Introduction

Stability refers to the ability of a pharmaceutical formulation to maintain its original quality and effectiveness over time, under specified conditions. It is a critical parameter in ensuring the safety, efficacy and therapeutic value of medicinal products. Stability encompasses a broad spectrum of characteristics including physical, chemical, biochemical, microbial, toxicological and medicinal properties. Stability studies are essential for understanding how a formulation behaves when exposed to various environmental factors, such as temperature, humidity, light and oxygen over time.

These studies provide valuable insights into the degradation pathways of active ingredients, helping to establish the product's shelf life and optimal storage conditions. As such, stability testing is fundamental in ensuring that the therapeutic benefits of a product are preserved throughout its shelf life and during use. In the context of pharmaceutical product development, stability testing is generally categorized into two types: real-time stability testing and accelerated stability testing. Real-time stability testing involves storing the product under standard, controlled conditions (e.g., at ambient temperature and humidity) and observing its performance over an extended period. This process allows for the observation of gradual degradation and the long-term behaviour of the product. In contrast, accelerated stability testing is designed to simulate the effects of long-term storage by exposing the product to elevated stress conditions, such as higher temperatures and humidity levels. These controlled stress conditions expedite the degradation processes, enabling the prediction of a product’s shelf life within a shorter duration.

While stability testing is well-established for conventional pharmaceutical products, traditional Ayurvedic formulations such as Dadrughni Vati (Lepa) and Dadrughna Malahara require similar attention to ensure their continued efficacy and safety. Ayurvedic preparations such as Lepa, Lepaguti, and Malahara have been known to retain their therapeutic potency for up to 3 years.

The aim of the present study is to conduct an accelerated stability assessment of Dadrughni Vati (Lepa) and Dadrughna Malahara.

Preparation of Dadrughni Vati (Lepa) and Dadrughna Malahara

All the batches of Dadrughni Vati (Lepa) were prepared as per the reference of Bheshaj Samhita^[2] and Dadrughna Malahara was a modified dosage form of Dadrughni Vati (Lepa) prepared in the Pharmaceutical Laboratory of Upgraded Department of Rasashastra and Bhaishajya Kalpana, Government Ayurved College, Vadodara, Gujarat.

Place of study

Vasu Research Centre, Division of Vasu Healthcare PVT. LTD. Vadodara, Gujarat.

Packing & Sample quantity

Both Dadrughni Vati (Lepa) (DL) and Dadrughna Malahara (DM), 100 g of each sample, were packed in three transparent, airtight, food-grade plastic containers and stored in an accelerated stability chamber.

Study period

The study was conducted from April 2023 to September 2024.

Storage condition

An accelerated stability study was conducted as per the ICH Guidelines. The samples were stored at 40℃ + 2℃, while the relative humidity was 75% + 5%. The products were analyzed for the accelerated stability study at 0, 3^rd and 6^th months.

Frequency of withdrawal of the samples

Samples were withdrawn from the chamber at an interval of 0, 3 and 6^thmonths.

Evaluation of Accelerated Stability Testing

Both samples were filled in containers and labelled properly including the drug name and date of preparation. At the six-month-long expedited stability investigation was conducted. Relative humidity (RH) was set at 75 % + 5 % with a controlled temperature of 40°C + 2°C.

The acceptable point was determined at 10 % degradation for extrapolating the accelerated stability data. Real-time ageing factor 3.3 are used for extrapolating shelf life for countries in climate zone IV.

Table 4: Details of HPTLC fingerprinting observed under UV light 254 nm, 366 nm and 540 nm of DL at the interval of 0 and 6 months

Data of organoleptic characters and physico-chemical parameters of DM at the interval of 0, 3^rd and 6^th months and microbial limit test, HPTLC fingerprinting of DM at the interval of 0 and 6^th months are tabulated in table no. 5 to 8.

Table 5: Details of organoleptic characters of PPA at the interval of 0, 3 and 6 months

Table 6: Details of Physico-chemical parameters of DM at the intervals of 0, 3 and 6 months

Table 10: The results of intercept and slope for shelf-life evaluation of DM

Table 11: The results of approximate period for 10 % degradation for shelf-life evaluation of DL

Table 12: The results of approximate period for 10 % degradation for shelf-life evaluation of DM

Table 13: The results of extrapolation of shelf life of DL and DM

Discussion

The shelf life or stability of a drug refers to the period from its manufacturing and packaging until its chemical or biological activity remains at or above the predetermined level of labeled potency,

High humidity can accelerate this process, fostering rancidity.^[10] Microbial growth, influenced by temperature and humidity, may further break down fats and oils, leading to a decrease in saponification value.^[11] Temperature variations can also affect viscosity and spreadability, with higher temperatures reducing product’s stability. Overall, both temperature and humidity significantly impact the physical and chemical properties of the DM sample.^[12]

Microbial contamination can impact the chemical composition of raw materials, diminishing the medicinal properties of herbal drugs. To assess this, a microbial limit test was performed^[13] on DL and DM, which included evaluations of total plate count, total yeast and mould count, as well as tests for four pathogens: E. coli, Salmonella, S. aureus, and P. aeruginosa. The absence of pathogens and total plate counts within acceptable limits at the initial stage and six months indicate that DL and DM were free from microbial contamination. This outcome demonstrates the diligence applied during the pharmaceutical process, including the careful selection of containers and storage duration, which ultimately contributes to reducing formulation degradation and extending shelf life.

HPTLC study of DL and DM was carried out in comparison. HPTLC tracks of both samples were scanned under UV light of 254 nm, 366 nm (after derivatization) and 540 nm (after derivatization) with their Rf value mentioned in Table No.5.21 and Table No.5.25. Track 1: Dadrughni Vati (Lepa) / Dadrughna Malahara (0 months) Track 2: Dadrughni Vati (Lepa) / Dadrughna Malahara (6 months). At 254 nm: 8 prominent spots bearing Rf value 0.20, 0.30, 0.41, 0.49, 0.59, 0.64, 0.71, 0.95 were spotted in track of DL. While in the track of DM, 3 prominent spots were observed at Rf values 0.35, 0.71, 0.95. At 366 nm (after derivatization): 5 prominent spots bearing Rf values 0.41, 0.49, 0.64, 0.71 and 0.95 were spotted in the track of DL. 6 prominent spots were observed at Rf values 0.17, 0.44, 0.46, 0.71, 0.76 and 0.84 in the track of DM. At 540 nm (after derivatization): 11 prominent spots were observed at Rf value 0.17, 0.20, 0.30, 0.41, 0.49, 0.56, 0.64, 0.71, 0.76, 0.84, 0.91 spotted in track of DL. While 12 prominent spots bearing Rf values 0.12, 0.20, 0.30, 0.38, 0.46, 0.56, 0.61, 0.71, 0.74, 0.76, 0.82 and 0.84 were spotted in track of DM.