.24.
Figure 5.24 Powder of seed of C. arborea
C. arborea was creamish white with dotted brown testa fragments
RESULTS AND DISCUSSION
104
presence of epidermal cells, mesocarp
reported in Figure
fruits
owed fibers, sclereids, mesocarp
red having pleasant
and unpleasant odour.
fragments
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HNGU Ph.D Thesis
Microscopy of C. arborea seed powder is given in Figure 5.25.
Figure 5.25 Powder characteristic of C. arborea Seed
Microscopy of C. arborea seed powder showed stone cells, endosperm cells, and
brownish matter and starch grains. Hence, the microscopical characters help in
distinguishing G. arborea and C. arborea each other and from other plants.
5.1.5 Quantitative Microscopy of leaf
The quantitative microscopy of G. arborea and C. arborea were determined by
evaluating leaf constants like stomatal number, stomatal index, vein-islet number, and
vein termination numbers. Leaf constants are given in Table 5.4.
Table 5.4 Quantitative Microscopy of C. arborea and G. arborea leaf
Parameter G. arborea C. arborea
Stomatal Number (per mm2)
Upper epidermis
Lower epidermis
--
205-225
36-39.5-43
110-117.5-125
Palisade Ratio 4.25-5.6-6.3 3.75-4.6-5.5
Vein termination Number (per mm2) 10-20 07-11
Vein islet number (per mm2) 30-40 11-17
Stomatal index Upper epidermis
Lower epidermis
--
8-18.7-27.8
2.86-4.35-5.88
12.5-17.9-21.9
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Result showed that the value of stomata index, palisade ratio, vein islet number and vein
termination number were less in C. arborea than G. arborea. The parameters for
quantitative microscopy like stomatal index, palisade ratio, vein islet number and vein
termination number remain constant, irrespective of age, the size and habitat of plant.
These parameters are very useful for identification of plant and detection of adulterant in
plant material.
5.2 Physicochemical Evaluation
Physicochemical parameters like ash value, extractive values, loss on drying and foam
index of leaves and fruits of G. arborea and C. arborea were determined as per WHO
guideline. The values are expressed as percentage of air dried material. Each value is the
average of three determinations. Physicochemical constant of G. arborea and C. arborea
leaf compiled in Table 5.5; physicochemical constant of fruits of both plants compiled in
Table 5.6, and Physicochemical constant of C. arborea seeds are assembled in Table 5.7.
Table 5.5 Physicochemical constant of G. arborea and C. arborea leaf
Parameter G. arborea C. arborea
Water soluble extractive value 17.5%w/w 17.1%w/w
Alcohol soluble extractive value 24%w/w 13.0%w/w
Loss on drying 0.5%w/w 0.6%w/w
Foreign matter 0.73%w/w 0.8%w/w
Total Ash 5.22%w/w 5.6%w/w
Acid-insoluble ash 0.75%w/w 1.5%w/w
Foaming index <100 > 100
Result had shown that the water soluble extractive value, loss on drying, foreign matter of
G. arborea and C. arborea leaf were almost same, whereas, alcohol soluble extractive
value was less in C. arborea leaf than G. arborea leaf. Total ash, foaming index and acid
insoluble ash was more in C. arborea leaf than in G. arborea leaf.
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Table 5.6 Physicochemical constant of G. arborea and C. arborea fruits
Parameter G. arborea C. arborea
Water soluble extractive value 37 %w/w 27.33%w/w
Alcohol soluble extractive value 26%w/w 36.5%w/w
Loss on drying 8%w/w 0.82%w/w
Foreign matter 0.8 %w/w 0.5%w/w
Total Ash 5.35 %w/w 2.40%w/w
Acid-insoluble ash 0.25%w/w 0.22%w/w
Physicochemical study revealed that G. arborea fruits contain more water soluble
extractive constituents, total ash, loss on drying and foreign matter, while alcohol soluble
constitutes were higher in C. arborea fruit. Acid-insoluble ash was almost same in both
the fruits.
Table 5.7 Physicochemical constant of C. arborea seed
Parameter %w/w
Water-soluble extractive 18.5
Alcohol-soluble extractive 9.25
Foreign matter 0.6
Total Ash 3.35
Acid-insoluble ash 0.32
Physicochemical constant showed that water soluble extractive was 18.5%w/w, alcohol-
soluble extractive 9.25%w/w, foreign matter 0.6%w/w, total ash %w/w and acid-
insoluble ash 0.32%w/w. Physicochemical parameters are useful for evaluation of plant
materials. E.g. Extractive value determines the amount of soluble matter in particular
solvent in given amount of plant material. This also gives preliminary idea about nature
of phytoconstituents present in plant materials. E.g water soluble phytoconstitutes are
extracted in aqueous solvent. Ash value represents inorganic content of the plants and
acid insoluble ash signifies the amount of acid insoluble matter like silica. Lower amount
of ash value and acid insoluble ash represented free from impurities of both the plants.
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Loss on drying less than 10%w/w in leaf and fruits of both plants indicated less chance of
fungal growth.
5.2.1 Fluorescence Analysis
Fluorescence analysis of G. arborea and C. arborea leaf and fruits with different reagents
are given in Table 5.8 and Table 5.9 respectively.
Table 5.8 Fluorescence analysis of G. arborea and C. arborea leaf
G. arborea C. arborea
Reagents Visible light UV Light Visible light UV Light
Drug powder Light green No Fluorescence Light green No Fluorescence
Aq. NaOH Brownish yellow Greenish yellow Brownish yellow Brownish yellow
NaOH (alcohol) Green Golden Yellowish Yellowish brown Greenish Yellowish
HCL Light green Light green Light green Light bluish- green
H2SO4 Light yellowish Greenish yellow Light yellowish Light blue
Nitric acid LYW Greenish yellow Pinkish -yellow Yellowish green
Picric acid Yellowish green Yellowish green Yellowish green Yellowish green
Acetic acid LYW Golden yellow LYG Light green
NH3 Yellowish green IYG Light green LYG
KOH(alcohol) Brownish yellow Greenish yellow LYW LYG
LYW = Light yellowish-brown LYG = Light yellowish-green IYG = Intense Yellowish green
Fluorescence analysis of G. arborea and C. arborea leaf showed that drug powder itself
and with picric acid had given similar fluorescence in visible and UV light whereas HCl,
H2SO4 and aqueous NaOH had shown the same florescence in visible light. Both the
plants had given distinguish fluorescence with NaOH (alcohol), nitric acid, acetic acid,
NH3 and KOH (alcohol) under visible and UV light. These reagents can be used for
differentiating G. arborea and C. arborea leaf.
