Correlation of Flavonoid and Phenolic Content with Antioxidant and Cytotoxic Activities of Centella asiatica L. Leaves

Jhuma Deb¹*, Soumya Saha¹,Doyel Mukherjee¹, Nilip Kanti Deb²

¹Netaji Subhas Chandra Bose Institute of Pharmacy, Tatla, Chakdaha, Nadia, West Bengal, Pin. – 741222, India.

²Global College of Pharmaceutical Technology, Kolkata, West Bengal, Pin. - 741102, India.

*Corresponding Author E-mail: jhumaniladi@gmail.com

ABSTRACT:

Oxidative stress plays a central role in the pathogenesis of numerous chronic diseases, including cancer, neurodegenerative disorders, cardiovascular dysfunction, diabetes, and inflammation. This biological imbalance occurs when the generation of reactive oxygen species exceeds endogenous antioxidant defenses, ultimately leading to cellular injury and cytotoxicity. Medicinal plants are widely recognized as rich sources of antioxidant and cytotoxic agents, largely attributed to their bioactive phytochemicals such as phenolics and flavonoids. Despite India’s extensive plant biodiversity, many traditionally used species remain insufficiently explored for their phytochemical composition and cytotoxic potential. Centella asiatica L. (Gotu kola), an important medicinal herb in Ayurvedic, African, and Chinese systems of medicine, has been historically valued for its cognitive, dermatological, anti-inflammatory, and wound-healing properties. However, limited information exists regarding the relationship between its phenolic and flavonoid content, antioxidant activity, and cytotoxic effects. The present study aimed to quantify the total phenolic and flavonoid content of C. asiatica leaf extracts (chloroform, ethanol, and aqueous), evaluate their antioxidant capacity using DPPH and hydrogen peroxide scavenging assays, and assess cytotoxicity via the brine shrimp lethality bioassay. The ethanolic extract contained the highest levels of flavonoids (77.01mg HE/g) and phenolics (28.05mg GAE/g), followed by the aqueous and chloroform extracts. Consistent with its higher phytochemical content, the ethanolic extract exhibited the strongest antioxidant properties, demonstrating an IC₅₀ of 51.66µg/mL in the DPPH assay and 4.88 µg/mL for hydrogen peroxide scavenging, approaching the activity of the standard ascorbic acid. Cytotoxic evaluation revealed a dose-dependent response, with the ethanolic extract again showing the greatest potency (LC₅₀ = 62.50µg/mL), followed by aqueous and chloroform extracts. Overall, the study establishes a clear correlation between phenolic/flavonoid concentration and both antioxidant and cytotoxic activities in C. asiatica leaves. These findings support the plant’s traditional medicinal applications and highlight its potential as a promising source of bioactive compounds for future fractionation, mechanistic investigations, and therapeutic development.

KEYWORDS: Centella asiatica, Phenolic content, DPPH, Hydrogen peroxide, Cytotoxicity.

INTRODUCTION:

Many human diseases like cancer, neurodegenerative disorders, cardiovascular disease, diabetes, and inflammation occur due to oxidative stress. This occurs when reactive oxygen species exceed the body’s antioxidant defense¹. Prolonged oxidative stress can cause cellular damage and cytotoxicity. A substantial body of evidence supports the antioxidant properties of various medicinal plants, like Amaranthus hybridus, Allium sativum, Mangifera indica, Garcinia kola²etc. Additionally, plants such as Catharanthus roseus, Taxus baccata, Annona squamosa, Salvia officinalis³ have shown significant cytotoxic properties in vitro and in vivo. These effects are frequently linked to polyphenols, phenolic acids, flavonoids, tannins, and lignans which neutralize free radicals, reduce lipid peroxidation, and enhance the body’s defenses⁴.

Despite the strong cytotoxic properties of numerous medicinal plants, many used in India’s rich biodiversity remain underexplored regarding their cytotoxic potential⁵ and the specific contributions of phenolics and flavonoids. Indigenous communities⁵ have traditionally utilized specific plants for treating tumors, skin infections, and inflammatory conditions, often linked to abnormal cell proliferation underscoring the value of validating traditional claims for drug discovery. With cancer emerging as a significant global health challenge and increasing resistance to current treatments, identifying affordable, plant-based cytotoxic agents is imperative. Among the ethnomedicinal plants with cytotoxic potential, Centella asiatica L. (Gotu kola) is notable for its traditional use in African, Chinese and Ayurvedic medicine⁶ with reported cognitive and dermatological benefits and broader anti-inflammatory and healing properties. Scientists have reported that plants with a complex phytochemical matrix, where phenolics and flavonoids are present contribute to antioxidant capacity and may influence cytotoxic outcomes⁷. This study therefore aims to quantify total phenolics and flavonoids in Centella asiatica L. leaves, assess antioxidant capacity and cytotoxic effects of leaf extracts, and establish correlations between phenolic/flavonoid contents and these bioactivities⁸. By clarifying the contributions of phenolics to antioxidant and cytotoxic responses, fractionation, mechanism studies, and potential applications of Centella asiatica L. may be further established as a source of bioactive agents.

