Further Pharmacognostical and Biological Studies on the ......Bull. Fac. Pharm. Cairo Univ., Vol. 45, No. 3 (2007) 185 FURTHER PHARMACOGNOSTICAL AND BIOLOGICAL STUDIES ON THE FLOWERS

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

185

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

The effect of the time of collection on the

phenolic content of the flowers of Oenothera

speciosa Nutt., cultivated in Egypt, was studied.

Samples collected at the early (March) and late

(July) flowering periods were analyzed.

Flavonoids (expressed as aglycones) and phenol

acids were determined by HPLC in the

hydrolyzed extracts. Samples gathered in March

showed a relatively high percentage of flavonoids

(407.05 mg/100g dry wt.) which decreased in the

July sample, amounting only to 180.20 mg/100g

dry wt. On the contrary, an increase was

observed in the phenol acid content which

reached 201.89 and 548.59 mg/100g dry wt. in

the two samples, respectively; however, the

qualitative pattern appeared the same for all

constituents. In addition, gravimetric and

spectrophotometric determinations of tannins

and proanthocyanidins revealed that both were

higher at the late flowering period (10.53 g%

and 23.35g%, respectively) than at the early one

(8.67g% and 19.03g%, respectively).

Two flavonol glycosides; hyperoside

(quercetin-3-O--β-D galactoside) and rutin

(quercetin-3-O-α-L-rhamnose-β-D-glucoside)

and the aglycone, quercetin, as well as a phenol

acid, chlorogenic acid, were isolated via

chromatographic fractionation of the ethyl

acetate extract. (+) Catechin, isolated from the

acetone extract, constituted the major component

of the tannin fraction. Characterization of the

isolated compounds was achieved through

physical, chemical, chromatographic and spectral

analyses, as well as, by comparison with available

authentic samples.

Moreover, the carbohydrate content was

investigated by PC and HPLC. Rhamnose,

arabinose and glucose were identified as free

sugars. Analysis of the hydrolyzate of the cold

extracted mucilage (CEM) revealed the presence

of galacturonic, glucuronic acids and galactose

in addition to the aforementioned sugars.

Likewise, the hydrolyzate of the hot extracted

mucilage (HEM) differed from that of (CEM) in

containing xylose and appearing free from

arabinose.

The aqueous and alcoholic extracts, as

well as, the hot extracted mucilage (HEM), were

subjected to biological evaluation as compared to

standard drugs. Their safety was ascertained

through determination of their LD50. The

alcoholic extract exhibited more potent anti-

inflammatory, analgesic, anti-oxidant and anti-

ulcer activities as compared to the aqueous

extract. Meanwhile, HEM exerted a more

pronounced anti-hyperglycemic action than the

alcoholic extract. The alcoholic extract,

moreover, revealed noticeable antibacterial and

antifungal activities, while those observed for the

aqueous extract were only moderate.

Finally, the macro- and micro-

morphological characters of the flowers are

described with the aim to provide useful data for

identification and differentiation of the plant from

other allied species either in the entire or

powdered form.

INTRODUCTION

Onagraceae (Evening Primrose family,

Oenotheraceae)(1,2) comprises a number of

popular flowering ornamentals, including

Oenothera species which are native to North and

South America. These are commonly known as

"Evening Primroses, Suncups or Sundrops" and

are widely cultivated in borders and gardens, the

most reputed being Oenothera speciosa Nutt.

(Hartmannia speciosa Small) (1, 2). The plant is

usually referred to as "White, Pink or Mexican

Evening Primrose" and "Showy Evening

Primrose or Sundrops" (speciosa meaning

spectacular or showy) (3, 4). The common name

"Day-flowering Evening Primrose" is attributed

to the plant due the opening of its flowers during

day light hours(5) (c.f. other "Evening

Primroses").

Previous reports focusing on the

pharmacological and clinical evaluation of O.

biennis L. (Evening primrose) are numerous and

claimed its potentialities for the treatment of

atopic eczema, rheumatoid arthritis, premenstrual

syndrome, mastalgia, diabetic neuropathy,

arteriosclerosis, asthma and psoriasis (6 , 7).

Although the Onagraceae plants are

known as accumulators of polyphenols (8), there

are relatively few reports on the phenolic

composition of Oenothera species (8- 13). Several


186

phenol acids and flavonol glycosides were

detected by two dimensional TLC and used as

chemotaxonomic markers, in addition delphinidin

and cyanidin were identified (8). This publication

dealt also with the variability in the phenolic

content among the various organs of three

Oenothera species (other than Oenothera

speciosa Nutt.). In a similar way, Quercitin-3-O-

rhamnoside, Myricetin-3 -O- rhamnoside and

Myricetin-3 -O-galactoside were detected in the

leaves of Oenothera speciosa Nutt.(9); meanwhile,

the unique available report on isolation and

structure elucidation was on Myricetin 3-O-

methyl ether-3'-O-β-D-glucoside from the leaves

of the plant (14). On the other hand, nothing could

be traced in the available literature concerning the

carbohydrate composition of any of the plant

organs including the flowers. In a previous

communication (15), the authors investigated the

floral volatiles and lipoids; the present work

focuses on the phenolic and carbohydrate

constituents of the same organ.

Furthermore, the bioactivities of the

aqueous and alcoholic extracts of the root, stem

and leaves (16); as well as, those of the total and

fractionated hexane extracts of the flowers (15),

were previously evaluated by the authors. The

present report includes a similar investigation of

the aqueous and alcoholic extracts of the flower.

Finally, the macro- and micro-

morphological characters of the flowers are

described with the aim to provide useful

additional data for identification and

differentiation of the plant from other allied

species either in the entire or powdered form as

those previously presented by the authors for the

other organs (16).

EXPERIMENTAL

Plant Material

Flowers of Oenothera speciosa Nutt. were

collected from plants cultivated in the Experimental

Station of Medicinal Plants, Pharmacognosy

Department, Faculty of Pharmacy, Cairo University,

Giza, Egypt, during the flowering stage from March

to July (2004-2006). The plant was kindly

authenticated by Mrs. Therese Labib, Herbarium

Section, Orman Garden, Giza, Egypt. Identity

was confirmed by Dr. Mohamed El Gebali (Plant

Taxonomy and Egyptian Flora Department,

National Research Center, Dokki, Giza, Egypt).

Voucher samples are kept at the Museum of the

Pharmacognosy Department, Faculty of

Pharmacy, Cairo University.

Fresh samples kept in 70% ethanol

containing 5% glycerin were used for the

botanical study and air-dried flowers (reduced to

powder No. 36) were saved in amber coloured

glass containers for further phytochemical and

microscopical examination.

Material for Phytochemical Investigation

Different types of silica gel (E. Merck,

Darmstadt, Germany) were used as stationary

phases including: precoated silica gel 60 F254

plates for thin layer chromatography (TLC);

Silica gel H for vacuum liquid chromatography

(VLC) and Silica gel 60 for column

chromatography (CC). Sephadex LH-20 for CC was

obtained from Pharmacia Fine Chemical AB

(Uppsala, Sweden).Whatman No 1 sheets for

paper chromatography (PC) were purchased from

Whatman Ltd. (Maidstone, Kent, England).

The following solvent systems were prepared

from analytical grade chemicals:

S1: n-Butanol-Acetic acid-Water (3:1:1 v/v/v)

[PC]; S2: Acetic acid -Water (15:85v/v) [PC]; S3:

Ethyl acetate-Acetic acid-Formic acid-Water

(100:11:11:26 v/v/v/v) [TLC]; S4: Chloroform-

Methanol-Water (65:35:10 v/v/v) [TLC]; S5:

Chloroform-Methanol (8:2 v/v) [TLC]; S6: n-

Butanol-Acetic acid-Water (4:1:5 v/v/v upper

phase) [PC]; S7: n- Butanol- Pyridine- Water

(6:4:3 v/v/v) [PC].

