BIOLOGICAL INVESTIGATION OF Polyalthia longifolia by regan ahmed

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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.

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ANNONACEAE

INCLUDES ABOUT

120-130 GENERA:

TABLE 1: 130 GENERA OF ANNONACEAE FAMILY: 1. 3. 5.

Artabotrys Deeringothamnus Oxandra

2. 4. 6.

Cananga Guatteria Rollinia

7. 9. 11. 13. 15. 17. 19. 21. 23. 25. 27. 29. 31. 33. 35. 37. 39. 41. 43. 45. 47. 49. 51. 53. 55. 57. 59. 61. 63.

Stelechocarpus Alphonsea Anaxagorea Annona Anonidium Asimina Balonga Bocageopsis Cananga Cleistochlamys Craibella Cyathocalyx Cymbopetalum Dasymaschalon Dendrokingstonia Desmopsis Diclinanona Disepalum Ellipeia Enicosanthum Exellia Fitzalania Froesiodendron Gilbertiella Greenwayodendron Guatteria Guatteriopsis Heteropetalum Hornschuchia

8. 10. 12. 14. 16. 18. 20. 22. 24. 26. 28. 30. 32. 34. 36. 38. 40. 42 44. 46. 48. 50. 52. 54. 56. 58. 60. 62. 64.

Afroguatteria Ambavia Annickia Anomianthus Artabotrys Asteranthe Bocagea Boutiquea Cardiopetalum Cleistopholis Cremastosperma Cyathostemma Dasoclema Deeringothamnus Dennettia Desmos Dielsiothamnus Duguetia Ellipeiopsis Ephedranthus Fissistigma Friesodielsia Fusaea Goniothalamus Guamia Guatteriella Haplostichanthus Hexalobus Isolona

65. 67. 69. 71. 73. 75. 77. 79. 81. 83. 85. 87. 89. 91. 93. 95. 97.

Letestudoxa Malmea Meiocarpidium Melodorum Mezzettiopsis Mischogyne Mitrephora Monanthotaxis Monocyclanthus Duckeanthus Neo-uvaria Ophrypetalum Orophea Pachypodanthium Petalolophus Phoenicanthus Platymitra

66. 68. 70. 72. 74. 76. 78. 80. 82. 84. 86. 88. 90. 92. 94. 96. 98.

Lettowianthus Marsypopetalum Meiogyne Mezzettia Miliusa Mitrella Mkilua Monocarpia Monodora Neostenanthera Onychopetalum Oreomitra Oxandra Papualthia Phaeanthus Piptostigma Polyalthia

99. 101. 103. 105. 107. 109. 111. 113. 115. 117. 119. 121. 123. 125. 127. 129.

Polyceratocarpus Porcelia Pseudephedranthus Pseuduvaria Raimondia Richella Ruizodendron Sanrafaelia Schefferomitra Stelechocarpus Tetrameranthus Tridimeris Trivalvaria Uvaria Uvariodendron Woodiellantha

100. 102. 104. 106. 108. 110. 112. 114. 116. 118. 120. 122. 124. 126. 128. 130.

Popowia Pseudartabotrys Pseudoxandra Pyramidanthe Reedrollinsia Rollinia Sageraea Sapranthus Sphaerocoryne Stenanona Toussaintia Trigynaea Unonopsis Uvariastrum Uvariopsis 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

1. Annona (a) Annona bullata

(b) Annona glabra

GENUS/SPECIES (c) Annona muricata

PLANT PARTS/ISOLATED PRODUCTS Bullataci Bullatacinone (Acetogenins)

Liriodenine (Alkaloid)

PLANT PARTS/ISOLATED PRODUCTS

MEDICINAL

OR OTHER

REF.

USES

& Selective cytotoxic agent Hui et al., 1989 in human tumor cell line. Bullatacin is a pesticidal at a concentration of 1 ppm Antibacterial, antifungal & Warthen et al., antitumor agent. 1969, Hufford et al., 1980

MEDICINAL

OR OTHER

REF.

