Further Pharmacognostical and Biological Studies on the Flowers of Oenothera Speciosa Nutt

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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

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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

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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 were dissolved in DMSO standard and DMSO-d6 as solvent. HPLC at concentrations of 200 mg/ml (so that each 50 analysis of phenolics was performed on an μl contained 10 mg of the extract). Agilent Series 1100 apparatus equipped with Quaternary pump, series 1100; degaser, series Animals used in the toxicological and 1100; column heater, series 1100 and a UV pharmacological evaluation were obtained from detector series 1100. The apparatus used for the animal house, of the National Research HPLC analysis of carbohydrates consisted of an Center, Dokki, Giza, Egypt; these included albino isocratic pump (Model Lc-l0As, Shimadzu, mice (25-30 g) and adult male albino rats Japan); refractive index detector (RID-6A, Shimadzu, (Sprague Dawley strain, 130-150g). They were Japan). A Rheodyne injector (Model 7161, Catati, kept in metabolic cages and fed on standard California, U.S.A.) equipped with 20 µl injector loop laboratory diet composed of vitamin mixture and an integrator (Model C-R 7A, Shimadzu, Japan), (1%), mineral mixture (4%), corn oil (10%), Phenom-Dim, 250x 460, Ser: 128854, 20 µg injected. sucrose (20%), cellulose (0.2%), casein (10.5%) The sensitivity was set as 0.001 AUFS. and starch (54.3%). Methodology Microorganisms used in the antimicrobial testing A. Investigation of the phenolic content were provided by the Microbiology Department, HPLC analysis of the phenolic constituents: Faculty of Pharmacy, Cairo University, Cairo, Egypt The phenolic composition of the flowers and included: Escherichia coli, Pseudomonas collected in March (sample A, early flowering aeruginosa, Proteus vulgaris, Staphylococcus period) was qualitatively and quantitatively aureus, Sarcina lutea, Bacillus subtilis, analyzed by HPLC as compared to a sample Mycobacterium phlei, Candida albicans and collected in July (sample B, late flowering Candida tropicalis. period). Phenolics including flavonoids (expressed as their aglycones) and phenol acids Standard drugs, kits and culture media were were determined in the hydrolyzed-flower supplied by their respective sources as follows: extracts adopting the procedure described by Indomethacin (EIPICO, Egyptian International Mattila et al. (2000) (21). One gram of the air- Pharmaceutical Industries Co., Egypt; as standard dried plant material was weighed into a 100 ml anti-inflammatory); Carrageenan (Sigma Co., conical flask then dispersed in 40 ml of aqueous Cairo, Egypt; for induction of oedema); Dipyrone methanol (62.5%). The mixture was then (Novalgin®, Hoechst (Sanofi Aventis), Orient, ultrasonicated for 5 min. To this extract 10 ml of Egypt; as standard analgesic); Paracetamol 6 M HCl were added. The flask containing the (Panadol®, Schering, Germany; as standard mixture was placed in a shaking water bath at antipyretic); Alloxan monohydrate (Sigma, USA ; 90°C for 2 hours. After hydrolysis, the sample for induction of diabetes); Glimepiride [Amaryl ®, was allowed to cool, then filtered, made up to Hoechst (Sanofi Aventis), Marion, Roussel, S.A.R.; 100 ml with methanol, and ultrasonicated again as standard antihyperglycemic) ; Glutathione kit for 5 min. Before injection into the HPLC (Wak-Chemie Medical, Germany; for assessement of apparatus, the sample was filtered through a 0.2 antioxidant activity) and Vitamin E (dl α-tocopheryl µm membrane filter into the sampler vial for acetate, Pharco Pharmaceutical Co.; as standard injection. HPLC separation was performed on a antioxidant). Trypticase soy agar (Oxoid, England) Hypersil-ODS (4.6x250 mm, 5µm) column. was used as nutrient medium, Ofloxacin (OFX) and Isocratic elution was employed using acetonitrile Amphotericin B (AMP.B), 5 μg/disc each (Oxoid, /15% acetic acid (40/60 v/v) as mobile phase. England); as standard antimicrobials. The flow rate of the mobile phase was 1 ml/min. and the injection volumes were 40 µl of the Apparatus standards and sample extracts. The standards Melting points were determined on a were made by diluting stock standards in MeOH Buchi 520 apparatus. UV lamp (254, 366 nm) to yield 50µg/ml. Detection was carried out by a Model ENF-260 CIF USA was used for UV detector set at 330 nm. localization of fluorescent spots on the The major components of the sample chromatograms. The UV spectra of flavonoids were identified by comparing their retention and tannins were determined on a Beckman Du-7 times and spectral data with those obtained for and Shimadzu 265 C spectrophotometers. Mass the standards (viz., quercetin, kaempferol, Spectra of the isolated compounds were recorded luteolin, naringenin, apigenin, chlorogenic acid, on Varian Mat 711, Finnigan SSQ 7000 and caffeic acid, ferulic acid and rosmarinic acid). OMM 7070t mass spectrometer. The 1H- and The identified flavonoids and phenol acids were 13C-NMR spectra were recorded at 300 and 100 quantified using the external standard method.

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Quantification was based on the peak areas of sugar and aglycone moieties were examined by both standards and samples. Stock standards PC using S6 and S7 as solvent systems and the (diluted in methanol, 20-600µg/ml) and samples chromatograms visualized by spraying with R5 were analyzed in duplicate. Results are recorded and R6. in Table (1). Determination of tannin content: The tannin Preparation and fractionation of the ethyl content was determined gravimetrically adopting acetate extract: An air-dried powdered flower the hide powder method (23). Furthermore, sample (1 Kg) was exhaustively extracted by cold proanthocyanidins (condensed tannins) were percolation with ethanol 70% at room determined adopting a chemical assay in which, temperature. The ethanol extract was proanthocyanidins are oxidatively depolymerized concentrated to dryness under reduced pressure at in butanol - HCI mixture to yield red a temperature not exceeding 50°C. The residue anthocyanidins that can be measured (116 g) was then suspended in water and spectrophotometrically at 550 nm (24). Results are partitioned with petroleum ether and then ethyl represented in Table (2). acetate. The solvents were, separately, evaporated under reduced pressure to yield 26 g and 47 g, Extraction and isolation of catechins: The respectively. The ethyl acetate extract was dried powdered flowers (500 g) were defatted investigated for its phenolic content using TLC with hexane; the marc was air dried and then and CC. TLC screening was achieved on exhaustively percolated with 50% acetone (v/v) precoated silica gel plates using different solvent on cold. The combined acetone extractives were systems (S1-S3) alongside with reference pooled and mixed, followed by removal of the samples. The chromatograms were visualized solvent under vacuum at a temperature not under visible and UV light (254 and 366 nm) exceeding 40°C to yield 11g. The obtained before and after exposure to ammonia vapour, as residue was investigated for its tannin content well as, after spraying with R1-R3 and R6. using TLC and CC. TLC-screening was carried Twenty g of the ethyl acetate extract was out using precoated Si-gel plates, two spots were fractionated on a vacuum liquid chromatography detected [major spot; Rf: 0.89 and 0.52; and column (VLC), packed with silica gel H. minor spot: Rf: 0.61 and 0.25 using S4 and S5 Gradient elution was performed using CHCl3, as solvent systems, respectively). The EtOAc and MeOH mixtures of increasing chromatograms were examined under UV light polarity. Fractions of 200 ml were collected and (254 and 366 nm) before and after exposure to monitored by TLC using precoated silica gel ammonia vapour, as well as, after spraying with plates and S2 and S3 as solvent systems. The R3 and R4. The crude acetone extractive (5 g) chromatograms were examined as previously was fractionated on a Sephadex LH-20 column mentioned. Fractions showing similar and eluted with methanol. Fractions (15 ml, chromatographic patterns were pooled to afford each), were collected and monitored by TLC three collective fractions (I-III). Fraction I (92 using S5 as solvent system and the mg) eluted with 90% -85% CHCl3 in EtOAC, was chromatograms were visualized as previously repeatedly purified on a sephadex LH-20 column, mentioned. The fractions (2.4 g), containing the using methanol as eluent and yielded compound major component (Rf: 0.52; TLC, S5) were 1 (52 mg). Fraction II (190 mg) eluted with 10- pooled and repeatedly chromatographed on 5% CHCl3 in EtOAC, was subjected to precoated silica gel plates (PTLC), elution was rechromatography on a sephadex LH-20 column performed with S4. Further purification on and eluted with methanol then followed by Sephadex LH-20 column, using methanol as purification using preparative TLC and S3 as eluent, afforded compound 5 as a white powder solvent system to afford compound 2 (41 mg). (61 mg). Structure elucidation was established Fraction III (260 mg) was eluted with 75%-60% on the basis of physical and spectral analyses EtOAC in MeOH; an aliquot of this fraction (130 (UV, 1H-NMR and 13C-NMR). mg) was rechromatographed on a sephadex column, eluted with MeOH: H2O mixtures (80%- B. Investigation of carbohydrate content (25): 50%) to afford compound 3 (23 mg) and A fresh sample of the flowers (100 g) was compound 4 (66 mg). minced and homogenized with ethanol 70% in a The isolated compounds were subjected to blender, filtered and the filtrate was concentrated tests for flavonoids, carbohydrates and /or under vacuum and then lyophilized. One gram of glycosides, as well as physical and spectral the residue was dissolved in pyridine and analyses (UV spectral data with different shift filtered. The filtrate was evaporated and saved reagents, IH-NMR, I3C-NMR and EIMS). for further analysis of the free sugars. The Compounds 2 and 4 gave positive Molisch's test mucilage was prepared by macerating the marc and were subjected to acid hydrolysis (22). The in cold water slightly acidified with hydrochloric

