1. INTRODUCTION:

A perusal of various authoritative Ayurvedic texts delves into the nuanced intricacies surrounding the shelf-life paradigm, uncovering a pre-existing acknowledgment of factors precipitating the degradation or unsuitability of formulations.

Remarkably, certain granthas not only recognize but also proffer guidance on these factors. In certain dosage forms, these ancient manuscripts prescribe a specific temporal window, delineating the period within which a compounded formulation should ideally be consumed for optimal efficacy. Notably, the Ayurvedic Formulary of India contributes to this temporal discourse, stipulating the recommended consumption window from the date of manufacture ¹. Within the labyrinth of Ayurvedic literature, the term "Saviryata avadhi" surfaces, intricately woven into the fabric of temporal considerations. This term, encapsulated in discussions surrounding the unadulterated potency or Virya of a drug, establishes its resilience against the relentless onslaught of environmental and microbial degradation ². India, with its rich repository of documented and time-honoured knowledge in Ayurvedic medicines, grapples with a formidable challenge emanating from the complex nature of these formulations. The paramount challenge arises from an inherent lack of exhaustive evaluation of constituents, a necessity dictated by the intricate and multifaceted composition of Ayurvedic formulations. The evaluation of these constituents assumes critical importance as it serves as the vanguard, ensuring the fortification of the finished product with the pillars of quality, purity, and stability. At the epicenter of this evaluative pursuit lies the stability study, a nuanced exploration providing empirical evidence on the temporal evolution of a drug substance or product.

The stability study unfolds as an elaborate narrative, unraveling the tapestry of a product's quality variation over time under the kaleidoscopic influence of environmental factors such as temperature, humidity, and light. This dynamic exploration is not merely an exercise in retrospection but, in fact, a visionary undertaking aimed at establishing a retest period and delineating recommended storage conditions for the drug substance or product. In essence, the stability study metamorphoses into a sagacious assessment of the very essence of product quality ³. Within the realm of stability studies, a dichotomy emerges in the form of accelerated stability and real-time stability. The former, a swift and incisive scrutiny, subjects pharmaceutical products to an environment of elevated temperature and humidity. The insights gleaned from this accelerated crucible offer a predictive vista, enabling the extrapolation of a reliable shelf-life or expiry date at standard room temperature. This extrapolation, however, hinges on the adoption of certain assumptions and criterions, transforming the accelerated stability study into a vital precursor for prognosticating the longevity of pharmaceutical formulations ^4-15. Every product, a fragile concoction of chemical, physical, environmental, and biological elements, bears an intrinsic shelf-life tethered to the capricious dance of these factors. Real-time stability studies, a protracted odyssey, present a conundrum for manufacturers who find themselves confronted with the arduous task of patiently awaiting the natural degradation of a drug at room temperature. In navigating the labyrinth of Ayurvedic formulations and their temporal dimensions, the stability study emerges not only as a scientific inquiry but also as a philosophical exploration, unraveling the enigmatic dance between time and the essence of therapeutic efficacy ^11-14.

The Mimosa pudica, an apothecary's treasure trove, harbors within its tissues an array of pharmacological activities, a veritable pharmacopeia embracing anti-diabetic, antitoxin, antihepatotoxin, antioxidant, and wound-healing virtues. A chemical constellation adorns its profile, featuring alkaloids, glycosides, flavonoids, and tannins, rendering it a nuanced palette in the painter's hands [Figure 1]. In the sacred scrolls of Ayurveda, this verdant alchemist finds its purpose—its bitter, astringent taste and cool temperament adept at quelling the fiery forces of kapha and pitta doshas.

Figure 1: Structures of phytochemicals analyzed in AGF by HPLC and GC.

