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Inter. J. of Phytotherapy / Vol 2 / Issue 1 / 2012 / 16-22.

e - ISSN - 2249-7722
Print ISSN - 2249-7730

International Journal of Phytotherapy


www.phytotherapyjournal.com

PHARMACOGNOSTIC STUDIES OF THE LEAVES OF Ficus nervosa


Heyne ex Roth (Moraceae)
*M. Himaja Trivedi, S. Mohana Lakshmi, Jyothi M. Joy
*Department of Pharmacognosy, Sree Vidyanikethan College of Pharmacy, Tirupati,
Andhra Pradesh-517102, India.

ABSTRACT
The present report is an investigation of anti-seizure activity of Dendrocnide harveyi (Family - Urticaceae)
which is common in wet primary and secondary forests, edge of forests,and along streams from sea-level to midmontane. The methanolic (90%) extract of Dendrocnide harveyi (MEDH) was subjected to acute toxicity and then
screened for antiepileptic activity on Maximal Electroshock (MES) and Pentylenetetrazole (PTZ) induced seizures
models in albino wistar rats. Acute toxicity of extract was non toxic up to the recommended dose 2000mg/kg body
weight orally as per OECD guidelines No.423. Animals were pretreated with MEDH at the doses of 200 and
400mg/kg body weight. The study reported the significant delay in clonic seizure induced by PTZ and dose
dependent decrease in duration of hindleg extensor phase in MES model. In MES model, MEDH showed
significant reduction in duration of hindleg extension with 200 mg/kg dose and effect was dramatically reduced
with 400mg/kg. Similar dose dependent results were obtained in PTZ model by delayed the onset of clonic
convulsions. The complete protective effect against mortality was reported in both the tests. This study predicted
possible mechanism of the formulation mediated through chloride channel of the GABA or benzodiazepine receptor
complex .However, the exact mechanism of action is not clear, but attributed to its antiepileptic effect. The
methanolic extract of Dendrocnide harveyi deserves further investigation for detailed elucidation of active
constituents and the mechanisms of action.
Keywords:-Anti seizureactivity, Traditional Medicine, Dendrocnide harveyi Maximal Electroshock,
Pentylenetetrazole.
following Kokate (1999) [1].
INTRODUCTION
Macroscopical & microscopical characters and
inorganic constituents present in a drug or plant play a
significant role in identification of crude drug.
Macroscopical and microscopical characters will help in
the identification of right variety and search for
adulterants. Physical constants like ash and extractive
values help in establishing the pharmacopoeial standards
of drug. Fluorescence analysis help to identify the drug in
powder form. Physical constants were determined

COLLECTION AND AUTHENTICATION


The leaves of Ficus nervosa Henye ex Roth
(Moraceae) were collected from Tirumala hills,
Tirupathi, India in the month of September 2009 and it
was identified and authenticated. The taxonomical
identification and authentication of the plant was done by
Dr. K. Madhava Chetty, Professor, Department of
Botany, Sri Venkateswara University, Tirupati. The
voucher specimen was preserved in our laboratory for
further reference.

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Inter. J. of Phytotherapy / Vol 2 / Issue 1 / 2012 / 16-22.

Corresponding Author:-K. Bhagya Deepthi


MATERIALS AND METHODS
Ethanol, formalin, glacial acetic acid, tannic
acid, ferric chloride, safrain, toluidine blue, benzoic acid,
sodium benzoate, Xylol, hydrochloric acid, chloroform,
alcohol, sodium hydroxide, sulfuric acid, nitric acid,
ammonia, acetic acid, iodine, petroleum ether, methanol,
distilled water.
MACROSCOPIC STUDIES
Macroscopical examination was carried out to
the freshly collected leaves and to the powder. In these
tests colour, odour, taste, size and shape of leaf and
powder were observed and noted and photographs were
taken in the original environment.
MICROSCOPIC STUDIES
Method
The fresh sample were cut into small pieces and
fixed in FAA solution (Formalin 5ml + Glacial acetic acid
5ml + 70% Ethanol 90ml). After fixing, the specimens
were dehydrated with graded series of tertiary butyl
alcohol (TBA) as per the standard procedure [2]. After
complete dehydration, the specimens were embedded in
paraffin wax. The paraffin embedded specimens were
sectioned with the help of Rotary microtome (thickness
10-12m). Dewaxing and staining of the sections were
done by customary procedure. Sections were stained
mostly with toluidine blue [3].
Staining
For anatomical studies the following staining
schedules were followed 1.Tannic Acid Ferric Chloride
counterstained with 0.5% alcoholic safrain. This schedule
was found to be quite satisfactory for all young plant
tissues in which the primary walls were stained.
Alcoholic safrain (0.5%) counterstained with
0.25% fast green. This schedule gives good result for
studying the histology of different tissues of the plant
organs especially the cell inclusions.
Toludine Blue O stain was prepared by
dissolving 0.25g of the stain in the mixture of benzoic
acid 0.25g, sodium benzoate 0.29g and distilled water
200ml with pH of 4.2 4.4. Since Toluidine blue is a
polychromatic stain, the staining results were remarkably
good and the dye render pink colour to the cellulose
walls, blue to the lignified cells, dark green to suberin,
violet to the mucilage, blue to the protein bodies, etc [4].
After dewaxing, the slides were stained for 5 10 minutes
and then dehydrated.
Photomicrograph
All permanent slides, after staining were dehydrated
by using graded series of Ethanol + Xylol and mounted in

