Phytochemical Screening, In Vitro Antioxidant And Apoptotic Potential Of Aqueous And Ethanolic Leaf Extracts Of Caesalpinia Bonducella (L.) Roxb

Medicinal plants remain a principal source of new therapeutic leads, particularly in the areas of antioxidant and anticancer drug discovery, owing to the structural diversity of their secondary metabolites and their generally favourable safety profile relative to synthetic agents. Caesalpinia bonducella (L.) Roxb. (family Fabaceae/Caesalpiniaceae), commonly known as fever nut or nicker nut, is a prickly, scandent shrub distributed throughout the coastal and semi-arid tracts of India, Sri Lanka and other tropical regions. Different parts of the plant – seeds, seed kernel, leaves, bark and root – are used extensively in Siddha, Ayurvedic and folk medicine systems for the management of fever, inflammation, diabetes, diarrhoea and parasitic infections, and the plant has been reported to possess antidiabetic, anti-inflammatory, antifilarial, anxiolytic, analgesic, antipyretic, immunomodulatory, antimicrobial, antifungal and anticonvulsant activities.

Phytochemical investigations of C. bonducella have consistently identified alkaloids, flavonoids, saponins, tannins, phytosterols and phenolic compounds as major constituent classes, and several of these metabolites have been correlated with the plant’s antioxidant and cytotoxic properties.¹⁻⁴ The seed kernel is the most extensively studied part of the plant, but the leaves – which are more sustainably harvestable than seeds – have received comparatively less systematic attention despite reports of comparable or superior antioxidant and flavonoid content in leaf tissue relative to other plant parts.⁴ Crude methanolic and ethanolic extracts of C. bonducella leaves have previously demonstrated antibacterial, antidiarrhoeal and preliminary cytotoxic activity in brine shrimp lethality assays,¹ lending further support to their pharmacological potential.

Reactive oxygen species (ROS)-mediated oxidative stress is implicated in the initiation and progression of chronic degenerative disease and carcinogenesis, and plant-derived antioxidants that additionally exhibit selective cytotoxicity toward malignant cells – while sparing normal tissue – are of particular pharmacological interest. Colorectal cancer remains one of the most frequently diagnosed malignancies worldwide, and screening of plant extracts against colorectal carcinoma cell lines such as HT-29 is a standard first step in the discovery of candidate chemopreventive or chemotherapeutic phytoconstituents. Against this background, the present study was undertaken with the following objectives: (i) to perform qualitative phytochemical screening of aqueous and ethanolic leaf extracts of C. bonducella; (ii) to evaluate the in vitro free-radical scavenging (antioxidant) activity of both extracts by the DPPH assay; and (iii) to assess the cytotoxic and apoptosis-inducing potential of the extract against the HT-29 human colorectal adenocarcinoma cell line relative to the non-cancerous Vero cell line, using the MTT viability assay and acridine orange/propidium iodide (AO/PI) dual fluorescence staining.

MATERIALS AND METHODS

Collection of plant material

Fresh leaves of Caesalpinia bonducella (L.) Roxb. were collected, cleaned to remove adhering debris, chopped into small pieces and shade-dried at room temperature. The dried material was powdered mechanically and stored in air-tight containers at room temperature until further use.

Preparation of extracts

The powdered leaf sample was extracted successively with ethanol using a Soxhlet apparatus, and separately with distilled water by the hot-water-bath method at 50–60°C. Each extract was concentrated to a semisolid mass by drying on a water bath at 40–50°C and stored in labelled, air-tight containers at 4°C. The aqueous and ethanolic extracts (Figure 1B, C) were subsequently used for qualitative phytochemical screening, DPPH radical scavenging assay, and cytotoxicity/apoptosis evaluation.

Qualitative phytochemical screening

Both extracts were subjected to standard qualitative chemical tests for the detection of alkaloids, carbohydrates, glycosides, saponins, proteins, amino acids, phenolic compounds, flavonoids, terpenoids and steroids, as detailed below.

