Chemicals
Gallic acid (GA), ascorbic acid (AA), DPPH and quercetin were purchased from Hi-Media Lab (Mumbai, India). FC reagent and aluminum chloride (AlCl3) were purchased from Thermo Fisher Scientific India Pvt. Ltd. (Mumbai, India). Reference standard Diclofenac was obtained from Lomus Pharmaceuticals Pvt. Ltd. (Kathmandu, Nepal). All other chemicals were of standard analytical grade.
Plant materials
The barley seeds were procured from the local market and were sown in soil from local nursery with daily watering. The Barley Grass were harvested on 7th day of sowing at the month of July. The samples were authenticated by Ganga Datt Bhatt, Research Officer, National Herbarium and Plant Laboratories (NHPL) (Godawari, Lalitpur, Nepal) Voucher number:217. The voucher specimen of this material has been deposited in National Herbarium and Plant Laboratories (NHPL) (Godawari, Lalitpur, Nepal).
Preparation of the extracts
The harvested BG were washed well using distilled water and shade dried for 21 days before grinding to fine powder. Three hundred grams of fine powder was subjected to successive maceration starting from hexane to ethyl acetate to methanol, 500 ml each for 48 h at room temperature (27 ± 1 °C). The extracts were filtered using a Buckner funnel and Whatman No. 1 filter paper. These extracts were dried in a rotary evaporator under reduced pressure until dryness and stored at 4 °C, protected from light and humidity for further analysis.
Determination of Total phenolic content
The total phenolic content (TPC) of the extracts was estimated by Folin-Ciocalteu reagent (FCR) method [20] with slight modifications. Briefly, 1 ml of various extracts (1 mg/ml) was mixed with FCR (5 ml, 1:10 v/v DW) and aq. sodium carbonate (4 ml, 7%) solution. The mixture was then incubated for 30 min at 40 °C in a water bath before measuring the absorbance at 760 nm using Microprocessor UV-Vis spectrophotometer-2371 (Electronics India, Himachal Pradesh, India). The phenolic contents were calculated using a standard curve for gallic acid (GA) (10-200 μg/ml), and the result was expressed as mg GAE per gram dry weight of fraction (mg GAE/g). All measurements were performed in triplicates.
Determination of Total flavonoid content
The total flavonoid content (TFC) was determined by AlCl3 coulometric method [21]. An aliquot of 1 ml of various extracts in methanol was added to 10 ml volumetric flask containing 4 ml of distilled water. At the zero-time, 0.3 ml, 5% sodium nitrite was added to the flask. After 5 min, 3 ml of 10% AlCl3 was added to the flask. At 6 min, 2 ml of 1 M sodium hydroxide was added to the mixture. Immediately, the total volume of the mixture was made up to 10 ml by the addition of 2.4 ml distilled water and mixed thoroughly. Absorbance of the pink colored mixture was determined at 510 nm against a blank containing using Microprocessor UV-Vis spectrophotometer-2371 (Electronics India, Himachal Pradesh, India). The flavonoid contents were calculated using a calibration curve prepared for Quercetin standards (10 to 100 μg/ml) and the result was expressed as mg of quercetin equivalent/g of extract (mg QE/g of extract).
Determination of anti-oxidant activity
The DPPH scavenging activity of different fractions was evaluated according to the method of Brand-Williams et al. [22] 1 mL of 0.1 mM DPPH solution in methanol was mixed with 1 mL of each extracts at varying concentrations (5, 10, 15, 20, 25 μg/ml). The corresponding blank sample was prepared, and ascorbic acid (AA) was used as reference standard. Mixture of 1 mL extract and 1 mL DPPH solution was used as control. The mixture was shaken well and incubated for 30 min in the dark. The reaction was carried out in triplicate, and the decrease in absorbance was measured at 517 nm after incubation using a using Microprocessor UV-Vis spectrophotometer-2371 (Electronics India, Himachal Pradesh, India). The scavenging activity was expressed as IC50 (μg/mL). The % scavenging was calculated using the formula:
$$ \%\mathrm{Scavenging}=\left[\left({\mathrm{A}}_0-\mathrm{A}1\right)/{\mathrm{A}}_0\right]\times \kern0.37em 100 $$
Where, A0 = absorbance of the control solution.