MATERIALS AND METHODS:^9-12

Plant materials:⁹

The fresh leaves of the plant Centella asiatica L. was collected from Nadia Nursery, Chakdaha, Nadia, West Bengal during the month of March 2025 and authenticated by Dr. Suchandra Samanta Mandal, Assistant Professor, M.Sc. (Botany), KU, West Bengal (Cert/03/25). The plant material after harvesting was thoroughly cleaned, dried and then powdered coarsely.

Experimental Chemicals and Reagents:

Grade chemicals, solvents, and reagents⁹ were used of Analytical grade from S. D. Fine Chem⁹ Ltd., Loba Chem, SRL, Mumbai. Brine shrimp (Artemia salina) eggs purchased online from “Pequon Capsule; U.S”. Artificial seawater was prepared under controlled laboratory conditions.

Preparation of Extract:⁹

Petroleum ether (40–60°C) was used to remove fats from the dried powdered plant material followed by cold maceration using chloroform, ethanol, and water. The extracts were concentrated and used⁹ for further studies.

Phytochemical Screening¹⁰

Screening of chemical substances present in the plant was carried out using standard established qualitative chemical tests.¹⁰

Spectrophotometric Determination of Total Flavonoids by Aluminium Chloride Colorimetric Method:^11-12

Preparation of calibration curve of Hesperidin:¹¹

A stock solution of Hesperidin¹¹ (1mg/mL)¹² was prepared using 10mL methanol where 10mg of the compound was dissolved. From this, standard solution, 20, 40, 60, 80, and 100µg/mL¹² were prepared by appropriate dilution with methanol. Absorbance of each sample was recorded at 415nm employing a UV-Vis spectrophotometer¹². A calibration curve was plotted by graphing absorbance against concentration.

Quantitative Determination of Total Flavonoid¹² Concentration:

The Total flavonoid content¹² was estimated using the method described by Alam et al. (2024)¹², with necessary alterations. 1mL of plant extract (1000µg/ mL)¹² was combined with 3mL methanol, 0.2mL 10% AlCl₃, 0.2mL 1M potassium acetate, and 5.6mL distilled water¹²; incubated for 30min at room temperature followed by measurement of absorbance at 415nm. Hesperidin (20–100µg/mL) was used as the standard and total flavonoid content¹² was expressed as mg Hesperidin¹¹ equivalents per gram of dry extract (mg HE/g).

Quantitative Estimation of Total Phenolic Concentration by Folin–Ciocalteu Method:^12-13

Preparation of Calibration Curve of Gallic Acid:¹³

A 1 mg/mL stock solution of Gallic acid¹³ was prepared in methanol using the method described by Swarna kumari et al. (2021)¹³. Standard solutions ranging from 20–100µg/mL were prepared by serial dilution¹². The absorbance¹³ of each solution was measured at 765nm¹³, and a calibration curve was constructed¹³ by plotting absorbance against concentration.

Estimation of Total Phenolic Content:¹²

Estimation of total phenolic compounds was carried out using the Folin–Ciocalteu method¹³. 1mL of plant extract (1000µg/mL) was mixed with 2.25mL of distilled water and 1.25mL of 0.2N Folin–Ciocalteu reagent. After 5 min, 1mL of saturated sodium carbonate was added¹³ and incubated for 90min with intermittent shaking at 30°C. Absorbance was recorded at 765nm¹³. Total phenolic content was expressed as mg gallic acid equivalents per gram of dry extract (mg GAE/g).

Anti-Oxidant Activity:^14-15

Preparation of Sample Solutions:

A 100 µg/mL stock solution of the extract¹⁴ was prepared in methanol. 20, 40, 60, 80, 100µg/ml were obtained by appropriate dilution¹⁴.

