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general summary and conclusion A Pharmacognostical Study of Calliandra haematocephala (Hassk.) Cultivated in Egypt Calliandra haematocephala (Hassk.) Family Mimosaceae (Fabaceae or Leguminosae) is an evergreen erect shrub, widely distributed in tropics. The genus Calliandra has many folk uses in malaria, fever, lumbago, gonorrhoea, diarrhea, infantile convulsion, kidney ache, cystitis, urethritis and gall stones. As for Calliandra haematocephala, it is known to be used as blood purifier. Literature survey showed that nothing could be traced concerning Calliandra haematocephala (Hassk.) cultivated in Egypt, this stimulates the present work which comprices the following parts:Part I:Botanical Study Macro- and micromorphology of leaf, stem and inflorescence of the plant under investigation in both entire and powdered forms.Part II:Phytochemical Study1-Preliminary phytochemical screening of the different constituents of the leaf, stem and inflorescence.2-Isolation and identification of lipids, sterols, phenolics and flavonoids to elucidate their chemical structures.Part III:Biological Study1)Determination of LD50 of different extracts of the leaves.2)Preliminary antibacterial study of different extracts of the leaves.3)Some preliminary pharmacological effects of different extacts of the leaves; which include the following:a)Anti-inflammatory effect.b)Antiepileptic effect.c)Effect on blood pressure.PART IBotanical Study of Calliandra haematocephala (Hassk.)Habitat:Calliandra haematocephala (Hassk.) is an erect, evergreen deciduous shrub, carried alternate compound leaves. The flowers bloom from October to March.1- The Leaf Macromorphology of the Leaf:The leaves are alternate bipinnately compound with one or two pairs of pinnae, each with 7-10 pairs of elliptic to oblong-lanceolate sessile leaflets. Each leaflet has obtuse to mucronate apex, obliquely rounded to subcordate base, entire margin and slightly hairy surface. It is green in colour and slightly astringent in taste.Micromorphology of the Leaf:The Lamina:The upper epidermis:Consists of polygonal cells, with sinuous anticlinal walls and covered with smooth cuticle. Numerous stomata of paracytic type and few covering trichomes are present.The lower epidermis:Is exactly similar to the upper epidermis but the cells possess more sinuous anticlinal wall and numerous paracytic stomata.The Mesophyll:Is isobilateral, showing palisade abutting on both the upper and lower epidermises enclosing a narrow spongy tissue. The upper palisade is continuous in the midrib region while the lower palisade is not continuous being interrupted by a collenchymatous mass.The Cortical Tissue:Is formed of few layers of parenchyma cells surrounding the vascular bundle some of them contain prisms of calcium oxalate specially those surrounding the pericyclic fibres forming crystal sheath; in addition, to a small mass of subepidermal collenchyma abutting on the lower epidermis.The Vascular System:Each vascular bundle is formed of upper xylem region and lower region of phloem, surrounded by more or less continuous band of pericyclic fibre. According to the leaflet midrib (upper or lower parts), an accessory inverted vascular bundle may appear embedded in the upper part of pericyclic fibre.The Leaf Rachis:The Epidermis:Consists of polygonal cells, with straight beaded anticlinal walls and covered with smooth cuticle. Stomata of paracytic type are observed. Numerous glandular and non-glandular trichomes are present.The Mesophyll:Is heterogenous, palisade-like layer is continuous beneath the upper epidermis. The spongy tissue forming a very narrow zone of collapsed chlorenchyma. Few prisms of calcium oxalate are observed.The Cortical Tissue:It surrounds the vascular system. It is formed of parenchymatous cells, contain prisms of calcium oxalate to form crystal sheath around the pericyclic fibres. The Vascular System:Consists of a large concentric structure with inner xylem and outer phloem, in addition to two small accessory inverted vascular bundles in the two prominent ridges.The main vascular bundle somewhat similar to that of the stem and it is also surrounded by a complete ring of lignified pericyclic fibres.The phloem consists of soft elements of sieve tube, companion cells and phloem parenchyma. Many tanniniferous cells are observed.The xylem formed of lignified vessels, fibres, wood parenchyma, tracheids and traversed by medullary rays.The Leaf Petiole:The Epidermis:Consists of polygonal axially elongated, with more or less straight anticlinal walls and covered with smooth cuticle. Numerous sunken stomata, of paracytic type are present.Both glandular and non-glandular hairs are observed and similar to those of the leaflets.The Cortical Tissue:Is formed of an outer collenchymatous zone and an inner parenchymatous one. The parenchymatous cells particularly around the pericyclic fibres contain prisms of calcium oxalate forming crystal sheathes.The Vascular System:Consists of two groups of vascular strands, the lower vascular strand which consists of 3-4 united collateral vascular bundles and the upper inverted vascular strand which consists of 2-3 united collateral vascular bundles and opposite to the