{"id":67994,"date":"2025-09-30T10:44:01","date_gmt":"2025-09-30T10:44:01","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=67994"},"modified":"2025-10-03T18:59:46","modified_gmt":"2025-10-03T18:59:46","slug":"in-vitro-antibacterial-efficacy-of-crude-leaf-extracts-from-catharanthus-roseus-a-natural-therapeutic-prospect","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol18no3\/in-vitro-antibacterial-efficacy-of-crude-leaf-extracts-from-catharanthus-roseus-a-natural-therapeutic-prospect\/","title":{"rendered":"In Vitro Antibacterial Efficacy of Crude Leaf Extracts from Catharanthus roseus: A Natural Therapeutic Prospect"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Antimicrobial resistance (AMR) has emerged as a major public health challenge. The rise of multidrug-resistant (MDR) bacteria is rendering many antimicrobial therapies ineffective, leading to prolonged hospitalizations and higher mortality rates.\u00b9\u02d2<\/sup>\u00b2 For centuries, herbs and medicinal plants have been utilized for their therapeutic properties. Compared to synthetic drugs, these natural remedies are often associated with fewer side effects and greater efficacy. Their diverse molecular structures and biological activities play a crucial role in the development of novel therapeutic agents.3<\/sup><\/p>\n

Apocynaceae family member Nayantara, scientifically known as C. roseus, is a perennial shrub of medicinal importance.<\/em> It is cultivated primarily for its valuable alkaloid compounds, which exhibit significant anticancer and antidiabetic activities.4-6<\/sup> Several studies have reported the antibacterial potential of crude extracts from different parts of C. roseus, including leaves, stems, roots, and flowers, against clinically significant single or multidrug-resistant bacteria.7,8<\/sup><\/em> The leaf extracts of C. roseus have been shown to be effective against several bacterial pathogens.9,10<\/sup><\/em> Additionally, leaf extracts are noted for their antihypertensive and antihyperlipidemic activity.11,12<\/sup> Furthermore, anti-Alzheimer\u2019s activity has also been shown by inhibiting the acetylcholine esterase enzyme and increasing cerebral blood flow by its constituent alkaloids, hence showing immense neuroprotective potential.13,14<\/sup> Even though these extracts appear to have potential health advantages, more study is required to completely comprehend their effects and determine the best ways to apply them in clinical settings.<\/p>\n

The entire plant is commonly utilized for alkaloid extraction, but it also comprises numerous other chemical constituents, such as tannins, phenolic compounds, steroids, and flavonoids, all of which possess therapeutic applications. Due to their polar nature, these compounds necessitate specific procedures and solvents for extraction, typically water or water-alcohol mixtures.<\/p>\n

The efficacy of plant extracts is primarily determined by the concentration and synergistic interactions of their bioactive compounds, both of which are significantly affected by the selection of extraction solvent, as it influences the chemical composition and overall biological activity of the resultant extract.15,16<\/sup> The current study aimed to investigate the phytochemical composition and antibacterial activity of\u00a0C. roseus<\/em> leaf extracts using three different solvents against\u00a0S. aureus, E. coli, S. typhi,<\/em> and P. aeruginosa<\/em>. This study also highlights the importance of using the appropriate solvent to ensure that the C. roseus <\/em>extract works effectively.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Processing and Extraction of Plant Leaves<\/strong><\/p>\n

Fresh leaves of C. roseus<\/em> were harvested, manually sorted, and left to wilt at room temperature for 15 days. After being cleaned with tap water, the leaves were rinsed with distilled water and allowed to dry. The dried leaves were then mechanically ground to obtain a fine powder. A total of 15 grams of powdered leaves was loaded into a Soxhlet apparatus with 300 mL of solvent. Extractions were performed separately using ethanol, methanol, and distilled water. The Soxhlet extraction was carried out at 65 \u00b0C for 24 hours.17<\/sup> It was noted that all three extracts had a dark green hue, sticky in consistency, and semi-solid in appearance.<\/p>\n

