Phytobioactive and in vitro antioxidant analysis in leaf extract of Ficus carica L.

Introduction

Worldwide, human beings use diverse forms of conventional medicinal plants to predict phytochemically bioactive substances and therapeutic effects of aging, which can be metabolizing the cellular antioxidants. The chemo preventive measure of in vitro antioxidants is to detect the neurodegeneration volatility during the period of aging, which leads to delineating cognitive decline (Kuriyama et al., 2006) [11] . A medicinal plant such as Ficus carica L. (Moraceae: Family) possesses foremost metabolic components such as vitamins A, E, ascorbic acid, flavonoids, polyphenols, which are phenolic compounds and found to be rich in antioxidant potentials. The antioxidant substrates help to mitigate the oxidative stress and protect against diseases like gastric ulceration, diabetes (T2D), and neurodegenerative diseases. The antioxidant compounds source are phytochemicals such as the phenolics, ascorbic acid, flavonoids, coumarins, carotenoids, amino acids, saponins, and polyphenols present in different herbal plant species related to promoting antioxidant capacities (Abdulhafiz et al., 2020) [1] . Up-to-date medicine, phytochemicals, and in vitro antioxidant profiling are essentially reliable for the significant or biological effect on food supplements. Traditional complementary alternative medicines are primarily obtained from plants (Swilam and Nematallah, 2020) [18] . Ficus carica (L.) comes under Moraceae family, commonly known as figs, local regionally in Tamil it's called as அத்திப்பழம் its origin of phenolic compounds; proanthocyanidins, however, phenolic chemicals found in red wine and tea have lower phenol content than figs. (Li et al., 2007) [12] . Its root, leaves and fruit stand used in historic medicine to treat gastrointestinal (gastritis), respiratory (bronchitis), and atherosclerosis and as antispasmodic and anti-inflammatory activity. Fig leaf extracts from various plants show significant affluence in phytochemicals like flavonoids, phenolic acids, saponins, coumarins, carotenoids and polyphenols. These compounds have been quantified and used for their antioxidants. Antioxidants neutralize free radicals according to (Re et al., 1999) [15] , which can cause cellular damage and contribute to chronic diseases. Medicinal benefits Fig are antispasmodic, metabolic, respiratory and cardiovascular, it has various phenolic compounds, which enhance the anti-inflammatory properties and also improve the neuroinflammation a putative marker for neurodegeneration. Phytochemicals retain a extensive assortment of biological functions, which includes antioxidant, antiapoptosis, anticarcinogen, antiatherosclerosis, antiinflammation properties, endothelial capacity improvement, including angiogenesis inhibition. Phenolic compounds confirm the antioxidant capacity, which is essential in reducing the effect of free radicals and neutralizing oxidative stress. It is a substantial source of neurodegenerative diseases in pathogenesis for dementia like Alzheimer's, Parkinson's, schizophrenia, and psychosomatic disease Prior et al., 2005 [14] . Recently, more attention has been persistently focused on determining the phytochemicals and their metabolic effects, showing more antioxidants like polyphenols, flavonoids, and phenolic substances, particularly in crude extracts from traditional medicinal herbs (Christova-Bagdassarian et al., 2014) [5] . Most antioxidant phytochemicals have been found to have high neuroinflammatory and antimicrobial properties (Alabri et al., 2014) [3] . The present study analyses the scavenging of free radicals and antioxidants, which are DPPH, ABTS, FRAP & total antioxidant capacity, and phenolic metabolites such as flavonoids and polyphenols. Phenolic content can facilitate and protect the neuronal cells from aging and longevity, which also leads to supporting neurodegeneration.

Materials and Methods

The plant material was collected from the farm of Dhanalakshmi Srinivasan Agriculture Farm, Perambalur, during February 2025. The leaves of F. carica L. were dried instantly subsequent to harvest in the shade and well aired in the Biochemistry laboratory, DSAC, Perambalur in two weeks. Next, they were packed in plastic stacks and kept in a dark room overnight. After that the leaves were managed using a Soxhlet extractor using dissimilar solvents subjected to ethanol, ethyl acetate, and methanol. 50 g of powdered sample of fig leaves were weighed and soaked in 50 ml of three different solvents. Then the mixture was incubated in the orbital shaker at 40°C with 140 rpm for 48 hours. Now, the mixture was filtered through Whatman filter paper, and the filtrates were evaporated, concentrated in room temperature and stored at 4°C until further use.