Spray reagents used for spot visualization were:

R1: Aluminum chloride: for flavonoids (17); R2:

Natural products-polyethylene glycol reagent

(NP/PEG) for phenolics (18); R3: Ferric chloride:

for phenolics (19); R4: Vanillin/HCl: for catechins (18); R5: Aniline phthalate: for sugars (17) and R6:

Anisaldehyde-Sulphuric acid (17).

Reference phenolics, sugars and uronic acids

were purchased from E. Merck (Darmstadt,

Germany) and included: luteolin, kaempferol,

quercetin, apigenin, naringenin, quercetin-3-O-

glucoside, quercetin-3-O-galactoside, quercetin-

3-O-rhamnoside, quercetin-3-O-rutinoside;

chlorogenic, caffeic, ferulic and rosmarinic acids;

(+) catechin and (-) epicatechin, in addition to

glucose, galactose, xylose, rhamnose, arabinose

and mannose; glucuronic and galacturonic acids.

Shift reagents for UV spectroscopic analysis of

flavonoids were prepared according to published

procedures (20).

Material for Biological Evaluation:

Plant extracts were, separately, prepared

each from a 200 gm air-dried powdered sample.

The aqueous and alcohol 70% extracts were

prepared by cold exhaustive percolation. The

solvent in the first case removed by lyophilization

and in the second by distillation under reduced

pressure. One gram of the solvent-free dried

residue was equivalent to 7.4 g and 7.8 g of the


187

air-dried powdered flowers for the aqueous and

alcoholic extracts, respectively. For antimicrobial

screening, the extracts were dissolved in DMSO

at concentrations of 200 mg/ml (so that each 50

μl contained 10 mg of the extract).

Animals used in the toxicological and

pharmacological evaluation were obtained from

the animal house, of the National Research

Center, Dokki, Giza, Egypt; these included albino

mice (25-30 g) and adult male albino rats

(Sprague Dawley strain, 130-150g). They were

kept in metabolic cages and fed on standard

laboratory diet composed of vitamin mixture

(1%), mineral mixture (4%), corn oil (10%),

sucrose (20%), cellulose (0.2%), casein (10.5%)

and starch (54.3%).

Microorganisms used in the antimicrobial testing

were provided by the Microbiology Department,

Faculty of Pharmacy, Cairo University, Cairo, Egypt

and included: Escherichia coli, Pseudomonas

aeruginosa, Proteus vulgaris, Staphylococcus

aureus, Sarcina lutea, Bacillus subtilis,

Mycobacterium phlei, Candida albicans and

Candida tropicalis.

Standard drugs, kits and culture media were

supplied by their respective sources as follows:

Indomethacin (EIPICO, Egyptian International

Pharmaceutical Industries Co., Egypt; as standard

anti-inflammatory); Carrageenan (Sigma Co.,

Cairo, Egypt; for induction of oedema); Dipyrone

(Novalgin®, Hoechst (Sanofi Aventis), Orient,

Egypt; as standard analgesic); Paracetamol

(Panadol®, Schering, Germany; as standard

antipyretic); Alloxan monohydrate (Sigma, USA ;

for induction of diabetes); Glimepiride [Amaryl ®,

Hoechst (Sanofi Aventis), Marion, Roussel, S.A.R.;

as standard antihyperglycemic) ; Glutathione kit

(Wak-Chemie Medical, Germany; for assessement of

antioxidant activity) and Vitamin E (dl α-tocopheryl

acetate, Pharco Pharmaceutical Co.; as standard

antioxidant). Trypticase soy agar (Oxoid, England)

was used as nutrient medium, Ofloxacin (OFX) and

Amphotericin B (AMP.B), 5 μg/disc each (Oxoid,

England); as standard antimicrobials.

Apparatus

Melting points were determined on a

Buchi 520 apparatus. UV lamp (254, 366 nm)

Model ENF-260 CIF USA was used for

localization of fluorescent spots on the

chromatograms. The UV spectra of flavonoids

and tannins were determined on a Beckman Du-7

and Shimadzu 265 C spectrophotometers. Mass

Spectra of the isolated compounds were recorded

on Varian Mat 711, Finnigan SSQ 7000 and

OMM 7070t mass spectrometer. The 1H- and 13C-NMR spectra were recorded at 300 and 100

MHz, respectively, on a Varian (Model L 900)

spectrometer (Germany), using TMS as internal

standard and DMSO-d6 as solvent. HPLC

analysis of phenolics was performed on an

Agilent Series 1100 apparatus equipped with

Quaternary pump, series 1100; degaser, series

1100; column heater, series 1100 and a UV

detector series 1100. The apparatus used for

HPLC analysis of carbohydrates consisted of an

isocratic pump (Model Lc-l0As, Shimadzu,

Japan); refractive index detector (RID-6A, Shimadzu,

Japan). A Rheodyne injector (Model 7161, Catati,

California, U.S.A.) equipped with 20 µl injector loop

and an integrator (Model C-R 7A, Shimadzu, Japan),

Phenom-Dim, 250x 460, Ser: 128854, 20 µg injected.

The sensitivity was set as 0.001 AUFS.

Methodology

A. Investigation of the phenolic content

HPLC analysis of the phenolic constituents: The phenolic composition of the flowers

collected in March (sample A, early flowering

period) was qualitatively and quantitatively

analyzed by HPLC as compared to a sample

collected in July (sample B, late flowering

period). Phenolics including flavonoids

(expressed as their aglycones) and phenol acids

were determined in the hydrolyzed-flower

extracts adopting the procedure described by

Mattila et al. (2000) (21). One gram of the air-

dried plant material was weighed into a 100 ml

conical flask then dispersed in 40 ml of aqueous

methanol (62.5%). The mixture was then

ultrasonicated for 5 min. To this extract 10 ml of

6 M HCl were added. The flask containing the

mixture was placed in a shaking water bath at

90°C for 2 hours. After hydrolysis, the sample

was allowed to cool, then filtered, made up to

100 ml with methanol, and ultrasonicated again

for 5 min. Before injection into the HPLC

apparatus, the sample was filtered through a 0.2

µm membrane filter into the sampler vial for

injection. HPLC separation was performed on a

Hypersil-ODS (4.6x250 mm, 5µm) column.

Isocratic elution was employed using acetonitrile

/15% acetic acid (40/60 v/v) as mobile phase.

The flow rate of the mobile phase was 1 ml/min.

and the injection volumes were 40 µl of the

standards and sample extracts. The standards

were made by diluting stock standards in MeOH

to yield 50µg/ml. Detection was carried out by a

UV detector set at 330 nm.

The major components of the sample

were identified by comparing their retention

times and spectral data with those obtained for

the standards (viz., quercetin, kaempferol,

luteolin, naringenin, apigenin, chlorogenic acid,

caffeic acid, ferulic acid and rosmarinic acid).

The identified flavonoids and phenol acids were

quantified using the external standard method.


188

Quantification was based on the peak areas of

both standards and samples. Stock standards

(diluted in methanol, 20-600µg/ml) and samples

were analyzed in duplicate. Results are recorded

in Table (1).

