INTRODUCTION:

The contemporary environment is replete with a multitude of poisons that have an impact on the human body. The presence of pollutants in the air, additives in food, household appliances, and everyday objects might generate free radicals that can harm the body. Unbeknownst to us, the body’s essential processes also generate free radicals^1,2.

Free radicals can oxidize other molecules, exhibiting oxidizing capabilities, leading to oxidative damage within the body. An instance of this is reactive oxygen species (ROS) that chemically interact with macromolecules in the body, including proteins, lipids, and nucleic acids, to acquire electron pairs and achieve atomic or molecular stability. Reactive oxygen species (ROS) can damage cells if their production surpasses the antioxidant defenses of the cells. Alterations in cellular morphology result in functional modifications that give rise to pathological disorders. Oxidative stress is the term used to describe an imbalance between free radicals (prooxidants) and antioxidants. This imbalance is caused by two main factors: a deficiency of antioxidants and an excessive generation of free radicals ^3,4.

Inflammation contributes to the production of free radicals throughout the body. It is a specific immune response in a particular body area due to tissue damage caused by physical, chemical, or microbial harm. The commonly used anti-inflammatory medications today are known to have significant adverse effects, including decreased immune response to infections, osteoporosis, hyperglycemia, gastrointestinal and renal disorders, and anemia. Therefore, there is increasing research on developing alternative medicines derived from plants ^5,6. Dioscorea hispida (Dennst.) is a plant with promising properties as an antioxidant and anti-inflammatory agent.

The tuber of Dioscorea hispida, a member of the Dioscorea genus. A significant amount of phenols in the Dioscorea genus suggests that this genus possesses antioxidant, anti-inflammatory, immune-boosting properties and the ability to modulate hormones ^7–9.

Determining antioxidant and anti-inflammatory activity can be evaluated in vitro and in vivo. In vitro, antioxidant testing can be carried out using several methods, including FIC, FRAP, ferrozine, DPPH, and ABTS. In the ABTS method, utilizing ABTS (2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid) is appropriate for assessing the antioxidant capacity of flavonoid and phenolic compounds. This approach has greater sensitivity compared to DPPH. Unlike DPPH, which exhibits sensitivity to acidic pH, the ABTS technique has greater versatility, allowing for its application across a range of pH values. This approach is appropriate for observing the impact of pH on the antioxidant activity of different substances. ABTS exhibits solubility in both organic and inorganic solvents ^10,11.

Investigation of anti-inflammatory properties can be carried out using serum albumin tests. This test aims to minimize the reliance on live specimens as much as feasible during the medication development procedure. Heating serum albumin causes it to undergo denaturation, exposing antigens linked to type three hypersensitivity reactions, which can lead to inflammation ¹². So far, there have been no reports regarding the relationship between the content of secondary metabolite compounds in Dioscorea hispida tuber and its antioxidant and anti-inflammatory activity.

MATERIALS AND METHODS:

Plant material collection:

Dioscorea hispida (Dennst.) specimens were collected from Deli Serdang District, North Sumatra. Sample identification was conducted at the Medanense Herbarium, specifically at the Herbarium Laboratory of the Faculty of Mathematics and Natural Sciences of Universitas Sumatera Utara (212/MEDA/2022).

Extract and fractions preparation:

Dioscorea hispida (Dennst.) specimens were collected, peeled, washed meticulously, sliced, and dried. The dried samples were then pulverized using a mechanical grinder and stored in a hermetically sealed plastic container. 4,500 g of dried plant materials powder underwent maceration with 45 L of 96% ethanol to obtain ethanol extract. The mixture was steeped for six initial hours with periodic stirring, then left undisturbed for 18 hours. The separation process entails the maceration of substances, followed by filtration. It is necessary to repeat the extraction process at least once using the same solvent while reducing the total volume of solvent to half of the starting volume of the filter. Utilizing a rotary evaporator, the maserate that had been gathered was evaporated until a dense extract of the intended viscosity was obtained ^9,13.

