BIOLOGICAL INVESTIGATION OF Polyalthia longifolia

VIEW WITH IMAGES AND CHARTS

BIOLOGICAL INVESTIGATION OF Polyalthia longifolia

CHAPTER: 1 INTRODUCTION

1.1. THE PLANT FAMILY: ANNONACEAE

The Annonaceae, also called custard apple family or soursop family, is a family of flowering

plants consisting of trees, shrubs or lianas. With about 2300 to 2500 species in 120 to 130

genera, it is the largest family in the Magnoliales. The family is concentrated in the Tropics,

with few species found in temperate regions. About 900 species are Neotropical, 450 are

African, and the other species Asian.

Members of the Annonaceae have simple, alternate, petiolate leaves with smooth, entire

margins. The leaves are arranged in two rows along the stems. There are no stipules. The

flowers are radially symmetrical and often bisexual. In most species the 3 sepals are united

at the base. There are 6 brown to yellow petals, many stamens in a spiral, and many pistils,

each with a one-chambered ovary containing many ovules. The pistils generally remain

distinct and develop into berry-like fruits but sometimes they coalesce into multiple fruits like

the custard apple. Flowers are sometimes borne directly on large branches or on the trunk.

CULTIVATION AND USES:

The large, pulpy fruits of some members are edible, including species of Annona (the

custard apple, the cherimoya, and the soursop), Asimina (the papaw), and Rollinia (the

biriba).

Besides bearing edible fruits, some members also have aromatic oil and are used for

perfumes or spices. The strong bark is used for carrying burdens in Amazonia. The wood is

valued as firewood.

The bark leaves and roots of some species are used in folk medicines. Besides,

pharmaceutic research has found antifungal, bacteriostatic, and especially cytostatic

capability of some chemical constituents of the leaves and bark.

Some species are also grown as ornamental plants, especially Polyalthia longifolia pendula.

ANNONACEAE INCLUDES ABOUT 120-130 GENERA: Table 1: 130 genera of Annonaceae family:

1. Artabotrys 2. Cananga

3. Deeringothamnus 4. Guatteria

5. Oxandra 6. Rollinia

7. Stelechocarpus 8. Afroguatteria

9. Alphonsea 10. Ambavia

11. Anaxagorea 12. Annickia

13. Annona 14. Anomianthus

15. Anonidium 16. Artabotrys

17. Asimina 18. Asteranthe

19. Balonga 20. Bocagea

21. Bocageopsis 22. Boutiquea

23. Cananga 24. Cardiopetalum

25. Cleistochlamys 26. Cleistopholis

27. Craibella 28. Cremastosperma

29. Cyathocalyx 30. Cyathostemma

31. Cymbopetalum 32. Dasoclema

33. Dasymaschalon 34. Deeringothamnus

35. Dendrokingstonia 36. Dennettia

37. Desmopsis 38. Desmos

39. Diclinanona 40. Dielsiothamnus

41. Disepalum 42 Duguetia

43. Ellipeia 44. Ellipeiopsis

45. Enicosanthum 46. Ephedranthus

47. Exellia 48. Fissistigma

49. Fitzalania 50. Friesodielsia

51. Froesiodendron 52. Fusaea

53. Gilbertiella 54. Goniothalamus

55. Greenwayodendron 56. Guamia

57. Guatteria 58. Guatteriella

59. Guatteriopsis 60. Haplostichanthus

61. Heteropetalum 62. Hexalobus

63. Hornschuchia 64. Isolona

65. Letestudoxa 66. Lettowianthus

67. Malmea 68. Marsypopetalum

69. Meiocarpidium 70. Meiogyne

71. Melodorum 72. Mezzettia

73. Mezzettiopsis 74. Miliusa

75. Mischogyne 76. Mitrella

77. Mitrephora 78. Mkilua

79. Monanthotaxis 80. Monocarpia

81. Monocyclanthus 82. Monodora

83. Duckeanthus 84. Neostenanthera

85. Neo-uvaria 86. Onychopetalum

87. Ophrypetalum 88. Oreomitra

89. Orophea 90. Oxandra

91. Pachypodanthium 92. Papualthia

93. Petalolophus 94. Phaeanthus

95. Phoenicanthus 96. Piptostigma

97. Platymitra 98. Polyalthia

99. Polyceratocarpus 100. Popowia

101. Porcelia 102. Pseudartabotrys

103. Pseudephedranthus 104. Pseudoxandra

105. Pseuduvaria 106. Pyramidanthe

107. Raimondia 108. Reedrollinsia

109. Richella 110. Rollinia

111. Ruizodendron 112. Sageraea

113. Sanrafaelia 114. Sapranthus

115. Schefferomitra 116. Sphaerocoryne

117. Stelechocarpus 118. Stenanona

119. Tetrameranthus 120. Toussaintia

121. Tridimeris 122. Trigynaea

123. Trivalvaria 124. Unonopsis

125. Uvaria 126. Uvariastrum

127. Uvariodendron 128. Uvariopsis

129. Woodiellantha 130. Xylopia

1.1.1. ANNONACEAE SPECIES AVAILABLE IN BANGLADESH:

Annonaceae plants grow well in Bangladesh. They are found in plain areas as well as in hilly

areas like Sylhet and Chittagong. According to the recent reports of Bangladesh National

Herbarium, the following Annonaceous plants are available in Bangladesh as shown in the

following Table:

TABLE 2: ANNONACEOUS PLANTS & THEIR MEDICINAL USES ARE LISTED BELOW:

GENUS/SPECIES PLANT PARTS/ISOLATED PRODUCTS

MEDICINAL OR OTHER USES

REF.

1. Annona

(a) Annona bullata

Bullataci & Bullatacinone(Acetogenins)

Selective cytotoxic agent in human tumor cell line.Bullatacin is a pesticidal at a concentration of 1 ppm

Hui et al., 1989

(b) Annona glabra Liriodenine (Alkaloid)

Antibacterial, antifungal & antitumor agent.

Warthen et al., 1969, Hufford et al., 1980

GENUS/SPECIESPLANT PARTS/ISOLATED PRODUCTS


REF.

(c) Annona muricata Flowers fruits, seeds & roots

Effective in cough & chronic dysentery, emetic, astringent , antispasmodic & parasiticidal

Hossain et al., 1991

(d) Annona reticulata Fruits Effective against biliousness & thirst (Ayurveda) & also used as anthelmentic

Kirtikar & Basu, 1980

(e)Annona senegalensis

Extracts of stem bark

Root bark

Showed good antibacterial activity

Antineoplastic activity against sarcoma 180 ascities tumor cells

Hasan et al., 1988

Adesogan & Durdola, 1976

(f) Annona squamosa

Leaves & fruits

Seeds

In ulcer. Tonic effect on the body which increases blood, muscular strength, relieve vomiting, lessen burning sensation & biliouness

Fatal to insects & worm

Ayurveda


2. Artabotrys Leave extract In treatment of cholera Ayurveda

(a) Artabotrys odorotissimus

Essential oils from flower

Alkaloidal mixtures

In perfumery

Showed antibacterial action

Chopra et al., 1953

Haider, 1988

(b) Artabotrys suaveolens

Leaves Used against cholera Kirtikar & Basu 1980

3. Cananga(a) Cananga odorata

Oils from flowers In treatment of gout, opthalmia & cephalagia

Kirtikar & Basu



REF.