Preparation and fractionation of the ethyl

acetate extract: An air-dried powdered flower

sample (1 Kg) was exhaustively extracted by cold

percolation with ethanol 70% at room

temperature. The ethanol extract was

concentrated to dryness under reduced pressure at

a temperature not exceeding 50°C. The residue

(116 g) was then suspended in water and

partitioned with petroleum ether and then ethyl

acetate. The solvents were, separately, evaporated

under reduced pressure to yield 26 g and 47 g,

respectively. The ethyl acetate extract was

investigated for its phenolic content using TLC

and CC. TLC screening was achieved on

precoated silica gel plates using different solvent

systems (S1-S3) alongside with reference

samples. The chromatograms were visualized

under visible and UV light (254 and 366 nm)

before and after exposure to ammonia vapour, as

well as, after spraying with R1-R3 and R6.

Twenty g of the ethyl acetate extract was

fractionated on a vacuum liquid chromatography

column (VLC), packed with silica gel H.

Gradient elution was performed using CHCl3,

EtOAc and MeOH mixtures of increasing

polarity. Fractions of 200 ml were collected and

monitored by TLC using precoated silica gel

plates and S2 and S3 as solvent systems. The

chromatograms were examined as previously

mentioned. Fractions showing similar

chromatographic patterns were pooled to afford

three collective fractions (I-III). Fraction I (92

mg) eluted with 90% -85% CHCl3 in EtOAC, was

repeatedly purified on a sephadex LH-20 column,

using methanol as eluent and yielded compound

1 (52 mg). Fraction II (190 mg) eluted with 10-

5% CHCl3 in EtOAC, was subjected to

rechromatography on a sephadex LH-20 column

and eluted with methanol then followed by

purification using preparative TLC and S3 as

solvent system to afford compound 2 (41 mg).

Fraction III (260 mg) was eluted with 75%-60%

EtOAC in MeOH; an aliquot of this fraction (130

mg) was rechromatographed on a sephadex

column, eluted with MeOH: H2O mixtures (80%-

50%) to afford compound 3 (23 mg) and

compound 4 (66 mg).

The isolated compounds were subjected to

tests for flavonoids, carbohydrates and /or

glycosides, as well as physical and spectral

analyses (UV spectral data with different shift

reagents, IH-NMR, I3C-NMR and EIMS).

Compounds 2 and 4 gave positive Molisch's test

and were subjected to acid hydrolysis (22). The

sugar and aglycone moieties were examined by

PC using S6 and S7 as solvent systems and the

chromatograms visualized by spraying with R5

and R6.

Determination of tannin content: The tannin

content was determined gravimetrically adopting

the hide powder method (23). Furthermore,

proanthocyanidins (condensed tannins) were

determined adopting a chemical assay in which,

proanthocyanidins are oxidatively depolymerized

in butanol - HCI mixture to yield red

anthocyanidins that can be measured

spectrophotometrically at 550 nm (24). Results are

represented in Table (2).

Extraction and isolation of catechins: The

dried powdered flowers (500 g) were defatted

with hexane; the marc was air dried and then

exhaustively percolated with 50% acetone (v/v)

on cold. The combined acetone extractives were

pooled and mixed, followed by removal of the

solvent under vacuum at a temperature not

exceeding 40°C to yield 11g. The obtained

residue was investigated for its tannin content

using TLC and CC. TLC-screening was carried

out using precoated Si-gel plates, two spots were

detected [major spot; Rf: 0.89 and 0.52; and

minor spot: Rf: 0.61 and 0.25 using S4 and S5

as solvent systems, respectively). The

chromatograms were examined under UV light

(254 and 366 nm) before and after exposure to

ammonia vapour, as well as, after spraying with

R3 and R4. The crude acetone extractive (5 g)

was fractionated on a Sephadex LH-20 column

and eluted with methanol. Fractions (15 ml,

each), were collected and monitored by TLC

using S5 as solvent system and the

chromatograms were visualized as previously

mentioned. The fractions (2.4 g), containing the

major component (Rf: 0.52; TLC, S5) were

pooled and repeatedly chromatographed on

precoated silica gel plates (PTLC), elution was

performed with S4. Further purification on

Sephadex LH-20 column, using methanol as

eluent, afforded compound 5 as a white powder

(61 mg). Structure elucidation was established

on the basis of physical and spectral analyses

(UV, 1H-NMR and 13C-NMR).

B. Investigation of carbohydrate content (25):

A fresh sample of the flowers (100 g) was

minced and homogenized with ethanol 70% in a

blender, filtered and the filtrate was concentrated

under vacuum and then lyophilized. One gram of

the residue was dissolved in pyridine and

filtered. The filtrate was evaporated and saved

for further analysis of the free sugars. The

mucilage was prepared by macerating the marc

in cold water slightly acidified with hydrochloric


189

acid (pH 3.5) till exhaustion, followed by

extraction with hot water (90-95°C) until

complete extraction of the mucilage. The

mucilage, in each case, was precipitated by

addition of 4 volumes of ethanol 95% to each

volume of the aqueous extract to yield two

different samples of mucilage; the cold

extracted-mucilage (CEM) and the hot extracted-

mucilage (HEM) which were carefully dried and

kept in a desiccator. About 0.5 g of each sample

(CEM and HEM) was separately hydrolyzed.

The mucilage hydrolyzates were evaporated to

dryness under vacuum. Analyses of the free

sugars and mucilage hydrolyzates were achieved

by PC (solvent systems S6 and S7, spray reagent

R5) and HPLC. The latter was carried out by

separately dissolving the free sugars and

mucilage hydrolyzates (0.2 g, each) in 2 ml of

acetonitrile-water (75:25), then 20 µl were

injected through the Rheodyne injector on a high

pressure Kromasil 10 NH2 column (250 x 4.6

mm). The sugars were eluted with acetonitrile-

water as a mobile phase, 1.5 ml/min as flow rate,

retention times and peak areas were determined

using refractive index detector and C-R7A

model integrator. Qualitative and quantitative

identifications of peaks were carried out by

comparison with authentic sugars analyzed under

the same experimental conditions. Results of PC

and HPLC analyses are recorded in Tables (3)

and (4), respectively.

C. Biological evaluation

Toxicological and Pharmacological

studies of the aqueous and alcoholic extracts, as

well as, the hot extracted mucilage (HEM) of the

flowers of Oenothera speciosa Nutt. were carried

out, including determination of the median lethal

dose (LD50) according to Karber's procedure

(1941) (26). Furthermore, the pharmacological

potentialities of the aqueous and alcoholic

extracts viz., anti-inflammatory (27), analgesic (28),

anti-ulcer (29), antipyretic (30) and antioxidant (31)

activities were assessed. In addition, the acute

and long-term anti-hyperglycemic activity (32, 33)

of HEM and alcoholic extract were evaluated.

The effects produced were, in each case,

compared to those of appropriate reference drugs.

Results obtained for the biological activities are

compiled in Tables (5-11).

The experimental data were, in each case,

statistically analyzed using student's "t" test

according to Snedecor and Chochran (1971) (34).

Moreover, the aqueous and alcoholic

extracts were subjected to antimicrobial

screening by applying the disc agar diffusion

method (35, 36). Aliquots (50 μl) of DMSO,

solutions of the different extracts (equivalent to

10 mg of each extract) were separately tested.

A disc impregnated with 50 μl of DMSO was

used as a negative control. Discs of Ofloxacin

(OFX) and Amphotericin B (AMP.B), 5 μg

g/disc each were used as positive controls. The

inhibition zones were measured and recorded in

Table (12).

RESULTS AND DISCUSSION

The preliminary phytochemical screening,

carried out in a previous publication (16)

ascertained the presence of flavonoids, tannins

and carbohydrates in the flowers of Oenothera

speciosa Nutt. , cultivated in Egypt.

