Amira Abdel Motaal

Anticancer and Antioxidant Activities of Standardized Whole Fruit, Pulp, and Peel Extracts of Egyptian Pomegranate

Different parts of the fruit of Punica granatum L. family Punicaceae cultivated in Egypt were extracted and standardized to be assessed for their anticancer and antioxidant properties. An HPLC method was modified and validated for standardization using ellagic acid (EA) as a marker. The 50% ethanol was proven to be a better solvent for extraction than the 70% ethanol, as the 50% hydroalcoholic fruit extract was standardized to contain 5.9 ± 0.15 % total polyphenols compared to 4.4 ± 0.35 % in the 70% hydroalcoholic extract. The 50% hydroalcoholic extracts of the whole fruit, pulp and peels were standardized to contain 0.3 ± 0.05, 0.02 ± 0.01 and 1.9 ± 0.1 % of EA, respectively. The peel extract showed the highest antioxidant activity (IC50 = 0.50 ± 0.9 mg/ml) compared to the other two extracts, as well as, a pronounced anticancer activity against MCF-7 human breast cancer cells and HCT-116 colon cancer cells with IC50 values of 7.7 ± 0.01 and 9.3 ± 0.06 � g/ml, respectively. The standardized peel extract was formulated into capsules. Here we report the possible use of pomegranate peels, a biological waste product, to develop natural pharmaceutical preparations.

In vivo anti-inflammatory activity of caffeoylquinic acid derivatives from Solidago virgaurea in rats

Abstract Context: Solidago virgaurea L. (Asteraceae) is traditionally used as an anti-inflammatory for the treatment of various symptoms including cystitis. However, little is known concerning the constituents responsible for this activity and the mechanism of their action. Objective: To assess the anti-inflammatory activity of the phenolic-rich fraction of S. virgaurea aerial parts in rats, isolate and assess the activity of the major compounds present. Materials and methods: An HPLC method was developed for the analysis of the phenolic-rich fraction (EtFr). The in vivo anti-inflammatory activity of the EtFr and four isolated compounds (at 25 and 50 mg/kg) were assessed in adult male rats using the carrageenan-induced rat paw oedema model. The levels of the pro-inflammatory cytokines (TNF-α and IL-1β) were measured using ELISA. Results: 3,5-O-Dicaffeoylquinic acid (1), 3,4-O-dicaffeoylquinic acid (2), 3,4,5-O-tricaffeoylquinic acid (3) and 4,5-O-dicaffeoylquinic acid (4) were isolated from EtFr. Compound 3 (50 mg/kg) showed a highly significant activity in inhibiting the oedema volume after 3 h (88% of the activity of indomethacin at 10 mg/kg). The EtFr and the isolated compounds largely inhibited the excessive production of the inflammatory mediators TNF-α and IL-1β. Discussion and conclusion: This is the first report of 3,4,5-tri-O-caffeoylquinic acid (3) in Solidago species. The tricaffeoylquinic acid (3) showed a significantly higher activity than the other three dicaffeoylquinic acids (1, 2, 4) and indomethacin in reduction of TNF-α and IL-1β concentrations (8.44 ± 0.62 and 5.83 ± 0.57 pg/mL compared to 12.60 ± 1.30 and 52.91 ± 5.20 pg/mL induced by indomethacin, respectively).

Aldose reductase inhibition of a saponin-rich fraction and new furostanol saponin derivatives from Balanites aegyptiaca