4. Desmos(a)Desmos longiflorus

Alkaloids from stembark

Good antibacterial agent & antifungal agent

Hossain, 1991

(b)Desmos chinensis Chloroform extract Strong inhibitor of tyrosine kinase enzyme

5.Goniothalamus (c) Goniothalamus macrophyllus

Plant constituents Cytotoxic to human tumor cell

Fang et al.,1990

(b)Goniothalamus giganteous

Acetogenius Selectively & significally cytotoxic to human tumor cell. Some of them active against murine leukemia. One of them was insecticidal & inhibited formation of crown gall tumor on potato discs, Antimitotic acetogenins was also isolated.

Alkokfahi et al.,1988; Fang et al.,1990, 1991

(c) Goniothalamus grifithi & Goniothalamus sesquipedalis

Powered leaves An embryotoxic and teratogenic compound was isolated

Sam et al., 1987

(d) G.malayanus G.montanus G.tapis

Different parts of these plants

Antibacterial agent Hasan et al.,1994b, 1994c

6. Miliusa

(a)Miliusa tomentosa Essential oil from this plant

Used as analgesic & possesses antibacterial activity

Menon & kar 1970, Kar & jain, 1971



REF.

(b)Miliusa cf. banacea

Oxoaporphine like alkaloids from root

Has been reported as good bioactive and cytotoxic compounds

(c)Miliusa velutina Sesquiterpenes (Spathuenol) and aromatic ester ( Benzyl benzoate) from stem bark

Enamul et al.,1998

7. Polyalthia

(a) Polyalthia longifolia

Volatile oils from this plant

Alkaloids from methanol extract of stem bark

Crude chloroform extract

Antibacterial agent

Good antibacterial & antifungal agents

Good antibacterial agent

Kar & Jain, 1971

Hasan et al., 1988b

Shaheen, 1986(b) Polyalthia longifolia var pendulla

Different plant parts and pure compound

Antimicrobial Ferdous et al.,1992 & Hasan et al.,1994, 1994a

(c) Polyalthia suaveolens

Extract of bark In black water fever & stomach disorder

Keay et al., 1964

(d) Polyalthia suberosa

Crude extract of stem bark plant parts

Good antibacterial activity

8. Uvaria

(a)Uvaria afzelli Plant parts Good activity against Bacillus subtilis, microbacterium semagmatis & Staph. Aureus

Hufford et al., 1981



REF.

(b)Uvaria chamae C-benzylated flavonoids

Cytotoxic against human carcinoma of the nasopharynx

in vitro Laswell & Hufford 1977a, Hufford & Oguntimein, 1980

(c)Uvaria duclis Root bark Astringent, stimulant & alternative properties


9. Xylopia(a) Xylopia aethiopica

Plant parts and isolated Diterpenes

Anticaugh, antifungal & antibacterial agent

Boakye, 1987

(b)Xylopia danguyell Plant parts CNS depressant & hypotensive

Cordell, 1981

1.1.2. CHEMISTRY OF THE ANNONACEAE:

Though there are about 120 genera and more than 2100 species (Trease & Evans, 1993) in

the family Annonaceae, chemical investigation has been very limited with only a few

GENERA, notably Annona, Ennantia, Goniothalamus, Uvaria and Xylopia have been

examined widely. Research carried out on Annonaceous paints till present time revealed that

the plants of this family posses many interesting, structurally varied secondary metabolites

including alkaloids. Terpenoids & steroids, flavonoids, coumarins, volatile oils, styryl

lactones, acetogenins and other Oxygen containing heterocycles. Alkaloids are most

probably the major and most widespread group of compounds isolated from the

Annonaceae.

A short description about the chemistry of Annonaceae is shown below:

1.1.2.1. TERPENOIDS:Terpenes consist of five carbon isoprene units, derived from mevalinic acid and are

classified according to the number of isoprene units involved. Terpenes are moderately

distributed in Annonaceae, Broadly terpenes are classified as:

I. Monoterpenes (C10)

II. Sesquiterpenes (C15)

III. Diterpenes (C20)

IV. Triterpenes (C30)

Almost every type of terpenes is isolated form various genus and species of Annonaceae.

Some of them are shown in table 3.

TABLE 3: TERPENOIDS FROM ANNONACEAE PLANTS:CLASS TERPENE ISOLATED SOURCE INVESTIGATOR

1. Monoterpenes

Camphor Borneol Annona squamosa Rao et al.,1978Chamanen (1) Uvaria chamae Hufford et

al.,19772. Diterpenes (-)-Kaur-16-en-19-ol (2)

(-)-Kaur-16-en-19-yl acetate (3)

Annona glabra Bohlmann et al.,1978

(-)-Kauran-17-ol-19-oc acid

Annona glabra Yarng et al.,1973

Stachanoic acid (6) Annona seegalensis

Adesogan et al., 1976

(-)-Kauran-16α-ol (7) Xylopia aethiopica Ekong et al.,1969

3. Triterpenes steroids

Sitosterol (8) Annona muricataAnnona SenegalensisAnnona squamosa

Ca. llan, 1911 Mackie, 1958 Farnsworth, 1974

Stigmasterol (9) Polyalthia longifolia

Beraz, 1976

Polycarpol (10) Fusaea longifolia Polyalthia oliveriXylopia longifolia

Cave et al.,1977Toeche, 1981

4. Sesquiterpenes

Β-caryophyllene (11) Annona squamosa Bohlmann et al.,1973

Yingzhaosu A (12) Artabotrys Uncniatus

Liang et al.,1979Yingzhaosu B (13)

Ishwarane (14) Cymbopetalum penduliflorum

[1] [4] [2] R= CH2OH [3]R= CH2OAC

[5] R= COOH

[6] [7] [8]

[10] [11] [12]

[13] [14]

FIG: STRUCTURAL TYPES OF TERPENOIDS AND STEROIDS FOUND IN ANNONACEAE

1.1.2.2. ALKALOIDS:

More than two hundreds alkaloids have been isolated from Annonaceous species. From

THE BIOGENETIC point of view, these alkaloids are classified in to two major classes:

i. Isoquinoline alkaloids

ii. Non-Isoquinoline alkaloids

i. Isoquinoline alkaloids:

Isoquinoline alkaloids are characterized by Isoquinoline skeleton. Some examples of this

type of alkaloids with their subclasses are given in the following Table:

TABLE 4: OCCURRENCE OF ISOQUINOLINE ALKALOIDS IN ANNONACEAE:

SUB CLASS ALKALOIDS SOURCE INVESTIGATORS 1. Simple isoquinolines Salsolinol (15) Annona reticulata Forgacs et

al.,1981

Corydaldine (16) Enantia polycarpa Jossang et

al.,1977

2.Benzyltetrahydro

isoquinolines

Reticuline (17) Annona montana Yang et al.,1979

Anomuricine Annona muricata Leboeuf et

al.,1980

3. Bisbenzylisoquinoline

& Bisbenzyltetrahydro

isoquinoline

Curine cycleanine Isolana pilosa Hocquemiller et

al.,1977 Isolana hexaloba

4. Protoberberines Berberine `Xylopia

polycarpa

Schermerhorn et

al.,1974

Oxypalmatine

(18)

