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 (H2O2), hydroxyl radicles (OH.-) and singlet oxygen (2O1).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.2 Sometimes 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.3 Under 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.4 Earlier 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.5,6 Natural 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 significant antioxidant properties and have an immense potential in pharmaceutical and food sectors.7 Buckwheat is among one of them that has gained a rapid momentum in the functional food sector due to its high neutraceutical properties. Buckwheat has attributed worldwide attention, especially from food scientists for its healthy effects over chronic diseases. In developing countries like India, majority of the population rely on traditional system of medicine, besides due to the population explosion the current food production is not sufficient to cater the food crisis so, it is the need of the hour to explore food crops that possess nutritional and medicine value. It is the only pseudocereal that contains a well-known glycoside “rutin”.8 Rutin is known to serve as anti-hypertensive, anti-inflammatory, anti-carcinogenic and vasoconstrictive.9 Other essential bioactive constituents of tartary buckwheat are phenols, fagopyrins, fagopyritols, resistant starch, dietary fibre, vitamins and lignans.10 Buckwheat is also an important source of macro-and micro-nutrients indispensable for human health.11,12 reported the Co/Sb/Ba/Se/Ag/Hg/Cr/Rb/Zn/Fe/Ni and Sn contents in the flour and bran of buckwheat, where most trace elements are concentrated mainly in the bran.13 reported the Mo/Ni/P/Co/Cu/Fe/Zn and Mn in extract of buckwheat flour.
To the best of our knowledge, there is scarcity of information regarding the antioxidant potential and elemental concentration analysis in buckwheat species grown in Kashmir region. Keeping in view of the above facts, the present study was conducted to evaluate the antioxidant potential and elemental analysis of two different buckwheat species (Fagopyrum tataricum Gaertn and F. kashmirianum Munshi) grown in Kashmir region.
MATERIALS AND METHODS
Seeds of Fagopyrum tataricum and F. kashmirianum 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 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 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 aqueous solvent following the protocol of14 with slight modifications. Briefly, aqueous leaf extract of F. tataricum and F. kashmirianum was prepared by mixing 5g dried fine powder in aqueous solvent and was constantly agitated on a rotatry shaker (200rpm, 25oC and 48h). Extract was then filtered through Whatman filter paper (No. 1) and the filtrate was centrifuged (8000rpm, 12oC, 15min) to get clear supernatant. Yield was calculated and 10 mg/mL was prepared as stock solution which was stored in dark coloured bottles at 4oC for further analysis.
Estimation of total phenol content (TPC) and total flavonoid content (TFC)
The TPC was estimated by Folin-Ciocalteau reagent following the method of.15 TFC were investigated by a method described by.16 A gallic acid standard (R2=0.998) was used to determine the TPC. For the determination of TFC, rutin was used as standard (R2=0.99).
Ferric Reducing Antioxidant Potential – FRAP assay
FRAP assay was done using a modified protocol of17 based on color (blue) development due to the reduction of the ferric iron (Fe3+) to ferrous form (Fe2+). FRAP-reagent was freshly prepared by mixing 25mL CH3COONa buffer (300mM, pH 3.6), 2.5 mL TPTZ solution (10mM TPTZ prepared in 40mM HCl) and 2.5 mL FeCl3 solution (20mM). The mixture was incubated at 37oC for 10 min before use. Different concentrations of the plant extract and standard (10-50μl) were allowed to react with 2mL FRAP-reagent for 30 min in dark. After incubation, the colored solution (ferrous tripyridyltriazine complex) formed was then read at 593 nm. Calibration standard was linear between 200 and 1000μM FeSO4 and the result were expressed in μM Fe (II)/g DW.
DPPH assay (1, 1-diphenyl 1-2-picryl-hydrazyl)
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.18 Briefly, various concentrations (5-100μg/mL) of the plant extract and standard (BHT) were added to the methanolic solution of DPPH (0.2mM) and the reaction mixture was throughly mixed and incubated in dark at room temperature for 10 minutes. The absorbance was read at 517nm using spectrophotometer (Shimadzu UV-1800, Japan) and the percent inhibition was calculated as:
In order to determine RSa50 value, i.e, amount of sample required to cause 50% inhibition of DPPH radical, the scavenging Percentage was plotted against logarithmic values of concentration and a linear regression equation, Y=mx + C was established.
