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 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 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 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. 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 ) is belonging to family . 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 .11 values (ppm) using tetramethylsilane as the internal standard and Codiaeum 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. 18 6.6) and 2.5ml (1%) potassium ferricyanide [K3Fe (CN)6]. The mixture was incubated at 50°C for 20 min with adding a 2.5 ml of 10% trichlo- + roacetic acid. Then the mixture was centrifuged for 10 min at 1000×g. Compound 2: white crystalline; EI-MS 70 eV m/z 164 [M ], which 1 The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 corresponds to the molecular formula C9H8O3. H NMR (DMSO-d6): d 7.3 (1H, d, J = 15.8 Hz, H-8), 7.2 (2H, d, J = 9.0 Hz, H-2 and H-6), 6.7 (2H, ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700 13 nm by a spectrophotometer (Schimadzu UV/Vis-240IPC). Higher ab- d, J = 9.0 Hz, H-3 and H-5), 6.5 (1H, d, J = 15.8 Hz, H-7). C NMR sorbance of the reaction mixture indicated greater reducing power. (DMSO-d6): d 170.6 (C-4), 160.1 (C-4), 146.1 (C-7), 131.3 (C-3,5), 120.8

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

OH COOH (C-1), 118.2 (C-8), 115.3 (C-2,6). Comparison of these data with litera- ture models led us to identify this compound as r-coumaric acid. 19 HO O

+ HO Compound 3: white powdered; EI-MS 70 eV m/z 180 [M ], which 1 corresponds to the molecular formula C9H8O4. H NMR (DMSO-d6): d OH O 7.8 (1H, d, J = 8.1 Hz, H-5), 7.4 (1H, d, J = 15.8 Hz, H-8), 7.0 (1H, d, J = Compound 1: apigenin Compound 2: r-coumaric acid 2.1 Hz, H-2), 6.9 (1H, dd, J = 2.1 Hz, H-6), 6.2 (1H, d, J = 18.8 Hz, H-7). 13 OH C NMR (DMSO-d ): d 168.1 (C-9), 148.2 (C-4), 145.4 (C-3), 144.4 (C- OH 6 7), 125.6 (C-1), 121.1 (C-6), 115.7 (C-5), 115.2 (C-8), 114.6 (C-2). The

OH 19 COOH HO OH data were found to be identical with caffeic acid.

O HO O Compound 4: light yellow amorphous powdered; EI-MS 70 eV m/z HO + 1 432 [M ], which corresponds to the molecular formula C21H20O10. H

OH NMR (DMSO-d6): d 6.8 (1H, s, H-3), 13.2 (OH, s, H-5), 6.4 (1H, s, H-6), 8.1 (2H, d, J = 8.5 Hz, H-2'), 6.9 (2H, d, J = 8.5 Hz, H-3' and H-5'), 8.6 (2H, OH O 13 d, J = 8.5 Hz, H-6'), 4.7 (1H, d, J = 9.9 Hz, H-1"). C NMR (DMSO-d6): Compound 3: caffeic acid Compound 4: vitexin d 163.9 (C-2), 102.5 (C-3), 182.2 (C-4), 104.1(C-4a), 160.4 (C-5), 89.2 (C- 6), 162.6 (C-7), 104.6 (C-8), 156.0 (C-8a), 121.6 (C-1'), 129.1(C-2'), 115.8 OH (C-3'), 161.2 (C-4'), 115.8 (C-5'), 129.0 (C-6'), 73.4 (C-1"), 70.8 (C-2"), 78.7 (C-3"), 70.6 (C-4"), 81.9 (C-5"), 61.3(C-6"). Comparison of these HO O HO data with literature models led us to identify this compound as O vitexin.20 HO HO OH Compound 5: light yellow amorphous powdered; EI-MS 70 eV m/z OH O + 1 432 [M ], which corresponds to the molecular formula C21H20O10. H

