Distribution

This species is frequently observed throughout the plains of India, and can even be sited at elevations of up to 2,400m in the Himalayas. It is primarily found in Bengal and Punjab but also in the southern regions of Sindh and the Deccan in India. This species typically inhabits sandy terrain.

Medicinal properties and applications of the plant

The L. procumbens plant possesses diuretic, soporific, and tonic properties, making it a valuable source of fodder for goats. In Bombay, it is also administered to buffaloes to enhance milk secretion. During times of scarcity, the leaves of the plant are consumed for their sand-binding properties. Locally, the leaves are added to curries, and the juice is used to alleviate rheumatic ailments. Moreover, the plant, with the exception of its roots, has insecticidal properties and is used to treat a variety of conditions, including inflammation, rheumatism, reproductive disorders, oxidative dysfunction in the kidney, and hormonal imbalances (Qureshi and Raza, 2008), and liver dysfunction (Khan, Khan, & Sahreen, 2011).

Research conducted on the topic of nutrition has discovered that it is comprised of a powerful blend of 2-methyl-resorcinol, synergic acid, salicylic acid, gallic acid, and vanillic acid (Shaukat, Siddiqui, & Nasim, 2003), which have anticancer, antioxidant, cardioprotective and neuroprotective effects (Balasundram et al., 2006; Middleton et al., 2000; Zhou, Luo, Li, & Luo, 2009). For centuries, people have used leaves for their numerous health benefits. They are believed to be effective in treating a variety of conditions, such as fever, toxemia, cancer, and swellings. A paste made from these leaves can be applied to rheumatism, boils, and other types of swellings. The entire plant, except for the root, is also used to treat kidney stones and body heat. In addition, the juice extracted from the leaves is known to be helpful in managing jaundice. Some people even mix the whole plant with ash and metal for even more potent effects against jaundice. Medicinal plants have been used for thousands of years as a natural source of medicine, contributing to the improvement of human life. Certain herbs, in particular, contain bioactive compounds like phenolic and polyphenolic compounds that are known to regulate the immune system. Herbs that are rich in flavonoids and phenolic content may also have anti-inflammatory and antioxidant properties (Bruneton, 1995; Tyler, 1994).

The World Health Organization (WHO) reports that herbal medicines are able to fulfill the health requirements of approximately 80% of the global population, particularly those residing in rural regions of developing nations. The literature cited in these articles focuses on the various pharmacological functions of phytochemicals (Khan et al., 2011; Khan, Khan, Sahreen, & Ahmed, 2012; Khan et al., 2012). This research on the pharmacognostic characteristics of Launaea procumbens (Roxb.) Amin can serve as a valuable tool in the identification of potential adulterants and in the standardization of future investigations. The study thoroughly explores the plant's phytochemical profiles, antioxidant potential, and GC-MS analysis of its aerial parts, drawing on literature review and ethnopharmacological significance. Further research and re-validation of its use would be advantageous.

Collection of plant sample

In July 2022, Launaea procumbens leaves and shoots were collected from the Banasthali Vidyapith campus and meticulously prepared both herbarium and voucher specimens. Subsequently, these specimens were officially deposited into the Department of Bioscience and Biotechnology and assigned the authentication number BURI-1629/2022. These valuable specimens are now housed within the Banasthali University Rajasthan India (BURI) Herbarium, which serves as a comprehensive resource for taxonomical and ethnobotanical information.

Preparation of plant material and extraction

The aerial parts of the plants were gathered and thoroughly washed with tap water and then autoclaved water to ensure they were free of any dirt. Next, the leaves and shoots were left to dry at room temperature for 10-15 days. Once completely dry, ground them up with an electronic grinder to create a fine plant powder. This powder was then stored in an airtight container at 4°C for future use (Mehra & De, 2017). An experiment was conducted using a Soxhlet assembly to extract compounds from 10 grams of powdered leaves and stems. Afterward, the sample was carefully placed in a Whatman filter paper thimble and inserted into the extraction chamber. Next, 150 ml of four different extraction solvents - methanol, chloroform, distilled water, and petroleum ether was added to the boiling flask and the Soxhlet apparatus was set to operate for 12 hours. Throughout the process, a constant ice-cold water flow was maintained in the condenser, and the heating mantle was set to keep the boiling flask at a temperature of 40-45 °C. Over the course of 20 to 25 cycles, the extraction solvent cyclically moved between the extraction chamber, the boiling flask, and the thimble. This movement was observed until the solvent in the extraction chamber became colorless.

