PharmacognosticalStudy ofPouteriacampechiana(Kunth) Baehni Family Sapotaceae A thesis presented by Rabab Mahrous AbdouHussein For The Degree of Master in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of Prof. Dr.Aly Mohamed El-Sayed Professor of Pharmacognosy- Faculty of Pharmacy Cairo University Prof. Dr.NebalDarwishElTanbouly Professor of Pharmacognosy - Faculty of Pharmacy Cairo University Dr. SherifaFahmy Aly Moustafa Lecturer of Pharmacognosy - Faculty of Pharmacy Cairo University Pharmacognosy Department Faculty of Pharmacy Cairo University 2016 1 Abstract A pharmacognostical study of Pouteria campechiana (Kunth) Baehni family Sapotaceae was carried out. Literature survey on several Pouteria species was reviewed. A botanical study including macro- and micro-morphological characters of the plant organs were presented for their identification in the entire or powdered forms. Secondary metabolites of Pouteria campechiana organs were analyzed for the first time via high resolution UPLC-PDA- qTOF-MSn which resulted into detection of sixty three chromatographic peaks belonging to various classes. The composition of lipoidal matter was analyzed using GLC technique. Furthermore, chemical analysis was conducted to isolate and identify the major constituents of Pouteria campechiana and resulted into the isolation and identification of ursolic acids, 2α, 3α, 19α, 23 tetrahydroxy ursenoic acid, quercitin, myrcitin, myricetin-3-O- β-galactoside and protocatechuic acid for the first time, beside myricetin-3-O-α-L- rhamnoside and gallic acid, which were detected before in Pouteria campechiana. Total phenolic and flavonoidal contents were estimated spectrophotometrically in addition to determination of tannin content by hide powder gravimetric method. Biological study was conducted including evaluation of the anti-inflammatory, analgesic, antiulcerogenic activities using carrageenan-induced paw oedema method, hot plate method and ethanol induced ulcer model, respectively. Moreover, antioxidant and antimicrobial properties were assessed by measuring free radical 1,1- diphenyl-2- picrylhydrazyl (DPPH) scavenging activity and agar well diffusion assay methods. Results revealed that the seeds ethanolic extract has higher anti-inflammatory effect than that of the leaves, while the leaves ethanolic extract exhibt higher analgesic activity. Both leaves and seeds ethanolic extracts exhibited a significant decrease in gastric ulcer number and ulcer severity. Antimicrobial screening revealed that leaves and seeds ethanolic extracts showed moderate to strong antimicrobial activity.on the other hand, leaves showed higher antioxidant activity than seeds. The nutritive values of the fruit were evaluated and found to be remarkable and encourage the propagation of plant in Egypt. Key words: Pouteria campechiana, botanical study, UPLC-PDA-ESI, metabolites, triterpenes, flavonoids, phenolics, anti-inflammatory, analgesic, antiulcerogenic, antimicrobial, in vitro antioxidant. 2 The study included five parts: Part I: Botanical Study of Pouteria campechiana (Kunth) Baehni. The following macro- and micromorphological characteristics were established for Pouteria campechiana (Kunth) Baehni stem, leaf, fruit and seed. Part II: UPLC-MS profiling of the secondary metabolites of Pouteria campechiana (Kunth) Baehni leaves seeds and fruits. The UPLC-PDA-qTOF-MSn technique was used to define chemical compositions of P. campechiana different organs and to explore any variations among them. A total of sixty three metabolic compounds were detected, seventeen compounds were phenolic, eighteen were fatty acids and ten were triterpenes. This work represent the first attempt to provide a map for the secondary metabolites distribution and variation in the leaves, seeds and pericarps using UPLC-PDA-ESI-qTOF-MSn technique. Part III: Phytochemical study of Pouteria campechiana (Kunth) Baehni leaves and seeds. Chapter I: investigation of the lipoidal content of the leaves and seeds of Pouteria campechiana (Kunth) Baehni. The saponifiable and unsaponifiable matters of P. campechiana leaves and seeds were investigated using GLC technique. Chapter II: Investigation of the terpenoidal content of Pouteria campechiana (Kunth) Baehni leaves. two triterpenes were isolated from the leaves chloroform fraction and were identified as 1- ursolic acid 2- 2α, 3α, 19α, 23 tetrahydroxyursolic acid This is the first report on the isolation of these compounds from P. campechiana (Kunth) Baehni. 