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Table 5.9 Fluorescence analysis of G. arborea and C. arborea fruits
G. arborea C. arborea
Reagent added Visible light U.V. light Visible light U.V. light
Drug powder Brownish No Fluorescence brownish No Fluorescence
Aq. NaOH Light Brownish LYG DGB Brownish yellow
NaOH (alcohol) LYG Yellowish-green DGB Brownish yellow
HCL LRB LYW Greenish yellow brownish yellow
H2SO4 Reddish brown Brownish red lemon yellow Light brownish
Nitric acid LYW Greenish yellow Brownish red Light brownish
Picric acid LYW Light brown Greenish yellow Yellowish green
Acetic acid Light brown Yellowish green LYW yellowish brown
NH3 Dark brown Brownish LYG Yellowish green
KOH(alcohol) LRB LYW DGB Brownish yellow
LYW = Light yellowish-brown LYG = Light yellowish-green LRB = Light reddish brown
DGB= Dark greenish brown
Fluorescence analysis of G. arborea and C. arborea fruits showed that drug powder itself
had given similar fluorescence in visible and UV light. Both the plants had given
distinguish fluorescence with, HCl, H2SO4 ,aqueous NaOH, NaOH (alcohol), nitric acid,
acetic acid, NH3 and KOH (alcohol) under visible and UV light. These reagents can be
used for differentiating G. arborea and C. arborea fruits.
5.3 Phytochemical Evaluation
5.3.1 Successive solvent extraction: Plant considered as biosynthetic factory contain
primary and secondary metabolites; alkaloid, glycoside, tannins, phenolic and
triterpenoids. Extraction methods involve the separation of medicinally active portions of
plant chemical from the inactive tissue by using selective solvents. During extraction,
solvents diffuse into the solid plant material and solubilize compounds with similar
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HNGU Ph.D Thesis
polarity. Preliminary screening is helpful to identify largest chemical constitute of plant
extracted in particular solvent and useful for selection of solvent. It gives idea about
nature of phytoconstituents e.g. if the plant contains non-polar phytoconstituents then
they get extracted in non polar solvent like petroleum ether, chloroform.
A successive solvent extracts of leaf of G. arborea and C. arborea was studied for their
phytochemical profile. Their % yield, colour and consistency are recorded in Table-5.10.
Table 5.10 Preliminary phytoprofile of G. arborea and C. arborea leaves
G. arborea C. arborea
Extracts Colour and consistency Yield
%w/w
Colour and consistency Yield
%w/w
PE Dark green, sticky, semisolid 3.76 Blackish green, semisolid, sticky 3.1
Benzene Dark green, sticky, semisolid 1.92 Dark greenish black, sticky solid 3.46
Chloroform Dark green, sticky, semisolid 1.56 Green, solid, non-sticky 2.30
acetone Dark green, sticky, semisolid 2.26 Light brown, solid, glassy, non-sticky 5.0
Methanol Green, sticky, semisolid 19.36 Reddish brown, shiny solid, non-
sticky
8.84
Water Brown, non sticky, solid 26.3 Brownish red, solid, non-sticky 7.64
PE = petroleum ether
Successive solvent extraction revealed that G. arborea and C. arborea leaves showed
higher yield in methanol and water indicating both plant contains more amount of polar
compounds whereas leaves showed higher yields in benzene, chloroform and acetone.
Almost similar yield was obtained in leaf both plants. Petroleum ether, benzene,
chloroform and acetone extracts of G. arborea was dark green, sticky and semisolid.
While methanol extract was green sticky and semisolid whereas water extract was brown,
non-sticky solid. The colour of C. arborea extract in petroleum ether was blackish green,
benzene was dark greenish black, chloroform was green, acetone was light brown, methanol was
reddish brown and aqueous was brownish red. The non-sticky solid extracts of C. arborea were
obtained in chloroform, acetone, methanol and water while petroleum ether was semisolid
and sticky whereas benzene was solid and sticky.
CHAPTER: 5 RESULTS AND DISCUSSION
111
HNGU Ph.D Thesis
The fruits powder of G. arborea and C. arborea were extracted with acetone and
methanol using soxhlet apparatus individually. The aqueous extracts of fruits of both
plants were prepared by maceration. Their % yield, colour and consistency are recorded
in Table-5.11.
Table 5.11 Phytoprofile of G. arborea and C. arborea fruits
G. arborea Yield C. arborea Yield
Extracts Colour and consistency %w/w Colour and consistency %w/w
Acetone Dark brownish, sticky 3.5 Light brownish-red, solid,
glassy , non-sticky
5.0
Methanol Dark Reddish brown,
solid, sticky
28.33 Reddish brown, shiny ,
solid, sticky
29.33
Water Dark brownish red,
solid, sticky
39.2 Dark brownish black,
solid, non-sticky
39.5
The percentage yield of methanol and water extracts of G. arborea and C. arborea fruits
were almost same and higher indicating more amount of polar compounds. Higher yield
was found in C. arborea than G. arborea in acetone extracts. Extracts of G. arborea in
acetone, methanol and water was dark brown to red, sticky and solid while C. arborea
were light brown to dark brownish black, shiny and solid.
5.3.2 Qualitative chemical examination
The extracts obtained from successive solvent extraction were then subjected to various
qualitative chemical tests for the identification of various plant constituents like steroids,
carbohydrates, alkaloids, glycosides, phenolics and tannins. It gives primary idea about
type of primary and secondary metabolite present in plants material.
CHAPTER: 5 RESULTS AND DISCUSSION
112
HNGU Ph.D Thesis
The results of qualitative chemical test of G. arborea and C. arborea leaves extracts are
reported in Table-5.12.
Table 5.12 Phytochemical screening of G. arborea and C. arborea leaves extracts
G. arborea C. arborea
C
hem
ical
co
nstit
uent
Pet
role
um e
ther
Tol
uene
Chl
orof
orm
Eth
yl a
ceta
te
Met
hano
l
Wat
er
P
etro
leum
eth
er
Ben
zene
Chl
orof
orm
Ace
tone
Met
hano
l
Wat
er
Carbohydrates - - - - + + - - - - + +
Proteins - - - - - + - - - - - +
Saponins - - - - + + - - - - + +
Alkaloids - - - + + - - - + + + -
Flavonoids - - - + + + - - - + + +
Tannin & phenolics
- - - + + + - - - + + +
Steroids & triterpens
+ + + + - - + + + + - -
(- = absent, + = positive)
G. arborea and C. arborea leaf extracts in various solvents showed similar pattern for
phytoconstituents. Carbohydrates and saponin were found present in methanol and water
extracts of both plants leaves. Protein was present in aqueous extracts of both the plants.
Ethyl acetate and methanol extracts of both the plants had shown presence of alkaloid,
while chloroform extract of C. arborea showed presence of alkaloids but was absent in
G. arborea. Flavonoid, phenolics and tannins were present in ethyl acetate, methanol and
water extracts of both plants. Steroid and triterpenoid were found in petroleum ether,
toluene, chloroform and ethyl acetate extracts of both plants. Phytochemical screening in
various solvents showed the presence of polar compounds like carbohydrates, saponins,
flavonoid, and phenolics in leaves of G. arborea and C. arborea, which supports finding
of preliminary screening.
CHAPTER: 5 RESULTS AND DISCUSSION
113
HNGU Ph.D Thesis
The results of qualitative chemical test of G. arborea and C. arborea fruits extracts are
reported in Table-5.13.