A. Investigation of the phenolic content The results of HPLC analysis of the

hydrolyzed methanol extracts of the flowers, as

displayed in table 1, revealed that its phenolic

composition was greatly influenced by the time

of collection. Although, qualitative variation was

not evident among the major phenol acids and

flavonoid components, yet, quantitative variation

was obvious. Constituents identified were

chlorogenic, caffeic, ferulic and rosemarinic

acids, as well as, quercetin, naringenin and

kampferol. The amount of phenol acids increased

from 201.89 to 548.59 mg/100g DW during the

flowering season with concomitant increase in the

amount of chlorogenic and caffeic acids which

reached up to 300.21 and 204.44 mg/100g DW,

respectively, at the end of the flowering stage

(sample B). On the contrary, that of ferulic and

rosemarinic acids was reduced. Concerning the

flavonoidal constituents (expressed as their

corresponding aglycones), they showed a marked

decrease, along the flowering season, to less than

half the total concentration in sample A, collected

at the early flowering period (from 407.07 to

180.02 mg/100g DW) with a parallel decrease in

the individual aglycone components which were

in both samples dominated by quercetin.

Chromatographic fractionation of the

ethyl acetate extract allowed the isolation of

four major compounds 1-4 which were

characterized through determination of their

physico-chemical and spectral data as follows:

Compound 1: 52 mg, yellow needle crystals

(CHCl3), 21 mg, m.p. 314 - 316o C, soluble in

methanol; Rf 0.75 and 0.64 (TLC, S5 and S6,

respectively), yellow (UV, UV/NH3 and AlCl3),

orange (NP/PEG). UV λ max nm (MeOH): 256,

269 (sh), 301 (sh), 372; (NaOMe) 247 (sh),

330(dec); (AlCl3) 269, 308 (sh), 335, 455;

(AlCl3/HCl) 272, 304 (sh), 352, 429 (NaOAc)

258 (sh), 268, 328, 398; (NaOAc /H3BO3) 259,

303 (sh), 386.

Compound 2: 41 mg, yellow powder; soluble in

methanol; m.p. 215-217°C; Rf: 0.43 (PC, S2);

and 0.61 (TLC, S3), respectively; purple (UV);


190

yellow (UV/NH3); orange (NP/PEG); UV λmax

nm, MeOH: 259, 267 sh, 299 sh, 369; NaOMe:

272, 325 sh, 412; AlCl3: 271, 303 sh,. 328 sh,

437; AlCl3/HCl: 272, 305 sh, 368, 408; NaOAc:

270, 324 sh, 375; NaOAc/ H3BO3: 260,304 sh,

378. Acid hydrolysis afforded the aglycone

moiety which was identified as quercetin by

direct comparison with an authentic sample (m.p.,

m. m. p. and co-chromatography). The sugar

moiety was identified as galactose by PC

alongside with authentic samples.

Compound 3: 23 mg, white needles; soluble in

methanol, ethanol and acetone; m.p. 207-209°C;

Rf: 0.68 (PC, S2) and 0.52 (TLC, S3),

respectively; blue fluorescence (UV); yellowish-

green (UV/NH3), blue (FeCl3); blue fluorescence

(NP/PEG); UV λ max nm, MeOH: 244, 298 sh,

328; NaOMe: 240, 265, 305 sh, 379. EIMS: m/z

(rel. inten.): 354[M+, 15%], 336[M+-18],

180[caffoeyl, 90%], 163[100%], 162[caffoeyl-18,

52%], 143[40%], 110[60%].

Compound 4: 66 mg, yellow powder; soluble in

methanol; m.p. 192-195°C; Rf: 0.56 (PC, S2)

and 0.42 (TLC, S3) , respectively; deep purple

(UV); yellow (UV/NH3 and UV/AlCl3); UV

λmax nm, MeOH: 258, 300 sh, 358; NaOMe:

270, 328 sh, 410; AlCl3: 277, 305 sh, 430;

AlCl3/HCl: 272, 298, 362 sh, 398; NaOAc: 268,

320 sh, 392; NaOAc/H3BO3: 262, 308 sh, 386.

Acid hydrolysis produced quercetin as aglycone

(direct comparison with authentic sample, m.p.,

m. m. p. and co-chromatography). The sugar

moiety consisted of glucose and rhamnose

(identity confirmed using PC alongside with

authentic reference samples).

Compounds 1-4 were identified as: quercetin,

hyperoside (quercetin-3-O--β-D galactoside),

chlorogenic acid and rutin (quercetin-3-O-α-L-

rhamnose-β-D-glucoside), respectively by direct

comparison with authentic samples and through

physical, chromatographic and UV spectral data

before and after addition of different shift

reagents, identification was confirmed by

comparison with published data (20, 22, 37-40).

Results of the gravimetric determination of

tannins by the Hide Powder method (23) and those

of the spectrophotometric estimation of the

proanthocyanidins (24), as recorded in table 2,

revealed that both were higher when the flowers

were collected at the end of the flowering season

(sample B, 10.53 g% and 23.35g%, respectively)

being present in lesser amounts in the early stage

(8.67 g % and 19.03 g%, respectively). The low

results obtained by the gravimetric method are

probably due to the inability of certain tannin

molecules to penetrate the hide and associate with

proteins (41).

Chromatographic fractionation of the acetone

extract afforded compound 5 which was

characterized as follows:

Compound 5: 61 mg; white powder; soluble in

methanol and acetone; m.p. 175-177°C; Rf: 0.89

and 0.52 (TLC, S4 and S5), respectively;

yellowish-brown (UV), dark brown (UV/NH3),

violet (FeCl3); pink (vanillin/HCl). UV λmax

nm, MeOH: 276, 288 sh; NaOMe: 300,310 sh;

AlCl3: 280, 300 sh; AlCl3/HCl: 275,288 sh;

NaOAc: 276, 310 sh; NaOAc/H3BO3: 282, 310

sh. 1H-NMR (DMSO-d6) δ ppm; 9.17 (1 H, s, 3'-

OH), 8.96 (1 H, s,4'-OH), 8.51 (2 H, s, 5,7-OH),

6.84 (1 H, d, J 2',6' =1.5 Hz, H-2'), 6.75 (1 H, d,

J 6',5'= 8.1 Hz, H-5'), 6.70 (1 H, dd, J 6',2'= 1.8,

J 6',5' = 8.0 Hz, H-6'), 5.93 (1 H, d, J 8,6=2.4 Hz,

H-8), 5.86 (1 H, d, J 6,8=2.2 Hz, H-6), 4.74 (1 H,

m, H-3), 4.56 (1 H, d, J 2,3 trans = 7.5 Hz, H-2),

3.94 (1 H, bs, 3-OH), 2.88 (1 H, dd, J=5.9,

16.2gem Hz, H-4 ax), 2.46 (1 H, dd, J=8.9,

16.5gem Hz,H-4 eq). 13C-NMR (DMSO-d6) δ

ppm, 157.8(C-7), 157.5 (C-5), 156.9 (C-9),

146.2 (C-3'), 146.2 (C-4'), 132.2 (C-l'), 120.0(C-

6'), 116.1 (C-5'), 115.3 (C-2'), 100.8 (C-10), 96.3

" (C-6), 95.6 (C-8), 82.9 (C-2), 68.8 (C-3), 28.5

(C-4). From the previous findings and through

comparison with published data (13, 20, 37, 39, 40, 42),

compound 5 could be identified as (+)Catechin.

B. Investigation of carbohydrate content The ethanolic extract obtained from the

fresh flowers yielded a residue (13.68%) which

gave a positive Fehling's test suggesting the

presence of reducing sugars.The polysaccharides

prepared by both cold and hot extraction

methods viz., CEM (7.48 %) and HEM (12.78

%) were soluble in water, insoluble in alcohol

and ether; they gave positive tests for mucilage

(red stain with ruthenium red) (43) and negative

tests for pectin (44). The percentage of mucilage

extracted by the hot process exceeded that

obtained by the cold extraction method.