BACKGROUND Balanites aegyptiaca Del. (Zygophyllaceae) fruits are used to treat hyperglycemia in Egyptian folk medicine and are sold by herbalists in the Egyptian open market for this purpose. Nevertheless, the fruits have not yet been incorporated into pharmaceutical dosage forms. The identity of the bioactive compounds and their possible mechanisms of action were not well understood until now. PURPOSE Aldose reductase inhibitors are considered vital therapeutic and preventive agents to address complications caused by hyperglycemia. The present study was carried out to identify the primary compounds responsible for the aldose reductase inhibitory activity of Balanites aegyptiaca fruits. STUDY DESIGN The 70% ethanolic extract of Balanites aegyptiaca fruit mesocarp and its fractions were screened for inhibition of the aldose reductase enzyme. Bio-guided fractionation of the active butanol fraction was performed and the primary compounds present in the saponin-rich fraction (D), which were responsible for the inhibitory activity, were characterized. HPLC chromatographic profiles were established for the different fractions, using the isolated compounds as biomarkers. METHODS Aldose reductase inhibition was tested in vitro on rat liver homogenate. The butanol fraction of the 70% ethanolic extract was fractionated using vacuum liquid chromatography (VLC, RP-18 column). The most active sub-fraction D, which was eluted with 75% methanol, was subjected to preparative HPLC to isolate the bioactive compounds. RESULTS The butanol fraction displayed inhibitory activity against the aldose reductase enzyme (IC50 = 55.0 ± 6 µg/ml). Sub-fraction D exhibited the highest inhibitory activity (IC50 = 12.8 ± 1 µg/ml). Five new steroidal saponin derivatives were isolated from this fraction. The isolated compounds were identified as compound 1a/b, a 7:3 mixture of the 25R:25S epimers of 26-O-β-D-glucopyranosyl-furost-5-ene-3,22,26-triol 3-O-[α-L-rhamnopyranosyl-(1→3)- β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 2, 26-O-β-D-glucopyranosyl-(25R)-furost-5-ene-3,22,26-triol 3-O-[ β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 3, 26-O-β-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[α-L-rhamnopyranosyl-(1→3)- β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; compound 4, 26-O-β-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[ β-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside; and compound 5, which is the 25S epimer of compound 4, by using various spectroscopic methods [MS,1D and 2D NMR (HSQC, HMBC, DQF-COSY, HSQC-TOCSY)]. Compounds 1a/b, 2, 3, 4, 5 exhibited highly significant aldose reductase inhibitory activities (IC50 values were 1.9 ± 0.2, 1.3 ± 0.5, 5.6 ± 0.2, 5.1 ± 0.4, 5.1 ± 0.6 µM, respectively) as compared to the activity of the reference standard quercetin (IC50 = 6.6 ± 0.3 µM). CONCLUSION The aldose reductase inhibitory activity of Balanites fruits is due to the steroidal saponins present. HPLC chromatographic profiles of the crude butanol fraction and its 4 sub-fractions showed that the most highly bioactive fraction D contained the highest amount of steroidal saponins (75%) as compared to the 21% present in the original butanol fraction. The isolated furostanol saponins proved to be highly active in an in vitro assay.

Isolation of new cytotoxic metabolites from Cleome droserifolia growing in Egypt.

The sulforhodamine B (SRB) assay was used to assess the cytotoxicity of the aqueous (AqEx) and ethanolic (AlEx) extracts, respectively, of the aerial parts of Cleome droserifolia (Forssk.) Del. against two human cancer cell lines, breast (MCF7) and colon (HCT116) adenocarcinoma. AqEx exhibited higher cytotoxic activity, thus its four subfractions, namely n-hexane (HxFr), chloroform (ClFr), ethyl acetate (EtFr), and n-butanol (BuFr) fractions, were also tested. Purifi cation of the more active ClFr and EtFr yielded nine compounds. Six terpenoids, guai-7(11),8-diene (C1), 1-hydroxy-guai-3,10(14)-diene (C2), 18-hydroxydollabela- 8(17)-ene (C3), (24E)-stigmasta-5,8-dien-3β-ol (C4), teucladiol [1α,5β-guai-10(14)- ene-4β,6β-diol] (C5), and buchariol (4,10-epoxy-6α-hydroxyguaiane) (C6), were isolated from ClFr and three fl avonol glycosides, isorhamnetin-3-O-β-D-glucoside (F1), quercetin- 3`-methoxy-3-O-(4``-acetylrhamnoside)-7-O-α-rhamnoside (F2), and kaempferol-4`-methoxy- 3,7-O-dirhamnoside (F3), were isolated from EtFr. Compounds C3 and F2 are new in nature. The isolated compounds were identifi ed using various spectroscopic methods (UV, IR, 1H NMR,13C NMR, HMQC, HMBC, and COSY). Compounds C1, C3, F2, and F3 showed significant cytotoxic activities against the two tested cell lines comparable to those of the anticancer drug doxorubicin®. The new compound C3 was the most active as it had the lowest IC50 values, (1.9 ± 0.08) and (1.6 ± 0.09) μg/ml corresponding to 6.5 and 5.4 μM, against MCF7 and HCT116 cells, respectively

Composition and Bioactivities of the Essential Oil from Leaves of Lippia citriodora Kunth Cultivated in Egypt

Abstract Analysis of the hydrodistilled essential oil from the fresh leaves of Lippia citriodora Kunth (Verbenaceae), cultivated in Egypt at two different growth stages, by gas chromatography-mass spectrometry (GC-MS) resulted in the identification of 22 and 25 components in the oils prepared during the vegetative (June) and flowering (October) stages, respectively. The composition of the essential oils differed quantitatively and qualitatively according to the time of collection. The four major detected constituents (neral, geranial, dl-limonene and α-curcumene) exhibited remarkable differences between the two stages. Total percentage of oxygenated monoterpene aldehydes, represented by neral and geranial, decreased from 41.2 % to 35.2 % in June and October, respectively. On the other hand, the percentage of α-curcumene increased from 5.6 % to 14.5 %, while dl-limonene, the major monoterpene hydrocarbon was found only in June (10.6 %). The essential oil exhibited variable anti-inflammatory, antipyretic, analgesic and antioxidant properties. It also showed a moderate antibacterial activity.

