Pharmacognosticalstudy Ofpouteriacampechiana(Kunth) Baehni Family Sapotaceae

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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

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.

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.

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.

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).

3- Methodology Material I- Plant material:

Plant material of Pouteria campechiana (Kunth) Baehni was collected in December 2010 from El-Mansouria, Giza governorate, Egypt and was kindly identified by Shahina A. Ghazanfar, Head of Temperate Regional Team, Royal Botanic Gardens Kew, UK and Dr. Mohammed El-Gibali former senior botanist at National Research Center, Cairo, Egypt. A voucher specimen (No. 19.4.2015) was deposited in the Herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University. a. Plant material for botanical study: Fresh samples of stem, leaf, seed and pericarp, as well as, samples preserved in ethanol 70% containing 5% glycerin were examined. Air-dried stem, leaves, seeds were finely powdered and packed in dark-colored, tightly closed containers and further used in addition to the fresh pericarp for examination of powdered organs. b. Plant material for UPLC-MS analysis: Dried plant organs (leaves, seeds and pericarps) were homogenized with a pestle and a mortar using liquid nitrogen. The powder (30 mg) was then mixed with 2.5 ml methanol containing 5 µg/ml umbelliferone (an internal standard for relative quantification) using Turrax mixer (11000 RPM) for five successive 20 second periods. To prevent heating, a period of 1 min. separated each mixing period. Extracts were then vortexed vigorously and centrifuged at 3000 g for 30 min to remove plant debris for UPLC-MS analysis, 500 µl were aliquoted and placed on a (500 mg) C18 cartridge preconditioned with methanol and water. Samples were then eluted using 3 ml 70% methanol and 3 ml 100% methanol, the eluent was evaporated under nitrogen gas and the obtained dry residue was resuspended in 500 µl methanol. Three µl samples were used for analysis (Farag et al. 2013). c. Plant material for phytochemical study: Samples of seeds and leaves of plant under investigation were separately air dried, powdered and saved in dark colored well-stoppered glass containers in a cool dark dry place prior to extraction. d. Plant material for determination of certain pharmacopoeial constants and the macro and micro elements: Fresh pericarp of Pouteria campechiana (Kunth) Baehni was separated from the seeds, chopped into pieces and packed in dark-colored, tightly closed containers for determination of certain pharmacopoeial constants, macro and microelements and carotenoids. II- Material for chromatographic study: a. Stationary phases: i. Pre-coated silica TLC plates 60 F254 (Macherey Nagel, Germany) (20 X 20 cm, 0.25 mm

7 thickness). ii. Silica gel H (E. Merck) type 60, employed for Vacuum Liquid Chromatography (VLC). iii. Silica gel 60 (Fluka, 70-230 mesh and 35-70 mesh, ASTM, Germany) for column chromatography (CC). iv. Sephadex LH-20 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) for column chromatography (CC). v. Polyamide (E-Merck Darmstadt, Germany) for column chromatography (CC). b. Solvent systems for thin layer chromatography: The following solvent systems were used:

 S1: [n -Hexane: chloroform (90:10 v/v)]

 S2 : [n -Hexane: ethyl acetate (95:15 v/v)]

 S3: [n -Hexane: ethyl acetate (80:20 v/v)]

 S4: [chloroform: methanol (98:2 v/v)]

 S5: [n -Hexane: ethyl acetate (60:40 v/v)]

 S6 : [n -Hexane: ethyl acetate (70:30 v/v)]

 S7: [chloroform: methanol (90:10 v/v)]

 S8: [chloroform: methanol: formic acid (85: 15: 0.01 v/v)]

 S9: [chloroform: methanol: formic acid (80: 20: 0.01 v/v)]

 S10: [Ethyl acetate: methanol: water: formic acid (100:15: 10: 0.01 v/v)] c. Reagents: 1. Spray reagents: i. p-anisaldehyde - sulphuric acid for sterols and triterpenes (Stahl, 1969): 0.5 ml p- anisaldehyde was dissolved in 10 ml glacial acetic acid. This was mixed with 85 ml methanol, then 5 ml of concentrated sulphuric acid were added cautiously. ii. Ferric chloride for phenolics: 5% of ferric chloride in 0.5 N hydrochloric acid. iii. Natural product- polyethylene glycol reagent (NP-PEG) for flavonoids (Wagner and Baldt, 2001): A: 1% methanolic solution of diphenylboric acid-β-ethyl-amino ester (diphenylboryloxyethylamine, NP). B: 5% ethanolic polyethylene glycol-4000 (PEG). The chromatogram was either sprayed with (A) followed by (B) or equal amount of (A) and (B) were first mixed together and the final solution used for spraying. The chromatogram was allowed to dry at room temperature and then examined under UV at 365 nm. iv. Aluminium chloride reagent (Markham, 1982), for detection of flavonoids: 1 %

