Chemical and Biological Investigation of Casuarina equisetifolia L.

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: Division: Class: Order:

Plantae Magnoliophyta Magnoliopsida 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 Species • Casuarina cristata Miq. Northeastern

•

Genera: Allocasuarina Species • Allocasuarina acuaria

Australia (Queensland, New South

•

Allocasuarina acutivalvis

Wales).

•

Allocasuarina brachystachya

Casuarina cunninghamiana Miq.

•

Allocasuarina campestris

•

Allocasuarina corniculata

•

Allocasuarina crassa

•

Allocasuarina decaisneana

•

Allocasuarina decussata

•

Allocasuarina defungens

•

Allocasuarina dielsiana

•

Allocasuarina diminuta

•

Allocasuarina distyla

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

(scrub sheoak)

Guinea. •

•

•

Casuarina junghuhniana Miq.

•

Allocasuarina drummondiana

Indonesia.

•

Allocasuarina duncanii (Duncan's sheoak)

Casuarina obesa Miq. Southern Australia (southwestern Western

•

Allocasuarina emuina

Australia, New South Wales [one site,

•

Allocasuarina eriochlamys

now extinct], Victoria).

•

Allocasuarina fibrosa

Casuarina oligodon L.A.S.Johnson.

•

Allocasuarina filidens

•

Allocasuarina fraseriana

New Guinea. Casuarina

pauper

F.Muell.

ex

(common sheoak)

L.A.S.Johnson. Interior Australia.

•

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 Species • Casuarina cunninghami

Genera: Allocasuarina Species • Allocasuarina acuaria

•

Casuarina equisetifolia L,

•

Allocasuarina defungens

•

Casuarina glauca

•

Allocasuarina distyla (scrub sheoak)

•

Casuarina junghuhniana

•

Allocasuarina luehmannii (bull-oak)

•

Casuarina oligodon.

•

Allocasuarina muelleriana (slaty sheoak)

•

Casuarina pauper

•

Allocasuarina striata (small bull-oak) Allocasuarina torulosa (forest

1.2 The Genus Casuarina L. – A brief discuss Kingdom: Order: Family: Genus:

Plantae Fagales Casuarinaceae 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 NAME

LOCAL

MEDICINAL USES

NAME

Casuarina equisetifolia Jhau tree

Effective

antibacterial,

anticancer,

L Casuarina cristata Casuarina glauca S. Casuarina

antitumor agent. Tonic,vulnerary,antisour,antiscorbutic. Used in colic pain, used as febge. Acrid,stimulant,diuretic,usedin

cunninghamiana.

uterinedisorder,dysentery,liver disease,pain,skindisease,tooth

and

disease,

Casuarina oligodon. Casuarina pauper F Casuarina obesa Casuarina

wound, fish poison. Used in dysentery. Used in stomach ache. Tonic, vulnerary. Used in colic.

junghuhniana. Casuarina grandis L Allocasuarina acuaria Allocasuarina

Applied to swelling. Effective against stomach debility Has mild depressant action on central nervous

defungens Allocasuarina

system Effective

luehmanni Allocasuarina torulosa Allocasuarina

antitumor agent. Effective against stomach debility Used in dysentery Tonic, vulnerary.

muelleriana Allocasuarina striata

Has mild depressant action on central nervous system

antibacterial,

anticancer,

and

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 β-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 3β-hydroxytriterpenoids which by oxidation and reduction can be transformed into 3α-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. β-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 lowdensity 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)

Casuarina oligodon.

Harborne & Mabry, 1982

Casuarina pauper F

Harborne & Mabry, 1982

Casuarina obesa

Harborne & Mabry, 1982

Casuarina junghuhniana.

Harborne & Mabry, 1982

Manihot Podocarpus flavone A(53) Podocarpus flavone B(54) Eriodictyol (55)

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 seems to work as a pesticide in the plants that produce it.