H2O2 radical scavenging activity
H2O2 scavenging activity of the various extracts was estimated followed the modified protocol of.19 Different extract concentrations (10-50μg/ml) was added to 600 µL of H2O2 solution (40mM) in phosphate buffer (0.1M, pH=7.4). Incubate the reaction mixture at 25oC for 10 min and then read at 230nm using UV- spectrophotometer against a solution blank containing only phosphate buffer. The H2O2 scavenging activity of the extract was calculated by using the formula:
Hydrogen peroxide scavenging activity (%) = [(A0 ̶ A1)/A0] × 100
Where A0 -control absorbance and A1-sample absorbance
Sample preparation for atomic absortion spectrophotometry (AAS)
Fresh seeds samples were dried at 55oC for 72h, mechanically grinded and sifted out with a mesh (178μm). Wet ashing was done following the protocol of20 by taking 0.25g of powdered groat samples in a separate 50 ml flask containing mixed acid solution [nitric acid (HNO3: sulfuric acid H2SO4: perchloric acid (HClO4)] in a ratio of 5:1:0.5. The samples were boiled in acid solution on hot plate under fume hood till the organic matter is completely digested as indicated by white fumes coming out from the flask. Thereafter, few drops of ultrapure water were added and allowed to cool. The volume of the digestion solution was adjusted to 50 ml with ultrapure water. The solution was filtered and submitted to AAS (Perkin Elmer USA) analysis.
RESULTS AND DISCUSSION
Total phenol and flavonoid content
The TPC and TFC of the F. tataricum and F. kashmirianum are presented in Figure 1a, b. From the results the aqueous extract of F. tataricum shows better TPC (159.51±10.3 mg GAE/g DW) and TFC (79.49±9.76 mg RE/g DW) as compared to F. kashmirianum. It has been reported that rich flavonoid and phenolic plants could be a vital source of therapeutic potential against the oxidative damages by scavenging free radicals.21-24 Previous study also reports that secondary metabolites act as strong chain breaking antioxidants.23 Our results are in accordance with Mann et al.25 using a comparative nutritional and antioxidant potential of two buckwheat species. Earlier reports also revealed that TPC of tartary buckwheat was much higher than that of cranberry, apple,26 raspberry,27 honey,28 corn, wheat, oats and rice29 suggesting that tartary buckwheat may serve as an excellent dietary source of phenolics. Earlier studies have also reported that the TPC and TFC of the plants are influenced by environmental factors as well as the type of species.30 The present study reveals that the buckwheat grown in Kashmir region is a potential source of phenolic and flavonoids bioactive constituents, thus could be used as an excellent source of functional food.
Total antioxidant activity
Total antioxidant activity of the plant extract was determined in terms of ferric reducing antioxidant power assay (FRAP) i.e, capability of the plant extract to convert Fe3+ to Fe2+. In this assay, formation of blue color due to the reduction of Fe (III)-TPTZ complex into Fe (II)-TPTZ complex that absorbs strongly at 593nm. The reducing property of extract is associated with the presence of metabolites that are involved in breaking the free radical chain reaction by donating H-atom.31,32 The present results reveal that the ferric reducing power of both the buckwheat species increased in a concentration-dependent manner (Figure 1c). Similar observation was also reported in Samac (Rhus coriaria L.) that shows an increase in ferric reducing power ability as the concentration increases.33 Results show that aqueous leaf extract of F. kashmirianum shows better ferric reducing power (350.68±15.89μM Fe (II)/g DW) as compared to F. tataricum (295.08±10.86μM Fe (II)/g DW). Technically, FRAP assay is simple to determine the total antioxidant potential of the plant extract and is a proven quantitative approach to determine potential of various phyto-foods.34 Our results are in accordance with earlier studies of Mann et al25
To evaluate the free radical scavenging power of the plant extract, DPPH assay is a widely accepted protocol and is based on the reduction of methanolic DPPH solution in the presence of antioxidant resulting in the formation of non-radical DPPH-H by the reaction and the degree of discoloration exhibited by the scavenging potential of the extract. DPPH radical scavenging activity of the aqueous leaf extract of two buckwheat species are depicted in Figure 1d. From the results, the data shows that in both the species the radical scavenging activity of leaf extract increases in a dose-dependent manner. Among the two buckwheat species, F. tataricum exhibits high scavenging activity (85.10%±10.78) at 40μL concentration as compared to F. kashmirianum (66.23%±8.76) over that same concentration. The present study also revealed that F. tataricum exhibits lower radical scavenging activity (RSa50=26.67μg/mL) as compared to F. kashmirianum (RSa50=34.15μg/mL) which is associated with the high percentage of scavenging of free radicals.35 reported that plants with antioxidant capacity exhibit better radical scavenging activity. The present study confirms that aqueous leaf extract of tartary buckwheat is a potent antioxidant as compared to F. kashmirianum. It also suggests that the plant extracts containing bioactive constituents that are able to donate H-atom to a free radical which in turn remove odd electron that is responsible for the radical’s reactivity.35 Our findings are in accordance with earlier reports.36,37
