Formulation and Evaluation of Picrorhiza Kurroa Based Polyherbal Syrup for Liver Cirrhosis

Liver cirrhosis is a chronic liver disorder characterized by progressive fibrosis, nodular regeneration, and distortion of hepatic architecture, leading to impaired liver function and increased mortality worldwide [1]. The major etiological factors include chronic alcohol consumption, viral hepatitis (hepatitis B and C), non-alcoholic fatty liver disease and autoimmune disorders [2]. The pathogenesis of liver cirrhosis involves oxidative stress and chronic inflammation, which play a central role in hepatocyte injury and fibrosis [3]. Excessive generation of reactive oxygen species (ROS) leads to lipid peroxidation, protein damage and DNA fragmentation, ultimately resulting in liver cell apoptosis and necrosis [4]. Inflammatory mediators such as cytokines and chemokines further exacerbate liver damage by activating hepatic stellate cells and promoting fibrogenesis [5].

Figure 1:  Liver Cirrhosis

Although conventional therapies such as antiviral drugs and liver transplantation are available, they are associated with several limitations including adverse effects, high cost, and limited accessibility [6]. Therefore, there is increasing interest in herbal medicines due to their safety, affordability, and multi-targeted therapeutic effects [7]. Picrorhiza kurroa (Kutki) is a well-known medicinal plant used in Ayurveda and Unani systems for the treatment of liver disorders [8]. It contains bioactive compounds such as picroside I, picroside II, and kutkoside, which exhibit hepatoprotective, antioxidant, and anti-inflammatory properties [9]. Similarly, Andrographis paniculata possesses andrographolide, which has hepatostimulant and anti-inflammatory effects [10]. Glycyrrhiza glabra contains glycyrrhizin, known for its hepatoprotective and immunomodulatory properties [11].

Figure 2:  Rhizomes of Picrorhiza kurroa

Figure 4: Glycyrrhiza glabra

The concept of polyherbal formulation is based on synergism, where multiple herbs act together to enhance therapeutic efficacy and reduce toxicity [12]. Piperine, a bioenhancer, improves the bioavailability of phytoconstituents by inhibiting metabolic enzymes and enhancing absorption [13]. The present study aims to develop and evaluate a polyherbal syrup for hepatoprotective activity. The objectives include formulation of different batches, physicochemical evaluation, phytochemical screening, antioxidant activity assessment, and stability studies.

MATERIALS AND METHODS

MATERIALS

The plant materials selected for the formulation included Picrorhiza kurroa (rhizomes), Andrographis paniculata (leaves) and Glycyrrhiza glabra (roots) all of which are well-known for their hepatoprotective potential. Piperine, obtained from Piper nigrum, was used as a bioavailability enhancer. Sucrose was used as a sweetening and viscosity-enhancing agent, while sodium benzoate served as a preservative. Purified water was used as the vehicle for the formulation. All chemicals and reagents used in the study were of analytical grade and procured from standard suppliers. Instruments such as a digital pH meter (Elico, India), Brookfield viscometer, UV-visible spectrophotometer and stability chamber were used for evaluation.

Preparation of Extracts

The plant materials were thoroughly washed with distilled water to remove adhering impurities and then shade-dried at room temperature (25–30°C) to preserve heat-sensitive phytoconstituents. The dried materials were coarsely powdered using a mechanical grinder and passed through a suitable sieve (No. 40) to obtain uniform particle size. Extraction was carried out using the Soxhlet extraction method with a hydroalcoholic solvent system (ethanol:water, 70:30 v/v) to ensure maximum extraction of both polar and non-polar phytoconstituents [14]. Approximately 200 g of powdered material was subjected to extraction for 6–8 hours until complete exhaustion of the drug. The obtained extracts were filtered and concentrated under reduced pressure using a rotary vacuum evaporator at a controlled temperature (below 50°C). The concentrated extracts were further dried in a desiccator to obtain a semi-solid mass. The dried extracts were stored in airtight containers at 4°C until further use [14,15].

Formulation of Polyherbal Syrup

The polyherbal syrup was prepared in three different batches, namely Batch I, Batch II and Batch III by varying the concentration of herbal extracts as per the formulation design [16-18].