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acid (pH 3.5) till exhaustion, followed by used as a negative control. Discs of Ofloxacin extraction with hot water (90-95°C) until (OFX) and Amphotericin B (AMP.B), 5 μg complete extraction of the mucilage. The g/disc each were used as positive controls. The mucilage, in each case, was precipitated by inhibition zones were measured and recorded in addition of 4 volumes of ethanol 95% to each Table (12). volume of the aqueous extract to yield two different samples of mucilage; the cold RESULTS AND DISCUSSION extracted-mucilage (CEM) and the hot extracted- mucilage (HEM) which were carefully dried and The preliminary phytochemical screening, kept in a desiccator. About 0.5 g of each sample carried out in a previous publication (16) (CEM and HEM) was separately hydrolyzed. ascertained the presence of flavonoids, tannins The mucilage hydrolyzates were evaporated to and carbohydrates in the flowers of Oenothera dryness under vacuum. Analyses of the free speciosa Nutt. , cultivated in Egypt. sugars and mucilage hydrolyzates were achieved by PC (solvent systems S6 and S7, spray reagent A. Investigation of the phenolic content R5) and HPLC. The latter was carried out by The results of HPLC analysis of the separately dissolving the free sugars and hydrolyzed methanol extracts of the flowers, as mucilage hydrolyzates (0.2 g, each) in 2 ml of displayed in table 1, revealed that its phenolic acetonitrile-water (75:25), then 20 µl were composition was greatly influenced by the time injected through the Rheodyne injector on a high of collection. Although, qualitative variation was pressure Kromasil 10 NH2 column (250 x 4.6 not evident among the major phenol acids and mm). The sugars were eluted with acetonitrile- flavonoid components, yet, quantitative variation water as a mobile phase, 1.5 ml/min as flow rate, was obvious. Constituents identified were retention times and peak areas were determined chlorogenic, caffeic, ferulic and rosemarinic using refractive index detector and C-R7A acids, as well as, quercetin, naringenin and model integrator. Qualitative and quantitative kampferol. The amount of phenol acids increased identifications of peaks were carried out by from 201.89 to 548.59 mg/100g DW during the comparison with authentic sugars analyzed under flowering season with concomitant increase in the the same experimental conditions. Results of PC amount of chlorogenic and caffeic acids which and HPLC analyses are recorded in Tables (3) reached up to 300.21 and 204.44 mg/100g DW, and (4), respectively. respectively, at the end of the flowering stage (sample B). On the contrary, that of ferulic and C. Biological evaluation rosemarinic acids was reduced. Concerning the Toxicological and Pharmacological flavonoidal constituents (expressed as their studies of the aqueous and alcoholic extracts, as corresponding aglycones), they showed a marked well as, the hot extracted mucilage (HEM) of the decrease, along the flowering season, to less than flowers of Oenothera speciosa Nutt. were carried half the total concentration in sample A, collected out, including determination of the median lethal at the early flowering period (from 407.07 to dose (LD50) according to Karber's procedure 180.02 mg/100g DW) with a parallel decrease in (1941) (26). Furthermore, the pharmacological the individual aglycone components which were potentialities of the aqueous and alcoholic in both samples dominated by quercetin. extracts viz., anti-inflammatory (27), analgesic (28), anti-ulcer (29), antipyretic (30) and antioxidant (31) Chromatographic fractionation of the activities were assessed. In addition, the acute ethyl acetate extract allowed the isolation of and long-term anti-hyperglycemic activity (32, 33) four major compounds 1-4 which were of HEM and alcoholic extract were evaluated. characterized through determination of their The effects produced were, in each case, physico-chemical and spectral data as follows: compared to those of appropriate reference drugs. Compound 1: 52 mg, yellow needle crystals o Results obtained for the biological activities are (CHCl3), 21 mg, m.p. 314 - 316 C, soluble in compiled in Tables (5-11). methanol; Rf 0.75 and 0.64 (TLC, S5 and S6, The experimental data were, in each case, respectively), yellow (UV, UV/NH3 and AlCl3), statistically analyzed using student's "t" test orange (NP/PEG). UV λ max nm (MeOH): 256, according to Snedecor and Chochran (1971) (34). 269 (sh), 301 (sh), 372; (NaOMe) 247 (sh), Moreover, the aqueous and alcoholic 330(dec); (AlCl3) 269, 308 (sh), 335, 455; extracts were subjected to antimicrobial (AlCl3/HCl) 272, 304 (sh), 352, 429 (NaOAc) screening by applying the disc agar diffusion 258 (sh), 268, 328, 398; (NaOAc /H3BO3) 259, method (35, 36). Aliquots (50 μl) of DMSO, 303 (sh), 386. solutions of the different extracts (equivalent to Compound 2: 41 mg, yellow powder; soluble in 10 mg of each extract) were separately tested. methanol; m.p. 215-217°C; Rf: 0.43 (PC, S2); A disc impregnated with 50 μl of DMSO was and 0.61 (TLC, S3), respectively; purple (UV);

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yellow (UV/NH3); orange (NP/PEG); UV λmax extract afforded compound 5 which was nm, MeOH: 259, 267 sh, 299 sh, 369; NaOMe: characterized as follows: 272, 325 sh, 412; AlCl3: 271, 303 sh,. 328 sh, Compound 5: 61 mg; white powder; soluble in 437; AlCl3/HCl: 272, 305 sh, 368, 408; NaOAc: methanol and acetone; m.p. 175-177°C; Rf: 0.89 270, 324 sh, 375; NaOAc/ H3BO3: 260,304 sh, and 0.52 (TLC, S4 and S5), respectively; 378. Acid hydrolysis afforded the aglycone yellowish-brown (UV), dark brown (UV/NH3), moiety which was identified as quercetin by violet (FeCl3); pink (vanillin/HCl). UV λmax direct comparison with an authentic sample (m.p., nm, MeOH: 276, 288 sh; NaOMe: 300,310 sh; m. m. p. and co-chromatography). The sugar AlCl3: 280, 300 sh; AlCl3/HCl: 275,288 sh; moiety was identified as galactose by PC NaOAc: 276, 310 sh; NaOAc/H3BO3: 282, 310 1 alongside with authentic samples. sh. H-NMR (DMSO-d6) δ ppm; 9.17 (1 H, s, 3'- Compound 3: 23 mg, white needles; soluble in OH), 8.96 (1 H, s,4'-OH), 8.51 (2 H, s, 5,7-OH), methanol, ethanol and acetone; m.p. 207-209°C; 6.84 (1 H, d, J 2',6' =1.5 Hz, H-2'), 6.75 (1 H, d, Rf: 0.68 (PC, S2) and 0.52 (TLC, S3), J 6',5'= 8.1 Hz, H-5'), 6.70 (1 H, dd, J 6',2'= 1.8, respectively; blue fluorescence (UV); yellowish- J 6',5' = 8.0 Hz, H-6'), 5.93 (1 H, d, J 8,6=2.4 Hz, green (UV/NH3), blue (FeCl3); blue fluorescence H-8), 5.86 (1 H, d, J 6,8=2.2 Hz, H-6), 4.74 (1 H, (NP/PEG); UV λ max nm, MeOH: 244, 298 sh, m, H-3), 4.56 (1 H, d, J 2,3 trans = 7.5 Hz, H-2), 328; NaOMe: 240, 265, 305 sh, 379. EIMS: m/z 3.94 (1 H, bs, 3-OH), 2.88 (1 H, dd, J=5.9, (rel. inten.): 354[M+, 15%], 336[M+-18], 16.2gem Hz, H-4 ax), 2.46 (1 H, dd, J=8.9, 13 180[caffoeyl, 90%], 163[100%], 162[caffoeyl-18, 16.5gem Hz,H-4 eq). C-NMR (DMSO-d6) δ 52%], 143[40%], 110[60%]. ppm, 157.8(C-7), 157.5 (C-5), 156.9 (C-9), Compound 4: 66 mg, yellow powder; soluble in 146.2 (C-3'), 146.2 (C-4'), 132.2 (C-l'), 120.0(C- methanol; m.p. 192-195°C; Rf: 0.56 (PC, S2) 6'), 116.1 (C-5'), 115.3 (C-2'), 100.8 (C-10), 96.3 and 0.42 (TLC, S3) , respectively; deep purple " (C-6), 95.6 (C-8), 82.9 (C-2), 68.8 (C-3), 28.5 (UV); yellow (UV/NH3 and UV/AlCl3); UV (C-4). From the previous findings and through (13, 20, 37, 39, 40, 42) λmax nm, MeOH: 258, 300 sh, 358; NaOMe: comparison with published data , 270, 328 sh, 410; AlCl3: 277, 305 sh, 430; compound 5 could be identified as (+)Catechin. AlCl3/HCl: 272, 298, 362 sh, 398; NaOAc: 268, 320 sh, 392; NaOAc/H3BO3: 262, 308 sh, 386. B. Investigation of carbohydrate content Acid hydrolysis produced quercetin as aglycone The ethanolic extract obtained from the (direct comparison with authentic sample, m.p., fresh flowers yielded a residue (13.68%) which m. m. p. and co-chromatography). The sugar gave a positive Fehling's test suggesting the moiety consisted of glucose and rhamnose presence of reducing sugars.The polysaccharides (identity confirmed using PC alongside with prepared by both cold and hot extraction authentic reference samples). methods viz., CEM (7.48 %) and HEM (12.78 Compounds 1-4 were identified as: quercetin, %) were soluble in water, insoluble in alcohol hyperoside (quercetin-3-O--β-D galactoside), and ether; they gave positive tests for mucilage chlorogenic acid and rutin (quercetin-3-O-α-L- (red stain with ruthenium red) (43) and negative rhamnose-β-D-glucoside), respectively by direct tests for pectin (44). The percentage of mucilage comparison with authentic samples and through extracted by the hot process exceeded that physical, chromatographic and UV spectral data obtained by the cold extraction method. before and after addition of different shift Paper chromatography and HPLC analysis reagents, identification was confirmed by (Tables 3 and 4) revealed the occurrence of comparison with published data (20, 22, 37-40). rhamnose, arabinose and glucose in the free state in the ethanolic extract. Rhamnose dominated the Results of the gravimetric determination of identified sugars in all the tested samples viz., tannins by the Hide Powder method (23) and those the ethanol extract, and both CEM and HEM of the spectrophotometric estimation of the hydrolyzates (55.63 %, 34.29 % and 29.67 %, proanthocyanidins (24), as recorded in table 2, respectively), followed by glucose (13.41 %, revealed that both were higher when the flowers 23.68 % and 14.77 %, respectively). Meanwhile, were collected at the end of the flowering season arabinose was detected in the ethanol extract and (sample B, 10.53 g% and 23.35g%, respectively) CEM hydrolyzate (15.47 % and% 8.12 %); and being present in lesser amounts in the early stage galactose in both CEM and HEM hydrolyzates (8.67 g % and 19.03 g%, respectively). The low (3.15 and 4.47% respectively). On the other results obtained by the gravimetric method are hand, xylose could be identified in the HEM probably due to the inability of certain tannin hydrolyzate only (13.12 %). Glucuronic and molecules to penetrate the hide and associate with galacturonic acids were detected in both proteins (41). hydrolyzates. Glucuronic being present in a noticeably higher amount in CEM hydrolyzate Chromatographic fractionation of the acetone (18.14%) than in that of HEM (8.09%).

