Chemical and Biological Investigation of Casuarina equisetifolia
L.
Chapter One
Introduction
1.1 THE PLANT FAMILY: Casuarinaceae
The Casuarinaceae are monoecious or dioecious trees and shrubs comprising four genus and
about 50 species with green, jointed, whorled photosynthetic branchlets. The leaves are
minute and whorled. The male flowers are minute and are clustered at the tips of branchlets
in catkin-like strobili. Each flower consists of a single stamen, a subtending bract and 2 pairs
of bracteoles. The female flowers are in ovoid clusters, each flower consists of a pistil, a
subtending bract and two bracteoles. The bicarpellate pistil has two long, filiform stigmas
from a short style. The ovary initially has two locules with two ovules in each but one locule
is generally completely aborted at anthesis. The bracts and bractlets enclosing the ovaries
persist and become woody, closely resembling a cone. Eventually, the bracts of individual
flowers separate, releasing the 1-seeded samaroid fruits
Classification of Kingdom Plantae down to family
Scientific classification
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Fagales
Family: Casuarinaceae
1.1.1 MEMBERS OF CASUARINACEAE FAMILY
The plants belonging to the family Casuarinaceae, which are available all over the world, are
shown in the Table 1.1.
Table 1.1 Casuarinaceae species available in the world.
Genera: Casuarina Genera: Allocasuarina
Species Species
Casuarina cristata Miq. Northeastern
Australia (Queensland, New South
Wales).
Casuarina cunninghamiana Miq.
Northern and eastern Australia
(Northern Territories to New South
Wales).
Casuarina equisetifolia L. Northern
Australia, southeastern Asia,
(Madagascar, doubtfully native).
Casuarina glauca Sieber ex Spreng.
New South Wales.
Casuarina grandis L.A.S.Johnson. New
Guinea.
Casuarina junghuhniana Miq.
Indonesia.
Casuarina obesa Miq. Southern
Australia (southwestern Western
Australia, New South Wales [one site,
now extinct], Victoria).
Casuarina oligodon L.A.S.Johnson.
New Guinea.
Allocasuarina acuaria
Allocasuarina acutivalvis
Allocasuarina brachystachya
Allocasuarina campestris
Allocasuarina corniculata
Allocasuarina crassa
Allocasuarina decaisneana
Allocasuarina decussata
Allocasuarina defungens
Allocasuarina dielsiana
Allocasuarina diminuta
Allocasuarina distyla
(scrub sheoak)
Allocasuarina drummondiana
Allocasuarina duncanii
(Duncan's sheoak)
Allocasuarina emuina
Allocasuarina eriochlamys
Allocasuarina fibrosa
Allocasuarina filidens
Casuarina pauper F.Muell. ex
L.A.S.Johnson. Interior Australia.
Allocasuarina fraseriana
(common sheoak)
Allocasuarina glareicola
Allocasuarina globosa
Allocasuarina grampiana
Allocasuarina grevilleoides
Allocasuarina gymnanthera
Allocasuarina helmsii
Allocasuarina huegeliana
(rock sheoak)
Allocasuarina humilis
Allocasuarina inophloia
Allocasuarina lehmanniana
(dune sheoak)
Allocasuarina littoralis
(black sheoak)
Allocasuarina microstachya
Allocasuarina misera
Allocasuarina monilifera
Allocasuarina muelleriana
(slaty sheoak)
Allocasuarina nana
Allocasuarina ophiolitica
Allocasuarina paludosa
(scrub sheoak)
Allocasuarina paradoxa
Allocasuarina pinaster
Allocasuarina portuensis[3]
Allocasuarina pusilla
Allocasuarina uehmannii
(bull-oak)
Allocasuarina mackliniana
(dwarf sheoak)
Allocasuarina ramosissima
Allocasuarina rigida
Allocasuarina robusta
Allocasuarina rupicola
Allocasuarina scleroclada
Allocasuarina simulans
Allocasuarina spinosissima
Allocasuarina striata (small bull-oak)
Allocasuarina tessellata
Allocasuarina thalassoscopica
Allocasuarina thuyoides
Allocasuarina tortiramula
Allocasuarina torulosa (forest sheoak)
Allocasuarina trichodon
Allocasuarina verticillata (drooping
sheoak)
Allocasuarina zephyrea
1.1.2 SOME EXAMPLE OF THIS FAMILY
Allocasuarina campestris Allocasuarina decaisneana
Allocasuarina distyla Allocasuarina nana
Allocasuarina torulosa Casuarina cristata
Casuarina equisetifolia Gymnostoma australianum
Figure 1.3: Different Kinds of Plants of Casuarinaceae Family
1.1.3: Casuarinaceae PLANTS AVAILABLE IN BANGLADESH :
Casuarinaceae plants are available in Bangladesh. They are found in sea shore areas as well
as in Chittagong. According to recent reports of Bangladesh National Herbarium, the
following Casuarinaceae plants are available in Bangladesh as shown in table 1.2.
Table 1.2: species of casuarinaceae available in Bangladesh
Genera: Casuarina Genera: Allocasuarina
Species Species
Casuarina cunninghami
Casuarina equisetifolia L,
Casuarina glauca
Casuarina junghuhniana
Casuarina oligodon.
Casuarina pauper
Allocasuarina acuaria
Allocasuarina defungens
Allocasuarina distyla (scrub sheoak)
Allocasuarina luehmannii (bull-oak)
Allocasuarina muelleriana (slaty sheoak)
Allocasuarina striata (small bull-oak)
Allocasuarina torulosa (forest
1.2 The Genus Casuarina L. – A brief discuss
Kingdom: Plantae
Order: Fagales
Family: Casuarinaceae
Genus: Casuarina
Casuarina is a genus of 17 species in the family Casuarinaceae, native to Australasia,
southeastern Asia, and islands of the western Pacific Ocean. It was once treated as the sole
genus in the family, but has been split into three genera
They are evergreen shrubs and trees growing to 35 m tall. The foliage consists of slender,
much-branched green to grey-green twigs bearing minute scale-leaves in whorls of 5–20. The
flowers are produced in small catkin-like inflorescences; the male flowers in simple spikes,
the female flowers on short peduncles. Most species are dioecious, but a few are monoecious.
The fruit is a woody, oval structure superficially resembling a conifer cone made up of
numerous carpels each containing a single seed with a small wing.[1][3]
Casuarina species are a food source of the larvae of hepialid moths; members of the genus
Aenetus, including A. lewinii and A. splendens, burrow horizontally into the trunk then
vertically down. Endoclita malabaricus also feeds on Casuarina. The noctuid Turnip Moth is
also recorded feeding on Casuarina.
1.3 MEDICINAL IMPORTANCE OF Casuarinaceae PLANTS
Among the 70 species of Casuarinaceae, only few are medicinally important. For many years
some species of this family are being medicinally used by the indigenous people of South
America, Brazil, Africa and India.
Table 1.3: Medicinal importance of Casuarinaceae plants
BOTANICAL NAMELOCAL
NAMEMEDICINAL USES
Casuarina equisetifolia
L
Jhau tree Effective antibacterial, anticancer, and
antitumor agent.
Casuarina cristata Tonic,vulnerary,antisour,antiscorbutic.
Casuarina glauca S. Used in colic pain, used as febge.
Casuarina
cunninghamiana.
Acrid,stimulant,diuretic,usedin
uterinedisorder,dysentery,liver
disease,pain,skindisease,tooth disease,
wound, fish poison.
Casuarina oligodon. Used in dysentery.
Casuarina pauper F Used in stomach ache.
Casuarina obesa Tonic, vulnerary.
Casuarina
junghuhniana.
Used in colic.
Casuarina grandis L Applied to swelling.
Allocasuarina acuaria Effective against stomach debility
Allocasuarina defungens Has mild depressant action on central
nervous system
Allocasuarina
luehmanni
Effective antibacterial, anticancer, and
antitumor agent.
Effective against stomach debility
Allocasuarina torulosa Used in dysentery
Allocasuarina
muelleriana
Tonic, vulnerary.
Allocasuarina striata Has mild depressant action on central
nervous system
1.4 CHEMISTRY OF CASUARINACEAE
Though there are about 4 genera and about 70 species in the family Casuarinaceae, chemical
investigation has been very limited with only species a few. Compounds isolated include
limonoids, mono-, di-, sesqui-, and triterpenoids, coumarins, chromones, lignans, flavonoids
and other phenolics.
1.4.1. Terpenoids
Terpenes consist of five carbon isoprene units, derived from mevalonate and are classified
broadly according to the number of isoprene units as follows:
I. Monoterpenes (C10)
II. Sesquiterpenes (C15)
III. Diterpenes (C20)
IV. Triterpenes (C30)
1.4.1.1. Biosynthesis of terpenoids
The terpenoids represent a large diverse class of secondary metabolites. They are constructed
from isoprene (2-methyl butadiene) units. The first set of reactions starts with the formation
of -hydroxy-p-methylglutaryl CoA (HMG COA) from acetyl CoA and acetoacetyl CoA.
HMG CoA is reduced to mevalonic acid which is then converted into
isopentenylpyrophosphate through 5-phosphomevalonate, 5- pyrophosphomevalonate and
3-phospho-5-pyrophosphomevalonate. Isopentenylpyrpohosphate is then isomerized into
dimethylallylpyrophosphate. Isopentenylpyrophosphate and dimethylallylpyrophosphate are
then condensed to form geranyl pyrophosphate. From the geranyl pyrophosphate
monoterpenes are formed. Geranyl pyrophosphate is condensed with another molecule of
dimethylallylpyrophosphate to form farnesyl pyrophosphate
Figure 1.2: Biosynthesis of mevalonate (IUBMB,2005)
Figure 1.3: Biosynthesis of Terpenoids (IUBMB, 2005)
Sesquiterpenes are formed from farnesyl pyrophosphate. A reductive condensation of two
molecules of farnesyl pyrophosphate leads to the synthesis of squalene (John D. Bu’lock,
1965).
Figure 1.4 : Biosynthesis of monoterpenes (IUBMB,2005)
Figure 1.5: Biosynthesis of diterpenes (IUBMB,2005)
1.4.1.2. BIOSYNTHESIS OF SESQUITERPENES:
The sesquiterpenes are C15 compounds biogenetically derived from farnesyl pyrophosphate,
and they are found mainly in plants and fungi. Some examples have been studied by tracer
methods which clarify the fact that farnesyl pyrophosphate undergoes cyclisation via
carbonium ions to form a complex series of cyclic sesquiterpenoids. The course of the
cyclisation depends on the geometry of the farnesyl pyrophosphate.
Figure 1.6: Biosynthesis of sesquiterpenes (IUBMB,2005)
1.4.1.3. BIOSYNTHESIS OF TRITERPENES:
Biosynthetically squalene or the 3S isomer of 2,3-epoxy-2,3-dihydrosqualene is the
immediate precursor of all triterpenoids (Newman, A.A. 1972). Triterpenoids are formed by
the cyclisation of these two precursors followed by rearrangement.
3(S)-2,3-epoxy-2,3-dihydrosqualene (squalene-2,3-epoxide) undergoes cyclisation to give
3-hydroxytriterpenoids which by oxidation and reduction can be transformed into
3-hydroxytriterpenoids.
Cyclisation of squalene-2,3-epoxide in a chair-boat-chair-boat conformation and by a
subsequent sequence of rearrangements leads to lanosterol, cycloartenol and cucurbitacin I
(J.D. Connolly and K.H. Overton, 1972). From cycloartenol, other terpenoids are formed.
Desmosterol is formed from lanosterol by a sequence of modification reactions. -Sitosterol
and stigmasterol are formed by the addition of extra carbon atoms to the side chain of
desmosterol in plants. Cyclisation of squalene-2,3epoxide in the chair-chair-chair-boat
conformation leads to the dammarane ring system. This cyclisation goes through a series of
carbonium ion intermediates to a cation from which dammaranes, euphanes and tirucallanes
are thought to be derived. According to the scheme suggested by Eschenmoser et al, 1955,
the transformation of the carbonium ion intermediates into euphol or tirucallol occurs either
by a concerted process or via the appropriate ethylenic intermediates.
