CHAPTER V RESULTS AND DISCUSSIONshodhganga.inflibnet.ac.in/bitstream/10603/43346/15/15_chapter5.pdf · The shape of Annona squamosa leaves is oblong, lanceolate or elliptic with entire

CHAPTER V RESULTS AND DISCUSSION

STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 148

5.1 PHARMACOGNOSTICAL STUDIES OF PLANT MATERIALS

The dried roots of Salacia prenoides, fresh leaves of Annona squamosa and

aerial parts of Coccinia indica were evaluated for complete

pharmacognostical parameters such macroscopy and microscopy.

Macroscopical description of roots of Salacia prenoides:

The pieces of roots are shown in fig 5.1 with following characteristics. The

root was rainbow colored when fresh and acquired golden yellow color

upon drying and has exfoliated bark. It had characteristic odor, was bitter in

taste. It had rootlets on its surface and its transverse section showed clear

annular rings. Thus, the roots of this shrubby scandent climber showed a

number of circular (annular), variously colored rings in cross section.

Fig 5.1 Morphology of Salacia prenoides roots



Microscopical description of roots of Salacia prenoides:

A portion of transverse section is shown in fig. 5.2 with following

characteristics. The average thickness of the section was 12-14μ. The

sections stained with toluidine blue. The section showed pink color when

stained with a mixture of 2 drops of phloroglucinol and one drop of

hydrochloric acid kept for 5 minutes. Iodine solution was used for detection

of starch. Its transverse section shows clear annular rings. The section of

root shows distinguishing characteristic having wavy cork. It consists of 6-7

layers. The cortex is a larger portion consisting of brown matter. It has

stellar region having secondary growth and the cells are mainly

parenchymatous in nature. Starch is present in the cortex region. The

vascular bundle consists mainly of secondary xylem and phloem. Distinct

xylem vessels and xylem parehchyma were observed. The section also

shows uniserriate and few biserriate medullary rays. Presence of starch is

detected in medullary rays. Pith is absent. Pericycle and endodermis is also

found to be absent.

Fig. 5.2a Microscopy of Salacia prenoides root bark (100×)



Fig. 5.2b Cork of Salacia prenoides bark (450×)

Fig. 5.2c Cortex of Salacia prenoides root (100×)

Fig. 5.2d Cortex of Salacia prenoides root (450×)



Fig. 5.2e Cortex of Salacia prenoides root (1000×)

Fig. 5.2f Cortex of Salacia prenoides root (oil immersion lens)

Fig. 5.2g Lower cortex of Salacia prenoides root (450×)



Fig. 5.2h T.S. of Salacia prenoides roots Showing Pith

Macroscopical description of leaves of Annona squamosa:

The shape of Annona squamosa leaves is oblong, lanceolate or elliptic with

entire margin, acute to subacute apex, glaucous surface, petiolate, pinnate

venation with 8-11 pairs of lateral nerves, pellucid-dotted, peculiarly

scented, length varying from 5 to 15cm and breadth is 1.9 to 3.8 cm. Leaves

are glaucous beneath, pubescent when young and turn black when dried.

Leaves gave pungent and offensive odor when crushed.

Fig 5.3 Upper and Lower Surface of Annona squamosa Leaves



Microscopical description of leaves of Annona squamosa:

In the microscopic studies, leaf is dorsiventral with thick prominent midrib

and thin lamina (fig. 5.4a). Single layer of rectangular epidermal cells

showing thin cuticle layer (fig 5.4c). In lamina, mesophyll shows presence

of single layer of palisade parenchyma followed by dark cells of spongy

parenchyma (fig. 5.4f). Ground tissue of midrib consists of a zone of

collenchyma below upper epidermis and above lower epidermis (fig. 5.4

d,e). It consists of curved vascular bundle. Lower side of vascular bundle is

wavy (fig 5.4b). It consists of layer of lignified pericyclic fibres above and

below vascular bundle (fig. 5.4g). Midrib also shows presence of abnormal

vascular bundle. Some parenchymatous cells of midrib region shows

presence of yellow oil cells (fig 5.4b). Surface preparation shows vein-islets

and vein terminations along with wavy epidermal cells and anomocytic

stomata (fig.5.5).

Microscopic study of Annona squamosa leaf powder shows the presence of

broken fragments of lamina, lignified pericyclic fibres, epidermal cells in

surface view and fragments of veinlets (fig. 5.6).

Fig.5.4a T.S. Showing Dorsiventral Lamina of Annona squamosa Leaf



Fig. 5.4b T.S. of Annona squamosa Leaf showing Oil cells

Fig. 5.4c Upper Epidermis of Annona squamosa Leaf (450x)

Fig. 5.4d Collenchyma below Upper Epidermis (450×)



Fig. 5.4e Collenchyma above Lower Epidermis (450×)

Fig. 5.4f T.S. Showing Mesophyll of Annona squamosa Leaf (450×)

Fig. 5.4g Pericycyclic fibres below Vascular bundle in Midrib (450×)



5.5a Type of Veination (100×) 5.5b Spongy cells (450×)

5.5c Type of Stomata and Epidermal cell

Fig. 5.5 Surface Preparation of Annona squamosa leaf

Fig. 5.6a Epidermis in Surface View(100×)



Fig. 5.6b Striated Cuticle (450×)

Fig. 5.6c Fragment of Lamina showing fibre (100×)

Fig. 5.6 Powder study of Annona squamosa Leaves Powder



Results of quantitative microscopy like stomatal no., stomatal index, vein-

islet number vein-termination number, palisade ratio are quoted in table 5.1

Table 5.1 Quantitative Microscopy of Annona squamosa Leaf

PARAMETERS RESULTS

Stomatal Number

1. Upper Epidermis

2. Lower Epidermis

2.5

5.0

Stomatal Index (%)

1. Upper Epidermis

2. Lower Epidermis

5.25

9.52

Vein-islet Number 03

Vein-termination Number 02

Palisade Ratio 8.50

Macroscopical description of aerial parts of Coccinia indica:

Leaves 5-10cm. long and broad, lamina bright green above, paler beneath,

surface studded and sometimes rough with papillae, obtusely 3 to 5 angled

or sometimes deeply 5-lobed, the lobes broad, obtuse or acute, apiculate,

more or less sinuate-toothed; petioles 2-3.2 cm. long, cylindrical. Palmately

5-nerved from a cordate base, often with circular glands between the nerves

near the petiole and nervules usually ending in glandular distant

denticulations; delicately venose beneath, ovate or orbicular. Simple and

slender tendrils (fig. 5.7a)

Stem Slender, soft, 0.3-1.5cm in diameter, branched, longitudinally

grooved, glabrous, nodes swollen, whitish dots over external surface, a few



tendrils attached with nodes (fig. 5.7a), grayish colored externally and

cream to light yellow internally, fracture, fibrous; no odor and taste.

Flower Ebracteate, pedicellate, incomplete, unisexual, actinomorphic,

pentamerous. They are large and white about 4cm in diameter and contains

five long tubular petals. It is a dioecious creeper of which male and female

flowers grow separately. Male flowers: Peduncles 1-flowered, 2-3.8 cm.

long, subfiliform. Calyx-tube glabrous, broadly campanulate, 4-5 mm. long;

teeth 2.5cm long, linear. Corolla 2.5cm long, veined, pubescent inside,

glabrous outside; segments 4.5-7.5mm long, triangular, acute. Staminal

column glabrous; capitulum of anthers subglobose. Female flowers:

Peduncles 1.3-2.5cm long, calyx and corolla as in male flowers;

Staminodes 3, subulate, 3 mm long, ovary fusiform, glabrous, slightly

ribbed, stigma 3, bifid (fig 5.7b).

Fruit A pepo, ovoid, glabrous, fusiform, ellipsoid, slightly beaked, 2.5-5cm

long and 1.3-2.5cm thick, greenish brown to yellowish brown, marked when

immature with white streaks, bright scarlet when fully ripe. No odor and

taste (fig. 5.7b)

Seeds Somewhat obovoid, 0.7cm long and 0.2-0.3cm wide, rounded at the

apex, slightly papillose, much compressed, yellowish grey (fig 5.7b).

Fig 5.7a A twig showing aerial parts of Coccinia indica



Fig 5.7b Flower and Edible Fruits of Coccinia indica

Microscopical description of aerial parts of Coccinia indica:

Leaf

Midrib Single layered epidermis, on either side, externally covered with

striated cuticle, followed by 1-3 layers of well developed more or less

isodiametric collenchyma bordering the epidermal cells on the dorsal side

and 3-5 layers on the ventral side; vascular bundles, bicollateral, three,

ventral larger and two dorsal smaller forming a prominent ridge, xylem is

well developed and the phloem consists of strands of sieve tubes and small

celled parenchyma; layers of collenchymatous cells gradually reduce to 2 or

3 towards dorsal side, 1 or 2 on ventral side and ultimately towards apex of

leaf, collenchyma reduces to 1 layer on ventral side and 2 layers on dorsal

side; parenchyma 2-3 layered on both sides; vascular bundles single,

semicircular; vessels arranged in radial rows.

Lamina Dorsiventral structure with single layered upper and lower

epidermis with a layer of polygonal cells having wavy walls, externally

covered with striated cuticles; epidermal cells show almost straight walls

and anomocytic (ranunculaceous) stomata in surface view; below upper

epidermis palisade single layered which is discontinuous where the glands



occur in the depressions of the upper epidermis; spongy parenchyma

represented by 3-6 layers of loosely arranged cells, a number of veins

surrounded by parenchyma, which contain chloroplasts and numerous

intercellular spaces present in mesophyll (fig. 5.8a-f)

Fig. 5.8a (100×)

Fig. 5.8b (450×)



Fig. 5.8d Lamina of Coccinia indica leaf

Fig. 5.8e Gland in Upper and Lower epidermis of lamina

Fig. 5.8f

Fig. 5.8(a-f) T.S. and surface preparation of leaf of Coccinia indica



Table 5.2 Quantitative Microscopy of Coccinia indica Leaf

PARAMETERS RESULTS

Stomatal Number

1. Upper Epidermis

2. Lower Epidermis

20

34

Stomatal Index

211Upper Epidermis

3. Lower Epidermis

14.70

16.66

Vein-islet Number 15.62

Vein-termination Number 37.5

Palisade Ratio 4.15

Stem

Mature stem with ridges and furrows, shows a single layered epidermis

composed of tabular cells externally covered with cuticle, or the epidermis

interrupted at certain places due to formation of cork cells; collenchyma 2-4

layered consisting of isodiametric cells; secondary cortex narrow, consisting

of thin-walled, parenchymatous cells; pericycle present in the form of

discontinuous ring of pericyclic fibres; vascular bundle 10 in number,

bicollateral, widely separated by broad strips of ground tissue arranged in a

single ring, inner part of which almost meeting at centre of stem; secondary

phloem semi-lunar in shape; secondary xylem in the centre of each bundle,

consists of vessels, tracheids, fibres and xylem parenchyma; vessels

numerous uniformly scattered throughout xylem, lignified, pitted and with

spiral thickening; tracheids pitted; pith small, composed of thin walled

parenchymatous cells (fig. 5.8g-k)



Fig. 5.8g (100×)

Fig. 5.8h (100×)



Fig. 5.8i (450×)

Fig. 5.8j (450×)



Fig. 5.8k (450×)

Fig. 5.8(g-k) T.S. of Coccinia indica Stem

Powder

Greyish-brown; shows groups of round to polygonal parenchymatous cells,

reticulate, spiral and pitted vessels, aseptate fibres, palisade cells, stone cells,

simple and compound, round to oval, starch grains, measuring 3-11μ in

diameter, fragments of epidermis with straight walled cells and anomocytic

stomata.

