13 CHAPTER 4 PHARMACOGNOSTICAL STUDIES The study of plant drugs from the pharmacognostical stand point would include the study of the habitat, general characters of the plant from which the drug is derived, its place in the botanical system, the organ or the organs of the plant used, their gross, minute structures in the whole and in the powdered conditions and the chemistry of the constituents especially of those which may be used in therapeutics. The macroscopic and microscopic description of a medicinal plant is the first step towards establishing the identity and the degree of purity of such materials. This should be carried out before any tests are undertaken. Lack of proper standards of medicinal plants may result in the usage of improper drugs which in turn will cause damage not only to the individual using it, but also to respect gained by the well known ancient system of medicine and the entire work on the plant becomes invalid. Thus, in recent years there has been an emphasis in pharmacognostical standardization of medicinal plants of therapeutic potential. So, the present study is undertaken to standardize Symplocos cochinchinensis (Lour.) S.Moore ssp. laurina (Retz.) Nooteb. pharmacognostically which will help in the correct identification of the drug.
Transcript
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CHAPTER 4
PHARMACOGNOSTICAL STUDIES
The study of plant drugs from the pharmacognostical stand point
would include the study of the habitat, general characters of the plant from
which the drug is derived, its place in the botanical system, the organ or the
organs of the plant used, their gross, minute structures in the whole and in the
powdered conditions and the chemistry of the constituents especially of those
which may be used in therapeutics.
The macroscopic and microscopic description of a medicinal plant
is the first step towards establishing the identity and the degree of purity of
such materials. This should be carried out before any tests are undertaken.
Lack of proper standards of medicinal plants may result in the usage of
improper drugs which in turn will cause damage not only to the individual
using it, but also to respect gained by the well known ancient system of
medicine and the entire work on the plant becomes invalid. Thus, in recent
years there has been an emphasis in pharmacognostical standardization of
medicinal plants of therapeutic potential. So, the present study is undertaken
to standardize Symplocos cochinchinensis (Lour.) S.Moore ssp. laurina
(Retz.) Nooteb. pharmacognostically which will help in the correct
identification of the drug.
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4.1 MATERIALS AND METHODS
4.1.1 Collection of Plant Materials
The fresh healthy plant leaves of Symplocos cochinchinensis
(Lour.) were collected from the Nilgiri Hills, TamilNadu, India during the
month of July 2007. The plant was identified and authenticated by Botanical
Survey of India and Dr.S.Rajan, Field Botanist, Survey of medicinal plants
and collection unit, Ooty and a voucher specimen has been reserved in the
Department of Pharmacognosy. The fresh leaves, petiole, stem and bark were
also collected and fixed immediately using FAA (formalin: acetic acid: ethyl
alcohol) as fixative agent for anatomical studies. The materials were cut into
small pieces before fixing.
4.1.2 Procedure for Anatomical Studies and Staining Methods
Fixation of plant organ – the collected materials were cut and left
in FAA solution for more than two days.
Dehydration of specimen – was done with graded series of tertiary
butyl alcohol and ethyl alcohol mixtures as per the schedule given by Sass
[26].
Infiltration of the specimens – was carried by gradual addition of
58-60O C melting pointed paraffin wax until tertiary butyl alcohol solution
attained super saturation. The specimens were transferred to pure paraffin wax
and they were cast into paraffin blocks.
Sectioning
The paraffin embedded specimens were sectioned with the help of
rotary microtome. The thicknesses of the sections were 10-12mcg. Sections
15
were stained with toludine blue (0.25%, pH of 4.7). Since, toludine blue is a
polychromatic stain, different colours of the cells were obtained depending
upon the chemical nature of the cells [27].
Leaf clearing
The following method was used for studying the leaf constants like
stomatal types, palisade ratio and venation patterns. Paraffin embedded
leaflets were used for paradermal sections. From these sections, the epidermal
layers as well as vein islets were studied. The leaf fragments were cleared by
immersing the material in warm alcohol (to remove chlorophyll) followed by
treating with 5 to 10% sodium hydroxide. The materials were rendered
transparent, washed thoroughly and stained with safranin for further studies.
The elements of xylem were studied by macerating small fragments
of stem and bark with Jeffery’s maceration fluid [28]. The separated vessel
elements and fibres were used to study the lateral wall characteristic and
dimensional variations.
Photomicrographs
Microscopic descriptions of tissues were supplemented with
photographs wherever necessary. Photographs of different magnifications
were taken with Nikon lab photo 2 microscopic unit. For normal observations
bright field was used.
The study of crystals, starch grains and lignified cells were done by
using polarized light. Since, these structures have birefringent property, under
polarized light they appear bright against dark background. Magnifications of
the figures were indicated by the scale bars. Descriptive terms of the
anatomical features are as given in the standard anatomy books [29, 30].
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4.1.3 Quantitative Microscopy [31, 32]
The cleared materials were washed thoroughly and stained with
safranin for quantitative microscopic studies.
Stomatal Number
It is the average number of stomata per square mm of epidermis of
the leaf. A minimum of ten readings were taken from different locations of
the leaf and the average value was calculated.
Stomatal Index
It is the percentage in which the number of stomata to the total
number of epidermal cells, each stoma being counted as one cell.
