0% READ
FULL TEXT
INTRODUCTION
Chamaecostus cuspidatus, commonly referred to as the Insulin plant, Spiral flag, or Step ladder, originates from Central and South America. In India, particularly in the southern regions, it is cultivated widely as an ornamental plant and is also found growing in the wild. Belonging to the Costaceae family, this plant is recognized in traditional Siddha medicine for its antidiabetic properties. People often chew its leaves twice daily or consume 0.5 to 1 g of dried leaves in powdered form. Indigenous communities, such as those in Tamil Nadu’s Kolli Hills, also use this plant for diabetes management, utilizing its fresh, dried, or powdered leaves. The Costaceae family, formally recognized by Nakai, is distinguished by spirally arranged leaves and rhizomes that lack aromatic essential oils. The family consists of four genera and approximately 200 species, with the genus Costus being the most prominent, encompassing around 150 species. In Indian traditional medicine, the leaves of Chamaecostus cuspidatus are commonly consumed to regulate blood glucose levels, with diabetics often taking a leaf per day to help manage their condition.[1] An overaccumulation of free radicals has been linked to various chronic health conditions. These unstable molecules can be neutralized by antioxidants, which help reduce their harmful effects. Oxidative stress, which results from an imbalance between the production of Reactive Oxygen Species (ROS) and the body’s antioxidant defenses, contributes to cellular damage over time. This type of damage is implicated in aging and numerous diseases, including cancer, cardiovascular ailments, and neurological disorders. ROS encompass both oxygen-based radicals, such as hydroxyl, superoxide, peroxyl, and alkoxyl radicals-and non-radicals like hydrogen peroxide, hypochlorous acid, singlet oxygen, and ozone. Additionally, nitrogen-based reactive species, like nitric oxide and peroxynitrite, fall under the broader category of Reactive Nitrogen Species (RNS). Despite the body’s natural antioxidant defense mechanisms, they may not always suffice to counteract these radicals effectively.[2] A compromised antioxidant system can elevate the frequency of radical-induced cell damage. Excessive free radical activity has been associated with various diseases, including cancer,[3] heart diseases,[4] neurological disorders,[5] Alzheimer’s disease,[6] mild cognitive impairment,[7] Parkinson’s disease,[8] alcohol-related liver damage,[9] ulcerative colitis,[10] aging,[11] and atherosclerosis.[12] This highlights the urgent need to explore plant-based antioxidants. Free radicals are atoms or molecules with an unpaired electron, rendering them unstable. In an attempt to stabilize, they interact with vital cellular components such as proteins, lipids, and DNA, potentially leading to structural and functional damage.[13] Although oxygen is vital for life, its metabolism in the mitochondria during energy production also gives rise to these reactive species. Lipids, proteins, nucleic acids, and carbohydrates are particularly vulnerable to oxidative attacks. To defend against this, antioxidants play a crucial role by neutralizing free radicals. These compounds donate electrons to unstable molecules, helping to stabilize them and prevent further cellular damage. In doing so, antioxidants convert harmful radicals into excretable byproducts, thereby protecting the body. Scientific research supports that a diet rich in antioxidant-containing fruits and vegetables can lower the risk of diseases associated with oxidative stress.[14] This protective effect is largely attributed to the presence of phytochemicals like polyphenols, carotenoids, and vitamins C and E.[15] Although many endemic plant species are used by local communities for medicinal purposes, there remains a significant gap in scientific data on their antioxidant potential. Yet, phenolic compounds-commonly found in edible and non-edible herbs, grains, fruits, vegetables, spices, and oils—are known for their strong antioxidant properties. Hence, identifying and evaluating these properties in underexplored plant species remains a valuable pursuit, especially in the context of developing functional foods or nutraceuticals.[16] This study, therefore, outlines commonly used methods to assess antioxidant activity in plant materials. The principal assays include:
- DPPH assay (2,2-diphenyl-1-picrylhydrazyl),
- Hydrogen peroxide scavenging assay (H₂O₂),
- Phosphomolybdenum assay,
- Nitric oxide scavenging activity,
- Superoxide radical scavenging assay,
- Metal ion chelation assay,
- Hydroxyl radical scavenging assay,
- These assays are fundamental tools used in antioxidant research to evaluate the free radical-scavenging potential of various plant extracts.
MATERIALS AND METHODS
Plant material
The fresh leaves of Chamaecostus cuspidatus were collected from Nadiad, Gujarat. The leaves were dried, powdered, passed through a sieve, and stored in an airtight container.