Fig. 5.8(l) (100×)



Fig. 5.8m (450×)

Fig. 5.8n (100×)

Fig. 5.8o (450×)



Fig. 5.8p (450×)

Fig. 5.8(l-p) Powder study for aerial part of Coccinia indica

5.2 PHYSICO-CHEMICAL EVALUATION

Powder of all the three samples studied for ash values, extractive values, loss

on drying and heavy metal analysis. Results are tabulated in following table

5.3 & table 5.4.

Table 5.3 Physico-chemical Parameters for all the Drugs

Sr.No. Parameters % w/w

S1 S2 S3

1 Total Ash Value 4.28 9.50 15.25

2 Water soluble ash value 2.75 3.00 7.66

3 Acid insoluble ash value 3.50 2.05 1.75

4 Water soluble extractive value 9.27 6.20 21.80

5 Alcohol soluble extractive value 5.80 12.00 14.25

6 Loss on drying (Moisture

content)

8.32 10.87 15.50

S1 = Salacia prenoides root

S2 = Annona squamosa leaf

S3 = Coccinia indica aerial part



Table 5.4 Heavy metal Analysis for all the Drugs

Sr.No. Sample name Heavy metals

Cd Pb As

1 Salacia prenoides BDL BDL BDL

2 Annona squamosa BDL BDL BDL

3 Coccinia indica BDL BDL BDL

BDL= Below Detection Limit

Detection limit for Cadmium (Cd) is 0.0027, Detection limit for Lead (Pb) is

0.0420 and Detection limit for Arsenic (As) is 0.0530.

5.3 PREPARATION OF SUCCESSIVE SOLVENT EXTRACTS

For each sample, successive solvent extract obtained was weighed and its

physical appearance and yields are reported in table 5.5 to table 5.7.

Table 5.5 Percentage yield of Dried extract of Salacia prenoides roots

Solvent Color of the

extract

Consistency Dry extract

(%w/w)

Petroleum ether

(60-80º)

Yellowish Moderately

sticky powder 4.04%

Toluene Yellowish brown Slightly sticky 3.34%

Chloroform Brown Granular

powder 1.14%

Methanol Blackish brown

(Shining surface)

Granular

powder 2.76%

Chloroform

Water

Blackish

(Shining surface)

Fine powder 4.51%



Table 5.6 Percentage yield of dried extract of Annona squamosa leaves


extract


(%w/w)

Petroleum ether

(60-80ºC)

Greenish brown Semi solid 3.98%

Toluene Light greenish Solid 3.21%

Chloroform Light greenish Solid 1.05%

Methanol Greenish Solid 9.58%

Chloroform

Water

Yellowish green Solid 6.20%

Table 5.7 Percentage yield of dried extract of Coccinia indica aerial parts


extract


(%w/w)

Petroleum ether

(60-80º)

Light green Solid

2.75%

Toluene Green Solid 2.91%

Chloroform Light green Solid 1.85%

Methanol Yellowish green Solid 4.64%

Chloroform

Water

Brown Solid 8.95%

5.4 PHYTOCHEMICAL STUDIES

Phytochemical analysis was done for presence of various phytoconstituents

in successive solvent extracts of all the three drugs. Preliminary chemical

tests indicated presence of various phytoconstituents and results are shown

in tables 5.8 to table 5.10 and results of TLC analysis are shown in table

5.11 to table 5.13.



Table 5.8 Qualitative tests for Different extracts of Salacia prenoides roots

Constituents P T C M W

1. Alkaloids a. Dragendorff‟s test

b. Hager‟s test

c. Wagner‟s test

-

-

-

-

-

-

-

-

-

+

+

+

-

-

-

2. Flavonoids a. Shinoda test

b. Fluorescence test

-

-

-

-

-

-

+

+

+

+

3. Saponins a. Foam test

b. Haemolytic test -

-

-

-

-

-

-

-

-

-

4. Carbohydrates & Glycosides a. Molisch‟s test

b. Fehling‟s test

c. Benedict‟s test

d. Legal‟s test

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

5. Phytosterols & Triterpenes a. Libermann Burchard test

b. Salkowski test

+

+

+

+

-

-

-

-

-

-

6. Tannins a. Gelatin test

b. Lead acetate test

-

-

-

-

-

-

-

+

-

+

7. Phenolic a. Ferric chloride test

b. Folin ciocalteu test

-

-

-

-

-

-

+

+

+

+

8. Coumarins a. Ammonia test

b. Hydroxylamine HCl test

-

-

-

-

-

-

+

+

+

+

9. Anthraquinones a. Borntrager‟s test

b. Modified borntrager‟s test

-

-

-

-

-

-

-

-

-

-

10. Cardiac glycoside a. Keller-killiani test

b. Legal‟s test

-

-

-

-

-

-

-

-

-

-

11. Fixed oil & Fats a. Spot test

b. Saponification test

-

-

-

-

-

-

-

-

-

-

12. Volatile oil a. Hydrodistillation

- - - - -

P: Pet.Ether extract; T:Toluene extract; C:Chloroform extract; M:Methanol

extract; W:Aqueous extract; (+):present; (-):absent



Table 5.9 Qualitative tests for Different extracts of Annona squamosa leaves



b. Hager‟s test

c. Wagner‟s test

-

-

-

-

-

-

+

+

+

-

-

-

-

-

-



-

-

-

-

-

-

+

-

-

-



-

-

-

-

-

-

-

-

-


b. Fehling‟s test


d. Legal‟s test

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

+

-

-

-

-


b. Salkowski test

+

+

-

-

-

-

-

-

-

-



-

-

-

-

-

-

-

+

-

+



-

-

-

-

-

-

+

+

-

-



-

-

-

-

-

-

-

-

-

-



-

-

-

-

-

-

-

-

-

-


b. Legal‟s test

-

-

-

-

-

-

-

-

-

-



-

-

-

-

-

-

-

-

-

-


- - - - +





Table 5.10 Qualitative tests for Different extracts of Coccinia indica aerial parts



b. Hager‟s test

c. Wagner‟s test

-

-

-

-

-

-

-

-

-

+

+

+

-

-

-



-

-

-

-

-

-

+

-

+

-



-

-

-

-

-

-

-

+

-


b. Fehling‟s test


d. Legal‟s test

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+


b. Salkowski test

-

-

-

-

-

-

-

-

-

-



-

-

-

-

-

-

-

-

-

-



-

-

-

-

-

-

+

-

+

-



-

-

-

-

-

-

-

-

-

-



-

-

-

-

-

-

-

-

-

-


b. Legal‟s test

-

-

-

-

-

-

-

-

-

-



+

+

-

-

-

-

-

-

-

-


- - - - -





Table 5.11 Thin Layer Chromatographic pattern of Successive solvent

extracts of Salacia prenoides roots

Successive

solvent

extract

Solvent system

Spraying

agent/

Visualization

No.

of

Spot

s

Rf

value

Petroleum

ether

Toluene:Ethyl-acetate

(93:7)

Anisaldehyde

H2SO4

2 0.39,

0.45

Toluene Benzene:Ethyl acetate

(8:2)

Same 1 0.35

Chloroform Chloroform:Methanol:

Ammonia (9.5:0.5:0.1)

Dragendorff‟s

reagent

Nil --

Methanol Ethyl acetate:Formic

acid: Glacial acetic acid:

Methanol

(10:1.1:1.1:2.7)

1) 10% H2SO4

2) UV light

3) FeCl3

4

3

0.09, 0.16,

0.38, 0.48

Same 0.16,

0.39, 0.48

Water Acetone:Water (9:1) Anisaldehyde

H2SO4

2 0.12,

0.36


extracts of Annona squamosa leaves:

Successive

solvent

extract

Solvent system

Spraying

agent/

Visualization

No.

of

Spot

s

Rf value

Petroleum

ether

Toluene:Ethyl acetate

(93:7)

1. Anisaldehyde

H2SO4

2. UV Light

4

4

0.23,0.32

,0.4, 0.55

Same

Toluene Benzene:Ethyl acetate

(8:2)

Vanillin H2SO4 3 0.35,0.48

,0.50

Chloroform Chloroform:Methanol:

Ammonia

(9.5:0.5:0.1)

Dragendorff‟s

reagent

2 0.49,0.87

Methanol Toluene:Ethyl acetate:

Glacial acetic acid

(15:4:1)

Dragendorff‟s

reagent

3 0.4, 0.49,

0.87

Water Acetone:Water (9:1) Anisaldehyde

H2SO4

3 0.42,0.48

, 0.55




extracts of Coccinia indica aerial parts

Successive

solvent

extract

Solvent system

Spraying

agent/

Visualization

No.

of

Spot

s

Rf value

Petroleum

ether

Toluene:Ethyl acetate

(70:30)

Vanillin H2SO4

2 0.24,

0.48

Toluene Hexane:Ethyl acetate

(6:3)

Same 2 0.24,

0.63

Chlorofor

m

Pet.ether:Diethyl ether:

Acetic acid (70:30:2)

Dragendorff‟

s reagent

1 0.37

Methanol Chloroform:Methano

l: Ammonia

(90:18:2)

UV 366nm

Vanillin H2SO4

Dragendorf‟s

3

1

0.23,0.47

0.61

0.38

Water Ethyl acetate:Methanol

:Water (100:13.5:10)

Anisaldehyde

H2SO4

4 0.58,0.70

0.83,

0.94

5.5 SEPARATION AND ANALYSIS OF PHYTOMARKERS

5.5.1 Isolation and identification of phytomarker from Salacia prenoides

Preliminary phytochemical examinations revealed that xanthone-C-

glucoside constituents of Salacia prenoides can be extracted with methanol.