1.4.2 FLAVONOIDS.
1.4.2.1 Properties:
Flavonoids have antioxidant activity. Flavonoids are becoming very popular because they
have many health promoting effects. Some of the activities attributed to flavonoids include:
anti-allergic, anti-cancer, antioxidant, anti-inflammatory and anti-viral. The flavonoids
quercetin is known for its ability to relieve hay fever, eszema, sinusitis and asthma.
Epidemiological studies have illustrated that heart diseases are inversely related to flavonoid
intake. Studies have shown that flavonoids prevent the oxidation of low-density lipoprotein
thereby reducing the risk for the development of atherosclerosis.
The contribution of flavonoids to the total antioxidant activity of components in food can be
very high because daily intake can vary between 50 to 500 mg.
Red wine contains high levels of flavonoids, mainly quercetin and rutin. The high intake of
red wine (and flavonoids) by the French might explain why they suffer less from coronary
heart disease then other Europeans, although their consumption of cholesterol rich foods is
higher (French paradox). Many studies have confirmed that one or two glasses of red wine
daily can protect against heart disease.
Tea flavonoids have many health benefits. Tea flavonoids reduce the oxidation of low-
density lipoprotein, lowers the blood levels of cholesterol and triglycerides. Soy flavonoids
(isoflavones) can also reduce blood cholesterol and can help to prevent osteoporis. Soy
flavonoids are also used to ease menopausal symptoms.
1.4.2.2 Description:
Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms. Flavonoids
belong to the polyphenol family. Flavanoids can be visualized as two benzene rings which
are joined together with a short three carbon chain. One of the carbons of the short chain is
always connected to a carbon of one of the benzene rings, either directly or through an
oxygen bridge, thereby forming a third middle ring, which can be five or six-membered. The
flavonoids consist of 6 major subgroups: chalcone, flavone, flavonol, flavanone,
anthocyanins and isoflavonoids.
Together with carotenes, flavanoids are also responsible for the coloring of fruits, vegetables
and herbs.
1.4.2.3 Distribution:
Flavonoids are found in most plant material. The most important dietary sources are fruits,
tea and soybean. Green and black tea contains about 25% percent flavonoids. Other important
sources of flavonoids are apple (quercetin), citrus fruits (rutin and hesperidin),
Table 1.4: Flavonoids from Casuarinaceae plants
Coumpound Source Reference
Quercetin (47) Allocasuarina striata Harborne & Mabry, 1982
Quercitol (48) Casuarina equisetifolia L Harborne & Mabry, 1982
Hyperin (49) Casuarina cristata Harborne & Mabry, 1982
Kaempferol (50) Casuarina glauca S. Harborne & Mabry, 1982
Heveaflavone (51) Casuarina cunninghamiana. Harborne & Mabry, 1982
Amentoflavone (52)
Manihot
Casuarina oligodon. Harborne & Mabry, 1982
Podocarpus flavone
A(53)
Casuarina pauper F Harborne & Mabry, 1982
Podocarpus flavone
B(54)
Casuarina obesa Harborne & Mabry, 1982
Eriodictyol (55) Casuarina junghuhniana. Harborne & Mabry, 1982
Fig. 1.7: Structural types of Flavonoids from Casuarineceae
1.4.3: Coumarin from Casuarinaceae plants
Coumarin
Synonyms: 1,2-Benzopyrone, 2H-1-Benzopyran-2-one
Properties: Coumarin has blood-thinning, anti-fungicidal and anti-tumor activities.
Coumarin should not be taken while using anticoagulants. Coumarin
increases the blood flow in the veins and decreases capillary
permeability. Coumarin can be toxic when used at high doses for a long
period
Facts about
Coumarin:
Coumarin seems to work as a pesticide in the plants that produce it.
Coumarin is responsible for the sweet smell of new mown hay.
Description: Coumarin is a phytochemical with a vanilla like flavour. Coumarin is a
oxygen heterocycle. Coumarin can occur either free or combined with
the sugar glucose (coumarin glycoside).
Distribution: Coumarin is found in several plants, including tonka beans, lavender,
licorice, strawberries, apricots, cherries, cinnamon, and sweet clover.
1.5 INFORMATIONS ABOUT THE INVESTIGATED PLANT
1.5.1 DESCRIPTION OF THE PLANT Casuarina equiseifolia
Preferred scientific name:
Casuarina equisetifolia L.
Family:
Casuarinaceae (casuarina family)
Non-preferred scientific names
Casuarina litorea L.
1.5.2 Taxonomic hierarchy of the investigated Casuarinaceae species
Kingdom : Plantae ( Plants)
Subkingdom : Tracheobionta (Vascular plants)
Superdivision : Spermatophyta (Seed plants)
Division : Magnoliophyta (Flowering plants)
Class : Magnoliopsida (Dicotyledons)
Subclass : Hamamelididae
Order : Casuarinales
Family : Casuarinaceae (She-oak family)
Genus : Casuarina Rumph. ex L. (sheoak)
Species : Casuarina equisetifolia L. (beach sheoak)
1.5.3 COMMON NAMES
English : Australian beefwood, Australian pine, beach she-oak,
beefwood tree, casuarina, coast she-oak, common ru,
horsetail casuarina, horsetail tree, ironwood, sea pine, she
oak, swamp she oak, wild pepper
Amharic : arzelibanos, shewshewe
Arabic : casuarina
Bengali : belaiti jhao, jau, jhau
Burmese : pink-tinyu, tin-yu
Cantonese : sarve
Creole : filao, pich pin
Fijian : nokonoko
Filipino : agoho
French : bois de fer, fialo, filao, pich pin, pin d'Australie
German : Eisenholz, Keulenbaum
Hindi: jangli saru, jungli jhao, vilayati saru
Indonesian : ai samara, aru, cemara laut, eru, tjemara laut
Japanese : mokumao, ogasawara-matsu
Khmer : snga:w
Malay: aru, ru, ru / rhu laut, ru laut
Pidgin English: yar
Sinhala : kasa ghas
Spanish : pino, pino d'Australia
Swahili : moinga, mvinje
Tamil : chouk sabuku, savukku
Thai : ku, son-thale
Tongan : toa
Trade name : beaf-wood
Vietnamese : c[aa]y phi lao, duong, filao, phi-lao
1.5.4 GENERAL DESCRIPTION OF Casuarina equisetifolia L.
Horsetail casuarina is the species most commonly planted in Hawaii and in other tropical and
subtropical regions around the world, where it has become naturalized. A rapidly growing
medium to large tree becoming 50–100 ft (15–30 m) tall and 1–11⁄2 ft (0.3–0.5 m) in trunk
diameter, with thin crown of drooping twigs. The bark is light gray brown, smoothish on
small trunks, becoming rough, thick, furrowed and shaggy, and splitting into thin strips and
flakes exposing a reddish brown layer. Inner bark is reddish and bitter or astringent. The wiry
drooping twigs mostly 9–º15 inches (23–38 cm) long, are dark green, becoming paler, with
6–8 long fine lines or ridges ending in scale leaves, shedding gradually like pine needles. A
few main twigs, gray and finely hairy, become rough and stout and develop into brownish
branches. Scale leaves less than 1⁄32 inch (1 mm) long, 6–8 in a ring (whorled) at joints or
nodes 1⁄4–3⁄8 inch (6–10 mm) apart. Leaves on main twigs in rings as close as 1⁄8 inch (3
mm), to 1⁄8 inch (3 mm) long and curved back. Flower clusters inconspicuous, light brown,
male and female on same tree (monoecious). Male flower clusters (like spikes or catkins)
terminal, narrowly cylindrical, 3⁄8–3⁄4 inch (10–19 mm) long and as much as 1⁄8 inch (3 mm)
across stamens, minute and crowded in rings among grayish scales, consisting of one
protruding brownish stamen less than 1⁄8 inch (3 mm) long with two minute brown sepal
scales at base. Female flower clusters are short-stalked lateral balls (heads) less than 1⁄8 inch
(3 mm) in diameter or 5⁄16 inch (8 mm) across spreading styles, consisting of pistil 3⁄16 inch
(5 mm) long including small ovary and long threadlike dark red style. The multiple fruit is a
light brown hard warty ball 1⁄2–3⁄4 inch (13–19 mm) in diameter, often longer than broad and
slightly cylindrical, composed of points less than 1⁄8 inch (3 mm) long and broad, each from
a flower. An individual fruit splits open in two parts at maturity to release one winged light
brown seed (nutlet) 1⁄4 inch (6 mm) long.
The sapwood is pinkish to light brown, the heartwood dark brown. The fine-textured wood is
very hard, heavy (sp. gr. 0.81), and very susceptible to attack by dry-wood termites. Tests of
the wood have been made in Puerto Rico. It is strong, tough, difficult to saw, but cracks and
splits, and is not durable in the ground. Rate of air-seasoning is moderate, and amount of
degrade is considerable. Machining characteristics are as follows: planing and turning are
fair; and shaping, boring, mortising, sanding, and resistance to screw splitting are good. In
Hawaii, the wood is used only as fuel. Elsewhere, the wood is used in the round. Uses include
fenceposts and poles, beams (not underground), oxcart tongues, and charcoal. The bark has
been employed in tanning, in medicine, and in the extraction of a red or blue-black dye. In
southern Florida, the fruits have been made into novelties and Christmas decorations. Often
propagated by cuttings for street, park, ornamental, and windbreak plantings, it can also be
trimmed into hedges. It is used for reforestation because of its rapid growth and adaptability
to degraded sites. This tree grows rapidly, reportedly as much as 80 ft (24 m) in height in 10
years, and adapts to sandy seacoasts, where ft becomes naturalized. It is very salt tolerant.
Common and naturalized along sandy coasts of Hawaii and up to more than 3000 ft (914 m).
It is used as windbreaks, such as along the Kohala Mountain Road, Hawaii; at Waimanalo,
Oahu; and Hanalei, Kauai, near the pier. More than 70,000 trees were planted on the Forest
Reserves and many others on private lands. The species was successfully established on
severely eroded Kahoolawe where it was to be a windbreak for other tree species. However,
goats broke through a fence and ate all the trees. The same system was used in the 1890’s to
plant the extremely windy Nuuanu Valley near the Pali.
Figure 1.9 Casuarina equisetifolia tree
Figure 1.10 Leaf of Casuarina equisetifolia
1.5.5 BOTANIC DESCRIPTION
Casuarina equisetifolia is an evergreen, dioecious or monoecious tree 6-35 (60) m tall, with a
finely branched crown. Crown shape initially conical but tends to flatten with age. Trunk
straight, cylindrical, usually branchless for up to 10 m, up to 100 (max. 150) cm in diameter,
occasionally with buttresses. Bark light greyish-brown, smooth on young trunks, rough, thick,
furrowed and flaking into oblong pieces on older trees; inner bark reddish or deep dirty
brown, astringent. The branchlets are deciduous, drooping, needlelike, terete but with
prominent angular ribs, 23-38 cm x 0.5-1 mm, greyish-green, articles 5-8 mm long, glabrous
to densely pubescent, dimorphic, either deciduous or persistent. Twigs deciduous, entirely
green or green only at their tips. The minute, reduced, toothlike leaves are in whorls of 7-8
per node. Flowers unisexual; perianth absent, replaced by 2 bracteoles. Male flowers in a
terminal, simple, elongated spike, 7-40 mm long, borne in whorls with 7-11.5 whorls/cm of
spike, with a single stamen. Female inflorescence on a short lateral branchlet, cylindrical,
cone-shaped or globose, 10-24 x 9-13 mm; bracteoles more acute, more or less protruding
from the surface of the cone. Infructescence a woody, conelike structure. Fruit a grey or
yellow-brown winged nut (samara). Seed solitary. Casuarina is from the Malay word
‘kasuari’, from the supposed resemblance of the twigs to the plumage of the cassowary bird.
One of the common names of Casuarina species, ‘she-oak’, widely used in Australia, refers to
the attractive wood pattern of large lines or rays similar to oak but weaker. The specific name
is derived from the Latin ‘equinus’, pertaining to horses, and ‘folium’, a leaf, in reference to
the fine, drooping twigs, which are reminiscent of coarse horse hair.