lower one. Both showing an outer phloem and an inner xylem.In addition to 2-4 accessory collateral vascular bundles present under the two prominent ridges, all bundles are crowned with arcs of sinuated pericycle.2- The Stem Macromorphology of the Stem:The main trunk of the plant is hard, woody, erect, cylindrical, monopodially branched. It has a faint odour and astringent taste.Micromorphology of the Stem:The Epidermis:It consists of polygonal, axially elongated cells with more or less straight and beaded anticlinal walls. The cells are covered with thick smooth cuticle. Numerous slightly sunken stomata of paracytic type are present. Few covering trichomes are observed.The Cortex:The cortex is formed of collenchymatous cells, followed by an inner parenchymatous region. The cells adjacent to the pericycle form a crystal sheath of calcium oxalate.The Pericycle:The pericycle consists of more or less complete ring of lignified fibres.The Phloem:It consists of thin walled, cellulosic soft elements of sieve tube, companion cells and phloem parenchyma, colourless tanniniferous cells are scattered.The Cambium:Forms a wide band of cambiform thin-walled cellulosic cells which are subrectangular, tangenitally elongated and radially arranged.The Xylem:The secondary xylem is the main constituent and consists of lignified, thick-walled elements, arranged in radial rows. Vessels show pitted, scalariform, reticulated and spiral thickening.The Medullary Rays:The secondary medullary rays are usually uni- to biserriate formed of elongated subrectangular cells.The Pith:It is formed of round parenchymatous cells mostly with slightly lignified and pitted walls, but no calcium oxalate crystals or starch are observed.3- The Inflorescence Macromorphology of the Inflorescence:The inflorescence is a capitulum (flower-head). Florets are sessile, bracteate, actinomorphic, bisexual, pentamerous and hypogynous. The flower has a faint odour and a slight astringent taste. The floral formula is:The Bract:Green in colour, very small in size, membraneous, hairy, triangular in shape with acute apex, entire margin and arranged in one row.The Calyx:Consists of five sepals, persistant, gamosepalous, tubular, valvate with 5 lobes or teeth, faint pinkish green in colour.The Corolla:Consists of five petals, gamopetalous, with 5 teeth, each has an acute apex, connate at the base into a short tube, white in colour with pink lobes.The Androecium:It is polyandrous of about 25 stamens per each floret, extent beyond the petals and provide a fluffy soft appearance to the floral cluster.The Gynaecium:The ovary is superior, monocarpellary, unilocular, greenish white in colour, slightly hairy, elongated and carrying at its upper end an elongated filiform style ending in stigma.Micromorphology of the Inflorescence:The Bract:It is formed of polygonal isodiametric cells with straight anticlinal walls. Both glandular and non-glandular hairs are present. Stomata of paracytic types are rarely observed.The Calyx:Upper epidermis:The cells are polygonal, slightly elongated, subrectangular, with somewhat straight anticlinal walls. They are covered with thin smooth cuticle. Stomata of paracytic types, non-glandular and glandular hairs are present.Lower epidermis:It is exactly identical with the upper epidermis.The Corolla:Upper epidermis:The cells are polygonal, slightly elongated, sometimes isodiametric with slightly curved (wavy) anticlinial walls and covered with smooth cuticle. Stomata of paracytic type are rarely observed. Both glandular and non-glandular hairs are present and similar to those shown in calyx.Lower epidermis:The cells are more or less identical in surface view in all aspects with those of the upper ones except that the stomata are more observed.Androecium:The filament:Axially elongated, polygonal, subrectangular cells having straight anticlinal walls. They covered with faintly striated cuticle. Trichomes and stomata are not observed.The anther:The epidermis of anther:Is of glandular-hairy polygonal cells, covered with faintly striated cuticle. Trichomes and stomata are not observed.The fibrous layer of anther:Polygonal cells always isodiametric with distinctly beaded walls.Pollinia:Pollinia of 8 united pollen grains are observed.The Gynaecium:The ovary:Epidermal cells of the ovary are polygonal isodiametric cells with straight anticlinal walls. Stomata are not observed, while numerous, nonglandular hairs are present.The style:Epidermal cells of the style are axially elongated subrectangular with straight anticlinal walls and covered with smooth cuticle. Stomata and hairs are not observed.The stigma:Polygonal elongated epidermal cells showing short papilla at the tip of the stigma, and covered with smooth cuticle.The Peduncle:The Epidermis:It is formed of polygonal, axially elongated cells with straight anticlinal walls and covered with smooth cuticle. Few paracytic stomata are present. The hairs are of non-glandular type.The Cortex:Consists of collenchymatous zone followed by parenchymatous one, some parenchyma cells contain prisms of calcium oxalate forming crystal sheathes around the pericyclic fibres.The Pericycle:It is formed of groups of lignified pericyclic fibres above the vascular bundles.The Vascular System:The