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.