Qualitative and quantitative estimation of Diosgenin in coded ayurvedic formulation and its ingredient Trigonella foenum-graecum Linn. seeds used in diabetics

Trigonella foenum-graecum (Methi) is a leguminous plant and botanically known as Trigonella foenum-graecum Linn, belong to the family Fabaceae. Trigonella foenum-graecum is used for a variety of health conditions, including digestive problems, bronchitis, tuberculosis, fevers, sore throats, wounds, arthritis, abscesses, swollen glands, skin irritations, diabetes, loss of appetite, ulcers, and menopausal symptoms, as well as in the treatment of cancer. Trigonella foenum-graecum seeds mainly contain Diosgenin [(3β,25R)-spirost-5-en3-ol], a plant-derived steroid sapogenin. The identification and quantification results by HPTLC and HPLC studies of Trigonella foenum-graecum seeds hydrolysed Trigonella foenum-graecum seeds, coded formulation, hydrolysed coded formulation extract with standard Diosgenin biomarker showed a significant highest peak in hydrolysed Trigonella foenum-graecum seeds and hydrolysed coded formulation. The standard Diosgenin is observed in the hydrolysed form of hydrolysed Trigonella foenum-graecum seeds and hydrolysed coded formulation. The literature on Trigonella foenum-graecum confirms its activity as antidiabetic, and the peak of standard biomarker Diosgenin is seen after derivatization with anisaldehyde sulphuric agent, which possesses medicinal phytoconstituents value. Further related to future scientific aspects, more studies on its potent antidiabetic activity and multipurpose action need to be carried out with medicinal composition and its effects on the human body. This study aims to establish the qualitative and quantitative estimation of standard Diosgenin in reliable with coded ayurvedic formulation and Trigonella foenum-graecum seeds and its activity as antidiabetic by HPTLC and HPLC.


Background
India was enriched with a variety of herbs, shrubs, trees, and seeds used in the ancient Indian system of medicine and reported to possess beneficial medicinal effects in curing illness. Trigonella foenum-graecum is used in both forms in food and medicine in Asia. Seeds are eaten as sprouts and rich in secondary metabolites, potential sources of drugs, and essential oils of therapeutic importance. Due to their bitter taste, seeds are a good source of resin, protein, fibre, and mucilage. The critical advantages of medicinal plants' therapeutic uses in various ailments are their safety besides being economical, effective, and easy availability [1][2][3]. Our ayurvedic system praised this herb and documented its healing capacity in its ancient texts.

Procurement of samples
CCRAS, New Delhi, supplied coded formulation and Trigonella foenum-graecum seeds.

Reagents and standards
All chemicals and solvents were used analytical grade or HPLC grade and obtained from E-Merck and other renowned companies. The Diosgenin reference standard was procured from Natural remedies, Bengaluru, India.

Extraction procedure Preparation of Trigonella foenum-graecum seed extract
The dried powdered seed 10.4896 g of Trigonella foenumgraecum was extracted with 200 ml of methanol using Soxhlet for 24 h. The extract was evaporated to dryness under reduced pressure. The obtained extract was collected, dried, weighed, and stored separately for further studies. The obtained extracted residue weight of extraction was 2.1845 g.

Preparation of formulation extract
The powdered 10.3038 g coded ayurvedic formulation was extracted with 200 ml of methanol using Soxhlet for 24 h. 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 1.2939 g.

Preparation of standard Diosgenin solution
Stock solutions of Diosgenin (0.52 mg /ml) were prepared by dissolving 5.2 mg accurately weighed standards in a small amount of HPLC grade methanol and made up the volume to 10 ml in a standard volumetric flask. The stock solution was further diluted as per the requirement for the preparation of working solutions.

Preparation of test solutions
The residues obtained from coded ayurvedic formulation and Trigonella foenum-graecum seeds were accurately weighed 50 mg each and dissolved in methanol using 10-ml

Preparation of hydrolysed formulation and Trigonella foenum-graecum test solution
433.4 mg of Trigonella foenum-graecum seeds were weighed and extracted in 5 ml of methanol, and 4 ml of 3 N HCl was added. The above extract was refluxed for 1 h on a water bath. The solution was cooled and adjusted to pH 7 using aqueous ammonia. Make up the solution by using methanol in a 10-ml standard flask. Similarly, Ayush D was hydrolysed by weighing 500 mg extract in 5 ml of methanol, and 4 ml of 3 N HCl was added refluxed for 1 h on a water bath. The above solution was cooled and adjusted to pH 7 using aqueous ammonia. Made up to the mark in a 10-ml standard flask using methanol, filtered through 0.22 μ membrane filters, and used for HPTLC fingerprint profiling and identification of reference standard Diosgenin.

