Chemical & Biological Investigation of Acacia auriculiformis
Chapter 1 1.1
INTRODUCTION
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
Random Screening
Phylogenetic
Ecological survey
Targeted screening
Isolated compounds
Unmodified natural plant products where ethno medical uses suggested clinical efficacy, e.g., digitalis.
Unmodified natural
Modified natural or
products of which
synthetic
the therapeutic
based on a natural
efficacy was only
product used in folk
remotely suggested
medicine, e.g., aspirin
substances
by indigenous plant use, e.g., vincristine
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: ď “ Successive cold extraction of the powdered leaves of the plant with methanol respectively. ď “ 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). 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 1
H 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 Division
Spermatophyta– Seed plants Magnoliophyta– Flowering plants
Class
Magnoliopsida– Dicotyledons
Subclass
Rosidae
Order
Fabales
Family Genus Species
Fabaceae– Pea family Acacia Mill.– acacia 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 Medicinal
Latin Name
Common Name
Synonyms
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
Rating
smallii,
Mimosa 2
farnesiana Acacia longifolia
Sidney Golden Wattle
Mimosa longifolia
0
Acacia melanoxylon
Blackwood
1
Acacia mucronata
Narrow-Leaf Wattle
0
Acacia paradoxa
Kangaroo Thorn
Acacia podalyriifolia
Queensland Silver Wattle
0
Acacia pycnantha
Golden Wattle
0
Acacia retinodes
Swamp Wattle
0
Acacia saligna
Blue-Leaved Wattle
Acacia sophorae
Coastal Wattle
0
Acacia verticillata
Prickly Moses
0
Acacia armata
0
Acacia cyanophylla
0
Adesmia lotoides
0
Albizia julibrissin
Mimosa
Acacia julibrissin
2
Alhagi mannifera
Manna Tree
Hedysarum alhagi
2
Alhagi maurorum
Camel Thorn
Alhagi
camelorum,
Alhagi 2
persarum, Alhagi pseudalhagi, Hedysarum pseudalhagi Amorpha canescens
Lead Plant
2
Amorpha fruticosa
False Indigo
0
Amorpha nana
Dwarf Indigobush
Amorpha microphylla
1
Amphicarpaea
Hog Peanut
Amphicarpaea monoica, Falcata 1
bracteata
comosa
Amphicarpaea
Amphicarpaea japonica, Falcata 0
edgeworthii
japonica
Amphicarpaea pitcheri Hog Peanut
Amphicarpaea
bracteata 0
comosa, Falcata pitcheri Anthyllis vulneraria
Kidney Vetch
Apios americana
Ground Nut
2 Apios tuberosa
1
Apios fortunei
1
Apios priceana
Glycine priceana
Arachis hypogaea
Peanut
Aspalathus linearis
Rooibos
0 2
Aspalathus
contaminatus, 3
Borbonia pinifolia Astragalus
Indian Milkvetch
Astragalus australis
0
Astracantha adscendens
0
aboriginorum Astragalus adscendens Persian Manna Astragalus boeticus
Swedish Coffee
0
Astragalus
0
brachycalyx Astragalus canadensis
Canadian Milkvetch
Astragalus carolinianus
Astragalus
2 0
carduchorum Astragalus
0
chartostegius Astragalus chinensis
Hua Huang Qi
2
Astragalus christianus Astragalus
0 Bei Bian Huang Qi
2
complanatus Astragalus crassicarpus
Ground Plum
Astragalus
caryocarpus, 1
Astragalus
mexicanus,
Astragalus
succulentus,
Geoprumnon succulentum 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 Astragalus floridus
1 Duo Hua Huang Qi
Astragalus florulentus
2 Astracantha florulenta
Astragalus garbancillo
0 0
Astragalus globiflorus
Astracantha
globiflora, 0
Astragalus elymaiticus Astragalus
Milk Vetch
0
glycyphyllos Astragalus gummifer
Tragacanth
Astracantha gummifera
Astragalus hamosus
3 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 subtropical 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
Width
Foliage
Flowering
A. acinacea
metres 2.5
metres 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
Cream or gold aut-late spring
A. alleniana
5
3
phyllodes Thread
Golden balls mar to ma
3-4
like/pendulous green Sprays yellow
A. araneosa A. argyrophylla
5-8 3-5
3
Silver-grey
throughout year Golden-yellow balls in
A. aneura
5-10
5
green
Winter 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.
