INTRODUCTION:

TLC or Thin layer chromatography is technique in chromatography that separate the given mixture components by employing a stationary phase i.e. thin and held by a backing which is inert in nature. Being highly sensitive technique TLC can perform analytical study like monitoring the reaction progress, or on preparative scale to decontaminate even traces of contaminants from given analyte. For this since long time, TLC has successfully demonstrated description profile of impurity (identified or unidentified), an important requirement in safer and effective formulation of drug, making it most eligible in processes of development of drug.

Being a technique for isolation, TLC can perform multiple detections owing to its enhanced sensitivity ability. extensively used tool in analysis TLC has gained preference for being simple, of lower cost, faster separation in less time with high sensitivity. Furthermore, requiring comparatively smaller sample volume, TLC microscopic method being simple takes very less time for development. Various tools used to perform TLC are handy and reasonably priced which suitable for labs running on low budget^1-3. Methods for screening those utilize TLC every so often with aim to isolate varied chemical compounds present in medicinal plants specifically target in its search, plants of therapeutic interest. Due to inexpensive methods, it is mostly used for the identification of the bioactive component present in the medicinal plants. It attract the researchers to explore the standardization of the herbs and developed monograph for them. Further, based on the rules of various regulatory units regarding the control in quality and verification of herbs meant for therapeutics, nutrition and spices, the clinical or actual usage are determined. These also consider the ethnobotanicals psychoactive with unlawful or unauthorized highs under forensic investigation based on criminal laws. Above mentioned are the added areas for TLC screening usage involving illicit category of products that lie under law of criminal consideration⁴.

Medicinal plant Cassia tora is widely distributed and well known in Indian continent. According to a study various plant parts of this species i.e. seeds, leaves, roots etc. are reported for use in native therapy in different clinical conditions⁵. According to traditional Indian Ayurvedic medicine, this plant is known for its use in many ailments such as sepsis, diabetes and menstrual disorders. It is also used to treat liver, eye and heart diseases and for its laxative and liver-strengthening properties^6,7. Components bioactive in Cassia tora along with anthocyanins, and antioxidants are also chiefly flavonoids and phenolics. Naphthalene and anthraquinone are also among active and most thought-about compounds in Cassia tora⁸. Furthermore, large number of antioxidant active ingredients like chrysarobin, campesterol, apigenin, achrosin, chrysophanol, emodin, and chryso-obtusin etc. have been separated from the mentioned species⁹. Additionally, flavonoids namely quercetin and gallic acid are responsible for the healing of the different diseases. Studies have shown that Cassia tora contain higher concentration of flavonoids and polyphenols compounds. However, no specific low-cost methods reported for the determination of the quercetin and gallic acid present in Cassia tora. Therefore, utilizing TLC, it was planned to separate and identify the gallic acid and quercetin found in the hydroalcoholic extract of Cassia tora.

MATERIALS AND METHODS:

Collection of plant material:

The aerial parts of the Cassia tora was collected for the present work. The plant parts were shade dried and made coarsely powdered for further studies.

Preparation of extract:

250 gm of drug powdered was used for the preparation of petroleum ether and hydroalcoholic (ethanol: water: 70: 30) extract employing maceration method.

Phytochemical investigation:

The phytochemical study of the petroleum ether extract and the hydroalcoholic extract was carried out. The existence of a number of components mentioning few i.e. phenolics, alkaloids, carbohydrates, flavonoids, saponins, fats and oils etc. was studied in the extract^10-12.