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From the above results, it could be flavonols (50) in both extracts. concluded that there is an obvious quantitative Concerning the anti-hyperglycemic variation, although slight qualitative differences activity (Table 10), HEM and the alcoholic are observed, in the composition of the mucilage extract exhibited significant effects in alloxan- hydrolyzates prepared by the two different induced diabetic rats, which was time dependant. methods. Finally, the reasonable detected The potency of HEM was higher than that of amounts of free sugars and mucilage could alcoholic extract after four hours oral drug suggest the use of the flower extracts as an administration (62%and 57%) and was more adjuvant in nutraceuticals. pronounced after six hours (67% and 64%), respectively, compared to glimepiride. Likewise, C. Biological evaluation daily administration (long-term effect) of HEM The aqueous and alcoholic extracts, as and alcoholic extract, at a dose of 100 mg/kg. well as, HEM of the flowers were found to be b.wt. (Table 11), revealed that HEM reduced non toxic at the tested doses as no mortality was blood glucose levels in alloxan-induced diabetic observed when given to mice orally in doses up rats with a potency of 77%, while the alcoholic to 9.1, 9.3 and 7.4 g/Kg. b. wt, respectively and extract exhibited a weaker action, being 62% as are considered to be safe according to Buck et al. potent as that of glimepiride after eight weeks of (1976) (45). treatment, respectively. Thus, we may conclude Both aqueous and alcoholic extracts exerted that HEM was more effective regarding the acute (Table 5) a remarkable anti- inflammatory action and long-term activity in the tested dose-level. reaching up to 89% and 95% that of indomethacin, The results obtained here are in agreement with respectively). The alcoholic extract appeared almost previous reports confirming that polysaccharides as effective as indomethacin, the standard anti- (51) (HEM) and polyphenolic compounds (52) inflammatory. (alcoholic extract) showed anti-hyperglycemic The analgesic effect of the aqueous and effect. alcoholic extracts of the flowers (Table 6) was Results obtained on evaluation of the time dependent. The aqueous extract produced a antimicrobial activity (Table 12) showed that marked action (with a potency 66% that of the alcoholic extract of the flowers of Oenothera Novalgin after one hour, and 77 % after two speciosa Nutt. exerted significant inhibitory hours, respectively), meanwhile, a higher action effect towards Staphylococcus aureus, was shown by the alcoholic extract after one and Mycobacterium phlei, Pseudomonas aeruginosa, two hours (with a potency 76 % and 83 % that of a marked effect on both Candida albicans and Novalgin, respectively). The anti-inflammatory Candida tropicalis and little effect on the other and analgesic properties may be attributed to the tested microorganisms. Meanwhile, the aqueous presence of flavonoids (46) and tannins (47). extract showed weak effect against the tested Results obtained from Table (7) showed microorganisms and no effect on Escherichia that both extracts had marked anti-ulcer activity. coli. From the previous findings, it is evident that The alcohol extract significantly decreased the the alcoholic extract is more powerful than the number of ulcers by 72.49% protection, while the aqueous extract. aqueous extract showed a moderate protection of It is worthy to note that the biological percentage 67.73% compared to indomethacin. potentialities of the flower exceeded those of the The flavonoids (48) and (+) catechin (49) may other organs when similarly assessed (16). contribute to the anti-ulcer activity of both From the aforementioned data, the hot extracts. extracted mucilage (HEM), aqueous and The aqueous and alcoholic extracts exhibited alcoholic extracts could be expected after further a pronounced and powerful antipyretic action of laboratory and clinical trials, as successful potency comparable to that of paracetamol (84%, candidates for herbal formulations prescribed for 100% after one hour and 89%, 88% after two hours, complementary treatment of a wide variety of respectively, Table 8). ailments such as inflammations, arthritis, Both tested extracts possessed a powerful diabetes mellitus and microbial infections, as antioxidant activity (Table 9). Animals treated well as, in management of painful processes, with Vitamin E (7.5 mg/Kg b.wt.) restored the prevention of stress ulcer formation, and as level of blood glutathione in diabetic rats (% powerful antioxidants. change from control= 1.4%). The level of blood glutathione in diabetic rats was restored after the BOTANICAL DESCRIPTION oral administration (10 mg/kg b.wt.) of the ethanolic and aqueous extracts of the flowers (% Oenothera speciosa Nutt. is a perennial change from control=1.6% and 2.7%, herb, reaching up to 65 cm in height and respectively). This effect may be due to the cultivated for its showy flowers. The plant synergestic action of proanthocyanidins and blooms from March to July. Flowers are solitary,

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axillary, sessile, few to several in the upper axils, The filaments (Fig. 1B) are yellow in colour; with a showy, white to pink corolla. The buds in filiform, glabrous; the free parts are unequal in the stem tips are nodding, lanceolate to lance- length (with the shorter ones inwards, measuring elliptic in shape and showing acute to acuminate 0.9-1.3 cm in L. and the longer ones outwards apices. and measuring 1.2-1.8 cm in L.).

Macro-morphology The Hypanthium (Fig.1B) is slender, The flower (Fig. 1A&B) is cup-shaped quadrangular and light green in colour measuring and appears pinkish- white to pink in colour, 1.0 - 2.7 cm in L. and 0.15 - 0.3 cm in D. The actinomorphic, hermaphrodite, epigynous and hypanthium adnates to the ovary and is tetramerous. The flower has a hypanthium prolonged above it forming a funnel-shaped tube, (Bailey, 1958) which adnates to an inferior ovary which is as long as or sometimes longer than the and is prolonged beyond its apex, bearing the ovary, getting wider towards the upper third and sepals, petals and stamens. It has a delicate measures 0.5-0.9 cm in L. and about 0.2-0.4 cm fragrant odour and an astringent, mucilaginous at the widest part. This tube bears the fused bases taste. The flower measures from about 4.0- 6.0 of the sepals, petals and stamens. cm in D. The ovary (Fig.1B) is inferior, tetracarpellary, The bract (Fig.1B) is small, persistent, sessile, tetralocular, syncarpous and fused with the green, lanceolate and rather rhomboidal in shape hypanthium. It measures 0.5 - 0.8 cm in L. and with broadly dentate margin, acuminate apex and 0.27 - 0.39 cm in W. at the prominent ridges pinnate venation. It has faint characteristic odour (widest part). The ovules are numerous, having and a slight astringent, mucilaginous taste. It an axile placentation. measures 0.7 - 1.6 cm in L. and 0.4 – 0.6 cm in W. The style (Fig.1B) is simple, cylindrical, long, filiform, greenish near the base, whitish near the The calyx (Fig. 1B) is yellow-green in colour apex and passing through the hypanthium. It and often with reddish margins. It consists of four measures 2.5 - 3.5 cm L. and 0.5-1.0 mm D. narrow lobes which cohere together to form the calyx tube. The calyx tube (1.5-3.2cm in L. and The stigma (Fig.1B) is creamy white in colour, 0.4-0.5cm in D.) shows closely parallel lobes composed of 4 linear radiating lobes, each with free acuminate tips (0.1–0.5 cm in L.) in the measuring 4 to 7 mm in L. unexpanded buds but is strongly reflexed to the The flower has the following floral formula: upper surface and pulled to one side in the open , ♀, K (4), C 4, A 4+4, G (4). flower. It has a faint characteristic odour and an astringent mucilaginous taste. Micromorphology

