FURTHER PHARMACOGNOSTICAL AND BIOLOGICAL STUDIES ON THE FLOWERS OF OENOTHERA SPECIOSA NUTT. CULTIVATED IN EGYPT Taha S.M. El-Alfy, Hanaa H. Eid and Amany A. Sleem* Pharmagocnosy Department, Faculty of Pharmacy, Cairo University, Cairo. * Pharmacology Department, National Research Center, Giza, Egyt. Received:20-11-2007 Accepted:31-12-2007 Abstract subjected to biological evaluation as compared to The effect of the time of collection on the standard drugs. Their safety was ascertained phenolic content of the flowers of Oenothera through determination of their LD50. The speciosa Nutt., cultivated in Egypt, was studied. alcoholic extract exhibited more potent anti- Samples collected at the early (March) and late inflammatory, analgesic, anti-oxidant and anti- (July) flowering periods were analyzed. ulcer activities as compared to the aqueous Flavonoids (expressed as aglycones) and phenol extract. Meanwhile, HEM exerted a more acids were determined by HPLC in the pronounced anti-hyperglycemic action than the hydrolyzed extracts. Samples gathered in March alcoholic extract. The alcoholic extract, showed a relatively high percentage of flavonoids moreover, revealed noticeable antibacterial and (407.05 mg/100g dry wt.) which decreased in the antifungal activities, while those observed for the July sample, amounting only to 180.20 mg/100g aqueous extract were only moderate. dry wt. On the contrary, an increase was Finally, the macro- and micro- observed in the phenol acid content which morphological characters of the flowers are reached 201.89 and 548.59 mg/100g dry wt. in described with the aim to provide useful data for the two samples, respectively; however, the identification and differentiation of the plant from qualitative pattern appeared the same for all other allied species either in the entire or constituents. In addition, gravimetric and powdered form. spectrophotometric determinations of tannins and proanthocyanidins revealed that both were INTRODUCTION higher at the late flowering period (10.53 g% and 23.35g%, respectively) than at the early one Onagraceae (Evening Primrose family, (8.67g% and 19.03g%, respectively). Oenotheraceae)(1,2) comprises a number of Two flavonol glycosides; hyperoside popular flowering ornamentals, including (quercetin-3-O--β-D galactoside) and rutin Oenothera species which are native to North and (quercetin-3-O-α-L-rhamnose-β-D-glucoside) South America. These are commonly known as and the aglycone, quercetin, as well as a phenol "Evening Primroses, Suncups or Sundrops" and acid, chlorogenic acid, were isolated via are widely cultivated in borders and gardens, the chromatographic fractionation of the ethyl most reputed being Oenothera speciosa Nutt. acetate extract. (+) Catechin, isolated from the (Hartmannia speciosa Small) (1, 2). The plant is acetone extract, constituted the major component usually referred to as "White, Pink or Mexican of the tannin fraction. Characterization of the Evening Primrose" and "Showy Evening isolated compounds was achieved through Primrose or Sundrops" (speciosa meaning physical, chemical, chromatographic and spectral spectacular or showy) (3, 4). The common name analyses, as well as, by comparison with available "Day-flowering Evening Primrose" is attributed authentic samples. to the plant due the opening of its flowers during Moreover, the carbohydrate content was day light hours(5) (c.f. other "Evening investigated by PC and HPLC. Rhamnose, Primroses"). arabinose and glucose were identified as free Previous reports focusing on the sugars. Analysis of the hydrolyzate of the cold pharmacological and clinical evaluation of O. extracted mucilage (CEM) revealed the presence biennis L. (Evening primrose) are numerous and of galacturonic, glucuronic acids and galactose claimed its potentialities for the treatment of in addition to the aforementioned sugars. atopic eczema, rheumatoid arthritis, premenstrual Likewise, the hydrolyzate of the hot extracted syndrome, mastalgia, diabetic neuropathy, mucilage (HEM) differed from that of (CEM) in arteriosclerosis, asthma and psoriasis (6 , 7). containing xylose and appearing free from Although the Onagraceae plants are arabinose. known as accumulators of polyphenols (8), there The aqueous and alcoholic extracts, as are relatively few reports on the phenolic well as, the hot extracted mucilage (HEM), were composition of Oenothera species (8- 13). Several Bull. Fac. Pharm. Cairo Univ., Vol. 45, No. 3 (2007) 185 phenol acids and flavonol glycosides were glass containers for further phytochemical and detected by two dimensional TLC and used as microscopical examination. chemotaxonomic markers, in addition delphinidin and cyanidin were identified (8). This publication Material for Phytochemical Investigation dealt also with the variability in the phenolic Different types of silica gel (E. Merck, content among the various organs of three Darmstadt, Germany) were used as stationary Oenothera species (other than Oenothera phases including: precoated silica gel 60 F254 speciosa Nutt.). In a similar way, Quercitin-3-O- plates for thin layer chromatography (TLC); rhamnoside, Myricetin-3 -O- rhamnoside and Silica gel H for vacuum liquid chromatography Myricetin-3 -O-galactoside were detected in the (VLC) and Silica gel 60 for column leaves of Oenothera speciosa Nutt.