Basic Informations

C.V

Master Title

Pharmacognostical study on Cicer arietinum family Leguminosae cultivated in Egypt”

Master Abstract

Cicer arietinum family Leguminosae is known as chickpea. It may be called Bengal gram. It is the only cultivated species of the genus Cicer. It is a cheap source of high-quality protein in the diets of millions in developing countries, in addition to carbohydrates, minerals, and trace elements. It is an important crop for direct human consumption and for animal feed. The present study includes the following: I ) - Phytochemical study of the different plant parts. II) -Isolation, identification & structure elucidation of the isolated compounds. III) -Biological study of different extracts of the plant. Part I: Phytochemical study of Cicer arietinum plant. Chapter I: Preliminary phytochemical screening of the different organs. Preliminary phytochemical screening of the different organs of the plant under investigation revealed revealed the prominent presence of carbohydrates and /or glycosides, in all organs,the presence of sterol and /or triterpenes in all organs, the absence of saponins in all organs and the presence of flavonoids and tannins mainly in aerial parts. Chapter III: Quantitative estimation of total flavonoids in different organs: Estimation of total flavonoids in different organs revealed that the aerial parts have the largest percent of total flavonoids followed by roots, pericarps, and seeds. ? So we completed the phytochemical study on the aerial parts: Chapter IV: TLC screening of the different extracts of aerial parts It revealed a number of major spots. Flavonoids were detected in the chloroform and ethyl acetate extracts.The major spots of the flavonoids present in the chloroform extract of the aerial parts. Chapter V: Investigation of petroleum ether fraction: A-Gas liquid chromatographic analysis Extraction: The air-dried powdered aerial parts were extracted with methanol by percolation. After solvent evaporation, the residue fractionated using petroleum ether, chloroform and ethyl acetate. The petroleum ether fraction contains the lipoidal matters. Saponification of the lipoidal matters: The petroleum ether extract was saponified with 10% alcoholic KOH. The unsaponifiable matters were extracted with ether, GLC investigation of unsaponifiable matter revealed that hexacosane is the major hydrocarbon and ß-sitosterol is the major sterol. On the other hand the aqueous solution left after the separation of the unsaponifiable matters was acidified and extracted with ether to obtain the fatty acids. Methyl esters of fatty acids were prepared and investigated by GLC, which revealed that the major fatty acid was linoeic acid followed by oleic acid. B- Column chromatography Isolation: the unsaponifiable matter was chromatographed on silica gel columns using hexane-ethyl acetate to yield compounds L1,L2 and L3. the identification of the compounds via their physico-chemical data, spectral data, comparison with previously published data and by comparison with authentic samples. The isolated compounds was identified as ß-amyrine, Stigmasterol and ß-sitosterol. Chapter VI: Investigation of the Chloroform extract The chloroform extract was chromatogaphed using silica gel columns, to yield four compounds, C1, C2, C3 and C4.. The identification of the compounds was via their physico-chemical data, spectral data, and comparison with previously published data and by comparison with authentic samples. The isolated compounds were identified as biochanin A, formononetin, genistein and ß-Sitosterol 3-O-glucoside. Chapter VII: Investigation of the ethyl acetate extract The ethyl acetate extract was fractionated on silica gel using gradient elution with ethyl acetate and increasing polarity with methanol, sephadex and preparative HPLC using acetonitrile-water as mobile phase to isolate three flavonoid-O-glycosides (C5, C6, C7) that were identified based on their physico-chemical data, spectral data and comparison with previously published data as trifolirhizin, medicocarpin and kaempferol 3-O-glucoside. Part II: Biological study of the different extracts of the plant. 