New Antioxidant Flavonoids from the Aerial Parts of Secamone Afzelii

A bioassay-guided fractionation of petroleum ether, EtOAc and n-BuOH soluble parts of the 80% hydromethanol extract was performed to investigate the antioxidant activity of Secamone afzelii aerial parts using DPPH free radical scavenging assay. The results revealed that EtOAc and n-BuOH soluble parts have moderate to good DPPH radical scavenging activity (EC50 = 139.3 and 30.5 μg/mL, respectively). Therefore, from the most active fractions of EtOAc and n-BuOH soluble parts were isolated two new flavonoid diglycosides quercetin-3-O-β-D-apiofuranosyl-(1→2)-α-L-rhamnopyranoside and genkwanin-8-C-β-Dapiofuranosyl-(1→2)-β-D-glucopyranoside in addition to nine known compounds (2-10). Their structures were elucidated on the basis of spectroscopic data including 1Dand 2D-NMR and ESI-MS. The ability of the isolated compounds to scavenge the DPPH was evaluated. The new compound 1, quercitrin (3) and rutin (6) have antioxidant potential with EC50 values ranging from 8.4 to 13.6 μg/mL, compared to the standard ascorbic acid (EC50 7.4 μg/mL). DOI : 10.14302/issn.2471-2140.jaa-15-887 Corresponding author. Tel.: +33-3-26-91-82-08; fax: +33-3-26-91-35-96. E-mail address: abdulmagid.alabdulmagid@univ-reims.fr (A. Alabdul Magid)


Introduction
Antioxidants are compounds that inhibit or delay the oxidation process by blocking the initiation or propagation of oxidizing chain reaction due to oxidative stress [1].Antioxidants were known as free radical scavengers, reducing agents, chelators of pro-oxidant metals, or as quenchers of singlet oxygen.Described as an imbalance between free radicals and the body's ability to detoxify these molecules or repair the resulting damage, oxidative stress causes extensive damage to biological molecules such as DNA, lipids and proteins [2,3].Thus, it is the cause of several diseases including cancer, cataracts, amyotrophic lateral sclerosis, accelerated aging, the acute respiratory distress syndrome and pulmonary oedema [4,5].In order to provide a solution to this problem, we propose to contribute to the search for new antioxidants from indigenous natural sources [6][7][8].In our continued search for new bioactive compounds from Ivory Coast medicinal plants [8], we investigated Secamone afzelii (Roem.& Schult.)K. Schum, a creeping woody climber belonging to the family Apocynaceae [9].S. afzelii is used in traditional medicine for stomach problems, diabetes, colic, dysentery and also for kidney problems [10].The decoction of the entire plant is prescribed for cough, catarrhal conditions and as galactogogue.For the treatment of gonorrhoea, the whole plant is crushed with fresh palm nuts and oil [9].Previous studies have shown that S. afzelli has antioxidant, anti-inflammatory and antimicrobial properties [11][12][13].A phytochemical screening of the methanol extract of the leaves and the stems of S. afzelii showed the presence of high amount of flavonoids, but none, to the best of our knowledge, have reported their structural elucidation [14].Lack of chemical scientific data about the flavonoids contents of S. afzelii and the antioxidant property of this plant, prompted us to make this study.A bioassay-guided fractionation of the hydromethanol extract using DPPH free radical scavenging assay was used to investigate the antioxidant activity and isolate flavonoids from the most active fractions.
The EtOAcfraction was also fractionated by VLC over RP-       The DPPH radical scavenging activity of compounds 1-11 was measured (Table 1).The new compound 1, quercitrin OH pattern is most favorable for the activity than 3',4',5'-tri-OH pattern (Table 1).Comparison of the antioxidant activity of the quercetin glycosides 1, 3, 5, and 6 showed that the saccharide chain linked at C-3 might have an influence on the antioxidant activity (Table 1).The antioxidant activity of these quercetin glycosides may justify the use of this plant in the treatment of diseases due to oxidative stress.

Plant Material
The aerial parts of S.

