INTRODUCTION:

Diabetes mellitus is a significant, intricate condition with multiple factors, marked by elevated blood glucose levels (hyperglycemia) and an inability to tolerate glucose¹. This can occur due to inadequate insulin production or impaired insulin function, which hampers the absorption of glucose. Failure to address this condition can result in serious complications such as high blood lipid levels (hyperlipidemia), oxidative stress, and protein glycation caused by enzymes².

Diabetes poses a significant health concern globally, affecting both developed and developing nations. In 2030, diabetes is projected to rank as the 7^th major cause of mortality according to the World Health Organization (WHO)³.

While various therapies exist to manage the condition, but they do not provide a complete cure and often come with a range of side effects⁴. Inflammation is the body's natural reaction to injury, infection, or damage, and it is characterized by symptoms such as heat, redness, pain, swelling, and disrupted physiological functions⁵. This response serves as a protective mechanism against tissue damage caused by physical trauma, harmful chemicals, or microbial agents. The body activates inflammation to neutralize or eliminate invading organisms, eliminate irritants, and create an environment conducive to tissue healing. Chemical mediators released from injured tissues and migrating cells play a crucial role in triggering this inflammatory response⁶. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly employed to alleviate inflammation. However, prolonged usage of these medications can result in significant negative consequences, such as gastrointestinal issues, hepatitis, nephritis, and other adverse effects⁷.

Significant contributions are made by medicinal plants in the advancement of contemporary herbal medicines due to the inability of allopathy to provide complete cures for various ailments such as liver diseases and arthritis. The bioactive components found in medicinal plants are utilized as remedies for conditions like diabetes, inflammation and arthritis when modern medicine fails to offer satisfactory solutions. Medications derived from plants are generally considered to be less toxic and have fewer side effects compared to synthetic drugs⁸. The development of modern medicine has greatly benefited from the contributions of natural products. Medicinal plants that possess therapeutic applications contain bioactive compounds such as alkaloids, glycosides, tannins, flavonoids, saponins, phenolics and vitamins⁹. The composition of bioactive compounds and medicinal properties varies across different parts of the plants. Secondary metabolites, commonly referred to as phytochemicals, are chemical compounds that are produced during normal metabolic processes in plants. These secondary metabolites exhibit a wide range of structural arrangements and properties, making them a valuable source of exploration¹⁰.

The increasing demand for green solvents as a substitute for organic solvents has gained significant attention due to the drawbacks associated with organic solvents, including their high volatility, flammability, and toxicity. The preferred alternative solvents for environmentally friendly extraction should possess characteristics such as easy recyclability without harming the environment, excellent solvency, high flash points combined with low toxicity and minimal environmental impact. These solvents should also be readily biodegradable, sourced from renewable resources, and available at a reasonable cost¹¹. The plant under study is Ficus drupacea growing in the Dehradun region of Uttarakhand. The literature review indicates that Ficus drupacea belongs to the Moraceae family, which comprises a total of 800 species within the Ficus genus. This plant is native to India and is recognized for its versatile nature¹²such as stembark of Ficus drupacea, have been used in treatment of cancer, anti-fungal and anti-bacterial infections by the local people of Canada¹³ whereas latex of this plant has been used in the treatment of wounds in Mysuru, India¹⁴. During the exploration of natural therapeutic substances, the researchers noticed a lack of documented research on Ficus drupacea leaves. Therefore, the current study was conducted to establish the biological activities i.e., anti-diabetic and anti-inflammatory activities of Ficus drupacea leaves and aiming to support its widespread utilization as a medicinal plant.

MATERIALS AND METHODS:

Plant Collection and Identification:

The Ficus drupacea plant leaves were collected from the Dehradun region of Uttarakhand. The plant was identified and verified as Ficus drupacea var. pubescens (Roemer and Schultes) Corner by the botanist from systematic botany discipline at FRI Dehradun and voucher specimen was deposited for reference. The collected plant material was shade dried, powdered, and preserved for further experimentation.

Sequential Extraction of Plant Samples:

The leaves were washed and rinsed with distilled water, followed by shade drying and grinding into a coarse powder. The powdered material was stored in an airtight container. The shade dried powdered plant samples was sequentially extracted using dimethyl carbonate, isopropyl alcohol, hydro alcohol and water by hot Soxhlet extraction technique. The resulting extracts were then filtered, concentrated under reduced pressure and controlled temperature using a rotary evaporator and were stored at 4°C in a refrigerator for further analysis¹⁵(Fig 01).