Compound 5: isovitexin NMR (DMSO-d6): d 6.8 (1H, s, H-3), 13.5 (OH, s, H-5), 6.5 (1H, s, H-8), 8.0 (2H, d, J = 8.8 Hz, H-2' and H-6'), 7.0 (2H, d, J = 8.8 Hz, H-3' and H- OH 13 OH 5'), 4.6 (1H, d, J = 9.9 Hz, H-1"). C NMR (DMSO-d6): d 163.4 (C-2), OH 102.7 (C-3), 181.8 (C-4), 103.1(C-4a), 160.6 (C-5), 108.9 (C-6), 93.7 (C-8), O H H O O H 156.3 (C-8a), 121.0 (C-1'), 128.4 (C-2'), 115.9 (C-3'), 160.7 (C-4'), 115.9 (C- 5'), 129.5 (C-6'), 73.0 (C-1"), 70.7 (C-2"), 78.9 (C-3"), 70.2 (C-4''), 81.6 (C- O HO O 5"), 61.5 (C-6"). These spectral data are in agreement with isovitexin .20

Compound 6: yellow amorphous powdered; EI-MS 70 eV m/z 448 [M+], which corresponds to the molecular formula C H O . 1H NMR O H O 21 20 11 (DMSO-d6): d 6.5 (1H, s, H-3), 13.1 (OH, s, H-5), 6.2 (1H, s, H-8), 7.5 (2H, Compound 6: orientin d, J = 1.6 Hz, H-2'), 5.8 (1H, d, J = 8.3 Hz, H-5'), 7.7 (2H, d, J = 8.3 Hz, H- 13 OH 6'), 4.6 (1H, d, J = 9.9 Hz, H-1"). C NMR (DMSO-d6): d 164.0 (C-2), OH 102.4 (C-3), 182.0 (C-4), 104.0 (C-4a), 160.4 (C-5), 98.1 (C-6), 162.5 (C-7), 104.4 (C-8), 156.0 (C-8a), 122.0 (C-1'), 114.1(C-2'), 145.8 (C-3'), 149.6 (C- OH HO OH 4'), 115.6 (C-5'), 119.4 (C-6'), 73.4 (C-1"), 70.7 (C-2"), 79.0 (C-3"), 70.7 (C-4"), 82.0 (C-5"), 61.6 (C-6"). Compound 6 was identified as orientin O HO O HO according to the above mentioned data which was confirmed by the 20 O literature.