Every twig, leaf, and root, a repository of medicinal prowess, unfolds a saga of utility. A diuretic, antispasmodic, emetic, and an elixir for ailments ranging from vaginopathy to jaundice, it stands as a sentinel against afflictions. The leaves, akin to nature's apothecary, prescribe healing for hydrocele, fistula, and conjunctivitis, while simultaneously orchestrating a dance of coagulation for the lifeblood. Internally, it wages war against vesical calculi, while externally, it dons the mantle of an herbal knight combating edema, rheumatism, myalgia, and the elusive tumor of the uterine realm. A symphony of knowledge reverberates through the corridors of Ayurvedic samhitas and the annals of modern literature, encapsulating the Mimosa pudica's essence. Vernacular names echo in harmonious resonance, synonyms unveil linguistic tapestries, geographical distributions map its botanical diaspora, and morphological nuances paint a canvas of its living form. Shlokas from antiquity whisper their ancient wisdom, elucidating its pharmacological ballet and its revered presence in formulations that stand as time-tested tributes to the perennial power of botanical healing ^16-18.

This research delves into a comprehensive stability study of AGF. It meticulously assesses physical, chemical, and microbial stability under diverse storage conditions, tracking changes in attributes like color, odor, texture, and pH. The investigation ensures the enduring physico-chemical integrity of AGF, using parameters like loss on drying, total ash, acid insoluble ash, water extractive value, alcohol extractive value while also evaluating microbial stability to safeguard topical product safety. The impact of storage conditions is scrutinized, aiding recommendations for optimal preservation. Shelf life determination and method development enhance the study's reliability, benefitting manufacturers and regulatory bodies.

The study may involve the development and validation of analytical methods used for stability testing. This ensures the reliability and accuracy of the data collected during the stability study, enhancing the overall robustness of the research findings.

2. MATERIALS AND METHODS:

2.1 Chemical and Reagents:

Coded Ayurvedic formulation and its ingredients were procured from Central Council for Research in Ayurvedic Sciences (CCRAS), Ministry of AYUSH, Government of India, New Delhi. All the chemicals and solvents used in this study were analytical grade and HPLC grade. The solvents and chemicals were procured from E-Merck, Sigma Aldrich and other renowned companies from GEM Portal and local market of Gwalior, Madhya Pradesh. The reference standard of markers was procured from Natural remedies, Bengaluru and Sigma Aldrich, India.

2.2 Sample extraction

(a) Soxhlet extraction:

The Mimosa pudica (w) plant was extracted with 200 ml of required appropriate solvent by using soxhlet apparatus for 24 hrs. The extracts were evaporated to dryness under reduced pressure. Calculate the obtained residue weights. The process at a temperature approximately that of the boiling point of the solvent soxhlet apparatus permits the uniform percolation of the drug and the continuous flow of vapor of the solvent around the percolator is best for this type of extraction. The obtained extracts were collected, dried, weighed and stored separately for further studies. The same extracts were used for HPTLC and HPLC ^19-21.

(b) Cold maceration extraction for HPTLC:

AGF, and coconut oil each 4.0 g were soaked in 40 ml methanol for 24 hours. These solutions were sonicated for 15 min, centrifuged for a further 15 min, and the transparent layer was stored for HPTLC analysis.

(c) Cold maceration extraction for HPLC:

500 mg of AGF was soaked for 24 hours in 10 ml of HPLC grade methanol. These solutions were sonicated for 15 min, centrifuged for a further 15 min, and the transparent layer was stored for HPLC analysis.

(d) Cold maceration extraction for GC:

5.0 gm of AGF and 10.0 gm of Coconut oil were soaked for 24 hours in 10 ml of HPLC grade methanol. These solutions were sonicated for 15 min, centrifuged for a further 15 min, and the transparent layer was stored for GC analysis.

