Screening of phenolics and flavonoids using FTIR and UV-Vis: Antioxidant activity and HPLC quantification of gallic acid and ellagic acid

Tarun Kumar Patle¹, Kamlesh Shrivas^1,*, Reena Jamunkar², Antresh Kumar³, Khushali Tandey¹

¹School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur-492010, CG, India

²Department of Chemistry, Government Nagarjuna Post Graduate College of Science, Raipur-492010, CG, India

³Department of Biochemistry, Central University of Haryana, Jant-Pali Mahendergarh-123029, HR, India

*Corresponding author: kshrivas@gmail.com

Abstract. Green leafy vegetables play a crucial role in the diet of the tribal communities in Central India, offering significant nutritional and medicinal benefits. This study investigates the phytochemical composition of Cordia dichotoma, Hibiscus sabdariffa, Marsilea vestita, and Portulaca oleracea, focusing on phenolic and flavonoid content using ultrasound-assisted extraction (UAE). Various UAE parameters, including vessel position, depth, frequency, time, and solvent ratio, were optimized to enhance extraction efficiency. Antioxidant activity was assessed using UV-Vis spectroscopy, while gallic acid and ellagic acid were quantified via reverse phase-high performance liquid chromatography-diode array detector (RP-HPLC-DAD). Fourier-transform infrared (FTIR) spectroscopy identified functional groups and analyzed polyphenol composition in the extracts. The results showed that UAE significantly improved the yield of total phenolic content (TPC) and total flavonoid content (TFC). H. sabdariffa exhibited the highest TPC (16.897±0.052 mg GAE/g) and TFC (92.522±0.081 mg RUE/g), while C. dichotoma had the lowest levels. DPPH radical scavenging activity ranged from 80.648±1.332% to 89.416±1.753% at 10 µg/mL extract, confirming strong antioxidant potential. This study highlights UAE as a sustainable method for extracting bioactive compounds, reinforcing the nutritional and pharmacological value of these vegetables as natural antioxidants for health benefits.

Keywords: Ultrasound-Assisted Extraction, Phenolics and Flavonoids, Green Leafy Vegetables, Antioxidant Activity, Phytochemical Profiling

Introduction

Green leafy vegetables are frequently classified based on their edible parts like leaves, stems, flowers, or fruits. The leaf, aerial, and whole part of green leafy vegetables of tribal region of Chhattisgarh region, which is locally known as “bhaji” are eaten as a food. Some leafy vegetables from the Chhattisgarh region are Chenopodium album (bathua bhaji), Cordia dichotoma (bohar bhaji), Hibiscus sabdariffa (amari bhaji), Ipomoea aquatic (karmatta bhaji), Ipomoea batats (kandabhaji), Marsileavestita (chunchunia bhaji), Portulaca oleracea (nuniya bhaji), etc. widely consumed in the regular diet (Chauhan, Shrivastava, & Patra, 2014; Sharma, Sangeeta Bajpai, Swati Shrivastava, & Kanungo, 2014; Shrivastava, Patra, & Chauhan, 2017; Tamrakar, Arora, & Arora, 2017). These plants are enriched with alkaloids, flavonoids, phenolics, terpenoids, and having the antidiabetic, antioxidant, antibacterial activity, anthelmintic activity, anti-inflammatory, antihypertensive, hemostatic activity, immunomodulatory activity, anti-analgesic activity, antiulcer activity, and cardiovascular activity (Chauhan, Shrivastava, & Patra, 2014; Tamrakar, Arora, & Arora, 2017). But here is lack of evidence regarding phenolic and flavonoids contents in green leafy vegetables of Chhattisgarh region.

     The criteria of consuming food energy are now shifted towards food that provides natural phytochemicals (phenolic compounds, flavonoids, alkaloids, etc.) along with energy and several health benefits including antioxidants (Bayrambaş, Çakır, & Gülseren, 2019; Suwanwong & Boonpangrak, 2021). The green leafy vegetables are an important source of natural components of a healthy diet, and their regular consumption has a significant effect on human health; which is due to the presence of antioxidant, vitamins, minerals and fibers (Alarcón-Flores, Romero-González, Martínez Vidal, Egea González, & Garrido Frenich, 2014; Sun, Mu, Xi, & Song, 2014). Nowadays, synthetic and artificial antioxidants have been widely supplemented in food and vegetables which have several short- and long-term side effects on human health (Zahid, Ahmed, & Khan, 2018). So, there is a need to find an alternative source of natural antioxidants which plays a significant role in elimination of body oxidants. In this circumstance, the consumers are looking towards the bioactive rich green leafy vegetables in their regular diet to reduce the body toxins. The active ingredient of green vegetables gains intense attention as a subject of research to discover the potential benefits of green leafy vegetables.

