Total phenolics and flavonoids content , antioxidant activity and GC / MS analyses of Euphorbia grandialata

Article history: Received on: 24/02/2017 Accepted on: 13/04/2017 Available online: 30/06/2017 Objective: This study has been designed to study phenolic and flavonoid contents quantitatively; screen antioxidant activity and investigate unsaponifiable and saponifiable matters of Euphorbia grandialata R. aerial parts. Methods: The phenolics and flavonoids content were assayed by Folin-Ciocalteu reagent and aluminium chloride reagent respectively, while the anti-oxidant activity was done by 2, 2-diphenyl-1-picrylhydrazyl (DPPH).GC/MS analysis was used to analyze unsaponifiable and saponifiable matters. Results: Total phenolics result was calculated as (17.61 ± 1.2 μg gallic acid/g), total flavonoids as (4.495 ± 0.39 μg rutin/g) and anti-oxidants activity as (140.6 % μg ascorbic acid/g). The identified compounds in unsaponifiable matter were hydrocarbons (51.1 %), steroids and triterpenes (35.92%). The saponifiable matter showed fourteen components from which six were identified as methyl esters of saturated fatty acids (46.26%) and eight as methyl esters of unsaturated fatty acids (53.74 %). Conclusion: Euphorbia grandialata could be considered as a valuable source of natural antioxidants. This is the first report for phenolics and flavonoids content, antioxidant activity and GC/MS analysis of this plant.

It prefers well-drained soil, during the active growing season from March to September and the flowering stage during Summer (Dyer, 1937).Free radicals, which are produced by the chemical reaction of organic compounds, could damage the body's tissues and cells, leading to human aging and causing a variety of diseases.Therefore, it is important to find the antioxidants for scavenging these free radicals.Generally, two methods have been developed for the assessment of antioxidative activities including in vitro and in vivo screening.1,1-Diphenyl-2-pierylhydrazyl (DPPH) method is one of the universal in vitro tools in which DPPH radical, a very stable nitrogen-centered radical, can be used to determine the free radical scavenging ability, which is related to their antioxidative activities.This method depends on spectrophotometric measurement of changes in concentration of DPPH • which results from the reaction of the free radicals with an antioxidant (Meng et al., 2016).
All the reported studies about Euphorbias were related to polar constituents and nothing concerned their fixed oil, although fatty acids were known to have anticancer, antimutagenic and antimicrobial activities (Al Ashaal et al., 2010).Therefore, it was interesting to quantitatively study phenolic and flavonoid contents; screen the antioxidant activity and find its correlation with the phenolics and flavonoids content; in addition to the investigation of unsaponifiable and saponifiable matters of E. grandialata aerial parts growing in Egypt.

General
UV-visible spectrophotometer (Shimadzu UV-1601 PC) was used.Analysis using GC/MS was carried out using a Thermo Scientific, Trace GC Ultra / ISQ Single Quadrupole MS and an electron ionization system with energy of 70 eV for the ionization process.For GC of unsaponifiable matter, capillary column (30 m x 0.25 mm) packed with TG-5MS fused silica was used.The temperature of the oven was programmed at an initial temperature 50 ºC (hold 2 min.) to 150 ºC at an increasing rate of 7 ºC /min.then to 270 ºC at an increasing rate 5 ºC /min.(hold 2 min.)then to 310 ºC as a final temperature at an increasing rate of 3.5ºC /min.(hold 10 min.).The carrier gas was helium gas and used at a constant flow rate of 1mL/min.For GC of saponifiable matter, capillary column (30 m x 0.25 mm) packed with 70% cyanopropyl-polysilphenylenesiloxane was used.The temperature of the oven was programmed at an initial temperature 80 C increased to 230 C by the rate of 3 C/min, then kept isothermal for 20 min.The carrier gas was helium gas and used at a constant flow rate of 1.5 mL/min.2, 2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagents, gallic acid, rutin and ascorbic acid were purchased from E-Merck.Other chemicals used were of analytical grade.

The Plant material
Euphorbia grandialata R. was collected in October 2014 (from Shebin El-Qanater, Qalubiya, Egypt).It was identified by Dr. Abdelhalim Mohamed (Plant Taxonomy Department, Agricultural Research Institute, Egypt).A voucher specimen was placed at the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Beni-suef University under registration number BUPD-44.The fresh aerial parts were cut into small pieces and used in the subsequent experiments.