Paper chromatography and HPLC analysis

(Tables 3 and 4) revealed the occurrence of

rhamnose, arabinose and glucose in the free state

in the ethanolic extract. Rhamnose dominated the

identified sugars in all the tested samples viz.,

the ethanol extract, and both CEM and HEM

hydrolyzates (55.63 %, 34.29 % and 29.67 %,

respectively), followed by glucose (13.41 %,

23.68 % and 14.77 %, respectively). Meanwhile,

arabinose was detected in the ethanol extract and

CEM hydrolyzate (15.47 % and% 8.12 %); and

galactose in both CEM and HEM hydrolyzates

(3.15 and 4.47% respectively). On the other

hand, xylose could be identified in the HEM

hydrolyzate only (13.12 %). Glucuronic and

galacturonic acids were detected in both

hydrolyzates. Glucuronic being present in a

noticeably higher amount in CEM hydrolyzate

(18.14%) than in that of HEM (8.09%).


191

From the above results, it could be

concluded that there is an obvious quantitative

variation, although slight qualitative differences

are observed, in the composition of the mucilage

hydrolyzates prepared by the two different

methods. Finally, the reasonable detected

amounts of free sugars and mucilage could

suggest the use of the flower extracts as an

adjuvant in nutraceuticals.

C. Biological evaluation

The aqueous and alcoholic extracts, as

well as, HEM of the flowers were found to be

non toxic at the tested doses as no mortality was

observed when given to mice orally in doses up

to 9.1, 9.3 and 7.4 g/Kg. b. wt, respectively and

are considered to be safe according to Buck et al.

(1976) (45).

Both aqueous and alcoholic extracts exerted

(Table 5) a remarkable anti- inflammatory action

reaching up to 89% and 95% that of indomethacin,

respectively). The alcoholic extract appeared almost

as effective as indomethacin, the standard anti-

inflammatory.

The analgesic effect of the aqueous and

alcoholic extracts of the flowers (Table 6) was

time dependent. The aqueous extract produced a

marked action (with a potency 66% that of

Novalgin after one hour, and 77 % after two

hours, respectively), meanwhile, a higher action

was shown by the alcoholic extract after one and

two hours (with a potency 76 % and 83 % that of

Novalgin, respectively). The anti-inflammatory

and analgesic properties may be attributed to the

presence of flavonoids (46) and tannins (47).

Results obtained from Table (7) showed

that both extracts had marked anti-ulcer activity.

The alcohol extract significantly decreased the

number of ulcers by 72.49% protection, while the

aqueous extract showed a moderate protection of

percentage 67.73% compared to indomethacin.

The flavonoids (48) and (+) catechin (49) may

contribute to the anti-ulcer activity of both

extracts.

The aqueous and alcoholic extracts exhibited

a pronounced and powerful antipyretic action of

potency comparable to that of paracetamol (84%,

100% after one hour and 89%, 88% after two hours,

respectively, Table 8).

Both tested extracts possessed a powerful

antioxidant activity (Table 9). Animals treated

with Vitamin E (7.5 mg/Kg b.wt.) restored the

level of blood glutathione in diabetic rats (%

change from control= 1.4%). The level of blood

glutathione in diabetic rats was restored after the

oral administration (10 mg/kg b.wt.) of the

ethanolic and aqueous extracts of the flowers (%

change from control=1.6% and 2.7%,

respectively). This effect may be due to the

synergestic action of proanthocyanidins and

flavonols (50) in both extracts.

Concerning the anti-hyperglycemic

activity (Table 10), HEM and the alcoholic

extract exhibited significant effects in alloxan-

induced diabetic rats, which was time dependant.

The potency of HEM was higher than that of

alcoholic extract after four hours oral drug

administration (62%and 57%) and was more

pronounced after six hours (67% and 64%),

respectively, compared to glimepiride. Likewise,

daily administration (long-term effect) of HEM

and alcoholic extract, at a dose of 100 mg/kg.

b.wt. (Table 11), revealed that HEM reduced

blood glucose levels in alloxan-induced diabetic

rats with a potency of 77%, while the alcoholic

extract exhibited a weaker action, being 62% as

potent as that of glimepiride after eight weeks of

treatment, respectively. Thus, we may conclude

that HEM was more effective regarding the acute

and long-term activity in the tested dose-level.

The results obtained here are in agreement with

previous reports confirming that polysaccharides (51) (HEM) and polyphenolic compounds (52)

(alcoholic extract) showed anti-hyperglycemic

effect.

Results obtained on evaluation of the

antimicrobial activity (Table 12) showed that

the alcoholic extract of the flowers of Oenothera

speciosa Nutt. exerted significant inhibitory

effect towards Staphylococcus aureus,

Mycobacterium phlei, Pseudomonas aeruginosa,

a marked effect on both Candida albicans and

Candida tropicalis and little effect on the other

tested microorganisms. Meanwhile, the aqueous

extract showed weak effect against the tested

microorganisms and no effect on Escherichia

coli. From the previous findings, it is evident that

the alcoholic extract is more powerful than the

aqueous extract.

It is worthy to note that the biological

potentialities of the flower exceeded those of the

other organs when similarly assessed (16).

From the aforementioned data, the hot

extracted mucilage (HEM), aqueous and

alcoholic extracts could be expected after further

laboratory and clinical trials, as successful

candidates for herbal formulations prescribed for

complementary treatment of a wide variety of

ailments such as inflammations, arthritis,

diabetes mellitus and microbial infections, as

well as, in management of painful processes,

prevention of stress ulcer formation, and as

powerful antioxidants.

BOTANICAL DESCRIPTION

Oenothera speciosa Nutt. is a perennial

herb, reaching up to 65 cm in height and

cultivated for its showy flowers. The plant

blooms from March to July. Flowers are solitary,


192

axillary, sessile, few to several in the upper axils,

with a showy, white to pink corolla. The buds in

the stem tips are nodding, lanceolate to lance-

elliptic in shape and showing acute to acuminate

apices.

Macro-morphology

The flower (Fig. 1A&B) is cup-shaped

and appears pinkish- white to pink in colour,

actinomorphic, hermaphrodite, epigynous and

tetramerous. The flower has a hypanthium

(Bailey, 1958) which adnates to an inferior ovary

and is prolonged beyond its apex, bearing the

sepals, petals and stamens. It has a delicate

fragrant odour and an astringent, mucilaginous

taste. The flower measures from about 4.0- 6.0

cm in D.

The bract (Fig.1B) is small, persistent, sessile,

green, lanceolate and rather rhomboidal in shape

with broadly dentate margin, acuminate apex and

pinnate venation. It has faint characteristic odour

and a slight astringent, mucilaginous taste. It

measures 0.7 - 1.6 cm in L. and 0.4 – 0.6 cm in

W.

The calyx (Fig. 1B) is yellow-green in colour

and often with reddish margins. It consists of four

narrow lobes which cohere together to form the

calyx tube. The calyx tube (1.5-3.2cm in L. and

0.4-0.5cm in D.) shows closely parallel lobes

with free acuminate tips (0.1–0.5 cm in L.) in the

unexpanded buds but is strongly reflexed to the

upper surface and pulled to one side in the open

flower. It has a faint characteristic odour and an

astringent mucilaginous taste.

The corolla (Fig. 1B) is epigynous, consisting of

four free petals (convolute in the bud), alternating

with the sepals. The petals are pinkish- white to

pink in colour, yellowish at the base, obcordate or

broadly obovate, smooth with entire margins,

emarginated apices, arising from the rim of the

hypanthium and fused at the base into a very

short tube which is in turn joined to the sepals.

