Chemical & Biological Investigation of Acacia auriculiformis

Chapter 1 INTRODUCTION

1.1 Rationale of the work

Medicinal plant formed the basis and foundation stone of diseases from the very beginning of

human civilization. Medicinal component from plants play many important roles in

traditional medicine. People in all around the worlds have long been applied poultices and

imbibed infusions of hundreds, if not thousands, of indigenous plants, dating back to

prehistory (cowan, 1999). It is estimated that there are about 2,500,000 species of higher

plants and the majority of these have not been investigated in details for their

pharmacological activities (ram et al., 2003). In developing countries, about 80% of the

population relies on traditional medicine for their primary health care needs (matu and

Staden, 2003).

Previously the plant medications in the crude form exhibited many unwanted effects due to

the presence of some toxic compounds beyond the active constituents. So, the purpose of

extensive phytochemical study is to isolate the active constituents in the pure form to avoid

unwanted effect and to ensure safe use of herbal medicines.

A medicinal plant represents a rich source of new molecules with pharmacological properties,

which are lead compounds for the development of new drugs. The importance of plants in

search of new drugs is increasing with the technological advancement of medicinal sciences.

Many chemical compounds of diversified nature from plants often played an important role

to give a new direction for laboratory synthesis of many new classes of drug molecules

(Avram et al., 1974). In some cases, the plant components become the starting material in the

synthetic process of industrial production of many drug molecules. As for example, the use of

sterol diosgenin isolated from Mexican Yam for laboratory synthesis of oral contraceptive

progesterone reduced the cost of progesterone from a value of $80 per gm to $1.75 per gm

(Avram et al., 1974). Sometimes the crude drug containing several constituents was found

to be ineffective in case of therapy for which it was used traditionally. The phytochemical

investigation of periwinkle plant Vinca rosea (Avram et al., 1974), once used traditionally

as an anti-diabetic drug was found to contain hypoglycemic alkaloid principles in minute

quantities but it was found to contain anticancer principle vinca alkaloid in a high yield. The

dried seeds of the plant Amni visanaga was used as a diuretic and antispasmodic in renal colic

in the Eastern Mediterranean countries and in Arabia, but the research carried out by G. V.

Anrep and coworkers (Anrep et al., 1949) resulted in the isolation Khelin, a component

having the vasodilator effect. Khelin appeared as an anti-anginal drug after subsequent

clinical trial. The research on Rauwlfia serpentine, which was traditionally used as an

antidote for snake bite, revealed the presence of an antihypertensive agent reserpine (Vakil et

al., 1949).

Ricin, a toxin ( one milligram of ricin toxin can kill an adult) produced by the beans of

Ricinous Communis, had been found to be effectively couple tumor targeted monoclonal

antibiotics and had proved to be a very potent antitumor drug (Gupta, 1992). Further HIV

inhibitory activity has been observed in some novel coumarins (complex angular

pyranocoumarins) isolated from calophyllum lanigerum and glycerrhizin (from Glycerrhiza

species). Hypericin from Hypercium species is an anti cancer agent. Taxol is another exemple

of one of the most potent anti tumor agent found from Taxus bravifolia. Thus phytochemical

research on medicinal plants might open the door for many unknown therapeutic choices.

The isolated plant constituents having pharmacologic interest may be used as a model for

synthesizing that compound or a series of its derivatives for finding out an ideal drug to

improve selectivity of action. Such a model was cocaine, an alkaloid having anesthetic

activity. Cocaine was isolated from coca leaves. Extensive pharmacological screening of this

plant constituent led to recognize its central stimulant and addictive properties later on. Using

the model of cocaine, several synthetic dialkylaminoalkyl-aminobenzoates were synthesized;

one of these synthetic compounds was procaine, which displaced cocaine due to lack of

addictive properties shown by cocaine. Due to relatively low therapeutic index of procaine,

search of new synthetic products lead to the synthesis of lidocaine, tetracaine and dibucaine,

which seem to better than procaine. But the basis of this search of ideal anesthetic having

high therapeutic index and free from addictive properties, though still not fruitful, was

isolation of cocaine from coca leaves (Avram et al., 1974).

Since chemical constituents of medicinal plants, particularly the secondary metabolites

(alkaloids sterols, terpenes, flavonoids, saponins, glycosides, cyanogenics, tannins, resins,

lactones, quinines, volatile oils etc.) have profound pharmacological action on animal

systems and organs; they are capable of mitigating sufferings, curing ailments, and healing

wounds cuts burns.

It is evident from the above discussion that pharmacological studies of crude extract are

required after phytochemical investigation. Without pharmacological studies phytochemical

studies alone can provide the chemical constituents of plants that may or may not have the

therapeutic value. That is why Dr. Kurt Hosttetmann (Irvine, 1995) of University of

Lausanne, Switzerland gave emphasis on the biological and pharmacological analysis might

be a rational approach. According to world health organization (WHO), herbal medicines

composed mainly of medicinal plants are still curing diseases of estimated 1.5 billion

(currently it is said to be 3.5 billion, i.e, 88%) of the world population (said, 1995). Natural

products and related drugs are used to treat 87% of all categorized human diseases including

bacterial infections cancer and immunological disorders (Newman et, al; 2007). About 25%

of prescribed drugs in the world originate from plants (Rates SMK; 2001) and over 3000

species of plant have been reported to have anti cancer properties. In developing countries,

about 80% of the population relies on traditional medicine for their primary health care (matu

and Staden, 2003). Whatever progress science might have made in the field of medicine over

the years, plants still remain the primary source of many important drugs used in modern

medicine & contributing to the development of synthetic drugs & medicine in a numerous of

ways as stated below:

Novel structures of biologically active chemical compounds, isolated from plant

sources, often prompt the chemists to carry out their total synthesis.

Synthetic drugs with similar or more potent therapeutic activity are often prepared by

molecular modification of the plant-derived compounds with known biological activities.

Various analogues and derivatives of plant constituents are synthesized to study SAR

for getting better drugs.

In fact some of the plant constituent possessing a wide range of pharmacological are their

impossible or to difficult to synthesize in the laboratory. A phytochemist uncovering these

resources is producing useful material for screening programs for drug discovery. Outgrowth

of newer diseases is also leading the scientists to go back to nature for producing newer

effective drug molecules.

Recently developed genetic engineering in plants has further increased their importance, in

the field of medicine for example in the production of antibiotics by expression of an

appropriate gene in the plant. By using these techniques it is possible to modify the activity or

regulates the properties of the key enzymes responsible for the production of secondary

metabolites. Thus by knowing the potential resources it is possible to increase the content of

the active compounds (owen et al., 1992) and in the future genes responsible for very specific

biosynthetic processes may be encoded into host organism to facilitate difficult synthetic

transformation.

Thus plants are considered as one of the most important and interesting subjects that should

be explored for the discovery and development of newer and safer drug candidates.

1.2 PURPOSE OF THE STUDY

Tropical Bangladesh is blessed with numerous kinds of medicinal plants and many of them

have medicinal value. Majority of our population has to rely upon indigenous system of

medication from economic point of view. The high cost of imported conventional drugs and

inaccessibility to western health care facility, imply that traditional mode of health care that is

affordable and available to rural people. On the other hand, even when western health care

facilities are available traditional medicine is viewed as an efficient and an acceptable system

from a cultural perspective (munguti, 1997) and as a result traditional medicine usually exist

side by side with western form of health care.

Medicinal plants are rich sources of bioactive compounds and thus serve as important raw

materials for drug development. However a very little are known about the chemical

constituents of these plants. Identification and isolation of the active constituents from

traditionally used phytotherapy can ensure the health care of the poor people. In addition,

herbal medicine could be scientifically modified for better pharmacological activity and to

establish safe and effective drugs and the rationality of the present study lies in meeting the

challenges in developing herbal medicine which needs a systematic research on indigenous

medicinal plants for the welfare of the humanity. Phytochemical investigation and isolation

of active components in the pure form thus become necessary to avoid untoward effects and

to ensure safe use of herbal medicines.

Therefore, studies on the isolation and characterization of the medicinally active compounds

from these plants are very important for the well being of human society.

Bangladesh is a good repository of medicinal plants belonging to various families including

Leguminosae. The Leguminosae Species contain a wide range of pharmacologically active

compounds which are very useful and effective as astringent, anti-dysenteric, anti-protozoal,

anthelmintic and antipyretic. These compounds are also useful in itching, eczema, diarrhea,

hemorrhage, psoriasis, inflammation, leprosy, ulcer, sore throat, leucorrhoea, diabetes

mellitus, impotency, piles and syphilitic affections of mouth and are effective in urinogenital

disorders. Although uses of some of these species are based on old and new experiences and

clinical data, many of them have no foundation whatsoever.

There are several familiar approaches for lead searching from plants (Fig:1.1) and isolate

bioactive compounds utilized in three basic ways (Cox, P.A.,1994):

Unmodified natural plant products where ethno-medial uses suggested clinical

efficacy, e.g.,digitalis.

Unmodified natural plant products of which the therapeutic efficacy was only

remotely suggested by indigenous plant use, e.g.,vincristine.

Modified natural or synthetic substances based on a natural product used in folk

medicine, e.g, aspirin.

Plants

Figure 1.1: Lead compound search & utilization from plants.

The work described in this dissertation is an attempt to isolate and characterize the chemical

constituents of an indigenous medicinal plant Acacia auriculiformis (family: liguminosae)

and to evaluate the possible microbiological and toxicological profiles of the crude extracts is

the primary objective of the present study

1.2.1 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

respectively.

Fractionation of the crude methanol extract by solvent-solvent extraction process into

Petroleum ether fraction, carbon tetrachloride fraction and chloroform fraction.

Fractionation of the carbon tetrachloride soluble fraction by column chromatography (CC).

Modified natural or

synthetic substances based on a natural

product used in folk medicine, e.g., aspirin

Unmodified natural

products of which the therapeutic

efficacy was only remotely suggested

by indigenous plant use, e.g., vincristine

Unmodified natural plant products where ethno medical uses suggested clinical efficacy, e.g., digitalis.

Isolated compounds

Random Screening Targeted screeningEcological surveyPhylogenetic

Fractionation of the Chloroform soluble fraction by column chromatography (CC).

Isolation and purification of compounds from the selected column fractions

Determination of the structure of the isolated compounds with the help of

1H NMR, 13C NMR, COSY, HSQC and HMBC spectroscopy.

Observation of in vitro antimicrobial activity of crude extracts, fractions and

column fractions.

Brine shrimp lethality bioassay and determination of LC50 for crude extract,

fractions and column fractions.

Evaluation of Assaying free radical scavenging activity & determination of IC50 for crude

extract, fractions and compounds.

1.2.2 The plant family: Leguminosae

Any of about 18,000 species in about 650 genera of flowering plants that make up the order

Fabales, consisting of the single family Leguminosae, or Fabaceae (the pea family).

The term also refers to their characteristic fruit, also called a pod. Legumes are widespread on

all habitable continents. Leaves of many members appear feathery, and flowers are almost

universally showy. In economic importance, this order is surpassed only by the grass and

sedge order (Cyperales). In the production of food, the legume family is the most important

of any family. The pods are part of the diet of nearly all humans and supply most dietary

protein in regions of high population density. In addition, legumes perform the invaluable act

of nitrogen fixation . Because they contain many of the essential amino acids, legume seeds

can balance the deficiencies of cereal protein. Legumes also provide edible oils, gums ,

fibers, and raw material for plastics, and some are ornamentals. Included in this family are

acacia, alfalfa, beans, broom, carob, clover, cowpea, lupine, mimosa, peas, peanuts,

soyabeans, tarmarind and vetch.

1.2.3 Classification of Kingdom Plantae down to family leguminosae.

Kingdom Plantae– Plants

Subkingdom Tracheobionta– Vascular plants

Superdivision Spermatophyta– Seed plants

Division Magnoliophyta– Flowering plants

Class Magnoliopsida– Dicotyledons

Subclass Rosidae

Order Fabales

Family Fabaceae– Pea family

Genus Acacia Mill.– acacia

Species Acacia auriculiformis A. Cunn. ex Benth . – earleaf acacia

The plant under investigation is Acacia auriculiformis belonging to the family Leguminosae.

This is one of the largest and most useful plant families. - 18,000 species, distributed almost

throughout the world. It includes many well-known vegetables particularly of temperate

regions (Beans, Peas), ornamental trees in tropical regions (Bauhinia, Flamboyant, Cassia),

fodder crops (Clover, Lucerne) and weeds (Vetches and Trefoils), and their growth habits

vary from ground cover and aquatic to shrubs, climbers and trees. Many species of trees in

this family are important for their timber.

Leguminosae, pea family- a large family of trees, shrubs, vines, and herbs bearing bean pods;

divided for convenience into the subfamilies Caesalpiniaceae; Mimosaceae; Papilionaceae.

These Families have been formed by splitting the old Leguminosae Family on the basis of

flower shape, type of leaves, and number of stamens.

The Papilionaceae Family is found in temperate, sub-tropical and tropical areas. Members of

this Family are mostly herbs, but with some trees and shrubs, and have irregular flowers

forming a butterfly or pea-flower shape, with the lateral petals enclosed by the standard when

in bud, with ten stamens. The family Papilionaceae includes the following genera:

Amorpha, Anthyllis, Astragalus, Baptisia, Caragana, Clianthus, Colutea, Cytisus, Dolichos,

Erythrina, Genista, Glycyrrhiza, Hardenbergia, Indigofera, Kennedia, Laburnum, Lathyrus,

Lotus, Lupinus, Medicago, Mucuna, Ononis, Oxytropis, Parochetus, Phaseolus, Pueraria,

Robinia, Sesbania, Sophora, Sutherlandia, Trifolium, Trigonella, Vicia, Wisteria.

The Mimosaceae Family contains mainly tropical and sub-tropical trees and shrubs, with

regular flowers with ten or more stamens. The Mimosoideae are characterised by their small,

regular (actinomorphic) flowers crowded together, generally into spikes or heads which

resemble a pom-pom. The stamens have become the most attractive part of the flower, the

five petals inconspicuous. The leaves are predominately bipinnate. The family Mimosaceae

includes the following genera:

Acacia, Albizia, Calliandra, Mimosa, Paraserianthes.

Certain Acacia species are extremely important economically. An extract from the bark of the

Golden Wattle (Acacia pycnantha) is used in tanning, several species, such as Australian

Blackwood (e.g. Acacia melanoxylon) provide useful timbers and some (e.g. Acacia senegal)

yield commercial gum arabic, which is used in a wide range of industrial processes.

The Caesalpiniaceae Family is also mainly tropical and sub-tropical trees and shrubs, with

irregular flowers and ten or fewer stamens. The family Caesalpiniaceae includes the

following genera:

Bauhinia, Caesalpinia, Cassia, Ceratonia, Cercis, Delonix, Gleditsia, Schizolobium, Schotia,

Tamarindus.

The seedpods of all these Families are the same - they are all legumes - pods, formed from a

superior ovary, usually containing several seeds, which splits along both sides. In some

tropical species, the seedpods are very large and woody.

The seeds of many members of these Families are the distinctive kidney-shape generally

referred to as 'beans', with a visible scar where the seed was attached to the seedpod. Many

are quite large, and some are brightly-coloured.

1.2.4 Members of Leguminosae family

The plants belonging to the family Leguminosae, which are available all over the world, are

shown in the Table 1.1.

Table 1.1 Leguminosae species available in the world.

Leguminosae

Latin Name Common Name SynonymsMedicinal

Rating

Acacia aneura Mulga Acacia 0

Acacia coriacea Wiry Wattle 0

Acacia cultriformis Knife-Leaf Wattle 0

Acacia dealbata Mimosa Acacia decurrens dealbata 0

Acacia decurrens Green Wattle Mimosa decurrens 1

Acacia farnesiana Sweet Acacia Acacia smallii, Mimosa

farnesiana

2

Acacia longifolia Sidney Golden Wattle Mimosa longifolia 0

Acacia melanoxylon Blackwood 1

Acacia mucronata Narrow-Leaf Wattle 0

Acacia paradoxa Kangaroo Thorn Acacia armata 0

Acacia podalyriifolia Queensland Silver Wattle 0

Acacia pycnantha Golden Wattle 0

Acacia retinodes Swamp Wattle 0

Acacia saligna Blue-Leaved Wattle Acacia cyanophylla 0

Acacia sophorae Coastal Wattle 0

Acacia verticillata Prickly Moses 0

Adesmia lotoides 0

Albizia julibrissin Mimosa Acacia julibrissin 2

Alhagi mannifera Manna Tree Hedysarum alhagi 2

Alhagi maurorum Camel Thorn Alhagi camelorum, Alhagi

persarum, Alhagi pseudalhagi,

Hedysarum pseudalhagi

2

Amorpha canescens Lead Plant 2

Amorpha fruticosa False Indigo 0

Amorpha nana Dwarf Indigobush Amorpha microphylla 1

Amphicarpaea

bracteata

Hog Peanut Amphicarpaea monoica, Falcata

comosa

1

Amphicarpaea

edgeworthii

Amphicarpaea japonica, Falcata

japonica

0

Amphicarpaea pitcheri Hog Peanut Amphicarpaea bracteata

comosa, Falcata pitcheri

0

Anthyllis vulneraria Kidney Vetch 2

Apios americana Ground Nut Apios tuberosa 1

Apios fortunei 1

Apios priceana Glycine priceana 0

Arachis hypogaea Peanut 2

Aspalathus linearis Rooibos Aspalathus contaminatus,

Borbonia pinifolia

3

Astragalus

aboriginorum

Indian Milkvetch Astragalus australis 0

Astragalus adscendens Persian Manna Astracantha adscendens 0

Astragalus boeticus Swedish Coffee 0

Astragalus brachycalyx 0

Astragalus canadensis Canadian Milkvetch Astragalus carolinianus 2

Astragalus

carduchorum

0

Astragalus

chartostegius

0

Astragalus chinensis Hua Huang Qi 2

Astragalus christianus 0

Astragalus

complanatus

Bei Bian Huang Qi 2

Astragalus

crassicarpus

Ground Plum Astragalus caryocarpus,

Astragalus mexicanus,

Astragalus succulentus,

Geoprumnon succulentum

1

Astragalus creticus 0

Astragalus densissimus 0

Astragalus diphysus Specklepod Milkvetch Astragalus lentignosus diphysus 0

Astragalus echinus Astracantha echinus 0

Astragalus edulis Tragacantha edulis 0

Astragalus exscapus 1

Astragalus floridus Duo Hua Huang Qi 2

Astragalus florulentus Astracantha florulenta 0

Astragalus garbancillo 0

Astragalus globiflorus Astracantha globiflora,

Astragalus elymaiticus

0

Astragalus

glycyphyllos

Milk Vetch 0

Astragalus gummifer Tragacanth Astracantha gummifera 3

Astragalus hamosus 2

Astragalus henryi Qin Ling Huang Qi 0

Astragalus hoantchy Wu La Te Huang Qi 1

Astragalus kurdicus Astracantha kurdica 0

Astragalus leioclados Astracantha leioclados 0

1.2.5 The Genus Acacia A. – A brief discussion

Acacia is the common name for the plants of genus Acacia of the family Leguminosae.

