All the chemicals used were of analytical grade and obtained from E. Merck Limited, India and Hi-Media Laboratories, Mumbai, India.

Fresh aerial parts of U. dioica were collected from Sankari, Uttarakhand, India. The plant was authenticated by Dr. AKS Rawat, NBRI, Lucknow. A voucher specimen (262543) has been submitted in institute’s repository.

The macroscopic evaluations involve study of morphological characters and organoleptic studies like colour, odour, taste, texture, etc.

In microscopic evaluation, studies were conducted qualitatively. All the microscopic evaluations were performed under Olympus CX-31 microscope. Photomicrographs were taken using Olympus digital camera Model No. E-420 [20].

Physicochemical and Phytochemical studies like extractive values, total ash, acid insoluble ash, total sugar, starch, tannin, and phenols were calculated from the shade-dried and powdered (60 mesh) plant material [21-24].

The dried plant material was powdered using electric grinder. Extraction was performed by soxhlation process in two steps. Firstly the powdered plant material was treated under soxhlet assembly using 250 mL of 98% petroleum ether for 9 hours. This is followed by 6 hours soxhlation of plant material by using 250 mL of 50% hydro-alcohol. The final extract was passed through Whatman No. 1 filter paper. The filtrate obtained was concentrated under vacuum in a rotary evaporator at 40˚C and stored at 4˚C for further use. The crude extract was obtained by dissolving a known amount of dry extract in 98% methanol to obtain a stock solution of 1000 μg/ml. The stock solutions were serially diluted with methanol to obtain lower dilutions of 20, 40, 60, 80, 100, 150 and 200 μg/ml.

Antioxidant activity of the plant extracts and standard was assessed on the basis of the radical scavenging effect of the stable DPPH free radical. Diluted working solutions of the test extracts were prepared in methanol. Ascorbic acid was used as the standard in solutions ranging from 1 to 50 μg/ml. 0.002% DPPH solution in methanol was prepared, further 2 ml of this solution was mixed with 2 ml of sample solutions (ranging from 50 µg/ml to 250 µg/ml), however, the standard solution to be tested separately. These solution mixtures were kept in dark for 30 min and optical density was measured at 517 nm using double beam spectrophotometer (make: Thermo) against methanol. Blank used was 2 ml of methanol with 2 ml of DPPH solution (0.002%). The optical density was recorded and percentage of inhibition was calculated using the formula given below [25]:

% of inhibition of DPPH activity = (A – B/A) × 100where, A is optical density of the blank and B is optical density of the sample.

Reagents used were from Merk (Germany) and standard ferulic acid was procured from Sigma-Aldrich (Steinheim).

Chromatography was performed on Merk HPTLC precoated silica gel 60 GF₂₅₄ (20 × 20 cm) plates. Methanolic solutions of samples and standard compound ferulic acid of known concentrations were applied to the layers as 6 mm-wide bands positioned 15 mm from the bottom and 15 mm from side of the plate, using Camag Linomat V automated TLC applicator with nitrogen flow providing a delivery speed of 150 nl/s from application syringe. These conditions were kept constant throughout the analysis of samples.

Air dried (45˚C - 55˚C) powdered sample of U. dioica (2.0 g) in triplicate were extracted separately with 3 × 10 ml 50% hydro-alcoholic solution. Extracts were concentrated under vacuum and redissolved in methanol, filtered and finally made up to 100 ml with methanol prior to HPTLC analysis.

Following sample application, layers were developed in a Camag twin trough glass chamber pre-saturated with mobile phase of toluene: ethyl acetate: formic acid (5:4:1) till proper separation of bands up to 8 cm height. After development, layers were dried with an air dryer and ferulic acid was simultaneously quantified using Camag TLC scanner model 3 equipped with Camag Wincats IV software. Following scan conditions were applied:

Slit width: 5 mm × 0.45 mm; wavelength: 325 nm; and absorption-reflection mode.

In order to prepare calibration curves, stock solution of ferulic acid (1 mg/ml) was prepared and various volumes of these solutions were analyzed through HPTLC, calibration curves of peak area vs. concentration were also prepared.

The organoleptic features of the leaves indicate dark green colour on upper surface and light green colour on lower surface. The powder of leaves appeared green in colour, coarse in texture, slightly aromatic and unpleasant in odour with tingling and burning taste. The stem was brown in colour with bitter taste and smooth texture.

The morphological characters of leaves were observed as deciduous, erect-ascending, annual or perennial, herbaceous, monoecious or dioecious, pubescent, stinging hairs copious. Leaves opposite, 3 to 15 cm long, leaf blades are elliptic, ovate, lanceolate or oblong, leaf bases may be sub-cordate, truncate or acuminate, leaf margin usually crenate or serrate, glabrous except sparsely pubescent below; leaf blades 3 - 7 nerved, stipule lateral, free. Flower mono or dioecious, axillary, uni or bisexual, in cymes which are combined into a paniculi form inflorescence, tapel segment 4, oblong obtuse hairy (Figure 1).

Parameters such as moisture content, extractive values (water, alcohol and ether soluble), total ash and acid insoluble ash values, total sugar, starch, and phenolics were determined. Results are shown in Figure 7.

In-vitro antioxidant study of U. dioica was performed using hydro-alcoholic extract. Ascorbic acid (AA) was used as standard. In this study U. dioica extract showed IC₅₀ value of 88.33 ± 2.88 µg/ml. Standard ascorbic acid showed IC₅₀ value of 2.8 ± 0.62 µg/ml (Figure 8).

Quantitative HPTLC analysis of ferulic acid in hydroalcoholic extract of U. dioica has been performed. A

densitogram and banding pattern obtained from extract shows ferulic acid 0.73%. During the analysis ferulic acid showed R_f 0.61 ± 0.01; and r² of 0.98 (Figures 9-11).