It was therefore decided to extract the marker from methanol extract. TLC

study of the methanol extract was done according to procedure given in

standard book and it showed presence of Mangiferin.329

Even the roots of

Salacia prenoides have been used as an antidiabetic drug in the indigenous

system of medicine, and clinical tests are said to have substantiated their

efficacy. The root bark contains two 1,3-diketones (C30H48O3 and C30H46O3),



fatty matter, rubber, dulcitol, mangiferin, phlobatannin and glycosidal

tannins. Further, the methanolic extract of stem of S.chinensis and its

constituents viz., 3β, 22β-dihydroxyolean-12-en-oic-acid, tingenone,

tingenine B, regeol A, triptocalline A, mangiferin showed an inhibitory

effect on rat lens aldose reductase and mangiferin (a xanthone from the

roots) and sulfonium ion derivatives, kotalanol and salacinol (from the roots

and stems), have been identified as the antidiabetic principles of S.reticulata

through pharmacological studies, therefore it was thought worthwhile to

separate the same by preparative TLC.

Isolated compound (USP-I) was identified and confirmed by melting point,

mix melting point (table 5.14), co-TLC (fig. 5.9), ultraviolet spectra (fig.

5.10) and overlain IR spectra with standard mangiferin (fig. 5.11) and data

are tabulated in table 5.15.

Table 5.14 Data of Melting point and Mixed m.p. of USP-I and Standard

Mangiferin

Sample Melting Point Mix m.p.

USP-I (a) Literature Standard(b) (a) + (b)

Mangiferin 267-272 ºC 271-274 ºC 267-272 ºC 267-272 ºC

On TLC both USP-I and standard mangiferin resolved at Rf 0.48 as blue

fluorescence and derivatized by ferric chloride reagent.



Fig 5.9 CO-TLC study of Standard Mangiferin & Isolated Compound

(USP-I)

Fig. 5.10 Ultraviolet spectra omparison of USP-I and Standard Mangiferin



Table 5.15 Data of IR spectroscopy of Isolated compound (USP-I) and

Standard Mangiferin

Standard Mangiferin Isolated Compound

(USP-I) Assigned

grouping Frequency Intensity Frequency Intensity

3449.25 cm-1

3260.55 cm-1

m,s

3448.50 cm-1

3259.49 cm-1

m,s

C=O stretching &

O-H stretching of

2º hydroxyl group

2922.50 cm-1

w 2922.48 cm-1

w presence of C-H

antisymmetric

stretching

2831 cm-1

w 2832 cm-1

w presence of C-H

symmetric

stretching

1640 cm-1

w 1639 cm-1

w C=O stretching of

aromatic ketones

1578 cm-1

m 1577 cm-1

m C=C stretch of

aromatic group

1485 cm-1

1450 cm-1

m,s 1485 cm-1

1449 cm-1

m,s CH-CH bend in

plane

1235 cm-1

m,w 1234 cm-1

m,w -OH bend

1115 cm-1

w 1114 cm-1

m C-H in plane

bending

810 cm-1

m-w 813cm-1

m-w C-H plane out of

bend



Fig 5.11 Overlain IR of Standard Mangiferin and Isolated compound (USP-I)



The results of melting point, co-TLC, ultraviolet spectra and IR spectrum

of isolated compound (USP-I) matched with that of standard mangiferin,

so the isolated compound (USP-I) is to be identified as mangiferin

having following structure.

Structure of USP-I (Mangiferin)

(2β-D-glucopyranosyl – 1,3,6,7-tetrahydroxy-9H-Xanthen-9-one)

5.5.2 Estimation of mangiferin in roots of Salacia prenoides by HPTLC

method

Calibration curve of Mangiferin: The calibration data for standard

mangiferin was obtained using standard solution (100μg/ml) of

mangiferin and diluted to different concentration as mentioned in table

5.16. The calibration curve was obtained by plotting concentration Vs

peak area.

Table 5.16 Calibration data of Standard mangiferin Concentration

Vs Peak area

Sr. No.

Conc.of

mangiferin

ng/spot

Mean peak area ±

S.D. (n=5) % C.V.

1 100 464.6±2.23 0.48

2 200 1186.5±4.86 0.41

3 300 1910.0±8.02 0.42

4 400 2678.8±14.19 0.53

5 500 3359.9±9.07 0.27

Correlation coefficient: 0.9997, Slope: 728.29, Intercept: 264.91



Estimation of Mangiferin: The root of Salacia prenoides was analysed

by proposed method mentioned in 4.8.2. The amount of mangiferin was

computed from calibration curve shown in fig. 5.12 and table 1 & table

5.17. The spectral comparison for various concentration of standard

mangiferin is shown in fig.3. The Overlain 3D HPTLC chromatogram of

Standard and test mangiferin and HPTLC chromatogram scanned in

fluorescence light is shown in fig. 5.16 & fig. 5.18. The HPTLC

densito-matric scan at 258nm is shown in fig. 5.19 for standard and test

solution.

CALIBRATION CURVE OF STANDARD

MANGIFERIN

y = 728.29x - 264.91

R2 = 0.9997

0

1000

2000

3000

4000

1 2 3 4 5

Concentration (μg/ml)

Mean

peak a

rea

Fig 5.12 Calibration curve for Standard Mangiferin



Fig 5.13 A three dimentional Calibration curve for Standard Mangiferin

Fig 5.14 HPTLC Chromatogram for track 1-5 of Standard

Mangiferin



Fig 5.15 Spectral comparison for Various concentration of Standard

Mangiferin

Fig 5.16 Overlain 3D HPTLC chromatogram of Standard

Mangiferin and Test sample containing USP-I



Fig 5.17 HPTLC chromatogram of Test sample containing USP-I

Fig 5.18 HPTLC chromatogram scanned in Fluorescence light



Fig 5.19 HPTLC densitometric scan at 258 nm A: Test solution of methanol extract of Salacia prenoides B: Mangiferin standard solution

Table 5.17 Estimation of USP-I (Mangiferin) in root of Salacia prenoides

Sample

Mean peak

area ± S.D.

(n=5)

Average

amount of

mangiferin

(ng/spot)

Average

%w/w of

mangiferin

± S.D.

% C.V.

S.prenoides 2690.96±6.52 4.27 0.16±0.024 0.24

Validation of HPTLC method:

Linearity:

A calibration curve of mangiferin was obtained by plotting the peak area

of mangiferin against the concentration of mangiferin over the range of

100-500 ng/spot. The correlation coefficient was found to be 0.9997 and

relative standard deviation ranged from 0.27-0.53 (table 5.16).

Precision:

The interday and intraday coefficient of variation for mangiferin varied

from 0.29-0.52 and 0.34-0.67 respectively (table 5.18).



Table 5.18 Data for Inter-day and Intraday precision for Mangiferin

Concentration

(ng/spot)

Inter-day Precision

(n=5)

Intraday Precision (n=5)

Peak area

(Mean±S.D.)

% C.V. Peak area

(Mean±S.D.)

% C.V.

100 461.6±2.07 0.45 463.5±2.13 0.46

200 1187.5±5.10 0.43 1185.5±5.21 0.44

300 1911.0±8.02 0.42 1910.1±8.02 0.42

400 2679.8±13.93 0.52 2677.8±17.94 0.67

500 3358.9±9.74 0.29 3358.2±11.41 0.34

Repeatability:

A. Relative standard deviation for repeatability of measurement of

peak area based on 7 times measurement of the same spot was

found to be 0.72 (table 5.19).

B. Relative standard Deviation for repeatability of sample application

of peak area based on 7 times measurement of the same spot was

found to be 0.71 (table 5.20).

Table 5.19 Data of Repeatability of Measurement of Peak area of

Mangiferin

No. of measurement Area

1 2679

2 2657

3 2685

4 2667

5 2685

6 2648



7 2635

Mean 2665.14

S.D. 19.37

R.S.D. (% C.V.) 0.72

Table 5.20 Data of Repeatability of Sample application of Mangiferin

No. of measurement Area

1 2669

2 2671

3 2681

4 2662

5 2695

6 2643

7 2710

Mean 2675.85

S.D. 19.14

R.S.D. (% C.V.) 0.71

Accuracy:

The % recovery of mangiferin was found to be 99.72-100.34% which was

satisfactory as shown in table 5.21.



Table 5.21 Data of Accuracy for Mangiferin

Concentration

(ng/spot) Mean peak

area (n=3)

Amount of

mangiferin

found

Mean±S.D.

(n=3)

% Recovery

(n=3) Taken

(Sample)

Added

(Standard)

300 0 1934.3 4.03±0.046 -

300 150 2898.5 4.29±0.032 99.72

300 300 4180.4 5.72±0.075 99.83

300 450 5523.7 7.15±0.12 100.34

Specificity:

It was observed that the other constituents present in the crude drugs did

not interfere with the peak of mangiferin. Therefore, the method was

specific. The chromatogram of standard mangiferin and isolated

mangiferin in roots of Salacia prenoides was found to be identical.

The minimal detectable limit found to be 1.34 ng/spot and limit of

quantitation was 4.78 ng/spot.

Table 5.22 Summary of Validation parameters for Mangiferin

Sr. No. Parameters Results

1 Linearity 0.9997

2 Precision (% CV.)

Repeatability of Measurement

Repeatability of Application

Interday

Intraday

0.72

0.71

0.29-0.52

0.34-0.67

3 Range 100-500 ng/spot



4 Limit of Detection 1.34 ng/spot

5 Limit of Quantitation 4.78 ng/spot

6 Accuracy 99.72-100.34%

7 Specificity Specific

5.5.3 Isolation and identification of phytomarker from Annona

squamosa:

The tribals and villagers of various places in India extensively use the

young leaves of Annona squamosa along with seeds of Piper nigrum for

the management of diabetes.195,197

The ethanolic extract from leaves of

Annona squamosa have shown hypoglycemic and antidiabetic effect330

and clinical tests are said to have substantiated their efficacy. Preliminary

phytochemical examinations revealed that ethanolic extract from the

leaves of Annona squamosa contain phytosterols, along with other

constituents like flavonoids & alkaloids, which are very good

antioxidants and many of them like β-sitosterol may be responsible for

hypoglycemic action.360

So attempt was made to isolate phytosterol as a

marker compound.

The sterol fraction was finally isolated on elution with toluene and

chloroform (75:25, v/v). Upon purification with methanol, it gave white

waxy powder with characteristic odour of sterol derivative which was

identified having melting point 139-140ºC (table 5.23), Liebermann

Burchard‟s positive test (a greenish color upon treatment in chloroform

with conc. sulfuric acid and acetic anhydride). It was further confirmed

by UV spectra (peak ) and prominent IR peaks at 3250, 1070 (-OH), and

1640 cm-1

(C=C), cumulatively indicating it to be a sterol derivative (table

5.24).



