NON-ENZYMATIC ASSAY BASED IN-VITRO ANTIOXIDANT ACTIVITY AND PHYTOCHEMICAL SCREENING OF FREEZE DRIED WHEAT (TRITICUM AESTIVUM L.) SEEDLINGS JUICE POWDER: NATURE’S FINEST MEDICINE-PART-II

NON-ENZYMATIC ASSAY BASED IN-VITRO ANTIOXIDANT ACTIVITY AND PHYTOCHEMICAL SCREENING OF FREEZE DRIED WHEAT (TRITICUM AESTIVUM L.) SEEDLINGS JUICE POWDER: NATURE’S FINEST MEDICINE-PART-II

H. S. Dhaliwal, N. Sharma, A. Bano, S. Kumar and V. Sharma^*

Akal College of Agriculture, Eternal University, Baru Sahib-173101 (H.P.) India

ABSTRACT: Free radical scavenging potential and phytochemical analysis of the two different extracts of lyophilized wheat seedling juice powder (WSJP) Triticum aestivum L.(Gramineae),“Nature’s Finest Medicine” was evaluated in vitro by using diphenyl-picryl-hydrazyl (DPPH) assay and preliminary phytochemical analysis. In this method the antioxidants present in WSJP extracts reacted with DPPH, which is a stable free radical and converted it to 1,1-diphenyl-1,2-picryl, hydrazine. The scavenging effect of plant extracts and standard (L-ascorbic acid) on the DPPH radical decreased in the following order: L-ascorbic acid>Methanolic extract (I) > Water extract (II) and it was found to be 97.3%, 58.6% and 35.1% at concentration of 100µg/mL, respectively. The results were expressed as IC₅₀. Ascorbic acid which was used as a standard showed an IC₅₀ of 19.2µg/mL, whereas, the crude methanolic (I) and water (II) extracts of lyophilized wheat seedlings juice powder showed antioxidant activity with IC₅₀ values of 81.4 and131.3, respectively at 1-100µg/mL concentration. None of the extracts were found to be more active than the standard i.e. ascorbic acid. But still, free radical scavenging activity of methanolic (I) and water (II) extracts were confirmed in the present investigation. In the present study, different extracts of lyophilized wheat grass juice powder showed concentration dependent free radical scavenging activity. Significant results were recorded first time in the given estimated parameters. Therefore, this is a pioneer attempt towards the prevention and treatment of various ailments and especially antiaging properties and better understanding of therapeutic potential of WSJP not described earlier

Triticum aestivum (L.);

Wheat Seedling Juice Powder (WSJP); Freeze Dried; In-vitro Antioxidant Activity; 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity;

Non-Enzymatic Assay.

INTRODUCTION: Young seedlings of wheat (Triticum aestivum L.) of family Gramineae also known as Gehun, Kanak, Godhuma, Gandham ¹, popularly called as wheat grass/wheat seedings are being used for extraction of crude juice for herbal medicine. The common wheat or bread wheat widely cultivated in almost all parts of the world, is native to South-West Asia and the Mediterranean region. The wheat plant is believed to have multifarious pharmacological activities in addition to its numerous nutritional values.

Wheat seedlings stimulates metabolism, restores alkalinity of blood and is also a de-toxificant which helps to restore healthy cells ². The use of wheatgrass juice for therapeutic purposes was developed and popularized by Dr. Ann Wig more (1909-1996), as a part of herbal therapeutic nutritional approach ³.

The therapeutic qualities of wheatgrass juice have been attributed to its nutritional content, including chlorophyll, enzymes, Vitamins (A, C and E), bioflavonoids, substantial amount of micronutrients andamino acids ⁴. During germination, vitamins, minerals, and phenolic compounds including flavonoids are synthesized in wheat sprouts, with the maximum antioxidant potential ⁵. The concentration of Vitamins such as C, E, β-carotene, in addition to ferulic acid and vanilic acid increases with the germination period ⁶. The wheat seedlings also contain chlorophyll, which was found to be responsible for inhibiting the metabolic activation of carcinogens ^{7, 8}. It is also reported that 50 pounds of wheatgrass is equivalent to 450 pounds of garden vegetables ⁹. In Asia and Europe, wheatgrass based products are consumed in the form of juices, powders and extracts for healthy growth of human body. Wheat grass has been identified as a complete food providing all the nutrients. Wheatgrass juice provides manifold health and wellness benefits by curing problems related with digestive system and anaemia. Wheat seedlings act as a blood purifier and strengthen the immune system. Anti-carcinogenetic activity of wheatgrass juice has already been reported ¹⁰.

