Most contemporary research on herbal medicine has focused on traditional folk remedies. Modern medicine has introduced a wide range of pharmaceuticals, none of which are non-toxic and safe for human consumption. Hundreds of medicinal plants have a long history of curative properties against various diseases and conditions. Nonetheless, it is crucial to thoroughly screen these plants for their efficacy, which requires immediate attention to determine their value. The screening process involves examining the biological activity of plants based on either chemotaxonomic analysis or ethnobotanical knowledge of a specific disease. Identifying a compound that is effective against a particular disease is complex and time-consuming. In India, approximately 2,500 plant species are utilised for medicinal purposes, with approximately 90% of these medicinal plants serving as the primary source of raw materials for herbal pharmaceuticals that are harvested from their natural habitats 1.

Coccinia indica (C. indica), commonly known as ivy gourd, belongs to the family Cucurbitaceae and grows abundantly and widely across India. Various parts of the plant have been used to relieve diabetes mellitus 2. In the Indian subcontinent, C. indica has been utilised in both Ayurveda and Unani medicine 3. It is also used as a wild vegetable by indigenous people of Southeast Asia and India. Every part of C. indica has valuable medicinal resources and is effective in treating ringworms, psoriasis, smallpox, scabies 4, and other itchy skin eruptions and ulcers. C. indica has antidiabetic 5, 6, 7, 8, 9, hypoglycaemic 8, 10, 11, anti-inflammatory 12, 13, 14, hepatoprotective 15, 16, 17, antioxidant 18, 19, antibacterial20, 21, antitussive 22, and analgesic 13 activities. The juice extracted from the roots is used to treat diabetes, whereas a tincture made from leaves is used to treat gonorrhoea. Additionally, a paste derived from the leaves is applied to the skin to treat various diseases 23. The dried bark of this plant has cathartic properties. Furthermore, the leaves and stems possess antispasmodic and expectorant properties. The fleshy green fruit of this plant is known for its bitter taste. It is also used to treat tongue sores by chewing 21, 24. The plant's active components are linked to its compounds, which are directly responsible for the plant's therapeutic properties, and are called active pharmaceutical ingredients (API). They are mainly secondary metabolites, such as alkaloids, steroids, tannins, and phenolic compounds, as well as flavonoids, resins, fatty acids, and gums, which can elicit specific physiological responses in the body 25.

The pharmaceutical industry is concentrating on the creation of novel medications and plant-based treatments by examining leads from the traditional Ayurveda medical system, which has been practiced for thousands of years. As the pharmacological importance and active phytochemicals involved have not been thoroughly investigated, our primary objective was to isolate the active compounds from the aerial parts of C. indica.

Extraction of Plant Material: The C. indica leaves were successively extracted with chloroform, methanol, and ethyl acetate in an extractor. Preliminary small-scale extraction was performed to determine the yield prior to pilot-scale extraction.

Extraction Process: Powdered leaves (200 g) were macerated with ethyl acetate (200 ml) for 3 h. This process was repeated three times, and the mixture was filtered to remove marc. The filtrate was concentrated to 2.5%. Similarly, powdered leaves were extracted with methanol (200 ml) and chloroform (200 ml) under the same conditions, yielding 3.0% and 5.0%, respectively. The column loading required extract was 100–200 g, and pilot-scale extraction was calculated as a percentage yield.

Purification: Dried aerial parts of C. indica (200 g) were extracted four times with methanol (500 ml) in a Soxhlet apparatus. The combined extract was filtered and the solvent was evaporated under reduced pressure, yielding 6 g of crude residue. The whole residue was extracted with chloroform (200 ml) four times using a Soxhlet apparatus, filtered, and concentrated to yield 4 g of crude residue. Because these compounds were more soluble in chloroform, column packing of the methanolic extract was not performed directly.

Column Chromatography: Column chromatography on a silica gel (60–200 mesh) stationary phase was performed on the chloroform extract. A glass column 120 cm in length and 10 cm in diameter was wet-packed with hexane. Dry hexane extract (4 g) was added to the column and elution was performed using a gradient of increasing polarity (hexane, benzene, chloroform, and methanol). The fractions were pooled and analysed using TLC. Fraction (58–61) gave a pale yellow crystalline solid (470 mg), which was further purified using HPTLC.

