Phytochemical and Antioxidant Evaluation of the Flavonoids and Tannins from Synadenium grantii Hook f, (Ephorbiaceae)

Plants are an significant source of natural antioxidants that counter oxidative stress induced by an increase in free radicals / ROS such as superoxide O2− anion, hydroxyl radical HO− and peroxide as well1. The phytoconstituents research of these bioactive compounds in a plant is therefore of paramount importance, resulting to further biological and pharmacological studies. This study can be presented to develop herbal medicines that require the isolation of bioactive compounds from medicinal plants2. In recent research advances around the world the properties of medicinal plants have been studied because of no side effects, safety in contrast to the synthetics that are unsafe to human and environment. As a result people are more favourable to use natural compounds obtained from plants. It is great interest for scientists to focus on the search of biologically active compounds from plants as a natural source of useful drugs against infectious diseases3. Biological studies of herbal products (active ingredient) may be due to a variety of chemical components including flavonoids, phenolics, glycosides, anthocyanins, tannins, etc.4. Natural products are considered a drug source for several years, and have been chosen to interact with biological studies as a target5. The bioactive constituents in plant Phenolic compounds (PCs) for example, flavonoids are greater antioxidant activity6. Several studies have shown that many plants are rich source of antioxidants. One of these plants is S. grantii (Ephorbiaceae) that a plant species which belongs to a small genus of about 15 species. Plants from this genus for example (S. umbellatum, S. glaucescens, S.compactum, S.claycinum ) have different biological activities as anti-ulcer activity7, antiasthmatic effect8, antitumoral and anti-angiogenic effects9, antinoceptive anti-inflammatory, anti-plasmodial activity10. The aim of this work is to study the antioxidant activity of the isolated pure compounds from MeOH-H2O extract (70:30) of S. grantii for the first time.


INTRODUCTION
Plants are an significant source of natural antioxidants that counter oxidative stress induced by an increase in free radicals / ROS such as superoxide O2− anion, hydroxyl radical HO− and peroxide as well 1 . The phytoconstituents research of these bioactive compounds in a plant is therefore of paramount importance, resulting to further biological and pharmacological studies. This study can be presented to develop herbal medicines that require the isolation of bioactive compounds from medicinal plants 2 . In recent research advances around the world the properties of medicinal plants have been studied because of no side effects, safety in contrast to the synthetics that are unsafe to human and environment. As a result people are more favourable to use natural compounds obtained from plants. It is great interest for scientists to focus on the search of biologically active compounds from plants as a natural source of useful drugs against infectious diseases 3 . Biological studies of herbal products (active ingredient) may be due to a variety of chemical components including flavonoids, phenolics, glycosides, anthocyanins, tannins, etc. 4 . Natural products are considered a drug source for several years, and have been chosen to interact with biological studies as a target 5 . The bioactive constituents in plant Phenolic compounds (PCs) for example, flavonoids are greater antioxidant activity 6 . Several studies have shown that many plants are rich source of antioxidants. One of these plants is S. grantii (Ephorbiaceae) that a plant species which belongs to a small genus of about 15 species. Plants from this genus for example (S. umbellatum, S. glaucescens, S.compactum, S.claycinum ) have different biological activities as anti-ulcer activity 7 , antiasthmatic effect 8 , antitumoral and anti-angiogenic effects 9 , antinoceptive anti-inflammatory, anti-plasmodial activity 10 . The aim of this work is to study the antioxidant activity of the isolated pure compounds from MeOH-H 2 O extract (70:30) of S. grantii for the first time.

