INTRODUCTION:

The oldest known method of treatment of a disease is the use of herbs, which has historically been practiced by all societies. Currently, more than 80% of people worldwide use herbal remedies. Although the composition of herbal medicines varies, it is believed that their active ingredients are directly related to their quality.

Therefore, it is important to use extracts in which the chemical composition and amounts of each active ingredient are precisely determined.^1,2

The chemicals produced by plants are known as phytoconstituents. They act as a defense system for plants and are, therefore, considered the main source of healing. Among all the plant components, flavonoids have many pharmacological properties and are, therefore, currently the subject of research. Their properties include antibacterial, antiviral, anticancer, anti-inflammatory, and antioxidant.^3,4,5

This vegetable plant has been consumed as "Rajma" in India from ancient times. The seeds are well known for their carbohydrate, protein, and vitamin contents. The seeds of the plant exhibit pharmacological properties, including anti-inflammatory, anti-cancer, anti-diabetic, and antioxidant properties.⁶ Flavonoids possess pharmacological activities. Hence, researchers are currently interested in the quantification and isolation of flavonoids, as they help in formulation and drug development studies.

The aqueous extraction procedure seems to be a more environmentally friendly choice because it eliminates the need for organic solvents and requires less energy and costs. Water-soluble phytoconstituents are beneficial because of their bioavailability. Water-based plant phytoconstituents have diverse applications in various industries and offer various health benefits. Their health-promoting properties make them valuable for nutraceuticals, pharmaceuticals, cosmetics, food preservation, and agriculture. By harnessing these natural compounds, we can develop effective and sustainable health and wellness solutions.⁷While there are many different chromatographic methods, HPLC, or high-performance liquid chromatography, is the newest, most effective, and user-friendly.⁸ This technique involves UV-visible light and can identify substances that are within the UV-visible range makes it useful. The best advantage of this chromatographic technique is that the separation, quantification, and isolation of polar compounds take place by RP-HPLC.^9,10,

Antioxidants, vitamins, and trace elements are well established to be useful in preventing cancer, cardiovascular disease, eye diseases, and illnesses associated with aging.^{11,12,13,14,15} As the demand for herbal medication continues to increase due to fewer side effects, the current research aims to establish a method for quantifying a flavonoid and to evaluate the DPPH radical scavenging activity of a crude aqueous extract of Phaseolus vulgaris Linn. leaves.

MATERIALS AND METHODS:

Authentication of plant:

Procurement and verification of plant material Specimens of Phaseolus vulgaris L. were harvested from Dapoli, Maharashtra. Authentication of the plant was conducted at St. Xavier's College, Mumbai, which assigned the voucher number D.P.2275. The harvested leaves underwent a cleaning process with water and were subsequently air-dried in a sheltered environment. Following complete dehydration, the leaves were reduced to a fine powder using a mechanical grinder. The resulting powdered plant material was then transferred to an airtight container for storage.

Extraction:¹⁶

A beaker was filled with 50g of powdered dried leaves and 1 liter of solvent was added. The plant powder was dissolved in the solvent by placing the beaker in a sonicator for approximately ten minutes. Solvents from the non-polar to polar range were used as it is a sequential method of extraction. Each extract was evaporated using a rotary evaporator to obtain the dry extract. The aqueous extract was lyophilized to obtain a dry crude aqueous extract. The extract was examined by RP-HPLC.

HPLC Instrumentation and Method¹⁷^,¹⁸

The ether, dichloromethane, ethyl acetate, methanol, and aqueous extracts were analyzed by RP-HPLC using a Waters 2695 separation module coupled to a photodiode array (PDA) detector (2996). A Hemochrom C18 column (150mm × 4.6mm, 5-micron dimension column is employed. The gradient program used was solvent A-Acetonitrile(ACN), solvent B-0.05% Trifluoroacetic acid (TFA) as mobile phase, run time 24 min, delay time 6 min, and gradient program used,0-20 Min- 0-100 % ACN, 20-24 min 100% ACN, 24-25 Min 0 %ACN, 25-30Min 0% ACN, the maximum wavelength of absorption (λ_max) was set at 210nm. The flow rate was set at 1 mL/min. The current goal of the study was to quantify flavonoids in the aqueous extract; therefore, the UV absorbance of each peak in the aqueous extract was measured at 210nm. The chromatographic data were recorded and processed using the Empower 1 software. The High-Performance Liquid Chromatography (HPLC) instrumentation is depicted in Figure 1.

