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Department of Pharmaceutical Sciences, Kumaun University, Nainital
CHAPTER: 4
PHARMACOGNOSTIC
EVALUATION
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INTRODUCTION
Plant products are used throughout the developed and developing countries as
home remedies, over-the-counter drug products and raw materials for the
pharmaceutical industry and represent a substantial proportion of the global drug
market. It is, therefore, essential to establish internationally recognized guidelines
for assessing their quality (Anonymous, 1998). The function of quality control
and drug evaluation is to assess the value of raw materials and to ensure that the
final product is of the required standard (Brain & Turner, 1975).
MATERIALS AND METHODS
a) Chemicals and apparatus
Chloral hydrate solution, iodine solution, phloroglucinol (Loba Chemie),
concentrated hydrochloric acid, glycerine water and safranine.
Compound microscope- Magnus MLX; eyepiece-10X; objectives- 4X, 10X, 40X
and 45X; stage micrometer, eyepiece micrometer, camera lucida and drawing
board. Camera-Sony cyber-shot (14.1 mega pixels).
b) Macroscopic characteristics
Macroscopic characteristics of leaf, root, bark, flower, fruit, peel and seed were
determined organoleptically (Anonymous, 2003).
c) Microscopic characteristics
Microscopy of leaf and root drugs were carried out.
Determination of leaf constants
A number of leaf measurements are used to distinguish some closely related
species not easily characterized by general microscopy (Evans, 2002).
• Stomatal number- Stomatal number is defined as the average number of
stomata per sq mm of epidermis. Fragments of leaf from the middle of the
lamina were cleaned with chloral hydrate. Upper and lower epidermis were
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peeled off separately by means of a forcep and mounted in glycerine water.
A square of known dimensions (1 sq mm) was drawn by means of a stage
micrometer and camera lucida and number of stomata was counted on that
area (Evans, 2002; Kokate, 2006). Type of stomata was also observed on
the same preparation. Ten determinations were carried out and the average
was calculated (Anonymous, 1998). Observed under 10X (eye piece) and
40X (objective).
• Stomatal index- Stomatal index is the percentage which the numbers of
stomata form to the total number of epidermal cells, each stoma being
counted as one cell. Whilst stomatal number varies considerably with the
age of the leaf and due to changes in environmental conditions, stomatal
index is relatively constant and, therefore, of diagnostic significance for a
given species. Same leaf preparations were used as for stomatal number.
Number of epidermal cells and stomata (the two guard cells and ostiole
was considered as one unit) were counted within the square (Evans, 2002;
Kokate, 2006). Ten determinations were carried out and the average was
calculated (Anonymous, 1998). Observed under 10X (eye piece) and 40X
(objective). Stomatal index was calculated by using the following equation.
Stomatal index (S.I.) = (S/ E+S) × 100
Where,
S = number of stomata per unit area
E = number of epidermal cells in the same unit area
• Palisade ratio- Palisade ratio is defined as the average number of palisade
cells beneath each epidermal cell. Pieces of leaf about 2 mm square were
cleared by boiling with chloral hydrate solution. A camera lucida was
arranged so that the epidermal cells and the palisade cells lying below them
may be traced. First a number of groups each of four epidermal cells were
traced and their outlines made more conspicuous. The palisade cells lying
beneath each group were then focused and traced. The palisade cells in
each group were counted, cells which were more than half covered by the
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epidermal cells were also counted; the figure obtained was divided by 4 to
obtain palisade ratio of that group (Evans, 2002). Twenty five groups from
different leaf samples were determined for the calculation of range and
average (Kokate, 2006). Observed under 10X (eye piece) and 40X
(objective).
• Vein-islet number- The vein-islet is the minute area of photosynthetic
tissue encircled by the ultimate divisions of the conducting strands. Vein-
islet number is defined as the number of vein-islets per sq mm of the leaf
surface, midway between the midrib and the margin. The leaf sample, after
soaking in water, was treated successively with sodium hypochlorite to
bleach, 10% hydrochloric acid to remove Ca oxalate and finally chloral
hydrate. The camera lucida was set up and by means of a stage micrometer
the paper was divided into squares of 1 sq mm. In the cleared preparation
veins were traced in four continuous squares, in a square of 2 mm×2 mm.
