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Abstract
Pharmacognostical Study of Flacourti cataphracta Roxb. Family Flacourtiaceae, Cultivated in Egypt.
Flacourtia cataphracta Roxb. Family Flacourtiaceae is a shrub or low branched tree growing in tropical and subtropical regions. It is used in the Indian folk medicine as tonic, diaphoretic, stomachic, in the treatment of diarrhea and distended stomach and to relieve bronchitis and cough. The leaves and bark are applied on bleeding gums and aching teeth. The bark infusion is gargled to alleviate hoarseness.
Literature survey showed that nothing could be traced concerning the botanical and phytochemical studies of Flacourtia cataphracta except few notes about taxonomical characters, this stimulates the present work which includes:
Part I: Botanical Study
Macro- and micromorpholgy of the organs of the plant under investigation in both entire and powdered forms.
Part II: Phytochemical Study
1- Preliminary phytochemical screening of the different organs of the plant.
2- Isolation and identification of the possible different constituents of the plant.
Part III: Biological Study
1- Preliminary antimicrobial study of different extracts of the leaves.
2- Toxicological effects of different extracts of the leaves.
3- Preliminary Pharmacological investigation of the leaves extract (a) Anti-diarrheal activity (b) Anti-inflammatory activity
(c) Anti-pyretic activity
Part I: Botanical Study
Chapter I
Macromorphology of Flacourtia cataphracta Roxb
Flacourtia cataphracta Roxb is a shrub or an erect, low branch tree. It has young spiny branches and flaking bark. The male and female flowers are on separate trees; they are greenish and has faint characteristic odour. Flower buds begin to appear in February and open in March-April. The fruits are round or slightly oval having dark-marron colour.
A) Macromorphology of the Stem:
The main trunk of the plant is erect, cylindrical, woody monopodially branched, about 8-10 meters in height with faint odour and slight bitter taste.
B) Macromorphology of the Leaf:
The leaf is simple, petiolate, exstipulate, varying in shape and, size being ovate, obovate to ovate-acuminate, alternate or spirally arranged. The leaf has a serrate margin, symmetric base and acute to acuminate apices. It is green in colour with faint odour and slight bitter taste.
C) Macromorphology of the Female Flower:
The flowers are solitary, axillary, pedicellate, yellowish-green in colour with faint odour and slight bitter taste. They are zygomorphic, pistillate polymerous, apetalous and having the floral formula:
% , P 5-7 , G (5).
The flower is sepaloid, with greenish, polysepalous perianth. It consists of one whorl of 5-7 free greenish sepaloid which is nearly ovate in shape with acute apex, entire margin and covered with hairs, it measures about 0.2-0.5 cm in length and 0.2-0.4 cm in width. The gynaeceium is superior, globular in shape measuring about 0.25-0.4 cm in length and 0.2-0.5 cm in diameter. It has hairy surface, with 5-7 short free styles and stigmaes.
D) Macromorphology of the Stem bark:
The stem bark consists of flat to slightly curved pieces. The outer surface is covered with rough dark-brown cork; while the inner surface is finely longitudinally striated having brown colour. It has faint odour, slight bitter taste and the fracture is short.
Chapter II
Micromorphology of Flacourtia cataphracta Roxb
A) Micromorphology of the stem:
A transverse section in the young stem appears more or less circular in outline. It shows an outer epidermis carrys few hairs, a few layers of collenchyma followed by a comparatively narrow parenchymatous cortex. The pericycle is represented by a nearly complete ring of pericyclic fibers interrupted by few sclerenchymatous cells, surround a continuous ring of vascular tissue and a wide parenchymatous central pith. The endodermis is indistinct.
The Epidermis:
In surface view it consists of polygonal square to subrectangular cells with straight anticlinal walls covered with thick smooth cuticle. Few non-glandular trichomes of unicellular type are present being conical in shape with bent elongated blunt apices. Stomata are almost absent.
The Cortex:
The primary cortex consists of few rows of collenchymatous cells, followed by wide zone of parenchymatous cells containing prismatic and clusters crystals of calcium oxalate.
The Pericycle:
The pericycle is formed of more or less continuous ring of lignified fibers interrupted by few sclerenchymatous cells.
The Phloem:
The phloem consists of sieve tubes, companion cells and phloem parenchyma which contain small cluster crystals of calcium oxalate.