Extraction and Isolation
The powdered dry aerial part of S. afzelii (

DPPH free Radical Scavenging Assay
All tested compounds 1-11 showed on HPLC purity of more than 95%.The free radical scavenging activity of the extracts and isolated compounds against DPPH was investigated by spectrophotometric methodology, as previously described [8].Briefly, 5 µL of the standard or sample solutions (dissolved in DMSO) was mixed with 95 µL of DPPH solution (158 µM, dissolved in absolute EtOH).After mixing gently and incubating for 30 min at 37 °C, the optical density was measured at l 515 nm using a Fluostar omega microplate reader (BMG labtech).The percentage of absorbance inhibition at l 515 nm was calculated using the following equation: % afzelii was concentrated and partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc) and nbutanol (n-BuOH), followed by concentrating.The crude hydromethanol extract, the PE soluble part, EtOAc soluble part and n-BuOH soluble part were tested for their radical scavenging activity by DPPH assay.The results indicated that the n-BuOH soluble part has significant activity with IC 50 value of 30.5 μg/mL, whereas the hydromethanol extract and EtOAc soluble part exhibited a moderate activity with IC 50 value of 150.5 and 139.3 μg/mL, respectively.In order to isolate the compounds involved in this antioxidant action, a bioassay-guided fractionation strategy was applied throughout the separation procedure.Four fractions (from B1 to B4) were obtained from the n -BuOH soluble part by vacuum liquid chromatography (VLC) over RP-18.The fraction B2 exhibited the highest DPPH radical scavenging activity with an EC 50 of 30.3
, and rutin (6) had antioxidant potential (EC 50 values ranging from 8.1 to 13.6 µg/mL) comparable to ascorbic acid, used as positive control (EC 50 7.4 µg/mL).The ten other compounds showed low or no antioxidant activity.Generally, substitution patterns on the B-ring especially affected antioxidant potencies of the flavonoids [28].The 3',4'-dihydroxy pattern is particularly important to the antiradical activity of a flavonoid.These trends are consistent with less active flavonoids (2, 4, and 7-11) possessing 3'-OH pattern.The active compounds 1, 3, and 6, shared a common aglycone di-OH substituted in the B-ring (quercetin) whereas compound 7 is tri-OH substituted in the B-ring (myricetin).Comparison of the antioxidant activity of compounds 6 and 7 sharing the same disaccharide [rha-(1→6)-glc-] indicated that the 3',4'-di- for the bioassay were purchased from Sigma-Aldrich, Chemical Co.(Germany).NMR spectra were carried in CD 3 OD on Bruker Avance DRX III 500 instruments ( 1 H at 500 MHz and 13 C-Jmod at 125 MHz).HR-ESI-MS experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK).Optical rotations were determined in MeOH with a Perkin-Elmer 341 polarimeter.TLC was performed on pre-coated silica-gel 60 F 254 Merck.CC was carried out on Kieselgel 60 (63-200 mesh), or LiChroprep RP-18 (40-63 µm) Merck.HPLC was performed on a Dionex apparatus equipped with an ASI-100 autosampler, an Ultimate 3000 pump, a diode array detector UVD 340S and Chromeleon software.C 18 reversed phase column (Phenomenex 250x15 mm, Luna 5µ) was used for semipreparative HPLC with binary gradient eluent (H 2 O (pH 2.4 with TFA); MeCN) and a flow rate of 4 mL/min; the chromatogram was monitored at 205, 225, 250, and 350 nm.Absorbance (A) values in the DPPH free radical scavenging assay were read on a Fluostar omega microplate reader (BMG labtech).UV spectra were recorded on Shimadzu UV-2450 spectrophotometer in MeOH.
afzelii were collected from Cocody-Abidjan, Ivory Coast, in December 2010.The plant was identified by Pr.Laurent AKE-ASSI of FHB University and a voucher specimen (No Aké-Assi 21253) has been deposited in the herbarium of the National Center of Floristic of FHB University of Cocody (Ivory Coast).
inhibition [(A control -A sample )/A control ] × 100.DPPH solution in EtOH was used as a control.The curve of the % scavenging activity against the concentration of sample was prepared by an MSExcel based program to obtain
b Used as positive control.