1.5.6 ECOLOGY AND DISTRIBUTION
C. equisetifolia has the widest distribution of all Casuarina species and occurs naturally on
subtropical and tropical coastlines from northern Australia throughout Malaysia, southern
Myanmar and the Kra Isthmus of Thailand, Melanesia and Polynesia. It is doubtfully
indigenous to the Mekong Delta in Vietnam and to Myanmar and possibly also to
Madagascar. It has also been introduced to a number of countries, where it is often
naturalized. By 1954 South China had established an estimated 1 million hectares.
1.5.6.1 Natural Habitat
The climate in its natural range is semi-arid to subhumid. In most regions there is a distinct
dry period of 4-6 months, although this seasonality decreases towards the equator in
Southeast Asia and in the southern parts of its range in Australia. C. equisetifolia is
commonly confined to a narrow strip adjacent to sandy coasts, rarely extending inland to
lower hills, as in Fiji. Found on sand dunes, in sands alongside estuaries and behind fore-
dunes and gentle slopes near the sea. It may be at the leading edge of dune vegetation, subject
to salt spray and inundation with seawater at extremely high tides. C. equisetifolia may be the
only woody species growing over a ground cover of dune grasses and salt-tolerant
broadleaved herbs; it can also be part of a richer association of trees and shrubs collectively
termed the Indo-Pacific strand flora.
1.5.6.2 Geographic distribution
Native :AustraliaBangladesh BruneiCambodiaFijiIndonesia MalaysiaNew ZealandPapua New Guinea Philippines SamoaSolomon Islands Thailand TongaVanuatuVietnam
Exotic : Antigua and BarbudaBahamasBeninBurkina FasoCameroonCentral African RepublicChad ChinaCongoCote d'Ivoire CubaDemocratic Republic of CongoDjiboutiDominicaDominican Republic EritreaEthiopia GabonGambiaGhanaGrenadaGuadeloupeGuineaGuinea-Bissau Haiti
India IsraelJamaicaKenya LiberiaMadagascar MaliMartiniqueMauritaniaMontserrat MyanmarNetherlands Antilles NigerNigeriaPakistanPuerto Rico SenegalSierra LeoneSomaliaSouth Africa Sri LankaSt Kitts and NevisSt LuciaSt Vincent and the Grenadines SudanTanzaniaTogoTrinidad and Tobago UgandaUnited States of America Virgin Islands (US)Zanziba
1.5.7 USES
Extensively cultivated for fuel, erosion control, and as a windbreak. It can be trimmed
and used as a hedge. The bark, used for tanning, penetrates the hide quickly,
furnishing a fairly plump, pliant, soft leather of pale reddish-brown color. With the
neutral sulfite semichemical process, wood yields a good pulp. The wood is used for
beams, boatbuilding, electric poles, fences, furniture, gates, house posts, mine props,
oars, pavings, pilings, rafters, roofing shingles, tool handles, wagon wheels, and
yokes. The needles have been employed in preparing active carbon by the zinc
chloride method (C.S.I.R., 1948–1976). Hill tribes of New Guinea use Casuarina in
rotation to restore nitrogen to the soil. They even use Casuarina oligodon as a cover
crop for coffee. Considering its unique ability to grow well, even in highly eroded
areas, Aspiras (1981) recommends it for Philippine barren hills and watersheds. "It is
not known to deplete the soil of important nutrients unlike other fast-growing species
now being grown in the countryside. Aside from its ability to raise the N status of the
soil when grown in rotational agriculture or in stabilizing road embankments, it also
produces good quality timber of high energy value. It may even be raised as a nurse
plant to pine, just like Myrica, or planted between coconut trees for its nitrogen and
timber." (Aspiras, 1981). In the Philippines, this is recognized as one of the best trees
for planting in sites covered by Imperata grass (NAS, 1983e). In Thailand it is planted
along coastlines to produce the poles used in building fish traps as well as fuelwood.
In the Dominican Republic, it has been used to reclaim stripmine lands. Egyptians
plant the trees along the coast to Protect houses from the wind and salt spray.
1.5.7.1 Medicinal Importance
Parts used : Leaf, stem, fruit
Therapeutic use:
Reported to be astringent, diuretic, ecbolic, emmenagogue, laxative, and tonic,
beefwood is a remedy for beri-beri, colic, cough, diarrhea, dysentery, headache,
nerves, pimples, sores, sorethroat, stomachache, swellings, and toothache (Duke and
Wain, 1981). In Ternate, the seeds are used for passing blood in diarrhea (Burkill,
1966).
1.5.8 CHEMISTRY OF Casuarina equisetifolia
Asparagine and glutamine accounted for 92% of the total amino acid in the nodules.
The bark contains 10% catchol tannin, the root 15%.
1.5.8.1 Constituents:
Ellagic acid,
beta-sitosterol,
kaempferol and glycosides,
quercetin,
cupressuflavone,
isoquercitrin,
several common triterpenoids,
trifolin,
catechin and epicatechin,
cholesterol,
stigmasterol,
campesterol,
cholest-5-en-3-beta-ol derivatives,
tannin,
proantho-cyanidins,
juglanin,
citrulline and amino acids,
afzelin,
casuarine,
gallicin,
catechol derivatives,
gentisic acid,
hydroquinone,
nictoflorin,
rutin,
trifolin.
1.5.8.2 Essential oils
Essential oils were obtained by separate hydrodistillation and analysed comprehensively for
their constituents by means of gas chromatography (GC) and gas chromatography-mass
spectrometry (GC–MS). The leaf essential oil of Casuarina equisetifolia L. (Casuarinaceae)
comprised mainly of pentadecanal (32.0%) and 1,8-cineole (13.1%), with significant amounts
of apiole (7.2%), α-phellandrene (7.0%) and α-terpinene (6.9%), while the fruit oil was
dominated by caryophyllene-oxide (11.7%), trans-linalool oxide (11.5%), 1,8-cineole (9.7%),
α-terpineol (8.8%) and α-pinene (8.5%).
1.5.8.3 Condensed tannins
Condensed tannins are a class of secondary metabolites with pronounced biological activities
found in many plants. Condensed tannins are formed of flavan-3-ol units, which are linked
together through C4–C6 or C4–C8 bonds to oligomers and high molecular weight polymers.
The diversity of condensed tannins is given by the structural variability of the monomer units:
different hydroxylation patterns of the aromatic rings A and B, different stereochemistry at
the chiral centers C2 and C3, and the distinct location and stereochemistry of the
interflavanoid bond. Condensed tannins, a major group with antioxidant properties, and act
against allergies, ulcers, tumours, platelet aggregation, cardiovascular diseases and can
reduce the risk of cancer. The bioactivity capacity of plant tannins is generally recognized to
be largely dependent on their structure and particularly the degree of polymerization.
However, tannins are diverse compounds with great variation in structure and concentration
within and among plant species. Due to the diversity and structural complexity of highly
polymerized tannins, the analysis and characterization of condensed tannins is a difficult task,
and less is known regarding structure-activity relationships. Various techniques including
NMR, acid-catalyzed depolymerization of the polymers in the presence of nucleophilic
reagents, and MALDI-TOF MS have been used to characterize condensed tannins. Casuarina
equisetifolia is traditionally used as a medicinal plant. The phenolic compounds from
branchlets (leaf) and bark showed the significant antioxidant activity. Therefore, this plant
might be a good candidate for further development as a nutraceutical or for its antioxidant
remedies. However, the structures of the condensed tannins from C. equisetifolia were rarely
studied, and detailed information on the condensed tannins profiles, especially with respect to
polymer chain length, chemical constitution of individual chains, and the sequential
succession of monomer units in individual chains present in C. equisetifolia is currently
lacking. In this study, contents of total phenolics and extractable condensed tannins of stem
bark and fine root of C. equisetifolia were determined.
tannins from stem bark and fine root are composed of catechin and epicatechin, afzelechin,
epiafzelechin, gallocatechin, epigallocatechin. it was further suggested that the condensed
tannins from stem bark and fine root contain procyanidin, prodelphindin and propelargonidin,
both with the procyanidin dominating.
1.5.8.4 Chemical Analysis of Biomass Fuels
Analysing 62 kinds of biomass for heating value, Jenkins and Ebeling (1985) reported a
spread of 19.44 to 18.26 MJ/kg, compared to 13.76 for weathered rice straw to 23.28 MJ/kg
for prune pits. On a % DM basis, the wh. plant contained 78.94% volatiles, 1.40% ash,
19.66% fixed carbon, 48.61% C, 5.83% H, 43.36% O, 0.59% N, 0.02% S, 0.16% Cl, and
undertermined residue.
1.6 LITERATURE SURVEY ON PHYTOCHEMICAL STUDY OF Casuarina
equisetifolia
The most frequently encountered natural organic compounds in Casuarina equisetifolia. The
results of previous investigations are summarized in Table1.3.
Table 1.5: Results of previous chemical work on Casuarina equisetifolia
Species Isolated compounds Part of
species
References
Casuarina
equisetifolia
Gallic acid
Protocatechuic acid
Hydroquinons
Fuglanin
Afzelin (+)
Catechin (-)
Cpicatechin (+)
Gallacatechin
Tittle fruits
and wood.
Khan et al., 1990;
Bhattacharyya et
al., 1984;
Talapatra et al.,
1969;
Paul et al., 1968,
1969;
Das et al., 1963;
Aher et al.,2009;
Roux, 1957;
Madhulata
et al., 1985;
Tryptophen
Leucin
Valine
Tyrosine
Glycine
Quercetin
Leaves.
Catechin
Gallic acid
Ellagic acid
Bark
The following phytoconstituents were also isolated from the plant so far, kaempferol (El-
Ansary et al., 1977), alicyclic acids: shikimik acid and quinic acid, amino acids
(Madhusudanamma et al.,1978) taraxerol, lupenone, lupeol, sitosterol (Rastogi and
Mehrotra, 1998).
(a) (b)
Figure 1.11: (a) Catechin (b) epicatechin
Biological activity:
The biological activities, viz, anti cancer, antibacterial (Wealth of India, 1992),
hypoglycemic, antifungal (Han, 1998) of the leaf has been reported.
1.7 RATIONALE OF THE WORK
Throughout the ages humans has relied on nature for their basic needs for the production
foodstuffs, shelters, clothing, means of transportation, fertilizer, flavors and not least,
medicines for treating various types of diseases in humans and animals for many years.
Plants are the important sources of a diverse range of chemical compounds. Some of these
compounds possessing a wide range of pharmacological activities are either impossible or to
difficult to synthesize in the laboratory. A Phytochemist uncovering these resources is
producing useful materials for screening programs for drug discovery. Emergency of newer
disease also leading the scientist to go back to nature for newer effective molecules.
Plants have formed the basis for traditional medicine system which have been used for
thousands of years in countries such as china (Chang et al., 1986 ) and India (Kapoor et
al.,1990). The use of plants in the traditional medicine of many other cultures has been
extensively documented. These plant – based system continue to play an essential role in
health care, and it has been estimated by the world health organization that approximately
80% of the world‘s inhabitants rely mainly on traditional medicines f0r their primary health
care (Schultes et al., 1990). Plant products also play an important role in the health care
system of the remaining 20% of the population, mainly residing in developed countries
(Arvigo et al. , 1993). In the study it has been shown that at least 119 chemical substances,
drive from 90 plant species, can be considered as important drugs that are in use in one or
more countries. Of these 119 drugs 74% were discovered as a result of chemical studies
directed at a isolation of the active substances from plants of traditional medicine (Arvigo et
al.,1993 ).
Examples of traditional medicine providing leads to bioactive natural products abound.
Suffice it to point to some recent confirmation of the wealth of this resource. Artimisine
(qinghaosu) (1 ,fig 1.1 ) is he antimalerial sesquiterpene from a Chinese medicinal herb
Artemisia annua (worm wood) used in herbal remedies since ancient times. Forskolin (2,
figure 1.1 ) is the antihypertensive agent from coleus forskohlii Briq. (Labiatae) , a plant
whose use was described in ancient Hindu Ayurvadic text (Bhat et al., 1977).