(9); meanwhile, chromatography (CC). Sephadex LH-20 for CC was the unique available report on isolation and obtained from Pharmacia Fine Chemical AB structure elucidation was on Myricetin 3-O- (Uppsala, Sweden).Whatman No 1 sheets for methyl ether-3'-O-β-D-glucoside from the leaves paper chromatography (PC) were purchased from of the plant (14). On the other hand, nothing could Whatman Ltd. (Maidstone, Kent, England). be traced in the available literature concerning the carbohydrate composition of any of the plant The following solvent systems were prepared organs including the flowers. In a previous from analytical grade chemicals: communication (15), the authors investigated the S1: n-Butanol-Acetic acid-Water (3:1:1 v/v/v) floral volatiles and lipoids; the present work [PC]; S2: Acetic acid -Water (15:85v/v) [PC]; S3: focuses on the phenolic and carbohydrate Ethyl acetate-Acetic acid-Formic acid-Water constituents of the same organ. (100:11:11:26 v/v/v/v) [TLC]; S4: Chloroform- Furthermore, the bioactivities of the Methanol-Water (65:35:10 v/v/v) [TLC]; S5: aqueous and alcoholic extracts of the root, stem Chloroform-Methanol (8:2 v/v) [TLC]; S6: n- and leaves (16); as well as, those of the total and Butanol-Acetic acid-Water (4:1:5 v/v/v upper fractionated hexane extracts of the flowers (15), phase) [PC]; S7: n- Butanol- Pyridine- Water were previously evaluated by the authors. The (6:4:3 v/v/v) [PC]. present report includes a similar investigation of the aqueous and alcoholic extracts of the flower. Spray reagents used for spot visualization were: Finally, the macro- and micro- R1: Aluminum chloride: for flavonoids (17); R2: morphological characters of the flowers are Natural products-polyethylene glycol reagent described with the aim to provide useful (NP/PEG) for phenolics (18); R3: Ferric chloride: additional data for identification and for phenolics (19); R4: Vanillin/HCl: for catechins differentiation of the plant from other allied (18); R5: Aniline phthalate: for sugars (17) and R6: species either in the entire or powdered form as Anisaldehyde-Sulphuric acid (17). those previously presented by the authors for the other organs (16). Reference phenolics, sugars and uronic acids were purchased from E. Merck (Darmstadt, EXPERIMENTAL Germany) and included: luteolin, kaempferol, quercetin, apigenin, naringenin, quercetin-3-O- Plant Material glucoside, quercetin-3-O-galactoside, quercetin- Flowers of Oenothera speciosa Nutt. were 3-O-rhamnoside, quercetin-3-O-rutinoside; collected from plants cultivated in the Experimental chlorogenic, caffeic, ferulic and rosmarinic acids; Station of Medicinal Plants, Pharmacognosy (+) catechin and (-) epicatechin, in addition to Department, Faculty of Pharmacy, Cairo University, glucose, galactose, xylose, rhamnose, arabinose Giza, Egypt, during the flowering stage from March and mannose; glucuronic and galacturonic acids. to July (2004-2006). The plant was kindly authenticated by Mrs. Therese Labib, Herbarium Shift reagents for UV spectroscopic analysis of Section, Orman Garden, Giza, Egypt. Identity flavonoids were prepared according to published was confirmed by Dr. Mohamed El Gebali (Plant procedures (20). Taxonomy and Egyptian Flora Department, National Research Center, Dokki, Giza, Egypt). Material for Biological Evaluation: Voucher samples are kept at the Museum of the Plant extracts were, separately, prepared Pharmacognosy Department, Faculty of each from a 200 gm air-dried powdered sample. Pharmacy, Cairo University. The aqueous and alcohol 70% extracts were Fresh samples kept in 70% ethanol prepared by cold exhaustive percolation. The containing 5% glycerin were used for the solvent in the first case removed by lyophilization botanical study and air-dried flowers (reduced to and in the second by distillation under reduced powder No. 36) were saved in amber coloured pressure. One gram of the solvent-free dried residue was equivalent to 7.4 g and 7.8 g of the Bull. Fac. Pharm. Cairo Univ., Vol. 45, No. 3 (2007) 186 air-dried powdered flowers for the aqueous and MHz, respectively, on a Varian (Model L 900) alcoholic extracts, respectively. For antimicrobial spectrometer (Germany), using TMS as internal screening, the extracts