Table 5.13 Preliminary phytochemical screening of G. arborea and C. arborea fruits extracts
G. arborea C. arborea
Chemical
constituent
Ethyl
acetate
Methanol Water Ethyl
acetate
Methanol Water
Carbohydrates + + + + + +
Proteins _ _ _ _ + _
Saponins _ + + + + +
Alkaloids + + _ _ + _
Flavonoids + + + + + +
Tannin &
phenolics + + + + + +
Steroids &
triterpens _ _ _ _ + _
(- = absent, + = positive)
The result showed that carbohydrates, flavonoids and phenolics were found present in
ethyl acetate, methanol and water extracts of both plants fruits. Protein and steroid were
present in methanol extract of C. arborea while absent in G. arborea. Methanol extracts
of both the plants had shown presence of alkaloid. While ethyl acetate of G. arborea
showed presence of alkaloids but was absent in C. arborea. Saponin was present in
methanol and water extracts of fruits in both plants. While ethyl acetate extract of C.
arborea showed presence of saponins but was absent in G. arborea. Phytochemical
screening in various solvents showed the presence of polar compounds like
carbohydrates, saponins, flavonoid, phenolics in fruits of G. arborea and C. arborea,
which supports finding of preliminary screening.
CHAPTER: 5 RESULTS AND DISCUSSION
114
HNGU Ph.D Thesis
5.3.3 TLC profile of the extracts
The quality of raw material is basic requirement for quality of final product. Thin layer
chreomatography is an important tool for evaluation of quality of plant materials. Plant
can be distinguished by finger print. The fruits and leaves extract of G. arborea and C.
arborea were subjected to Thin Layer Chromatography (TLC) to detect and confirm the
presence of phytoconstituents. Silica gel G was used as an adsorbent. TLC profile like
solvent system for saponin, steroid & triterpenoids and phenolics; reagent used for
detection and Rf value of spots for successive solvents extracts of G. arborea and C.
arborea leaves are summarized in Table 5.14 and Table 5.15 respectively.
Table 5.14 TLC profile of successive solvents extracts of G. arborea leaves
Chemical
Constituents
Saponins Steroids& Triterpenoid Phenolic
Compound
Mobile phase CHCl3 :MeOH:
Water(7:3:0.4)
CHCl3:Tol:EA (6:3:1) EA: TOl:M:FA
(8:2:1:0.2)
Detection *AS reagent *AS reagent Alcoholic FeCl3
Petroleum
Ether
0.05, 0.27, 0.55, 0.63,
0.72, 0.96, 0.98 (Purple)
0.76 (Green)
Toluene 0.04, 0.62 (Pink)
0.83 (Greenish yellow)
0.87, 0.97 (Purple)
CHCl 3 0.3,0.40 (Pink)
0.50, 0.60, 0.90 (Purple)
Ethyl acetate 0.11, 0.36, 0.74, 0.85, 0.95
(Purple), 0.79 (Yellow)
0.06, 0.52 (Pink)
0.86, 0.98 (Purple)
0.39, 0.83, 0.9
(Black)
Methanol 0.26, 0.84, 0.95, 0.97 (Purple)
0.55, 0.80 (Yellowish green)
0.39, 0.83, 0.9
(Black)
Water 0.09, 0.8, 0.96 (Purple),
0.27 (Greenish-brown)
0.52,0.85
(Black)
EA= Ethyl acetate, M = Methanol, Tol; Toluene, W = Water, FA = Formic acid, *AS
reagent = Anisaldehyde sulphuric acid
CHAPTER: 5 RESULTS AND DISCUSSION
115
HNGU Ph.D Thesis
Table 5.15 TLC profile of successive solvents extracts of C. arborea leaves
Chemical
Constituents
Saponins Steroids& Triterpenoid Phenolic
Compound
Mobile
phase
CHCl3 :MeOH: water
(7:3:0.4)
CHCl3:Tol:EA
(6:3:1)
EA: MeOH:FA
(9:1:0.4)
Detection AS reagent AS reagent Alcoholic FeCl3
Petroleum
Ether
0.22, 0.34, 0.4, 0.72,
0.78, 0.80, 0.90 (Pink)
0.36, 0.50, 0.66
(Greenish-brown)
Toluene 0.55 (Purple)
0.80, 0.97 (Brownish-
purple)
CHCl 3 0.03, 0.56, 0.63, 0.70,
0.96 (Purple) 0.73 (Green)
Ethyl
acetate
0.2, 0.58, 0.75, 0.81, 0.95,
0.97 (Purple), 0.37 (Green)
0.06, 0.52, 0.86, 0.98
(Pinkish-Purple)
0.06, 0.21, 0.44,
0.76 (Black)
Methanol 0.06, 0.21, 0.78, 0.83,
0.97 (Purple), 0.11 (Green)
0.23 (Brownish- purple
0.45, 0.59, 0.84, 097
(Pinkish-Purple)
0.06, 0.21, 0.44,
0.76 (Black)
Water 0.06, 0.95, 0.97 (Purple)
0.19, 0.35 (Greenish-brown)
0.06, 0.21, 0.44,
0.7 (Black)
EA= Ethyl acetate, M = Methanol, W = Water, DEA = Diethyl amine, FA = Formic acid,
GAA = Glacial acetic acid, AS reagent = Anisaldehyde sulphuric acid
Thin layer chromatography gives reproducible result for method for qualitative analysis
of phytoconstituents. TLC study gives primary idea of simple composition and helpful in
identification of separated compound.
Result showed that petroleum ether extract of G. arborea leaf had demonstrated seven
purple spots and one green spot; while, C. arborea demonstrated seven pink spots and
three greenish-brown spots in solvent system for steroid and triterpenoids after
derivatization with anisaldehyde sulphuric acid reagent. Toluene extracts of G. arborea
CHAPTER: 5 RESULTS AND DISCUSSION
116
HNGU Ph.D Thesis
leaf had demonstrated two pink, one greenish yellow and two purple spots; while, C.
arborea demonstrated one purple and two brownish purple spots in solvent system for
steroid and triterpenoids after derivatization with anisaldehyde sulphuric acid reagent.
Chloroform extracts of G. arborea leaf had demonstrated two pink and three purple
spots; while, C. arborea demonstrated five purple and one green spots in solvent system
for steroid and triterpenoids after derivatization with anisaldehyde sulphuric acid reagent.
Ethyl acetate extracts of G. arborea leaf had demonstrated five purple and one yellow
spot while, C. arborea demonstrated six purple and one green in solvent system for
saponin after derivatization with anisaldehyde sulphuric acid reagent.
Ethyl acetate extracts of G. arborea leaf had demonstrated two pink and two purple spots
while, C. arborea demonstrated four pinkish-purple in solvent system for steroid and
triterpenoids after derivatization with anisaldehyde sulphuric acid reagent.
Ethyl acetate extracts of G. arborea leaf had demonstrated three black spots while, C.
arborea demonstrated four black in solvent system for phenolics after derivatization with
alcoholic FeCl3 reagent.
Methanol extracts of G. arborea leaf had demonstrated four purple and two yellowish
green spot while, C. arborea demonstrated five purple and one green in solvent system
for saponin after derivatization with anisaldehyde sulphuric acid reagent.
Methanol extracts of G. arborea leaf had not demonstrated any spots while, C. arborea
demonstrated one brownish purple and four pinkish-purple in solvent system for steroid
and triterpenoids after derivatization with anisaldehyde sulphuric acid reagent.
Methanol extracts of G. arborea leaf had demonstrated three black spots while, C.
arborea demonstrated four black in solvent system for phenolics after derivatization with
alcoholic FeCl3 reagent.
