INTRODUCTION:

Antioxidants play a vital role in both food systems as well as in the human body to reduce oxidative processes and harmful effects of reactive oxygen species (ROS)¹. Some of these free radicals include reactive oxygen free radicals species (ROS), reactive hydroxyl radicals (OH^•), the superoxide anion radical (O₂^•), hydrogen peroxides (H₂O₂) and peroxyl (ROO^•) radical¹. There are many synthetic antioxidants in use. It is reported, however, they have several side effects², such as risk of liver damage and carcinogenesis in laboratory animals^{3,4,5,6,7,8,9}. There is therefore a need for more effective, less toxic and cost effective antioxidants. Plants are a rich natural sources of antioxidants with least or no side effect. Plants produce the secondary metabolites like phenolics and flavonoids, most of which probably evolved as a chemical defense against predation or infection¹⁰. They are found in all parts of plants such as leaves, fruits, fruits seeds, roots and bark. The increasing use of plant extracts in the food, cosmetic and pharmaceutical industries suggests that in order to find active compounds, a systematic study of medicinal plants is very important¹⁰. Epidemiological evidence indicates an inverse relationship between the intake of food rich in phenolic compounds and the reduction of certain chronic diseases and coronary heart disease mortality¹⁰.

Nerium indicum, belongs to the family Apocynaceae, is a large evergreen shrub with milky juice. It is commonly cultivated for its sweet-smelling flowers which have medicinal uses^2,9,10,11,12. Line shaped leaves are mostly in whorls of 3 to 4, sometimes two, linear-lanceolate, thick, about 1cm stemmed somewhat bent with maternal bone protruding. Flowers are showy, sweet-scented, and single or double, Panicle-shaped at the end of the branch with funnel-shaped crown. The whole plant of Nerium indicum is very poisonous, containing a powerful cardiac toxin^2,9,10,11,12. Ingestion of only one leaf may lead to death in children, while skin contact with the plant can cause irritation. The plant may be medicinal only if used with extreme care. Nerium indicum has been reported with a broad spectrum of activities. Seed, bark, and root of this plant contain cardiac glycosides. It is reported for antibacterial, antileprotic, anticancer, and cardiotonic activities, skin disease, epilepsy, and central nervous system depression^2,9,10,11,12. However, there is few systematic studies of the phytochemical profile and antioxidant activity of different parts (leaves and branches) of Nerium indicum. In particular, unlike other regions, there has been no specific research on Nerium indicum that inhabits Jeju island, which has a unique climate and soil, so scientific research is deemed urgent.

Therefore, we undertook the present investigation to estimate the total phenolic content as well as total flavonoids and to evaluate the antioxidant potential of Nerium indicum grown in Jeju Island, which will be useful for the production of functional nutraceutical and pharmaceutical sources.

MATERIALS AND METHODS:

Plant material and extraction procedure:

The whole plant of Nerium indicum, collected in the field at Seogwipo (Jeju Island, Korea) located Seogwipo-si, Jeju-do Province, South Korea, under the coordinates 33° 17' 30" N, 126° 29' 59" E during July and August, 2022 and the voucher specimens (JNI-2022) have been deposited in the herbarium of College of Applied Life Science, Jeju National University. The fresh leaves and branches were washed and air-dried under shade at room temperature. To prepare the solvent extracts of Nerium indicum, samples were freeze-dried. The powder (100g) was extracted with ethanol (70%) by constant shaking for 72h, and then purified with a Sep‑Pak C18 cartridge and a 0.45μm membrane filter (Waters Corporation). The extracted liquid was concentrated using a rotary vacuum evaporator (Buchi Rotavapor R-200, New Castle, DE, US), and the yield of solvent extracts was determined. The residues were taken to the laboratory for further analysis.