In vitro and in vivo antidiabetic potential of extracts and a furostanol saponin from Balanites aegyptiaca

Abstract Context: Balanites aegyptiaca Del. (Zygophyllaceae) fruits are well-known antidiabetic drug in Egyptian folk medicine. Nevertheless, its mechanism of action is still unclear. Objectives: Searching for the possible mechanisms of action of the plant and identification of its bioactive compounds. Materials and methods: A bio-guided protocol based on the evaluation of α‐glucosidase (AG) and aldose reductase (AR) inhibitory activities was adopted to isolate the biologically active compounds from the methanol extract (MeEx). An in vivo antidiabetic study was conducted for the active extract, fraction and compound using streptozotocin-induced diabetic male albino Wistar rats at two dose levels (100 and 200 mg/kg.b/wt) for 2 weeks. Results: Three compounds were isolated and identified: a sterol, (1) stigmasterol-3-O-β-d-glucopyranoside; a pregnane glucoside, (2) pregn-5-ene-3β,16β,20(R)-trio1-3-O-β-d-glucopyranoside; a furostanol saponin, (3) 26-(O-β-d-glucopyranosyl)-22-O-methylfurost-5-ene-3β,26-diol-3-O-β-d-glucopyranosyl-(1 → 4)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside. Only compound 3 possessed significant AG and AR inhibitory activities (IC50 = 3.12 ± 0.17 and 1.04 ± 0.02 μg/mL, respectively), while compounds 1 and 2 were inactive. The in vivo antidiabetic study revealed that MeEx and furostanol saponin 3 possessed significant activities at a dose of 200 mg/kg through reducing the fasting plasma glucose level by 46.14% and 51.39%, respectively, as well as reducing the total cholesterol by 24.44% and 31.90%, respectively. Compound 3 also caused increment in insulin and C-peptide levels by 63.56% and 65%, respectively. Discussion and conclusions: We presented a scientific base for using Balanites aegyptiaca, and shed the light on one of its saponins, as an antidiabetic agent in fasting and postprandial hyperglycaemia along with the improvement of diabetic complications.

Randomized double-blinded pilot clinical study of the antidiabetic activity of Balanites aegyptiaca and UPLC-ESI-MS/MS identification of its metabolites

Abstract Context: Balanites aegyptiaca Del. (Zygophyllaceae) fruits are traditionally known for the treatment of hyperglycaemia. Several in vitro and in vivo studies proposed some mechanisms of action. However, clinical trials in human beings were never reported to date. Objectives: To investigate the antidiabetic efficacy of the 70% ethanol extract of the pericarps of B. aegyptiaca (BE) within a nutritional intervention in elderly people. Materials and methods: Ultra-performance electrospray ionization-mass spectroscopy (UPLC-ESI-MS/MS) analysis was used for metabolic profiling of BE which was incorporated in hard gelatine capsules (400 mg/day) and tested on 30 type 2 diabetes (T2D) Egyptian patients for 8 weeks. According to sex, age and body mass index participants were divided into two equivalent groups, placebo and treatment. Results: Thirteen compounds were identified in BE using UPLC-ESI-MS/MS analysis among which five steroidal saponins, seven phenolic compounds and a sterol glucoside. At the end of the 8-week treatment, the treated group showed 26.88% decrease in 2 h postprandial plasma glucose relative to 2.6% increase in the placebo group, while fasting plasma glucose was reduced to 10.3%. Treatment with BE capsules for 8 weeks produced significant reduction in the plasma triglyceride, total cholesterol and low-density lipoprotein cholesterol by 9.0, 12.76 and 21.35%, respectively, with 29.8% increase in the high-density lipoprotein cholesterol. Plasma alanine transaminase and aspartate transaminase were reduced by 42.6 and 43.3%, respectively. Discussion and conclusion: Administration of the BE capsules to T2D resulted in significant improvements in the glycaemic markers and the lipid profile, without adverse effects or hypoglycaemia.