Preliminary Phytochemical Screening<\/strong><\/p>\n

Qualitative Detection of Alkaloids<\/strong><\/p>\n

A few milliliters of diluted HCl were added to 50 milligrams of the solvent-free extract, and the mixture was filtered. The resulting filtrate was then analyzed using a variety of alkaloidal reagents, including Mayer\u2019s, Wagner\u2019s, and Hager\u2019s tests for alkaloid detection.18,19<\/sup><\/p>\n

Test for Carbohydrates<\/strong><\/p>\n

Benedict’s qualitative test, Fehling’s test, and Molisch’s test were performed to detect carbohydrates.20<\/sup><\/p>\n

Test for Proteins and Amino Acids<\/strong><\/p>\n

A 100 milligrams of the extract was dissolved in 10 mL of distilled water and filtered using Whatman filter paper. The resulting filtrate was then subjected to protein and amino acid analysis, including the Ninhydrin test for amino acid detection.21<\/sup><\/p>\n

Test for Sterols and Triterpenoids <\/strong><\/p>\n

Sterols and triterpenoids were detected using the sulfur powder test and Salkowski’s assay.22<\/sup><\/p>\n

Test for Tannins and Phenol Compounds<\/strong><\/p>\n

The ferric chloride test was used to identify the presence of phenolic compounds.23<\/sup><\/p>\n

Antimicrobial Activity Test<\/strong><\/p>\n

Four bacterial cultures obtained from the Microbial Type Culture Collection (MTCC), each with distinct characteristics and identification numbers, were used in the study. E. coli<\/em> was designated as MTCC#43, S. aureus<\/em> as MTCC#87, P. aeruginosa<\/em> as MTCC#2488, and S. typhi<\/em> as MTCC#96. To evaluate antibacterial activity, a loopful of each culture was inoculated into sterile nutrient broth and incubated overnight at 37 \u00b0C.<\/p>\n

Preparation of Culture Media<\/strong><\/p>\n

For antimicrobial testing, 38 grams of Muller-Hinton agar (MHA) in 1000 mL was sterilized for 15\u201330 minutes at 121\u00b0C.<\/p>\n

Concentration of Extract<\/strong><\/p>\n

Stock extract concentrations were prepared in dilutions of 100 mg\/mL, 200 mg\/mL, and 300 mg\/mL in ethanol, methanol, and water.<\/p>\n

Use of Antibiotics<\/strong><\/p>\n

Penicillin (10 U), Tetracycline (10 \u00b5g), and Ampicillin (25 \u00b5g) were used to identify standard zones of inhibition. Antibiotic discs (3 mm) were placed on MHA agar and incubated at 37\u00b0C for 24 hours, and zones of inhibition were noted.<\/p>\n

Antimicrobial Activity via Well Diffusion Method<\/strong><\/p>\n

The agar well diffusion method was employed for the assay. Bacterial strains were cultured in nutrient broth for 24 hours, after which 100 \u03bcL of the cell suspension was spread onto Mueller\u2013Hinton Agar (MHA) plates. Wells with a diameter of 9 mm were filled with 200 \u03bcL of the respective extracts: 100 mg\/mL ethanol extract, 200 mg\/mL methanol extract, and 300 mg\/mL aqueous extract, which also served as controls. Following incubation at 37 \u00b0C for 24 hours, the plates were examined for zones of inhibition.22<\/sup><\/p>\n

Minimum Inhibitory Concentration and <\/strong>Minimum Bactericidal Concentration <\/strong><\/p>\n

The antibacterial activity of C. roseus<\/em> leaf extracts prepared in ethanol, methanol, and water against E. coli<\/em>, S. typhi<\/em>, P. aeruginosa<\/em>, and S. aureus<\/em> was evaluated using the broth dilution method. Except for the first well, each well of the microtiter plate was filled with 95 \u03bcL of Mueller\u2013Hinton Broth (MHB) and 5.0 \u03bcL of bacterial inoculum, with each column assigned to a different bacterium. A 100\u03bcL aliquot of the test extract was added to the first and second wells, followed by serial dilution from the second to the eleventh well, using the first and twelfth wells as controls. After incubation at 37 \u00b0C for 16\u201318 hours, 20 \u03bcL of resazurin dye was added to each well and incubated for an additional 2\u20133 hours to assess bacterial growth. For MBC determination, 50 \u03bcL from each MIC well was inoculated onto Mueller\u2013Hinton Agar (MHA) plates using the lawn culture method and incubated at 37 \u00b0C for 24 hours. The lowest concentration showing no visible growth was recorded as the MBC.24-26<\/sup>\u00a0<\/strong><\/p>\n

