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CHAPTER 5 DISCUSSION
DISCUSSIONAaatoiEical and phytocliemical studies
In India as well as in other countries, adulteration and low
standards arc common drawbacks as far as the purity of crude drugs is
concerned (Qadry, 1 9 6 3 ; Wallis, 1 9 6 9 ; Afaq, 1 9 7 0 , 1 9 7 1 ; Dutt et a l,
1 9 7 2 , 197v3; Trease and Evans, 1 9 7 2 ; Afaq et al, 1 9 7 4 and Zafarulla et
al, 1 9 7 7 ) . For detection of adulteration, many procedures are applied and
it is always a good policy to obtain confirmatory results by using different
means of detection as iar as possible. The processes which are used, may
be classified into two categories viz.; (1) visual observation for
establishing the identity and (2) experimental analysis to determine the
qualities of the drugs of plant origin.
To establish the identity gross morphology, anatomy and
quantitative microscopy play an important role (P'oster, 1 9 3 4 ;
Chowdhary, 1 9 4 0 ; Metcalfee and Chalk, 1 9 5 0 ; Zahur, 1 9 5 9 ; Esau, 1 9 6 5 ;
WaUis, 1 9 6 9 ; Fahn, 1 9 7 4 ; Esau, 1 9 7 7 ; Khan and Ghouse, 1 9 7 7 ; Fahn,
1 9 8 2 ; Solereder, 1 9 9 5 and Dickison, 2 0 0 0 ) . The anatomical studies are
also very useful, especially for those plants which have similar characters
and controversies (Metcalfe, 1968).
Morphology is the most important tool in the classification of
higher plants. It is one of the important modern trends in the taxonomy
of angiosperm. The modern trends also include the macro and
microscopic features, so called tribal features such as spines, trichomes,
stomata and epidermal structures etc. which are used as diagnostic
features in the identification of genera and species. The development of
macro-microscopic photography also leads to detailed examination and
higher magnification. This has helped a lot to morphologists and
pharmacognoists. Through the photographic studies, the detailed
features may be used to separate closely related species. Anatomical
features are of great importance to the taxonomists in the identification
90
CHAF'l’ER 5 DISCUSSmN
of anomalous genera in a satisfactory position in classification and in
indicating^ the relationships that may have been observed by superficial
morphological features (Metcalfe and Chalk, 1950), The anatomy of
various parts (root, stem, leaf, flower, seed and fruit) of the plant received
a great impetus by the development of rapid maceration techniques for
the study of its different components. Extensive studies conducted in the
recent past have revealed a detailed knowledge of the behaviour of the
individual cell types and tissues like parenchyma and rays, their width
and length, cell wall lignification, sclerotic cell inclusions such as tannin,
starch, oil, resin, gum and crystals etc. (Khan, 1977). The anatomical
data and many conclusions are attaining a very useful role in the
taxonomic revisions of plant groups.
Phytochemical studies are also worth mentioning in the recognition
of qualitative phytochemical constituents and their elements. Thin layer
chromatography, paper chromatography, column chromatography,
physicochemical constants and the fluorescence characters are some
important criteria for determining the characters of plant drugs (Wallis,
1969 and Tariq, 1975).
hi the present study, the parts of six Indian medicinal plants have
been investigated on the basis of morphological, histological and
phytochemical techniques. The main diagnostic characters, supported by
chemical standards have been discussed below:
Acacia leucophloea (flowers):
A careful examination of the results shows various details of
macroscopic, microscopic and phytochemical characters of the flowers of
A. leucophloea for the identification purpose. There are certain diagnostic
characters by which the plant part can be differentiated from the other
parts of the plants. These diagnostic characters are given below which
are useful for identification, characterization and standardization of the
flower of the investigated species.
91
CHAPTERS DISCUSSION
The flower heads are numerouvs, globose, small, pale yellow, sweet-
scented, in large terminal, leafless, dense tomentose panicles, 6-9 mm in
diameter. Anthers are small, bilocular, dehiscing in longitudinal manner.
Ovary is monocarpellary, unilocular with two ovules having marginal
placcntaLion. 'Fhe above findings are also confirmed by Maheshwari
(1997), Anonymous (1985) Shetty and Singh (1987) and Kirtikar and
Basu (1993). Peduncle is found long with a toothed ring of bracts which
are below the middle portion of the peduncle, The above finding is not in
confirmity with the findings of Nadkarni (1998) and Hooker (1979) about
the position of the bracts on the peduncle. Peduncle shows a single layer
of epidermis with short, hemispherical papillae with glandular and non-
glandular triehomes. The non-glandular trichomes have been found with
tappering and unbranched ends. Metcalfe and Chalk (1950) and
Solereder (1995) have not mentioned the specific measurements of the
cell types and the c[uantitative microscopy in the investigated species.