It is well known and reported widely that, wheat grass possesses anti-mutagenic and antioxidant activity because of the redox enzymes including catalase, peroxidase and other antioxidant compounds such as phenolic acids, alkyresorcinols and amino phenols ^{5, 11}. The products of wheatgrass are also reported to cure many dreadful diseases such as thalassemia and distal ulcerative colitis ^{12, 13}.

The research work by Bar-Sela and co-workers reported that the breast cancer patients who took wheatgrass juice daily showed a decreased need for blood and bone marrow building medications during chemotherapy, without diminishing the effects of the therapy ¹⁴. Cytotoxic effects of commercial wheatgrass and fibre towards human acute promyelocytic leukemia cells were also reported recently¹⁵. Wheatgrass has been recommended as a supplement for the last four to five decades as a treatment for various diseases, but till today very little clinical data exists to support its therapeutic uses. Research scientists and physicians from all over world are conducting extensive studies on animal models and human systems to develop herbal medicines, dietary supplements as alternative or complimentary therapy for the treatment of chronic diseases. Epidemiological studies have shown that wheatgrass products are protective against cardiovascular disease, diabetes and cancer ^16-19.

There are reports on the antimutagenic effect of oxidative DNA damage towards benzo(a)pyrene induced mutagenicity ¹¹. Falcioniand co-workers have demonstrated the inhibition effect of wheatgrass on oxidative DNA damage ¹⁰. It has been shown that wheatgrass extracts contain significant amounts of phenolic compounds including flavonoids ²⁰. Phenolic compounds of plant products are mainly responsible for the antioxidant activity which reverse the effect of ROS mechanism by various pathways, and also reduce incidence of cancer ²¹. Reactive oxygen species (ROS) are produced as a by-product of various metabolic processes, in living organisms mainly during respiration.

Normal physiological concentrations of ROS usually have a role of regulation of cell activities, whereas higher concentrations cause oxidative damage. Oxidative stress has been implicated in the pathophysiology of many diseases including cancers. The generation of ROS is prevented by ROS interacting enzymes (catalase, superoxide dismutase) and additional antioxidants containing medicinal plants (green tea, wheat). Recently, investigators have focused on the antioxidant potential of plant tissues. There are some reports on inhibition of in vitro metabolic activation of carcinogens by wheat sprout extracts ^{7, 8} and the antimutagenic effect of oxidative DNA damage towards benzo(a)pyrene induced mutagenicity ¹¹.

We have developed organic wheat seedling juice powder, which has been used for analysis various in-vitro and in-vivo bioactivities. In the present research, attempts have been used and approved non-enzymatic in-vitro antioxidant activity analysis on different extracts of wheat seedling juice powder (T. aestivum L.). This is a pioneer attempt towards the prevention and treatment of various ailments especially antiaging properties of this herb for better understanding of therapeutic potential of this medicinal grass which was previously not described on freeze dried wheatgrass juice powder.

A very little information has been available on the antioxidant profile of the wheatgrass and its different products such as wheatgrass powder, wheatgrass juice powder, etc. Therefore, the present study was designed to estimate the antioxidant activity of lyophilized Wheat Seedling Juice Powder (WSJP). We know that, wheatgrass is not significantly popular due to little convincing research on its different indoor or outdoor growing conditions, low characteristic features corresponding to its taste, aroma, colour, poor shelf-life and its cost.  Our main objective for future study is to improve the different indoor or outdoor growing conditions for wheat grass and also to optimize its taste, aroma and shelf-life of wheat grass juice powder for supreme recognition worldwide without modification in its originality.

MATERIALS AND METHODS:

Plant Material:

The plant material comprises of (Triticum aestivum L.), lyophilized Wheat Seedling Juice Powder (WSJP), was taken for determination of antioxidant activity. The wheat seedlings were grown in the green house and prepared in the laboratory of Akal College of Agriculture, Eternal University, Himachal Pradesh.

Which comes under the foot hills of North-Indian Himalayan region (at the altitude range of 1400m) on the banks of the famous Giri River. Plants specimens were identified and were deposited in the Herbarium, with accession number (V. Sharma-20129).