    Several extraction methods are employed to isolate phenolics and flavonoids from leafy vegetables, each with distinct advantages and limitations. Conventional methods such as Soxhlet extraction and maceration are widely used due to their simplicity and effectiveness (Alara, Abdurahman, & Ukaegbu, 2018; Chua, Abd Wahab, & Soo, 2023). Maceration involves soaking plant materials in solvents at room temperature for an extended period, allowing the diffusion of bioactive compounds. However, it requires long extraction times and high solvent consumption. Soxhlet extraction, on the other hand, continuously cycles fresh solvent through the sample, enhancing extraction efficiency but requiring prolonged heating, which may lead to the degradation of heat-sensitive phenolics and flavonoids. Modern techniques like pressurized liquid extraction (PLE) (Machado, Portugal, Kodel, Fathi, Fathi, Oliveira, et al., 2024) and supercritical fluid extraction (SFE) (He, Shao, Liu, & Ru, 2012) offer improved efficiency and sustainability. PLE uses high temperatures and pressures to enhance solubility and diffusion, leading to faster and more efficient extraction with reduced solvent usage. SFE, particularly with supercritical CO₂, provides a green alternative by avoiding toxic solvents while maintaining high selectivity and preserving bioactive compounds.

   Among these, ultrasound-assisted extraction (UAE) is gaining popularity due to its efficiency, reduced processing time, and lower solvent consumption. UAE of phenolics and flavonoids relies on acoustic cavitation, which generates localized high pressure and temperature, disrupting plant cell walls and enhancing solvent penetration. This accelerates mass transfer, facilitating the release of bioactive compounds while minimizing thermal degradation and solvent consumption. UAE is particularly effective for extracting phenolics and flavonoids due to its rapid processing, high yield, and preservation of antioxidant properties (Khursheed, Khalil, Akhtar, Khalid, Tariq, Alsulami, et al., 2024; Nawawi, Khushairi, Ijod, & Azman, 2025; Saeed Abadi, Eghlima, Mirjalili, & Ghorbanpour, 2025). Khursheed et al. highlighted ultrasonication as an effective technique for separation bioactive biomolecules from medicinal plants. This study examined its impact using five solvents (water, hexane, methanol, chloroform, and ethyl acetate) on total phenolic and flavonoid contents, antioxidant activities, and triterpenoids in C. asiatica leaves. Methanol-based ultrasound extraction yielded the highest bioactive content. Ultrasonication with methanol significantly improves the separation of valuable molecules from C. asiatica (Khursheed, et al., 2024). Abadi et al. explored UAE of phenolics from Equisetum arvense L., analyzing the effects of temperature (20, 40, and 60 °C), sonication time (6, 10, and 14 min), and ethanol concentration (0, 50, and 100%). The response surface methodology optimized the process, identifying ideal conditions: 6.91 min, 27.88 °C, and 56.62% ethanol. These parameters yielded total phenolic content (3.938 mg GAE/g DW), isoquercitroside (2.567 mg/g DW), and FRAP antioxidant activity (0.007 mmol Fe²⁺/g DW), demonstrating enhanced bioactive compound extraction (Saeed Abadi, Eghlima, Mirjalili, & Ghorbanpour, 2025). A study examined the impact of water bath and ultrasonication extraction time on TPC, TFC and anthocyanins. Using 50% ethanol, ultrasonication at 50% amplitude for 10 min yielded 27% more anthocyanins than water bath at 20 min. The highest TPC and TFC were observed at 20 min, while ultrasonication for 25 min resulted in significantly higher antioxidant activity (DPPH: 52.76%, FRAP: 352.60 µmol TE/g) (Nawawi, Khushairi, Ijod, & Azman, 2025). Thus, ultrasonication showed an efficient technique for extracting bioactive substances, with methanol showing superior results. Optimized sonication conditions enhance phenolic and flavonoid yields, demonstrating its potential for improved antioxidant extraction across various plant sources.

      This study investigated the phenolic and flavonoid content in green leafy vegetables (Bhaji) from the tribal region of Central India using UAE. The research optimized UAE parameters, including vessel position, depth, frequency, time, and solvent ratio, to improve the separation efficiency of bioactive compounds. Furthermore, the study evaluated the antioxidant activity of extracts using UV-Vis and FTIR spectrophotometry and quantified gallic acid and ellagic acid via RP-HPLC-DAD. This work aimed to assess the potential health benefits of these traditionally consumed leafy vegetables and highlight their role as natural sources of antioxidants. By developing an eco-friendly extraction method, this study contributed to the field of natural product chemistry, providing insights into the nutritional and pharmacological significance of these indigenous plants.

Materials and methods

Materials

Gallic acid, ellagic acid, methanol, dimethyl sulfoxide (DMSO), and acids (HCl, H2SO4) were purchased from Merck Inc. (Mumbai, India). The aluminum chloride (AlCl₃), ascorbic acid, 1,1-diphenyl picrylhydrazyl (DPPH), ferric chloride (FeCl3), Folin-Ciocalteu reagent (FCR), lead acetate (Pb(OAc)₂, mercuric chloride (HgCl₂), sodium hydroxide (NaOH), potassium iodide (KI), sodium carbonate (Na₂CO₃) and sodium nitrite (NaNO₂) were obtained from Hi Media (Mumbai, India). The HPLC grade solvents such as acetic acid, methanol and water were used as a mobile phase obtained from Avantor Performance Materials Ltd. (Gurgaon, India).