Email:- bhagyadeepthik@gmail.com
DPX. Photomicrographs were done on NIKON Labphot
2 microscope using Konica colour film (100 ASA). For
normal observations bright field was used. For the study
of crystals and starch grains, the sections were
photographed under polarized light. Magnifications of the
figures are indicated by scale bars. Descriptive terms of
various observations are as found in standard Anatomy
books [5].
PHYSICO CHEMICAL PARAMETERS
Determination of moisture content
Moisture is an inevitable component of crude
drugs, which must be eliminated as far as practicable.
Drying plays a very important role in the quality as well
as purity of the material. Moisture will lead to the
activation of enzymes and gives suitable condition, to the
proliferation of microorganisms.
Method
About 2 g of the drug was weighed in a watch
glass, kept in hot air oven at 1050C and dried for a period
until constant weight was obtained. Weight loss on drying
was noted and difference in weight gives the moisture
content of powdered drug. Total moisture content of root
was noted.
Determination of ash values
Ash value aids in determination of quality and
purity of crude drug in powdered form. The ash content of
a crude drug is generally considered as a residue
remaining after maceration. Ash contains inorganic salts
like phosphates, carbonates and silicates of sodium,
potassium, magnesium, calcium are adhere to it or may
also be added to for the purpose of adulteration. There is a
considerable difference (varies with in narrow limits) in
the case of same individual drug. Hence ash
determination furnishes a basis for judging the identity
and quality of the drug gives information to its
adulteration with inorganic matter. Ash standards have
been established for a number of drugs in the
pharmacopoeias. The acid insoluble ash is a part of a ash
is imposed, especially in case where silica and calcium
oxalate content of the drug is very high. In most of the
cases inorganic matter is present in small amounts which
are not objectionable if only traces are present. Procedure
is given in Indian pharmacopoeia were used to determine
the different ash values such as total ash, acid insoluble
ash.
Determination of total ash value
Weigh accurately 3gms of the powdered material in
a silica crucible which was previously ignited and
weighed. The powdered material was spread as a fine
even layer at the bottom of the crucible. The crucible was
incinerated until a red hot material was obtained not

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Inter. J. of Phytotherapy / Vol 2 / Issue 1 / 2012 / 16-22.

exceeding 450C temperature and it is free from carbon.


The crucible was cooled and weighed. The procedure was
repeated until the constant weights. The percentage of the
total ash was calculated with reference to the air dried
powdered sample.
Determination of acid insoluble ash value
The obtained total ash was boiled with 25 ml of
2N Hcl for 5 min. The insoluble ash was collected on ash
less filter and washed with hot water. The insoluble ash
was transferred into pre-weighed silica crucible, ignited,
cold and weighed. The procedure was repeated till the
constant weight was obtained. The percentage of acid
insoluble ash was calculated with reference to the air
dried drugs.
Determination of water soluble ash value
The total ash obtained was boiled with 25 ml of
chloroform water for five min. The insoluble matter was
collected on a ash less filter paper & and washed with hot
water. The insoluble ash was transferred into pre-weighed
silica crucible, ignited for 15 min at a temperature not
exceeding 450, cooled and weighed .The procedure was
repeated to get the constant weight. The weight of the
insoluble matter was subtracted from the weight of total
ash. The percentage of water soluble ash was calculated
with reference to the air-dried sample drug.
Determination of extractive values
Extraction values are useful for determination of
crude drugs & it gives an idea about the nature of the
chemical constituents present. The solvent used for the
extraction should be in position to dissolve appropriate
quantities of desired substances.