Aluminium chloride in ethanol. 2. Reagents for UV spectroscopic analysis of flavonoids (Mabry et al., 1996): i. Sodium methoxide: 2.5 g of freshly-cut metallic sodium were cautiously added, in small pieces, to 100 ml cooled spectroscopic methanol and the resulting solution used. ii. Aluminium chloride solution: 5 g anhydrous aluminium chloride was cautiously dissolved in 100 ml of spectroscopic methanol and the resulting solution was used. iii. Hydrochloric acid: 50 ml concentrated HCl was mixed with 100 ml distilled water. iv. Sodium acetate: used as anhydrous powder of analytical grade obtained from E. Merck, Darmstadt, Germany. v. Boric acid: used as anhydrous powder of analytical grade obtained from E. Merck, Darmstadt, Germany. 3. Reagents for spectrophotometric determination: i. Folin-Ciocalteu reagent: used for determination of total polyphenolic content obtained from Loba-Chemie (Mumbai, India). ii. Gallic acid: gallic acid was purchased from Sigma-Aldrich Chemicals (Germany). iii. Sodium carbonate: Sodium carbonate (290 g) was dissolved in 1000 ml of distilled water with sonication. iv. Aluminium chloride solution (0.1 M): anhydrous aluminium chloride (0.6625 g) was cautiously dissolved in 50 ml of spectroscopic methanol. v. Rutin: rutin was purchased from Sigma-Aldrich Chemicals (Germany). 4. Solvents and test solutions: Solvents used in this work, viz., n-hexane, chloroform, ethyl acetate, n-butanol, absolute ethanol and methanol were of commercial grade and supplied through El Gomhouria Company for Trading Pharmaceutical Chemicals Medical Appliances, Cairo, Egypt. Methanol used for spectrophotometric analysis was of analytical grade and supplied by Merck Co., Darmstadt, Germany.

Diluted acids (HCl and H2SO4), alkalies (diluted NaOH, KOH) and ferric chloride were prepared according to Egyptian Pharmacopoeia (2005) monographs. 6. Authentic reference materials: Authentic reference samples for GLC analysis (Hydrocarbons, sterols and fatty acids) were obtained from National Research Centre, Dokki, Egypt. III- Material for pharmacological study: a. Experimental animals:

Adult male albino Wistar rats of 130-150 g body weight (aged 8 weeks) were used to study acute toxicity, anti-inflammatory, analgesic and anti-ulcerogenic activities. All rats were obtained from the animal facility, Pharmacology Department, National Research Center, Dokki, Giza, Egypt and were housed under standardized conditions of temperature and humidity. They were randomized into groups (each of 6-8 rats). Animals were kept under the same hygienic conditions and on a standard laboratory diet which consisted of vitamin mixture (1%), mineral mixture (4%), corn oil (10%), sucrose (20%), cellulose (0.2%), casein (95% pure) (10.5%) and starch (54.3%). Water was supplied ad libitum. Animal usage in the experiments was approved by Research Ethics Committee, Faculty of pharmacy, Cairo University in Egypt, approval number (MP 13). b. Reagents, drugs and chemicals: 1. Carrageenan: (sigma, USA) solution (10 mg/ 0.1 ml) was used for induction of inflammation. 2. DPPH (1,1-diphenyl-2- picrylhydrazyl): (sigma, USA) was purchased for evaluation of in vitro antioxidant activity and prepared of 0.004% w/v in methanol. 3. Standard drugs used as positive controls during the biological evaluation were: • Indomethacin as anti-inflammatory and analgesic • Ranitidine as antiulcerogenic • Ascorbic acid as antioxidant • Amphotericin B, ampicillin and gentamicin (Sigma –Aldrich, Germany) as antifungal, antibacterial for gram positive and gram negative bacteria, respectively. DPPH, ranitidine, ascorbic acid, indomethacin were purchased from Misr Company for Pharmaceutical Industry (Mataria, Cairo, Egypt) and Sedico Pharmaceutical Co. (6 October City, Egypt). 4. Agar and nutrient broth: (Oxoid laboratories, UK) was used for antimicrobial activity. c. Microorganisms: Microorganisms; Streptococcus pneumoniae (RCMB 010010), Bacillis subtilis (RCMB 010067), Pseudomonas aeruginosa (RCMB 010043), Escherichia coli (RCMB 010052), Aspergillus fumigatus (RCMB 02568), Syncephalastrum racemosum (RCMB 05922), Geotricum candidum (RCMB 05097) and Candida albicans (RCMB 05036) were supplied from culture collection of the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar university, Cairo, Egypt. IV- Material for quantitative estimation of tannin content:

Hide powder obtained from E-Merck, Darmstadt, Germany. V- Reagents for phosphorous determination: Nitrovanado molybdate reagent was prepared by mixing the following solutions: 100 ml of a 5 % ammonium molybdate solution + 100 ml of a 0.25 % ammonium vanadate solution +