2.3 High Performance Thin Layer Chromatography (HPTLC):

HPTLC stands as a pivotal method for discerning marker compounds in extracts and conducting chemical profiling across diverse samples. Precision in sample application was achieved through the CAMAG Linomat V applicator, while plate development occurred in CAMAG Twin trough glass chambers of varying dimensions (20 × 10 cm and 10 x 10 cm). Utilizing the CAMAG TLC Visualizer, images of HPTLC plates were captured. Employing pre-coated silica gel aluminium plates 60 F254 (E. Merck, Darmstadt, Germany) at a thickness of 0.2 mm ensured accuracy. The HPTLC process involved weighing and dissolving residues from Mimosa pudica whole plants and other samples (AGF) in appropriate solvents. After filtration through a 0.22 μ membrane filter, these solutions were used for HPTLC fingerprint profiling and reference standard identification. The CAMAG Linomat V applicator facilitated the application of sample solutions onto E. Merck aluminium plates pre-coated with Silica gel 60 F254. Subsequent plate development, drying, and observation through CAMAG TLC Visualizer under UV at 254 nm and 366 nm were systematically documented. Enhanced spot visualization involved plate immersion in Iodine reagent/Anisaldehyde-sulphuric acid reagent, followed by heating in a hot air oven at 105°C until distinct spot colors appeared. The entire process, including Retention Factor (R_F) value calculation, contributed to a quantitative dimension in this robust HPTLC methodology ^19-21.

2.4 High Performance Liquid Chromatography (HPLC):

This technique is used to identify and quantify the bioactive compounds in the AGF formulations and its ingredients extracts. The identified marker compound in the plant extracts was quantified using Agilent 1260/Agilent 1200 Infinity 2 HPLC system with auto sample injector. It has quaternary pump, degasser unit with DAD detector ^22-24. Chemstation software was used for data generation. A calibration graph was prepared with reference standards purchased from TCI, Natural remedies and Sigma Aldrich were used to quantify the percentage of the maker compound present in the AGF and its ingredients extracts.

2.5 Gas Chromatography:

Gas chromatography involved the utilization of an Agilent GC 8890 series equipped with a manual sampler. The chromatographic column employed was an HP-5 with dimensions of 30 mm x 0.25 mm. Hydrogen served as the fuel gas, while Nitrogen functioned as the carrier gas. Detection was achieved using Flame Ionization Detection (FID). The temperature settings ranged from 80 to 240 ºC, with a flow rate of 1 ml/min. The total run time for the gas chromatography analysis was 44.333 minutes. A minimal injection volume of 1 μl was used, and the retention time for the targeted compounds was observed at 32.441 minutes. These meticulously defined gas chromatography conditions were crucial for obtaining precise and reliable results in the analysis ^25-26.

2.6 Shelf-life study:

Coded Ayurvedic Formulation (AGF) was rigorously packaged in a containers following ICH guidelines Q1A (R2) to undergo extended shelf-life studies at 30°C ± 2 °C/60% RH ± 5% RH. A comprehensive assessment of AGF's shelf-life dynamics included continuous physico-chemical. Regular withdrawals, initiated at zero days and continued at 3-month intervals, ensured a thorough examination of the formulation's stability over the prescribed duration. Safety parameters, such as microbial load, specific pathogens, and aflatoxin analysis, were systematically monitored. Additionally, Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) were quantified to provide a comprehensive understanding of the formulation's composition and stability ^27-30.

2.7 Safety Parameters like heavy metals studies, Pesticides Residues, Microbial load and Aflatoxins Analysis

(a) Estimation of heavy metals:

AGF sample was sent to ARBRO Pvt. Ltd. for heavy metals i.e. Lead (Pb), Cadmium (Cd), Arsenic (As) and Mercury (Hg) analysis at zero day by using AAS/ ICP-OES (Inductively Coupled Plasma - Optical Emission Spectrometry) techniques.

(b) Pesticides residues analysis:

The extraction and quantification of the pesticide residues including organochlorine, organophosphate, and pyrethroid pesticides were done as described by Beneta et. al., 2018. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure combined with gas chromatography coupled with tandem mass spectrometry method (GC-MS/MS) was used to analyze the pesticides residues at zero day analysis ³⁴.

(c) Microbial load, specific pathogens and aflatoxin analysis:

Toxicity assays like microbial load, specific pathogens (Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Staphylococcus aureus), and aflatoxins (B1, B2, G1, G2) were analysed as per protocols mentioned in the Ayurvedic Pharmacopoeia of India (API) ^31-32. The analysis was performed at regular time intervals i.e., at 0th, 3rd, 6th, 9th, 12th, 15th, 18th, 21th, 24th, 27th, and 30th month.

The extraction and quantification of aflatoxin (B1, B2, G1 and G2) were done by a high performance liquid chromatography (HPLC) method, as prescribed by Muller and Basedow 2007 ³³.