The corolla (Fig. 1B) is epigynous, consisting of The Bract (Fig. 2): four free petals (convolute in the bud), alternating A transverse section in the bract (Fig. 2A) with the sepals. The petals are pinkish- white to is plano-convex, consisting of an inner and outer pink in colour, yellowish at the base, obcordate or epidermis, enclosing the mesophyll which broadly obovate, smooth with entire margins, consists of palisade-like and oval emarginated apices, arising from the rim of the parenchymatous cells with relatively wide hypanthium and fused at the base into a very intercellular spaces and is traversed by 4 to 5 short tube which is in turn joined to the sepals. small vascular strands. The midrib is prominent Each petal measures 2.1 – 4.2 cm L. and 2.2-3.6 on the lower surface and shows a collateral cm W. at the widest part. It has a faint aromatic vascular bundle which is accompanied by a odour and an astringent mucilaginous taste. scanty perimedullary (intraxylary) phloem. Some parenchyma cells contain mucilage (red stain The andrœcium (Fig. 1B) consists of 8 with ruthenium red) in which are embedded yellowish, unequal stamens distinctly arising near bundles of raphides of calcium oxalate. the hypanthium rim, arranged in two whorls. The outer stamens are longer, alternating with the The inner (upper) epidermis (Fig. 2B&D) petals, while the inner ones are shorter, being consists of polygonal, axially elongated cells, antepetalous and adnate to the base of corolla. having wavy anticlinal walls covered with thin smooth cuticle. Stomata of anomocytic types The anther (Fig. 1B) is yellow in colour, oblong (rarely anisocytic) are present. Non-glandular to linear, versatile, opening longitudinally hairs are few, unicellular, straight or curved, measuring 0.7-1.2 cm in L. and 1.0-1.5 mm in D. arising from the cicatrices; they have thick walls, moderately wide lumina and tapering apices and

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are covered with a warty cuticle. Non-glandular, unicellular, club-shaped hairs are rare and The mesophyll (Fig. 4D) is homogeneous and covered with a thin, smooth cuticle. The outer consists of several rows of polygonal, nearly (lower) epidermis (Fig. 2C&D) is more or less isodiametric, thin-walled parenchymatous cells. similar to the inner one but showing less Raphides of calcium oxalate are embedded in the elongated cells with strongly wavy anticlinal mucilage and scattered in the mesophyll. The walls and more stomata. vascular strands are formed of soft phloem tissue and lignified xylem vessels. The mesophyll (Fig. 2D) is wide, homogeneous, formed of thin walled, elongated, more or less The Andrœcium (Fig. 5): oval cells and showing wide intercellular spaces; The Anther (Fig. 5A-C & H): A transverse some of them contain mucilage in which raphides section through the anther (Fig. 5A) shows two of calcium oxalate are embedded (Fig. 2A and anther lobes attached together by a connective D). The vascular strands show small lignified tissue which is traversed by a small vascular vessels, delicate phloem elements and scanty strand. Each anther lobe has two pollen sacs perimedullary phloem. containing numerous pollen grains. The anther wall (Fig. 5A& B) is thin and consists of an The Calyx (Fig. 3): epidermis followed by a fibrous layer then the A transverse section in the calyx-tube (Fig. remains of tapetal layer. The epidermal cells 3A) is circular in outline; consisting of four (Fig. 5B&C) are tabular, polygonal, more or less segments. Each segment comprising outer and isodiametric with straight anticlinal walls and inner epidermises, enclosing a ground tissue covered with thin smooth cuticle, devoid of containing raphides of calcium oxalate and stomata and hairs. The fibrous layer (Fig. traversed by numerous small vascular strands. 5B&H) consists of a single row of polygonal, rectangular-shaped, thick-walled, lignified cells The cells of the inner (upper) and outer (lower) and showing bar-like thickening. They appear in epidermises (Fig. 3 A-D) are more or less surface view as polygonal, axially elongated cells similar; they are polygonal with more or less with lignified, beaded walls. The tapetal layer straight or slightly wavy anticlinal walls and (Fig. 5B) is represented by delicate, flattened, covered with smooth cuticle and some of them thin-walled, parenchymatous cells. The containing oil droplets. Anisocytic and connective tissue encloses a central vascular anomocytic stomata are few on the inner surface bundle. The pollen grains (Fig. 5G) are large; and numerous on the outer one. Non-glandular triangular with smooth thick exine and have three hairs, similar to those of the bract, are present. prominent germ pores which occupy the angles; each one is covered by a dome formed by the The mesophyll (Fig. 3D) is homogenous thickening of the intine and some having consisting of thin-walled, polygonal, more or less protruding pollen tubes. The pollen grains are isodiametric parenchymatous cells. Raphides of connected by viscid threads. calcium oxalate similar to those of the bract are present. The Filament (Fig. 5D-F): A transverse section in the filament (Fig. 5E& F) is nearly oval to The Corolla (Fig. 4): elliptical in outline. It consists of an epidermis A transverse section in the petal (Fig. 4A) covered with thin cuticle, followed by the ground is more or less crescent-shape in outline and tissue that is traversed by a small vascular strand consists of inner and outer epidermises enclosing and shows raphides of calcium oxalate. The a homogenous parenchymatous mesophyll, which epidermis (Fig. 5D&F) is formed of polygonal is traversed by numerous small vascular strands. thin-walled axially elongated cells covered with Raphides of calcium oxalate similar to those of smooth cuticle and few of them containing oil the bract and calyx are present. droplets. Stomata and hairs are absent. The The inner (upper) epidermis (Fig. 4B&D) is cortex (Fig. 5F) consists of thin-walled, formed of polygonal, axially elongated cells with parenchymatous cells containing scattered minute slightly wavy anticlinal walls and covered with raphides of calcium oxalate. smooth cuticle and some of them containing oil droplets (stained red with Sudan III). Stomata are The Hypanthium and Gynæcium (Fig. 6 A-F): absent. Few non-glandular hairs, similar to those A transverse section in the hypanthium of the bract and calyx tube, are present. The cells below the level of the ovary (Fig. 6A) is of the outer (lower) epidermis (Fig. 4C&D) are quadrangular in outline and consists of hairy similar to those of the upper but with more wavy epidermis having numerous unicellular non- anticlinal walls and few anomocytic stomata are glandular hairs, followed by narrow present. parenchymatous cortex. The pericycle is

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parenchymatous and followed by a continuous a parenchymatous mesophyll, which is traversed ring of collateral vascular bundle, which is by numerous small vascular strands. Raphides of traversed by medullary rays and surrounding a calcium oxalate are scattered all over the ground comparatively wide quadrangular pith showing tissue. The style appears occupying the centre as few small patches of perimedullary (intraxylary) it passes through the hypanthium and the calyx phloem near its periphery. Raphides of calcium tube. These serial sections illustrate the oxalate are scattered throughout the cortex and arrangement of the different floral parts in the pith, being more abundant in the pith. flower as follows:

A transverse section in the hypanthium passing A transverse section in the hypanthium tube through the base of the ovary (Fig. 6B) is above the ovary (the free prolonged part of the quadrangular in outline, showing slight hypanthium) (Fig. 7A) is quite similar to that prominences and numerous unicellular non- illustrated in (Fig. 6D) but non-glandular hairs of glandular hairs. It consists of an epidermis the inner epidermis are much fewer. The rim of followed by a wide, parenchymatous ground the hypanthium tube (Fig. 7B) show the fused tissue and shows four cavities of the ovary bases of the sepals, petals and stamens to the locules at the center. The outer part of the ground summit of the hypanthium and four anthers of the tissue is traversed by collateral vascular bundles shorter stamens. which appear next to the prominent regions. Raphides of calcium oxalate are scattered all over Moreover, a transverse section in the bud passing the ground tissue. through the lower part of the calyx-tube (Fig. 7C) shows that the calyx-tube, petals, and A transverse section in the hypanthium passing stamens begin to separate from each other. The through the middle of the ovary (Fig.6C), is outer stamens are alternate with the petals while also quadrangular and wedged in outline (8 the inner stamens are antepetalous and adnate to wedges or ridges, the four wedges at the corners the base of the corolla. are strongly prominent), showing numerous unicellular non-glandular hairs. It consists of an The unequal length of the stamens has been epidermis followed by a parenchymatous ground illustrated in the transverse sections passing tissue. The outer part of the ground tissue shows through the middle, upper parts of the bud collateral vascular bundles which appear next to and the free tips of the calyx-tube (Fig.7D-F). the prominent regions and accompanied by small The style appeared at the center of the flower bud groups of perimedullary (intraxylary) phloem and as shown in (Fig. 7 A-E). In addition, a T. S. at raphides of calcium oxalate scattered all over the the free tips of the calyx-tube (Fig. 7F) showed ground tissue. Centrally four locules appear, each four branches of the stigma which occupying the one showing two ovules of an axile placentation. center of the flower.