Total phenol and flavonoid contents:

The total phenol and flavonoid content were quantified according to the protocol previously described¹³. To measure the total polyphenol contents, 30μL of the extracts, 30μL of 95% ethanol, 150μL of distilled water, and 15μL of Folin-Ciocalteu reagent were added to 96 well microplates. The reaction was allowed to proceed for 5min at room temperature, followed by an additional 1h reaction with 15μL of Folin-Ciocalteu reagent. The optical density was measured at 725nm using a microplate reader (Dynex Technologies, Inc., Chantilly, VA, US). A gallic acid standard curve was generated from the concentration range of 0-400μg/mL. Total phenol values were expressed as gallic acid equivalents (mg/100g of dry mass).

The total flavonoid content of Nerium indicum extracts was measured using the aluminum chloride colormetric assay. An aliquot (15μL) of each extract was mixed with 4.5μL of 5% sodium nitrite, 60μL of distilled water, and 4.5μL of 10% aluminum chloride and left at room temperature for 5min. Two milliliters of 1M sodium hydroxide were added to the mixture and then filled up to 150μL with distilled water. The absorbance of reaction mixture was measured at 510nm. A rutin standard curve was used to calculate the flavonoid content.

Radical scavenging activity:

The free radical scavenging activity of plant extracts was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) following the method described by Kim and Kim¹¹. Briefly, 100μL of the plant extracts were mixed with 100μL of 0.4mM DPPH solution in a glass tube and allowed to react for 10min in the dark at room temperature. The absorbance was measured at 517nm.

The superoxide anion scavenging activity of Nerium indicum extracts was measured using a method previously described¹³. Superoxide radicals were generated in a mixture containing 50mM sodium carbonate buffer (pH 10.5), 3 mM xanthine, 3mM ethylenediamine tetraacetic acid (EDTA), 0.5mM nitroblue tetrazolium (NBT), and 0.15% bovine serum albumin. The solution of Nerium indicum extracts was added to the mixture, and the reaction was initiated by the addition of xanthine oxidase (XO) (0.25 units/mL). The reaction mixture was incubated at room temperature for 25min, and the absorbance was measured at 560 nm using a microplate reader.

The ability of Nerium indicum extracts to scavenge hydrogen peroxide was determined according to a previously described method¹³. Briefly, a solution of hydrogen peroxide (20µL, 10mM) and 80µL of extract were mixed with 100µL of 100mM phosphate buffer (pH 5.0). After incubation at 37°C for 5 min, a freshly prepared solution of 1.25mM ABTS (30µL) and 1U/mL peroxidase (30µL) were added to the reaction mixture. The absorbance of the hydrogen peroxide at 405nm was determined spectrophotometrically 10min later at 37°C against a phosphate buffer blank without hydrogen peroxide.

The results for the scavenging activity of the radicals, obtained from triplicate analyses, were expressed as IC₅₀ values (mg/mL), which is the dose required to cause 50% inhibition. All samples were analyzed in triplicate, and L-ascorbic acid was used as a positive control.

Ferrous ion chelating activity:

The ferrous ion chelating activity was determined as previously described¹³. Freshly prepared FeCl₂ (2mM) was mixed with 250μL of Nerium indicum extracts. Then, 10μL of 5mM ferrozine was added to the mixture, and absorbance readings were taken after exactly 10min at 25ºC.

Reducing power assay:

The Fe³⁺ reducing power of Nerium indicum extracts was carried out as described previously¹³. Different concentrations of the extract (200μL, 0.125-2mg/mL) were mixed with 200 μL of 200mM phosphate buffer (pH 6.6) and K₃Fe(CN)₆ (200μL, 1%). After incubation for 20min at 50°C, 200 μL of 10% trichloroacetic acid solution was added to the mixture and then centrifuged at 800 × g for 10min. One hundred μL of the upper layer of solution was mixed with deionized water (100μL) and FeCl₃ solution (20μL, 0.1%), and the absorbance was measured at 700nm.

Statistical analysis:

All data are presented as mean±standard deviation of triplicate values. Significant differences between the groups were performed by using SPSS program (SPSS Inc. Chicago, IL, USA) using two-tailed Student’s t-test. A dose-response curve was plotted to determine IC₅₀ values. Correlations among data obtained were analyzed using Pearson’s correlation coefficient. A p-value less than 0.05 and 0.01 was considered statistically significant.