Chemicals and Reagent
All reagents used in this study were of analytical grade and obtained from reputable suppliers. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) was sourced from Natraj Scientific, while methanol, sodium nitroprusside, and potassium ferricyanide were obtained from Rankem. Hydrogen peroxide (H₂O₂), Dimethyl Sulphoxide (DMSO), and Butylated Hydroxytoluene (BHT) were purchased from Finar. Ascorbic acid was procured from Neelam Enterprise. Chemicals such as sulphuric acid, ferrous ammonium sulphate, and ammonium acetate were supplied by Loba Laboratories. Sodium phosphate, Ethylenediaminetetraacetic Acid (EDTA), and glacial acetic acid were obtained from SDFCL, while ammonium molybdate came from Laser Laboratories. Sulphadiazine and phosphoric acid were supplied by Zenith, and Trichloroacetic Acid (TCA) was procured from Himedia. Additional chemicals including ferric chloride, riboflavin, and acetone were purchased from Nilam Chemicals, Ahmedabad. All other chemicals used in the experiments were of analytical grade and acquired from established manufacturers.
Preparation of Plant Extract
The extraction of bioactive compounds from the plant material was carried out using a Soxhlet extraction method. A total of 50 g of powdered leaves were accurately weighed and placed in a filter paper thimble. Methanol was used as the extraction solvent. The Soxhlet apparatus was set up with the solvent heated to a boil, causing it to vaporize, condense in the cooling unit, and drip into the thimble containing the plant powder. This cyclic process was continued until the solvent in the thimble became colorless, indicating that exhaustive extraction had been achieved. The methanolic extract was then concentrated by evaporating the solvent over a hot plate. The resulting concentrated extract was transferred to a labeled container and stored under appropriate conditions for subsequent analysis.
Antioxidant Assay
DPPH Radical Scavenging Assay
Principle
The foundation of this technique is the antioxidants' ability to reduce the DPPH radical, a stable free radical. When DPPH comes into contact with an antioxidant, it takes up an electron or hydrogen atom, which causes a noticeable change in color from deep purple to yellow.[17]
Procedure
Free radical scavenging was evaluated by mixing 3 mL of plant extract at different doses (50-150 µg/mL) with 1 mL of 0.1 mM DPPH solution made in methanol. After being vortexed, the samples were left to stand at room temperature for half an hour in the dark. The wavelength of absorbance was 517 nm. The baseline was a control sample with all reagents except the extract. An antioxidant reference was vitamin E.
Antioxidant activity was assessed using:
Hydrogen Peroxide (H₂O₂) Scavenging Assay
Principle
This method evaluates the ability of antioxidants in plant extracts to reduce hydrogen peroxide, monitored through UV absorbance at 230 nm, where H₂O₂ absorbs strongly.
Procedure
Different concentrations (300-900 µg/mL) of the plant extract were prepared. To each test tube, 600 µL of H₂O₂ was added, followed by 400 µL of either the extract or ascorbic acid (standard). Phosphate buffer (pH 7.4) was added to bring the total volume to 2 mL. Samples were incubated at room temperature for 10 min, and absorbance was measured at 230 nm against a blank.
Scavenging efficiency was calculated using the same formula as above.[18]
Where, A1= the absorbance for the crude plant extract or standard, and
Ao= the absorbance of the control (without extract).[19]
Phosphomolybdenum Assay
Principle
This assay evaluates the total antioxidant capacity based on the reduction of Mo (VI) to Mo(V) by antioxidants, forming a green-colored phosphate/Mo(V) complex, which is measured spectrophotometrically.
Procedure
A total of 3 mL of reagent (consisting of 1 mL each of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) was mixed with 0.3 mL of plant extract (20-100 µg/mL). The mixture was incubated at 95ºC for 90 min. After cooling, the absorbance was read at 695 nm against a blank, using BHT as a reference standard.[20]
Nitric Oxide Scavenging Activity
Principle
Nitric Oxide (NO) generated from sodium nitroprusside interacts with oxygen to form nitrite ions, which can be quantified via the Griess reaction. Antioxidants inhibit NO formation by reducing nitrite accumulation.
Procedure
2 mL of 10 mM sodium nitroprusside was mixed with 0.5 mL phosphate buffer saline (pH 7.4) and 0.5 mL of extract at different concentrations. The mixture was incubated for 150 min at 25ºC. Then, 1 mL of the incubated sample was treated with 3 mL of Griess reagent (1% sulfanilamide, 2% phosphoric acid, 0.1% NED dihydrochloride). After 30 min at room temperature, absorbance was recorded at 540 nm. Ascorbic acid was used as the standard.