2.8 Thermal Analysis of AGF formulation:

(a) Thermo-gravimetric analysis of AGF formulation:

Thermo-gravimetric analysis (TGA) is an analytical technique used to determine a material’s thermal stability and its fraction of volatile components by monitoring the weight change that occurs when a sample is heated at a constant rate. The Thermo-gravimetric analysis (TGA) profile of the AGF formulation clearly gives an approximation about the weight loss with respect to temperature due to the release of surface bounded water and volatile matter ³⁵.

(b) Differential Scanning Calorimetry of AGF formulation:

Differential Scanning Calorimetry (DSC) is a thermal analysis technique that looks at how a material’s heat capacity (C_p) is changed by temperature. This allows the detection of transitions like melts, glass transitions, phase changes, and curing. The biggest advantage of DSC is the ease and speed with which it can be used to see transitions in materials i.e. glass transition, phase changes or polymorphs and study the degree of purity in materials ³⁵.

2.9 Method for assays:

(a) Total Phenolic Content (TPC):

Quantification of total phenolic content (TPC) was carried out by Folin Ciocalteu method ³⁶ with slight modifications. The FCR reagent oxidizes phenols in extract and changes in the dark blue colour (k = 765 nm) which is monitored by UV-Visible spectrophotometer. Briefly 5 ml of 10% (FC reagent in distilled water) Folin Ciocalteu reagent was mixed with 1 ml of aliquot (1mg/ml). The test tube was shaken up to mix the solutions thoroughly, followed by addition of 3 ml Na2CO3 (1M). The solutions were mixed again and left at room temperature for 15 min. The phenolic content was expressed as Gallic acid equivalents (μg /ml) using the calibration curve. The standard curve was prepared by using 10, 20, 30, 40, 50, 60, 70 and 80 μg/ml solutions of Gallic acid (standard phenolic compound) in ethanol.

(b) Total Flavonoid Content (TFC):

The methanolic extracts were used to determine the total flavonoids contents by aluminium chloride colorimetric method as described in the method ³⁷, sodium nitrate (2.5 g) was taken in a volumetric flash (50 mL) and water was added to prepare a solution of 5 % sodium nitrate. Sodium hydroxide (2.5 g) was taken in another volumetric flash (50 mL) and added water up to the mark that was 5 %sodium hydroxide. Then 10 % aluminium chloride solution was prepared based on the same procedure. After that, the extracts (MEF B) were taken in a test tube and mixed with water (4mL) and sodium nitrate (0.3 mL). Then, the test tubes were kept in the dark place for 5 min. Then, 10 % aluminium chlorides (0.3 mL) were added into the test tube and were maintained for 5 min in the dark condition to complete the reaction. Finally, the solution of 5 % sodium hydroxide (02 mL) and water were added to the test tube. The absorbance of the samples was measured at a fixed wavelength 510 nm. In this method, quercetin standard was used for the calibration curve by preparing quercetin solutions at concentrations of 10, 20, 30, 40, 50, 60, 70 and 80 μg/ml. The results of total flavonoids contents were expressed as quercetin equivalent per gram weight of sample.

3. RESULTS AND DISCUSSION:

3.1 Soxhlet Extraction:

The dried powdered Mimosa pudica (10.0627 g) was extracted with 200 ml of methanol by using soxhlet for 24 hrs. The extract was evaporated to dryness under reduced pressure. The obtained extract was collected, dried, weighed and stored separately for further studies. The obtained residue weight of extraction was 0.3993g.

3.1 HPTLC Fingerprint profile of Mimosa pudica plant and AGF:

(a) Standard solution:

10.0 mg of linoleic acid was accurately weighed and dissolved in HPLC grade methanol and the volume was made up to 10 ml to obtain 1.0 mg/ml linoleic acid stock solution.

(b) Test solution:

4.0g of AGF in 40 ml methanol and 99.5 mg of Mimosa pudica plant extract were accurately weighed and dissolved in methanol using 10 ml volumetric flask, filtered through 0.22μ membrane filters and used for HPTLC fingerprint profiling and identification of linoleic acid reference standard.