Stomatal Index was calculated by using the following equation,
S.I = (S/E+S) × 100 (4.1)
Where,
S.I = Stomatal Index
S = Number of stomata per unit area
E = Number of epidermal cells in the same unit area.
Vein-Islet and Vein termination number
It is the average number of vein-islets per square mm of the leaf
surface. It is determined by counting the number of vein-islets in an area of 4
sq.mm of the central part of the leaf mid way between the midrib and the
margin.
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Vein termination number is the average number of veinlet
termination per sq.mm of the leaf surface. It is determined by counting the
number of vein terminations in an area of 4 sq.mm of the central part of the
leaf mid way between the midrib and the margin.
Palisade Ratio
It is the average number of palisade cells beneath each epidermal
cells of a leaf. It is determined by counting the palisade cells beneath four
continuous epidermal cells.
4.1.4 Powder Microscopic Observations [33, 34]
Well shade dried, chopped crude leaves were powdered, sieved and
used for powder organoleptic and microscopic analysis. The powder was
stained with safranin and observed through microscope. Starch grains were
observed by staining with iodine in potassium iodide (IKI) and calcium
oxalate crystals of the powder was observed under the polarized microscope.
4.1.5 Histochemical Analysis [35, 36]
The sections embedded in paraffin were used for histochemical
studies. The sections were deparaffinised and hydrated before staining them
for the tests. The chemicals localised in leaf, petiole, stem and bark were
studied using the standard procedure.
Lignin
The sections were placed in a saturated aqueous solution of
phloroglucinol in hydrochloric acid.
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Fatty acids
The sections were placed first in 50% alcohol and then in a
saturated solution of Neutral red and finally in 50% alcohol for 1min.
Proteins
The sections were stained in 0.02% Coomassie brilliant blue R 250
in Clark’s solution (pH 2) and rinsed in Clarke’s solution. It was destained in
fresh solution for 20min and then dehydrated in absolute alcohol for 5min.
Mucilages
The sections were stained with ruthenium red.
Starch
The deparaffinised sections were placed in iodine and potassium
iodide. This solution was made by dissolving 2gms of potassium iodide and
0.2gm of iodine in 100ml water.
Alkaloids
The deparaffinised sections were placed in Dragendroff’s reagent.
Flavanoids
The sections were stained with O-Toludine blue solution.
4.1.6 Physico-chemical Constants [37, 38]
The procedures recommended in Indian Pharmacopoeia and WHO
guidelines were followed to calculate the physico-chemical constants.
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Ash values
Total ash value
The total ash was determined by incinerating 2-3gms of accurately
weighed air dried coarsely powdered drug in a tarred silica crucible which
was previously ignited and cooled before weighing, at a temperature not
exceeding 4500C. The ignition was repeated and the percentage of ash with
reference to air-dried drug was calculated.
Water soluble ash
The total ash was boiled for 5min with 25 ml of water. The residue
was washed with hot water, ignited for 15min at a temperature not exceeding
4500C, cooled and weighed. This weight was subtracted from the weight of
ash, the difference in weight represents the water soluble ash. The percentage
of water soluble ash was calculated with reference to air-dried drug.
Acid insoluble ash
The ash obtained was boiled with 25 ml of dilute hydrochloric acid
for 5min and filtered through an ashless filter paper. The residue was washed
with hot water, ignited, cooled in a dessiccator and weighed. The percentage
of acid insoluble ash was calculated with reference to air dried drug.
Sulphated ash
The sulphated ash was determined by incinerating 1 gm of
accurately weighed air dried coarsely powdered drug in a tarred silica crucible
which was previously ignited and cooled before weighing at a temperature not
exceeding 4500C. The residue was moistened with 1 ml of concentrated
sulphuric acid, ignited at 800±250C until all black particles have disappeared.
20
It was then cooled, again sulphuric acid was added and ignited. It was cooled
and the percentage of sulphated ash was calculated with reference to air dried
drug.
Extractive values
Ethanol soluble extractive
5gms of dried coarse powder of Symplocos cochinchinensis
(Lour.) was macerated with 100ml of 90% ethanol in a closed flask for 24hrs,
shaken frequently during 6 hours and allowed to stand for 18hrs. Filtered
immediately taking precautions against loss of ethanol. 25ml of the filtrate
was evaporated to dryness in a tarred flat bottomed shallow dish. The residue
was dried at 1050C and weighed. The percentage of ethanol soluble extractive
was calculated with reference to air dried drug.
Water soluble extractive
5gms of coarse powder was weighed and dissolved in 100ml of
water in a stoppered flask, heated at 800C, shaken well and allowed to stand
for 10min. It was cooled, 2gms of kieselghur was added and filtered. 5ml of
the filtrate was transferred to a tarred evaporating dish and the solvent was
evaporated on a water bath. The percentage of water soluble extractive was
calculated with reference to air dried drug.
Determination of volatile oil in drug
50gms of the drug was boiled with water in a Clavenger’s
apparatus. The process was continued till no more oil was collected in the
graduated tube. The volume of oil was measured and expressed in percentage.
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Determination of crude fibre content
About 2gms of the drug was accurately weighed and extracted with
ether. Then 200ml of 1.25% sulphuric acid was added and boiled for 30min
under reflux. It was filtered and washed with boiling water until free of acid.
