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INTRODUCTION
Diabetes mellitus and inflammatory disorders represent two of the most widespread chronic health conditions worldwide and pose a significant burden on healthcare systems due to their long-term complications and management costs. These disorders are closely interconnected, sharing common pathological pathways such as oxidative stress, chronic low-grade inflammation, insulin resistance, and metabolic imbalance. Although several synthetic antidiabetic and anti-inflammatory drugs are currently available and effective in managing these conditions, their prolonged use is often associated with adverse effects, reduced patient compliance, and high economic costs. This has intensified global interest in the identification of safer, cost-effective, and plant-based therapeutic alternatives (Corathers et al., 2013).
Medicinal plants have long been recognized as valuable reservoirs of bioactive phytochemicals, including flavonoids, phenolic compounds, tannins, glycosides, and alkaloids, which exhibit diverse pharmacological properties. These secondary metabolites play a crucial role in modulating oxidative stress, regulating glucose metabolism, and suppressing inflammatory mediators. Notably, unripe fruits are reported to contain higher concentrations of these phytoconstituents compared to their ripe counterparts, owing to active metabolic processes during early stages of fruit development. This enhanced phytochemical richness makes unripe fruits particularly attractive candidates for pharmacological and nutraceutical research (Tran et al., 2020).
Ficus carica (fig) and Phyllanthus emblica (Indian gooseberry) are well-established medicinal plants extensively used in traditional systems of medicine for the management of metabolic and inflammatory disorders. The fruits of F. carica are rich in phenolics and flavonoids that influence carbohydrate-digesting enzymes, improve glycemic control, and attenuate inflammatory responses. Similarly, P. emblica is renowned for its high content of hydrolysable tannins, flavonoids, and vitamin C, which collectively contribute to its potent antioxidant, antidiabetic, and anti-inflammatory activities (Chakraborty et al., 2026; Sharma, 2022; Huang et al., 2023).
The phytochemical composition and biological activity of medicinal plants are influenced by geographical and climatic conditions. West Bengal offers a diverse environment that may affect the phytochemical profile of these fruits; however, limited studies have focused on unripe fruits grown in this region (Acharya, 2016).
Therefore, the present study aims to perform comprehensive phytochemical profiling of unripe fruits of Ficus carica and Phyllanthus emblica collected from West Bengal and to evaluate their antidiabetic and anti-inflammatory potential using relevant in vitro assays.
MATERIALS AND METHODS
Chemicals required
All chemicals used were of analytical grade and were procured from authorized suppliers. The chemicals were procured from Emplura and Emparta (Chennai). The standard chemicals were procured from Sigma-Aldrich, including quercetin, gallic acid, tannic acid, aspirin.
Instruments used
The instruments used included a UV-Visible Spectrophotometer (JASCO Corporation, V-630, Tokyo, Japan); Digital Weighing Balance (Wensar Weighing Scales Ltd., PGB-200, Maharashtra, India); pH meter (SYSTRONICS, 335, Ahmedabad, India); Incubator (Remi Elektrotechnik Ltd., Mumbai, India) and Hot Water Bath (SSFW, LWB-12H/D, Kolkata, India).
Fruit sample collection
About 25 fresh unripe fruits of each Fig (Ficus carica) and Amla (Phyllanthus emblica) procured from local market of Kanchrapara, West Bengal, India in September 2025. The unripe fruits washed properly, kept in shade dry and powdered.
Fruit extract preparation
About 2 g of the powdered sample of each unripe fruit was taken and macerated in 15 mL of ethanol for 7 days at a controlled temperature of 10±1ºC with occasional shaking. The macerated product was filtered and dried to a residue at room temperature and stored.
Preliminary phytochemical testing
The presence or absence of phytoconstituents was determined using different testing procedures. Alkaloid was estimated by Dragendroff’s test, Wagner’s test, Carbohydrates by Iodine test, Molisch test, Fehling’s test. The lead acetate test was performed for the identification of Tannin and Phenolics. Frothing’s test was used for the identification of Saponin. Glycoside, Steroids test was also performed. Identification of proteins was done by performing Ninhydrin test (Kancherla et al., 2019).
Total Flavonoid Content determination
A calibration curve was prepared at 20, 40, 60, 80, and 100 mg/mL using Quercetin as reference standard, and the absorbance was recorded at 510 nm. The TFC of the plant extract was subsequently estimated at concentration of 0.1 mg/mL.
Total Phenolic content determination
The Total Phenolic Content (TPC) was determined using an established method (Martins et al., 2021). A calibration curve was constructed using gallic acid at concentrations of 20, 40, 60, and 100 mg/mL by reacting with 5 mL of 10% Folin-Ciocalteu reagent and 4 mL of 7% sodium carbonate (Na₂CO₃), followed by measurement of absorbance at 760 nm. The TPC of the plant extract was similarly evaluated at concentrations of 0.1 mg/mL.