Results<\/strong><\/p>\n

Phytochemical Screening of C. roseus<\/em> Leaf Extracts<\/strong><\/p>\n

The phytochemical profiles of the ethanol, methanol, and aqueous extracts of C. roseus<\/em> leaves are presented in Table 1. Qualitative tests were performed to detect carbohydrates, proteins, amino acids, alkaloids, glycosides, saponins, tannins, phenolic compounds, and steroids. The presence (+) or absence (\u2013) of each compound was recorded for each extract type.<\/p>\n

Table 1: Phytochemical analysis of ethanol, methanol, and aqueous extracts of C. roseus<\/em> leaves.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Name of test<\/strong><\/td>\nEthanol<\/strong><\/td>\nMethanol<\/strong><\/td>\nAqueous<\/strong><\/td>\n<\/tr>\n
1. Alkaloids<\/em>a)\u00a0\u00a0\u00a0\u00a0 Mayer\u2019s test<\/p>\n

b)\u00a0\u00a0\u00a0\u00a0 Wagner\u2019s test<\/p>\n

c)\u00a0\u00a0\u00a0\u00a0 Hager\u2019s test<\/td>\n

\u00a0<\/strong>a)\u00a0 \u2013ve<\/p>\n

b)\u00a0 \u2013ve<\/p>\n

c)\u00a0 +ve<\/td>\n

a)\u00a0 \u2013ve<\/p>\n

b)\u00a0 \u2013ve<\/p>\n

c)\u00a0 +ve<\/td>\n

\u00a0<\/strong>a)\u00a0 \u2013ve<\/p>\n

b)\u00a0 \u2013ve<\/p>\n

c)\u00a0 +ve<\/td>\n<\/tr>\n

2. Carbohydrates and glycosides<\/em>a)\u00a0\u00a0\u00a0\u00a0 Fehling\u2019s test<\/p>\n

b)\u00a0\u00a0\u00a0\u00a0 Molisch\u2019s test<\/p>\n

c)\u00a0\u00a0\u00a0\u00a0 Benedict’s test<\/td>\n

\u00a0<\/strong>a)\u00a0\u00a0 -ve<\/p>\n

b)\u00a0 +ve<\/p>\n

c)\u00a0\u00a0 -ve<\/td>\n

\u00a0<\/strong>a)\u00a0 -ve<\/p>\n

b)\u00a0 +ve<\/p>\n

c)\u00a0 +ve<\/td>\n

\u00a0<\/strong>a)\u00a0\u00a0 -ve<\/p>\n

b)\u00a0 +ve<\/p>\n

c)\u00a0\u00a0 -ve<\/td>\n<\/tr>\n

3. Proteins and amino acids<\/em>Ninhydrin test<\/td>\n+ve<\/td>\n+ve<\/td>\n+ve<\/td>\n<\/tr>\n
4. Saponins<\/em>Foam test<\/td>\n\u2013ve<\/td>\n\u2013ve<\/td>\n\u2013ve<\/td>\n<\/tr>\n
5. Tannins & Phenol compounds<\/em>Ferric chloride test<\/td>\n\u00a0<\/strong>+ve<\/p>\n

\u00a0<\/strong><\/td>\n

\u00a0<\/strong>\u2013ve<\/p>\n

\u00a0<\/strong><\/td>\n

\u00a0<\/strong>\u2013ve<\/p>\n

\u00a0<\/strong><\/td>\n<\/tr>\n

6. Steroids<\/em>a)\u00a0\u00a0\u00a0\u00a0 Salkowasky\u2019s test<\/p>\n

b)\u00a0\u00a0\u00a0\u00a0 Sulphur powder test<\/td>\n

a)\u00a0\u00a0\u00a0\u00a0 +veb)\u00a0\u00a0\u00a0\u00a0 +ve<\/td>\na)\u00a0\u00a0\u00a0\u00a0 +veb)\u00a0\u00a0\u00a0\u00a0 +ve<\/td>\na)\u00a0 +veb)\u00a0 +ve<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Screening of C. roseus<\/em> leaf extracts for antibacterial activity<\/strong><\/p>\n