Hypoderm is absent. Similar findings are given by Metcalfe and Chalk
(1950). Round to oval parenchymatous cells are observed in several
layers in the pith area of the peduncle. Such findings are not mentioned
by Metcalfe and Chalk (1950) and Solereder (1995). Solitary
rhombohedron crystals of calcium oxalate are distributed in various
tissues of the peduncle. Tanniniferous cells are also observed in the
cortex and other regions. The present findings are similar to that of
Metcalfe and Chalk (1950) and Solereder (1995). A distinguishing
character is the composite and continues sclerenchyma ring in the
pericycle. Similar observations are quoted by Metcalfe and Chalk (1950)
and Solereder (1995), Mesophyll is undifferentiated and is composed of
several layers of slightly thick walled polygonal or rectangular
parenchymatous cells. The stomata are parasitic type and have been
found on the outer surface of the sepals, Vessels are with simple
perforations. Fibers are found elongated. Trachieds are very small.
The quantitative microscopy of the flower parts is given below,
92
CHAPTERS DISCUSSION
The epidermal cells are measured from 9-18 |,im in width.
Glandular trichornes are measured from 31.5-54.0 (,mi in length and
from 4.8-13.5 },irn in width, having glandular heads and uniseriate stalk.
Non-glandular trichornes var}'' from 27-90 (.im in length and from 6.8-9.0
(.ini in width, having tappering ends, mostly unicellular and unbranciied.
The cortical parenchymatous cells are ranged between 18-27 |im in
length and from 13.5-22.5 |,tm in width. The hexagonal, thick walled
sclerenchymatous cells are from 9.0-13.5 ).tm in length and from 6.7-9.9
f irn in width. The crystals are solitary rhornbohedron and vary from 13.5-
45.0 pm in length and from 9,0-22.5 |.im in width. The size of stomata’s is
measured from 13-18 |,Lm in length and from 9.0-13.9 (.irn in width. The
stoma ranges from 6.0-9.1 |:im in length and from 40-65 pm in width
respectively. On analysis it was found that on drying at 105 °C the loss
in weight (60.5%), solid contents (55.24%), water soluble matter
(30.08%), alcoholic soluble matter (45,30%) and crude fibres (25.95%).
pH values are noted in 1% aqueous solution (7.23) and in 10% aqueous
solution (7.18). The total percentage of ash (8.50 %), water soluble ash
(3.60%), alcohol soluble ash (1.0%) and sulfated ash (0.30%) are noted
while earlier workers reported the results of the ash analysis of the hard
wood of this plant. The percentage of total alkaloids is found to be 0.03%.
Extractive values of the flowers in different solvents are as follows:
hexane, petroleum ether, benzene, chloroform, acetone, methanol and
water have been found to be 2.90%, 0.22%, 0.38%, 0.16% 2.37%, 7.08%
and 8.39%). The thin layer chromatographic study of the flowers of this
plant has given the highest spots (4) in acetone extract. The Rf values are
0.18, 0.36, 0.40 and 0.72 respectively. Qualitative studies have shown
the presence o f alkaloids, tannins, carbohydrates, saponins, flavonoids,
resins, steroids in the water and ethanolic extracts of the flowers of A.
leucophloea. Fluorescence analysis of the powder as such and after giving
93
CHAF'I'ER 5 DISCUSSION
differciiL treatments liavc l:)ccn noted in ultra-violet light and presented
in Table-5.
The acetone extract was evaporated to dryness to give a pale green
viscous mass. After crystallization it gave rhomboic crystals having m.p.
186~188°C obtained in acetone extract, v^hich gave a positive
Liebermann-Burchard test. After paper chromatography examination
followed by heating at 120 °C a single brown spot of pure single
compound was obtained. The acetylation of the parent compounds (100
mg) gave fine needles of its acetate having m,.p 72 '’C. The compound is
identified as sucrose on the basis of its physical properties and
acetylation. In order to stabilize the identity of this compound beyond
any doubt it was subjected to acid hydrolysis. After completion of
hydrolysis a small quantity of syrupy mass left in china dish, which has
reduced a mixture of Fehling’s solution A and B as well as Benedict
solution. The sugar has been hydrolyzed to give reducing sugar. On
performing the paper chromatography of the hydrolysis product, it was
found that two spots, corresponding with the spots of glucose and
fructose which confirmed the present compound is sucrose. Further it
was confirmed by preparation of glucosazone of the compound that the
parent compound is only sucrose. As fructose also gave same osazone
(glucosazone) like the parent compound in the present study.