Enantia polycarpa Leboeuf et al.,

1977

5. Tetrahydropro- Coreximine Annona montana Leboeuf et al.,

toberberine 1982

6. Proaporphines Stepharine Annona muricata Leboeuf et

al.,1981

Crotsparine Monodora

angolensis

Leboeuf et

al.,1974

7. Aporphines- Anolobine Annona

squamosa

SUB CLASS ALKALOIDS SOURCE INVESTIGATORS

a. Simple aporphines Oliveridine Enantia pilosa

b. 7-Substituted

aporphines

Liriodenine (19) Annona

Squamosa

Yang et al.,1970

c. Oxoaporphines

8. Phenanthrenes Argentinine (20)

Uvariopsine

Annona montana

Uvariopsis

congolana

Yang et al.,1979Bouquet et al.,1972

[15] [16] [17]

[18] [19] [20]

FIG: STRUCTURAL TYPES OF VARIOUS ISOQUINOLINE ALKALOIDS FROM ANNONACEAE

1.1.2.3. FLAVONOIDS:

The flavonoid compounds can be regarded as C6-C3-C6 compounds, in which each C6

moiety is a benzene ring, the variation in the state of oxidation of the connecting C3 moiety

determining the properties and class of each such compound.

Flavonoid compounds usually occur in plants as glycosides in which one or more of the

phenolic hydroxyl groups are combines with sugar residues. The hydroxyl groups are nearly

always found in positions 5 and 7 in ring A, while B ring commonly carries hydroxyl or alkoxyl

groups at the 4’ position, or at 4’-position, or at both 3’-and 4’-positions. Glycosides of

flavonoid compounds may bear the sugar on any of the available hydroxyl groups.

TABLE 5: FLAVONOIDS FROM ANNONACEAE PLANTS:

COMPOUNDS SOURCE INVESTIGATOR

1. Quercetin Annona glabra

A.senegalensis

Asimia triloba

Heganuer, 1964Mackie et al.,1958

2. Quercetrin rutin Annona senegalensis Mackie et al.,1958

3. Nicotiflorin Cananga latifolia Siv et al.,1972

4. Pachypodol (33) Pachypodanthium confine Cave et al.,1973

5. 5,6,7-trimethoxyflavone

(34) Monanthotaxis Cauliflora Waterman et al.,1979

5-hydroxy-6,7-

dimethoxyflavon (35)

5,7,8-trimethoxyflavanone

(36)

5,6,7,8-

tetramethoxyflavanone (37)

6. Dependensin U.dependens Nkhunya et al.,1993

7. Triuvaretin U.leptocladon Nkhunya et al.,1993

8. Triuvaretin U.scheffleri Chantrapromma et al.,1989

9. Tetochrysin (38) U. rufa Chantrapromma et al.,1989

COMPOUNDS SOURCE INVESTIGATOR

10. Angoluvarin U. angolensis Hufford et al.,1987

U.leptocladon Nkhunyet al.,1993

11. Uvangoletin (39) U. angolensis Hufford et al.,1980

12.Chamanetin (40) U. chamae Hufford et al.,1980

13. Isochamaetin U. lucida lucida Achenbach et al.,1997

Okorie et al.,1977

Weenen et al.,1990Dichamanetin

14. Isovaretin U. angelonsis Hufford et al.,1980

15. Uvaretin (41)

Diuvaretin (42)

U. chamae

U. lucida lucida

U. kirkii

Nkunya et al.,1985

16. Isovaretin U. angelonsis Hufford et al.,1980

17. Pinocembrin (43)

Pinostrobin (44)

Chamuvaritin

Uvarinol

U. chamae Hufford et al.,1978,1979

18. Vafzelin (45)

Uvafzelin

Syncarpic acid

U. afzelii Hufford et al.,1980

[33] [34] R= ME [35} R=H

[36] R=H [38] [37] R= OMe

[39] [40]

[44] [42] R1= R2= H

[43] R1=R2=

[45]

FIG: STRUCTURAL OF FLAVONOIDS ISOLATED FROM ANNONACEAE

1.1.3. TAXONOMY OF ANNONACEAE:

On the basis of morphology and habit Annonaceae is a very homogenous plant family.They

are trees or shurbs, sometimes climbing, usually evergreen, with resin canals septate pith in

the stems. The leaves are alternate, entire and exsipulate.

The leaves are simple, alternate, lack stipules, and generally are distichously arranged in flat

sprays. The flowers are bisexual and the fragrant flowers frequently open before all parts are

fully developed. The elongated floral axis also bears many helically disposed stamens and

several to many simple pistils. All of the floral parts are distinct. The stamens are very short,

consisting of the fertile central anther portion, a distal pad of fleshy connective tissue, and a

short fleshy basal portion. The stamens are generally so tightly packed on the receptacle

that often only the fleshy connective tissue of each is exposed. The pistils each have a

superior ovary with one locule and 1-many parietal ovules. Sectioned seeds reveal channels

or partitions in the ruminate endosperm. The pistils generally remain distinct and develop

into berry-like fruits but sometimes they coalesce into multiple fruits like the custard apple.

1.2. INFORMATION ABOUT: Polyalthia longifolia

Polyalthia longifolia (Sonn.) Thw (=Unona longifolia (Sonn.) Dunal, Uvaria longifolia Sonn.)

Family: Annonaceae

COMMON NAMES: Debdaru, Saralgachh (Beng.);Mast tree (Eng.), Ashoka (Hindi).

1.2.1. PLANT DESCRIPTION:

A tall evergreen tree with undulate-margined narrow lanceolate leaves, axillary solitary

flowers, and an etaerio of distintc and separate berries, grows wild as well as planted

throughout the country. It is most commonly used as an ornamental street tree due to its

effectiveness in combating noise pollution.

FIG: 1 Polyalthia longifolia: TREE, FRUIT & LEAVE

LEAVES: Each leaf is a foot long, having 3-7 pairs of wavy-edged leaflets. Young leaves are

dropping, coppery, limp and remain pendent even after attaining full maturity. The leaves

grow alternately on the branches. Fresh leaves are a coppery brown color and are soft and

delicate to touch; as the leaves grow older the color becomes a light green and finally a dark

green.

FLOWERS: The flowers are star-shaped, yellowish-green in colors, inconspicuous borne on

long slender stalks, appearing from February to April.

FRUITS: The fruiting season is July and the fruits are egg-shaped. Fruit are borne in clusters

of 10-20. Initially green but turning purple or black when ripe. These are loved by bats

including the flying foxes.

1.2.2. COMPOUNDS ISOLATED FROM THE PLANT POLYALTHIA LONGIFOLIA

TABLE 6: COMPOUNDS ISOLATED FROM THE PLANT POLYALTHIA LONGIFOLIA

PLANT PART COMPOUND ISOLATED REF.Leaves Azafluorene alkaloid

Polylongine

3-aporphine

N-oxide alkaloids

(+)-O methyl bulbocapnine-α-N-

oxide

(+)-O methyl bulbocapnine-β-N-

oxide

Goyal & Gupta

Stem bark Tetrahydroprotoberine

(-)-Stepholidine

Oxychine

Darienine

6,7-dimethoxychime

Aporphine alkaloid

Liriodenine (cytotoxic)

Noroliveroline

oliveroline oxide

Azofluorene alakloid

Polyfothine

Iso-oncodine

Wu, 1989; Chakrabarty &

Patra, 1990; Wu et al.,1990

Bark & Seeds Clerodane diterpenoids (Phadnis et al.,1988;

Chakrabarty & Nath 1992;

Hara et al.,1995; Hasan et

al.,1995b; Rashid et al.,1996

1.3. BIOLOGICAL INVESTIGATION OF P.longifolia

ANTI-INFLAMMATORY AND CYTOTOXIC DITERPENES FROM FORMOSAN P. longifolia VAR. pendula:

Chang FR, Hwang TL, Yang YL, Li CE, Wu CC, Issa HH, Hsieh WB, Wu YC.

Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC.

PMID: 17022008 [PubMed - indexed for MEDLINE]

NEW ANTIMICROBIAL ALKALOIDS FROM THE ROOTS OF P. longifolia VAR. pendula.

Faizi S, Khan RA, Azher S, Khan SA, Tauseef S, Ahmad A.

H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi, Pakistan. [email protected]


HYPOTENSIVE ACTIVITY AND TOXICOLOGY OF CONSTITUENTS FROM ROOT BARK OF P. longifolia VAR. pendula.

Saleem R, Ahmed M, Ahmed SI, Azeem M, Khan RA, Rasool N, Saleem H, Noor F, Faizi S.

Dr. HMI Institute of Pharmacology and Herbal Sciences, Hamdard University, Karachi-74600, Pakistan. [email protected]


CYTOTOXIC CONSTITUENTS OF P. longifolia VAR. pendulla.

Chen CY, Chang FR, Shih YC, Hsieh TJ, Chia YC, Tseng HY, Chen HC, Chen SJ, Hsu MC, Wu YC.

Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan


CYTOTOXIC CLERODANE DITERPENES FROM P. longifolia

Phadnis et al.,1988; Chakrabarty & Nath 1992; Hara et al.,1995; Hasan et al.,1995b; Rashid et al.,1996

SECTION: 2 MATERIALS AND METHODS

2.1. CHEMICAL INVESTIGATION OF THE EXPERIMENTAL PLANTS

The plant species belonging to Annonaceae is investigated in this study.

Name of plant Family Plant part

Polyalthia longifolia Annonaceae Stem Bark

TAXONOMIC HIERARCHY OF THE INVESTIGATED PLANTS (WEKEPEDIA)

TABLE7: TAXONOMIC HIERARCHY OF THE INVESTIGATED PLANT

P.longifolia

KINGDOM Plantae

PHYLUM Magnoliophyta

CLASS Magnoliopsida

ORDER Magnoliales

FAMILY Annonaceae

GENUS Polyalthia

SPECIES Polyalthia longifolia

2.2. CHEMICAL INVESTIGATION OF Polyalthia longifolia:

2.2.1. COLLECTION OF PLANT MATERIAL:

The plant was collected from BCSIR, Dhaka on 20th February 2007. The stem bark was collected.

2.2.2. DRYING AND GRINDING:

The stem bark collected was grounded in to powder in University of Dhaka. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced.

2.2.3. METHODS:

EXTRACTION CAN BE DONE IN TWO WAYS:

A. Cold extraction.B. Hot extraction.

A. Cold Extraction:

In cold extraction the powdered plant material is submerged in a suitable solvent or solvent system in an air-tight flat bottom container for several days, with occasional shaking and stirring. The major portion of the extractable compounds of the plant material will be dissolving 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 soxhiet at an elevated temperature with several solvents of increasing polarity. The individual extractives are then filtered through several means, e.g., cotton, cloth, filter paper etc. All the extractives are concentrated with a rotary evaporator at low temperature (40°-50°) and reduced pressure. The concentrated extract thus obtained is termed as crude extract.

2.3. EXTRACTION OF THE PLANT MATERIAL:

About 350gm of the powdered material was taken in a clean, round bottom flask and soaked in 1300ml of methanol. The container with its content was sealed and kept for a period of 10 days accompanying occasional shaking and stirring. The whole mixture then filtered through filter paper and filtrate thus obtained was concentrated at 50°C using airflow.

2.3.1. SOLVENT-SOLVENT PARTITION (MODIFIED KUPCHAN PARTITION) OF CRUDE EXTRACT:

2.3.1.1. PRINCIPLE OF MODIFIED KUPCHAN PARTITION:

The crude extract is diluted with 100ml 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) that is immiscible with aqueous alcohol. The mixture is kept undistributed 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 sample. 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 the detection and identification of the antibacterial activity of the compound.

2.3.1.2. PREPARATION OF AQUEOUS METHANOL SOLUTION:

3gm of methanol extract was triturated with 50ml of methanol containing 5ml of distilled water. (45ml CH3OH + 5ml H2O). The crude extract went to the solution completely. This is called mother solution, which was partitioned off successively by three solvent of different polarity.

2.3.1.3. PET ETHER EXTRACT:

The mother solution was taken in a separating funnel. 75ml of Pet ether was added to it and the funnel was shaken and then kept undistributed. The organic portion was collected. The process was repeated thrice. The fractions were collected together and evaporated to dryness and kept for further analysis. The aqueous fraction was preserved for the next step.

2.3.1.4. CARBON TETRACHLORIDE EXTRACT:

The aqueous mother solution left after washing with pet ether, 6ml water was added and mixed. The mother solution was taken in a separating funnel and extracted with 75ml of CCl4. This process was repeated thrice. The fractions were collected together and evaporated to dryness and kept for further analysis. The aqueous fraction was preserved for the next step.

CRUDE EXTRACT (3 GM)

THE WHOLE PARTITIONING PROCESS IS SCHEMATICALLY SHOWN IN THE FOLLOWING FLOW CHART:

SOLVENT-SOLVENT PARTITIONING OF METHANOL EXTRACT:

METHANOL (45ML) + WATER (5 ML)

EXTRACTION WITH PET ETHER (75 ML X 3) ML

+ WATER (6 ML)

EXTRACTION WITH CCl4 (75 ML X 3) ML

+WATER (8 ML)

EXTRACTION WITH CH2Cl2 (75 ML X3 ML)

AQUEOUS METHANOL SOLUTION

PET ETHER SOLUBLE FRACTION

AQUEOUS FRACTION

CCl4 SOLUBLE FRACTION AQUEOUS FRACTION

FIGURE: 2 SCHEMATIC REPRESENTATION OF THE MODIFIED KUPCHAN PARTIONING OF METHANOLIC CRUDE EXTRACT OF Polyalthia longifolia.SECTION: 3 MICROBIOLOGICAL INVESTIGATION:

3.1. INTRODUCTION:

Herbal medicines in developing countries are commonly used for the traditional treatment of health problems (Martinez et al., 1996). It is estimated, in developing countries, 80% of the population rely on traditional medicine for their primary health care (Esther and Staden, 2003). Owing to hot temperature and high humidity, the infections due to wounds are common in Bangladesh. For a developing country like Bangladesh, the therapy with synthetic antibiotic is not always possible due to their high cost. Additionally, the rapid development of drug resistant microbes has lead to the search of new antimicrobial agents especially from plant extracts to discover new chemical structures. The antimicrobial compounds from plants may inhibit bacterial growth by different mechanisms than those presently used antimicrobials and may have a significant clinical value in treatment of resistant microbial strains. In recent times, traditional medicine has served as an alternative form of health care and to overcome microbial resistance has led the researchers to investigate the antimicrobial activity of medicinal plants (Austin et al., 1999).