Each vein-islet was numbered during counting (Evans, 2002). The range
and average was determined in 10 sets of 2 mm×2 mm area (Kokate,
2006). Observed under 10X (eye piece) and 4X (objective).
• Veinlet termination number- Veinlet termination number is defined as
the number of veinlet terminations per sq mm of the leaf surface. A vein
termination is the ultimate free termination of a veinlet or branch of a
veinlet (Evans, 2002). Veinlet termination was counted in the same
preparation as for vein-islet number. The total number of vein-islets and
veinlet terminations in four adjoining squares was divided by four in order
to get the value in 1 sq mm. The range and average was determined in 10
sets of 2 mm×2 mm area (Kokate, 2006). Observed under 10X (eye piece)
and 4X (objective).
T.S. of leaf and root
Thinnest possible section of root and leaf (through midrib) was taken on a slide,
mounted in a solution of chloral hydrate and warmed slightly to decolorize.
Glycerine was used as a mounting medium (Iyengar & Nayak, 2006). Another
section mounted in water was treated with dilute iodine solution to identify starch
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grains. Cleared sections in chloral hydrate were also mounted in acetic acid,
hydrochloric acid and sulphuric acid to confirm the presence of Ca oxalate
crystals (Kokate, 2006).
Powder study of leaf and root
For microscopic examination of powder drug, three slides were prepared; one in
chloral hydrate, one in water and one in phloroglucinol + hydrochloric acid.
• A judicious quantity of powder was taken on a slide to which few drops of
chloral hydrate were added and heated for 1-2 minutes after placing the
cover slip.
• To observe starch grains, powder was mounted in water and 1-2 drops of
dilute iodine were added to stain starch grains blue.
• To the cleared powder in chloral hydrate as above, few drops of 1:1
phloroglucinol and concentrated hydrochloric acid were added and after 3-
4 minutes mounted in glycerine. Lignified tissues stained pink (Iyengar,
2007).
Cleared sections in chloral hydrate were also mounted in acetic acid, hydrochloric
acid and sulphuric acid to confirm the presence of Ca oxalate crystals (Kokate,
2006).
d) Determination of physicochemical parameters
• Total ash- 4 g of the ground air-dried material was accurately weighed in a
previously ignited and tared silica crucible. The material was ignited by
gradually increasing the temperature to 500-600ºC until it was white,
cooled in a desiccator and weighed (Anonymous, 1998). Total ash was
calculated as % w/w of air-dried material.
• Acid-insoluble ash- To the crucible containing the total ash, 25 ml of
hydrochloric acid was added, covered with a watch glass and boiled gently
for 5 minutes. Watch glass was washed with 5 ml of hot water and this
liquid was added to the crucible. The insoluble matter was collected on an
ashless filter paper and washed with hot water until the filtrate was neutral.
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The filter paper containing the insoluble matter was transferred to the
original crucible, dried and ignited to constant weight. Residue was cooled
in a dessicator, weighed (Anonymous, 1998) and calculated as % w/w of
air-dried material.
• Loss on drying- A completely dried glass-stoppered weighing bottle was
weighed. 10 g of powdered drug was taken in to the weighing bottle,
covered and accurately weighed the bottle with the sample. After removing
the stopper, the bottle was placed in an oven, sample was dried to constant
weight. Bottle was covered promptly and allowed to cool in a desiccator
and weighed with contents (Anonymous, 2003). Loss on drying was
calculated as % w/w.
• Water-soluble extractive- 4 g of coarsely powdered air-dried material was
accurately weighed and placed in a glass-stoppered conical flask.
Macerated with 100 ml of water for 6 hours, shaked frequently and allowed
to stand for 18 hours. Filtered rapidly without loss of any solvent, 25 ml of
the filtrate was transferred to a tared flat-bottomed dish and evaporated to
dryness on a water bath. Content was dried at 105ºC for 6 hours, cooled in
a desiccator for 30 minutes and weighed without delay (Anonymous,
1998). Content of extractable matter was calculated as % w/w of air-dried
material.
• Ethanol-soluble extractive- Same procedure was followed as for water-
soluble extractive; instead of water ethanol (70%) was used as a solvent.
