Comparative Analysis of Phenolic and Flavonoid Content in Ashwagandharishta, Dashmularishta and Maharasnadi Kadha and their Anti-Inflammatory Activity

Inflammation represents a complex cascade of physiological and biochemical responses that the human body initiates as a protective mechanism against a diverse range of endogenous and exogenous insults. Based on its temporal progression, inflammatory responses are broadly categorized into acute and chronic forms. It constitutes an integral component of the immune-mediated healing process, facilitating the elimination of foreign agents — whether biological, chemical, or physical in nature — from host tissues. The cardinal clinical manifestations associated with inflammatory conditions include localized pain, edema, erythema, elevated tissue temperature, and restricted mobility. ¹ Accumulating epidemiological evidence has consistently demonstrated an inverse relationship between the regular dietary intake of polyphenol- and flavonoid-rich foods — including fruits, vegetables, and herbal preparations — and the prevalence of chronic non-communicable disorders such as malignancies, cardiovascular complications, and persistent inflammatory conditions. Commercially available polyherbal formulations have garnered considerable attention as therapeutic agents owing to their substantial content of bioactive secondary metabolites, including polyphenols, flavonoids, and antioxidant vitamins, which collectively contribute to their anti-inflammatory efficacy. ² Contemporary pharmaceutical research continues to grapple with the fundamental challenge of developing therapeutic agents that simultaneously satisfy the criteria of safety, economic accessibility, and clinical effectiveness. The limitations inherent to synthetic pharmacotherapy — including adverse effect profiles, prohibitive costs, and suboptimal therapeutic outcomes — have compelled researchers to reconsider and revisit traditional medicinal frameworks. A particularly relevant consideration is that the majority of chronic pathological conditions are polygenic in origin, thereby necessitating a multi-target pharmacological strategy rather than a single-molecule approach. This paradigm has directed scientific attention toward Ayurveda, an ancient holistic healing tradition with origins in the Indian subcontinent dating back approximately six thousand years. ³ A fundamental tenet of Ayurveda therapeutics is that no single herb or isolated compound can adequately restore physiological equilibrium or correct "Doshic" imbalances. Consequently, Ayurveda practitioners have historically prescribed synergistic combinations of botanicals, representing what may arguably be regarded as one of the earliest conceptualizations of combinatorial and multi-targeted therapy. Within such formulations, individual phyto-constituents may function to potentiate the pharmacological activity of co-present compounds, enhance their systemic bioavailability, or attenuate associated toxicity. A well-documented illustration of this principle is Ashwagandha (Withania somnifera), which exerts significant analgesic activity by modulating nociceptive signaling within the nervous system.⁴ Its potent anti-arthritic, antipyretic, and adapt genic properties have been extensively validated in contemporary literature, and classical Ayurveda scholars have further attributed to it the capacity to restore physiological vigor, alleviate rheumatic manifestations, and counteract physical debilitation.⁵ Against this background, the polyherbal Ayurvedic formulations — Ashwagandharishta, Dashmularishta, and Maharasnadi Kadha — represent therapeutically significant preparations with a well-established ethno pharmacological basis for use in inflammatory disorders. All three formulations are known to harbor appreciable quantities of phenolic and flavonoid compounds, which are recognized as principal contributors to their biological activity.⁶Accordingly, the present study was designed to comparatively assess the total phenolic and flavonoid content alongside the in vitro anti-inflammatory potential of these three preparations. Phenolic quantification was accomplished through the Folin–Ciocalteu colorimetric assay, flavonoid estimation was performed via the aluminum chloride spectrophotometric method, and anti-inflammatory activity was evaluated through inhibition of heat-induced protein denaturation and erythrocyte hemolysis assays.⁷

MATERIALS AND METHODS:

2.1 MATERIALS:

All three polyherbal Ayurvedic formulations under investigation, namely Ashwagandharishta, Dashmularishta, and Maharasnadi Kadha, were procured from a locally authorized Ayurvedic pharmaceutical outlet. All additional reagents and chemicals utilized throughout the experimental procedures were obtained from the institutional laboratory.