HO Compound 7: yellow amorphous powdered; EI-MS 70 eV m/z 594 HO OH + 1 OH O [M ], which corresponds to the molecular formula C27H30O15. H NMR (DMSO-d ): d 6.6 (1H, s, H-3), 13.6 (OH, s, H-5), 8.0 (2H, d, J = 8.9 Hz, Compound 7: vicenin-2 6 H-2' and H-6'), 6.9 (2H, d, J = 8.9 Hz, H-3'and H-5'), 4.88 (1H, d, J = 9.8 Fig. (1): Structure of the isolated compounds 13 Hz, H-1"), 4.8 (1H, d, J = 9.8 Hz, H-1"'). C NMR (DMSO-d6): d 163.9 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 (C-2), 102.4 (C-3), 182.0 (C-4), 158.3 (C-5), 108.3 (C-6), 161.2 (C-7), 105.2 increasing the transformation of Fe3+ to Fe2+ in presence of test sample (C-8), 155.0 (C-9), 102.3 (C-10), 121.4 (C-1'), 128.8 (C-2'), 115.9 (C-3'), implies that sample is electron donor and thus can cause reduction of 161.3 (C-4'), 115.9 (C-5'), 128.8 (C-6'), 73.9 (C-1"), 71.9 (C-2"), 78.8 (C- the oxidized intermediates of lipid peroxidation process. In this assay, 3"), 70.5 (C-4"), 81.8 (C-5"), 60.7 (C-6"), 74.3 (C-1"'), 71.9 (C-2"'), 78.8 the yellow color of the test solution changes to various shades of (C-3"'), 70.3 (C-4"'), 81.4 (C-5"'), 61.1 (C-6"'). From these spectral data, green and blue depending on the reducing power of antioxidant compound 7 could be identified as vicenin-2.21 samples. The reducing capacity of a compound may serve as a sig- nificant indicator of its potential antioxidant activity. However, the It is important to declare that all of these compounds were isolated antioxidant activity of an antioxidant compound have been attributed from this plant cultivar for the first time. However, most of them were to various mechanisms, among which are prevention of chain initia- found in different plant species belonging to family Euphorbiaceae. tion, binding of transition metal ion catalysts, decomposition of per- Apigenin, vitexin and isovitexin were isolated from Jatropha oxides, prevention of continued hydrogen abstraction, reductive ca- gossypiifolia leaf.22 While, orientin and vitexin were identified in Cro- pacity and radical scavenging.26 ton zambezicus.23 Also, vitexin was detected in Croton lechleri.24 Table (1): DPPH radical scavenging activity (IC50, µg/ml), total reduction capability (RP , µg/ml) and inhibition of lipid peroxidation 3.2. Antioxidant activity: 50 (IC50, µg/ml) of apigenin, vitexin and isovitexin of Codiaeum In vitro antioxidant activity of the main isolated compounds of variegatum cv. spirale and standards. Codiaeum variegatum cv. spirale was investigated in the present study by DPPH radical scavenging, total reduction capability and Compound Test DPPH radical Total reduction Inhibition of inhibition of lipid peroxidation assays. These methods have proven scavenging capability lipid the effectiveness of the tested samples in comparison to that of the activity peroxidation (IC , µg/ml) (RP , µg/ml) (IC , µg/ml) reference standard antioxidants, L-ascorbic acid and BHT. 50 50 50 Apigenin 12.25±0.01 14.48±0.02 31.82±0.03 3.2.1. DPPH radical scavenging antioxidant activity: Vitexin 8.95±0.04 8.65±0.03 41.55±0.07 Table (1) represents the radical scavenging activity of the main iso- Isovitexin 13.30±0.02 9.27±0.03 39.04±0.06 L- ascorbic acid 18.47±0.06 15.74±0.02 14.89±0.03 lated compounds from Codiaeum variegatum cv. spirale. The anti- BHT 20.34±0.07 19.32±0.04 16.32±0.01 oxidant activity of these compounds was tested by measuring their Data are presented as mean± SD. Standards are L.ascorbic acid and Butylated capacity to scavenge DPPH radical. Vitexin showed the highest anti- hydroytoluene (BHT). -1 oxidant activity with an IC50 value of 8.95 µg ml , followed by apige- nin and isovitexin (12.25 and 13.30 µg ml-1, respectively). The stan- 3.3.3. Inhibition of lipid peroxidation: dards antioxidant L-ascorbic acid and BHT showed an IC50 value of The thiobarbituric acid reactive substances (TBARS) assay is sensi- 18.47 and 20.34 µg ml-1, respectively. The tested samples reduced the tive, requires small sample amounts and provides reproducible re- stable radical DPPH to the yellow-colored diphenylpicrylhydrazine. sults. This method is preferable for obtaining useful data in an envi- DPPH radicals have been widely used to evaluate the antioxidant ronment similar to the real-life situation and allows testing of both properties of natural products as well as plant extracts.25 lipophilic and hydrophilic substances.27 Data presented in Table (1) show good activity of all tested compounds on inhibition of lipid 3.2.2. Total Reduction Capability: peroxidation process. Apigenin is the most effective compound with As shown in Table (1), vitexin and isovitexin showed a promising -1 an IC50 value of 31.82 µg ml . While, IC50 values of isovitexin and -1 effect as reducers for ferric ions with RP50 values of 8.65 and 9.27 µg vitexin are 39.04 and 41.55 µg ml , respectively. On the other hand, ml-1, respectively. While, apigenin showed RP of 14.48 µg ml-1. How- 50 The standards, L-ascorbic acid and BHT, showed IC50 values of 14.89 - -1 ever, L-ascorbic and BHT gave RP50 values of 15.74 and 19.32 µg ml and 16.32 µg ml , respectively. 1, respectively. The total reduction capability reflects the electron donating capacity of bioactive compounds, which is associated with Many reports have been available that the antioxidant properties in antioxidant activity. So, antioxidant can block oxidants. The reducing Euphorbiaceae plants are mainly due to the presence of high content capacity of a compound can be measured by the direct reduction of of secondary metabolites such as flavonoids.28,29 3+ Fe[(CN)6]3 to Fe[(CN)6]2. Addition of free Fe to the reduced product leads to the formation of intense Perl’s Prussian blue complex, 4. CONCLUSION: - Thus, it could be considered that Codiaeum variegatum cv. spirale Fe4[Fe(CN )6]3, which has a strong absorbance at 700 nm. The reduc- ing ability of a compound greatly depends on the presence of plant is a promising natural source of antioxidants which can be used reductones, which have exhibit antioxidative potential by breaking in nutritional or pharmaceutical fields for the prevention of free-radi- the free radical chain by donating a hydrogen atom. In this regarded, cal-mediated diseases.

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.8 Issue 5.May 2014 619-623