Phytochemical Screening

To screen for phytochemicals, various solvents including methanol, chloroform, petroleum ether, and distilled water were used to prepare the extracts. Each solvent was evaporated at 40 ºC with a heating mantle. Using standard methods, some of the extracts were screened from each solvent to identify the different types of active chemical components. The findings were categorized as (++) indicating a significant amount of phytochemicals, (+) for a small amount, and (-) for the complete absence of such a compound (Table 1).

Total phenolic content (TPC)

The estimation of Total phenolic contents (TPC) was done using the specific method of Singleton and Rossi (1965). To thoroughly evaluate each fraction, 200 microliters of it (dissolved in its respective solvent with a concentration of 1-5 mg/ml) were combined with ten milliliters of 1:10 folin-ciocalteu reagent. Following a 5-minute incubation, 7 ml of 0.115 mg/ml Na₂CO₃ was introduced, and the solution was incubated for an additional two hours. To determine the absorbance readings at 765 nm, gallic acid was implemented as the standard in the calibration curve. The results were then reported in mg gallic acid (GAE)/gram of dried plant extract, and the data for each fraction was meticulously recorded three times.

Total flavonoid content (TFC)

To determine the Total flavonoid content (TFC) method described by Sakanaka et al., 2005 was used. To prepare the samples, 0.25 ml of each fraction was combined (at a concentration of 1-5 mg/ml dissolved in its respective solvent) and Rutin standard solution (15-250 μg/ml) with 1.25 ml of distilled water in a test tube. Next, 75 μl of a 5% (w/v) sodium nitrite solution was added and waited for 6 minutes. Then added 150 μl of 10% (w/v) aluminum chloride solution and allowed the mixture to sit for another 5 minutes. Finally, we added 0.5 ml of 1 M NaOH and thoroughly mixed the contents. The mixture was adjusted to 2.5 ml with distilled water before use. The absorbance was promptly taken at a wavelength of 510 nm, and the outcomes of the samples were reported in milligrams of Rutin equivalents for the complete dried fractions. In order to ensure accuracy, each fraction was subjected to three tests.

Antioxidant Assays

Statistical analysis

The experiments were performed thrice and the average along with the standard deviation was presented. To evaluate the variations in IC₅₀ of different antioxidant tests for various fractions, the ANOVA test was used with a minimum significance level (LSD) of P < 0.01. The experimental data was also examined for the correlation coefficient of phenolics and flavonoids with different antioxidant tests, using the student's test to establish significance (P < 0.05; P < 0.01). The graph pad prism software was used to compute the IC₅₀ values.

GC-MS analysis

The analysis of the Methanol extract of the leaf of L. procumbens was done by performing GC-MS analysis. The carrier gas used was helium at a consistent flow rate of 3.2ml/min, and an injection volume of 1μl was employed with a split ratio of 10:1. The scan mass range was 50m/z - 650m/z. To conduct the GC-MS analysis of methanol extracts of Launaea procumbens (Roxb.) Amin leaves, Thermo Scientific Triple quadruple GC-MS (trace 1300 GC, Tsq 8000 triple quadrupole MS) equipped with TG 5 MS (30 m 0.25 mm, 0.25 m) column was used. The ion source temperature was set at 230 °C, and the injector temperature was maintained at 25 °C. We kept the temperature in the oven at 50 °C. (Mehdi et al., 2020). An extensive review of the literature was conducted to identify the therapeutic benefits of phytoconstituents that were discovered through GC-MS analysis.

Phytochemical analysis

Several phytoconstituents were present in L. procumbens plant extracts are produced in different solvents. Methanolic plant extracts contain the highest amount of phenols, tannins, alkaloids, and flavonoids. At the same time, extracts made in chloroform had a lower quantity of tannins, flavonoids, steroids, phenols, and the least number of alkaloids. The cardiac glycosides are negligible in all the solvents. Little to no traces of selected variables were found in leaf and shoot extracts prepared with distilled water and petroleum ether (Table 2 ). Finally, the ideal outcome was achieved in the methanolic extract of plants.