6
4
Bluish
bancroftiorum
20cm Yellow ball sprays late
long
aut. to late winter
Green/grey
Bright yellow late aut. to
as known as A. bancroftii A. beckleri
1-3
1-2
mid winter A. bivenosa A. boormanii
1-3 3-5
1-3 4
Green-
Yellow mid aut. to late
glaucous
spring
grey/green
Bright yellow, early spring.
A.
2-6
2-6
Glaucous
brachystachya A. browiniana
A. brownii A. buxifolia A calamifolia
Yellow in axis of phyllodes aut – late
2
1 3 2-4
2
1 2 2-4
winter Tiny bipinnate Golden ball flowers with oblong
larger then the leaves in
leaflets Prickly
spring Golden balls in slim
phyllodes
peduncles
Green to
Golden balls in late
glaucous
winter to spring
Grey-green
Pale yellow to golden
with
bent tip A. cardiophylla
1-3
1 To 3
Pale green
Bright yellow balls in spring
A. cognata
1- 10
1-6
Yellow- green Pale lemon/cream in spring to dark green
A. colletioides
1.5 or
3
more A. complanata
To 5m
pendulous Prickly with yellow
3
Orange or yellow in spring
stem
projections Light green phyllodes
A. conferta
2
2
0cm green
A. continua
1-2
To 1
Hooked
Deep yellow spring to autumn to Bright yellow autumn to mid spring – Large golden balls early
spiky
spring
A.
1 To 4
1 To 2.5
blue-green Grey
Golden spikes mainly in
craspedocarpa A. cultriformis
3-4
3
blue/green
spring Golden spring,
A. cyclops
1-6
1-6
Blue/green
Yellow in spring and
blue/green
showy Bright yellow, late winter
A. dealbata
5-20
8
to spring A. deanei
5-10
3-5
green
Pale yellow, all year
A. decora
3-5
4
grey/green
Golden yellow –early
A. denticulosa
1-4
2-4
Dark green
spring. Rod shaped golden in
A. dimidiata
1 To 7
3
Curved to one
spring Terminal sprays of
side.
golden flowers in
Blue/green
autumn Cream/yellow early –
Green
mid spring Golden spikes in spring
A. dentifera A. doratoxylon
2-4 6-10m
3 6
A. drummondii
1.5
1
Green
Golden, late winter – mid spring
A. dunii
To 7m
Long
30cm Golden balls year round
glaucous
to
A. elata
10-20
8
20cm wide Brown/ green
A. elongata
3
1.5
Pale green
Yellow to gold balls in
Cream, summer
A. erinacea
1
1.5
Grey-green
late winter - spring Yellow balls in winter-
A. extensa
2 -3
2
Long 20cm
spring Light golden - yellow
phyllodes pale balls in spring A.A. falciformis
1 To 12
3
green Grey-green
A. falcata
To 4
1-2
Early summer Grey- green to Cream in winter
Cream - yellow globes
glaucous sickle A. falvescens A. flexifolia
4 - 20 1 – 1.5
1-3 .5-1
shaped Pale green
Creamy globes late
Grey- green
autumn to early winter Small yellow balls in
A. floribunda
4-8
4-6
Green
winter 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 winter – early spring
To 3
Yellow tinge Green
Golden in sprays winter
Green
and spring Yellow rods in early
A. hakeoides A. hemsleyi
To 4 To 7m
3-5
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
Bright golden spikes
2-5
blue/green Silvery
A. kettlewelliae
2 -10
mid summer to spring - Light bright gold in late spring
green to A. lanigera
1
1
glaucous Woolly
Small balls in spring
narrow A. latescens
3 - 10
3-5
green-bluish Sickle shaped
Cream balls in autumn
long leaves to 20cm A. leprosa
2-4
To 2
green Green
A. leptostachya
1-5
1-5
Green
pale Yellow- orange in spring to Golden rods in winter
slightly A. longifolia
4-10
4-8
glaucous Green
A. macradenia
3-6
3-6
Green with
Yellow, July-Sept Bright yellow winter and spring
reddish new A. mearnsii
10-25
10
growth Grey/green
Pale yellow, spring
A. melanoxylon
5-30
5-15
Grey/green
Cream, July-Oct
A.
