Metabolite Fingerprinting and Antioxidant Potential of Tartary Buckwheat-an Underutilized Pseudocereal crop from Kashmir Region

Objective: Buckwheat is an underutilized pseudocereal crop used as a staple food especially in Himalayan regions. The aim of the present study was to evaluate the phytochemical screening, antioxidant potential and metabolite profiling of tartary buckwheat extract. Methods: Tartary buckwheat leaf samples were tested for total phenols, flavonoids and in vitro antioxidant potential in terms of total antioxidant activity, free radical scavenging (superoxide, hydrogen peroxide) and DPPH assay. GC-MS profiling was done to identify and quantify the various metabolites from the methanolic leaf and groat extract. Results: Preliminary phytochemical screening of methanolic extract revealed the presence of alkaloids, flavonoids, phenols, tannins, saponins, phlobatannins, coumarins, glycosides and anthoquinones. Methanolic extract exhibits higher TPC (28.32 ± 5.31 mg gallic acid equivalent g-1 DW) and TFC (25.18 ± 3.5 mg rutin equivalent g-1 DW). DPPH radical (EC50=1.8 μg ml -1) and H2O2 scavenging (EC50=0.103 μg ml -1) potential of tartary buckwheat leaf extract shows promising results. From GC-MS metabolite fingerprinting, over 111 and 24 metabolites were identified among leaf and groat extract respectively. The major compounds present in the extracts were fatty acids, hydrocarbons, steroids, terpenoids, esters, organic acids and aldehydes with excellent pharmaceutical properties. Conclusion: The tartary buckwheat extract were found to contain numerous metabolites with potent antioxidant and other pharmacological actions. Thus, tartary buckwheat could be a promising alternative in functional food sector to improve social well-being and neutraceutical to diminish malnutrition especially for the impoverished community.


INTRODUCTION
Generation of free radicals in the form of active oxygen species (AOS) in biological system is a normal phenomenon.These AOS include; superoxide anions (O .-),hydrogen peroxide (H 2 O 2 ), hydroxyl radicles (OH .-)and singlet oxygen ( 2 O 1 ). 1 Previously, AOS were considered as dangerous molecules which must be maintained at low levels in cells.However, this perception has been changed because these serve as important signaling molecules. 2Sometimes these free radicles are produced to such an extent that the body's defence system is not able to expel them out and thus leads to oxidative stress. 3Under such conditions these AOS cause damage to various cell organelles, cell death, DNA damage and gene mutation which often leads to chronic ailments like neurodegenerative diseases, cardiovascular dysfunctions, aging, weakening of immune system. 4arlier reports suggests that there exists strong association between dietary intake of these natural products and the disease prevention and such wonderful properties of these botanicals is due to the presence of secondary metabolites with healthcare properties. 5,6Natural antioxidants are interesting green alternatives to artificial antioxidants because of the safety concerns and limitation of usage.Plants contain plethora of secondary metabolites (e.g, flavonoids, glycosides, terpenoids, tannins etc) with potential antioxidant properties and have an immense potential in pharmaceutical and food sectors. 7Isolation and structural analysis of these secondary metabolites from medicinal plants is a main thrust of natural product chemistry to identify and evaluate their therapeutic potential.GC-MS is a robust approach for the qualitative and quantative analysis of metabolites of plant origin. 8gopyrum tataricum (tartary buckwheat) -a dicot pseudocereal belongs to family Polygonaceae is a potential candidate due to its high neutraceutical properties.It is the only pseudocereal that contains a well-known glycoside "rutin". 9Rutin is known to serve as anti-hypertensive, antiinflammatory, anti-carcinogenic and vasoconstrictive. 10Other essential bioactive constituents of tartary buckwheat are phenols, fagopyrins, fagopyritols, resistant starch, dietary fibre, vitamins and lignans. 11Buckwheat has attributed worldwide attention, especially from food scientists for its healthy effects over chronic diseases.In developing countries like India, majority of the population relay on traditional system of medicine besides due to the population explosion the current food production is not sufficient to cater the food crises so, it is the need of the hour to explore food crops that possess nutritional and medicine value.In view of the above facts, the current study was focussed to evaluate the phytochemical screening and the antioxidant potential of tartary buckwheat extracts by using various assays like FRAP, DPPH, RP, SOD, TPC and TFC.Besides, we performed metabolite profiling by GC-MS to identify and quantify the essential metabolites present in the extract of tartary buckwheat.