Phytochemical analysis a) Total Phenolic Content (TPC)

The Folin-Ciocalteu reagent technique was used to determine the total phenolic content (Azhagu Madhavan et al., 2024) [16] . Its reagent 250 µl was combined with 100 µl of leaf extract. Let it sit at room temperature for five minutes. After adding 1.5 cc of 20% sodium bicarbonate, incubate for two hours. The UV spectrophotometer was used to detect the maximum absorption at 765 nm. μg of gallic acid equivalents (GAE)/mg of dry extract was used to express the results. As recommended by Azhagu Madhavan (et al., 2024) [16] , the Folin-Ciocalteu reagent was used to measure the total phenolic content [16] . Its reagent 250 µl was was mixed with an aliquot of 100 µl of the leaf extract. For five minutes, the mixture was left to stand at room temperature. Following the addition of 1.5 ml of a 20% sodium bicarbonate solution, the mixture was incubated for two hours. The UV spectrophotometer was used to record the highest absorption at 765 nm, for the dry extract, the results were reported as μg of gallic acid equivalents (GAE)/mg.

b) Total flavonoid contents (TFCs)

The total flavonoid content (TFCs) was measured based to aluminium chloride colorimetric technique Azhagu Madhavan (et al., 2024) [7] and catechin equivalents expressed as mg (CE)/g of the dried extract. The standard of catechin was used for the determination of total flavonoids and analysed at 510 nm, total TFCs were declared in terms of catechin equivalents (CE) in µg/mg.

c) Total Polyphenols

The Li et al. (2007) [12] technique was used to calculate the total amount of polyphenols, the 100 µL of each of the extract was taken and mixed Folin-Ciocalteu reagent 500 µL (10%). Afterward, four min, the 7.5% sodium carbonate (Na₂CO₃) solution 400 µL was added. Prior to the estimation of polyphenol content, the chemicals was adjusted through responding at room temperature (25 + 1 °C) for 2 hours to estimate the absorbance at 760 nm, total polyphenols was stated in terms of mg of gallic acid equivalents (mg GAE/g extract).

In vitro Antioxidant Assay a) ABTS radical scavenging assay

The total capacity of antioxidant of the extracts was analyzed according to Sumathy et al., 2024 [6] . ABTS [2, 2azinobis-3-ethylbenzothiazoline-6-sulfonic acid] radicals were generated through an oxidation reaction with potassium persulfate. The mixing of the ABTS + radical cation with potassium persulfate was kept for 16 hrs. under incubation at room temperature until further use. The ABTS assay was performed with leaf extract in addition to the reagent, which was diluted with 95% ethanol until the absorption measured reached maximum at 734 nm. The ABTS radical scavenging activity of the extract, compared to ascorbic acid, was used as standard and calculated.

b). Ferric reducing antioxidant power (FRAP)

According to Sumathy et al., 2023 [4] , plant leaf extract is assayed with FRAP reagent, which consists of TPTZ 10 mM in HCl 40 mM, FeCl₃20 mM, and sodium acetate buffer (pH 3.6) 250 mM. FRAP reagent was newly prepared through TPTZ solution mixing. A solution of FeCl₃, with acetate buffer, was prepared in a volumetric 1:1:10 a ratio, an aliquot of 100 µl of the extract solution was varied with FRAP reagent 900 µl, following a the incubation period at 37°C of 4 min in room temperature, the absorbance measurement was carried out at a wavelength of 593 nm, with the results compared to a blank control. The standard Butylated hydroxytoluene (BHT) was used as reference solution for FRAP assay, the results existed quantified and expressed as µg of BHT corresponding per mg of sample.

c). DPPH Radical Scavenging Assay

The potential antioxidant of leaf extracts of Ficus carica L. was evaluated in relation to hydrogen donation or free radical scavenging capacity, using stable radical DPPH (2,2'-diphenyl-1-picrylhydrazyl) as a reagent at 4 mg/100 mL concentration, the interaction with a proton donor, such as an antioxidant, this radical exhibits a colour change from purple to yellow, accompanied by a reduction in absorbance (Vinothini et al., 2017) [13] . The samples 50 µL in different concentrations obtained from DPPH solution 850 µL were added, in ten different test tubes. The readings at 517 nm were recorded after 20 minutes of incubation in dark, at room temperature of (25±1°C). Quercetin solution was used as positive control, the inhibition percentage of DPPH radical was determined using the following formula:

Where, AB denotes the absorbance of the control (-), and AE signifies the absorbance of the extract.