Total Tannin content determination
The Total Tannin Content (TTC) was determined using an established procedure (Ononamadu et al., 2019). Tannic acid was employed as the reference standard to construct a calibration curve at concentrations of 20, 40, 60, 80, and 100 mg/mL, and the absorbance was recorded at 725 nm. The TTC of the plant extract was subsequently estimated at concentrations of 0.01, 0.05, and 0.1 mg/mL.
Total pigment content
Chlorophyll a, chlorophyll b, total chlorophyll content, and total carotenoid content were determined using 80% acetone as the solvent system. Anthocyanin content was estimated using a methanol-HCl-water mixture (90:1:9, v/v/v) according to the reported method and expressed as μg/g Fresh Weight (FW) (Chazaux et al., 2022). All measurements were performed in triplicate.
(i)Anthocyanin Content
(ii)Chlorophyll a Content
(iii)Chlorophyll b Content
(iv)Total Chlorophyll Content
(v)Total Carotenoid Content
Anti diabetic potential
The α-amylase inhibitory activity was evaluated using an established method (Wickramaratne., 2016). The sample solution was prepared at a concentration of 0.1 mg/mL and further diluted to obtain 0.05 and 0.01 mg/mL. Reaction mixtures containing different concentrations of the plant extract (test), acarbose (positive control), or ethanol (control) were incubated with α-amylase solution (0.5 mg/mL), 1% starch solution, and 0.2 M phosphate buffer (pH 6.9) for 5 min. The reaction was terminated by the addition of 1% 3,5-dinitrosalicylic acid, and the absorbance was measured at 540 nm. The antidiabetic percentage was calculated by
(vi) % Antidiabetic Potential
Where Ac=Absorbance of control solution and At= Absorbance of test Solution.
Anti-inflammatory potential
The anti-inflammatory activity was assessed using an egg albumin denaturation assay following an established method. The reaction mixture consisted of 0.2 mL egg albumin, 2.8 mL phosphate buffer (pH 6.4), and 0.2 mL of the sample at concentrations of 0.1, 0.05, and 0.01 mg/mL (aspirin as the positive control, plant extract as the test sample, and appropriate control). The mixtures were incubated at 27ºC for 10 min, followed by heating in a water bath at 70ºC for 10 min. The absorbance was measured at 660 nm (Mustafa, 2023). Anti-inflammatory potential was estimated by using the formula:
(vii) % Anti-inflammatory Activity
Where, AC=Absorbance of control solution, AT=Absorbance of test solution.
Statistical Analysis
All experiments were performed in triplicate. Data are presented as Mean±Standard Deviation (SD). Linear regression analysis was used to calculate IC₅₀ values and calibration curves.
Ethical Statement
As this study involved the use of commercially available plant materials and did not involve human or animal subjects, ethical approval was not required.
RESULTS
Preliminary Phytochemical test
The phytochemical tests were conducted to determine the presence or absence of different classes of phytoconstituents. The results are shown in Table 1.
| Sl. No. | Identification | Test Name | F. carica | P. emblica |
|---|---|---|---|---|
| 1 | Alkaloid | Dragendroff’s Test | - | - |
| Wagner’s Test | + | + | ||
| 2 | Carbohydrate | Iodine Test | + | + |
| Molisch Test | + | + | ||
| Fehling’s Test | + | + | ||
| 3 | Protein | Ninhydrin Test | - | - |
| 4 | Terpenes Test | + | - | |
| 5 | Tannin and Phenolics | Lead Acetate Test | + | - |
| 6 | Glycoside Test | - | + | |
| 7 | Test for Tannin | + | + | |
| 8 | Steroids Test | - | + | |
| 9 | Saponin | Frothing’s Test | + | + |
Phytochemical quantification
Standard curve of quercetin, gallic acid and Tannic acid was prepared at different concentration. Calibration curves of standard quercetin, gallic acid and tannic acid and demonstrated high regression correlation(R2) of 0.9044, 0.9395,0.9987 with linear equations:
Respectively, as presented in Figures 1-3.
The estimated value of TFC, TPC and TTC content of the unripe fruits is represented in Table 2.
| Sl. No. | Fruit Name | TFC (mg QE/g) | TPC (mg GAE/g) | TTC (mg TAE/g) |
|---|---|---|---|---|
| 1 | F. carica | 0.5296±0.001 | 0.100±0.0140 | 0.9982±0.0001 |
| 2 | P. emblica | 0.2608±0.3388 | 0.445±0.0004 | 0.991±0.0048 |
Pigment content estimation
Pigment content estimation identified that F. carica has higher amount of Chlorophyll b, Total Chlorophyll content and Total carotenoid content, whereas P. emblica is rich in Chlorophyll a and anthocyanin.