Plant material
Seeds of Fagopyrum tataricum (buckwheat) were procured from Department of Botany, University of Kashmir, Hazratbal, Srinagar.Later these seeds were sown during the month of April-2014 in the Botanical garden of Free Radicals and Antioxidants, Vol 7, Issue 1, Jan-Jun, 2017 Kashmir University.Harvesting of the leaf sample was done at the pre-flowering stage.

Collection and preparation of sample material
Fresh and healthy leaves of tartary buckwheat were collected and washed gently with distilled water (without squeezing) to remove debris and dust particles.The plant material is then air-dried under shade at room temperature for 15 days and ground into a powdered form using a surface sterilized mortar and pestle which was further used for extraction.

Solvent extraction procedure
Preparation of leaf extract was done in methanol and ethanol solvents following the protocol of Okogun. 12

Phytochemical screening
Phytochemical analysis for antioxidants was done following the method of Bruneton. 13

Estimation of TPC and TFC
The TPC was estimated by Folin-Ciocalteau reagent following the method of Malick and Singh. 14TFC were investigated by a method described by Hung and Morita. 15A gallic acid standard (R 2 =0.998) was used to determine the TPC.For the determination of TFC, rutin was used as standard (R 2 =0.99).

Antioxidant assays Total reducing power
The reducing activity of the extracts was determined followed the protocol of Yen and Duh. 16rric Reducing Antioxidant Potential-FRAP assay.
FRAP assay was done using a modified protocol of Benzie and Strain 17 based on color (blue) development due to the reduction of the ferric iron (Fe 3+ ) to ferrous form(Fe 2+ ).

Superoxide radical scavenging activity
Superoxide radical scavenging activity of the leaf extracts was determined following the method of Fontana et al. 18

H 2 O 2 radical scavenging activity
The scavenging activity of the extracts towards hydrogen peroxide radicals was determined by the method of Ebrahimzadeh et al. 19

DPPH assay
DPPH activity was measured by determining the hydrogen donating or radical scavenging ability of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical followed the method of Braca et al. 20

Metabolite fingerprinting Sample preparation for GC-MS analysis
The 0.2 g of dried extract powder of tartary buckwheat leaf and groat samples were dissolved in 10 ml of methanol solvent properly mixed and kept for 72 hrs, then filtered through 0.45 μm syringe filter (Millipore Corp., Bedford, MA, USA). 1 μl aliquot of the sample was then injected into the GC-MS port for the metabolite analysis (Shimadzu QP-2010 Plus with Thermal Desorption System TD 20).GC-MS analysis was performed according to the method of Dhar et al. 21

Statistical analysis
Results are presented as mean ± standard deviation (SD) of three replicates.Data were subjected to analysis of variance using Graph pad prism 6.07 software and was considered significant at p ≤ 0.05.IC 50 values were calculated by using linear regression plots.

Total phenol and flavonoid contents
The present investigation reports that the total phenol content was high in the methanolic extract (28.32 ± 5.31 mg GAE g -1 dry powder) as compared to ethanolic extract (25.64 ± 3.41 mg GAE g -1 dry powder).Total flavonoid content follows the similar trend, methanolic extract contains 25.18 ± 3.5 mg RE g -1 dry fraction as compared to ethanolic extract (19.3 ± 2.7 mg RE g -1 dry fraction) (Figure 1A, B).

Antioxidant assays Reducing power
The present result shows that reducing power increase in a concentrationdependent manner and is highest in the methanolic extract (0.50 ± 0.11 µg/ml) as compared to ethanolic extract (0.432 ± 0.2 µg/ml) (Figure 1C).The antioxidant present in the extract donates an electron to stabilize the radicals and also causes chain termination. 22The capability of extract to exhibit the reducing power in this study may be due to the presence of antioxidant metabolites.