Acacia is a large genus with 900 species (Hatchinson 1964, Nasir et al., 1973) approximately

700 of which are native to Australia. The remainder occurs mainly in tropical and sub-

tropical regions of Africa, Asia and America. Acacia have the capabilities to grow under the

xerophytic conditions and to survive under extreme droughts, is an important feature of the

genus Acacia. The trees of Acacia are exceedingly hardy and they prefer to grow under the

severest natural conditions than in the cultivated places. These can grow in sandy, saline and

even on water–logged soils (PCSIR 1987). In deserts of Asia and Africa, goats and camel

browse on leaves and young shoots of Acacias. In Australia some species also serve as forage

for cattle and sheep.

The name is derive from the Greek AKAZO which means “I Sharpen”, in allusion to the

species of thorny bushes or small trees also called Mimosas are known as “thorn”, as “Kikar”

in Indo-Pak and as Acacias in Asia and America. In Australia Acacias are known as many

popular names, the principal one applying to the whole genus being the “Wattle”. Australian

gave many names to different species of Acacias such as Myall, Mulga, Boree, Brigalow,

Miljee, Windi, Cooba, Gidgee, Euonung and Yarram. In Pakistan different Acacias were

known as babul, phulai, khair, katha, khor and raru etc.

The wood of Acacia trees is in some cases very valuable, though usually small in making

railway carriage, wheels, handles, furniture and is the best of making charcoal (Gohl 1975,

Lexicon Universal Encyclopedia 1987). The bark of some Acacia is extensively used for

tanning leather (Olivannan et al., 1966). In Australia and some parts of Africa and Asia,

seeds and pods are used by human for food (Tanaka 1976). Tanaka reported the edible uses

of 56 species of Acacias (Nironala et al., 1984). Due to their large uptake of salts, Acacias are

used for soil reclamation and to increase fertility through their high nitrogen fixation

capability (Stravge 1977). The medicinal uses of the Acacias species are also known since

time immemorial (Chowdhury et al., 1948, Lir 1936, Perry 1980 etc).

A large number of Acacias yield gum in greater lesser quantities. It exudes naturally from the

trunk of the trees of wild, although this is often encouraged by making incisions in the trunk.

The more the cuts, the more the gum is expels, which on exposure to air hardens into

yellowish white transparent beads. The finest gum Acacia or gum arabic is known as

kordofan gum which comes from A.senegal, a small tree native to Africa, from Ethopia to

sudan. Acacia gum is also has medicinal properties.

Table 1.2 .6Acacia species available in the world

Acacia species Height

metres

Width

metres

Foliage Flowering

A. acinacea 2.5 1.5 green Golden balls in spring

A. acuminata 5 2 green Golden in spikes spring

A. adunca 6 2 green Very showy late winter

A. alata 2 1 Spined

phyllodes

Cream or gold aut-late spring

A. alleniana 5 3 Thread

like/pendulous

Golden balls mar to ma

A. araneosa 5-8 3-4 green Sprays yellow

throughout year

A. argyrophylla 3-5 3 Silver-grey Golden-yellow balls in

Winter

A. aneura 5-10 5 green Bright yellow spikes

A. armata 2 2 green Gold balls spring

A. aulacocarpa .5 - 8 .5 - 8 Blue/green Midsummer to winter

A. baileyana 5-8 5 blue/green Yellow, late winter

A. bancroftiorum

as known as A.

bancroftii

6 4 Bluish 20cm

long

Yellow ball sprays late

aut. to late winter

A. beckleri 1-3 1-2 Green/grey Bright yellow late aut. to

mid winter

A. bivenosa 1-3 1-3 Green-

glaucous

Yellow mid aut. to late

spring

A. boormanii 3-5 4 grey/green Bright yellow, early

spring.

A. brachystachya 2 - 6 2 - 6 Glaucous Yellow in axis of

phyllodes aut – late

winter

A. browiniana 2 2 Tiny bipinnate

with oblong

leaflets

Golden ball flowers

larger then the leaves in

spring

A. brownii 1 1 Prickly

phyllodes

Golden balls in slim

peduncles

A. buxifolia 3 2 Green to

glaucous

Golden balls in late

winter to spring

A calamifolia 2-4 2-4 Grey-green

with

bent tip

Pale yellow to golden

A. cardiophylla 1-3 1 To 3 Pale green Bright yellow balls in

spring

A. cognata 1- 10 1 - 6 Yellow- green

to dark green

pendulous

Pale lemon/cream in spring

A. colletioides 1.5 or

more

3 Prickly with

yellow stem

projections

Orange or yellow in spring

A. complanata To 5m 3 Light green Deep yellow spring to autumn

phyllodes to

0cm

A. conferta 2 2 green Bright yellow autumn to

mid spring

A. continua 1-2 To 1 Hooked –

spiky

blue-green

Large golden balls early

spring

A.

craspedocarpa

1 To 4 1 To 2.5 Grey Golden spikes mainly in

spring

A. cultriformis 3-4 3 blue/green Golden spring,

A. cyclops 1-6 1-6 Blue/green Yellow in spring and

showy

A. dealbata 5-20 8 blue/green Bright yellow, late winter

to spring

A. deanei 5-10 3-5 green Pale yellow, all year

A. decora 3-5 4 grey/green Golden yellow –early

spring.

A. denticulosa 1-4 2-4 Dark green Rod shaped golden in

spring

A. dimidiata 1 To 7 3 Curved to one

side.

Terminal sprays of

golden flowers in

autumn

A. dentifera 2-4 3 Blue/green Cream/yellow early –

mid spring

A. doratoxylon 6-10m 6 Green Golden spikes in spring

A. drummondii 1.5 1 Green Golden, late winter –

mid spring

A. dunii To 7m Long 30cm

glaucous to

20cm wide

Golden balls year round

A. elata 10-20 8 Brown/ green Cream, summer

A. elongata 3 1.5 Pale green Yellow to gold balls in

late winter - spring

A. erinacea 1 1.5 Grey-green Yellow balls in winter-

spring

A. extensa 2 -3 2 Long 20cm

phyllodes pale

green

Light golden - yellow

balls in spring

A.A. falciformis 1 To 12 3 Grey-green Cream - yellow globes

Early summer

A. falcata To 4 1-2 Grey- green to

glaucous

sickle

shaped

Cream in winter

A. falvescens 4 - 20 1-3 Pale green Creamy globes late

autumn to early winter

A. flexifolia 1 – 1.5 .5-1 Grey- green Small yellow balls in

winter

A. floribunda 4-8 4-6 Green Yellow, July-Sept

A. genistifolia .6 - 3 1 To 2 Green Large cream balls winter to early spring

A. glaucoptera To 1.5 To 1.5 Glaucous Yellow globes in spring

A. gonocarpa .6 – 3.5 .6 - 2 Green Pale cream rods summer and again in winter

A. gracilifolia 1-2 1-2 Narrow green Pale gold in spring

A. guinetii .5 2.5 Pale green-

Yellow tinge

Yellow winter – early spring

A. hakeoides To 4 To 3 Green Golden in sprays winter

and spring

A. hemsleyi To 7m 3-5 Green Yellow rods in early

spring

A. hispidula 1-2m 1-2 Green Yellow balls all year

A. howittii 4-8 4 Green Pale yellow, Spring

A. inophloia 1 – 3.5 1- 2 Greyish-green Bright yellow rods late winter to mid spring

A. iteaphylla 4-5 4 Blue/green Pale yellow, Mar-Aug

A. kempeana

2- 5

2-5

Grey to

blue/green

Bright golden spikes

mid summer to spring

A. kettlewelliae 2 -10 2-5 Silvery -

green to

glaucous

Light bright gold in late spring

A. lanigera 1 1 Woolly

narrow

green-bluish

Small balls in spring

A. latescens 3 - 10 3-5 Sickle shaped

long leaves to

20cm pale

green

Cream balls in autumn

A. leprosa 2-4 To 2 Green Yellow- orange in spring

A. leptostachya 1-5 1-5 Green to

slightly

glaucous

Golden rods in winter

A. longifolia 4-10 4-8 Green Yellow, July-Sept

A. macradenia 3 - 6 3 - 6 Green with

reddish new

growth

Bright yellow winter and spring

A. mearnsii 10-25 10 Grey/green Pale yellow, spring

A. melanoxylon 5-30 5-15 Grey/green Cream, July-Oct

A.

merinthophora

4m To 3m Grey/green Creamy yellow rods in the leaf axis late

autumn to early spring

A. montana 1 -4 1-4 Bright green

and

sticky

Golden balls in spring

A. muelleriana 1 - 8 1 - 8 Dark green Cream balls in spring

A. myrtifolia .5 - 3 .5 - 3 Dark green Creamy/yellow

A. notabilis To 3 m To 3-4m Grey/green -

Glaucous

Golden balls in spring

A. obliquinervia To 15 2-5m Grey/green to

slightly

glaucous

Lemon to golden globes Late winter to early

summer

A. oncinocarpa To 5 To 4 Mid green Pale yellow rods in autumn

A. papyrocarpa 3-4 2-3 Grey Yellow in spring

A. paradoxa 2-4 3-4 Prickly Yellow to bright yellow balls late winter to

late spring

A. pendula 5-13 3-13 Glaucous/grey Yellow balls in spring

A. phasmoides 1-4 To 4 Glaucous/grey Golden-yellow rods in

spring

A. podalyriifolia 4 3 blue Golden, July-Oct.

A. pravissima 4-8 5-7 Olive green Yellow, Sept.

A. prominens 5-15 7 Blue/green Lemon, Sept.

A. pycnantha 4-10 4 Green Yellow, July-Oct.

A. retinodes 5- 8 5 Grey Cream-yellow balls in

winter-spring

A. rigens 2 3 Grey-green

sticky, glossy.

Golden balls in spring

A. rubida 1.5 - 5 1-4 Green to

Glaucous

Yellow in spring

A. saligna 4-10 5 Green Yellow, Aug-Nov.

A sophorae - see

A. longifolia

A sclerophylla To 2 3 Glossy, sticky

green

Golden balls borne in the leaf axis in spring

A. siculiformis To 2 - 3 2-3 Dark green Cream balls in spring

A. spectabilis 2 -4 up

to 6

2-4 Blue-green to

Glaucous

Golden balls in spring

A. stricta 1- 5 Suckering Dullish green Stem clasping balls in spring

habit 1-5

A. suaveolens 3 4 Blue/green Pale, April-Sept

A. terminalis 3 2 Dark green Cream to yellow balls in autumn - winter

A. torulosa 1.5 - 15 1-10 Yellowish-

green

Bright yellow in winter

A. triptera 3 To 7 Bluish-green,

sickle shaped

Golden rods in spring

A. ulcifolia 1-2 1-2 Green Cream, Mar-Sept

A. umbellata 2-6 3 -6 Light green Golden rods in summer

A. uncinata 3 3 Grey-green Golden rods in summer

A. verniciflua 1-8 1-5 Green Cream- yellow balls in

spring

A. verticilata 2-7 1-3 Green Yellow, June-Dec.

1.2.6.1 Medicinal importance of Acacia species

Many Acacia species have important uses in traditional medicine. Most all of the uses have

been shown to have a scientific basis, since chemical compounds found in the various species

have medicinal effects. In Ayurvedic medicine , Acacia nilotica is considered a remedy that

is helpful for treating premature ejaculation . A 19th century Ethiopian medical text

describes a potion made from an Ethiopian species of Acacia (known as grar) mixed with the

root of the tacha, then boiled, as a cure for rabies . An astringent medicine, called catechu

or cutch, is procured from several species, but more especially from Acacia catechu, by

boiling down the wood and evaporating the solution so as to get an extract.

Table 1.2.6.2 Medicinal plants of Leguminosae family available in Bangladesh

BOTANIC NAME LOCAL NAME

Abrus precatorius L. Kunch, Rati, Chanyi, Kaich, Gungchi, Gujna

Agati grandif lora Desv.

(Sesbania grandiflora (L.) Pers.)

Bakphul, Agasta, Buko, Bak, Agati

Caesalpinia crista L.

(C. nuga L.)

Let Kanta

Cassia alata L. Dad Mardan, Dadmari

Clitoria ternatea L. Aparajita, Nila Aparajita

Mimosa pudica L. Lajjabati, Lajak

Saraca indica L. Ashoke

Tephrosia purpurea Pers. Bannil, Lohamori, Sarpunkha

1.2.6.3 Taxonomy of Acacia

Family: Fabaceae (Pea family) (Wagner et al. 1999).

Latin name: Acacia auriculiformis Cunn. ex Benth. (PIER 2002).

Synonyms: Racosperma auriculiforme (Benth.) Pedley (Randall 2002).

Common names: Earpod wattle, Papuan wattle, auri, earleaf acacia, northern black wattle,

Darwin black wattle (GRIN 2002, PIER 2002).

Taxonomic notes: The genus Acacia is made up of about 1,200 species that are

widespread but with a large number in Australia (Wagner et al. 1999).

Nomenclature: The genus name is derived from akakia, the Greek name for Acacia arabica

(Lam.) Willd., which is derived from akis, a Greek word meaning sharp point, in reference to

the thorns of the plant (Wagner et al. 1999).

Acacias belong to the legume family (Fabaceae), the third largest family of flowering plants,

including three subfamilies, 650 genera and 18,000 described species. All three subfamilies

produce typical legume seed pods that either split open or remained closed at maturity, but

their flowers are quite different. Acacia blossoms are not pea-like, and for this reason the

genus is placed in the subfamily Mimosoideae, along with silk tree (Albizia), fairy duster

(Calliandra) and mesquite (Prosopis). The flowers consist of an inconspicuous calyx and

greatly reduced or no petals, with numerous, showy stamens. Acacia flowers are clustered

together in small yellow or white globose heads, or in cylindrical spikes. In some species (A.

baileyana) the flower clusters are produced in spectacular yellow masses, and in others (A.

farnesiana) they are very fragrant, attracting numerous insect pollinators. The latter species is

a spiny shrub native to the southwestern United States and Mexico. The flowers contain an

essential oil used for perfumery in France

One of the most intriguing taxonomic features of the genus Acacia is its divergence into two

major groups with entirely different leaf types. One group has fern-like, bipinnate leaves

subdivided into numerous minute leaflets. It includes hundreds of species throughout

Australia, Africa and the Americas. Another group has "simple" leaves that are not divided

into leaflets. The leaves of this group are called phyllodes, and they are actually expanded or

broadened petioles (leaf stalks) which have lost the upper pinnate portion. Seedlings of this

group produce the ancestral pinnate leaf, gradually replaced by phyllodes. Pruned branches of

some species often develop phyllodes bearing bipinnate leaves at their tips. The phyllode

group also contains hundreds of species distributed throughout Australia and the Pacific

Islands. In fact, one of these species is the magnificent "koa" tree (Acacia koa) native to

Hawaii. The following chart shows the vegetative divergence in the genus Acacia:

Information about the investigated plant

1.2.6.4 General botanical data of Acacia auriculiformis

Botanical Name: Acacia auriculiformis.

