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Assiut university researches Pharmacognostical Study Of Juglans Nigra L Black Walnut Family Cultivated In Egypt درا سة ع قاق يري ة ل ن بات ال جوز األ سود ال جوج الن س ن يجرا ال تاب ع ل ل عائ لة ال جوزي ة ال جوج الن دي سى وال م نزرع ف ى م صر Iman Sayed Ahmed Khallaf إي مان س يد أحمد خ الف dawood w. bishy , ahmed a. attia , samia a. youssef سام ية ع باس ي و سف، أحمد عاب دي ن محمد،داود ون يس ب شاي Abstract: A pharmacognostical study of Juglans nigra L. cultivated in Egypt. Juglans nigra L. (Black walnut) family Juglandaceae (walnut family) is a deciduous tree, growing abundantly through north temperate zone, South Europe and mountain’s regions of North Africa. In folk medicine the plant is used as hypotensive, hypoglycemic, for treatment of syphilis and eczema. The stem and root bark are use as a stringent and anthelmentic, while the dried nut used as kidney and stomach stimulant. Literature survey showed that nothing could be traced concerning leaves of Juglans nigra L. However, naphthoquinones and ellagic acid are reported to be isolated from the fruit of the plant. The present work includes : Part I : Botanical study : Macro and micromorphology of the leaves, stem, root, stem bark and inflorenscence are studied in both the entire and powdered form. Part II : Phytochemical study : That includes: 1.Preliminary phytochemical screening of the leaves and stem bark. 2.Isolation and identification of the flavonoids, lipids, tannins and volatile contents to elucidate their chemical conctitution. Part III : Biological screening : That includes : 1.Preliminary antibacterial study of different extracts of the leaves . 2.Pharmacological study that include the hypotensive activity of the leaves. 3.Toxicological effects of the total extract of the leaves. Part I : Botanical study of Juglans nigra L. Habitat : Juglans nigra L. is an erect, monoecious, decidious tree. The male inflorescence occurs as drooping catkins, while female flowers are erect. The fruit is a large drupe like nut. The plant prefers temperate temperature, alluvial soil with moderate moisture content. I. Macromorphological study of Juglans nigra L.: a.The leaves : The leaves are alternate, compound, imparipinnate and exstipulate. Each leaflet is formed of about 7-15 leaflets. The leaflet is ovate to lanceolate in the outline, with entire sinuate margin, acute to acuminate apex and symmetric base. Both surfaces are hairy especially the lower one. The petide and rachis are cylinderical is shape, green in colour with hairy surface. b.The stem : The main trunk is solid, erect, cylinderical in shape and monopodially branched. The terminal and lateral branches are narrower with short internodes, covered with brown cork except the young portion just below the apical buds. c.The root system : It was obtained from nurseling plant that attains about 50-60cm in height and cultivated in a flower pot. It consists of a long fusiform tap root, bearing few lateral roots and numerous rootlets. d.The stem bark : It occurs in flat or curved pieces. The outer surface is greyish black to brownish grey bearning numerous lenticeles, while the inner surface is yellowish green in colour and longitudinally striated. e.The flower : The flowers are unisexual, monoecious, pedicellate, apetalous and greenish yellow in colour. Both male and female flowers are subtended by numerous bracts and possessing the following floral formula : P4 A3- (male) (female) f.Fruit : Large, fleshy, indhescent, glabrous drupe like nut. It contain one exalbuminous hard seed. II. Micromorphological study of Juglans nigra L. 1. The lamina : •The upper epidermis : It consists of polygonal cells with straight beaded anticlinal walls. Stomata of anomocytic type are rare. Non glandular trichomes are present. •The lower epidermis : It consists of polygonal cells with wavy beaded anticlinal walls, covered with striated cuticle. Numerous anomocytic stomata and nonglandular hair exactly similar to the upper epidermis are abundant. •The mesophyll : heterogenous, formed of one row of columnar palisade cells, some are large containing cluster crystals of calcium oxalate. The spongy parenchyma contain numerous resinous canals, in addition to schizogenous glands at its upper part where the pericyclic fibres form a crown over them. The pericycle consists of fibres and parenchyma. 