Coumarin:

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

Distribution:

sugar glucose (coumarin glycoside). 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 foredunes 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 : Australia Bangladesh Brunei Cambodia Fiji Indonesia Malaysia New Zealand Papua New Guinea Philippines Samoa Solomon Islands Thailand Tonga Vanuatu Vietnam Exotic : Antigua and Barbuda Bahamas Benin Burkina Faso Cameroon Central African Republic Chad China Congo Cote d'Ivoire Cuba Democratic Republic of Congo Djibouti Dominica Dominican Republic Eritrea Ethiopia Gabon Gambia Ghana Grenada Guadeloupe

Guinea Guinea-Bissau Haiti India Israel Jamaica Kenya Liberia Madagascar Mali Martinique Mauritania Montserrat Myanmar Netherlands Antilles Niger Nigeria Pakistan Puerto Rico Senegal Sierra Leone Somalia South Africa Sri Lanka St Kitts and Nevis St Lucia St Vincent and the Grenadines Sudan Tanzania Togo Trinidad and Tobago Uganda United 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,

tannin,

beta-sitosterol,

proantho-cyanidins,

kaempferol and glycosides,

juglanin,

quercetin,

citrulline and amino acids,

cupressuflavone,

afzelin,

isoquercitrin,

casuarine,

several common triterpenoids,

gallicin,

trifolin,

catechol derivatives,

catechin and epicatechin,

gentisic acid,

cholesterol,

hydroquinone,

stigmasterol,

nictoflorin,

campesterol,

rutin,

cholest-5-en-3-beta-ol derivatives,

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

References

Casuarina

Gallic acid

species Tittle fruits

Khan et al., 1990;

equisetifolia

Protocatechuic acid

and wood.

Bhattacharyya et

Hydroquinons

al., 1984;

Fuglanin Afzelin (+)

Talapatra et al.,

Catechin (-)

1969;

Cpicatechin (+) Gallacatechin

Paul et al., 1968, 1969;

Tryptophen

Leaves.

Das et al., 1963;

Leucin Valine

Aher et al.,2009;

Tyrosine Glycine

Roux, 1957;

Quercetin Catechin Gallic acid

Bark

Madhulata et al., 1985;

Ellagic acid

The following phytoconstituents were also isolated from the plant so far, kaempferol (ElAnsary 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) Figure 1.11: (a) Catechin

(b) (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).

H

HO

O O

O O

O

OH

H O

OCOCH3 OH

O 1

2 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 ).

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, antidiarrhea, 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 costeffective 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 LC 50 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 below2.2 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) 째C 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 40 0 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, 40째-60째C), 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)

20 x 5

Thickness (mm)

Amount of silica gel/plate (gm)

0.3

0.9

0.4

1.2

0.5

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 110 oC 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. Rf =

Distance from sample front Distance from solvent front

Usually, the Rf value is constant for any given compound and it corresponds to a physical property of that compound. Solvent front Distance from solvent front, A

Compound

Distance from sample front, B

Compound Baseline

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 (25-100Âľm, Pharmacia, Sweden) was suspended in petroleum ether and the column was packed with this suspended gel.

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:

Leaves of C. equisetifolia

Dried leaves

Leaves Powder

Extraction with Methanol

Extract

Concentrated Mass

Subjected CC & eluted with PE, EA, MeOH

01A

01B

02A

19A

19B

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 Solvent systems no.

Volume collected (ml)

1

Petroleum ether : ethyl acetate = 80:20

100

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 further investigation.

Table 2.3 List of new fraction codes

for

Wt. of the extracts Column fractions

New code (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

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.

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. TA1102 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 PF 254) 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 (Âľg/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 Refrigerator

Incubator

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

Gram negative Fungi

Bacteria Bacillus cereus Bacillus megaterium

Bacteria Escherichia coli

Candida albicans

Pseudomonas aeruginosa

Aspergillus niger

Bacillus subtilis Staphylococcus aureus Sarcina lutea

Salmonella paratyphi

Sacharomyces cerevacae

Salmonella typhi Shigella 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 P H (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 (30Âľg/disc) and amoxycillin (30Âľg/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.