The Cambium:
It forms a zone of cambiform cells which are subrectangular tangentially elongated and radially arranged.
The Xylem:
The xylem consists of lignified vessels, wood fibers, tracheids and wood parenchyma. The vessels are with spiral and reticulate thickenings. The medullary rays are usually uni-to triseriate; formed of elongated subrectangular thick walled lignified cells in xylem region but in the phloem region they are of thin-walled cellulosic cells.
The Pith:
It is formed of parenchymatous cells with pitted and slightly lignified walls, they contain prismatic crystals of calcium oxalate.
The Moderate-aged Stem:
A transverse section in moderate-aged stem is nearly circular in outline with an outer cork. The phellogen originates superficially in the subepidermal region. The cork is followed by a moderately narrow secondary cortex, a narrow collapsed remaining of the primary cortex, then a continuous zone of pericycle which surrounds the vascular cylinder. The vascular system is formed of a complete ring of secondary elements enclosing comparatively narrow pith. Both phloem and xylem are traversed by uni- to triseriate medullary rays.
B) Micromorphology of the leaf:
The Lamina:
A transverse section in the lamina through the midrib region appears more or less slightly bioconvex in outline. It consists of upper and lower epidermises enclosing in between the mesophyll tissue which is replaced in the midrib region by the main vascular bundle, in addition to several small bundles at the small veins. The cortical tissue shows an isobilateral structure. The palisade layers are interrupted in the midrib region by collenchyma, abutting on both epidermises. The upper epidermis only carries few non-glandular unicellular hairs. The mesophyll contains scattered prisms and cluster crystals of calcium oxalate.
The upper epidermis:
In surface view, it is formed of polygonal, square to subrectangular, rarely isodiammetric axially elongated cells, having wavy anticlinal walls and covered with smooth cuticle. They carry non-glandular unicellular conical hairs. Stomata are almost absent.
The upper neural epidermal cells:
In surface view it consists of polygonal, square to subrectangular axially elongated cells with straight anticlinal walls and covered with smooth cuticle.
Lower Epidermis:
It is formed of polygonal, square to subrectangular axially elongated cells with sinuous anticlinal walls, covered with smooth cuticle. The stomata are abundant of paracytic type .Hairs are almost absent.
Lower Neural Epidermal Cells:
It is similar to that of the upper neural epidermal cells.
The Mesophyll:
The mesophyll is isobilateral. The upper palisade is formed of two rows of columnar cells, but the lower palisade is of one row. The spongy tissue consists of parenchymatous cells contain chloroplast, prisms and cluster crystals of calcium oxalate.
The Cortical Tissue:
The main vascular bundle is surrounded by an upper & lower arc of collenchyma cells. The remaining cortical tissue is of parenchymatous cells, some of them containing cluster and prismatic crystals of calcium oxalate.
The Vascular System:
It is consists of radiating xylem and phloem surrounded by lignified pericyclic fibers. The xylem is formed of lignified spiral & reticulate thickened vessels, wood fibers and wood parenchyma. The phloem is formed of sieve tubes, companion cells and phloem parenchyma containing small cluster crystals of calcium oxalate.
The Petiole:
A transverse section in the petiole is nearly triangular in shape with rounded angles. It has an outer epidermis carry few hairs followed by a wide parenchymatous cortex enclosing a central vascular system.
The Epidermis:
The epidermis in surface view, consists of polygonal, nearly isodiammetric sometimes subrectangular cells with thin straight anticlinal walls, covered with smooth cuticle and carrying few conical non-glandular unicellular hairs. Stomata are almost absent.
The cortical tissue:
Formed of several rows of parenchymatous cells containing cluster and prismatic crystals of calcium oxalate.
The vascular system:
It is represented by one main crescent shaped vascular bundle consists of an upper arc of xylem followed by a lower arc of phloem and surrounded by pericyclic fibers except in front of upper parenchyma. The xylem consists of lignified spiral & reticulate vessels, wood parenchyma and tracheids. The phloem consists of sieve tubes, companion cells and phloem parenchyma containing small clusters of calcium oxalate. The medullary rays are uni-to biserriate polygonal to subrectangular cells.
C) Micromorphology of the Stem bark:
A transverse section in the bark is formed of three distinct regions; the cork, the cortex and the phloem.