[6]. The extractive values are presented in the table no.2.


FLUORESCENCE ANALYSIS
Fluorescence provided by a drug is one of the
several methods used for analyzing crude drugs.
Fluorescence is a type of luminescence in which the
molecule emits visible radiation passing from higher to
lower electronic state. The molecules absorbs light
usually over a specific range of wavelength, get excited
from ground state to a high energy level and many of
them emit such radiations while coming back to the
ground state. Such a phenomenon of re-emission of
absorbed light that occurs only when the substance is
receiving the exciting rays is known as Fluorescence.
For fluorescence analysis, powdered drug was sieved
through 60 mesh and observations were made following
[7-9].
Method
About 10 g of powdered drug was taken in
petridish and treated separately with different reagents
viz., methanol, 1N methanolic sodium hydroxide, ethanol
(70% v/v), 1N ethanolic sodium hydroxide, 1N HCl, 50%
sulphuric acid, 50% nitric acid and 5% potassium
hydroxide.
These were observed under short UV (254 nm),
long UV (365 nm) and visible light.
RESULT AND DISCUSSION
MACROSCOPIC STUDIES
Colour: Pale green; Odour: Characteristic; Taste: Bitter
to astringent; Size: 8-20cm in length; Shape: Coriaceous;
Apex: Acute; Base: Narrow; Margin: Entire.

Determination of alcohol soluble extractive value


About 5gms of air dried coarse powdered drug
was weighed and macerated with 100ml of 90%alcohol in
a closed flask for 24 hrs, shaking frequently during the
first 6 hrs &these allowed to stand for 18 hrs .There after
it was filtered rapidly taking precautions against loss of
the solvent.25 ml of the filtrate was evaporated to dryness
in a tarred flat bottomed swallowed dish, dried at 105C
& weighed. The %of the alcohol soluble extractive values
was calculated with reference to the air-dried drug.

MICROSCOPIC STUDIES
Microscopical examination of the leaf showed
the following: The leaf is dorsiventral with distinct
differentiation of the mesophyll tissue and smooth
surfaces (fig 4). The lamina is 240m thick. The adaxial
epidermis is fairly thick and the epidermal cells are
rectangular to square shaped. There is a hypodermal layer
of slightly larger cells beneath the epidermis. The
epidermal and hypodermal layer together measured 50m
in thickness. The abaxial epidermis is slightly thinner and
the cells are tabular in shape, stomata are seen on the
abaxial epidermis.

Determination of water soluble extractive value


About 5gm of air-dried powdered drug was taken
& macerated with 100 ml of chloroform water in a closed
flask for 24 hrs shaking frequently during the first 6 hrs
&then allowed to stand for 18 hrs. Thereafter, it was
filtered rapidly taking precautions against loss of the
solvent.25 ml of the filtrate was evaporated to dryness in
a tarred flat bottomed shallow dish, dried at 105
&weighed .The percentage of the water soluble extractive
values was calculated with reference to the air-dried drug

The mesophyll tissue is differentiated into


narrow abaxial band of short, cylindrical palisade cells
with dark contents. The palisade zone is 70-80m in
height. The lower part of the lamina consists of several
layers of small, lobed and loosely introverts spongy
parenchyma cells (fig 5). The lateral veinlets are
prominent, but do not protrude beyond the surface of the
lamina. The vascular bundle of the lateral vein consists of

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Inter. J. of Phytotherapy / Vol 2 / Issue 1 / 2012 / 16-22.