3.1.1. ANTIMICROBIAL SCREENING:

The antimicrobial potency of the plant can be visualized by antimicrobial screening which measures the ability of a test sample to inhibit the in vitro microbial growth by any of the following three methods:

A) Disc diffusion method.B) Serial dilution method.

CH2Cl2 SOLUBLE FRACTION

AQUEOUS FRACTION

C) Bio autographic method.

In 1966, Bauer et al. published a detailed description of a standardized single-disk method for performing the anti-microbial susceptibility test. This procedure has been widely accepted as the preferred reference method for bacterial susceptibility screening.

A. DIFFUSION METHODS:

Diffusion technique does not require homogenous dispersion in water and the agar overlay method require disc, hole or cylinder as reservoir. The reservoir containing the test sample is bought in to contact with an inoculated medium and after incubation the diameter of the clear zone around the reservoir (inhibition diameter) is measured. In order to increase the precision the inoculated system can be kept at a low temperature before incubation, which favors diffusion through the culture medium, and this increase the inhibition diameter. The aqueous solubility of lipophilic samples, such as essential oils or non-polar extracts, makes it difficult to use an aqueous medium in the study of microbial activity (Allergini et al., 1973; Pellecuer et al., 1976). Therefore, the use of other solvents or the aqueous dispersions or emulsions using a surface-active agent may be helpful. Several solvents including alcohols, acetone, chloroform, dimethylsulfoxide, dioxane, glycerol, and others and different emulsifiers such as macrogol ethers, sorbitan, and cellulose derivative etc., have been used (leven et al., 1979, Janssen et al., 1987). Solvents other than water should always be tested simultaneously with the extracts to make sure that they have no antimicrobial properties in the test system. Diffusion methods are not the best choice for testing non-polar or other samples, which are difficult to diffuse in media; however there is no relation between diffusion process and antimicrobial activity (Rios et al., 1988). Also aqueous dispersions containing high molecular weight solubilizer (mol. wt.>100,000) should be avoided in diffusion methods since they cannot diffuse in to 1% agar medium. The pH should be adjusted to neutrality (between pH 6.0 and 8.0) (Berghr and Vlietinck 1990) for this assay.

B. DILUTION METHODS:

Dilution technique requires a homogenous dispersion of the sample in water. They are used to determine, principally, the minimum inhibitory concentration (MIC) values of an extract, essential oils or pure substance but can also be used in the preliminary screening of antimicrobial activity. The physicochemical properties of dispersions are important for observing the activity, and surface active agents, such as Tween 80 or Span 80 can improve the dispersion of test substances.

In the liquid dilution method, turbidity is taken as a measure of bacterial density. When no growth takes place, the medium remains clear, when the sample is inactive against the organism used in the test as there is growth, it appears turbid. The grade of inhibition is related to the turbidity of the medium and is measured spectrophotometrically (Rios et al., 1988). This method is simple and speedy and i.e. is possible to study the antibacterial activity of water soluble or insoluble samples such as essential oils using this technique.

C. BIOAUTOGRAPHIC METHODS:

According to Betina (1973), bioautography is the most important detection method for new or unidentified antimicrobial compounds. It is based on the biological (antibacterial, antiprotozoal, antitumoral, etc.) effects of the substances under study. Both paper chromatography (PC) and thin-layer chromatography (TLC) are utilized in bioautographic technique, although the later has greater resolving power and is more rapid of the two techniques (Rios et al., 1988). The typical bioautographic procedure is based on the so called agar diffusion technique, where the bacterial compounds are transferred from the chromatographic layer to an inoculated agar plate. Inhibition zones are visualized by dehydrogenase activity detecting reagents (Begit and Kline, 1972).

3.1.2. PRINCIPLE OF DISC DIFFUSION METHOD:

In this classical method, antibiotics diffuse from a confined source through the nutrient agar gel and create a concentration gradient. Dried and sterilized filter paper discs (6 mm diameter) containing the test samples of known amounts are

placed on nutrient agar medium uniformly seeded with the test microorganisms. Standard antibiotic (kanamycin) discs and blank discs are used as positive and negative control. These plates are kept at low temperature (4°C) for 24 hours to allow maximum diffusion of the test materials to the surrounding media (Barry, 1976). The plates are then inverted and incubated at 37°C for 24 hours for optimum growth of the organisms. The test materials having antimicrobial property inhibit microbial growth in the media surrounding the discs and thereby yield a clear, distinct area defined as zone of inhibition. The antimicrobial activity of the test agent is then determined by measuring the diameter of zone of inhibition expressed in millimeter (Bary, 1976; Bauer et al, 1966).

3.2. EXPERIMENTAL:3.2.1. APPARATUS AND REAGENTS:

1. Filter paper discs. 9. Screw cap test tubes2. Sterile cotton. 10. Autoclave3. Micropipette 11. Nutrient Agar Medium4. Laminar air flow hood 12. Inoculating loop5. Refrigerator 13. Spirit burner6. Chloroform 14. Nose mask and Hand gloves7. Petri dishes 15. Incubator8. Sterile forceps 16. Ethanol

3.2.2: TEST ORGANISMS:

THE MICROBIAL STRAINS USED FOR THE EXPERIMENT WERE LISTED IN THE TABLE:

TABLE 8: LIST OF TEST BACTERIA:

1. Bacillus cereus

2. Bacillus megaterium

3. Bacillus subtilis

4. Salmonella paratyphi

5. Salmonella typhi

6. Vibrio parahemolyticus

7. Vibrio mimicus

8. Staphylococcus

9. E.coli

10. Shigella dysenteriae

11. Pseudomonas aureus

12. Sarcina lutea

13. Shigella boydii

14. Saccharromyces cerevaceae

15. Candida albicans

16. Aspergillus niger

3.2.3. TEST MATERIALS:

TABLE 9: LIST OF TEST MATERIALS

PLANT TEST SAMPLES SAMPLE

CODE

Polyalthia longifolia

1. Pet ether soluble fraction of methanolic extract

PE

2. CCl4 (Carbon tetrachloride) soluble fraction of methanolic extract

CCl4

3.2.4. CULTURE MEDIA:

The following media are used normally to demonstrate the antibacterial activity and to make subculture of the test organism.

a. Nutrient agar mediab. Nutrient broth mediac. Muellar-Hinton agar mediad. Tryptic soya broth (TSB)

Among these, the first one is most frequently used which was also used in the present study for testing the sensitivity of the organisms to the test materials and to prepare fresh cultures.

3.2.5. COMPOSITION OF MEDIA:

INGREDIENTS AMOUNTS

Bacto peptone 0.5 gmSodium chloride 0.5 gmBacto yeast extract 1.0 gmBacto agar 2.0 gmDistilled water q.s. 100 mlpH 7.2-7.6 at 25°C

3.2.6. PREPARATION OF MEDIUM:

To prepare required volume of this medium calculated amount of each of the constituents was taken in a conical flask & distilled water was added to it to make a clear solution. 10 ml and 5 ml of the medium were 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 121°C for 15 minutes. The slants were used for making fresh culture of microorganisms that were in turn used for sensitivity study.