Each extract’s zone of inhibition (diameter in millimeters) was assessed and contrasted with that of common antibiotics such as ampicillin, tetracycline, and penicillin. The maximum inhibitory zone was observed with a concentration of 300 mg\/mL of ethanolic extract against E. coli<\/em> (23\u00b11.02 mm) and S. typhi<\/em> (21\u00b11 mm). Similarly, the maximum inhibitory zone was observed against P. aeruginosa<\/em> (22.3\u00b11.52 mm) with the concentration of 300 mg\/mL of aqueous extract. In the case of S. aureus<\/em>, the maximum inhibitory zone was recorded (20.3\u00b11.5 mm) with the concentration of 300 mg\/mL of methanolic extract.\u00a0 The details of antibacterial activities of ethanol, methanol, and aqueous extracts against E. coli, P. aeruginosa, S. aureus<\/em>, and S. typhi<\/em> are given in Table 2 and Figure 1.<\/p>\n

Screening of C. roseus<\/em> Leaf Extracts for Antibacterial Activity<\/strong><\/p>\n

The antibacterial activity of each extract was evaluated by measuring the zone of inhibition (diameter in millimeters) and comparing it with standard antibiotics such as ampicillin, tetracycline, and penicillin. The largest inhibitory zones were observed with 300 mg\/mL concentrations of the extracts: ethanolic extract showed maximum inhibition against E. coli<\/em> (23 \u00b1 1.02 mm) and S. typhi<\/em> (21 \u00b1 1 mm), the aqueous extract was most effective against P. aeruginosa<\/em> (22.3 \u00b1 1.52 mm), and the methanolic extract exhibited the highest inhibition against S. aureus<\/em> (20.3 \u00b1 1.5 mm). Detailed antibacterial activity data for ethanol, methanol, and aqueous extracts against E. coli<\/em>, P. aeruginosa<\/em>, S. aureus<\/em>, and S. typhi<\/em> are presented in Table 2 and Figure 1.<\/p>\n\n\n\n
\"\"<\/td>\nTable 2:\u00a0 Antibacterial activity of aqueous, ethanol, and methanolic extracts of C. roseus<\/em> leaf.<\/strong>Click here to view Table<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

a<\/sup>P – Penicillin, <\/span>b<\/sup>TE – Tetracycline, <\/span>c<\/sup>AMP \u2013 Ampicillin<\/span><\/p>\n\n\n\n
\"\"<\/td>\nFigure 1: Antibacterial activity of C. roseus<\/em> leaf extracts (aqueous, ethanol, and methanol) against various bacterial pathogens, with standard antibiotic discs as positive controls.<\/strong>Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

In this study, following the initial assessment of antibacterial activity using the well diffusion method, the MIC and MBC of C. roseus<\/em> leaf extracts were determined for the four bacterial strains via the broth dilution method (Table 3). For S. aureus<\/em> and E. coli<\/em>, the ethanolic extract exhibited MBCs of 50 and 55 mg\/mL, respectively, while the MIC for both was 25 mg\/mL. The same extract showed MICs of 45 and 55 mg\/mL and MBCs of 65 and 75 mg\/mL against P. aeruginosa<\/em> and S. typhi<\/em>, respectively. The methanolic extract demonstrated MICs and MBCs of 40 and 60 mg\/mL for P. aeruginosa<\/em>, 20 and 45 mg\/mL for S. typhi<\/em>, and 50 and 75 mg\/mL for E. coli<\/em> and S. aureus<\/em>. The aqueous extract showed an MIC of 20 mg\/mL and an MBC of 45 mg\/mL against E. coli<\/em>, P. aeruginosa<\/em>, and S. typhi<\/em>, while for S. aureus<\/em>, the MIC and MBC were 40 mg\/mL and 65 mg\/mL, respectively (Figure 2). In all cases, the MBC values were higher than the MIC values, indicating that a higher concentration of extract is required to completely kill the bacteria compared to the amount needed to inhibit their growth.<\/p>\n