 Sample collection and preparation for analysis of phytochemicals 

The different plant samples such as C. dichotoma, H. sabdariffa, M. vestita, and P. oleracea were collected from Raipur district, Chhattisgarh, India. The plants are identified according to their physical characteristics as reported earlier (Chauhan, Shrivastava, & Patra, 2014; Sharma, Sangeeta Bajpai, Swati Shrivastava, & Kanungo, 2014; Shrivastava, Patra, & Chauhan, 2017; Tamrakar, Arora, & Arora, 2017). Additionally, Table 1 presents plant identification data obtained from the Integrated Taxonomic Information System and Plant List (Guala, 2019; List, 2019) and International Plant Names Index (IPNI). The collected plant samples were dried at room temperature for several days to evaporate the moisture contents and samples were grinded into fine powder using mortar and pestle.

    The phytochemical extraction was performed using the ultrasound-assisted method described earlier (Patle, Shrivas, Kurrey, Upadhyay, Jangde, & Chauhan, 2020). Briefly, 1 g dry powder of plant samples was dissolved in 10 mL methanol for extraction at 29±2.3 °C using ultrasound-assisted extraction. The filtrate was recovered after passing through the Whatman paper (No. 42) and aspirated at 40 °C. The dried crude extract (phytochemicals) was stored at 4 °C for further analysis. The percentage yield of crude extract is calculated as follows:

UV-Vis and FTIR analysis of phytochemicals in extract of leafy vegetables

Phenolics, flavonoids, and other phytochemicals in crude extract of plant samples were monitored using UV-Vis in 200-800 nm wavelength range. In addition, FTIR spectral analysis of the crude extracts of C. dichotomaI, H. sabdariffa, M. vestita, and P. oleracea plant samples was performed using Bruker Alpha Eco-ATR (Bruker India Scientific Pvt. Ltd.) with zinc selenide (ZnSe) crystal cell in the range of 400-4000 cm^-1.

Estimation of total phenolic compounds (TPC)

The Folin-Ciocalteu (FCR) method was employed for the estimation of TPC in selected vegetables (Patle, Shrivas, Kurrey, Upadhyay, Jangde, & Chauhan, 2020). Briefly, 1 mL sample extract and 1 mL FCR (two-fold diluted) were mixed at room for 5 min, followed by the introduction of 2 mL 7.5% Na₂CO₃ into the solution. The reaction mixture volume was brought to a total of 10 mL using distilled water (Wallace, Chapman, Sullivan, & Bhardwaja). The absorbance of phenolics was measured at 765 nm after 1 h. of incubation time. The total phenolics content was measured with respect to the absorbance of gallic acid (standard). All measurements were taken in triplicates and total phenolic content was represented in mg of gallic acid corresponding to per gram of the dry plant sample (mg GAE/g samples).

Analysis of total flavonoids contents

The aluminum chloride assay was used to determine the total TFC in the selected green vegetable samples (Patle, Shrivas, Kurrey, Upadhyay, Jangde, & Chauhan, 2020). In this method, 1 mL of plant extract was combined with 0.3 mL of 5% NaNO₂ and allowed to react for 5 min. Subsequently, 3 mL of 10% AlCl₃ and 1 M NaOH were added, and the total volume was adjusted to 10 mL using distilled water. After 30 min of incubation, the absorbance was measured at 415 nm.

DPPH radical scavenging activity

To assess the antioxidant nature of active phytochemicals, DPPH free radical scavenging method was employed (Patle, Shrivas, Kurrey, Upadhyay, Jangde, & Chauhan, 2020). To evaluate free radical scavenging activity, varying concentrations (2–10 μg/mL) of plant extracts or ascorbic acid (1 mL) were combined with 2 mL of 0.004% (w/v) DPPH solution prepared in methanol. The mixture was then incubated in the dark for 30 min. After incubation, the absorbance was recorded at 515 nm. The antioxidant activity of the samples was determined using the following formula, and a graph between free radical scavenging and extract concentration was plotted for calculation of IC₅₀.

Chromatographic conditions for determination of gallic acid and ellagic acid

The methanol extract of plant samples was purified using silica gel column chromatography to isolate gallic acid and ellagic acid. A C-32 chromatography glass column, plain with sintered disk PTFE stopcock (10 x 150 mm) was used for fractionation of phytochemicals as the described method with small modification (Gini & Jeya Jothi, 2018). Briefly, the acetone rinsed column was packed with silica slurry prepared in hexane solvent. A thin layer of sand was added on top of the silica slurry before loading a 1 g crude extract sample. Low-to-high polar solvents (hexane, ethyl acetate and methanol) were used as a mobile phase with a flow rate of 5 mL/min by varying the ratio of hexane: ethyl acetate (100:0; 80:20; 50:50; and 30:70) and ethyl acetate: methanol (100:0; 80:20; 50:50; and 30:70). Eight fractions were collected from each solvent system for determination of gallic acid and ellagic acid.