FRAP assay
The total antioxidant potential of the botanical extracts was calculated from their capability to reduce TPZR-Fe (III) complex to TPTZ-Fe (II).FRAP assay is a cost-effective approach and has become a valuable protocol to measure total antioxidant/ reducing power of the extract.In our study, the total antioxidant potential was higher in the methanolic extract (375.75 ± 36.74 µmol Fe-II/g DW) at 50 µg/ml concentration as compared to ethanolic extract (365.20 ± 40.12 µmol Fe-II/g DW) that signifies its high antioxidant potential (Figure 1D).

Superoxide radicle scavenging activity
Tartary buckwheat leaf extract shows a superoxide radicle scavenging activity ina dose-dependent manner (Figure 1E).Methanolic extract exhibits highest activity (88.05 ± 16.44 % inhibition) at 50 µg/ml concentration as compared to ethanolic extract (78.81 ± 15.76 % inhibition) over the same concentration.Besides, methanolic extract exhibits lowest EC 50 value (EC 50 =5.86µg/ml) which means that the metabolites present in the methanolic extract are potent scavengers of O 2 .-radicles at low concentration.

Hydrogen peroxide radical scavenging activity
From the results, the H 2 O 2 scavenging activity increases with increase in concentration of extract and are high in methanolic extract (98.59%) at 50 µg/ml concentration as compared to ethanolic extract (85%) over the same concentration (Figure 1F).The EC 50 value of methanolic extract was found to be 0.103 µg/ml compared to ethanolic extract (24.54 µg/ml).The strong H 2 O 2 scavenging activity of the buckwheat leaf extract may be due to the presence of bioactive constituentssuch as phenolic compounds and other metabolites (tannins, anthocyanins etc) which donates electron to H 2 O 2 radicles thus neutralizing their effect. 23PH radicle scavenging activity DPPH-radicle scavenging activity of tartary buckwheat is presented in Figure 1G.The results shows DPPH radicle scavenging activity of both the extracts was found to enhance in a dose-dependent manner.The methanolic extract shows high activity (93.84 ± 14.21) at the concentration of 40 µg/ml as compared to ethanolic extract (78.38 ± 12.11) over the same concentration.A higher DPPH radicle scavenging activity is linked with  a lower EC 50 value (EC 50 =1.8µg/ml for methanolic extract; EC 50 =11.13µg/ml for ethanolic extract).