Synonym: Acacia auriculaeformis

Local name: Akashmoni, sonazhuri

Family: Leguminosae

Description: Evergreen, unarmed tree to 15 m (50 ft) tall, with compact spread, often multi-

stemmed; young growth glaucous. Quickly reaching a height of 40 feet and a spread of 25

feet, it becomes a loose, rounded, evergreen, open shade tree. It is often planted for its

abundance of small, beautiful, bright yellow flowers and fast growth. Leaves alternate,

simple, reduced to phyllodes (flattened leaf stalks), these blade-like, slightly curved, 11-20

cm (5-8 in) long, with 3-7 main parallel veins and a marginal gland near the base; surfaces

dark green. The flattened, curved branchlets, which look like leaves, are joined by twisted,

brown, ear-shaped seed pods. Flowers in loose, yellow-orange spikes at leaf axils or in

clusters of spikes at stem tips; flowers mimosa-like, with numerous free stamens. Growing 6

to 8 feet per year, Acacia auriculiformis quickly grows into a medium-sized shade tree. This

makes it a popular tree. However, it has brittle wood and weak branch crotches, and the tree

can be badly damaged during wind storms. Prune branches so there is a wide angle of

attachment to help them from splitting from the tree. Also be sure to keep the major branches

pruned back so they stay less than half the diameter of the trunk. These techniques might

increase the longevity of existing trees.

Fruit and seed description:

Fruit: Flat, dehiscent, somewhat woody pod, 6.5 cm long, 1.5 cm wide, strongly curved and

with undulate margins. Fruits are twisted at maturity, splitting to reveal flat black seeds

attached by orange, string like arils.

Seed: Shiny black or brown, encircled by a long, red or yellow funicle. There is 55,000-

75,000 seeds/kg.

Flowering and fruiting habit:

The yellow flower spikes can be found on individual trees throughout the year but there is

usually a distinct peak flowering season which may vary considerably with location.

Pollination is carried out by a wide range of insects. Seed is produced at an early age and

normally in large quantities.

Distribution:

Planted widely in the Old World for pulp and fuel wood, particularly in India and Southeast

Asia; undergoing forestry trials in Africa and Central and South America (Pinyopusarerk

1990, Boland et al. 1991).

Figure 1.1 Seed with funicle, flowering branch and pod.

Figure 1.2 Forest tree form of Acacia auriculiformis. Bensbach River, Balamuk, Western

Provenance, Papua New Guinea.

Figure 1.3 Leaves of Acacia auriculiformis

Importance:

This plant is raised as an ornamental plant, as a shade tree and it is also raised on plantations

for fuel wood throughout south-east Asia Oceania and in Sudan. Its wood is good for making

paper, furniture and tools. It contains tannin useful in animal hide tanning. In India, its wood

and charcoal are widely used for fuel. Gum from the tree is sold commercially, but it is said

not to be as useful as gum arabic. The tree is used to make an analgesic by indigenous

Australians. A decoction of the root is used to treat aches and pains and sore eyes; an infusion

of the bark treated rheumatism (aborigines of Australia).

Extracts of Acacia auriculiformis heartwood inhibit fungi that attack wood. Aborigines of

Australia have traditionally harvested the seeds of some acacia species as food as paste or

baked into a cake because it assumed to be contains 25% more protein than common cereals

like rice or wheat etc. Acacias were purposely introduced and planted in Southeast Asia and

Oceania as a source of firewood and good quality charcoal (does not smoke), as well as

timber for furniture and pulp for making paper (acacia produces high yields of pulp and

produces strong paper. In India, the tree was cultivated to feed the lac insect, which produces

a resinous secretion that is harvested to produce lacquer. Acacia has the potential to protect

poor soils from erosion by its long root and revive their mineral content. Acacia can grow on

poor soils including clay, limestone and unstable sand dunes, even soil tainted with uranium

wastes.

Acacias recover wastelands, returning nutrients to poor soils and providing shade for other

plants to take hold. They do not produce a lot of pollen or nectar as food, but their plentiful

seed supply is a valuable food source for animals (mainly birds and also small mammals),

particularly in dry places. Various insects eat their leaves and wood, and sugar gliders and

squirrels may eat their sap.

1.2.6.5Previous phytochemical studies of the genus Acacia

All or a combination of the compounds below may be found in many flowering plants,

including acacias. This is however a rather simplified treatment of a very complex subject,

there being literally thousands of different compounds and metabolites in plants. The role or

function, if any, is still debatable, protection against predation, end metabolites, plant

hormones, pheromones, anti-fungal/ viral etc.

Carbohydrates, sugars and gums - Carbohydrates (sugars) are the products of

photosynthesis that plants use as starting material for most of the other compounds in plants.

Cellulose is a carbohydrate that most plants make and contain that gives plants their structure

and strength; some parts of plants may be more than 50% cellulose. Gums are

polysaccharidic (made from sugars) compounds, where various different sugars are joined

together to form polymer like structures. Some acacias produce quite large amounts of gum

from injuries or insect attack, some are edible; they can vary greatly in their water solubility,

some becoming gelatinous and not really dissolving.

Terpenes, oils and resins

Generally water insoluble organic compounds, originally applied to substances made up of

two 5-carbon units, the so called isoprene unit. Mono-terpenes are two units, sesquiterpenes

are three units, diterpenes are four units, triterpenes six units etc. Different oils and terpenes

may be found in the flowers and foliage, some acacia flower essential oils are used in

perfumery. Most essential oils are mono or sesqui terpenes, resins are often more complex

terpenoid mixtures that may also contain gums. There are some new terpinoids has been

invented from Acacia auriculiformis. These includes-

Three new triterpenoid saponins, proacaciaside-I, proacaciaside-II and acaciamine isolated

from the fruits of Acacia auriculiformis, were identified as acacic acid.

(1 → 6)-β- -glucopyranoside, acacic acid

(1 → 2)-β- -glucopyranoside and acacic acid

*(1 → 6)-2-acetamido-2-deoxy-β- -glucopyranoside

* Acaciasides A and B, two novel acylated triterpenoid bisglycosides isolated from the

fruits of Acacia auriculiformis, were respectively defined to be 3- -[β-D-glucopyranosyl

(1→6) &{;α-L-arabinopyranosyl (1→2)&};- β-D-glucopyranosyl]-21- -&{;6′S)-2′-trans-

2′,6′-dimethyl-6′- -β-D-glucopyranosyl-2′,7′-octadienoyl&}; acacic acid 28- -α-L-

rhamnopyranosyl (1→6) [β-D-xylopyranosyl (1→6) &{;α-L-arabinopyranosyl (1→2)&};-β-

D-glucopyranosyl]-21- -[(6′S)-2′-trans-2′,6′- -&{;β-D-xylopyranosyl (1→2)-β-D-

glucopyranosyl&};- 2′,7′-octadienoyl] acacic acid 28- -α-L-rhamnopyranosyl (1→6) [β-D-

xylopyranosyl (1→2)]-β-D-glucopyranoside (2). The structural details were elucidated by a

combination of fast-atom-bombardment mass spectrometry, 1H-, and 13C NMR spectroscopy,

and some chemical transformations.

Fig : acaciaside-B

* The structure of a new triterpenoid trisaccharide isolated from the seeds of Acacia

auriculiformis has been elucidated as acacic acid lactone-3-O-β-d-glucopyranosyl (1 → 6)-

[α-l-arabinopyranosyl (1 → 2)]-β-d-glucopyranoside based on its spectral properties and

some chemical transformations.

* The structural elucidation of auriculoside, a new flavan glucoside named -7,3′,5′-

trihydroxy-4′-methoxyflavan 3′-glucoside; α-spinasterol.from Acacia auriculiformis has been

done, This is the third report of a flavan glycoside unsubstituted in the heterocyclic ring.

Inventor-Shashi B. Mahatoa, Bikas C. Pala and Keith R. a Indian Institute of Chemical

Biology 4, Raja S. C. Mullick Road, Jadavpur Calcutta-700032, India

FIG: list of New triterpinoid compound from isolated from Acacia Acacia mellifera-Inventor-

-

Constituent from Acacia cedilloi and Acacia gaumeri.–Inventor-Gwendeli G. pech,

Gonjalo.j.mena

And leuvigillido.,mexico

Tannins - tannins are complex compounds based on tannic and gallic acid, very common in

the wood, bark and foliage that are water soluble but react with proteins, this is what causes

the astringency of many plants and is utilized to preserve leather in the tanning process.

Acacia bark has been used as a source of tannins, some species having large amounts in the

bark.

Glycoside - Is a general term for substances made up of a sugar residue (glucose unit) and

another compound, such as a flavanoid, coumarin, steroid or terpene, collectively known as

the aglycone. Glycosides are common in plants, there are quite a few that have a strong action

on the body, including the heart, digestive and peripheral nervous system. ‘Cyanogenetic

glycosides’ produce free HCN (cyanide) when reduced (digested?), and along with other

glycosides, like the cardioactive glycosides can produce toxic even fatal results if enough is

ingested, which may not be very much. About forty species from sub-genus Phyllodineae

have been recorded as being cyanogenetic.

1.2.6.6 Glycoside reported from the acacia species

The glycoside kaempferol has been isolated from the flowers of A. discolour, A. linifolia, A.

decurrens and A. longifolia, kaempferol is water soluble and yellow, and in these cases

responsible for the color of the flowers ( J Petrie, Proc. Linn. Soc. NSW, #48: 356-67, 1923),

and this may be the case with many, if not most acacia flowers. This compound has been

found to be a diuretic (promotes urination) and natriuretic (causes sodium loss), increasing

urine secretions and the functioning of the kid nay cells, increasing in turn, their permeability

and circulation. The general result is that kidney function improves which helps the body to

positively react to water retention and excessive blood glucose levels, both of which are

secondary symptoms of diabetes (Winkelman, Ethnobotanical treatments of diabetes in Baja

California norte. unpublished report, Arizona state uni, Tempe, Ariz. 1991). Some of the new

glycosidic compounds isolated from these species are listed below-

Myricetin -3,7-diglucoside

Kaempferol -7-glucoside , 3-glucoside (9) etc.

Quercetin -3’-methyl ether (12) & 7-glucoside (13

Flavanoids

This is a term that is applied to compounds common in many plants and quite often

responsible for the colours in wood, fruit and flowers.

1.2.7Flavanoids present in different species of acacia

The flavanoids of the heartwoods of Australian acacias has been the subject of some study.

The studies have found that Australian acacias can be broadly divided into different groups

depending on the flavanoids present in the wood. These groupings did not correspond exactly

with the classification based on morphological differences. There were however some

correlations with the Botrycephaleae forming a distinct group and Phyllodineae species

with flowers in racemes having a similar flavanoid pattern. The Juliflorae and Plurinerves

had a similar flavanoid pattern, the Juliflorae being a fairly well defined group, with a

further small group in the Juliflorae having unique but related flavanoids. There was also a

distinct group in the Phyllodineae that had unique flavanoids that give members of this

group distinctively purple heartwood. There were some mixed results for some species in

sections Phyllodineae , Plurinerves and Juliflorae , especially the tropical northern species.

Other studies of the free amino acids in the seeds of different species found that sub-genus

Acacia was a distinct group different to sub-genus Phyllodineae and Acueiliferum, a sub-

genus of mostly Asian, African and Central American species. There seemed to be some

relationship between sub-genus Phyllodineae and Acueiliferum, with the addition of two

more amino acids, one toxic, in the Acueiliferum species seeds compared to sub-genus

Phyllodineae. Three extra Australian species of sub-genus Phyllodineae, a. confusa, a.

simplex and a. kuauiensis also have been found contain these extra amino acids.

(2,3-trans-3,4′,7,8-tetrahydroxyflavanone,

Teracacidin,

4′,7,8,-trihydroxyflavanone)

Fig : structure of compound isolated from Acacia auriculiformis and other acacia species.

.

Figure 1.4 : Structures of falvonoids isolated from Acacia auriculiformis

( Leucodelphinidin ^ A new flavan-3,4-diol from Acacia auriculiformis by

paper ionophoresis, S. E. Drewes and D. G. Roux, 1966)

Alkaloids - is a general term for basic (alkaline) nitrogen containing organic compounds,

generally bitter in taste and strong physiological action, many plant derived drugs and

medicines are alkaloids, eg quinine, scopolomine, codiene, morphine, ephedrine, tryptamines

etc. A lot of them can be potentially toxic, even fatal, especially when in the form of purified

alkaloids extracted from plants, quite often only a small amount of the alkaloids can have a

strong effect. Obviously some or at least the plants that contain them have proved immensely

useful to people for disease and illness, for thousands of years.

1.2.8Alkaloids from the species of acacia

Alkaloids are relatively common in the leguminosae as a whole, and within the genus acacia

in Australia alkaloids that have been reported include N, N-dimethyltryptamine, N-

methyltryptamine, tryptamine, tetrahydroharman, N-methyl-tetrahydroharman, b-

phenethylamine, N-methyl-b-phenethylamine, hordenine (N, N-dimethyl-4-hydroxy-b-

phenethylamine), N-cinnamoylhistamine..... For the number of species, there has been little

research on the alkaloids of Australian acacias, and like many studies of Australian plants

there has been quite a lot of variability in the results. For example the root bark of Acacia

holoserica is reported in a few publications as containing the B-phenethylamine alkaloid

hordenine, up to 1.22% of the dry weight. Yet in a recent study of aboriginal medicinal plants

all parts of this species were found to give a negative result for alkaloids. It was still used

medicinally and another species, Acacia auriculiformis, which was used in a similar way, was

found to give a positive test for alkaloids, both are members of section Juliflorae . Other

studies have found that there can not only be variation in the amount, but also in the type of

alkaloids present, eg A. baileyana has been found to contain both B-carboline and tryptamine

alkaloids at different times of the year. Qualitative studies of the alkaloids have found that B-

phenethylamine alkaloids are quite common in the uninerved members of section

Phyllodineae with flowers in racemes, with some specimens found to contain more than 1%

alkaloids. B-phenethylamines have been found in other species from section Phyllodineae .

N-cinnamoylhistamine has been isolated from at least one member of section Juliflorae .

Tryptamine or its N-methyl and N, N-dimethyl derivatives have been found in a number of

members of section Juliflorae , and a single species from the Botrycephalae . An extra-

Australian member of sub-genus Phyllodineae is recorded as containing methylated

tryptamine and B-carboline alkaloids together. A member of section plurinerves is reported to

contain B-carboline alkaloids.

So the picture regarding alkaloids seems complex, with much variation from different areas

or amongst types or chemical races. Other plants in the Australian flora exhibit this sort of

phenomena, with great variation in the amount and even the constitu ents of the volatile oils

(Eucalyptus, Melaleuca ), alkaloids (Duboisia) or other compounds between types or

localities. Many Aboriginal people recognised this trait in the Australian bush by using plants

from one

area, and claim that the same plant from a different spot would not be effective, or may even

be toxic.

Fig: list of al alkaloid isolated from acacia species.

Fig : List of alkaloids isolated from acacia species.

Table 1.2.8.1: Alkaloids in different acacia species

Acacias Known to Contain Psychoactive Alkaloids

Acacia acuminata Up to 1.5% alkaloids, mainly consisting of tryptamine in leaf.

Acacia adunca β-methyl-phenethylamine, 2.4% in leaves.

Acacia alpina Active principles in leaf.

Acacia aneura Psychoactive. Ash used in Pituri. Ether extracts about 2-6% of the dried leaf mass.

Acacia angustifolia Psychoactive, Tryptamines.

Acacia angustissima β-methyl-phenethylamine, NMT and DMT in leaf (1.1-10.2 ppm).

Acacia aroma Tryptamine alkaloids. Significant amount of tryptamine in the seeds.

Acacia auriculiformis 5-MeO-DMT in stem bark.

Acacia baileyana 0.02% tryptamine and β-carbolines, in the leaf, Tetrahydroharman.

Acacia beauverdiana Psychoactive Ash used in Pituri.

Acacia berlandieri DMT, amphetamines, mescaline, nicotine.

Acacia catechu DMT and other tryptamines in leaf, bark.

Acacia caven Psychoactive.

Acacia chundra DMT and other tryptamines in leaf, bark.

Acacia colei DMT

Acacia complanata 0.3% alkaloids in leaf and stem, almost all N-methyl-tetrahydroharman, with traces of tetrahydroharman, some of tryptamine.

Acacia concinna Nicotine.

Acacia confusa DMT & NMT in leaf, stem & bark 0.04% NMT and 0.02% DMT in stem. Also N,N-dimethyltryptamine N-oxide.

Acacia constricta β-methyl-phenethylamine.

Acacia coriacea Psychoactive Ash used in Pituri.

Acacia cornigera Psychoactive, Tryptamines

Acacia cultriformis Tryptamine, in the leaf, stem and seeds. Phenethylamine in leaf and seeds

Acacia cuthbertsonii Psychoactive

Acacia decurrens Psychoactive, but less than 0.02% alkaloids

Acacia delibrata Psychoactive

Acacia falcata Psychoactive, but less than 0.02% alkaloids

Acacia farnesiana Traces of 5-MeO-DMT in fruit. β-methyl-phenethylamine, flower. Ether extracts about 2-6% of the dried leaf mass. Alkaloids are present in the bark and leaves. Amphetamines and mescaline also found in tree.

Acacia filiciana Psychoactive

Acacia floribunda Tryptamine, phenethylamine, in flowers other tryptamines,phenethylamines

Acacia georginae Psychoactive, plus deadly toxins

Acacia greggii N-methyl-β-phenethylamine, phenethylamine

Acacia harpophylla Phenethylamine, hordenine at a ratio of 2:3 in dried leaves, 0.6% total

Acacia holoserica Hordenine, 1.2% in bark

Acacia horrida Psychoactive

Acacia implexa Psychoactive

Acacia jurema DMT, NMT

Acacia karroo Psychoactive

Acacia kempeana Psychoactive

Acacia kettlewelliae 1.5-1.88%alkaloids, 92% consisting of phenylethylamine. 0.9% N-methyl-2-phenylethylamine found a different time.