Data and statistical analysis

The plant extracts of Ficus carica L. were analysed in triplicate for phytochemicals, in vitro antioxidants, phenolic, flavonoid, and polyphenol content. The results of antioxidant activity and phenolic-related content are shown as mean±standard error (SE). The mean (n = 3) of phenolic compounds are shown with Duncan Multiple Range Test (DMRT) result for the probability value of less than 0.05. Using SPSS Statistics 21.0 software (SPSS Inc., Chicago, USA), data from phenolic content and in vitro antioxidant analysis were subjected to multiple range test (Tukey's test) and analysis of variance (ANOVA) (Daoud et al., 2021) [17] .

Results and Discussion

Phytochemical Analysis

Fig. 1 shows phytochemical analysis using different solvent methods evaluated based on the different assays against in vitro antioxidants, which are found to be more in ethanol extract (90%) compared to other solvents. Macerated or powdered leaves were treated with ethanol in herbal plants of Ficus carica L. Different concentrations were found to facilitate those with the maximum antioxidant activity, such as polyphenols, and also the DPPH and FRAP assay concentrations reveal the strongest anti-inflammatory and neurodegeneration effects in relation to brain health. Glutamine, polyphenols, total phenolic and flavonoid content are compounds that are more beneficial from the perspective of neuroinflammation due to their antiinflammatory and antioxidant properties (Fig. 2). DPPH activity is found to be as 98.8% and ABTS activity is 94.7% in fig leaf extracts, different analyte concentrations in vitro antioxidants showed higher concentration (Fig. 3). The restorative efficiency of these cellular oxidative stress molecules targets scavenging the free radicals and depends on their bio accessibility. , supporting the immune response, which scavenges the free radicals and helps in improving the neuroinflammatory, antidiabetic, antiarthritic, and antimicrobial properties. This phytochemical profile, potential neuroprotective effects and antioxidant properties of Ficus carica L was investigated during this study. The extract was found to be rich in bio-active compounds like flavonoids, phenolic acids, alkaloids and tannins (Christova-Bagdassarian et al., 2014) [5] . These phytobioactive constituents are widely known for its antioxidant and neuroprotective properties that contribute towards cognitive health and mitigate neurodegenerative processes (Agati et al., 2012) [2] . The antioxidant assays (e.g., DPPH, ABTS, FRAP) demonstrated significant free radical scavenging activity, suggesting that the extract can effectively neutralize oxidative stress-a key contributor to cognitive decline and neuronal damage according to (Prior et al., 2005; Re et al., 1999; Kahkonen et al., 1999) [14,15,10] . High total polyphenolic, phenolic, and flavonoid contents were correlated positively with antioxidant activity, supporting the concept that polyphenolic compounds are important contributors for the pragmatic effects in phytoactive substances (Ivanov et al., 2015) [8] . In the milieu of brain health, oxidative stress is ascertained with various neurological disorders, which include cognitive capability gradually impairing and having an affinity to turn down dementia-type Alzheimer's disease, schizophrenia, Parkinson's disease, and stroke. The bioactive and volatile compounds (Jun et al., 2011) [9] in the plant extract might be helpful for protecting neuronal integrity in reducing free radicals, reactive oxygen species (ROS), improving inner mitochondrial function, and varying in cellular antioxidants. Likewise, a few phytochemicals recognized in the leaf extract (e.g., saponin, coumarin, glutamine, polyphenols, flavonoids, quercetin, kaempferol, or phytocatechins) are known to be involved along the blood-brain barrier and make use of an undeviating effect on nerve cells. Further, assays in nerve cells using plant-based treatments followed by in vivo or in vitro (e.g., neuronal cytotoxic cell activity, lipid peroxidation, or acetylcholinesterase inhibition) support its potential as a neuroprotective agent.

Acknowledgement

International Journal of Advanced Biochemistry Research https://www.biochemjournal.com

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