100 ml diluted HNO3 + 5% ammonium molybdate solution. Technique

I-Method for botanical study: Different organs were separately imbedded in paraffin and serial transverse sections, 10- 15 μm thick, were cut with a manual microtome and stained with Safranin and Fast Green (Ruzin, 1999) except for testa transverse section, which was stained with phloroglucine and conc. HCl. Sections were mounted in synthetic balsam. Epidermis was obtained by scraping fresh material. II- Ultra performance liquid chromatography (UPLC): Chromatographic separation was performed on an Acquity UPLC system (Waters) equipped with a HSS T3 column (100£1.0mm, particle size 1.8mm; Waters). The analysis was carried out by applying the following binary gradient at a ﬂow rate of 150mLmin21: 0 to 1min, isocratic 95% A (water/formic acid, 99.9/0.1 [v/v]), 5% B (acetonitrile/formic acid, 99.9/0.1 [v/ v]); 1 to 16 min, linear from 5 % to 95% B; 16 to 18min, isocratic 95% B; 18 to 20 min, isocratic 5% B. The injection volume was 3.1 mL (full loop injection). Eluted compounds were detected from m/z 100 to 700 in negative ion mode using the following instrument settings: nebuliser gas, nitrogen, 1.6bar; dry gas, nitrogen, 6Lmin21, 1908C; capillary, 25500V; in-source CID energy, 0V; hexapole RF, 100Vpp; quadrupole ion energy, 5eV; collision gas, argon; collision energy, 10eV; collision RF 200/400Vpp (timing 50/50); transfer time, 70ms; prepulse storage, 5ms; pulser frequency, 10kHz; spectra rate, 3Hz. Internal mass calibration of each analysis was performed by infusion of 20mL 10M lithium formate in isopropanol: water, 1:1 (v/v), at a gradient time of 18min using a diverter valve. III- Chromatographic techniques: a. Successive extraction of the leaves and seeds of Pouteria campechiana (Kunth) Baehni The ethanolic extracts were obtained by cold maceration of 1.3 kg and 900 g of the air dried powdered leaves and seeds, respectively in 70% ethyl alcohol till exhaustion. The combined ethanol extracts were separately filtered and evaporated under reduced pressure, at temperature not exceeding 50 °C, to yield 145 g of solid dark green residue and 67 g semisolid brown residue of the leaves and seeds ethanolic extracts’, respectively. The residue,

11 in each case, was suspended in water and extracted by n-Hexane till exhaustion. The n- hexane and defatted ethanol fractions were evaporated separately at 50 °C under vacuum using rotary evaporator. Defatted ethanol fractions of leaves (80 g) and seeds (42 g) were separately suspended in distilled water and successively extracted, by liquid-liquid fractionation, with chloroform, ethyl acetate and n-butanol. Solvents were evaporated under vacuum and the dried residues were weighed. The percentage yields of the different extractives were calculated in respect to plant dry weight. . b. Isolation and identification of the constituents of chloroform fraction of Pouteria campechiana (Kunth) Baehni leaves. The chloroform fraction (10 g) was subjected to fractionation by vacuum liquid chromatography (VLC) on a 150 g silica gel H column (10 cm × 7 cm). Gradient elution was performed using chloroform, chloroform/ethyl acetate mixtures until reaching 100%, ethyl acetate then ethyl acetate/methanol mixtures were used. The polarity was increased by 10% every 400 ml till 50% methanol was attained. Fractions (400 ml, each) were collected and monitored by TLC using solvent system S4. Spots were examined in visible and UV light before and after exposure to concentrated ammonia, AlCl3 and p-anisaldehyde/H2SO4 spray reagents. Similar fractions were pooled, evaporated under reduced pressure, weighed and saved in a desiccator. For isolation and purification of individual compounds, pooled fraction I (eluted with 60-70% chloroform in ethyl acetate) and fraction II (eluted with 20% chloroform in ethyl acetate) were subjected to further chromatographic separation.

Fraction I: (0.65 g) showed three spots one of which was major (Rf =0.46 in solvent system S4, violet with p-anisaldehyde/H2SO4 /heat) was subjected to further chromatography on a Silica gel column (15 cm × 3 cm). Elution was done using n-hexane with increasing polarity with ethyl acetate. Fractions, (20 ml, each), were collected and monitored by TLC using solvent system S4. Fractions eluted with 0.5% ethyl acetate in n-hexane yielded on concentration under vacuum 130 mg of an impure residue which was rechromatographed on a Silica gel column (20 cm × 1 cm). Isocratic elution was performed with n-hexane- ethyl acetate (95:5 v/v). Fractions, (10 ml, each), were collected and monitored by TLC (S4). Similar fractions were pooled and yielded on concentration under vacuum 50 mg white powder of compound C1 which was kept for further investigation.

Fraction II: (1.4 g) showed three spots one of which was major (Rf = 0.23 in solvent system S7, violet with p-anisaldehyde/H2SO4 /heat) was subjected to further chromatography on Silica gel column (18 cm × 3 cm). Elution was done using n-hexane and increasing polarity with ethyl acetate. Fractions, (20 ml, each), were collected and monitored by TLC