Where, A1= the absorbance for the crude plant extract or standard, and
Ao= the absorbance of the control (without extract).[21]
Reducing Power Assay
Principle
This test assesses an antioxidant's ability to reduce Fe³⁺ to Fe²⁺. In an acidic medium (pH 3.6), the increased absorbance at 700 nm indicates higher reducing capacity due to electron transfer activity.[22]
Procedure
2.5 mL of 0.2 M phosphate buffer (pH 6.6), 2.5 mL of 1% potassium ferricyanide, and 1 mL of extract were mixed and incubated at 50ºC for 20 min. After adding 2.5 mL of 10% TCA, the mixture was centrifuged. The supernatant (2.5 mL) was combined with 0.5 mL of 0.1% FeCl₃ and 2.5 mL distilled water. Absorbance was measured at 700 nm.[23]
Superoxide Radical Scavenging Assay
Principle
Riboflavin in the presence of light initiates the generation of superoxide radicals, which reduce NBT to formazan. Antioxidants inhibit this reaction by scavenging the superoxide radicals.
Procedure
A reaction mixture (1 mL) containing 50 mM phosphate buffer (pH 7.4), 10 µM riboflavin, 56 µM NBT, 12 µM EDTA, and different concentrations of the extract (10-200 µg) was prepared. The mixture was exposed to light for 2 min, and absorbance was measured at 590 nm at 30-second intervals. Vitamin E was used for comparison.[24]
Metal Chelating Assay
Principle
Ferrozine forms a red complex with Fe²⁺ ions. When a chelating agent is present, it binds Fe²⁺, reducing complex formation and absorbance at 562 nm.
Procedure
0.5 mL of 0.2 mM FeCl₂ was added to 0.1 mL of extract, followed by 0.2 mL of 5 mM ferrozine. The mixture was incubated for 10 min at room temperature. Absorbance was measured at 562 nm. Citric acid or EDTA served as positive controls.[25]
Hydroxyl Radical Scavenging Assay
Principle
Hydroxyl radicals generated by the reaction between Fe²⁺ and H₂O₂ react with DMSO to form formaldehyde. The amount of formaldehyde, detected by Nash reagent, reflects hydroxyl radical levels.
Procedure
Various extract concentrations (100-500 µg) were combined with 1 mL of Fe-EDTA, 0.5 mL EDTA (0.018%), and 1 mL DMSO (0.85% in 0.1 M phosphate buffer, pH 7.4). The reaction was initiated by adding 0.5 mL of 0.22% ascorbic acid and incubated at 80-90ºC for 15 min. After cooling, 1 mL of cold 17.5% TCA and 3 mL of Nash reagent were added. After 15 min at room temperature, absorbance was recorded at 412 nm.
Each assay used the same formula to calculate radical scavenging or inhibitory activity:
Where, A1= the absorbance for the crude plant extract or standard, and
Ao= the absorbance of the control (without extract).[26]
RESULTS AND DISCUSSION
The antioxidant efficacy of the plant extract was thoroughly assessed using a comprehensive set of well-established in vitro assays. The extract exhibited notable free radical scavenging and antioxidant properties in the following assays: DPPH (2,2-Diphenyl-1-picrylhydrazyl), hydrogen peroxide scavenging, phosphomolybdenum reduction, nitric oxide scavenging, reducing power, superoxide radical scavenging, metal chelation, and hydroxyl radical scavenging. Figures 1-3 represents picture of Chamaecostus cuspidatus plant, Graphs of DPPH and Reducing power assay and Graph of Hydrogen Peroxide radical, ferrous iron chelating, phosphomolybdenum. Hydroxyl radical, Nitric oxide, superoxide radical scavenging assay respectively. While Tables 1-3 represents Observation of DPPH, H2O2, Nitric oxide Assay, Observation of Superoxide, Metal chelating, Hydroxyl radical Assay and Comparison of IC50 between sample and standard extract respectively.