Acacia laeta DMT, in the leaf

Acacia lingulata Psychoactive

Acacia longifolia 0.2% tryptamine in bark, leaves, some in flowers, phenylethylamine in flowers, 0.2% DMT in plant Histamine alkaloids.

Acacia longifolia

var. sophorae

Tryptamine in leaves, bark

Acacia macradenia Tryptamine

Acacia maidenii 0.6% NMT and DMT in about a 2:3 ratio in the stem bark, both present in leaves

Acacia mangium Psychoactive

Acacia melanoxylon DMT, in the bark and leaf, but less than 0.02% total alkaloids

Acacia mellifera DMT, in the leaf

Acacia nilotica DMT, in the leaf

Acacia nilotica

subsp. adstringens

Psychoactive, DMT in the leaf

Acacia obtusifolia Tryptamine, DMT, NMT, other tryptamines, 0.4-0.5% in dried bark, 0.07% in branch tips.

Acacia oerfota Less than 0.1% DMT in leaf, NMT

Acacia penninervis Psychoactive

Acacia phlebophylla 0.3% DMT in leaf, NMT

Acacia platensis Psychoactive

Acacia podalyriaefolia

Tryptamine in the leaf 0.5% to 2% DMT in fresh bark, phenethylamine, trace amounts.

Acacia polyacantha DMT in leaf and other tryptamines in leaf, bark

Acacia polyacantha

ssp. campylacantha

Less than 0.2% DMT in leaf, NMT; DMT and other tryptamines in leaf, bark.

Acacia prominens phenylethylamine, β-methyl-phenethylamine.

Acacia pruinocarpa Psychoactive, Ash used in Pituri.

Acacia pycnantha Psychoactive, but less than 0.02% total alkaloids.

Acacia retinodes DMT, NMT, nicotine, but less than 0.02% total alkaloids found.

Acacia rigidula DMT, NMT, tryptamine, amphetamines, mescaline, nicotine and

others.

Acacia roemeriana β-methyl-phenethylamine.

Acacia salicina Psychoactive Ash used in Pituri.

Acacia sassa Psychoactive.

Acacia schaffneri β-methyl-phenethylamine, Phenethylamine.Amphetamines and mescaline also found.

Acacia schottii β-methyl-phenethylamine.

Acacia senegal Less than 0.1% DMT in leaf, NMT, other tryptamines. DMT in plant, DMT in bark.

Acacia seyal DMT, in the leaf.Ether extracts about 1-7% of the dried leaf mass.

Acacia sieberiana DMT, in the leaf.

Acacia simplex DMT and NMT, in the leaf, stem and trunk bark, 0.81% DMT in bark, MMT.

Acacia taxensis β-methyl-phenethylamine.

Acacia tenuifolia Psychoactive.

Acacia tenuifolia

var. producta

Psychoactive.

Acacia tortilis DMT, NMT, and other tryptamines.

Acacia verek Psychoactive, Less than 0.1% DMT in leaf, NMT, other tryptamines

Acacia vestita Tryptamine, in the leaf and stem, but less than 0.02% total alkaloids.

Acacia victoriae Tryptamines, 5-MeO-alkyltryptamine.

Acacia visco Psychoactive.

1.2.9Possible biosynthetic pathways of secondary metabolites

Biosynthesis of triterpenoids and phytosterols (Trease and Evans, 1996)

Biosynthetically squalene or the 3S isomer of 2, 3-epoxy-2,3-dihydrosqualene is the

immediate precursor of all triterpenoids (Newman, 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

(Connolly and 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-2,3 epoxide 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.

1.11.2 Biosynthesis of napthoquinones anthraquinones

C

H

A

P

T

E

R

2.1 Methods

The chemical investigation of a plant can be divided roughly into the following major steps:

a) Collection and proper identification of the plant materials

b) Preparation of plant sample

c) Extraction

d) Fractionation and isolation of compounds

e) Structural characterization of purified compounds

2 2 ExperimentalExperimental

- Chemical- Chemical

The last step will be discussed in Chapter - 3. However, other steps will be presented here

initially as general procedure and then in connection with concerned plants.

2.2.1 Collection and proper identification of the plant 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 a bona fide 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 Plant material preparation

The leaves of the plant were collected in fresh condition. The leaves were sun-dried and then,

dried in an oven at reduced temperature (not more than 50C) to make it suitable for grinding

purpose. 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 Extraction procedures

2.2.3.1 Initial extraction

Extraction can be done in two ways such as

a) Cold extraction

b) Hot extraction

a) 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.

b) 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 individual extractive is then filtered through several means, e.g., cotton, cloth, filter

paper etc.

All the extractives are concentrated with a rotary evaporator at low temperature (40 -50C)

and reduced pressure. The concentrated extract thus obtained is termed as crude extract.

2.2.4 Solvent-solvent partitioning of crude extract

The crude extract is diluted with sufficient amount of aqueous alcohol (90%) and then gently

shaken in a separating funnel with almost equal volume of a suitable organic solvent (such as

petroleum ether) which is immiscible with aqueous alcohol. The mixture is kept undisturbed

for several minutes for separation of the organic layer from the aqueous phase. The materials

of the crude extract will be partitioned between the two phases depending on their affinity for

the respective solvents. The organic layer is separated and this process is carried out thrice for

maximum extraction of the samples. After separating of the organic phase, the aqueous phase

thus obtained is successively extracted with other organic solvents, usually of the increasing

polarity (such as carbon tetrachloride, dichloromethane, chloroform, ethyl acetate, butanol

etc). Finally, all the fractions (organic phases as well as the aqueous phase) are collected

separately and evaporated to dryness. These fractions are used for isolation of compounds.

2.2.5 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.

2.2.5.1 Chromatographic techniques

Chromatographic techniques are the most useful in the isolation and purification of

compounds from plant extracts. The advent of relatively new chromatographic media e.g.

Sephadex and Polyamide, have improved the range of separations that can be performed.

2.2.6 Column Chromatography

Column Chromatography is the most common separation technique based on the principle of

distribution (partition/adsorption) of compounds between a stationary and mobile phase.

A normal Chromatographic column is packed with silica gel (Kieselgel 60, mesh 70-230).

Slurry of silica gel in a suitable solvent is added into a glass column of appropriate height and

diameter. When the desired height of adsorbent bed is obtained, a few hundred milliliter of

solvent is run through the column for proper packing of the column. After packing, the

sample to be separated is applied as a concentrated solution in a suitable solvent or the

sample is adsorbed onto silica gel (Kieselgel 60, mesh 70-230), allowed to dry and

subsequently applied on top of the adsorbent layer. Then the column is developed with

suitable solvent mixtures of increasing polarity. The elutes are collected either in test tubes or

in conical flasks.

2.2.7 Vacuum Liquid Chromatography (VLC)

Vacuum Liquid Chromatography is a relatively recent separation technique which involves

short column chromatography under reduced pressure, the column being packed with fine

TLC grade silica (Kieselgel 60H). Details of the method have been published by Pelletier et

al (1986) and by Coll and Bowden (1986). This technique is used for the initial rapid

fractionation of crude extracts.

The column is packed with silica gel (Kieselgel 60H) under vacuum. The size of the column

and the height of the adsorbent layer are dependent upon the amount of extract to be

analyzed. The column is initially washed with a non-polar solvent (petroleum ether) to

facilitate compact packing. The sample to be separated was adsorbed onto silica gel

(Kieselgel 60, mesh 70-230), allowed to dry and subsequently applied on top of the adsorbent

layer. The column is then eluted with a number of organic solvents of increasing polarity and

the fractions are collected.

2.3.1 Thin Layer Chromatography (TLC)

Ascending one-dimensional thin layer chromatographic technique is used for the initial

screening of the extracts and column fractions and checking the purity of isolated

compounds. For the latter purpose commercially available pre-coated silica gel (Kieselgel 60

PF254) plates are usually used. For initial screening, TLC plates are made on glass plates with

silica gel (Kieselgel 60 PF254).

A number of glass plates measuring 20cm x 5cm are thoroughly washed and dried in an oven.

The dried plates are then swabbed with acetone-soaked cotton in order to remove any fatty

residue. To make the slurry required amount of silica gel 60 PF254 and appropriate volume of

distilled water (2 ml/gm of silica gel) are mixed in a conical flask and the flask is gently

shaken. The slurry is then evenly distributed over the plates using TLC spreader. After air

drying the coated plates are subjected to activation by heating in an oven at 110C for 70

minutes (Stahl, 1969; Remington Pharmaceutical sciences, 1988). Table 2.1 shows the

amount of silica gel required for preparing plates of varying thicknesses.

Table 2.1: Amount of silica gel required preparing TLC plates of various thicknesses

Size

(cm x cm)Thickness (mm)

Amount of silica gel/plate

(gm)

20 x 50.3

0.4

0.9

1.2

Cylindrical glass chamber (TLC tank) with airtight lid is used for the development of

chromatoplates. The selected solvent system is poured in sufficient quantity into the tank. A

smooth sheet of filter paper is introduced into the tank and allowed to soak in the solvent. The

tank is then made airtight and kept for few minutes to saturate the internal atmosphere with

the solvent vapour. A small amount of dried extract is dissolved in a suitable solvent to get a

solution (approximately 1%) (Harborne, 1976; Touchstone and Dobbins, 1978). A small spot

of the solution is applied on the activated silica plate with a capillary tube just 1 cm above the

lower edge of the plate. The spot is dried with a hot air blower and a straight line is drawn 2

cm below the upper edge of the activated plate which marks the upper limit of the solvent

flow.

The spotted plate is then placed in the tank in such a way as to keep the applied spot above

the surface of the solvent system and the cap/lid is placed again. The plate is left for

development. When the solvent front reaches up to the given mark, the plate is taken out and

air-dried. The properly developed plates are viewed under UV light of various wavelengths as

well as treated with suitable reagents to detect the compounds.

Preparative thin layer chromatographic technique is routinely used in separating and for final

purification of the compounds. The principle of preparative TLC is same as that of TLC.

Here larger plates (20cm x 20cm) are used. Table 2.2 shows the amount of silica gel required

for preparing plates of varying thicknesses.

Table 2.2: Amount of silica gel required preparing PTLC plates of various thicknesses

Size Thickness (mm) Amount of silica gel/plate

(gm)

(cm x cm)

20 x 200.3

0.4

3.6

4.8

The sample to be analyzed is dissolved in a suitable solvent and applied as a narrow uniform

band rather than spot. The plates are then developed in an appropriate solvent system

previously determined by TLC. In some cases multiple development technique was adopted

for improved separation. After development, the plates are allowed to dry and the bands of

compounds are visualized under UV light (254 nm and 366 nm) or with appropriate spray

reagents on both edges of the plates. The required bands are scraped from the plates and the

compounds are eluted from the silica gel by treating with suitable solvent or solvent mixtures.

2.3.2 Solvent treatment

Solvent treatment is a process by which a compound consisting of the major portion of a

mixture of compounds can be purified utilizing selective solvent washing. Initially, a solvent

or a solvent mixture in which the desired compound is practically insoluble and other

components are soluble is chosen. The undesired components are separated with repeated

washing with this solvent or solvent mixture. If required other solvent or solvent mixture can

be used until a pure compound is obtained.

2.3.3 Visualization / detection of compounds

Detection of compounds in TLC plates is a very important topic in analyzing extractives to

isolate pure compounds. The following techniques are used for detecting the compounds in

TLC/PTLC plates.

Visual detection

The developed chromatogram is viewed visually to detect the presence of colored

compounds.

UV light

The developed and dried plates are observed under UV light of both long and short

wavelength (254 nm and 366 nm) to detect the spot/band of any compound. Some of the

compounds appear as fluorescent spots while the others as dark spots under UV light.

Iodine chamber

The developed chromatogram is placed in a closed chamber containing crystals of iodine and

kept for few minutes. The compounds that appeared as brown spots are marked. Unsaturated

compounds absorb iodine. Bound iodine is removed from the plate by air blowing.

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.

a. Vanillin/H2SO4 (Stahl, 1966):

1% vanillin in concentrated sulfuric acid is used as a general spray reagent followed by

heating the plates to 100C for 10 minutes.

b. Modified Dragendorff’s reagent (Touchstone and Dobbins, 1977):

Modified Dragendorff’s reagent was used to detect alkaloids. Some coumarins also give a

positive test with modified Dragendorff’s reagent. The reagent is prepared by mixing equal

parts (v/v) of 1.7% bismuth sub-nitrate dissolved in 20% acetic acid in water and a 40%

aqueous solution of potassium iodide.

c. Ferric chloride/EtOH (Dyeing Reagents for TLC and PC, 1974):

Some of the phenolic compounds were detected by spraying the plates with ferric chloride

(5% ferric chloride in absolute ethanol) reagent.

d. Perchloric acid reagent (Touchstone and Dobbins, 1978):

2% aqueous perchloric acid produces brown spots with steroids after heating at 150 0C for 10

minutes.

e. Potassium permanganate reagent

Only the oxidizable compounds were detected by this reagent. After spraying with the

reagent the compound appeared as yellow or pale yellow spot on the colored (color of

permanganate) plate.

Determination of Rf (retardation factor) values

Rf value is characteristic of a compound in a specific solvent system. It helps in the

identification of compounds. Rf value of a compound can be calculated by the following

formula:

Rf value =

2.3.4 Chemical Investigation of Acacia auriculiformis

In this study, leaf of Acacia auriculiformis belonging to the family Leguminosae was

chemically investigated.

Taxonomic hierarchy of the investigated Leguminosae species

Kingdom Plantae

Division Magnoliophyta

Distance traveled by the compoundDistance traveled by the solvent

system

Class Magnoliopsida

Order Fabales

Family Fabaceae

Genus Acacia

Species Acacia auriculiformis.

Collection and preparation of plant material

Fresh leaves of Acacia auriculiformis was collected from Chitagong. It was identified by,

Sorker Nasir Uddin, Principal Scientific officer, Bangladesh National Herbarium, Dhaka . A

voucher specimen has been deposited in the Bangladesh National Herbarium, Dhaka

(DACB Accession no. 32,416), for the collection. All the leaves were cut into small pieces

and then air dried for several days. The pieces were then oven dried for 24 hours at

considerably low temperature to effect grinding. The plant was then ground into a coarse

powder using a grinding machine.

2.3.5 Extraction of the plant material

About 600gm of the powdered material was taken in a clean, round bottomed flask (2.5 liters)

and soaked in about 2.25 liter of methanol. The container with its content was sealed by

cotton and foil and kept for a period of 15 days accompanying occasional shaking and

stirring. The major portion of the extractable compounds of the plant material was dissolved

in the solvent during that time and hence extracted as solution. The extractive was filtered

through fresh cotton bed and finally with Whatman no.1 filters paper. The volume of the

filtrate was concentrated with a rotary evaporator at low temperature (40-50C) and reduced

pressure. Thus, one methanol extract was prepared. The same procedure was done twice with

the residue, remained after the first filtration. Hence another methanol extract was found.

Thus, two extract was found.

i) First crude methanol extract (20.82 gm).

ii) Second crude methanol extract (15.20 gm).

Investigation of the methanol-soluble extract

Both the methanol-soluble crude extracts were subjected to TLC screening to see the type of

compounds present in the extracts. From the TLC screening it was found that both the

extracts were similar and may be mixtures of some compounds. Hence, it was decided to

undergo fractionation by taking a portion of the extract from any of the two methanol-

extracts.

Solvent-solvent partition of crude extract

Solvent-solvent partition of crude extract was done to specialize the extract in order with

their selectivity, polarity etc. to separate the polar, non polar, semi polar compound to

similar group. For this purpose different solvent system of different polarity was used e.g.,

methanol, chloroform, ethyl acetate, petroleum ether, carbon tetra chloride etc . Solvent-

solvent partition of crude extract was done by Modified Kupchan method (Beckett

and Stenlake, 1986.).

Preparation of mother solution

Crude methanol extract (10.0935gm) was triturated with 100 ml of aqueous methanol (90%).

The crude extract went to the solution completely. This is the mother solution, which was

partitioned off successively by three solvents of different polarity. In subsequent stages each

of the fractions was analyzed separately for the detection and identification of antibacterial

and anticancer activity of the compound.

2.3.6 Partitioning techniques:

Partitioning with Petroleum ether

The mother solution was taken in a separating funnel. 100 ml of the Petroleum ether was

added to it and the funnel was shaken and then kept undisturbed. The organic portion was

collected. The process was repeated thrice; Petroleum ether soluble fractions (300 ml) were

collected together and evaporated. The aqueous fraction was preserved for the next step.

Partitioning with Carbontetrachloride

The aqueous fraction from the previous step was taken into the separating funnel. To the

mother solution left after washing with petroleum ether, 12.5 ml of distilled water was

added and mixed. The mother solution was then added into the aqueous fraction in the

separating funnel and extracted with CCl4 (100ml × 3). The CCl4 soluble fractions were

collected together and evaporated. The aqueous fraction was preserved for the next step.

Partitioning with chloroform

The aqueous fraction from the previous step was taken into the separation funnel. To the

mother solution that left after washing with petroleum ether and CCl 4, 16 ml of distilled

water was added and mixed uniformly. The mother solution was then added into the

aqueous fraction in a separating funnel and extracted with CHCl 3 (100 ml X 3). The CHCl3

soluble fractions were collected together and evaporated. The aqueous methanolic fraction

was preserved as aqueous fraction.