Preparation of Plant Extracts:

The 7-10 days old (500g) wheat seedlings were washed thoroughly with running tap water, followed by washing with deionised autoclaved water to remove the dust particles, possible parasites and then used for extraction of juice with the help of a manual extractor/juicer (MJ-445, Miracle Company, Taiwan). 310mL juice was collected from 500g seedlings and stored in the deep freezer for pre-freezing at -40^oC for 1h. The stored juice was then freeze dried (lyophilized) to form powder (18.3g). The freeze dried powdered sample was then stored in closed air tight bottles for further experimentation. 10g of freeze dried juice powder was extracted simultaneously with methanol and water.

Then 2mg each extract was dissolved in 10mL of methanol and water to form two separate extracts. Extracts were then filtered with Whatman’s filter paper No.1. The filtrate was collected and stored in air tight tubes under 4^oC till further analysis (Flow chart 1). Finally, different concentrations of each extract were prepared for the free radical scavenging activity.

FLOW CHART 1: EXTRACTION PROCEDURE APPLIED ON WHEAT SEEDLINGS JUICE POWDER (T.AESTIVUM L.).

Preparation of Chemical Solutions:

For antioxidant activity, chemical solution of L-ascorbic acid as a control was used and prepared by dissolving 2mg of L-ascorbic acid in 10mL of methanol. For Diphenyl-picryl-hydrazyl assay, 0.1mM stock solution of DPPH in methanol was prepared. All the chemicals, including solvents, were of analytical grade from E. Merck, India and Sigma (New Delhi, India).

Reaction Time:

In the original method a reaction time of 30min was recommended ²². There were so many different views for reaction time, given by workers all over the world. Shorter time had also been used for the reaction, such as 10min ²³. However, in view of the fact that the rate of reaction varied widely among substrates, the best practice seemed to be to follow the reaction until it had gone to completion. In present attempt of research work, 30min reaction time was followed, to check the antioxidant activity.

Absorbance Measurements - Wavelength and Instrument Used:

During evaluation of antioxidant activity for these two extracts (I, II), we followed the methodology given by Blois in 1958 ²⁴. Absorbance was measured at 517nm by using Labtronics (LT-2900) Double Beam (UV-Vis) spectrophotometer. The working wavelength of maximum absorbance, to be used for the absorbance measurements were given by various workers such as 515nm by Brand-Williams, 516nm by Schwarz, 517nm by Blois and 518nm by Leit²⁵.

Free Radical Scavenging Activity (Diphenyl-picryl-hydrazyl Assay):

The free-radical scavenging capacity of the freeze dried juice powder (2mg each) dissolved with 10mL of methanol and water extracts were evaluated with the DPPH stable radical following the methodology described by Blois ²⁴. Briefly, 0.1mM alcoholic solution of DPPH in methanol was prepared and 2mL of this solution was added to 0.3mL of different extract concentrations (1-100mg/mL) and allowed to react at room temperature. After 30min, the absorbance values were measured at 517nm against the blank, which did not contain the extract. One synthetic antioxidant, L-ascorbic acid was used as control.

The radical scavenging activity (percent(%) inhibition) was expressed as percentage of DPPH radical elimination calculated according to the following equation:

Where A_control is the absorbance of the control (L-ascorbic acid) and A_sample is the absorbance of reaction mixture (in the presence of sample). All tests were run in triplicates (n = 3), and the average values were calculated.

IC₅₀ Value:

One parameter (IC₅₀) inhibition concentration was introduced for the interpretation of the results from DPPH method. The discoloration of sample was plotted against the sample concentration in order to calculate the IC₅₀ value. It was the amount of sample necessary to decrease the absorbance of DPPH by 50%. This parameter was introduced by Brand-Williams and his colleagues ²⁶.

Phytochemical Screening:

Preliminary qualitative phytochemical screening was carried out on two different freeze dried juice powdered extracts of Wheat Seedling Juice Powder (T. aestivum L.) with the following methods to find out the exact relationship between secondary metabolites and antioxidant activity. These two methanolic and water extracts were tested for the presence of active principles such as: phenols, tannins, flavonoids, alkaloids, steroids, sterols, quinones, saponins, proteins, carbohydrate, coumarins, sugars, polysaccharides, organic acid and fatty acid. Following standard procedures were used.

Test for Phenols:

Ferric chloride Test:

Take 1mL for the solution in a test tube, add acetic acid and then a few drops of 1% ferric chloride reagent. If it turns brownish green or blue black, then it may contain phenolic composition or tannin.

Test for Tannins:

Braymer’s Test:

5mL of extract was treated with 10% alcoholic ferric chloride solution and observed for formation of blue or greenish colour solution²⁷.

