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Department of Pharmaceutical Sciences, Kumaun University, Nainital

CHAPTER: 4

PHARMACOGNOSTIC

EVALUATION

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Pharmacognostic Evaluation

Department of Pharmaceutical Sciences, Kumaun University, Nainital Page 72

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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Department of Pharmaceutical Sciences, Kumaun University, Nainital 7

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

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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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