A transverse section in the hypanthium passing The Style (Fig. 8A, A1& C): through the top of the ovary (Fig. 6D) shows A transverse section in the style is nearly that the hypanthium appear as a hollow short tube round in outline. It consists of an epidermis surrounding the pubescent base of the style. The followed by a narrow parenchymatous ground outer surface is more or less circular in outline, tissue traversed by four small vascular strands while the inner surface is pubescent and irregular. (representing the four united individual styles), showing few raphides of calcium oxalates. The outer epidermis of hypanthium (Fig. 6E) The epidermis (Fig. 8C) is formed of consists of polygonal, slightly axially elongated polygonal, axially elongated, thick-walled cells cells with straight anticlinal walls and covered with straight anticlinal walls and covered with with smooth cuticle and some of them containing smooth cuticle and some of them containing oil oil droplets. Anisocytic stomata and numerous droplets. Anomocytic stomata are rare and hairs non-glandular hairs similar to those of the bract are absent. and calyx tube are present. The inner epidermis (Fig. 6F) of the hypanthium tube above the The stigma (Fig. 8B, B1& C1): ovary is formed of polygonal, slightly axially A transverse section in the stigma is more elongated cells similar to those of the outer or less circular in outline. It consists of an epidermis (Fig. 6 E) but the cells are larger in epidermis followed by parenchymatous ground size. tissue, traversed by a small vascular strand and few raphides of calcium oxalates are present. Serial Sections through the flower bud (Fig. 7 The epidermis (Fig. 8C1) is formed of A-F) are more or less rounded in outline. Each polygonal isodiametric, thin-walled cells, slightly consists of outer and inner epidermises enclosing papillose with short, blunt, papillae at the tip and

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covered with smooth cuticle. with straight, thin anticlinal walls and The different tissues of the flower showing covered with smooth cuticle. Stomata and parenchyma cells containing tannins (bluish-black hairs are absent. with ferric chloride T.S.). 9. Large triangular pollen grains with Powdered Flower: smooth thick exine, three prominent pores, The powdered flower is greyish-brown in color, each covered by a dome and having with slightly fragrant odour and astringent, protruding pollen tubes. mucilaginous taste. It is microscopically 10. Fragments of the stigmatic surface with characterized by the following: short papillae and smooth cuticle. 1. Fragments of the inner and outer 11. Fragments of epidermal cells of the style epidermises of the bract with wavy to with thick straight walls, smooth cuticle, strongly wavy anticlinal walls and covered rare anisocytic stomata and hairs are with smooth cuticle. Anisocytic, anomocytic absent. stomata and few non-glandular hairs are 12. Numerous raphides of calcium oxalate of present different sizes. 2. Fragments of the inner and outer 13. Fragments of parenchyma cells containing epidermises of the calyx tube with cells mucilage (red stain with ruthenium red). having more or less straight to slightly wavy 14. Fragments of parenchyma cells containing walls, smooth cuticle, few anisocytic, tannins (bluish-black colour with ferric numerous anomocytic stomata and non- chloride T.S.). glandular hairs. 15. Fragments of epidermal cells containing oil 3. Fragments of the inner epidermis of the droplets (stained red with Sudan III). corolla, with slightly wavy anticlinal walls, no stomata but those of the outer, showing From the previous botanical study we can strongly wavy anticlinal walls, few conclude that: anomocytic stomata and both are covered  Oenothera speciosa Nutt. (Onagraceae), with smooth cuticle. cultivated in Egypt can be identified in the 4. Fragments of the inner epidermal cells of entire form from the morphological the hypanthium tube above the ovary characters of its flowers which are in which are polygonal, having straight concordance with published descriptions (1, 2, anticlinal walls, covered with smooth 53, 54, 55). cuticle. Anisocytic, anomocytic stomata and  In the powdered form the most diagnostic non-glandular hairs are present. Also, elements are: fragments of the outer epidermis of a. Large triangular pollen grains with smooth hypanthium at the united part with the thick exine, three prominent pores, each ovary which are similar to those of the inner covered by a dome and having protruding but with smaller cells and more hairs. pollen tubes. 5. Numerous non-glandular, unicellular, b. Numerous, non-glandular, unicellular hairs straight or curved hairs, covered with a arising from cicatrices; straight or curved, granular cuticle and few club-shaped ones short or long, thick walled and covered with smooth cuticle. with a granular cuticle. 6. Fragments of epidermal cells of anther, c. Non-glandular, unicellular, club-shaped polygonal nearly isodiametric, with straight hairs and covered with thin smooth cuticle. walls and smooth cuticle. d. Abundant raphides of calcium oxalate with 7. Fragments of fibrous layer of anther with varying sizes. lignified bar-like thickenings and appearing in surface view elongated with lignified, The microscopical measurements of the beaded walls. different elements of the flower of Oenothera 8. Fragments of elongated epidermal cells of speciosa Nutt. are listed in Table (13). the filament, polygonal axially elongated,

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Table (1): Phenolic constituents identified by HPLC in the hydrolyzed flower extracts of Oenothera speciosa Nutt. Concentration (mg/100g DW) Rt Constituent A B 6.52 Chlorogenic acid 45.68 300.21 7.04 Caffeic acid 75.32 204.44 8.69 Ferulic acid 42.07 21.13 9.07 Rosmarinic acid 38.82 22.81 Total identified phenol acids 201.89 548.59 10.66 Quercetin 212.23 77.80 11.37 Naringenin 132.34 76.51 11.81 Kaempferol 62.48 25.89 Total identified flavonoids 407.05 180.2 Rt: retention time in minutes. DW: dry weight. (A): sample collected at the early flowering stage. (B): sample collected at the late flowering stage.

Table (2): Determination of tannins of the flowers of Oenothera speciosa Nutt. Average % Method A B Gravimetric determination 8.67 10.53

Colourimetric determination 19.03 23.35

(A): sample collected at the early flowering stage (B): sample collected at the late flowering stage (  ): gravimetric determination of tannin content. (): colourimetric determination of proanthcyanidins.

Table (3): Results of PC analysis of free sugars and mucilage hydrolyzates of the flowers of Oenothera speciosa Nutt. Mucilage Solvent system Free hydrolyzate Authentic sugars R5 sugars S6 S7 CEM HEM Rhamnose 0.42 0.66 Yellowish-brown + + + Xylose 0.30 0.55 Reddish-violet - - + Arabinose 0.26 0.50 Reddish-violet + + - Mannose 0.23 0.45 Brown - - - Glucose 0.23 0.40 Pale brown + + + Galactose 0.18 0.38 Pale brown - + + Glucuronic acid 0.05 0.14 Pale brown - + + Galacturonic acid 0.03 0.11 Pale brown - + + S6: n-butanol-acetic acid-water (4:1:5) S7: n-butanol-pyridine-water (6:4:3). CEM: cold extracted mucilage. HEM: hot extracted mucilage. R5: aniline phthalate reagent. (+): present, (-): absent

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Table (4): Results of HPLC analysis of the free sugars and mucilage hydrolyzates of the flowers of Oenothera speciosa Nutt. Relative percentage Peak No. RRt* Component Mucilage hydrolyzate Free sugars CEM HEM 1 0.67 Galacturonic acid - 3.73 9.19 2 0.89 Glucuronic acid - 18.14 8.09 3 1.00 Rhamnose 55.63 34.29 29.67 4 1.19 Arabinose 15.47 8.12 - 5 1.30 Xylose - - 13.12 6 1.37 Glucose 13.41 23.68 14.77 7 1.67 Galactose - 3.15 4.47

RRt*: relative retention time to rhamnose (Rt in min: 3.50). CEM: cold extracted mucilage. HEM: hot extracted mucilage. (-): absent

Table (5): Results of testing the acute anti-inflammatory activity of the aqueous and alcoholic extracts of the flowers of Oenothera speciosa Nutt. in male albino rats (n=6) % Oedema Dose in mg/kg Group B.Wt. Mean ± S.E. % change Potency Control 1 ml saline 62.2±1.8 - - Aqueous extract 100 26.7±1.8* 57.07 89 Alcoholic extract 100 24.6±0.9* 60.45 95 Indomethacin 20 22.4±0.7* 64 100 * Significantly different from the control group at P <0.01 % change is calculated as regard the control group.

Table (6): Results of testing the analgesic activity of of the aqueous and alcoholic extracts of the flowers of Oenothera speciosa Nutt. in male albino rats (n=6) Volts needed Volts needed after single oral dose Dose before in treatment Group After 1 hour After 2 hours mg/kg (zero time) b.wt. % % Mean ± S.E. Mean±S.E. Potency Mean±S.E. Potency change change Control(saline) 1 ml 78.6± 1.9 78.7 ± 1.6 0.13 - 78.4 ± 1.4 0.26 - Aqueous extract 100 79.2±1.5 142.7±4.1* 80.2 66 165.5±5.2* 109.3 77 Alcoholic extract 100 78.9±1.8 151.2±5.3* 91.6 76 172.4±6.3* 118.5 83 Novalgin 50 76.1 ± 2.2 168.9±6.1* 122.0 100 184.3±5.1* 142.2 100 * Significantly different from the zero time at P<0.01 % change is calculated as regard the zero time.