Chemicals and Reagents:

Analytical grade chemicals were used in the study. Bovine serum albumin, tris buffer (Merck), p-nitrophenyl-α-D-glucopyranoside (SRL Pvt. Ltd), α-glucosidase (SRL Pvt. Ltd), soluble starch, sodium potassium tartrate, 3,5-dinitrosalicylic acid (DNSA) (SRL Pvt. Ltd), α-amylase (SRL Pvt. Ltd), dimethyl carbonate, ethanol, isopropyl alcohol, dimethyl sulphoxide (Loba chemie Pvt. Ltd), sodium chloride, sodium phosphate dihydrate and sodium hydroxide were obtained from various vendors. Reference standards including diclofenac sodium and acarbose were obtained from Cipla Ltd, Bangalore. UV-Visible spectrophotometer (Systronics 118) was used for the estimation of anti-diabetic and anti-inflammatory activity.

Assessment of Antidiabetic Activity Using In-Vitro Assays:

Alpha-Amylase Inhibitory Assay:

In-vitro amylase inhibition was performed with slight modifications¹⁶. The test extract (1ml) was incubated with α-amylase enzyme (1ml) and phosphate buffer (20 mM, pH 6.9). After a 30-minute incubation at 37°C, 1% starch solution (1ml) was added following a 15-minute incubation at 37°C. To stop the reaction 1 ml of color reagent called 3,5-dinitrosalicylic acid was added to both the control and test samples and tubes were placed in boiling water bath for 5 minutes and then diluted by adding 10ml of distilled water after cooling. Finally, the absorbance was measured at 540 nm using a UV-Visible spectrophotometer. Acarbose was used as a standard at various concentrations. The percentage of α-amylase inhibitory activity was determined using the following formula:

Inhibition (%) = [(A_C⁺ − A_C^-) – (A_S- A_B)]/ (A_C⁺ − A_C^-) × 100

Where, A_C⁺ represents 100% enzyme activity, A_C^- represents 0% enzyme activity, A_Srepresents test sample and A_Brepresents blank. The effectiveness of enzymatic inhibition was quantified through IC₅₀values (µg/mL), which denote the concentration of the sample required to inhibit 50% of enzyme activity.

Alpha-Glucosidase Inhibitory Assay:

The inhibitory activity of α-glucosidase was assessed using a modified method¹⁷. Plant extracts (1ml) at different concentrations were incubated with α-glucosidase enzyme solution for 30minutes at 37°C, along with 1ml of 100 mM phosphate buffer (pH 6.8). After 20minutes, the reaction was initiated by adding 1 ml of 5mM p-NPG, and the mixture was further incubated for 15minutes. The reaction was stopped by adding 4ml of 0.5M Tris buffer, and the final absorbance was measured at 410 nm using a UV-Visible spectrophotometer. Acarbose was used as a standard at various concentrations.

Inhibition (%) = [(A_C⁺ − A_C^-) – (A_S- A_B)]/ (A_C⁺ − A_C^-) × 100

Where, A_C⁺ represents 100% enzyme activity, A_C^- represents 0% enzyme activity, A_Srepresents test sample and A_Brepresents blank. The effectiveness of enzymatic inhibition was quantified through IC₅₀ values (µg/mL), which denote the concentration of the sample required to inhibit 50% of enzyme activity.

Assessment of Anti-inflammatory Activity Using In-vitro Assay:

The anti-inflammatory activity was evaluated using an in-vitro assay known as the protein denaturation method, with some modifications¹⁸. The plant extracts were prepared at different concentrations (μg/mL). The assay mixtures were created by combining 2.8ml of 50mM potassium phosphate buffer (pH 7.5), 2ml of sample solutions, and 0.2ml of freshly prepared bovine serum albumin solution. These mixtures were then incubated at 72°C for 10 minutes. The inhibitory activities were measured spectrophotometrically at 660nm using a UV-Visible spectrophotometer. Diclofenac sodium was used as a standard at various concentrations. The percent inhibition of protein denaturation was estimated by using formula:

% Inhibition = 100 × [A_B– (A_S – A_C)]/A_B

Where, A_B represents absorbance of blank, A_S represents absorbance of sample and A_C represents absorbance of control.

DATA ANALYSIS:

To assess the strength of inhibition by the extracts, the concentration required to inhibit 50% of enzyme activity (IC₅₀) was determined. The extract/standard concentration for 50% inhibition (IC₅₀) was analyzed by plotting percentage inhibition with respect to control against the concentration of the inhibitor.