(c) Solvent system:

n-hexane: Ethyl acetate: Formic acid (6:4:0.1 v/v)

(d) Procedure:

Applied 5µL of each linoleic acid reference standard and test solution of Mimosa pudica plant and AGF respectively on different tracks on a precoated silica gel 60 F₂₅₄ TLC plate (E. Merck) of 0.2 mm thickness by using CAMAG Linomat V applicator. The plate was developed in the suitable solvent system till the solvent rises to a distance of 8 cm.

(e) Detection:

The plate was observed through TLC Visualizer under UV at 254 nm and 366 nm and photos were documented. Further, the plate was derivatized via iodine vapors and photos were documented. Finally, the plate was dipped in anisaldehyde-sulphuric acid reagent (ASR) and heated in hot air oven at 105⁰C until the colour of the spots were appeared and photo was documented under white light, further calculated the R_fvalues given in Figure 2 and Table 1.

At UV 254 nm	At UV 366 nm	After Derivatization with iodine vapors at 540nm	After Derivatization with ASR at UV 366nm

1 2 3	1 2 3	1 2 3	1 2 3
Track 1: Linoleic acid; Track 2: Mimosa pudica; plant Track 3: AGF
*Figure 2. HPTLC fingerprint Profiling of Linoleic acid, Mimosa pudica* plant and AGF extract**

Table 1. R_f values of Linoleic acid, Mimosa pudica plant extract and AGF formulation

Wavelength	Track 1: Linoleic acid		Track 2: *Mimosa pudica* plant extract		Track 3: AGF
At UV 254 nm	R_f	Color	R_f	Color	R_f	Color
	-	-	0.07	Green	-	-
	-	-	0.11	Green	-	-
At UV 366 nm	-	-	0.07	Red	-	-
	-	-	0.12	Red	-	-
	-	-	0.15	Red	-	-
	-	-	0.26	Blue	-	-
After Derivatization with iodine vapors	-	-	0.07	Yellow	-	-
	-	-	0.12	Yellow	0.12	Yellow
	-	-	-	-	0.16	Yellow
	-	-	0.20	Yellow	0.21	Yellow
	0.26	Yellow	0.26	Yellow	0.26	Yellow
	-	-	-	-	0.60	Yellow
After Derivatization with anisaldehyde sulphuric acid reagent at UV 366 nm	-	-	0.08	Red	-	-
	-	-	0.13	Red	0.13	Orange
	-	-	0.17	Orange	0.17	Orange
	-	-	0.21	Green	0.21	Green
	0.26	Orange	0.26	Orange	0.26	Orange
	-	-	0.32	Orange	0.32	Orange
	-	-	0.37	Orange	0.65	Orange
	-	-	0.96	Orange	-	-

Calibration curve
Palmitic acid
AGF Formulation
Coconut oil

Figure 4. Calibration curve and HPLC Chromatogram of palmitic acid biomarker compound in AGF Formulation and Coconut oil

Table 3. Estimation of palmitic acid biomarker compound in AGF Formulation and Coconut oil

S. No.	Sample Name	Weight of the sample	Amount of palmitic acid present in samples	Percentage of palmitic acid present in sample
S. No.	Sample Name	(mg/10ml)	(mg/ml)	Result	Mean
1.	AGF	5000	0.1835	0.0367	0.0351
		5000	0.1725	0.0345
		5000	0.1705	0.0341
2.	Coconut oil	10000	0.106	0.0106	0.0119
		10000	0.106	0.0106
		10000	0.146	0.0146

Table 4. Shelf-life studies of Ayurvedic formulation (Zero day to 11^th quarter)