4m
To 3m
Grey/green
Creamy yellow rods in the leaf axis late
merinthophora A. montana
autumn to early spring 1 -4
1-4
Bright
green Golden balls in spring
and A. muelleriana
1-8
1-8
sticky 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 -
Golden balls in spring
2-5m
Glaucous Grey/green to
Lemon to golden globes Late winter to early
slightly
summer
A. obliquinervia
To 15
A. oncinocarpa
To 5
To 4
glaucous 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
A. podalyriifolia
4
3
blue
spring 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
3
Grey-green
winter-spring Golden balls in spring
1-4
sticky, glossy. Green to Yellow in spring
4-10
5
Glaucous Green
Yellow, Aug-Nov.
To 2
3
Glossy, sticky
Golden balls borne in the leaf axis in spring Cream balls in spring Golden balls in spring
A. rigens A. rubida A. saligna
2 1.5 - 5
A sophorae - see A. longifolia A sclerophylla A. siculiformis
To 2 - 3 2-3
green Dark green
A. spectabilis
2 -4 up 2-4
Blue-green to
A. stricta
to 6 1- 5
Glaucous Suckering Dullish green
Stem clasping balls in spring
3
habit 1-5 4
Pale, April-Sept
A. suaveolens
Blue/green
A. terminalis
3
2
Dark green
Cream to yellow balls in autumn - winter
A. torulosa
1.5 - 15
1-10
Yellowish-
Bright yellow in winter
To 7
green Bluish-green,
Golden rods in spring Cream, Mar-Sept
A. triptera
3
A. ulcifolia
1-2
1-2
sickle shaped Green
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
A. verticilata
2-7
1-3
Green
spring 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 Abrus precatorius L.
LOCAL NAME Kunch, Rati, Chanyi, Kaich, Gungchi, Gujna
Agati grandif lora Desv.
Bakphul, Agasta, Buko, Bak, Agati
(Sesbania grandiflora (L.) Pers.) Caesalpinia crista L.
Let Kanta
(C. nuga L.) 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 multistemmed; 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 55,00075,000 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 includesThree 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]-212′,6′-dimethyl-6′-
-&{;6′S)-2′-trans-
-β-D-glucopyranosyl-2′,7′-octadienoyl&}; acacic acid 28-
-α-L-
rhamnopyranosyl (1→6) [β-D-xylopyranosyl (1→6) &{;α-L-arabinopyranosyl (1→2)&};-βD-glucopyranosyl]-21-
-[(6′S)-2′-trans-2′,6′-
glucopyranosyl&};- 2′,7′-octadienoyl] acacic acid 28-
-&{;β-D-xylopyranosyl
(1→2)-β-D-
-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 melliferaInventor-
-
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 subgenus 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, Nmethyltryptamine,
tryptamine,
tetrahydroharman,
N-methyl-tetrahydroharman,
b-
phenethylamine, N-methyl-b-phenethylamine, hordenine (N, N-dimethyl-4-hydroxy-bphenethylamine), 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 Bphenethylamine 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 extraAustralian 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-methyltetrahydroharman, 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% Nmethyl-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
Tryptamine in leaves, bark
var. sophorae 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
Psychoactive, DMT in the leaf
subsp. adstringens 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
Less than 0.2% DMT in leaf, NMT; DMT and other tryptamines in leaf, bark.