Each petal measures 2.1 – 4.2 cm L. and 2.2-3.6

cm W. at the widest part. It has a faint aromatic

odour and an astringent mucilaginous taste.

The andrœcium (Fig. 1B) consists of 8

yellowish, unequal stamens distinctly arising near

the hypanthium rim, arranged in two whorls. The

outer stamens are longer, alternating with the

petals, while the inner ones are shorter, being

antepetalous and adnate to the base of corolla.

The anther (Fig. 1B) is yellow in colour, oblong

to linear, versatile, opening longitudinally

measuring 0.7-1.2 cm in L. and 1.0-1.5 mm in D.

The filaments (Fig. 1B) are yellow in colour;

filiform, glabrous; the free parts are unequal in

length (with the shorter ones inwards, measuring

0.9-1.3 cm in L. and the longer ones outwards

and measuring 1.2-1.8 cm in L.).

The Hypanthium (Fig.1B) is slender,

quadrangular and light green in colour measuring

1.0 - 2.7 cm in L. and 0.15 - 0.3 cm in D. The

hypanthium adnates to the ovary and is

prolonged above it forming a funnel-shaped tube,

which is as long as or sometimes longer than the

ovary, getting wider towards the upper third and

measures 0.5-0.9 cm in L. and about 0.2-0.4 cm

at the widest part. This tube bears the fused bases

of the sepals, petals and stamens.

The ovary (Fig.1B) is inferior, tetracarpellary,

tetralocular, syncarpous and fused with the

hypanthium. It measures 0.5 - 0.8 cm in L. and

0.27 - 0.39 cm in W. at the prominent ridges

(widest part). The ovules are numerous, having

an axile placentation.

The style (Fig.1B) is simple, cylindrical, long,

filiform, greenish near the base, whitish near the

apex and passing through the hypanthium. It

measures 2.5 - 3.5 cm L. and 0.5-1.0 mm D.

The stigma (Fig.1B) is creamy white in colour,

composed of 4 linear radiating lobes, each

measuring 4 to 7 mm in L.

The flower has the following floral formula:

, ♀, K (4), C 4, A 4+4, G (4).

Micromorphology

The Bract (Fig. 2): A transverse section in the bract (Fig. 2A)

is plano-convex, consisting of an inner and outer

epidermis, enclosing the mesophyll which

consists of palisade-like and oval

parenchymatous cells with relatively wide

intercellular spaces and is traversed by 4 to 5

small vascular strands. The midrib is prominent

on the lower surface and shows a collateral

vascular bundle which is accompanied by a

scanty perimedullary (intraxylary) phloem. Some

parenchyma cells contain mucilage (red stain

with ruthenium red) in which are embedded

bundles of raphides of calcium oxalate.

The inner (upper) epidermis (Fig. 2B&D)

consists of polygonal, axially elongated cells,

having wavy anticlinal walls covered with thin

smooth cuticle. Stomata of anomocytic types

(rarely anisocytic) are present. Non-glandular

hairs are few, unicellular, straight or curved,

arising from the cicatrices; they have thick walls,

moderately wide lumina and tapering apices and


193

are covered with a warty cuticle. Non-glandular,

unicellular, club-shaped hairs are rare and

covered with a thin, smooth cuticle. The outer

(lower) epidermis (Fig. 2C&D) is more or less

similar to the inner one but showing less

elongated cells with strongly wavy anticlinal

walls and more stomata.

The mesophyll (Fig. 2D) is wide, homogeneous,

formed of thin walled, elongated, more or less

oval cells and showing wide intercellular spaces;

some of them contain mucilage in which raphides

of calcium oxalate are embedded (Fig. 2A and

D). The vascular strands show small lignified

vessels, delicate phloem elements and scanty

perimedullary phloem.

The Calyx (Fig. 3): A transverse section in the calyx-tube (Fig.

3A) is circular in outline; consisting of four

segments. Each segment comprising outer and

inner epidermises, enclosing a ground tissue

containing raphides of calcium oxalate and

traversed by numerous small vascular strands.

The cells of the inner (upper) and outer (lower)

epidermises (Fig. 3 A-D) are more or less

similar; they are polygonal with more or less

straight or slightly wavy anticlinal walls and

covered with smooth cuticle and some of them

containing oil droplets. Anisocytic and

anomocytic stomata are few on the inner surface

and numerous on the outer one. Non-glandular

hairs, similar to those of the bract, are present.

The mesophyll (Fig. 3D) is homogenous

consisting of thin-walled, polygonal, more or less

isodiametric parenchymatous cells. Raphides of

calcium oxalate similar to those of the bract are

present.

The Corolla (Fig. 4): A transverse section in the petal (Fig. 4A)

is more or less crescent-shape in outline and

consists of inner and outer epidermises enclosing

a homogenous parenchymatous mesophyll, which

is traversed by numerous small vascular strands.

Raphides of calcium oxalate similar to those of

the bract and calyx are present.

The inner (upper) epidermis (Fig. 4B&D) is

formed of polygonal, axially elongated cells with

slightly wavy anticlinal walls and covered with

smooth cuticle and some of them containing oil

droplets (stained red with Sudan III). Stomata are

absent. Few non-glandular hairs, similar to those

of the bract and calyx tube, are present. The cells

of the outer (lower) epidermis (Fig. 4C&D) are

similar to those of the upper but with more wavy

anticlinal walls and few anomocytic stomata are

present.

The mesophyll (Fig. 4D) is homogeneous and

consists of several rows of polygonal, nearly

isodiametric, thin-walled parenchymatous cells.

Raphides of calcium oxalate are embedded in the

mucilage and scattered in the mesophyll. The

vascular strands are formed of soft phloem tissue

and lignified xylem vessels.

The Andrœcium (Fig. 5): The Anther (Fig. 5A-C & H): A transverse

section through the anther (Fig. 5A) shows two

anther lobes attached together by a connective

tissue which is traversed by a small vascular

strand. Each anther lobe has two pollen sacs

containing numerous pollen grains. The anther

wall (Fig. 5A& B) is thin and consists of an

epidermis followed by a fibrous layer then the

remains of tapetal layer. The epidermal cells

(Fig. 5B&C) are tabular, polygonal, more or less

isodiametric with straight anticlinal walls and

covered with thin smooth cuticle, devoid of

stomata and hairs. The fibrous layer (Fig.

5B&H) consists of a single row of polygonal,

rectangular-shaped, thick-walled, lignified cells

and showing bar-like thickening. They appear in

surface view as polygonal, axially elongated cells

with lignified, beaded walls. The tapetal layer

(Fig. 5B) is represented by delicate, flattened,

thin-walled, parenchymatous cells. The

connective tissue encloses a central vascular

bundle. The pollen grains (Fig. 5G) are large;

triangular with smooth thick exine and have three

prominent germ pores which occupy the angles;

each one is covered by a dome formed by the

thickening of the intine and some having

protruding pollen tubes. The pollen grains are

connected by viscid threads.

The Filament (Fig. 5D-F): A transverse section

in the filament (Fig. 5E& F) is nearly oval to

elliptical in outline. It consists of an epidermis

covered with thin cuticle, followed by the ground

tissue that is traversed by a small vascular strand

and shows raphides of calcium oxalate. The

epidermis (Fig. 5D&F) is formed of polygonal

thin-walled axially elongated cells covered with

smooth cuticle and few of them containing oil

droplets. Stomata and hairs are absent. The

cortex (Fig. 5F) consists of thin-walled,

parenchymatous cells containing scattered minute

raphides of calcium oxalate.