For quantitative analysis of ellagic acid and gallic acid in plant extract, HPLC (Ultimate 3000, Thermo Fisher Scientific, Madison, USA) with DAD was used. A 20 µL extract sample/standard was injected into a C-18 reverse phase column (250×4.6 mm x 5µm; Thermo Fisher Scientific, Madison, USA) for purification and quantification of individual phenolics and flavonoids as per method described earlier with slight modifications. The purification was carried out using two different mobile phases termed as A (methanol: acetic acid: water: 10:2:88) and B (methanol: aceticacid: water:: 90:2:8) with 1 mL/min flow rate. These two mobile phase solvents were run for 42 min with a gradient program as follows: 100 % A (0-5 min), 15 % A (5-20 min), 50 % A (20-25 min), 30 % A (25-30 min), 0 % A (30-40 min), and for next 40-42 min 100 % A. The chromatogram of target molecules was observed at 280, 320, and 360 nm. The peaks for different concentrations of gallic acid and ellagic acid were used to draw the standard calibration curve for estimation of these phytochemicals in green leafy vegetables. 

Result and discussion

Optimization of ultrasound-assisted extraction of TFC and TPC

The physical factors such as ultrasound exposure time, frequency, position and depth of vessel are responsible for extraction of TFC and TPC from the plant source. These physical factors were optimized for efficient extraction of TFC and TPC from different plant samples such as C. dichotoma H. sabdariffa, C. dichotoma, M. vestita, and P. oleracea.

Position of ultrasonic vessel

The impact of the position of the tested sample within the ultrasonic bath on the cavitation effect, generated by the multiple transducers present in the ultrasonic bath, was systematically investigated in this study. To assess this phenomenon, five different positions within the ultrasonic bath were carefully selected. A 1:20 ratio of 1 gram of powdered sample mixed with the solvent was subjected to ultrasonic treatment at a frequency of 33 kHz for 10 minutes, with the sample tube positioned vertically 1 cm away from the bottom of the ultrasonic bath. The results revealed intriguing variations in the TPC and TFC based on the different sample positions. Specifically, the sample tubes positioned at 2 and 4 exhibited TPC values of 8.876±0.025 and 8.753±0.001 mg GAE/g sample, respectively, along with corresponding TFC values of 16.987±0.033 and 16.435±0.028 mgRUE/g sample, as detailed in Table 2.

    A noteworthy observation was the synergetic cavitation effect generated in the two vertical positions located within the zones of two transducers. This synergetic cavitation effect was found to create highly conducive conditions, leading to a more efficient extraction process and resulting in higher yields of both TFC and TPC. This finding aligned with earlier reports suggesting that vertical positions within the ultrasonic bath exhibit superior cavitation effects compared to horizontal and radial positions (Kulkarni & Rathod, 2014; Rao & Rathod, 2015). The strategic selection of the ultrasonic bath positions not only sheds light on the significance of cavitation effects but also provides a practical insight into optimizing extraction processes for achieving enhanced yields of phenolic and flavonoid compounds. The findings enhance the understanding of how ultrasonic bath parameters impact extraction efficiency, offering valuable information for researchers and practitioners in the field of sample preparation and extraction techniques.

Ultrasonic waves

The pivotal role of ultrasonic waves in the extraction of phytochemicals from plant samples was explored in this study, with a focus on two operational frequencies, namely 33 kHz and 44 kHz. The results, detailed in Table 2, showcase the impact of these frequencies on the extraction of TPC and TFC from the plant samples. Notably, the application of the 33 kHz frequency demonstrated a higher extraction efficiency, yielding a TPC of 8.876±0.025 mg GAE/g sample and a TFC of 16.987±0.033 mg RUE/g sample. In contrast, the use of the 44 kHz frequency resulted in lower concentrations, with TPC and TFC estimated at 7.065±0.035 mg GAE/g sample and 13.868±0.029 mg RUE/g sample, respectively.

    The superior extraction efficiency observed at 33 kHz can be attributed to the formation of a highly energetic state characterized by larger cavitation bubbles. These bubbles can readily penetrate and rupture the cell wall of the plant sample, facilitating the abundant release of phytochemicals. In contrast, at 44 kHz, the sound waves are scattered and attenuated, leading to the formation of weaker and smaller cavitation bubbles. These smaller bubbles, containing a limited amount of solvent, face challenges in penetrating the cell wall effectively, resulting in lower concentrations of both TPC and TFC. This finding aligns with previous research by Kulkarni and their research group (Kulkarni & Rathod, 2014), highlighting the frequency-dependent impact on cavitation and subsequent extraction efficiency. Understanding the influence of ultrasonic frequencies on the extraction process provides valuable insights for optimizing extraction conditions and enhancing the yield of phytochemicals. The results underscore the importance of selecting appropriate frequencies to achieve optimal cavitation effects and maximize the extraction of bioactive compounds from plant samples.

Effect of vessel depth

In the pursuit of optimizing the extraction of TFC and TPC from plant sources, the depth of the ultrasonic bath emerged as a critical parameter in this study. Different sample vessel depths (1.5 cm, 2.5 cm, and 3.5 cm) from the bottom of the ultrasonic cleaner were systematically investigated. The results, as detailed in Table 2, revealed that the most favorable yield of TPC (8.876±0.025 mg GAE/g sample) and TFC (16.987±0.033 mg RUE/g sample) was achieved when the sample vessel was positioned at a depth of 2.5 cm, compared to depths of 1.5 cm and 3.5 cm.

