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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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 149
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 150
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 151
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 152
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 153
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 154
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 155
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 156
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 157
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 158
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 159
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 160
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 161
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 162
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 163
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)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 164
Fig. 5.8g (100×)
Fig. 5.8h (100×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 165
Fig. 5.8i (450×)
Fig. 5.8j (450×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 166
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×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 167
Fig. 5.8m (450×)
Fig. 5.8n (100×)
Fig. 5.8o (450×)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 168
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 169
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%
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 170
Table 5.6 Percentage yield of dried extract of Annona squamosa leaves
Solvent Color of the
extract
Consistency Dry 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
Solvent Color of the
extract
Consistency Dry 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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 171
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 172
Table 5.9 Qualitative tests for Different extracts of Annona squamosa leaves
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 173
Table 5.10 Qualitative tests for Different extracts of Coccinia indica aerial parts
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 174
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
Table 5.12 Thin Layer Chromatographic pattern of Successive solvent
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 175
Table 5.13 Thin Layer Chromatographic pattern of Successive solvent
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),
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 176
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 177
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 178
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 179
Fig 5.11 Overlain IR of Standard Mangiferin and Isolated compound (USP-I)
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 180
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 181
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 182
Fig 5.13 A three dimentional Calibration curve for Standard Mangiferin
Fig 5.14 HPTLC Chromatogram for track 1-5 of Standard
Mangiferin
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 183
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 184
Fig 5.17 HPTLC chromatogram of Test sample containing USP-I
Fig 5.18 HPTLC chromatogram scanned in Fluorescence light
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 185
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 186
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 187
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 188
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 189
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 190
Table 5.23 Data of Melting point and Mixed m.p. of β-sitosterol
& UAS-II
Sample Melting Point Mix m.p.
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 191
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
grouping Frequency Intensity Frequency Intensity
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 192
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 193
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:
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 194
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
Correlation coefficient: 0.9971, Slope: 166.34, Intercept: 848.44
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 195
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 196
Fig 5.25 HPTLC scanned in Fluorescence and UV light
Fig 5.26 HPTLC densitometric chromatogram of Test solution
containing UAS-II
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 197
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
Validation of HPTLC method:
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-
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 198
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 199
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
No. of Measurement Area
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 200
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 201
Table 5.31 Summary of Validation Parameters of β-sitosterol
Sr. No. Parameters Results
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
7 Specificity Specific
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 202
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
Sample Melting Point Mix m.p.
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 203
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
grouping Frequency Intensity Frequency Intensity
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 204
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:
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 205
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
Correlation coefficient: 0.9937, Slope: 935.45, Intercept: 518.59
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 206
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 207
Fig 5.32 HPTLC scanned in Ultra-Violet light
Fig 5.33 HPTLC densitometric chromatogram of Test solution
containing UCI-III
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 208
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
Validation of HPTLC method:
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 209
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
No. of Measurement Area
1 2347
2 2386
3 2357
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 210
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
No. of Measurement Area
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 211
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 212
Table 5.40 Summary of Validation Parameters of Cucurbitacin B
Sr. No. Parameters Results
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
7 Specificity Specific
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 213
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
2. 2000 163 161 No sign Nil Nil Nil
3. 2000 149 148 No sign Nil Nil Nil
2. Ethanolic extract of
Annona squamosa
(EAS)
1. 2000 162 162 No sign Nil Nil Nil
2. 2000 129 128 No sign Nil Nil Nil
3. 2000 158 157 No sign Nil Nil Nil
3. Ethanolic extract of
Coccinia indica (ECI) 1. 2000 187 186 No sign Nil Nil Nil
2. 2000 140 140 No sign Nil Nil Nil
3. 2000 150 149 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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 214
Salacia prenoides
(ESP) 2. 2000 161 160 No sign Nil Nil Nil
3. 2000 148 148 No sign Nil Nil Nil
5. Ethanolic extract of
Annona squamosa
(EAS)
1. 2000 162 161 No sign Nil Nil Nil
2. 2000 128 128 No sign Nil Nil Nil
3. 2000 157 156 No sign Nil Nil Nil
6.
Ethanolic extract of
Coccinia indica (ECI) 1. 2000 186 185 No sign Nil Nil Nil
2. 2000 140 139 No sign Nil Nil Nil
3. 2000 149 149 No sign Nil Nil Nil
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 215
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 - - - - - - - - - - - - - - - - -
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 216
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*
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 217
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 218
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
extract of roots of Salacia prenoides, leaves of Annona squamosa and
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 219
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
I Normal control 72.48±3.35a 72.04±4.12
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 220
Fig 5.35 Effects of various extracts on Fasting plasma glucose level in
Normal and Alloxan Induced Experimental Animals
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.
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 221
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)
Day 1 Day 7 Day 15 Day 21
I Normal control 83.17±4.139a 81.14±6.73
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 222
Fig 5.36 Effects of Various Extracts on Blood Glucose in Normal and
Alloxan induced Experimental Animals
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.
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 223
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.
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.
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 224
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)
I Normal control 5.7±0.15a
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*
VII Diabetic+ Std Glibenclamide (600 μg/kg) 6.1±0.44a**
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.
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 225
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)
I Normal control 4.7±0.36a
II Diabetic control 13.6±1.02b
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*
VII Diabetic+ Std Glibenclamide (600 μg/kg) 5.2±0.62a**
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.
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 226
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
extract of roots of Salacia prenoides, leaves of Annona squamosa and
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)
Day 1 Day 7 Day 15 Day 21
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 227
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.
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.
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 228
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)
Day 1 Day 7 Day 15 Day 21
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.
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 229
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
I Normal control 14.02±2.45a
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
VII Diabetic+ Std Glibenclamide (600 μg/kg) 13.19±2.31a**
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 230
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.
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.
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).
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 231
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)
I Normal control 5.9±0.18a
II Diabetic control 12.4±0.58b
III Diabetic + ESP(250mg/kg) 7.1±0.34c*
IV Diabetic + EAS (250mg/kg) 7.9±0.52c
V Diabetic + ECI (250mg/kg) 8.2±0.18c
VI Diabetic + CEE (250mg/kg) 6.9±0.37a*
VII Diabetic+ Std Glibenclamide (600 μg/kg) 6.3±0.59a**
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.
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.
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 232
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)
I Normal control 4.5±0.45a
II Diabetic control 12.9±1.32b
III Diabetic + ESP (250mg/kg) 7.8±0.79c*
IV Diabetic + EAS (250mg/kg) 9.1±0.58c
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
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 233
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 234
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 235
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 236
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 237
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.
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 238
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 239
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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 240
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
4 No change Powder Brown Strong
6 No change Powder Brown Strong
8 No change Powder Brown Strong
10 No change Agglomeration Brown Strong
12 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
2 No change Powder Brown Strong
4 No change Powder Brown Strong
6 No change Agglomeration Brown Strong
8 No change Agglomeration Brown Strong
10 No change Agglomeration Brown Strong
12 No change Agglomeration 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
CHAPTER V RESULTS AND DISCUSSION
STANDARDIZATION AND FORMULATION OF ANTI-DIABETIC HERBAL DRUGS 241
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.
Recommended