| DPPH Assay | H2O2 Assay | Nitric oxide assay | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample | Standard | Sample | Standard | Sample | Standard | ||||||
| Conc. (μg) | %Scavenging | Conc. (μg) | %Scavenging | Conc. (μg) | %Scavenging | Conc. (μg) | % Scavenging | Conc. (μg) | % Scavenging | Conc. (μg) | % Scavenging |
| 100 | 20.43 | 5 | 1.2 | 200 | 24.67 | 100 | 26.26 | 100 | 5.7 | 100 | 15.19 |
| 200 | 52.52 | 10 | 4.6 | 300 | 40.74 | 200 | 29.33 | 300 | 7.9 | 200 | 23.86 |
| 300 | 62.74 | 20 | 12.74 | 600 | 58.9 | 300 | 33.61 | 500 | 10.2 | 300 | 28.11 |
| 400 | 71.03 | 25 | 21.63 | 700 | 68.72 | 400 | 35.36 | 700 | 14.7 | 400 | 31.37 |
| 500 | 72.83 | 30 | 31.73 | 800 | 78.85 | 500 | 40.95 | 900 | 24.6 | 500 | 44.58 |
| 900 | 97.9 | 1500 | 35.7 | ||||||||
| 2000 | 42.66 |
| Superoxide Assay | Metal chelating assay | Hydroxyl radical assay | ||||||
|---|---|---|---|---|---|---|---|---|
| Conc. (μg) | %Scavenging (Sample) | %Scavenging (Standard) | Conc. (μg) | %Scavenging (Sample) | %Scavenging (Standard) | Conc. (μg) | %Scavenging (Sample) | %Scavenging (Standard) |
| 10 | 6 | 14.28 | 10 | 21.9 | 31.8 | 100 | 22.22 | 28.8 |
| 25 | 23.57 | 43.57 | 20 | 35 | 44.5 | 200 | 33.95 | 36.6 |
| 50 | 67.5 | 54.64 | 30 | 38.8 | 49.6 | 300 | 38.68 | 43.2 |
| 100 | 74.64 | 72.5 | 40 | 42.1 | 54.6 | 400 | 48.35 | 54.52 |
| 200 | 75.71 | 77.85 | 50 | 50.9 | 60 | 500 | 55.76 | 59.87 |
| Assay | Sample extract IC50 (µg) | Standard IC50 (µg) |
|---|---|---|
| DPPH radical scavenging assay | 190.4 | 47.3 (Vitamin E) |
| H2O2 radical scavenging assay | 509.3 | 610.5 (Ascorbic acid) |
| Nitric oxide scavenging assay | 2300 | 560 (Ascorbic acid) |
| Superoxide radical scavenging assay | 37 | 45.83 (Ascorbic acid) |
| Metal chelating assay | 50 | 30 (BHT) |
| Hydroxyl radical scavenging assay | 413 | 366 (BHT) |
DPPH Radical Scavenging Assay: The extract showed effective free radical inhibition in the DPPH assay. It exhibited an IC₅₀ value of 190.4 µg, which, while indicative of strong antioxidant potential, was less potent than the standard (47.3 µg). The comparatively higher IC₅₀ value suggests that a greater quantity of the extract is needed to achieve similar radical scavenging effects.
Hydrogen Peroxide Scavenging Assay: The extract demonstrated superior antioxidant capacity with an IC₅₀ of 509 µg, outperforming the standard (610 µg). These findings highlight the potential of the extract as a natural alternative to synthetic antioxidants.
Phosphomolybdenum Assay: This assay, which assesses the total antioxidant capacity through molybdenum reduction, revealed that increasing the concentration of the extract correspondingly increased absorbance at 695 nm, indicating a dose-dependent rise in reducing power.
Nitric Oxide Scavenging Activity: The extract showed nitric oxide scavenging activity with an IC₅₀ of 2.3 mg. However, its efficacy was lower compared to the reference standard, ascorbic acid (560 µg), suggesting a relatively moderate capacity in quenching nitric oxide radicals.
Reducing Power Assay: The extract displayed dose-dependent reducing potential, albeit lower than that of the standard antioxidant, BHT. The observed reducing activity is likely due to the complex mixture of phytochemicals present in the crude extract, each contributing differently to the antioxidant mechanism.
Superoxide Radical Scavenging Assay: The extract exhibited stronger activity than the standard, ascorbic acid, with an IC₅₀ of 37 µg compared to 45.83 µg. This indicates that the extract has potent superoxide scavenging ability, likely owing to its intrinsic antioxidant components.
Metal Chelating Activity: The extract (50 µg) demonstrated noteworthy metal ion chelating ability, closely comparable to that of the standard (30 µg). This suggests that the extract can effectively bind ferrous ions and reduce metal-catalyzed oxidative reactions.
Hydroxyl Radical Scavenging Assay: The hydroxyl radical scavenging capability of the extract was moderate, with an IC₅₀ of 413 µg, which was slightly less effective than that of the standard antioxidant BHT (366 µg). Despite being less potent, the extract still showed significant radical scavenging potential.
CONCLUSION
The current investigation highlights Chamaecostus cuspidatus as a potent natural source of antioxidants, exhibiting significant efficacy in neutralizing hydrogen peroxide, superoxide, and hydroxyl radicals. Notably, the extract outperformed standard antioxidants in hydrogen peroxide and superoxide scavenging assays, suggesting the presence of phytochemicals with strong radical-quenching properties that can effectively reduce oxidative stress. These findings support the traditional medicinal uses of C. cuspidatus and point to its potential application in the development of pharmaceutical, nutraceutical, and functional food products aimed at preventing or managing oxidative stress-related conditions. The plant has demonstrated therapeutic promise in the management of diabetes, protection of pancreatic cells from age-related damage, and in mitigating neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, as well as inflammatory diseases like rheumatoid arthritis. Its effectiveness, coupled with a favorable safety profile, positions C. cuspidatus as a valuable candidate for the development of plant-based oral antidiabetic formulations.