USES

Flowers fruits, Effective in cough & Hossain seeds & roots chronic dysentery, 1991 emetic, astringent , antispasmodic &

al.,

parasiticidal

(d) Annona reticulata

Fruits

(e)Annona senegalensis

Extracts bark

Effective against Kirtikar & Basu, biliousness & thirst 1980 (Ayurveda) & also used as anthelmentic stem Showed good Hasan et al., antibacterial activity 1988 Antineoplastic activity against sarcoma 180 Adesogan & ascities tumor cells Durdola, 1976

Root bark (f) squamosa

Annona

Leaves & fruits

Seeds

In ulcer. Tonic effect on Ayurveda the body which increases blood, muscular strength, relieve vomiting, lessen burning sensation & biliouness Kirtikar & Basu, 1980 Fatal to insects & worm

2. Artabotrys

Leave extract

In treatment of cholera

(a) Artabotrys odorotissimus

Essential oils from In perfumery flower

Ayurveda Chopra 1953

al.,

Alkaloidal mixtures

Showed action

(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

PLANT PARTS/ISOLATED PRODUCTS

MEDICINAL

REF.

GENUS/SPECIES 4. Desmos (a)Desmos longiflorus (b)Desmos chinensis 5.Goniothalamus (c) Goniothalamus macrophyllus

Alkaloids stembark

antibacterial Haider, 1988

OR OTHER

USES

from Good antibacterial agent Hossain, 1991 & antifungal agent

Chloroform extract

Strong inhibitor of tyrosine kinase enzyme

Plant constituents

Cytotoxic to human tumor cell

Fang et al.,1990

(b)Goniothalamus giganteous

Acetogenius

Powered leaves

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

Different parts these plants

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. An embryotoxic and teratogenic compound was isolated

of Antibacterial agent

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

Sam et al., 1987

Hasan et al.,1994b, 1994c

6. Miliusa (a)Miliusa tomentosa

PLANT PARTS/ISOLATED PRODUCTS

GENUS/SPECIES (b)Miliusa banacea

Essential oil from Used as analgesic & Menon & kar this plant possesses antibacterial 1970, Kar & jain, activity 1971

cf.

(c)Miliusa velutina

MEDICINAL

OR OTHER

REF.

USES

Oxoaporphine like Has been reported as alkaloids from root good bioactive and cytotoxic compounds

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

Enamul al.,1998

Volatile oils this plant

Kar & Jain, 1971

7. Polyalthia (a) longifolia

Polyalthia

from Antibacterial agent

Alkaloids from Good antibacterial methanol extract of antifungal agents stem bark Crude extract

& Hasan 1988b

al.,

chloroform Good antibacterial agent Shaheen, 1986

(b) longifolia pendulla

Polyalthia var

Different plant parts Antimicrobial and pure compound

Ferdous et al.,1992 & Hasan et al.,1994, 1994a In black water fever & Keay et al., 1964 stomach disorder

Extract of bark

(d) Polyalthia suberosa

Crude extract of Good stem bark plant activity parts

antibacterial

8. Uvaria (a)Uvaria afzelli

Plant parts

Good activity against Hufford et al., Bacillus subtilis, 1981 microbacterium semagmatis & Staph. Aureus

PLANT PARTS/ISOLATED PRODUCTS

MEDICINAL

GENUS/SPECIES (b)Uvaria chamae

C-benzylated flavonoids

(c)Uvaria duclis

Root bark

Cytotoxic against human in vitro Laswell & carcinoma of the Hufford 1977a, nasopharynx Hufford & Oguntimein, 1980 Kirtikar & Basu, Astringent, stimulant & 1980 alternative properties

9. Xylopia (a) Xylopia aethiopica (b)Xylopia danguyell

OR OTHER

REF.

USES

Plant parts and Anticaugh, antifungal & Boakye, 1987 isolated Diterpenes antibacterial agent Plant parts CNS depressant & Cordell, 1981 hypotensive

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 Chamanen (1)

Annona squamosa Uvaria chamae

Rao et al.,1978 Hufford et

Annona glabra

al.,1977 Bohlmann

2. Diterpenes

(-)-Kaur-16-en-19-ol (2) (-)-Kaur-16-en-19-yl

(-)-Kauran-16Îą-ol (7) Triterpenes Sitosterol (8)

steroids Stigmasterol (9) Polycarpol (10)

al.,1978

acetate

(3) (-)-Kauran-17-ol-19-oc acid Stachanoic acid (6)

Annona glabra Annona seegalensis

Yarng et al.,1973 Adesogan et al.,

Xylopia aethiopica Annona muricata

1976 Ekong et al.,1969 Ca. llan, 1911

Annona Senegalensis

Mackie,

Annona squamosa Polyalthia longifolia Fusaea longifolia

Farnsworth, 1974 Beraz, 1976 Cave et al.,1977

Polyalthia oliveri

Toeche, 1981

1958

Xylopia longifolia 4.