The ethanol extract was then fractionated by liquid-liquid extraction using ethyl acetate, n-hexane, and water sequentially. The solution was prepared by dissolving 10 g of ethanol extract obtained from Dioscorea hispida (Dennst.) tubers in a mixture of ethanol and water in a 1:1 ratio, with a total volume of 100 mL. The solution was separated into two distinct layers using a separating funnel and 100 mL of ethyl acetate. The ethyl acetate fraction was subsequently evaporated utilizing a rotary evaporator ¹³.

Phytochemical screening and characterization:

Qualitative phytochemical methods were used to evaluate dried plant materials, extracts, and fractions for secondary metabolites. Dried plant materials analysis included water, water-soluble essence, ethanol-soluble essence, total ash, and acid-insoluble ash content and functional group analysis using Fourier-transform infrared spectroscopy ¹³.

Antioxidant Activity Assay:

Antioxidant activity testing using the method ABTS. Antioxidant activity testing was carried out on the ethanol extract and fraction (n-hexane, ethyl acetate, and water of Dioscorea hispida tubers with varying concentrations of 20, 40, 60, 80, 100, 120 μg/mL and a blank solution as a control negative. Quercetin was used as a positive control in the test with varying concentrations of 5, 10, 15, and 20 μg/mL. The presence of antioxidant activity is measured by calculating the IC₅₀value, obtained by observing changes in the absorbance of the sample solution, which will give different color intensities after reacting with ABTS at each concentration, using a UV-visible spectrophotometer ^14,15. Using the following formula, one can determine the percent inhibition, which in turn shows free radical scavenger activity:

Control absorbance – Sample absobance

% Inhibition = -------------------------------- × 100%

Control absorbance

After obtaining a calibration curve by plotting the percent dampening value on the Y axis and the sample concentration on the X axis, the results of the percent dampening are then calculated using the linear regression equation y = ax+b. ¹⁴. The interpretation of IC50 values is as follows: ¹⁶

- Below 50 μg/mL: very strong

- 50-100 μg/mL: strong

- 101-150 μg/mL: medium

- 150-200 μg/mL: weak

Anti-Inflammatory Activity Assay:

Anti-inflammatory activity testing was carried out on four groups of solutions, namely negative control solution 50 μL of solvent in 0.2% Bovine Serum Albumin (BSA) in a volumetric flask up to 5 mL and three test solutions with varying concentrations of 1, 10, and 100 μg/mL. Additionally, there were three test solutions with 1, 10, and 100 μg/mL concentrations. The test included using a positive control, specifically diclofenac sodium, to evaluate its anti-inflammatory effectiveness at concentrations ranging from 1.3-40 μg/mL. The sample was placed in an incubator at a temperature of 25˚C for 30 minutes. Subsequently, it was heated at 72˚C for 5 minutes, followed by leaving it for 25 minutes at room temperature. Following the chilling process, the solution was vigorously mixed using a vortexer, and the absorbance was quantified using UV-visible spectrophotometry at a specific wavelength of 660 nm. The anti-inflammatory activity test was conducted thrice. ^12,17. The percentage of protein denaturation inhibition was calculated using the following formula:

Negative control absorbance – Sample absorbance

% Inhibition = ------------------------------------ × 100%

Negative control absorbance

The IC50 value was determined by doing a linear regression analysis on the relationship between concentration (X) and percent inhibition (Y) for the extract and each fraction of Dioscorea hispida tuber and diclofenac sodium^12,17. The interpretation of IC50 values is similar to the antioxidant assay. As a note, the anti-inflammatory activities of secondary metabolites if they could inhibit protein denaturation by a magnitude of more than 20 %. Moreover, this result can serve as a reference for further drug development.

Statistical analysis:

The research findings are presented in the mean ± standard deviation format. The statistical analysis was performed using SPSS software version 22.0. Shapiro-Wilk test was used to do normality testing. The parametric data was analyzed using one-way ANOVA, and the non-parametric data was analyzed using the Kruskal-Wallis test. The difference is considered significant if the p-value is less than 0.05 (p<0.05).

RESULT AND DISCUSSION:

Characteristics of Dried plant materials:

The result of extraction with ethanol solvent was 96% using the maseration method, which obtained a yield value of 3.5%. The yield percentage result for each fraction was as follows: 37.15% for the n-hexane fraction, 12.85% for the ethyl acetate fraction, and 50% for the water fraction.

