1. 2321-7871
Weekly Science Research Journal
Vol-1, Issue-10, 26th September 2013
Primary Article
Histochemical Studies Of Cochlospermum
Religiosum (l) Alston
Sasikala A, Linga Rao M And Savithramma N
ABSTRACT
Traditional medicines are prepared from a single plant or combination of
plants. Histochemical studies are helpful in drug adulteration and
biosystematics arrangement. The present study deals with the location or
identification of phytochemicals like tannins, polyphenols, crystals and
starch grains in various regions of leaf, stem and stembark of
Cochlospermum religiosum by using different chemicals or reagents (FeCl3,
Iodine solution, Toluidine blue reagent and HCl). The results showed that
the bluish black, purple or blue, bluish green and dark black colour
indicates the presence of tannins, starch grains, polyphenols and crystals
in various regions like epidermis, endodermis, midrib, cortex and vascular
bundle of leaf, stem and stembark of Cochlospermum religiosum. These
observations could be of immense value in the botanical identification and
standardization of crude drug.
formulations were found to possess
KEY WORDS:
good antibacterial and antifungal
Cochlospermum Religiosum,
activity (Cecilie et al., 2005).
Stembark, Phenols, Tannins.
Sasikala and Savithramma (2012)
studied the antimicrobial activity of
INTRODUCTION
Plants are the great sources biological synthesis of silver
o f m e d i c i n e s , e s p e c i a l l y i n nanoparticles from leaves of C.
religiosum, preliminary
traditional system of medicine,
which are useful in the treatment of phytochemical screening (Sasikala
various diseases. Indian and Savithramma, 2012),
contribution to herbal market and quantification of phytochemical
emphasis on novel research is (Sasikala and Savithramma, 2013)
c o n t i n u o u s l y i n c r e a s i n g . and also studied the invitro
Cochlospermum religiosum (L) p r o p a g a t i o n ( S a s i k a l a a n d
Alston is a sparsely branched small Savithramma, 2012).
Histochemistry or
tree, belonging to the family
Cochlospermaceae. It is commonly c y t o c h e m i s t r y d e a l s w i t h
called as Yellow Silk Cotton, localization of chemical compounds
Buttercup Tree and Torchwood within the cells by means of specific
Tree because of flowers are large, colors of the compounds. Staining
bright golden yellow and seeds the cells with different stains or
covered with silky hairs.C. dyes, which render the compounds
religiosum stem bark and root visible under the microscope,
powder is traditionally used for makes the specific color reaction
fertility and ash of fruit mixed with compounds. The importance of
coconut is used for the treatment of histochemistry in solving critical
scabies (Goud et al., 2005). The biosystematic problems is as
gum of C. religiosum is also found to popular as the use of other
be an ingredient of unani medicine markers. According to botanical
Qurs-e-Sartaan Kafoori which is l i t e r a t u r e s , t h e u s e o f
used for Styptic, Antipyretic, histochemical characters in
Phthisis, Tuberculosis, Hectic fever taxonomic conclusions is now a
and Qurs-e-Suzak Cicatrizant, common practice. For example, the
D i u r e t i c , G o n o r r h e a . T h e s e presences of calcium oxalate
crystals in various plant families
Page No-1
Sasikala A, Linga Rao M And
Savithramma N
From
1,2,3
Research Scholar
Department of Botany,
Sri Venkateswara University,
Tirupati – 517 502.
Andhra Pradesh, INDIA.