Table 3:\u00a0MIC and MBC of C. roseus<\/em> leaf extracts prepared in ethanol, methanol, and aqueous solvents against various bacterial pathogens.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
Plant extract<\/strong><\/td>\nBacterial pathogens<\/strong><\/td>\n<\/tr>\n
S. aureus<\/em><\/strong><\/td>\nE. coli<\/em><\/strong><\/td>\nP. aeruginosa<\/em><\/strong><\/td>\nS. typhi<\/em><\/strong><\/td>\n<\/tr>\n
MIC<\/strong>(mg\/mL)<\/strong><\/td>\nMBC<\/strong>(mg\/mL)<\/strong><\/td>\nMIC<\/strong>(mg\/mL)<\/strong><\/td>\nMBC<\/strong>(mg\/mL)<\/strong><\/td>\nMIC<\/strong>(mg\/mL)<\/strong><\/td>\nMBC<\/strong>(mg\/mL)<\/strong><\/td>\nMIC<\/strong>(mg\/mL)<\/strong><\/td>\nMBC<\/strong>(mg\/mL)<\/strong><\/td>\n<\/tr>\n
Aqueous extract<\/td>\n40<\/td>\n65<\/td>\n20<\/td>\n45<\/td>\n20<\/td>\n45<\/td>\n20<\/td>\n45<\/td>\n<\/tr>\n
Ethanolic extract<\/td>\n50<\/td>\n75<\/td>\n50<\/td>\n75<\/td>\n40<\/td>\n60<\/td>\n20<\/td>\n45<\/td>\n<\/tr>\n
Methanolic extract<\/td>\n25<\/td>\n55<\/td>\n25<\/td>\n50<\/td>\n45<\/td>\n65<\/td>\n55<\/td>\n75<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 2: Radar plot showing the MIC and MBC of C. roseus<\/em> leaf extracts against different bacterial pathogens.<\/strong>Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Discussion<\/strong><\/p>\n

The current study makes a substantial addition to the fields of phytotherapeutics and natural product science by advancing our knowledge of the antibacterial properties of C. roseus<\/em> leaf extracts. Examining different extracts (ethanol, methanol, and aqueous) and their efficacy against the nosocomial pathogens like E. coli, S. typhi<\/em>, P. aeruginosa, and S. aureus<\/em>. The thorough chemical analysis of the C. roseus<\/em> leaf extracts is a crucial component of this study. The diverse composition of these extracts is evidenced by the presence of a wide range of bioactive constituents, including alkaloids, phenols, glycosides, flavonoids, terpenoids, steroids, carbohydrates, proteins, and amino acids. These results highlight the possible therapeutic use of C. roseus<\/em> extracts and are in line with other studies of a similar nature.14,27,28<\/sup><\/p>\n

The study demonstrates that the ethanolic extract of C. roseus<\/em> leaves exhibits strong antibacterial activity, as evidenced by the largest zones of inhibition, which reflect its antimicrobial potency. This significant finding is in line with previous research findings and supports the potential of C. roseus<\/em> leaf extracts as strong substitutes for traditional antibiotics.17,29,30 <\/sup>The bacteria P. aeruginosa, S. typhi, <\/em>and E. coli <\/em>are Gram-negative, while S. aureus<\/em> has a Gram-positive cell wall structure and is naturally sensitive to the active ingredients of the extract. There are variances in how different bacteria react to C. roseus<\/em> leaf extracts. The main method these extracts fight bacteria is by breaking down the structure of their cell walls, including peptidoglycan and other cell wall components, which prevents the bacterial cells’ vital enzymatic functions.9,10,31 <\/sup>Polyphenols, a class of chemicals renowned for their capacity to bind with and destroy important bacterial components such as enzymes, proteins, and adhesins involved in cell transport, are primarily responsible for this impact. Additionally, by interfering with bacterial DNA, these polyphenols can prevent bacterial growth and multiplication.32<\/sup> Furthermore, it has been discovered that the extraction solvents such as methanol and ethanol, have an impact on the efficacy.33,34 <\/sup>These solvents are more effective than water at dissolving the active ingredients, which emphasizes how crucial the extraction technique is in establishing the extracts’ efficacy. This highlights how important it is to select the right solvent when removing different advantageous compounds from plant materials and figuring out how they work against bacteria. Ethanol extracts may be more effective than water at solubilizing these bioactive chemicals, which could explain their enhanced potency in this study. Given that it affects the kinds and amounts of phytochemicals that are extracted from any plant material, it is clear that the extraction solvent can also have a significant impact on the mechanism of action.35,36<\/sup> This study is notable for its comprehensive analysis of C. roseus<\/em> leaf extracts’ antibacterial properties.<\/p>\n