3 Chapter III: Investigation of the phenolic content of Pouteria campechiana (Kunth) Baehni leaves and seeds. A. Quantitative estimation of the total phenolic content. B. Quantitative estimation of the total flavonoidal content.. C. Quantitative estimation of the tannin content. D. Investigation of the ethyl acetate fractions of Pouteria campechiana leaves and seeds. Chromatographic isolation of four flavonoids and two phenolic acids was carried out. The compounds were identified through co-TLC with authentic reference samples, and comparison of 1H-NMR and 13C-NMR data with those previously reported. They were identified as: 1- quercitin 2- myrcitin 3- myricetin-3-O-α-L-rhamnoside 4- myricetin-3-O-β-galactoside. 5- protocatechuic acid 6- gallic acid This is the first report on the isolation of quercitin, myrcitin, myricetin-3-O-β- galactoside and protocatechuic acid from P. campechiana (Kunth) Baehni. Part IV: Biological activity of Pouteria campechiana (Kunth) Baehni leaves and seeds. The study included: A. Determination of median lethal dose (LD50). B. Acute anti-inflammatory activity. C. Analgesic activity. 4 D. antiulcerogenic activity. E. In vitro Antioxidant activity. F. Antimicrobial activity. Part V: Nutritional evaluation of Pouteria campechiana (Kunth) Baehni fruit (Canistel). Moisture, lipid, protein and carbohydrates contents together with ash and crude fiber values were estimated for Pouteria campechiana (Kunth) Baehni. fruits (Canistel). Macroelements (Ca, Na, Mg and P); and microelements (Fe, Co, Cu and Zn) were determined besides amino acids, vitamin C, vitamin E and carotenoid content. 1- Introduction Healing with medicinal plants is as old as mankind itself. Despite the great world wide spread of synthetic drugs, adverse effects of synthetic drugs encourage many researchers to find natural substitutes which enjoy safety, efficacy and low cost. Patients are also aware with the painful and sometimes the dangerous side effects of these drugs when severe diseases such as cancer, HIV and other viral infections are involved. Nowadays, huge efforts are devoted to plant research to explore their constituents and screen them for biological effects. The employment of bioassay-guided isolation techniques have added another dimensions to the research to discover new drugs. The structure diversity of the natural constituents in herbs make them valuable source for novel lead compounds against diseases. The family Sapotaceae (Evans, 1998) includes 35-75 well-defined genera and about 800 species, worldwide distributed in the tropical and subtropical regions. The genus Pouteria includes 9 sections and about 325 species (Triono et al., 2007). Members of the genus Pouteria produce edible fruits of great economical value. They are not only commercially important, but are also valuable in folk medicine and are recommended for the treatment of inflammation, ulcers, diabetes and diarrhea (Silva et al., 2009). However, only few research papers reported the chemical composition and biological properties of these plants. Pouteria campechiana (Kunth) Baehni is native to northern South Americas (Morton, 1987). Its name is originated from its native Mexican town of Campeche. The fruit of P. campechiana (Kunth) Baehni is reported as a rich source for carotenoids (Costa et al., 2010) 5 and is well known as an antioxidant and a hepatoprotective (Ma et al., 2004; Suda et al., 2005; Kubola et al., 2010; Aseervatham et al., 2013; Kong et al., 2013). P. campechiana is also valued as an ornamental tree and its wood has found commercial application for construction of buildings. In its native range, it has been a source of latex used to adulterate gum (Morton, 1987). A decoction of P. campechiana (Kunth) Baehni bark is taken as a febrifuge in Mexico and is applied on skin eruptions in Cuba. A preparation of the seeds is employed as a remedy for ulcers (Morton, 1987). To the best of our knowledge, few repots were traced about P. campechiana (Kunth) Baehni leaves and seeds, as most of the research work done was about the fruits. Therefore, this study has found it of interest to discover any possible medicinal uses of P. campechiana leaves and seeds. 2- Plan of work 1. Literature survey of periodicals and reference books about Pouteria campechiana (Kunth) Baehni as well as different Pouteria species. 2. Examination of macro- and micromorphological characters of Pouteria campechiana (Kunth) Baehni. 3. Providing well characterized screening profile using UPLC-PDA-qTOF-MS technique and give a map for various classes of compounds contained and their distribution in different organs viz, leaves, seeds and pericarps. 4. Quantitative determination of the main active constituents of Pouteria campechiana (Kunth) Baehni. 5. Isolation, purification and characterization of the possible chemical constituents of the plant using different chromatographic and spectral methods. 6. Biological study of the different extracts prepared from the plant. 7. Nutritional evaluation of Pouteria campechiana (Kunth) Baehni fruits (Canistel).