Calliandra haematocephala (flowers):
The flowers are characterized by the presence of attractive odour,
astringent taste, pinkish-red colour, actinomorphic, pentamerous and
are measured from 3-5 cm long. Pollens agglutinated into 8 -celled
pollinia. Gynoecium is monocarpellary, unilocular with one ovule and
having marginal placentation.
A transverse section of the peduncle shows a single layer of
spherical to rectangular epidermal cells and are measured from 13.5-
94
CHAPTER 5 DISCUSSWN
15.5 j-im in length and from 12.5-13.0 ).im in width. This is not reported
by Metcalfe and Chalk (1950) and Solereder (1995) in C. haematocephala.
Subpapillose differentiation of the ejDidermal cells especially of those on
the lower side of the leaf, is not uncommon, Solereder (1995) has
described it in various species of CaUiandra. The stomata are paracytic
type and ranged from 20-30 (im in length and from 13-15 fim in width.
The stoma are ranged from 12-17.5 i-im in length and from 4-5.5 |,im in
width. The occurrence of two subsidiary cells which are parallel to the
pore, is characteristic of the stomata in the species investigated,
Subsidiary cells are also measured from 22-34.7 [,im in length and from
20-32.5 im in width. The above findings arc additional from the findings
of Metcalfe and Chalk (1950). Only non-glandular trichomes have been
obsei'ved on the outer surface of the peduncle. Non glandular trichomes
have tapering ends, slightly curved on the tips unbranched, unseptate
and unicellular. While Metcalfe and Chalk (1950) have reported the
occurrence of unicellular, bracked-shaped, hooked in the species of
CaUiandra. The hypodermis is absent. The cortical parenchymatous cells
below the epidermis are 7-13 layered. These cells are oval shaped. The
cells are measured from 10-40 |.im in length and from 9-35 .im in width.
Metcalfe and Chalk (1950) have not mentioned the specific
measurements of cells types. Tlie cortical cells are found having brown
tanniniferous contents. Pericycle usually containing a continuous and
frequently composite ring of selerenchyma. The fibres are unlignified;
strands of fibres are found less common, Vascular bundles in the
peduncle are arranged in a ring. Two rows of vascular bundles, which lie
in pairs opposite to one another with their xylem portions turned towards
one another. Similar findings have been mentioned by Solereder (1995).
The large solitary rhombohedral crystals have been observed in the
cortex, between vascular bundles and pith region. Tanniniferous cells are
also observed in cortex. The medullary rays are, 1-2 celled thick,
95
CHAPTER S DISCUSSION
elongated to rectangular. The spherical, oval to hexagonal types of
parenchymatous cells constitute the pith. No much information is
provided by Metcalf and Chalk (1950) and Solereder (1995) on these
aspects.
The quantitative microscopy of the isolated parts of the flower has
show the following results:
The epidermal cells are ranged from 1 3 . 5 - - 1 5 . 5 ).im in length and
from 1 2 , 5 - 1 3 . 0 f i m in width. Non-glandular trichomes are measured from
7 3 . 5 - 8 0 (am in length and from 2 5 - 3 5 |im in width. Calcium oxalate
crystals are also measured from 1 3 . 5 - 4 5 )im in length and from 9 . 0 - 2 2 . 5
|,tm in width in peduncle. Pollinium is measured from 165-180 |.im in
length and from 1 1 0 - 1 2 0 ).im in width. No much anatomical work has
been reported by earlier workers.
It is concluded that on drying at 1 0 5 ° C : the loss in weight
( 1 0 . 6 9 % ) , solid contents ( 5 3 . 7 0 % ) , water soluble matters ( 2 7 . 5 2 % ) ,
alcoholic soluble matters ( 9 . 6 0 % ) , crude fibres ' ( 2 7 . 7 2 % ) , pH values in 1 %
aqueous solution ( 7 , 2 1 % ) and 1 0 % aqueous solution ( 7 . 6 P /o ) . The total
percentage of ash ( 5 . 5 6 % ) , water-soluble ash ( 1 , 5 7 % ) ) , acid insoluble ash
( 0 . 8 2 % ) and sulfated ash ( 0 . 0 2 % ) are also noted. The quantitative
elemental analysis of the ash shows the presence of phosphorous
( 0 .3 3 % ) ) , iron (0 ,1 4 % o ) , Zinc ( 0 . 3 5 % ) , cadmium (not detected), potassium
( 0 . 9 5 % ) , calcium (0 .4 0 % o ) , magnesium ( 0 , 2 0 % ) ) , sodium ( 0 . 8 7 % ) and
aluminium ( 0 . 0 9 % ) respectively. The percentage of total alkaloid is found
to be 0 . 6 8 % . The percentage of successive extractive values are: viz.