Detection of alkaloids (Wagner’s test): One hundred millilitres of extract was stirred with 3 mL of dilute hydrochloric acid and filtered. Two millilitres of the filtrate was treated with Wagner’s reagent (1.27 g iodine and 2 g potassium iodide dissolved in 5 mL water and made up to 100 mL with distilled water), added along the sides of the test tube; a reddish-brown precipitate indicated a positive reaction.⁵

Detection of carbohydrates: The extract (100 mg) was dissolved in 5 mL water and filtered. (a) Fehling’s test – 1 mL filtrate was boiled with 1 mL each of Fehling’s solution I (copper sulphate, 34.66 g/500 mL) and II (potassium sodium tartrate 173 g and sodium hydroxide 50 g/500 mL); a red precipitate indicated the presence of reducing sugars. (b) Benedict’s test – 0.5 mL filtrate was heated with 0.5 mL Benedict’s reagent (sodium citrate 173 g and sodium carbonate 100 g in 800 mL water, with copper sulphate 17.3 g in 100 mL water added) on a boiling water bath for 2 min; a characteristic coloured precipitate indicated a positive reaction.

Detection of glycosides (Borntrager’s test): Fifty millilitres of extract was hydrolysed with concentrated HCl for 2 h on a water bath and filtered. To 2 mL of the hydrolysate, 3 mL chloroform was added and shaken; the chloroform layer was separated and treated with 10% ammonia solution. A pink colour indicated the presence of glycosides.⁵

Detection of saponins (foam test): One milligram of extract was dissolved in 2 mL distilled water and filtered through Whatman No.1 filter paper; persistent froth on vigorous shaking indicated the presence of saponins.

Detection of proteins and amino acids: (a) Biuret test – 2 mL filtrate was treated with 2% copper sulphate solution followed by 1 mL of 95% ethanol and excess potassium hydroxide pellets; a pink colour in the ethanolic layer indicated proteins. (b) Ninhydrin test – two drops of ninhydrin reagent (10 mg ninhydrin in 200 mL acetone) were added to 2 mL extract; a characteristic purple colour indicated amino acids.

Detection of phenolic compounds (ferric chloride test): The extract (50 mg) was dissolved in 5 mL distilled water and treated with a few drops of neutral 5% ferric chloride solution; a dark green colour indicated the presence of phenolic compounds.

Detection of terpenoids: To 5 mL methanolic extract, 2 mL chloroform was added and mixed, followed by careful addition of concentrated H₂SO₄; formation of a reddish-brown interfacial layer indicated the presence of terpenoids.

Detection of steroids: Two millilitres of chloroform was added to the extract along with a few drops of acetic anhydride, followed by concentrated H₂SO₄; development of a blue-green colour indicated the presence of steroids.

DPPH radical scavenging assay

The free-radical scavenging activity of both extracts was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. Briefly, a 0.4 mM solution of DPPH in methanol was prepared, and 2 mL of this solution was added to increasing volumes (20–100 µL) of each extract. The reaction mixtures were incubated in the dark at room temperature for 15 min, and the absorbance was recorded at 517 nm against a blank. A lower absorbance of the reaction mixture indicated a higher free-radical scavenging activity. Percentage inhibition (radical scavenging activity) was calculated as:

Inhibition (%) = [(Abs control − Abs sample) / Abs control] × 100

Cell culture maintenance

Vero (African green monkey kidney, normal epithelial) and HT-29 (human colorectal adenocarcinoma, epithelial) cell lines were procured from the National Centre for Cell Science (NCCS), Pune, India. Cells were maintained in the logarithmic growth phase in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin, and incubated at 37°C in a humidified atmosphere of 5% CO₂ in air.

Cytotoxicity assay (MTT)

Cytotoxicity of the ethanolic leaf extract against Vero and HT-29 cell lines was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.⁷ Cells were seeded in 96-well microplates (1 × 10⁶ cells/well) and incubated at 37°C for 48 h in 5% CO₂ until 70–80% confluence was attained. The medium was then replaced, and cells were treated with the extract at concentrations of 20, 40, 60, 80 and 100 µg/mL for 24 h; untreated cells served as controls. Morphological changes were recorded under a digital inverted microscope (20×magnification) after 24 h. Cells were then washed with phosphate-buffered saline (PBS, pH 7.4), and 20 µL of MTT solution (5 mg/mL in PBS) was added to each well. Plates were incubated at 37°C in the dark for 2 h, the resulting formazan crystals were dissolved in 100 µL DMSO, and absorbance was read at 570 nm. Percentage cell viability was calculated as:

Cell viability (%) = (Absorbance of sample / Absorbance of control) × 100

All absorbance readings were recorded in duplicate (I, II) and the mean value used to calculate percentage viability; the half-maximal inhibitory concentration (IC₅₀) was estimated by linear interpolation between the two concentrations bracketing 50% viability.

