A Pharmacognostical Study of Cuphea ignea A. DC. Family: Lythraceae, Cultivated in Egypt A Thesis Presented By Walaa Maged Ismail El-Kot Assistant Lecturer Pharmacognosy Department Faculty of Pharmacy Cairo University For the Degree of Doctor of Philosophy in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of Prof. Dr. Ahlam M. El-Fishawy Professor of Pharmacognosy Faculty of Pharmacy Cairo University Prof. Dr. Kadriya S. El Deeb Professor of Pharmacognosy Faculty of Pharmacy Cairo University Prof. Dr. Shahira M. Ezzat Professor of Pharmacognosy Faculty of Pharmacy Cairo University Pharmacognosy Department Faculty of Pharmacy Cairo University Egypt 2020 1 Abstract Cuphea is the largest genus of family Lythraceae with about 260 species of herbaceous perennials and small shrubs. It is distributed from North America in Western and Southern Mexico to South America in Eastern Brazil. Some Cuphea members are widely used in the traditional herbal medicine in treatment of arterial hypertension, atherosclerosis, congestive heart failure, and cardiac arrhythmias. Cuphea ignea A. DC. is an ornamental shrub native to Mexico and West Indies. The plant is widely grown in the Egyptian gardens and streets for ornamental purposes. Macro- and micromorphological studies were done for the different plant parts. Phytochemical study of the leaves, stems and flowers was performed including preliminary phytochemical screening, investigation of the volatile constituents using headspace solid-phase microextraction analysis, quantitative estimation of the total phenolic and flavonoid contents and UHPLC-Orbitrap HRMS analysis of the secondary metabolites. A biologically-guided study of the in vitro antihypertensive activity was done on the leaves, stems and flowers together with the fractions of the leaves and the stems and revealed the potent inhibition activities of the ethyl acetate fraction of the leaves and the methylene chloride fraction of the stems against angiotensin-converting enzyme (ACE) and renin enzyme. Chemical investigation of the two active fractions led to the isolation of six compounds which are quercetin, gallic acid, myricetin-3-O-α-rhamnopyranoside, quercetin-3-O-α- rhamnopyranoside, 3,4,3′-tri-O-methyl ellagic acid and β-sitosterol-3-O-β-D-glucoside. ACE and renin inhibition activities of the isolated compounds showed their high potency which was correlated with the activity of their fractions. In order to confirm results from the in vitro biological screening, the previously prepared 95 % ethanolic extract of leaves was chosen for the in vivo biological assessment of its antihypertensive activity due to its high in vitro ACE and renin inhibition activities, antioxidant activity and high phenolic and flavonoid contents. The results showed its potential to decrease the blood pressure and to restore the changes in ACE and nitric oxide levels in addition to the oxidative stress parameters in L-NAME-induced hypertensive rats. Cuphea ignea A. DC. can be used as a complementary treatment in hypertension and other cardiovascular complications. Keywords: Cuphea ignea A. DC., UHPLC-Orbitrap HRMS, antihypertensive, angiotensin- converting enzyme. 2 General summary “A Pharmacognostical Study of Cuphea ignea A. DC. Family: Lythraceae, Cultivated in Egypt” Lythraceae is a family of flowering plants, including about 620 species of 31 genera of mostly herbs. They are distributed worldwide in the tropic and subtropic regions. Cuphea is the largest genus of family Lythraceae with about 260 species of herbaceous perennials and small shrubs. It is distributed from North America in Western and Southern Mexico to South America in Eastern Brazil. Some Cuphea members are widely used in the traditional herbal medicine in treatment of arterial hypertension, atherosclerosis, congestive heart failure and cardiac arrhythmias. Cuphea ignea A. DC. is an ornamental plant native to Mexico and West Indies. It is composed of shrubs of about 30 to 90 cm height which can be used as a bedding plant in landscaping and gardens. This study aimed to evaluate the biological activities of Cuphea ignea A. DC. and their relation with the phytochemical composition and the biologically active compounds in the plant. The present study included: Part 1: Botanical profiling I- Macromorphological study of the different plant organs. II-Micromorphological study of the different plant organs. Part 2: Phytochemical study I- Preliminary phytochemical screening. II- Investigation of the volatile constituents of the flowers. III- Quantitative estimation of the total phenolic and flavonoid contents. 