S. No.	Test parameter	Zero day	11^th quarter
1.	Loss on drying at 105^ºC (%w/w)	81.6054	83.9811
2.	Total ash (%w/w)	0.1077	0.1364
3.	Acid insoluble ash (%w/w)	0.0444	0.0433
4.	Water soluble extractive (%w/w)	5.6359	6.3776
5.	Alcohol soluble extractive (%w/w)	17.0516	15.9600
6.	pH (10% w/v aqueous solution)	6.44	6.46
7.	Locking length (cm)	1.0285	1.0594
8.	Total phenolic content (%GAE)	1.46	1.46
9.	Total flavonoid content (%QE)	NO	NO
10.	Total Viable Aerobic Count (cfu/g)	1.5088	1.2822
11.	Total Fungal Count (cfu/g)	26.2275	23.3332
12.	Entero-bacteriaceae (cfu/g	6.7569	6.2857
13.	E. coli per/gm	24.7187	22.0510
14.	Salmonella spp. per/gm	0.3087	0.3057 %
15.	Staphylococcus aureus per/gm	0.1828	0.1839 %
16.	Pseudomonas aeruginosa per/gm	4500	210
17.	Aflatoxins B₁	25 Absent Absent Absent	15 <10 Absent Absent
18.	Aflatoxins B₂
19.	Aflatoxins G₁
20	Aflatoxins G₂

3.4 Shelf life studies:

Physico-chemical parameters play a pivotal role in determining the shelf-life of pharmaceutical products, particularly in the herbal industry where high moisture content, indicated by elevated loss on drying values, can jeopardize the integrity of the formulation²⁸. Table 4 presents detailed shelf-life analysis data collected at regular intervals (0^th, 3^rd, 6^th, 9^th, 12^th, 15^th, 18^th, 21^st, 24^th, 27^th, and 30^th month) for CPF. Analysis reveals significant changes (at p<0.001) between the zero day and the 30^th month, notably in loss on drying (0.018%), pH (0.31%), water extractive value (7.76%), alcohol extractive value (6.99%), total ash (27.0%), and acid-insoluble ash (16.0%). The pH value exhibited a marginal increase from 6.44 to 6.46 over the 30-month period. (Table-4).

3.6 Safety Parameters like heavy metals studies, Pesticides Residues, Microbial load and Aflatoxins Analysis:

(a) Estimation of heavy metals:

The heavy metal analysis of the sample, conducted in accordance with API permissible limits, revealed that Lead, Cadmium, Mercury, and Arsenic were not detected, indicating concentrations below the permissible limits of 10.0 ppm, 0.3 ppm, 1.0 ppm, and 3.0 ppm, respectively. These results affirm the sample's compliance with regulatory standards for heavy metal concentrations, ensuring its safety within established limits. The absence of lead, a toxic heavy metal, is particularly noteworthy, emphasizing the sample's strict adherence to stringent safety criteria, making it suitable for consumption.

(b) Microbial load, and specific pathogens:

The microbiological quality of the sample was assessed through various parameters, revealing satisfactory results. The total viable aerobic count was determined to be 4500 colony-forming units per gram (cfu/g), indicating a moderate level of aerobic microorganisms present. The total fungal count was found to be low, with only 25 cfu/g observed. Notably, the absence of Entero-bacteriaceae, E. coli, Salmonella spp., Staphylococcus spp., and Pseudomonas aeruginosa signifies a high level of hygiene and safety in the sample. These findings suggest that the sample meets the microbiological criteria set for safe consumption, demonstrating a lack of pathogenic bacteria and fungi that could pose a health risk. The absence of specific indicator organisms such as E. coli and Salmonella spp. further attests to the product's adherence to quality and safety standards.

(c) Aflatoxin analysis:

The analysis for aflatoxin levels in the sample was conducted in accordance with API permissible limits. Aflatoxin B1, B2, G1, and G2 was found to be below the quantifiable limit (BQL) with a concentration lower than the limit of quantification (LLOQ) of 1.0 microgram per kilogram (mcg/kg). These results indicate that the sample complies with the established standards for aflatoxin levels, ensuring the safety and quality of the product within the accepted regulatory thresholds.