hexane (2 .1 0 %), petroleum ether (0 .2 0 %>), benzene (0 .3 5 %), chloroform
( 0 ,5 5 % ) ) , acetone ( 3 . 0 % ) , methanol (8 .0% o) and water ( 8 . 9 3 % ) . Among the
various extracts, methanol has given the best yield. Alkaloids, tannins,
carbohydrates, saponins and fixed oil have given positive tests in
methanol and water. Fluorescence analysis of the powder has shown
diagnostic colours. When treated with different reagents, it has given very
96
CHAPTER S DISCUSSION
interesting colours, which are worth noting for the detection of the purity
of genuine drug. No such work has been reported on this part by earlier
workers.
Compoimd CH
Compound CH, designated octacosane, obtained in petroleum
ether (6 O-8 O0 C), m.p. 60-62°C, had a molecular formula of C28H58 on the
basis of -' C NMR spectrum data. The NMR spectrum of the compound
has shown a triplet for methyl group at 5 0.88, a strong methylene signal
at 5 1.27 at another small signal for CH2 group around 5 1.56. These
data have suggested that the compound must be a hydrocarbon. The
ratio of methyl group to methylene groups has suggested that the
compound is a, C-28 li3-'drocarbon. On the basis of physical properties it
has been characterized as octacosane having the molecular formula
C28H58.
Ehretia aspera fleaves|;
The leaves of E. aspera are elliptical-oblong, simple, opposite,
entire, exstipulate, petiolate, hairy above, puberulent beneath, base
rounded or slightly cuneate, lateral nerves 4-6 pairs, midrib prominent
and lateral nerves raised below; petioles 5-20 mm long. Similar
description has been given by Hooker (1885), Bailey (1954), Cooke
(1958), Shetty and Singh (1991), Bhandari (1995) and Maheshwari
(1997). Microscopically the leaf shows a dorsiventral structure, The
epidermal cells of the leaf have straight walls. The hypoderm is not seen
in the leaf of this species. Similar findings are recorded by Solereder
(1995). Stomata of anomocytic type have been observed on the abaxial as
well as adaxial surfaces of the leaf. Similar findings are also quoted by
Metcalfe and Chalk (1950). Mesophyll cells are heterogenous in nature
and are divided into palisade and spongy parenchymatous cells. A single
layer of palisade cells is present below epidermis. Each palisade cell is
elongated, unbranched and compactly arranged, The present findings are
97
not mentioned by Metcalfe and Chalk (1950) and Solereder (1995).
Spongy parenchymatous cells occur in colourless, polygonal types and in
abundance next to the adaxial epidermis. Clusters crystals of calcium
oxalate are present below the palisade cells. Solereder (1995) has also
mentioned the presence of cluster crystals below palisade layer. Vascular
bundles are arranged in an arc form and the veins vertically transcurrent
by sclerenchyma in the investigated species. Tcinniniferous sacs occur in
the mesophyll around the vascular bundles. A number of small
medullary strands have also been observed in the wings of the petiole.
The occurrence of two additional pairs of vascular bundles has been
reported by Solereder (1995),
Quantitative microscopy of the leaf has given the following results;
The stomatal index is noted between 15-18. Stomatal frequency
varies from 40 - 6 6 / mm^. The stomata are measured from 12.5-17.3 fim
in length and from 7.5 - 15.1 }im in width. The palisade ratio vaiies from
5-10 and spongy parenchymatous cells are measured from 10.5-13.5 |j,m
in length and from 14.5-19.2 |,tm in width. Vein islet and vein
termination numbers have been recorded from 10- 18 and 9-15
respectively.
Preliminary phytochemical studies have established the following
facts: The loss in weight (85.3%) on drying at 105"C: solid contents
(55,3%), water soluble matters (63.1%), alcoholic soluble matters
(35.9%) and crude fibers (49.5%), pH values in 1% aqueous solution
(6.95) and in 10% aqueous solution (6,90 ) have been recorded. The
percentage of total ash (10.67 %), water-soluble ash (2.57 %), acid
insoluble ash (1.06%) and sulphated ash (0.51 %) are also calculated.