Phytochemical screening and characterization:

Phytochemical screening was performed on dried plant materials, ethanolic extract, and the three fractions. The outcomes acquired are visible in Table 1.

The screening of simplisia and extracts showed alkaloids, flavonoids, glycosides, tannins, saponins, and triterpenoids/steroids. Whereas in the fraction obtained, the result that the n-hexane fraction contains the secondary metabolite triterpenoids/steroids, in the ethyl acetate fraction it contains alkaloids, flavonoid, glycoside, tannins and the water fraction of the alkaloids, flavonoids, glycosides, tannins, saponins. The difference in the content of secondary metabolites in each fraction is due to the principle of fractionation that the compounds will be extracted according to their polarity ¹⁸.

The results of the characterization examination that have been obtained show that dried plant materials from Dioscorea hispida tubers have met the minimum requirements that have been set ¹⁹. The results of the characterization of dried plant materials can be seen in Table 2.

Table 2. Characterization of dried plant materials

Characterization	Content (%)	Requirement (%)
Water content	6,67	< 10
Water-soluble Essence Content	10,10	≥ 6
Ethanol-soluble Essence Content	4,05	≥ 4
Total Ash Content	2,29	< 6
Acid-insoluble Ash Content	0,463	< 1

Figure 2 presents the Fourier Transform Infrared (FTIR) spectra of Dioscorea hispida (Dennst.) tuber extract, which was used to characterize its functional groups and establish a correlation with the phytochemical screening results. The absorption wavenumbers observed at 3456, 2931, 1639, and 1233 cm⁻¹ correspond to different functional groups relevant to the bioactive compounds present in the extract. The absorption peak at 3456 cm⁻¹ is associated with the hydroxyl (-OH) functional group, which is commonly found in phenolic and flavonoid compounds. These compounds are well known for their antioxidant activity, as their hydroxyl groups play a key role in scavenging free radicals. The band at 2931 cm⁻¹, corresponding to the C-H stretching vibration, may indicate the presence of aliphatic compounds or terpenoids, which have been reported to exhibit various pharmacological activities. The strong absorption peak at 1639 cm⁻¹, attributed to the C=O (carbonyl) functional group, indicates the presence of flavonoids, tannins, or other polyphenolic compounds. These secondary metabolites have been associated with anti-inflammatory properties by modulating oxidative stress and inhibiting inflammatory pathways. Additionally, the peak at 1233 cm⁻¹, corresponding to the C-O-C functional group, suggests glycosidic bonds, often found in flavonoid glycosides and other biologically active compounds.

Figure 1. FTIR Spectra of extract of Dioscorea hispida (Dennst.) tubers.

Antioxidant Activity Assay:

In antioxidant testing, the results showed that each concentration of ethanol extract and its fractions and the quercetin comparison standard provided antioxidant activity, as seen in Table 3.

Table 3. Antioxidant Activity Test Results

Samples	IC₅₀(μg/mL)	Category
Ethanol Extract	105.06	Medium
n-Hexane Fraction	147.78	Medium
Ethyl Acetate Fraction	129.08	Medium
Water Fraction	132.43	Medium
Quercetin	14.41	Very strong

Table 3 displays the IC50 values for the antioxidant activity of the ethanol extract and all fractions, all falling within the medium category. This class of metabolite compounds can be expected to have a relationship between metabolites and ani-inflammatory activity as protein denaturation. Alkaloid metabolite compounds can exhibit anti-inflammatory properties by inhibiting the release of histamine from mast cells, decreasing the secretion of interleukin-1 from monocytes, and reducing PAF in platelets. Flavonoid compounds can prevent protein denaturation by interacting with albumin through hydrogen bonds. This interaction is facilitated by the hydroxyl groups and aromatic rings present in flavonoids, allowing them to bond with specific amino acid residues in the protein chain. ^20–22.