Table 5.23 Data of Melting point and Mixed m.p. of β-sitosterol

& UAS-II


UAS-II (a) Literature Standard(b) (a) + (b)

β-sitosterol 139-140 ºC 136-140 °C 136-140 °C 136-140 °C

Fig 5.20 CO-TLC study of Standard β-sitosterol & Isolated UAS-II



Fig. 5.21 Ultraviolet Spectral Comparison of UAS-II and Standard

β-sitosterol

Table 5.24 Data of IR spectroscopy of Isolated compound UAS-II and

Standard β-sitosterol

Standard β-sitosterol Isolated Compound

UAS-II Assigned


3414.56 cm-1

1646.65 cm-1

m

s

3433 cm-1

m C=O stretching

1060.15 cm-1

m-s 1098 cm-1

m-s C-C stretch



1468.05 cm-1

1379.90 cm-1

m-s 1345 cm-1

m-s C-H bend in plane

Oleafin double

bond

Aromatic group

2936.72 cm-1

2871.72 cm-1

s 2882 cm-1

s C-H stretching

-CH2,-CH3 group

2362.57 m-w 2302 m-w C≡C stretching

959.78 m-s 941 m-s C-O stretching

836.54

804.98

w 790 w C-H bend out of

plane



Fig 5.22 Overlain Infra Red spectra of Standard β-sitosterol and Isolated

compound UAS-II

On TLC both isolated compound (UAS-II) and standard β-sitosterol

resolved at Rf 0.46 as blue fluorescence and derivatized by ferric chloride

reagent.

The results of melting point, co-TLC, ultraviolet spectra and IR spectrum

of isolated compound UAS-II matched with that of standard β-sitosterol,

so the isolated compound UAS-II is to be identified as β-sitosterol and its

structure is as follows:



Structure of UAS-II (β-sitosterol)

17-(5-Ethyl-6-methyl heptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,

11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a] phenanthren-3-ol

5.5.4 Estimation of β-sitosterol in roots of Annona squamosa by

HPTLC method

Calibration curve of β-sitosterol

The calibration data for standard β-sitosterol was obtained using

graded concentration of standard solution of β-sitosterol (table 5.25).

The calibration curve was obtained by plotting concentration Vs peak

area as in fig. 5.23.

Table 5.25 Calibration data of Standard β-sitosterol Concentration Vs

Mean peak area

Sr. No. Concentration

(μg/ml)

Mean peak

area

±S.D. (n=5)

% C.V.

1 60 997.7±6.48 0.65

2 80 1199.8±10.48 0.87

3 100 1357.2±5.70 0.42

4 120 1506.6±14.91 0.99

5 140 1676.0±11.89 0.71




Estimation of β-sitosterol: The leaves of Annona squamosa was

analysed by proposed method mentioned in 4.8.4. The amount of

β-sitosterol was computed from calibration curve shown in fig.

5.23 and table 5.26. The 3D spectral comparison for various

concentration of standard β-sitosterol and test solution is shown in

fig. 5.24. HPTLC chromatogram scanned in fluorescence & UV

light is shown in fig. 5.25 & the HPTLC densitomatric scanned at

258nm for test solution is shown in fig. 5.26.

CALIBRATION CURVE OF β-sitosterol

y = 166.34x + 848.44

R2 = 0.9971

0

200

400

600

800

1000

1200

1400

1600

1800

60 80 100 120 140

CONCENTRATION (μg/ml)

ME

AN

PE

AK

AR

EA

Series1

Linear (Series1)

Fig 5.23 Calibration curve for Standard β-sitosterol

Fig 5.24 3D spectral comparison for various concentration of Standard

β-sitosterol and Test solution containing UAS-II



Fig 5.25 HPTLC scanned in Fluorescence and UV light

Fig 5.26 HPTLC densitometric chromatogram of Test solution

containing UAS-II



Table 5.26 Estimation of UAS-II (β-sitosterol) in Leaves of Annona

squamosa

Sample Mean peak

area

±S.D.

(n=5)

Average

amount of

β-sitosterol

(μg/ml)

Average

%w/w of

β-sitosterol

±S.D.

% C.V.

A.squamosa 1415.23±47 107.85 0.63±0.01 1.58


Linearity:

The linearity of the method was tested by applying standard β-

sitosterol solution from 60µg/ml to 140µg/ml using above

chromatographic condition. The densitograms were recorded and the

peak areas of β-sitosterol for each applied concentration were noted.

The response factors were calculated for each concentration of β-

sitosterol by dividing peak areas by corresponding concentration of β-

sitosterol. The correlation coefficient was found to be 0.9971 and

relative standard deviation ranged from 0.42-0.99 (table 5.25) which

showed that within the concentration range indicated, there was a

good correlation between mean peak area and concentration of β -

sitosterol.

Precision:

The method was validated in terms of intraday precision and

intermediate (Inter-day) precision. The intraday precision method was

studied by analyzing five different concentration of sample solution i.e

methanolic extract of dried leaf powder of Annona squamosa in

triplicates. The Intermediate (Inter-day) precision of the method was

evaluated by analyzing the sample solution in triplicates on three

different days, in the chromatographic system, under the specified

conditions. The results expressed as % C.V. of peak area of β-



sitosterol, are listed in table 5.27. The intraday and inter-day

coefficient of variation for β-sitosterol varied from 0.37-0.92 and 0.44-

0.94 respectively. The results indicate that the method is precise and

reproducible.

Table 5.27 Data for Intraday and Inter-day Precision for β-sitosterol

Concentration

(µg/ml)

Intraday Precision

(n=5)

Inter-day Precision

(n=5)

Peak area

(Mean±S.D.) % C.V.

Peak area

(Mean±S.D.) % C.V.

60 995.9±6.44 0.64 994.7±5.89 0.59

80 1201.3±11.09 0.92 1267.8±9.56 0.75

100 1337.2±4.99 0.37 1367.2±6.10 0.44

120 1584.6±14.57 0.91 1601.6±15.11 0.94

140 1671.0±11.34 0.67 1687.0±11.19 0.66

Repeatability:

A. Relative Standard Deviation for repeatability test was carried out

by applying 10μl of standard solution of β-sitosterol of

concentration 120μg/mL on TLC plate in five replicates and

analyzing under specified chromatographic conditions. The values

of mean peak area of β-sitosterol of the same spot was found to be

1531.60 and R.S.D. 1.26. (R.S.D. value less than 2) (table 5.28)

Table 5.28 Data for Repeatability of Measurement of Peak area of

β-sitosterol

No. of Measurement Area

1 1550

2 1524

3 1502



4 1546

5 1536

Mean 1531.60

S.D. 19.35

R.S.D. (% C.V.) 1.26

B. Relative Standard Deviation for repeatability of sample application

of peak area was carried out by applying 10μl of standard solution

of β-sitosterol of concentration 140μg/mL on TLC plate in five

replicates and analyzing under specified chromatographic

conditions. The values of mean peak area of β-sitosterol of the

same spot was found to be 1673.80 and R.S.D. 0.60. (R.S.D. value

less than 2) (table 5.29)

Table 5.29 Data for Repeatability of Sample application of Peak area

of β-sitosterol


1 1678

2 1689

3 1663

4 1668

5 1671

Mean 1673.80

S.D. 10.08

R.S.D. (% C.V.) 0.60

Accuracy (% Recovery):

The accuracy of the method was established by performing recovery

experiments, using the standard addition method, at three different



levels. About 80µg/ml test solution of powdered leaves of Annona

squamosa was accurately weighed into each of the four stoppered test

tubes. Known concentration (0,20,40, 60 µg/ml) of standard β-

sitosterol were added in solution form to each of the stoppered test

tubes respectively. The stoppered test tubes were then shaken at 20

rpm on rotary shaker for overnight at room temperature (28ºC ± 2ºC).

The contents of the test tube were filtered separately and each solution

was analyzed under optimized chromatographic conditions. Value of

percentage recovery for β-sitosterol was then determined. The results

of the recovery experiment are given in table 5.30. The value of

percentage recovery of β-sitosterol found was 99.28-101.66, indicating

good accuracy of the method.

Table 5.30 Data of Accuracy for β-sitosterol

Concentration

(µg/ml) Mean

Peak Area

(n=3)

Amount of β-

sitosterol

Found Mean±S.D.

(n=3)

%

Recovery

(n=3) Taken

(Sample)

Added

(Std)

80 0 1201.3 82.03±2.35 -

80 20 1337.2 99.23±4.67 99.25

80 40 1584.6 122±5.86 101.66

80 60 1671.0 139±3.35 99.28

Specificity:

It was observed that the other constituents present in the crude drugs

did not interfere with the peak of β-sitosterol. Therefore, the method

was specific. The spectrum of β-sitosterol and β-sitosterol in leaves of

Annona squamosa was found to be similar.

The minimum detectable limit was found to be 40.00 µg/ml and limit

of quantitation was 83.57 µg/ml (table 5.31).



Table 5.31 Summary of Validation Parameters of β-sitosterol


1 Linearity 0.9971

2 Presision (% C.V.

Repeatability of measurement

Repeatability of application

Intraday

Inter-day

1.26

0.60

0.37-0.92

0.44-0.94

3 Range 60-140 µg/ml

4 Limit of Detection (LOD) 40.00 µg/ml

5 Limit of Quantitatin (LOQ) 83.57 µg/ml

6 Accuracy 99.28-101.66


5.5.5 Isolation and identification of phytomarker from aerial parts

of Coccinia indica

Coccinia indica has not been studied as much as some of the other

traditional remedies for diabetes, but the available data on this plant are

chemically and pharmacologically interesting. The plant has been used

since ancient times for treating diabetes mellitus in the Indian system of

medicine known as ayurvedha.361

Mukherjee and co-workers362

showed

that the plant had hypoglycaemic activity in alloxan-diabetic rats,

concluding that the active principle of the plant may have insulin-like

activity. Hyperglycaemia induced by somatotrophin and corticotrophin in

albino rats can be reduced by administration of the alcoholic extract of

the plant. Isolation of the active principle would open up many

possibilities, especially as the plant is not toxic.