vascular system consists of numerous collateral vascular bundles arranged in a circle, each being capped by a group of pericyclic fibres.The pith is formed of a wide zone of thin-walled parenchyma cells. The cells are free from contents.PART IIPhytochemical Study of Calliandra haematocephala (Hassk.) Cultivated in EgyptChapter IPreliminary Phytochemical Screening of the Leaf, Stem and Inflorescence of Calliandra haematocephala (Hassk.) The preliminary phytochemical screening of the air dried-powdered leaf, stem and inflorescence revealed:1-Powdered leaf contains sublimable substances, carbohydrates and/or glycosides, unsaturated sterols and/or triterpenes, tannins, flavonoids (aglycones, glycosides) and saponins. It contains traces of lactones and/or esters, alkaloids and/or basic nitrogenous substances and coumarins.2-Powdered stem contains carbohydrates and/or glycosides, unsaturated sterols and/or triterpenes, flavonoids (aglycones, glycosides) and saponins. It contains traces of sublimable substances, tannins, lactones and/or esters, alkaloids and/or basic nitrogenous substances and coumarins.3-Powdered inflorescence contains sublimable substances, carbohydrates and/or glycosides, unsaturated sterols and/or triterpenes, tannins, flavonoids (aglycones, glycosides) and saponins. It contains traces of lactones and/or esters, alkaloids and/or basic nitrogenous substances and coumarins. Chapter IIExtraction, Fractionation and Isolation of the Constituents of the Leaves of Calliandra haematocephala (Hassk.)1. Extraction and Fractionation:Preparation of Ethanolic Extract of the Leaves:The air-dried powdered leaves (4 kg) were extracted till exhaustion with ethanol.Fractionation of the Ethanolic Extract:The concentrated ethanolic extract partitioned with successive portions of n-hexane, chloroform, ethyl acetate and lastly n-butanol, separately concentrated under reduced pressure.2.Isolation of the Main Constituents of the n-Hexane Fraction (F1) of the Leaves of Calliandra haematocephala (Hassk.):n-Hexane fraction (F1) was saponified, about 5 g of the unsaponifiable fraction were chromatographed over silica gel column and eluted with gradient system of n-hexane and ethyl acetate. Compound Ca-1 and mixture of compounds Ca-2 and Ca-3 were isolated.3.Isolation of the Main Constituents of the Chloroformic Fraction (F2) of the Leaves of Calliandra haematocephala (Hassk.):About 16 g of the chloroformic fraction were chromatographed over silica gel column and eluted using chloroform followed by chloroform and methanol in an increasing polarities to isolate two pure compounds Ca-4 and Ca-5.4.Isolation of the Main Constituents of the Ethyl Acetate Fraction (F3) of the Leaves of Calliandra haematocephala (Hassk.):About 30 g of the ethyl acetate soluble fraction were chromatographed over silica gel column and eluted using n-hexane, ethyl acetate gradient, then ethyl acetate with increasing gradient of methanol to isolate eight pure compounds from Ca-6 to Ca-13.Chapter IIIIdentification of the Isolated Compounds of the Leaves of Calliandra haematocephala (Hassk.)Investigation of Fatty Acids Composition:The methyl ester of fatty acids fraction, obtained from the saponifiable fraction was analysed alongside with authentic samples by gas-liquid chromatography. Ten saturated and four unsaturated fatty acids were identified (54.736% and 26.31% respectively) in addition to the unidentified ones (18.954%).The major fatty acids are palmitic (17.445%), linolenic (14.653%) and lauric (13.772%) while caprylic (0.965%) and pelargonic acids (1.378%) are minors.Investigation of Unsaponifiable Matter Composition:The unsaponifiable matter was analysed alongside with authentic samples by gas-liquid chromatography. The major hydrocarbons identified were n-Eicosane (17.3%), n-docosane (11.7%) in addition to two major sterols, stigmasterol (10.7%) and -sitosterol (6.6%).PART IIIBiological Study of Calliandra haematocephala (Hassk.) Cultivated in EgyptChapter IDetermination of LD50 of Different Extracts of the Leaves The total ethanolic, non-polar and polar extracts have LD50 of 3, 1.5 and 2.4 g/kg, respectively, so the plant must be used cautiously at doses less than these doses.Chapter IIPreliminary Antibacterial Study of Different Extract of the Leaves The different extracts of the leaves showed significant antibacterial activity against both Gram +ve and Gram -ve bacteria under investigation.Chapter IIISome Preliminary Pharmacological Effects of Different Extracts of the LeavesA)Anti-inflammatory Effect:The different leaves extracts exhibited anti-inflammatory effect; against yeast induced inflammation in rats; ranging from the weakest one (total ethanolic extract), intermediate effect (non-polar extract) and the most potent (polar extract).B)Antiepileptic Effect:Total ethanolic, non-polar and polar extracts exhibit antiepileptic effect against pentylene tetrazole-induced convulsions in mice. It was found that polar extract is the most potent one.C) Effect on Blood Pressure:It was found that total ethanolic and non-polar extracts can reduce blood pressure of normotensive anesthesized rabbit, while polar extract cause elevation in the blood pressure.
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