Standard Solution Preparation:

A 100 µg/ml of stock solution of Ascorbic acid¹⁵ was prepared in distilled water for use as standard. Working standard solutions of 20-100µg/ml were then obtained by appropriate dilution.

Evaluation of Free Radical Scavenging Activity by DPPH Method:¹⁴

A 50µM DPPH solution in methanol was prepared¹⁴. Aliquots (2mL) of DPPH¹⁴ were mixed with 2mL of test samples or standards at varying concentrations. The mixtures were incubated in the dark at room temperature for 30minutes¹⁴, and absorbance was measured at 517 nm using a UV–Visible spectrophotometer¹⁴. A control (DPPH in methanol) and a blank (methanol) were included. The percentage inhibition of DPPH radicals was calculated as:

% Inhibition = [(A₀ - A₁) / A₀] × 100, where A₀ and A₁ represent the absorbance of the control and samples, respectively. A dose–response curve was plotted, and the IC₅₀ value was determined.

Hydrogen Peroxide (H₂O₂) Radical Scavenging Activity:¹⁶

The H₂O₂ scavenging activity¹⁶ of the extracts was assessed spectrophotometrically (Karthik et al., 2019)¹⁶. A 4mM H₂O₂ solution in 0.1 M phosphate buffer (pH 7.4)¹⁶ was prepared. Extracts dissolved in DMSO (1 mL) were added to H₂O₂ at final concentrations¹⁶ of 0.5, 5, and 50µg/mL (DMSO 0.1% as control). After 10min at 20°C, absorbance was measured at 230nm¹⁶. Phosphate buffer without H₂O₂ served as the blank, and ascorbic acid was the reference. Scavenging activity (%) was calculated as: [(Absorbance _Control – Absorbance _Sample) / Absorbance _Control] × 100¹⁶.

Cytotoxic Activity:^17-20

Brine Shrimp (Artemia salina) Bioassay:¹⁷

Primary evaluation of the extracts for cytotoxic screening was evaluated using Brine shrimp cytotoxicity assay with Podophyllotoxin as reference standard and Dimethyl sulfoxide (DMSO) as control.

Hatching of Brine Shrimp:¹⁷

Brine shrimp eggs were hatched in a rectangular jar containing 1L of distilled water, 25g of sea salt, and sodium bicarbonate to maintain salinity and pH. Aeration was provided using an air pump, and a 60-100 W incandescent bulb facilitated continuous illumination at 25–30°C. After 20–24hours, nauplii were collected using a Pasteur capillary tube. Ten nauplii were placed in each 100mL plastic container for cytotoxicity testing.

Preparation of Test Solutions:¹⁷

A stock solution of each test sample (1000µg/mL) was prepared by dissolving 1mg in 1mL of DMSO¹⁷. “Serial dilutions of this stock were subsequently prepared to obtain concentrations of 500, 250, 125, 62.5, 31.25, 15.625, 7.82, and 3.9µg/mL.¹⁷ For the cytotoxicity assay, 1mL of each test concentration was added to vials containing 10 Brine shrimp nauplii along with 1mL of seawater. The control group received 1mL of DMSO and 1mL of seawater¹⁷ (1% NaCl solution).

Brine Shrimp Lethality Bioassay for Toxicity Testing:¹⁸

The nauplii were exposed to different concentrations of the extracts¹⁸. After 24 hours of exposure, the number of surviving nauplii was counted, and the mortality rate was calculated¹⁸ as percentage of dead nauplii relative to the total organisms in each group.

RESULTS AND DISCUSSION:

Statistical Analysis:¹⁸

All experiments were conducted in triplicate¹⁸, and the results are expressed as mean values±standard deviation. Statistical analysis was performed using “GraphPad Prism software¹⁸ [version 8.0.2 (263), 1992-2019 GraphPad Software, Inc. USA].” Mean comparisons were carried out by “one-way ANOVA”¹⁸, followed by “Tukey’s t-test”, with significance set at P<0.05.¹⁸

Plant Extraction:

The yield of the Chloroform, Ethanolic and Aqueous extract was 3.5% (w/w), 18.87% (w/w) and 15.6% (w/w), respectively.