STATISTICAL ANALYSIS:

All the experiments were performed in triplicates and the results were expressed as Mean±standard deviation. Statistical analysis was carried out using one-way ANOVA followed by Duncan's test. Mean values were considered statistically significant if the p-value was less than 0.05.

RESULTS AND DISCUSSION:

α-Amylase and α-Glucosidase Assays:

The in-vitro method was used to assess the anti-diabetic inhibitory effects of various fractions of green extracts on the α-amylase enzyme. All the extracts demonstrated concentration-dependent inhibition of the enzyme (Table 01). The dimethyl carbonate, isopropyl alcohol, hydro alcohol and water extracts exhibited good inhibitory activity against α-amylase, with percentages of 47.36%, 62.98%, 76.63%, and 72.47%, respectively, at a concentration of 1500μg/ml when compared to standarda carbose which exhibited 65.09% inhibition at same concentration (Fig 02). The results indicated that the hydro alcohol extract had the strongest α-amylase activity compared to other extracts at all tested concentrations.

Table 01: IC₅₀ values of α-amylase inhibition assay

IC₅₀ values of α-amylase inhibition assay
S.No.	Extracts	IC₅₀ Value (μg/mL)
1.	Dimethyl Carbonate (DMC)	1588.29 ± 0.02
2.	Isopropyl Alcohol (IPA)	1200.65 ± 0.05
3.	Hydro alcohol	649.05 ± 0.18
4.	Water	877.17 ± 0.01
5.	Acarbose (Standard)	964.18 ± 0.09

Fig 02: Correlation between extracts concentration and the percentage of inhibition of α-amylase enzyme of Ficus drupacea leaves.

Similarly, the α-glucosidase inhibitory activity of different extracts of Ficus drupacea leaves was evaluated using the same in-vitro method. All the extracts displayed dose-dependent inhibitory effects on α-glucosidase (Table 02). The dimethyl carbonate, isopropyl alcohol, hydro alcohol and water fractions exhibited significant α-glucosidase inhibitory activity, with percentages of 50.08%, 39.62%, 82.95%, and 77.93%, respectively, when compared to standard acarbose which exhibited 95.58% inhibition at a concentration of 1500μg/ml (Fig 03). The results indicated that the hydro alcohol extract had the strongest α-glucosidase activity compared to other extracts at all tested concentrations.

Table 02: IC₅₀ values of α-glucosidase inhibition assay

IC₅₀ values of α-glucosidase inhibition assay
S.No.	Extracts	IC₅₀ Value (μg/mL)
1.	Dimethyl Carbonate (DMC)	1494.25 ± 0.14
2.	Isopropyl Alcohol (IPA)	1915.14 ± 0.08
3.	Hydro alcohol	688.46 ± 0.23
4.	Water	902.01 ± 0.05
5.	Acarbose (Standard)	562.21 ± 0.05

Fig 03: Correlation between extracts concentration and the percentage of inhibition of α-glucosidase enzyme of Ficus drupacea leaves.

In the inhibitory assays, acarbose was used as a standard reference drug for both α-amylase and α-glucosidase enzymes. The standard (acarbose) enzyme inhibitor displayed an IC₅₀ value of 964.18±0.09μg/ml for α-amylase inhibitory activity and 562.21±0.05 μg/ml for α-glucosidase inhibitory activity. Among all the extracts, hydro alcohol extracts exhibited the highest inhibitory activity against α-amylase with IC₅₀ values of 649.05± 0.18μg/ml and with α-glucosidase enzymes IC₅₀ values of 688.46±0.23μg/ml, respectively, which were comparable to that of acarbose. The lowest IC₅₀values corresponded to the highest inhibitory activity.

Protein Denaturation Assay:

The results of the Bovine Serum Albumin Assay (BSA) for anti-inflammatory activity are depicted in (Table 03 and Fig 04). Among all the tested green solvent extracts (dimethyl carbonate, isopropyl alcohol, hydro alcohol and water), the hydro alcohol extract obtained from the leaves exhibited a dose-dependent anti-inflammatory effect increasing as the sample concentration rose. At a concentration of 1000μg/ml, the hydro alcoholic extract of leaves demonstrated inhibitions of 52.63% against bovine serum albumin, whereas the standard drug diclofenac sodium produced an inhibition of 98.83%. The data indicates that Ficus drupacea leaves exhibited an anti-inflammatory property.