Total phenol and flavonoid content

The presence of phenolics and flavonoid contents in different L. procumbens fractions are shown in Table 4; Table 3 . The maximum total phenolics concentration was found in the leaf methanolic extract (40 ± 0.0098) mg GAE/ whereas the lowest total phenolics content was found in the petroleum ether extract (14.81 ± 0.0015) mg GAE/g extract Methanolic extracts had the highest total flavonoid contents (33.1 ± 0.007) while petroleum ether extracts had the lowest concentration (16.51 ± 0.0075) mg equivalent Rutin/g of dry fraction.

Similarly in the case of shoot extracts the methanolic extracts possessed the highest total phenolics contents (37.24 ± 0.0084) mg GAE/g while petroleum ether comprised of lowest total phenolics content (8.5 ± 0.003) mg GAE/g extract. The highest amount of total flavonoids was found in methanolic extracts (27.41 ± 0.003) while the lowest concentration was found in extracts of petroleum ether (5.4 ± 0.002) mg equivalent Rutin/g of dry fraction. The extraction yield of these samples varied in descending order of methanol> chloroform > distilled water> petroleum ether fraction. For both leaves and shoots, the extraction yield with distilled water and petroleum ether was substantially lower (P<0.01) than that of the methanol and chloroform fractions, which produced the most significant amount of total extractable components.

DPPH assays

The evaluation of the scavenging activities of bioactive fractions in phytomedicine has frequently used the stable free radical DPPH. Using 1, 1-diphenyl 1-2-picryl-hydrazyl (DPPH) free radicals, the scavenging capabilities of different fractions of leaf and shoot extracts were assessed. (Table 5 ). Results showed that the methanolic extract of leaf (IC₅₀ 57.7 ± 0.06 μg/ml) possessed the highest antioxidant activity while extracts of petroleum ether had the lowest scavenging effect (IC₅₀ 95.08 ± 0.8 μg/ml). Similarly in the case of shoot extracts the methanol extract of leaf (IC₅₀ 52.74 ± 0.02 μg/ml) possessed the highest antioxidant activity while extracts of petroleum ether had the lowest scavenging effect (IC₅₀ 87.05 ± 0.95 μg/ml). The extracts of leaf and shoot prepared in other solvents i.e., chloroform and distilled water also didn’t show promising results. The more will be the IC₅₀ value lesser the antioxidant activity. Since the IC₅₀ value of methanolic extracts of leaf and shoot are less in comparison to other extracts, hence they show more antioxidant activity. The DPPH radical scavenging activities of the methanolic extracts were even less (P < 0.01) than those of ascorbic acid.

Correlation between total phenol and flavonoid contents, as well as the IC₅₀ values for antioxidant activity

Shows the association (Pearson correlation coefficient = r) between the IC₅₀ values and the extracts' total phenol and flavonoid content. TPC and TFC have a positive association, but IC₅₀ values and TPC/TFC have a negative correlation, indicating that a low IC₅₀ value indicates a high antioxidant capacity and vice versa.

GC-MS analysis of methanol leaf extract of Launaea procumbens (Roxb Amin)

Gas chromatography-mass spectrometry (GC-MS) stands as the most widely employed analytical technique for identifying organic substances in complex matrices. Chemo-profiling of leaf methanol extract of L. procumbens was done by GC-MS analysis. Peaks, phytoconstituents name, area %, retention time (RT), phytoconstituents class, and individual fragmentation patterns of phytoconstituents identified in methanol extract of Launaea procumbens by GC-MS analysis are tabulated in Table 6 . GC-MS analysis revealed the presence of a total of 9 phytoconstituents in leaf methanol extract of Launaea procumbens, which belongs to a different class of phytoconstituents like - aldehyde, ketone, phthalate, carbohydrate, and organic acid anhydride. Methyl stearate and trihydroxy boron were the main compounds in higher concentrations (pyridine). Hexadecanoic acid was the compound that was present in the least amount.