Preparation of sample discs with test samples C.equisetifolia

1 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 Âľg. 3.3.10.

Preparation and application of the test samples

2 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

05. Lamp to attract shrimps

eggs) 02. Sea salt (NaCl) 03. Small tank with perforated dividing

06. Micropipette 07. Pipettes

dam to hatch the shrimp 04. Test samples of experimental plants:

08. Glass vials

CTA, PETA, CTTA, CFTA, EATA

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.

PROCEDURE

4 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 µl of pure dimethyl sulfoxide (DMSO) in vials to get stock solutions. Then 100 µl 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 µg/ml.

Then a series of solutions of varying concentrations were prepared from the stock solution by serial dilution method. In each case 100 µl sample was added to test tube and fresh 100µl 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

Test tube No.

µg/ml

Concentration µg/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 µg/ml from which serial dilutions are made using DMSO to get 10 µg/ml, 5 µg/ml, 2.5µg/ml, 1.25 µg/ml, 0.625 µg/ml, 0.3125 µg/ml, 0.15625 µg/ml, 0.078125 µg/ml, 0.0390 µg/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 µl 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 noncharacteristics 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 NMR Spectrum For Compound TA-1101

FigNMR 5.2 (b) : 1H NMR ForTA-1101 Compound TA-1101 Fig 5.2 (c) : 1H Spectrum ForSpectrum Compound

Fig 5.2 (d) : 1H NMR Spectrum For Compound TA-1101 Fig 5.2 (c) : 1H NMR Spectrum For Compound TA-1101

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) TA-1101

β-amyrin (12-Oleanen-3-beta-ol)

(δH in ppm)

(δH in ppm)

H-12

δ 5.18(2H, t)

δ 5.18 (1H, t, J = 3.7 Hz)

H-3

δ 3.21(1H, dd, J = 1.2, 5.2 Hz)

δ 3.23 (1H, dd, J = 11.0,5.0 Hz)

H3-27

δ 1.13 (3H )

δ 1.07(3H ),

H3-23, H3-26

δ 0.99 (6H )

δ 1.00(3H ), 0.99(3H )

H3-25

δ 0.93 (3H )

δ 0.92 (3H )

H3-29, H3- 30

δ 0.82 (6H )

δ 0.80(6H )

H3-28, H3-24

δ 0.79 (6H )

δ 0.79 (6H )

Protons

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 δ 4.63 (1H, dd, J=8.4,8.0 Hz)

and a doublet δ 3.56 (1H, d,

J=11.2 Hz)

characteristic of H-29 and H-28 protons respectively of betulin. The presence of doublet at δ 7.43 and δ 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 NMR Spectrum For Compound TA-1101

Fig 5.4 (a) : 1H NMR Spectrum For Compound TA-1101

Fig 5.4 (c): 1H NMR Spectrum For Compound TA-1101

Table 5.2: Comparison between the

1

H 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

(δH in ppm)

betulin (δH in ppm)

H-29

δ 4.63 ( 2H, d, J = 9.6 Hz)

δ 4.81 ( 2H, m)

H-28

δ 3.56 ( 2H, d, J = 11.2.6 Hz)

δ 3.52( 2H, d, J = 10.7 Hz)

H-3

……………

δ 3.18 ( 1H, d, J = 4.4,10.4 Hz)

30-CH3

δ 1.72 ( 3H, s)

δ 1.75 ( 3H, s)

26-CH3

δ 1.18 ( 3H, s)

δ 1.08 (3H, s)

25-CH3, 27-CH3

δ 0.99 ( 3H, s)

δ 1.02 (3H, s)

24-CH3

δ 0.92 ( 3H, s)

δ 0.92 (3H, s)

23-CH3

δ 0.88 ( 3H, s)