Tannins exhibit antioxidant properties and function as anti-inflammatory agents by suppressing the generation of oxidants by neutrophils, monocytes, and macrophages. Inhibiting the production of the oxidant will reduce the formation of H₂O₂, which results in the inhibition of the production of hypochlorous acid (HOCl) and OH. Directly inhibits reactive oxidants such as hydroxy radicals (OH) and hypochlorous acid. Phenolic compounds inhibit inflammation by capturing free radicals, which can cause tissue damage andrigger the biosynthesis of arachidonic acid into inflammatory mediators, namely prostaglandins, and inhibit the cyclooxygenase enzyme ^23–25.

Steroid compounds can activate glucocorticoid receptors by increasing or decreasing the transcription of genes involved in the inflammatory process. Saponins’ anti-inflammatory mechanism hinders the release of pro-inflammatory chemicals triggered by LPS, including TNF-α, iNOS, and iL. Saponin molecules can interact with various lipid membranes, including phospholipids, precursors of prostaglandins, and other inflammatory mediators. Terpenoids generally work by inhibiting the phospholipase enzyme via the arachidonic acid pathway ^26,27. Statistical analysis of the IC50 value shows a significant difference in antioxidant activity between extracts and fractions for quercetin (0.050).

Anti-Inflammatory Activity Assay:

The test showed that each concentration of ethanol extract and the positive control, namely diclofenac sodium, provided anti-inflammatory activity, as seen in Table 4.

Table 4. Anti-Inflammatory Activity Test Results

Samples	IC₅₀(μg/mL)	Category
Ethanol Extract	27.35	Very strong
n-Hexane Fraction	-	-
Ethyl Acetate Fraction	67.49	Strong
Water Fraction	101.11	Medium
Sodium diclofenac	13,05	Very strong

Based on the results of anti-inflammatory testing, the IC50 value of ethanol extract is classified as very strong. However, the IC50 value is not less than 20μg/mL, so it is not yet a reference value for anti-inflammatory activity for drug development. Only the ethyl acetate fraction has an IC50 value in the strong category, while the water fraction has an IC50 value in the medium category. The n-hexane fraction has no absorbance and % inhibition values, so the IC50 value cannot be calculated. The presence of secondary metabolites in Dioscorea hispida tuber extract is believed to be responsible for inhibiting protein denaturation. This extract has the potential to function as an anti-inflammatory agent²⁸.

Flavonoid compounds can inhibit protein denaturation caused by the presence of hydroxyl groups and aromatic rings in the structure of these compounds so that flavonoids can interact with albumin in the form of hydrogen bond interactions formed between the H atoms of the hydroxyl groups on flavonoids and the N atoms on amino acid residues and O from the carbonyl group on flavonoids with the H atom on amino acid residues in the protein chain, thus making the protein structure more stable and not easily denatured^29,30.

Tannins can influence the inflammatory response with their activity as free radical scavengers. Steroids can inhibit the release of prostaglandins from their source cells so that the formation of histamine, prostaglandins, and other chemical mediators that cause inflammation can be inhibited^29,31.

CONCLUSION:

The secondary metabolite components contained in the ethanol extract and fractions influence the antioxidant and anti-inflammatory activity of the four samples. Because many secondary metabolites are synergistic, the ethanol extract has a more substantial IC50 value than the n-hexane, ethyl acetate, and water fractions. Meanwhile, the n-hexane, ethyl acetate, and water fractions have secondary metabolite components drawn based on the polarity of the solvent.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We thank Universitas Sumatera Utara for funding this TALENTA-supported research project.

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Received on 16.11.2024 Revised on 15.03.2025

Accepted on 21.05.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4689-4694.

DOI: 10.52711/0974-360X.2025.00674

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

Group	Dried plant materials	Extract	Fractions			Observations (indicating positive test)
Group	Dried plant materials	Extract	n-hexane	Ethyl acetate	Water	Observations (indicating positive test)
Alkaloids	+	+	-	+	+	Dragendroff: reddish brown precipitate Mayer’s: Creamy white/ yellow precipitate Bouchardat: reddish brown color
Flavonoids	+	+	-	+	+	Reddish brown/Orange color precipitate
Glycosides	+	+	-	+	+	Purple ring
Tannins	+	+	-	+	+	Blue-black
Saponins	+	+	-	-	+	Stable persistent
Triterpenoids/steroids	+	+	+	-	-	Reddish/ purple color