The triterpenoidal fraction was finally isolated on elution with mixture of

n-hexane and toluene and chloroform (75:25, v/v). Upon purification with

preparative TLC, it gave crystals from absolute ethanol with

characteristic odor (UCI-III) which was partially identified having

melting point 139-140ºC (table 5.32), Liebermann Burchard‟s positive

test (a greenish color upon treatment in chloroform with conc. sulfuric

acid and acetic anhydride). Isolated compound UCI-III was further

confirmed by UV spectra (peak 230nm) and intense somewhat broadened

IR band at 1710 cm-1

, a weak band at 1670 cm-1

, and strong absorption at

3600 cm-1

and 3470 cm-1

indicating it to be triterpene derivative.

Table 5.32 Data of Melting point and mixed m.p. of Cucurbitacin B &

Isolated compound UCI-III


UCI-III (a) Literature Standard(b) (a) + (b)

Cucurbitacin B 183-186 ºC 184-186 °C 184-186 °C 183-186 °C

Fig 5.27 CO-TLC of Standard Cucurbitacin B & Isolated Compound

UCI-III



Fig. 5.28 Ultraviolet spectrum of Isolated Compound UCI-III and

Standard Cucurbitacin B

Table 5.33 Data of IR spectroscopy of Isolated compound (UCI-III)

and Standard Cucurbitacin B

Standard β-sitosterol Isolated Compound

(UCI-III) Assigned


3605.23 cm-1

S 3612.65 cm-1

S C=O stretching

3423.52 cm-1

S 3430.25 cm-1

s O-H stretching

2923.83 cm-1

2853.36 cm-1

s

m

2918.78 cm-1

2840.12 cm-1

s

m

C-H stretching

1710.78 cm-1

1675.48 cm-1

s

w

1709.14 cm-1

1670.25 cm-1

s

w

C=O stretching

1384.09 cm-1

S 1382.10 cm-1

S C-H bend in plane

1290.93 cm-1

M 1283.45 cm-1

M O-H bending

1097.99 cm-1

S 1095.36 cm-1

S C-O stretching

791.56 cm-1

W 792.74 cm-1

W C-C stretching

667.68 W 670.45 W C-H rocking



Fig 5.29 Overlain spectra of Standard and Isolated compound UCI-III

On TLC both isolated compound UCI-III and standard cucurbitacin B

resolved at Rf 0.90 with vanillin-phosphoric acid reagent as weak yellow

brown and blue violet zones (vis.) (fig. 5.28).

The results of melting point, co-TLC, ultraviolet spectrum and IR

spectrum of isolated compound (UCI-III) matched with that of standard

cucurbitacin B (fig. 5.29), so isolated compound UCI-III is to be

identified as cucurbitacin B and its structure is as follows:



Structure of UCI-III (Cucurbitacin B) 25-(ACETYLOXY)-2,16,20-TRIHYDROXY-9-METHYL-

19-NORLANOSTA-5,23-DIENE-3,11,22-TRIONE

5.5.6 Estimation of Cucurbitacin B in aerial parts of Coccinia indica

by HPTLC method

Calibratin curve of Cucurbitacin B

The calibration data for standard Cucurbitacin B was obtained using

graded concentration of standard solution of Cucurbitacin B (table

5.34). The calibration curve was obtained by plotting concentration Vs

mean peak area (fig. 5.30).

Table 5.34 Calibration data of Standard Cucurbitacin B concentration

Vs Mean peak area

Sr. No. Concentration

(μg/ml)

Mean peak

area

±S.D. (n=5)

% C.V.

1 10 1359.3±12.74 0.93

2 20 2384.7±15.01 0.62

3 30 3501.1±18.70 0.53

4 40 4301.4±19.95 0.46

5 50 5078.2±13.39 0.26




Estimation of Cucurbitacin B: The aerial parts of Coccinia indica

was analysed by proposed method mentioned in 4.8.6. The amount

of Cucurbitacin B was computed from calibration curve shown in

fig.5.30 and table 5.35. The spectral comparison for various

concentration of standard Cucurbitacin B and test is shown in

fig.5.31. HPTLC chromatogram scanned in fluorescence & UV

light is shown in fig.5.32 & the HPTLC densitomatric scanned at

230nm for Cucurbitacin B is shown in fig. 5.33.

CALIBRATIN CURVE FOR CUCURBITACIN B

y = 935.45x + 518.59

R2 = 0.9937

0

1000

2000

3000

4000

5000

6000

10 20 30 40 50

CONCENTRATION (μg/ml)

ME

AN

PE

AK

AR

EA

Series1

Linear (Series1)

Fig 5.30 Calibration curve for Standard Cucurbitacin B

Fig 5.31 Spectral comparison for Various concentration of Standard

Cucurbitacin B and Test solution containing UCI-III



Fig 5.32 HPTLC scanned in Ultra-Violet light

Fig 5.33 HPTLC densitometric chromatogram of Test solution

containing UCI-III



Table 5.35 Estimation of UCI-III (Cucurbitacin B) in aerial parts of

Coccinia indica

Sample Mean peak

area

±S.D. (n=5)

Average

amount of

Cucurbitacin

B (μg/ml)

Average

%w/w of

Cucurbitacin B

±S.D.

% C.V.

C.indica 2653.1±18.35 22.60 0.56±0.01 1.78


Linearity:

The linearity of the method was tested by applying standard

Cucurbitacin B solution from 10µg/ml to 50µg/ml using above

chromatographic condition. The densitograms were recorded and the

peak areas of Cucurbitacin B for each applied concentration were

noted. The response factors were calculated for each concentration of

Cucurbitacin B by dividing peak areas by corresponding concentration

of Cucurbitacin B. The correlation coefficient was found to be 0.9937

and relative standard deviation ranged from 0.26-0.93 (table 5.34)

which showed that within the concentration range indicated, there was

a good correlation between mean peak area and concentration of

Cucurbitacin B.

Precision:

The method was validated in terms of intraday precision and

intermediate (Inter-day) precision. The intraday precision method was

studied by analyzing five different concentration of sample solution i.e

methanolic extract of dried powder of aerial parts of Coccinia indica

in triplicates. The Intermediate (Inter-day) precision of the method

was evaluated by analyzing the sample solution in triplicates on three

different days, in the chromatographic system, under the specified

conditions. The results expressed as % C.V. of peak area of



Cucurbitacin B, are listed in table 5.36. The intraday and inter-day

coefficient of variation for Cucurbitacin B varied from 0.37-0.90 and

0.43-0.94 respectively. The results indicate that the method is precise

and reproducible.

Table 5.36 Data for Intraday and Inter-day Precision for Cucurbitacin B

Concentration

(µg/ml)

Intraday Precision

(n=5)

Inter-day Precision

(n=5)

Peak area

(Mean±S.D.) % C.V.

Peak area

(Mean±S.D.) % C.V.

10 1348.3±11.74 0.87 1320.3±12.43 0.94

20 2383.7±15.01 0.62 2312.7±11.56 0.49

30 3489.1±31.69 0.90 3535.1±28.70 0.81

40 4320.4±21.57 0.49 4489.4±19.68 0.43

50 5102.2±19.37 0.37 5034.2±17.95 0.35

Repeatability:

A. Relative Standard Deviation for repeatability test was carried out

by applying 10μl of standard solution of Cucurbitacin B of

concentration 20μg/mL on TLC plate in five replicates and analyzing

under specified chromatographic conditions. The values of mean peak

area of Cucurbitacin B of the same spot was found to be 2350.80 and

R.S.D. 0.96. (R.S.D. value less than 2) (table 5.37)

Table 5.37 Data for Repeatability of Measurement of Peak area of

Cucurbitacin B


1 2347

2 2386

3 2357



4 2326

5 2338

Mean 2350.80

S.D. 22.75

R.S.D. (% C.V.) 0.96

C. Relative Standard Deviation for repeatability of sample application

of peak area was carried out by applying 10μl of standard solution

of cucurbitacin B of concentration 50μg/mL on TLC plate in five

replicates and analyzing under specified chromatographic

conditions. The values of mean peak area of cucurbitacin B of the

same spot was found to be 5106.80 and R.S.D 0.36 (R.S.D. value

less than 2). (table 5.38)

Table 5.38 Data for Repeatability of Sample application of Peak area

of Cucurbitacin B


1 5102

2 5134

3 5089

4 5116

5 5093

Mean 5106.8

S.D. 18.40

R.S.D. (% C.V.) 0.36

Accuracy (% Recovery):

The accuracy of the method was established by performing recovery

experiments, using the standard addition method, at three different



levels. About 20µg/ml test solution of powdered aerial parts of

Coccinia indica was accurately weighed into each of the four

stoppered test tubes. Known concentration (0, 10, 20, 30µg/ml) of

powdered cucurbitacin B standard were added in solution form to each

of the stoppered test tubes respectively. The stoppered test tubes were

then shaken at 20 rpm on rotary shaker for overnight at room

temperature (28ºC ± 2ºC). The contents of the test tube were filtered

separately and each solution was analyzed under optimized

chromatographic conditions. Value of percentage recovery for

cucurbitacin B was then determined. The results of the recovery

experiment are given in table 5.39. The value of percentage recovery

of cucurbitacin B found was 96.40-99.92, indicating good accuracy of

the method.

Table 5.39 Data of Accuracy for Cucurbitacin B

Concentration

(µg/ml) Mean

Peak

Area

(n=3)

Amount of

cucurbitacin B

Found Mean±S.D.

(n=3)

%

Recovery

(n=3) Taken

(Sample)

Added

(Std)

20 0 2373.47 20.11±0.17 -

20 10 3468.35 29.23±0.28 97.43

20 20 4329.74 38.56±0.31 96.40

20 30 5131.26 49.96±0.25 99.92

Specificity:

It was observed that the other constituents present in the crude drugs

did not interfere with the peak of Cucurbitacin B. Therefore, the

method was specific. The spectrum of standard cucurbitacin B and

cucurbitacin B in aerial parts of Coccinia indica was found to be

similar.

The minimum detectable limit was found to be 10.00 µg/ml and limit

of quantitation was 49.96 µg/ml (table 5.40).



Table 5.40 Summary of Validation Parameters of Cucurbitacin B


1 Linearity 0.9937

2 Presision (% C.V.)

Repeatability of measurement

Repeatability of application

Intraday

Inter-day

0.96

0.36

0.37-0.90

0.43-0.94

3 Range 10-50 µg/ml

4 Limit of Detection (LOD) 10.00 µg/ml

5 Limit of Quantitatin (LOQ) 49.96 µg/ml

6 Accuracy 96.40-99.92


5.6 PHARMACOLOGICAL EVALUATION

Acute toxicity study

Ethanolic extract of Salacia prenoides, Annona squamosa and Coccinia

indica were fed orally for its acute toxicity and behavioral changes in

overnight starved male albino rats, and here we had followed the Acute

Toxic Class Method (OECD Guidelines 423).



Table 5.41 Study of Various ethanolic extracts using Acute Toxic Class Method (OECD GUIDELINES 423)

Sl.