The entire residue was rinsed back into flask with 200ml of boiling 1.25%
sodium hydroxide solution and again boiled under reflux for 30min. The
liquid was quickly filtered and the residue was washed with boiling water
until neutral, dried at 1100C to constant weight. It was then ignited to 30min
at 6000C, cooled and weighed. The percentage of crude fibre content was
calculated with reference to the air dried drug.
Determination of loss on drying
Glass stoppered shallow bottle was weighed that had been dried in
the same conditions to be employed in the determination. About 1gm of the
sample was transferred to the bottle and distributed evenly by gently side wise
shaking to a depth not exceeding 10mm. Place the loaded bottle in a drying
chamber (the stopper was removed and left in the chamber). The sample was
dried to a constant weight and allowed to cool. The bottle along with the
content was weighed. The process was repeated until the successive weights
differed not more than 0.5mg (drying to constant weight). The percentage loss
of weight was calculated with reference to the air dried drug.
Determination of foaming Index
1gm of the coarsely powdered drug was weighed and transferred to
500ml conical flask containing 100ml of boiling water. The flask was
maintained at moderate boiling at 80-900C for about 30min. It was cooled,
filtered into a volumetric flask and sufficient water was added through the
filter to make up the volume to 100ml.
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Ten stoppered test tubes were cleaned (height 16cm, diameter 1-
6cm) and marked from 1 to 10. 1, 2, 3ml up to 10ml of the filtrate was
measured and transferred to each tube and adjusted the volume of the liquid
with water to 10ml. Then the tubes were stoppered and shaken lengthwise for
15sec uniformly, allowed to stand for 15min the length of the foam was
measured in each tube.
If the height of the foam in each tube is more than 1cm, the
foaming index is more than 1000. In this case, 10ml of the first decoction of
the plant material is measured and transferred to a 100ml volumetric flask
(V2) and the volume is made to 100ml and followed the same procedure.
4.1.7 Fluorescence Analysis
The fluorescence analysis of the drug powder as well as various
extracts were carried out by using the method of Chase and Pratt [39,40]. The
behavior of the powder with different chemical reagents was also carried out.
4.1.8 Inorganic Mineral Analysis
Chemical analysis of higher plants in general has revealed the
presence of 40 or more elements. Plant physiologists have proved that 18 of
these elements are indispensable to plants and human beings require 28 or
more elements. Of these elements, carbon, hydrogen, oxygen and nitrogen are
present in larger quantities than others. Sulphur and phosphorous are present
in protoplasm and has constituents of proteins or other important organic
compounds. A study of inorganic constituents of plants is of interest to
research workers in several fields, such as nutrition medicine and others
because plants constitute direct or indirect sources of many of the elements,
which are essential to animals including man[41]. Therefore, the plant
material was subjected to inorganic mineral analysis.
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Preparation of sample solution for inorganic mineral analysis [42]
The plant material (10gms) was digested with 10 ml of nitric acid
and left over night. It was then heated on a hot plate until the reddish brown
colour ceased and cooled. A small volume of perchloric acid was added and
transferred to a 50ml standard flask and made up to volume with double
distilled water.
Determination of sodium and potassium by flame photometry [43]
Flame photometry is based on the measurement of intensity of light
emitted when a metal is introduced into the flame. The material when
introduced into the flame is converted into gaseous state. The gaseous
molecules are progressively dissociated to give free neutral atoms or radicals,
which are excited by thermal energy of the flame. The excited atoms which
are unstable quickly emits photons and return to the lower energy state,
evenly reaching the unexcited state. The measurement of the emitted photons
forms the basis of flame photometry. A plot of emission versus concentration
in micrograms is prepared. The given unknown solution was diluted suitably
and aspirated into the instrument. From the emission intensity produced,
concentration of metal is determined by the interpolation of calibration curve.
The instrument used was Systronics Flame Photometer.
Procedure
A series of standard solutions containing the element to be
determined in increasing concentrations within the concentration range
recommended for the instrument were prepared. Nitric acid and perchloric
acid used for the preparation of sample solution of the plant material were
also added in the same concentration to the standard solution. The
appropriate filter was chosen, water was sprayed into the flame and the
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galvanometer reading was adjusted to zero. The most concentrated solution
was then sprayed into the flame and the galvanometer reading was recorded.
Again, water was sprayed till the galvanometer reading was zero. Then the
standard solution was sprayed into the flame and the procedure was repeated
three times for each concentration. A calibration curve was prepared by
plotting the mean of three readings of each standard against the concentration.
The sample solution prepared as above was then aspirated into the flame three
times, the galvanometer reading was recorded and the apparatus was washed
thoroughly with water after each aspiration. Using the mean of three readings,
the concentration of the element being examined was determined from the
calibration curve. To confirm the concentration thus obtained, the operation
was repeated with the standard solution of the same concentration as that of
the solution being examined.
Determination of calcium, cobalt, iron, copper, magnesium and
manganese by atomic absorption spectroscopy
This technique is based on the fact that when atoms, ions or ion
complexes of an element in the ground state are atomized in a flame, the
absorbed light has the characteristic wavelength of that element. If the
absorption process takes place under reproducible conditions the absorption is
proportional to the number of absorbing atoms. The concentration of the
metal is determined by interpolation of the calibration curve. The instrument
used was Perkin Elmer Atomic Absorption Spectrophotometer.