Extraction and isolation
The aerial parts of S. grantii (1.75 kg) were crushed and extracted by macération with 70% aqueous CH 3 OH, the methanol water extract was evaporated under reduced pressure and lyophilized (250gm). A sample (150gm) of the dry extract was fractionated by chromatography on polyamide 6S column (60x4cm). The column was eluted with H 2 O and H 2 O-MeOH step gradient by decreasing polarities at a flow rate 1ml/ minute was then carried out. The bands migrated along the column were traced under UV light during elution to note their characteristics and to control the fractionation process as well. The obtained fractions (1litre, each) were subjected to paper chromatography using BAW and 15% AcOH/H2O as a developing solvents, and the similar fractions were collected together to give four major fractions (I-IV). The subfractions were then subjected to different chromatographic techniques including, cellulose and repeated sephadex LH-20 column using eluents of different polarities. This led to the isolation and purification of six flavonoid compounds (1)(2)(3)(4)(5)(6). Some sub fractions precipitated with diethyl ether, then subjected to sephadex LH-20 column with absolute ethanol to give compounds (7)(8)(9)(10)(11)(12)(13)(14).
Separation of fraction I (8g) on sephadex column LH-20 CC with saturated n-Bu-H 2 O gave three sub-fractions, then the first and third sub-fractions were purified on sephadex LH-20CC using EtOH-H2O (1:1) as solvent system and gave a pure samples of 1 (28 mg) and 2 (35 mg). The second sub-fraction was subjected to sephadex LH-20CC with EtOH-H2O (30:70) as an eluent to give two major compounds which were further purified on sephadex CC using MeOH (HPLC) as solvent to give pure samples of 3 (30 mg) and 4 (35 mg).
Fraction II (6.5 gm) was chromatographed on sephadex LH-20CC and eluted with EtOH for further times to afford pure compounds of 5 (25 mg) and 6 (18 mg) Fraction III (13 gm) has been separated on cellulose column chromatography with n-BIW as eluent to give six sub-fractions. Four of them subjected to sephadex LH-20CC with absolute EtOH to give four pure samples of 7(20 mg), 8(22 mg) and 9(18 mg). The last two subfracions were purified by precipitation with diethyl ether the filtrate and precipitate were applied on sephadex CC with HPLC methanol to afford pure compounds of 10(20 mg), 11(16 mg) and 12(18 mg).
Fraction IV (3.5 gm) was fractionated by the same way of fraction I to give two pure samples 13(1.5 gm) and 14(1.1gm) which identified by compared with authentic samples on paper chromatography (PC).
Structure elucidation established by using NMR data 1 H NMR (400MHz, DMSO-d 6 ) and 13    Ellagic acid (7) White amorphous powder, UV λ max (MeOH)nm: 364, 255 characteristic for ellagic acid (Tanaka et al., 1986). 1     3, 4, 5-trihydroxy benzoic acid (gallic acid). (13) and methyl gallate (14) UV λ max (MeOH) nm: 272 characteristic for phenolic acids, were proved by co chromatography with authentic samples Quantification of total phenolic content (TPC) TPC was carried out using the Folin-Ciocalteu colorimetric method described by (Saboo et al 2010). Five mgs of MeOH extract dissolved in 5 ml of 50% MeOH to prepare a solution of final concentration, 1 mg/ ml. An aliquot (2 ml) of the extract and standard solution of gallic acid (8-100 µg/ml) was added to 25 ml volumetric flask containing 1.5 ml of Folin-Ciocalteureagent and 4ml of 20% Na 2 CO 3 solution, then the solution was diluted to 25 ml with distilled water. The absorbance was measured, after 30 min at 765 nm using spectrophotometer, against a blank prepared at the same time using 2 ml of distilled H 2 O instead of the standard solution. All concentrations were carried out in triplicate.

Antioxidant activity (DPPH Assay)
The free radical scavenging activity using the 1.1-diphenyl-2-picrylhydrazil (DPPH) reagent was determined according to Brand-Williams 21 . The isolated compounds (1-9) were dissolved in 85% methanol: water. To 0.5 ml of each, 1.0 ml of freshly prepared methanolic DPPH solution (20 µg/ ml-1) was added and stirred. The discoloration processes was recorded after 5 min of reaction at 517 nm and compared with a blank control.
Antioxidant activity = [(control absorbance -sample absorbance) / control absorbance] × 100% In this test, data can only be compared when obtained under identical settings.

Reducing power Assay (RPA)
The reducing power of the examined compounds was determined according to the method of Oyaizu 1986 22 . 0.5ml of each isolated compound were added to Phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1% potassium ferricyanide (2.5ml). The mixture was incubated at 50°C for 20 min. Aliquots of trichloroacetic acid (2.5 ml,10%) were added to the mixture, which was then centrifuged at 1000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared FeCl 3 solution (0.5 ml, 0.1%). The absorbance was measured at 700 nm. Increased absorbance of their reaction mixture indicated increased reducing power.

ABTS radical scavenging assay
ABTS assay was carried out using the method of Re et al. 23 . The stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted by mixing 1 ml ABTS . + Solution with 60 ml methanol to obtain an absorbance of 0.706 ± 0.001 units at 734 nm using the spectrophotometer. ABTS . + Solution was freshly prepared for each assay. The isolated compounds were allowed to react with 3 ml of the ABTS . + Solution and the absorbance were taken at 734 nm after 7 min using the spectrophotometer. The ABTS . + scavenging capacity of different concentrations percentage inhibition calculated as ABTS radical scavenging activity using the following equation.
(%) = Abs.control -Abs.sample)] / (Abs.control)] × 100 Where Abs. control is the absorbance of ABTS radical + methanol; Abs. sample is the absorbance of ABTS radical + sample extract. Each of the above assays was carried out in triplicate.
This test does not distinguish between the kinetics of radical trapping and stoichiometry.