collateral xylem and phloem with thick adaxial and


abaxial masses of sclerenchyma elements.
The leaf-margin is blunt and semicircular and
slightly bent dowers (fig. 4 & 5). It is 250m thick. It
consists of thick walled epidermal cells and the entire
mesophyll tissue is connected into homogeneous compact
thick walled cells.
Midrib: The midrib is quite prominent,
projecting much beyond the surface of the lamina. The
adaxial part is protruding more than the abaxial part. It is
1.5mm thick; the adaxial conical part is 900m wide and
the abaxial part is nearly 1mm wide. The epidermis of the
midrib consists of the layer of papillate cells. The outer
ground tissue is collenchymatous and the remaining inner
zone is parenchymatous.
The vascular system of the midrib is complex. It
consists of shallow wide abaxial arc and a narrow
adaxial arc of xylem and phloem strewels. These arcs
comprise short radial, parallel lines of xylem elements
and thick zone of phloem elements (fig. 6, 7 & 8). Outer
to the vascular arcs is thin sheath of sclerenchyma cells
abutting the phloem zone.
In the medullary part of midrib these are wide, circular
masses of phloem tissue. No xylem elements are evident
among the phloem masses. Fairly wider sieve elements
are seen mixed with parenchyma cells.
Cystolith:- Calcium carbonate crystals of
cystoliths are very common especially on the adaxial part
of the lamina. The cystoliths are spherical bodies with
spiny surface. They have fairly long, slender stalk with
which they are attached to the roof a wide chamber which
is known as litho cysts. The epidermis in the region of
1.3 PHYSICO-CHEMICAL PARAMETERS

the litho cyst is thin where the stalk of the cystolith is


hanging (fig. 9, 10 & 11). The cystolith is 30-40m in
diameter and 50m in length.
Epidermal cells and Stomata:- As seen in Para
dermal sections, the epidermal cells appear polygonal in
outline. They have fairly thick, straight anticline walls.
Stomata are densely distributed on the abaxial side of the
lamina. The stomata are actinocytic type; that is, stoma
is surrounded by a radiating circle of 5-7 subsidiary cells.
The guard cells are 30m long and 20m thickness (Fig
12 & 13).
Venation pattern:- Cleaned lamina was slidied
for the venation pattern. The lateral veins and veinlets
gradually decrease in size and form dense reticular
venation. The vein-islets are fairly distinct; they are
rectangular to polygonal in outline. The vein-terminations
are less prominent. When distinct, they are long and
slender, simple or branched (Fig. 14 & 15).
The taxonomical identification of plant material
and Pharmacognostical evaluation is important to provide
the standards and to avoid adulteration of drugs.
Macroscopical and Microscopical characters of the plant
used for the identification of the drug. The
physicochemical evaluation helps in formulating
pharmacopoeial standards, while fluorescence analysis
helps in distinguishing the drug in powder form. The
physico chemical constants like moisture content, ash
values, melting point, extractive values and fluorescence
analysis are rarely constant for crude drugs, but they may
help in evaluation. Ash value is a criterion to judge the
identity and purity of crude drug. The extract obtained by
exhausting crude drug is indicative of approximate
measure of their chemical constituents. Melting point is
one of the parameter to judge the purity of crude drug. To
check the moisture content helps prevent degradation.

Table 2: Physico-Chemical parameters of powdered leaves of Ficus nervosa Heyne ex Roth (Moraceae)
S. No.
1.

2.

3.

Parameters

Average % W/W

Ash values
a) Total ash

7.13

b) Acid insoluble ash

1.14

c) Water soluble ash

1.41

Extractive values
a) Alcohol soluble Extractive

1.08

b) Water soluble extractive

1.10

Moisture content

2.14

Loss on drying

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Inter. J. of Phytotherapy / Vol 2 / Issue 1 / 2012 / 16-22.

FLUORESCENCE ANALYSIS
Table 3: Fluorescence analysis of powdered leaves of Ficus nervosa Heyne ex Roth (Moraceae)
Treatments
Powder as such
Powder + 1N NaOH (aqueous)
Powder + 1N NaOH (alcoholic)
Powder + 1N H2SO4
Powder + 1N HNO3
Powder + 1N HCl
Powder + Ammonia
Powder + Acetic acid
Powder + Iodine
Powder + FeCl3
Powder + water

Observations
Long UV
Light green
Green Fluorescence
Yellow
Yellow Fluorescence
Light green
Brownish
Dark green
Brown
Green
Dark green
Pale yellow

Day light
green
Orange
Green
Yellow
Pale yellow
Green
Brown
Light green
Pale yellow
Light green
Pale yellow

Short UV
Green
Brown
Brown
Light green
Pale green
Orange
Green
Green Fluorescence
Light green
Yellowish
Light green

Table 4: Fluorescent analysis of various extracts of Ficus nervosa Heyne ex Roth (Moraceae)
Extract
Petroleum ether
Chloroform
Methanol
Distilled water