3.2.7. Sterilization procedures:

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 strictly maintained. UV light was switched on an hour before working in the Laminar Hood. Petri dishes and other glassware were sterilized by autoclaving at a temperature of 121°C and a pressure of 15-lbs./sq.inch for 15 minutes. Micropipette tips, cotton, forceps, blank discs were also sterilized by autoclave.

3.2.8. 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°C for their optimum growth. These fresh cultures were used for the sensitivity test.

3.2.9. PREPARATION OF THE TEST PLATES:

The test organisms were transferred from the subculture to the test tubes containing about 10 ml of melted and sterilized agar medium with the help of a sterilized transfer loop in an aseptic area. The test tubes were shaken by rotation to get a uniform suspension of the organisms. The microbial suspension was immediately transferred to the sterilized Petri dishes. The Petri dishes were rotated several times clockwise and anticlockwise to assure homogenous distribution of the test organisms in the media.

3.2.10. PREPARATION OF DISCS:

Measured amount of each test sample (specified in table 4.4) was dissolved in specific volume of solvent (methanol) to obtain the desired concentrations in an aseptic condition. Sterilized metrical (BBL, Cocksville, USA) filter paper discs were taken in a blank Petri dish under the laminar hood. Then discs were soaked with solutions of test samples and dried.

TABLE 10: PREPARATION OF SAMPLE DISCS

Plant Sample code

SampleDose (g/disc)

Dose (g/disc)

Poly

alth

ia

long

ifolia

PE Pet ether soluble fraction of methanolic extract 200 400

CCl4 CCl4 (Carbon tetrachloride) soluble fraction of methanolic extract 200 400

Standard Kanamycin (30 g/disc) discs were used as positive control to ensure the activity of standard antibiotic against the test organisms as well as for comparison of the response produced by the known antimicrobial agent with that of the test sample. Blank discs were used as negative controls which ensure that the residual solvents (left over the discs even after air-drying) and the filter paper were not active themselves.

3.2.11. DIFFUSION AND INCUBATION:

The sample, standard antibiotic and control discs were placed gently on the previously marked zones in the agar plates pre-inoculated with test microorganisms. The plates were then kept in a refrigerator at 4°C for about 24 hours 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 37°C for 24 hours.

3.2.12. Determination of the Zone of Inhibition:

After incubation, the antimicrobial activity of the test materials was determined by measuring the diameter of the zones of inhibition in millimeter using vernier calliper.

3.3. Results and Discussion of the test samples of Polyalthia longifolia:Various fraction of methanolic extract of the plant Polyalthia longifolia obtained by solvent-solvent partitioning, were tested for antibacterial against a number of

both gram positive and gram negative bacteria. Standard antibiotic disc of Kanamycin was used for comparison purpose.

The antimicrobial activities of extracts from Polyalthia longifolia were examined in the present study. The results were given in the following table. The zone of inhibition produced by Pet. Ether, carbon tetrachloride soluble fraction of methanolic extract ranged from 27-30mm and 25-30mm respectively.

However, at a concentration of 400µg/disc the result of Pet ether soluble fraction of methanolic extract (PE) showed significant activity against most of the test microorganisms. At a concentration 200µg/disc the activity is well correlated with the activity at 400µg/disc. The growth of Pseudomonas aureus and Aspergillus niger was moderately inhibited (zone diameter 22mm both).

The carbon tetrachloride soluble fraction of the methanolic extract at a concentration of 400 µg/ disc showed significant activity against all of the test micriorganisms. However at a concentration of 200µg/disc the activity was almost similar to the activity of 400µg/disc. Only the growth of Bacillius cereus & Aspergillus niger was moderately inhibited (Zone of inhibition 24mm & 21mm respectively).

Out of all the samples, Pet ether soluble fraction of the methanolic extract showed best result in terms of zone size. If we compare the antibacterial activities of the extracts of the plant Polyalthia longifolia we find that the overall activity is very promising. The results indicate the possible presence of some important antibacterial compounds in the extracts. Through further research some pure compounds can be isolated and from them a therapeutically useful compound might be found.

TABLE 11: ANTIMICROBIAL ACTIVITY OF TEST SAMPLES OF Polyalthia longifolia AT A CONCENTRATION OF 400µg/disc:

TEST MICROORGANISMS DIAMETER OF ZONE OF INHIBITION (mm)Kanamycin(30µg/disc)

PE CCl4

1. Bacillius cereus 34 28 28

2. Bacillius megaterium 34 28 28

3. Bacillius subtilis 35 27 28

4. Salmonella paratyphi 35 27 28

5. Salmonella typhi 32 28 28

6. Vibrio parahemolyticus 35 28 28

7. Vibrio mimicus 35 28 30

8. Staphylococcus 35 27 30

9. E.coli 35 30 30

10. Shigella dysenteriae 35 28 30

11. Pseudomonas aureus 35 27 30

12. Sarcina lutea 35 27 30

13. Shigella boydii 35 27 30

14. Saccharromyces

cerevaceae

35 27 30

15. Candida albicans 35 28 30

16. Aspergillus niger 32 29 30

TABLE 12: ANTIMICROBIAL ACTIVITY OF TEST SAMPLES OF Polyalthia longifolia AT A CONCENTRATION OF 200µg/disc:

TEST MICROORGANISMS DIAMETER OF ZONE OF INHIBITION (mm)

Kanamycin(30µg/disc)

PE CCl4

1. Bacillius cereus 36 27 27 2. Bacillius megaterium 38 25 25 3. Bacillius subtilis 40 30 27 4. Salmonella paratyphi 37 26 27 5. Salmonella typhi 40 27 30 6. Vibrio parahemolyticus 40 26 27 7. Vibrio mimicus 40 29 29

8. Staphylococcus 35 24 25 9. E.coli 38 25 26 10. Shigella dysenteriae 35 26 26 11. Pseudomonas aureus 34 22 24 12. Sarcina lutea 35 27 27 13. Shigella boydii 35 30 30 14. Saccharromyces cerevaceae

40 27 27

15. Candida albicans 36 25 25 16. Aspergillus niger 30 22 24

SECTION: 4 BRINE SHRIMP LETHALITY BIOASSAY

4.1. INTRODUCTION:

Bioactive compounds are always toxic to living body at some higher doses and it justifies the statement ‘Pharmacology is simply toxicology at a lower doses, and toxicology is simply pharmacology at a higher doses. Brine shrimp lethality bioassay (McLaughlin, 1990; Persoone, 1980) is a rapid and comprehensive bioassay for the bioactive compound of natural and synthetic origin. By this method, natural product extracts, fractions as well as pure compounds can be tested for their bioactivity. In this method, in vivo lethality in a simple zoological organism can be used as a convenient monitor for screening and fractionation in the discovery and monitoring of bioactive natural products.

This bioassay indicates cytotoxicity as well as wide range of pharmacological activities such as antimicrobial, antiviral, pesticidal and anti-tumor etc of the compounds (Meyer, 1982; McLaughlin, 1988). Generally the LC50 values for cytotoxicities are one tenth of LC50 values in the Brine Shrimp Lethality Test.

Brine shrimp lethality bioassay technique stands superior to other cytotoxicity testing procedures because it is a rapid process, inexpensive and requires no special equipment or aseptic technique. It utilizes a large number of organisms for statistical validation and a relatively small amount of sample. Furthermore, unlike other methods, it does not require animal serum.