Thin Layer Chromatography (TLC): TLC was carried out using various solvent systems. The chloroform extract was analysed with a mobile phase of chloroform: methanol: ammonia (13.5:2.7:0.3), and spots were detected at 366 nm using an anisaldehyde-sulfuric acid spray reagent.

High-Performance Thin-Layer Chromatography (HPTLC): HPTLC analysis of the C. indica extract was performed on a CAMAG HPTLC system with a pre-coated silica plate (silica gel 60 F254, 10 × 10 cm, Merck). The three sample preparations were methanol-extracted, filtered, concentrated, and spotted. A mobile phase consisting of Chloroform: Methanol: Ammonia (13.5:2.7:0.3) was prepared and the chromatographic tank was saturated for 30 min. Samples were applied using a Linomat applicator, and the plate was left to evaporate the solvent prior to development. The plate was removed after ascending to 1.5 cm below the top, dried, and detected under UV 254 nm and anisaldehyde-sulfuric acid. (Figure 1 A and Figure 1 B)

The synthesised compounds were identified and characterised as follows.

Melting Point Determination: Conducted via Thiel’s tube method to assess purity.

Thin Layer Chromatography (TLC): Used for compound identification and reaction monitoring. Pre-coated silica plates with a Chloroform:Methanol:Ammonia (13.5:2.7:0.3) mobile phase were analysed under UV light.

Infrared (IR) Spectroscopy: Determined functional groups based on molecular vibrations. The spectra were recorded using a SHIMADZU FTIR 8400S spectrometer. (Figure 2 )

Nuclear Magnetic Resonance (NMR) Spectroscopy: Structural analysis was performed using a Bruker Spectrospin-400 NMR spectrometer with chloroform and DMSO as the solvents.

Mass Spectroscopy: This analysis provided molecular weight, structural, and fragmentation insights through electron bombardment ionisation. (Figure 3 )

The isolated compound was found to have a molecular formula of C₂₉H₅₀O with a molecular weight of 414.71 g/mol. It was a pale yellow crystalline solid that melted at 145°C and was soluble in both alcohol and chloroform. The Rf value was 0.55, and the yield was 470 mg.

The compound was characterised using melting point determination, thin-layer chromatography (TLC), infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and mass spectroscopy. The melting point was obtained using the Thiel melting point tube method to ensure purity. TLC analysis was conducted using chloroform and methanol (7:3) as the mobile phase to ensure the presence of the compound.

The IR spectral scan, which was done with KBr pellets, showed characteristic absorption bands at 3439.6 cm⁻¹ (O-H stretching), 2924.7 cm⁻¹ and 2867.9 cm⁻¹ (C-H stretching), 1631.6 cm⁻¹ (C=C absorption), 1410.3 cm⁻¹ (CH₂ bending), 1043.7 cm⁻¹ (cycloalkane), and 881.6 cm⁻¹, verifying the presence of functional groups in accordance with the expected structure.

The ¹H-NMR spectrum, which was obtained in CDCl₃, exhibited signals at δ 5.26 (H-6), 5.19 (H-23), 4.68 (H-22), 3.63 (H-3), and other typical peaks, confirming the suggested molecular structure. Other signals appeared between δ 0.69-2.00 ppm due to alkyl groups.

Mass spectroscopy scanning by Electron Spray Ionization (ESI) also validated the molecular ion peak at m/z 414 according to the molecular formula C₂₉H₅₀O. The fragmentation ion peaks at m/z 367, 271, 255, 229, 189, 175, 161, 133, 121, 105, 95, 81, 69, 55, and 41 provided additional structural confirmation.

Phytochemical screening of the C. indica extract showed the presence of carbohydrates, amino acids, phytosterols, triterpenoids, alkaloids, fats and oils, and flavonoids; however, tannins, phenols, vitamin C, and saponins were not detected.

