2.1. Materials

HPLC and analytical grade chemicals were purchased from the commercial supplier and used in the study.

2.2. Procurement of Plant Material

The dried plant materials was purchased from verified commercial suppliers. The samples were visually inspected and validated for their morphological traits, then powdered using a mechanical blender at very low speed. The powdered samples were stored in airtight containers and kept in a dry place away from sunlight.

2.3. Preparation of Prasaranyadi Kashayam

The dried powder of plants was mixed in a previously described proportion [Table 1] to obtain the dried powder of Prasaranyadi Kashyam and the aqueous decoction (Kashyam/Kwath) was prepared as described by Nisteshwar and Vidyanath [5]. The prepared kwath solution was concentrated under vacuum to obtain a semisolid mass which was further dried at 40?C to obtain a powdered formulation and stored in airtight containers in a dry place away from sunlight until further use.

2.4. Physicochemical Evaluation

Physicochemical parameters such as total ash content, water-soluble ash, acid insoluble ash, loss on drying of the extract, pH was carried out on the dried extract according to the standard protocols [6,7].

2.5. Total Phenolic Contents

Total phenolic content was spectrophotometrically estimated using Folin-Ciocalteu reagent assays at 750 nm [8]. Gallic acid was used as a standard and the total phenolic contents were expressed as Gallic acid equivalents.

2.6. DPPH Radical Scavenging Assay

DPPH radical scavenging assay was performed as per the method described by Molole et al. [9]. In brief, 0.4 mL of test sample (PK) and gallic acid (50–400 μg/mL) were mixed with 2.6 mL of a methanolic solution of DPPH (0.1 mM). An equal amount of methanol (0.4 mL) and solution (2.6 mL) was used as a blank. The samples were all produced in triplicate, vortexed for one minute, and then incubated at 37°C in the dark for thirty minutes. The decrease in absorbance of each sample was measured against methanol as a blank on UV-Visible spectrophotometer at 517 nm.

All the experiments were performed in triplicate, and the results were expressed as mean ± SD.

2.7. HPTLC Method

The dried aqueous extract of Prasaranyadi kashayam was dissolved in water and subjected to HPTLC analysis. 10 uL of the extract was spotted on HPTLC silica gel 60F 254 (Merck) plate as bands of length 6 mm. The plates were developed using toluene, ethyl acetate, methanol, and formic acid (16, 14, 1, and 4) in the CAMAG twin-trough glass chamber previously saturated with the solvent for 30 min. After development, the plates were dried in an oven at 60°C and scanned at 254 nm and 366 nm.

3.1. Physicochemical Evaluation and Total Phenolic Contents

The physiochemical parameters give the primary quality validation data; for example, the ash value of herbal drugs represents the total mineral content and helps in the identification of possible adulteration with inorganic materials. The loss on drying refers to moisture content, which has to be foremost in avoiding microbial growth, thus conferring stability to the drug during storage. Phenolic content is a key parameter for estimating antioxidant capacity and demonstrating the therapeutic potential of the herbal drug. These are the parameters whose exact measurements determine the quality, purity, and efficacy of herbal medicines [7]. Physicochemical parameters, such as total ash content, water-soluble ash, acid-insoluble ash, loss on drying, pH, and total phenolic content, were evaluated for PK and the results have been summarized in Table 2.

Table 2: Physicochemical analysis of Prasaranyadi Kashayam.

3.2. DPPH Radical Scavenging Assay

PK indicated good antioxidant activity in terms of percentage inhibition (72.30%) of DPPH radical [Figure 1].

Figure 1: DPPH Free Radical Scavenging Effect of Prasaranyadi Kashayam. [Click here to view]

3.3. HPTLC Figure Printing

HPTLC fingerprinting is a preferred analytical technique for the quality control and authentication of herbal drugs. It does not provide brief details about the chemical constituents present and helps in the identification and comparison of complex herbal formulations. Which, in turn, helps to ensure the consistency, and purity of herbal products. The HPTLC finger printing profile was developed in toluene, ethyl acetate, methanol, and formic acid (16, 14, 1, and 4) and the HPTLC chromatogram showed a total eight bands and corresponding peaks were recorded at respective Rf values as depicted in Figure 2.

Figure 2: HPTLC fingerprint profile of tannins in the leaf extract of Prasaranyadi Kashayam. [Click here to view]