1- Chapter I: : Analgesic activity This activity was evaluated by using an electric current as anoxious stimulus wheren electrical stimulation was applied to the rat's tail. The minimum voltage required for the animal to emit a cry was recorded The alcoholic extract is more potent than aqueous extract and is nearly similar (77.6%) to the reference drug (novalgin). 2- Chapter II: : Antipyretic activity The normal rectal temperature was recorded before the start of the experiment. Pyrexia was induced by intramuscular injection of yeast suspension. The alcoholic and aqueous extracts of the aerial parts of Cicer arietinum showed variable antipyretic activity, which increases after two hours.The alcoholic extract showed highly significant activity which was about 80.9% of paracetamol after one hour and higher than paracetamol after two hours. 3- Chapter III: : Anti-inflammatory activity The alcoholic extract is more potent than aqueous extract and is nearly similar (76.4%) to the reference drug (Indomethacin). Since ulcer formation is one of the most serious side effects of most anti-inflammatory drugs in market, and our studies revealed that extracts of Cicer arietinum have significant anti-inflammatory activity, we were interested in investigating ulcerogenic activity of these extracts. aqueous extract,showed less ulcer number than alcoholic extract , and both were less than that of the reference drug, (indomethacin). 4-Chapter IV: : Antihyperlipidemic activity This was assessed by measuring serum cholesterol level, triglycerides, cholesterol fractions HDL-c and LDL-c Alcoholic and aqueous extracts showed significantly high decrease in serum cholesterol and triglycerides level, and showed significant decrease in risk factor (LDL / HDL ratio), which is near to that of the reference drug, simvastatin. 5-Chapter V: : Cytotoxic activity For this work, we used human breast carcinoma (MCF7) and liver carcinoma (HEPG2) cell lines to evaluate the cytotoxic activities of chickpea hydroalcoholic extract. The extract showed mild activity against breast carcinoma cell line (MCF7) using the tested Concentrations and showed significant cytotoxic activity against liver carcinoma cell line (HEPG2) with IC50 = 9.93 µg and need more investigation. 6-Chapter VI: : Antioxidant activity To explore the antioxidant and free radical scavenger properties of the plant, the antioxidant activities of the different chickpea extracts; hydroalcoholic, chloroform and ethyl acetate obtained from aerial parts as well as pure isolated flavonoids were evaluated using DPPH radical. The ethanolic extract has lower activity in DPPH quenching than chloroform extract, while the ethyl acetate extract has the highest antioxidant activity, which is nearly similar to that of ascorbic acid. Among the isolated flavonoids, kaempferol 3-O-glucoside showed high antioxidant activity followed by genistein, biochanin A and formononetin. 7-Chapter VII: : Antibacterial activity The alcoholic, chloroformic, and ethyl acetate extracts have a broad spectrum antibacterial activity against gram negative bacteria, while they have moderate activity against Bacillus sphaericus bacteria, while the extracts have no activity against Bacillus pumlus bacteria. The alcoholic extract has moderate activity against Brevibacterium otitidis bacteria.