ssp. campylacantha 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
Psychoactive.
var. producta 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-2,3dihydrosqualene
(squalene-2,3-epoxide)
undergoes
cyclisation
to
give
3β-
hydroxytriterpenoids which by oxidation and reduction can be transformed into 3αhydroxytriterpenoids. 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
2
H A P T E
Experimental
R
- Chemical 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
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 50째C) 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 (40째-50째C) 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 PF 254 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 110째C 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 Thickness (mm) (cm x cm)
Amount of silica gel/plate (gm)
0.3 20 x 5
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 20
0.3
3.6
0.4
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 100°C 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:
Distance traveled by the compound
Rf value =
Distance traveled by the solvent system
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
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 (40째-50째C) 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 methanolextracts. 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 . Solventsolvent 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 CCl 4 (100ml Ă— 3). The CCl 4 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 CHCl 3 soluble fractions were collected together and evaporated. The aqueous methanolic fraction was preserved as aqueous fraction.
Crude extract (12.09 gm)
Methanol (90 ml) + Water (10 ml) Aqueous methanol solution
Extraction with Petroleum ether (100 ml x 3)
Aqueous fraction
Petroleum ether soluble fraction (300 ml)
+ Distilled Water (12.5 ml)
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)
CHCl3 soluble fraction (300 ml)
Aqueous fraction
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:
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 110째C. 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 1, 2, 3, 4,5, 6
New Codes F-1
7, 8, 9 10, 11 12,13,14,15 16,17,18,19 20,21,22
F-2 F-3 F-4 F-5 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 110°C. 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
Chloroform : methanol (50 : 50)
100
21 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 110째C. 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 tableFraction 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
100%
100
26-34
Methanol
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 110째C. 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 CDCl 3 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
3
T E R
Results & Discussion - Chemical
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 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, CDCl 3) 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
(-CH 2OH) 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). 30 29
20 19
21
H 12
18
11 1
13
9
2
14 8
10 3
5
H
4
HO
OH
17
H 16 15
7
6 23
H
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) Betulin
Position AA-2
(Muhammad Riaz et al., 2001)
no.
δ H (mult., J in Hz)
δ 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 Lupen3β, 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 4.682 and 4.561 (2S, 1H
Leupeol 4.68 and 4.56 (2s, 1H each, H-
each)
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),
H-29
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
Lupeol
1HNMR, CDCl3, 400 MHz 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 Side chain linkage
0.749 (s, 3H) AA-5
1HNMR, CDCl3, 400 MHz 3-beta glucosidic multiplet
∂ 3.84-∂4.24
0.75 (s, 3H),
O-β-glucopyranosyl-βsitosterol,1HNMR, CDCl3, 300 MHz ∂ 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 Hatoms (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 R1
C
O 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
Lupeol
1HNMR,
CDCl3,
400
H-29
MHz 4.682 and 4.561 (2S, 1H 4.71and 4.59 (2s, 1H each, H-
H-3
each) 3.63(m, 1H, H-3)
29 3.59 (m, 1H, H-3)
H-19
2.361 (m, 1H, H-2)
2.36 (m, 1H, H-2).
3H-30 3H-26
1.67 (s, 3H, H-30) 1.019 (s, 3H),
1.68 (s, 3H, H-30) 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, isobutyl-3,4-dihydroxy
400 MHz)
cinnamate.(1H NMR, CDCl3,
H-2
7.43(1H,d,j=8.4 Hz)
400 MHz) 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 H-8
7.59 (1H, d, j=16Hz) 6.28 (1H, d, j=16Hz).