O
OH
O
O
O
H
Fig: 1.12: Artemisinin (1) and Forskolin (2)
Figure 1.3: Paclitaxel
Paclitaxel (figure 1.2 ) is the most recent example of an important natural product that has
made an enormous impact on medicine. It is interact with tubulin during the mitotic phase of
the cell cycle, and thus prevents the disassembly of the microtubules and their by interrupts
the cell division (wani et al., 1991). The original target diseases for the compound were
ovarian and breast cancers, but now it is used to treat a number of other human tissue
proliferating diseases as well (Strobel et al., 2004).
A case of serendipity is the discovery of the so called vinca alkaloids, vincristine (4) and
vinblastin (5) in catharanthus roseus. A random screening program (conducted at Eli Lilly
and company) of plants with antineoplastic activity found these anticancer agent in the 40 th of
200 plants examined. Ethno medicinal information attributed an anorexigenic effect (I,e.
causing anorexia ) to an infusion from plant (tyler , 1986 ).
1 2
Fig. 1.4: Vincristine (4)
Within the next quarter century, the achievement of science and technology will be so great
that, when brought to bear upon the mysteries of nature that have long puzzled us those
mysteries will yield their secrets wing amazing rapidity. It will be a fascinating and eventful
period. We will not know only the causes of disease but the cures for most. Significant new
drugs of plant origin and new methods of producing them will continue to be important parts
of that service and thus plants are considered as are of the most important and subjects that
should be explored for the discovery and development of newer and safer drug candidates.
1.8 PURPOSE OF THE STUDY
Bangladesh is a good repository of medicinal plants belonging to various families, including
casuainaceae. The casuainaceous plants contain a wide range chemical and unique
pharmacologically active compounds, including anticancer, in colic, in stomach ache, anti-
diarrhea, dysentery, beriberi, coughs, ulcer, and nervous disorders activities.
Casuarinaceae is a family of dicotyledonous flowering plants placed in the order Fagales,
consisting of 3 or 4 genera and approximately 70 species of trees .In Bangladesh, there are
more than 13 species and 02 varieties of the genus Casuarina available (Khan & Hasan, 1979)
including Casuarina equisetifolia. Though a large number of Casuarina species have been
investigated, little attention was given to it. Therefore, an attempt has been taken to study the
chemical constituents and biological activities of Casuarina equisetifolia.
These investigations may provide some interesting compounds, which may be
pharmacologically active. If significant results are obtained these can be used remedies for
the treatment of some diseases. Since this plant is available in Bangladesh, this may be a cost-
effective treatment.
So, the objective is to explore the possibility of developing new drug candidates from this
plant for the treatment of various diseases.
1.9 PRESENT STUDY PROTOCOL
The present study was designed to isolate pure compounds as well as to observe biological
activities of the isolated pure compounds with crude extract and their different fractions. The
study protocol consisted of the following steps:
Successive cold extraction of the powdered leaves of the plant
with methanol.
Fractionation of the crude concentrated methanolic extract by
column chromatography.
Isolation and purification of the pure compounds from different
column fractions by Thin layer chromatography (TLC).
Determination of the structure of the isolated compounds with the
help of 1H NMR.
Solvent-solvent partitioning of the crude concentrated methanolic
extract and collect four fractions (petroleum ether, carbon tetrachloride, ethyl
acetate and chloroform fractions).
Observation of in vitro antimicrobial activity of crude extract,
fractions.
Brine shrimp lethality bioassay and determination of LC50 for
crude extract and fractions.
Chapter Two
MATERIALS AND METHODS
2.1 METHODS OF PHYTOCHEMICAL SCREENING
The aim of Phytochemical analysis is to detect, isolate, characterize and identify the chemical
constituents. The chemical compounds present in the fruits and plants are of diverse and
varied nature. They usually include simple hydrocarbons to different classes of compounds.
To far the knowledge goes, there is no single method to accomplish this task. Thus large
numbers of different physiochemical methods and physiochemical techniques have to be
employed to study of those plants. The working methodology and experimental are given
below-
2.2 GENERAL METHODS
The chemical investigation of a sample can be divided roughly into the following
major steps:
a) Collection and proper identification of the sample materials
b) Preparation of sample materials
c) Extraction
d) Isolation of compounds
e) Structural characterization of purified compounds
2.2.1 COLECTION AND PROPER IDENTIFICATION OF THE SAMPLE
At first with the help of a comprehensive literature review a plant was selected for
investigation and then the whole plant/plant part(s) was collected from an authentic source
and was identified by a taxonomist. A voucher specimen that contains the identification
characteristics of the plant was submitted to the herbarium for future reference.
2.2.2 SAMPLE PREPARATION
The plants were collected in fresh condition and the leaves were separated. After the
separation, the leaves were cleaned with water, sun-dried and then, dried in an oven at
reduced temperature (not more than 400C) to make it suitable for grinding purpose. Then the
dried leaves were ground to obtain powder using cyclotec grinding machine (200mesh). The
coarse powder was then stored in air-tight container with marking for identification and kept
in cool, dark and dry place for future use.
2.2.3 SOLVENTS AND CHEMICALS
Analytical grade solvents and chemicals used in the experiments. All solvents and reagent
used in the experiments were purchased from E. Merk (Germany), BDH (England). The
analytical grade solvents (n-hexane, Pet-Ether, Ehtyl acetate, Absolute ethanol, Chloroform
and methanol) were used.
2.2.4 DISTILLATION OF THE SOLVENTS
The commercial grade solvents (petrol, ethyl acetate, chloroform and methanol) were
distilled. Petroleum ether (b.p 40-60) °C was obtained by distilling petrol. Distilled solvents
were used through the investigation.
Figure 2.1 Distillation Pump
2.2.5 EVAPORATION
All evaporations were carried out under reduced pressure using a rotary evaporator at a bath
temperature of 450C. The residual solvent in the extract and compounds were removed under
high vacuum.
Figure 2.2 Vacuum Rotary Evaporator
2.2.6 PREPARATION OF EXTRACTS
The sample was collected and washed with water to remove mud and dust particles. Then
dried in room temperature and in the oven at 400 C. The dried leaves were grind to powder by
a grinder. The powder was stored for extracts in air tight bottle.
2.2.7 EXTRACTION PROCEDURES
2.2.7.1 INITIAL EXTRACTION
Extraction can be done in two ways such as
a) Cold extraction
b) Hot extraction
2.2.7.1.1 COLD EXTRACTION
In cold extraction the powdered plant materials is submerged in a suitable solvent or solvent
systems in an air-tight flat bottomed container for several days, with occasional shaking and
stirring. The major portion of the extractable compounds of the plant material will be
dissolved in the solvent during this time and hence extracted as solution.
2.2.7.1.2 HOT EXTRACTION
In hot extraction the powdered plant material is successively extracted to exhaustion in a
Soxhlet at elevated temperature with several solvents of increasing polarity.
The plant material extracted exhaustively in Soxhlet apparatus first with petroleum ether (boiling
point, 40°-60°C), then with ethyl acetate (EA) and last with methanol (MeOH). All the extracts
were filtered individually and then concentrated with a rotary evaporator (Buchi) at low temperature
(400-500C) under reduced pressure.
2.2.8 DETECTION / VISUALIZATION
2.2.8.1 UV-LIGHT
The fluorescent compounds on the plates were observed under UV- light at 254 and 350 mm.
Some of the compounds appeared as fluorescing spots while the others are dark spots under
the UV-light.
The developed chromatogram is viewed visually to detect the presence of colored
compounds.
Figure 2.3 Visualization/Detection of Compounds in UV Lamp
2.2.8.2 IODINE CHAMBER
Iodine vapour has also used as a general reagent to detect spots in the TLC plates. A closed
jar or tank with powdered iodine was used to identify the spots. The compounds that
appeared as brown spots are marked. Unsaturated compounds absorb iodine. Bound iodine is
removed from the plate by air blowing.
2.2.8.3 SPRAY REAGENTS
Different types of spray reagents are used depending upon the nature of compounds expected
to be present in the fractions or the crude extracts.
Figure 2.4 Vanillin-Sulphuric Acid Spray
Vanillin/H2SO4: 1% vanillin in concentrated sulfuric acid is used as a general spray reagent
followed by heating the plates to 1000C for 10 minutes.
2.2.9 PREPARATION OF THE REAGENTS
2.2.9.1 VANILLIN-SULPHURIC ACID REAGENT
Vanillin (1.0 g) was added to the sulfuric acid (100 ml) (kept in ice bath), cooled and used for
spraying the TLC plates.
2.2.10 SEPARATION AND ISOLATION OF COMPOUNDS
Pure compounds are isolated from the crude and fractionated extracts using different
chromatographic and other techniques. A brief and general description of these is given
below.
2.2.10.1 CHROMATOGRAPHIC TECHNIQUES
Two types of chromatographic techniques were used such as thin layer chromatography
(TLC) and vacuum liquid chromatographic chromatography (VLC).
2.2.10.1.1 THIN LAYER CHROMATOGRAPHY (TLC)
Two types of TLC plates were used throughout the experiment;
1. Precoated TLC plates: 0.2mm thin coatings of silica gel on glass plates or aluminum
sheets were used.
2. Manually prepared silica gel coated glass plates were used.
Table 2.1 Amount of Silica Gel Required for Preparing TLC Plates of Various Thicknesses
Size (cm x cm) Thickness (mm) Amount of silica gel/plate (gm)
20 x 5
0.3
0.4
0.5
0.9
1.2
1.5
2.2.10.1.2 PREPARATION OF PLATES
Thin layer chromatographic plates were prepared by spreading a film of an aqueous slurry
(gel: water = 1:2 w/v) of silica gel G-60 PF254 (E, Merck 7731) over the entire surface of the
glass plates (6cm x 12 cm) by means of spreader. This thickness of the silica gel layer was
0.2 mm. The plates were dried the air and finally activated by heating at 110oC for 1 hour
followed by cooling at room temperature for few hours.
Figure 2.5 Some TLC Plates
2.2.10.1.3 PREPARATIVE THIN LAYER CHROMATOGRAPHY (PTLC)
Silica gel (Merck 60 PE 254) was used to prepare PTLC plates. The 20cm X 20cm glass plates
were cleaned and dried. The Slurry was prepared by mixing 32g of silica gel with 64mL of
distilled water. The slurry spreaded on the plates to yield a thin layer of 0.50mm thickness.
The prepared plates were allowed to set in air dried for some time and then heated in the
oven at 1100C for about half an hour. The samples were dissolved in a small amount of a
suitable solvent and applied on to plates as a thin band near the base line. The plates were
then developed in the appropriate solvent system previously ascertained by TLC. In some
cases, double or triple developments were visualized by the use of either spray reagent orUV
light, scrape the individuals’ band of the plate with the help of a spatula and the compound
was eluted with a solvent, usually slightly more polar than the solvent used for developing the
plates. These elutes were concentrated by evaporating the excess solvent under reduced
pressure in rotary evaporator keeping the bath temperature below 40 oC.
2.2.10.1.4 SAMPLE APPLICATION (SPOTTING THE PLATES)
The TLC plates were spotted with a small amount of the crude extract by using a narrow
glass capillary. The capillary was washed with either acetone or ethanol before each sample
was applied.
Figure 2.6 Process of Spotting
2.2.10.1.5 SOLVENT SYSTEMS
The solvents of different polarity used for TLC are given below:
n-Hexane
Pet-Ether
Ethyl acetate
Methanol
n-Hexane / Pet-Ether : Ethyl acetate (in different ratio)
n-Hexane / Pet-Ether: Methanol (in different ratio)
Ethyl acetate: Methanol (in different ratio)
Figure 2.7 Developing of TLC Plate
2.2.10.1.6 PREPARATION OF TLC TANK
The ascending technique in glass jars or tanks were used to develop TLC plates. A suitable
solvent system was poured into glass jar or tank in a sufficient amount. The tank was then
covered with a lid and kept for a certain period allowing it to achieve saturation. A filter
paper was usually introduced into the tank to promote the saturation process. The solvent at
the bottom of the tank must not be above the line of spot where the sample solution was
applied to the plate. As the solvent rises upward, the plate becomes moistened. The plate was
then taken out and dried. The solvent front was not allowed to travel beyond the end of the
silica-coated surface.