Table 1: Formulation of Polyherbal Syrup

Preparation of Polyherbal Syrup

A calculated quantity of sucrose was dissolved in purified water with gentle heating (60–70°C) to prepare a clear syrup base. The solution was filtered to remove any impurities and allowed to cool to room temperature. The accurately weighed extracts of Picrorhiza kurroa, Andrographis paniculata, and Glycyrrhiza glabra were dissolved separately in a small quantity of warm purified water and then added to the syrup base with continuous stirring to ensure uniform distribution [17]. Piperine was dissolved in a small amount of ethanol and incorporated into the formulation to enhance the bioavailability of phytoconstituents. Sodium benzoate was added as a preservative to prevent microbial growth. The final volume of the formulation was adjusted to 1000 mL using purified water, followed by thorough mixing to ensure homogeneity. The prepared syrup was filtered and stored in amber-colored bottles [18,19].

Evaluation of Different Parameters for Formulation

Organoleptic Properties

The organoleptic properties of the formulated polyherbal syrups (Batch I, II and III) were evaluated to assess their sensory characteristics and patient acceptability. Approximately 5 mL of each formulation was taken in a clean, transparent glass container and visually inspected under natural daylight for color, clarity and presence of any particulate matter. The odor was evaluated by gently shaking the sample and noting the characteristic aroma, while the taste was assessed by placing a small quantity (1–2 mL) on the tongue under controlled conditions. The evaluation was carried out by a small panel of volunteers to minimize subjective bias. Parameters such as color uniformity, pleasant odor, palatability, and absence of grittiness were carefully recorded [20,21].

pH Determination

The pH of the polyherbal syrup formulations was determined to ensure their suitability for oral administration and stability of active constituents. The measurement was carried out using a calibrated digital pH meter. Prior to analysis, the instrument was standardized using buffer solutions of pH 4.0 and 7.0. About 10 mL of each formulation was taken in a clean beaker, and the electrode was immersed into the sample. The pH reading was recorded once it stabilized. All measurements were performed in triplicate at room temperature (25 ± 2°C), and the mean value was calculated [21,22].

Viscosity (cP)

The viscosity of the formulations was determined to evaluate their flow properties and pourability, which are critical for oral liquid dosage forms. The measurement was carried out using a Brookfield viscometer. Approximately 50 mL of the syrup was transferred into a suitable container, and the appropriate spindle (such as spindle No. 2 or 3) was selected based on the expected viscosity range. The spindle was immersed in the sample and rotated at a constant speed (typically 50 rpm). The viscosity reading was recorded in centipoise (cP) once the dial reading stabilized. The analysis was performed at a controlled temperature of 25 ± 1°C in triplicate, and the average value was reported [22].

Specific Gravity

The specific gravity of the formulations was determined using a pycnometer to assess the density of the syrup relative to water. Initially, the clean and dry pycnometer was weighed empty (W₁). It was then filled with the formulation and weighed again (W₂). After cleaning and drying, the pycnometer was filled with distilled water and weighed (W₃). The specific gravity was calculated using the ratio of the weight of the formulation to that of water. All measurements were carried out at room temperature and performed in triplicate to ensure accuracy [23].

Total Solid Content (%)

The total solid content of the formulations was determined to estimate the amount of dissolved solids present, which influences viscosity and stability. A measured volume (10 mL) of the syrup was transferred into a previously weighed evaporating dish. The sample was evaporated to dryness on a water bath and then dried in a hot air oven at 105°C until a constant weight was obtained. The residue obtained represented the total solid content of the formulation. The percentage of total solids was calculated based on the weight of the dried residue relative to the initial volume of the sample [24]. The experiment was performed in triplicate.

Microbial Load – Total Aerobic Microbial Count (TAMC)

The total aerobic microbial count was determined to evaluate the bacterial contamination in the formulation. The syrup samples were serially diluted using sterile saline solution under aseptic conditions. A known volume (1 mL) of the diluted sample was transferred into sterile Petri plates, followed by the addition of molten nutrient agar medium using the pour plate technique. The plates were allowed to solidify and then incubated at 37°C for 24–48 hours. After incubation, the number of bacterial colonies formed was counted and expressed as colony forming units per milliliter (CFU/mL). The test was performed in accordance with standard microbiological procedures [25].

Microbial Load – Total Yeast and Mold Count (TYMC)

The total yeast and mold count was determined to assess fungal contamination in the formulations. The samples were serially diluted and inoculated onto Sabouraud dextrose agar plates using the pour plate method. The inoculated plates were incubated at 25°C for 3–5 days. After the incubation period, visible fungal colonies were counted, and the results were expressed as CFU/mL. This test ensured that the formulation complied with acceptable microbiological limits for oral preparations [25,26].