4.1.1. ANTI-TUMOR ACTIVITY OF NATURAL COMPOUNDS:

Tumor or cancer, name of terrifying disease against which yet we have almost nothing to do. In the United States of America one out of every three or four persons experiences cancer in there life time. Cancer caused 20% of all deaths in 1983, with 440,000 mortalities in America. If these trends continue, the American Cancer Society projects that 510000 people will die of cancer by the year of 2010. Even in China, a developing country cancer is in top position along with heart disease as killer of human being. In our country the exact picture of cancer incidence is not found but from the data of patients, being admitted in the hospital for the treatment of cancer, it can be assumed that cancer is at the top of the list of fatal diseases.In the era of science and technology, no treatment with 100% accuracy has been developed. Cancer research and treatment are extremely complex fields of study, as the exact nature of the single cancer cell is not identified. Problem is still now that no specific receptor in cancer cell is discovered through which an anti cancer drug can act. Thus anti tumor therapy is site non-specific.Chemotherapy with existing anti-tumor drugs is associated with severe toxicity. That’s why throughout the world scientists are engaged in search for new anti-tumor agents of low toxic profile with better accuracy. Anti-tumor drugs with synthetic origin are mostly cell cycle non-specific whereas the natural products are cell cycle specific. Thus they are in a sense little bit selective. This inspires the scientists to concentrate on the higher plant products having anti-tumor activity and screening on plants is continued to get lead compounds.

4.1.2. LOCAL SOURCE OF ANTI-TUMOR DRUGS:

In the ancient time majority of the people were dependent on plants for remedy of diseases. Still now a significant portion of the patients in the third world, including Bangladesh, India, Pakistan depends on traditional medicine in the

form of Ayurvedic and Unani formulations, which are derived from plants extract or juice. There are a huge number of plants in Bangladesh traditionally known to have cytotoxic and anti-tumor properties. Some have folkloric reputation of being used in different types of diseases. Therefore it may be expected that compounds active against cancer may be isolated from these plants.This realization is emphasized from the fact that some of these plants (Table: 11) has already been screened and have been proven to be cytotoxic and anti-tumor.

PLANTS WITH PROVEN CYTOTOXIC AND ANTI-TUMOR PROPERTIES:TABLE 13: LOCAL PLANTS WITH PROVEN CYTOTOXIC AND ANTI-TUMOR PROPERTIES:

Family Botanical Name Plant Part Used

Availability in Bangladesh

Leguminosae Albizzaia lebbeck Linn.B Seed All over the countryApocynaceae Vinca rosea Linn Leaf Grown in gardensLauraceae Dehaasia kurzii Leaf Grown in gardensAnnonaceae Desmos chinensis Linn Leaf Grown in gardensApocynaceae Ervatamia divaricata Linn Root All over the countryRutaceae Mangifera indiaca Linn Seed All over the countryRutaceae Murraya koenigii Linn.S Leaf All over the countryNymphaceae Nymphae rubra Tuber,

FlowerAll over the country

4.1.3. ANTI-TUMOR DRUGS FROM TERRESTRIAL PLANTS:

Terrestrial plants are recently considered to be the new frontier for searching new anti-tumor drugs, capable of being used in different types of cancer. Numerous compounds have already been isolated from plants, which are employed to combat various diseases. Even the pre-existing compounds (phodopyllotoxins) effective as drugs, are modified by designing to the molecular level with the help of synthetic organic chemistry and used as more effective, selective and safer therapeutic agent.

4.1.4. MODERN MEDICINE FROM FOLKLORE:

Occasionally, native lore provides clues to plants with pharmacological activity. Digitalis, Opiates and Cinchona alkaloids (Quinine and Quinidine) came into

modern medicine by this route. Curare was obtained from a South American plant long used by natives to prepare arrow poison. Rawolfia serpentina was used for centuries in India as native remedy for a variety of illness. Only in recent years in tranquilizing property was recognized in western medicine and the active principle Reserpine is isolated.

4.1.5. UNRELIABILITY OF FOLK MEDICINE:

Despite much useful contribution to the modern pharmacopoeia, folk medicine is a notoriously unreliable guide in search of biologically active products. There has been intensive interest, for example, in discovering anti-fertility agents. According to the natives of certain Pacific Islands, about 200 different local plants are efficacious in reducing male fertility. Extracts made from 80 of these were fed at high dose levels to rats for periods up to four weeks without any effect upon pregnancy and litter size.

4.2. PRINCIPLE OF BRINE SHRIMP LETHALITY BIOASSAY:

Brine shrimp eggs are hatched in simulated sea water to get nauplii. Sample solutions are prepared by dissolving the test materials in pre-calculated amount of DMSO. Ten nauplii are taken in test tubes containing 5 ml of simulated sea water. The samples of different concentrations are added to the pre-marked vials with a micropipette. The assay is performed using three replicates. Survivors are counted after 24 hours. These data are processed in a simple program for probate analysis to estimate LC50 values with 95% confidence intervals for statistically significant comparisons of potencies.

4.2.1. MATERIALS:

Artemia salina leach (brine shrimp eggs) Sea salt (NaCl) Small tank with perforated dividing dam to hatch the shrimp Lamp to attract shrimps Pipettes (5, 10, 25ml) and Micropipette (5-100l) Glass vials

Magnifying glass Pasteur pipette Test samples of experimental plants

TABLE 14: TEST SAMPLES OF EXPERIMENTAL PLANT

PLANT TEST SAMPLES MEASURED

AMOUNT(mg)

Polyalthia

longifolia

Pet. Ether soluble fraction of methanolic extract 4.0

CCl4 soluble fraction of methanolic extract 4.0

4.2.2. PROCEDURE:4.2.1.1 PREPARATION OF SEA WATER:76 gm sea salt (pure NaCl) was weighed, dissolved in 2000ml of distilled water and filtered off to get clear solution.

4.2.1.2. HATCHING OF BRINE SHRIMPS:

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 that side was covered. 24 hours were allowed to hatch the shrimp and to be matured as nauplii. Constant oxygen supply and warm of lamp was provided throughout the hatching time. The hatched shrimps were attracted to the lamp through the perforated dam and with the help of a Pasteur pipette 10 living shrimps were added to each of the vials containing 5 ml of seawater.

4.2.1.3. PREPARATION OF TEST SOLUTIONS:

Measured amount of each sample was dissolved in 100l of DMSO. A series of solutions of lower concentrations were prepared by serial dilution with DMSO. From each of these test solutions 50 l were added to pre-marked glass vials/test tubes containing 5 ml of seawater and 10 shrimp nauplii. So, the final concentration of samples in the vials/test tubes were 400 g/ml, 200 g/ml, 100 g/ml, 50 g/ml, 25 g/ml, 12.5 g/ml, 6.25 g/ml, 3.125 g/ml, 1.5625g/ml,

0.78125g/ml for 10 dilutions. Only 50µg of DMSO was added to the 11 th test tube containing the same amount of nauplii to act as a standard.

4.2.1.4. COUNTING OF NAUPLII AND ANALYSIS OF DATA:

After 24 hours, the vials were inspected using a magnifying glass and the number of survivors were counted. The percent (%) mortality was calculated for each dilution. The concentration-mortality data were analyzed by using Microsoft Excel. The effectiveness or the concentration-mortality relationship of plant product is usually expressed as a median lethal concentration (LC50) value. This represents the concentration of the chemical that produces death in half of the test subjects after a certain exposure period.