Î&#x2019;-caryophyllene (11)

Annona squamosa

Bohlmann

al.,1973

Sesquiterpenes

Artabotrys Uncniatus

Yingzhaosu A (12)

Liang et al.,1979

Yingzhaosu B (13)

Cymbopetalum

Ishwarane (14)

penduliflorum

H CH2OH OMe CHO [1]

[4]

[2] R=

CH2OH [3]R= CH2OAC [5] R= COOH

OH COOH HO [6]

[7]

[8]

OH O HO

[10]

[11]

OH OH OH

[12]

[13]

FIG: STRUCTURAL

[14]

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 1. Simple isoquinolines

ALKALOIDS Salsolinol (15)

SOURCE Annona reticulata

INVESTIGATORS Forgacs et al.,1981

2.Benzyltetrahydro isoquinolines

Corydaldine (16)

Enantia

Jossang

Reticuline (17) Anomuricine

polycarpa Annona montana Annona muricata

al.,1977 Yang et al.,1979

Leboeuf

al.,1980

3. &

Bisbenzylisoquinoline Curine cycleanine Bisbenzyltetrahydro

isoquinoline

Isolana pilosa

Hocquemiller

Isolana hexaloba

al.,1977

4. Protoberberines

Berberine

`Xylopia

Schermerhorn al.,1974

Oxypalmatine

polycarpa Enantia

Leboeuf

al.,

polycarpa Annona montana

1977 Leboeuf

al.,

Annona muricata

1982 Leboeuf

(18) Tetrahydropro- Coreximine

toberberine 6. Proaporphines

Stepharine

al.,1981

Crotsparine

Monodora

Leboeuf al.,1974

Anolobine

angolensis Annona

SUB CLASS

ALKALOIDS

squamosa SOURCE

a. Simple aporphines

Oliveridine

Enantia pilosa

7. Aporphines-

7-Substituted Liriodenine (19)

aporphines

INVESTIGATORS

Annona

Yang et al.,1970

Squamosa

c. Oxoaporphines 8. Phenanthrenes

Argentinine (20)

Annona montana

Uvariopsine

Uvariopsis

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

congolana

OMe

MeO

OMe NH

OMe Me

O OMe OH

[15]

[16]

OMe OMe

[17]

O N

O OMe OMe

N O

OMe

N Me

[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 C 3 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

Heganuer, 1964 Mackie et al.,1958

A.senegalensis 2. Quercetrin rutin 3. Nicotiflorin 4. Pachypodol (33) 5. 5,6,7-trimethoxyflavone (34)

Asimia triloba Annona senegalensis Cananga latifolia Pachypodanthium confine Monanthotaxis Cauliflora

Mackie et al.,1958 Siv et al.,1972 Cave et al.,1973 Waterman et al.,1979

5-hydroxy-6,7dimethoxyflavon (35) 5,7,8-trimethoxyflavanone (36) 5,6,7,8tetramethoxyflavanone (37) 6. Dependensin 7. Triuvaretin 8. Triuvaretin

U.dependens U.leptocladon U.scheffleri

Nkhunya et al.,1993 Nkhunya et al.,1993 Chantrapromma et al.,1989

9. Tetochrysin (38)

U. rufa

Chantrapromma et al.,1989

COMPOUNDS

SOURCE

INVESTIGATOR

10. Angoluvarin

U. angolensis U.leptocladon U. angolensis U. chamae U. lucida lucida

Hufford et al.,1987 Nkhunyet al.,1993 Hufford et al.,1980 Hufford et al.,1980 Achenbach et al.,1997

11. Uvangoletin (39) 12.Chamanetin (40) 13. Isochamaetin Dichamanetin

Okorie et al.,1977

14. Isovaretin

U. angelonsis

Weenen et al.,1990 Hufford et al.,1980

15. Uvaretin (41)

U. chamae

Nkunya et al.,1985

Diuvaretin (42)