    The improved extraction efficiency at a vessel depth of 2.5 cm can be attributed to the heightened impact of sound waves, leading to the effective rupture of cell walls and subsequent release of phytochemicals. According to literature, the sound velocity in water is approximately 1500 m/s, and the wavelength is a product of velocity and frequency (33 kHz), as reported by Rao et al. (Rao & Rathod, 2015). The wavelength of the sound wave at a frequency of 33 kHz is determined to be 4.5 cm from the transducer. Importantly, sound waves are significantly influential at approximately half of the wavelength, which equates to around 2.3 cm above the transducer. Therefore, maintaining the sample vessel at a height of 2.5 cm ensures maximum separation of TFC and TPC, facilitating an efficient extraction process. This knowledge of how sample vessel depth impacts extraction efficiency offers a useful framework for optimizing experimental conditions. The findings underscore the importance of selecting an appropriate vessel depth to harness the full potential of ultrasonic waves in breaking down cell walls and extracting bioactive compounds from plant samples effectively.

Solvents and their ratio

The selection of solvents and their corresponding volumetric ratios in the extraction process is crucial in determining the efficiency of phytochemical extraction from plant samples. The permeability of phytochemicals from the cellular structure to the extracting solvent is directly influenced by the solvent polarity, and an increase in polarity enhances this permeability, as reported in studies by Rao and Kulkarni (Kulkarni & Rathod, 2014; Rao & Rathod, 2015). In this study, methanol emerged as the preferred solvent for extracting TFC and TPC from plant samples, given its optimal polarity for effective phytochemical extraction. The solute-to-solvent ratio is another critical factor influencing the extraction process. The concentration of TFC and TPC was observed to increase when the solute-to-solvent ratio was raised from 1:10 to 1:20, particularly at 33 kHz and positions 2 and 4, while maintaining the extraction vessel depth at 2.3 cm, as detailed in Table 2.

     Previous studies have consistently highlighted the direct dependence of phytochemical extraction on the solvent ratio. This dependence arises from the nature, specifically the polarity, of the solvent and its volumetric concentration, resulting in an increased yield of solute transfer (Kulkarni & Rathod, 2014). The concentration of solute in the solvent can influence the ultrasound wave scattering, potentially interrupting the formation of strong cavitation bubbles. As a general guideline, it is recommended that the ratio of solvent-to-solute be several folds higher for optimal recovery of TFC and TPC, emphasizing the importance of maintaining an appropriate balance between solvent and solute concentrations in the extraction process (Kulkarni & Rathod, 2014; Rao & Rathod, 2015). This detailed investigation into solvent nature and ratio provides crucial insights for refining extraction protocols. The findings underscore the significance of careful selection and optimization of solvents and their volumetric ratios to ensure efficient recovery of phytochemicals from plant samples, contributing to the overall success of ultrasonic-assisted extraction processes.

Time dependent effect of ultrasonic waves

The exposure period of ultrasonic waves is considered to be an important factor in extraction of bioactive compounds (Saeed Abadi, Eghlima, Mirjalili, & Ghorbanpour, 2025). The extraction of TFC and TPC from plant samples was carried out for 5, 10, 15, and 20 min of exposure time. As shown in Table 2, higher extraction of phenolics and flavonoids were obtained when extraction was kept for 10 min and further increase did not show any significant change. All the extractions were performed using 1:20 volumetric ratio of solute and solvent by placing the extraction vessel at 2.5-cm height at 33 kHz for 10 min of extraction time.

Table 2. Optimization parameter of ultrasonic bath for the extraction of TPC and TFC from green leafy vegetables.

± is standard deviation for three replicate results

    The optimized conditions and percentage yield (%) for extraction of TPC, TFC from the selected plant samples is summarized in Table 3. The crude extract yield was obtained in the range of 3.352±0.013 to 10.816±0.112%. The higher phenolic (16.897±0.052 mgGAE/g sample) and flavonoids content (92.522±0.081 mgRUE/g sample) were observed in H. sabdariffa. The concentrations of TPC in M. vestita, P. oleracea, and C. dichotoma were 12.112±0.036, 10.210±0.012, and 8.876±0.025 gGAE/g sample respectively; and the concentration of TFC were 40.131±0.054, 34.608±0.042, and 16.987±0.033 mgRUE/g, respectively. Earlier study revealed that calyx of H. sabdariffa is rich in phenolic and flavonoids showing various pharmacological effects and used as a traditional medicine (Borrás-Linares, Fernández-Arroyo, Arráez-Roman, Palmeros-Suárez, Del Val-Díaz, Andrade-Gonzáles, et al., 2015; Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Formagio, Ramos, Vieira, Ramalho, Silva, Zárate, et al., 2015; Gini & Jeya Jothi, 2018; Izquierdo-Vega, Arteaga-Badillo, Sánchez-Gutiérrez, Morales-González, Vargas-Mendoza, Gómez-Aldapa, et al., 2020; Wang, Cao, Jiang, Qi, Chin, & Yue, 2014).

