Abutilon indicum leaves were processed into aqueous, ethanol, and chloroform extracts following the methodology outlined in the Indian Pharmacopoeia . After washing the leaves with running tap water, they were air-dried in the shade and then underwent Soxhlet extraction. A total of 50 grams of leaf powder was subjected to 24-hour extraction with aqueous, ethanol, and chloroform solvents using the aforementioned apparatus. The obtained extracts were safely store in sealed containers and kept in a refrigerator at 4°C for further investigation.

Thin-layer chromatography of various extracts involves the following steps: Creating the TLC plate involved making slurry of gel 'G' with distilled water, applying a 0.25 mm thick layer to glass plates. Following air-drying for 10 minutes, the plates are then heated in a hot air oven at 105°C for 30 proceedings to activate them.

Preparing the sample solution involved creating a 0.1% solution of the aqueous extract, and any suspended impurities were filtered off. Choosing the solvent system:The choice of the solvent system or mobile phase influences the separation process. Solvents, selected based on their different polarity, were prepared as binary, ternary, or quaternary systems with a constant composition to achieve effective separation. Saturation of TLC chamber: Solvents were filled in a chamber up to a height of approximately 15 cm and marked. The chamber was lined with filter paper soaked in solvent, ensuring uniform distribution of solvent vapors throughout the vapor space, thereby saturating the chamber.

Application of spots: The prepared sample solution of the extract was applied to one end of the plate, approximately 1.5 cm from the bottom 3, 4.

The plates were carefully placed into a pre-saturated TLC chamber, ensuring that the solvent saturated the TLC layer below the starting line. Capillary forces drove the solvents upward, carrying the substance mixture to be separated until it reached approximately ¾ of the TLC plate's height. Subsequently, the plate was taken out of the chamber, and the solvent was allowable to evaporate at room high temperature. The TLC-separated substances shown in Figure 2 detected at 245 nm and 366 nm with RF values 0.09, 0.37, 0.54, and 0.64.

The leaves of Abutilon indicum (Malvaceae) were air-dried and crushed into powder form. 500 grams of the pulverized substance were enclosed in muslin fabric furthermore underwent continuous hot extraction with water for 72 hours using a Soxhlet extractor. Following this, the aqueous extracts of Abutilon indicum leaves (EA) (Malvaceae) were filtered through Whatman paper No. 42, and the resulting filtrates were determined under reduced pressure and ultimately vacuum desiccated. The ethanolic extract yielded 5.2% w/w. The preparation of sample solutions was meticulously optimized for first-class fingerprinting and efficient extraction of marker compounds.

For each sample, 50 mg was weighed and dissolve in 5 ml of methanol and water (1:1) at a concentration of 10 mg/ml. The consequential way out was sonicated for 10 minutes, followed by centrifugation for 2 minutes at 2000 rpm. The clear supernatant solution was then applied to the plate, with 5, 2.0, and 5.0 microliters of each sample being applied to separate 10x10 plates. This corresponded to on-plate amounts of 5, 20, and 50 micrograms, respectively, shown in Figure 3, Figure 4, Figure 5.

Figure 3

Figure 4

Figure 5

Derivatization: 10% Sulphuric acid in methanol is used as a developing reagent. The developed plate is dipped in 10% Sulphuric acid for 2 seconds and heated at 110 °C for 3 minutes, as shown in Figure 3, Figure 4, Figure 5 and tracks 1, 2, and 3 with respective wavelengths of 254 and 366 nm.

Figure 6

Figure 7

Figure 8

Figure 9

Ethanol extracts of Abutilon indicum by High-Performance Thin Layer Chromatography: chromatographic parameters are shown in Table 2.

To create standard and QC samples, catechin, gallic acid, and quercetin stock solutions (10 mg/mL) in methanol were diluted to prepare standard solutions (0.1-1.0 mg/mL). Calibration involved applying GA (1-10 µL) and QE (0.5-5 µL) to HPTLC plates. Linear least squares weakening was used, with each amount apply 6 times. Quality control samples (150, 300, 600 ng band-1 for GA; 200, 400, 800 for QE) validated the method.

Applying a properly diluted ethanolic extract, 10 µL of the sample solution was spotted on an HPTLC plate for High-Performance Thin Layer Chromatography UV 530 nm fingerprinting and image analysis and results are shown in Figure 6, Figure 7, Figure 8. This process was replicated six times for each amount. Linear least-squares regression correlated peak area with applied amounts. Following plate development and scanning, peak areas were quantified, and the known amounts of catechin, gallic acid, and quercetin were determined using the calibration curve.

Figure 11

Utilizing methanol as the solvent system and under optimized chamber saturation conditions, a chromatogram for catechin, gallic acid, and quercetin was successfully generated. The ideal saturation time was determined to be 10 minutes are shown in Figure 10. Notably, for catechin, the spots exhibited the highest absorbance at approximately 276 nm, for gallic acid at 271 nm, and for quercetin at 366 nm. Consequently, HPTLC spectra analysis was conducted at these specific wavelengths in the reflectance method, denoted as High-Performance Thin Layer Chromatography-UV 276 nm, 271 nm, and 366 nm. At Rf (0.67), the standard sample of catechin, gallic acid, and quercetin displayed condensed bands with sharp, proportioned, and high-resolution characteristics, particularly at Rf (0.63).