Thin layer chromatography or TLC:

Hydroalcoholic extract was employed for separating gallic acid and quercetin. Preparative silica gel 60F254, 7X6 cm plate was used as a stationary phase. The mobile phase Ethyl acetate: toluene: formic acid (5:7:1) and Ethyl acetate: toluene: formic acid (4:5:1) were used for separation of, gallic acid along with quercetin, respectively. Then pure quercetin with extract were spotted on the first plates using a capillary tube, while second plate was spotted with gallic acid and extract. The plates were immersed in the mobile phase chamber, and after the samples had passed through, the plates were dried. The dried plates were observed under normal light, short and long UV light^13-16. The distance travelled by the active components on the plates was detected by their R_f i.e. retention factor. For above samples, the value of R_f calculated using the following formula:

Distance traveled by solute

Rf = -------------------------------------

Distance traveled by solvent

RESULTS AND DISCUSSIONS:

Phytochemical screening:

In table 1, detailed information regarding the phytochemical properties of petroleum ether and hydroalcoholic extract is offered. The hydroalcoholic extract was found to contain glycosides, alkaloids, phenolic compounds, carbohydrates, saponins, tannins, and flavonoids, as were revealed by the findings of the phytochemical screening. Steroids, lipids and oils were found in the petroleum ether extract (Table 1).

It is well known that number of phytochemicals advantageous for health are procured from plants eg. carbohydrates, alkaloids minerals, phenols, vitamins, flavonoids, etc. Health care systems native and essential, majorly depend of medicines from plant origin owing to their ampleness in secondary metabolites and phytochemicals, across the globe. Based on various pharmacological studies of the plant obtained phytochemicals, now and then in past, eg., anthocyanins, and terpenoids, phenolics, and flavonoids, etc. have been recognized and separated^17,18. The phytochemical profile of a plant was investigated to determine its active components. They help guide further investigations and isolation of specific compounds for detailed characterization and bioactivity studies. Additionally, these studies can aid in the QC (quality control) and calibration of medicines that are herbal including natural products, ensuring their safety and efficacy.

In order to overcome the issues of side effects, in present modern time, detailed study of different plant based phytoconstituents with their respective significance can be very useful and fascinating stream. Based on the increased demand for straightforwardly available saponin sources, that can be studied in labs and used as a source for synthesizing therapeutics purpose sterols, number of plants have been researched^19,20. Hypoglycemic conditions can be affected by certain phytochemicals eg. polyphenolics, alkaloids, saponins, flavonoids etc. existing in plants.

Polyphenolic compounds are known for their diversity of biological properties, which could explain the possible antioxidant. Positive impact on Health by flavonoids and poly-phenolic consist of activity damaging free radicals, contrasting cardiovascular (CV) ailments, diabetes, hyperlipidemia, hypertension, geriatric issues, and hindering inflammation and allergies^21,22. Hence, the phytochemical study of the present study suggested significant medicinal uses of Cassia tora particularly hydroalcoholic extract.

Table 1: Phytochemical screening of the Cassia tora extract

Test	Petroleum ether extract	Hydroalcoholic extract
Glycosides	-	+
Alkaloids	-	+
Phenolic compound	-	+
Carbohydrates	-	+
Tannins	-	+
Flavonoids	-	+
Saponin	-	+
Proteins	-	-
Steroid	+	-
Fats and oil	+	-

Thin layer chromatography or TLC:

TLC has been used for separating quercetin and also gallic acid from the Cassia tora hydroalcoholic extract. Quercetin was separated using system of solvent composed of ethyl acetate, toluene, and formic acid in 4:5:1 ratio. Under extended UV light, the extract showed nine distinct spots for values of R_f at 0.10, 0.46, 0.50, 0.56, 0.62, 0.68, 0.74, 0.84 and 0.96. The seven different spots for values of R_f at 0.10, 0.50, 0.56, 0.62, 0.68, 0.74 and 0.96 were observed under short UV light. Similarly, five spots for values of R_f at 0.56, 0.62, 0.68, 0.74 and 0.96 were seen under normal light (Table 2). The pure quercetin showed single spot at R_fvalue 0.62. The R_fvalue of standard quercetin established existence of quercetin in extract examined.