7.56 (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 triterpenoid 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 AA-15
Wave Number (cm-1) of peaks 3440
Vibrations (a) OH str.(H bond)
Intensity Strong
2950 2380 1680
Strong low Medium
1360-1420
SP2 –C-H stretch Para group Substitution at aromatic ting Aromatic c=c stretch CH3 bend
1020-1250
C-O-C stretch
medium
Ortho beta linkage
medium
1450-1600
815-880
medium medium
Table 3.2. IR data for AA-2 Sample
Wave Number (cm-1) of peaks
Vibrations (a)
Intensity
3420
OH str.(H bond)
Strong
2950 2380 1680
Strong Low Medium
1450-1600 1360-1460
SP2 –C-H stretch Para group Substitution at aromatic ting Aromatic c=c stretch CH2,CH3 bend
1035
CH2OH para linkage
High
880
Para linkage
Medium
AA-2
Low 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
2950 2380 1720
Strong Low Low
1450-1600 1360-1460
SP2 –C-H stretch Para group Substitution at aromatic ting Aromatic c=c stretch CH2,CH3 bend
1160&1180
-CO-stretch
low
AA-3
Table 3.2. IR data for AA-5
Low low
Sample AA-5
Wave Number (cm-1) of peaks 3420 2950 2380 1720 1450-1600 1360-1460 1160
Intensity
Vibrations (a) OH str.(H bond) SP2 –C-H stretch Para group Substitution at aromatic ting Aromatic c=c stretch CH2,CH3 bend
Strong Strong Low Low
-CO-stretch
low
(Powdered leaves) Acacia auriculiformis
Cold extraction with methanol
Low low
Petroleum ether Extract
Partitioning by Modified Kupchan Method
Chloroform Extract
Carbontetrachloride Extract
CF-(11-14) CF-(1620)
Column fraction 12-15 Isolation of pure crystals
AA-3
Column fraction 21
Isolation of pure crystals
AA-2
AA-5
Fig 3.1: Schematic diagram of the chemical investigation of Acacia auriculiformis
C H
4
AA-15
A P T E
Design of Biological Investigations
R
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, propranolol and other β-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). ED 50 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.
C H A
P T E R
Evaluation of Antioxidant Activity
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 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 Liyannapathiranan 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 (20Îźg/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.
N N N
NH
.
O2N
O2N NO2
NO2
+ RH Antioxidant
NO2
NO2
*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
2.0
A. auriculiformis leaves. 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
Extract in methanol-2.0 ml
(conc.- 20μg/ml)
(conc.- 500 to 0.977μg/ml)
+
+
Reaction allowed for 30 minutes in absence of light at room temperature
De-colorization of purple color of DPPH
Absorbance measured at 517 nm using methanol as blank
Calculation of IC50 value from the graph plotted inhibition percentage against extract concentration
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.
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 antioxidative scavenging activity (IC50=cannot be determined). .Table 5.1: IC50 values of standard and different fractions of A. auriculiformis. IC50 (μg/ml)
Code
Sample
ASA AsAAL
Ascorbic acid Aqueous extract of the leaves of the plant
5.8 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 1 2 3 4 5 6 7
Absorbance of blank 0 .525
8 9 10
Concentration (μg/ml) 500 250 125 62.5 31.125 15.625 7.813
Absorbance of %inhibition sample 0.005 98.46 0.006 98.15 0.015 95.38 0.024 92.61 0.068 79.07 0.098 69.84 0.139 57.23
3.906 1.953 0.977
0.186 0.175 0.098
IC50
5.8
42.76 46.15 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-1hydroxytoluene (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 IC 50 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 IC 50 value 160.50μg/ml.