Qualitative Chemical Tests:^21,22

Qualitative phytochemical screening of chloroform, ethanolic, and aqueous extracts of Centella asiatica L. leaves revealed various bioactive secondary metabolites (Table 1). All extracts consistently contained alkaloids, flavonoids, glycosides, tannins, and terpenoids, highlighting the diverse chemical profile of the plant. The ethanolic extract displayed a broad spectrum of constituents, including “alkaloids, carbohydrates, flavonoids, glycosides, phenolic compounds, saponins, tannins, terpenoids, coumarins, and reducing sugars”²², indicating its effectiveness in extracting both polar and moderately non-polar compounds²². The aqueous extract contained carbohydrates, phenolics, flavonoids, tannins, coumarins, and reducing sugars, while the chloroform extract included alkaloids, flavonoids, glycosides, tannins, and terpenoids. Notably, proteins, quinones, steroids, and anthraquinones were absent or present in negligible amounts across all extracts, suggesting their limited presence in C. asiatica leaves.

Total Flavonoid Content:¹²

“The total flavonoid content¹²in plant extracts were determined with the help of spectroscopic method¹²using Hesperidin¹² as standard.” A calibration curve was plotted for Hesperidin in the concentration range¹² of “20-100µg./ml” (Figure 1). The regression equation (y = 0.0067x+0.2221) for the standard curve for Hesperidin¹² showed an excellent coefficient of correlation (R² = 0.9995) with low intercept value. “The total flavonoid content was found to be 7.75±0.21mg. HE/g. (Chloroform) 77.01±0.38mg. HE/g (Ethanol) 49.84± 0.63mg. HE/g (Aqueous) (Figure 2).”^23,24 The values are depicted in Table 2.

Figure 1. Calibration curve of Hesperidin

Figure 2. Total Flavonoid Content in different leaves extracts

he values indicate significant differences at P < 0.05^18.

Total phenolic¹³ content:

“The total Phenolic content¹³ in plant extract was examined by using Folin - Ciocalteu’s reagent.”¹³ A calibration curve was plotted for Gallic acid¹³ in the concentration range¹³ of “20-100µg./ml” (Figure 3). The regression equation (y = 0.0105x - 0.1552) for the standard curve for Gallic acid showed an excellent coefficient of correlation (R² = 0.9985). “The total Phenolic content was found¹³ to be 8.34±0.72mg. of GAE/g (Chloroform); 28.05±0.21mg. of GAE/g (Ethanol), 11.20±0.56mg. of GAE/g¹³ (Aqueous) (Figure 4).”^25,26 The values are given in Table 2.

Figure 3. Calibration curve of Gallic acid

Figure 4. Total Phenolic Content in different leaves extracts

The values indicate significant differences at P < 0.05¹⁸

Table 2. Quantitative screening of phytochemicals (Flavonoids, Phenols) in different leaves extracts of C. asiatica L.

Leaves extract	Flavonoids (mg. HE/g. of Extract)	Phenols (mg. GAE/g. of Extract)
Chloroform	7.75 ± 0.21	8.34 ± 0.72
Ethanol	77.01 ± 0.38	28.05 ± 0.21
Aqueous	49.84 ± 0.63	11.20 ± 0.56

Significant differences were found at P < 0.05¹⁸ [HE: Hesperidin equivalent, GAE: Gallic acid equivalent]

Anti-Oxidant Activity:¹⁴

The antioxidant potential of Centella asiatica L. leaf extracts¹⁵ was determined by DPPH assay¹⁵, with Ascorbic acid¹⁵ as a standard (20–100µg/mL). All extracts showed concentration-dependent radical scavenging, with the Ethanolic extract (AE) exhibiting the highest activity, followed by the water (WE) and chloroform extract (CE)⁹. At 100µg/mL, AE achieved 74.15±0.11% inhibition, comparable to Ascorbic acid (77.45±0.38%), while WE and CE showed 70.33± 0.08% and 66.90±0.21%, respectively. IC₅₀ values confirmed AE as the most potent (51.66±0.31µg/mL), followed by WE (60.06±0.61µg/mL) and CE (71.93± 0.75µg/mL), compared to the standard (47.49±0.22 µg/mL). Statistical analysis showed significant differences (P<0.05)¹⁸, highlighting the strong antioxidant potential of AE, likely due to phenolic and flavonoid compounds.^27-30The results are depicted in Table 3; Figure 5 and 6.

Brine Shrimp Lethality Bioassay for Toxicity Testing:

Table 5. Cytotoxic Potential of leaf extracts of C. Asiatica L.