Te Article is published on
September 2013 issue &
available at
www.weeklyscience.org
DOI : 10.9780/ 2321-7871/1102013/29

2. 2321-7871
Vol-1, Issue-10, 26th September 2013
Histochemical Studies Of Cochlospermum Religiosum (l) Alston
have been reported by various scientists
(Mbagwu et al., 2009) reported that the
size and shape of calcium oxalate crystals
though variable in each species showed
enough interspecific differences that may
be used for taxonomic references in Vigna
species. This has been done in other
groups of plants such as Dioscoreaceae,
(Edeoga and Okoli, 1995). Icacinaceae,
(Heintzelman and Howard, 1948)
Nyctaginaceae, (Edeoga and Ikem, 2002)
and Verbenaceae (Mathew and Shah,
2006). The biosystematic importance
and implications of histochemical
features of ergastics, calcium oxalate
crystals, nature of tannins and saponins
have been investigated in various plants
families such as Dioscoreaceae (Edeoga
and Ogbegbor, 1999), Leguminosaepapilionoideae (Mbagwu and Edeoga,
2006). Identification of localization of
secondary metabolites in plant parts
which are using in the preparation of
drug is an immense importance to
prevent adulteration and also helpful in
taxonomic hierarchy.
Hence in the
present study an attempt has been made
to identification and localization of
secondary metabolites in the medicinal
plants.
MATERIAL AND METHODS
Fresh and healthy leaf, stem and
sembark of Cochlospermum religiosum
were collected from Tirumal hills of
Chittoor district, Andhra Pradesh, India
during the year May, 2012.
These
specimens were initially fixed in FAA
(1:1:18) glacial acetic: 40% formaldehyde:
70% ethanol (v/v) for 48-72 h. after 72 h
the specimens were transverse (T.S)
section were taken using a rotary
microtome (RMT-30).
Anatomical
staining was done by initially staining
with few drops of alcian blue for 5 min
and counter stained with safranin
solution for 2 min. The specimens were
treated with FeCl3, Iodine solution,
Toluidine blue reagent and HCl for
identification of polyphenols, tannins,
crystals and starch grains.
Photomicrographs of the anatomical
features were then taken from the slides
using Nikhon Labhot 2 microscopic unit
(Asokan, 2006).
Fluorescence Studies
The fluorescence studies were
carried out as per the method of
Bhattacharya and Zaman (2009).
A
small quantity of the leaf powder was
placed on a grease free clean microscopic
slide and added 1-2 drops of the freshly
prepared reagent solution, mixed by
gentle tilting the slide and waited for 1-2
min. Then the slides were placed inside
the UV-viewer chamber and viewed in day
light short (254 nm) and long (365 nm)
ultraviolet radiations.
The colours
observed by application of different
reagents in different radiations were
recorded.
Behavior of the leaf powder with
different chemical reagents was studied
to detect the presence of major
phytoconstituents with color changes
under day light by reported method of
Pratt and Chase (1949)
RESULTS AND DISCUSSION
Histochemical Studies
Present day's adulteration of the
drugs is a common phenomenon. It is
inevitable to study the pharmacognostic
studies to avoid adulteration.Hence the
following experiments were carried out.
Histochemical color reactions
were carried out through transverse
sections of leaf, stem and bark of
Cochlospermum religiosum. The results
were showed in Table-1. Fig-1 showed
various secondary metabolites present in
leaf with treatment of different reagents.
The presence of tannins was indicated by
the development of bluish black colour,
when treated with ferric chloride (FeCl3).
The tannins were found mainly in the
parenchyma tissue of the midrib region,
whereas the starch grains were indicated
by the development of blue color or
purple, when treated with iodine
solution. The starch grains were located
in the mesophyll and parenchymatous
region of midrib. The presence of
polyphenols was indicated by the
development of bluish green color, when
treated with toluidine blue reagent. The
polyphenols were found surrounding the
vascular bundle sheath. The presences of
crystals were indicated by the
development of dark black color, when
treated with HCl, the crystals were
present in the midrib region and vascular
bundles.
Fig-2 showed the histochemical
studies of stem, the presence of tannins
were indicated by the development of
bluish black color, when treated with
ferric chloride (FeCl3). The tannins were
present in endodermis and cortex region.
The starch grains were indicated by the
development of blue color or purple, when
treated with iodine solution found in
epidermis, cortex and vascular bundles.
Page No-2

3. 2321-7871
Vol-1, Issue-10, 26th September 2013
Histochemical Studies Of Cochlospermum Religiosum (l) Alston
The polyphenols were found mainly in the
endodermis and cortex region, which
were indicated by the development of
bluish green colour, when treated with
toluidine blue reagent. The presences of
crystals were indicated by the
development of dark black color, when
treated with HCl the crystals were present
mainly in the cortex.