2.2 Quantification of Total Phenolic Content:

Total phenolic content (TPC) was estimated employing the Folin–Ciocalteu colorimetric method, with Gallic acid serving as the reference standard.

2.2.1 Preparation of Primary Standard Stock Solution;

A primary stock solution of Gallic acid at a concentration of 1 mg/mL (1000 µg/mL) was prepared by accurately dissolving 100 mg of Gallic acid in 10 mL of 80% methanol, followed by volumetric adjustment to 100 mL using distilled water in a calibrated volumetric flask.⁸

2.2.2 Construction of Calibration Curve:

Working standard solutions of varying concentrations — 50, 100, 150, 250, and 500 µg/mL — were prepared through serial dilution of the primary stock solution with distilled water. Optical absorbance of each solution was recorded at λ max 725 nm against a reagent blank, and a standard calibration curve was subsequently constructed.⁹

2.2.3 Sample Solution Preparation:

An accurately weighed quantity of 100 mg of dried extract from each formulation was dissolved in 10 mL of 80% methanol and subsequently diluted to 100 mL with the same solvent to yield a working concentration of 1 mg/ml.¹⁰

2.2.4 Analytical Procedure:

An aliquot of 1 mL of each methanolic sample solution was transferred into a 25 mL volumetric flask containing 9 mL of distilled water. Subsequently, 1 mL of Folin–Ciocalteu phenol reagent was introduced and the contents were thoroughly agitated. Following a 5-minute reaction interval, 10 mL of 7% aqueous sodium carbonate solution was incorporated into the mixture. The total volume was then adjusted to 25 mL with distilled water, and the resulting solution was allowed to incubate at ambient temperature for 90 minutes. Absorbance measurements were recorded at 765 nm against a reagent blank. All analyses were conducted in triplicate, and the mean absorbance values were applied to the calibration curve for concentration determination. Results were expressed as milligrams of Gallic acid equivalents per gram of dry extract (mg GAE/g).¹¹

TPC was computed using the following expression:

A = (C × V) / m

Where, A = total phenolic content (mg GAE/g dry extract); C = Gallic acid concentration (mg/mL) derived from the calibration curve; V = total volume of extract (mL); m = mass of dried extract (g).¹²

2.3 Quantification of Total Flavonoid Content:

Total flavonoid content (TFC) was determined spectrophotometrically utilizing the aluminum chloride complexation colorimetric method, with quercetin as the reference standard.

2.3.1 Preparation of Primary Standard Stock Solution:

A stock solution of quercetin at 1 mg/mL (1000 µg/mL) was prepared by dissolving 10 mg of quercetin in 2 mL of 80% methanol and subsequently bringing the volume to 10 mL with the same solvent in a calibrated volumetric flask.¹³

2.3.2 Construction of Calibration Curve:

Calibration standard solutions at concentrations of 12.5, 25, 50, 75, 100, 125, and 150 µg/mL were obtained by appropriate dilution of the stock solution with 80% methanol. Absorbance of each solution was measured at λ max 415 nm against a reagent blank to establish the standard curve.¹⁴

2.3.3 Sample Solution Preparation:

A quantity of 100 mg of dried extract was dissolved in 10 mL of 80% ethanol and diluted to a final volume of 100 mL with the same solvent to achieve a concentration of 1 mg/ml¹⁵.

2.3.4 Analytical Procedure:

One milliliter of each ethanolic sample solution was pipetted into a 10 mL volumetric flask containing 3 mL of 95% ethanol (v/v). To this, 0.2 mL of 10% aluminum chloride solution was added, and after a 5-minute interval, 0.2 mL of 1M potassium acetate was incorporated. The total volume was made up to 10 mL with distilled water, and the mixture was incubated at room temperature for 30 minutes. Absorbance was subsequently recorded at 415 nm. In the blank preparation, aluminum chloride was substituted with an equivalent volume of distilled water. Triplicate analyses were performed, and mean values were utilized for concentration interpolation from the calibration curve. Results were expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g).¹⁶

TFC was computed using the following expression:

A = (C × V) / m

Where, A = total flavonoid content (mg QE/g dry extract); C = quercetin concentration (mg/mL) from the calibration curve; V = total extract volume (mL); m = mass of dried extract (g).