Hydrogen peroxide radical scavenging activity
H2O2 itself is not very toxic to cellular system but sometimes it becomes toxic as it is directly involved in the Fenton’s reactions that leads to the production of OH.- radicals.38,39 From the results, the H2O2 scavenging activity of the aqueous extract of both buckwheat species increases with increase in concentration and was found high in F. tataricum (76.28±8.785) at 50µg/ml concentration as compared to F. kashmirianum (61.34±7.67%) over the same concentration (Figure 1e). The RSa50 value of aqueous extract of F. tataricum was found to be 21.32µg/ml compared to F. kashmirianum (34.81µg/ml). The strong H2O2 scavenging activity of the buckwheat leaf extract may be due to the presence of secondary metabolites like phenolic compounds and other metabolites such as, tannins, anthocyanins which donates electron to H2O2 radicles thus neutralizing their effect.40 These results suggest that aqueous extract can be a better antioxidant for removing H2O2 and thus protecting living systems under oxidative stress.
Macro-and micro-nutrient analysis
The various mineral element concentrations in the groat samples of two buckwheat species are presented in Figure 2a-k. A comparative macro-and micro-nutrient analysis of the groat samples was done among two buckwheat species (F. tataricum and F. kashmirianum) and the results revealed that F. tataricum contains highest Ca level (5125±56.76ppm) as compared to F. kashmirianum (4055±45.67ppm). Ca plays a significant role in muscular contraction, provides strength to bones and reduces the risks of osteoporosis.41 Fe was more abundant in the groat samples of F. kashmirianum (1122.5±25.77ppm) as compared to F.tataricum (875±10.86ppm). Fe constitutes an important part of the hemoglobin, thus is necessary to overcome the problems of anemia, besides it also maintains the function of central nervous system (CNS).42 Fe is also important to prevent cough linked with angiotensin-converting enzyme inhibitors.43 The micro-nutrient Zn constitutes an important co-factor of various enzymes. Deficiency of Zn especially in children leads to retardation in growth, loss of appetite, general indisposition and skin related disorders.44 Our results show that F. kashmirianum contains more amount of Zn (122.75±12.34ppm). Another micro-nutrient manganese (Mn) is very essential to improve insulin sensitivity and is the structural component of many enzymes.45 The present study shows that Mn was high in F. kashmirianum (127.27±11.55ppm). Copper (Cu) also takes part in various metabolisms and the deficiency of this mineral element leads to microcytic anemia, neutropenia and deformation of bones.46 Among two buckwheat species Cu was found high in F. tataricum (31.25±1.89ppm). Cr plays a vital role regulating blood-glucose level, hunger, cholesterol level and also protects DNA.41 Results reveal that Cr was found high in F. tataricum (1.5±0.15ppm). Other micro-nutrients such as nickle (Ni) and cobalt (Co) are required by human body in little amount. Co is an essential component of vitamin B12 and thyroid metabolism.47 In the present study, F. tataricum contains higher Co-content (1.75±0.2ppm). Lead (Pb), Aluminium (Al) and Cadmium (Cd) are considered as toxic elements and their presence in the groat samples of buckwheat is due to the degraded quality of the soil.
From the present investigation, it was concluded that the aqueous extract of tartary buckwheat possesses high antioxidant and free radical scavenging activity. These in vitro assays indicate that the buckwheat plant extract is a significant source of natural antioxidant which might be useful in preventing the progress of various oxidative stresses. Further, the macro-and micro-nutrient analysis of buckwheat groat samples revealed that it is rich in calcium, iron and zinc and thus could be used as a potential biofortified crop for reducing mal-nutrition especially among impoverished regions of the world. The study also revealed that among two buckwheat species, tartary buckwheat is efficient in terms of antioxidant potential and mineral element analysis.