Water extracts of G. arborea leaf had demonstrated three purple and one greenish brwon
green spot while, C. arborea demonstrated three purple and two greenish brown in
solvent system for saponin after derivatization with anisaldehyde sulphuric acid reagent.
CHAPTER: 5 RESULTS AND DISCUSSION
117
HNGU Ph.D Thesis
Water extracts of G. arborea leaf had demonstrated three black spots while, C. arborea
demonstrated two black in solvent system for phenolics after derivatization with
alcoholic FeCl3 reagent.
Ethyl acetate, methanol and water extract of C. arborea leaf had shown four black spots
at Rf 0.06, 0.21, 0.44, 0.76 in solvent system for phenolics after derivatization; while
ethyl acetate and methanol extract of G. arborea leaf had shown three black spots at Rf
0.39, 0.83, 0.9 in solvent system for phenolics after derivatization.
The TLC profile of fruits extracts of G. arborea and C. arborea are compiled in Table
5.16.
Table 5.16 TLC profile of various extracts G. arborea and C. arborea fruits
Chemical Saponin Steroids & Triterpenoid Phenolic Compound
Mobile
phase
CHCl3 :MeOH: water
(7:3:0.4)
CHCl3:Tol:EA
(6:3:1)
EA: MeOH:FA
(9:1:0.4) (8:2:1:0.2)
Detection AS reagent AS reagent Alcoholic FeCl3
G. arborea C. arborea G. arborea C. arborea G. arborea C. arborea
Ethyl
acetate
0.15, 0.35
(Brownish)
0.68 (yellowish)
0.87 (pink),
0.11, 0.19,
0.30, 0.83,
0.92, 0.98
0.23, 0.47,
0.65 0.96
(Purple),
0.82 (Pink),
0.21, 0.47,
0.67,0.81
(Violet),
0.84, 0.97
(Purple)
0.9,0.11
(Black)
0.1, 0.28
0.57, 0.88
(Black)
Methanol
0.16, 0.30
0.61 (yellowish)
0.87 0.93, 0.97,
0.98 (Purple)
0.06, 0.86,
0.97 (Purple)
0.22, 032,
0.54, 0.82
(brown)
0.23, 0.47,
0.65 ,0.84 ,
0.97 (Purple)
0.22, 0.88,
0.97 (Purple)
0.9, 0.11
(Black)
0.1, 0.28,
0.57, 0.88
(Black)
Water
0.19, 0.78,
0.98 (Purple)
0.34
(Yellowish
brown) 0.91,
0.95 (Purple)
0.12, 0.32
(Black)
0.1, 0.28
0.57, 0.88
(Black)
Result showed that ethyl acetate extracts of G. arborea fruits had demonstrated two
brownish, one pink and one yellowish spot while, C. arborea demonstrated six spots in
solvent system for saponin after derivatization with anisaldehyde sulphuric acid reagent.
CHAPTER: 5 RESULTS AND DISCUSSION
118
HNGU Ph.D Thesis
Ethyl acetate extracts of G. arborea fruits had demonstrated four purple and one pink
spots while, C. arborea demonstrated four violet and two purple spots in steroid and
triterpenoids solvent system after derivatization with anisaldehyde sulphuric acid reagent.
Ethyl acetate extracts of G. arborea fruits had demonstrated two black spots while, C.
arborea demonstrated four black spots in solvent system for phenolics after
derivatization with alcoholic FeCl3 reagent.
Methanol extracts of G. arborea fruit had demonstrated four purple and three yellowish
spot while, C. arborea demonstrated three purple and four brown spots in solvent system
for saponin after derivatization with anisaldehyde sulphuric acid reagent.
Methanol extracts of G. arborea fruit had shown five purple spots while, C. arborea
demonstrated threes purple spots in solvent system for steroid and triterpenoids after
derivatization with anisaldehyde sulphuric acid reagent.
Methanol extracts of G. arborea fruit had demonstrated two black spots while, C.
arborea demonstrated four black spots in solvent system for phenolics after
derivatization with alcoholic FeCl3 reagent.
Water extracts of G. arborea fruits had demonstrated three purple spots while, C. arborea
demonstrated two purple and one yellowish brown spots in solvent system for saponin
after derivatization with anisaldehyde sulphuric acid reagent.
Water extracts of G. arborea fruits had demonstrated two black spots while, C. arborea
demonstrated four black spots in solvent system for phenolics after derivatization with
alcoholic FeCl3 reagent.
5.4 Estimation of secondary metabolites
The estimation of total phenolic, flavonoids, saponin and alkaloids were carried out in
various extract of leaf and fruits of C. arborea and G. arborea.
5.4.1 Total phenolics content
Folin-Ciocalteu reagent is mixture of phosphomolybdate and phosphotungstate. Phenolic
compound reduce phosphotungstate-phosphomolybdate complex to blue reaction
products in alkaline conditions. The amount of the substance (phenolics) need to inhibit
CHAPTER: 5 RESULTS AND DISCUSSION
119
HNGU Ph.D Thesis
the oxidation of the reagent was estimated by measurement of the absorbance of coloured
complex at 765nm. Gallic acid was use as reference standard. The calibration curve is
reported in Figure 5.26.
Figure 5.26 Calibration curve of gallic acid
The calibration curve was found to be linear with correlation coefficient (R2= 0.9803) and Y=0.0106x-0.3154.
Total phenolic content in aqueous, methanol and ethyl acetate extract of leaf and fruits of
G. arborea and C. arborea were determined using calibration curve of gallic acid and
results are given in Table 5.17. Values are represented mean ± standard deviation (n=3)
Table 5.17 Total Phenolic content in G. arborea and C. arborea leaves and fruits
extracts
G. arborea C. arborea
Extract %w/w %w/w
Aqueous leaf extract 1.702±0.210 3.892±0.2663
Methanol leaf extract 1.766±0.197 4.298±0.3079
Ethyl acetate leaf extract 2.376±0.4 4.52±0.5508
Aqueous fruit extract 2.304±0.264 2.70±0.2804
Methanol fruit extract 1.876±0.55 3.956±0.3606
Ethyl acetate fruit extract ----- 4.55±0.5292
y = 0.010x - 0.315
R² = 0.980
0
0.4
0.8
1.2
1.6
2
0 50 100 150 200 250
Ab
sorb
an
ce
Conc.(mcg/ml)
Calibration curve of Gallic acid
CHAPTER: 5 RESULTS AND DISCUSSION
120
HNGU Ph.D Thesis
Result showed that higher amount of phenolic was found in ethyl acetate extract of leaves
of G. arborea than methanol and water extract; whereas, higher amount of phenolic were
present in water extract than methanol extract of G. arborea fruit. Ethyl acetate extracts
contain higher amount of phenolic than methanol and water extract of C. arborea leaf.
Ethyl acetate extract of fruits contain higher amount of phenolic than water and methanol
extract. The highest amount of phenolic was found in ethyl acetate extract of C. arborea
fruits while the lowest phenolic content in aqueous extract of leaf of G. arborea. The
higher amounts of phenolic contents were observed in all three extracts of fruits and
leaves of C. arborea than in G. arborea.
5.4.2 Flavonoid content
Flavonoid content was determined in aqueous, methanol and ethyl acetate extract of leaf,
and fruits of G. arborea and C. arborea using aluminium chloride method at 415nm.