The Cork:
It is formed of several rows of radially elongated and tangentially arranged cells with thin non-lignified walls containing yellowish-brown pigments.
The Cortex:
It is formed of few rows of collenchymatous cells followed by parenchyma cells containing few prisms of calcium oxalate. Few sclerenchymatous cells are scattered in the cortical region.
The Pericycle:
Consists of few groups of lignified pericyclic fibers associated with sclerenchymatous cells which are polygonal, subrectangular to fusiform with thick lignified pitted walls. The parenchyma cells surrounding the fibers contain prisms of calcium oxalate forming a crystal sheath.
The Phloem:
It is comparatively wide zone, formed of few phloem fibers accompanied by stone cells. The phloem parenchyma consists of thick walled non-lignified cells containing prisms of calcium oxalate.
Part II : Phytochemical Study of Flacourtia cataphracta Roxb.
Chapter I
Preliminary Phytochemical Screening of The Leaves and Stem bark of Flacourtia cataphracta Roxb.
1- Powdered leaves and stem bark contain carbohydrates, unsaturated sterols and/or triterpenes, tannins, flavonoids (aglycone and/or glycosides) and crystalline sublimates.
2- Powdered leaves and stem bark contain traces of saponins, lactones and/or esters, cyanogenic glycosides and coumarins.
3- Powdered leaves and stem bark do not contain any volatile substances, cardenolides, oxidase enzyme, alkaloids and / or basic nitrogenous substances, anthraquinones and iridoids.
Chapter II
Extraction, Fractionation and Isolation of
the Constituents of the Leaves of Flacourtia cataphracta Roxb.
1. Extraction and Fractionation:
About (3 kg) of the air-dried powdered leaves were extracted with ethanol 70% by maceration in a glass percolator for 24 hours at room temperature. The ethanolic extract was concentrated to give 200 g residue and successively partitionated between n-hexane, chloroform and ethyl acetate to give the corresponding soluble fractions, n-hexane (60 g), chloroform (40 g) and ethyl acetate (35).
2. Isolation of the Main Constituents of the n-Hexane Fraction of the Leaves of Flacourtia cataphracta Roxb.:
2.1. Thin Layer Chromatographic Screening:
The n-Hexane fraction was subjected to TLC screening which revealed the presence of 7 major spots and other minor spots.
2.2. Column Chromatographic Fractionation of the n-Hexane Fraction:
About 30 g of the n-hexane fraction was chromatogrphed over silica gel column and eluted with n-hexane and ethyl acetate in a manner of increasing polarity to isolate three compounds (F.C.1-F.C.3).
3. Isolation of the Main Constituents of the Chloroform Fraction of the Leaves of Flacourtia cataphracta Roxb.:
3.1. Thin Layer Chromatographic Screening:
The chloroform fraction of the leaves was subjected to TLC screening which revealed the presence of 5 major spots and other minor spots
3.2. Column Chromatographic Fractionation of the Chloroform Fraction:
About 25 g of the chloroform fraction was chromatogrphed over silica gel column and eluted with gradient system of chloroform: methanol to isolate five pure compounds (F.C.4-F.C.8).
4. Isolation of the Main Constituents of the Ethyl acetate Fraction of the Leaves of Flacourtia cataphracta Roxb.:
4.1. Thin Layer Chromatographic Screening:
The ethyl acetate fraction was subjected to TLC screening which revealed the presence of two major spots and other minor spots
4.2. Column Chromatographic Fractionation of the Ethyl acetate Fraction:
About 20 g of the ethyl actate fraction was chromatogrphed over silica gel column and eluted with gradient system of chloroform: methanol to isolate two pure compounds (F.C.9, F.C.10).
Chapter III
Extraction, Fractionation and Isolation of The Constituents of the Stem bark of Flacourtia cataphracta Roxb.
1. Extraction and Fractionation:
About (1/2 kg) the air-dried powdered stem bark was extracted with ethanol 70% by maceration in a glass percolator for 24 hours at room temperature. The ethanolic extract was concentrated to give 30 g residue and successively partitionated between n-hexane, chloroform and ethyl acetate to give the corresponding soluble fractions, n-hexane (8 g), chloroform (5 g) and ethyl acetate (12).