The histochemical analysis of
bark showed in Fig-3 the presence of
tannins were indicated by the
development of bluish black color, when
treated with ferric chloride (FeCl3) and
these were found in cortex and vascular
bundle region. The polyphenols were
found mainly in the cortex region which
was indicated by the development of
bluish green color, when treated with
toluidine blue reagent. The presence of
crystal indicated by the development of
dark black color, when treated with HCl
the crystals were present mainly in the
cortex. The starch grains were indicated
by the development of blue or purple
colour, when treated with iodine solution
the starch grains were present mainly in
the cortex and vascular bundle region.
Toluidine blue is a cationic dye
that binds to negatively charged groups.
An aqueous solution of this dye is blue,
but different colors are generated when
the dye binds with different anionic
groups in the cell for example, a pinkish
purple colour will appear when the dye
reacts with carboxylated polysaccharides
such as pectic acid; green, greenish blue
or bright blue with polyphenolic
substances such as ligning and tannins;
and purplish or greenish blue with
nucleic acids (Baker, 1966). Plants store
glucose as the polysaccharide starch, it
can be separated into two fractionsamylose and amylopectin. Amylose forms
a colloidal dispersion in hot water,
whereas amylopectin is completely
insoluble. The structure of amylose
consists of long polymer chains of glucose
units connected by an alpha acetyl
linkage. Amylose in starch is responsible
for the formation of a deep blue colour in
the presence of iodine.
The iodine
molecule slips inside of the amylose coil.
When added the Iodine-KI reagent to a
solution or other materials blue colour is
present. If starch amylose is not present,
then the colour will stay orange or yellow.
Leaf Powder Behavior
Behavior of the leaf powder with
different chemical reagents was studied
Page No-3
to detect the presence of major
phytoconstituents with color changes
under day light by reported method (Pratt
and Chase, 1949). And the results were
showed in Table-2.
Fluorescence Analysis
The fluorescence characteristics
of leaf powder with different chemical
reagents were summarized in Table-3.
Although a change in color was observed
by the addition of various reagents under
day light, none of the reagents induced
any fluorescence to the leaf powder under
both short and long UV radiations.
Under UV light dark brown and black
colors were prominent. Fluorescence is
the phenomenon exhibited by various
chemical constituents present in the
plant material. Some constituents
showed fluorescence in the visible range
in the day light. The natural products
(Alkaloids) produce fluorescence in UV
light but do not produce fluorescence in
visible day light.
If the substances
themselves are not fluorescent, they often
be converted fluorescent derivatives or
decomposition products by applying
different reagents (Mukherjee, 2002).
The pharmacognostical studies
and histochemical studies and behavior
analysis of fluorescence studies of
Cochlospermum religiosum are useful to
supplement the information with regard
to its botanical identification and drug
standardization. Moreover, it also helps
in distinction from other allied species
and adulteration.
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Vol-1, Issue-10, 26th September 2013
Histochemical Studies Of Cochlospermum Religiosum (l) Alston
Fig-1: Histochemical studies of leaf of Cochlospermum religiosum
(a) Tannins, (b) Starch, (c) Poly phenols and (d) Crystals
Fig-2: Histochemical studies of stem of Cochlospermum religiosum
(a) Polyphenols, (b) Crystals, (c), (d) Starch and (e) (f) Tannins
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Histochemical Studies Of Cochlospermum Religiosum (l) Alston
Fig-3: Histochemical studies of stembark of Cochlospermum religiosum
(a) Tannins, (b) Crystals, (c) Poly phenols and (d) Starch
Table-1: Histochemical analysis of leaf, stem and bark of Cochlospermum religiosum
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Histochemical Studies Of Cochlospermum Religiosum (l) Alston
Table-2: Leaf powder behavior of the Cochlospermum religiosum with
different chemical reagents
Table-3: Fluorescence analysis of leaf powder of Cochlospermum religiosum
Page No-7