Crude extract (12.09 gm)

Aqueous methanol solution

Methanol (90 ml) + Water (10 ml)

Extraction with Petroleum ether (100 ml x 3)

Scheme-2.1: Schematic representations of the modified Kupchan partioning of

methanolic crude extract of Acacia auriculiformis.

Thus three types of crude extracts were found:

Aqueous fractionPetroleum ether soluble fraction (300 ml)

+ Distilled Water (12.5

Extraction with CCl4 (100 ml x 3)

CCl4 soluble fraction (300 ml)

Aqueous fraction

+ Distilled Water (16 ml)

Extraction with CHCl3 (100 ml x3 ml)

Aqueous fractionCHCl3 soluble fraction (300 ml)

I. Petroleum ether fraction ( 3.25gm)

II. Carbon tetrachloride fraction (1.72 gm)

III. Chloroform fraction (3.04 gm)

IV. Aqueous fraction (3.38gm)

2.3.7. Investigation of the carbontetrachloride soluble fraction

The carbon tetrachloride fraction was subjected to TLC screening to see the type of

compounds present in the extract. The whole portion of the carbon tetrachloride fraction

(1.72 gm) was subjected to Column Chromatography (CC) for rapid fractionation. The VLC

fractions were screened by TLC to find out interesting fractions.

2.3.8 Column Chromatography (CC) of carbontetrachloride fraction

The normal chromatographic column was packed with silica gel (Kieselgel 60, mesh 70-230)

as the packing material. Slurry of silica gel in a suitable solvent was added into the glass

column of appropriate height and diameter. When the desired height of adsorbent bed is

obtained, a few hundred milliliter of solvent was run through the column for proper packing

of the column. After packing, the sample was prepared by adsorbing about 1.0362 gm of

carbontetrachloride soluble fraction onto 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 chloroform and then chloroform

and then chloroform and methanol. The polarity was gradually increased by adding

increasing proportions of chloroform and methanol. Solvent systems used as mobile phases in

the CC analysis of carbon tetrachloride soluble fraction are listed in Table 2.3. A total of 27

fractions were collected.

Table 2.3: Different solvent systems used for CC analysis of carbontetrachloride

fraction

Fraction no. Solvent system Volume collected (ml)

1 Petroleum ether 100% 100

2 Petroleum ether : chloroform (97.5 : 2. 5) 100

3 Petroleum ether : chloroform (95 : 5) 100

4 Petroleum ether: chloroform (92.5 : 7.5) 100

5 Petroleum ether: chloroform (90 : 10) 100

6 Petroleum ether : chloroform (85 : 15) 100

7 Petroleum ether: chloroform (80 : 20) 100

8 Petroleum ether: chloroform (75 : 25) 100

9 Petroleum ether: chloroform (70 : 30) 100

10 Petroleum ether : chloroform (60 :40) 100

11 Petroleum ether : chloroform (50 :50) 100

12 Petroleum ether : chloroform (40 :60) 100

13 Petroleum ether : chloroform (30 : 70) 100

14 Petroleum ether : chloroform (20 : 80) 100

15 Petroleum ether : chloroform (10 : 90) 100

16 Chloroform ( 100% ) 100

17 Chloroform : methanol (99 : 1) 100

18 Chloroform : methanol (98 : 2) 100

19 Chloroform : methanol (95 : 5) 100

20 Chloroform : methanol (90 : 10) 100

21 Chloroform : methanol (50 : 50) 100

22 Methanol (100%) 100

2.3.8.1 Analysis of CC fractions by TLC

All the column fractions were screened by TLC under UV light and by spraying with

vanillin-sulfuric acid reagent followed by heating at 110C. Depending on the TLC behavior

a number of column fractions were mixed together and the rest are kept unchanged. All the

fractions were then identified by a new code which is summarized in the following table.

Table :2.3.8.1 List of new fraction codes

Column fractions

New Codes1, 2, 3, 4,5, 6 F-1

7, 8, 9 F-210, 11 F-312,13,14,15 F-416,17,18,19 F-520,21,22 F-6

2.2.3.8.2 Isolation and purification of compounds from the selected column fractions

All the column fractions were screened by TLC under UV light and by spraying with

vanillin-sulfuric acid reagent followed by heating at 110C. Depending on the TLC behavior

fractions (12-15), (16-19) &(20-22) were selected for further investigation.

Isolation and purification of compound AA-3

The column fractions12-15were screened on TLC plate and were found to be give identical

spots. So these four fractions were mixed together. The combined were subjected to

preparative thin layer chromatography (PTLC) (stationary phase: silica gel PF254, mobile

phase ethyl acetate: tolune (10:90), thickness of the plates 0.5 mm).from the developed plates

one band was visible under UV lamp at 254nm but after spraying with vanillin –sulfuric acid

reagent followed by heating at 110°C. there was an appearance of another band of purple

color . The bands were then scrapped on to a Aluminum foil and eluted using ethyl acetate.

The UV inactive compound was checked for purity and named as AA-3.

Isolation and purification of compound AA-2

F-21 was found to yield colored crystal. The crystals were first washed with petroleum ether

carefully. As a result, green colored solution was obtained leaving back the white colored

needles. After several washing by pure petroleum ether, mixtures of petroleum ether and

ethyl acetate with increased polarity were used for the washing purpose. As soon as the

crystals were started to dissolve at a certain polarity, washing was stopped. After completion

of the washing, the beaker containing the crystals was designated as AA-2. Compound AA-2

was also obtained from carbontetrachloride fraction by CC (Stationary phase:-Silica gel

(Kieselgel 60, mesh 70-230), Mobile phase:-Chloroform, 100%).

2.3.8.3Investigation of the CHCL3 soluble fraction:

The chloroform soluble fraction was subjected to TLC screening to see the type of

compounds present in the extract. The whole portion of the carbon tetrachloride fraction

(3.04 gm) was subjected to Column Chromatography (CC) for rapid fractionation. The VLC

fractions were screened by TLC to find out interesting fractions.

Solvent system used for VLC analysis of CHCL3 fraction:

Fraction no. Solvent system Volume collected (ml)

1 Petroleum ether 100% 100

2 Petroleum ether : chloroform (98 : 2) 100

3 Petroleum ether : chloroform (95 : 5) 100

4 Petroleum ether: chloroform (90 : 10) 100

5 Petroleum ether : chloroform (85 : 15) 100

6 Petroleum ether: chloroform (80 : 20) 100

7 Petroleum ether: chloroform (75 : 25) 100

8 Petroleum ether: chloroform (70 : 30) 100

9 Petroleum ether : chloroform (60 :40) 100

10 Petroleum ether : chloroform (50 :50) 100

11 Petroleum ether : chloroform (40 :60) 100

13 Petroleum ether : chloroform (30 : 70) 100

14 Petroleum ether : chloroform (20 : 80) 100

15 Petroleum ether : chloroform (10 : 90) 100

16 Chloroform ( 100% ) 100

17 Chloroform : methanol (99 : 1) 100

18 Chloroform : methanol (98 : 2) 100

19 Chloroform : methanol (95 : 5) 100

20 Chloroform : methanol (90 : 10) 100

21 Chloroform : methanol (50 : 50) 100

22 Methanol (100%) 100

Analysis of VLC fraction by TLC

All the column fractions were screened by TLC under UV light and by spraying with

vanillin-sulfuric acid reagent followed by heating at 110C. Depending on the TLC behavior

fractions they were selected for further investigation.

2.3.8.4Gel permission chromatography of chloroform soluble fraction of leaves of A.

auriculiformis

The column was packed with sephadex(LH-20). At first sephadex was soaked in amixture of solvent

with a ratio of n-hexane : Dichloromethane : Methanol = 2:5:1 for at least 12 hours for proper

swelling. After that, slurry of sephadex was made and added into a glass column having the length &

diameter of 55cm&1.1cm respectively. When sufficient height of the adsorbent bed was obtained ,

afew hundred milliliter of solvent mixture with the same ratio was run through the column for

proper packing of the column. The sample was dissolved in this solvent mixture and subsequently

applied on the top of the adsorbent layer with the help of pasture pipette. The column was then

eluted with the same solvent mixture and finally the column was washed with dichloromethane

&methanol mixture of increasing polarity. The column fraction were collected in test tubes each

containing 2ml approximately. The solvent used as mobile phases in this analysis of the fraction are

listed in the table-

Fraction No. Solvent system Proportion Volume collected( ml)

1-12 n-Hexane: Dichloromethane: Methanol 2:5:1 100

13-19 Dichloromethane: Methanol 9:1 50

20-25 Dichloromethane: Methanol 1:1 50

26-34 Methanol 100% 100

2.3.8.5Analysis of column fractions by TLC

All the fractions were screened by TLC under UV light and by spraying with vanillin sulfuric

acid reagent. Depending on the TLC behavior sub-fractions 11-14 were taken for further

investigation.

2.3.8.6 Isolation and purification of compounds from the selected CC fractions

Isolation and purification of compound AA-5

The column fractions 11-14 were screened on TLC plate and were found to be give identical

spots. So these four fractions were mixed together. The combined were subjected to

preparative thin layer chromatography (PTLC)(stationary phase: silica gel PF254,mobile

phase ethyl acetate : tolune (10:90), thickness of the plates 0.5 mm).from the developed

plates a band was visible under UV lamp at 254nm and shown a purple color after spraying at

two sides of the plate with vanillin sulfuric acid spray followed by heating at 110°C. The

band was then scrapped on to a Aluminum foil and eluted using ethyl acetate. The material

was checked for purity and named as AA-3.

Isolation and purification of compound AA-15

The column fractions16-20 were screened on TLC plate and were found to be give identical

spots. So these four fractions were mixed together. The combined were subjected to

preparative thin layer chromatography (PTLC)(stationary phase: silica gel PF254,mobile

phase ethyl acetate : tolune (10:90), thickness of the plates 0.5 mm).from the developed

plates a band was visible under UV lamp at 254nm and shown a violet color after spraying at

two sides of the plate with vanillin sulfuric acid spray followed by heating at 110°C. The

band was then scrapped on to a Aluminum foil and eluted using ethyl acetate. The material

was checked for purity and named as AA-3.

2.2.9. Instrumentation for isolation and characterization of compounds

The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were acquired in CDCl3 on an

Ultra Shield Bruker DPX 400 spectrometer and the chemical shifts are reported in parts per

million (ppm) relative to the residual nondeuterated solvent signals. Follow up of the

reactions and checking the homogenicity of the compounds were made by TLC on Kieselgel

60 PF254 pre-coated sheets (E.Merck) and the spots were detected by exposure to UV-lamp at

254 nm. Column chromatography was done on silica gel (70 – 230 mesh ASTM).

C

H

A

P

T

E

R

Preliminary investigation of the plant material

Plant material

A species of the Leguminosae family, Acacia auriculiformis, has been investigated in this

work. The plant part used was the leaves.

Extraction of the plant material

Fresh leaves of A. auriculiformis were collected, dried and ground to a coarse powder. The

powder sample (1300gm) was subjected to cold extraction with methanol for about 7 days

3 3 Results & DiscussionResults & Discussion

- Chemical- Chemical

and then filtered and the residue was further subjected to cold extraction for about a week.

Thus two separate crude methanol extractives were obtained.

Isolation and characterization of compounds

From the extractives pure compounds were isolated applying various chromatographic

techniques according to the following scheme (Figure-3.1). The isolated pure compounds

were then characterized using various spectroscopic techniques.

3.1 Characterization of isolated compounds from Acacia auriculiformis

3.1.1Characterization of AA-2 as Lupen-3β, 28-diol (Betulin)

Physical characteristics:

Color: white

Physical state: Amorphous solid.

UV sensitivity: Yes

Rf value: 44 (10% Ethyl acetate in tolune)

Chemical characteristics:

Solubility: soluble in n-hexane, Ethyl acetate and CHCl3 & methanol.

Compound AA-2 (Figure-3.2) was isolated as white crystals from the column fraction of

carbon tetrachloride fraction by elution with 10% Ethyl acetate in tolune. It appeared as a

dark quenching spot on the TLC plate (90% Toluene) under UV light at 254 nm. Spraying the

developed plate with Vanillin-sulfuric acid spray reagent followed by heating at 110°C for

several minutes, gave a magenta colour. It was found to be soluble in ethyl acetate and

chloroform.

The 1H NMR spectrums (400 MHz, CDCl3) of AA-2 (Table-3.1, Figure-3.3, 3.4, 3.5 and 3.6)

revealed signals for five tertiary methyl [δH : 0.75, 0.82, 0.96, 0.97 and 1.01] and also one

vinyl methyl [δH : 1.67]. The 1H NMR spectrums also displayed two olefinic proton as

singlets at δH 4.57 and 4.67; In addition two methylene protons (-CH2OH) appeared at δH

3.32 (1H, d, J=10.6 Hz) and 3.78 (1H, d, J=10.6 Hz) a secondary carbinol at δH 3.18 (1H, dd,

J=4.4, 10.4 Hz).

This data indicated a penta cyclic triterpenoid of lupen-3β, 28-diol (Betulin) and comparing

its 1H NMR spectral data with the literature values of reported compounds (Mahato et al.,

1994, Rosenel et al., 1998, Marina et al., 1997), the structure of compound AA-2 was

confirmed as lupen-3β, 28-diol (Betulin).

Figure 3.2: Structure of AA-2

Table 3.1 Comparison of 1H (400 MHz, CDCl3) NMR data for AA-2 with previously

published data for Betulin ((Muhammad Riaz et al., 2001)).

δm in ppm in CDCL3 (400Mz)

Position

AA-2

Betulin

(Muhammad Riaz et al., 2001)

no. H (mult., J in Hz) H(mult.,J in Hz)

H-29 4.67 (1H, s ) 4.68 (1H, s )

H-29 4.57 1(1H, s) 4.57 (1H, s)

H-28 3.78 (1H, d, J=10.6 ) 3.78(1H,d, J=10.6 )

H-28 3.32 (1H , d, J =10.6 ) 3.32 (1H , d, J =10.6 )

H-3 3.183 (1H , d, J = 4.4, 10.4 ) 3.18 (1H , d, J = 4.4, 10.4 )

H-19 2.38 (1H, m) 2.37 (1H, m)

30-CH3 1.671 (3H, s ) 1.67 (3H, s )

24-CH3 1.014(3H, s ) 1.02 (3H, s )

23-CH3 0.97 1(3H, s ) 0.97 (3H, s )

26-CH3 0.959 (3H, s ) 0.95(3H, s )

27-CH3 0.816 (3H, s) 0.82 (3H, s)

25-CH3 0.75 (3H, s) 0.75 (3H, s)

From the above discussion and data interpretation we can say that this compound is Lupen-

3β, 28-diol (Betulin). Though this a common natural product but extensive literature

survey suggest no finding of this compound from this plant. So AA-2 is the first time report

from this plant.

Characterization of AA-5 as Lupeol.

Physical characteristics:

Color: white

Physical state: Amorphous solid.

UV sensitivity: Yes

Rf value: 0.38 (5% Ethyl acetate in tolune)

Chemical characteristics:

Solubility: soluble in n-hexane, Ethyl acetate and CHCl3 & methanol.

Compound AA-5 was isolated as white amorphous powder from the leaves of Acacia

auriculiformis. TLC examination showed it as a single compound. It was found to be UV

active in short wave length on TLC (silica gel PF254). It appeared as purple color on TLC

after spraying the developed plate with vanillin-sulfuric acid followed by heating at 110°C

several minutes.

The compound was soluble in n-Hexane, Ethyl acetate and CHCl3, and sparingly soluble in

methanol. So the spectral data were taken in CDCL3. Thus it was moderately non polar in

nature.

The 1H NMR spectrum (400 MHz,CDCL3) of table- and figure showed one double doublet

of one 3.20 (j=11.2, 4.8) proton intensity at ∂ typical for H-3. The spectrum displayed two

singlet at ∂ 4.56 and ∂ 4.68 (1H each) assignable to protons at C-29. Doublet of double

doublet at ∂ 2.36 assignable to protons at C-19. The spectrum displayed seven singlet at ∂

0.755, ∂ 0.783, ∂ 0.825, ∂ 0.94, ∂ o.96, ∂ 1.03 and ∂ 1.675 (3 H) each suggestive of the

presence of seven methyl groups in this compound. This were attributed to H3-28, H3-24,

H3-25, H3-27, H3-23, H3-26, H3-30 respectively.

By comparing the 1H NMR data of AA-5 with that previous published data (Aratanechemuge

et, al, 2004) it was confirmed as Lupeol.

Though this is a common natural product but extensive literature survey suggest that this is

first time report from this plant.

Position δm in ppm in CDCL3

AA-5 Leupeol

H-29 4.682 and 4.561 (2S, 1H

each)

4.68 and 4.56 (2s, 1H each, H-

29

H-3 3.20 (m, 1H, H-3) 3.23 (m, 1H, H-3)

H-19 2.361 (m, 1H, H-2) 2.36 (m, 1H, H-2).