The quantitative elemental analysis of the avSh has confirmed the
presence of phosphorous (0.41%), iron (0.19%), zinc (0.09%), potassium
(2.35 %), calcium (1.25%), magnesium (0.29%), sodium (2.95%) and
aluminum (0.50 %) respectively. The total alkaloid percentage is found to
CHAPTER 5 _ _ _ _ _ DISCUSSION
98
be 0.06%. The percentage of successive extractive values of leaf viz.:
hexane (10,12%), petroleum ether (5.92 %), benzene (4.63 %),
chloroform (15.10 %), acetone (13.95%), rnetlianol (16.35%)) and water
(18.32%). Among the above solvents, methanol lia s given the higher
yield. Alkaloids, carbohydrates, tannins and vsaponins are found present
in methanol and water extracts of the leaf. Florescence characters of the
powdered leaf have been found noteworthy for the identification purpose.
Isolation and cltaracterizatioE of conipo«nd SA;Compound EA, designated p-arnyrin, has been obtained as cream
coloured crystals having m.p. 196-198 "C from ethyl alcohol (95%)
eluants. The compound was characterized by NMR and mass spectrum.
According to NMR spectrum of the compound which has shown
characteristics of the triterpenes, which has shown the presence of eight
tertiaiy methyl groups, spread from 5 0.8 to 5 1.05. Besides, there are
two multiplets countered at 8 4.5 for a proton a to the hydroxyl and a
multiple at 5 5.2 characteristic of the olefinic proton. The above data and
m.p. have confirmed that the compound (EA) may be p amyrin.
These findings are supported by the mass spectral data, which
have shown the molecular ion peak at m/z 4 to 6 , The peaks at m/z 207
and 218 are due to fragments (a) and (b). The fragment arising from A/B
rings and the fragment (b) arising from D/E rings. The parent compound
m.p. 196 to 198 °C on crystallization has given the colorless needles m.p,
238-240 °C, The NMR spectrum of this compound has shown all the
characteristic features of the parent compound except that there was an
additional singlet at 6 2.16 which may be accounted by one acetyl
function. Hence, after taking into consideration all the above data, it may
be concluded that the present compound is p-amyrin. However Suri et al.
(1980) have investigated the presence of ehretinine, a novel pyrrolizidine
alkaloid from E. aspera leaves. No much findings are available regarding
CHAPTER 5 ^ ^ DISCUSSION
99
CHAPTER 5 MSCUSSION
its pharmacognostical and phytochemical studies on. this part of the
pkiiit.
Leucaena leticocephala {leavesf:
The leaves are characterized by the presence of characteristic
odour, bitter in taste and fast greenish in colour. The leaves are
bipinnate, 10-25 cm long, cauline and ramal. The epidermal (lamina)
cells have been observed short in size, sub-papilose on the lower surface
and are measured from 8-15 [.im in length and from 6.8-12.3 i im in
width. Such observations are not mentioned by Metcalfe and Chalk
(1950). The vStornata are found paracytic type on both the surfaces of the
leaf. The abaxial surface of the leaf shows less number of stomata than
the adaxial surface and are measured from 10.1-20.2 jiim in length and
from 8,1-14.1 iiim in width. Only non-glandular trichomes have been
found on the margins of the leaflet while Metcalfe and Chalk (1950) and
Solereder (1995) have recorded the presence of unicellular and hooked
hairs. The hypoderrn is absent in the leaf of the investigated species. The
same is reported by Solereder (1995). The rnesophyll cells are
differentiated into palisade and spongy parenchymatous cells. The
palisade consist of 1 or 2 layers, made up of radially elongated cells. The
spongy parenchymatous cells are found polygonal having intercellular
spaces among them. The solitary rhombohedral crystals are frequently
found in the mesophyll tissues. The brown tanniniferous inclusions are
observed in the cells of the mesophyll. This is also reported by Metcalfe
and Chalk (1950) and Solereder (1995). The cross- section of the rachis
shows a single layered epidermis which is thickly cutinised externally,
Many covering trichomes are present on external surface and are
measured from 40.3-150,2 (am in length and from 8.2-15.3 jum in width.
Below the epidermis a wide zone of cortex is composed of slightly thick
walled parenchyma cells. Such detailes are also given by Metcalfe and
Chalk (1950), The pericycle is found in a continuous ring of a few layered
cells. The vascular bundles are arranged closely in horseshoe shaped
100
CHAPTER 5 DISCUSSION
manner. A pair of small bundles in the adaxial cortex region form the
cortical accessory bundles. The present findingvS are also mentioned by
Metcalfe and Chalk (1950).
The quantitative microscopy of the leaf has given the following results:
Tlie stomatal frequency varies from 25-40/mm2. The stomatal
index is ranged between 12-20. The vein termination and vein islet
numbers are recorded from 18-28 and 13-20 respectively. The pallisade
ratio is from 3-8. Calcium oxalate ciystals are measured from 15.2-52.2
pm in length and from 12.3-25.5 |im in width, Stone cells are also
measured from 13.5-72.2 jdrn in length and from 9.5-45.3 nm in width.
