Biological Investigation of Polyalthia longifolia

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Biological Investigation of Polyalthia longifolia

Chapter: 1


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


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/Species Plant parts/Isolated Products Medicinal or other uses 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


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

Fatal to insects & worm


Kirtikar & Basu, 1980

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
Genus/Species Plant parts/Isolated Products Medicinal or other uses 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

(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



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
Genus/Species Plant parts/Isolated Products Medicinal or other uses 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
Genus/Species Plant parts/Isolated Products Medicinal or other uses 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 Kirtikar & Basu, 1980
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: 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.,1978
Chamanen (1) Uvaria chamae Hufford et al.,1977
2. 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-16?-ol (7) Xylopia aethiopica Ekong et al.,1969
3. Triterpenes steroids Sitosterol (8) Annona muricata

Annona Senegalensis

Annona squamosa

Ca. llan, 1911 Mackie, 1958 Farnsworth, 1974
Stigmasterol (9) Polyalthia longifolia Beraz, 1976
Polycarpol (10) Fusaea longifolia

Polyalthia oliveri

Xylopia longifolia

Cave et al.,1977

Toeche, 1981

4. Sesquiterpenes ?-caryophyllene (11) Annona squamosa Bohlmann et al.,1973
Yingzhaosu A (12) Artabotrys Uncniatus Liang et al.,1979
Yingzhaosu 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 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:

  1. Isoquinoline alkaloids
  2. 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-toberberine Coreximine Annona montana Leboeuf et al., 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)


Annona montana

Uvariopsis congolana

Yang et al.,1979

Bouquet et al.,1972

[15] [16] [17]

[18] [19] [20]

FIG: Structural types of various isoquinoline alkaloids from Annonaceae 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


Asimia triloba

Heganuer, 1964

Mackie 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.,1990

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)



U. chamae Hufford et al.,1978,1979
18. Vafzelin (45)


Syncarpic acid

U. afzelii Hufford et al.,1980


[34] R= Me [35} R=H

[36] R=H [38]

[37] R= OMe

[39] [40]


[42] R1= R2= H

[43] R1=R2=


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-?-N-oxide

§ (+)-O methyl bulbocapnine-?-N-oxide

Goyal & Gupta
Stem bark Tetrahydroprotoberine





Aporphine alkaloid

Liriodenine (cytotoxic)


oliveroline oxide

Azofluorene alakloid



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.

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.

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

Theplant species belonging to Annonaceae is investigated in this study.

Name of plant


Plant part

Polyalthia longifolia Annonaceae Stem Bark

Taxonomic hierarchy of the investigated plants (Wekepedia)

Table7: Taxonomic hierarchy of the investigated plant

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:

  1. Cold extraction.
  2. 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: 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. 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. 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. 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:

Methanol (45ml) + Water (5 ml)

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 (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 tubes
2. Sterile cotton. 10. Autoclave
3. Micropipette 11. Nutrient Agar Medium
4. Laminar air flow hood 12. Inoculating loop
5. Refrigerator 13. Spirit burner
6. Chloroform 14. Nose mask and Hand gloves
7. Petri dishes 15. Incubator
8. 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
  1. CCl4 (Carbon tetrachloride) soluble 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.

  1. Nutrient agar media
  2. Nutrient broth media
  3. Muellar-Hinton agar media
  4. 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 gm
Sodium chloride 0.5 gm
Bacto yeast extract 1.0 gm
Bacto agar 2.0 gm
Distilled water q.s. 100 ml
pH 7.2-7.6 at 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