H-30 1.675 (s, 3H, H-30) 1.68 (s, 3H, H-30)

3H-30 1.675(s, 3H), 1.68 (s, 3H),

3H-26 1.026 (s, 3H), 1.02 (s, 3H),

3H-23 0.962 (d, 3H), 0.96 (s, 3H),

3H-27 0.939 (s, 3H) 0.94 (s, 3H)

3H-25 0.825 (s, 3H) 0.82 (s, 3H)

3H-24 0.783 (s, 3H), 0.78 (s, 3H),

3H-28 0.755 (s, 3H) 0.75 (s, 3H),

Characterization of AA-5 as Lupeol glucoside and a minor impurity.

Physical characteristics:

Color: Light green

Physical state: Amorphous solid.

UV sensitivity: no

Rf value: 0.64 (10% Ethyl acetate in tolune)

Chemical characteristics:

Solubility: soluble in n-hexane, Ethyl acetate and CHCl3 & methanol.

Color on vanillin sulfuric acid spray: purple

Compound AA-5 was isolated as slightly greenish amorphous powder from the leaves of

Acacia auriculiformis. TLC examination showed it as a single compound. It was found to be

UV inctive in short wave length on TLC (silica gel PF254). It appeared as purple color on

TLC after spraying the developed plate with vanillin-sulfuric acid followed by heating at

110°C several minutes.

The compound was soluble in n-Hexane, Ethyl acetate and CHCl3, and sparingly soluble in

methanol. So the spectral data were taken in CDCL3. Thus it was moderately non polar in

nature.

Though visual appearance confirmed it as a single compound but the 1H NMR spectrum (400

MHz, CDCL3) of that compound proves that the compound is lupeol glucoside with minor

trace impurity which cannot be confirmed from the spectrum.

The 1H NMR spectrum (400 MHz,CDCL3) of table- and figure displayed no double doublet

of one 3.20 (j=11.2, 4.8) proton intensity at ∂ typical for H-3 which is characteristics for

lupeol likewise a spectrum of four triplet at ∂3.84 to ∂4.24 which represent a glucose

molecule in that structure. The spectrum displayed two singlet at ∂ 4.54 and ∂ 4.65 (1H each)

assignable to protons at C-29 and one D-shielded proton at ∂4.60 assignable to C-4. Doublet

of double doublet at ∂ 2.34 assignable to protons at C-19. The spectrum displayed seven

singlet at ∂ 0.75, ∂ 0.78 (2 singlet ), ∂ 0.82, ∂ o.93, ∂ o.96, ∂ 1.03 and ∂ 1.68 (3 H) each

suggestive of the presence of seven methyl groups in this compound. This were attributed to

H3-28, H3-24, H3-25, H3-27, H3-23, H3-26, H3-30 respectively.

By comparing the 1H NMR data of AA-5 with that previous published data (Aratanechemuge

et, al, 2004) it was confirmed that it has a Lupeol structure. Hence the molecule has a

multiplet at ∂ 3.64-∂ 4.24 which is characteristics for linkage of β-glucoside the compound is

confirmed as Lupeol glucoside. Although it is a known natural product but this is the first time

isolation from this plant.

Position δm in ppm in CDCL3

AA-5

1HNMR, CDCl3, 400 MHz

Lupeol

H-29 4.682 and 4.561 (2S, 1H each) 4.71and 4.59 (2s, 1H each, H-29

H-3 3.63(m, 1H, H-3) 3.59 (m, 1H, H-3)

H-19 2.361 (m, 1H, H-2) 2.36 (m, 1H, H-2).

3H-30 1.67 (s, 3H, H-30) 1.68 (s, 3H, H-30)

1.65 (s, 3H, H-30) 1.64(s, 3H, H-30)

3H-26 1.019 (s, 3H), 1.02 (s, 3H),

3H-23 0.95 (d, 3H), 0.96 (s, 3H),

3H-27 0.93 (s, 3H) 0.94 (s, 3H)

3H-25 0.825 (s, 3H) 0.82 (s, 3H)

3H-24 0.776 (s, 3H), 0.78 (s, 3H),

3H-28 0.749 (s, 3H) 0.75 (s, 3H),

Side chain linkage AA-5

1HNMR, CDCl3, 400 MHz

O-β-glucopyranosyl-β-sitosterol,1HNMR, CDCl3, 300 MHz

3-beta glucosidic multiplet ∂ 3.84-∂4.24 ∂ 3.21-∂ 4.36

Characterization of AA-15 as-Para hydroxyl Lupeol-3-o-β Cinnamate.

Physical characteristics:

Color: Greenish blue

Physical state: Amorphous solid.

UV sensitivity: yes

Rf value: 0.42 (10% Ethyl acetate in tolune)

Chemical characteristics:

Solubility: soluble in n-hexane, Ethyl acetate and CHCl3 & methanol.

Color on vanillin sulfuric acid spray: violet

Compound AA-15 was isolated as light greenish blue amorphous powder from the leaves of

Acacia auriculiformis. TLC examination showed it as a single compound. It was found to be

UV active in short wave length on TLC (silica gel PF254). It appeared as violet color on TLC

after spraying the developed plate with vanillin-sulfuric acid followed by heating at 110°C

several minutes.

The compound was soluble in n-Hexane, Ethyl acetate and CHCl3, and sparingly soluble in

methanol. So the spectral data were taken in CDCL3. Thus it was moderately non polar in

nature.

The 1H NMR spectrum (400 MHz,CDCL3) of table- and figure showed one double doublet

of one 3.20 (j=11.2, 4.8) proton intensity at ∂ typical for H-3. The spectrum displayed two

singlet at ∂ 4.56 and ∂ 4.68 (1H each) assignable to protons at C-29 and one D-shielded

proton at ∂4.60 assignable to C-4. Doublet of double doublet at ∂ 2.36 assignable to protons

at C-19. The spectrum displayed seven singlet at ∂ 0.784, ∂ 0.88 (2 singlet ), ∂ 0.91, ∂ o.95, ∂

1.03 and ∂ 1.68 (3 H) each suggestive of the presence of seven methyl groups in this

compound. This were attributed to H3-28, H3-24, H3-25, H3-27, H3-23, H3-26, H3-30

respectively.

1H NMR spectra also reveals two sets signals of double distributed benzene rings, one at ∂

7.43(1H,d,j=8.4 Hz) & ∂ 6.84(1H,d,j=8.4 Hz) and the other one at 7.41(1H,d,j=8.4 Hz) &

6.82(1H,d,j=8.4 Hz). Furthermore, the spectra showed the signals for the (Z)-olefinic H-

atoms (H-7& H-8) at ∂7.59 (1H, d, j=16Hz) & ∂6.28 (1H, d, j=16Hz).

The two protons ∂7.59 (1H, d, j=16Hz) & ∂6.28 (1H, d, j=16Hz) are trans-coupled. The

proton having ∂6.28 is more shielded then ∂7.59 suggesting its connection with a easter (-

COO) moiety & the proton having ∂7.59 must be attached to an aromatic ring. So the

structure of the compound should contain the following pattern:

H O

R1 C C C O R2

H

By comparing the 1H NMR data of AA-15 with that previous published data (Aratanechemuge et, al,

2004) of Lupeol we see that R2 section is a triterpenoid having lupeol structure.

Position δm in ppm in CDCL3

AA-15

1HNMR, CDCl3, 400

MHz

Lupeol

H-29 4.682 and 4.561 (2S, 1H

each)

4.71and 4.59 (2s, 1H each, H-

29

H-3 3.63(m, 1H, H-3) 3.59 (m, 1H, H-3)

H-19 2.361 (m, 1H, H-2) 2.36 (m, 1H, H-2).

3H-30 1.67 (s, 3H, H-30) 1.68 (s, 3H, H-30)

3H-26 1.019 (s, 3H), 1.02 (s, 3H),

3H-23 0.95 (d, 3H), 0.96 (s, 3H),

3H-27 0.93 (s, 3H) 0.94 (s, 3H)

3H-25 0.825 (s, 3H) 0.82 (s, 3H)

3H-24 0.776 (s, 3H), 0.78 (s, 3H),

3H-28 0.749 (s, 3H) 0.75 (s, 3H),

Again further comparing the rest of data with isobutyl-3, 4-dihydroxy cinnamate (Hoeneisen et al.,

2003) it is evident that there is a cinnamate group linked with lupeol molecule.

Position δm in ppm in CDCL3

AA-15(1HNMR, CDCl3,

400 MHz)

isobutyl-3,4-dihydroxy

cinnamate.(1H NMR, CDCl3,

400 MHz)

H-2 7.43(1H,d,j=8.4 Hz) 7.09(d, j=2Hz)

H-5 6.84(1H,d,j=8.4 Hz) 6.87(1H,d,j=8.4 Hz)

H-6 7.41(1H,d,j=8.4 Hz) 6.95(dd,j=8.4,1.8Hz)

H-7 7.59 (1H, d, j=16Hz) 7.56 (1H, d, j=16Hz)

H-8 6.28 (1H, d, j=16Hz). 6.24 (1H, d, j=16Hz).

H-3 6.82(1H,d,j=8.4 Hz)

From the above discussion we can say that this compound is Para hydroxyl Lupeol-3-o-β

Cinnamate which is very rare in nature. In my best knowledge, there is no certain finding of

this compound from this species and also this is the first report from this plant.

Infrared spectroscopy

IR spectroscopy is the subset of spectroscopy that deals with the infrared region of the

electromagnetic spectrum. It covers a range of techniques, the most common being a form of

absorption spectroscopy. As with all spectroscopic techniques, it can be used to identify

compounds and investigate sample composition. A common laboratory instrument that uses

this technique is an infrared spectrophotometer.

Characterization of the isolated compounds with FTIR spectroscopy:

FTIR of the compounds Betulin(AA-2), Lupeol glycoside(AA-3), Lupeol(AA-5) and para

hydroxyl Lupeol-3β-O-cinnamate(AA-15) was done which is shown in the Figure .Their

positions, relative intensity of the observed bands together with their assignments to different

vibrational modes are also recorded in Table.

The common feature in IR for Betulin(AA-2), Lupeol glycoside(AA-3), Lupeol(AA-5) and

para hydroxyl Lupeol-3β-O-cinnamate(AA-15)is the presence of band near 3400 cm-1 due to

presence of strong H-bonded –OH group which confirms the presence of OH group strongly

bonded in that compound. A long sharp band near 2900 to 2950 cm -1 represents SP2-CH

stretch in all the compound which correspond to characteristic pick of aromatic –CH

stretching vibration, furthermore small band near 2380 cm-1 is a characteristic peak of para

linkage confirms that a -OH is present in these compounds with para linkage. All of the four

compounds shows two medium sharp peak at 1450-1600 cm-1 which corresponds to the

characteristics band of multiple aromatic rings present in these compounds. Some small

peaks near the aromatic bands (1360-1420cm-1) reveals that a multiple –CH3 group present in

these compound. There is some common small peak like long chain bend (780 to 800 cm-1 ), -

CO- stretch (1100-1170 cm-1) and Cl- (440-450 cm-1 due to presence of some solvent CHCl3

in KBr Plate) and From the above discussion it is clear that all the compound have a same

characteristic main body and literature survey shows that they have long aromatic tri-

terpenoid rings with para hydroxyl group. Compound AA-5 is confirmed as lupeol by H

NMR spectral with no other anomaly.

The FTIR data of compound AA-3 shows there is a peak near 1720 cm which is indicative of

a substitution at the 3-beta position of the molecule. 1H NMR spectra suggests that there is a

glucosidic substitution at para position of lupeol. So the described compound is Lupeol

glucoside. (characteristics for -CH2OH) and a another para group at near 880 cm-1 suggest

that this is a trans-coupled dihydroxy triterpenoid. With the reference of 1H NMR spectral

data it can be easily concluded that this compound is lupen-3β, 28-diol (Betulin).

The FTIR data of compound AA-15 represents a sharp peak at around 1680 cm-1 suggestive

of a substitution at para position of aromatic ring. Furthermore three sharp peak in the region

of 1000-1280c cm-1 suggest a C-O-C stretch and a peak ortho group (820-880 cm-1)

suggestive of presence of ortho-beta linkage in the substitute ring.1H NMR data & spectral

FTIR data reveals same compound with no anomaly and confirmed the structure of the

compound AA-15 as para hydroxyl Lupeol-3β-O-cinnamate.

Table 3.1. IR data for AA-15

Sample Wave Number (cm-1) of peaks Vibrations (a)Intensity

AA-153440 OH str.(H bond) Strong

2950 SP2 –C-H stretch Strong2380 Para group low1680 Substitution at

aromatic tingMedium

1450-1600 Aromatic c=c stretch

medium

1360-1420 CH3 bend medium

1020-1250 C-O-C stretch medium

815-880 Ortho beta linkage medium

Table 3.2. IR data for AA-2

Sample Wave Number (cm-1) of peaks Vibrations (a)Intensity

AA-23420 OH str.(H bond) Strong

2950 SP2 –C-H stretch Strong2380 Para group Low1680 Substitution at

aromatic tingMedium

1450-1600 Aromatic c=c stretch Low1360-1460 CH2,CH3 bend Medium

1035 CH2OH para linkage High

880 Para linkage Medium

Table 3.2. IR data for AA-3

Sample Wave Number (cm-1) of peaks Vibrations (a)Intensity

3420 OH str.(H bond) Strong

AA-32950 SP2 –C-H stretch Strong2380 Para group Low1720 Substitution at Low

aromatic ting1450-1600 Aromatic c=c stretch Low1360-1460 CH2,CH3 bend low

1160&1180 -CO-stretch low

Table 3.2. IR data for AA-5

Sample Wave Number (cm-1) of peaks Vibrations (a)Intensity

AA-53420 OH str.(H bond) Strong2950 SP2 –C-H stretch Strong2380 Para group Low1720 Substitution at

aromatic tingLow

1450-1600 Aromatic c=c stretch

Low

1360-1460 CH2,CH3 bend low

1160 -CO-stretch low

CF-(11-14) CF-(16-20)

(Powdered leaves)

Acacia auriculiformis

Cold extraction with methanol

Fig 3.1: Schematic diagram of the chemical investigation of Acacia auriculiformis

Isolation of pure crystals

Isolation of pure crystals

Carbontetrachloride Extract

Partitioning by Modified Kupchan Method Chloroform Extract

Petroleum ether Extract

AA-3

Column fraction 12-15 Column fraction 21

AA-2 AA-5AA-15

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CHAPTER 4 DESIGN OF BIOLOGICAL INVESTIGATION

4.1 General Approaches to Drug Discovery from Natural Sources

New medicines have been discovered with traditional, empirical and molecular approaches

(Harvey, 1999). The traditional approach makes use of drug that has been found by trial and

error over many years in different cultures and systems of medicine (Cotton, 1996).

Examples include drugs like morphine, quinine and ephedrine that have been in widespread

use for a long time, and more recently adopted compounds such as the antimalarial

artemisinin. The empirical approach builds on an understanding of a relevant physiological

process and often develops a therapeutic agent from a naturally occurring lead molecule

(Verpoorte, 1989, 2000). Examples include tubocurarine and other muscle relaxants,

4 4 Design of BiologicalDesign of Biological

InvestigationsInvestigations

propranolol and other -adrenoceptor antagonists, and cimetidine and other H2 receptor

blockers. The molecular approach is based on the availability or understanding of a molecular

target for the medicinal agent (Harvey, 1999). With the development of molecular biological

techniques and the advances in genomics, the majority of drug discovery is currently based

on the molecular approach.

The major advantage of natural products for random screening is the structural diversity

(Cleason and Bohlin, 1997; Harvey, 1999). Bioactive natural products often occur as a part of

a family of related molecules so that it is possible to isolate a number of homologues and

obtain structure-activity relationship. Of course, lead compounds found from screening of

natural products can be optimized by traditional medicinal chemistry or by application of

combinatorial approaches. Overall, when faced with molecular targets in screening assays for

which there is no information about low molecular weight leads, use of a natural products

library seems more likely to provide the chemical diversity to yield a hit than a library of

similar numbers of compounds made by combinatorial synthesis. Since only a small fraction

of the world’s biodiversity has been tested for biological activity, it can be assumed that

natural products will continue to offer novel leads for novel therapeutic agents.

4.2 Design of Biological Investigations

In earlier times, all drugs and medicinal agents were derived from natural substances, and most of these remedies were obtained from higher plants. Today, many new chemotherapeutic agents are synthetically derived, based on "rational" drug design. The study of natural products has advantages over synthetic drug design in that it leads optimally to materials having new structural features with novel biological activity. Not only do plants continue to serve as important sources of new drugs, but phytochemicals derived from them are also extremely useful as lead structures for synthetic modification and optimization of bioactivity. The starting materials for about one-half of the medicines we use today come from natural sources. Virtually every pharmacological class of drugs includes a natural product prototype. The future of plants as sources of medicinal agents for use in investigation, prevention, and treatment of diseases is very promising. (Setzer, W.N., 1999)

Natural products are naturally derived metabolites and/or by products from microorganisms,

plants, or animals (Baker et al., 2000). The major advantage of natural products for random

screening is the structural diversity .Bioactive natural products often occur as a part of a

family of related molecules so that it is possible to isolate a number of homologues and

obtain structure-activity relationship. Of course, lead compounds found from screening of

natural products can be optimised by traditional medicinal chemistry or by application of

combinatorial approaches. Overall, when faced with molecular targets in screening assays for

which there is no information about low molecular weight leads, use of a natural products

library seems more likely to provide the chemical diversity to yield a hit than a library of

similar numbers of compounds made by combinatorial synthesis. Since only a small fraction

of the world’s biodiversity has been tested for biological activity, it can be assumed that

natural products will continue to offer novel leads for novel therapeutic agents.