Procedure
Three standard solutions of the element to be determined covering
the concentration range recommended for the instrument were prepared.
Nitric acid and perchloric acid used in the preparation of the substance being
examined were also added to the standard solutions in the same concentration.
25
After calibration of the instrument, each standard solution was introduced into
the flame three times and the steady reading was recorded. The apparatus was
thoroughly washed after each introduction. A calibration curve was prepared
by plotting the mean of each group of three readings against concentration.
The plant extract prepared above was then introduced into the flame and the
reading was recorded. The sequence was then repeated twice. Using the mean
of the three readings, the concentration of the element was determined from
the calibration curve. The process was repeated for the determination of other
elements using different lamps.
4.2 RESULTS
4.2.1 Macroscopy
The plant Symplocos cochinchinensis (Lour.) S.Moore ssp.
laurina (Retz.) Nooteb. is a small evergreen tree up to seven meter in height
with thin, smooth, light grey bark and white wood. The branchlets are
generally glabrous. It is present throughout India in evergreen forests up to an
altitude of 900m. About 68 species are found in India.
Leaves are simple, alternate, very thick, lanceolate or oblong,
shortly acuminate at the apex, irregularly crenate or serrate, 8-18cm× 3.5-6cm
and symmetric base. The colour is dark green on the upper side and light
green at the lower side with strong tea like odour and slightly sweet taste
[Figure 4.1]. The flowers are yellowish white, fragrant, in close clusters and
in auxiliary spikes. Fruits are globose, purple, ribbed drupes with 1- 3 seeds.
The bark is greyish green to slightly grey with patches of crustose lichens.
The outer bark is thin and the inner bark is greenish or light brown, the cut
surface of the thick bark of a matured tree is reddish brown on drying, when
broken short thin fibre tips are seen. The root is cylindrical, slightly tapering
and posses longitudinal wrinkles. The wood is white, soft and even grained.
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4.2.2 Microscopic Features
Transverse section of leaves, petiole, stem and bark were studied.
4.2.2.1 Leaf
The leaf has fairly prominent midrib, lateral veins and dorsiventral
lamina [Figure.4.2].
Mid rib
The mid rib is planoconvex in transactional view with flat adaxial
side and broadly semicircular abaxial side. It is 1 mm in both vertical as well
as horizontal planes. The epidermal layer along the midrib is narrow
comprising of small slightly appellate thick walled cells with thick cuticle
[Figure 4.2(a)] the cells are 15µm thick.
The ground tissue of the midrib is homogenous and
parenchymatous. The cells are fairly thick walled, circular and compact. No
specific cell inclusions are evident in the ground parenchyma cells
[Figure 4.3].
The vascular strand is single fairly large and omega shaped. It has
an abaxial arc with two lateral out curved wings. The xylem part of the strand
has several long parallel compact lines of xylem elements. The elements are
narrow, elliptical and thin walled [Figure 4.3]. Phloem occurs in broad and
continuous arc along the abxial side of the xylem. External to the phloem is a
thin layer of discontinuous sclerenchyma cells which contains dark contents.
The vascular strand (xylem and phloem) is 550µm wide and 300µm thick.
27
Lamina
The lamina has smooth and even surfaces. It is 350µm in the
middle part and 200µm thick along the marginal part. The epidermal layer of
the adaxial side has small squarish cells with thick cuticle; the cells are 20-
25µm thick. The abaxial epidermis is thin and has narrow rectangular thick
cuticularised cells. [Figure 4.2(b)].
The palisade tissues of the lamina are distinct; the palisade cells are
wide, cylindrical and compact. There are two layers of palisade cells which
are 70- 100µm in height and the individual cells are 20µm in wide. The
spongy mesophyll has about seven layers of large, lobed loosely arranged
cells with wide air spaces [Figure 4.3(b)]
The lateral vein has similar structure as that of the midrib
[Figure 4.2(b)]. It is planoconvex in sectional view measuring 440µm thick. It
has parenchymatous ground tissue and single, large collateral, bowl shaped
vascular strand with abaxial dark sclerenchymatous bundle sheath.
The marginal part of the lamina is curved down; it is bluntly
conical [Figure 4.2(c)]. It has thick and prominent epidermal layer. The
mesophyll consists of undifferentiated, compact circular thick walled
parenchyma cells.
Epidermal cells [Figure 4.4]
The adaxial epidermis has polyhedral thick walled cells. Their
anticlincal walls are thick and straight.
The abaxial epidermis has similar size of cells as those of the
adaxial epidermis. But, the anticlinal walls are thin and slightly wavy. The
layer is stomatiferous. The stomata are paracytic type with two narrow
28
parallel subsidiary cells [Figure 4.4(b)]. The guard cells are circular or
elliptical measuring 20 to 30µm in size.
Venation pattern
The lateral veins are reduced gradually in thickness and the
ultimate veinlets are thin and wavy [Figure 4.5(a)]. The vein-islets are not
well defined. When the vein-islet is distinct, it is wide and not having distinct
outline.
The vein terminations are well defined and elaborate dendroid
appearance [Figure 4.5(b)]. They are formed once or twice having two of four
branchlets. The vein endings are unique in having a cluster of short, thick
tracheids. These terminal tracheids have spiral or recticulate thickenings
[Figure 4.5(c)]. They are short and thick. The numbers of tracheids vary from
one to five per terminal.