Characterisation confirmed the successful isolation of the compound and provided clues to its structure and composition.

Structural Elucidation

The IR absorption spectrum exhibited absorption peaks at 3439.6cm‐1(O‐H stretching.); 2924.7 cm‐1and 2867.9 cm‐1 (aliphatic C-H stretching); 1631.6 cm‐1 (C=C absorption peak); additional absorption peaks are 1410.3 cm‐1 (CH2); 1043.7 cm‐1(cycloalkane) and 881.6 cm‐1. (Figure 4 )

1HNMR (CDCl3) of isolated compound: 1HNMR has given signals at δ3.2(1H, m, H‐3), 5.26 (1H, m, H‐6), 5.19(1H, m, H‐23), 4.68(1H,m,H‐22), 3.638( 1H, m, H‐3), 2.38(1H, m, H‐20), 1.8‐2.0 (5H, m) ppm. Other peaks are observed at δ 0.76‐0.89 (m, 9H), 0.91‐1.05 (m, 5H),1.35‐1.42 (m, 4H), 0.69‐0.73 (m, 3H), 1.8‐2.00 (m, 5H), 1.07‐1.13 (m,3H), 1.35‐1.6 (m, 9H) ppm.

¹H-NMR: 7.69-7.67 (2H, m, 2OH), 4.82-4.64 (1H, s, alkyl), 4.38 (1H, d, J=8 Hz), 3.86 (1H, m, alkyl), 3.74,61 (2H, m, alkyl), 3.52-3.11 (7H, m, alkyl), 2.75 (2H, t, J= 16 Hz), 2.51-1.55 (4H, m, alkyl), 1.22 (2H, s, alkyl), 1.11-0.75 (5H, m, alkyl).

FAB‐MS showed molecular ion peaks at 414, corresponding to the molecular formula C₂₉H₅₀O. Ion peaks were also observed at m/z 367, 271, 255, 229,189, 175, 161, 133, 121, 105, 107, 95, 81, 69, 55, 41.

Phytochemical analyses confirmed the presence of saponins, flavonoids, glycosides, steroids, tannins, and alkaloids. The varied pharmacological activities of the plant are due to its bioactive components, which are found to exert a variety of beneficial effects, including blood sugar control, prevention of cancer, pain, fever reduction, parasite inhibition, reduction of inflammation, relaxation of muscles, healing of wounds, protection against stomach, seizure prevention, liver protection, and immunomodulation 26 . Traditional healers in Kerala, India, normally use plant leaves and fruits to treat various medical problems, ranging from skin conditions to muscle weakness, infertility issues, and diabetes. The residents commonly use a paste made of C. Indica fruits and leaves are combined with milk to treat diabetes and with sugar to treat jaundice 1.

Several clinical trials have been conducted to prove the efficacy of C. indica in humans, and most of these trials focused on its antidiabetic activity due to the extensive history of the plant as a natural antidiabetic agent. Venkateswaran et al. 6 had an experiment to identify the effect of C. indica on Blood glucose and insulin. Administration of C. indica leaf ethanol extract to diabetic animals for 45 days resulted in a remarkable reduction in blood glucose and glycosylated hemoglobin content, with concomitant increases in total hemoglobin and plasma insulin levels. Therefore, the initial phytochemical tests are useful in identifying chemical constituents in plant material that might lead to their quantitative estimation, as well as for identifying the site of pharmacologically active chemical compounds. Quantitative estimation of proteins, phenols, and flavonoids provides an insight into their chemical nature, which might provide rich data for understanding some elementary patterns of growth and metabolism. Phytochemical indicators of C. indica, such as proteins, phenols, and flavonoids, can also act as chemical markers in taxonomic studies. They were separated via column chromatography/HPLC and characterised using analytical data through MASS and NMR spectroscopy.

This study was able to isolate and identify the bioactive phytoconstituents of Coccinia indica using chromatographic and spectroscopic methods. Structural elucidation was performed to validate the molecular structures and functional groups. These results enrich the knowledge of Coccinia indica phytochemistry to further support its future pharmacological applications and potential drug development.