The gallic acid was separated from the Cassia tora hydroalcoholic extract using system of solvent composed of ethyl acetate, toluene, and formic acid in 5:7:1 ratio. The extract demonstrated eight spots with R_f values of 0.08, 0.34, 0.40, 0.46, 0.62, 0.70, 0.82 and 0.94 under long UV light. The six different spots with R_f values of 0.48, 0.52, 0.62, 0.70, 0.90 and 0.94 under short UV light, while five spots with R_f values of 0.52, 0.54, 0.62, 0.70 and 0.94 under normal light (Table 3). The standard gallic acid showed spot at R_f 0.34 under long UV, short UV and normal light, confirming gallic acid in examined extract.

The TLC technique is considered the most effective approach for the identification and quantification of the bioactive constituent inside a plant extract. The process of isolating components from a mixture by TLC is straightforward and facilitates the interpretation of results. This approach is cost-effective and user-friendly. The chromatographic analysis of the extract of Leea indica, Tinospora crispa and Phyllanthus niruri was conducted by Patel et al., Mahalle et al. and Chhawari et al, respectively. Using same mobile phase solvent both the investigations noted existence of quercetin and gallic acid^23-26. Two bands with values of R_f 0.11 and 0.38 were obtained for flavonoids from C. bispinosa, two with values of R_f 0.63 and 0.81 for G. bicolar and two with values of R_f 0.094 and 0.81 for F. sycomorus (Gwatidzo). Applying TLC, from various Alchemilla species flavonoids with R_f: 0.77, 0.70, 0.72, 0.44, 0.84, 0.65, found were vitexin, orientin, isoquercetin, rutin, quercitrin, hyperoside, (Kaya). Enhydrae fluctuans extract in ethanol when studied by TLC confirmend flavonoid with R_f0.65 (Pahari). Henceforth, the results above support existence of quercetin and gallic acid in Cassia tora.

Table 2: R_fof hydroalcoholic extract of Cassia tora extract in quercetin estimation

Mobile phase	Samples	Number of spots	R_f value
Ethyl acetate: Toluene: Formic acid (4:5:1)	Pure quercetin	Long UV light = 1	0.62
		Short UV light = 1	0.62
		Normal light = 1	0.62
	Hydroalcoholic extract	Long UV light = 9	0.10, 0.46, 0.50, 0.56, 0.62, 0.68, 0.74, 0.84, and 0.96
		Short UV light = 7	0.10, 0.50, 0.56, 0.62, 0.68, 0.74, and 0.96
		Normal light = 5	0.56, 0.62, 0.68, 0.74, and 0.96

Table 3: R_fof hydro-alcoholic extract of Cassia tora extract in gallic acid estimation

Mobile phase	Samples	Number of spots	R_f value
Ethyl acetate: Toluene: Formic acid (5:7:1)	Pure gallic acid	Long UV light = 1	0.34
		Short UV light = 1	0.34
		Normal light = 1	0.34
	Hydroalcoholic extract	Long UV light = 8	0.08, 0.34, 0.40, 0.46, 0.62, 0.70, 0.82, and 0.94
		Short UV light = 6	0.48, 0.52, 0.62, 0.70, 0.90, and 0.94
		Normal light = 5	0.52, 0.54, 0.62, 0.70, and 0.94

CONCLUSION:

The phytochemical study found that the hydroalcoholic extract of Cassia tora contained flavonoids and polyphenol compounds, while petroleum ether extract did not. The TLC was performed on hydroalcoholic extract and it confirmed the presence of gallic acid and quercetin. Due to its cost-effectiveness and ease of use, TLC is the optimal choice for separating gallic acid and quercetin. The potential of TLC to be used in the coming years depends on its ability to identify other flavonoids found in medicinal plants. In the future, Cassia tora will undergo extensive in vitro and in vivo pharmacological studies to explore its antioxidant effects.

CONFLICT OF INTEREST:

None.

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Received on 11.03.2024 Modified on 29.05.2024

Research J. Pharm. and Tech 2024; 17(10):4847-4850.

DOI: 10.52711/0974-360X.2024.00745