No. Group

Dose

mg/kg (P.O.)

Weight of animals

Sign of

Toxicity

Onset of

Toxi-city

Reversi-

ble/Irre-

versible

Dura-tion of

obser-vation Before

test

After test

(on 4th

day)

1. Ethanolic extract of

Salacia prenoides

(ESP)

1. 2000 155 155 No sign Nil Nil Nil




Annona squamosa

(EAS)





Coccinia indica (ECI) 1. 2000 187 186 No sign Nil Nil Nil



As no toxicity or death was observed for these dose levels, the same dose level was repeated.

4. Ethanolic extract of 1. 2000 155 154 No sign Nil Nil Nil



Salacia prenoides

(ESP) 2. 2000 161 160 No sign Nil Nil Nil



Annona squamosa

(EAS)




6.

Ethanolic extract of

Coccinia indica (ECI) 1. 2000 186 185 No sign Nil Nil Nil





Table 5.42 Behavioral studies of rats during Acute Toxic Class Method (OECD guidelines 423) for ESP, EAS and ECI

Time

Ale

rt

Pa

ssiv

e

Gro

om

ing

Res

tles

snes

s

Agg

ress

ive

nes

s

To

uch

res

po

nse

Pa

in r

esp

on

se

Con

vu

lsio

n

Gri

p s

tren

gth

Pin

ref

lux

Corn

eal

refl

ux

Wri

thin

g

Pu

pil

siz

e

Sa

liv

ati

on

Sk

in c

olo

r

La

chri

mati

on

Res

pir

ati

on

1st hour - - - - - - - - - - - - - - - - -

2nd

hour - - - - - - - - - - - - - - - - -

3rd

hour - - - - - - - - - - - - - - - - -

4th

hour - - - - - - - - - - - - - - - - -

24th

hour - - - - - - - - - - - - - - - - -



According to this, the starting dose was 2000mg/kg body weight for

all the extracts which showed no mortality when it was observed for

72 hours. And at the end of 3 days, as it showed no mortality the same

dose was repeated for the same set of experiment and again it showed

no mortality. So, the LD50 cut off was grouped under category X and it

was classified under group X (unclassified group) according to GHS

system for classification of toxic chemicals.

Thus, acute toxicity revealed the non-toxic nature of the ethanolic

extract of roots of Salacia prenoides, leaves of Annona squamosa and

aerial parts of Coccinia indica. There was no lethality or any toxic

reactions found at any of the doses selected until the end of the study

period.

Oral Glucose Tolerance Test (OGTT)

The oral glucose tolerance test was performed in overnight fasted (18

h) normal rats and the results were estimated using a glucose oxidase-

peroxidase (POD) glucose estimation kit which are tabulated as below

in table 5.43.

Table 5.43 Effects of Various extracts on Oral Glucose Tolerance Test

on Experimental Animals

Group

(n=6) Treatment

Plasma glucose concentration (mg/dl)

30 min 60 min 90 min

I Normal control 74.54±1.29a 99.24±3.48

b 98.86±4.12

b

II Normal+ ESP

(250mg/kg)

60.42±3.83 87.79±2.98c 69.48±1.45

a

III Normal + ESP

(500mg/kg)

60.19±2.47 85.25±1.87c**

65.71±1.67a**

IV Normal + EAS

(250mg/kg)

69.37±4.56a 92.36±1.76

b* 71.12±2.92

a*



V Normal + EAS

(500mg/kg)

61.65±3.65 86.47±2.65c*

67.53±3.36a

VI Normal + ECI

(250mg/kg)

68.23±1.74a 91.58±2.54

b** 70.91±3.97

a

VII Normal + ECI

(500mg/kg)

59.71±2.38 85.69±3.43c**

68.64±2.84a**

ESP: Ethanolic extract of roots of Salacia prenoides

EAS: Ethanolic extract of leaves of Annona squamosa

ECI: Ethanolic extract of aerial parts of Coccinia indica

In OGTT, the ethanolic extract of roots of Salacia prenoides, from 30

minutes onwards, showed significant decrease in plasma glucose level at

dose level of 250mg/kg while ethanolic extract of leaves of Annona

squamosa and aerial parts of Coccinia indica in dose level of 500mg/kg

showed the same activity from 30 minutes onwards (fig. 5.34).

Fig 5.34 Effects of Various extracts on Oral Glucose Tolerance Test

The values were expressed as mean ± standard deviation for six animals in each group.

Values not sharing a common superscript letter differ significantly at p 0.05.

Values are statistically significant at *p 0.05 from normal control group and highly significant at

** p 0.001 from normal control group.



Alloxan induced diabetes

Induction of diabetes in the experimental rats was confirmed by the

presence of a high fasting plasma glucose level. The effect of ethanolic


aerial parts of Coccinia indica in 250mg/kg dose level on fasting plasma

glucose levels of normal and alloxan induced diabetic rats are presented

in table 5.44 and fig. 5.35.



Table 5.44 Effects of various extracts on Fasting plasma glucose level in Normal and Alloxan Induced Experimental

Animals

Group

(n=6) Treatment

Fasting Plasma glucose concentration (mg/dl)

Day 1 Day 7 Day 15 Day 21


a 71.08±2.45

a 72.67±3.93

a

II Diabetic control 182.54±9.13b*

188.54±8.45b 203.54±9.88

b 205.54±8.65

b

III Diabetic+ESP

(250mg/kg) 179.27±7.38

b** 107.47±4.45

c* 102.48±3.76 95.47±2.67

a

IV Diabetic+EAS

(250mg/kg) 180.76±7.98

b** 112.61±4.09

c 105.86±3.10

c** 96.23±2.29

a

V Diabetic+ECI

(250mg/kg) 180.83±7.54

b** 109.98±3.54

c 106.75±3.24 97.64±3.19

VI Diabetic+CEE

(250mg/kg) 183.75±8.23

b 103.57±2.76 101.38±2.43 91.64±2.81

a*

VII Diabetic+Std.

Glibenclamide

(600μg/kg)

180.38±7.17b**

103.86±3.68 99.93±5.64

91.38±3.73a**

CEE: combined ethanolic extract of Salacia prenoides, Annona squamosa, Coccinia indica in ratio of 1:2:2



Fig 5.35 Effects of various extracts on Fasting plasma glucose level in

Normal and Alloxan Induced Experimental Animals





In all the groups prior to alloxan administration, the basal levels of blood

glucose of the rats were not significantly different. However, 15 days

after alloxan administration, blood glucose levels were significantly

higher in the rats selected for the study. Table 5.45 shows the effect of

ethanolic extract of roots of Salacia prenoides, leaves of Annona

squamosa, aerial parts of Coccinia indica and combined extract in

250mg/kg dose and glibenclamide (600 μg/kg) on blood glucose levels.

Although a significant antihyperglycemic effect was evident from the

first week onwards, the decrease in blood glucose was maximum on third

week in group receiving 250 mg kg−1

BW of compound extract (fig.

5.36).



Table 5.45 Effects of Various Extracts on Blood Glucose in Normal and Alloxan induced Experimental Animals

Group

(n=6) Treatment

Blood glucose (mg/dl)



a 79.67±5.54

a 79.98±5.90

a

II Diabetic control 264.04±17.45b 278.44±13.47

b 284.62±11.80

b 296.12±14.24

b

III Diabetic+ESP

(250mg/kg) 257.24±13.45

b 200.76±15.68

c 150.20±7.34

c 115.36±6.42

a

IV Diabetic+EAS

(250mg/kg) 248.26±12.54

b 214.82±11.73

c 179.37±8.46

c 140.35±6.94

c*

V Diabetic+ECI (

(250mg/kg) 254.76±16.23

b 231.45±12.83

b* 191.39±5.76

c 145.37±6.78

c

VI Diabetic+CEE

(250mg/kg) 253.65±15.55

b 195.65±11.45

c 149.97±9.92

c 104.78±8.72

a

VII Diabetic+

Std Glibenclamide

(600 μg/kg)

246.47±14.07b 180.30±11.34

c 132.56±8.96

c** 86.58±4.56

a*

CEE: combined ethanolic extract of Salacia prenoides, Annona squamosa, Coccinia indica in ratio of 1:2:2.



Fig 5.36 Effects of Various Extracts on Blood Glucose in Normal and

Alloxan induced Experimental Animals





The levels of plasma insulin in control and experimental rats are

represented in table 5.46. The diabetic rats showed a significant decrease

in plasma insulin compared to control rats. Effect of ethanolic extract of

roots of Salacia prenoides, leaves of Annona squamosa, aerial parts of

Coccinia indica and compound extract in 250mg/kg dose showed nearly

same effect as shown by standard drug and increased plasma insulin

level similar to normal control after 3 weeks of treatment as shown

below in fig. 5.37.



Table 5.46 Changes in Level of Plasma Insulin of Normal and Alloxan

Induced Experimental Animals

SR.

No. Groups

Plasma Insulin

(μU/ml)

after 3 weeks of

treatment

I Normal control 13.63±2.52a

II Diabetic control 5.50±1.13b*

III Diabetic + ESP(250mg/kg) 10.07±2.23c

IV Diabetic + EAS (250mg/kg) 8.16±1.67c*

V Diabetic + ECI(250mg/kg) 7.34±1.50b

VI Diabetic +CEE (250mg/kg) 11.50±2.12a*

VII Diabetic+ Std Glibenclamide (600 μg/kg) 12.45±2.21a**

Fig 5.37 Changes in Level of Plasma Insulin of Normal and Alloxan

Induced Experimental Animals The values were expressed as mean ± standard deviation for six animals in each group.




The glycosylated hemoglobin in control and experimental animals

after 3 weeks of treatment are represented in table 5.47. The levels of

glycosylated hemoglobin are significantly increased in diabetic control

and a concomitant decrease in the level of glycosylated hemoglobin in



the diabetic rats, when compared with normal control. The compound

extract and standard glibenclamide treated diabetic rats significantly

decreased the glycosylated hemoglobin level as compared to ethanolic

extract of roots of Salacia prenoides, leaves of Annona squamosa,

aerial parts of Coccinia indica and compound extract in 250mg/kg

dose (fig.5.38).

Table 5.47 Levels of Glycosylated Hemoglobin in Control and

Alloxan Induced Experimental Animals after 3 weeks

of Treatment

SR.

No. Groups

Glycosylated

Hemoglobin

(%Hb)


II Diabetic control 12.8±0.46b

III Diabetic + ESP(250mg/kg) 6.8±0.21a

IV Diabetic + EAS (250mg/kg) 7.3±0.19c*

V Diabetic + ECI (250mg/kg) 7.9±0.11c

VI Diabetic + CEE(250mg/kg) 6.2±0.31a*


Fig 5.38 Levels of Glycosylated Hemoglobin in Control and Alloxan

Induced Experimental Animals after 3 weeks of Treatment The values were expressed as mean ± standard deviation for six animals in each group.






