Pharmacognostical Study of Some Species of Tradescantia Family Commelinaceae cultivated in Egypt A THESIS SUBMITTED BY Aya Mohamed Faisal Mohamed Teaching Assistant Faculty of Pharmacy – Ahram Canadian University For the Degree of Master in Pharmaceutical Sciences (PHARMACOGNOSY) Under the Supervision of Prof. Dr. Prof. Dr. Seham Salah El-Din El-Hawary Ibrahim Ibrahim Mahmoud Professor of Pharmacognosy Professor of Pharmacognosy Faculty of Pharmacy Dean of Faculty of Pharmacy Cairo University Ahram Canadian University Dr. Manal Mahmoud Sabry Lecturer of Pharmacognosy Faculty of Pharmacy Cairo University PHARMACOGNOSY DEPARTMENT FACULTY OF PHARMACY CAIRO UNIVERSITY 2018 Abstract A preliminary phytochemical screening of the aerial partsof T. pallida (Rose) D.R.Hunt, T. zebrinaHeynh ex.Bosse and T. spathaceaSwartz.(Commelinaceae) cultivating in Egypt was carried out. Lipoidal contents was investigated in the petroleum ether extractive of T. pallida (Rose) D.R.Hunt. The phytochemical investigation of the flavonoidal and phenolic compounds using HPLC of the three species indicated that T. zebrinaHeynh ex. Bosse possessed the highest percentage of flavonoid and phenolic content where Kamp3, (2-p-comaroyl) glucoside was the most abundant compound (2740 mg/100g). Analysis of the secondary metabolites of the three Tradescantia species using HPLC-PDA-MS/MS led to the identification of 27 phenolic compounds. The phytochemical investigation of T. pallida (Rose) D.R.Hunt led to the isolation of 1 steroidal compound. β-sitosterol compound was isolated from the petroleum ether extractive. In addition to the separation and identification of seven phenolic compounds from the n-butanol fraction by using LC-ESI-MS/MS (trans-cinnamic acid, caffeic-O-pentoside, p- coumaroyl-O-pentoside, quercetin, syringic-O-hexoside, naringenin and apigenin). Using the in-vivo and in-vitro biological studies, the total ethanolic extracts of the three species were safe, exhibiting potent anti-inflammatory, antihyperglycemic, hepatoprotective and antioxidant activities. The ethanolic extracts of the threeTradescantiaspecies showed no cytotoxic activity against the tested carcinoma cell lines (HEPG-2, MCF-7 and HCT-116). The botanical features of the stems and leaves of the three species have been studied in order to find out the diagnostic characters that help in identification of the plant either in the entire or the powdered form. Introduction Commelinaceae: dayflower or spiderwort family, is a monocotyledon family. Members of this family are ornamentals. The stems of these plants are generally well-developed, and often swollen at the nodes. Flowers are often short-lived, lasting for a day or less. The family includes about 40-50 genera and 731 species. Commelina(170 species), Tradescantia (75-80 species), Murdannia (55 species), are well-known genera of the family. The family is diverse in both the Old-World tropics and the New World tropics, with some genera present in both. In traditional medicine, plants of genus Tradescantiawere used for the treatment of mycosal infections, venereal diseases , urinary tract infections, hemorrhoids, tuberculosis and cough (Other traditional uses viz; anti-inflammatory, anti-toxic supplement and improve blood circulation, antidiarrheal, expectorant, hypoglycemic agent and against snakebiteswere also reported. Pharmacological activities of different extracts of various Tradescantiaspecies had been experimentally proved viz; antibacterial, anti-hyperuricemic, analgesic and anti-inflammatory effects. Moreover, the cytotoxic and hepatoprotective activitiesof T. spathaceaSw. were reported. Aim of Work Tracing the current literature, there was a little reported about the active constituents regarding genus Tradescantiaand their biological activity, thus the present study includes a pharmacognostical study of three species of genus Tradescantia: T. pallida (Rose) D.R.Hunt., T. zebrinaHeynh. ex Bosse and T. spathaceaSw., their main active constituents and to find the way to differentiate between them. This work comprises four parts: Part I: A detailed genetic profiling of the three Tradescantia species, as well as, macro and micromorphological studies of the aerial parts of the three species. Part II: Includes Phytochemical investigation, involving Preliminary Phytochemical Screening, Qualitative and Quantitative determination of Flavonoids and Phenolic compounds using spectrophotometric and HPLC analyses. Part III: Extraction, Fractionation, as well as, Isolation and Identification of the main constituents of Tradescantia pallida (Rose) D.R. Hunt Part IV: Includes Biological Study, including Toxicological study (LD50 determination), in vitro studies (Cytotoxic and Antioxidant screening) and in vivo studies (Hepatoprotective, Anti-inflammatory and Anti-hyperglycemic activities) of the alcohol extracts of the three species. Review of Literature Tradescantia species (syn. Spiderwort, Family: Commelinaceae) are succulent perennial plants. There are about eighty species of Tradescantia, native to the New World from southern Canada to northern Argentina including the West Indies. The genus possesses numerous medicinal properties such as antimicrobial, hepatoprotective, anti-oxidant, antiviral, analgesic, and anti-inflammatory activities. In herbal medicine, they are used to treat sore throats, skin problems, stomach problems, expectorant, and anti-diarrheal. Bioactive constituents as phenolics, flavonoids, glycosides, sterols were reported about the genus. General Summary Genus Tradescantia (syn. Spiderwort, Fam: Commelinaceae) comprises succulent perennial plants. There are over seventy-five species of Tradescantia, native to the New World, but have naturalized throughout the tropics. In traditional medicine, plants belonging to genus Tradescantiawere used for the treatment of mucosal infections, venereal diseases, urinary tract infections, hemorrhoids, tuberculosis and cough. Other traditional uses viz; anti-inflammatory, anti-toxic supplement and improve blood circulation, antidiarrheal, expectorant, hypoglycemic agent and against snakebites were also reported. The present study has been planned with the aim to evaluate the potentialities of three Egyptian Tradescantia species: T. pallida (Rose) D.R. Hunt, T. zebrinaHenyh. ex Bosse and T. spathacea Sw. to illucidate their main active constituents and to find the way to differentiate between them. This study comprises three parts. Part I: Genetic and Botanical Study Chapter I DNA Fingerprinting The extracted DNA from each of the three Tradescantiaspecies was amplified using ten decamer primers to detect their genetic variability. The ten primers of arbitrary sequences generated a total of 55 fragments in T. pallida (Rose) D.R. Hunt and 55 in T. spathacea Sw., while 64 fragments were generated in T. zebrinaHeynh. ex Bosse The highest degree of similarities (92%) was recorded using primer A1 while the lowest degree of similarity (71.5%) was recorded using primer A2. The genetic characterization of the three Tradescantiaspecies showed that primer A1 could be used as an indicator for obtaining genetic markers for genus Tradescantia. In addition, primers A7 and A10 were found to be the most effective in generating unique bands on application of the RAPD technique to both plants, and therefore can act as species-specific markers in addition to the morphological and anatomical characteristics. Chapter II Botanical Study A.Macromorphology I. Macromorphology of Tradescantia pallida (Rose) D.R. Hunt. Tradescantia pallida (Rose) D.R. Hunt. is an ornamental, succulent, perennial herb, native to Mexico. It reproduces by sprouting (when a leaf is removed from the plant and placed in a warm moist place, young plants will very soon appear in the notches around the margin of the leaf) or by cutting. It carries lanceolate, fleshy leaves and pink, small, petaled flowers in the leaf axills. The Stem: The stem is thick, succulent and cylindrical. The stem is smooth, glabrous, violet purple in color. The stem has no odor but has an astringent taste. The Leaf: The leaf is simple, alternate, sessile, fleshy, green in color and turned purple when exposed to sunlight. The lamina is lanceolate, glabrous, with an acute apex and asymmetric base. It has an entire margin with parallel venation. It measures from 5-12 cm in length, 2 cm in width. The leaf has no odor but has an astringent taste. II. Macromorphology of TradescantiazebrinaHeynh. ex Bosse TradescantiazebrinaHeynh. ex Bosse is an ornamental, succulent, perennial herb, native to southern Mexico. It reproduces by sprouting or by cutting. The plant cultivated in Egypt, reaches up to 7 cm in height. The Stem: short, easily produce roots at their nodes, succulent. Branching stems are glabrous and usually tinged with purplish red. It has no odor but has an astringent taste. The Leaf: simple, opposite, sessile, exstipulate. The upper surface of the leaf is green and/or purplish in color with broad silvery stripes and the lower surface is dark purple. The lamina is ovate, glabrous, acute to acuminate apex. It has an entire margin and parallel venation. Lamina measures from 1- 3-7 cm in length, 1.5-3 cm in width. The leaf has no odor but has an astringent taste. III. Macromorphology of Tradescantiaspathacea Swartz Tradescantiaspathacea Sw. is an ornamental, succulent, perennial herb native to Central America, Mexico and the Caribbean. It is reproduced by division or by leaf cutting. The plant cultivated in Egypt reaches up to 16 cm in height. The Stem: erect,