Figure 2.8 TLC Tank & Iodine Chamber
2.2.10.1.7 DETECTION OF SPOTS
For the location of the separated components, the plates were examined by the following
methods:
1. Examination under UV lights in different wavelength, 254 and 350 nm.
2. The plates were exposed to iodine vapor for several minutes.
3. The plates were sprayed with vanillin-sulfuric acid regent (1.0%) followed by heating
in an oven at 1200C for 15 minutes.
2.2.10.1.8 THE Rf VALUE
Retardation factor (Rf) is the ratio of the distance the compound travel to the distance the
solvent front moves.
Usually, the Rf value is constant for any given compound and it corresponds to a physical property
of that compound.
Figure .2.9 A Plate for the Calculation of Rf value
2.3 COLUMN CHROMATOGRAPHY
2.3.1 Vacuum Liquid Chromatography (VLC)
For normal phase column chromatography, silica gel of particle size 230-400 mesh from
(Merck) was used and separation was performed by gravitational flow with solvents of
increasing polarity. The sample was applied into the column either as a solution or in a
powdered form. The eluted samples were collected in several test tubes and were monitored
by TLC to make different fractions on the basis of Rf values. .
For preparation of Sephadex LH-20 column, the required amount of Sephadex LH-20 gel
(25-100mm, Pharmacia, Sweden) was suspended in petroleum ether and the column was
packed with this suspended gel.
Compound
Compound
Baseline
Solvent front
Distance from solvent front, A
Distance from sample front, B
Figure 2.10 Various Part of a Column
2.3.2 PROCEDURE FOR MICRO SCALE FLASH COLUMN CHROMATOGRAPHY
In micro scale flash chromatography, the column does not need either a pinch clamp or a
stopcock at the bottom of the column to control the flow, nor does it need air-pressure
connections at the top of the column. Instead, the solvent flows very slowly through the
column by gravity until we apply air pressure at the top of the column with an ordinary
Pasteur pipet bulb.
Figure 2.11 Various stages in micro scale column.
2.3.3 PREPARATION OF COLUMN (FOR MICRO SCALE OPERATION)
A Pasteur pipet was plugged with a small amount of cotton to prevent the adsorbent from
leaking. The Pasteur pipet was filled with the slurry of column grade silica gel with a stream
of solvent using a dropper. It was ensured that the “Sub Column” is free from air bubbles by
recycling the solvents several times. The samples were applied at the top of the column.
Elution was started with petroleum ether followed by increasing polarity.
2.4 SPECTROSCOPIC TECHNIQUES
Nuclear magnetic resonance (nmr) spectroscopy
NMR spectra of pure sample were recorded by using 1H-NMR (400 MHz) and C-13 NMR
spectrometer. The spectra were record using CDCl3 with tetramethyl silane (TMS) as standard
reference.
2.5 CHEMICAL INVESTIGATION OF Casuarina equisetifolia
In this study, Leaves of Casuarina equisetifolia belonging to the family Casuarinaceae was
chemically investigated.
Taxonomic hierarchy of the investigated Casuarinaceae species
Kingdom Plantae – Plants
Subkingdom Tracheobionta
Superdivision Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Subclass Hamamelididae
Order Casuarinales
Family Casuarinaceae
Genus Casuarina Rumph. ex L.
Species Casuarina equisetifolia L.
2.5.1 Collection and preparation of plant material
The plant Casuarina equisetifolia grows naturally coastlines in Bangladesh. The plants were
collected from Bangladesh Council for Science and Industrial Research Garden, Chittagong,
in the month of February 2010. It has been submitted for identification in Bangladesh
National Herbarium, Dhaka.
2.5.1.1 Identification by Bangladesh National Herbarium, Dhaka:
DACB Accession Number: 35545
Botanical name: Casuarina equisetifolia L.
Local name: Jhau
Family: Casuarinaceae.
The collected plants were made free from dust. The leaves were then separated from the stems and air dried. Finally they were grounded to yield powder (1.2 kg) by a cyclotec grinder and then was stored for extraction.
2.5.2 Extraction of the plant material
The air dried and powdered plant material ( 1200 gm) was suspended in 2.5 litre of methanol
for eight days for the purpose of cold extraction. The extract was filtered through fresh cotton
bed and finally with Whatman No.1 filter paper. The volume of the filtrate was concentrated
with a rotary evaporator at low temperature (400-500C) and reduced pressure. The weight of
the crude extract was24.312 gm.
2.5.2.1 EXTRACTION SCHEME OF Casuarina equisetifolia:
Extract
Concentrated Mass
Leaves of C. equisetifolia
Dried leaves
Leaves Powder
Extraction with Methanol
Figure 2.12 Extraction Scheme of Casuarina equisetifolia
2.5.3 Investigation of the crude extract
A portion of the crude extract soluble fraction (2.855gm) was subjected to column
chromatography for fractionation. Then the chromatographic fractions were analysed by
TLC.
2.5.3.1 Column chromatography of crude extract
The column was packed with silica gel (Kieselgel 60, mesh 70-230). Slurry of silica gel in
petroleum ether 600-800 was added into a glass column having the length and diameter 33 cm
and 2.8 cm respectively. When the desired height of the adsorbent bed was obtained, a few
hundred millilitre of petroleum ether was run through the column for proper packing of the
column. The sample was prepared by adsorbing 2.855g of crude extract into silica gel
(Kieselgel 60, mesh 70-230), allowed to dry and subsequently applied on top of the adsorbent
layer. The column was then eluted with petroleum ether, followed by mixtures of petroleum
ether and ethyl acetate of increasing polarity, then by ethyl acetate and finally with ethyl
acetate and methanol mixtures of increasing polarity. Solvent systems used as mobile phases
in the analysis of crude extract were listed in Table 2.4. A total of 38 fractions were collected.
Table 2.2 : Different solvent systems us ed for column chromatogr8aphy of crude extract
Fraction
no.Solvent systems
Volume collected
(ml)
1 Petroleum ether : ethyl acetate = 80:20 100
Subjected CC & eluted with PE, EA, MeOH
19B19A02A01A 01B
2 Petroleum ether : ethyl acetate = 77.5:22.5 100
3 Petroleum ether : ethyl acetate = 75:25 100
4 Petroleum ether : ethyl acetate = 72.5:27.5 100
5 Petroleum ether : ethyl acetate = 70:30 100
6 Petroleum ether : ethyl acetate = 67.5:32.5 100
7 Petroleum ether : ethyl acetate = 65:35 100
8 Petroleum ether : ethyl acetate = 62.5:37.5 100
9 Petroleum ether : ethyl acetate = 60:40 100
10 Petroleum ether : ethyl acetate = 57.5:42.5 100
11 Petroleum ether : ethyl acetate = 55:45 100
12 Petroleum ether : ethyl acetate = 52.5:47.5 100
13 Petroleum ether : ethyl acetate = 50:50 100
14 Petroleum ether : ethyl acetate = 47.5:52.5 100
15 Petroleum ether : ethyl acetate = 45:55 100
16 Petroleum ether : ethyl acetate = 42.5:57.5 100
17 Petroleum ether : ethyl acetate = 40:60 100
18 Petroleum ether : ethyl acetate = 37.5:62.5 100
19 Petroleum ether : ethyl acetate = 35:65 100
20 Petroleum ether : ethyl acetate = 32.5:67.5 100
21 Petroleum ether : ethyl acetate = 30:70 100
22 Petroleum ether : ethyl acetate = 25:75 100
23 Petroleum ether : ethyl acetate = 20:80 100
24 Petroleum ether : ethyl acetate = 17.5:82.5 100
25 Petroleum ether : ethyl acetate = 15:85 100
26 Petroleum ether : ethyl acetate = 12.5:87.7 100
27 Petroleum ether : ethyl acetate = 10:90 100
28 Petroleum ether : ethyl acetate = 7.5:92.5 100
29 Petroleum ether : ethyl acetate = 5:95 100
30 Petroleum ether : ethyl acetate = 2.5:97.5 100
31 Petroleum ether : ethyl acetate = 0:100 100
32 ethyl acetate: methanol = 99.5:0.5 100
33 ethyl acetate: methanol = 99:1.0 100
34 ethyl acetate: methanol = 98:2.0 100
35 ethyl acetate: methanol = 95:5.0 100
36 ethyl acetate: methanol = 90:10 100
37 ethyl acetate: methanol = 50:50 100
38 ethyl acetate: methanol = 0.0:100 100
2.5.3.2 Analysis of column fractions by TLC
All the column fractions were screened by TLC under UV light and by spraying with Dragendorffs
reagent. Depending on the TLC behaviour fractions were mixed and list of new fraction codes for
further investigation.
Table 2.3 List of new fraction codes
2.5.3.3
Analysis of new column fraction codes by TLC
All the new column fractions codes were screened by TLC under UV light and by spraying with
Dragendorffs reagent. Depending on the TLC behaviour new fractions codes F fractions showed
satisfactory resolution of components. For this, further chemical investigation was concised only for the
latter one fraction.
Column fractions New codeWt. of the extracts
(in gm)
1-4A A 0.0234
4B-6A B 0.0245
6B-8B C 0.1000
9A-10A D 0.0843
10B E 0.1109
11A-14A F 0.1364
14B-15B G O.0124
16A-16B H 0.0198
17A I 0.0090
17B-18A J 0.0035
18B-19B K 0.0080
Fig 13:Analysis of column fraction codes by TLC
2.5.4 Isolation and purification of compounds from selected fractions
2.5.4.1 Isolation and purification of compound TA-1101
Compound TA-1101 was found to yield colorless mass. It was isolated from the column
fraction of methanol crude extract by elution with petroleum ether 80-20% ethyl acetate. The
crystals were washed with dichloromethane carefully. These crystals were dissolved in
chloroform and transferred to a vial and was designated as TA-1101. It appeared in the
preparative thin layer chromatography using 5% Ethyl acetate in Toluene.
2.5.4.2 Isolation and purification of compound TA-1102
Compound TA-1102 was isolated from the column fraction of methanol crude extract by
elution with petroleum ether/ ethyl acetate 52.5-47.5%. It was obtained as white gum. TA-
1102 was washed with dichloromethane carefully. As a result colored solution was obtained
leaving back the white gum. These white gum was dissolved in chloroform and transferred to
a vial and was designated as TA-1102.
2.5.5 Test for purity of the isolated compounds
The purity of each of the isolated compounds was monitored by TLC using different solvent
systems. Commercially available plates pre-coated with silica gel (Kieselgel 60 PF254) on
plastic and aluminium sheets were used for this purpose. Moreover, purity was also tested by
spraying the developed plates with different spray-reagents followed by heating at 1100C for
several minutes.
Chapter Three
ANTIMICROBIAL SCREENING
3.1 INTRODUCTION
Plants are the natural reservoir of many antimicrobial agents. In recent times traditional
medicine has served as an alternative form of health care and to overcome microbial
resistance has led the researchers to to investigate the antimicrobial activity of medicinal
plants (Austin et al.. 1999).
Owing to high temperature and high humidity, the infectious diseasesare very common in
Bangladesh. Bacteria and fungi are responsible for many infectious diseases. The increasing
clinical implications of drug resistant fungal and bacterial pathogens have lent additional
urgency to antimicrobial drug research. The antimicrobial screening which is the first stage of
antimicrobial drug research is performed to ascertain the susceptibility of various fungi and
bacteria to any agent. This test measures the ability of each test sample to inhibit the in vitro
fungal and bacterial growth. This ability may be estimated by either of the following three
methods.
i) Disc diffusion method
ii) Serial dilution method
iii) Bioautographic method
But there is no standardized method for expressing the results of antimicrobial screening
(Ayafor et. al; 1982). Some investigators use the diameter of zone of inhibition and/or the
minimum weight of extract to inhibit the growth of microorganisms. However, a great
number of factors viz., the extraction methods (Nadir et al., 1986), inoculum volume, culture
medium composition (Bayer et al., 1966), PH (Leven et al., 1979), and incubation temperature
(Lorian, 1991) can influence the results.