Chloroform stock solutions of quercetin, gallic acid, and catechin (10 mg/mL) were prepared for standard and QC samples. Diluting these solutions yielded standard solutions (0.1-1.0 mg/mL). Calibration involved applying GA (1-10 µL) and QE (0.5-5 µL) to an HPTLC plate, with linear least squares regression applied. QC samples at different concentrations (150, 300, 600 ng band-1 for GA and 200, 400, 800 ng band-1 for QE) ensured method reliability.picture analysis involved subjecting air-dried and powdered leaves of Abutilon indicum (Malvaceae) to continuous hot water extraction for 72 hours using a Soxhlet extractor. The resulting chloroform extracts were filtered, determined under reduced pressure, and vacuum dried, yielding a chloroform extract with a 4.6% w/w. The protocol for preparing test solutions was fine-tuned to optimize high-quality fingerprinting and efficient extraction of marker compounds shown in Figure 11.

Figure 13

To fingerprint the chloroform extracts of leaves from Abutilon indicum (Malvaceae), 10 µL of an appropriately diluted test solution of the aqueous extract was spotted on a High-Performance Thin Layer Chromatographyplate. This application was repeated 6 times for each amount. Linear least-squares regression was employed, considering the peak area and applied amounts. Subsequently, the plates underwent development and scanning, following the procedure discussed earlier. The recorded peak areas were then utilized to calculate the amounts of catechin, gallic acid, and quercetin, employing the calibration curve 5, 6.

The GC-MS analysis was performed using a Perkin Elmer, USA, and System XL with NIST Library. The system was operated by Analyzer: Single Quadrupole with prefilter. GC-MS parameters are shown in Table 4. Abutilon indicum commonly referred to as Indian mallow or "Atibala" in Ayurveda, has a rich history of traditional medicinal use. Seeking to unravel its therapeutic potential, aqueous extracts of this plant underwent GC-MS profiling to identify the Phytochemical compounds present. This analytical technique enables the separation and characterization of a diverse array of compounds within the extract.

Table 4

Sr. No.	Instrument use	GC-MS
1.	Model	Auto System Xl with Turbo Mass
2.	Make	Perkin Elmer
3.	Column Use	ELITE-5MS (30METERX0.250MMX0.) 250 Micro mm
4.	Carrier gas	Helium
5.	Flow rate	01ml/min
6.	Injector temp.	260 OC
7.	Oven temp	75 OC Hold for 5min, rate 10 OC per min up to 280 OC hold for10 mint
8.	EI Source temp	220 OC
9.	Scan Range	20 to 610 (amu)
10.	Injection Volume	2 micro liters

The GC-MS analysis of Abutilon indicum aqueous extract unveiled a spectrum of phytochemicals, encompassing alkaloids, flavonoids, saponins, terpenoids, and other bioactive substances are shown in Figure 14, Figure 15. Among the noteworthy compounds identified are quercetin, kaempferol, and various alkaloids like vasicine.

These compounds collectively contribute to the plant's medicinal properties, offering benefits such as anti-inflammatory, antioxidant, and antimicrobial effects.

Figure 15

The gas chromatography-mass spectrometry profiling serves as valuable scientific evidence supporting the presence of these bioactive compounds, shedding light on its pharmacological and medicinal significance shown in Figure 16, Figure 17. Researchers continue to explore the plant's potential applications in modern medicine, aiming to harness its diverse phytochemical profile for the development of new pharmaceuticals or nutraceuticals products.

Examining the chloroform extract of Abutilon indicum through gas chromatography-mass spectrometry (GC-MS) offers valuable insights into the specific phytochemical compounds within this extract. This analytical technique facilitates the separation and identification of diverse compounds based on their mass and chemical properties, providing a comprehensive understanding of the constituents.

The GC-MS profiling of the chloroform extract of Abutilon indicum has uncovered the presence of several noteworthy phytochemicals. Chloroform extraction, known for enriching lipophilic compounds, has highlighted terpenoids, alkaloids, and other non-polar substances. Among the significant compounds identified in this extract are:

Figure 17

Abutilon indicum as a source of bioactive compounds for pharmaceutical, nutraceuticals, or traditional medicine applications.

Utilizing GC-MS to analyze the ethanol extract of Abutilon indicum proves to be an efficient approach for identifying the phytochemical compounds present in this particular extract. Ethanol is a versatile solvent that can extract a wide range of both polar and non-polar compounds from plant material, making it particularly valuable for a comprehensive phytochemical analysis.

The GC-MS profiling of the ethanol extract from Abutilon indicum unveils a diverse array of bioactive compounds are shown in Figure 18, Figure 19 :

Figure 19

The ethanol extract of Abutilon indicum rich in such diverse phytochemical underscores the plant's medicinal value. It serves as a potential source for developing herbal medicines, dietary supplements, or functional foods. Further investigate is essential to elucidate the exact therapeutic reimbursement and applications of these compounds and their interactions within the plant's overall chemical profile 7, 8.

I extend my sincere appreciation to Dr. Ravindra B. Patil, a professor at DCS's A. R. A. College of Pharmacy in Nagaon, Dhule, for his invaluable mentorship and unwavering support. Heartfelt gratitude is also extended to Professor Ravindra Nikam and the College of Pharmacy in Gondur, Dhule, for generously providing essential research facilities. Additionally, I would like to convey my thanks to Dr. A. V. Patil, the Principal of Prof. Ravindra Nikam College of Pharmacy in Gondur and Dhule, for their consistent encouragement and support throughout this endeavor.