DISCUSSION
Phytochemical analysis is an essential parameter which provides basic information regarding medicinal importance of the plant extract.In the present investigation, phytochemical screening of chemical constituents of tartary buckwheat exhibits the presence of distinct metabolites such as coumarins, alkaloids, flavonoids, phenols, tannins, saponins, phlobatannins, glycosides and anthoquinones.Our results corroborates with earlier reports. 24In the present study, the qualitative and quantitative estimation of total phenolic and flavonoid contents of tartary buckwheat revealed that methanolic extract exhibits the highest concentration of total phenol and flavonoid content.Our results are in accordance with earlier reports where total flavonoid and phenolic content are found in higher amount in different parts. 25,24It has been reported that plants rich in phenolic and flavonoids could be a potential source of therapeutics against oxidative stress. 26Reducing power is often an indicator of antioxidant activity.In reducing power, antioxidants present in the extract cause reduction of Fe 3+ to Fe 2+ this in turn can be observed by measuring the formation of Prussian blue at λ=700 nm.It is known that increasing absorbance means increasing reducing ability. 27The present study shows that methanolic extract of tartary buckwheat possesses high reducing power.It is believed that the antioxidant property is primarily due to the redox potential 28 that plays a significant role in scavenging the free radicals.0][31] Technically, FRAP assay is simple to determine the total antioxidant potential and is a proven quantitative approach to determine potential of various phyto-foods. 32Present results revealed that both the extracts of tartary buckwheat shows higher FRAP value, however it was more pronounced in methanolic extract as compared to ethanolic extract.Our results are in accordance with earlier reports of Jeong et al. 33 PMS-NADPH oxidation reaction system generates the superoxide radicals (O 2 .-)which can be determined by their ability to reduce NBT.In the presence of plant extract, the absorbance at 560nm decreases indicating the ability of the plant extract to scavenge the O 2 .
-present in the reaction mixture.In the present study, the O 2 .
-radical scavenging activity of different fractions was increased dose-dependently.Low levels of IC 50 values suggested that the chemical constituents found in the methanolic extract and its fractions are potent scavengers of superoxide radicals at low concentration.Superoxide radicle is regarded as a precursor of hydroxyl radicle (OH .-)that is more dangerous and causes lipid peroxidation thus damages the cell membrane and often leads to apoptosis.The presence of potent electron quenching activity of tartary buckwheat extract may be due to the presence of various secondary metabolites especially phenols and flavonoids. 24H 2 O 2 itself is not very toxic to cellular system but sometimes it becomes toxic as it is directly involved in the production of OH .-radicals. 34n this study methanolic extract exhibits strong potential to scavenge the H 2 O 2 potential hazard indicating the antioxidant capacity of the plant.Our results coincides with Liu et al. 35 who compares the antioxidant potential of tartary and common buckwheat and concluded that tartary buckwheat possesses potent radical scavenging activity.DPPH assay is a cost-effective procedure to evaluate the antioxidant potential of botanical extract, where DPPH is consumed as a stable free radical.In the present study, DPPH radical scavenging ability of the tartary buckwheat leaf extract increases in a concentration-dependent manner.IC 50 value of methanolic extract for DPPH radical was found lower as compared to ethanolic extract.Our results are in accordance with the studies on Phodopyyllum hexandrum 36 and Acalypha manniana. 37etabolite profiling of tartary buckwheat leaf and groat methanolic extract was done by GC-MS and a single metabolic profile can be thought of as a snapshot of the metabolic state of an organism at a given moment.In medicinal chemistry, metabolite profiling is of paramount importance to ascertain the chemo-typing of natural products that will not only allow us to scientifically determine but validate their traditional uses, pharmacological activities and therapeutic potential. 38From the present investigation, the methanolic leaf and groat extract of tartary buckwheat revealed the presence of 135 metabolites.Rutin (3, 3' , 4' , 5, 7-pentahydroflavone-3-rhamnoglucoside-71.94%) a major flavonoid of buckwheat found in the groat extract possesses desirable physiological and biological properties such as anti-hypertensive, anti-carcinogenic, vasoconstrictive, anti-inflammatory properties. 10,11Rutin is known to keep capillaries and arteries strong and flexible, besides it acts as a shield against gastric lesions, improve eyesight and hearing, protects against UV-light, X-rays and oxidative stress, 39,40 lowers plasma cholesterol and also suppresses gallstone formation. 41Phytol (5.22%) found in the leaf extract is having anticancer, antioxidant, antitumor, diuretic and chemopreventative properties and used in vaccine formulation. 42,43The other metabolites such as linoleic acid ester, 9-octadecanoic acid (Z)-, methyl ester is also having anti-inflammatory, anti-androgenic and anemiagenic properties. 44The metabolite profiling by GC-MS from different botanicals that possess various pharmacological properties have been studied earlier that supports our current study. 45,46The metabolites identified during GC-MS profiling were further investigated for their biological properties using Dr. Duke's database 47 that revealed that tartary buckwheat possess immense pharmaceutical properties, as the identified   metabolites possess potent anti-diabetic, anti-hypertensive, antioxidant, anti-carcinogenic, hypocholesterolemic and 5-α-reductase inhibitor activity.Thus from the metabolite fingerprinting reveals that the tartary buckwheat can be used as an important source of neutraceutical food.

CONCLUSION
From the current study, it is concluded that tartary buckwheat possesses a potent antioxidant potential as revealed by its higher scavenging activity.Methanolic extract exhibits higher activity, thus could be the optimal solvent for the extraction of bioactive constituents.Metabolite fingerprinting of tartary buckwheat revealed its potential in the neutraceutical and functional food sector to diminish malnutrition and improve social well-being especially for the impoverished community.