Among the above mentioned techniques the disc diffusion (Bauer et al., 1966) is a widely
accepted in vitro investigation for preliminary screening of test agents which may possess
antimicrobial activity. It is essentially a quantitative or qualitative test indicating the sensitivity or
resistance of the microorganisms to the test materials. However, no distinction between
bacteriostatic and bacteriocidal activity can be made by this method (Roland, R., 1982).
3.2 PRINCIPLE OF DISC DIFFUSION METHOD
Solutions of known concentration (mg/ml) of the test samples are made by dissolving
measured amount of the samples in calculated volume of solvents. Dried and sterilized filter
paper discs (6 mm diameter) are then impregnated with known amounts of the test substances
using micropipette. Discs containing the test material are placed on nutrient agar medium
uniformly seeded with the test microorganisms. Standard antibiotic discs and blank discs
(impregnated with solvents) are used as positive and negative control. These plates are then
kept at low temperature (4 0C) for 24 hours to allow maximum diffusion. During this time
dried discs absorb water from the surrounding media and then the test materials are dissolved
and diffused out of the sample disc. The diffusion occurs according to the physical law that
controls the diffusion of molecules through agar gel (Barry, 1976). As a result there is a
gradual change of test materials concentration in the media surrounding the discs.
The plates are then incubated at 37 0C for 24 hours to allow maximum growth of the
organisms. If the test materials have any antimicrobial activity, it will inhibit the growth of
the microorganisms and a clear, distinct zone of inhibition will be visualized surrounding the
medium. The antimicrobial activity of the test agent is determined by measuring the diameter
of zone of inhibition expressed in millimeter.
The experiment is carried out more than once and the mean of the readings is required (Bayer
et al., 1966).
In the present study all the crude extracts and fractions, some column ractions as well as some
purified compounds were tested for antimicrobial activity by disc diffusion method. Some pure
compounds could not be tested due to scarcity of samples.
3.3 EXPERIMENTAL
3.3.1 Apparatus and Reagents
Filter paper discs Petridishes Inoculating loop
Sterile cotton Sterile forceps Spirit burner
Micropipette Screw cap test tubes Nosemask and Hand gloves
Laminar air flow hood Autoclave Incubator
Refrigerator Nutrient Agar Medium Ethanol
Chloroform
3.3.2 Test materials
3.3.2.1 Test materials of Casuarina equisetifolia
Code no. Test sample Amount (mg)
CTA Methanol Crude extract 8.0
PETA Petroleum ether fraction of methanol extract 8.0
CTTA Carbon tetrachloride fraction of methanol extract 8.0
CFTA Chloroform fraction of methanol extract 8.0
EATA Ethyl acetate fraction of methanol extract 8.0
3.3.3 Test Organisms
The bacterial and fungal strains used for the experiment were collected as pure cultures from
the Institute of Nutrition and Food Science (INFS), University of Dhaka. Both Gram positive
and Gram-negative organisms were taken for the test and they are listed in the Table 4.1.
Table 3.1: List of Test Bacteria and fungi
Gram positive
Bacteria
Gram negative
BacteriaFungi
Bacillus cereusEscherichia coli Candida albicans
Bacillus megaterium Pseudomonas aeruginosa Aspergillus niger
Bacillus subtilis Salmonella paratyphi Sacharomyces cerevacae
Staphylococcus aureusSalmonella typhi
Sarcina luteaShigella boydii
Shigella dysenteriae
Vibrio mimicus
Vibrio parahemolyticus
3.3.4 Culture medium and their composition
The following media is used normally to demonstrate the antimicrobial activity and to make
subculture of the test organisms.
Nutrient agar medium
Ingredients Amounts
Bacto peptone 0.5 gm
Sodium chloride 0.5 gm
Bacto yeast extract 1.0 gm
Bacto agar 2.0 gm
Distilled water q.s. to 100 ml
PH 7.2 0.1 at 250C
Nutrient broth medium
Ingredients Amounts
Bacto beef extract 0.3 gm
Bacto peptone 0.5 gm
Distilled water q.s.to 100 ml
PH 7.2 0.1 at 250C
Muller – Hunton medium
Ingredients Amounts
Beef infusion 30 gm
Casamino acid 1.75 gm
Starch 0.15 gm
Bacto agar 1.70 gm
Distilled water q.s. to 100 ml
PH 7.3 0.2 at 250 C
d. Tryptic soya broth medium (TSB)
Ingredients Amounts
Bacto tryptone 1.7 gm
Bacto soytone 0.3 gm
Bacto dextrose 0.25 gm
Sodium chloride 0.5 gm
Di potassium hydrogen Phosphate 0.25 gm
Distilled water q.s. to 100 ml
PH 7.3 0.2 at 250c
Nutrient agar medium (DIFCO) used most frequently for testing the sensitivity of the organisms to
the test materials and to prepare fresh cultures.
3.3.5 Preparation of medium
To prepare required volume of this medium, calculated amount of each of the constituents
was taken in a conical flask and distilled water was added to it to make the required volume.
The contents were heated in a water bath to make a clear solution. The PH (at 25 0C) was
adjusted at 7.2 – 7.6 using NaOH or HCl. 10 ml and 5 ml of the medium was then transferred
in screw cap test tubes to prepare plates and slants respectively. The test tubes were then
capped and sterilized by autoclaving at 15-lbs. pressure/ sq. inch at 121 0C for 20 minutes. The
slants were used for making fresh culture of bacteria and fungi that were in turn used for sensitivity
study.
3.3.6 Sterilization procedures
In order to avoid any type of contamination and cross contamination by the test organisms the
antimicrobial screening was done in Laminar Hood and all types of precautions were highly
maintained. UV light was switched on one hour before working in the Laminar Hood.
Petridishes and other glasswares were sterilized by autoclaving at a temperature of 121 0C and
a pressure of 15-lbs./sq. inch for 20 minutes. Micropipette tips, cotton, forceps, blank discs etc. were
also sterilized.
3.3.7 Preparation of subculture
In an aseptic condition under laminar air cabinet, the test organisms were transferred from the
pure cultures to the agar slants with the help of a transfer loop to have fresh pure cultures.
The inoculated strains were then incubated for 24 hours at 37 0C for their optimum growth.
These fresh cultures were used for the sensitivity test.
3.3.8 Preparation of the test plates
The test organisms were transferred from the subculture to the test tubes containing about 10 ml of
melted and sterilized agar medium with the help of a sterilized transfer loop in an aseptic area. The
test tubes were shaken by rotation to get a uniform suspension of the organisms. The bacterial and
fungal suspension was immediately transferred to the sterilized petridishes. The petridishes were
rotated several times clockwise and anticlockwise to assure homogenous distribution of the test
organisms in the media.
3.3.9 Preparation of discs
Three types of discs were used for antimicrobial screening.
3.3.9.1 Standard discs
These were used as positive control to ensure the activity of standard antibiotic against the
test organisms as well as for comparison of the response produced by the known
antimicrobial agent with that of the test sample. In this investigation, kanamycin (30mg/disc)
and amoxycillin (30mg/disc) standard disc was used as the reference.
3.3.9.2 Blank discs
These were used as negative controls which ensure that the residual solvent (left over
the discs even after air-drying) and the filter paper were not active themselves.
3.3.10 Preparation of sample discs with test samples
Measured amount of each test sample was dissolved in specific volume of solvent to obtain
the desired concentrations in an aseptic condition. Sterilized metrical (BBL, Cocksville, USA) filter
paper discs were taken in a blank petridish under the laminar hood. Then discs were soaked with
solutions of test samples and dried.
3.3.10.1 Preparation of sample discs with test samples C.equisetifolia
Methanol crude extract(CTA), pet ether fraction of methanol extract(PETA), carbon tetra
chloride fraction of methanol extract(CTTA), chloroform fraction of methanol extract
(CFTA), ethyl acetate fraction of methanol extract (EATA) were tested for antimicrobial
activity against a number of both gram positive and gram negative bacteria and fungi.
The amount of sample per disc was 500 mg.
3.3.10.2 Preparation and application of the test samples
The test samples were weighed accurately and calculated amounts of the solvents were added
accordingly using micropipette to the dried samples to get desired concentrations. The test samples
were applied to previously sterilized discs using adjustable micropipette under aseptic conditions.
3.3.11 Diffusion and Incubation
The sample discs, the standard antibiotic discs and the control discs were placed gently on the
previously marked zones in the agar plates pre-inoculated with test bacteria and fungi. The
plates were then kept in a refrigerator at 4 0C for about 24 hours upside down to allow
sufficient diffusion of the materials from the discs to the surrounding agar medium. The
plates were then inverted and kept in an incubator at 370C for 24 hours.
3.3.12 Determination of antimicrobial activity by the zone of inhibition
The antimicrobial potency of the test agents are measured by their activity to prevent
the growth of the microorganisms surrounding the discs which gives clear zone of
inhibition. After incubation, the Antimicrobial activities of the test materials were
determined by measuring the diameter of the zones of inhibition in millimeter with a
transparent scale.
Chapter Four
BRINE SHRIMP LETHALITY BIOASSAY
4.1 INTRODUCTION
Bioactive compounds are always toxic to living body at some higher doses and it justifies the
statement that 'Pharmacology is simply toxicology at higher doses and toxicology is simply
pharmacology at lower doses. Brine shrimp lethality bioassay (McLaughlin, 1990; Persoone,
1980) is a rapid and comprehensive bioassay for the bioactive compound of the natural and
synthetic origin. By this method, natural product extarcts, fractions as well as the pure
compounds can be tested for their bioactivity. In this method, in vivo lethality in a simple
zoological organism (Brine shrimp nauplii) is used as a favorable monitor for screening and
fractionation in the discovery of new bioactive natural products.
This bioassay indicates cytotoxicity as well as a wide range of pharmacological activities
such as antimicrobial, antiviral, pesticidal & anti-tumor etc. of the compounds (Meyer, 1982;
McLaughlin, 1988).
Brine shrimp lethality bioassay technique stands superior to other
cytotoxicity testing procedures because it is rapid in process, inexpensive
and requires no special equipment or aseptic technique. It utilizes a large
number of organisms for statistical validation and a relatively small
amount of sample. Furthermore, unlike other methods, it does not require
animal serum.
4.2 PRINCIPLE
Brine shrimp eggs are hatched in simulated sea water to get nauplii. Test
samples are prepared by dissolving in DMSO and by the addition of
calculated amount of DMSO, desired concentration of the test sample is
prepared. The nauplii are counted by visual inspection and are taken in
vials containing 5 ml of simulated sea water. Then samples of different
concentrations are added to the marked vials through micropipette. The
vials are then left for 24 hours and then the nauplli are counted again to
find out the cytotoxicity of the test agents.4.3 MATERIALS
01. Artemia salina leach (brine shrimp
eggs)
05. Lamp to attract shrimps
02. Sea salt (NaCl) 06. Micropipette
03. Small tank with perforated dividing
dam to hatch the shrimp
07. Pipettes
04. Test samples of experimental plants:
CTA, PETA, CTTA, CFTA, EATA
08. Glass vials
09. Magnifying glass
4.3.1 Test Samples
Table 4.1: Test samples of Casuarina equisetifolia:
Code no. Test sample Amount (mg)
CTA Methanol Crude extract 4.0
PETA Pet ether fraction of methanol extract 4.0
CTTA Carbon tetra chloride fraction of methanol extract 4.0
CFTA Chloroform fraction of methanol extract 4.0
EATA Ethyl acetate fraction of methanol extract 4.0
4.4 PROCEDURE
4.4.1 Preparation of sea water
76 gm sea salt (pure NaCl) was weighed, dissolved in two liter of distilled water and filtered
off to get clear solution.