Table 03: IC₅₀ values of bovine serum denaturation inhibition assay

IC₅₀ values of Protein denaturation assay
S. No.	Extracts	IC₅₀ Value (μg/mL)
1.	Dimethyl Carbonate (DMC)	1443.72 ± 0.17
2.	Isopropyl Alcohol (IPA)	1371.41 ± 0.19
3.	Hydro alcohol	831.84 ± 0.32
4.	Water	1231.98 ± 0.12
5.	DiclofenacSodium(Standard)	440.78 ± 0.15

Fig 04: Correlation between extracts concentration and the percentage of protein inhibition of Ficus drupacea leaves.

DISCUSSION:

Modern medicine was initially derived from plants, but nowadays, it is often synthesized artificially, including many other drugs that still originate from plant materials. With an abundance of medicinal plants accessible, traditional healing remedies are commonly employed in rural areas^19,20. The efficient extraction of different phytochemicals using environmentally friendly solvents offers a viable alternative to traditional solvents²¹. Moreover, the reduced toxicity of these solvents contributes to the economic and ecological sustainability of chemical processes, making them environmentally friendly and economically viable. The use of green solvents shows no signs of slowing down and holds great potential for further advancements²². Numerous plant extracts have been documented for their hypoglycemic and anti-inflammatory effects^{23,24,25,26,27,} ²⁸. In a controlled environment, the study investigated the anti-diabetic inhibitory properties of different Ficus drupacea leaves extracts on porcine pancreatic amylase and alpha-glucosidase activities with the standard drug acarbose for the comparison. Using two in-vitro assays-alpha-amylase and alpha-glucosidase, the study examined the anti-diabetic potential of various solvent extracts (dimethyl carbonate, isopropyl alcohol, hydro alcohol and water) of Ficus drupacea leaves. Out of all the green solvent extracts of leaves (dimethyl carbonate, isopropyl alcohol, hydro alcohol and water), the strongest inhibitory action was seen in the hydro alcoholic extract. This extract effectively lowered blood glucose levels, due to elevated insulin levels in the bloodstream. The chemical constituents present in the extracts, including terpenes, quercetin, rutin, flavonoids and alkaloids, may be responsible for their activity²⁹.

Protein denaturation is a biochemical process that takes place in prolonged inflammatory responses, leading to impaired tissue function. Moreover, chronic inflammation triggers the breakdown of lysosomal membranes, releasing pro-inflammatory substances like activated neutrophils, proteases, and histamines at the site of tissue damage. Therefore, medicinal plant extracts that can hinder protein denaturation and protect cell membranes from lysis hold promise as potential candidates for effective anti-inflammatory drugs. The research investigated the anti-inflammatory properties of various green solvents extracts (dimethyl carbonate, isopropyl alcohol, hydro alcohol and water) of Ficus drupacea leaves using bovine serum albumin method³⁰ with the standard drug diclofenac sodium for the comparison. The hydroalcoholic extracts were found to exhibit significant anti-proteinase activity, which likely contributes to their anti-inflammatory effects. Hydroalcoholic extract produced a notable stabilizing impact in our research. Therefore, any phytonutrient that can prevent protein denaturation during an inflammatory reaction may be crucial in decreasing inflammation^31-33. Thus, Ficus drupacea leaves extract exhibited better anti-diabetic activity and significant anti-inflammatory property when compared with the standard drug in dose dependent pattern.

CONCLUSION:

This study presents the potential application of environmentally friendly solvents, including dimethyl carbonate, isopropyl alcohol, hydro alcohol, and water for extracting Ficus drupacea leaves. The use of a Soxhlet extraction technique was found to be a suitable technology for extraction using these green solvents. The results of the study revealed that all the green solvent extracts of Ficus drupacea leaves exhibited anti-inflammatory and anti-diabetic properties. Among the various green solvent extracts (dimethyl carbonate, isopropyl alcohol, hydro alcohol and water extracts), hydroalcoholic extract of Ficus drupacea leaves exhibited a better inhibition of heat-induced albumin denaturation, non-enzymatic glucosidase inhibitory activity, and amylase inhibitory activity. These findings suggest that the hydroalcoholic extracts of Ficus drupacea leaves could serve in development of potent drugs targeting inflammation and diabetes.

CONFLICTS OF INTEREST:

The authors have no conflicts of interest regarding this study.

ACKNOWLEDGMENTS:

The authors are grateful to Department of Chemistry, Kanya Gurukul, Gurukula Kangri (Deemed to be University), Haridwar for providing all the necessary facilities.

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Received on 03.10.2023 Revised on 23.04.2024

Accepted on 28.09.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):619-624.

DOI: 10.52711/0974-360X.2025.00092

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.