Extraction
The extraction of the fresh cut aerial parts of E. grandialata (1g) was carried out at room temperature using 50 mL methanol (80%) for 2h on an orbital shaker adjusted at 200 rpm.The supernatant produced from mixture centrifugation for 20 min.
was transferred to a 100 mL volumetric flask.The procedure was repeated and respective supernatant was pooled.The final volume was adjusted to 100 mL.

Total phenolics content
Using Folin-Ciocalteu reagent for determination of total phenolics content (Abu Bakar et al., 2009).The methanol extract (300 µl) was added to 2.25 mL of Folin-Ciocalteu reagent and wait for 5 min at room temperature then add to the mixture2.25mL of sodium carbonate (60 g/L) solution and stand at room temperature for 90 min then measure the absorbance at 725 nm.The standard curve was prepared using gallic acid for quantification purpose and the results were calculated as µg of gallic acid equivalent per g fresh weight.

Total flavonoids content
Using aluminium chloride method for determination of total flavonoids content (Abu Bakar et al., 2009).In a test tube, methanol extract (0.5 ml) was added to 2.25 mL of distilled water then 0.15 mL of 5% NaNO 2 solution was added to the mixture.Wait for 6 min.then add to the mixture 0.3 mL of 10% AlCl 3 .6H 2 O solution.Stand for another 5 minutes then add 1.0 mL of 1 M NaOH.Mix the mixture well using vortex.Immediately, the absorbance was measured spectrophotometrically at 510 nm.The standard curve was prepared using rutin for quantification purpose and the results were calculated as µg of rutin equivalent per g fresh weight.

Antioxidant activity
Using DPPH free radical model to determine the antioxidant activity (Abu Bakar et al., 2009).An aliquot of 300 µl of samples were added to 3.0 mL of 500 µM DPPH in absolute ethanol.Shake the mixture vigorously and wait for 30 min in the dark at room temperature.The absorbance was measured spectrophotometrically at 517 nm.Ascorbic acid was used as a positive control.Using the following equation to calculate the free radical scavenging activity Scavenging effect (%) = [1 -(absorbance of sample/absorbance of control)] x 100

Statistical analysis
All experiments were carried out three times and the result was represented as mean ± standard errors.Microsoft Excel Windows 2013 was used to calculate the linear regression analysis and Pearson's correlation coefficient

Preparation of the unsaponifiable matter (USM), the saponifiable (SM) and the fatty acid methyl esters (FAME) derivatives of SM
The fresh cut aerial parts of E. grandialata (7 kg) were exhaustively extracted by cold maceration (2000 ml, x4, each 48 h) with aqueous ethanol (70%).The extract was evaporated under reduced pressure using a rotary evaporator.Distilled water (1L) was used to suspend the obtained residue (200g) and fractionated successively with n-hexane (500 mL x 4) followed by evaporation of hexane under reduced pressure.The n-hexane extract (10 g) was saponified by subjecting it to heat under reflux with 100 mL 10% alcoholic potassium hydroxide together with 40 mL benzene for 24 h to make sure that hydrolysis had been completed.The saponified solution was concentrated under reduced pressure and suspended in 100 mL distilled water followed by extraction with diethyl ether (10 x 50 mL) till exhaustion.Distilled water was used to wash the combined ether extract several times to remove alkalinity.Anhydrous sodium sulphate was used to dry ether extract, followed by evaporation till dryness.The solvent-free residue obtained representing the unsaponifiable matters (USM), was orange-yellow in color and amounted to 8 g calculated as 80% of the total lipoidal composition of the corresponding n-hexane extract.The dried residue was kept in a desiccator for GC/MS analysis (Vogel, 1975).Liberation of the free fatty acids (FA) from the aqueous alkaline solution left after separation of the unsaponifiable matter was done by acidification with dilute HCl (10%).The FA was extracted with diethyl ether (5 x 50 mL).Distilled water was used to wash the combined ether extract to remove acidity.Anhydrous sodium sulphate was used to dry ether extract, followed by evaporation till dryness.The oily residue obtained representing the saponifiable matters (SM), amounted to 2 g calculated as 20% of the total lipoidal composition of the corresponding n-hexane extract (Vogel, 1975).SM was subjected to methylation to afford fatty acid methyl esters (FAME) derivatives of SM (Vogel, 1975).Chemical investigation using GC/MS technique was conducted to identify components of USM and FAME.A tentative identification of both USM and FAME components was doneby comparison between their relative retention time and mass spectra with those of the NIST, WILLY library data of the GC/MS system.The quantification of all identified components was based on the integration of the area under the peak.