4.4.2 Hatching of brine shrimp
Artemia salina leach (brine shrimp eggs) collected from pet shops was used as the test
organism. Seawater was taken in the small tank and shrimp eggs were added to one side of
the tank and then this side was covered. Two days were allowed to hatch the shrimp and to be
matured as nauplii. Constant oxygen supply was carried out through the hatching time. The
hatched shrimps were attracted to the lamp through the perforated dam and they were taken
for experiment.
With the help of a pasteur pipette 10 living shrimps were added to each of the test tubes
containing 5 ml of seawater.
4.4.3 Preparation of test solutions with samples of experimental plants
Clean test tubes were taken. These test tubes were used for ten different concentrations (one
test tube for each concentration) of test samples and ten test tubes were taken for standard
drug Vincristine for ten concentrations of it and another one test tubes for control test.
All the test samples (CTA, PETA, CTTA, CFTA, EATA) of 4 mg were taken and dissolved
in 200 ml of pure dimethyl sulfoxide (DMSO) in vials to get stock solutions. Then 100 ml of
solution was taken in test tube each containing 5ml of simulated seawater and 10 shrimp
nauplii. Thus, final concentration of the prepared solution in the first test tube was 400 mg/ml.
Then a series of solutions of varying concentrations were prepared from the stock solution by
serial dilution method. In each case 100 ml sample was added to test tube and fresh 100ml
DMSO was added to vial. Thus the concentrations of the obtained solution in each test tube
shown in the table.
Table 4.2: Concentrations of the obtained solution in each test tube
Test tube No. Concentration
mg/ml
Test tube No. Concentration
mg/ml
01 400 06 12.5
02 200 07 6.25
03 100 08 3.125
04 50 09 1.5625
05 25 10 0.7813
4.4.4 Preparation of control group
Control groups are used in cytotoxicity study to validate the test method and ensure that the
results obtained are only due to the activity of the test agent and the effects of the other
possible factors are nullified. Usually two types of control groups are used
i) Positive control
ii) Negative control
4.4.4.1 Preparation of positive control group
Positive control in a cytotoxicty study is a widely accepted cytotoxic agent and the result of
the test agent is compared with the result obtained for the positive control. In the present
study vincristine sulphate is used as the positive control. Measured amount of the vincristine
sulphate is dissolved in DMSO to get an initial concentration of 20 mg/ml from which serial
dilutions are made using DMSO to get 10 mg/ml, 5 mg/ml, 2.5mg/ml, 1.25 mg/ml, 0.625
mg/ml, 0.3125 mg/ml, 0.15625 mg/ml, 0.078125 mg/ml, 0.0390 mg/ml. Then the positive
control solutions are added to the premarked vials containing ten living brine shrimp nauplii
in 5 ml simulated sea water to get the positive control groups.
4.4.4.2 Preparation of negative control group
100 ml of DMSO was added to each of three pre-marked glass vials containing 5 ml of
simulated sea water and 10 shrimp nauplii to use as control groups. If the brine shrimps in
these vials show a rapid mortality rate, then the test is considered as invalid as the nauplii
died due to some reason other than the cytotoxicity of the compounds.
4.4.5 Counting of nauplii
After 24 hours, the vials were inspected using a magnifying glass and the number of survived
nauplii in each vial was counted. From this data, the percent (%) of lethality of the brine
shrimp nauplii was calculated for each concentration.
Chapter Five
RESULTS AND DISCUSSION
5.1 RESULTS AND DISCUSSION OF CHEMICAL INVESTIGATION OF THE
PLANT MATERIAL
5.1.1 Plant material
A species of the Casuarinaceae family, Casuarina equisetifolia, has been investigated in this
work. The plant part used was the leaves.
5.1.2 Extraction of the plant material
Fresh leaves of Casuarina equisetifolia was collected, dried and ground to a coarse powder.
The powder sample (1200 g) was subjected to cold extraction with methanol for about 8 days.
The methanol extract was then subjected to column chromatography for isolation of
compounds.
5.1.3 Isolation and characterization of compounds
From the extractives pure compounds were isolated applying various chromatographic
techniques. The isolated pure compounds were then characterized using various
spectroscopic techniques.
5.2 CHARACTERIZATION OF ISOLATED COMPOUNDS FROM Casuarina
equisetifolia
Characterization of the isolated compound is made with the help of NMR spectroscopy.
5.2.1 Characterization of TA-1101 as β-amyrin (12-Oleanen-3-beta-ol).
Compound TA-1101 (Fig. 5.1) was isolated from the column fraction of methanol crude
extract by elution with petroleum ether 80-20% Ethyl acetate. It was obtained as colorless
mass. It appeared in the preparative thin layer chromatography using 5% Ethyl acetate in
Toluene. Under UV light at 365 nm it is detected. The 1H NMR spectrum exhibited few non-
characteristics signals due to the presence of some impurities. Compound TA-1101 was
soluble in dichloromethane, chloroform and ethyl acetate. Spraying the developed plate with
Vanillin/H2SO4 spray reagent, followed by heating gave a purple color.
Figure 5.1: TA-1101
The 1H NMR spectrum (400 MHz , CDCl3) of TA-1101 (Table-5.1, Fig:5.1 ) the 1HNMR
chemical shifts (δ) are shown in table 5.1.
The 1H NMR of TA-1101 in CDCl3 displayed the characteristic olefinic proton resonance as a
triplet (J=3.7 Hz) at δ 5.18 and the oxymethine proton signal as a double doublet (J= 11.0,
5.0) at δ 3.21. In addition, the 1H NMR spectrum showed signals for eight methyl groups at δ
1.13 (3H), 0.99 (3H), 0.99 (3H), 0.93(3H), 0.82 (3H ×2) and 0.79 (3H ×2).
The 1H NMR spectrum are found to identical to those reported for the compound was
previously reported from the plant Bursera serrata (Ereil et al., 2004), Gentiana straminea
(www.paper.edu.cn, 2009) and from more other plants (Dictionary of natural plants,
Chapman and Hall, 2001) . On this basis TA-1101 was identified as β-amyrin (12-Oleanen-3-
beta-ol). Although it is known natural product, this is the first report of its occurrence from
the family of Casuarinaceae, Casuarina equisetifolia on the best of available information.
Fig
5.2
(a):
1H N
MR
Spec
trum
For
Com
poun
d TA
-110
1
Fig
5.2
(b) :
1H
NM
R Sp
ectr
um F
or C
ompo
und
TA-1
101
Fig
5.2
(c) :
1H
NM
R Sp
ectr
um F
or C
ompo
und
TA-1
101
Table 5.1: Comparison between the 1H NMR spectral data of TA-1101 (400 MHz,
CDCl3) and β-amyrin (12-Oleanen-3-beta-ol). (400 MHz, CDCl3) (Muhammad Riaz et
al. 2001)
ProtonsTA-1101
(H in ppm)
β-amyrin (12-Oleanen-3-beta-ol)
(H in ppm)
H-12 5.18(2H, t) 5.18 (1H, t, J = 3.7 Hz)
H-3 3.21(1H, dd, J = 1.2, 5.2 Hz) 3.23 (1H, dd, J = 11.0,5.0 Hz)
H3-27 1.13 (3H ) 1.07(3H ),
H3-23, H3-26 0.99 (6H ) 1.00(3H ), 0.99(3H )
H3-25 0.93 (3H ) 0.92 (3H )
H3-29, H3- 30 0.82 (6H ) 0.80(6H )
H3-28, H3-24 0.79 (6H ) 0.79 (6H )
5.2.2 Characterization of TA-1102 as 3-(p-hydroxycinnamyl)-betulin.
Compound TA-1102 (Figure-5.3) was isolated from the column fraction of methanol crude
extract by elution with petroleum ether/ ethyl acetate 52.5-47.5%. It was obtained as white
gum. It appeared as a blue spot on the TLC plate ( 80% toluene/ ethyl acetate) under UV light
at 254 nm. It exhibited a blue fluorescence under UV light at 365 nm. The 1H NMR spectrum
exhibited few non-characteristics signals due to the presence of some impurities. The
compound was identified as 3-(p-hydroxycinnamyl)-betulin by comparing the 1H NMR data
(Table 5.2) with those published for this betulin (Muhammad Riaz et al., 2001) and para
hydroxycinnamic acid (Varadarassou Mouttaya Mounnissamy et al.2010).
Fig 5.3: 3-(p-hydroxycinnamyl)-betulin.
The 1H NMR spectrum (400 MHz, CDCl3) of TA-1102 (Table 5.2, Figure 5.2) displayed
signals characteristics of a 3-(p-hydroxycinnamyl)-betulin. The spectrum revealed a double
doublet at 4.63 (1H, dd, J=8.4,8.0 Hz) and a doublet 3.56 (1H, d, J=11.2 Hz)
characteristic of H-29 and H-28 protons respectively of betulin. The presence of doublet at
7.43 and 6.83 were attributable to H- α and H-6 of cinnamyl group. Absence of H-3 proton
suggest that cinnamyl group is attached with betulin at this carbon.
Finally, the structure of TA-1102 was confirmed by comparing its 1H NMR data to those
reported for betulin (Muhammad Riaz et al., 2001) and para hydroxycinnamic acid
(Varadarassou Mouttaya Mounnissamy et al.2010). On this basis TA-1102 was identified as
3-(p-hydroxycinnamyl)-betulin. This is the first report of TA-1102 from Casuarinaceae
family.
Fig
5.4
(a) :
1H
NM
R Sp
ectr
um F
or C
ompo
und
TA-1
101
Fig
5.4
(a) :
1H
NM
R Sp
ectr
um F
or C
ompo
und
TA-1
101
Table 5.2: Comparison between the 1H NMR spectral data of TA-1102 (400 MHz,
CDCl3) and p-hydroxycinnmic acid (Varadarassou Mouttaya Mounnissamy et al. 2010)
and betulin (Muhammad Riaz et al., 2001). (500 MHz, CDCl3)
Protons (H in ppm) betulin (H in ppm)
H-29 4.63 ( 2H, d, J = 9.6 Hz) 4.81 ( 2H, m)
H-28 3.56 ( 2H, d, J = 11.2.6 Hz) 3.52( 2H, d, J = 10.7 Hz)
H-3 …………… 3.18 ( 1H, d, J = 4.4,10.4 Hz)
30-CH3 1.72 ( 3H, s) 1.75 ( 3H, s)
26-CH3 1.18 ( 3H, s) 1.08 (3H, s)
25-CH3, 27-CH3 0.99 ( 3H, s) 1.02 (3H, s)
24-CH3 0.92 ( 3H, s) 0.92 (3H, s)
23-CH3 0.88 ( 3H, s) 0.87 (3H, s)
p-hydroxycinnmic acid
H-α 7.43 ( 2H, d, J = 11.2.6 Hz) 7.38 (d, J=16.0 Hz, 1H,)
H-2 6.84 ( 2H, d, J = 8.4 Hz) 6.99 (d, J=2.3 Hz, 1H,)
H-6 6.82 ( 2H, d, J = 8.4 Hz) 6.92 (dd, J=8.4, 2.3 Hz, 1H,)
H-ß. 6.27 ( 1H, d, J = 16.0 Hz) 6.15 (d, J=16.05 Hz, 1H, )
5.3 RESULTS AND DISCUSSION OF IN VITRO ANTIMICROBIAL
SCREENING OF Casuarina equisetifolia
Methanol crude extract (CTA), petrolium ether fraction of methanol extract (PETA), carbon
tetra chloride fraction of methanol extract (CTTA), chloroform fraction of methanol extract
(CFTA), ethyl acetate fraction of methanol extract (EATA) were tested for antibacterial and
antifungal activities against a number of Gram positive bacteria, Gram negative bacteria and
fungi respectively. Standard disc of kanamycin (30 μg/disc) and amoxycillin (30 μg/disc)
were used for comparison purpose.
Methanol crude extract, pet ether fraction, carbon tetrachloride, ethyl acetate fraction and
chloroform fractions exhibited poor and mild antimicrobial activity against most of the test
organisms (Table-5.3).The zone of inhibition produced by Methanol, pet ether fraction,
carbon tetrachloride, chloroform and ethyl acetate fractions were found to be 07 – 8 mm, 07 –
9 mm 08 – 11 mm and 7-10 mm respectively at a concentration of 500 μg/disc.