RESULTS AND DISCUSSION
The plant of Citrus medica Linn. is an evergreen thorny shrub or small tree, 1.8-
3.6 m high with stems up to 10 cm in diameter.
a) Macroscopic characteristics
Leaf- Leaves were fragrant, large, 7.5-18 cm long and 3-9.5 cm wide, oblong to
elliptic with crenate to serrate margins, rounded apex, glabrous, dark green above
and light green below with short narrowly winged petiole. Large number of oil
glands were visible on the surface of leaves.
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Root- Root cylindrical, 5-20 cm in diameter; dried pieces were unbranched with
scars of rootlets; freshly collected roots were having many rootlets. The outer
surface was brownish yellow with faint longitudinal ridges while inner woody
part was pale yellow in colour. The fracture was fibrous; root bark was only up to
1 mm in width, most of the part was occupied by the compact wood; odour
characteristic.
Bark- Bark was about 1-4 mm in thickness, hard, yellowish brown in colour;
thick barks were channeled while others were quilled; rough outer surface with
irregular yellow linings; fibrous fracture.
Flower- The flowers were fragrant, large, white with purple-tinged outside (buds
also), unisexual often higher proportion of male flowers. Flowering takes place in
the month of March-April.
Fruit- Fruit hesperidium, 10-15 cm long, ovoid to oblong, small nipple at the end,
thick rind; dark green when unripe and yellow when ripe; pulp pale yellow with
abundant acidic juice. The fruit is edible and the inner rind (albedo) is also edible.
Peel- Dried peels of the ripe fruit were fragrant, curved to inner side; yellow
coloured outer surface (flavedo) with white inner surface (albedo); large number
of oil glands were clearly visible on the surface of fresh peels.
Seeds- Seeds were cone shaped 10-13 mm long, pointed at the apex and broaden
downwords. Seeds were cream coloured outside with a brown coloured inner seed
coat; dark brown to purple chalazal spot at the base. Seeds were bitter in taste and
non-edible.
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A) A fruiting tree
B) Ripe fruit C) Leaf
Fig 4.1: Photographs of a (A)-tree, (B)-fruit and (C)-leaf of C. medica L.
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A) Half fruit
B) Dried peels C) Seeds
D) Roots E) Flower with buds
Fig 4.2: Photographs of different parts of C. medica L. (A-E).
albedo
flavedo
juicy vesicles
seed
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b) Microscopic characteristics
Leaf constants- The microscopical measurements of leaf constants are mentioned
in Table 4.1.
Table 4.1: Leaf constants of C. medica L.
S.No. Leaf constant Range Average
1 Stomatal number (SN)
Upper epidermis
Lower epidermis
Nil
4-14
Nil
12.1
2 Stomatal index (SI)
Upper epidermis
Lower epidermis
Nil
3.96-16.66
Nil
9.69
3 Palisade ratio (PR) Upper surface
Lower surface
1.25-5.0
Nil
3.30
Nil
4 Vein-islet number (VIN) - 3.5-9.25 5.68
5 Veinlet termination
number (VTN)
- 3.75-10 7.55
SN & SI- average of 10 determinations (each of 1 sq mm); PR- average of 25 groups
(each of 4 epidermal cells); VIN & VTN- average of 10 sets of 2 mm × 2 mm area
(having 4 squares each of 1sq mm).
Fig 4.3: Lower epidermis of C. medica L. leaf.
anomocytic stomata
beaded wall of epidermal cells
epidermal cell
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Fig 4.4: Leaf surface of C. medica L.
T. S. of leaf- T. S. of leaf exhibits a dorsi-ventral nature. Upper epidermis was
single layered, cells more or less rectangular with outer wall cuticularized.
Trichomes were absent, large numbers of Ca oxalate crystals were observed in
epidermal cells. Being a dorsi-ventral leaf, mesophyll was differentiated into
upper palisade layer and lower spongy parenchyma. Schizolysigenous oil glands,
Ca oxalate crystals, oil droplets and vascular bundles were seen in the mesophyll.
Prismatic Ca oxalate crystals were polygonal; mostly hexagonal and tetragonal.
Upper palisade single to double layered, cells thin walled, compact and not so
elongated. More or less circular schizolysigenous oil glands, opening towards the
upper epidermis were seen in the palisade region. Irregular parenchyma cells were
observed with few Ca oxalate crystals. Lower palisade was absent; compared to
upper epidermis lower epidermal cells were small and Ca oxalate crystals were
lesser in number.