Test for Flavonoids:

Ferric chloride Test:

Test solution when treated with few drops of ferric chloride solution would result in the formation of blackish red colour indicating the presence of flavonoids.

Alkaline reagent Test:

Test solution when treated with sodium hydroxide solution, shows increase in the intensity of yellow colour which would become colourless on addition of few drops of dilute hydrochloric acid, indicates the presence of flavonoids.

NaOH Test:

A small amount of extract was treated with aqueous NaOH and HCl, observed for the formation of yellow orange colour.

H₂SO₄ Test:

A fraction of extract was treated with concentrated H₂SO₄ and observed for the formation of orange colour.

Test for Alkaloids:

Hager's Test:

Test solution was treated with few drops of Hager's reagent (saturated picric acid solution). Formation of yellow precipitate would show a positive result for the presence of alkaloids.

Wagner’s Test:

A fraction of extract was treated with Wagner’s test reagent (1.27g of iodine and 2g of potassium iodide in 100 mL of water) and observed for the formation of reddish brown colour precipitate ²⁸.

Test for Steroids:

Steroids:

1ml of the extract was dissolved in 10mL of chloroform and equal volume of concentrated sulphuric acid was added by sides of the test tube. The upper layer turns red and sulphuric acid layer showed yellow with green fluorescence. This indicated the presence of steroids ²⁹.

Test for Sterols:

H₂SO₄ Test:

The fraction of extract was treated with ethanol and H₂SO₄ and observed for the formation of violet blue or green colour.

Test for Quinones:

A small amount of extract was treated with concentrated HCl and observed for the formation of yellow colour precipitate.

Test for Saponins:

Foam Test: Test solution was mixed with water and shaken and observed for the formation of froth, which is stable for 15 minutes for a positive result³⁰.

Bubble Test:

5mL aqueous extract was shaken vigorously for 2 min. The appearance of foam that persisted for at least 15 min or the forming of an emulsion when olive oil was added confirmed the presence of saponins.

Test for Proteins:

Ninhydrin Test (aqueous):

The extract was treated with aqueous ninhydrin, purple colour indicates the presence of protein.

Test for Carbohydrate:

Fehling’s Test:

Each extract was dissolved in distilled water and filtered. The filtrate was heated with 5mL of equal volumes of Fehling's solution A and B. Formation of a red precipitate of cuprous oxide was an indication of the presence of reducing sugars.

Test for Coumarin:

1mL of ethanol extract was stirred and placed separately in a test tube. The result of the solution was seen in the following experiments.

Opened loop - closed loop response:

In the test tube, add 2 drops of 1% sodium hydroxide solution, and heat in boiling water for 3 min to get clear solution. Also add 4 drops of 2% hydrochloric acid to the solution. If the solution becomes cloudy, that means it contains coumarins ^{31, 32}.

Test for Sugar and Polysaccharides:

The extracts were taken to do the following experiments separately.

Fehling Reaction:

4mL of the aqueous extract was placed in test tube and 5 ml mixture of equal volumes of Fehling’s solutions A and B was added and boiled in a water bath for 5 min. It resulted in brick- red cuprous oxide precipitate that contains sugar. The solution will remove sediment filtration. To the filtrate, add 1 ml and 10% hydrochloric acid in boiling water bath for home heating hydrolysis for few minutes. Let it cool. Then drop 10% sodium hydroxide solution adjusted to neutral pH, and repeats the Fehling’s reaction. If still produces the brick-red cuprous oxide precipitates, then it contains polysaccharides.

Iodine Test:

1mL of the aqueous extract was taken in a test tube and 1 drop mixture of iodine solution was added. The change in colour denotes the presence of polysaccharide.

Test for Organic acid:

Take the water extract and do the following experiment separately.

pH Test Strip Reaction:

If the solution is put in a wide range of pH test paper, and found to be acidic, then it may contain organic acids or phenolics.

Fatty Acids:

0.5mL of extract was mixed with 5mL of ether. These extract was allow it for evaporation on filter paper and dried the filter paper. The appearance of transparence on filter paper indicates the presence of fatty acids ³³.

RESULTS AND DISCUSSION:

The wheat seedling juice powder is believed to have manifold pharmacological properties in addition to its important nutraceutical uses. The present work includes screening of antioxidant properties of two different extracts of lyophilized wheat seedling juice powder along with their preliminary phytochemical evaluations. It is well known fact that, a diet rich in vegetables and fruits provides protection against cardiovascular and other chronic diseases originating from oxidative stress. Therefore, suitable antioxidant therapies to control oxidative damage have already attracted the worldwide attention in recent years.