RESULT AND DISCUSSION:

The leaves and branches of Nerium indicum were extracted using ethanol solvent. The extraction yield of leaves and brances was 26.1 and 17.4%. respectively. The extractive yield is influenced by the polarity of the solvent used for extraction and the solubility of particular phytoconsituent in the solvent used for extraction^14,15. The polar phytoconstituents dominate in the leaves and brance, which can mean that the non-phenolic polar compounds are also solved in the polar ethanolic solvent.

Determination of total phenol and flavonoid:

Phenolic compounds act as free radical terminator, and mostly include flavonoids, phenolic acids, stilbenes, coumarins and tannins¹⁶. In many cases, flavonoids may increase antioxidant activity as part of a general stress response¹⁶. The mechanisms of action of flavonoids are through scavenging or chelating process¹⁷. In the present study, leaves extract (774 and 162mg/100g) had significantly higher total phenolic and flavonoid contents than the branches extract of Nerium indicum (534 and 116mg/100g), respectively (p < 0.05) (Figure 1).

Figure 1. Total phenolic and flavonoid contents of ethanolic extracts from Nerium indicum leaves and branches.

Values are the mean of three replications (n=3).

Antioxidant capacities of the Nerium indicum extracts:

Free radicals are closely associated with oxidative damage, and antioxidants act as reducing agents to limit this damage by donating electrons to free radicals and passivating them^18,19,20,21. Eventually, these antioxidants can neutralize the free radicals before they cause damage^18,19,20. Many secondary metabolites synthesized by plants act as antioxidants²². Therefore, the current study aimed to investigate the free-radical scavenging ability of Nerium indicum in vitro.

The antioxidant activity of ethanolic extracts from leaves and branches of Nerium indicum is expressed in terms of IC₅₀ (mg/mL) values (Table 1). As shown in Table 1, leaves extract of Nerium indicum exhibited significantly higher DPPH, superoxide and hydrogen peroxide scavenging activities than branches extract (p< 0.05). DPPH is a dark-colored crystalline powder composed of stable free-radical molecules²³. It is a common antioxidant assay and a well-known radical²⁴. Many plant extracts have been reported to scavenge DPPH radicals in vitro²⁴. DPPH is usually used as a substrate to evaluate antioxidative activity of antioxidants²³. The method is based on the reduction of methanolic DPPH solution in the presence of a hydrogen donating antioxidant, due to the formation of the non-radical form DPPH-H by the reaction. Superoxide is generated during cellular respiration and is less toxic, but it can be converted into highly reactive hydroxyl radicals in the presence of iron²⁵. Additionally, superoxide anions produced as a result of incomplete oxygen metabolism damage biomolecules directly or indirectly by forming hydrogen peroxide, hydroxyl radical, peroxynitrite, or singlet oxygen^{26, 27}. Therefore, it is necessary to remove or neutralize superoxide radicals to protect cells from their deleterious effects^{26, 27}. The IC₅₀ concentrations (concentrations at which 50% of DPPH, superoxide and hydrogen peroxide are scavenged) were higher in leaves extract (0.63, 0.77 and 0.10 mg/mL, respectively) than in branches extract (1.32, 1.63 and 0.42 mg/mL, respectively). Leaves extract had a higher total phenolic content (Figure 1), which increased the DPPH, superoxide and hydrogen peroxide scavenging activities. The DPPH, superoxide and hydrogen peroxide scavenging activities of Nerium indicum may be due to the presence of flavonoids and other polyphenols in the extracts, as indicated in the current study (Figure 1).