Epidermal layers of lamina were continuous in the midrib region. A well
developed vascular bundle was observed in the centre of midrib. Above lower
epidermis more number of oil glands was visible. Anomocytic stomata were
observed only in lower epidermis.
prismatic crystals
of Ca oxalate
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A)
B) C)
D)
Fig. 4.5: T. S. of leaf of C. medica L. (A-D). le, lower epidermis; pal,palisade; sc, secretory cell; ue, upper epidermis; vb, vascular bundle; xy, xylem.
vb
le
ue
pal
vb
sc
le
xy sc
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Leaf powder- Leaf powder was green with characteristic odour and taste. The diagnostic characters observed were:
• Prisms of Ca oxalate, both free and within epidermal cells. The crystals
were polygonal, mostly hexagonal.
• Anomocytic stomata.
• Epidermal walls were beaded and polyhedral.
• Many fragments of spiral vessels were observed.
• Vessels were lignified.
• Fibres present.
T. S. of root- T.S. of root represented a circular outline and the following tissues:
• Cork cells
• Cortex with abundant starch grains, almost every cell of the cortical
parenchyma was heavily loaded with starch grains which were both simple
and compound. Some parenchymatous cells contain prismatic crystals of
Ca oxalate, mostly polyhedral.
• Medullary rays run radially from the centre to the cortex.
• Xylem well represented, divided by large medullary rays at regular
intervals. The xylem vessels were relatively wide.
• Pith was absent.
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A) Diagrammatic
B) Enlarged portion of T.S. (stained with iodine)
Fig 4.6: T.S. of root of C. medica L. (A-B). ck, cork; ct, cortex; mr, medullary ray; ph, phloem; sg, starch grain, xy, xylem.
ct
ph
mr
xy
ck
xy
sg
mr Estelar
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Root powder- Root powder was yellowish-brown with characteristic odour. The
diagnostic characters observed were:
• Presence of cork cells.
• Fragments of pitted vessels.
• Presence of fibres and fibres with crystal sheath.
• Starch grains, both simple and compound (often 2-3) were observed. Starch
grains were abundant, mostly spherical with hilum at the centre. Size of
starch grains varied from 1.75-10.5 µm (from 30 observations).
• Prismatic (often polygonal) crystals of Ca oxalate were abundant. Size of
crystals varied from 7-32 µm in length and 7-25 µm in width (from 30
observations).
Cork cells starch grains prisms of Ca oxalate
Fragment of pitted vessels
Fig 4.7: Powder Characteristics of C. medica L. root.
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c) Physicochemical parameters
Physicochemical parameters of leaf and root powder are shown in Table 4.2.
Table 4.2: Physicochemical parameters of leaf and root powder of C. medica L.
S. No. Physicochemical
parameter
Leaf powder Root powder
1
2
3
4
5
Total ash
Acid insoluble ash
Loss on drying
Water-soluble extractive
Ethanol-soluble extractive
10.67% w/w
3.46% w/w
9.67% w/w
7.2% w/w
10.29% w/w
11.12% w/w
1.47% w/w
10.76% w/w
9.96% w/w
7.64% w/w
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REFERENCES
Anonymous. (1998). Quality Control Methods for Medicinal Plant Materials.
Geneva: World Health Organization, p. 20, 28-30.
Anonymous. (2003). Quality Standards of Indian Medicinal Plants, vol I. New
Delhi, India: Indian Council of Medical Research, p. 237.
Brain KR, Turner TD. (1975). The Practical Evaluation of Phytopharmaceuticals.
Great Britain: John Wright & Sons Ltd., p. 2.
Evans WC. (2002). Trease and Evans Pharmacognosy, 15th ed. London, United
Kingdom: Saunders, p. 245-47.
Iyengar MA, Nayak SGK. (2006). Anatomy of Crude Drugs, 10th ed. Manipal,
India: Manipal Press Ltd., p. 8.
Iyengar MA. (2007). Pharmacognosy of Powdered Crude Drugs, 8th ed. Manipal,
India: Manipal Press Ltd.
Jackson BP, Snowdon DW. (2000). Atlas of Microscopy of Medicinal Plants,
Culinary Herbs and spices. New Delhi, India: CBS Publishers and
Distributors.
Kokate CK. (2006). Practical Pharmacognosy, 4th ed. Delhi, India: Vallabh
Prakashan, p. 26, 115-21.
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