PHD Title

Chemical studies and bioactivities of Eugenia rigida DC

PHD Abstract

Family Myrtaceae is economically fairly important. Its plants are grown as ornamentals; others produce edible fruits rich in vitamins and are of medicinal value. The genus Eugenia is the largest in the Myrtaceae family with up to 2,000 species distributed from the south of Mexico, Cuba and the Antilles to Uruguay and Argentina, with a small number of species in Africa. Eugenia rigida DC. is a shrub or small tree containing characteristic brown and green fruits, which turn black when mature. It was used traditionally in Argentina for leukemia. This plant has not previously been subjected to either chemical or biological investigations, but the genus exhibits a wide array of secondary metabolites. The work is composed of two parts Part I: Chemical study Part II: Biological study Part I: Chemical study includes three chapters Chapter 1: Phytochemical study Chemical investigation of the constituents of Eugenia rigida DC. leaves afforded 16 compounds; among which five compounds were new in nature; and one of which (Er-5) was previously synthesized but for the first time from nature. The compounds are: two new flavanones; 5,7-dihydroxy-6-formylflavanone (Er-1) and 5,7-dihydroxy-8-formylflavanone (Er-2); two stilbenes Z-3,4,3?,5?-tetramethoxystilbene (Er-5) and E-3,4,3?,5?-tetramethoxystilbene (Er-6), which were found to be positional isomers to each other, one new dihydrochalcone; 2'-methoxy- 3'-methyl - 4',6'- dihydroxydihydrochalcone (Er-3), and one chalcone: 2',6'-dihydroxy-4'-methoxy-5'-methylchalcone (Er-14). In addition to ten known compounds were previously isolated from other plants and new in the species: 3,4?,5-trimethoxy-trans-stilbene (Er-4), betulinal (Er-7), a-betulinic acid (Er-8), betulonic acid (Er-9), ß-betulinic acid (Er-10), comptonin (Er-11), barbinervic acid (Er-12), diospyric acid (Er-13), 3?-formyl-2?,4?,6?-trihydroxy-5?-methyldihydrochalcone (Er-15) and 3'-formyl-2',4',6'-trihydroxydihydrochalcone (Er-16). Compounds isolated from Eugenia rigida DC. Leaves Code Nature IUPAC name Common name Isolation Er-1 natural 5,7-dihydroxy-6-formylflavanone 6-formylpinocembrin new in nature Er-2 natural 5,7-dihydroxy-8-formylflavanone 8-formylpinocembrin new in nature Er-3 natural 2'-methoxy- 3'-methyl - 4',6'- dihydroxydihydrochalcone - new in nature Er-4 natural 3,4?,5-trimethoxy-trans-stilbene - New in species Er-5 natural Z-3,4,3?,5?-tetramethoxystilbene - New in nature but previously synthesized Er-6 natural E-3,4,3?,5?-tetramethoxystilbene - New in species Er-7 natural 3ß-Hydroxy-lup-20(29)-en-28-al Betulinal New in species Er-8 natural 3a-Hydroxy-lup-20(29)-en-28-oic acid a- Betulinic acid New in species Er-9 natural Lup-20(29)-en-3-on-28-oic acid Betulonic acid New in species Er-10 natural 3ß-Hydroxy-lup-20(29)-en-28-oic acid ß- Betulinic acid New in species Er-11 natural 7-Hydroxy-5-mthoxy-6-methylflavanone Comptonin New in species Er-12 natural 3a,19-dhydroxyurs-12-en-24,28-dicarboxyliic acid Diospyric acid New in species Er-13 natural 3a,19,24-trihydroxyurs-12-en-28-oic acid Barbinervic acid New in species Er-14 natural 2',6'-dihydroxy-4'-methoxy-5'-methylchalcone - New in nature Er-15 natural 3?-formyl-2?,4?,6?-trihydroxy-5?-methyldihydrochalcone - New in species Er-16 natural 3'-formyl-2',4',6'-trihydroxydihydrochalcone - New in species 5S synthetic (S) 5,7-dihydroxy-6-formylflavanone (S) 6-formylpinocembrin - 5R synthetic (R) 5,7-dihydroxy-6-formylflavanone (R) 6-formylpinocembrin - 6S synthetic (S) 5,7-dihydroxy-8-formylflavanone (S) 8-formylpinocembrin - 6R synthetic (R) 5,7-dihydroxy-8-formylflavanone (R) 8-formylpinocembrin - 7 synthetic 2?,4?,6?-trihydroxydihydrochalcone - - 7a synthetic 3?-formyl-2?,4?,6?-trihydroxydihydrochalcone - - 8 synthetic 2?,4?,6?-trihydroxychalcone - - 8a synthetic 3?-formyl-2?,4?,6?-trihydroxychalcone - - 1a synthetic 6-formyl-5,7-dihydroxyflavone 6-formylchrysin - 1b synthetic 8-formyl-5,7-dihydroxyflavone 8-formylchrysin - Chapter 2: Chemical modification of 18 flavonoid derivatives Each of the two flavanone derivatives 6-formylpinocembrin (Er-1) and 8-formylpinocembrin (Er-2), flavanone derivatives, each has a chiral center at C2, and was isolated during the phytochemical study of Eugenia rigida DC. leaves as a racemic mixture. They exhibited antifungal activity, herein, the synthesis of their enantiomers (R) and (S) of both Er-1 and Er-2 along with other products (2?,4?,6?