Flavonoid content was expressed as % quercetin using calibration curve of quercetin. The
calibration curve is reported in Figure 5.27.
Figure 5.27 Calibration curve of Quercetin
The calibration curve was found to be linear with correlation coefficient (R2= 0.9994) and Y=0.0344x-0.0112.
The Flavonoid content in aqueous, methanol and ethyl acetate extract of leaves and fruits
of G. arborea and C. arborea were determined and are tabulated in Table 5.18.
y = 0.034x - 0.011
R² = 0.999
0
0.2
0.4
0.6
0 5 10 15 20
Ab
sorb
an
ce
Conc.(mcg/ml)
Calibration curve of Quercetin
CHAPTER: 5 RESULTS AND DISCUSSION
121
HNGU Ph.D Thesis
Table 5.18 Flavonoid content in leaves and fruits extracts of G. arborea and C.
arborea
Extracts G. arborea C. arborea
%W/W %W/W
Aqueous leaf extract 0.159 ±0.01 0.196±0.011
Methanol leaf extract 0.468 ±0.007 0.192±0.011
Ethyl acetate leaf extract 0.617 ±0.004 0.229±0.021
Aqueous fruit extract 0.044 ±0.021 0.254±0.012
Methanol fruit extract 0.209 ±0.006 0.128±0.002
Ethyl acetate fruit extract 0.34±0.004 0.258±0.011
Result showed that highest amount of flavonoid showed in ethyl acetate extract of leaf of
G. arborea than all other extracts. Higher amount of flavonoid was present in ethyl
acetate extract than methanol and water extract of G. arborea leaf. Whereas higher
amount of flavonoid were observed in ethyl acetate than methanol extract and water
extract of G. arborea fruit. Ethyl acetate extract contain higher amount of flavonoid than
methanol and water extract of leaf and fruits of C. arborea. The methanol and ethyl
acetate extract of leaves and fruits of G. arborea showed higher amount of flavonoid than
C. arborea. The aqueous extract of leaves and fruits of C. arborea showed higher amount
of flavonoid than G. arborea.
5.4.3 Total alkaloid content
Alkaloids are basic compound and found in salt form with plant acid. Alkaloid is soluble
in acidic methanol. On addition of concentrated ammonia, alkaloids get precipitate. The
precipitate was dried and weighed to obtain % yields. Results are given in table 5. 19.
Table 5.19 Alkloidal content in G. arborea and C. arborea
Parts G. arborea C. arborea
Leaf 0.12%w/w 0.2 %w/w
Fruits 0.05%w/w ---
CHAPTER: 5 RESULTS AND DISCUSSION
122
HNGU Ph.D Thesis
G. arborea leaf and fruits contained 0.12%w/w and 0.05%w/w total alkaloid
respectively. Highest amount of alkaloid was found in C. arborea leaf (0.2 %w/w).
5.4.4 Total saponin content
Saponin content was determined in aqueous and methanol extract of leaf and fruits of G.
arborea and C. arborea. Total saponin was determined using calibration curve of
diosgenin and expressed as %w/w of diosgenin. The calibration curve of diosgenin is
given in Figure 5.28.
Figure 5.28 Calibration curve of diosgenin
The calibration curve was found to be linear with correlation coefficient (R2= 0.990) and
Y=0.008x+0.519. The total saponin content in different extracts of leaves and fruits of G.
arborea and C. arborea are tabulated in Table 5.20.
Table 5.20 Saponin content of leaves and fruits extracts of C. arborea and G. arborea
y = 0.008x - 0.519
R² = 0.990
0
0.4
0.8
1.2
1.6
2
0 50 100 150 200 250 300
Ab
sorb
an
ce
Conc(µg/ml)
Calibration curve of diosgenin for total saponin
Extracts G. arborea C. arborea %W/W %W/W
Methanol leaf extract 15.09 ±0.95 19.35 ± 0.70 Aqueous leaf extract 7.79 ±0.26 17.79±0.30 Methanol fruit extract 7.82 ±0.26 10.60 ±0.87 Aqueous fruit extract 7.22 ±0.72 6.37 ± 1.00
CHAPTER: 5
HNGU
Result showed that the highest amount of saponin
arborea leaf. Methanol and
arborea contain higher amount of saponin than
arborea fruits contain higher amount of saponin than
5.5 Estimation of Gallic Acid by HPTLC Method
Quantification of gallic acid was carried out in methanol, ethyl acetate extract of leaves
and fruits of C. arborea.
5.5.1 Development of HPTLC method
The solvent system were tried and checked for band separation using varying
concentration of mobile phase viz. Ethyl acetate: Methanol: Formic acid: Gallic acid
(9:1:0.4:0.2), Ethyl acetate: toluene: methanol: formic acid (8:2
Methanol: Formic acid: Gallic acid (9:1:0.2:0.2) and Ethyl acetate: toluene: formic acid
(8:2:0.3) were tried. Optimised mobile phase (Ethyl acetate: toluene: formic acid
(8:2:0.3)) was selected on the basis of the best separation a
and error. The calibration curve of gallic acid was
concentration of gallic acid of
curve of gallic acid is given in Figure 5.
Figure 5.29
RESULTS AND DISCUSSION
Ph.D Thesis
highest amount of saponin was found in methanol
and aqueous extract of leaf and methanol extract of fruit
contain higher amount of saponin than G. arborea. Aqueous extracts of
fruits contain higher amount of saponin than C. arborea.
Gallic Acid by HPTLC Method
Quantification of gallic acid was carried out in methanol, ethyl acetate extract of leaves
.5.1 Development of HPTLC method for estimation of gallic acid
The solvent system were tried and checked for band separation using varying
concentration of mobile phase viz. Ethyl acetate: Methanol: Formic acid: Gallic acid
(9:1:0.4:0.2), Ethyl acetate: toluene: methanol: formic acid (8:2:1:0.2), Ethyl acetate:
Methanol: Formic acid: Gallic acid (9:1:0.2:0.2) and Ethyl acetate: toluene: formic acid
(8:2:0.3) were tried. Optimised mobile phase (Ethyl acetate: toluene: formic acid
(8:2:0.3)) was selected on the basis of the best separation and sharpness of bands by trial
The calibration curve of gallic acid was prepared by plotting area verses
gallic acid of each peak corresponding to the respective spot.
curve of gallic acid is given in Figure 5.29.
29 Calibration curve of Gallic acid using HPTLC
RESULTS AND DISCUSSION
123
methanol extract of C.
of leaf and methanol extract of fruit of C.
Aqueous extracts of G.
Quantification of gallic acid was carried out in methanol, ethyl acetate extract of leaves
The solvent system were tried and checked for band separation using varying
concentration of mobile phase viz. Ethyl acetate: Methanol: Formic acid: Gallic acid
:1:0.2), Ethyl acetate:
Methanol: Formic acid: Gallic acid (9:1:0.2:0.2) and Ethyl acetate: toluene: formic acid
(8:2:0.3) were tried. Optimised mobile phase (Ethyl acetate: toluene: formic acid
nd sharpness of bands by trial
by plotting area verses
each peak corresponding to the respective spot. Calibration
Calibration curve of Gallic acid using HPTLC
CHAPTER: 5 RESULTS AND DISCUSSION
124
HNGU Ph.D Thesis
5.5.2 Validation of developed HPTLC method for estimation of gallic acid
The developed HPTLC method for estimation of gallic acid was validated for linearity,
precision, repetability, acuuraccy, limit of detection, limit of quantitation and range has
shown in Table 5.21.