Day light
Light green
Green
Green
Brown

Fig 1:- Plant Ficus nervosa Heyne ex Roth (Moraceae)

Long UV
Yellow
Bluish green
Green Fluorescence
Light Green

Short UV
Green
Green fluorescence
Green
Light green

Fig 2:- Twig of the Plant Ficus nervosa Heyne ex Roth


(Moraceae)

Fig 3:- Leaf of the Plant Ficus nervosa Heyne ex Roth


(Moraceae)

Fig 4 : T.S. of lamina of leaf

AbE Abaxial epidermis; AdE : Adaxial epidermis; Hd


Hypodesmis; SP Spongy Parenchyma; Cy Cystolith;

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Inter. J. of Phytotherapy / Vol 2 / Issue 1 / 2012 / 16-22.

PM Palisade Mesophyll; LC Lithocyst.


Fig 5: T.S. of leaf margin

Fig 6 : T.S. of leaf through midrib with lamina

AbS Abaxial strand; LV Laferal vein; LM Leaf margin;


SC Sclerenchyna.

AdH Adaxial hump; AdS Adaxial strand; AbS


Abaxial strand; La Lamina.

Fig 7 : T.S. of midrib ground tissue showing laticiferous


medullary phloem, and tanniniferous

Fig 8 : T.S. of midrib of leaf

MPh Medullary phloem, EP Epidermis; Col


Collenchyma; Ph Phloem; Pa Parenchymatous ground
tissue; Sc Sclerenehyma; X Xylem.

MPh Medullary Phloem; Lf Laticifer; Ta


Tanniniferous cells.

Fig 9 : T.S. of lamina showing cystolith on the adaxial side

Fig 10 : T.S. of lamina showing cystolith on the adaxial


side enlarged

Ep Epidermis; St Stalk, Cy Cystolith; SM


Spongy mesophyll; Hd Hypodermis; PM Palisade
mesophyll.

St Stalk; Cy Cystolith; Lc Lithocysth

Fig 11 : Paradermal section showing cystrolith distribution


in the mesophyll tissue

Fig 12 : Abaxial epidermis with stomata

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Inter. J. of Phytotherapy / Vol 2 / Issue 1 / 2012 / 16-22.

LC, Li Lithocyst; Cy Cystolith; AdE Adaxial epidermis

St Stoma; Ec Epidermal cell.

Fig 13 : Stomata of leaf

Fig 14 : Cleared leaf showing vein-islets and veintermination

Ec Epidermal cell, Gc Guard cell; SC Subsidary cell.

Vi Vein-islets; VT Vein-termination.

Fig 15: Cleared leaf showing vein-islets and vein-termination enlarge

Vi Vein-islets; VT Vein-termination.
REFERENCES
1. Kokate CK. Practical Pharmacognosy. Vallabh Prakashan; New Delhi, 1999. p.10721.
2. Sass JE. Elements of Botanical Microtechnique, Mc.Graw Hill Book. Co., New York, 1940, 222.
3. Johanson DA. Plant Microtechnique, Mc.Graw Hill Book. Co. New York, 1940, 523.
4. OBrien TP, Feder N, Mc Cull ME. Polychromatic Staining of Plant Cell walls by Toluidine blue-O, Protolpasma,
1964, 364-373.
5. Esau K. Plant Anatomy, John Wiley & Sons, New York, 1979, 550.
6. Indian Pharmacopoeia, 4th edition, Vol. 2, Government of India, Ministry of Health and Family, Controller of
Publication, New Delhi, 1996, A53-A57 & A100-A107.
7. Chase CR, Pratt RJ. Fluprescence of Powdered vegetable drugs with particular reference to development of a system
of identificaltion. Journal of American Pharmaceutical Assoiation, 38, 1949, 324-331.
8. Kokoshi J, Kokoshi R, Slama FJ. Fluorescence of powdered vegetable drugs under ultra violet radiation. Journal of
American Pharmaceutical Assoiation, 47 (10), 1958, 715.
9. Pimenta AM, Montenegro MC, Ara ujo AN, Martiez JC. Application of Sequential injections analysis to
Pharmaceutical Analysis. Journal of Pharm.Biomed.Anul, 40, 2006, 16-34.
10. Usha kumari J, Navas M, Dan M. Pharmacognostic Studies on Pellionia heyneana. Journal of Trop Med Plant, 5,
2004, 259-261.

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