-trihydroxydihydrochalcone (7), 3?-formyl-2?,4?,6?-trihydroxydihydrochalcone (7a), 6-formylchrysin (1a), 8-formylchrysin (1b), 2?,4?,6?-trihydroxychalcone (8), 3?-formyl-2?,4?,6?-trihydroxychalcone (8a) has been achieved. Chapter 3: Analytical study • Analysis of photoisomerization of E isomer (Er-6) to Z isomer (Er-5) of 3,4,3?,5?-tetramethoxystilbene In order to incresase the low yield of the sterically hindered Z-isomer of 3,4,3?,5?-tetramethoxystilbene (Er-5), an additional quantity of this compound was prepared from the more stable E isomer (Er-6) by photoisomerization at ?254 nm. A time dependent UV irradiation was carried out to study the conversion of E- (Er-6) to Z- (Er-5) isomers over a time range of 0 - 1000 min, where the yield of product Er-5 was quantified by UHPLC/APCI-MS. • Quantification of 10 standard compounds in Eugenia rigida DC. plant samples UPLC method was applied for the quantification of the ten selected compounds (Er-1, Er-2, Er-4, Er-5, Er-6, Er-8, Er-9, Er-10, Er-14 and Er-17) using two types of detectors: UV and MS. Results indicated that Er-6 (E)-3,3´,4´,5 tetramethoxystilbene is the major component in both Euginia rigida DC. leaves (4.98 µg/mg plant sample, methanol extract) and stems (0.35 µg/mg plant sample, methanol extract). The contents of the other compounds are in the range of (0.06-3.56 µg/mg leaves, methanol) and (0.05-0.35µg/mg stems, methanol). Compounds Er-4, Er-5, Er-8, and Er-14 were not detected in the stems. The low recovery rate of Er-8, Er-9 and Er-10 might be due to the low solubility of these compounds in methanol. Part II: Biological study The isolated and synthetic compounds were subjected to screening for potential antimicrobial, cytotoxic and antioxidant activities. • Antimicrobial activity The new compounds Er-1 and Er-2 were isolated in minute amounts, showed high antifungal activity against Candida glabrata, therefore synthesis of certain derivatives, in addition to their isomers was carried out to be able to study their structure-activity relationship. The results revealed that both the addition of a formyl group affected the antifungal activity of the tested compounds. • Cytotoxic activity . The results showed cytotoxic activity of Er-5 against HL-60, of KB, BT-459, HeLa and SK-MEL cell lines with IC50 values of 4.3, 4.3, 4.0, 3.6 and 4.3 µM, respectively. Compound Er-6 on the other hand, was weakly active against HL-60, HeLa and SK-MEL (IC50 33.3, 8.0 and 60.0 µM) . In addition, Er-4 was also weakly active as cytotoxic towards KB, BT-459, Hela and SK-MEL cells (IC50 17.7, 15.6, 13.3 and 12.2 µM, respectively). Compound, Er-5 was inactive against a noncancerous cell line (monkey kidney fibroblast; VERO) up to 33.3 µM, thus exhibiting selectivity toward the tumor cells. Er-5 was more potent than Er-6 as inhibitor of Stat3, Smad2/3, myc, Ets, Notch, and Wnt signaling. Ap-1 and NF-?B signalings were inhibited by both compounds Er-5 and Er-6 similarly and neither of them inhibited E2F or Hedgehog pathway activation at the tested concentrations. Similarly, the activation of the apoptotic mediator FoxO was not observed with either compound. Compound Er-5 was similar in potency to resveratrol for inhibiting signaling mediated by Stat3, Smad2/3, myc, Ets, and Notch while resveratrol was more potent for NF-?B and Hedgehog. Compound Er-4 was also tested and found to be inactive up to 100 µM. None of the compounds at the tested concentrations inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity or luciferase enzyme inhibition. Activity of synthetic compounds revealed that both presence and position of the formyl group in the flavanone compound is crucial for the activity, but there is no difference in activity between the different enantiomers. • Antioxidant activity: The inhibition of intercellular generation of reactive oxygen and nitrite species was measured to determine the potentials of the test compounds against oxidative and inflammatory stress in the cellular environment. The results obtained showed that compound Er-6 (the E-isomer of 3,4,3?,5?-tetramethoxystilbene) exhibited an inhibition of 50% in ROS generation at 33.3µM in PMA-induced HL-60 cells, while the Z-isomer did not exhibit any effect at the dose tested (100 µM), which might be due to the enhanced conjugation of aromatic ring in E, compared to Z, olefinic bond. However, they did not show any effect on iNOS activity in LPS induced macrophages (RAW264.7) up to 33.3 µM.