Image 1: A picture depicting HPLC

Chromatograms:

Sample preparation:

Each extract was dissolved in high-performance liquid chromatography (HPLC) water at a concentration of 1 mg/mL and used for HPLC analysis. The solution was then injected into the system. The injection volume was approximately 20µL. The chromatogram obtained for each extract provided information about the retention time and % area. All that is represented by the region beneath the percentage is the amount of the component in the extract. Samples were filtered through a 0.45µm micro syringe filter before injection¹⁷.The peaks obtained in the chromatogram of each extract were resolved and clear.

Fig 1: represents the chromatogram produced from the PET ether extract of Phaseolus vulgaris Linn.

Fig 2: represents the chromatogram produced from the DCM extract of Phaseolus vulgaris Linn.

Fig 3: represents the chromatogram produced from the EAA extract of Phaseolus vulgaris Linn.

Fig 4: represents the chromatogram produced from the methanol extract of Phaseolus vulgaris Linn.

Fig 5: represents the chromatogram produced from the aqueous extract of Phaseolus vulgaris Linn.

DPPH assay: in vitro

Evaluating the extract's antioxidant potential can be done quickly, affordably, and effectively with the DPPH assay.¹⁹^,²⁰^,²¹This assay is the most commonly used assay to test the antioxidant potential of plant extracts. DPPH assay performed at room temperature DPPH assay performed at room temperature.²²^,²³^,²⁴^,²⁵^,²⁶

Procedure:

0.1ml of 0.1mM DPPH solution was mixed with 5 μl of a separate stock of the test drug (0, 1, 10, 50, 100, 250, 500, 1000)µg/mL in a 96-well plate. The reaction was set up in triplicate, and blank duplicates containing 0.2 ml DMSO/Methanol and 5μl of various chemical concentrations (0, 0.1, 0.5, 1, 5, 10, 25, 50) µg/mL were made. The plate was left in the dark for thirty minutes. Using a microplate reader (iMark, Bio-Rad), the decolorization was measured at 495nm at the conclusion of the incubation. The control was a reaction mixture with 20μl of deionized water in it. For the control, the scavenging activity was displayed as "% inhibition." IC-50 was computed using Software Graph Pad Prism 6. The following equation was used to compute the DPPH scavenging activity.

Radical scavenging activity = ((Abs Control- Abs Sample)/Abs Control) ×100

Where, Abs. control – Absorbance of blank without extract, Abs. Sample- Absorbance value of test compound (with extract)

Statistical data:

ANOVA or one-way analysis of variance was used to analyze the data. Statistical significance was set at p< 0.05. The mean±standard error of the mean (SEM) was used to express the results of four replicated experiments.

RESULTS:

HPLC chromatogram (Fig1) of PET ether extract of Phaseolus vulgaris Linn. at 210nm showed two peaks with retention times of 21.970 and 23.264 with percent areas of 90.83% and 9.17%, respectively. The chromatogram (Fig2) of dichloromethane extract (DCM) shows four main peaks with retention time of (20.994, 21.314, 22.025, 23.321) and 7.88% 4.47%,79.93%, and 7.73%. The chromatogram (Fig3) of the ethyl acetate extract showed seven main constituents with retention times (11.279, 11.394, 20.931, 21.254, 21.951, 23.225, and 23.932) and percent area (5.95%, 15.95%, 4.22%, 2.53%, 41.87%, 4.61%, and 24.86%). The chromatogram (Fig4) of methanol extract showed seven main constituents with retention times (6.003, 6.286, 6.503, 6.757, 6.954, 7.456, 8.760) and percent area (5.26 %, 0.74%, 17.52%, 4.16 %, 5.33%, 3.03%, 63. 94%). The chromatogram (Fig5) of the aqueous extract showed four well-resolved peaks with retention times (5.816, 6.607, 8.218, and 8.864) with percent area (11.09% 16.12%, 45.89%, and 26.89%, respectively), and the four peaks were analyzed for the presence of flavonoids using a PDA detector at 210nm. Peak 1, at a retention time of 5.816, showed an absorption band at 313nm. Alpha-amino acids or proteins in aqueous media show electronic absorption in the UV region from 185 to 320 nm. Hence, it can be said that peak 1 is an alpha amino acid.²⁷ Peak 2 at a retention time of 6.607 min shows an absorption maximum at 313nm. Most of the substituted coumarin derivative shows absorbance from 302nm to 350nm.²⁸ It is observed that peak no.3 having a retention time of 8.218 and a % area of 45.89% shows UV absorbance shows two maximum absorption bands I at 255nm and band II at 354nm, respectively.²⁹ The basic structure of flavonoids consists of three heterocycles. As shown in Fig.6. A heterocyclic ring system connects rings A and B to ring C. Flavonoids show two absorption bands I and II at 255nm and 355nm due to the cinnamoyl and benzoyl system.³⁰ Peak 4, which has a retention time of 8.864 min and a peak area of 26.89%, shows two absorption bands at 220nm and 279nm, respectively.³¹ Flavones are a subgroup of flavonoids and are mostly found in the leaves of the plant in the form of glycosides.