The Hypanthium and Gynæcium (Fig. 6 A-F): A transverse section in the hypanthium

below the level of the ovary (Fig. 6A) is

quadrangular in outline and consists of hairy

epidermis having numerous unicellular non-

glandular hairs, followed by narrow

parenchymatous cortex. The pericycle is


194

parenchymatous and followed by a continuous

ring of collateral vascular bundle, which is

traversed by medullary rays and surrounding a

comparatively wide quadrangular pith showing

few small patches of perimedullary (intraxylary)

phloem near its periphery. Raphides of calcium

oxalate are scattered throughout the cortex and

pith, being more abundant in the pith.

A transverse section in the hypanthium passing

through the base of the ovary (Fig. 6B) is

quadrangular in outline, showing slight

prominences and numerous unicellular non-

glandular hairs. It consists of an epidermis

followed by a wide, parenchymatous ground

tissue and shows four cavities of the ovary

locules at the center. The outer part of the ground

tissue is traversed by collateral vascular bundles

which appear next to the prominent regions.

Raphides of calcium oxalate are scattered all over

the ground tissue.


through the middle of the ovary (Fig.6C), is

also quadrangular and wedged in outline (8

wedges or ridges, the four wedges at the corners

are strongly prominent), showing numerous

unicellular non-glandular hairs. It consists of an

epidermis followed by a parenchymatous ground

tissue. The outer part of the ground tissue shows

collateral vascular bundles which appear next to

the prominent regions and accompanied by small

groups of perimedullary (intraxylary) phloem and

raphides of calcium oxalate scattered all over the

ground tissue. Centrally four locules appear, each

one showing two ovules of an axile placentation.


through the top of the ovary (Fig. 6D) shows

that the hypanthium appear as a hollow short tube

surrounding the pubescent base of the style. The

outer surface is more or less circular in outline,

while the inner surface is pubescent and irregular.

The outer epidermis of hypanthium (Fig. 6E)

consists of polygonal, slightly axially elongated

cells with straight anticlinal walls and covered

with smooth cuticle and some of them containing

oil droplets. Anisocytic stomata and numerous

non-glandular hairs similar to those of the bract

and calyx tube are present. The inner epidermis

(Fig. 6F) of the hypanthium tube above the

ovary is formed of polygonal, slightly axially

elongated cells similar to those of the outer

epidermis (Fig. 6 E) but the cells are larger in

size.

Serial Sections through the flower bud (Fig. 7

A-F) are more or less rounded in outline. Each

consists of outer and inner epidermises enclosing

a parenchymatous mesophyll, which is traversed

by numerous small vascular strands. Raphides of

calcium oxalate are scattered all over the ground

tissue. The style appears occupying the centre as

it passes through the hypanthium and the calyx

tube. These serial sections illustrate the

arrangement of the different floral parts in the

flower as follows:

A transverse section in the hypanthium tube

above the ovary (the free prolonged part of the

hypanthium) (Fig. 7A) is quite similar to that

illustrated in (Fig. 6D) but non-glandular hairs of

the inner epidermis are much fewer. The rim of

the hypanthium tube (Fig. 7B) show the fused

bases of the sepals, petals and stamens to the

summit of the hypanthium and four anthers of the

shorter stamens.

Moreover, a transverse section in the bud passing

through the lower part of the calyx-tube (Fig.

7C) shows that the calyx-tube, petals, and

stamens begin to separate from each other. The

outer stamens are alternate with the petals while

the inner stamens are antepetalous and adnate to

the base of the corolla.

The unequal length of the stamens has been

illustrated in the transverse sections passing

through the middle, upper parts of the bud

and the free tips of the calyx-tube (Fig.7D-F).

The style appeared at the center of the flower bud

as shown in (Fig. 7 A-E). In addition, a T. S. at

the free tips of the calyx-tube (Fig. 7F) showed

four branches of the stigma which occupying the

center of the flower.

The Style (Fig. 8A, A1& C):

A transverse section in the style is nearly

round in outline. It consists of an epidermis

followed by a narrow parenchymatous ground

tissue traversed by four small vascular strands

(representing the four united individual styles),

showing few raphides of calcium oxalates.

The epidermis (Fig. 8C) is formed of

polygonal, axially elongated, thick-walled cells

with straight anticlinal walls and covered with

smooth cuticle and some of them containing oil

droplets. Anomocytic stomata are rare and hairs

are absent.

The stigma (Fig. 8B, B1& C1): A transverse section in the stigma is more

or less circular in outline. It consists of an

epidermis followed by parenchymatous ground

tissue, traversed by a small vascular strand and

few raphides of calcium oxalates are present.

The epidermis (Fig. 8C1) is formed of

polygonal isodiametric, thin-walled cells, slightly

papillose with short, blunt, papillae at the tip and


195

covered with smooth cuticle.

The different tissues of the flower showing

parenchyma cells containing tannins (bluish-black

with ferric chloride T.S.).

Powdered Flower:

The powdered flower is greyish-brown in color,

with slightly fragrant odour and astringent,

mucilaginous taste. It is microscopically

characterized by the following:

1. Fragments of the inner and outer

epidermises of the bract with wavy to

strongly wavy anticlinal walls and covered

with smooth cuticle. Anisocytic, anomocytic

stomata and few non-glandular hairs are

present

2. Fragments of the inner and outer

epidermises of the calyx tube with cells

having more or less straight to slightly wavy

walls, smooth cuticle, few anisocytic,

numerous anomocytic stomata and non-

glandular hairs.

3. Fragments of the inner epidermis of the

corolla, with slightly wavy anticlinal walls,

no stomata but those of the outer, showing

strongly wavy anticlinal walls, few

anomocytic stomata and both are covered

with smooth cuticle.

4. Fragments of the inner epidermal cells of

the hypanthium tube above the ovary

which are polygonal, having straight

anticlinal walls, covered with smooth

cuticle. Anisocytic, anomocytic stomata and

non-glandular hairs are present. Also,

fragments of the outer epidermis of

hypanthium at the united part with the

ovary which are similar to those of the inner

but with smaller cells and more hairs.

5. Numerous non-glandular, unicellular,

straight or curved hairs, covered with a

granular cuticle and few club-shaped ones

with smooth cuticle.

6. Fragments of epidermal cells of anther,

polygonal nearly isodiametric, with straight

walls and smooth cuticle.

7. Fragments of fibrous layer of anther with

lignified bar-like thickenings and appearing

in surface view elongated with lignified,

beaded walls.

8. Fragments of elongated epidermal cells of

the filament, polygonal axially elongated,

with straight, thin anticlinal walls and

covered with smooth cuticle. Stomata and

hairs are absent.

9. Large triangular pollen grains with

smooth thick exine, three prominent pores,

each covered by a dome and having

protruding pollen tubes.

10. Fragments of the stigmatic surface with

short papillae and smooth cuticle.

11. Fragments of epidermal cells of the style

with thick straight walls, smooth cuticle,

rare anisocytic stomata and hairs are

absent.

12. Numerous raphides of calcium oxalate of

different sizes.

13. Fragments of parenchyma cells containing

mucilage (red stain with ruthenium red).

14. Fragments of parenchyma cells containing

tannins (bluish-black colour with ferric

chloride T.S.).

15. Fragments of epidermal cells containing oil

droplets (stained red with Sudan III).

From the previous botanical study we can

conclude that:

Oenothera speciosa Nutt. (Onagraceae),

cultivated in Egypt can be identified in the

entire form from the morphological

characters of its flowers which are in

concordance with published descriptions (1, 2,

53, 54, 55).

In the powdered form the most diagnostic

elements are:

a. Large triangular pollen grains with smooth

thick exine, three prominent pores, each

covered by a dome and having protruding

pollen tubes.

b. Numerous, non-glandular, unicellular hairs

arising from cicatrices; straight or curved,

short or long, thick walled and covered

with a granular cuticle.

c. Non-glandular, unicellular, club-shaped

hairs and covered with thin smooth cuticle.

d. Abundant raphides of calcium oxalate with

varying sizes.