Apoptosis assay by dual AO/PI staining

HT-29 cells were seeded in 6-well plates (1 × 10⁴ cells/well) and treated with the ethanolic extract for 24 h. Treated cells were washed with PBS and stained with 5 µL acridine orange (AO, 100 µg/mL) and 5 µL propidium iodide (PI, 100 µg/mL). AO permeates both viable and non-viable cells and fluoresces green, whereas PI is excluded by intact plasma membranes and stains only non-viable/late-apoptotic and necrotic cells red; the dual-staining protocol therefore allows discrimination of viable, early apoptotic, late apoptotic and necrotic cell populations. Morphological hallmarks of apoptosis were visualised by fluorescence microscopy (FLoid Cell Imaging Station) and compared between untreated control and extract-treated cells.

Statistical treatment

All cytotoxicity readings represent the mean of duplicate absorbance measurements. Dose–response data were plotted as mean percentage viability/inhibition against concentration, and IC₅₀ values were derived by linear interpolation between the two flanking data points using GraphPad-style regression in Python (SciPy/Matplotlib, v3.x).

RESULTS AND DISCUSSION

Extraction yield and preliminary observations

Successive extraction of shade-dried, powdered C. bonducella leaves (Figure 1A) with ethanol (Soxhlet) and water (hot-water bath) afforded dark brown, semisolid extracts (Figure 1B, C). The ethanolic extract was visibly darker and more viscous than the aqueous extract, consistent with more efficient extraction of moderately non-polar and amphiphilic secondary metabolites (flavonoid aglycones, terpenoids) by ethanol relative to water.

 

Figure 1. Plant material and extracts of Caesalpinia bonducella. (A) Shade-dried, powdered leaf sample. (B) Aqueous extract. (C) Ethanolic (Soxhlet) extract.

 

Figure 2. Colour reactions observed during qualitative phytochemical screening. (A) Aqueous extract (tubes 1–10 correspond to alkaloids, carbohydrates, glycosides, saponins, proteins, amino acids, phenolic compounds, flavonoids, terpenoids and steroids, respectively). (B) Ethanolic extract, tested in the same sequence.

Table 2. Dpph Radical Scavenging Activity Of Aqueous And Ethanolic Leaf Extracts

Extract volume (µL)	Aqueous – OD (517 nm)	Aqueous – Inhibition (%)	Ethanol – OD (517 nm)	Ethanol – Inhibition (%)
Control (0)	0.936	–	0.936	–
20	0.920	1.70	0.251	73.18
40	0.901	3.73	0.236	74.78
60	0.843	9.93	0.202	78.41
80	0.796	14.95	0.185	80.23
100	0.665	28.95	0.164	82.47

 

 

Figure 3. DPPH radical scavenging assay. (A) Colour change of the DPPH chromogen with increasing volume of aqueous extract (C = control; 20–100 µL). (B) Colour change with increasing volume of ethanolic extract. (C) Percentage DPPH radical scavenging activity of aqueous and ethanolic extracts as a function of extract volume.

Table 4. Mtt Cytotoxicity Of Ethanolic Extract On Ht-29 (Colorectal Adenocarcinoma) Cell Line

Concentration (µg/mL)	Absorbance I	Absorbance II	Mean Abs.	Cell viability (%)
Control	0.743	0.751	0.7470	100.00
20	0.641	0.634	0.6375	85.34
40	0.454	0.443	0.4485	60.04
60	0.253	0.241	0.2470	33.07
80	0.126	0.123	0.1245	16.67
100	0.098	0.092	0.0950	12.72

 

 

Figure 4. Dose-response curves showing percentage cell viability of Vero (normal) and HT-29 (colorectal adenocarcinoma) cell lines following 24 h treatment with the ethanolic leaf extract of C. bonducella (MTT assay). The dashed line indicates 50% viability

 

Figure 5. Phase-contrast photomicrographs (20×) of HT-29 cells following 24 h treatment with the ethanolic leaf extract of C. bonducella at 0 (control), 20, 40, 60, 80 and 100 µg/mL, showing progressive loss of monolayer confluence, cell rounding and membrane blebbing with increasing concentration. Scale bar, 200 µm.

 

Figure 6. Phase-contrast photomicrographs (20×) of Vero cells following 24 h treatment with the ethanolic leaf extract of C. bonducella at 0 (control), 20, 40, 60, 80 and 100 µg/mL, showing largely preserved monolayer morphology across the tested concentration range. Scale bar, 200 µm.