Ferric reducing antioxidant power assay (FRAP)
The FRAP assay was done according to Benzie and Strain 24 with some modifications. The stock solutions included 300 mM acetate buffer, pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40 m MHCl, and 20 mM FeCl 3 •6H2O solution. The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution, and 2.5 ml FeCl 3 •6H2O solution and then warmed at 37 °C before using. Compounds (1-9) were allowed to react with 2850 μl of the FRAP solution for 30 min in the dark condition. Readings of the colored product [ferrous tripyridyltriazine complex] were then taken at 593 nm. Results are expressed in μmolTrolox / g dry matter. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve. This method essentially provides the stoichiometry of antioxidants.

Flavonoids isolated from 70% (CH 3 OH/H 2 O) extract
The purification of the aerial parts extract from S. grantii afforded 14 known compounds were separated for the first time by different chromatographic technique, their structures established based on NMR spectroscopic data and by comparing them to those reported in the literature (Figure 1) Figure 1: Chemical structure of isolated compounds from S. grantii. and aglycon, obtained as yellow solid powder, appear as dark purple spot under UV light on PC, changed to bright yellow colour and green fluorescence when exposed to ammonia vapour, after spraying with aluminium chloride. 1 H NMR spectrum of compound 1 showed in its aromatic region an ABX-spin coupling system, each of one proton, at δppm7.67, 7.36 and 6.87 assignable to H-6', 2' and 5', respectively. In addition, an AM-spin coupling system described in the form of two meta coupled protons at 6.41, 6.21 for H-8 and 6, respectively. Thus, the aglycone moiety was confirmed as a quercetin 3-O-substituted. In the aliphatic region one anomeric proton with large J value was assigned at 5.34 (d, J = 8Hz) for β-configuratioin and the appearance of H-3'' at 3.23 (1H, t, J = 8.4 Hz, H-3'') established compound 1 as quercetin3-O-β-D-glucopyranoside. Compounds 2, 3 were very similar to 1. The difference between them was in the aliphatic region (sugar moiety). In compound 2 two singlet signals appeared at δppm 5.29 and 1.07 assignable for the anomeric and methyl rhamnose, revealed rhamnose sugar with α-linkage. Compound 2 was confirmed to be quercetin3-O-α-L-rhamnoside. In compound 3 the appearance of H-3'', J = 6 Hz and H-4'' as br s differentiated the glycoside moiety as galactoside rather than glucoside (J = 9.5-10 Hz), so compound 3 was identified as quercetin3-O-β-D-galactopyranoside. 1 H NMR spectrum of compound 4 showed in its aromatic region an A 2 X 2 -spin coupling system as two o-doublets, (J = 9.6 Hz), each of two protons, at δ 8.05 and 6.90 assignable to H-2'/6' and H-3'/5', respectively. In addition, an AM-spin coupling system described in the form of two m-coupled protons (J = 2 Hz), at 6.44 and 6.22 for H-8 and H-6, respectively. Thus, the glycone moiety was confirmed as a kaempferol 3-O-substituted. In the aliphatic region the anomeric proton was assigned at 5.37(d, J=7.5 Hz) exactly the same pattern in previous mono glycoside. Accordingly, compound 4 was finally identified as Kaempferol3-O-β-D-glucoside.
Compound 5 has the same chemical structure of 4, but its aliphatic region showed signal at δ ppm 5.30 s, J=1.5 was attributed to α-anomeric proton and a singlet at 1.18 for the methyl rhamnose protons, indicating presence of rhamnosyl moiety, and confirmed compound 5 to be Kaempferol3-O-α-L-rhamnoside. The disappearance of the anomeric in the aliphatic region exhibited that compound 6 is aglycone not glycoside and identified as Kaempferol 11 .

Tannins isolated from methanol water extract
Six ellagic acid derivatives were separated. Compound 7 obtained as white amorphous powder that displayed shiny buff fluorescence spot on PC under UV light, changed to dull yellow fluorescence with ammonia vapours. Spraying with FeCl3 gave blue colour indicating its phenolic nature. The UV spectral data exhibited two absorption bands (λmax364, 255) characteristic for ellagic acid. 1 H-NMR spectrum showed a singlet signal at δ ppm 7.49 integrated for two equivalent protons in aromatic region assigned for H-5 and H-5`, so the compound 7 elucidated as ellagic acid 12 .
Compound 8, isolated as yellow powder exhibited dark violet fluorescence under short and long UV light turned greenish-yellow fluorescence with NH 3 vapours. Spraying with FeCl3 gave blue colour, R f (x100)56 (15%HOAc) 30 (BAW) indicating its phenolic nature. The UV spectral data exhibited two absorption bands at (λmax 247, 375) characteristic for ellagic acid derivatives 13 . The molecular formula C 16   The gallic acid 13 and methyl gallate 14 were identified from chromatographic properties, by comparing UV spectra with reported data and by co-chromatography on TLC with authentic sample 15,16 .
The existence of this kind of compounds in the aerial parts of S. grantii was confirmed by the TPC assessment, by the Folin-Ciocalteu method and TFC. S. grantii, 258.04. ± 15.67mg/g content and 380.48 ± 31.07 mg/g content such results confirm the significance of assessing antioxidant activity, as it depends on the structural features of organic compounds, mainly due to the presence of phenolics and flavonoids so the antioxidant properties of this species can be responsible. Analyzes of NMR revealed the presence of phenolics and flavonoids compounds in the extract of the aerial parts. Hence its ability to prevent the development of free radicals assessed.