U. lucida lucida

16. Isovaretin

U. kirkii U. angelonsis

Hufford et al.,1980

17. Pinocembrin (43)

U. chamae

Hufford et al.,1978,1979

U. afzelii

Hufford et al.,1980

Pinostrobin (44) Chamuvaritin Uvarinol 18. Vafzelin (45) Uvafzelin Syncarpic acid

MeO

OMe O

[33]

[34] R= ME

[35} R=H

OMe OH

OMe

CH2 O

OMe OH

[36] R=H

[38]

[37] R= OMe

OMe OMe

OMe [39]

OMe OR

[40]

OMe O

OMe

[44] [42] R1= R2= H

HO [43] R1=R2=

CH2

O [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 â&#x20AC;˘

Polyalthia longifolia (Sonn.) Thw

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

Family: Annonaceae

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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 Leaves

COMPOUND ISOLATED

REF.

 Azafluorene alkaloid  Polylongine  3-aporphine  N-oxide alkaloids

Goyal & Gupta

 (+)-O methyl bulbocapnine-α-Noxide  (+)-O methyl bulbocapnine-β-NStem bark

oxide Tetrahydroprotoberine (-)-Stepholidine Oxychine Darienine 6,7-dimethoxychime Aporphine alkaloid

Wu, 1989; Chakrabarty &

Liriodenine (cytotoxic)

Patra, 1990; Wu et al.,1990

Noroliveroline oliveroline oxide Azofluorene alakloid Polyfothine Bark & Seeds

Iso-oncodine Clerodane diterpenoids

(Phadnis

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 VAR.

P. longifolia

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. shaheen@khi.comsats.net.pk PMID: 12709903 [PubMed - indexed for MEDLINE]

•

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. rs127pk@yahoo.com PMID: 16261519 [PubMed - indexed for MEDLINE]

•

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 PMID: 11087586 [PubMed - indexed for MEDLINE]

•

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 Polyalthia longifolia

Family

Plant part

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 20 th 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 (40째50째) 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 50째C 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.

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

SOLVENT-SOLVENT PARTITIONING OF METHANOL EXTRACT:

Crude extract (3 gm) METHANOL (45ML) + WATER (5 ML) Aqueous methanol solution EXTRACTION

WITH

PET ETHER (75 ML X 3) ML

Aqueous fraction

Pet ether soluble fraction

+ WATER (6 ML)

EXTRACTION WITH CCl4 (75 ML X 3) ML

CCl4 soluble fraction

Aqueous fraction

+WATER (8 ML)

EXTRACTION

CH2Cl2 soluble fraction

WITH

CH2Cl2 (75 ML X3 ML)

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. 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 (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 37 째C 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. 2. Sterile cotton.

9. Screw cap test tubes 10. Autoclave

3. 4. 5. 6. 7. 8.

Micropipette Laminar air flow hood Refrigerator Chloroform Petri dishes Sterile forceps

11. 12. 13. 14. 15. 16.

Nutrient Agar Medium Inoculating loop Spirit burner Nose mask and Hand gloves Incubator 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. 2. 3.

Bacillus cereus Bacillus megaterium Bacillus subtilis

4. 5. 6. 7.

Salmonella paratyphi Salmonella typhi Vibrio parahemolyticus Vibrio mimicus

Staphylococcus

9. 10. 11. 12. 13. 14. 15. 16.

E.coli Shigella dysenteriae Pseudomonas aureus Sarcina lutea Shigella boydii Saccharromyces cerevaceae Candida albicans Aspergillus niger

3.2.3. TEST MATERIALS: TABLE 9: LIST OF TEST MATERIALS PLANT

TEST SAMPLES

SAMPLE CODE

1. Pet ether soluble fraction of methanolic PE

Polyalthi a longifolia

extract 2. CCl4 (Carbon

tetrachloride)

soluble CCl4

fraction of methanolic extract

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 media b. Nutrient broth media c. Muellar-Hinton agar media d. 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 Sodium chloride Bacto yeast extract Bacto agar Distilled water q.s. pH

0.5 gm 0.5 gm 1.0 gm 2.0 gm 100 ml 7.2-7.6 at 25째C

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 121째C 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 121째C 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 37째C 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

longifolia Polyalthia

Plant

OF SAMPLE DISCS

Sample code

Sample

Pet

CCl4

methanolic extract CCl4 (Carbon tetrachloride) soluble

ether

soluble

fraction

Dose (µg/disc)