HPTLC analysis
HPTLC analysis was performed on a CAMAG HPTLC system (Muttenz, Switzerland) equipped with an automatic TLC sampler IV, twin trough development chamber, TLC Scanner 3 linked with WINCATS software version 1.4.4 [20][21][22][23][24][25][26].   anisaldehyde sulphuric acid reagent and heated in a hot air oven at 105 °C until the colour of the spots appeared and the photograph was documented under white light. Before derivatization, the plate was scanned using CAMAG TLC Scanner with WINCATS software at a wavelength of UV 254 and 366 nm using deuterium lamps. After derivatization, the plate was scanned at 540 nm using a tungsten lamp.

HPLC analysis HPLC instrumentation and chromatographic conditions
HPLC analysis was performed on an Agilent 1200 series high-performance liquid chromatographic system equipped with a quaternary pump, manual sample  injector using Chemstation HPLC software. All samples and standards were filtered through 0.22 μm filters. Separation was achieved on C 18 Eclipse, XDB, 4.6 mm × 50 mm, 5 μm particle size Agilent Column. The mobile phase has consisted of acetonitrile/water (95:5) (v/ v) in an isocratic elution with a flow rate of 2 ml/min, and 10 μl of the test sample (triplicate) was injected into the HPLC system. The column temperature was kept at 32 °C. The detection of analytes at 210 nm was carried out using Variable Wavelength Detector (VWD) [26,27].   system. Recorded the chromatogram and determined the area of the peak of the test solution corresponding to that of Diosgenin as described above from the calibration curve. The amount of Diosgenin present in the residues of coded ayurvedic formulation and Trigonella foenumgraecum seeds extract was calculated.

Results
HPTLC chromatogram gives a clear picture of standard biomarker whether it is present in ayurvedic formulations and their ingredients. R F values were calculated, and photograph documentation is recorded in Fig. 3 and Table 1. The fingerprint profile data were recorded by WIN CATS software. Details of HPTLC fingerprint profiling are given in Figs. 4, 5 and 6.

Quantification by HPLC Calibration curve
A linear regression analysis has an equation of the form y = mx + b. The standard calibration curve was established for peak area Vs concentration of Diosgenin applied is shown in Fig. 7

Quantitative analysis
Recorded the chromatogram and determined the area of the peak of the test solution corresponding to that of Diosgenin as described above from the calibration curve given in Fig. 8. The amount of Diosgenin present in the residues of coded ayurvedic formulation and Trigonella foenum-graecum seeds extract was calculated and tabulated in Table 2. HPLC chromatogram of standard Diosgenin and calibration curve, Ayush D tab, and Trigonella foenum-graecum seeds are given in Figs. 7 and 8.

Discussion
In the current study, qualitative Diosgenin identification was carried out using the HPTLC method, and quantitative estimation of specific biologically active Diosgenin compound was conducted in the ayurvedic coded formulation and its ingredient Trigonella foenum-graecum seed using the HPLC method [14,15]. The HPTLC fingerprinting method is an accurate, simple, and specific method for quantifiable bioactive markers [20,21]. HPTLC chromatogram gives a clear picture of standard biomarker whether it is present in ayurvedic formulations and their ingredients.
The HPTLC study evaluated that [11], a band in wavelength 540 nm (Green, R F 0.26) corresponding to Diosgenin is visible in both standard and test solution tracks of Trigonella foenum-graecum seeds and coded ayurvedic formulation extract.
Estimated standard Diosgenin content in coded ayurvedic formulation and Trigonella foenum-graecum seeds extracts was performed using the HPLC method. All the samples showed characteristic peaks of Diosgenin at the same retention time as that of standard Diosgenin.
All the samples showed characteristic peaks of Diosgenin at the same retention time as that of standard Diosgenin. Retention time is 5.728 min detected at 210 nm. The results of HPLC showed that standard Diosgenin are present in Trigonella foenum-graecum seeds are 0.0109 mg/ml, hydrolysed Trigonella foenum-graecum 0.433 mg/ml, Ayush D (0.0716 mg/ml) hydrolysed coded ayurvedic formulation (0.0892 mg/ml). The amount of standard Diosgenin is tabulated in Table 2.

Conclusions
Traditional medicine plays a vital role in the healing of many ailments. Interest in herbal resources is growing day by day due to their efficacy and lesser