The Methanol crude extract was screened against 08 (eight) test bacteria and 02 (two) fungii.
This fraction showed poor activity against the test bacteria Bacillus subtilis,, , Escherichia
coli, Salmonella typhi, Vibrio mimicus and the fungi Candida albicans and Aspergillus niger.
On the other hand, Bacillus cereus, Bacillus megaterium, Shigella boydii and Staphylococcus
aureus bacteria was found to be resistant to it.
The pet ether fraction of methanol extract (PETA) was screened against 08 (eight) test
bacteria and 02 (two) fungii. This fraction showed poor activity against the test bacteria
Bacillus megaterium, Salmonella typhi. On the other hand, Bacillus cereus, Bacillus subtilis,
Staphylococcus aureus, Shigella boydii, Vibrio mimicus and Escherichia coli bacteria and
the fungi Candida albicans and Aspergillus niger was found to be resistant to it.
The carbon tetra chloride fraction of methanol extract(CTTA) was screened against 08 (eight)
test bacteria and 02 (two) fungii. This fraction showed poor activity against the test bacteria
Bacillus cereus, Bacillus megaterium, , Escherichia coli, Salmonella typhi, Shigella boydii,
Vibrio mimicus and the fungi Candida albicans and On the other hand Bacillus subtilis,
Staphylococcus aureus and the fungi Aspergillus niger was found to be resistant to it.
The chloroform fraction of methanol extract (CFTA) was screened against 08 (eight) test
bacteria and 02 (two) fungii. This fraction showed poor activity against the test bacteria
Bacillus cereus, Bacillus megaterium, Bacillus subtilis,, Staphylococcus aureus, Escherichia
coli, Salmonella typhi, Shigella boydii, Vibrio mimicus and the fungi Candida albicans and
Aspergillus niger.
The ethyl acetate fraction of methanol extract was screened against 06 (six) test bacteria and
01 (one) fungus. This fraction showed poor activity against the test bacteria Bacillus cereus,
Bacillus megaterium, Staphylococcus aureus, Escherichia coli, Shigella boydii, Vibrio
mimicus and the fungi Candida albicans.
Table 5.3 Antimicrobial activity of different fractions of Methanol crude extract of
Casuarina equisetifolia
Test bacteria and fungi
Diameter of Zone of inhibition (mm)
CTA PETA CTTA CFTA EATA
Kanam
ycin
µg/disc
500 500 500 500 500 30
Gram Positive bacteria
Bacillus cereus (BTCC-19) NA NA 7 9 10 39
Bacillus megaterium (BTCC-
18)
NA 7 7 10 7 32
Bacillus subtilis 7 NA NA 9 ND 20
Staphylococcus aureus
(BTCC-43)
NA NA NA 8 8 22
Gram Negative bacteria
Escherichia coli (BTCC-172) 7 NA 7 9 8 23
Salmonella typhi 7 8 7 9 ND 20
Shigella boydii NA NA 9 10 7 26
Vibrio mimicus 8 NA 7 11 9 24
Fungi
Candida albicans 7 NA 7 9 8 24
Aspergillus niger 7 NA NA 9 ND 32
“NA” Indicates ‘No activity’, “ND” Indicates ‘Not done’
5.4 RESULTS AND DISCUSSION OF BRINE SHRIMP LETHALITY
BIOASSAY
Bioactive compounds are almost always toxic at higher dose. Thus, in vivo lethality in a
simple zoological organism can be used as a convenient informant for screening and
fractionation in the discovery of new bioactive natural products.
In the present bioactivity study all the crude extracts, column fractions and pure compounds
showed positive results indicating that the test samples are biologically active. Each of the
test sample showed different mortality rates at different concentrations. Plotting of log of
concentration versus percent mortality for all test samples showed an approximate linear
correlation. From the graphs, the median lethal concentration (LC50, the concentration at
which 50% mortality of brine shrimp nauplii occurred) was determined for the samples. The
positive control groups showed non linear mortality rates at lower concentrations and linear
rates at higher concentrations. There was no mortality in the negative control groups
indicating the test as a valid one and the results obtained are only due to the activity of the
test agents.
5.5 Results and Discussion of the test samples of Casuarina equisetifolia
Methanol Crude extract(CTA), pet ether fraction of methanol extract(PETA), carbon
tetrachloride fraction of methanol extract(CTTA), chloroform fraction of methanol extract
(CFTA), ethyl acetate fraction of methanol extract (EATA) were screened by brine shrimp
lethality bioassay.
From the bioassay the LC50 value for the methanol crude extract(CTA), pet ether fraction of
methanol extract(PETA), carbon tetrachloride fraction of methanol extract(CTTA),
chloroform fraction of methanol extract (CFTA), ethyl acetate fraction of methanol extract
(EATA) were found to be 6.02 μg/ml (Table-5.5, Figure-5.6), 630.96 μg/ml (Table-5.6,
Figure-5.7), 3.72 μg/ml (Table-5.7, Figure-5.8), 17.78 μg/ml (Table-5.8, Figure-5.9), 2.51
μg/ml (Table-5.9, Figure-5.10) respectively. It is evident that all the test samples were lethal
to brine shrimp nauplii. However, methanol crude extract(CTA), carbon tetrchloride fraction
of methanol extract(CTTA), ethyl acetate fraction of methanol extract (EATA) were
moderately active and the pet ether fraction of methanol extract(PETA) was less active.
Carbon tetrachloride fraction of methanol extract and ethyl acetate fraction of methanol
extract quite potent activity in brine shrimp lethality bioassay. This positive result suggests
that these fractions may contain antitumor or pesticidal compounds. However, this cannot be
confirmed without further higher and specific tests.
5.5.1 VINCRISTINE SULPHATE
Table 5.4: Effects of Vincristine Sulphate on brine shrimp nauplii
Sl. No. Conc (C)
(mg/ml)
Log C % Mortality
LC50 (mg/ml)
01 20 1.30 100
0.33
02 10 1 100
03 5 0.698 90
04 2.5 0.397 80
05 1.25 0.096 70
06 0.625 -0.204 60
07 0.3125 -0.488 40
08 0.15625 -0.806 40
09 0.07812 -1.10723 30
10 0.0390 -1.4089 20
Figure 5.5: Effects of Positive control on brine shrimp nauplii
Calculation:
LC50 (mg/ml) = antilog (-0.48)
= 0.33 g/ml
5.5.2 Samples Code: CTA
Table 5.5: Effects of methanol crude extract of Casuarina equisetifolia on brine shrimp nauplii
Sl. No. Conc (C)
(mg/ml)
Log C % Mortality
LC50 (mg/ml)
01 400 2.60 100
6.02
02 200 2.30 100
03 100 2.00 100
04 50 1.70 80
05 25 1.40 70
06 12.5 1.10 60
100
90
80
70
60
50
40
30
20
10
0
% M
orta
lity
Log C
07 6.25 0.80 60
08 3.125 0.50 40
09 1.5625 0.20 30
10 0.78 -0.10 20
Figure 5.6: Effects of methanol crude extract of Casuarina equisetifolia on brine shrimp nauplii
Calculation:
LC50 (mg/ml) = antilog (0.78)
= 6.02 g/ml
5.5.3 Samples Code: PETA
Table 5.6: Effects of petroleum ether fraction of methanol extract of Casuarina equisetifolia on brine shrimp naupliiSl. No. Conc (C)
(mg/ml)
Log C % Mortality
LC50 (mg/ml)
100
90
80
70
60
50
40
30
20
10
0
Log C
% M
orta
lity
01 400 2.60 50
630.96
02 200 2.30 40
03 100 2.00 30
04 50 1.70 30
05 25 1.40 20
06 12.5 1.10 30
07 6.25 0.80 20
08 3.125 0.50 10
09 1.5625 0.20 0
10 0.78 -0.10 0
Figure 5.7: Effects of Petroleum ether fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii
Calculation:
LC50 (mg/ml) = antilog (2.8)
100
90
80
70
60
50
40
30
20
10
0 Log C
% M
orta
lity
= 630.96 mg/ml
5.5.4 Samples Code: CTTA
Table 5.7: Effects of carbon tetra chloride fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii
Sl. No. Conc (C)
(mg/ml)
Log C % Mortality
LC50 (mg/ml)
01 400 2.60 100
3.72
02 200 2.30 100
03 100 2.00 80
04 50 1.70 70
05 25 1.40 70
06 12.5 1.10 60
07 6.25 0.80 50
08 3.125 0.50 50
09 1.5625 0.20 40
10 0.78 -0.10 40
Figure 5.8: Effects of Carbon tetra chloride fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii
Calculation:
LC50 (mg/ml) = antilog (0.57)
= 3.72 g/ml
5.5.5 Samples Code: CFTA
Table 5.8: Effects of chloroform fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii
Sl. No. Conc (C)
(mg/ml)
Log C % Mortality
LC50 (mg/ml)
01 400 2.60 100
17.78
02 200 2.30 80
03 100 2.00 70
04 50 1.70 60
05 25 1.40 50
06 12.5 1.10 40
100
90
80
70
60
50
40
30
20
10
0
Log C
% M
orta
lity
07 6.25 0.80 40
08 3.125 0.50 30
09 1.5625 0.20 20
10 0.78 -0.10 20
Figure 5.9: Effects of chloroform fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii
Calculation:
LC50 (mg/ml) = antilog (1.25)
= 17.78 g/ml
5.5.6 Samples Code: EATA
Table 5.9: Effects of ethyl acetate fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii
100
90
80
70
60
50
40
30
20
10
0Log C
% M
orta
lity
Sl. No. Conc (C)
(mg/ml)
Log C % Mortality
LC50 (mg/ml)
01 400 2.60 100
2.51
02 200 2.30 100
03 100 2.00 100
04 50 1.70 100
05 25 1.40 90
06 12.5 1.10 80
07 6.25 0.80 70
08 3.125 0.50 60
09 1.5625 0.20 40
10 0.78 -0.10 30
Figure 5.10: Effects of ethyl acetate fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii
100
90
80
70
60
50
40
30
20
10
0Log C
% M
orta
lity
Calculation:
LC50 (mg/ml) = antilog (0.4)
= 2.51 mg/ml
Table 5.10: Regression line equation and Value of R2 for different test samples:
Code no. Test sample Regression line equation Value of R2
Vincristine Sulphate y = 32.03x + 64.67 0.979
CTA Methanol Crude extract y = 31.91x + 26.10 0.964
PETA Pet ether fraction of methanol
extract
y = 17.17x + 1.535 0.908
CTTA Carbon tetra chloride fraction
of methanol extract
y = 23.83x + 36.20 0.950
CFTA Chloroform fraction of
methanol extract
y = 28.48x + 15.39 0.957
EATA Ethyl acetate fraction of
methanol extract
y = 27.27x + 42.90 0.889
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52. CONCLUSION
53. Two compounds were isolated in my work ‘Chemical and biological investigations of
one species of Casuarinaceae, Casuarina equisetifolia’. One has been characterized
as β-amyrin (12-Oleanen-3-beta-ol) and the other as 3-(p-hydroxycinnamyl)-betulin.
54. From literature information, beta-amyrin have high hepatoprotective potential against
toxic liver injury and suggest that it’s isolation from the plant may be implemented to
obtain medicinal agent and developing drugs for treatment of liver disorders.
Compounds synthesized from Betulinic and Betulinic acid are now a days being used
as anti-AIDS and anti-cancer agents. So further investigation of this compound is
recommended against tumor cell lines of different histogenic origins.
55. Different fractions of the crude methanol extract of the plant show moderate activities
against antibacterial and antifungal agents. The evidence of cytotoxicity suggests the
presence of anti-tumor and pesticidal agents, which encourages further antitumor
investigation of the plant constituents.
56. So, advanced research on the constituents obtained in my work might have effect on
the antitumor and antiAIDS treatment. It can be hoped that its high potential soon be
realized and it will contribute in the medicinal sector.