Calcium oxalate crystals are abundant in the mesophyll tissues of
the leaf. The crystals are rosette type. They are circular, flat and plate like
margin [Figure 4.5(b)]. The crystal has a central core of dark spot, which is
the organic part; the outer part is the calcium oxalate. They are 30µm in
diameter.
4.2.2.2 Petiole
The petiole is circular in cross sectional outline without external
differentiation of the adaxial- abaxial sides [Figure 4.6]. The surface is
smooth and even. The petiole is 2.2 mm in diameter. It consists of a thin layer
of epidermis, homogenous ground tissue and a deep bowl shaped vascular
strand with two lateral smaller accessory strands [Figure 4.6(a)].
29
The epidermal layer consists of small, thick walled squarish cells.
The ground tissue is parenchymatous and the cells are angular, thin walled
and compact [Figure 4.6(b)]. The main vascular strand consists of several
thin, long parallel lines of xylem elements with intervening fibres and outer
broad zone of phloem elements.
Proximal part
The proximal part of the petiole is planoconvex with distinct
slightly concave adaxial side [Figure 4.6(c)]. It is 1.8mm thick and 2.5mm
wide. It consists of thick walled, small radially oblong epidermal layer of cells
and loosely arranged parenchymatous ground tissue [Figure.4.6(d)]. The main
vascular strand is bowl shaped with wavy outline. The strand has short
parallel lines of angular thick walled xylem elements and wide zone of
phloem elements [Figure 4.6(e)].
There are two pairs of small circular accessory strands, one pair on
either corner of the adaxial wings. These wing bundles are circular with
central mass of xylem surrounded by eccentric phloem and thin layer of fibres
[Figure 4.6(d)].
Cell inclusions
Calcium oxalate crystals of druses are fairly abundant in the central
and outer ground tissues. The druses are either scattered or arranged in short
rows. The crystals are up to 20µm in size.
4.2.2.3 Stem
Young stem measuring 2.5mm thick was studied. The stem is
circular in cross sectional view with even and smooth surface [Figure 4.7(a)].
30
The stem has thin continuous epidermal layer of squarish cells with thick
cuticle. The epidermis with cuticle is 30µm thick.
The cortex is wide and has homogenous layers of parenchyma cells.
The cortical zone is 300µm wide. The cells are circular, thick walled and
compact. No specific cell inclusions are evident in the cortical cells.
The vascular cylinder is 250µm thick. It consists of a thin,
discontinuous layer of sclerenchyma abutting in phloem and a wider zone of
xylem elements. Phloem has dialated rays and parallel lines of phloem
elements [Figure 4.7(a)]. Xylem consists of a narrow compact secondary
xylem with vessels, fibres and primary xylem strands. Pith is wide and
parenchymatous cells; they are circular to angular, compact and no
intercellular spaces.
4.2.2.4 Bark
The bark is 3.5mm in thickness. It has narrow periderm, fairly wide
cortex and wide granular secondary phloem [Figure 4.8].
Periderm is narrow and superficial. It has thin dark broken layer on
the surface. The inner part has about five layers of tangentially elongated
narrow phellem cells. The periderm zone is 80µm wide.
The cortex has several layers of tangentially elongated narrow, thin
walled compact cells. The cells are radially compressed and have small nests
of scattered sclereides and sparsely distributed calcium oxalate crystals.
Secondary phloem is wider part of the bark. It is differentiated into
outer zone of collapsed phloem (crushed phloem) and inner zone of non
collapsed or intact phloem.
31
The collapsed phloem is several times wider than the non collapsed
phloem. It consists of tangential thin dark streaks of crushed phloem
elements, broad highly dialated rays and large masses of sclereides. The
dialated rays have tangential rows of rectangular cells forming a ladder like
arrangement of the cells [Figure 4.8(b)]
Non collapsed phloem (Intact phloem) has well preserved phloem
elements including parenchyma cells, narrow undialated rays and sparsely
distributed sclereides [Figure 4.10]. The sieve elements are rectangular or
polygonal, thick walled and are in radial rows [Figure 4.10(a)]. They have
distinct companion cells situated at the corner of the sieve element
[Figure 4.10(a,b)]. The sieve elements are 25-30µm in tangential diameter.
The cells of the phloem rays are radially rectangular wide and have wide,
simple pits.
In tangential longitudinal sections of the bark, the characters of the
phloem rays and the sieve tubes were studied. The rays are uniseriate,
biseriate or multiseriate [Figure 4.11]. The rays are heterocellular; the rays
have polygonal central cells and vertical elongated triangular marginal cells.
The central cells are called proaembent cells and the marginal cells are
upright cells. The rays are 550-950µm in height and the multiseriate rays are
100-150µm wide and the uniseriate rays are 40µm wide. Ray frequency is
9/mm.
The sieve tubes are straight. They have wide and oblique sieve
plates. The sieve tube is up to 900µm long. Phloem parenchyma cells are
narrowed and vertically elongated. They occur in vertical strands
[Figure 4.10(b)]. They are 70µm long and 30µm wide.
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In radial longitudinal sections (RLS), the cells of the rays appear in
horizontal ribbon like bands [Figure 4.12(a,b)]. The cells are vertically
rectangular and appear in regular horizontal series. Some of the cells are
squarish. The cells are up to 70µm in height and 40µm in breadth.