3 IV- UHPLC-Orbitrap-HRMS analysis of the secondary metabolites. Part 3: Biologically-guided study I- In vitro evaluation of the antioxidant and antihypertensive activities. II- Isolation of the major phytoconstituents from the main active fractions. III- In vitro antihypertensive activity of the isolated compounds. Part 4: In vivo biological study I- Determination of the safety of the ethanolic extract of the leaves. II- Assessment of the in vivo antihypertensive activity. Part 1: Botanical profiling I- Macromorphological study: Cuphea ignea A. DC. is an evergreen perennial small shrub, widely grown in Egyptian gardens and streets for ornamental purposes. The flowering period starts in January and continues to June. The plant cultivated in Egypt doesn’t produce fruits or seeds. 1- The stem: The old stems are pale brown in colour, hard, cylindrical with a rough exfoliating surface. Young stems are green to purplish green in colour, erect, herbaceous, cylindrical, showing longitudinal fine striations and are hairy to naked eye. 2- The leaf: The leaves are opposite, exstipulate and petiolate. The leaf lamina is simple, green, ovate to ovate lanceolate in shape with an acute apex. The petiole is very short, cylindrical, pale green sometimes with purplish tinge. The leaf is odourless and possesses a mucilagenous taste. 3- The flower: Flowers are bright orange red in colour and hermaphrodite with faint odour. Flowers are solitary and pedicellate. The calyx is persistent, elongated, tubular in shape and formed of six 4 orange red coloured sepals forming a calyx tube. Petals are very small, inserted at the rim of the floral tube. The androecium shows 11 stamens. Gynoecium shows 2 united carpels. 4- The root: The root is dark brown in colour. The surface is rough, showing longitudinal fissures. The fracture of the root is fibrous on the inner part and smooth on the outer part. II- Micromorphological study: 1- The stem: a- The old stem: A transverse section in the old stem is circular in outline. It is formed of a narrow cork which is composed of brown radially arranged, tangentially elongated cells, having suberized walls. The cortex is formed of 2-6 rows of thin walled oval parenchyma cells containing starch granules and some cells contain tannins. Few parenchyma cells showed scattered calcium oxalate clusters. The pericycle is parenchymatous followed by a ring of comparatively wide vascular tissue. The vascular tissue is radially traversed by numerous uni- or rarely biseriate medullary rays. The phloem is present above and below the xylem. It consists of thin walled elements with phloem parenchyma cells that contain starch granules. The cambium is formed of 2 to 6 rows of tangentially elongated thin walled cellulosic cells. The vessels show lignified spiral, annular or pitted thickenings. Wood fibres have wide lumina and straight thin lignified walls. Wood parenchyma consists of rectangular elongated cells with pitted lignified walls. The pith consists of large parenchyma cells containing calcium oxalate clusters. Few cells contain tannins and starch granules. b- The young stem: The structure of the young stem is almost similar to that of the old stem with the following differences: - The epidermal cells are polygonal with straight anticlinal walls and covered with a thin smooth cuticle. Stomata are rare and mostly anomocytic. - The presence of non-glandular unicellular hairs. - The vascular tissue is narrower. - The pith is wider. 5 2- The leaf: a- The lamina: The epidermises of both surfaces are nearly similar in shape, polygonal, elongated cells with straight to slightly wavy anticlinal walls. Stomata are anomocytic with few diacytic and anisocytic. Hairs are non-glandular unicellular or multicellular, uniseriate hairs. The epidermal cells contain mucilage. The mesophyll is composed of the palisade which is formed of one row of columnar, closely packed cells. The spongy tissue contains calcium oxalate clusters and simple starch granules. The cortical tissue of the midrib consists of rows of collenchymatous cells abutting the upper and lower epidermises, followed by parenchyma cells. The parenchymatous cells contain calcium oxalate clusters and few cells contain tannins. The pericycle is composed of parenchymatous cells. The vascular tissue consists of a bicollateral vascular bundle. The xylem vessels show spiral and annular thickenings. The cambium is followed by the phloem tissue consists of soft sieve elements and phloem parenchyma. b- The petiole: A transverse section in the petiole is nearly circular in shape. It is formed of an outer epidermis with non-glandular hairs, followed by the cortex, which shows a layer of collenchyma then a parenchymatous tissue in which clusters of calcium oxalate are scattered. The cortex is followed by a crescent shaped bicollateral vascular bundle. 3-The root: A transverse section in the root is formed of an outer cork of brown radially arranged,