In vitro Antioxidant Activity
The antioxidant was determined by using four assays DPPH, RPC, ABTS, and FRAP.

DPPH Radical Scavenging Activity
Free radicals of (DPPH) are widely used for screening of medicinal plants to investigate their antioxidant potential. The DPPH radical scavenging activity was measured and compared with that of BHA. Results in (Figure 2) showed that DPPH radical scavenging activity of compounds (1-9) ranged from 24.83 ± 0.32% for compound 5 (500 µg/ ml) to 88.47 ± 0.37% for compound 2 (100 µg/ml). Compound 4 also exhibited a very high DPPH radical antioxidant activity.

Reducing power capacity (RPC)
In reducing power assay, the presence of the antioxidant agents can improvement the reduction of Fe (III)/ferric cyanide complex on the Pharmacognosy Journal, Vol 12, Issue 6, Nov-Dec, 2020  ferrous form [Fe (II)] by donating an electron. The occurrence of reluctant in the sample would cause the reduction of (Fe3+) to (Fe2+) ion through the donation of an electron and the creation of the Perl Prussian blue complex. Results illustrated in (Figure 3) indicated that all the examined compounds have good reducing power ability. Compounds 2, and 4 have the superiority their absorbance at 700 nm found to be 1.63 ± 0.03 and 1.51 ± 0.01 respectively at the concentration 500 µg/ml. Compound 7 recorded the lowest reducing power capacity at the same concentration (0.43 ± 0.02).

ABTS scavenging activity
The isolated flavonoids and tannins as well as the synthetic antioxidant BHA revealed considerably different in their ABTS radical scavenging activities. All the isolated compounds caused an inhibition in ABTS+ activity (Figure 4). The highest ABTS scavenging activity obtained corresponded to compound 2 (71.65 ± 0.42% at the concentration 100 ug / ml), followed by compound 6. (70.77 ± 0.34%) but at a higher concentration 250 µg/ml. Also compound 4 exhibited a good ABTS radical scavenging activity (60.89 ± 0.18%) at a low concentration 100µg/ml .Compounds 8, 9 and 1 recorded 63.15 ± 0.14, 59.82 ± 0.26 and 55.42 ± 0.20 respectively at the same concentration (500 µg/ml), while compound 5 showed the lowest activity (31.85 ± 0.34) compared to the standard BHA (92.38 ± 0.26.The highest activity may be attributed to the presence of the high molecular weight flavonoids which are responsible for quenching of ABTS cation as reported by 17 .

Ferric reducing power ability (FRAP)
The iron reducing power test based on the reduction of Fe3+ iron to Fe2+ iron was used in this study to highlight the antioxidant potential of the isolated compounds (1-9). Similar trend was noticed in the obtained results of ferric reducing power ability as compound 2 exhibited the highest ferric reducing power ability, it found to be 3574 ± 19.14 µmolTrolox/100g DW at the concentration 100 ug / ml followed by compound 6 where the compound is achieved 3080 ± 17.43µmol Trolox/100g DW 500 ug / ml also compounds 8, 5 and 3 recorded good ferric reducing power ability they recorded 1352 ± 10.02, 1149 ± 13.50 and 1129 ± 11.93 µmol Trolox/100g DW respectively at the same concentration ( Figure 5).
Analyzes of NMR showed that extract of aerial parts from S. Grantii has a high level of flavonoids and tannins, so its ability to inhibit free radical formation was assessed. All of the compounds tested possessed strong antioxidant activity across all assays used. Compounds 2,  4 and often 6 consistently displayed higher antioxidant activity at lower concentrations than the majority of the compounds used. This high capacity can be attributed for their chemical configuration and contains multiple effective groups which were the main reason for the radical scavenging operation. Antioxidant property is widely used as a parameter for medicinal bioactive components [4]. Phenolic compounds and flavonoids may be responsible for the alcoholic extract's antioxidant properties 18,19 .

CONCLUSIONS
The compounds tested had strong antioxidant function as a natural source of antioxidant medicinal products.