Stability Study

Short-term stability studies were conducted to evaluate the stability of the formulations over a period of three month. The prepared optimised syrups were filled in airtight, amber-colored bottles and stored under two different conditions: room temperature (25 ± 2°C) and accelerated conditions (40 ± 2°C and 75% RH ± 5%). Samples were withdrawn at initial (0 month) and after every month and evaluated for changes in organoleptic properties, pH, viscosity, specific gravity and physical appearance and other parameters. The stability of the formulation was determined based on the absence of significant changes in these parameters over the study period [27].

RESULTS AND DISCUSSION

Organoleptic Properties

The present investigation focused on the formulation and evaluation of a polyherbal syrup using hepatoprotective medicinal plants. The results obtained from organoleptic, physicochemical, microbiological, and stability studies clearly demonstrate the influence of extract concentration on formulation performance. The organoleptic evaluation indicated that all batches possessed acceptable sensory characteristics; however, Batch II exhibited the most favorable taste and appearance. The balanced concentration of herbal extracts and sucrose in Batch II contributed to improved palatability, whereas Batch III showed increased bitterness due to higher phytoconstituent concentration.

Table 2: Organoleptic Evaluation of Polyherbal Syrup

Physicochemical Parameters

The pH of all formulations remained within the acceptable range for oral liquid preparations, ensuring stability and compatibility. Batch II showed an optimal pH, which is essential for maintaining the integrity of phytoconstituents and preventing degradation. Viscosity plays a crucial role in determining the flow properties and patient compliance of syrups. The results showed a direct relationship between extract concentration and viscosity. Batch II exhibited ideal viscosity, ensuring ease of administration while maintaining adequate consistency. Specific gravity and total solid content increased progressively from Batch I to Batch III, reflecting the increasing concentration of herbal extracts. While higher solid content enhances therapeutic potential, excessive levels, as observed in Batch III, may lead to formulation instability and poor acceptability.

Figure 5: Physicochemical Evaluation of Formulations

Table 3: Physicochemical Evaluation of Formulations

Microbial Load

The microbiological evaluation of the polyherbal syrup formulations was performed to assess their microbial quality and safety for oral administration. The results obtained for Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC) demonstrated that all three batches (Batch I, Batch II, and Batch III) complied with the acceptable pharmacopeial limits (TAMC < 10³ CFU/mL and TYMC < 10² CFU/mL), indicating that the formulations were microbiologically safe. Among the three batches, Batch II exhibited the lowest microbial load, with TAMC recorded at 90 CFU/mL and TYMC at 30 CFU/mL. This suggests superior microbial stability, which may be attributed to the optimal balance of herbal extracts and excipients, as well as effective preservation by sodium benzoate. The reduced microbial count in Batch II also indicates better formulation homogeneity and minimal contamination during processing. In comparison, Batch I showed slightly higher microbial counts (TAMC: 120 CFU/mL; TYMC: 40 CFU/mL), which may be due to lower extract concentration, resulting in reduced inherent antimicrobial activity of phytoconstituents. Batch III exhibited the highest microbial load (TAMC: 140 CFU/mL; TYMC: 50 CFU/mL), possibly due to increased solid content and viscosity, which may create a more favorable environment for microbial retention or reduced preservative efficiency. The presence of phytochemicals such as flavonoids, tannins, and glycosides in the formulation may contribute to inherent antimicrobial activity; however, the primary microbial control is achieved through the addition of sodium benzoate. The effectiveness of this preservative is evident from the fact that microbial counts remained well within permissible limits across all batches.

Table 4: Microbiological Evaluation

Figure 6: Microbiological evaluation of polyherbal syrup formulations showing TAMC and TYMC

Stability Study (1 Month)

The optimized formulation (Batch II) remained physicochemically stable throughout the 3-month stability study. No significant changes were observed in pH, viscosity, or specific gravity. The formulation maintained its clarity and homogeneity without any signs of precipitation or phase separation. Microbial analysis indicated a slight increase in TAMC and TYMC values over time; however, the values remained within acceptable pharmacopeial limits, confirming the effectiveness of the preservative system. Overall, the results demonstrate that Batch II possesses excellent stability under accelerated conditions, making it suitable for further development and potential commercialization.

Table 5: Stability Study Results of Optimized Formulation (Batch II)