 

Taken together, the phytochemical, antioxidant and cytotoxicity/apoptosis data indicate that the phenolic-, flavonoid- and terpenoid-rich ethanolic leaf extract of C. bonducella combines meaningful free-radical scavenging capacity with selective, apoptosis-associated cytotoxicity toward HT-29 colorectal cancer cells, while showing comparatively limited toxicity toward the non-cancerous Vero cell line at the same concentrations. Phenolic and flavonoid compounds are well recognised for their dual capacity to scavenge free radicals and to modulate apoptotic signalling pathways (e.g., via mitochondrial membrane depolarisation and caspase activation) in malignant cells, and it is plausible that the constituents responsible for the strong DPPH scavenging activity of the ethanolic extract also contribute to its pro-apoptotic action on HT-29 cells; however, bioassay-guided fractionation and mechanistic (caspase activity, mitochondrial membrane potential, gene expression) studies would be required to establish this link directly.

CONCLUSION

The present study demonstrates that the ethanolic leaf extract of Caesalpinia bonducella is richer in alkaloids, carbohydrates, glycosides, saponins, phenolic compounds, flavonoids and terpenoids than the corresponding aqueous extract, and correspondingly exhibits substantially stronger DPPH radical scavenging activity. In vitro cytotoxicity screening further showed that the ethanolic extract selectively reduced the viability of HT-29 colorectal adenocarcinoma cells (IC₅₀ ≈ 47.4 µg/mL) while largely sparing the non-cancerous Vero cell line, and AO/PI dual staining confirmed that this cytotoxicity was accompanied by classical morphological features of apoptosis. These findings support the traditional use of C. bonducella and warrant further bioassay-guided isolation of the active constituents, together with mechanistic studies (caspase activation, mitochondrial membrane potential, cell cycle analysis) and in vivo validation, to fully establish its potential as a source of antioxidant and colorectal-cancer-selective cytotoxic agents.

ACKNOWLEDGEMENT

The authors thank the Department of Microbiology and the Department of Bio-Engineering, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Chennai, for laboratory facilities and support, and the National Centre for Cell Science (NCCS), Pune, for supply of the Vero and HT-29 cell lines.

ETHICAL STATEMENT

This study did not involve any human participants or live animal experimentation. The Vero and HT-29 cell lines used are established, commercially procured cell lines obtained from the National Centre for Cell Science (NCCS), Pune, India, and their use does not require institutional ethics committee approval.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest relevant to the content of this article.

REFERENCES

1. Billah MM, Islam R, Khatun H, Parvin S, Islam E, Islam SMA, Mia AA. Antibacterial, antidiarrhoeal, and cytotoxic activities of methanol extract and its fractions of Caesalpinia bonducella (L.) Roxb leaves. BMC Complement Altern Med. 2013; 13:101.
2. Bhandary SK, Kumari NS. Traditional medicinal applications and pharmacological activities of Caesalpinia bonduc. Journal of Applied Pharmaceutical Science 2012; 2(8):25-29.
3. Saha P, Mazumder K, Gupta M. Isolation and characterisation of bioactive compounds from Caesalpinia bonducella seeds. Phytotherapy Research 2004; 18(5):392-395.
4. Sembiring EN, Elya B, Sauriasari R. Phytochemical screening, total flavonoid and total phenolic content and antioxidant activity of different parts of Caesalpinia bonduc (L.) Roxb. Pharmacognosy Journal 2018; 10(1):123-127.
5. Evans WC: Trease and Evans' Pharmacognosy. WB Saunders, Edinburgh, Edition 14, 1996.
6. Kokate CK: Practical Pharmacognosy. Vallabh Prakashan, New Delhi, Edition 4, 1999.
7. Mossmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 1983; 65:55-63.
8. Kannur DM, Paranjpe MP, Sonavane LV, Dongre PP, Khandelwal KR. Evaluation of Caesalpinia bonduc seed coat extract for anti-inflammatory and analgesic activity. Journal of Advanced Pharmaceutical Technology and Research 2012; 3(3):171-175.
9. Wagner H, Bladt S, Zgainski EM: Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer-Verlag, Berlin, 1984.
10. Manzocco L, Anese M, Nicoli MC. Antioxidant properties of tea extracts as affected by processing. Lebensmittel-Wissenschaft und-Technologie 1998; 31(7-8):694-698