4.3 Experimental Design

4.3.1 Bioassay

Two “bench top” bioassays were adopted which do not require higher animals to screen and

direct the fractionation of botanical extracts in drug discovery efforts. These are:

1. The brine shrimp lethality test (BST) (a general bioassay).

2. The inhibition of crown gall tumors on discs of potato tubers (an antitumor

bioassay)

4.3.2 Brine shrimp lethality test: A Rapid Bioassay

Brine shrimp lethality bioassay (Mclaughlin et al., 1976; Meyer et al., 1986) is a rapid and

comprehensive bioassay for the bioactive compounds of natural and synthetic origin and is

considered a useful tool for preliminary assessment of toxicity. It has also been suggested for

screening pharmacological activities in plant extracts. The method utilizes in vivo lethality in

a simple zoological organism (Brine shrimp nauplii) as a convenient monitor for screening

and fractionation in the discovery of new bioactive natural products.

Brine shrimp toxicity is closely correlated with 9KB (human nasopharyngeal carcinoma)

cytotoxicity (p=0.036 and kappa = 0.56). ED50 values for cytotoxicities are generally about

one-tenth the LC50 values found in the brine shrimp test. Thus, it is possible to detect and then

monitor the fractionation of cytotoxic, as well as 3PS (P388) (in vivo murine leukaemia)

active extracts using the brine shrimp lethality bioassay.

The brine shrimp assay has advantages of being rapid (24 hours), inexpensive, and simple

(e.g., no aseptic techniques are required). It easily utilizes a large number of organisms for

statistical validation and requires no special equipment and a relatively small amount of

sample (2-20 mg or less). Furthermore, it does not require animal serum as is needed for

cytotoxicities

4.3.3 Microbiological Investigations

The in vitro antimicrobial study was designed to investigate the antibacterial as well as

antifungal spectrum of the crude extracts by observing the growth response. The rationale for

these experiments is based on the fact that bacteria and fungi are responsible for many

infectious diseases, and if the test materials inhibit bacterial or fungal growth then they may

be used in those particular diseases. However, a number of factors viz. the extraction method

inocula volume, culture medium composition, pH and incubation temperature can influence

the results.

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CHAPTER-5

EVALUATION OF ANTIOXIDANT ACTIVITY

5.1 Rationale and Objective

There is considerable recent evidence that free radical induce oxidative damage to

biomolecules. This damage causes cancer, aging, neurodegenerative diseases, atherosclerosis,

malaria and several other pathological events in living organisms (Halliwell et al., 1992).

Antioxidants which scavenge free radicals are known to posse an important role in preventing

these free radical induced-diseases. There is an increasing interest in the antioxidants effects

of compounds derived from plants, which could be relevant in relations to their nutritional

incidence and their role in health and diseases (Steinmetz and Potter, 1996; Aruoma, 1998;

Bandoniene et al., 2000; Pieroni et al., 2002; Couladis et al., 2003). A number of reports on

the isolation and testing of plant derived antioxidants have been described during the past

decade. Natural antioxidants constitute a broad range of substances including phenolic or

5 5 Evaluation ofEvaluation of

Antioxidant ActivityAntioxidant Activity

nitrogen containing compounds and carotenoids (Shahidi et al., 1992; Velioglu et al., 1998;

Pietta et al., 1998).

Lipid peroxidation is one of the main reasons for deterioration of food products during

processing and storage. Synthetic antioxidant such as tert-butyl-1-hydroxytoluene (BHT),

butylated hydroxyanisole (BHA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ)

are widely used as food additives to increase shelf life, especially lipid and lipid containing

products by retarding the process of lipid peroxidation. However, BHT and BHA are known

to have not only toxic and carcinogenic effects on humans (Ito et al., 1986; Wichi, 1988), but

abnormal effects on enzyme systems (Inatani et al., 1983). Therefore, the interest in natural

antioxidant, especially of plant origin, has greatly increased in recent years (Jayaprakasha &

Jaganmohan Rao, 2000).

5.2 Principle

The free radical scavenging activities (antioxidant capacity) of the plant extracts on the stable

radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were estimated by the method of Liyanna-

pathiranan and shahidi (2005). 2.0 ml of a methanol solution of the extract at different

concentration were mixed with 3.0 ml of a DPPH methanol solution (20μg/ml). The

antioxidant potential was assayed from the bleaching of purple colored methanol solution of

DPPH radical by the plant extract as compared to that of tert-butyl-1-hydroxytoluene (BHT)

and ascorbic acid (ASA) by UV spectrophotometer.

+ RH

Antioxidant

*DPPH (oxidized form) DPPH (reduced form)

* DPPH = 1, 1-diphenyl-2-picrylhydrazyl

Control preparation for antioxidant activity measurement:

Ascorbic acid (ASA) was used as positive control. Calculated amount of ASA was dissolved

in methanol to get a mother solution having a concentration 1000 µg/ml. serial dilution was

made using the mother solution to get different concentration ranging from 500.0 to 0.977

µg/ml.

Test sample preparation:

Calculated amount of different extractives were measured and dissolved in methanol to get

mother solution having a concentration 1000 µg/ml. serial dilution of the mother solution gave

different concentration ranging from 500.0 to 0.977 µg/ml which were kept in the marked flasks.

Plant part Sample code Test sample Concentration

Mg/ml

Acacia auriculiformis

(leaves)

MEAAL Methanolic extract of

A. auriculiformis leaves.

2.0

CTAAL Carbon tetrachloride soluble partitionate of AA leaves

2.0

CHAAL Chloroform soluble partitionate of AA leaves

2.0

PEAAL Pet ether soluble partitionate of AA leaves 2.0

Compound AA-2

Synthesized compound Betulin 2.0

DPPH solution preparation

20 mg DPPH powder was weighed and dissolved in methanol to get a DPPH solution having

a concentration 20 µg/ml. The solution was prepared in amber reagent bottle and kept in the

light proof box.

5.3 Materials & Methods

DPPH was used to evaluate the free radical scavenging activity (antioxidant potential) of

various compounds and medicinal plants (Choi et al., 2000; Desmarchelier et al., 1997).

5.3.1 Materials

1,1-diphenyl-2-picrylhydrazyl Test tube

Ascorbic acid (ASA) Light-proof box

Distilled water Pipette (5ml)

Methanol Micropipette (50-200 µl)

UV-spectrophotometer Amber reagent bottle

Beaker (100 & 200ml) -

5.3.2 Assay of free radical scavenging activity

2.0 ml of a methanol solution of the extract at different concentration (500 to

0.977μg/ml) were mixed with 3.0 ml of a DPPH methanol solution (20μg/ml).

After 30 min reaction period at room temperature in dark place the absorbance was

measured against at 517 nm against methanol as blank by UV spetrophotometer.

Inhibition free radical DPPH in percent (I%) was calculated as follows:

(I%) = (1 – Asample/Ablank) X 100

Where Ablank is the absorbance of the control reaction (containing all reagents except

the test material).

Extract concentration providing 50% inhibition (IC50) was calculated from the graph

plotted inhibition percentage against extract concentration.

ASA was used as positive control.

DPPH in methanol-3.0 ml

(conc.- 20μg/ml)

Extract in methanol-2.0 ml

(conc.- 500 to 0.977μg/ml)

+ +

Figure: 5.1: Schematic representation of the method of Assaying free radical scavenging

activity.

5.4 Results & Discussion of the test samples of Acacia auriculiformis.

Reaction allowed for 30 minutes

in absence of light at room

temperature

Absorbance measured at 517 nm

using methanol as blank

De-colorization of purple color of DPPH

Calculation of IC50 value from the graph plotted inhibition percentage against extract concentration

Different partitionates of methanolic extract of A. auriculiformis were subjected to free

radical scavenging activity by the method of Liyanna-pathiranan and shahidi (2005). Here,

Ascorbic acid was used as reference standard.

In this investigation, the aqueous soluble fraction showed the highest free radical scavenging

activity with IC90 value 4.47μg/ml which was also evident by changing of color on 5-6 test

tube in the course of reaction between DPPH and extracts in dark.At the same time the

Carbon tetrachloride soluble fractions of leaves and Pet ether soluble fraction also exhibited

strong antioxidant potential having IC50 value 1.78μg/ml and 3.509 μg/ml respectively which

are much which exhibits excellent anti-oxidative property in the investigated plant.

Chloroform soluble fractions of leaves (CHAAL) also revealed moderate scavenging activity

(IC50=98.56 μg/ml) whereas isolated compound AA-2 (Betulin) failed to exhibit any anti-

oxidative scavenging activity (IC50=cannot be determined).

.Table 5.1: IC50 values of standard and different fractions of A. auriculiformis.

Code Sample IC50 (μg/ml)

ASA Ascorbic acid 5.8

AsAAL Aqueous extract of the leaves of the plant 4.47(IC90)

CTAAL Carbon tetrachloride soluble fractions of leaves 1.78

CHAAL Chloroform soluble fractions of leaves 98.56

PEAAL Pet ether soluble fractions of leaves 3.509

AA-2 Isolated compound AA-2 ---------

Table: IC50 value of Ascorbic acid (ASA)

SL Absorbanceof blank

Concentration (μg/ml)

Absorbance of sample

%inhibition IC50

1

0 .525

500 0.005 98.46

5.8

2 250 0.006 98.153 125 0.015 95.384 62.5 0.024 92.615 31.125 0.068 79.076 15.625 0.098 69.847 7.813 0.139 57.23

8 3.906 0.186 42.769 1.953 0.175 46.1510 0.977 0.098 98.46

Figure 5.2: IC 50 values of the standard ASA.

5.5 Results & Discussion of the test samples of A. auriculiformis.

Different partitionates of methanolic extract of C. longa were subjected to free radical

scavenging activity by the method of Brand-Williams et al., 1995. Here, tert-butyl-1-

hydroxytoluene (BHT) was used as reference standard.

In this investigation, the carbon tetrachloride soluble fractions showed the highest free radical

scavenging activity with IC50 value 62.50μg/ml. At the same time the dichloromethane

soluble fractions also exhibited strong antioxidant potential having IC50 value 71.50μg/ml.

Pet-ether soluble fractions showed moderate antioxidant potential having IC50 value

100.50μg/ml. Crude methanolic extract exhibited very weak antioxidant potential having IC50

value 160.50μg/ml.

Table 5.12: IC50 value of aqueous soluble fraction of leaves of Acacia auriculiformis.

Concentration

C μg/ml

Absorbance of blank

Absorbance of sample Inhibition %Inhibition IC90

500 0.428 0.048 0.88785 88.7850467

4.47

250 0.428 0.025 0.941589 94.1588785

125 0.428 0.027 0.936916 93.6915888

62.5 0.428 0.008 0.981308 98.1308411

31.25 0.428 0.021 0.950935 95.0934579

15.625 0.428 0 1 100

7.8125 0.428 0.012 0.971963 97.1962617

3.90625 0.428 0.013 0.969626 96.9626168

1.953125 0.428 0.018 0.957944 95.7943925

0.9765625 0.428 0.148 0.654206 65.4205607

DPPH radical scavenging activity of CHCl3 Soluble fraction of leaves of A.auriculiformis.

Concentration

C μg/ml

Absorbance of blank

Absorbance of sample Inhibition %Inhibition

IC 50

500 0.428 0.119 0.721963 72.1962617

98.56

250 0.428 0.187 0.563084 56.3084112

125 0.428 0.224 0.476636 47.6635514

62.5 0.428 0.266 0.378505 37.8504673

31.25 0.428 0.273 0.36215 36.2149533

15.625 0.428 0.246 0.425234 42.5233645

7.8125 0.428 0.312 0.271028 27.1028037

3.90625 0.428 0.281 0.343458 34.3457944

1.953125 0.428 0.292 0.317757 31.7757009

Table 5.13: IC50 value of CHCl3 Soluble fraction leaves of A. auriculiformis

DPPH radical scavenging activity of Pet ether Soluble fraction of leaves of A.auriculiformis

Concentration

C μg/ml

Absorbance of blank

Absorbance of sample Inhibition %Inhibition

IC 50

500 0.428 0.023 0.946262 94.6261682

3.509

250 0.428 0.015 0.964953 96.4953271

125 0.428 0.006 0.985981 98.5981308

62.5 0.428 -0.02 1.046729 104.672897

31.25 0.428 0.034 0.920561 92.0560748

15.625 0.428 0.119 0.721963 72.1962617

7.8125 0.428 0.249 0.418224 41.8224299

3.90625 0.428 0.253 0.408879 40.8878505

1.953125 0.428 0.255 0.404206 40.4205607

0.9765625 0.428 0.256 0.401869 40.1869159

DPPH radical scavenging activity of CCL4 Soluble fraction of leaves of A.auriculiformis

Concentration

C μg/ml

Absorbance of blank

Absorbance of sample Inhibition %Inhibition LC50

500 0.428 0.026 0.93.9252336 93.9252336

1.78

250 0.428 0.014 0.96.728972 96.728972

125 0.428 0.018 0.95.7943925 95.7943925

62.5 0.428 -0.009 1.02.102804 102.102804

31.25 0.428 0.077 0.82.0093458 82.0093458

15.625 0.428 0.187 0.56.3084112 56.3084112

7.8125 0.428 0.193 54.9065421 54.9065421

3.90625 0.428 0.176 58.8785047 58.8785047

1.953125 0.428 0.214 50 50

0.9765625 0.428 0.223 47.8971963 47.8971963

DPPH radical scavenging activity of isolated compound AA-2 fraction of leaves of A.auriculiformis

Concentration

C μg/ml

Absorbance of blank

Absorbance of sample %Inhibition IC50

500 0.428 0.22 48.5981308

2534.31

250 0.428 0.224 47.6635514

125 0.428 0.25 41.588785

62.5 0.428 0.253 40.8878505

31.25 0.428 0.255 40.4205607

15.625 0.428 0.259 39.4859813

7.8125 0.428 0.264 38.317757

3.90625 0.428 0.265 38.0841121

1.953125 0.428 0.268 37.3831776

0.9765625 0.428 0.28 34.5794393

DPPH scavenging activity of AA-2

From the above data & explanation it is evident that the plant contains huge anti oxidative

property especially aqueous fraction shows highest potentiality which is elucidated by its

LC90 value which is very minimal. Two other fraction i.e., carbon tetrachloride and pet ether

also exhibits very good anti oxidant property, (IC50 below 25) the activity may be due to the

presence of potent antioxidant principles in the extract.

6 6

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6.1 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

extracts, 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.

Brine ShrimpBrine Shrimp

Lethality BioassayLethality Bioassay

This bioassay indicates cytotoxicity as well as a wide range of pharmacological activities

such as cytotoxicity antimicrobial, antiviral, pesticidal & anti-tumor, anticancer and different

other pharmacological actions and is used as a screening tool for the determination of

bioactivity of different 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.

6.2 Materials

a. Artemia salina leach (brine shrimp eggs)

b. Sea salt (NaCl)

c. Small tank with perforated dividing dam to hatch the shrimp

d. Lamp to attract shrimps

e. Pipettes

f. Micropipette

g. Glass vials

h. Magnifying glass

i. Test samples of experimental plants.

Test samples of Acacia auriculiformis:

One crude extracts (Methanol extract)

Three fractions (Petroleum ether, Carbontetrachloride and chloroform fractions)

6.3 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 test-tubes containing 5 ml of simulated sea water. Then

samples of different concentrations are added to the premarked test-tubes through

micropipette. The test-tubes are then left for 24 hours and then the nauplli are counted

again to find out the cytotoxicity of the test agents.

6.4. Experimental Procedure

6.4.1 Preparation of sea water

72 gm sea salt (pure NaCl) was weighed, dissolved in two liters of distilled water and

filtered off to get clear solution.

6.4.2 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.

6.4.3 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 sulphate for ten concentrations of it and another one test tubes

for control test.

As the test samples crude methanol extract of 4mg and three fractions (Petroleum ether,

Carbontetrachloride and chloroform fractions) of it were taken and dissolved in 60 l of

pure dimethyl sulfoxide (DMSO) in vials to get stock solutions. Then 30 l 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 g/ml.

Then a series of solutions of varying concentrations were prepared from the stock

solution by serial dilution method. In each case 30 l sample was added to test tube and

fresh 30l DMSO was added to vial. Thus the

concentrations of the obtained solution

in each test tube were as-

1. 400 g/ml 6. 12.5 g/ml

2. 200 g/ml 7. 6.25 g/ml

3. 100 g/ml 8. 3.125 g/ml

4. 50 g/ml 9. 1.5625 g/ml

5. 25 g/ml 10. 0.78125 g/ml

6.4.4 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

6.4.4.1 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 g/ml

from which serial dilutions are made using DMSO to get 10 g/ml, 5 g/ml, 2.5g/ml,

1.25 g/ml, 0.625 g/ml, 0.3125 g/ml, 0.15625 g/ml, 0.078125 g/ml, 0.0390 g/ml.

Then the positive control solutions are added to the premarked test-tubes containing ten

living brine shrimp nauplii in 5 ml simulated sea water to get the positive control groups

64.4.2 Preparation of negative control group

30l of DMSO was added to each of three pre-marked glass test-tubes containing 5ml 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.

6.4.5 Counting of nauplii

After 24 hours, the test-tubes were inspected using a magnifying glass and the number of

survived nauplii in each test-tube was counted. From this data, the percent (%) of

lethality of the brine shrimp nauplii was calculated for each concentration.