    Similar studied has also been carried out in the fruit of C. dichotoma called as an "Indian cherry" showed the 5.52±0.37 gGAE/100g of TPC and 2.47 ±0.33 g quercetin equivalent/100g of TFC, but is found absent in leaf sample (El-Newary, Sulieman, El-Attar, & Sitohy, 2016). The TPC (8.876±0.025 mgGAE/g sample) and TFC (16.987±0.033 mgRUE/g sample) in the leaf of C. dichotomais reported higher than its fruit part. In contrast, TPC and TFC concentration in P. oleracea were found to be 10.210±0.012 mgGAE/g sample and 34.608±0.042 mgRUE/g sample, respectively which is much higher than the reported literature (Siriamornpun & Suttajit, 2010). The selected leafy vegetables are enriched with phenolics and flavonoids are suggested to be responsible for antioxidant activity.

Table 3. Concentration of TPC, TFC, gallic acid, and ellagic acid in selected green leafy vegetables of Chhattisgarh, India and its antioxidant activity.

± is standard deviation for three replicate results

UV-Vis analysis of phenolic and flavonoids in plant extract

The UV-Vis absorption spectra analysis provides a detailed glimpse into the intricate chemical composition of the tested samples, offering valuable insights into the existence of various substances such as aromatic rings, chromophoric groups, polyphenols, amino acids, and lipids. These findings are crucial for understanding the molecular makeup and potential bioactive components within the plant extracts. The absorption peaks observed in the UV-Vis region were attributed to electronic transitions, specifically involving lone pair (n) electrons and pi (π) electrons. The distinctive absorption peaks of pure gallic acid at 265 nm and ellagic acid at 280 nm served as key markers, aligning with established literature references, including studies by Patle et al and Beshbishy et al. (Beshbishy, Batiha, Yokoyama, & Igarashi, 2019; Patle, Shrivas, Kurrey, Upadhyay, Jangde, & Chauhan, 2020). Additionally, the absorption peak at 320 nm indicated the presence of aromatic rings and chromophoric groups, shedding light on the molecular structures contributing to the absorbance characteristics in this spectral region.

   Further exploration of the electronic transitions within aromatic rings reveals a π -π* transition at 280 nm, with subsequent transitions in other rings manifesting in UV-Vis spectra between 300 and 600 nm. This nuanced understanding of the electronic transitions enhances our comprehension of the intricate molecular arrangements contributing to the observed absorption peaks. Specifically focusing on the extracts of C. dichotoma, M. vestita, and P. oleracea, the absorption band between 260 and 280 nm strongly suggested the presence of gallic acid. This is visually represented in Fig. 1, emphasizing the reliability of the spectral data. Notably, the absorption bands between 400 and 450 nm in these extracts point towards the concurrent presence of carotenoids, expanding the scope of identified compounds within the samples. The absorption peak at 676 nm across all four plant samples indicated the abundance of phenolic compounds. Phenolic molecules are renowned for their antioxidant properties, and their presence in the extracts underscores the potential health-related benefits associated with these plant samples.

   Consistency was observed in the absorption bands at 280 and 325 nm across all four plant samples, strongly indicating the presence of ellagic acid. This adds another layer of detail to the characterization of the chemical profile of the selected plant extracts, highlighting the reproducibility of results across different plant sources. Thus, the UV-Vis absorption spectra analysis hasd provided a comprehensive understanding of the chemical constituents within the tested plant samples. The identification of ellagic acid, gallic acid, aromatic rings, chromophoric groups, carotenoids, and phenolic compounds, contributes to the knowledge of the potential bioactivity and applications of these plant extracts in various fields, including medicine, nutrition, and agriculture.

Fig. 1. UV-Vis spectra of plant extracts and band in the range of 260-280 nm indicating the presence of gallic acid and 280 and 325 nm indicating the presence of ellagic acid in H. sabdariffa, C. dichotoma, M. vestita and P. oleracea

FTIR analysis of phenolic and flavonoids in plant extract

The FTIR study conducted in this research serves as a pivotal tool for gaining fundamental insights and chemical characterization of organic compounds, specifically phenolics and flavonoids present in plant extracts. This analytical approach facilitates the analysis of these bioactive compounds by examining their distinct vibrational spectra. To establish a reference for the identification of analogous vibrations in the tested samples, the FTIR spectra of gallic acid and ellagic acid were measured in the range of 4000-400 cm⁻¹, providing a control basis for comparative analysis, illustrated in Fig. 2(a) and 2(b).

