CHAPTER - V RESULTS & DISCUSSIONshodhganga.inflibnet.ac.in/bitstream/10603/44238/12... · Type...

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


, 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


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


. 5.2.

C. arborea (DC-CA-

Degree Pharmacy

3 and Figure 5.4

CHAPTER: 5

HNGU

Figure 5.

Figure 5.


Ph.D Thesis

Figure 5.2 Certificate of authentication of plants

Figure 5.3 Herbarium of Gmelina arborea


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CHAPTER: 5 RESULTS AND DISCUSSION

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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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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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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


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


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


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


104

presence of epidermal cells, mesocarp

reported in Figure

fruits

owed fibers, sclereids, mesocarp

red having pleasant

and unpleasant odour.

fragments


105

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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HNGU Ph.D Thesis

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


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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HNGU Ph.D Thesis

Table 5.6 Physicochemical constant of G. arborea and C. arborea fruits


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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HNGU Ph.D Thesis

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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HNGU Ph.D Thesis

Table 5.9 Fluorescence analysis of G. arborea and C. arborea fruits


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


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.


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.


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


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.


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


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.


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


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


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


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.


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


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.


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


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


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


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


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


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 ---


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


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


.5.1 Development of HPTLC method for estimation of gallic acid



(9:1:0.4:0.2), Ethyl acetate: toluene: methanol: formic acid (8:2:1:0.2), Ethyl acetate:



(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


123

methanol extract of C.

of leaf and methanol extract of fruit of C.

Aqueous extracts of G.




:1:0.2), Ethyl acetate:



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


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


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]


T3 T4 T5 T6 T7 T8 T9 T10 T11

T4 T5 T6 T7 T8 T9 T10 T11 T12


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


[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


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


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.


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


Ph.D Thesis


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


127


UV spectra of quantifying gallic acid in methanol and ethyl acetate extract of leaf and

of quantifying gallic acid in methanol and ethyl acetate


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.


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.


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


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


131

HNGU Ph.D Thesis

Table 5.25 Effect of G. arborea extract on isolated rat ileum preparation


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.


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


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



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


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.


135

HNGU Ph.D Thesis

5.6.3.2 Reducing power by FeCl3



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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