12 using solvent system S4. Fraction eluted with 10% ethyl acetate in n-hexane yielded upon concentration under vacuum 170 mg of impure residue which was rechromatographed on a Silica gel column (20 cm ×1 cm). Isocratic elution was performed using n-hexane: ethyl acetate (80:20 v/v). Fractions, (10 ml, each), were collected and monitored by TLC using solvent system S4. Similar fraction yielded upon concentration under vacuum 25 mg of white powder of compound C2 which was kept for further investigation. c. Isolation and identification of the constituents of ethyl acetate fraction of Pouteria campechiana (Kunth) Baehni leaves. A weighed amount (10 g) of the ethyl acetate fraction of P. campechiana leaves was subjected to fractionation by vacuum liquid chromatography (VLC) on 200 g silica gel H column (12 cm× 7 cm). Gradient elution was performed using chloroform/ethyl acetate mixtures until reaching 100%, ethyl acetate then ethyl acetate/methanol mixtures were used. The polarity was increased by 10 % every 300 ml till 100% methanol. Fractions (300 ml, each) were collected and monitored by TLC using solvent system S8. Spots were examined in visible and UV light before and after exposure to concentrated ammonia, AlCl3 and NP-PEG spray reagents. Similar fractions were pooled, evaporated under reduced pressure, weighed and saved in a desiccator. Pooled fractions I, II, III and IV were subjected to further chromatography. Fraction I (eluted with 60% Ethyl acetate in chloroform) showed three spots one of which was major (Rf = 0.35 in solvent system S7, yellow with NP-PEG spray reagent) and subjected to column chromatography on a Sephadex LH-20 (18 cm × 2.5 cm). Isocratic elution was performed using methanol. Fractions, (10 ml, each), were collected and monitored by TLC using solvent system (S7). Pooled fractions (450 mg) were further purified using a Sephadex LH-20 column (20 cm × 1.5 cm) and eluted with water-methanol (50:50 v/v). An impure fraction showing one major spot was obtained and further chromatographed on Sephadex LH-20 column (20 cm × 1 cm) using methanol- water (90:10 v/v) as eluting system to get compound E1 which was kept for further investigation. Fraction II (eluted with 70 % Ethyl acetate in chloroform) showed three spots one of which was major (Rf = 0.17 in solvent system S7, orange with NP-PEG spray reagent) and subjected to column chromatography on Sephadex LH-20 (18 cm × 2.5 cm). Isocratic elution was performed with methanol. Fractions, (10 ml, each), were collected and monitored by

TLC using solvent system S7. Pooled fractions (220 mg) were further purified using Sephadex LH-20 column (20 cm × 1 cm); eluted with water-methanol (50:50 v/v) yielding yellow powder of compound E2 which was kept for further investigation.

Fraction lll (eluted with 10% Methanol in ethyl acetate) showed four spots one of which

was major (Rf = 0.38 in solvent system S9, orange with NP-PEG spray reagent) was subjected to column chromatography on a Silica gel column (17 cm × 3 cm). Elution was done using chloroform with increasing polarity using methanol. Fractions, (20 ml, each), were collected

and monitored by TLC using solvent system S9. Similar pooled fractions yielded on concentration under vacuum 45 mg of pure yellow powder (compound E3) which was kept for further investigation. Fraction IV: (eluted with 20-40% Methanol in ethyl acetate) showed two spots one of

which was major (Rf = 0.31 in solvent system S9, pale orange with NP-PEG spray reagent) was subjected to column chromatography on a polyamide column (20 cm × 2.5 cm) using gradient elution with water - methanol. Fractions, (10 ml, each), were collected and

monitored by TLC using solvent system S9. Pooled fractions (200 mg) were further purified using a Sephadex LH-20 column (20 cm × 1 cm); eluted with chloroform -methanol (50:50 v/v) yielding yellow powder of compound E4 which was kept for further investigation. d. Isolation and identification of the constituents of ethyl acetate fraction of Pouteria

campechiana (Kunth) Baehni seeds. Ethyl acetate fraction of P. campechiana seeds (3 g) were subjected to column chromatography using Sephadex LH-20 (22 cm× 3 cm). Isocratic elution was performed using methanol: water (90:10 v/v). Fractions (20 ml, each) were collected and monitored

using TLC and solvent system S8. Spots were examined under visible and UV light before

and after exposure to concentrated ammonia, AlCl3 and NP-PEG spray reagents. Subrfation

A (0.98 g) showed 2 major spots (Rf = 0.55 and 0.4) in solvent system S8, dark blue color

with FeCl3 spray reagent) and several minor spots. Subfraction A was further chromatographed using a Silica gel 40 column (15 cm × 1.5 cm) and chloroform: methanol: water (98:2:0.5v/v) as eluting system to obtain compounds E5 and E6 which was kept for

further investigation. e. GLC analysis of unsaponifiable matter and fatty acid methyl esters (Kovats, 1965): 1. Preparation of unsaponifiable matter: One g of the n-hexane fraction (page 58) was saponified by refluxing with 10 ml alcoholic potassium hydroxide (10%). The saponified solution was concentrated under reduced pressure and the residue was mixed with distilled water (100 ml) then exhaustively extracted with diethyl ether. The combined ethereal extract was washed several times with distilled water till free from alkalinity and dehydrated over anhydrous sodium sulphate and filtered. The residues left after evaporation of ether represented 4.97% and 9.01% of the total