2. Thin Layer Chromatographic Screening of the Main Constituents of the n-Hexane Fraction of the Stem bark of Flacourtia cataphracta Roxb.:
The n-hexane fraction of the stem bark was subjected to TLC screening using precoated silica gel plates and systems I&II as developers. Location of the spots was done by spraying with 10% v/v sulphuric acid followed by heating at 110°C for 10 minutes. The chromatogram revealed the presence of 7 major spots and other minor spots.
According to Rf values and colour reaction with sulphuric acid reagent, the major compounds of the n-hexane fraction of the stem bark were similar to the compounds have been isolated from the n-hexane fraction of the leaves. So the n-hexane fraction of the stem bark contain compounds F.C.1-F.C.3.
3. Thin Layer Chromatographic Screening of the Main Constituents of the Chloroform Fraction of the Stem bark of Flacourtia cataphracta Roxb.:
The chloroform fraction of the stem bark was subjected to TLC screening using precoated silica gel plates as adsorbent and systems I&II as developers. Location of the spots was done by spraying with 10% v/v sulphuric acid followed by heating at 110°C for 10 minutes. The chromatogram of the chloroform fraction showed 4 major spots and other minor spots. According to Rf values and colour reaction with sulphuric acid reagents, the major compounds of the chloroform fraction of the stem bark were similar to the compounds have been isolated from the chloroform fraction of the leaves. So the chloroform fraction of the stem bark contains compounds F.C.4, F.C. 6 - F.C.8.
4. Isolation of the Main Constituents of the Ethyl acetate Fraction of the Stem bark of Flacourtia cataphracta Roxb.:
4.1. Thin Layer Chromatographic Screening:
The ethyl acetate fraction of the stem bark was subjected to TLC screening which showed 4 major spots and other minor spots.
4.2. Column Chromatographic Fractionation of the Ethyl acetate Fraction:
About 12 g of the ethyl actate fraction was chromatographed over silica gel column and eluted with gradient system of chloroform and methanol to isolate four pure compounds (F.C.11-F.C.14).
Chapter IV
Identification of the Isolated Compounds of Flacourtia cataphracta Leaves
Investigation of the Lipids of the Leaves
Investigation of the Fatty Acids Composition:
The methyl ester of fatty acids were prepared from the saponifiable fraction of the n-hexane extract then analysed together with authentic samples by gas-liquid chromatography. It was found that the major fatty acids identified in the leaves are butyric, oleic, behenic, stearic, linolenic, palmitoleic and palmitic acids.
Investigation of Unsaponifiable Matter Composition:
The unsaponifiable matter was analysed by gas-liquid chromatography with authentic samples. It was found that the major hydrocarbons identified in the unsaponifiable matter are n-octacosane, n-hexacosane, n-tetracosane and n-docosane in addition to cholesterol, β-sitosterol and stigmasterol.
Structure Elucidation of the Isolated Compounds:
The structure elucidation of the isolated compounds was deduced on the basis of spectroscopic methods: (IR, UV, 1H-NMR, 13C-NMR and MS), in addition to comparison of the physical, chemical and chromatographic characters of these compounds with those of the
PART III
Biological Study of Flacourtia cataphracta Roxb.
Chapter I
Antimicrobial Activity of the Different Extracts of the Leaves
The different extracts of the leaves showed significant antibacterial activity against both gram +ve and gram -ve bacteria but it did not show any antifungal activity against candida albicans.
Chapter II
Toxicological Study of the Different Extracts of the Leaves
The n-hexane, chloroform and ethyl acetate extracts of the leaves are safe to be used as anti-diarrheal, anti-inflammatory and anti-pyretic.
Chapter III
Pharmacological Activities of the Different Extracts of the Leaves
A) Anti-diarrheal Activity:
The methanolic extract of the leaves showed a significant and dose dependant anti-diarrheal activity.
B) Anti-inflammatory Activity:
The different extracts of the leaves exhibited significant anti-inflammatory activity on yeast induced oedema in a dose dependant way ranged from intermediate potency with ( n-hexane, chloroform) to high potency with ethyl acetate.
C) Anti-pyretic Activity:
The chloroform fraction showed a well marked anti-pyretic activity while ethyl acetate showed a mild anti-pyretic activity. This effect was observed at doses 200 and 400 mg/kg for yeast induced fever.