g#''-W'.;y-!? I " . - . - •■ ’ .k S..; • 'y v ■. :■-

Discussion

. f ' ; ^ - : v- .i^ -i . - v • • j > - ; ^ 'g J g , j « g v ! K ; ^ ab*’; y ; y »

m

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).

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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