Fig 6: Basic ring skeleton of flavonoid.

DPPH assay:

Image 2: Micro plate reader comparison of standard ascorbic acid and crude aqueous extract.

Fig: 8 The graph shows the concentration of ascorbic acid in µg/mL required for radical scavenging activity.

Fig: 9 The graph shows of the concentration of crude aqueous extract of the leaves in µg/mL required for radical scavenging activity

The concentration in µg/mL was plotted on the X-axis and percentage inhibition was plotted on the Y-axis to obtain the graph. Ascorbic acid at very low concentrations (0, 0.1, 0.5, 1, 5, 10, 25, 50) µg/mL shows a significant difference from crude aqueous extract having concentrations (0, 1, 10, 50, 100, 250, 500, 1000) µg/mL as shown in above Fig:8 and Fig.9. Ascorbic acid and aqueous extracts were compared for their respective DPPH radical scavenging capacities, which were calculated using IC50 values. The final extract concentration, measured in µg/mL of the dry extract, which is required to reduce the initial DPPH concentration by half, is called the IC50 value. By graphing % inhibition vs. concentration, the IC50 of the crude aqueous extract and ascorbic acid standard was determined graphically.

DISCUSSION:

There are numerous benefits of using water as an extraction solvent for flavonoids, including environmental friendliness, cost-effectiveness, safety, and ease of regulation. Quantification of a class of phytoconstituents is highly dependent on the extraction technique used. The sequential extraction method follows the rule “likes to dissolve in likes” and hence, the targeted class of phytoconstituents can be easily detected. HPLC helps quantify the phytoconstituents present in a particular extract.

The analysis of flavonoids was carried out using UV-Vis spectrophotometry because their conjugated aromatic systems exhibit substantial absorption bands in both the visible and ultraviolet light spectra. While flavonoids can be easily identified using UV data, further methods need to be used to characterize a compound, and spectroscopic techniques, including Mass, NMR, and IR, can be employed.

DPPH is a purple radical, but when it reacts with antioxidants, its color changes to yellow. The reaction was performed in triplicate. The extent of the reaction depends on the ability of the antioxidant to release hydrogen. By observing the color change in the microplate, it is clear that when ascorbic acid combines with the DPPH reagent, its original purple color transforms to a dark yellow color. Similarly, when the crude aqueous extract reacted with the DPPH reagent, it changed to a pale yellow color. This demonstrated that the crude aqueous extract had antioxidant potential. Compared to ascorbic acid, the crude extract had a lower radical scavenging activity. This may be due to the synergetic effects of other phytoconstituents.

CONCLUSION:

Therefore, it is essential to measure flavonoids to maximize their health benefits. Supporting research, standardization, and application in disease prevention and treatment is imperative to improve public health and therapeutic outcomes. It has been verified from the literature that peak 3, which has a percentage area of 45.89% and a retention period of 8.218 minutes, has a UV absorption band that is identical to a flavonoid. Therefore, in comparison to other types of phytochemicals, it can be inferred that flavonoids are present in significant concentrations in the aqueous extract of Phaseolus vulgaris Linn.

Studies have reported a crude aqueous extract from the leaves of Phaseolus vulgaris Linn. has an antioxidant effect, but is weaker than that of conventional ascorbic acid. A lower IC50 value indicates better antioxidant capacity in a product. The aqueous crude extract had an IC50 value of 129.7±0.031µg/ml, while ascorbic acid had an IC50 value of 14.27±0.068µg/ml.

Thus, the involvement of other phytoconstituents may be responsible for the low antioxidant activity of plant leaves. Therefore, plant components need to be isolated and purified from aqueous extracts, as they are eco-friendly and inexpensive solvents, to assess their antioxidant capacity. The isolated compound can be used in the new drug formulation hence its quantification, isolation, and characterization of flavonoid is essential. Furthermore, to understand the antioxidant capacity of an isolated compound, it is necessary to investigate its DPPH radical scavenging activity.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

ACKNOWLEDGEMENT:

The authors acknowledge the continuous support and guidance provided by research guide Dr. Madhavi Badole and Principal Dr. Anushree Lokur of Ramnarain Ruia Autonomous College, Matunga, Mumbai.

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Received on 20.05.2024 Revised on 11.09.2024

Accepted on 19.12.2024 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2633-2638.

DOI: 10.52711/0974-360X.2025.00378

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