200

400

200

400

fraction of methanolic extract

Standard Kanamycin (30 µg/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 solventsolvent 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 2730mm 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 CONCENTRATION OF

ACTIVITY OF TEST SAMPLES OF

Polyalthia longifolia

400µg/disc:

TEST MICROORGANISMS 1. Bacillius cereus 2. Bacillius megaterium 3. Bacillius subtilis 4. Salmonella paratyphi 5. Salmonella typhi

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

CCl4

34 34 35 35 32

28 28 27 27 28

28 28 28 28 28

AT A

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

35 35 35 35 35 35 35 35 35

28 28 27 30 28 27 27 27 27

28 30 30 30 30 30 30 30 30

cerevaceae 15. Candida albicans 16. Aspergillus niger

35 32

28 29

30 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)

CCl4

1. Bacillius cereus 2. Bacillius megaterium 3. Bacillius 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

36 38 40 37 40 40 40 35 38 35 34 35 35 40

27 25 30 26 27 26 29 24 25 26 22 27 30 27

27 25 27 27 30 27 29 25 26 26 24 27 30 27

cerevaceae 15. Candida albicans 16. Aspergillus niger

36 30

25 22

25 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 â&#x20AC;&#x2DC;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 LC 50 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. Thatâ&#x20AC;&#x2122;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

Leguminosae Apocynaceae Lauraceae Annonaceae Apocynaceae

Albizzaia lebbeck Linn.B Vinca rosea Linn Dehaasia kurzii Desmos chinensis Linn Ervatamia divaricata Linn

Plant Used Seed Leaf Leaf Leaf Root

Part Availability Bangladesh All over the country Grown in gardens Grown in gardens Grown in gardens All over the country

Rutaceae Rutaceae Nymphaceae

Mangifera indiaca Linn Murraya koenigii Linn.S Nymphae rubra

Seed Leaf Tuber, Flower

All over the country All over the country All 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 antitumor 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 LC 50 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 (5-100µl)  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 100µl of DMSO. A series of solutions of lower concentrations were prepared by serial dilution with DMSO. From each of these test solutions 50 µl 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 µg/ml, 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3.125 µg/ml, 1.5625µg/ml, 0.78125µg/ml for 10 dilutions. Only 50µg of DMSO was added to the 11th 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

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

PET. ETHER

SOLUBLE FRACTION OF METHANOLIC EXTRACT ON

BRINE SHRIMP LETHALITY BIOASSAY CONCENTRATION

LOG C (CONC.)

% MORTALITY

(ΜG/ML) 400 200 100 50 25 12.5

2.602 2.301 2 1.7 1.4 1.1

100 100 100 100 90 90

LC50 ΜG/ML

0.434

6.125 3.125 1.5625 0.78125

0.79 0.49 0.19 -0.107

80 80 60 60

Effect of Pet.Ether soluble fraction of methanolic extract 120 110 100

% Mortality

90 80 70 60 50 40 30

y = 16.314x + 66.652 2 R = 0.826

20 10 0 -0.5

0.5

1.5

Log C

2.5

FIGURE: 3 EFFECT OF PET. ETHER SOLUBLE FRACTION OF METHANOLIC EXTRACT. TABLE 17: EFFECT

OF CARBON TETRACHLORIDE

METHANOLIC EXTRACT ON

(CCl4)

SOLUBLE FRACTION OF

BRINE SHRIMP LETHALITY BIOASSAY

CONCENTRATION

LOG C (CONC.)

% MORTALITY

(ΜG/ML) 400 200 100 50 25 12.5 6.125 3.125 1.5625 0.78125

2.602 2.301 2 1.7 1.4 1.1 0.79 0.49 0.19 -0.107

100 100 100 100 100 90 80 90 70 60

LC50 ΜG/ML

0.16

Effect of carbontetrachloride soluble fraction of methanolic extract 120 110 100

% Mortality

90 80 70 60 50 40

y = 13.874x + 71.705 R2 = 0.7635

30 20 10 0 -0.5

FIGURE: 4 EFFECT

0.5

Log C

1.5

2.5

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.

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