Crystal distribution [Figure 4.13]
Calcium oxalate crystals are abundant in the lamina, midrib and
bark. The crystals are of two types: prismatic crystals and sphaero crystals or
druses.
Prismatic crystals of cuboidal shape are more common in the bark
[Figure 4.13(a)]. The prismatic crystals are located in the phloem rays and
axial parenchymatous cells.
The druses are abundant in the lamina, midrib, petiole and stem. In
the leaf and petiole the druses occur in continuous chains [Figure 4.13(a,b)].
These crystal strands are seen scattered in the powder of the leaf. The
individual druses are 15µm wide.
The druses are seen scattered diffusely in ground parenchyma of
the midrib [Figure 4.13(a)] or petiole [Figure 4.13(b)].
4.2.3 Powder Microscopy
The leaf powder is dark green in colour with strong tea like odour
and slightly sweet in taste. On microscopical examination the powder showed
numerous uniseriate multicellular covering trichomes which are slightly
curved and paracytic stomata. Fibres are occasionally seen in the powder.
They are long, thick walled cells with reduced luman [Figure 4.14(a)]. The
fibres are nearly 1mm long.
33
4.2.4 Histochemical Colour Reactions
Localisation of lignin stained with phloroglucinol and Hydrochloric acid
Lignin was found as red coloured cells in xylem elements of the
elements of the stem [Figure 4.15(c)] and in the sclereide masses of the
collapsed phloem in the bark [Figure 4.15(d)].
Localisation of mucilage stained with ruthenium red
The mucilage was found as red coloured bodies in the fibres
ensheathing the vascular arc, two or three subepidermal layers along the
adaxial side and some isolated solitary thick walled cells of the ground
parenchyma of the midrib turned reddish brown [Figure 4.16(a)],
collenchyma cells along the periphery and thick walled cells around the
vascular bundles of the petiole turned reddish orange [Figure 4.16(b)], in the
collenchyma cells outside the cortex and phloem fibres of the stem.
Localisation of proteins stained with CBB solution
Protein was found as blue coloured bodies in the epidermal cells,
mesophyll, phloem fibres and bundle sheath fibres of the mid rib [Figure 4.17(a)],
epidermal cells and mesophyll of the leaf [Figure 4.17(b)], the cells of the
epidermis, outer ground tissue and phloem elements around the vascular
strands of the petiole [Figure 4.17(c)], hypodermal cells, phloem parenchyma
and xylem parenchyma cells stained blue [Figure 4.17(d)], outer collapsed
phloem cells, especially dialated ray cells of the bark [Figure 4.17(e)].
Localisation of alkaloids by Dragendroff’s reagent
Alkaloids were found in the sclerenchyma bundle sheath and xylemfibres of the mid rib [Figure 4.18(a)], both palisade parenchyma and spongy
34
parenchyma cells [Figure 4.18(b)], epidermal cells and xylem fibres of thepetiole [Figure 4.18(c)], fibre clusters outside the phloem and xylem fibres ofthe stem [Figure 4.18(d)] and the sclereides of the bark [Figure 4.18(e)] asbrownish orange colouration.
Localisation of lipids stained with neutral red
The lipids were found as red coloured bodies in the ground parenchymacells, phloem fibre sheath, mesophyll cells on the lateral sides of the vascularstrand of the mid rib [Figure 4.19(a)], both palisade and spongy mesophyll cells ofthe lamina [Figure 4.19(b)], scattered ground parenchyma cells and xylemelements of the petiole [Figure 4.19(c)], the epidermal layer, cortical fibres nearphloem and xylem fibres of the stem [Figure 4.19(d)], phloem sclerenchymaespecially phloem sclereids are stained deeply in the bark [Figure 4.19(e)].
Localisation of starch stained with Iodine in potassium iodide(IKI)
Starch was localised by using IKI. Starch was found as dark purplebodies in the pith of the stem [Figure 4.20(d)], in the dialated ray cells of thephloem [Figure 4.20(d)], outer and central ground cells of the petiole [Figure4.20(c)] and spongy mesophyll of the lamina [Figure 4.20(b)].
Localisation of flavanoids with TBO solution
The xylem elements and sclerenchyma sheath of the vascular strandin the mid rib [Figure 4.21(a)], the fibres and xylem elements of the petiole[Figure 4.21(b)], cortical and pith parenchyma cells of the stem [Figure 4.21(c)],ray parenchyma of the bark [Figure 4.21(d)] were showed the presence offlavanoids.