The Glucose-6-Phosphate Dehydrogenase (G-6-PDH) activity of control

and experimental groups are represented in table 5.48. There is a

significant decrease in activity of Glucose-6-Phosphate Dehydrogenase

(G-6-PDH) in diabetic rats as compared to normal control. The

compound and glibenclamide (600 μg/kg) treated diabetic rats showed

significant restoration of normal value as compared to ethanolic extract of

roots of Salacia prenoides, leaves of Annona squamosa, aerial parts of

Coccinia indica 250mg/kg body weight dose level (fig. 5.39).

Table 5.48 Activity of G-6-PDH enzyme in Control and Alloxan Induced

Experimental Animals after 3 weeks of Treatment

SR.

No. Groups

G-6-PDH

(U/g Hb)



III Diabetic + ESP (250mg/kg) 7.9±0.81c

IV Diabetic + EAS (250mg/kg) 9.4±0.79b*

V Diabetic + ECI (250mg/kg) 10.5±0.87b**

VI Diabetic + CEE(250mg/kg) 6.3±0.48a*


Fig 5.39 Activity of G-6-PDH enzyme in Control and Alloxan

Induced Experimental Animals after 3 weeks of Treatment The values were expressed as mean ± standard deviation for six animals in each group.






Streptozotocin (STZ) induced diabetes

Induction of diabetes in the experimental rats was confirmed by the

presence of a high fasting plasma glucose level. The effect of ethanolic


aerial parts of Coccinia indica in 250mg/kg dose level on fasting plasma

glucose levels of normal and neonatal STZ (nSTZ) induced wistar rats

are presented in table 5.49 and fig. 5.40.

Table 5.49 Effects of Various Extracts on Fasting Plasma Glucose Level

in Normal and nSTZ Induced Experimental Animals

Group

(n=6) Treatment

Fasting Plasma glucose concentration (mg/dl)


I Normal

control 75.36±4.02

a 77.95±3.97

a 73.76±3.14

a 75.93±4.52

a

II Diabetic

control 191.31±8.43

b 199.52±9.23

b 214.65±9.54

b 221.73±9.82

b

III Diabetic+ESP

(250mg/kg) 182.54±6.34

b 125.65±5.34 113.62±9.83

c 99.47±2.67

a*

IV Diabetic+EAS

(250mg/kg) 184.51±7.43

b 129.59±4.82 118.86±2.98

c 108.23±3.04

c*

V Diabetic+ECI

( (250mg/kg) 183.67±6.98

b 130.01±2.97 121.75±3.21 109.64±2.85

c*

VI Diabetic+CEE

(250mg/kg) 182.43±7.87

b 119.45±3.54

c 102.56±2.56

c** 95.62±2.96

a*

VII Diabetic+

Std

Glibenclamide

(600 μg/kg)

183.23±6.78b 114.76±3.71

c 98.03±4.31

a* 92.63±2.51

a**

Compound extract: combined ethanolic extract of Salacia prenoides, Annona

squamosa, Coccinia indica in ratio of 1:2:2.



Fig 5.40 Effects of Various Extracts on Fasting Plasma Glucose Level in

Normal and nSTZ Induced Experimental Animals The values were expressed as mean ± standard deviation for six animals in each group.




In all the groups prior to STZ administration, the basal levels of blood

glucose of the rats were not significantly different. However, 15 days

after STZ administration, blood glucose levels were significantly higher

in the neonatal rats selected for the study. Table 5.50 shows the effect of

ethanolic extract of roots of Salacia prenoides, leaves of Annona

squamosa, aerial parts of Coccinia indica and compound extract in

250mg/kg dose and glibenclamide (600 μg/kg) on blood glucose levels.

Although a significant antihyperglycemic effect was evident from the

first week onwards, the decrease in blood glucose was nearly equivalent

to standard on third week in group receiving 250 mg kg−1

body weight of

combined extract (fig. 5.41).



Table 5.50 Effects of Various Extracts on Blood Glucose in Normal and

nSTZ Induced Experimental Animals

Group

(n=6) Treatment

Blood Glucose (mg/dl)


I Normal

control 80.34±3.25

a 79.26±3.16

a 81.28±4.01

a 82.86±3.98

a

II Diabetic

control 259.16±15.65

b 271.65±14.82

b 278.43±12.76

b 289.45±13.97

b

III Diabetic+ESP

(250mg/kg) 258.13±14.76

b 203.38±11.75 149.34±8.57

c 113.69±7.43

c**

IV Diabetic+EAS

(250mg/kg) 260.90±14.87

b 235.82±12.98

b* 189.56±9.67 159.35±6.94

V Diabetic+ECI

( (250mg/kg) 257.48±15.84

b 239.57±11.73

b* 195.78±8.54 160.89±7.69

VI Diabetic+CEE

(250mg/kg) 258.60±13.94

b 200.12±10.75

b** 145.37±8.87

c 108.65±7.64

a

VII Diabetic+

Std

Glibenclamide

(600 μg/kg)

256.56±13.18b 189.45±10.57 139.73±9.35

c* 85.84±5.76

a*

Compound extract: combined ethanolic extract of Salacia prenoides, Annona

squamosa, Coccinia indica in ratio of 1:2:2.

Fig 5.41 Effects of Various Extracts on Blood Glucose in Normal and

nSTZ Induced Experimental Animals The values were expressed as mean ± standard deviation for six animals in each group.






The levels of plasma insulin in control and nSTZ induced experimental

animals are represented in table 5.51. The diabetic rats showed a

significant decrease in plasma insulin compared to control rats. Effect of

compound ethanolic extract of roots of Salacia prenoides, leaves of

Annona squamosa, aerial parts of Coccinia indica and compound extract

in 250mg/kg dose showed nearly same effect as shown by standard drug

and increased plasma insulin level similar to normal control after 3

weeks of treatment as shown below (fig. 5.42).

Table 5.51 Changes in level of Plasma Insulin of Normal and nSTZ

Induced Experimental Animals

SR.

No. Groups

Plasma Insulin

(μU/ml)

after 3 weeks of

treatment


II Diabetic control 6.11±1.24c**

III Diabetic + ESP (250mg/kg) 9.56±2.21b*

IV Diabetic + EAS (250mg/kg) 7.86±1.36c

V Diabetic + ECI (250mg/kg) 7.25±1.548c*

VI Diabetic + CEE (250mg/kg) 10.42±2.04b




Fig 5.42 Changes in level of Plasma Insulin of Normal and nSTZ

Induced Experimental Animals The values were expressed as mean ± standard deviation for six animals in each group.




The glycosylated hemoglobin in control and nSTZ induced

experimental animals after 3 weeks of treatment are represented in

table 5.52. The level of glycosylated hemoglobin are significantly

increased in diabetic control and a concomitant decrease in the level of

glycosylated hemoglobin in the diabetic rats, when compared with

normal control. The compound extract and standard glibenclamide

treated diabetic rats significantly decreased the glycosylated

hemoglobin level as compared to ethanolic extract of roots of Salacia

prenoides, leaves of Annona squamosa, aerial parts of Coccinia indica

and compound extract in 250mg/kg dose (fig. 5.43).



Table 5.52 Levels of Glycosylated Hemoglobin in Control and nSTZ

Induced Experimental Animals after 3 weeks of Treatment

SR.

No. Groups

Glycosylated

Hemoglobin

(%Hb)



III Diabetic + ESP(250mg/kg) 7.1±0.34c*


V Diabetic + ECI (250mg/kg) 8.2±0.18c

VI Diabetic + CEE (250mg/kg) 6.9±0.37a*


Fig. 5.43 Levels of Glycosylated Hemoglobin in Control and nSTZ

Induced Experimental Animals after 3 weeks of treatment The values were expressed as mean ± standard deviation for six animals in each group.




The Glucose-6-Phosphate Dehydrogenase (G-6-PDH) activity of

control and nSTZ induced experiment animals in different groups are

represented in table 5.53. There is a significant decrease in activity of

Glucose-6-Phosphate Dehydrogenase (G-6-PDH) in diabetic rats as



compared to normal control. The compound extract (250 mg/kg) and

glibenclamide (600 μg/kg) treated groups showed significant

restoration of normal value as compared to individual ethanolic extract

of roots of Salacia prenoides, leaves of Annona squamosa, aerial parts

of Coccinia indica 250mg/kg body weight dose level (fig. 5.44).

Table 5.53 Activity of G-6-PDH enzyme in Control and nSTZ

Induced Experimental Animals after 3 weeks of Treatment

SR.

No. Groups

G-6-PDH

(U/g Hb)



III Diabetic + ESP (250mg/kg) 7.8±0.79c*


V Diabetic + ECI (250mg/kg) 10.3±0.61b*

VI Diabetic + CEE (250mg/kg) 6.9±0.27a

VII Diabetic+ Std Glibenclamide (600 μg/kg) 5.7±0.53a*

Fig 5.44 Activity of G-6-PDH enzyme in Control and nSTZ induced

experimental animals after 3 weeks of treatment







5.7 DEVELOPMENT OF FORMULATION

5.7.1 PREFORMULATION STUDY

A summary of the pre-formulation testing results is shown in table 5.54.

Table 5.54 The Summary of Pre-formulation Testing results of

S.prenoides, A.squamosa and C.indica

Testing S.prenoides A.squamosa C.indica

Yield of freeze-

dried extracts

(%)

17.53±0.9432 11.35±1.281 14.58±1.436

The solubility

of extracts

(g/ml)

0.0253±0.0065 0.0473±0.0042 0.0381±0.0039

Degree of

sphericity

0.6050±0.1173 0.6913±0.1119 0.6423±0.1146

Particle size Moderately

fine

Moderately fine Moderately fine

Carr‟s index

(%)

7.193±1.805% 13.24±1.015% 14.51±1.117

Angle of

repose(°)

33.72±1.630° 37.32±5.883° 36.74±3.116

Total ash (%) 18.3±0.570% 21.56±0.368% 20.4±0.480%

Acid-insoluble

ash(%)

1.32±0.180% 2.79±0.488% 2.01±0.270%

Moisture

content (%)

3.78±0.1256% 7.28±0.1468% 6.378±0.1389%

The contact

angle (°)

6.64±1.492° 6.78±1.545° 7.30±1.581°

5.7.1.1 Organoleptic properties of the plant extracts

The picture of the freeze-dried aqueous extracts (fig. 5.45) and a

summary of the organoleptic properties (table 5.55) of the freeze-dried

ethanolic extract of S.prenoides, A.squamosa and C.indica are given.