4.3. RESULTS AND DISCUSSION OF THE TEST SAMPLES OF Polyalthia longifolia:

Following the procedure of Meyer (Meyer et al, 1982) the lethality of Petroleum Ether and CCl4 (CT) of the methanolic extract to brine shrimp were investigated.

The following table gives the results of the brine shrimp lethality after 24 hours exposure to all the samples and the standard (only DMSO). The standard compared with the negative control (Test samples) was lethal, giving significant mortality to the shrimp. The lethal concentration LC50 of the test samples after 24 hr. was obtained by a plot of percentage of the shrimps killed against the logarithm of the sample concentration (toxicant concentration) and the best-fit line was obtained from the curve data by means of regression analysis.

TABLE 15: RESULTS OF THE TEST SAMPLES OF Polyalthia longifolia

SAMPLE LC50 (ΜG/ML) REGRESSION EQUATION R2

Pet. Ether 0.434 y = 20.66x + 57.496 0.747

CCl4 0.160 y = 17.907x + 64.252 0.7014

Each of the test samples showed different mortality rate at different concentrations. The degree of lethality was directly proportional to the concentration of the extract ranging from significant with the lowest concentration (0.78125µg/ml) to highly significant with the highest concentration (400µg/ml). Maximum mortalities took place at a concentration of 400µg/ml, whereas least mortalities were at 0.78125 µg/ml concentration. In other words, mortality increased gradually with the increase in concentration of the test samples. LC50 obtained from the best-fit line slope were 0.434µg/ml and 0.16µg/ml for Pet. Ether and CCl4 respectively.

The partitionates of Polyalthia longifolia were found active against the Brine Shrimp nauplii which indicate the presence of anti-tumor and pesticidal compound. Further research can be conducted to isolate bioactive component.

TABLE 16: Effect of Pet. Ether soluble fraction of methanolic extract on Brine Shrimp

Lethality Bioassay

CONCENTRATION

(ΜG/ML)

LOG C (CONC.) % MORTALITY LC50 ΜG/ML

400 2.602 100

0.434

200 2.301 100100 2 10050 1.7 10025 1.4 9012.5 1.1 906.125 0.79 803.125 0.49 801.5625 0.19 600.78125 -0.107 60

Effect of Pet.Ether soluble fraction of methanolic extract

y = 16.314x + 66.652R2 = 0.826

0

10

20

30

40

50

60

70

80

90

100

110

120

-0.5 0 0.5 1 1.5 2 2.5 3Log C

% M

orta

lity

FIGURE: 3 EFFECT OF PET. ETHER SOLUBLE FRACTION OF METHANOLIC EXTRACT.

TABLE 17: EFFECT OF CARBON TETRACHLORIDE (CCL4) soluble fraction of methanolic

extract on Brine Shrimp Lethality Bioassay

CONCENTRATION

(ΜG/ML)

LOG C (CONC.) % MORTALITY LC50 ΜG/ML

400 2.602 100

0.16

200 2.301 100100 2 10050 1.7 10025 1.4 10012.5 1.1 906.125 0.79 803.125 0.49 901.5625 0.19 700.78125 -0.107 60

Effect of carbontetrachloride soluble fraction of methanolic extract

y = 13.874x + 71.705R2 = 0.7635

0

10

20

30

40

50

60

70

80

90

100

110

120

-0.5 0 0.5 1 1.5 2 2.5 3Log C

% M

orta

lity

FIGURE: 4 EFFECT OF CARBON TETRACHLORIDE SOLUBLE FRACTION OF METHANOLIC EXTRACT.

Conclusion

The methanolic extract of the plant Polyalthia longifolia i.e. Carbon tetrachloride and Pet. Ether soluble fractions showed significant antimicrobial activities, which support the traditional use of this plant in various infectious diseases.

The cytotoxicity study by brine shrimp lethality bioassay provided strong activity. Pet. Ether and Carbon tetrachloride fraction of the plant showed very interesting activity in cytotoxicity study.

The plant 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:

1. Achenbach. Hans; Hoehn, Matthias; Waibel, Reiner; Nkunya, Maymga. H.H;

Jonkar, Stephen A; Muhie, Seid (1997). Phytochemistry. 44(2), 359-364

(Eng).

2. Baner, A W, Kirby, W.M.M., Sherrus, J.C and Truck, M (1966) Antibiotic

susceptibility testing by a standardized single disc method. Am.j.Clin.

Pathol. 45, 493-496.

3. Fang, X,P, Alkofahi, A and Mclaughlin, J.L. (1991) Heterocycles 32,11

4. Fang, X, P, Anderson, J.E and Mclaughlin, J.L. (1991) J.Chem. Soc. Perkin

Trans.1, 1655.

5. Ferdous, A.J.; Islam, O.M. and Hassan, C. M. and Nazrul, S.N. (1992) in vitro

antimicrobial activity of Languinosine and Oxostephanine. Fitoterapia LXII

(No.6), 549-550.

6. Ghani, A., 1998, Medicinal Plants of Bangladesh: Chemical constituents

and uses, Asiatic Society of Bangladesh, Dhaka.

7. Heywood, V. H, 1978

8. Hutchison, J., 1964

9. Hasan, C.M.; Islam, S.N. and Ahsan, M. (1988) Antimicrobial activity of

Polyalthia longifolia stem bark, Dhaka University Studies 4(1), 63-66.

10.Hasan, C.M.; Islam, O.M. and Rashid, M.A. (1995) Clerodane diterpene

from Polyalthia longifolia var. Pendlla. Pharmabiz 50, H.2,227-228

11.Hasan, C.M.; Islam, O.M. and Rashid, M.A. (1995) Clerodane diterpene

from Polyalthia longifolia var. Pendlla.Biochem. Syst. Ecol. 23 (no.3), 331-

332

12.Hasan, C.M.; J.D.Connolly; Mia, M.Y.; M.A.Rashid. (1994c) Phytochemistry,

37(6), 1763-1764.

13.Hasan, C.M.; Hossain M.A. and Connoly, J.D (1994) Constituents of

Polyalthia longifolia var. Pendlla. Fitoterapia LXV (No.3), 283-284

14.Hossain, M.S. (1991) M.Pharm. Thesis. Department of Pharmacy,

University of Dhaka.

15.Internet: www.pubmed.com, www.wikipedia.com , www.banglapedia.com,

www.google.com.

16.Kirtikar and Basu (1980) Indian Medicinal plants, Vol-1, second ed. Pg: 60-

73. Published by B.sing and M.P. Singh, India.

17.Leboeuf, M., Cave, A., Bhaumik, P.K., Mukharjee, R. (1982.

Phytochemistry.21, 2783

18.Munguti, K., 1997. Indigenous knowledge in the management of malaria

and visceral leishmaniasiss among the Tugen of Kenya. Indigenous

Knowledge and Development Monitory 5, 10-12

19. Talapatra, Sunil. K., Basu, Dipankar. Chattopadhayay, Pratap. And

Talapatra, Bani. (1988). Phytochemistry, Vol.27 No.3, Pg: 903-906.

20.Trease, G.E. and Evans, W.C., 1972, Pharmacognosy, Bailliere Tindall,

London.

21.Takhajan, 1969.

Documents

BIOLOGICAL INVESTIGATION OF Polyalthia longifolia