These findings underscore the considerable therapeutic potential of the extracts, suggesting their use as natural agents against nosocomial infections, particularly in chronic wounds and bedsores in long-term hospitalized patients. The results provide a foundation for the development of plant-based antimicrobial agents and support further research into the underlying mechanisms and clinical efficacy of these extracts.<\/p>\n

Stdy Limitations<\/strong><\/p>\n

In this study, C. roseus<\/em> leaves were used; other parts of the plant, such as the shoot, root, and stem, were not utilized. To analyze the phytochemical components and their structure, and identity of the molecules, the following techniques, such as High-Performance Liquid Chromatography (HPLC), High-Performance Thin-Layer Chromatography (HPTLC), and Nuclear Magnetic Resonance (NMR) spectroscopy, were not used in this study. The molecular level of structural interactions of the leaf extracts with various cellular components of the bacterial pathogens has not been studied. The other drug-resistant nosocomial pathogens, Enterobacter aerogenes, Streptococcus pyogenes, Klebsiella pneumoniae, <\/em>and Acinetobacter baumannii,<\/em> are not used in this study to determine the antimicrobial activity of different solvent extractions of the plant products.\u00a0 These important limitations should be addressed in future studies.<\/p>\n

Conclusion<\/strong><\/p>\n

This study highlights the significant potential of C. roseus<\/em> leaf extracts as a sustainable source of bioactive compounds with potent antibacterial properties against common nosocomial pathogens. The presence of diverse phytochemicals such as alkaloids, flavonoids, steroids, tannins, cardiac glycosides, and terpenoids highlights the plant\u2019s rich chemical profile and the pivotal role of solvent selection in extracting targeted compounds. By transforming an often-overlooked plant into a valuable natural resource, this research aligns with the Sustainable Development Goals (SDGs), particularly Goal 3 (Good Health and Well-being) and Goal 12 (Responsible Consumption and Production), by promoting both public health and resource efficiency. These findings open doors to customized extraction methods and the exploration of other parts of C. roseus<\/em> for broader health applications, offering a sustainable pathway for drug discovery that harnesses natural resources to promote both global health and environmental well-being.<\/p>\n

Acknowledgement<\/strong><\/p>\n

We thank the staff and faculty of Dolphin Institute of Biomedical and Natural Sciences, Dehradun, India, for their continuous support and encouragement. <\/em><\/p>\n

Funding source<\/strong><\/p>\n

The author(s) received no financial support for the research, authorship, and\/or publication of this article.<\/p>\n

Conflict of interest<\/strong><\/p>\n

The author(s) do not have any conflict of interest<\/p>\n

Data Availability<\/strong><\/p>\n

This statement does not apply to this article.<\/p>\n

Ethics Statement<\/strong><\/p>\n

This research did not involve human participants, animal subjects, or any material that requires ethical approval.<\/p>\n

Informed Consent Statement<\/strong><\/p>\n

This study did not involve human participants, and therefore, informed consent was not required.<\/p>\n

Clinical Trial Registration<\/strong><\/p>\n

This research does not involve any clinical trials.<\/p>\n

Permission to reproduce material from other sources<\/strong><\/p>\n

Not applicable<\/p>\n

Authors\u2019 Contribution<\/strong><\/p>\n