Phenolic Metaboloites and Antioxidant Activity of Codiaeum Variegatum CV
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Emad M. Hassan et al. / Journal of Pharmacy Research 2014,8(5),619-623 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Phenolic Metaboloites and Antioxidant Activity of Codiaeum variegatum CV. spirale Emad M. Hassan*a, Rasmia A. Hassan b, Sayed A. El-Toumy c, Samy M. Mohamed a, Elsayed A. Omer a a Medicinal and Aromatic Plants Research Department, National Research Centre, Dokki-12311, Cairo, Egypt. b Chemistry of Medicinal Plants Department, National Research Centre, Dokki-12311, Cairo, Egypt. c Chemistry of Tannins Department, National Research Centre, Dokki-12311, Cairo, Egypt. Received on:07-04-2014; Revised on: 28-04-2014; Accepted on:06-05-2014 ABSTRACT The methanol extract of Codiaeum variegatum cv. spirale yielded five flavonoids; apigenin, orientin, vitexin, isovitexin and vicenin-2 along with phenolic acids known as caffeic and r-coumaric acids. Structures of the isolated compounds were determined by using chromatography, MS and 1H, 13C NMR spectroscopy. The antioxidant activity of the main isolated compounds; apigenin, vitexin and isovitexin was investi- gated using the DPPH, total reduction capability and inhibition of lipid peroxidation assay. Vitexin exhibited the highest DPPH radical -1 scavenging antioxidant activity (IC50 = 8.95 µg ml ). In addition, vitexin and isovitexin showed a significant reducing power (RP50 = 8.65 and -1 -1 9.27 µg ml , respectively). While, apigenin was found to be the active compound against the lipid peroxidation process (IC50 = 31.82 µg ml ). KEY WORDS: Codiaeum variegatum cv. spirale; phenolic compounds; antioxidant activity. 1. INTRODUCTION In recent years, it has been observed that oxidative stress, induced 2. MATERIALS AND METHODS by oxygen radicals, is found to be the main reason in several human diseases.1,2 The formation of the reactive oxygen species (ROS) dur- 2.1. Plant Material: ing metabolism may generate peroxidation of lipid membranes fol- The leaves of Codiaeum variegatum cv. spirale were collected from lowed by increasing lipid peroxide levels and damage nucleic acids.3.4 National Research Centre garden, Cairo, Egypt, in July 2012, and iden- So, antioxidants have a good effect in the protection of human body tified by Dr. Mohamed Al-Gebaly. A voucher specimen is deposited against damage by reactive oxygen species.5 Flavonoids are consid- at National research Centre herbarium, under the number of C 168. ered as an important chemical group possessing antioxidant proper- The plant leaves were dried and finely powdered. ties which may be attributed to their classical hydrogen-donating mechanism.6,7 Therefore, many studies proved the possibility of us- 2.2. General Experimental Conditions: ing plants as a source of antioxidants.8,9 Codiaeum variegatum Column chromatography was performed on sephadex LH-20 (Fluka) L. (garden croton) is belonging to family Euphorbiaceae. It is a native and polyamide 6S (Riedel-De Haen, Hannover, Germany). Paper chro- to southern India, Sri Lanka, Indonesia, Malaysia, and the western matography Whatman No. 1 and 3 MM were carried out using sol- Pacific Ocean islands. 10 Glaucine, oxoglaucine and hemiargyrine are vent systems: A (15% AcOH) and B (n-BuOH-AcOH-H2O, 4:2:1). Mass alkaloids which have been isolated from the methanolic extract of spectra were achieved on Finnigan SS Q 700 spectrometer, 70 eV. Codiaeum variegatum cv. petra and two diterpenoids, ent- NMR experiments were performed on a Jeol EX-500 spectrometer: 500 trachyloban-3-one and ent-18-OH-trachyloban-3-one in addition to MHz (1H NMR), 125 MHz (13C NMR). Chemical shifts are given in d a-amyrin and ß-sitosterol, were identified in the same plant cultivar.11 values (ppm) using tetramethylsilane as the internal standard and Codiaeum genus exhibited some biological activities as DMSO-d6 as solvent at room temperature. antimycobacterial, antifungal and mosquitocidal.12-14 2.3. Extraction and Isolation: So, the objective of the present work is to investigate the phenolic Dried leaves of Codiaeum variegatum cv. spirale (1 kg) were ex- compounds and the antioxidant properties of Codiaeum variegatum tracted with MeOH (80%) four times. The concentrated extract (127 g) cv. spirale growing in Egypt. was suspended into water and successively extracted with CHCl3, EtOAc, and n-BuOH. The EtOAC extract (0.4 g) was subjected to *Corresponding author. paper chromatography 3 MM, eluting with solvent system B (n-BuOH- Emad M. Hassan AcOH-H O, 4:2:1) to afford 2 bands. The first band was eluted with Medicinal and Aromatic Plants Research Department, 2 