Total phenolics, flavonoids content and antioxidant activity
Spectrophotometric determination of total phenolics content in E. grandialata alcoholic extract was done according to Folin-Ciocalteu method using gallic acid as the standard.Results showed that 17.61 ± 1.2 µg of gallic acid were equivalent to 1g of fresh weight, and the results of total flavonoids content determination by aluminium chloride method was 4.495 ± 0.39 µg rutin were equivalent to 1g fresh weight.Antioxidant assay of the methanol extract showed scavenging capacity to the free radical DPPH 140.6 % μg ascorbic acid/gin comparison to standard ascorbic acid as positive control.
Plants with high contents of secondary metabolites especially phenolics and flavonoids were known to have antioxidant efficacy because of their redox properties and chemical structures.Phenolic compounds are one of the most abundant phytochemicals in plants.They play important role since they safeguard against ultraviolet rays and ensure security against microbial attack and predators (Naczk,2006).The abilities of phenolic compounds for scavenging free radical are due to their hydroxyl groups (Soobrattee et al., 2005).It showed antioxidant efficacy by different strategies like donating hydrogen atoms to free radicals or through binding to transition metal ions resulting in more stable forms (Kumar and Sandhir, 2014).Accordingly, polyphenol showed some physiological activities such as the protection against neurodegenerative and cardiovascular diseases and cancer since they have a high antioxidant capacity (Wootton-Beard and Moran,2011).Also, flavonoids showed in vitro and in vivo antioxidant activities which depends on the free OH groups, particularly 3-OH (Geetha et al., 2003;Shimoi et al., 1996).
DPPH assay has been used worldwide as a method to determine the free radical scavenging capacity of various plant extracts due to its simplicity and relatively short time compared to other methods (Majewska et al., 2011).E. grandialata alcoholic extract showed high antioxidant activity.According to previous studies (Pietta 2000;Michalak, 2006) this activity was thought to be due to the high content of phenolics and flavonoids in this plant.The high content of phenolics and flavonoids is the accountable for the antioxidant efficacy of the plant extract.Flavonoids are considered as the highly efficacious scavengers of majority of oxidizing molecules and free radicals involved in many diseases (Bravo, 1998).They have the ability to inhibit the formation of reactive oxygen , bind to trace elements implicated the production of the free radical and clean out reactive species (Agati et al., 2012).Crude extracts of plant materials rich in phenolics were known have antioxidative properties and health benefits, so far it could used in the food industry for (Baba and Malik 2015) This study has been conducted for other Euphorbia species as E. hirta (Asha et al., 2016), E. helioscopia (Maoulainine et al., 2012), E. echinus (Lahlou et al., 2014)and E. heterophylla (Abbasi et al., 2013), but this is the first report for antioxidant study of E. grandialata.

CONCLUSION
The analysis of the aerial parts of E. grandialata using GC/MS displayed that the saponifiable matter revealed the presence of14 components.The major saturated and unsaturated fatty acids were methyl isohexadecanoate and 8, 11octadecadienoic acid respectively, while USM showed 29 . components; 1-nonanolwas the major hydrocarbon, lanosta-8, 24dien-3-olwas the major sterol and A'-neogammacer-5-en-3-ol and α-amyrin were the major triterpenes.Euphorbia grandialata could be counted as a valuable exporter for natural antioxidants.These antioxidant phytochemicals may be helpful in treatment or prevention of many degenerative diseases such as cancer and other  (3β,15α,16α,21β,15,16,21,22,  human diseases related to oxidative stress.Further work is needed for full characterization of its active principles.
Financial support and sponsorship: Nil.

Conflict of Interests:
There are no conflicts of interest.

Table 1 :
GC/MS analysis of the saponifiable matters of the n-hexane extract of Euphorbia grandialata R.aerial parts.

Table 2 :
GC/MS analysis of the unsaponifiable matters of the n-hexane extract of Euphorbia grandialata R.aerial parts.