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Table (7): Results of testing the anti-ulcer activity of the aqueous and alcoholic extracts of the flowers of Oenothera speciosa Nutt. in male albino rats (n=6) Number of Dose in Group gastric ulcers % protection mg/kg B.Wt. (Mean±S.E.) Indomethacin 20 18.9 ± 0.4 - Indomethacin +aqueous extract 100 6.1±0.1* 67.73 Indomethacin + alcoholic extract 100 5.2 ±0.04* 72.49 * Statistically significant from the control at P< 0. 01

Table (8): Results of testing the antipyretic activity of the aqueous and alcoholic extracts of the flowers of Oenothera speciosa Nutt. in male albino rats (n=6) Body temperature change Dose Induced rise After 1 hour After 2 hours in in Group mg/kg temperature % % Mean±S.E. Potency Mean±S.E. Potency b.wt. (Mean±S.E.) change change

Control (saline) 1 ml 38.4± 0.3 39.1± 0.5 - - 39.3± 0.4 - - Aqueous extract 100 39.5± 0.5 37.8± 0.2* 4.3 84 36.9± 0.1* 6.6 89 Alcoholic extract 100 39.3 ± 0.5 37.3± 0.2* 5.1 100 36.8± 0.2* 6.5 88 Paracetamol 20 39.2±0.3 37.2±0.03* 5.1 100 36.3±0.02* 7.4 100 * P <0.01 corresponding induced rise in temperature of the tested group. % change is calculated as regard the temperature before treatment.

Table (9): Results of testing the antioxidant effect of the aqueous and alcoholic extracts of the flowers of Oenothera speciosa Nutt and vitamin E drug in male albino rats (n=6) Blood Glutathione Group % change (mg %) 36.7 ± 1.2 - Control (1 ml saline)

Diabetic non- treated 23.4 ± 0.6* 36.2 35.7 ± 1.1 2.7 Diabetic + aqueous extract (10 mg/kg)

Diabetic+ alcoholic extract (10 mg/kg) 36.1 ± 0.8 1.6

Diabetic + Vitamin E (7.5mg/kg) 36.2 ± 0.9 1.4 * Statistically significant from control group at P<0.01

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Table (10): Acute effect of the hot extracted mucilage (HEM) and alcoholic extracts of the flowers of Oenothera speciosa Nutt on blood glucose level in diabetic rats (n=10) Blood glucose level (mg/dl) Time Dose in At zero time After four hours After six hours

mg/kg b.wt. Group M.±S.E. M.±S.E. % change M.±S.E. % change

Diabetic non- treated 1ml saline 261.5±9.7 262.9±10.2 - 265.1±12.6 - Diabetic treated with 25.86 43.61 100 246.3±8.2 182.6±6.7* 138.9±3.5* HEM (62) (67) Diabetic treated with 23.73 41.40 100 258.7± 8.4 197.3±7.2* 151.6±4.1* alcoholic extract (57) (64) 41.75 64.85 Glimepiride 0.2 254.6±9.7 148.3±4.2* 89.5 ±2.1* (100) (100) * Significantly different from the zero time at P <0.01, % change is calculated as regard the zero time, ( ): Values in parenthesis are the potencies

Table (11): Long-term effect of the hot extracted mucilage (HEM) and alcoholic extracts of the flowers of Oenothera speciosa Nutt on blood glucose level in diabetic rats (n=10) Blood glucose level (mg/dl) Time Dose in At zero time After four weeks After eight weeks mg/kg b.wt. Group M.±S.E. M.±S.E. % change M.±S.E. % change

Control 1ml saline 83.1±2.5 80.9±2.4 - 82.4±2.1 -

Diabetic non- treated - 231.6±8.5 247.8±9.4 - 248.9±9.6 - Diabetic treated with 38.5 52.1 100 247.8±9.3 152.9±6.5* 118.7±4.2* HEM (68) (77) Diabetic treated with 28.84 42.08 100 256.2±11.3 183.4±6.7* 148.4±5.1* alcoholic extract (51) (62) 56.5 67.5 Glimepiride 0.2 226.7±8.1 98.6±3.9* 73.8±2.4* (100) (100) * Significantly different from the zero time at P <0.01, % change is calculated as regard the zero time, ( ): Values in parenthesis are the potencies

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Table (12): Anti-microbial Activity of aqueous and alcoholic extracts of the flowers of Oenothera speciosa Nutt.

Diameter of zone of inhibition (mm) (Potency)

Microorganism

Aqueous Alcoholic OFX. AMP. B extract extract

Gram–negative:

14 31 Escherichia coli - (45%) (100%) 14 20 25 Pseudomonas aeruginosa (66%) (80%) (100%) 14 14 29 Proteus vulgaris (48%) (48%) (100%)

Gram–positive:

15 28 31 Staphylococcus aureus (48%) (90%) (100%) 17 16 36 Sarcina lutea* (47%) (44%) (100%) 10 16 32 Bacillus subtilis (31%) (50%) (100%)

Acid fast bacilli :

14 24 27 Mycobacterium phlei* (52%) (89%) (100%)

Fungi:

12 17 21 Candida albicans (57%) (81%) (100%) 11 16 25 Candida tropicalis* (44%) (64%) (100%) OFX: Ofloxacin (5μg/disc) AMP.B: Amphotericin B (5μg/disc) (-): No inhibition zone. (*): Laboratory collection strains.

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Table (13): Microscopical measurements of the different elements of the flower of oenothera speciosa nutt. in microns (µ) Item Measurements (µ) Item Measurements (µ) Epidermal cells: Petals L.: 6 – 11 -18 Bract Filament L.: 5 – 10 - 16 Inner epidermis L.: 39 – 57 - 74 Hypanthium L.: 24 – 51 - 71 W.: 9 – 16 - 34 Style L.: 6 – 10 - 15 H.: 6 – 11 - 17 Stigma L.: 9 - 12 - 17 Outer epidermis L.: 25 – 43 - 62 Stomata: W.: 8 – 19 - 30 Bract H.: 4 – 8 - 14 Inner epidermis L.: 11 – 18 – 23 Calyx- tube W.: 6 – 10 – 14 Inner epidermis L.: 29 – 55 - 83 Outer epidermis L.: 10 – 14 – 19 W.: 9 -29 - 42 W.: 7 – 12 – 17 H.: 4 – 6 - 8 Calyx- tube Outer epidermis L.: 25 – 45 - 65 Inner epidermis L.: 12 – 19 – 25 W.: 8 – 24 - 42 W.: 8 – 13 – 18 H.: 5 – 10 - 15 Outer epidermis L.: 16 – 21 – 27 Petal W.: 11 – 15 – 20 Inner epidermis L.: 44 – 72 - 103 Petal W.: 9 – 21 - 30 Outer epidermis L.: 12 – 15 – 19 H.: 8 – 10 - 14 W.: 11 – 14 – 18 Outer epidermis L.: 40 – 52 - 66 Hypanthium W.: 6 – 12 – 20 Inner epidermis L.: 19 – 23 – 27 H.: 5 – 9 - 15 W.: 11 – 15 – 19 Filament L.: 51 – 64 - 79 Outer epidermis L.: 8 – 11 – 15 W.: 8 – 18 - 25 W.: 7 – 10 - 14 H.: 4 – 7 - 12 Style L.: 20 – 23 - 26 Anther L.: 20 – 37 – 50 W.: 15 – 17 - 20 W.: 16 – 39 – 40 Non -glandular hairs: (n. g. h.) H.: 9 – 16 – 25 Bract L.: 89 – 101 – 149 Hypanthium W.: 8 - 12 - 14 Inner epidermis L.: 21 – 45 – 70 Calyx- tube L.: 26 – 116 - 205 W.: 9 – 23 – 38 W.: 9 – 11 - 15 H.: 7 – 9 - 13 Petal L.: 15 - 67 - 82 Outer epidermis W.: 6 – 8 - 11 L.: 14 – 29 – 45 Hypanthium L.: 40 – 74 - 110 W.: 8 – 15 – 25 W.: 6 – 13 25 H.: 6 – 10 – 12 Club-shaped (n. g. h.): Style L.: 60 – 99 – 137 Bract L.: 19 – 33 - 42 W.: 8 – 17 - 22 D.: 8 – 13 - 22 H.: 9 - 12 - 16 Calyx- tube L.: 25 – 37 - 50 Stigma L.: 12 – 23 – 35 D.: 5 – 10 - 15 W.: 6 – 15 -21 Petal L.: 14 – 25 – 32 H.: 9 – 13 - 19 D.: 5 – 9 - 12 Raphides: Hypanthium L.: 18 – 27 – 47 Bract L.: 8 – 20 - 32 D.: 5 – 8 - 17 Calyx- tube L.: 9 – 15 -25 Pollen grains: L.: 70 – 90 - 115 D.: Diameter. H.: Height. L.: Length. W.: Width.

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Fig. (1A) A Photograph of the flowering branch of Oenothera speciosa Nutt. (X=0.8)

Fig. (1B) Macromorphology of the Flower of Oenothera speciosa Nutt. (X=0.9)

Fig. (2) Micromorphology of the Bract: A: Diagrammatic T.S. in the bract. (X=62 ) B: Inner epidermis of the bract (X=380) C: Outer epidermis of the bract. (X=380) D: Detailed T.S. in the bract. (X=380) cu., cuticle; l.ep., lower epidermis; n.g.h., non-glandular hairs; p. ph., perimedullary phloem; ph., phloem; raph., raphides; st., stomata; u.ep., upper epidermis; xy., xylem; xy.v., xylem vessels.

Fig. (3) Micromorphology of the Calyx: A: Diagrammatic T.S. of the calyx tube segment. (X=75 ) B: Inner epidermis of the calyx tube. (X=259) C: Outer epidermis of the calyx tube. (X=259) D: Detailed sector of the calyx tube. (X=426) cic., cicatrix; cu., cuticle; i.ep., inner epidermis; n.g.h., non-glandular hairs; o.ep., outer epidermis; ph., phloem; raph., raphides; st., stomata; v.b., vascular bundle; xy., xylem.