It has been already reported that phytochemicals used to study in cell culture system and animal models have provided a wealth of information on the mechanism by which such nutraceuticals show their beneficial effects ^{34, 35}. The preliminary phytochemical studies indicated the presence or absence of phenols, flavonoids, tannins, alkaloids, steroids, sterols, saponins, coumarines, sugars, carbohydrate, polysaccharides, proteins, organic acids and fatty acid in one or both the extracts (Table 1).

TABLE 1: PRELIMINARY QUALITATIVE PHYTOCHEMICAL SCREENING OF TWO DIFFERENT FREEZE DRIED JUICE POWDERED EXTRACTS OF T. AESTIVUM (L.).

Like other medicinal plants, it is hypothesized that different extracts of wheat seedling juice powder showed antioxidative effect, which can be attributed to the presence of phenolic compounds such as alkaloids, flavonoids, and tannins in the seedlings of T. aestivum L. These phenolic components possess many hydroxyl groups including o-dihydroxy group which have very strong radical scavenging effect and antioxidant power. In the DPPH assay, the antioxidants were able to reduce the stable radical DPPH to the yellow colored 1, 1-diphenyl-1, 2-picryl hydrazine.

The molecule of 1, 1-diphenyl-1, 2-picryl hydrazine is characterised as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole. The delocalisation also gives rise to the deep violet colour, characterised by an absorption band in methanol solution at 517 nm.

The scavenging effect of plant extracts and standards on the DPPH radical decreases in descending order: L-ascorbic acid >Methanolic extract (I) > Water extract (II). These were 97.3%, 58.6% and 35.1% at concentration of 100mg/mL, respectively (Table 2).

TABLE 2: MEASUREMENT OF ABSORBANCE AT 517NM AND INHIBITION EFFECT (%) OF DIFFERENT EXTRACTS WITH STANDARD L-ASCORBIC ACID FROM CONCENTRATION RANGE OF 1-100 mg/mL.

(Given values are average of triplicate i.e. n=3; DPPH= 1.125).

The potential of L-ascorbic acid to scavenge DPPH radical became almost stable after 70 or 80 mg/mL (Fig.1).

FIG.1: FREE RADICAL SCAVENGING ACTIVITY OF DIFFERENT EXTRACTS OF WHEAT SEEDLING JUICE POWDER (T. AESTIVUM L.) AND L-ASCORBIC ACID (CONTROL) WAS SPECTROPHOTOMETRICALLY MEASURED AT 517 nm USING DPPH ASSAY.

(Given values are average of triplicate i.e. n=3; DPPH= 1.125).

The experimental data which obtained from all two extracts, it is clear that methanolic extract (I) showed maximum percentage of inhibitory effect i.e. 58.6% at 100mg/mL of concentration. Among these two extracts and standard tested for in vitro antioxidant activity, using the DPPH method, the L-ascorbic acid, crude methanolic (I) and water (II) extracts of lyophilized wheatgrass juice powder (T. aestivum L.) showed antioxidant activity with IC₅₀ values of 19.2, 81.4 and 131.3 respectively at 1-100 mg/mL concentration (Tables 3-5). However, none of the extracts were found to be more active than the standard i.e. (L-ascorbic acid with IC₅₀ value of 19.2 at 1-100 mg/mL) since their IC₅₀ value found to be higher. Free radical scavenging activity of methanolic (I) and water (II) extracts was confirmed in the present investigation (Fig. 2-4).

TABLE 3: IN VITRO DPPH ANTIOXIDANT ACTIVITY SHOWING IC₅₀ VALUES OF STANDARD L-ASCORBIC ACID.

 TABLE 4: IN VITRO DPPH ANTIOXIDANT ACTIVITY SHOWING IC50 VALUES FOR METHANOLIC EXTRACT OF WHEAT SEEDLING JUICE POWDER (T. AESTIVUM L.).

 TABLE 5: IN VITRO DPPH ANTIOXIDANT ACTIVITY SHOWING IC₅₀ VALUES FOR WATER EXTRACT OF WHEAT SEEDLING JUICE POWDER (T. AESTIVUM L.).

FIG. 2: IN VITRO DPPH ANTIOXIDANT ACTIVITY SHOWING GRAPH FOR IC₅₀ VALUES OF STANDARD L-ASCORBIC ACID.