35
Figure 4.1 Morphology of the leaves
36
Figure 4.2 Anatomy of the leaf(a) Transverse section of the leaf through midrib (b) Lamina and lateral veinand (c) Leaf margin
Figure 4.5 Venation pattern as seen in based leaf surface(a)Venation- Low magnification, (b) One vein-islet and vein terminationenlarged, rosettes of calcium oxalates and (c) Terminal tracheids occurringat the end of a vein termination
Figure 4.6 (Continued) (d) TS of proximal part of the petiole and (e) One corner of the petiole –enlarged (Ads: Adaxial side; Ep: Epidermis; GT: Ground tissue; Ph: Phloem; X: Xylem; MS: Medium strand; WB: Wing bundles )
(e)
43
Figure 4.6 (Continued) (f) TS of proximal part of the petiole a sector enlarged and (g) Crystals in the ground tissue (CR: Crystals; Ep: Epidermis; GT: Ground tissue; Ph: Phloem; X: Xylem)
(f)
(g)
44
Figure 4.7 Anatomy of the stem(a) TS of stem and (b) TS of stem- a sector
Figure 4.14 Powder microscopy (a) Fibres, (b) Druses in several continuous chains and (c) Druses- chains of druses enlarged (Dr: Druses; Fi: Fibres)
(c)
(b)
(a)
52
Figure 4.15 Localisation of Lignin (Phloroglucinol and HCl)(a) TS of the mid rib showing the lignin in the xylem elements and thevascular strand (b) TS of petiole showing the presence of lignin in thevascular arc
(a)
(b)
53
Figure 4.15 (Continued) (c) TS of stem showing the lignin in the xylem elements and (d) TS of the
bark showing the lignin in the sclereid masses of the collapsed phloem
(c)
(d)
54
Figure 4.16 Localisation of mucilage (a) TS of the mid rib showing the mucilage in the adaxial epidermis, fibres
ensheathing the vascular arc and in the ground parenchyma, (b)TS ofpetiole showing the presence of mucilage in the collenchyma cells and(c)TS of stem showing the mucilage in the collenchyma cells and phloemfibres
(a)
(b)
(c)
55
Figure 4.17 Localisation of Proteins (CBB SOLUTION) (a) TS of the mid rib showing the proteins in phloem and bundle sheath and
(b) In the lamina the protein is present in the epidermal cells and mesophyll
(a)
(b)
56
Figure 4.17 (Continued) (c) TS of petiole showing the presence of proteins in the epidermis, outer
ground tissue and phloem elements in the collenchyma cells, (d) TS of stemshowing the protein in the hypodermal cells, xylem and phloemparenchyma and (e)TS of the bark showing the protein in the outercollapsed phloem cells
(c)
(d) (e)
57
Figure 4.18 Localisation of alkaloids (Dragendroff’s reagent)(a) TS of the mid rib showing the alkaloids in sclerenchyma bundle sheathand xylem fibres and (b)In the lamina the alkaloid is present in the palisadeparenchyma cells
58
Figure 4.18 (Continued) (c)TS of the petiole showing the alkaloids in the epidermal cells and xylem
fibres (d) TS of stem showing the alkaloid in the fibre cluster outside thephloem and xylem and (e) TS of the bark showing the alkaloid in thesclereides
(c)
(d)
(e)
59
Figure 4.19 Localisation of fatty acids (Neutral red) (a) TS of the mid rib showing the fatty acids in the ground parenchyma,
phloem fibres and mesophyll cells and (b)In the lamina the fatty acids ispresent in the palisade cells and spongy parenchyma
(a)
(b)
60
Figure 4.19 (Continued)
(c) TS of the petiole showing the fatty acids in the ground parenchyma andxylem and (d) TS of stem showing the fatty acids in the epidermis, corticalfibres and xylem fibres
(c)
(d)
61
Figure 4.19 (Continued) (e)TS of bark showing the fatty acids in the phloem sclereides
(e)
62
Figure 4.20 Localisation of Starch (IKI) (a) TS of the mid rib showing starch in the ground tissue and (b) In the
lamina the starch is present in spongy mesophyll
(a)
(b)
63
Figure 4.20 (Continued) (c)TS of the petiole showing the starch in the outer and central ground cells
(c)
64
Figure 4.20 (Continued) (d) TS of stem showing the starch in pith cells and (e) TS of bark showing
the starch in the dialated ray cells
(d)
(e)
65
Figure 4.21 Localisation of Flavanoids (TBO solution) (a) TS of the mid rib showing the falvanoids in the xylem, sclerenchyma sheath and (b) TS of the petiole showing the flavanoids in the fibres and xylem elements
(a)
(b)
66
Figure 4.21 (Continued)(c) TS of stem showing the flavanoids in the epidermis and sclerenchyma
cells and (d) TS of bark showing the flavanoids the dialated ray cells
(c)
(d)
67
4.2.5 Quantitative Microscopy
The observed values for stomatal number, stomatal index, vein-
islets, vein termination number and palisade ratio are given in Table 4.1.
Table 4.1.Quantitative Microscopic data
S.No ParametersValue in 1 sq.mm
(average of 10 fields)1. Stomatal number
Abaxial
Adaxial
35.87
20.46
2. Stomatal index
Abaxial
Adaxial
10.23
5.08
3. Vein-islets number 5.35
4. Vein termination number 5.12
5. Palisade ratio 9.52
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4.2.6 Physico-chemical Constants
The observed values for the physico-chemical constants are given
in Table 4.2.