Fig 5.45 Freeze-dried extracts of S.prenoides, A.squamosa and C.indica

Table 5.55 The Organoleptic properties of the Freeze-dried extracts of

S.prenoides, A.squamosa and C.indica

Properties S.prenoides A.squamosa C.indica

Physical

appearance

Brittle, free-

flowing, small

particulate

powder

smooth, free-

flowing, fine

powder

Brittle, free-

flowing, fine

particulate

powder

Colour

Brown, darker

than ground

stem

Greenish

brown, darker

than ground

leaves

Mud brown

lighter than

aerial part

powder

Odour Characteristic

odour

Slight aromatic

odour

Characteristic

odour

Taste Bitter and

astringent

Characteristic Bitter

The bitter taste and unpleasant odors normally result in poor patient

acceptance of dosage forms. Hopefully these negative characteristics still

present in the extracts can be masked when incorporated in capsule form.

5.7.1.2 The solubility of various extracts

The results obtained in the solubility testing of the various extracts of

S.prenoides, A.squamosa and C.indica are summarized in table 5.54. At

room temperature 1g of S.prenoides extract powder dissolved in

39.39±5.134 ml of water, 1g of A.squamosa extract powder dissolved in

30.54±2.342 ml of water and 1g of C.indica extract powder dissolved in



25.76±1.549 ml of water. All the extracts could thus be described as

being sparingly soluble.

5.7.1.3 The size and shape of particles of various extracts

Particle size and shape are crucial parameter. They are important for the

manufacture of the dosage forms, influence dissolution and

bioavailability. The results of the particle size study are given in tables

5.56 and fig. 5.46.

Table 5.56 Particle size of S.prenoides, A.squamosa and C.indica Freeze-

dried extract powders

Sieve S.prenoides

extract(g)

A.squamosa

extract(g)

C.indica

extract(g)

1400 0.000 0.000 0.000

355 0.144 2.0055 0.187

180 2.329 2.1880 2.763

125 2.258 0.8746 3.105

90 2.289 0.9853 1.037

Pass through

90 sieve

2.980 2.6317 2.908

Salacia prenoides

0

0.5

1

1.5

2

2.5

3

3.5

>355 355-180 180-125 125-90 <90

The particle size

Th

e w

eig

ht (g

m)

Series1

Annona squamosa

0

0.5

1

1.5

2

2.5

3

>355 355-180 180-125 125-90 <90

The particle size

Th

e w

eig

ht (g

m)

Series1

Coccinia indica

0

0.5

1

1.5

2

2.5

3

3.5

>355 355-180 180-125 125-90 <90

The particle size

Th

e w

eig

ht (g

m)

Series1

Fig 5.46 Particle size of S.prenoides, A.squamosa and C.indica Freeze-

dried extract powders



According to the above results, the S.prenoides, A.squamosa and C.indica

freeze-dried extract powders were all categorized as moderately fine

powders based on the British Pharmacopoeia (British Pharmacopoeia

2000) standard. Their particle shapes were irregular. The degree of

sphericity (θ) of S.prenoides freeze-dried extract powder was

0.6050±0.1173, A.squamosa freeze-dried extract powder was

0.6913±0.1119 and C.indica freeze-dried extract powder was

0.6423±0.1146. Generally, if θ approaches 1, the particle is close to

sphericity. The results obtained showed that the S.prenoides, A.squamosa

and C.indica freeze-dried extract powders possessed appropriate

flowability for the manufacture of the capsule dosage form, and the

„moderately fine powder‟ classification implied that the powder

possessed a relatively bigger surface area and could be easily dissolved

by water.

5.7.1.4 The densities of freeze-dried extract powders

The results of the determinations for the extracts are summarized in Table

34. The Carr‟s index of Compressibility for S.prenoides extract is

7.193±1.805%, for A.squamosa is 13.24±1.015% and for C.indica is

14.51±1.117. The density study results shows that the extract of

S.prenoides having excellent while A.squamosa and C.indica freeze-dried

extract powders can all be categorized as having good flow properties.

5.7.1.5 The flowability of freeze-dried extract powders

The results of the determinations on the powders are given in table 34.

The S.prenoides, A.squamosa and C.indica freeze-dried extract powders

had angles of repose of 33.72±1.630°, 37.32±5.883° and 36.74±3.116°

respectively. Therefore all the powder had passable flow properties. This

implicated that all the above mentioned freeze-dried extract powders



possessed appropriate flowability for the manufacture of capsule dosage

form.

5.7.1.6 The ash values for the extract powders

The total ash and acid insoluble ash values for the freeze-dried extract

powders of S. prenoides, A. squamosa and C. indica are mentioned in

table 5.54. For S. prenoides extract the total and acid insoluble ash values

are 18.3±0.570% and 1.32±0.180% respectively, for A. squamosa was

21.56±0.368% and 2.79±0.488% and for C. indica the values were

20.4±0.480% and 2.01±0.270% .This ash testing was the first ash testing

for S.prenoides, A.squamosa and C.indica extracts. And this result could

be used for future reference.

5.7.1.7 The moisture content of the extracts

The percentage of moisture content of S.prenoides, A.squamosa and

C.indica extracts are given in table 5.54. The results were 3.78±0.1256%,

7.28±0.1468% and 6.378±0.1389% respectively. Moisture content is an

important parameter for capsule dosage form and it is also important for

herbal medicines, which are hygroscopic. Moisture content cannot

represent the hygroscopic property of the powder, but it can serve as a

reference for quality control (e.g. stability study).

5.7.1.8 The wet ability of the extract powders

The contact angle of S.prenoides, A.squamosa and C.indica extract

powders were 6.64±1.492°, 6.78±1.545° and 7.30±1.581°. All of them

<90°(table 34), and this implied that they all could be wetted

spontaneously.



5.7.2 EVALUATION OF FORMULATION

5.7.2.1 Uniformity of weight and content of capsules

The results of the uniformity of weight and content of the formulated

capsules are mentioned as the average deviation in weight from average

for prepared capsules was 1.58±1.24% and the average total content per

capsule 102.8±2.14% respectively. According to the British

Pharmacopoeia,363

the limit on the acceptable deviation in weight from

average for capsules is ±7.5% and the limits on the amount of content in

the capsules 90% to 110%. The afore-mentioned results thus indicated

that both the formulated capsules met the British Pharmacopoeia

specifications.

5.7.2.2 Moisture level of the content of capsules

After the capsules were filled the moisture level of its contents were again

(within 5 days) tested just to ascertain if there had been changes in

moisture level during the manufacturing procedure. The results of these

tests indicated that the moisture level of the contents of the formulated

capsules was 7.94±0.38%. When analysed in the pre-formulation study,

the highest moisture content was 7.28±0.1468% for the A.squamosa

extract (as in table 5.54).

There thus appeared to have been a slight increase in the moisture level of

the formulated capsules after encapsulation. This suggested that the

compound extract formulation absorbed some moisture during the filling

procedure, presumably because it was more hygroscopic than the

individual extract. Since the moisture absorbed may speed up

degradation, the humidity conditions during the manufacture of the



capsules can thus be a crucial factor and these capsules should preferably

be manufactured under more tightly controlled humidity conditions.

5.7.2.3 Stability of capsules

For the study of the organoleptic properties four batches of capsules were

stored under the different conditions as follows:

Table 5.57 The storage conditions for the Stability study of the Capsules

Batch

No.

Temperature(ºC) Relative Humidity

(RH)

Container

1 *Room

temperature

**Room relative

humidity

Outside

container

2 Room

temperature

Room relative

humidity

In container

3 40±2 ºC 75±5% (RH) In container

4 40±2 ºC 75±5% (RH) Outside

container

*25±2 ºC **40±5% (RH)

A summary of the changes in the organoleptic properties that occurred

during storage of the formulated capsules at 40±2º / 75±5% relative

humidity (RH) and at room temperature and relative humidity (RH) over

12 weeks period are given in table 5.58.

Table 5.58 The Stability results of Formulated capsule Organoleptic

properties after 12 weeks storage

Batch 1: Room temperature and relative humidity (Outside container)

Week Size and

shape of

capsule

Gross nature

of powder in

capsule

Color of

powder

(seen

through

capsule)

Odor of

powder

0 Regular “0”

size and

shape

Powder Brown Strong



2 No change Hard and crisp Deep brown Faint

4 No change Hard but not

crisp

Deep brown Faint

6 No change Hard like

rubber

Deep brown Faint

8 No change Hard like

rubber

Deep brown Faint

10 No change Slightly sticky Deep brown Faint

12 No change Slightly sticky Deep brown Faint

Batch 2: Room temperature and relative humidity ( In container )

0 Regular “0”

size and

shape

Powder Brown Strong

2 No change Powder Brown Strong




10 No change Agglomeration Brown Strong


Batch 3: 40±2 ºC 75±5% Relative Humidity ( In container )

0 Regular “0”

size and

shape

Powder Brown Strong







The formulated capsules that were not in a plastic container and stored at

high temperature (40±2º) and relative humidity (75%±5% RH) underwent

drastic changes in physical integrity within 2 days (i.e. the capsule shell

broke and the capsule content liquefied). So, this batch of capsules could



not be further evaluated for their organoleptic properties and moisture

content.

For the capsules stored at room temperature and room humidity and

outside the container (batch 1 in table 5.57), the size and shape of the

capsules were maintained but very obvious changes occurred in most of

the organoleptic properties (i.e. gross nature, colour and odours) of the

capsule content in the first 2 weeks of storage. The compound powder

first became hard and crispy, then hard and rubbery and eventually

slightly sticky, changed in colour from brown to deep brown and quickly

lost most of its strong odour. Clearly, when these capsules were stored at

room temperature and relative humidity unprotected from light (i.e.

outside a plastic container) significant deterioration occurred in the plant

materials.

When the capsules were stored in the plastic container, whether at room

temperature and relative humidity or high temperature and relative

humidity i.e. 25±2ºC / 40%±5% RH and 40±2ºC / 75%±5% RH (batches

2 and 3 in table 5.57), the organoleptic properties of the plant material

remained relatively unchanged during the 12 weeks storage.

Collectively, the above results suggested that the capsules of compound

extract of S.prenoides, A.squamosa and C.indica freeze-dried extract

were very stable upon storage in proper container.

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CHAPTER V RESULTS AND DISCUSSIONshodhganga.inflibnet.ac.in/bitstream/10603/43346/15/15_chapter5.pdf · The shape of Annona squamosa leaves is oblong, lanceolate or elliptic with entire