     The FTIR spectra of gallic acid and ellagic acid reveal characteristic vibrational bands associated with specific functional groups. Notably, the hydroxyl (-OH) group exhibits a stretching band at 3500-3300 cm⁻¹, while the C-H stretching band is observed at 2900-2800 cm⁻¹. The carbonyl (C=O) group manifests a vibration band at 1690-1800 cm⁻¹. Further, phenols C-H and -OH deformations are represented at 1500-1150 cm⁻¹, and the vibrational spectra of Ar-H substituted -OH and C-O are evident at 1500-1400 cm⁻¹ and 1000 cm⁻¹, respectively. These observed vibrations are consistent with previous studies by Okur et al. (Okur, Baltacıoğlu, Ağçam, Baltacıoğlu, & Alpas, 2019), Lu et al. (Lu, Ross, Powers, Aston, & Rasco, 2011), da Silva et al. (da Silva, Prasniewski, Calegari, de Lima, & Oldoni, 2018), and Silva et al. (Silva, Feliciano, Boas, & Bronze, 2014), validating the reliability of the FTIR technique for the identification of functional groups.

Fig. 2(a). FTIR analysis of gallic acid (Standard) and (b) FTIR analysis of ellagic acid (standard).

     Moving to the FTIR spectra of the plant extracts of C. dichotoma, H. sabdarifa, M. vestita, and P. oleracea (Fig. 3), the selected spectral region of 1800-800 cm⁻¹ is highlighted, which is attributed to polyphenols based on earlier studies. This region is particularly selected for the spectral characterization of phytochemicals, such as gallic acid and ellagic acid, in the plant extract samples. The results exhibit vibrational spectra in the range of 1800-600 cm⁻¹, with prominent bands at around 1790-1700 cm⁻¹ (C=O), 1600-1500 cm⁻¹ (-OH deformation), 1400 cm⁻¹ (C-H phenols), and 1200-1000 cm⁻¹ (C-O). The presence of these characteristic bands in both the standard solutions and plant extracts strongly suggests the extraction of phenolic and flavonoid compounds from the selected plant samples. The observed consistency in the vibrational spectra between standard compounds and plant extracts provided the confidence in the accurate identification and characterization of these bioactive compounds using FTIR spectroscopy. Thus, the FTIR study offered a robust means of chemical characterization, revealing the presence of phenolic and flavonoid compounds in the plant extracts. The identified vibrational bands in the selected plant samples aligned with those of standard solutions, affirming the reliability of FTIR for the qualitative analysis of bioactive compounds in botanical extracts.

Fig 3. FTIR analysis of plant extract isolated from (a) H. sabdariffa (b) C. dichotoma (c) M. vestita, and (d) P. oleracea

 Determination of antioxidant activity of plant extracts

Antioxidant activity is closely linked to the concentration of phenolic compounds and flavonoids in the plant extract. To assess the antioxidant potential, the plant extract was tested for DPPH radical scavenging activity. The results demonstrated that the selected vegetable extract exhibited notable antioxidant activity. The optimum radical scavenging activity of standard ascorbic acid was found to be 5.04±0.25 µg/mL (Fig. 4) and equivalent to IC₅₀ value 5.43±0.28 µg/mL to the plant extract of H. sabdariffa extract.  Fig 5 (a-d) shows the UV-Vis spectra of different plant extracts (C. dichotoma, H. sabdariffa, M. vestita, P. oleracea) with their DPPH radical scavenging activity. The concentration of 10 µg/mL extract of C. dichotoma, H. sabdariffa, M. vestita, P. oleracea, and standard ascorbic acid scavenged the oxidative free radicals of 82.14±2.676 %, 89.41±1.753 %, 82.86±2.482 %, 80.64±1.332 %, 93.93±3.219 %, respectively (Table 3, Fig 6(a) and Fig. 7). The IC₅₀ value of plant extracts samples are shown in Fig 6(b).  IC₅₀ can be defined as the amount of extract/standard that inhibits the 50% of DPPH radicals. This indicated that the vegetable extracts had a good antioxidant activity against oxidative threats. The phenolics and flavonoids play a very important role in discrimination of different oxygen species (singlet and triplet), inactivation of radicals, and breakdown of peroxides. The antioxidants inhibit free radicals via formation of complex or hydrogenation compounds (Yan, Huang, & Zhu, 2020). 

    The antioxidant activity of the selected green leafy vegetables can be attributed to their high content of phenolic compounds and flavonoids (gallic acid, ellagic acid). Earlier studies showed that 1.6 mg/mL extract concentration of P. oleracea inhibits only 76.71±0.42 % DPPH.²⁷ The total antioxidant activity of fruit extract of C. dichotoma was 1 mg/mL to scavenge 88.73±2.50 % as well as IC₅₀ was 86.60 µg/mL, was less significant than leaf extract of the present investigation (Ibrahim, El-Newary, & Ibrahim, 2019). They also studied the superoxide scavenging capability from fruit which was able to scavenge 90.97±2.80 % at 1 mg/mL fruit extract. Similarly, the IC₅₀ value of different samples to inhibit 0.068 µM DPPH was 154.65 to 207.73 µg/mL (Wang, Cao, Jiang, Qi, Chin, & Yue, 2014). The different accessions were collected and tested for antioxidant activity, and there is a huge difference in antioxidant potential of the same plant due to the difference in accessions as well as differences in the concentration of antioxidant compounds.

Fig. 4. DPPH radical scavenging assay for antioxidant activity of ascorbic acid (standard) which ranges from 2-10 µg for DPPH radical scavenging.