lipoidal matter of the leaves and seeds, respectively. The residues were kept for studying the unsaponifiable content by GLC technique. 2. Isolation of fatty acids from the saponifiable fraction: The aqueous mother liquor remaining after extraction of the unsaponifiable matter was acidified with 10% hydrochloric acid to liberate free fatty acids which were extracted with diethyl ether (4× 50 ml). The combined ethereal extract was washed several times with distilled water till free from acidity and dehydrated over anhydrous sodium sulphate and filtered. The residue left after evaporation of ether representing 95.02% and 84.42% of the total lipoidal matter of the leaves and seeds, respectively. The residues were kept for further studying of the fatty acids. The unsaponifiable matter and the fatty acid methyl esters were subjected to GLC analysis. VI- Quantitative estimation of the total phenolic content in Pouteria campechiana (Kunth) Baehni leaves and seeds. Spectrophotometric determination of the total phenolic content was carried out according to the procedure reported in the European Pharmacopopeia (Druckerei, 2002), using the Folin-Ciocalteu colorimetric method. Total phenols were expressed as µg of gallic acid equivalents/ mg of dry extract. a. Establishment of the standard calibration curve: The standard stock solution of gallic acid in distilled water was prepared at a concentration of 250 µg/ml. The stock solution was serially diluted with 50% methanol to obtain the required concentrations equivalent to 25-150 µg/ml. An aliquot (0.8 ml) of each standard solution was mixed with 0.4 ml of Folin-Ciocalteu reagent and 4 ml distilled water, then diluted to 10 ml with 290 g/L sodium carbonate solution. The absorbance of the

resulting blue color was measured after 30 min at λmax 760 nm using UV spectrophotometer against blank similarly prepared except for replacing test solution by 50% methanol. All sample determinations were performed protected from light. For each concentration, three replicates were carried out and the average of the obtained absorbances was plotted versus the concentration. b. Estimation of the phenolic content: Defatted ethanol extracts of P. campechiana leaves and seeds were separately dissolved in 50% methanol and water in a concentration of 1 mg/ml. Each test solution was treated as mentioned before for the standard gallic acid solution, then the total phenolic content of each extract was deduced from the pre-established standard calibration curve.

V- Quantitative estimation of the total flavonoidal content in Pouteria campechiana (Kunth) Baehni leaves and seeds. The method adopted was based on measuring the intensity of the colour developed when flavonoids were complexed with aluminium chloride reagent (Geissman, 1962). The percentage of flavonoids was calculated as rutin with reference to a pre-established standard calibration curve. a. Establishment of the calibration curve: Different aliquot amounts (0.5 - 4 ml) of a 0.004% solution of rutin in 95% ethanol (equivalent to 20-160 μg) were separately transferred to test tubes and evaporated to dryness on a water bath at a temperature not exceeding 40°C. The residues obtained were, separately, treated with 5ml of 0.1M aluminium chloride

(page 49). The intensity of the developed yellow colour was measured at λmax 420 nm immediately against a blank prepared in the same way but replacing the rutin solution by ethanol (95%), using a UV spectrophotometer. For each concentration, three determinations were carried out and the average of the obtained absorbencies was plotted versus the rutin concentrations. b. Estimation of the flavonoid content: Defatted ethanol extracts of P. campechiana leaves and seeds (100 mg, each) were separately transferred to a measuring flask 100 ml capacity and completed to volume with ethanol (95%). From the ethanolic extract, 2 ml were transferred to a test tube and evaporated to dryness. To the residue, 5 ml of 0.1M aluminium chloride were added and the procedure continued as mentioned before. The flavonoid content was deduced from the pre-established standard calibration curve and calculated using the following equation: Y= 0.0044X - 0.0119 , Where: Y= absorbance, X= corresponding concentration (μg/ml). VI- Quantitative estimation of the tannin content in Pouteria campechiana (Kunth) Baehni leaves and seeds. The tannin content of the leaves and seeds of P. campechiana (Kunth) Baehni was estimated adopting the hide powder method (Schofield et al., 2001) in which the difference in dry weight of the extract before and after treatment with standard hide powder was taken as an approximate measure of the tannin content. Dried powdered leaves and seeds (0.75 g each) of P. campechiana (Kunth) Baehni were separately boiled with 150 ml distilled water in 250 ml Erlenmeyer flask. The extract in each case was left on a boiling water bath for 30 minutes.

Extracts were separately cooled and transferred to a 250 ml measuring flask. The volume was completed to 250 ml with distilled water, left to sediment for 2 hours and then filtered. The first 50 ml of the filtrate was rejected in each case. a. Determination of the total water soluble extractives (G1): 25 ml of each filtrate was transferred separately to a pre-weighed 50 ml beaker, allowed to evaporate and dried in a hot air oven at 70 0C. after cooling, the beakers were kept in a desiccator till a constant weight was recorded. The residue in each beaker was weighed (G1) b. Determination of substances not adsorbed by hide powder (G2): Another 50 ml of each filtrate were mixed with 0.5 g hide powder and left for one hour with continuous shaking. The mixture was filtered; 25 ml of each filtrate were transferred to a pre-weighed 50 ml beaker and treated as above. The residue left in each beaker was weighed

(G2). c. Blank experiment: A blank determination was carried out twice using distilled water. The residue left was weighed (G0). The tannin percentage was calculated according to the following equation:

Tannin % = [G1- (G2-G0) × 1000 × 100 ×10] / W

Where: G1 = weight of the residue containing tannin in g

G2 = weight of the residue after removal of tannin in g

G0 = blank W = weight of powdered drug in mg VII- Techniques for pharmacological study: a. Determination of median lethal dose (LD50):

The LD50 of leaves and seeds ethanolic extracts were estimated according to Karber procedure (1931). Preliminary experiments were done to determine the minimal dose that killed all animals

(LD100) and the maximum dose that failed to kill any animal. Several doses at equal logarithmic intervals were chosen in between these two doses; each dose was orally administrated in a group of 6 rats. The rats were then observed for 24 hours and symptoms of toxicity and mortality in each group were recorded. The LD50 was calculated. b. Acute anti-inflammatory activity: Carrageenan-induced rat paw oedema method described by Winter et al. (1962) was used for evaluating anti-inflammatory activity. The rats were divided into 14 groups (each of 6 rats). Group I, received saline 0.9% (control); Group II, indomethacin ( 20 mg Kg-1 ) serving as a positive control and the other twelve groups were treated with ethanol extract, defatted ethanol fraction and hexane fraction of the leaves and seeds each at two doses 100 and 200

17 mg Kg-1. Oedema was induced in the right hind paw of each rat by subplantar injection of 100 µl of 1% carrageenan. The plant extracts were administered orally one hour before induction of inflammation. Paw volume of both control and plant extract treated rats was measured at 1, 2, 3, and 4 hours after carrageenan injection by water displacement method (Chattopadhyay et al., 2002 and Li et al, 2003). The percentage of oedema inhibition in treated animals versus control was calculated according to the following formula of Chu and Kovacs (1977).

% inhibition in oedema volume ×100

Where: Vc =Oedema volume of control Vt =Oedema volume of test Statistical analysis was carried out using repeated measures one way ANOVA followed by least significant test for multiple comparisons. c. Analgesic activity: Hot plate method was carried out according to procedures of Woolfe and McDonald (1944). The rats were divided into 14 groups (each of 8 rats). Saline was administrated in Group I as a control, Indomethacin was administrated (20 mg Kg -1) in Group II as positive control and the other 12 groups were given ethanol extract, defatted ethanol fraction and hexane fraction of the leaves and seeds each at two doses 100 and 200 mg Kg-1. The reaction time between the moment when the rat reached the plate and that when the animal licked its hand paw was measured after 0, 30, 60 and 90 minutes following the administration of tested plant extracts or saline. Analgesic activity was expressed as the prolongation of the reaction time. The statistical analysis was carried out using repeated measures one way ANOVA followed by least significant test for multiple comparisons. d. Anti-ulcerogenic activity: Ethanol-induced ulcer model described by Robert et al. (1979) was used for evaluating anti-ulcerogenic activity. Acute erosion of the gastric mucosa was induced in rats, which had been fasting for 18 hours, by intragastric administration of 1 ml of absolute ethanol. The rats were divided into 14 groups (each of 6 rats). Ranitidine (20 mg/kg, 60 min prior to ethanol) was used in one group as a reference drug; the control group was given vehicle and the other twelve groups were given ethanol extract, defatted ethanol fraction and hexane fraction of the leaves and seeds, each at two doses 100 and 200 mg Kg-1. The animals were sacrificed one hour after giving ethanol and the gastric lesions were examined under an illuminated magnifier according to Adami et al. (1964). The intensity of gastric lesions was assessed according to the following scoring system:

0: normal mucosa 1: < 1 mm (spot ulcers) 2: ulcers ≤1mm but <2mm 3: ulcers ≤2mm but <3mm 4: ulcers ≤3 mm but <4 mm 5: ulcers ≤4mm but < 5 mm Statistical analysis was carried out using Kruskal-Wallis non parametric one way ANOVA. e. Antimicrobial activity: Antimicrobial activity was determined using the agar well diffusion assay method as described by Holder and Boyce (1994). Eight pathogenic bacteria and fungi were used in this study; Streptococcus pneumoniae (RCMB 010010), Bacillis subtilis (RCMB 010067). Pseudomonas aeruginosa (RCMB 010043), Escherichia coli (RCMB 010052), Aspergillus fumigatus (RCMB 02568), Syncephalastrum racemosum (RCMB 05922), Geotricum candidum (RCMB 05097) and Candida albicans (RCMB 05036). The tested microorganism were subcultured on nutrient agar medium. Amphotericin B, ampicillin and gentamicin were used as a positive control (standard reference drugs). The plates were done in triplicate. Bacterial and fungal cultures were incubated at 37°C for 24 h. Antimicrobial activity was determined by measuring zone of inhibition (Agwa et al., 2000). Determination of MIC: The minimum inhibitory concentrations (MIC) of the samples were estimated for each of the tested organism in triplicates. Variable concentrations of the samples (1000-0.007µg/ml) was introduced to the tubes, nutrient broth was added and then a loopful of the organism of choice previously diluted to 0.5 McFarland turbidity standard was introduced too. A tube containing broth media only was seeded with the same test organisms to serve as control. Tubes containing tested organisms cultures were then incubated at 37°C for 24 h. The tubes were then examined for growth by observing turbidity (Doughari, 2006).