2. The petiole : •The epidermis : It consists of polygonal cells with straight beaded anticlinal walls covered with smooth cuticle and carrying non-glandular trichomes. •The cortical tissue : It consists of outer collenchymatous cells followed by wide zone of parenchyma cells containing clusters of calcium oxalate. The endodermis is indistinct. •The pericycle : it consists of isolated groups of lignified fibers that interrupted by parenchyma cells. •The phloem : consists of sieve tubes, companion cells, phloem parenchyma and phloem fibers. The phloem shows a ring of schizogenous glands at its outer part where pericyclic fibres form an arc over them. Also it contains numerous resinous canals. •The cambium is a wide zone of cambiform cells. •The xylem it is formed of lignified vessels, fibres, wood parenchyma, tracheids, tracheidial vessels and traversed by uniserriate medullary rays. •The pith : consists of comparatively wide zone of parenchymatous cells containing cluster crystals of calcium oxalate . 3. The rachis : Exactly similar to the petiole but smaller in size. 4. The stem : •Cork : It is formed of several layers of yellowish brown suberized tabular cells. The phellogen originates early and subepidermally. It is collapsed and indistinct. •The cortex : formed of several layers of thin walled parenchyma cells, containing cluster crystals of calcium oxalate and minute starch granules. The endodermis is indistinct. •The pericycle : It is formed of continuos zone of lignified fibres interrupted by parenchyma. The parenchyma surrounding the fibres contain prisms of calcium oxalate forming crystal sheath. •The phloem : consists of soft element in addition to phloem fibres that form crystal sheath. At the periphery of the phloem large schizogenous glands are present where the phloem fibres form a crown over them . •The cambium : wide zone of cambiform cells . •The xylem : consists of lignified vessels, fibres, parenchyma, tracheids, trachedial vessels and traversed by lignified uni-or sometimes biserriate medullary rays. •The pith : wide zone of parenchymatous cells containing prisms and large clusters of calcium oxate, in addition to numerous schizogenous glands. 5. The root : •The cork : it originates early in the pericyclic region. It consists of several layers of tabular cells containing reddish brown pigment. •The phelloderm : consists of wide zone of thin walled parenchymatous cells containing numerous starch granules, prisms of calcium oxalate, few schizogenous glands and isolated group of lignified fibres. •The phloem : consists of soft elements in addition to phloem fibres and traversed by numerous uniserriate or sometimes biserriate medullary rays. Phloem parenchyma and meelullary rays contain numerous strach granules and prisms of calcium oxalate. •The cambium : it is formed of several rows of thin walled tangentially elongated cells. •The xylem, it is formed of lignified xylem vessels, wood parenchyma, fibres, tracheids, trachedial vessels and traversed by uni- or biserriate medullary rays. 6. The stem bark : •The cork : it is formed of several layers of tangentially elongated cells. In surface view, the cells are polygonal containing dark brown colouring matter. •The cortex : consists of several layers of parenchyma cells containing numerous stach granules. Groups of the sclereids are scattered in the cortex. •The pericycle : it is formed of continous ring of lignified fibres interrupted by parenchyma cells. The fibres form crystal sheath. •The phloem : consists of sieve tube, companion cells, phloem parenchyma and phloem fibres. The fibres form crystal sheath with the surrounding parenchyma that contain prisms of calcium oxalate. The phloem parenchyma contains starch granules. Tannin cells are present. The phloem is traversed by uni or biserriate non-lignified medullary rays. 7. The inflorescence : A. The male flower : •The bract : the inner epidermis is polygonal with straight beaded anticlinal walls, covered with smooth cuticle. The outer epidermis is similar to those of the inner showing anomocytic stomata. •The perianth : the inner epidermis, the cells are polygonal, slightly elongated with straight beaded antclinal walls and covered with smooth cuticle. The outer epidermis similar to the inner and showing anomocytic stomata. •Androecium : the filament consists of polygonal cells, with straight anticlinal walls and covered with faintly striated cuticle. The anther : •Epidermis of anther : it is formed of polygonal cells with straight beaded anticlinal walls. The cells are papillosed and covered with smooth cuticle. •The fibrous layer : it consist of a single layer of beaded bar-like thickened walls. •The pollen grains : spherical in sphape, yellow in color with three germ-pores . B. The female flower : •The bract : The outer epidermis : polygonal cells with straight beaded anticlinal walls. Hairs are not observed. The inner epidermis : similar to those of the outer. •The perianth : The outer epidermes : polygonal with