Table 5.21 Validation parameter for developed HPTLC method of gallic acid
Validation parameter Result
Accuracy (average % recovery) 98.97%
Precision -Coefficient of variance (CV %) 1.794
Limit of detection (µg) 0.12µg
Limit of quantification (µg) 0.365µg
Linearity (correlation coefficient) 0.98
Range (µg/spot) 1.6-8µg
Specificity Specific
The accuracy of method was determined by performing recovery studies at three level
(50%, 100%, 150% addition) and average % recovery was found 98.97% indicating good
accuracy. The repeatability was checked by repeated scanning (n=7) of same spot of
Gallic acid (1.6µg) and expressed as coefficient of variance (1.794%CV). The limit of
detection and limit of quantification were determined by comparing the peak height of
sample with methanol as a blank on the basis of signal to noise ratio. Limit of detection
was found 0.12µg and limit of quantification was found 0.365µg indicating developed
method was precise and accurate. The correlation coefficient was found 0.98 and linearity
equation was found Y=1845.55x+7306.117 indicating developed method was linear
within a 1.6-8µg range. Thus the developed method was accurate, precise, reproducible,
linear and specific.
5.5.3 Quantification of Gallic acid using HPTLC method in extracts
Gallic acid content in methanol and ethyl acetate extract of leaf and a fruit of C. arborea
was carried out. The photographs of TLC plate for estimation of gallic acid in extracts at
254nm and visible mode are given in Figure 5.30 and Figure5.31 respectively.
CHAPTER: 5
HNGU
[TI=1.6µg gallic acid, T2=3.2µg gallic acid, T3=4.8µg gallic acid, T4=6.4µg gallic acid and T5= 8µg gallic acid, T6, T7; ethyl acetate extracts of fruit of fruit of C. arborea, T10, T11; ethyl acetate extracts leavesextract of leaves of C. arborea
Figure 5.30 The photographs of TLC plate for estimation of gallic acid in extracts at 254nm
[TI=1.6µg gallic acid, T2=3.2µg gallic acid, T3=4.8µg gallic acid, T4=6.4µg gallic acid and T5= 8µg gallic acid, T6, T7; ethyl acetate extracts of fruit of fruit of C. arborea, T10, T11; ethyl acetate extracts leaves of extract of leaves of C. arborea
Figure 5.31 The photographs of TLC plate for estimation of gallic acid in extracts at visible mode
T1 T2 T3 T4
T1 T2 T3 T4
RESULTS AND DISCUSSION
Ph.D Thesis
[TI=1.6µg gallic acid, T2=3.2µg gallic acid, T3=4.8µg gallic acid, T4=6.4µg gallic acid and T5= T6, T7; ethyl acetate extracts of fruit of C. arborea, T8, T9; methanol extract of
, T10, T11; ethyl acetate extracts leaves of C. arborea T12, T13: methanol C. arborea]
The photographs of TLC plate for estimation of gallic acid in extracts at
[TI=1.6µg gallic acid, T2=3.2µg gallic acid, T3=4.8µg gallic acid, T4=6.4µg gallic acid and T5= T6, T7; ethyl acetate extracts of fruit of C. arborea, T8, T9; methanol extract of
, T10, T11; ethyl acetate extracts leaves of C. arborea T12, T13: methanol C. arborea]
The photographs of TLC plate for estimation of gallic acid in extracts at
T3 T4 T5 T6 T7 T8 T9 T10 T11
T4 T5 T6 T7 T8 T9 T10 T11 T12
RESULTS AND DISCUSSION
125
[TI=1.6µg gallic acid, T2=3.2µg gallic acid, T3=4.8µg gallic acid, T4=6.4µg gallic acid and T5= , T8, T9; methanol extract of
T12, T13: methanol
The photographs of TLC plate for estimation of gallic acid in extracts at
[TI=1.6µg gallic acid, T2=3.2µg gallic acid, T3=4.8µg gallic acid, T4=6.4µg gallic acid and T5= , T8, T9; methanol extract of
T12, T13: methanol
The photographs of TLC plate for estimation of gallic acid in extracts at
T11 T12 T13
T11 T12 T13
CHAPTER: 5
HNGU
HPTLC chromatogram quantifying gallic
leaf and fruits of C. arborea
Ethyl acetate extract of
Ethyl acetate extract of
Figure 5.32 HPTLC chromatogram showing gallic acid in methanol and ethyl acetate extracts leaf and fruits of
3-D HPTLC chromatogram quantifying gallic acid in methanol and ethyl acetate extracts
of leaf and fruits of C. arborea
RESULTS AND DISCUSSION
Ph.D Thesis
quantifying gallic acid in methanol and ethyl acetate extracts of
C. arborea is given in Figure 5.32.
Gallic acid
Ethyl acetate extract of C. arborea fruit Alcohol extract of C. arborea
Ethyl acetate extract of C. arborea leaf Alcohol extract of C. arborea
HPTLC chromatogram showing gallic acid in methanol and ethyl acetate extracts leaf and fruits of C. arborea
D HPTLC chromatogram quantifying gallic acid in methanol and ethyl acetate extracts
C. arborea is given in Figure 5.33.
RESULTS AND DISCUSSION
126
acid in methanol and ethyl acetate extracts of
C. arborea fruit
C. arborea leaf
HPTLC chromatogram showing gallic acid in methanol and ethyl
D HPTLC chromatogram quantifying gallic acid in methanol and ethyl acetate extracts
CHAPTER: 5
HNGU
Figure 5.33 3-D HPTLC chromatogram showing gallic acid in methanol and ethyl
acetate extracts leaf and fruits of
UV spectra of quantifying gallic acid in methanol and ethyl acetate extract
fruits of C. arborea are given in Figure 5.34.
Figure 5.34 UV spectra
extracts of leaf and fruits of
RESULTS AND DISCUSSION
Ph.D Thesis
HPTLC chromatogram showing gallic acid in methanol and ethyl
acetate extracts leaf and fruits of C. arborea
UV spectra of quantifying gallic acid in methanol and ethyl acetate extract
given in Figure 5.34.
of quantifying gallic acid in methanol and ethyl acetate
extracts of leaf and fruits of C. arborea
RESULTS AND DISCUSSION
127
HPTLC chromatogram showing gallic acid in methanol and ethyl
UV spectra of quantifying gallic acid in methanol and ethyl acetate extract of leaf and
of quantifying gallic acid in methanol and ethyl acetate
CHAPTER: 5 RESULTS AND DISCUSSION
128
HNGU Ph.D Thesis
The gallic acid content determined by HPTLC method in methanol and ethyl acetate
extracts of leaf and fruits of C. arborea reported in Table 5.22.
Table 5.22 Gallic acid content in leaves and fruits extract of C. arborea
Extract of C. arborea Gallic acid (%w/w) Ethyl acetate extract of Fruit 2.11
Methanol extract of Fruit 1.2 Ethyl acetate extract of leaves 0.54
Methanol extract of leaves 0.48
Result showed that gallic acid content in ethyl acetate extract of C. arborea fruit was
found to be containing highest, whereas lowest was found in methanol extract of C.
arborea leaves. The ethyl acetate and methanol extract of C. arborea fruits contain more
amount of gallic acid than leaves extracts.