The chelating effect of various concentrations (0.125-2 mg/mL) of Nerium indicum extracts on Fe²⁺ and ferrozine complex formation is shown in Table 1. Chelating abilities of both leaves and branches extracts were increased with an increase in concentration. The IC₅₀ value of the chelating effect of leaves extract was 0.82mg/mL, which was similar with that of branches extract (0.77mg/mL) (Table 1). Ethylenediaminetetraacetic acid (EDTA) used as positive control showed 99% at 0.5mg/mL. The results suggest that Nerium indicum extracts can inactivate free radicals and reduce the rate of production of radical species by chelating the metal catalysts involved in this production. The ferric (Fe³⁺) reduction capacity is an important characteristic property of phenolic antioxidant, which serves as a significant indicator of potential antioxidant activity²⁸. For the measurements of the reducing ability, the Fe³⁺-Fe²⁺ transformation was investigated in the presence of Nerium indicum extracts. The antioxidant properties expressed as EC₅₀ are summarized in Table 1. Similar to the ferrous ion chelating activity, the reducing power of the extracts increased with increasing dosage. However, leaves and branches extracts showed similar reducing power activity (Table 1). Ascorbic acid, used as a positive control, showed 0.64 at 0.25mg/mL.

Correlation between antioxidant components and antioxidant activity:

Phenolic compounds in plants have been reported to effectively scavenge free radicals²⁹. However, there is no information available on the contribution of individual phenolics to the overall antioxidant capacity of Nerium indicum. To further investigate the contribution of the predominant individual phenolic compounds to the antioxidant capacity of phenolic extracts of Nerium indicum, a correlation analysis was conducted (Table 2). Significant positive correlations (p <0.01) were observed between the TPC and the antioxidant activities in both leaves and branches extracts (r² = 0.900-1.000, p<0.05), implying that the antioxidant capacity of Korean fir primarily originates from its phenolic substances. This positive relationship between total phenolic content and antioxidant activity has been reported previously³⁰. However, no significant correlation was found between the total phenolic content and the reducing power activity in the branches extract (r² = 0.761).

Table 1. Antioxidant activity of ethanolic extracts from Nerium indicum leaves and branches

Part	Radical scavenging (IC₅₀, mg/mL)			Ferrous ion chelating (IC₅₀, mg/mL)	Reducing power (EC₅₀, mg/mL)
Part	DPPH	Superoxide	Hydrogen peroxide	Ferrous ion chelating (IC₅₀, mg/mL)	Reducing power (EC₅₀, mg/mL)
Leaves	0.63 ± 0.022^*	0.77 ± 0.047^*	0.10 ± 0.016^*	0.82 ± 0.665	1.49 ± 0.081
Branches	1.32 ± 0.061	1.63 ± 0.037	0.42 ± 0.054	0.77 ± 0.636	1.60 ± 0.028

IC₅₀ and EC₅₀ mean the effective concentration at which the antioxidant activity was 50% and at which the absorbance was 0.5, respectively, which was obtained by interpolation from linear regression analysis. Each value is expressed as mean ± standard deviation (n = 3). ^*p < 0.05 compared to branches extract by Student’s t-test.

Table 2. Coefficients of correlation between total phenolics and antioxidant activities of ethanolic extracts from Nerium indicum leaves and branches

	Part	Radical scavenging (IC₅₀, mg/mL)			Ferrous ion chelating (IC₅₀, mg/mL)	Reducing power (EC₅₀, mg/mL)
	Part	DPPH	Superoxide	Hydrogen peroxide	Ferrous ion chelating (IC₅₀, mg/mL)	Reducing power (EC₅₀, mg/mL)
Total phenolics	Leaves	0.987^*	0.964^*	0.989^*	0.981^*	0.963^*
Total phenolics	Branches	0.991^*	0.987^*	0.977^*	0.988^*	0.761

All values are absolute value of correlation coefficients: p < 0.05 is considered statistically significant.

CONCLUSION:

The result of present study provides information on the potential medicinal uses of this plant. Overall, Nerium indicum possessed the higher total phenolic content and exhibited strong antioxidant activity and it, therefore, may be developed as natural antioxidant for food industry and other fields. The bioactive components and antioxidative actions of Nerium indicum extract warrant further studies in both in vitro and in vivo models.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This research was supported by Basic Science Research Program (2020R1F1A1048429 and 2017R1D1A1B03028849) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea.

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Received on 16.12.2024 Revised on 14.04.2025

Accepted on 20.06.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4897-4901.

DOI: 10.52711/0974-360X.2025.00706

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