FIG. 3: IN VITRO DPPH ANTIOXIDANT ACTIVITY SHOWING GRAPH OF IC₅₀ VALUES FOR METHANOLIC EXTRACT OF WHEAT SEEDLING JUICE POWDER (T. AESTIVUM L.).

FIG.4: IN VITRO DPPH ANTIOXIDANT ACTIVITY SHOWING GRAPH OF IC50 VALUES FOR WATER EXTRACT OF WHEAT SEEDLING JUICE POWDER (T. AESTIVUM L.).

The methanolic and water extracts have been chosen because of its expected flavonoid contents that were reported to have antioxidant activity in many other plants ³⁶. The activity of phenolics having antioxidant activity further confirmed this view ³⁷. The phytochemical analysis of wheatgrass showed the presence of tannins, flavonoids, saponins, and sterols. The antioxidant activity of the phenolic, tannins, and flavonoid compounds are attributed to its redox properties which can act as reducing agents, hydrogen donators, and singlet oxygen quenchers³⁸. Polyphenolic shaving hydroxyl groups are very important plant constituents which can protect body from oxidative stress ³⁹.

The major clinical utility of wheat grass in diseased conditions might be due to the presence of biologically active compounds and minerals in it and due to its antioxidant potential which is derived from its high content of bioflavonoids. Furthermore, few indole compounds present in it might be also responsible for its therapeutic potential. The presence of chlorophyll, which is almost chemically identical to haemoglobin, in wheat grass makes it more useful in various clinical conditions involving haemoglobin deficiency and other chronic disorders ⁴⁰.

Phytochemical studies are in progress to evaluate the therapeutic efficacy of wheat grass, grown through the concept of organic farming and free from pesticides, herbicides, insecticides, heavy metals and other harmful chemicals and toxins, in various disease conditions and the patients are being benefitted by the multitude potential of wheat grass.

Considering the facts that very little scientific and clinical studies have been done on the use of wheat seedlings in various diseases, efforts are needed to conduct extensive studies on the wheat seedlings both in experimental models and human subjects to develop wheat seedling therapy. Since the production of organic wheat grass is cheap and it does not require the involvement of pharmaceutical industry, wheat grass therapy could be easily developed as an alternative or complimentary therapy for the benefit of large number of people suffering from chronic diseases in developing countries.

CONCLUSION: In the present research, attempts have been made to provide the scientifically and clinically approved studies on the freeze dried wheatgrass juice powder (T. Aestivum L.) for better understanding of therapeutic potential of this medicinal grass. The present study clearly concluded that methanol extract of wheatgrass possesses the ability to control oxidative damage and acts as antioxidant. Its free radical scavenging property has potential to prevent many complications. Our current investigation supports the traditional use of wheatgrass in the treatment of various diseases.

Wheatgrass naturally rich in essential vitamins, minerals, enzymes, amino acids, dietary fibres and world’s best antioxidant “chlorophyll” may be useful for all age groups. Thus wheatgrass therapy may serve as a preventive and curative measure for such health problems which may lead to serious diseases like diabetes mellitus, anaemia, ulcers, arthritis, cancer, thalassemia and others.Thus it is clear that polyphenolic antioxidants in wheatgrass juice powder play an important role as bioactive principles. However, polyphenolic constituents present in these extracts which are responsible for this activity need to be investigated in detail.           

ACKNOWLEDGEMENT: The authors are grateful to His Holiness Baba Iqbal Singh Ji President, The Kalgidhar Trust & Founder Chancellor of Eternal University (H.P.), Hon’ble Vice Chancellor, Akal School of Biotechnology, Eternal University, Baru Sahib (Himachal Pradesh) India; Director, IHBT (CSIR), Palampur (Himachal Pradesh) India and Head, Department of Botany, Punjabi University, Patiala (Punjab) India for providing chemicals, necessary facilities and logistic support for carrying out the study.

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    How to cite this article:

    Dhaliwal HS, Sharma N, Bano A, Kumar S and Sharma V: Non-Enzymatic Assay Based in-vitro Antioxidant Activity and Phytochemical Screening of Freeze Dried Wheat (Triticum Aestivum L.) Seedlings Juice Powder: Nature’s Finest Medicine-Part-II. Int J Pharm Sci Res 2015; 6(9): 4036-46. doi: 10.13040/IJPSR.0975-8232.6(9).4036-46.

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