5.5 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 the crude extracts and all the fractions showed positive

results indicating that the test samples are biologically active. Each of the test samples

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.

6.6 Results and Discussion of the test samples of Acacia auriculiformis

Crude methanol extract, three fractions (Petroleum ether, Carbontetrachloride and

Chloroform fractions) of crude methanol extract were screened by brine shrimp lethality

bioassay for probable cytotoxic activity.

The LC50 values of crude methanol extract and three fractions (Petroleum ether, carbon

tetrachloride and chloroform fractions) were found to be 100μg/ml, (Table-5.2, Figure-

5.2), 3.16 μg/ml(Table-5.3, Figure-5.3),1.55μg/ml, (Table-5.4, Figure-5.4), 50.12 μg/ml

(Table-5.5, Figure-5.5) respectively.

It is evident that all the test samples were lethal to brine shrimp nauplii. However,

petroleum ether fraction and carbon tetrachloride fraction shows very high lethality

having LC50 values as low as 3.16 μg/ml and 1.55 μg/ml respectively. On the other hand

chloroform soluble fraction and crude methanol extract exhibits moderate to low

lethality on brine shrimp nauplii, having LC50 values as high as 50.12μg/ml & 100μg/ml

respectively.

From the above result on brine shrimp lethality bioassay, it can be easily predicted that

the polar compounds are moderately bioactive (CHCl3 & crude methanol extract) but

non polar fractions of leaves of investigated plant is highly bioactive (Pet ether & CCl4

fraction) suggestive of further investigation on these fractions which may lead to find

new bioactive potent drug having antitumor, anticancer or pesticidal

compounds.However, this cannot be confirmed without further higher studies and

specific tests.

Table 6.1: Effects of Vincristine sulfate on brine shrimp nauplii

Vincristine Sulfate

Concentration

C μg/ml Log CNo. of Nauplii taken

No. of Nauplii alive

No. of Nauplii dead

%Mortality

LC50

400 2.60206 10 0 10 100 0.33

200 2.30103 10 0 10 100

100 2 10 1 9 90

50 1.69897 10 2 8 80

25 1.39794 10 3 7 70

12.5 1.09691 10 4 6 60

6.25 0.79588 10 6 4 40

3.125 0.49485 10 6 4 40

1.563 0.193959 10 30

0.781 -0.10735 10 20

Figure 6.1 Effects of Positive control on brine shrimp nauplii

Table 6.2: Effect of methanol extract of Acacia auriculiformis on brine shrimp nauplii

Concentration C μg/ml

Log C

No. of Nauplii taken

No. of Nauplii alive

No. of Nauplii dead

% Mortality LC50

400 2.60206 10 3 7 70

100

200 2.30103 10 3 7 70 100 2 10 5 5 50 50 1.69897 10 6 4 40 25 1.39794 10 7 3 30 12.5 1.09691 10 7 3 30 6.25 0.79588 10 8 2 20 3.125 0.49485 10 8 2 20

1.563 0.193959 10 9 1 10 0.781 -0.10735 10 9 1 10

Figure 6.2 Effects of methanol extract on brine shrimp nauplii

Table 6.3: Effect of Petroleum ether fraction of Acacia auriculiformis on brine

shrimp nauplii

Concentration C μg/ml

Log C

No. of Nauplii taken

No. of Nauplii alive

No. of Nauplii dead

% Mortality

LC50

400 2.60206 10 0 10 100

3.16

200 2.30103 10 1 9 90 100 2 10 2 8 80 50 1.69897 10 3 7 70 25 1.39794 10 3 7 70 12.5 1.09691 10 4 6 60 6.25 0.79588 10 4 6 60 3.125 0.49485 10 5 5 50 1.563 0.193959 10 6 4 40 0.781 -0.10735 10 7 3 30

Figure 5.3 Effects of Petroleum ether fraction on brine shrimp nauplii

Table 6.4: Effect of carbontetrachloride fraction of Acacia auriculiformis on

brine shrimp nauplii

Concentration C μg/ml

Log C

No. of Nauplii taken

No. of Nauplii alive

No. of Nauplii dead

% Mortality

LC50

400 2.60206 10 0 10 100

1.55

200 2.30103 10 0 10 100 100 2 10 0 10 100 50 1.69897 10 1 9 90 25 1.39794 10 2 8 80 12.5 1.09691 10 3 7 70 6.25 0.79588 10 4 6 60

3.125 0.49485 10 4 6 60 1.563 0.193959 10 5 5 50 0.781 -0.10735 10 5 5 50

Fig: Effect of carbon tetrachloride fraction of Acacia auriculiformis on brine shrimp

nauplii

Table 5.5: Effect of chloroform fraction of Acacia auriculiformis on brine

shrimp nauplii

Concentration C μg/ml

Log C

No. of Nauplii taken

No. of Nauplii alive

No. of Nauplii dead

% Mortality

LC50

400 2.60206 10 0 10 100 200 2.30103 10 2 8 80 100 2 10 3 7 70

50.12

50 1.69897 10 5 5 50 25 1.39794 10 6 4 40 12.5 1.09691 10 7 3 30 6.25 0.79588 10 7 3 30 3.125 0.49485 10 8 2 20 1.563 0.193959 10 8 2 20 0.781 -0.10735 10 9 1 10

Figure 5.4 Effects of CHCl3 soluble fraction on brine shrimp nauplii

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CHAPTER 7

ANTIMICROBIAL SCREENING

7.1 Introduction

Worldwide infectious disease is one of main causes of death accounting for approximately

one-half of all deaths in tropical countries. Perhaps it is not surprising to see these statistics in

developing nations, but what may be remarkable is that infectious disease mortality rates are

actually increasing in developed countries, such as the United States. Death from infectious

disease, ranked 5th in 1981, has become the 3rd leading cause of death in 1992, an increase

of 58% .It is estimated that infectious disease is the underlying cause of death in 8% of the

deaths occurring in the US (Pinner et al., 1996). This is alarming given that it was once

believed that we would eliminate infectious disease by the end of the millennium. The

7 7 Antimicrobial Antimicrobial

ScreeningScreening

increases are attributed to increases in respiratory tract infections and HIV/AIDS. Other

contributing factors are an increase in antibiotic resistance in nosicomial and community

acquired infections. Furthermore, the most dramatic increases are occurring in the 25–44 year

old age group (Pinner et al., 1996).

These negative health trends call for a renewed interest in infectious disease in the medical

and public health communities and renewed strategies on treatment and prevention. It is this

last solution that would encompass the development of new antimicrobials (Fauci, 1998).

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 any 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, inoculum volume, culture medium

composition (Bayer et al., 1966), pH, and incubation temperature can influence the results.

Among the above mentioned techniques the disc diffusion (Bayer 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 bactericidal activity can be made by this method (Roland R,

1982).

7.2 Principle of Disc Diffusion Method

In this classical method, antibiotics diffuse from a confined source through the nutrient agar

gel and create a concentration gradient. Dried and sterilized filter paper discs (6 mm

diameter) containing the test samples of known amounts are placed on nutrient agar medium

uniformly seeded with the test microorganisms. Standard antibiotic (kanamycin) discs and

blank discs are used as positive and negative control. These plates are kept at low temperature

(4°C) for 24 hours to allow maximum diffusion of the test materials to the surrounding media

(Barry, 1976). The plates are then inverted and incubated at 37°C for 24 hours for optimum

growth of the organisms. The test materials having antimicrobial property inhibit microbial

growth in the media surrounding the discs and thereby yield a clear, distinct area defined as

zone of inhibition. The antimicrobial activity of the test agent is then determined by

measuring the diameter of zone of inhibition expressed in millimeter (Barry, 1976; Bayer et

al., 1966.)

In the present study the crude extracts as well as fractions were tested for antimicrobial

activity by disc diffusion method. The experiment is carried out more than once and the mean

of the readings is required (Bayer et al., 1966).

7.3 Experimental

7.3.1 Apparatus and Reagents

Filter paper discs Autoclave

Nutrient Agar Medium Laminar air flow hood

Petridishes Spirit burner

Sterile cotton Refrigerator

Micropipette Incubator

Inoculating loop Chloroform

Sterile forceps Ethanol

Screw cap test tubes Nose mask and Hand gloves

7.3.2 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 7.1.

Table7.1: List of Test Bacteria and Fungi

Gram positive Bacteria Gram negative Bacteria Fungi

Bacillus cereusEscherichia coli Aspergillus niger

Bacillus megaterium Salmonella paratyphi Candida albicans

Bacillus subtilisSalmonella typhi

Sacharomyces cerevacae

Sarcina luteaShigella boydii

Staphylococcus aureus Shigella dysenteriae

Pseudomonas aeruginosa

Vibrio mimicus

Vibrio parahemolyticus

7.3.3 Test Materials

Table 7.2: List of Test materials

Plant Test samples Code

Acacia

auriculiformis

(leaves)

1. Methanolic extract fraction of plant leaves. MEFP

2. Pet ether soluble fraction of methanolic extract PESF

3. Carbon tetrachloride soluble fraction of methanolic extract CTSF

4. chloroform soluble fraction of methanolic extract CHSF

7.3.4 Culture medium and their compositions

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 25°C

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 25°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 250°C

Composition of Nutrient agar medium

Ingredients Amount

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. 100 ml

pH 7.2 -7.6 at 250C

Nutrient agar medium (DIFCO) is used most frequently for testing the sensitivity of the

organisms to the test materials and to prepare fresh cultures.

So DIFCO is used in the present study for testing the sensitivity of the organisms to the test

materials and to prepare fresh cultures.

7.3.5 Preparation of the Medium

To prepare required volume of this medium, calculated amount of each of the constituents

was taken in a conical flask and distilled water was added to it to make the required volume.

The contents were heated in a water bath to make a clear solution. The pH (at 250C) 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 at 1210C for 20 minutes. The slants

were used for making fresh culture of bacteria and fungi that were in turn used for sensitivity

study.

7.3.6 Sterilization Procedure

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 glassware were sterilized by autoclaving at a temperature of 1210C and

a pressure of 15-lbs/sq. inch for 20 minutes. Micropipette tips, cotton, forceps, blank discs

etc. were also sterilized by UV light.

7.3.7 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 370C for their optimum growth.

These fresh cultures were used for the sensitivity test.

7.3.8 Preparation of the Test Plate

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.

7.3.9 Preparation of Discs

Measured amount of each test sample (specified in table 7.3) was dissolved in specific

volume of solvent (Chloroform or methanol) 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.

Table 7.3: Preparation of sample Discs

Plant Samples Code

Dose

(μg/disc)

Required

amount

for 20

disc (mg)

Acacia

auriculiformis

1. Methanolic extract of leaves of plant MEFP 400 8.0

2. Pet ether soluble fraction PESF 400 8.0

3. Carbon tetrachloride soluble fraction CTSF 400 8.0

4. Chloroform soluble fraction CHSF 400 8.0

Standard doxycycline (400 g/disc) discs were used as positive control to ensure the activity

of standard antibiotic against the test bacteria and standard griseofulvin (400 g/disc) discs

were used as positive control to ensure the activity of standard antibiotic against the test

fungal organisms as well as for comparison of the response produced by the known

antimicrobial agent with that of produced by the test sample. Blank discs were used as

negative controls which ensure that the residual solvents (left over the discs even after air-

drying) and the filter paper were not active themselves.

7.3.10 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 40C 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.

7.3.11 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.

Fig. 7.1: Clear zone of inhibition Fig. 7.2: Determination of clear zone of

inhibition

Result and Discussion:

Result and Discussion of in vitro antibacterial activity of the test samples of Acacia

auriculiformis

Crude methanol extract and three other fractions (petroleum ether, carbon tetrachloride and

chloroform fractions) were tested for antibacterial activities against a number of Gram

positive bacteria and Gram negative bacteria. Standard disc of doxycycline (400μg/disc) was

used for comparison purpose.

The methanolic crude extract of the leaves (MEFP) exhibited mild activity against some

bacteria, but chloroform soluble fractions (CHSF) exhibited moderate activity against most of

the test organisms. Pet ether soluble fraction (PESF) and carbon tetrachloride soluble (CTSF)

fraction did not show any activity against any of the test organisms.

The crude methanolic extract (MEFP) exhibited very mild activity against some bacteria such

as Staphylococcus aureus (gram +ve), S. lutea (gram –ve), Escherichia coli (gram –ve), etc.

The chloroform soluble fractions (CHSF) showed moderate activity against most of the test

gram positive and gram negative bacteria which are almost insensitive to carbon tetrachloride

(CTSF) and pet ether soluble fractions (PESF).

Table7.4: Antimicrobial activity of test samples of Acacia auriculiformis.

Test microorganismsDiameter of zone of inhibition (mm)

MEFP PESF CHSF CTSF doxycycline

Gram positive bacteria

Bacillus sereus - - 8 - 40

Bacillus megaterium - - 8 - 41

Bacillus subtilis - - 8 - 40

Staphylococcus aureus 7 - 9 44

Sarcina lutea - - 9 - 44

Gram negative bacteria

Escherichia coli 7 - 10 - 44

Pseudomonas aeruginosa - - 9 - 44

Salmonella paratyphi - - 9 - 41

Salmonella typhi - - 9 - 45

Shigella boydii 7 - 10 - 45

Shigella dysenteriae - - 9 - 44

Vibrio mimicus - - 9 - 44

Vibrio parahemolyticus - - 9 - 44

From the above figure we see that the zones of inhibition produced by methanolic crude

extract, and chloroform fractions were found to be 07 mm, and 08-10 mm respectively at a

concentration of 400μg/disc.

Most of the test organisms are insensitive to Pet ether soluble fraction (PESF) and carbon

tetrachloride soluble (CTSF) fraction of the methanolic extract (MEFP).

From the above discussion it is clear that chloroform soluble fraction of crude methanolic

extract exhibits moderately well anti-bacterial activity and has a good future perspective of

further research on this fraction to develop new antibacterial agent from this fraction.

Result and Discussion of in vitro of antifungal activity of the test samples of Acacia

auriculiformis

Crude methanol extract and three other fractions (petroleum ether, carbon tetrachloride and

chloroform fractions) were tested for antifungal activities against three types of fungi.

Standard disc of griseofulvin (400μg/disc) was used for comparison purpose

Test microorganismsDiameter of zone of inhibition (mm)

MEFP PESF CHSF CTSF griseofulvin

Fungi

Candida albicans - - 10 - 45

Aspergillus niger 7 - 10 - 45

Sacharomyces cerevacae - 10 45

The methanolic crude extract of the leaves (MEFP) exhibited mild antifungal activity only

against Aspergillus niger, but chloroform soluble fractions (CHSF) exhibited moderate

activity against all of the three test organisms. Pet ether soluble fraction (PESF) and carbon

tetrachloride soluble (CTSF) fraction did not show any activity against any of the test

organisms.

From the above figure we see that the zones of inhibition produced by methanolic crude

extract, and chloroform fractions were found to be 07 mm, and 10 mm respectively at a

concentration of 400μg/disc.

Most of the test organisms are insensitive to Pet ether soluble fraction (PESF) and carbon

tetrachloride soluble (CTSF) fraction of the methanolic extract (MEFP).

From the above discussion it is clear that chloroform fraction exhibits, moderately good anti-

fungal activity and has a future perspective of further research on this fraction to create good

antifungal drugs.

CONCLUSION

Different partitionates of the methanolic extract of leaves of Acacia auriculiformis were

investigated for isolation the potent secondary metabolites of this plant. Successive

chromatographic separation and purification of the carbon tetrachloride and chloroform

soluble partitionate of the crude methanolic extract and chloroform fraction yielded a total of

four compounds. The structures of these compounds were elucidated as Lupeol, Lupeol-

glucoside, Betulin, and para hydroxyl-Lupeol-3β-ortho cinnamate.

Most of the fraction of leaves of A. auriculiformis showed potent antioxidant activity but the

aqueous soluble fraction of methanolic extract is superior among them having a low IC90

value (4.47)reflects its excellent anti-oxidant property. LC50 values of Pet ether(3.509) and

CCL4(1.78) fraction are also notable which is indicating that further investigation of bio-

active compound having anti-cancer, anti-tumor etc. property can be done on this plant.

Antimicrobial activity test has been carried out in four fraction of methanolic extract of A.

auriculiformis. The chloroform soluble parts of investigated plant showed moderate anti-

bacterial activity against all the test micro-organism (08-10mm), aqueous fraction showed

little anti-microbial activity against some test micro-organism whereas other fraction failed to

prove any anti-bacterial activity. Anti-fungal activity test has been done against three test

fungi Candida albicans, Aspergillus niger, and Sacharomyces cerevacae and the result was

similar to that anti-bacterial test having a moderate value for CHCL3 soluble portion. So

further investigation of bio-active compound having anti-microbial property on this parts can

be carried out.

In the brine shrimp lethality bioassay, pet-ether and Carbon tetrachloride soluble fraction of

methanolic extract of leaves of A. auriculiformis exhibited significant cytotoxic activity while

the chloroform and the crude methanolic extract of leaves of A. auriculiformis showed

moderate cytotoxic activity.

From the above investigation it is evident that the plant is very bio-active having good

antimicrobial, antioxidant, and cytotoxic property hence it can be further screened against

various diseases in order to find out its unexplored efficacy and can be a potential source of

chemically interesting and biologically important drug candidates.

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