Effect of Seasonal Variation on the Composition of the Essential Oil of Solidago canadensis Cultivated in Egypt

Abstract The hydrodistilled essential oils of the fresh flowers and the remaining green aerial parts of Solidago canadensis L., family Asteraceae, recently introduced into Egypt were investigated by GC-MS analysis. A comparative study on the composition of the essential oils obtained in the four seasons of the year was carried out to assess the effect of seasonal variation on the collected oil samples. The major compounds detected in the oil samples of all seasons were germacrene D (9.86-29.47 %), α-pinene (3.38-29.17 %), γ-cadinene (0.39-20.36 %), myrecene (2.98-13.74 %) and limonene (4.81-11.47 %). Summer samples contained the highest percentage of monoterpene hydrocarbons, while winter samples showed the highest percentage of sesquiterpene hydrocarbons. Oil samples collected in summer and winter showed potential cytotoxic activity against human liver, breast and cervix carcinoma; Hepg2, MCF7 and Hela respectively. Winter samples showed a relatively higher cytotoxic activity compared to the summer samples.

A novel role of sONE/NOS3/NO signaling cascade in mediating hydrogen sulphide bilateral effects on triple negative breast cancer progression

Hydrogen sulphide (H2S) gas has been recognized as an intracellular mediator influencing an array of signaling pathways. Yet, the role of H2S in cancer progression has been controversial. This study aims to unravel the role of exogenous H2S in triple negative breast cancer (TNBC) and to further investigate any possible association of H2S mediated actions with the endogenous production of nitric oxide (NO) gas. A wide concentration range of NaHS (20-2000 μM) and a variable reaction time (2-72 h) were probed. A bell-shaped impact of H2S on TNBC cellular viability, proliferation, migration, invasion and colony forming ability was repeatedly observed in the aggressive TNBC cell lines, MDA-MB-231 but not in hormone receptor positive, MCF-7 cells. This bell-shaped effect was found to be shifted towards the left upon increasing the reaction time within the range of 2-24 h. However, this was totally opposed in case of continuous exposure (72 h) to exogenous H2S. An inverted bell-shaped effect of H2S on TNBC cellular growth, migration, proliferation and colony forming ability was shown. Moreover, this study provided the first evidence of a possible involvement of NO in mediating H2S actions in TNBC. Such intricate cross-talk was found to be orchestrated by the novel lncRNA, sONE and its down-stream target NOS3 building up a novel axis, sONE/NOS3/NO, that was shown to play a pivotal role in plotting the bilateral effect of H2S on TNBC progression. Finally, this study showed that low and continuous exposure of H2S serves as a novel, selective and effective strategy in harnessing TNBC oncogenic profile through cGMP dependent and independent pathways where alterations of cell cycle regulatory proteins such as TP53 and c-Myc was observed. Moreover, NaHS could repress TNBC migration and invasion capacities through repressing the intracellular adhesion molecule, ICAM-1. In conclusion, this study provides an insight about the role of exogenous H2S in TNBC cell lines highlighting a novel crosstalk between H2S and NO orchestrated by sONE/NOS3 axis.

An in vivo study of Hypericum perforatum in a niosomal topical drug delivery system

Abstract The active compounds present in Hypericum perforatum L. (Hypericaceae) include hyperforin, hypericins and flavonoids, which are assumed to be responsible for the activity of the extract in the treatment of wounds and scars. The present study aimed to incorporate H. perforatum extract standardized to a known content of phloroglucinols, naphthodianthrones and polyphenolic compounds into an effective transdermal drug delivery system capable of entrapping both lipophilic and hydrophilic constituents in the form of niosomal gels for wound treatment. An 80% ethanol extract (HE) was prepared on a pilot scale using DIG-MAZ. An HPLC-DAD holistic profile was established for HE and was standardized to contain 3.4 ± 4 rutin, 1.1 ± 3 chlorogenic acid, 0.5 ± 2 quercitrin, 2.8 ± 2 hyperforin, and 0.51 ± 3% w/w total hypericins. Niosomes were prepared using the modified reverse phase evaporation technique (REV). The wound healing effect of the gel was tested on 16 adult mongrel dogs. A significant decrease in the inflammatory cell count (18.4 ± 5.3) was recorded in the niosomal gel 1.5% NaCMC-treated group at the 7th day post wounding. It induced a marked regression in the inflammatory phase and enhanced the early beginning of the proliferative phase of wound healing. After 21 days, it showed complete re-epithelization, formation of new matrix fibers and significant reduction in the wound size, compared to the control and the Panthenol® 2% cream treated groups. This is the first study of H. perforatum in a niosomal topical drug delivery system.