δ 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

Diameter of Zone of inhibition (mm) Kanam EATA ycin µg/disc 500 30

CTA

PETA

CTTA CFTA

500

500

500

NA

NA

7

9

10

39

7

7

10

7

32

NA

NA

9

ND

20

NA

NA

8

8

22

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

Candida albicans

7

NA

7

9

8

24

Aspergillus niger

7

NA

NA

9

ND

32

Test bacteria and fungi

500

Gram Positive bacteria Bacillus cereus (BTCC-19)

Bacillus megaterium (BTCC- NA 18) Bacillus subtilis Staphylococcus

7 aureus NA

(BTCC-43) Gram Negative bacteria

Fungi

“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 (LC 50, 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)

Log C

% Mortality

(µg/ml) 01

20

1.30

100

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

100 90 80

% Mortality

70 60 50 40 30 20 10 0

Log C

Figure 5.5: Effects of Positive control on brine shrimp nauplii Calculation: LC50 (µg/ml) = antilog (-0.48) = 0.33 µg/ml

LC50 (µg/ml)

0.33

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)

Log C

(µg/ml) 01

400

2.60

100

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

07

6.25

0.80

60

08

3.125

0.50

40

09

1.5625

0.20

30

10

0.78

-0.10

20

100 90 80 70 60 % Mortality

% Mortality

50 40 30 20 10 0 Log C

LC50 (µg/ml)

6.02

Figure 5.6: Effects of methanol crude extract of Casuarina equisetifolia on brine shrimp nauplii Calculation: LC50 (µg/ml) = antilog (0.78) = 6.02 µg/ml 5.5.3 Samples Code: PETA Table 5.6: Effects of petroleum ether fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii Sl. No.

Conc (C)

Log C

(µg/ml)

% Mortality

01

400

2.60

50

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

LC50 (µg/ml)

630.96

100 90 80 % Mortality

70 60 50 40 30 20 10 0

Log C

Figure 5.7: Effects of Petroleum ether fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii Calculation: LC50 (µg/ml) = antilog (2.8) = 630.96 µg/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)

Log C

(µg/ml)

% Mortality

01

400

2.60

100

02

200

2.30

100

03

100

2.00

80

04

50

1.70

70

05

25

1.40

70

LC50 (µg/ml)

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

3.72

100 90 80

% Mortality

70 60 50 40 30 20 10 0 Log C

Figure 5.8: Effects of Carbon tetra chloride fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii Calculation: LC50 (µg/ml) = antilog (0.57) = 3.72 µg/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)

Log C

(µg/ml) 01

400

2.60

% Mortality 100

LC50 (µg/ml)

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

07

6.25

0.80

40

08

3.125

0.50

30

09

1.5625

0.20

20

10

0.78

-0.10

20

17.78

100 90 80 % Mortality

70 60 50 40 30 20 10 0

Log C

Figure 5.9: Effects of chloroform fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii Calculation: LC50 (Âľg/ml) = antilog (1.25) = 17.78 Âľg/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 Sl. No.

Conc (C)

Log C

(Âľg/ml)

% Mortality

01

400

2.60

100

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

LC50 (Âľg/ml)

2.51

100 90 80

% Mortality

70 60 50 40 30 20 10 0

Log C

Figure 5.10: Effects of ethyl acetate fraction of methanol extract of Casuarina equisetifolia on brine shrimp nauplii

Calculation:

LC50 (µg/ml) = antilog (0.4) = 2.51 µg/ml Table 5.10: Regression line equation and Value of R2 for different test samples:

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

y = 17.17x + 1.535

0.908

y = 23.83x + 36.20

0.950

y = 28.48x + 15.39

0.957

y = 27.27x + 42.90

0.889

Code no. Test sample

extract CTTA

Carbon tetra chloride fraction of methanol extract

CFTA

Chloroform fraction of methanol extract

EATA

Ethyl acetate fraction of methanol extract

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347-356. 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.