'rhc pliytochtnnical standards are also summed up in the following
paragraph:
The physicochemical constants on drying at 105 “C: the loss in
weight (91.4%), solid contents (49.9%), water-soluble matter (41,16%),
alcoholic soluble matter (27.64%) and crude fibres (37.39%). pH values
in 1% aqueous solution (6.93) and in 10% aqueous solution (6.58) are
also recorded. The total percentage of ash (11.27 %}, water soluble ash
(4.87%), acid in soluble (1.02%) and sulfated ash (0.53%) have been
recorded. Chopra et al (1996) reported that leaf and twigs are rich in
nitrogen and potassium salts, The qualitative and quantitative elemental
analysis of the ash are: Phosphorous (0.34%), iron (0,15%), zinc (0.19%),
cadmium (less than 0.005 PPm), potassium (1.52%), calcium (1.43%),
magnesium (0.39%), sodium (2.13%).The percentage of total alkaloids is
found to be 0.05%. Extractive values of the leaves in different solvents
viz: hexane, petroleum ether, benzene, chloroform, acetone, methanol
and water have been found to be 5,07%, 16.51%, 4.55%, 12.55%
14.35%, 17,01% and 13.91% respectively. This has shown that 'the
maximum extractive values are recorded in methanol extract, The thin
layer chromatographic study has given the maximum spot (7) in
chloroform extract. The Rf values are 0.08, 0,18, 0.45, 0.59, 0.68, 0.84
101
and 0.87 respectively, Qua.lita.tive studies have shown the presence of
alkaloids, tannins, carbohydrates, saponins, flavonoids, resins, steroids
in the methanolic extract. Fluorescence analysis of the powder as such
and after giving different treatments have given diagnostic colours.
A brick red compound separated out from petroleum ether (60-
80®C), m.p. 1.78-80'’C and its yield has been recorded 10 mg. On TLC
examination in benzene:ethyl acetate (1:1) solvent system, a single spot
(Rf value 0.63) was obtained. The NMR spectra revealed that it is an
impure compound and hence it could not be characterized.
Trianthema portulacastrum fleaf, stem and root|:
The following anatomical and phytochemical standards are laid
down for the leaf, stem and root of T. portulacastrum on the basis of the
present investigations. The leaves are sub fleshy, opposite and are
measured from 3.0-4,7X1.6-4.2 cm. Stem is angular and solid (young).
The root is small, fusiform measuring between 8-18 cm in length and
0.2-0.5 cm in width. The odour is pungent and taste is bitter.
The leaves are dorsiventral in structure in the investigated species.
The epidermal cells of the leaves are composed of large bladder like water
storage cells interrelated between much smaller ones. However, Metcalfe
and Chalk (1950) and Solereder (1995) have reported the presence of
epidermal cells like water storage cells. Stomata are found mostly
anomocytic type which are confined on abaxial and adaxial surfaces of
the leaf. While Metcalfe and Chalk (1950) have recorded the
ranunculacious as well as rubiaceous types of stomata in few species.
Deposition of small cluster crystals of calcium oxalate are found in the
mesophyll and epidermal cells. The mesophyll is differentiated into
palisade and spongy parenchymatous cells. The midrib is protruding on
the adaxial side. Stomatal index, frequency of stomata, vein termination
and vein islets numbers are also recorded. In cross section of the petiole,
below the epidermis spherical cortical cells are present in 6-7 layers.
CHAPTER 5 DISCUSSION
102
The transverse section of stem shows a single layer of epidermis
which is composed of tabular cells and are covered externally by thick
cuticle. Such details have not been given by Metcalfe and Chalk (1950).
Pericycle is devoid of sclerenchyma, Vascular bundles are arranged in a
ring, F ith is present and is made up of parenchymatous cells. The most
important feature of the axis is the presence of anomalous secondary
growth in the stem. Such details have also been recorded by earlier
workers.
The transverse section of the root is circular in outline. The
epidermis is composed of large bladder-like, water storage cells
interrelated between much smaller cells. The cork is absent however,
Surange et a! (1973) have recorded same observations in this species.
Anomalous structure is also observed in the root. However, Metcalfe and
Chalk (1950), Surange et al (1973) and Solereder (1995) have reported
same observations in the root as well as in the stem. The cortex is a
noteworthy feature of the root.
The quantitative microscopy of the leaf, stem and root have given the
following results:
The stomatal index is 18. Vein termination and vein islet numbers
vary from 6-8 and 3-5 respectively. The palisade ratio is in the range of
4-6.