5.6 Pharmacological Evaluation
5.6.1 Acute toxicity study: The animals were kept on fasting for overnight providing
only water. They were divided in four groups each containing three animals. The
methanol extract (300mg/kg, 2000mg/kg doses) of fruits and leaf of G. arborea and C.
arborea were given orally. The animals were periodically observed after dosing
continuously for 30min, for during the first 24hrs and thereafter daily for 14 days. The
observations like convulsion, salivation, sleep, movement, body weight, death etc. were
recorded and results are given in Table 5.23.
Table 5.23 Acute toxicity of methanol extract of leaf and fruits of G. arborea and C. arborea on rat
Parameters Up to 30min 24hrs 14days Convulsion NC NC NC
Salivation NC NC NC
Movement NC NC NC
Body weight NC NC NC
Death NC NC NC
NC = no change
Result showed that no change was recorded in convulsion, salivation, sleep, movement,
body weight, death etc. until the end of the study period, revealed the non-toxic nature of
the methanol extract fruit and leaf of G. arborea and C. arborea.
CHAPTER: 5 RESULTS AND DISCUSSION
129
HNGU Ph.D Thesis
5.6.2 Antiallergic activity
Anti-allergic activity was carried out on methanol extract of leaves (GLA) & fruits
(GFA) of G. arborea and methanol extract of leaves (CLA) & fruits (CFA) of C. arborea
using isolated guinea pig ileum, isolated rat ileum and passive paw anaphylaxis in rats.
5.6.2.1 In vitro antiallergic activity on isolated guinea pig ileum
The inhibitory effect of contractile response of histamine on isolated guinea pig ileum by
100, 200µg/ml methanol extract of leaves and fruits of G. arborea and C. arborea were
recorded. Results are reported in Figure 5.35 and Table 5.24.
Figure 5.35 Effect of G. arborea and C. arborea on isolated guinea pig ileum
Table 5.24 Effect of G. arborea and C. arborea on isolated guinea pig ileum.
G. arborea C. arborea
Treatment % inhibition Treatment % inhibition Histamine (10µg/ml) -- Histamine (10µg/ml) --
H1+ GLA (100µg/ml) 71.63±0.15* H1 + CLA (100µg/ml) 35.14±0.12*
H1 + GLA (200µg/ml) 82.03±0.06 H1 + CLA (200µg/ml) 62.98±0.02**
H1 + GFA (100µg/ml) 43.94±0.11* H1 + CFA (100µg/ml) 43.25± 0.12*
H1 + GFA (200µg/ml) 64.20±0.03** H1 + CFA (200µg/ml) 68.79±0.04**
*P < 0.01, ** P < 0.005
CHAPTER: 5 RESULTS AND DISCUSSION
130
HNGU Ph.D Thesis
Result showed that the methanol extract of fruit and leaves of G. arborea and C. arborea
had exhibited significant the dose dependant inhibition on histamine induced contraction
of guinea pig ileum. Methanol extract of G. arborea leaves showed more inhibition on
histamine induced contraction of guinea pig ileum than methanol extract of fruits.
Methanol extract of C. arborea leaves showed less inhibition on histamine induced
contraction of guinea pig ileum than methanol extract of fruits.
The methanol extract of leaves of G. arborea showed the highest inhibition on histamine
induced contraction of guinea pig ileum than C. arborea while methanol extract of fruits
of G. arborea and C. arborea showed almost identical inhibition on histamine induced
contraction of guinea pig ileum.
5.6.2.2 In vitro antiallergic activity on isolated rat ileum
Antiallergic activity by inhibition on contractile response of acetylcholine on isolated rat
ileum by varying dose of methanol extract of leaves and fruits of G. arborea and C.
arborea were carried out and results are reported in Figure 5.36 and Table 5.25.
Figure 5.36 Effect of G. arborea extract on isolated rat ileum preparation
CHAPTER: 5 RESULTS AND DISCUSSION
131
HNGU Ph.D Thesis
Table 5.25 Effect of G. arborea extract on isolated rat ileum preparation
G. arborea C. arborea
Treatment % Inhibition Treatment % Inhibition
ACH (10 µg/ml) -- ACH (10 µg/ml) --
ACH + GLA(100µg/ml) 38.90± 0.044 ** ACH + CLA (100µg/ml) - 4.76± 0.058**
ACH + GLA(200µg/ml) 68.89± 0.061* ACH + CLA (200µg/ml) -31.74± 0.29**
ACH + GFA(100µg/ml) 34.14± 0.060** ACH + CFA (100µg/ml) 42.06± 0.058**
ACH+ GFA (200µg/ml) 60.63± 0.035** ACH + CFA (200µg/ml) 62.7± 0.078*
*P < 0.01, ** P < 0.005
Result showed that the methanol extract of fruit and leaves of G. arborea and fruits of C.
arborea had exhibited significant dose dependant similar inhibition on acetylcholine
induced contractile response on rat ileum.
Methanol extract of C. arborea leaf showed significant dose dependant increase on
acetylcholine induced contractile response on rat ileum. This may be due to cholinergic
activity or cholinesterase inhibitory activity or direct contractile activity of
phytoconstituents present in methanol extract of C. arborea leaf. Qualitative and
quantitative phytochemical study revealed that C. arborea leaf contained more amount
saponin and alkaloid and hence, may be responsible for increasing contractile response of
acetyl choline on rat ileum.
5.6.2.3 Antiallergic activity by passive paw anaphylaxis in rats
Antiallergic activity of methanol extract of leaf and fruits of G. arborea and C. arborea
was carried out using passive paw anaphylaxis model. Sensitised blood serum was
prepared by injecting egg albumin adsorbed on aluminium hydroxide gel in saline.
Albino Wistar rats were passively sensitized with serum into the left hind paw of animals.
An equal volume of saline was administered to contra lateral paw. Standard and plant
extracts were given orally 24hr after sensitization. After 1hr of drug treatment, Animals
were again challenged with egg albumin in the left hind paw, and the paw volume was
measured using a plethysmometer. The percentage of oedema volume was calculated by
the difference between prior and after antigen challenge. Results are given in Figure 5.37.
CHAPTER: 5 RESULTS AND DISCUSSION
132
HNGU Ph.D Thesis
Figure 5.37 Antiallergic activity of G. arborea and C. arborea extracts by passive paw anaphylaxis in rats
The inhibition paw volume by G. arborea and C. arborea extracts on passive anaphylaxis
rats is given in Table 5.26.
Table 5.26 The inhibition paw volume by G. arborea and C. arborea extracts on
passive anaphylaxis rats.