f. Antioxidant activity: The antioxidant activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) reagent and ascorbic acid as standard. Ten mg of the ethanolic extract was dissolved in 1 ml methanol then the volume was completed to 20 ml by methanol to the final concentration of 0.5 mg/ml. different dilutions ( 50, 200, 400, 600 and 800 μg/ml) were prepared. Plant extract (0.3 ml) was added to 2 ml of a 0.004% methanol solution of DPPH. The mixture was left in dark for 30 min at room temperature then the absorbance was measured spectrophotometrically against blank at

λmax 495 nm. Ascorbic acid was used as a positive control at a concentration (1-50 μg/ml). Antioxidant activity was expressed as the concentration of the extract inhibiting DPPH

19 formation by 50% relative to methanol (IC50). Percentage inhibition was calculated from the equation: % Inhibition = [(A0 – A1) /A0] x100 Where: A0 is the absorbance of the control (containing all reagents except the extract) and A1 is the absorbance of the extract. Each measurement was performed in triplicate. VIII- Determination of some pharmacopoeial constants: a. Determination of total ash: Total ash value was estimated according to AOAC (1990). Two-four g powdered material was weighed. The material was spread in an even layer in a crucible which was ignited by gradually increasing the heat to 500-600 C until free from carbon. It was allowed to cool in a desiccator then weighed. If carbon-free ash cannot be obtained the crucible was cooled and the residue was moistened with about 2 ml of water or a saturated solution of ammonium nitrate. The crucible was dried on water bath, then on a hot plate and ignited to a constant weight. The content of total ash was calculated in mg per g of air dried material. c. Determination of crude fiber: Its value was estimated according to AOAC (1990). Certain weight of the powdered drug was treated with ether. Then refluxed with 1.25 % sulphuric acid followed by 1.25 % of sodium hydroxide. The residue left was washed with water, dried and weighed. This residue represented the crude fibers and minerals which are insoluble in the acid and alkali. The residue is incinerated and the ash was weighed again. The weight of the crude fiber equaled the weight of the residue after treatment with alkali minus the weight of the ash. d. Determination of moisture content: Its value was estimated according to AOAC (2000). This involved drying to a constant weight at 100 0C and calculating moisture as the loss in weight of the dried samples. The percentage moisture content was calculated from the equation:

Where: W1= weight (g) of sample before drying, W2= weight (g) of sample after drying. IX. Determination of the total carbohydrates: It was carried out according to phenol-sulfuric acid method described by Dubios et al. (1956). This method is based on complete hydrolysis of the pericarp by boiling with 1M

H2SO4 for 10 hrs in a boiling water bath, neutralization by barium carbonate, then filtration. The clear solution was made up to known volume; followed by the addition of 0.5 mls of

phenol (5%) and 2.5 mls of H2SO4 (96%) to the known volume of the hydrolysate (0.5 ml).

The optical density of the obtained yellow orange color was measured at λmax 490 nm using Perkin-Elemer spectrophotometer against blank. Graphic plot of the optical density values

20 against various standard solutions of different concentrations of glucose was used as standard curve. X- Techniques for determination of macro and micro elements: a. Flame photometric determination of potassium: After dilution of the original ash solution to 1/5, the K-emission was measured in an air-propane flame at 768 nm (Cotennie, 1980). A calibration curve was made with a standard series of 0-200 ppm K. b. Determination of calcium: Calcium was determined by atomic absorption in an air- acetylene flame after addition of strontium to produce a concentration of 1000 ppm strontium in the analyte solution (Cotennie, 1980). c. Determination of phosphorus (El-Merzabani et al., 1977): In presence of V5+ and Mo6+, orthophosphates formed a yellow colored phosphovanado-molybdate complex.

Fifty g (NH4)6Mo7O24.4H2O was dissolved in 500 ml warm distilled water (50 C) then transferred quantitatively into a 1 L volumetric flask, cooled and completed to the mark. A standard series between 0 and 25 ppm phosphorus were prepared. Five ml of the original ash solution was added into a 50 ml volumetric flask. Ten ml nitrovanadomolybdate reagent was added and completed to the mark with distilled water. After one hour the absorbance was measured at λmax 430 nm in a spectrophotometer. XI- Methods for amino acids analysis The total protein was extracted from the pericarps according to AOAC (1995) using 50 mM Tris-HCl (pH=7.5). Acid hydrolysis of the protein was carried out according to Spackman et al. (1958) by adding one ml 6N HCI (HCI Suprapure® from Merck) and mixing with one mg protein in a hydrolysis tube. The solution was frozen in a mixture of dry ice/ethanol in a test tube. The tube was evacuated with a vacuum pump and sealed using gas- burner. The sealed tube was placed in an oven at 110 °C for 72 hours for hydrolysis, and then cooled to room temperature then kept in an ice-bath. The solution was centrifuged to precipitate insoluble components. The supernatant was evaporated at 40°C in a rotary evaporator. The residue was then dissolved in a diluting buffer. XII- Techniques for determination of vitamins a. Determination of vitamin E (Mohammad et al., 2009): Vitamin E was measured using Emmorie Engel reaction, which is based on reduction of ferric to ferrous ions by tocopherols, which then form red complex with α,α-dipyridyl. Fresh pericarp (1 g) was extracted with petroleum ether, the petroleum ether was then evaporated at room temperature and the residue dissolved in 1 ml of chloroform; 1 ml of 95

% ethanol was then added followed by 1 ml of 0.2 % α,α-dipyridyl and 0.1ml of 0.1 % FeCl3.