2.4 Evaluation of In Vitro Anti-Inflammatory Activity:

Anti-inflammatory potential was assessed through two complementary in vitro experimental models.

2.4.1 Inhibition of Heat-Induced Protein Denaturation:

The reaction mixture, with a total volume of 5 mL, was constituted by combining 0.2 mL of 1% bovine serum albumin solution, 4.78 mL of phosphate-buffered saline (pH 6.4), and 0.02 mL of the respective test formulation extract. The mixture was thoroughly blended and subjected to pre-incubation at 37°C for 15 minutes in a thermostatically regulated water bath, followed by thermal challenge at 70°C for 5 minutes. Upon cooling to ambient temperature, the resulting turbidity was quantified spectrophotometrically at 660 nm. Phosphate buffer saline without extract served as the experimental control.¹⁷

Percentage inhibition of protein denaturation was determined using the formula:

% Inhibition = 100 × (1 − A₂/A₁)

Where, A₁ = absorbance of control; A₂ = absorbance of test sample.

2.4.2 Inhibition of Heat-Induced Erythrocyte Hemolysis:

A reaction mixture was prepared by combining 0.05 mL of erythrocyte suspension with 0.05 mL of the test extract in 2.95 mL of phosphate buffer (pH 7.4). The resulting mixture was incubated at 54°C for 20 minutes under continuous agitation in a shaking water bath. Following incubation, the mixture was centrifuged at 2500 rpm for 3 minutes, and the absorbance of the clarified supernatant was recorded at 540 nm using a UV-Visible spectrophotometer. Phosphate buffer alone served as the experimental control.¹⁸

The degree of hemolytic inhibition was calculated as per the method of O. koli, using the equation:

% Inhibition = 100 × (1 − A₂/A₁)

Where, A₁ = absorbance of control; A₂ = absorbance of test sample.

3. RESULT AND DISCUSSION:

3.1 Determination of total phenolic content:

The total phenolic content was determined by using folin-ciocalteau assay-

Table No. 1: Absorbance table of standard gallic acid.

Table No. 2: The Percentage of Total Phenolic Content.

Fig. No. 1:  Standard curve graph.

Table No. 3: Total Phenolic Content (TPC) of Ashwagandharishta, Dashmularishta, and Maharasnadi Kadha Expressed as Gallic Acid Equivalents (mg GAE/g of Dry Extract).

The study shown that Ashwagandharishta formulation has maximum phenolic content compared to Dashmularishta and Maharasnadi kadha because, Ashwagandharishta contain a greater number of various and plant extract such an Amla, shyonaka, curcuma, etc. hence showing the maximum phenolic content as compared to Dashmularishta and Maharasnadi kadha.

3.2 Determination of Flavonoid Content

The total flavonoid content was determined by using aluminium chloride colorimetric assay.

Fig. No. 2: Standard curve graph of Quercetin.

Table No. 4: Total Flavonoid Content (TFC) of Ashwagandharishta, Dashmularishta, and Maharasnadi Kadha Expressed as Quercetin Equivalents (mg QE/g of Dry Extract).

The total flavonoid content of the ethanol extract of Ashwagandharishta, Dashmularishta and Maharasnadi kadha (herbal preparations) were found to be 2.91,2.88,2.73 mg quercetin equivalent/g. The study shown that Ashwagandharishta formulation has maximum flavonoid content compared to Dashmularishta and Maharasnadi kadha.

3.3 Determination of Anti-Inflammatory Activity

3.3.1 Effect of Protein Denaturation Method:

The absorbance of control was found to be 0.1955.

Table No. 5: Effect of Protein Denaturation Method.

Fig. No. 3: The % Inhibition of Protein Denaturation methods.

The study shown that Dashmularishta formulation has maximum anti-inflammatory as compared to Maharasnadi kadha and Ashwagandharishta by protein denaturation method.

3.3.2 Heat Induced Haemolysis Method:

The absorbance of control was found to be – 0.0389.

Table No. 6: Effect of Heat Induced Haemolysis method.

Fig. No. 6: The effect of % inhibition of hemolysis method.