Table 4. 2. Physico-chemical constants
S.N Parameters Percentage(%w/w)
1. Total ash 9.65
2. Acid insoluble ash 1.25
3. Water soluble ash 2.34
4. Sulphated ash 10.3
5. Solubility
Water soluble extractive
Alcohol soluble extractive
16.2
3.5
6 Crude fibre content 15.25
7 Loss on drying 8.56
8 Foaming index Less than 100
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4.2.7 Fluorescence Analysis
The fluorescence analysis of powder with various reagents and
extracts are given in the Tables 4.3 and 4.4
Table 4.3 Fluorescence analysis of powder
S.No Reagents Day light Short UV LongUV(365nm)
1. Powdered drug Green YellowishGreen Dark green
2 Powder + 1 N HCl Pale Yellow Pale Green Pale Green3 Powder + 1 N NaOH Red Brownish Green Pale Green
4 Powder + 50% HCl Pale Yellow Fluorescentgreen
Fluorescentgreen
5 Powder + 50% H2SO4 Dark green Dark green Dark green
6 Powder +50%HNO3 Dark brown Brown Greenishbrown
7 Powder + Methanol Green Fluorescentgreen Light green
8 Powder + Methanol + 1 NNaOH
Orangishgreen
Yellowishbrown Green
Table 4.4 Fluorescence analysis of various extracts
S.No Extracts Day light UV lightShort (254nm) Long (365 nm)
1. n-Hexane Green Green Dark green2. Chloroform Greenish brown Brown Dark green3. Ethyl acetate Yellowish green Greenish fluorescence Light green4. Methanol Brownish green Orangish green fluorescence Light green
70
4.2.8 Inorganic Mineral Analysis
The amount of sodium and potassium present in 1 gm of plant
material was estimated by flame photometry. The amount of other metals
present was estimated by Atomic absorption spectroscopy and the results are
given in Table 4.5.
Table 4.5. Inorganic mineral analysis
S.No Parameters Amount present(mg/Kg)
1. Total Iron 464
2 Cadmium (DB) < 0.0008
3 Copper (DB) 75
4 Chromium (DB) <0.01
5 Cyanide (DB) <0.005
6 Cobalt (DB) <0.009
7 Lead (DB) <0.015
8 Manganese (DB) 420
9 Nickel (DB) <0.006
10 Zinc (DB) 63
11 Sodium (WB) 268
12 Total phosphate (DB) 235DB-Dry basis WB –Wet basis
71
4.3 DISCUSSION
Pharmacognostical standardization was carried out on the basis of
detailed botanical evaluation of the leaves which includes morphology and
microscopy as well as WHO recommended physico-chemical studies. The
results of the standardization may throw an immense light on the botanical
identity of the leaves of Symplocos cochinchinensis (Lour.) S.Moore ssp.
laurina (Retz.) Nooteb. which may furnish a basis of judging the authenticity
of the plant and also to differentiate the drug from its adulterants and other
species.
The macroscopic characters were examined to identify the right
crude drug.
Microscopical features
Transverse section of the leaves showed a fairly prominent midrib,
lateral veins and dorsiventral lamina. The vascular strand is omega shaped
with an abaxial arc and two lateral out curved wings. The lamina has smooth
even surface with two layers of palisade cells, the marginal part of the lamina
is curved down and bluntly conical.
The petiole consists of a thin layer of epidermis which consists of
small, thick walled squarish cells, homogenous ground tissue and a deep bowl
shaped vascular strand with two lateral smaller accessory strands.
The stem has thin continuous epidermal layer, a wide cortex with
homogenous layers of parenchyma cells, vascular cylinder consists of a thin,
discontinuous layer of sclerenchyma abutting in phloem and a wider zone of
xylem elements.
72
Bark has narrow periderm, fairly wide cortex and wider granular
secondary phloem. Secondary phloem is the wider part of the bark. It is
differentiated into outer zone of wide collapsed phloem (crushed phloem) and
inner zone of non collapsed or intact phloem. The sieve elements are
rectangular or polygonal, thick walled and are in radial rows with distinct
companion cells situated at the corner of the sieve element. The cells of the
phloem rays are radially rectangular wide and have wide, simple pits.
The tangential longitudinal sections of the bark showed uniseriate,
biseriate or multiseriate rays which are heterocellular; the rays have polygonal
central cells and vertical elongated triangular marginal cells. The central cells
are called proaembent cells and the marginal cells are upright cells. The sieve
tubes are straight, wide, oblique sieve plates. Phloem parenchyma cells are
narrowed and vertically elongated.
In radial longitudinal sections (RLS), the cells of the rays appear in
horizontal ribbon like bands. The cells are vertically rectangular and appear in
regular horizontal series. Some of the cells are squarish.
Powder Characters
The powder characters of a drug are mainly used in the
identification of the drug in the powder form. The leaf powder is dark green
in colour with strong tea like odour and slightly sweet in taste. On
micrsocopical examination the powder showed numerous uniseriate
multicelluar covering trichomes (1-3 celled), which are slightly curved.
Paracytic stomata made up of rectangular or polygonal epidermal cells with
thin anticlinal walls are seen.
73
Histochemical analysis
Histochemistry is mainly used to localise the chemical compounds
with in the cells and tissues using some chemical reagents which will
selectively stain the compounds. The observations of histochemical analysis
showed that lignin was found in xylem elements, vascular strands and
sclereide masses of the petiole, mid rib, stem and bark. The lignified cells
offer mechanical strength to the organs and provides mechanical barrier
against micro organism entering the vascular elements.
The mucilage was found as red coloured bodies in the fibres, some
isolated solitary thick walled cells of the ground parenchyma, collenchyma
cells, vascular bundles and phloem fibres.
Protein is a predominant compound in different tissues of leaf,
petiole and root. Protein was found in the epidermal cells, mesophyll, phloem