National Research Centre, Dokki-12311, Cairo, Egypt. MeOH and purified on a small sephadex LH-20 CC eluted with MeOH (80%) to afford compound 1 (22 mg). The second band was eluted Journal of Pharmacy Research Vol.8 Issue 5.May 2014 619-623 Emad M. Hassan et al. / Journal of Pharmacy Research 2014,8(5),619-623 2.4.3. Inhibition of lipid peroxidation: with MeOH and subjected to sephadex LH-20 CC (MeOH-H2O, 7:3) to give compounds 2 and 3 (11 and 8 mg, respectively). The n-butanol The effect of FeCl2/H2O2 stimulated linoleic acid peroxidation was extract (7.5 g) was loaded on polyamide 6S column (50 × 5 cm). The determined by the method of 17. Compounds (10-80 µg ml-1) were added to a solution of 0.1 M l-1 of linoleic acid (0.2 ml), 2.0 mM l-1 FeCl column was eluted firstly with H2O, and then H2O-MeOH mixtures of 2 -1 -1 decreasing polarity. Five fractions (1 L, each) were collected. The (H2O)4 (0.2ml), 2.0 mM l H2O2 (0.2 ml) and 0.2 M l phosphate buffer major flavonoid fractions obtained were combined into two (pH 7, 5 ml). The reaction mixture was incubated at 37°C for 24h. After subfractions after PC analysis. Both subfractions were subjected to incubation 0.2 ml of BHT (20 mg ml-1), 1.0 ml thiobarbituric acid (10 mg column chromatography on Sephadex LH-20 with aqueous MeOH (0- ml-1) and 1.0 ml trichloroacetic acid (10 mg ml-1) were added to the 70 %) for elution to give compounds 4, 5, 6 and 7 (12, 26, 8 and 6 mg, mixture, which was heated for 30min in a boiling water path. After respectively). cooling 5ml of chloroform was added and the mixture was centrifuged at 1000×g to give a supernatant. The absorbance of supernatant was 2.4. Antioxidant activity: measured spectrophotometrically at 532 nm. All data are average of Tested compounds were prepared in methanol at different concentra- triplicates. The inhibition of FeCl2/H2O2 stimulated linoleic acid tions (10, 20, 40 and 80 µg ml-1). peroxidation (%) was calculated as follows: Chemicals: 1,1-diphenyl-2-picryl-hydrazyl (DPPH), potassium Inhibition (%) = [1-(A sample/A control)] × 100 hexacyanoferrate, trichloroacetic acid (TCA), L-ascorbic acid and butylated hydroxytoluene (BHT) were purchased from Sigma (Sigma- The 50% of inhibitory concentration (IC50) was also measured. BHT Aldrich GmbH, Germany). and L-ascorbic acid were used as standard anti-lipid peroxidation reference. 2.4.1. DPPH radical scavenging antioxidant activity determination: The effects of isolated compounds and positive controls (L-ascorbic Statistical Analysis acid and BHT) on DPPH radicals were estimated based on the method The results obtained in all analyses were expressed in mean ± SD 15 of . Aliquots (20µl) of the isolated compounds at various concentra- (standard deviation). The 50% of inhibitory concentration (IC50) was tions were each mixed with 100 mM Tris-HCl buffer (80 µl, pH 7.4) and also measured. then with 100 µl of DPPH in ethanol to a final concentration of 250 µl. The mixture was shaken vigorously and left to stand at room tempera- 3. RESULTS AND DISCUSSION: ture for 20 min in the dark. The absorbance of the reaction solution was measured spectrophotometrically at 517 nm. The percentage of 3.1. Identification of the isolated compounds: DPPH decolorization of the samples was calculated according to the The MeOH extract of Codiaeum variegatum cv. petra leaves was equation: fractionated into EtOAc and n-BuOH extracts. From EtOAc extract, compounds 1, 2 and 3 were isolated. While, n-BuOH extract were further purified on polyamide 6S and Sephadex LH-20 CC to yield % decolorization = [1- (A sample /A control)] ×100. compounds 4, 5, 6 and 7. IC50 value was the effective concentration at which DPPH radicals were scavenged by 50% and was obtained by interpolation from Compound 1: yellow amorphous powdered; EI-MS 70 eV m/z 270 [M+], which corresponds to the molecular formula C H O . 1H NMR linear regression analysis. A lower IC50 value indicated a greater anti- 15 10 5 oxidant activity. (DMSO-d6): d 7.7 (2H, d, J = 9 Hz, H-2' and H-6'), 6.7 (2H, d, J = 9 Hz, H-3' and H-5'), 6.5 (1H, s, H-3), 6.4 (1H, d, J = 1.3 Hz, H-8), 6.2 (1H, d, 13 2.4.2. Total Reduction Capability: J = 1.3 Hz, H-6). C NMR (DMSO-d6): d 181.7 (C-4), 164.0 (C-2), 163.7 Total reduction capability of plant components was estimated by (C-7), 161.4 (C-9), 161.1 (C-4'), 157.3 (C-5), 128.3 (C-6', C-2'), 121.3 using the method of 16. The tested compounds (10-80 µg ml-1) in 1ml (C-1'), 116.1 (C-3', C-5'), 103.7 (C-10), 99.2 (C-6), 94.3 (C-8). Hence, of distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH according to the above mentioned data and by comparison with the literature, compound 1 was identified as apigenin.