Conc. µ/ml		3.91	7.82	15.63	31.25	62.5	125	250	500	1000
% Mortality	Chloroform	20±0.21	20±0.33	30±0.41	30±0.14	40±0.24	50±0.36	70±0.21	80±0.45	100±0.01
	Ethanol	10±0.17	10±0.42	20±0.21	30±0.65	50±0.14	70±0.26	90±0.52	90±0.23	100±0.11
	Water	10±0.48	20±0.36	20±0.11	30±0.47	40±0.31	60±0.21	70±0.44	80±0.21	100±0.05

The values indicate significant differences by one-way ANOVA¹⁸, followed by “Tukey’s t-test”, P< 0.05¹⁸.

CONCLUSION:^13,16,17

The present study demonstrates clearly that the biological activities of Centella asiatica L. leaves are closely associated with their flavonoid and phenolic content.^13,16 The ethanolic extract showed the presence of high levels of flavonoids (77.01mg QE/g) and phenolics (28.05mg GAE/g), correlating with the strongest antioxidant potential both in the DPPH assay (IC₅₀ = 51.66µg/mL) and hydrogen peroxide scavenging activity (IC₅₀ = 4.88µg/mL) comparable to the standard antioxidant Ascorbic acid¹⁵ (IC₅₀=3.21µg/mL). Cytotoxicity analysis revealed a dose-dependent response for all extracts, with the ethanolic fraction displaying the greatest potency, indicating the presence of constituents with potential anticancer properties. Conversely chloroform and aqueous extracts, which contained lower flavonoid and phenolic levels, exhibited proportionally weaker biological activities.

Collectively, these findings confirm a notable correlation of the flavonoid and plant phenolics with the antioxidant and cytotoxic properties of C. asiatica L. The findings validate the ethnomedicinal use and highlight its application as a natural source of therapeutic agent¹⁷ to develop antioxidant and anticancer agents. Further studies may be focused on the isolation and characterization of active constituents, as well as elucidation of their mechanisms of action to advance its therapeutic application.

CONFLICT OF INTEREST:

The authors’ Dr. Jhuma Deb, Soumya Saha, Doyel Mukherjee and Dr. Nilip Kanti Deb declare no conflict of interest, financially or otherwise.

ACKNOWLEDGEMENTS:

The authors are grateful to Dr. Suchandra Samanta Mandal, M.Sc. (Botany), KU, M.Sc. (Education), KU, M Phil. (Education), KU, Assistant Professor, K. Bed College, Krishnanagar, West Bengal, India and management of Netaji Subhas Chandra Bose Institute of Pharmacy for providing the facilities to work.

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Received on 10.01.2026 Revised on 06.02.2026

Accepted on 04.03.2026 Published on 21.04.2026

Available online from April 24, 2026

Res. J. Pharmacognosy and Phytochem. 2026; 18(2):129-136.

DOI: 10.52711/0975-4385.2026.00018

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

Test	Alkaloid	Carbohydrate	Flavonoid	Glycoside	Phenolic compound	Saponin	Tannin	Terpenoid	Coumarin
Chloroform	+	-	+	+	-	+	+	+	-
Ethanol	+	+	+	+	+	+	+	+	+
Aqueous	+	+	+	-	+	-	+	-	+

Concentration (µg./ ml.)		20	40	60	80	100	IC 50
DPPH Radical Scavenging activity (% inhibition)	CE	23.13 ± 0.45	37.51 ± 0.69	41.96 ± 0.12	55.44 ± 0.35	66.90 ± 0.21	71.93 ± 0.75
	AE	29.11 ± 0.25	43.99 ± 0.74	54.30 ± 0.25	65.88 ± 0.47	74.15 ± 0.11	51.66 ± 0.31
	WE	27.84 ± 0.22	40.69 ± 0.58	49.97 ± 0.33	61.30 ± 0.68	70.33 ± 0.08	60.06 ± 0.61
	Ascorbic acid	35.85 ± 0.45	46.28 ± 0.23	56.21 ± 0.18	66.77 ± 0.64	77.45 ± 0.38	47.49 ± 0.22

Concentration µg/ml	Hydrogen peroxide free radical scavenging activity (%)
Concentration µg/ml	Chloroform extract	Ethanol extract	Water extract	Ascorbic acid¹⁵
0	-	-	-	-
0.5	17.21 ± 0.23	36.57 ± 0.34	29.22 ± 0.45	41.80 ± 0.14
5	35.12 ± 0.11	50.38 ± 0.17	42.19 ± 0.26	55.42 ± 0.23
50	48.23 ± 0.56	62.17 ± 0.14	52.60 ± 0.52	67.80 ± 0.11
IC 50	-	4.88 ± 0.41	38.76 ± 0.23	3.21 ± 0.36