A1 = absorbance of extract/standard.
Determination of RBC membrane stabilization activity
RBC membrane stabilization activity of three different extracts of BG was evaluated by using in vitro human red blood cell stability method. The membrane stabilizing activity of the sample was assessed according to the method described by Shinde et al [23] with slight modifications.
The assay mixture contained 1 ml phosphate buffer [PH 7.4, 0.15 M], 2 ml hypo saline [0.36%], 0.5 ml HRBC suspension [10% v/v] with 0.5 ml of plant extracts and standard drug diclofenac sodium of various concentrations (10, 20, 40, 80, 100 μg/ml). The control sample consisted of 0.5 mL of RBCs mixed with hypotonic-buffered saline alone. The mixture was incubated at 37 °C for 30 min and centrifuged at 3000 RCF. The hemoglobin content in the suspension was estimated using Microprocessor UV-Vis spectrophotometer-2371 (Electronics India, Himachal Pradesh, India).
$$ \%\mathrm{Protection}=1-\left[\mathrm{OD}\ \mathrm{of}\ \mathrm{Test}/\mathrm{OD}\ \mathrm{of}\ \mathrm{Control}\right]\ \mathrm{X}\ 100 $$
Determination of toxicity
The toxic activity of the plant was evaluated using Brine shrimp lethality bioassay (BSLA) method [8] where 6 graded doses (viz 1600 μg/mL, 800 μg/mL, 400 μg/mL, 200 μg/mL, 100 μg/mL, and 50 μg/mL) were used. Brine shrimps (Artemia salina Leach) nauplii were used as test organisms. For hatching, eggs were kept in artificial sea salt with a constant oxygen supply for 48 h. The mature nauplii were then used in the experiment. DMSO was used as a solvent and also as a negative control. Vincristine sulfate was used as a reference standard in this case. The numbers of survivors were counted after 24 h. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation. The larvae did not receive food. To ensure that the mortality observed in the bioassay could be attributed to bioactive compounds and not to starvation; we compared the dead larvae in each treatment to the dead larvae in the control.
The median lethal concentration (LC50) of the test samples were calculated using the Probit analysis method described by Finney [24], as the measure of toxicity of the plant extract.
$$ \mathrm{Mortality}\%=\left(\mathrm{No}.\mathrm{of}\ \mathrm{dead}\ \mathrm{larvae}/\mathrm{Total}\ \mathrm{no}.\mathrm{of}\ \mathrm{larvae}\right)\times 100. $$
Gas chromatography-mass spectroscopy analysis
GC-MS analysis was performed at Nepal Academy of Science & Technology (Khumaltar, Kathmandu, Nepal). For GC-MS analysis of plant extract, GC-MS QP2010 (Shimadzu, Kyoto, Japan) equipped with RTx-5MS fused silica capillary column of 30 m length X 0.25 mm diameter X 0.25 μm film thickness. Helium (> 99.99% purity) with 36.2 cm/sec linear velocity was employed as carrier gas. The system was programmed with 3.9 ml/min of total flow rate, 0.95 ml/min of column flow and 3.0 ml/min purge flow. The volume of injected sample was 1 μl. The injector was set in spitless mode having 280 °C of temperature. Oven temperature started from 100 °C and increased to 250 °C at 15 °C/min with holding time of 1 min, which afterwards increased to 280 °C at 30 °C/min with holding time of 1 min and again increased from 280 °C to 300 °C at 15 °C/min with holding time of 11 min.
The ion source temperature and interface temperature were set to 200 °C and 280 °C respectively with solvent cut time of 3.5 min. Total run time was 20 min with mass range scan of 40 to 500 m/z. Identification of compounds was performed by comparing their mass spectra with data from NIST08 mass spectral library.
Statistical analysis
Each sample analysis was performed in triplicate. All results presented are means (±SEM) of at least three independent experiments. Statistical analysis, ANOVA with a statistical significance level set at p < 0.05 with post-hoc Tukey procedure was carried out with SPSS 16 for Windows. Correlations between the total phenolic contents, flavonoid contents and antioxidant capacities were determined using the Pearson correlation.