Treatment % inhibition
1hr 2hr 3hr 4hr
Dexamethasone (0.27mg/kg)
57.99±0.042* 67.75±0.027** 65.41±0.030 61.17±0.017**
G. a
rbor
ea GLA (100mg/kg) 29.73±0.086* 35.50±0.062 31.45±0.067* 31.50±0.046*
GLA (300mg/kg) 46.44±0.045** 46.88±0.029** 44.03±0.041** 42.86±0.028**
GFA (100mg/kg) 20.88±0.055 25.20±0.075* 19.18±0.056** 12.82±0.046**
GFA (300mg/kg) 35.38±0.0522** 38.75±0.057* 33.65±0.068* 24.54±0.062*
C. a
rbor
ea CLA (100mg/kg) 13.02±0.061 17.62±0.072 14.78±0.085* 6.96±0.060*
CLA (300mg/kg) 24.82±0.083* 20.84±0.082* 18.55±0.063* 11.72±0.040**
CFA (100mg/kg) 20.64±0.079* 29.81±0.069* 26.42±0.046** 21.25±0.065*
CFA (300mg/kg) 32.92±0.064* 33.88±0.058* 34.91±0.051* 27.84±0.039** *P < 0.01, ** P < 0.001
Result revealed that the methanol extract of G. arborea leaves and fruits showed dose
dependent significant anti-allergic activity. Antiallergic activity in all extracts and
dexamethasone were increased up to 2hrs and then after decline. Methanol extract of leaf
of G. arborea have shown maximum antiallergic activity whereas methanol extract of
C. arborea leaf exhibited minimum antiallergic activity. The methanol extract of fruits of
CHAPTER: 5 RESULTS AND DISCUSSION
133
HNGU Ph.D Thesis
G. arborea and C. arborea had shown almost same antiallergic activity after 1hr, 2hr, 3hr
and 4hr.
Antiallergic activity of methanol extract of leaf and fruits of G. arborea and C. arborea
in isolated guinea pig ileum, isolated rat ileum and passive paw anaphylaxis in rats
models may be attributed due to presence phytoconstituents like carbohydrate, saponin,
alkaloid, flavonoid, tannin and phenolics.
5.6.3 Anti oxidant activity in vitro
Antioxidant activity of methanol extract of leaves (GLA) & fruits (GFA) of G. arborea,
leaf (CLA) & fruits (CFA) of C. arborea and ethyl acetate extract of leaves (GLE) &
fruits (GFE) of G. arborea, leaf (CLE) & fruits (CFE) of C. arborea using DPPH free
radical scavenging assay and reducing assay by FeCl3 models was carried out.
5.6.3.1 DPPH free radical scavenging activity
Antioxidant activity of methanol extract of leaves (GLA) & fruits (GFA) of G. arborea,
leaf (CLA) & fruits (CFA) of C. arborea and ethyl acetate extract of leaves (GLE) &
fruits (GFE) of G. arborea, leaf (CLE) & fruits (CFE) of C. arborea were performed
using DPPH free radical scavenging assay. Antioxidant activity was expressed as
decrease in absorbance of the samples at a different concentration levels. Results of
antioxidant activity of methanol and ethyl acetate extracts of leaves and fruits of G.
arborea and C. arborea are given in Figure 5.38.
Figure 5.38 DPPH free radical scavenging activity of G. arborea and C. arborea
CHAPTER: 5 RESULTS AND DISCUSSION
134
HNGU Ph.D Thesis
Result showed that methanol extract of leaves (GLA) & fruits (GFA) of G. arborea, leaf
(CLA) & fruits (CFA) of C. arborea and ethyl acetate extract of leaves (GLE) & fruits
(GFE) of G. arborea, leaf (CLE) & fruits (CFE) of C. arborea had exhibited dose
dependent antioxidant activity in DPPH free radical scavenging model. The ethyl acetate
extract of fruits and leaf of G. arborea and C. arborea showed more radical scavenging
activity than methanol extract of fruit and leaf of both the plants.
The IC50: radical-scavenging activity (concentration in µg required for 50% inhibition of
DPPH radical) was calculated from the graph. The IC50 (µg) value for DPPH free radical
scavenging activity of G. arborea and C. arborea are compiled in Table 5.32.
Table 5.27 The IC50 value of G. arborea and C. arborea extracts in DPPH model
G. arborea IC 50(µg) C. arborea IC 50(µg)
Ascorbic acid (AA) 53.43 ± 0.93 Ascorbic acid (AA) 53.43 ± 0.93
GLA (Methanol extract leaf) 145.2 ±0.65 CLA (Methanol extract leaf) 80.25 ±0.72
GLE(Ethyl acetate extract leaf) 103.82 ±0.81 CLE (Ethyl acetate extract leaf) 44.28 ± 1.16
GFA (Methanol extract fruit) 137.74 ±0.66 CFA (Methanol extract fruit) 91.96 ±0.91
GFE (Ethyl acetate extract fruit) 97.3 ±0.86 CFE (Ethyl acetate extract fruit) 32.93 ±0.98
Values represent mean ± standard deviation (n=3)
The lower IC50 value indicates the higher antioxidant activity. Result suggest that ethyl
acetate extract of both plants had shown higher antioxidant activity than methanol
extract. Methanol and ethyl acetate extract of G. arborea fruits were found more
antioxidant activity than leaf. Ethyl acetate extract of fruits of G. arborea showed the
higher radical scavenging activity than methanol extract of fruit, methanol and ethyl
acetate extract of leaves of G. arborea. Methanol extract of leaf of C. arborea was found
more antioxidant activity than fruit. While ethyl acetate extract of leaf of C. arborea was
found less antioxidant activity than fruit. Ethyl acetate and methanol extract of fruit and
leaves of C. arborea showed higher free radical scavenging activity than G. arborea fruit
and leaf extract. Ethyl acetate extract of fruits of C. arborea showed the highest radical
scavenging activity whereas methanol extract of G. arborea leaf exhibited the lowest
radical scavenging activity.
CHAPTER: 5 RESULTS AND DISCUSSION
135
HNGU Ph.D Thesis
5.6.3.2 Reducing power by FeCl3
Antioxidant activity of methanol extract of leaves (GLA) & fruits (GFA) of G. arborea,
leaf (CLA) & fruits (CFA) of C. arborea and ethyl acetate extract of leaves (GLE) &
fruits (GFE) of G. arborea, leaf (CLE) & fruits (CFE) of C. arborea were carried out
using Reducing power by FeCl3 model. Antioxidant activity was expressed as increase in
absorbance of the samples at a different concentration levels. Results of antioxidant
activity of methanol and ethyl acetate extracts of leaves and fruits of G. arborea and C.
arborea are given in Figure 5.39.
Figure 5.39 Reductive potential of G. arborea and C. arborea fruits and leaf extracts
Result showed that the ethyl acetate extract of fruits and leaf of G. arborea and C.
arborea showed more reductive potential than methanol extract of fruit and leaf of both
the plants. Methanol and ethyl acetate extract of leaves (GLA) & fruits (GFA) of G.
arborea and leaf (CLA) & fruits (CFA) of C. arborea had exhibited dose dependent
reductive potential by FeCl3 model.
Antioxidant activity of methanol and ethyl acetate extract of leaf and fruits of G. arborea
and C. arborea DPPH free radical scavenging and reducing assay by FeCl3 models, may
be attributed due to presence phytoconstituents like carbohydrate, saponin, alkaloid,
flavonoid, tannin and phenolics.
0
0.4
0.8
1.2
1.6
2
0 50 100 150
Abs
orba
nce
Conc.(mcg/ml)
Reducing power
Ascorbic acidGLECFACLACLEGFECFEGFAGLA
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