title: Aldose reductase inhibition of a saponin-rich fraction and new furostanol saponin derivatives from Balanites aegyptiaca.
abstract: 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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Faculty of Pharmacy, Cairo University, Kasr-El-Ainy St., Cairo 11562, Egypt; Faculty of Pharmacy and Drug Technology, Heliopolis University, 2834 El Horreya, Cairo, Egypt. Electronic address: a_motaal@hotmail.com.
authors.forename: Amira
authors.initials: A
authors.lastname: Abdel Motaal
authors.affiliation: Faculty of Pharmacy, Cairo University, Kasr-El-Ainy St., Cairo 11562, Egypt. Electronic address: heshamaskary@yahoo.com.
authors.forename: Hesham
authors.initials: H
authors.lastname: El-Askary
authors.affiliation: Institute of Pharmaceutical Sciences, University of Graz, A-8010 Graz, Austria. Electronic address: sara.crockett@uni-graz.at.
authors.forename: Sara
authors.initials: S
authors.lastname: Crockett
authors.affiliation: Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, University of Graz, A-8010 Graz, Austria. Electronic address: olaf.kunert@uni-graz.at.
authors.forename: Olaf
authors.initials: O
authors.lastname: Kunert
authors.affiliation: Faculty of Pharmacy and Drug Technology, Heliopolis University, 2834 El Horreya, Cairo, Egypt. Electronic address: basma.sakr@hu.edu.eg.
authors.forename: Basma
authors.initials: B
authors.lastname: Sakr
authors.affiliation: Faculty of Pharmacy and Drug Technology, Heliopolis University, 2834 El Horreya, Cairo, Egypt. Electronic address: sherif.shaker@hu.edu.eg.
authors.forename: Sherif
authors.initials: S
authors.lastname: Shaker
authors.affiliation: National Institute for Chemical - Pharmaceutical Research and Development (ICCF), Bucharest, Romania. Electronic address: alicella77@yahoo.com.
authors.forename: Alice
authors.initials: A
authors.lastname: Grigore
authors.affiliation: National Institute for Chemical - Pharmaceutical Research and Development (ICCF), Bucharest, Romania. Electronic address: radu_a1@hotmail.com.
authors.forename: Radu
authors.initials: R
authors.lastname: Albulescu
authors.affiliation: Institute of Pharmaceutical Sciences, University of Graz, A-8010 Graz, Austria. Electronic address: rudolf.bauer@uni-graz.at.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Bauer
mesh_terms: D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D000818:Animals; D032430:Balanites / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D005638:Fruit / Q000737:chemistry; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D010936:Plant Extracts / Q000737:chemistry*; D051381:Rats; D012503:Saponins / Q000737:chemistry*; D013261:Sterols / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D010936:Plant Extracts; D012503:Saponins; D013261:Sterols; D000449:Aldehyde Reductase
keywords: Aldose reductase; Balanites aegyptiaca; Diabetes; Furostane; Preparative HPLC; Zygophyllaceae