Fig. (4) Micromorphology of the corolla: A: Diagrammatic T.S. in the petal. (X=62 ) B: Inner epidermis of the petal. (X=380) C: Outer epidermis of the petal. (X=380) D: Detailed T.S. in the petal. (X=490) cu., cuticle; i.ep., inner epidermis; n.g.h., non-glandular hairs; o.ep., outer epidermis; ph., phloem; raph., raphides; st., stomata; v.st., vascular strand; xy.v., xylem vessels.

Fig. (5) Micromorphology of the Andrœcium: A: Diagrammatic T.S. in the anther. (X=79 ) B: Detailed T.S. in the anther. (X=342) C: Epidermis of the anther. (X=342) D: Epidermis of the filament. (X=342) E: Diagrammatic T.S. in the filament. (X=79 ) F: Detailed T.S. in the filament. (X=308) G: Pollen grains. (X=240) H: Fibrous layer of the anther. (X=342) con., connective tissue; cu., cuticle; ep., epidermis ; f.lay., fibrous layer of anther; p.gr., pollen grain; p.s., pollen sac; ph., phloem; raph., raphides; S.V., side view; T.V., top view; tap., tapetum; v.b., vascular bundle; xy., xylem.

Fig. (6) Micromorphology of the hypanthium and Gynæcium of the Flower: A: Diagrammatic T.S. in the hypanthium. (X=38 ) B: Diagrammatic T.S. in the hypanthium passing through the base of the ovary. (X=38 ) C: Diagrammatic T.S. in the hypanthium passing through the middle of the ovary. (X=38 ) D: Diagrammatic T.S. in the hypanthium passing through the top of the ovary. (X=38 ) E: Outer epidermis of the hypanthium. (X=300) F: Inner epidermis of the hypanthium tube above the ovary level. (X=300) loc., locule; n.g.h., non-glandular hairs; ovu., ovule; p.ph., perimedullary phloem; ph., phloem; pi., pith; pl., placenta ; raph., raphides; st., stomata; sty., style; v.b., vascular bundle; xy., xylem.

Fig. (7) Serial Section of the Flower Bud from the Base to the Apex (above the ovary): A: Diagrammatic T.S. in the hypanthium tube above the ovary. (X=44) B: Diagrammatic T.S. in the rim (summit) of hypanthium. (X=44) C: Diagrammatic T.S. in the lower part (third) of the flower bud. (X=44) D: Diagrammatic T.S. in the middle of the flower bud. (X=44) E: Diagrammatic T.S. in the upper part (third) of the flower bud. (X=44) F: Diagrammatic T.S. at the tip of the flower bud. (X=44) anth., anther; cal.t., calyx tube; fil., filament; hyp. hypanthium; n.g.h., non-glandular hairs; pet., petal; raph., raphides; stg., stigma; sty., style; v.b., vascular bundle.

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Fig. (8) Micromorphology of the Style and Stigma of the Flower: A: Diagrammatic T.S. in the style. (X=62 ) B: Diagrammatic T.S. in the stigma. (X=62 ) C: Epidermis of the style. (X=300) A1: Detailed T.S. in the style. (X=300) B1: Detailed T.S. in the stigma. (X=300) C1: Epidermis of the stigma. (X=300) cu., cuticle; ep., epidermis; ph., phloem; raph., raphides; st., stomata; v.b., vascular bundle; xy.v., xylem vessels.

A Photograph of the flowering branch of Oenothera speciosa Nutt. (X=0.8)

Fig. (1A)

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REFERENCES 16. Eid, H. H. and Sleem, A.A.; “Botanical and 1. Bailey, L. H.; “Manual of Cultivated Plants”, Biological Study of Oenothera Speciosa Nutt. The Macmillan Company, New York, 733- Cultivated In Egypt”, Egypt. J. Biomed. Sci., 739 (1958). 23, March, 91-120 (2007). 2. Lawrence, G. H. M; “Taxonomy of Vascular 17. Stahl, E.; “Thin Layer Chromatography”; 2nd Plants”, The Macmillan Company, New York, Edition, Springer Verlag, Berlin, Heidelberg, 637-639 (1958). New York (1969). 3. Fernald, M.L.; “Gray’s Manual of Botany”, 18. Wagner, H. and Bladt, S.; “Plant Drug D.Van Nostrand Company, London, Toronto, Analysis: A thin layer chromatography atlas”, Melbourne, 1051, 1063-1069 (1970). 2nd Ed., Springer - Verlag, Berlin, Heidelberg. 4. http://www.paghat.com/mexicanprimrose.htm New York (2001). l 19. Smith, L; “Chromatographic and 5. Bailey, L.H.; “Hortus Third”, The Macmillan Electrophoretic Techniques”, William Co. Inc., New York (Collier Macmillan Heinman, London (1960). Publishers, London), 979-981 (1976). 20. Mabry, T.J.; Markham, K.R. and Thomas, 6. Ebadi, M.; “ Pharmacodynamic Basis of M.B.; “The Systematic Identification of Herbal Medicine” Boca Raton, London, New Flavonoids”, 2nd Ed., Springer Verlag; New York, Washington D.C., CRC PRESS, 34, York (1970). 135, 145 ( 2002) 21. Mattila, P; Astola, J. and Kurnpulainen, J.; 7. WHO monographs on selected medicinal “Determination of Flavonoids in Plant plants; Vol. 2, 217-230 (2002). Material by HPLC with Diode-Array and 8. Zinsmeister, H. D. and Bartl, S., “The Electro-Array Detections”, Journal of phenolic compounds of Oenothera”, Agriculture and Food Chemistry, 48(12), Phytochemistry, 10, 3129-3132 (1971). 5834-5841(2000). 9. Howard, G.; Mabry, T. G. and Raven, P. H., 22. Markham, K.R.; “Techniques of Flavonoid “Distribution of Flavonoids in Twenty One Identification”; Academic Press, London, Species of Oenothera”, Phytochemistry, 11, New York, Paris, Tokyo, Toronto (1982). 289-291 (1972). 23. “Pharmacopoeia Eu.”, Band I - III, Deutscher, 10. Averett, J. E.; Huang, S. and Wagner, W. L.; Apotheker, Verlag, Stuttgart, Govi Verlag, “Flavonoid Survey of Oenothera Gmbh, Frankfurt / M (1974 -1975,1978). (Onagraceae) : Sects. Goropsis, Hartmannia, 24. Porter, L. J.; Hrstich, L. N. and Chan, B. G.; Kneiffia, Paradoxus and Xyloplorum”, Amer. “The conversion of procyanidins and J. Bot., 75(4), 476-483(1988). prodelphinidins to cyanidin and delphinidin”, 11. Nakanishi, T.; Inatomi, Y.; Murata, H.; Ishida, Phytochemistry, 25 (1), 223- 230 (1986). S. ; Fujino, Y.; Miura, K.; Yasuno, Y.; Inada, 25. Peach, K. and Tracy, M.V.; “Modern Methods A.; Lang, F. A. and Murata, J.; “Triterpenes of Plant Analysis”, Vol. II-IV, Springer- and Flavonol Glucuronides from Oenothera Verlag, Berlin (1955). cheiranthifolia”, Chem. Pharm. Bull., 55 (2), 26. Karber, G.; “Pharmacologische Methoden Zur 334—336 (2007). Auffindung Von Arzneimittein und Giffter 12. Taniguchi, S.; Imayoshi, Y.; Yabu-uchi, R.; und Analyse Seiner Wirking Sweise”, Von Dr. Ito, H.; Hatano T.and Yoshida, T.; “A Med. Leopold. Ther. Wissenschoftliche macrocyclic ellagitannin trimer, oenotherin T1, Verlag Gess. Gesellschaft, Gmbh (1941). from Oenothera species”; Phytochemistry; 27. Winter, G.A.; Risley, E.A. and Nuss, G.W.; 59(2), 191-195 (2002). “Carrageenin-induced Oedema in Hind Paw 13. Wettasinghe, M.; Shahidi, F. and Amarowicz, R.; of the Rat as an Assay of Anti-inflammatory “Identification and Quantification of Low Drugs”, Proc. Soc. Exp. Biol. Med., III, 1544- Molecular Weight Phenolic Antioxidants in Seeds 547(1962). of Evening Primrose (Oenothera biennis L.)”, J. 28. Charlier, R.; Prost, H.; Binon, F. and Dellous, Agric. Food Chem., 50 (5), 1267 -1271 (2002). G.; “Pharmacology of an Antitussive, 1- 14. Howard, G. and Mabry, T. J.; “Myricetin 3-O- phenethyl-4-(2-propenyl)-4- methyl ether 3′-O-β-D-glucoside, the major propinoxypipridine Acid Fumarate”, Arch. flavonoid of Oenothera speciosa Intern. Pharmacodynamic, 134, 306-327 (Onagraceae)”, Phytochemistry, 9(11), 2413- (1961). 2414 (1970) (Through ref 9). 29. Corell, T.K.; Jensen, K.M. and Splowinski, 15. Eid, H. H. and Sleem, A.A.; “Volatiles, J.B.; “A New Anti-Inflammatory Derivative Lipoids and Bioactivity of the Flowers of of Imidazol Which Is Less Ulcerogenic Than Oenothera Speciosa Nutt. Cultivated in Indomethacin in Rats”, Acta Pharmacol .et Egypt”, Egypt. J. Biomed. Sci., 25, November, Toxicol., 45, 232-239 (1979). 103-126 (2007). 30. Bush, J.E. and Alexander, R.W.; “An Improved Method for the Assay of Anti-