The percentage of the maximum extractive values of leaf, stem and
root in methanol are 2.10%, 2.15% and 2.06% respectively while the
minimum values in benzene are found to be 0.46%, 0.21%, and 0.04%
respectively. The loss in weight of leaf, stem and root on drying at 105°C
are found to be 79%, 6.2% and 5.5% respectively. The percentage of total
ash, water soluble ash, acid insoluble ash and sulphated ash of the leaf,
stem and root are found to be 2.6%, 7.5%, 10.5% ; 9.0%, 8.2%, 7.7% ;
0.6%, 0.08%, 1.0% and 0.39%, 0.73%>, 0.51% respectively. The pH values
of the root in 1% and 10 % aqueous solutions have been recorded 7.56
and 7.5 respectively. The total alkaloids of the leaf, stem and root are
CHAPTER 5 _ _ ^ DISCUSSION
103
CHAPTER 5 DISCUSSION
found to be 0.02%, 0.03% and 0.5%) respectively. Glycosides, saponins,
resins and tannins have given positive tests in methanol and water
extracts.
The compo’tiacls investigated in root are given belows
Isolation and characterization, of compcmii.€! TP-1
Compound TP-1, designated p-sitosterol, is obtained in petroleum
ether 60-80°C eluants, on the basis of N.M.R. and Mass spectral data.
The N.M.R spectrum of the compound has shovirn the presence of
6 rnetliyl functions in the range of 6 0.6-5 1.08, It has shown a multiplet
around 5 3.5, which could be described to proton to the hydroxyl group.
Another signal around 5 5.02 was characteristics of an olefinic proton of
sterols. These data suggest that the compound could be p-sitosterol.
In the Mass spectrum of the compound the molecular ion peak at
m/z 414 was absent, however, these are strong peaks located at m/z 396
which could arise by the loss of one molecule of water.
The other characteristic peaks could be seen at m/z 256 and 214.
The fragmentation pattern has been shown below. These data have
confirmed that the compound is p-sitosterol.
Isolation and characterization of compouad TP-2
Compound TP-2, named stearic acid, is obtained as oily mass from
petroleum ether (60-80“C) eluants, had a molecular formula of
C17H35COOH on the basis of i^C NMR mass spectral data.
An examination of the peaks in the NMR spectrum observed that it
could be carboxylic acid derivative. A signal at 5 0.90 may arise from the
protons of the CH3 group. This was a very strong signal at 8 1.25 which
accounted for the protons of the methyl group. The other two signals at 6
1.63 and 8 2.34 accounted for the protons of the two methylene groups p
to CO group and a to CO group respectively,
The integration ratio suggested between the peaks that this
compound could be stearic acid (C17H35COOH).
104
A further support to the above compound as stearic acid is
obtained by the mass spectral data wliich has shown the molecular ion
peak at in/z 380 could arise from molecular ion peak by the loss of 44
mass units. These data are in accordance with the proposed fjtructure of
the compound as stearic acid.
Isolation and characterization of compomicl TP-3:
Compound TP-3, designated palmitic acid, is obtained as oily mass
from petroleum:benzene:methanol eluants has a palmitic acid on the
basis of NMR and mass spectreil data.
The NMR spectrum of the compound has shown a signal at 5 0.82
arising from the CH3 group. There was a strong peak at 6 1,25 which
could arise from tlie protons of the methylene groups. Another two
signals at 5 1.60 and at 8 2.33 accounted for the two CH2 groups. The
former signal may be due to the protons of the CHa moeity f3 to the
carboxyl group, while the latter may arise from the protons of the CH2
group a to the carboxyl function. A correlation between the ratio of the
three peaks suggested that it is palmatic acid, hence this compound may
be palmatic acid.
A further support to the above compound as palmitic acid is
obtained by the mass spectral data which has shown the molecular ion
peak at m/z 424. A peak could arise from the molecular ion peak by the
loss of 14 atomic mass unit characteristic of the saturated fatty acid.
These data are in accordance with the proposed structure of the
compound as palmatic acid.
Isolation and ctiaractesrization of compound TP»4
Compound TP-4-, named as Potassium nitrate is obtained as
colourless sugar like crystals from methanol eluants, has a molecular
formula KNO3 on the basic radical test and acidic radical test.
Basic radical test: The crystals of the salts are made into the form of a
paste by mixing with 1 drop of concentrated HCl. The tip of platinum
CHAPTER 5 _ __ _ DfSCUSSION
105
CHAPTER 5 mSCUSSIO.
wire is touched into this paste and it is then heated on a non luminou
flame and is seen by naiced eye. The flame colour ha.s become pale vide
in the region where tiie platinum wire was being heated and when it wa
seen through a blue glass, it looked crimBon red in colour and indicate
that the compound may be potasvsium (K).