After 15 minutes, the absorbance at λmax 520 nm was measured in a colorimeter. b. Determination of vitamin C: Fresh pericarp (1 g in 25 ml conical flask) was treated with 10 ml oxalic acid (0.05M) and sample was placed in shade for 24 hrs for extraction of vitamin C content. After 24 hrs, sample was filtered through 0.45µm filter paper then 2.5 ml of the sample was transferred to 25 ml volumetric brown flask with 2.5 ml oxalic acid (0.05M), 0.5 ml meta phosphoric acid with acetic acid, 1 ml H2SO4 (5%v/v) and 2 ml ammonium molybdate.

The volume was completed to 25ml with distilled water. The absorbance was measured at λmax 760 nm (Hussain et al., 2010). XIII- Determination of total carotenoids (Ranganna, 1977): The pericarp (5 g) was extracted in a blender for 5 min with 40 ml acetone, 60 ml hexane, and 0.1 g magnesium carbonate. The residue was allowed to settle and was decanted into a separating funnel. The residue was washed twice with 25 ml portions of acetone, then with 25 ml hexane. The acetone was separated and removed from the extract by repeated washing with water. The upper layer was transferred into 100 ml volumetric flask containing 9 ml acetone and was diluted to volume with hexane, this solution now contain 9% acetone. A chromatographic column with 1:1 mixture of activated magnesia and diatomaceous earth (supercel) was prepared. One cm layer of anhydrous sodium sulphate was placed above the absorbent, with vacuum continuously to flask, then 50 ml of acetone-hexane extract of pigment was transferred into the column. Carotenes passed rapidly through the column. Bands of xanthophylls, carotene oxidation products and chlorophylls remain adsorbed on the column. The eluate was collected, concentrated under reduced pressure and made up to known volume with 9% acetone in hexane. The absorbance was measured at λmax 436 nm and the instrument was adjusted to 100% transmittance with 9% acetone in hexane. The concentration was calculated using pre-established standard calibration curve.

Conclusions

i. The botanical profiling of different organs of Pouteria campechiana (Kunth) Baehni was in agreement with the general characters of family Sapotaceae and genus Pouteria. ii. A total of sixty three metabolic compounds were detected using UPLC-PDA-qTOF- MSn technique, seventeen compounds were phenolic, eighteen were fatty acids and ten were triterpenes.

iii. The leaves ethanolic extract showed the highest total phenolic, flavonoidal and tannin contents of all tested samples. iv. eight compounds were isolated and their structures were elucidated; two triterpenoids (ursolic acids and 2α, 3α, 19α, 23 tetrahydroxy ursenoic acid), four flavonoids (quercitin, myrcitin, myricetin-3-O-α-L-rhamnoside and myricetin-3-O-β-galactoside) and two phenolic acids (protocatechuic acid and gallic acid), six of these compounds were isolated for the first time from Pouteria campechiana. v. Evaluation of the biological activities of Pouteria campechiana revealed significant anti-inflammatory and analgesic activities of Pouteria campechiana seeds and leaves ethanolic extrats together with their potent gastroprotective effect. vi. The antimicrobial screening support the medicinal use of Pouteria campechiana seeds and leaves for treatment of some bacterial and fungal infections. vii. The high flavonoidal and phenolic content of Pouteria campechiana leaves besides their antioxidant activity encourage possible medicinal use of the leaves as a natural protective support for free radical oxidation caused diseases. viii. It is recommended to use the plant for the evidenced biological activities as whole extracts rather than fractions to provide the potentiating effects of all constituents. ix. Correlation appeared to exist between the chemical compounds isolated or detected by UPLC-PDA-ESI-MSn and the investigated biological activities. x. Clinical trials should be performed in order to support all the investigations and to facilitate their pharmaceutical formulations. xi. The nutritive values of Canistel cultivated in Egypt was remarkable and encourages more propagation of the plant for its benefits.

5- Studying period تاريخ التسجيل: 2010/7/8 تاريخ التشكيل: 2016/1/19 فترة الدراسة: خمس سنىات و 5 أشهر  Plant samples were collected during the fruiting stage from El-Mansoureya, Giza, Egypt.  Botanical study including macro-and micro-morphological investigation: 1 year.  UPLC-PDA-ESI-MSn analysis of the leaves, seeds and pericarps ethanolic extracts: 1year.

 Phytochemical study including: extraction, isolation and identification of chemical constituents: 2 years and 2 months.  Biological activity study including in-vitro and in-vivo studies: 1 year.  Nutritional evaluation of the fruit’s pericarp: 3 months 6-Date of research: 2010 – 2016.