Fig. 5. Concentration dependant analysis of antioxidant activity of phytochemicals isolated from (a) H. sabdariffa (b) C. dichotoma (c) P. oleracea, and (d) M. vestita.

Fig. 6. Determination of (a) % of DPPH radical scavenging activity and (b) IC₅₀ of methanol extract of  H. sabdariffa, C. dichotoma, P. oleracea, and M. vestita.

Fig. 7. Antioxidant analysis of tested plant sample, 2-10 µg of plant extracts such as (a) H. sabdariffa (b) C. dichotoma (c) P. oleracea, and (d) M. vestita have been used for determination of antioxidant activity showing the color change from purple to yellow.

Chromatographic estimation of gallic acid and ellagic acid

RP-HPLC was employed in this study to confirm the presence of gallic acid and ellagic acid in extracts obtained from green vegetables. As illustrated in Fig. 8(a-e) and Fig. 9(a-e), the chromatograms display peaks at retention times of 2.080 and 23.363 min, which corresponded to those of the standard solutions of ellagic acid and gallic acid, respectively. The quantification of these compounds was achieved through standard calibration curves (y = 2.955x + 1.129, R² = 0.997 for gallic acid and y = 0.186x + 0.336, R² = 0.982 for ellagic acid), utilizing the prescribed retention times. The concentration analysis revealed varying amounts of gallic acid in the vegetable extracts. Notably, H. sabdariffa and P. oleracea displayed higher concentrations of gallic acid (0.462±0.002 and 0.396±0.001 mg/100g, respectively) compared to C. dichotoma and M. vestita (0.128±0.001 and 0.216±0.003 mg/100g, respectively). Gallic acid, known for its potent antioxidant, anti-HIV, anti-inflammatory, antimicrobial, and antifungal activities, finds applications in skin care products and the leather industry (Ibrahim, El-Newary, & Ibrahim, 2019).

     Ellagic acid concentrations were also determined, with H. sabdariffa exhibiting the highest concentration (1.154±0.001 mg/100g) compared to M. vestita and P. oleracea (Table 3). Intriguingly, ellagic acid was not detected in the C. dichotoma extract. Previous research highlighted the red calyx of H. sabdariffa as the most phytochemically rich part of the plant, surpassing the composition found in leaves (Borrás-Linares, et al., 2015; Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Formagio, et al., 2015; Gini & Jeya Jothi, 2018; Wang, Cao, Jiang, Qi, Chin, & Yue, 2014). Furthermore, while various studies have explored the phytochemicals in the fruits of C. dichotoma, limited attention has been given to the leafy parts of the plant (Hatware, Sharma, Patil, Shete, Karri, & Gupta, 2018; Ibrahim, El-Newary, & Ibrahim, 2019). The concentration of gallic acid in C. dichotoma was found to be 0.128±0.001 mg/100g, contributing to its pharmacological activities. A study reported the concentration of gallic acid, rutin, quercetin, and myricetin in P. oleracea to be 2.78±0.28 mg/L, 47.38±3.69, 5.35±0.27, and 10.46±0.04 µg/g, respectively (Okur, Baltacıoğlu, Ağçam, Baltacıoğlu, & Alpas, 2019). These concentrations aligned with the findings of the present study regarding gallic acid and ellagic acid content.

    For M. vestita, limited data is available regarding gallic acid and ellagic acid content. However, a study reported the presence of quercetin derivatives in the plant (Wallace, Chapman, Sullivan, & Bhardwaja, 1984). The current investigation presented a significant strategy for the extraction, separation, identification, and quantitative estimation of phytochemicals, specifically gallic acid and ellagic acid, in plant samples. The results contribute to the expanding knowledge of the chemical composition of these green vegetables and their potential applications in various industries.

Conclusion

This study successfully demonstrated the efficiency of ultrasound-assisted extraction in enhancing the yield of bioactive compounds, particularly phenolics and flavonoids, from green leafy vegetables (Cordia dichotoma, Hibiscus sabdariffa, Marsilea vestita, and Portulaca oleracea). Among the tested vegetables, H. sabdariffa exhibited the highest total phenolic content (16.897±0.052 mg GAE/g) and total flavonoid content (92.522±0.081 mg RUE/g), while C. dichotoma had the lowest. The antioxidant potential, evaluated using DPPH scavenging activity, ranged from 80.648±1.332% to 89.416±1.753%, confirming the strong radical scavenging properties of these vegetables. RP-HPLC analysis identified significant levels of gallic acid (0.128±0.001 to 0.462±0.002 mg/100g) and ellagic acid (0.767±0.002 to 1.154±0.001 mg/100g), reinforcing their health benefits.

   The study highlights UAE as an eco-friendly and efficient technique for extracting antioxidants, promoting the use of green leafy vegetables as natural health supplements. However, limitations include the lack of a comparative study with conventional extraction methods and the need for in vivo validation of bioactivity. Future research should explore the bioavailability, metabolic pathways, and therapeutic applications of these bioactive compounds. Overall, this work emphasizes the nutritional and pharmacological importance of tribal leafy vegetables, advocating their integration into functional foods and nutraceuticals for improved health outcomes.

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