Acid radical test: A few crystals were taken in a dry test tube. To it i
added concentrated H2SO4 about 1 ml when the light brown fume
evolved when copper turning were added.
It suggested the presence of nitrate. In order to confirm it, a fe\
crystals are dissolved in about 1/2 ml distilled water. To this solution i
added 2 mi of concentrated H2SO4 . The test tube is cooled and to it i
added saturated ferrous sulphate solution (1 rnl) slowly by the side c
this test tube when a brown ring is formed at the juncture of the twi
layer which confirmed the presence of nitrate.
Hence the compound under investigation is Postassium nitrat
(KNO3).
Mallotus phitippinensis |powder|:
Kamala is a controversial drug which is grossly adulterated witl
ferric oxide or with a ferruginous sand, or with brick dust, inferio:
qualitaties of the drug. Its quality may be roughly judged by throwing ?
small on to the surface of water, True kamala will float, but mos
adulterants will sink. Substitutes for kamala consisting of granc
safflower (florets of Cathamus tinctorius L.), dyed starch etc. have, beer
reported (Anonymous, 1992).
A careful examination of the results shows the details 0 :
macroscopical, microscopical and phytochemical characters of the
powder, of Mallotus philippensis which are helpful in the identification oi
the drug.
Kamala is a granular brick red powder having no specific taste and
odour, The powder consists of no cellular structure except the
characteristic stellate hairs which are thick walled, lignified, curved,
106
unicellular and arranged in a small radiating groups. It ranges from
30.5-145.9 j-un in length and from 13.7-19.5 [,tm in width. The present
findings are similar to the finding of other workers. The minute hairs are
observed by Kapoor and Rain (1977) in powder. The unicellular curved
trichomes have been mentioned b};- Trease and EJvans (1983) in the
powder. The powder is seen to consist of characteristic globular glands
which contain red resin under microscope and are measured from 31.9-
115.58 j-im in diameter. Similar observations are also given by Trease and
Evans (1983),
It is concluded that on drying at 105“C the loss in weight (4.31%),
solid contents (90,35%), water soluble matters (11,7%), acid insoluble
matter (4.6%). The percentage of the total ash (53.5%), water soluble ash
(0,17%), acid insoluble ash (45.72%) and sulphated ash (20.55%). Others
reported the results of the ash analysis of the powder of the plant
(Anonymous, 1962 and Anonymous, 1992). The percentage of the
extractives has been noted as hexane (11.37%), petroleum ether 60*-80°C
(5.31%), benzene (8.32%), chloroform (13,7%), acetone (12.2%), methanol
(3.12%) and water (4.51%). The thin layer chromatographic study of the
powder of the plant has given seven spots in petroleum ether (60-80°C
(5.31%), benzene (8.32%), chloroform (13.7%), acetone (12,2%), methanol
(3.12%) and water (4.51%). The thin layer chromatographic study of the
powder of the plant has given seven spots in petroleum ether (60-80°C).
The Rf values are 0.29, 0.36, 0.39, 0.48, 0.63, 0.77 and 0.83. Qualitative
studies have shown the presence of tannins, glucosides, resin and
saponins in the powder of Mallotus philippensis. The colour of the powder
as such under ultra-violet light has been observed chocolate colour.
Similar observations have been given by Anonymous (1992),
Isolation and characterization of the compound:
The dark brown rhombic crystals were separated out which were
filtered having m.p, 203-05“C. This compound was found to be pure on
CHAPTER 5 DISCIJSSJON
107
CH A PTEil 5 DISCUSSION
I'LC exEvrnination in toluene, ethyl formate, formic acid solvent system in
the ratio of 5:4:1.
In order to characterize it chemically its NMR spectrum was
recorded.
The NMR spectrum of the compound should a singlet at 8 1.5
integrating the 6 protons. There was three other singlets located at 8 2.1,
2.7 (both integrating in 3 protons) and at 5 3.8 (integrating in 2 protons)
which accounted for CH3 group, COCH3 group and CH2 group.
In the arorriat:ic region there was a doublet at 6 5.4 and another
doublet at 8 6 .6 which could arise from the proton H-3 and H-4 of the
chromene ring. The two multiplets at 5 7.4 an 7.6 could arise from the
three and two protons of the phenyl ring. The two doublets centred at 8
7.8 and 8.2 may arise from the a & p protons of the cinnamoyl moiety.
These data finally correspond to rottlerin which has been reported
to be a constituent of kamala and hence it could be concluded that the
powder under study is kamala.
108