Quantification of different pigment contents like anthocyanin, Chlorophyll, and Carotenoid is represented in Table 3.
| Sl. No. | Fruit Name | Chlorophyll a content (µg/g) | Chlorophyll b content (µg/g) | Total Chlorophyll content (µg/g) | Total anthocyanin content (µg/g) | Total carotenoid content (µg/g) |
|---|---|---|---|---|---|---|
| 1 | F. carica | 2.2707±0.3336 | 4.2013±0.2568 | 3.3165±2.1963 | 0.1093±0.0043 | 1.5451±0.0458 |
| 2 | P. emblica | 0.3316±0.1196 | 1.8559±1.3502 | 3.0482±0.0274 | 0.2199±0.0002 | 1.4191±0.0107 |
Anti diabetic activity
The antidiabetic activity of both fruits was calculated and compared against the standard compound (Acarbose). The antidiabetic potential of the fruits is represented in Table 4.
| Activity | Fruit Name | 0.01 mg/mL | 0.05 mg/mL | 0.1 mg/mL | IC50 (µg/mL) |
|---|---|---|---|---|---|
| Antidiabetic | F. carica | 35.489±0.0564 | 32.8934±0.0978 | 49.5922±0.0523 | 39.3249 |
| P. emblica | 13.6116±1.1999 | 27.0348±0.0499 | 70.1486±0.5721 | 36.9317 | |
| Anti-inflammatory | F. carica | 23.71±0.3019 | 20.2166±0.7746 | 61.79±0.1908 | 35.2588 |
| P. emblica | 32.02±0.1452 | 15.5766±0.0611 | 76.39±0.7313 | 41.3288 |
Anti-inflammatory activity
The egg albumin denaturation assay was performed to evaluate the ability of the fruit extract to inhibit protein denaturation, a key mechanism involved in inflammation. The anti-inflammatory activity of the plant samples was assessed at three different concentrations (0.1, 0.05, and 0.01 mg/mL) and compared with aspirin as the positive control. The results revealed a significant difference in anti-inflammatory activity among the tested concentrations and between the fruit extracts and aspirin. The anti-inflammatory potential of the fruits is represented in Table 4.
DISCUSSION
The present study provides a comprehensive evaluation of the phytochemical composition and in vitro pharmacological activities of unripe fruits of Ficus carica and Phyllanthus emblica cultivated in West Bengal. The findings demonstrate that unripe fruits are rich sources of bioactive secondary metabolites and exhibit significant antioxidant, antidiabetic, and anti-inflammatory potential, supporting their traditional use and emerging role as functional foods.
Preliminary phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, carbohydrates, glycosides, and saponins in both fruits, although variations in individual phytoconstituents were observed. These differences may be attributed to species-specific metabolic pathways and environmental factors such as soil composition and climatic conditions prevalent in West Bengal. The absence of proteins and alkaloids in both fruits suggests that the observed biological activities are primarily mediated by polyphenolic compounds rather than nitrogenous constituents.
Quantitative analysis revealed that F. carica possessed a higher total flavonoid content and higher tannin content, whereas P. emblica exhibited significantly greater total phenolic content. Phenolics and flavonoids are well-known for their ability to donate hydrogen atoms or electrons, thereby neutralizing free radicals and reducing oxidative stress. The appreciable pigment content, including anthocyanins, chlorophylls, and carotenoids, further contributes to the antioxidant potential of the extracts and may synergistically enhance their biological efficacy.
The α-amylase inhibitory assay demonstrated concentration-dependent antidiabetic activity for both fruits, indicating their ability to delay carbohydrate digestion and reduce postprandial glucose levels. Such enzyme inhibition is a validated therapeutic approach in the management of type 2 diabetes. The observed activity may be attributed to the interaction of phenolics and flavonoids with the enzyme’s active site, leading to reduced starch hydrolysis.
Anti-inflammatory activity assessed through the egg albumin denaturation assay showed significant inhibition of protein denaturation, a key mechanism involved in inflammation. Both fruit extracts exhibited notable activity, with F. carica showing comparatively lower IC₅₀ values than P. emblica. The anti-inflammatory effects can be linked to the stabilization of proteins and suppression of inflammatory mediators by polyphenolic compounds.
Overall, the results suggest that unripe fruits of F. carica and P. emblica possess multifunctional bioactivities mediated by their rich phytochemical profile. These findings highlight their potential application as natural therapeutic agents for managing oxidative stress-associated metabolic and inflammatory disorders. However, further in vivo studies, bioavailability assessments, and standardization protocols are necessary to validate their clinical relevance and support their development into nutraceutical or pharmaceutical products.
CONCLUSION
The diverse phytochemical composition of unripe fruits, including phenolics, flavonoids, anthocyanins, carotenoids, and chlorophyll, highlights their potential to modulate glucose metabolism, and inflammatory pathways in vitro. The findings provide strong evidence supporting the inclusion of unripe fruits in the daily diet to promote overall human health and well-being. This study pioneers the quantitative assessment of phytoconstituents and explores the therapeutic potential of selected unripe fruits cultivated in the soil of West Bengal, India. F. carica and P. emblica are potent antidiabetic and anti-inflammatory agents respectively. Further research can be extended up to examine the in vivo pharmacological activities shown by these unripe fruits and conduct bioavailability studies, and assessment of challenges in standardization and commercialization.