Table 5.12: IC50 value of aqueous soluble fraction of leaves of Acacia auriculiformis. Concentration Absorba nce of C Îźg/ml blank
Absorbance of sample
Inhibition
%Inhibition
IC90
500
0.428
0.048
0.88785
88.7850467
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 4.47
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 Absorb ance of Absorbanc C Îźg/ml blank e of sample Inhibition
IC 50 %Inhibition
500
0.428
0.119
0.721963
72.1962617
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
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
36.2149533 98.56
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 Absorba nce of C Îźg/ml blank
IC 50 Absorbance of sample
Inhibition
%Inhibition
500
0.428
0.023
0.946262
94.6261682
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 3.509
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 Absorba nce of Absorbance C Îźg/ml blank of sample 500
0.428
0.026
Inhibition 0.93.9252336
%Inhibition 93.9252336
LC50
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 1.78
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 Absorb ance of Absorbanc C Îźg/ml blank e of sample %Inhibition
IC50
500
0.428
0.22
48.5981308
250
0.428
0.224
47.6635514
125
0.428
0.25
41.588785
62.5
0.428
0.253
40.8878505 2534.31
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, (IC 50
below 25)
presence of potent antioxidant principles in the extract.
C
6
the activity may be due to the
H A P T E R
Brine Shrimp Lethality Bioassay
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. 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 µl of pure dimethyl sulfoxide (DMSO) in vials to get stock solutions. Then 30 µl of solution was taken in test tube each containing 5ml of simulated seawater and 10 shrimp nauplii. Thus, final concentration of the prepared solution in the first test tube was 400 µg/ml. Then a series of solutions of varying concentrations were prepared from the stock solution by serial dilution method. In each case 30 µl sample was added to test tube and fresh 30µl DMSO was added to vial. Thus the concentrations of the obtained solution in each test tube were as1. 400 µg/ml
6. 12.5 µg/ml
2. 200 µg/ml
7. 6.25 µg/ml
3. 100 µg/ml
8. 3.125 µg/ml
4. 50 µg/ml
9. 1.5625 µg/ml
5. 25 µg/ml
10. 0.78125 µg/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 µg/ml from which serial dilutions are made using DMSO to get 10 µg/ml, 5 µg/ml, 2.5µg/ml, 1.25 µg/ml, 0.625 µg/ml, 0.3125 µg/ml, 0.15625 µg/ml, 0.078125 µg/ml, 0.0390 µg/ml. Then the positive control solutions are added to the premarked 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
30µl 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 LC 50 values of crude methanol extract and three fractions (Petroleum ether, carbon tetrachloride and chloroform fractions) were found to be 100μg/ml, (Table-5.2, Figure5.2), 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 LC 50 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
400
2.60206
No. Nauplii taken 10
200
2.30103
10
100
2
50
Log C
C Îźg/ml
of No. Nauplii alive 0
of No.
of %Mortalit Nauplii dead y 10
100
0
10
100
10
1
9
90
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
LC50
0.33
Table 6.2: Effect of methanol extract of Acacia auriculiformis on brine shrimp nauplii Concentratio n C Îźg/ml Log C
No. Nauplii taken
of No. Nauplii alive
of
No. of % LC50 Nauplii dead Mortality
400
2.60206
10
3
7
70
200
2.30103
10
3
7
70
100
2
10
5
5
50
50
1.69897
10
6
4
40
1.39794
10
7
3
30
12.5
1.09691
10
7
3
30
6.25
0.79588
10
8
2
20
0.49485
10
8
2
20
0.193959
10
9
1
10
-0.10735
10
9
1
10
25
3.125 1.563 0.781
100
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
Concentratio n C Îźg/ml Log C
No. Nauplii taken
of No. Nauplii alive
of No. of % LC50 Nauplii dead Mortality
400
2.60206
10
0
10
100
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
0.49485
10
5
5
50
0.193959
10
6
4
40
-0.10735
10
7
3
30
3.125 1.563 0.781
3.16
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 Îźg/ml Log C
No. Nauplii taken
of No. Nauplii alive