212 Bull. Fac. Pharm. Cairo Univ., Vol. 45, No. 3 (2007)

Inflammatory Substances in Rats”, Acta 46. Parveen, Z.; Deng, Y.; Saeed, M. K.; Dai, R.; Endocrinologica, 35, 268-276 (1960). Ahamad, W. And Yu, Y. H.; “Anti- 31. Beutler, E.; Duron, O. and Kelly, B.A.; inflammatory and Analgesic Activities of “Improved method for the determination of Thesium chinense Turcz Extracts and its blood glutathione”, J. Lab. Clin. Med., 61, Major Flavonoids, Kaempferol and 882-888 (1963). Kaempferol-3-O-glucoside”, YAKUGAKU 32. Eliasson, S.G. and Samet, T.M.; “Alloxan ZASSHI, Vol. 127, 1275-1279 (2007). induced neuropathies: Lipid changes in nerve 47. Dongmo, A. B.; Kamanyi, A.; Anchang, M. and root fragments”, Life Sciences, 8 (9), part S.; Chungag-Anye Nkeh, B.; Njamen, D.; (l), 493-98 (1969) Nguelefack, T. B.; Nole, T. and Wagner, H.; 33. Trinder, P.; “Determination of glucose in “Anti-inflammatory and analgesic properties blood using glucose oxidase with an of the stem bark extracts of Erythrophleum alternative oxygen acceptor”, Ann. Clin. suaveolens (Caesalpiniaceae), Guillemin & Biochem., 6, 24 (1969). Perrottet”, Journal of Ethnopharmacology, 34. Snedecor, W. G. and Chochran, G. W.; 77 ( 2-3), 137-141 ( 2001). “Statistical Methods”, 6th Ed. Iowa State, 48. Sannomiya, M.; Fonseca, V. B.; da Silva, University Press. Ames. USA, 362 -364 M.A.; Rocha, L.R.M.; dos Santos, L.C.; (1971). Hiruma-Lima, C.A.; Souza Brito A.R.M. and 35. Collins, C.H.; “Microbial Methods”, Vilegas, W.; “Flavonoids and antiulcerogenic Butterworths, London, 92 (1964). activity from Byrsonima crassa leaves 36. Lorian, V.; “Antibiotics in Laboratory extracts”, Journal of Ethnopharmacology, 97 Medicine”, Williams and Wilkins, Baltimore (1), 1-6 (2005). London, 1014 (1980). 49. Parmar, S.N. and Ghosh, M.N.; “Gastric anti- 37. Geissman, T.A.; "The Chemistry of ulcer activity of (+)-cyanidanol-3, a histidine Flavonoid Compounds", The Macmillan decarboxylase inhibitor”, Eur. J. Pharmacol., Company, New York (1962). 69 (1), 25-32 (1981). 38. Devon, T. K. and Scott, A. L.; “Handbook of 50. Škerget, M.; Kotnik, P.; Hadolin, M.; Hraš, A. Naturally Occurring Compounds”; I&II , R.; Simonič, M. and Knez, Ż.; “Phenols, Academic Press, New York, London (1975). proanthocyanidins, flavones and flavonols in 39. Harborne, J.B.; Mabry, T.J. and Mabry, H.; some plant materials and their antioxidant "The Flavonoids", Chapman and Hall Ltd, activities”, Food Chemistry, 89 (2), 191-198 London (1975). (2005). 40. The Merck Index; "An Encyclopedia of 51. Xie, J.T.; Wu, J.A.; Mehendale, S.; Aung, Chemicals, Drugs, and Biologicals", 13th Ed.; H.H. and Yuan, C.-S.; “Anti-hyperglycemic Merck Research Laboratories Division of effect of the polysaccharides fraction from Merck & Co., INC.; Whitehouse Station, Nj American ginseng berry extract in ob/ob (2001). mice”, Phytomedicine, 11(2-3), 182-187 41. Swain, T.; Harborne, J. B. and Sum ere, C. (2004). F.; “Biochemistry of Plant Phenolics”, Vol. 52. Aitani, M.; Kimura, H.; Abiru, Y.; Soyama, 12, Plenum Press, New York and London H.; Murakami, H; “Effect of an Extract from (1977). Evening-Primrose Seeds on Postprandial 42. Agrawal, P. K.; “Carbon-13 NMR of Blood Glucose Level and Its Active Flavonoids", Elsevier, Amsterdam, Oxford, Components”, Journal of the Japanese Society New York, Tokyo, 444-445 (1989). for Food Science and Technology, 50 (4), 43. Evans, W.C.; “Trease and Evans, 180-187 (2003). Pharmacognosy”, William Charles Evans and 53. McLean, R. C. and Ivimey-cook, W. R., revised by Daphne Evans; W.B Saunders, “Theoretical Botany”, Longmans, Green and Edinburgh, London, New York, Philadelphia, Co, London, New York, Toronto, Vol. II, St. Louis, Sydney, Toronto, 15th Ed., (2002). 1135,1172 (1956). 44. The National Formulary; “Official American 54. Lowson, J.M.; Howarth, W.O. and Warne, Pharmaceutical Associations”, Washington, L.G.G., “Textbook of Botany”, University 14 th Ed. (NF XIV), 534 (1975). Tutorial Press LTD., Clifton House, Euston 45. Buck, W.B; Osweiter, G.D. and Van Gelder, Road, London, N.Y., 9th edition, 341 (1949). A., "Clinical Diagnostic Veterinary 55. Metcalfe, C.R. and Chalk, L.; “Anatomy of Toxicology", 2nd Ed., 52011, Kendall/hunt the Dicotyledons”, Oxford at the Clarendon Iowa Publishing Company, Iowa (1976). Press, 658-662 (1950).

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دراسة عقاقيرية وبيولوجية إضافية ألزهار نبات أونوثيرا سبيسيوزا نت المنزرع فى مصر

أ.د. طه شحات محمد األلفى ، د. هناء حسن عيد وأ.د. أماني أمين سليم* قسم العقاقير- كلية الصيدلة- جامعة القاهرة - مصر. * قسم الفارماكولوجي- المركز القومي للبحوث- القاهرة -مصر.

تم فى هذا البحث تعيين نسبة الفالفننياا ن احأحاض الفيننلية ف أزهضر نبض أنننثيرا سبيسينزا ن الانزرع فى اصر فى بااية فترة اإلزهضر ن اقضرنة هذه النسب باثيلتهض ف فى نهضية تلك الفترة نذلك بضستخاام الكرناضتنجرافيض السضئلة ذا الكفضءة العضلية. كاض تم أيضض تعيين كاية الاحتنى العفص عن طريق حسضب الزيضاة ف النزن نالطرق الطيفية نقا نجا أن احأزهضر تحتنى على نسبة أعل ان احأحاض الفيننلية نالعفصيض ف نهضية فترة اإلزهضر أاض بضلنسبة للفالفننياا فتكنن كايتهض أعل ف بااية اإلزهضر. نان نضحية أخرى أا تجزئة الخالصة الكحنلية إلى فصل أربع اركبض نهى كنرسيتين)1( نكنرسيتين-3-جضالكتنسيا )2( ن حا الكلنرنجينيك )3( نكنرسيتين-3- رنتيننسيا )4( ان جزء خال اإلثيل. كاض تم فصل )+( كضتيشين ) ( 5ان خالصةاحأسيتنن. نقا تم التعرف على هذه الاركبض بضلنسضئل الطيفية الاختلفة ناقضرنتهض بعينض أصلية. نكذا تم اراسة الاناا الهالاية نتعيين كايتهض نتجزئتهض نالتعرف على السكضكرالحرة نالاتحاة بضستخاام الكرناضتنجرافيض السضئلة ذا الكفضءة العضلية نكرناضتنجرافيض النرق. كاض أ جري اراسة الساية للاناا الهالاية نالخالصتين الاضئية نالكحنلية حأزهضر النبض ناسفر عن أنهم آاننن. نكذلك أثبت الاراسة احأقربضزينية أن لكل ان الخالصتين الاضئية نالكحنلية تأثيراً ًً اضضااً لاللتهضب ناسكنض ًلآلالم ناضضااً لألكساة ناضضاا ً للقرح نخضفضضً للحرارة. نأن الاناا الهالاية نالخالصة الكحنلية لهاض تأثيراً ًً خضفضض ً لنسبة السكر ف الام على الااى البعيا نالقريب. كاض أظهر أيضض الاسح البينلنج أن الخالصتين الاضئية نالكحنلية لهاض تأثيرا ً اثبطض ً لبع أنناع البكتريض نالفطريض الاختبرة. هذا بضإلضضفة الى اراسة عيضنية ةناجهري حأزهضر النبض احل البحث بغر التعرف عليهض كضالة ان على هيئة اسحنق حتى ياكن التاييز بينهض نبين احأنناع احأخرى . نيعتبر هذا التقرير احأنل عن اراسة الاكننض الفيننلية نالاناا الكربنهياراتية ناإلختبضرا البينلنجية نكذلك الاراسة العيضنية نالاجهرية حأزهضر هذا النبض .

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