straight anticlinal walls, covered with smooth cuticle. Anomocytic stomata are present. Numerous non glandular unicellular or multicellular hairs are present, the inner epidermis are similar to those of the outer ones. The gynaecium : •The ovary : formed of polygonal cells with straight or slightly wavy anticlinal walls. Stomata are not observed. The wall is hairy carrying numerous glandular trichomes. •The style : consists of polygonal cells with straight anticlinal walls. Stomata are not observed. •The stigma : consists of polygonal cells with straight anticlinal walls and slightly papillosed at its tip. C. The peduncle : •The epidermis : polygonal cells with more or less straight beaded anticlinal walls, showing anomocytic stomata and carrying nonglandular hairs. •The cortex : consists of several layers of parenchyma cells containing cluster crystals of calcium oxalate . •The pericycle : separated groups of lignified fibres interupted by parenchyma. The fibres form an arc ove the schizogenous glands that present at the peripheral part of the phloem. •The phloem : it consists of sieve tubes, companion cells and phloem parenchyma. •The xylem : formed of lignified xylem vessels, wood parenchyma, wood fibres, tracheids and trachedial vessels. The medullara rays being uni-or biserriate with pitted lignified walls. However, being not lignified in the phloem region. •The pith : consists of wide parenchymatous zone containing cluster crystals of calcium oxalate . Part II : Phytochemical study of Juglans nigra L. cultivated in Egypt : Chapter I : Preliminary phytochemical screening of the leaves and stem bark of Juglans nigra L. The preliminary phytochemical screening of the air dried leaves and stem bark revealed : 1.The leaves showed the presence of carbohydrates and/or glycosides, unsaturated sterols and /or triterpenes, flavonoids (aglycone and glycosides), low concentration of coumarin and volatile oil and absence of alkaloids, cardenolides, iridoids, lactones. 2.The stem bark showed the presence of carbohydrates and/or glycosides, tannins, unsaturated sterols and/or triterpenes and absence of volatile oil, alkaloids, anthraquinones, iridoids, cardenolides and cyanogenitic glycosides. Chapter II : Extraction and isolation of the active cons-tituents of Juglans nirgra L. leaves : •The air-dried powdered leaves are extracted with ethanol. The ethandic extract was evaporated under reduced pressure till dryness. The residue was suspended in water and extracted with n-hexane, chloroform. The aqueous layer was applied to a column of Diaion Hp-20 and eluted with water, methanol and acetone respectively. •The n-hexane fraction was saponified with alc. KOH to obtain the unsaponifiable fraction, that further applied to silica gel column to isolate two compounds J1 and J3. •The saponifiable fraction was methylated and subjected to GLC analysis for detection of fatty acids. •The chloroformic fraction was subjected to silica gel column chromatography to obtain compound J4. •The methanolic fraction was subjected to silica gel column chromatography where three compounds J5 , J6 and J7 were isolated . •The air dried stem bark was extracted with ethanol and fractionated to obtain n-hexane, chloroformic and ethylacetate fractions. •The n-hexane fraction was subjected to silica gel column chromatography to obtain three major compounds J1, J2, J3 , while the fractionation of ethylacetate fraction on silica gel column two compounds J8 and J9. Chapter III : Identification of the isolated compounds : Investigation of fatty acids composition. The methylesters of fatty acids were analysed by GLC together with authentic samples. It was found that the major fatty acids are capric (23.8), oleic (19.3) and eicosenic (20.8) . Structure elucidation of the isolated compounds : With the help of physical, chemical and spectroscopic techniques . Chapter IV : Study of the volatile contents of Juglans nigra L. The steam distillation of the leaves of Juglans nigra L. revealed the presence of low percentage of volatile oil. The GC/MS revealed the presence of fourty three components, from them thirty tree components were identified. Part III : Biological study of Juglans nigra L. cultivated in Egypt : Chapter I : Antibacterial activity of the different extracts : The total and aqueous extracts show significant antibacterial activity against gram +ve and gram –ve bacteria. Chapter II : Pharmacological activates of the different extracts : Hypotensive activity : the total and aqueous extract of the leaves exhibited a significant hypotensive activity. Chapter III : Toxicological study of the extract : The leaves extract are found to be safe LD50 are found to be 14g/kg.