(d) Pesticides residues analysis:

The pesticide residue analysis of the sample revealed generally positive outcomes, with several substances, such as Aldrin, Dieldrin (linked to Aldrin), Heptachlor (including its epoxide), Cypermethrin, and certain isomers of Fenvalerate and Esfenvalerate, found below detectable limits, as indicated by a Detection Limit (DL) of 0.005 mg/kg. However, specific results for Chlordane cis and trans and oxychlorodane, Alachlor, 4, 4 – DDT, Ethion, Quintozene, 2, 4 – DDE, Endosulphan Sulphate, and various other pesticides were also found below detectable limits, as indicated by a Detection Limit (DL) of 0.005 mg/kg. This analysis underscores the importance of monitoring and controlling pesticide residues in AGF, ensuring that they comply with safety standards to safeguard public health. The absence of detectable levels for several pesticides is particularly reassuring, indicating the adherence of the sample to stringent safety criteria and contributing to the overall safety of the analyzed product for consumption.

3.7 Total phenolic and total flavonoid content assay

The provided equation is Y=0.0097x−0.0104, with a coefficient of determination (R²) value of 0.9985. The dataset includes the following pairs of x and Y values. The total phenolic content is measured at 0.3087 %.

The linear equation is Y=0.0016x−0.0032, exhibiting a high level of correlation with R² value of 0.9949. The dataset comprises the corresponding X and Y values: 10, 20, 30, 40, 50, 60, 70, 80, with Y values of 0.016, 0.028, 0.042, 0.061, 0.072, 0.089, 0.107, and 0.127, respectively. The total flavonoid content is determined to be 0.1828%.

3.8 Thermal Analysis of AGF formulation:

(a) Thermo-gravimetric analysis of AGF formulation:

To check thermal stability of AGF product we perform Thermo-gravimetric analysis under anaerobic condition by increasing temperature rate 10 °C/min from 25°C to 1000 °C. On the TGA profile, the initial step shows weight loss of 85% in the temperature range of 40°C to 115°C, which might have resulted from the release of moisture and volatile matter. The second step shows steep weight loss of 15% at the temperature range from 115°C to 345°C due to degradation of other organic compounds present in materials. After 345°C, there is no any residue of gel material is remains (Figure 5).

Figure 5. Thermo-gravimetric Analysis curve of gel formulation

(a) Differential Scanning Calorimetry of AGF formulation:

The analysis was performed using the dynamic option of the differential scanning calorimetry (DSC) on the AGF formulation with heating rates: 10°C/min from 0°C to 550.00°C. Decomposition curves of sample on DSC were characterized by endothermic behaviour (Figure 6).

Figure 6. Differential Scanning Calorimetry curve of gel formulation

4. CONCLUSION:

In conclusion, this research article has systematically explored the HPTLC fingerprint profiling of Mimosa pudica plant and AGF, employing a robust solvent system with Linoleic acid as the reference standard. The study employed various detection methods, including UV observation, iodine vapors derivatization, and anisaldehyde-sulphuric acid reagent, resulting in comprehensive HPTLC fingerprint profiles. The Coded Ayurvedic Formulation (AGF) underwent meticulous packaging and extended shelf-life studies, adhering to ICH guidelines. Continuous evaluations, including physico-chemical and safety parameter-based assessments, were conducted. The shelf-life dynamics were systematically scrutinized through withdrawals at various intervals. The quantified results disclosed linoleic acid and palmitic acid content in AGF and Mimosa pudica Plant, providing valuable insights into the formulation. Safety parameters, encompassing heavy metals, microbial load, specific pathogens, aflatoxin levels, and pesticide residues, were systematically examined. The findings demonstrated compliance with permissible limits for heavy metals, ensuring the safety of the formulation. Microbial analysis highlighted a low aerobic count and fungal count, affirming high hygiene standards. Aflatoxin levels and pesticide residues below quantifiable limits emphasized adherence to stringent safety standards. Furthermore, total phenolic and flavonoid content assays revealed significant percentages, contributing to the overall understanding of the formulation's composition. The thermal analysis confirmed the stability of AGF, providing crucial insights into its safety and quality attributes. In summary, the comprehensive analyses presented in this research article collectively signify the safety, quality, and compositional attributes of the studied Ayurvedic formulation. The adherence to stringent guidelines and the continuous evaluations contribute to the overall assurance of the safety and efficacy of AGF for consumption.

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Received on 29.02.2024 Revised on 22.08.2024

Accepted on 07.11.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):797-808.

DOI: 10.52711/0974-360X.2025.00118

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