The microscopical measurements of the

different elements of the flower of Oenothera

speciosa Nutt. are listed in Table (13).


196

Table (1): Phenolic constituents identified by HPLC in the hydrolyzed flower extracts

of Oenothera speciosa Nutt.

Rt Constituent Concentration (mg/100g DW)

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.

Method Average %

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.

Authentic sugars

Solvent system

R5 Free

sugars

Mucilage

hydrolyzate

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


197

Table (4): Results of HPLC analysis of the free sugars and mucilage hydrolyzates

of the flowers of Oenothera speciosa Nutt.

Peak No. RRt* Component

Relative percentage

Free sugars Mucilage hydrolyzate

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)

Group Dose in mg/kg

B.Wt.

% Oedema

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)

Group

Dose

in

mg/kg

b.wt.

Volts needed

before

treatment

(zero time)

Volts needed after single oral dose

After 1 hour After 2 hours

Mean ± S.E. Mean±S.E. %

change Potency Mean±S.E.

%

change Potency

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.


198

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)

Group Dose in

mg/kg B.Wt.

Number of

gastric ulcers

(Mean±S.E.)

% protection

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)

Group

Dose

in

mg/kg

b.wt.

Induced rise

in

temperature

(Mean±S.E.)

Body temperature change

After 1 hour After 2 hours

Mean±S.E. %

change Potency Mean±S.E.

%

change Potency

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)

Group Blood Glutathione

(mg %) % change

Control (1 ml saline) 36.7 ± 1.2 -

Diabetic non- treated 23.4 ± 0.6* 36.2

Diabetic + aqueous extract (10 mg/kg) 35.7 ± 1.1 2.7

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


199

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)

Time

Group

Dose in

mg/kg b.wt.

Blood glucose level (mg/dl)

At zero time After four hours After six hours

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

HEM 100 246.3±8.2 182.6±6.7*

25.86

(62) 138.9±3.5*

43.61

(67)


alcoholic extract 100 258.7± 8.4 197.3±7.2*

23.73

(57) 151.6±4.1*

41.40

(64)

Glimepiride 0.2 254.6±9.7 148.3±4.2* 41.75

(100) 89.5 ±2.1*

64.85

(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)

Time

Group

Dose in

mg/kg b.wt.

Blood glucose level (mg/dl)

At zero time After four weeks After eight weeks

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 -


HEM 100 247.8±9.3 152.9±6.5*

38.5

(68) 118.7±4.2*

52.1

(77)


alcoholic extract 100 256.2±11.3 183.4±6.7*

28.84

(51) 148.4±5.1*

42.08

(62)

Glimepiride 0.2 226.7±8.1 98.6±3.9* 56.5

(100) 73.8±2.4*

67.5

(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


200

Table (12): Anti-microbial Activity of aqueous and alcoholic extracts of the

flowers of Oenothera speciosa Nutt.

Microorganism

Diameter of zone of inhibition (mm)

(Potency)

Aqueous

extract

Alcoholic

extract OFX. AMP. B

Gram–negative:

Escherichia coli - 14

(45%)

31

(100%)

Pseudomonas aeruginosa 14

(66%)

20

(80%)

25

(100%)

Proteus vulgaris 14

(48%)

14

(48%)

29

(100%)

Gram–positive:

Staphylococcus aureus 15

(48%)

28

(90%)

31

(100%)

Sarcina lutea* 17

(47%)

16

(44%)

36

(100%)

Bacillus subtilis 10

(31%)

16

(50%)

32

(100%)

Acid fast bacilli :

Mycobacterium phlei* 14

(52%)

24

(89%)

27

(100%)

Fungi:

Candida albicans 12

(57%)

17

(81%)

21

(100%)

Candida tropicalis* 11

(44%)

16

(64%)

25

(100%)

OFX: Ofloxacin (5μg/disc) AMP.B: Amphotericin B (5μg/disc)

(-): No inhibition zone. (*): Laboratory collection strains.


201

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.


202

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.


203

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)


204


205


206


207


208


209


210


211


212

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214

ألزهار إضافية دراسة عقاقيرية وبيولوجية نبات أونوثيرا سبيسيوزا نت

المنزرع فى مصر

*وأ.د. أماني أمين سليم د. هناء حسن عيدطه شحات محمد األلفى ، أ.د. .مصر - جامعة القاهرة -كلية الصيدلة -قسم العقاقير

.مصر -القاهرة -المركز القومي للبحوث -قسم الفارماكولوجي *

أزهضر نبض أنننثيرا فننياا ن احأحاض الفيننلية ف تم فى هذا البحث تعيين نسبة الفال

النسب باثيلتهض ف فى هذهر ن اقضرنة ضزهإلن الانزرع فى اصر فى بااية فترة ا سبيسينزايضض تعيين سضئلة ذا الكفضءة العضلية. كاض تم ألنهضية تلك الفترة نذلك بضستخاام الكرناضتنجرافيض ا

كاية الاحتنى العفص عن طريق حسضب الزيضاة ف النزن نالطرق الطيفية نقا نجا أن أاض رضزهإلااحأزهضر تحتنى على نسبة أعل ان احأحاض الفيننلية نالعفصيض ف نهضية فترة

. رضزهإلابضلنسبة للفالفننياا فتكنن كايتهض أعل ف بااية ىنه اركبض لخالصة الكحنلية إلى فصل أربعنان نضحية أخرى أا تجزئة ا

-3-( نكنرسيتين3( ن حا الكلنرنجينيك )2جضالكتنسيا )-3-( نكنرسيتين1كنرسيتين) .( ان خالصةاحأسيتنن5كضتيشين ))+( . كاض تم فصل ان جزء خال اإلثيل (4رنتيننسيا )

تم انكذ ا تم التعرف على هذه الاركبض بضلنسضئل الطيفية الاختلفة ناقضرنتهض بعينض أصلية.قنبضستخاام نالاتحاة اراسة الاناا الهالاية نتعيين كايتهض نتجزئتهض نالتعرف على السكضكرالحرة

كرناضتنجرافيض النرق.الكرناضتنجرافيض السضئلة ذا الكفضءة العضلية ن نبض الزهضر حأ لخالصتين الاضئية نالكحنليةان الهالايةلاناا الساية ل سةاراجري كاض أ

لخالصتين الاضئية ا أن لكل انقربضزينية احأاراسة ال كذلك أثبت ن أنهم آاننن.ناسفر عن ًً نالكحنلية للقرح نخضفضضً ناضضااً لألكساة ناضضااً اسكنض ًلآلالمناضضااً لاللتهضب تأثيراًًً خضفضضً لهاض الكحنلية ةلخالصان ناا الهالايةلاانأن للحرارة. لنسبة السكر ف الام تأثيراً

الاضئية نالكحنليةلخالصتين الاسح البينلنج أن أيضض اكاض أظهر .على الااى البعيا نالقريب .الاختبرة لبع أنناع البكتريض نالفطريض اثبطضً تأثيراً لهاض

بغر التعرف عليهض احل البحث ة حأزهضر النبض هذا بضإلضضفة الى اراسة عيضنية ناجهري يز بينهض نبين احأنناع احأخرى .يكضالة ان على هيئة اسحنق حتى ياكن التا

ختبضرا إلانية كربنهياراتالاناا النالفيننلية راسة الاكننض نل عن احأيعتبر هذا التقرير ان لنبض .اهذا زهضرحأالبينلنجية نكذلك الاراسة العيضنية نالاجهرية

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