of No. of % LC50 Nauplii dead Mortality
400
2.60206
10
0
10
100
200
2.30103
10
0
10
100
100
2
10
0
10
100
50
1.69897
10
1
9
90
1.39794
10
2
8
80
12.5
1.09691
10
3
7
70
6.25
0.79588
10
4
6
60
0.49485
10
4
6
60
0.193959
10
5
5
50
-0.10735
10
5
5
50
25
3.125 1.563 0.781
1.55
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 Îźg/ml Log C
No. Nauplii taken
400
2.60206
10
0
10
100
200
2.30103
10
2
8
80
100
2
10
3
7
70
50
1.69897
10
5
5
50
25
of No. of Nauplii No. of % alive Nauplii dead Mortality
1.39794
10
6
4
40
12.5
1.09691
10
7
3
30
6.25
0.79588
10
7
3
30
0.49485
10
8
2
20
0.193959
10
8
2
20
-0.10735
10
9
1
10
3.125 1.563 0.781
Figure 5.4 Effects of CHCl3 soluble fraction on brine shrimp nauplii
LC50
50.12
C H A P T E R
7
Antimicrobial Screening
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 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 (4째C) 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 37째C 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 Bacillus cereus Bacillus megaterium Bacillus subtilis Sarcina lutea Staphylococcus aureus
Gram negative Bacteria
Fungi
Escherichia coli
Aspergillus niger
Salmonella paratyphi
Candida albicans
Salmonella typhi Shigella boydii Shigella dysenteriae Pseudomonas aeruginosa Vibrio mimicus Vibrio parahemolyticus
7.3.3 Test Materials Table 7.2: List of Test materials
Sacharomyces cerevacae
Plant
Acacia auriculiformis (leaves)
Test samples
Code
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 7.2 ± 0.1 at 25°C
PH Muller – Hunton medium
Ingredients
Amounts
Beef infusion Casamino acid
30 gm 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 7.3 ¹ 0.2 at 250°C
PH
Composition of Nutrient agar medium Ingredients Bacto peptone
Amount 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 25 0C) was adjusted at 7.2-7.6 using NaOH or HCl. 10 ml and 5 ml of the medium was then transferred in screw cap test tubes to prepare plates and slants respectively. The test tubes were then capped and sterilized by autoclaving at 15-lbs. pressure 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 121 0C 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 37 0C 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
Required
(Îźg/disc)
amount for 20 disc
Acacia
1. Methanolic extract of leaves of plant 2. Pet ether soluble fraction
MEFP PESF
400 400
(mg) 8.0 8.0
auriculiformis
3. Carbon tetrachloride soluble fraction
CTSF
400
8.0
4. Chloroform soluble fraction
CHSF
400
8.0
Standard doxycycline (400 Âľg/disc) discs were used as positive control to ensure the activity of standard antibiotic against the test bacteria and standard griseofulvin (400 Âľg/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 airdrying) 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 4 0C for about 24 hours upside down to allow sufficient diffusion of the materials from the discs to the surrounding agar medium. The plates were then inverted and kept in an incubator at 370C for 24 hours. 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 microorganisms
Diameter of zone of inhibition (mm)
MEFP
PESF
CHSF
CTSF
doxycycline
Bacillus sereus
-
-
8
-
40
Bacillus megaterium
-
-
8
-
41
Bacillus subtilis
-
-
8
-
40
Staphylococcus aureus
7
-
9
Sarcina lutea
-
-
9
-
44
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
Gram positive bacteria
44
Gram negative bacteria
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 400Îźg/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 microorganisms
Diameter of zone of inhibition (mm) MEFP
PESF
CHSF
CTSF
griseofulvin
Candida albicans
-
-
10
-
45
Aspergillus niger
7
-
10
-
45
Sacharomyces cerevacae
-
Fungi
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 antifungal 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, Lupeolglucoside, 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 bioactive 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 antibacterial 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. REFERENCES
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